Research ArticleSynthesis Radical-Scavenging Activities and ProtectiveEffects against AAPH-Induced Oxidative Damage in DNA andErythrocytes of Piperine Derivatives

Bei Qin 12 Kuan Yang23 and Ruijun Cao1

1School of Science Xirsquoan Jiaotong University Xirsquoan 710049 Shaanxi China2Department of Pharmacy Xirsquoan Medical University Xirsquoan 710021 Shaanxi China3College of Bioresources Chemical and Materials Engineering Shaanxi University of Science and Technology Xirsquoan 710021Shaanxi China

Correspondence should be addressed to Bei Qin qinbei0526163com

Received 29 December 2018 Revised 2 April 2019 Accepted 17 June 2019 Published 24 March 2020

Academic Editor Silvia Persichilli

Copyright copy 2020 Bei Qin et al-is is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Piperine amino acid derivatives containing phenolic hydroxyl groups were synthesized using piperine as the raw material byamide hydrolysis amidation ester hydrolysis and deacetalization -e obtained products were characterized by mass spec-trometry and nuclear magnetic resonance -e antioxidant activity of the piperine derivatives was evaluated by the DPPH andABTS scavenging rates and the total antioxidant capacity -e results showed that the piperine amino acid (4andash4d) had relativelyweak radical-scavenging ability while the piperine amino acid derivatives (5andash5d) containing phenolic hydroxyl groups hadsignificant radical-scavenging effects In addition the total reducing ability of 5andash5d was better than that of piperine -e studyalso found that piperine derivatives containing phenolic hydroxyl groups played an important role in inhibiting oxidative damagein DNA and erythrocytes

1 Introduction

Free radicals are the products of normal metabolism in thebody Production of excess free radicals induces immunedysfunction which causes lipid peroxidation damage in cellmembranes enzyme deactivation and oxidative DNAdamage leading to many oxidation-related diseases [1 2]Consequently there has been an increase in research activityfocusing on the synthesis and development of free radicalscavengers [2ndash4]

Many studies on the synthesis of antioxidants are based onthe structural modification of plant-derived substances in-cluding piperine an alkaloid derived from plants in thePiperaceae family [5ndash7] which has received an increasingamount of attention due to its excellent biological and phar-macological activity [6 8 9] Researchers have demonstratedthat the antioxidative activity of piperine is manifested throughfree radical-quenching effects and a reduction in GSH

consumption [10] -erefore piperine has been widely appliedin research on oxidation-related diseases [10ndash14] For instanceKhajuria et al found that piperine can inhibit carcinogen-induced oxidative damage in the intestinal mucosa increaseGSH levels and restore ATPase activity [15] Vijayakumar et alhave shown that piperine exerts significant antioxidative effectson the erythrocytes of high-fat diet- and antithyroid drug-induced hyperlipidemic rats [13] Other researchers have re-ported that piperine-amino conjugates exhibit enhanced bio-logical activity when comparedwith piperine [16] For instanceKoichi et al have reported the use of piperic acid amides as freeradical scavengers and α-glucosidase inhibitors [17] whileInder Pal et al reported the antileishmanial activity of piperoyl-amino acid conjugates [16] Furthermore studies have indi-cated the antioxidative performance of certain amino acids offood origin is due to characteristics such as the electron-do-nating and hydrogen bond-donating properties as well asmetal ion chelation and hydrophobic properties [18ndash20] On

HindawiJournal of ChemistryVolume 2020 Article ID 9026286 12 pageshttpsdoiorg10115520209026286

these grounds the present study has proposed a novel researchapproach based on the excellent biological activity of pipericacid amides and the possible antioxidative performance ofamino acids which involves the derivatization of piperineusing amino acids followed by the removal of the acetal moietyin the parent structure of piperine to form phenolic hydroxylgroups thereby obtaining phenolic hydroxyl-containing pip-erine-amino acid derivatives

-e most common in vitro experimental methods used forthe assessment of antioxidant activity can be classified aschemical methods simulated biological system methods andcell investigation methods Chemical methods (such as theDPPH assay [21] ABTS assay [22] and total reductive abilitytest) mainly assess the free radical-capturing ability and re-ductive ability of antioxidants Since such methods are easy tooperate and provide rapid accurate measurements they aremost commonly used in antioxidant prescreening studiesSimulated biological system methods assess the antioxidantperformance by measuring the effects of antioxidants on freeradical-induced oxidative damage in biomacromolecules (egAAPH- Cu2+GSH- and OHbull-induced oxidative DNAdamage) [23 24] Cell investigation methods provide an as-sessment of the antioxidant performance by evaluating theeffects of antioxidants on free radical-induced oxidativedamage and changes in the activity of antioxidant enzymes[20 25] To perform a comprehensive assessment of the an-tioxidant performance of our piperine-amino acid derivativeswe selected the most conventional methods used among thesethree categories namely the DPPH assay ABTS assay totalreductive ability test and AAPH-induced oxidative DNA anderythrocyte damage model

2 Materials and Methods

21 Materials Piperine (98) was purchased from ShaanxiSciphar Natural Products Co Ltd (China) Oxalyl chloride(95) boron tribromide (BBr3) (99) (2-methyl-propionamide)-dihydrochloride (AAPH) (98) 22prime-azi-nobis(3-ethylbenzothiazoline-6-sulfonic acid) diammoniumsalt (ABTS+) (98) potassium ferricyanide (K3[Fe(CN)6])(98) trichloroacetic acid (TCA) (98) L-alanine methylester hydrochloride (98) tertiary butylhydroquinone(TBHQ) (98) L-valine methyl ester hydrochloride (98)L-leucinate methyl ester hydrochloride (98) L-methioninemethyl ester hydrochloride (98) 46-dihydroxy-2-mer-captopyrimidine (TBA) (98) and ascorbic acid (VC)(98) were obtained from Energy Chemical (China) DNAfrom fish sperm pure was purchased from JampK Scientific(China) Ferric chloride (FeCl3) (98) was obtained fromTianjin Beilian Fine Chemicals Development Co Ltd

22 Synthesis of Piperine Derivatives -e synthesis of pip-erine derivatives 4a-4b and 5a-5b containing differentamino acids is shown in Figure 1

221 (2E4E)-5-(Benzo[d][13]dioxol-5-yl)penta-24-dien-oicAcid (1 C12H10O4) (2E4E)-5-(Benzo[d][13]dioxol-5-yl)penta-24-dien-oic acid (1) was prepared by the improved

alkaline hydrolysis method according to the method re-ported by Inder Pal et al [16] -e method reported in theliterature is to directly acidify with hydrochloric acid [16]and the consumption of hydrochloric acid is large In thisexperiment the potassium salt of the compound was sep-arated from the system firstly followed by the pH adjust-ment treatment and the recrystallization operation isomitted-e experimental yield has reached more than 90In brief piperine (1069 g 3751mmol) was dispersed in aKOH methanol solution (mass fraction 20 300mL) andrefluxed at 75degC for 24 h -e suspension was then cooledand a white solid was obtained after suction filtration -esolid was dispersed in a small amount of methanol -e pHwas adjusted to 1 using 6molL hydrochloric acid -esuspension was then filtered through suction and dried toafford compound 2 as a yellow solid (761 g 931) 1HNMR(400MHz DMSO-d6) δ 1221 (s 1H -COOH) 733-724 (m1H CHCH-CO) 725-724 (d 1H Ar-H) 703-679 (m3H Ar-H Ar-CHCH-) 694-692 (d 1H Ar-CHCH-)606 (s 2H -OCH2O-) 595-691 (d 1H CHCH-CO) 13CNMR (101MHz DMSO-d6) δ 16803 14856 14845 1450114021 13100 12533 12351 12163 10897 10620 10182

222 (2E4E)-5-(Benzo[d][13]dioxol-5-yl)penta-24-dien-oylChloride (2 C12H9ClO3) Inder Pal et al [16] prepared thepiperine amino acid methyl ester conjugate from the in-termediate product piperonic acid mesylate with a yield of40ndash75 In this experiment the corresponding acid chloridewas prepared by the reaction of compound 1 with oxalylchloride and the esterification reaction was carried out -eyield of the reaction was over 79 Compound 2 (691 g317mmol) was dispersed in 15mL of anhydrousdichloromethane (DCM) Oxalyl chloride DCM solution(12molL 270mL) was then added and the mixture wasstirred at room temperature for 2 h to afford an orangeliquid -e oxalyl chloride and DCMwere distilled off underreduced pressure to afford an orange acid chloride whichhad to be freshly prepared

223 Piperine L-Alanine Methyl Ester Conjugate (3aC16H17NO5) -e above-prepared acid chloride (2) wasdissolved in 10mL of anhydrous DCM and TEA (40mmol)was added with stirring at room temperature L-Alaninemethyl ester hydrochloride (441 g 317mmol) was dissolvedin 20mL of DCM and was then slowly added into the re-action system using a dropping funnel After 4 h of reactionat room temperature the product was washed with 30mL of5 NaHCO3 solution 30mL of 5 HCl solution and 30mLof water -e concentrated crude product was then purifiedby column chromatography on silica gel with a mixture ofEA-PE to afford 806 g of the product (3a) (yield 880) as apale yellow solid 1H NMR (400MHz CDCl3) δ 742-736(m 1H CHCH-CO) 701-700 (d 1H Ar-CH) 693-691(dd 1H Ar-H) 682-679 (m 2H Ar-H Ar-CHCH-)674-667 (m 1H Ar-H) 615-613 (d 1H CHCH-CO)600 (s 2H -OCH2O-) 478-471 (m 1H CONHCH) 380(s 3H -OCH3) 149-147 (d 3H -CH3) 13C NMR(101MHz CDCl3) δ 17321 16499 14782 14772 14128

2 Journal of Chemistry

13886 13027 12401 12220 12189 10800 10528 100835203 4761 1819 ESI mz 3041 [M+ 1]+

224 Piperine L-Valine Methyl Ester Conjugate (3bC18H21NO5) -e synthesis process of compound 3b wassimilar to that of compound 3a -e amount of L-valinemethyl ester hydrochloride used was 530 g (317mmol)-eproduct was a pale yellow solid (812 g 803) 1H NMR(400MHz CDCl3) δ 743-737 (m 1H CHCH-CO) 701(d 1H Ar-CH) 693-668 (m 4H Ar-H Ar-CHCH-)603 (s 1H CHCH-CO) 600 (s 2H -OCH2O-) 474-470(m 1H CONHCH) 378 (s 3H -OCH3) 225-220 (m 1HCH (CH3)2) 100-095 (m 6H CH (CH3)2)13CNMR(101MHz CDCl3) δ 17222 16541 14782 14772 1413613886 13028 12400 12222 12199 10801 10527 100835656 5171 3109 1846 1741 ESI mz 3321 [M+ 1]+

225 Piperine L-Leucinate Methyl Ester Conjugate (3cC19H23NO5) -e synthesis process of compound 3c wassimilar to that of compound 3a -e amount of L-leucinatemethyl ester hydrochloride used was 574 g (317mmol)-eproduct was a pale yellow solid (835 g 795) 1H NMR(400MHz CDCl3) δ 741ndash735 (m 1H CHCH-CO) 698-697 (d 1H Ar-CH) 690-665 (m 4H Ar-H Ar-CHCH-)616-614 (d 1H CHCH-CO) 599 (s 2H -OCH2O-) 482-476 (m 1H CONHCH) 377 (s 3H -OCH3) 173-1170 (m2H CH2CH (CH3)2) 162-158 (m 1H CH (CH3)2) 099-096 (m 6H CH (CH3)2)13C NMR (101MHz CDCl3) δ17387 16587 14829 14819 14187 13933 13078 1245312269 12239 10847 10578 10132 5234 5077 41822490 2284 2198 ESI mz 3461 [M+1]+

226 Piperine L-Methionine Methyl Ester Conjugate (3dC18H21NO5S) -e synthesis process of compound 3d wassimilar to that of compound 3a -e amount of L-methioninemethyl ester hydrochloride used was 514 g (317mmol) -eproduct was a pale yellow solid (853 g 856) 1H NMR(400MHz CDCl3) 743-736 (m 1H CHCH-CO) 700 (d1H Ar-CH) 693-667 (m 4H Ar-H Ar-CHCH-) 632-630 (d 1H CHCH-CO) 600 (s 2H -OCH2O-) 489-484

(m 1H CONHCH) 380 (s 3H -OCH3) 255-254 (m 2HSCH2) 206-205 (m 5H CHCH2 SCH3) 13C NMR(101MHz CDCl3) δ 17264 16582 14837 14823 1420513957 13073 12445 12276 12219 10851 10579 101325260 5163 3187 3000 1551

227 Piperine L-Alanine Conjugate (4a C15H15NO5)Compounds 4andash4d were synthesized by improving themethod reported by Inder Pal et al [16] -e experiment wascarried out by heating hydrolysis with some water in the solidmedium -e yield of compounds 4andash4d reached 71ndash86

Compound 3a (199 g 657mmol) and 974 g of KF-Al2O3 were thoroughly ground and mixed together -emixture was then transferred to a 100mL round-bottomedflask and 3mL of water was added After heating andstirring at 120degC for 1 h 10mL of water was added andstirred for another 10min before suction filtration -efiltrate pH was adjusted to 1 with hydrochloric acid whilestirring at room temperature to precipitate solids -e crudeproduct obtained after suction filtration was then purified bycolumn chromatography on silica gel (developing agentDCM ∶MeOH ∶HAc 60 ∶ 3 ∶ 01) to afford 142 g of theproduct (4a) (yield 751) as a pale yellow solid 1H NMR(400MHz DMSO-d6) δ 1254 (s 1H -COOH) 838-836 (d1H CHNHCO) 728-727 (d 1H CHCH-CO) 717-713(m 1H Ar-CH) 701-689 (m 4H Ar-H Ar-CHCH-)616-613 (d 1H CHCH-CO) 604 (s 2H -OCH2O-) 431-427 (m 1H CONHCH) 131-129 (d 3H -CH3) 13C NMR(101MHz DMSO-d6) δ 17424 16491 14789 1477113988 13818 13077 12512 12388 12273 10840 1056210123 4753 1721 ESI mz 2901 [M+ 1]+

228 Piperine L-Valine Conjugate (4b C17H19NO5) -esynthesis process of compound 4b was similar to that ofcompound 4a -e product was a pale yellow solid (yield789) 1H NMR (400MHz DMSO-d6) δ 1251 (s 1H-COOH) 826-820 (d 1H CHNHCO) 729 (d 1HCHCH-CO) 720-714 (m 1H Ar-CH) 702-690 (m4H Ar-H Ar-CHCH-) 631-628 (d 1H CHCH-CO)minus605 (s 2H -OCH2O-) 427-423 (m 1H CONHCH)211-204 (m 1H CH (CH3)2) 091-089 (m 6H CH

O

O

O

O

O

O

O

O

O OO

ONH

N

Piperine

20KOH in methanol

KF-Al2O3 (40)70 ~ 120degC 3hR

75degC 24h1 2

3andash3d

3a 4a 5a R = CH33b 4b 5b R = CH(CH3)23c 4c 5c R = CH2CH(CH3)23d 4d 5d R = CH2CH2SCH3

4andash4d5andash5d

OO

OOH

OHHO

HO

BBr3

OO

R

(COCI)2

OO

O O OH

R

ONH2 middot HCI

R

CI

NH

NH

Figure 1 Scheme for the preparation of piperine derivatives KOH potassium hydroxide (COCl)2 oxalyl chloride KF potassium fluorideAl2O3 aluminium oxide BBr3 boron tribromide

Journal of Chemistry 3

(CH3)2) 13C NMR (101MHz DMSO-d6) δ 17310 1653914789 14770 13985 13812 13078 12513 12407 1226910840 10564 10123 5729 2983 1917 1810 ESI mz3181 [M+ 1]+

229 Piperine L-Leucinate Conjugate (4c C18H21NO5)-e synthesis process of compound 4c was similar to that ofcompound 4a -e product was a white solid (yield 857)1H NMR (400MHz DMSO-d6) δ 1253 (s 1H -COOH)831-829 (d 1H CHNHCO) 728 (d 1H CHCH-CO)720-714 (m 1H Ar-CH) 702-686 (m 4H Ar-H Ar-CHCH-) 619-615 (d 1H CHCH-CO) 605 (s 2H-OCH2O-) 435-430 (m 1H CONHCH) 167-161 (m 1HCH (CH3)2) 158-153 (m 2H CH2CH (CH3)2) 092-086(dd 6H CH (CH3)2) 13C NMR (101MHz DMSO-d6) δ17417 16518 14789 14771 13986 13813 13078 1251312394 12268 10840 10564 10122 5029 4000 24372282 2127 ESI mz 3321[M+ 1]+

2210 Piperine L-Methionine Conjugate (4d C17H19NO5S)-e synthesis process of compound 4d was similar to that ofcompound 4a -e product was a pale yellow solid (yield713) 1H NMR (400MHz DMSO-d6) δ 1271 (s 1H-COOH) 837-835 (d 1H CHNHCO) 729-728 (d 1HCHCH-CO) 721-715 (m 1H Ar-CH) 702-686 (m 4HAr-H Ar-CHCH-) 618-615 (d 1H CHCH-CO) 605 (s2H -OCH2O-) 444-443 (m 1H CONHCH) 206-186 (m5H SCH2 SCH3) 13C NMR (101MHz DMSO-d6) δ 1739116582 14839 14823 14049 13875 13127 12561 1243212322 10891 10613 10174 5154 3123 3022 1503 ESImz 3501[M+ 1]+

2211 ((2E4E)-5-(34-Dihydroxyphenyl)penta-24-dienoyl)Alanine (5a C14H15NO5) Compound 4a (200mg069mmol) was dispersed in 10mL of anhydrous DCM ADCM (5mL) solution of boron tribromide (2609 μL276mmol) was added dropwise to the reaction mixture inan ice-bath condition After 4 h the reaction was quenchedby adding 10mL of ice-cold saturated ammonium chloridesolution in an ice-bath condition After stirring at roomtemperature for 05 h ethyl acetate (3times15mL) was used forthe extraction -e organic phase was dried with anhydroussodium sulfate for 24 h and then filtered to obtain theconcentrated crude product -e crude product was thenpurified by column chromatography on silica gel (devel-oping agent DCM MeOH HAc 75 1 01) to afford3652mg of the product (5a) (yield 191) as an orangesolid 1H NMR (400MHz DMSO-d6) 983 (s 2H -OH)805-803 (d 1H CHNHCO) 717-711 (m 1H CHCH-CO) 695 (s 1H Ar-CH) 684-6671 (m 4H Ar-H Ar-CHCH-) 618-614 (d 1H CHCH-CO) 430-427 (m1H CONHCH) 128-126 (d 3H -CH3) ESI mz 27819[M+ 1]+

2212 ((2E4E)-5-(34-Dihydroxyphenyl)penta-24-dienoyl)Valine (5b C16H19NO5) -e synthesis process of com-pound 5b was similar to that of compound 5a -e product

was a dark orange solid (yield 123) 1H NMR (400MHzDMSO-d6) δ 976 (s 2H -OH) 791 (s 1H CHNHCO)717-711 (m 1H CHCH-CO) 696 (s 1H Ar-CH) 684-674 (m 4H Ar-H Ar-CHCH-) 629-626 (d 1HCHCH-CO) 422-419 (m 1H CONHCH) 210 (s 1HCH (CH3)2) 088-081 (m 6H CH (CH3)2) ESI mz 3201[M+ 1]+

2213 ((2E4E)-5-(34-Dihydroxyphenyl)penta-24-dienoyl)Leucine (5c C17H21NO5) -e synthesis process of com-pound 5c was similar to that of compound 5a -e productwas a dark orange solid (yield 293) 1H NMR (400MHzDMSO-d6) δ 931 (s 2H -OH) 823-821 (d 1HCHNHCO) 719-712 (m 1H CHCH-CO) 695 (d 1HAr-CH) 686-671 (m 4H Ar-H Ar-CHCH-) 614-610(d 1H CHCH-CO) 432-430 (m 1H CONHCH) 165-160 (m 1H CH (CH3)2) 156-152 (m 2H CH2CH(CH3)2) 091-085 (dd 6H CH (CH3)2) ESI mz 3202[M+ 1]+

2214 ((2E4E)-5-(34-Dihydroxyphenyl)penta-24-dienoyl)Methionine (5d C16H19NO5S) -e synthesis process ofcompound 5d was similar to that of compound 5a -eproduct was a dark green solid (yield 204) 1H NMR(400MHz DMSO-d6) δ 1269 (s 1H -COOH) 931 (s 1H-OH) 902 (s 1H -OH) 831-826 (m 1H CHNHCO) 720-714 (m 1H CHCH-CO) 695 (m 1H Ar-CH) 787-672(m 4H Ar-H Ar-CHCH-) 614-610 (d 1H CHCH-CO) 443-437 (m 1H CONHCH) 207-202 (m 5H SCH2SCH3) ESI mz 3382[M+ 1]+

23 ABTS Radical-Scavenging Properties -e ABTS radical-scavenging activities were assayed according to the methodreported by-aipong et al [22] (1) ABTS+ aqueous solution(7mmolL) preparation 30mg ABTS+ was weighed 8mL ofultrapure water was added and it was dissolved by ultra-sonication (2) K2S2O8 solution preparation 10mg K2S2O8was weighed 15mL of ultrapure water was added and it wasdissolved by ultrasonication (3) ABTS working solutionpreparation (1) and (2) were mixed at the ratio of 1 1 andkept in the dark for 12 to 16 h-e abovemixture was dilutedfour to eight times with 95 ethanol such that it had anabsorbance of ca 07 Abs at 734 nm to obtain the ABTSworking solution After keeping the 300 μL ABTS workingsolution and 100 μL ethanol solution of compounds (4andash4d5andash5d and piperine) with a certain concentration gradientin the dark for 30min their absorbances at 734 nm weremeasured -e experiment was repeated three times inparallel

24 DPPH Radical-Scavenging Properties -e DPPH radi-cal-scavenging activities were assayed according to themethod reported by Nimse et al [26] -e DPPH workingsolution was prepared by dissolving 20mg DPPH in 250mLof 95 ethanol and diluting it until its absorbance was ca 08at 517 nm After keeping 200 μL DPPHmiddot working solutionand 200 μL ethanol solution of compounds (4andash4d 5andash5d

4 Journal of Chemistry

and piperine) with a certain concentration gradient in thedark for 30min their absorbances at 517 nmwere measured-e experiment was repeated three times in parallel

25 Measurement of Total Reducing Ability Total reducingability of piperine derivatives was detected according to themethod reported by Oyaizu [27] First 100μL of PBS (02MpH 66) solution 100μL of 1 potassium ferricyanide solu-tion and 20μL of compound solution (1600μM 800μM) weremixed together -e mixed solution was then heated in a 50degCwater bath for 20min and cooled in an ice bath Subsequently10 TCA solution was added to quench the reaction -emixture was then centrifuged at 3000 rmin for 10min and200μL of the supernatant was taken to add in 200μLH2O and40μL 01 FeCl3 solution-e solutionwas allowed to stand for10min before measuring its absorbance at 700nm

26 Inhibiting DNA Oxidation -e ability of piperine de-rivatives to inhibit DNA oxidation was detected by Gonget alrsquos reported methods [23] A DNA solution (224mgmL) and an AAPH solution (400 μM) were prepared usingPBS1 (NaH2PO4 19mM Na2HPO4 81mM and EDTA10 μM) as the solvent First 804mL of DNA solution and60 μL of compound solution (24mM 12mM 0) were mixedtogether and 900 μL AAPH solution was added -e well-mixed solution was then poured into 18 tubes (400 μLtube)which were placed in a 37degC water bath for 30min 6 h and8 h respectively After incubation 200 μL of TBA solution(TBA 025 g NaOH 01 g final volume brought to 25mLusing double-distilled water) and 200 μL of TCA solution(TCA 075 g final volume brought to 25mL using double-distilled water) were added to the tubes which were wellshaken and heated in a boiling water bath for 15min -eywere then cooled in an ice bath Subsequently 300 μL of n-butanol was added and a vortex was used to extract theresulting TBARS (thiobarbituric acid reactive material)Finally the tubes were centrifuged at 1500 rmin for 3minand 100 μL of the supernatant was taken out for the ab-sorbance measurement at 535 nm

27 Erythrocyte Hemolysis Assay -e inhibition of piperinederivatives on AAPH-induced erythrocyte hemolysis wasevaluated by the method reported by Zheng et al [20] SD ratswere anesthetized by injecting 20 urethane intraperitone-ally Blood was sampled from the heart and was collected inheparinized tubes -e tubes were then centrifuged at3500 rmin for 5min to separate the erythrocytes which werewashed four times using centrifugal washing with PBS (pH74 containing 137mM NaCl 27mM KCl 81mMNa2HPO4 and 15mM KH2PO4) -e erythrocytes weredispersed with PBS solution to obtain a 10 erythrocytesuspension Subsequently 100 μL of a 10 erythrocyte sus-pension with 50 μL of compound solution or PBS was in-cubated at 37degC for 30min -en 100 μL PBS solution of200mMAAPHwas added and themixture was incubated fora certain period of time at 37degC For the control group theAAPH solution was substituted with PBS After incubation

200 μL of the reaction solution was diluted with 1mL of PBSand was centrifuged at 3500 rmin for 5min -e absorbance(APBS) of the supernatant was measured at 540 nm using amicroplate reader -e same volume of the reaction solutionwas diluted with 1mL of distilled water to obtain a completehemolysis solution -e supernatant absorbance (Awater) wasmeasured under the same condition -e hemolysis rate wascalculated according to the following equation

erythrocyte hemolysis () Apbs

Awatertimes 100 (1)

28 Measurement of Hemoglobin Oxidation -e inhibitionof piperine derivatives on hemoglobin oxidation was alsoevaluated by the method reported by Zheng et al [20] First200 μL of erythrocyte suspension was diluted with 1mL ofdistilled water and centrifuged at 3500 rmin for 5min -esupernatant absorbance was measured at 630 nm and700 nm and recorded as A630 and A700 respectively Inaddition the supernatant was treated with 10 μL of 5potassium ferricyanide and the absorbances at 630 nm and700 nm were measured which were recorded as Ahb630 andAhb700 respectively -e hemoglobin oxidation rate wascalculated according to the following equation

Hemoglobin oxidation () A630 minus A700

Ahb630 minus Ahb7001113890 1113891 times 100

(2)

29 Influence of Piperine Derivatives on the AntioxidantEnzyme System of AAPH-Treated Rat Erythrocytes -ecompound solution (50μL) was added to 100μL of a 10erythrocyte suspension and incubated at 37degC for 30min -en100μL of 200mmolL AAPH was added and the mixture wasfurther incubated at 37degC for 1ndash4h After incubation ultrapurewater precooled to 4degCwas added to lyse all the erythrocytes-eresultant mixture was centrifuged and the sediment was re-moved andwashed three times in PBS 990μL of double-distilledwater precooled to 4degC was then added to 10μL of theerythrocyte sediment and the mixture was stored in a minus80degCfreezer before use-e glutathione peroxidase (GSH-Px) contentand the total superoxide dismutase and catalase (CAT) activitieswere determined using assay kits in accordance with themanufacturerrsquos instructions

210 Statistical Analysis Each experiment was repeated atleast three times in parallel -e results were reported asaverageplusmn standard deviation (SD) SPSS version 190 (SPSSInc Chicago IL) was used for the statistical analysis -edata were analyzed using one-way ANOVA-e significanceof difference was determined by the LSD range test(plt 005)

3 Results and Discussion

31 Radical-Scavenging Activities of 4andash4d and 5andash5d De-termined inDPPHandABTSAssays -e radical-scavenging

Journal of Chemistry 5

ability of piperine derivatives (4andash4d and 5andash5d) andpiperine was evaluated using the DPPH and ABTS assays-e results are shown in Figure 2 It can be seen from thefigure that compounds 4andash4d had no significant scavengingeffect on the DPPH radicals below the concentration of160 μM and the scavenging rate was below 20 Amongthem 4a 4b and 4c showed a stronger scavenging abilitythan the prototype compound (piperine) at a high con-centration (plt 005) It can be seen that after the amino acidderivatization of piperine its DPPH radical-scavengingability improved however the effect was not obviousCompounds containing phenolic hydroxyl groups (5andash5d)were obtained by removing the acetal from compounds4andash4d It can be observed from Figure 2 that the phenolichydroxyl groups played a significant role in improving theDPPH radical-scavenging ability of the compounds At aconcentration of 160 μM the scavenging rates of DPPH ofthe four compounds namely 5a 5b 5c 5d and VC werenot significantly different at 800plusmn 107 781plusmn 036766plusmn 609 787plusmn 078 (plt 005) and 9334plusmn 142respectively -e values of IC50 for 5a 5b 5c 5d and VCwere 575 μM 653 μM 664 μM 809 μM and 5940 μMrespectively It can be seen that the DPPH radical-scav-enging effect of 5a is similar to that of VC in the concen-tration range of 10ndash160 μm

-e scavenging effect of piperine derivatives on ABTSradicals was also evaluated in this experiment It can be seenfrom Figure 3 that at concentrations of 25 to 80 μM thecompounds 4andash4d showed a slightly stronger ABTS radical-scavenging effect than the prototype compound piperinebut the improvement was not significant It can also be seenthat the ABTS radical-scavenging rates of compounds 5a5b 5c 5d and VC at 80 μM concentration were882plusmn 304 7944plusmn 275 7636plusmn 289 5426plusmn 508and 7850plusmn 549 (plt 005) respectively and the values ofIC50 were 427 μM 490 μM 518 μM 518 μM and 493 μMrespectively Among them compounds 5a and 5b showedthe most significant ABTS radical-scavenging abilitieswhich were slightly stronger than that of VC It can be seenthat the presence of phenolic hydroxyl groups in thestructure of piperine derivatives had a significant effect onimproving the DPPH and ABTS radical-scavenging abilitiesof the compounds

32 Reducing Ability of Piperine Derivatives -e reducingability is an important indicator to evaluate the antioxidantability of compounds In this experiment the method de-scribed by Oyaizu [27] was used to test the reducing ability ofthe compound -e principle of reducing ability determi-nation is that a reducing substance reacts with potassiumferricyanide (K3Fe(CN)6) to form potassium ferrocyanide(K4Fe(CN)6) which then reacts with ferric chloride to formPrunbullrsquos Blue (KFe[Fe(CN)6]) that has the maximumabsorbance at 700 nm -erefore the larger the absorbanceat 700 nm the stronger the reducing ability of the sample Itcan be seen from Figure 4 that all compounds were sig-nificantly concentration-dependent such that the reducingability at a high concentration (160 μM) was stronger than

Concentration (μM)

DPP

H-s

cave

ngin

g ac

tivity

()

0 20 40 60 80 100 120 140 160 180

0

20

40

60

80

100

lowast

Figure 2 DPPH radical-scavenging activity of 4a () 4b () 4c() 4d () 5a () 5b () 5c () 5d () piperine () and VC() lowastindicates a significant difference from the piperine group(plt 005)

Concentration (μM)

ABT

S-sc

aven

ging

activ

ity (

)

0 20 40 60 80

0

20

40

60

80

100

lowast

Figure 3 ABTS radical-scavenging activity of 4a () 4b () 4c() 4d () 5a () 5b () 5c () 5d () piperine () and VC() lowastindicates a significant difference from the piperine group(plt 005)

4a 4b 4c 4d 5a 5b 5c 5d

TBH

Q

Pipe

rine

00

02

04

06

08

10

12

Abso

rban

ce at

700

nm

lowast

lowastlowast lowast lowast

lowast lowast lowast

Figure 4 Measurement of total reducing ability 80 μM ( )160 μM ( ) lowastindicates a significant difference from the piperinegroup (plt 005) indicates a significant difference from the TBHQgroup (plt 005)

6 Journal of Chemistry

that at a low concentration (80 μM) -e experimental re-sults showed that the reductive ability of compounds 4a 4b4c and 4d was similar to that of piperine -e reducingstrength of the compound has a certain correlation with thenumber of active hydrogen atoms supplied by the com-pound Compounds 5a 5b 5c and 5d all had three activehydrogen atoms TBHQ had two active hydrogen atoms andpiperine had no active hydrogen atom From Figure 4 it canbe seen that at a concentration of 160 μM the absorbancesof 5a 5b 5c and 5d at 700 nm were 0999plusmn 00100880plusmn 0022 0866plusmn 0021 and 0860plusmn 0036 respectively-e absorbances of piperine and TBHQ were 0247plusmn 0015and 0261plusmn 0010 respectively -is showed that the re-ducing ability of piperine was comparable to that of TBHQwhich was not strong However compounds 5andash5d had arelatively strong reducing ability which was related to theirelectron-donating ability (hydrogen-donating ability)

33 Protective Effects of 5andash5d on AAPH-Induced DNAOxidation AAPH can react with oxygen in the air to produceperoxy radicals (ROO_) at a steady rate ROO_ can capture thehydrogen atom supplied by the C-4prime atom in DNA whichcould result in the DNAmolecule unwinding and generating avariety of carbonyl-containing small molecular substances[23 28] -ese substances can react with TBA to producethiobarbituric acid reactive species (TBARS λmax 535nm)under acidic conditions-erefore in this study the content ofcarbonyl-containing small-molecule compounds produced byAAPH-oxidized DNA was quantitatively calculated by testingthe absorbance of TBARS thereby tracking the extent ofAAPH-induced oxidative damage in DNA As shown in thecontrol experiment in Figure 5 in the process of DNA oxi-dation induced by AAPH the absorbance of TBARS increasedwith time indicating that as the reaction time increased morecarbonyl-containing small-molecule compounds were pro-duced as a result of AAPH-induced oxidative damage in DNAIt can be seen from Figure 6 that the addition of piperine didnot affect the increase in absorbance of TBARS in the above-mentioned system -is indicated that neither the potentialantioxidant units N-H nor the conjugated double bonds in thepiperine molecule could significantly inhibit AAPH-inducedoxidative damage in DNA However when the other fivederivatives were added to the above system the increase in theabsorbance of TBARS was significantly reduced -is showedpreliminarily that phenolic hydroxyl groups could protectDNA from AAPH-induced oxidative damage -e inhibitioneffect of compounds on AAPH-induced oxidative damage inDNA is shown in Figure 5 from which it can be seen that theinhibition effect of compounds 5a-5b onAAPH-induced DNAoxidative damage was concentration-dependent such that theinhibition rate at 160μM was higher than that at 80μM Inaddition as AAPH could generate ROO_ continuously in theoxidative damage process the inhibition rate of compounds5a-5b on DNA oxidative damage first increased and thendecreased with time and was the highest at 360min

34 Protective Effects of 5andash5d on AAPH-Induced HemolysisinErythrocytes In this study compounds 5andash5dwith strong

radical-scavenging ability were studied -e protective effectof derivatives on the AAPH-induced hemolysis of raterythrocytes was discussed It can be seen from Figure 7(a)that the erythrocyte suspension was stable and the hemolysisrate was 1445 in the PBS solution (pH 74) (control group)after incubation at 37degC for 3 h No obvious hemolysis wasobserved After adding AAPH under the same condition thehemolysis of erythrocytes increased to 6208 (AAPHgroup) -e significant increase in hemolysis showed thatAAPH could induce damage in erythrocytes thereby in-ducing hemolysis When a low concentration (20 μM) ofpiperine derivatives was added to the erythrocytes thehemolysis rates all decreased significantly (plt 005) andshowed significant concentration dependency such that thehigher the compound concentration the lower the hemo-lysis rate All four types of piperine derivatives reduced thehemolysis rate of erythrocytes to less than 50 Amongthem compound 5d showed the most prominent effect andreduced the hemolysis rate to 2590 after 3 h of incubation

-e protection time of 5a-5d on the AAPH-inducedhemolysis of erythrocytes is shown in Figure 7(b) It can beseen that the erythrocytes were stable in the PBS solutionand no significant change in the hemolysis rate was observedwith time -e hemolysis of the erythrocyte suspensioncaused by AAPH-induced oxidative damage showed a sig-nificant time effect such that the hemolysis rate increasedwith increasing damage time -e hemolysis rate increasedfrom 3664 to 7475 from 2 h to 4 h-is was because theantioxidant system of erythrocytes (GSH SOD CAT etc)could inhibit the oxidative damage from ROO_ in the initialstage However due to the consumption of antioxidants andthe increase of free radical chain reactions over time theerythrocyte membrane became damaged and the hemolysisoccurred rapidly -e addition of piperine derivatives couldeffectively suppress hemolysis As shown in the figure thehemolysis rate of the AAPH group was 3664 after 2 h ofincubation while the hemolysis rates of 5a 5b 5c and 5dgroups were reduced to 2808 3371 1899 and1492 respectively -erefore the piperine derivatives hada certain protective effect on hemolysis caused by oxidativedamage Among all groups compound 5d showed the besteffect before 3 h while compound 5a had the most signif-icant effect at 4 h

35 Effects of 5andash5d on AAPH-Induced HemoglobinOxidation in Erythrocytes Hemoglobin is the main proteinin erythrocytes Excessive free radicals can cause hemoglobinto oxidize and form methemoglobin thereby leading to thedisorder of erythrocyte function [20 29] -is study furtherdemonstrated the inhibitory effect of derivatives on oxi-dative damage in erythrocytes by examining the content ofmethemoglobin in erythrocytes -e effect of piperine de-rivatives against AAPH-induced hemoglobin oxidation inerythrocytes is shown in Figure 8 After 3 h of incubation(Figure 8(a)) the methemoglobin content in the normalcontrol group was low (174) whereas it was relatively high(2565) after oxidation by AAPH In addition as shown inFigure 8(b) the methemoglobin content in the normal

Journal of Chemistry 7

group was relatively stable while that in the AAPH groupcontinued to increase with time -us it can be seen thatAAPH could induce the continuous oxidation ofhemoglobin

It can be seen from Figure 8(a) that after 3 h of in-cubation the compounds showed some inhibitory effect onthe production of methemoglobin Among them

compounds 5c and 5d showed the most significant effectsA certain concentration effect was also observed such thatthe inhibitory effect increased with concentration In ad-dition as shown in Figure 8(b) the inhibitory effect of thecompounds on hemoglobin oxidation weakened with time-is was because AAPH could continuously release ROO_

over time

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

Abso

rban

ce at

535

nm

Time (h)

(a)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

Time (h)

(b)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

07

08

09

Time (h)

(c)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 1400Time (h)

01

02

03

04

05

06

07

08

09

(d)

Abso

rban

ce at

535

nm

200 400 600 800 1000 1200 140001

02

03

04

05

06

07

08

09

Time (h)

(e)

Figure 6 Variation in the absorbance of piperine derivatives in AAPH-induced oxidative damage in DNA over time 0 μM () 80 μM ()160 μM () (a) 5a (b) 5b (c) 5c (d) 5d (e) Piperine

0 360 480 1080

0

10

20

30

40

50

Time (min)

AA

PH-in

duce

d D

NA

dam

age i

nhib

ition

rate

() lowast

lowast

lowast

Figure 5 Inhibition effect of piperine derivatives on AAPH-induced oxidative damage in DNA 5a 160 μM ( ) 5a 80 μM ( ) 5b 160 μM( ) 5b 80 μM ( ) 5c 160 μM ( ) 5c 80 μM ( ) 5d 160 μM ( ) 5d 80 μM ( ) piperine 80 μM ( ) lowastindicates a significant differencefrom the piperine group (plt 005)

8 Journal of Chemistry

36 Influence of Piperine Derivatives on GSH-Px T-SOD andCATActivities in AAPH-Treated Erythrocytes GSH-Px is anantioxidant enzyme present in cells that catalyzes the re-duction of glutathione (GSH) and the removal of peroxidesin the body such as H2O2 in cells causing antilipid oxidation[30] As shown in Figure 9(a) the GSH-Px content inerythrocytes belonging to the control group was stable whilethat in erythrocytes of the AAPH group showed a statisti-cally significant gradual decrease with increasing incubationtime Following the addition of 50 μMof piperine derivativesto erythrocytes prior to the induction of oxidative damage byAAPH the piperine derivatives did not significantly influ-ence the erythrocyte GSH-Px content after 1 h of incubationcompared with the AAPH group After 2 h of incubationcompounds 5a and 5b significantly increased the GSH-Pxcontent (plt 005) after 4 h of incubation only the GSH-Pxcontent in the 5b and 5d groups was increased compared

with that in the AAPH group -ese results indicate that theamino acid derivatives of piperine can increase GSH-Pxactivity in erythrocytes Particularly compounds 5b and 5dproduced the strongest effects with the differences beingstatistically insignificant compared with the positive drugie vitamin C (VC) (pgt 005)

SOD is a key enzyme for removing free radicals withinthe body -e SOD activity level serves as an indicator of thebodyrsquos ability to remove free radicals [30 31] Figure 9(b)shows the influence of amino acid derivatives of piperine onT-SOD activity in AAPH-treated erythrocytes In the AAPHgroup with increasing incubation time T-SOD activitydecreased gradually Following the addition of 50 μM ofpiperine derivatives T-SOD activity increased after 1 h ofincubation albeit insignificantly compared with the AAPHgroup (pgt 005) After 2 h of incubation compounds 5a 5b5c and 5d increased T-SOD activity significantly (plt 005)

0

5

10

15

20

25

30

35

40

Met

Hb

()

Control AAPH 10 20Concentration (μM)

5

lowast

lowast

(a)

Time (h)

Met

Hb

()

2 3 40

10

20

30

40

50

(b)

Figure 8 (a) Protective effects of piperine derivatives on AAPH-induced hemoglobin oxidation (3 h) (b) Kinetic curve of the protectiveeffect of 20 μMpiperine derivatives on AAPH-induced hemoglobin oxidation Control ( ) AAPH ( ) AAPH+ 5a ( ) AAPH+ 5b ( )AAPH+ 5c ( ) AAPH+ 5d ( ) lowastindicates a significant difference from the control group (plt 005) indicates a significant differencefrom the control group (plt 005)

0

10

20

30

40

50

60

70

80H

emol

ysis

()

Control AAPH 10 20Concentration (μM)

5

lowast

lowast

(a)

Hem

olys

is (

)

0

20

40

60

80

100

Time (h)2 3 4

(b)

Figure 7 (a) Protective effect of piperine derivatives on AAPH-induced hemolysis of erythrocytes (3 h) (b) Kinetic curve of the protectiveeffect of piperine derivatives on AAPH-induced hemolysis of erythrocytes at 20 μM Control ( ) AAPH ( ) AAPH+ 5a ( ) AAPH+ 5b( ) AAPH+ 5c ( ) AAPH+ 5d ( ) lowastindicates a significant difference from the control group (plt 005) indicates a significantdifference from the AAPH group

Journal of Chemistry 9

while the positive control and prototype drug (piperine) hadno significant influence on erythrocyte T-SOD activity(pgt 005) After 4 h of incubation compound 5d increasedT-SOD activity significantly (pgt 005) while other com-pounds had no significant influence on T-SOD activity(pgt 005) -ese results highlight the significant enhance-ment caused by piperine derivatives of T-SOD activity inerythrocytes with AAPH-induced oxidative damage com-pared to the relatively inferior effect of the prototypecompound and of the positive drug VC

CAT an abundant enzyme in erythrocytes catalyzesH2O2 decomposition which reduces oxidative damagecaused by OHbull an oxidation product formed from H2O2under catalysis by metal ions [30 31] As shown inFigure 9(c) CAT activity in the AAPH group decreasesgradually relative to that in the control group with in-creasing incubation time Following the addition of 50 μMofpiperine derivatives compound 5c and piperine increasederythrocyte CATactivity after 1 h of incubation After 2 h ofincubation compounds 5a and 5b and piperine increasedCAT activity in erythrocytes significantly (plt 005) whilethe positive control had no significant influence on CAT

activity (pgt 005) After 4 h of incubation compound 5bincreased CAT activity significantly (pgt 005) while othercompounds had no significant influence (pgt 005) -eseresults indicate that piperine derivatives produce signifi-cant differences in CAT activity in AAPH-treated raterythrocytes

4 Conclusion

In the present study piperine-amino acid derivatives weresynthesized and the results indicate that the free radicalremoval ability and total reductive ability of the phenolichydroxyl-containing piperine-amino acid derivatives werehigher than those of the parent compound Specificallycompounds 5a and 5b have free radical removal perfor-mances comparable to that of VC-e derivatives have goodinhibitory effects on AAPH-induced oxidative DNA dam-age while certain compounds also show inhibitory effects onAAPH-induced hemolysis in rat erythrocytes Particularlycompound 5b can significantly inhibit AAPH-inducederythrocyte hemolysis and hemoglobin oxidation In addi-tion the experiments further verified that the protection

Time

GSH

-Px

(U)

1h 2h 4h0

100

200

300

400

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

(a)

Time

SOD

activ

ity (U

mgH

b)

1h 2h 4h0

5

10

15

20

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

(b)

Time1h 2h 4h

CAT

activ

ity (U

mgH

b)

0

1

2

3

4

5

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast lowast

lowast

lowast

lowast

lowast

(c)

Figure 9 Influence of piperine derivatives on the (a) GSH-Px content (b) T-SOD activity and (c) CATactivity in AAPH-treated rat erythrocyteslowastindicates a significant difference from the control group (plt 005) Control ( ) AAPH ( ) AAPH+5a ( ) AAPH+5b ( ) AAPH+5c( ) AAPH+5d ( ) AAPH+piperine ( ) AAPH+VC ( ) indicates a significant difference from the AAPH group (plt 005)

10 Journal of Chemistry

provided by piperine derivatives against AAPH-inducedoxidative damage can be achieved by preserving the activityof the antioxidant enzyme system (GSH-Px T-SOD andCAT) within cells -e protection by piperine derivativeswas also found to be superior to that by piperine and vitaminC -is paper described a structural modification methodthat could effectively improve the antioxidant ability ofpiperine-is type of compound could be a candidate for thedevelopment and research of oxidative damage-relateddiseases (hyperlipidemia diabetes cancer whitening etc)

Data Availability

-e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

-is work was financially supported by the Shaanxi Pro-vincial Department of Education serving local (industrial-ization) research project (no 15JF028) and the ScientificResearch Fund of Xirsquoan Medical University(nos2016YXXK09 2018DC-12 and 201825012)

References

[1] J P Adjimani and P Asare ldquoAntioxidant and free radicalscavenging activity of iron chelatorsrdquo Toxicology Reportsvol 2 pp 721ndash728 2015

[2] I Dalle-Donne R Rossi R Colombo D Giustarini andA Milzani ldquoBiomarkers of oxidative damage in humandiseaserdquo Clinical Chemistry vol 52 no 4 pp 601ndash623 2006

[3] L J Machlin and A Bendich ldquoFree radical tissue damageprotective role of antioxidant nutrientsrdquo Fe FASEB Journalvol 1 no 6 pp 441ndash445 1987

[4] N SatoW Li H Takemoto et al ldquoComprehensive evaluationof antioxidant effects of Japanese Kampo medicines led toidentification of Tsudosan formulation as a potent antioxidantagentrdquo Journal of Natural Medicines vol 73 no 1pp 163ndash172 2018

[5] N Bao Z Sun H Baigude G Borjihan and Z Jin ldquoLightinduced isomerization of piperlonguminine to scutifoliamideA isopiperlonguminine and hoffmannseggiamide Ardquo Phy-tochemistry Letters vol 16 pp 324ndash327 2016

[6] T Velpandian R Jasuja R K Bhardwaj J Jaiswal andS K Gupta ldquoPiperine in food interference in the pharma-cokinetics of phenytoinrdquo European Journal of Drug Meta-bolism and Pharmacokinetics vol 26 no 4 pp 241ndash247 2001

[7] A Duangjai K Ingkaninan S Praputbut andN Limpeanchob ldquoBlack pepper and piperine reduce cho-lesterol uptake and enhance translocation of cholesteroltransporter proteinsrdquo Journal of Natural Medicines vol 67no 2 pp 303ndash310 2013

[8] D E Eigenmann C Durig E A Jahne et al ldquoIn vitro blood-brain barrier permeability predictions for GABAA receptormodulating piperine analogsrdquo European Journal of Phar-maceutics and Biopharmaceutics vol 103 pp 118ndash126 2016

[9] Y A Samra H S Said N M Elsherbiny G I Liou M M El-Shishtawy and L A Eissa ldquoCepharanthine and Piperineameliorate diabetic nephropathy in rats role of NF-κB andNLRP3 inflammasomerdquo Life Sciences vol 157 pp 187ndash1992016

[10] R Mittal and R L Gupta ldquoIn vitro antioxidant activity ofpiperinerdquoMethods and Findings in Experimental and ClinicalPharmacology vol 22 no 5 pp 271ndash274 2000

[11] Z Zarai E Boujelbene N Ben Salem Y Gargouri andA Sayari ldquoAntioxidant and antimicrobial activities of varioussolvent extracts piperine and piperic acid from Piper nig-rumrdquo LWT-Food Science and Technology vol 50 no 2pp 634ndash641 2013

[12] A Yasir S Ishtiaq M Jahangir et al ldquoBiology-orientedsynthesis (BIOS) of piperine derivatives and their comparativeanalgesic and antiinflammatory activitiesrdquo MedicinalChemistry vol 14 no 3 pp 269ndash280 2018

[13] R S Vijayakumar D Surya and N Nalini ldquoAntioxidantefficacy of black pepper (Piper nigrumL) and piperine in ratswith high fat diet induced oxidative stressrdquo Redox Reportvol 9 no 2 pp 105ndash110 2004

[14] T Miyazawa K Nakagawa S H Kim et al ldquoCurcumin andpiperine supplementation of obese mice under caloric re-striction modulates body fat and interleukin-1βrdquo Nutrition ampMetabolism vol 15 no 1 p 12 2018

[15] A Khajuria N -usu U Zutshi and K L Bedi ldquoPiperinemodulation of carcinogen induced oxidative stress in intes-tinal mucosardquo Molecular and Cellular Biochemistry vol 189no 12 pp 113ndash118 1998

[16] S Inder Pal J Shreyans Kumar K Amandeep et al ldquoSyn-thesis and antileishmanial activity of piperoyl-amino acidconjugatesrdquo European Journal of Medicinal Chemistry vol 45no 8 pp 3439ndash3445 2010

[17] T Koichi M Takaki and S Yoshiaki ldquoSynthesis and bio-logical evaluation of piperic acid amides as free radicalscavengers and α-glucosidase inhibitorsrdquo Chemical amp Phar-maceutical Bulletin vol 63 no 5 pp 326ndash333 2015

[18] J M Latorres D G Rios G Saggiomo W Wasielesky andC Prentice-Hernandez ldquoFunctional and antioxidant prop-erties of protein hydrolysates obtained from white shrimp(Litopenaeus vannamei)rdquo Journal of Food Science and Tech-nology vol 55 no 2 pp 721ndash729 2018

[19] A Moayedi L Mora M C Aristoy M Safari M Hashemiand F Toldra ldquoPeptidomic analysis of antioxidant and ACE-inhibitory peptides obtained from tomato waste proteinsfermented using Bacillus subtilisrdquo Food Chemistry vol 250pp 180ndash187 2018

[20] L Zheng H Dong G Su Q Zhao and M Zhao ldquoRadicalscavenging activities of Tyr- Trp- Cys- andMet-Gly and theirprotective effects against AAPH-induced oxidative damage inhuman erythrocytesrdquo Food Chemistry vol 197 no Part App 807ndash813 2016

[21] M Miceli E Roma P Rosa et al ldquoSynthesis of benzofuran-2-one derivatives and evaluation of their antioxidant capacity bycomparing DPPH assay and cyclic voltammetryrdquo Moleculesvol 23 no 4 p 710 2018

[22] K -aipong U Boonprakob K Crosby et al ldquoComparisonof ABTS DPPH FRAP and ORAC assays for estimatingantioxidant activity from guava fruit extractsrdquo Journal of FoodComposition amp Analysis vol 19 no 6-7 pp 669ndash675 2006

[23] X-R Gong G-L Xi and Z-Q Liu ldquoActivity of coumarin-oxadiazole-appended phenol in inhibiting DNA oxidationand scavenging radicalrdquo Tetrahedron Letters vol 56 no 45pp 6257ndash6261 2015

Journal of Chemistry 11

[24] G-L Xi and Z-Q Liu ldquoCoumarin sharing the benzene ringwith quinoline for quenching radicals and inhibiting DNAoxidationrdquo European Journal of Medicinal Chemistry vol 95pp 416ndash423 2015

[25] H R Shin B R You and W H Park ldquoPX-12-induced HeLacell death is associated with oxidative stress and GSH de-pletionrdquo Oncology Letters vol 6 no 6 pp 1804ndash1810 2013

[26] S B Nimse D Pal A Mazumder et al ldquoSynthesis of cin-namanilide derivatives and their antioxidant and antimi-crobial activityrdquo Journal of Chemistry vol 2015 Article ID208910 5 pages 2015

[27] M Oyaizu ldquoStudies on products of browning reactionAntioxidative activities of products of browning reactionprepared from glucosaminerdquo Fe Japanese Journal of Nu-trition and Dietetics vol 44 no 6 pp 307ndash315 1986

[28] Y Yoshioka X Li T Zhang et al ldquoBlack soybean seed coatpolyphenols prevent AAPH-induced oxidative DNA-damagein HepG2 cellsrdquo Journal of Clinical Biochemistry and Nu-trition vol 60 no 2 pp 108ndash114 2017

[29] R C Chiste M Freitas A Z Mercadante and E FernandesldquoCarotenoids inhibit lipid peroxidation and hemoglobinoxidation but not the depletion of glutathione induced byROS in human erythrocytesrdquo Life Sciences vol 99 no 1-2pp 52ndash60 2014

[30] R Ozturk-Urek L A Bozkaya and L Tarhan ldquo-e effects ofsome antioxidant vitamin-and trace element-supplementeddiets on activities of SOD CAT GSH-Px and LPO levels inchicken tissuesrdquo Cell Biochemistry and Function vol 19 no 2pp 125ndash132 2001

[31] O Levy Y Achituv Y Z Yacobi N Stambler andZ Dubinsky ldquo-e impact of spectral composition and lightperiodicity on the activity of two antioxidant enzymes (SODand CAT) in the coral Favia favusrdquo Journal of ExperimentalMarine Biology and Ecology vol 328 no 1 pp 35ndash46 2006

12 Journal of Chemistry

13886 13027 12401 12220 12189 10800 10528 100835203 4761 1819 ESI mz 3041 [M+ 1]+

224 Piperine L-Valine Methyl Ester Conjugate (3bC18H21NO5) -e synthesis process of compound 3b wassimilar to that of compound 3a -e amount of L-valinemethyl ester hydrochloride used was 530 g (317mmol)-eproduct was a pale yellow solid (812 g 803) 1H NMR(400MHz CDCl3) δ 743-737 (m 1H CHCH-CO) 701(d 1H Ar-CH) 693-668 (m 4H Ar-H Ar-CHCH-)603 (s 1H CHCH-CO) 600 (s 2H -OCH2O-) 474-470(m 1H CONHCH) 378 (s 3H -OCH3) 225-220 (m 1HCH (CH3)2) 100-095 (m 6H CH (CH3)2)13CNMR(101MHz CDCl3) δ 17222 16541 14782 14772 1413613886 13028 12400 12222 12199 10801 10527 100835656 5171 3109 1846 1741 ESI mz 3321 [M+ 1]+

225 Piperine L-Leucinate Methyl Ester Conjugate (3cC19H23NO5) -e synthesis process of compound 3c wassimilar to that of compound 3a -e amount of L-leucinatemethyl ester hydrochloride used was 574 g (317mmol)-eproduct was a pale yellow solid (835 g 795) 1H NMR(400MHz CDCl3) δ 741ndash735 (m 1H CHCH-CO) 698-697 (d 1H Ar-CH) 690-665 (m 4H Ar-H Ar-CHCH-)616-614 (d 1H CHCH-CO) 599 (s 2H -OCH2O-) 482-476 (m 1H CONHCH) 377 (s 3H -OCH3) 173-1170 (m2H CH2CH (CH3)2) 162-158 (m 1H CH (CH3)2) 099-096 (m 6H CH (CH3)2)13C NMR (101MHz CDCl3) δ17387 16587 14829 14819 14187 13933 13078 1245312269 12239 10847 10578 10132 5234 5077 41822490 2284 2198 ESI mz 3461 [M+1]+

226 Piperine L-Methionine Methyl Ester Conjugate (3dC18H21NO5S) -e synthesis process of compound 3d wassimilar to that of compound 3a -e amount of L-methioninemethyl ester hydrochloride used was 514 g (317mmol) -eproduct was a pale yellow solid (853 g 856) 1H NMR(400MHz CDCl3) 743-736 (m 1H CHCH-CO) 700 (d1H Ar-CH) 693-667 (m 4H Ar-H Ar-CHCH-) 632-630 (d 1H CHCH-CO) 600 (s 2H -OCH2O-) 489-484

(m 1H CONHCH) 380 (s 3H -OCH3) 255-254 (m 2HSCH2) 206-205 (m 5H CHCH2 SCH3) 13C NMR(101MHz CDCl3) δ 17264 16582 14837 14823 1420513957 13073 12445 12276 12219 10851 10579 101325260 5163 3187 3000 1551

227 Piperine L-Alanine Conjugate (4a C15H15NO5)Compounds 4andash4d were synthesized by improving themethod reported by Inder Pal et al [16] -e experiment wascarried out by heating hydrolysis with some water in the solidmedium -e yield of compounds 4andash4d reached 71ndash86

Compound 3a (199 g 657mmol) and 974 g of KF-Al2O3 were thoroughly ground and mixed together -emixture was then transferred to a 100mL round-bottomedflask and 3mL of water was added After heating andstirring at 120degC for 1 h 10mL of water was added andstirred for another 10min before suction filtration -efiltrate pH was adjusted to 1 with hydrochloric acid whilestirring at room temperature to precipitate solids -e crudeproduct obtained after suction filtration was then purified bycolumn chromatography on silica gel (developing agentDCM ∶MeOH ∶HAc 60 ∶ 3 ∶ 01) to afford 142 g of theproduct (4a) (yield 751) as a pale yellow solid 1H NMR(400MHz DMSO-d6) δ 1254 (s 1H -COOH) 838-836 (d1H CHNHCO) 728-727 (d 1H CHCH-CO) 717-713(m 1H Ar-CH) 701-689 (m 4H Ar-H Ar-CHCH-)616-613 (d 1H CHCH-CO) 604 (s 2H -OCH2O-) 431-427 (m 1H CONHCH) 131-129 (d 3H -CH3) 13C NMR(101MHz DMSO-d6) δ 17424 16491 14789 1477113988 13818 13077 12512 12388 12273 10840 1056210123 4753 1721 ESI mz 2901 [M+ 1]+

228 Piperine L-Valine Conjugate (4b C17H19NO5) -esynthesis process of compound 4b was similar to that ofcompound 4a -e product was a pale yellow solid (yield789) 1H NMR (400MHz DMSO-d6) δ 1251 (s 1H-COOH) 826-820 (d 1H CHNHCO) 729 (d 1HCHCH-CO) 720-714 (m 1H Ar-CH) 702-690 (m4H Ar-H Ar-CHCH-) 631-628 (d 1H CHCH-CO)minus605 (s 2H -OCH2O-) 427-423 (m 1H CONHCH)211-204 (m 1H CH (CH3)2) 091-089 (m 6H CH

O

O

O

O

O

O

O

O

O OO

ONH

N

Piperine

20KOH in methanol

KF-Al2O3 (40)70 ~ 120degC 3hR

75degC 24h1 2

3andash3d

3a 4a 5a R = CH33b 4b 5b R = CH(CH3)23c 4c 5c R = CH2CH(CH3)23d 4d 5d R = CH2CH2SCH3

4andash4d5andash5d

OO

OOH

OHHO

HO

BBr3

OO

R

(COCI)2

OO

O O OH

R

ONH2 middot HCI

R

CI

NH

NH

Figure 1 Scheme for the preparation of piperine derivatives KOH potassium hydroxide (COCl)2 oxalyl chloride KF potassium fluorideAl2O3 aluminium oxide BBr3 boron tribromide

Journal of Chemistry 3

(CH3)2) 13C NMR (101MHz DMSO-d6) δ 17310 1653914789 14770 13985 13812 13078 12513 12407 1226910840 10564 10123 5729 2983 1917 1810 ESI mz3181 [M+ 1]+

229 Piperine L-Leucinate Conjugate (4c C18H21NO5)-e synthesis process of compound 4c was similar to that ofcompound 4a -e product was a white solid (yield 857)1H NMR (400MHz DMSO-d6) δ 1253 (s 1H -COOH)831-829 (d 1H CHNHCO) 728 (d 1H CHCH-CO)720-714 (m 1H Ar-CH) 702-686 (m 4H Ar-H Ar-CHCH-) 619-615 (d 1H CHCH-CO) 605 (s 2H-OCH2O-) 435-430 (m 1H CONHCH) 167-161 (m 1HCH (CH3)2) 158-153 (m 2H CH2CH (CH3)2) 092-086(dd 6H CH (CH3)2) 13C NMR (101MHz DMSO-d6) δ17417 16518 14789 14771 13986 13813 13078 1251312394 12268 10840 10564 10122 5029 4000 24372282 2127 ESI mz 3321[M+ 1]+

2210 Piperine L-Methionine Conjugate (4d C17H19NO5S)-e synthesis process of compound 4d was similar to that ofcompound 4a -e product was a pale yellow solid (yield713) 1H NMR (400MHz DMSO-d6) δ 1271 (s 1H-COOH) 837-835 (d 1H CHNHCO) 729-728 (d 1HCHCH-CO) 721-715 (m 1H Ar-CH) 702-686 (m 4HAr-H Ar-CHCH-) 618-615 (d 1H CHCH-CO) 605 (s2H -OCH2O-) 444-443 (m 1H CONHCH) 206-186 (m5H SCH2 SCH3) 13C NMR (101MHz DMSO-d6) δ 1739116582 14839 14823 14049 13875 13127 12561 1243212322 10891 10613 10174 5154 3123 3022 1503 ESImz 3501[M+ 1]+

2211 ((2E4E)-5-(34-Dihydroxyphenyl)penta-24-dienoyl)Alanine (5a C14H15NO5) Compound 4a (200mg069mmol) was dispersed in 10mL of anhydrous DCM ADCM (5mL) solution of boron tribromide (2609 μL276mmol) was added dropwise to the reaction mixture inan ice-bath condition After 4 h the reaction was quenchedby adding 10mL of ice-cold saturated ammonium chloridesolution in an ice-bath condition After stirring at roomtemperature for 05 h ethyl acetate (3times15mL) was used forthe extraction -e organic phase was dried with anhydroussodium sulfate for 24 h and then filtered to obtain theconcentrated crude product -e crude product was thenpurified by column chromatography on silica gel (devel-oping agent DCM MeOH HAc 75 1 01) to afford3652mg of the product (5a) (yield 191) as an orangesolid 1H NMR (400MHz DMSO-d6) 983 (s 2H -OH)805-803 (d 1H CHNHCO) 717-711 (m 1H CHCH-CO) 695 (s 1H Ar-CH) 684-6671 (m 4H Ar-H Ar-CHCH-) 618-614 (d 1H CHCH-CO) 430-427 (m1H CONHCH) 128-126 (d 3H -CH3) ESI mz 27819[M+ 1]+

2212 ((2E4E)-5-(34-Dihydroxyphenyl)penta-24-dienoyl)Valine (5b C16H19NO5) -e synthesis process of com-pound 5b was similar to that of compound 5a -e product

was a dark orange solid (yield 123) 1H NMR (400MHzDMSO-d6) δ 976 (s 2H -OH) 791 (s 1H CHNHCO)717-711 (m 1H CHCH-CO) 696 (s 1H Ar-CH) 684-674 (m 4H Ar-H Ar-CHCH-) 629-626 (d 1HCHCH-CO) 422-419 (m 1H CONHCH) 210 (s 1HCH (CH3)2) 088-081 (m 6H CH (CH3)2) ESI mz 3201[M+ 1]+

2213 ((2E4E)-5-(34-Dihydroxyphenyl)penta-24-dienoyl)Leucine (5c C17H21NO5) -e synthesis process of com-pound 5c was similar to that of compound 5a -e productwas a dark orange solid (yield 293) 1H NMR (400MHzDMSO-d6) δ 931 (s 2H -OH) 823-821 (d 1HCHNHCO) 719-712 (m 1H CHCH-CO) 695 (d 1HAr-CH) 686-671 (m 4H Ar-H Ar-CHCH-) 614-610(d 1H CHCH-CO) 432-430 (m 1H CONHCH) 165-160 (m 1H CH (CH3)2) 156-152 (m 2H CH2CH(CH3)2) 091-085 (dd 6H CH (CH3)2) ESI mz 3202[M+ 1]+

2214 ((2E4E)-5-(34-Dihydroxyphenyl)penta-24-dienoyl)Methionine (5d C16H19NO5S) -e synthesis process ofcompound 5d was similar to that of compound 5a -eproduct was a dark green solid (yield 204) 1H NMR(400MHz DMSO-d6) δ 1269 (s 1H -COOH) 931 (s 1H-OH) 902 (s 1H -OH) 831-826 (m 1H CHNHCO) 720-714 (m 1H CHCH-CO) 695 (m 1H Ar-CH) 787-672(m 4H Ar-H Ar-CHCH-) 614-610 (d 1H CHCH-CO) 443-437 (m 1H CONHCH) 207-202 (m 5H SCH2SCH3) ESI mz 3382[M+ 1]+

23 ABTS Radical-Scavenging Properties -e ABTS radical-scavenging activities were assayed according to the methodreported by-aipong et al [22] (1) ABTS+ aqueous solution(7mmolL) preparation 30mg ABTS+ was weighed 8mL ofultrapure water was added and it was dissolved by ultra-sonication (2) K2S2O8 solution preparation 10mg K2S2O8was weighed 15mL of ultrapure water was added and it wasdissolved by ultrasonication (3) ABTS working solutionpreparation (1) and (2) were mixed at the ratio of 1 1 andkept in the dark for 12 to 16 h-e abovemixture was dilutedfour to eight times with 95 ethanol such that it had anabsorbance of ca 07 Abs at 734 nm to obtain the ABTSworking solution After keeping the 300 μL ABTS workingsolution and 100 μL ethanol solution of compounds (4andash4d5andash5d and piperine) with a certain concentration gradientin the dark for 30min their absorbances at 734 nm weremeasured -e experiment was repeated three times inparallel

24 DPPH Radical-Scavenging Properties -e DPPH radi-cal-scavenging activities were assayed according to themethod reported by Nimse et al [26] -e DPPH workingsolution was prepared by dissolving 20mg DPPH in 250mLof 95 ethanol and diluting it until its absorbance was ca 08at 517 nm After keeping 200 μL DPPHmiddot working solutionand 200 μL ethanol solution of compounds (4andash4d 5andash5d

4 Journal of Chemistry

and piperine) with a certain concentration gradient in thedark for 30min their absorbances at 517 nmwere measured-e experiment was repeated three times in parallel

25 Measurement of Total Reducing Ability Total reducingability of piperine derivatives was detected according to themethod reported by Oyaizu [27] First 100μL of PBS (02MpH 66) solution 100μL of 1 potassium ferricyanide solu-tion and 20μL of compound solution (1600μM 800μM) weremixed together -e mixed solution was then heated in a 50degCwater bath for 20min and cooled in an ice bath Subsequently10 TCA solution was added to quench the reaction -emixture was then centrifuged at 3000 rmin for 10min and200μL of the supernatant was taken to add in 200μLH2O and40μL 01 FeCl3 solution-e solutionwas allowed to stand for10min before measuring its absorbance at 700nm

26 Inhibiting DNA Oxidation -e ability of piperine de-rivatives to inhibit DNA oxidation was detected by Gonget alrsquos reported methods [23] A DNA solution (224mgmL) and an AAPH solution (400 μM) were prepared usingPBS1 (NaH2PO4 19mM Na2HPO4 81mM and EDTA10 μM) as the solvent First 804mL of DNA solution and60 μL of compound solution (24mM 12mM 0) were mixedtogether and 900 μL AAPH solution was added -e well-mixed solution was then poured into 18 tubes (400 μLtube)which were placed in a 37degC water bath for 30min 6 h and8 h respectively After incubation 200 μL of TBA solution(TBA 025 g NaOH 01 g final volume brought to 25mLusing double-distilled water) and 200 μL of TCA solution(TCA 075 g final volume brought to 25mL using double-distilled water) were added to the tubes which were wellshaken and heated in a boiling water bath for 15min -eywere then cooled in an ice bath Subsequently 300 μL of n-butanol was added and a vortex was used to extract theresulting TBARS (thiobarbituric acid reactive material)Finally the tubes were centrifuged at 1500 rmin for 3minand 100 μL of the supernatant was taken out for the ab-sorbance measurement at 535 nm

27 Erythrocyte Hemolysis Assay -e inhibition of piperinederivatives on AAPH-induced erythrocyte hemolysis wasevaluated by the method reported by Zheng et al [20] SD ratswere anesthetized by injecting 20 urethane intraperitone-ally Blood was sampled from the heart and was collected inheparinized tubes -e tubes were then centrifuged at3500 rmin for 5min to separate the erythrocytes which werewashed four times using centrifugal washing with PBS (pH74 containing 137mM NaCl 27mM KCl 81mMNa2HPO4 and 15mM KH2PO4) -e erythrocytes weredispersed with PBS solution to obtain a 10 erythrocytesuspension Subsequently 100 μL of a 10 erythrocyte sus-pension with 50 μL of compound solution or PBS was in-cubated at 37degC for 30min -en 100 μL PBS solution of200mMAAPHwas added and themixture was incubated fora certain period of time at 37degC For the control group theAAPH solution was substituted with PBS After incubation

200 μL of the reaction solution was diluted with 1mL of PBSand was centrifuged at 3500 rmin for 5min -e absorbance(APBS) of the supernatant was measured at 540 nm using amicroplate reader -e same volume of the reaction solutionwas diluted with 1mL of distilled water to obtain a completehemolysis solution -e supernatant absorbance (Awater) wasmeasured under the same condition -e hemolysis rate wascalculated according to the following equation

erythrocyte hemolysis () Apbs

Awatertimes 100 (1)

28 Measurement of Hemoglobin Oxidation -e inhibitionof piperine derivatives on hemoglobin oxidation was alsoevaluated by the method reported by Zheng et al [20] First200 μL of erythrocyte suspension was diluted with 1mL ofdistilled water and centrifuged at 3500 rmin for 5min -esupernatant absorbance was measured at 630 nm and700 nm and recorded as A630 and A700 respectively Inaddition the supernatant was treated with 10 μL of 5potassium ferricyanide and the absorbances at 630 nm and700 nm were measured which were recorded as Ahb630 andAhb700 respectively -e hemoglobin oxidation rate wascalculated according to the following equation

Hemoglobin oxidation () A630 minus A700

Ahb630 minus Ahb7001113890 1113891 times 100

(2)

29 Influence of Piperine Derivatives on the AntioxidantEnzyme System of AAPH-Treated Rat Erythrocytes -ecompound solution (50μL) was added to 100μL of a 10erythrocyte suspension and incubated at 37degC for 30min -en100μL of 200mmolL AAPH was added and the mixture wasfurther incubated at 37degC for 1ndash4h After incubation ultrapurewater precooled to 4degCwas added to lyse all the erythrocytes-eresultant mixture was centrifuged and the sediment was re-moved andwashed three times in PBS 990μL of double-distilledwater precooled to 4degC was then added to 10μL of theerythrocyte sediment and the mixture was stored in a minus80degCfreezer before use-e glutathione peroxidase (GSH-Px) contentand the total superoxide dismutase and catalase (CAT) activitieswere determined using assay kits in accordance with themanufacturerrsquos instructions

210 Statistical Analysis Each experiment was repeated atleast three times in parallel -e results were reported asaverageplusmn standard deviation (SD) SPSS version 190 (SPSSInc Chicago IL) was used for the statistical analysis -edata were analyzed using one-way ANOVA-e significanceof difference was determined by the LSD range test(plt 005)

3 Results and Discussion

31 Radical-Scavenging Activities of 4andash4d and 5andash5d De-termined inDPPHandABTSAssays -e radical-scavenging

Journal of Chemistry 5

ability of piperine derivatives (4andash4d and 5andash5d) andpiperine was evaluated using the DPPH and ABTS assays-e results are shown in Figure 2 It can be seen from thefigure that compounds 4andash4d had no significant scavengingeffect on the DPPH radicals below the concentration of160 μM and the scavenging rate was below 20 Amongthem 4a 4b and 4c showed a stronger scavenging abilitythan the prototype compound (piperine) at a high con-centration (plt 005) It can be seen that after the amino acidderivatization of piperine its DPPH radical-scavengingability improved however the effect was not obviousCompounds containing phenolic hydroxyl groups (5andash5d)were obtained by removing the acetal from compounds4andash4d It can be observed from Figure 2 that the phenolichydroxyl groups played a significant role in improving theDPPH radical-scavenging ability of the compounds At aconcentration of 160 μM the scavenging rates of DPPH ofthe four compounds namely 5a 5b 5c 5d and VC werenot significantly different at 800plusmn 107 781plusmn 036766plusmn 609 787plusmn 078 (plt 005) and 9334plusmn 142respectively -e values of IC50 for 5a 5b 5c 5d and VCwere 575 μM 653 μM 664 μM 809 μM and 5940 μMrespectively It can be seen that the DPPH radical-scav-enging effect of 5a is similar to that of VC in the concen-tration range of 10ndash160 μm

-e scavenging effect of piperine derivatives on ABTSradicals was also evaluated in this experiment It can be seenfrom Figure 3 that at concentrations of 25 to 80 μM thecompounds 4andash4d showed a slightly stronger ABTS radical-scavenging effect than the prototype compound piperinebut the improvement was not significant It can also be seenthat the ABTS radical-scavenging rates of compounds 5a5b 5c 5d and VC at 80 μM concentration were882plusmn 304 7944plusmn 275 7636plusmn 289 5426plusmn 508and 7850plusmn 549 (plt 005) respectively and the values ofIC50 were 427 μM 490 μM 518 μM 518 μM and 493 μMrespectively Among them compounds 5a and 5b showedthe most significant ABTS radical-scavenging abilitieswhich were slightly stronger than that of VC It can be seenthat the presence of phenolic hydroxyl groups in thestructure of piperine derivatives had a significant effect onimproving the DPPH and ABTS radical-scavenging abilitiesof the compounds

32 Reducing Ability of Piperine Derivatives -e reducingability is an important indicator to evaluate the antioxidantability of compounds In this experiment the method de-scribed by Oyaizu [27] was used to test the reducing ability ofthe compound -e principle of reducing ability determi-nation is that a reducing substance reacts with potassiumferricyanide (K3Fe(CN)6) to form potassium ferrocyanide(K4Fe(CN)6) which then reacts with ferric chloride to formPrunbullrsquos Blue (KFe[Fe(CN)6]) that has the maximumabsorbance at 700 nm -erefore the larger the absorbanceat 700 nm the stronger the reducing ability of the sample Itcan be seen from Figure 4 that all compounds were sig-nificantly concentration-dependent such that the reducingability at a high concentration (160 μM) was stronger than

Concentration (μM)

DPP

H-s

cave

ngin

g ac

tivity

()

0 20 40 60 80 100 120 140 160 180

0

20

40

60

80

100

lowast

Figure 2 DPPH radical-scavenging activity of 4a () 4b () 4c() 4d () 5a () 5b () 5c () 5d () piperine () and VC() lowastindicates a significant difference from the piperine group(plt 005)

Concentration (μM)

ABT

S-sc

aven

ging

activ

ity (

)

0 20 40 60 80

0

20

40

60

80

100

lowast

Figure 3 ABTS radical-scavenging activity of 4a () 4b () 4c() 4d () 5a () 5b () 5c () 5d () piperine () and VC() lowastindicates a significant difference from the piperine group(plt 005)

4a 4b 4c 4d 5a 5b 5c 5d

TBH

Q

Pipe

rine

00

02

04

06

08

10

12

Abso

rban

ce at

700

nm

lowast

lowastlowast lowast lowast

lowast lowast lowast

Figure 4 Measurement of total reducing ability 80 μM ( )160 μM ( ) lowastindicates a significant difference from the piperinegroup (plt 005) indicates a significant difference from the TBHQgroup (plt 005)

6 Journal of Chemistry

that at a low concentration (80 μM) -e experimental re-sults showed that the reductive ability of compounds 4a 4b4c and 4d was similar to that of piperine -e reducingstrength of the compound has a certain correlation with thenumber of active hydrogen atoms supplied by the com-pound Compounds 5a 5b 5c and 5d all had three activehydrogen atoms TBHQ had two active hydrogen atoms andpiperine had no active hydrogen atom From Figure 4 it canbe seen that at a concentration of 160 μM the absorbancesof 5a 5b 5c and 5d at 700 nm were 0999plusmn 00100880plusmn 0022 0866plusmn 0021 and 0860plusmn 0036 respectively-e absorbances of piperine and TBHQ were 0247plusmn 0015and 0261plusmn 0010 respectively -is showed that the re-ducing ability of piperine was comparable to that of TBHQwhich was not strong However compounds 5andash5d had arelatively strong reducing ability which was related to theirelectron-donating ability (hydrogen-donating ability)

33 Protective Effects of 5andash5d on AAPH-Induced DNAOxidation AAPH can react with oxygen in the air to produceperoxy radicals (ROO_) at a steady rate ROO_ can capture thehydrogen atom supplied by the C-4prime atom in DNA whichcould result in the DNAmolecule unwinding and generating avariety of carbonyl-containing small molecular substances[23 28] -ese substances can react with TBA to producethiobarbituric acid reactive species (TBARS λmax 535nm)under acidic conditions-erefore in this study the content ofcarbonyl-containing small-molecule compounds produced byAAPH-oxidized DNA was quantitatively calculated by testingthe absorbance of TBARS thereby tracking the extent ofAAPH-induced oxidative damage in DNA As shown in thecontrol experiment in Figure 5 in the process of DNA oxi-dation induced by AAPH the absorbance of TBARS increasedwith time indicating that as the reaction time increased morecarbonyl-containing small-molecule compounds were pro-duced as a result of AAPH-induced oxidative damage in DNAIt can be seen from Figure 6 that the addition of piperine didnot affect the increase in absorbance of TBARS in the above-mentioned system -is indicated that neither the potentialantioxidant units N-H nor the conjugated double bonds in thepiperine molecule could significantly inhibit AAPH-inducedoxidative damage in DNA However when the other fivederivatives were added to the above system the increase in theabsorbance of TBARS was significantly reduced -is showedpreliminarily that phenolic hydroxyl groups could protectDNA from AAPH-induced oxidative damage -e inhibitioneffect of compounds on AAPH-induced oxidative damage inDNA is shown in Figure 5 from which it can be seen that theinhibition effect of compounds 5a-5b onAAPH-induced DNAoxidative damage was concentration-dependent such that theinhibition rate at 160μM was higher than that at 80μM Inaddition as AAPH could generate ROO_ continuously in theoxidative damage process the inhibition rate of compounds5a-5b on DNA oxidative damage first increased and thendecreased with time and was the highest at 360min

34 Protective Effects of 5andash5d on AAPH-Induced HemolysisinErythrocytes In this study compounds 5andash5dwith strong

radical-scavenging ability were studied -e protective effectof derivatives on the AAPH-induced hemolysis of raterythrocytes was discussed It can be seen from Figure 7(a)that the erythrocyte suspension was stable and the hemolysisrate was 1445 in the PBS solution (pH 74) (control group)after incubation at 37degC for 3 h No obvious hemolysis wasobserved After adding AAPH under the same condition thehemolysis of erythrocytes increased to 6208 (AAPHgroup) -e significant increase in hemolysis showed thatAAPH could induce damage in erythrocytes thereby in-ducing hemolysis When a low concentration (20 μM) ofpiperine derivatives was added to the erythrocytes thehemolysis rates all decreased significantly (plt 005) andshowed significant concentration dependency such that thehigher the compound concentration the lower the hemo-lysis rate All four types of piperine derivatives reduced thehemolysis rate of erythrocytes to less than 50 Amongthem compound 5d showed the most prominent effect andreduced the hemolysis rate to 2590 after 3 h of incubation

-e protection time of 5a-5d on the AAPH-inducedhemolysis of erythrocytes is shown in Figure 7(b) It can beseen that the erythrocytes were stable in the PBS solutionand no significant change in the hemolysis rate was observedwith time -e hemolysis of the erythrocyte suspensioncaused by AAPH-induced oxidative damage showed a sig-nificant time effect such that the hemolysis rate increasedwith increasing damage time -e hemolysis rate increasedfrom 3664 to 7475 from 2 h to 4 h-is was because theantioxidant system of erythrocytes (GSH SOD CAT etc)could inhibit the oxidative damage from ROO_ in the initialstage However due to the consumption of antioxidants andthe increase of free radical chain reactions over time theerythrocyte membrane became damaged and the hemolysisoccurred rapidly -e addition of piperine derivatives couldeffectively suppress hemolysis As shown in the figure thehemolysis rate of the AAPH group was 3664 after 2 h ofincubation while the hemolysis rates of 5a 5b 5c and 5dgroups were reduced to 2808 3371 1899 and1492 respectively -erefore the piperine derivatives hada certain protective effect on hemolysis caused by oxidativedamage Among all groups compound 5d showed the besteffect before 3 h while compound 5a had the most signif-icant effect at 4 h

35 Effects of 5andash5d on AAPH-Induced HemoglobinOxidation in Erythrocytes Hemoglobin is the main proteinin erythrocytes Excessive free radicals can cause hemoglobinto oxidize and form methemoglobin thereby leading to thedisorder of erythrocyte function [20 29] -is study furtherdemonstrated the inhibitory effect of derivatives on oxi-dative damage in erythrocytes by examining the content ofmethemoglobin in erythrocytes -e effect of piperine de-rivatives against AAPH-induced hemoglobin oxidation inerythrocytes is shown in Figure 8 After 3 h of incubation(Figure 8(a)) the methemoglobin content in the normalcontrol group was low (174) whereas it was relatively high(2565) after oxidation by AAPH In addition as shown inFigure 8(b) the methemoglobin content in the normal

Journal of Chemistry 7

group was relatively stable while that in the AAPH groupcontinued to increase with time -us it can be seen thatAAPH could induce the continuous oxidation ofhemoglobin

It can be seen from Figure 8(a) that after 3 h of in-cubation the compounds showed some inhibitory effect onthe production of methemoglobin Among them

compounds 5c and 5d showed the most significant effectsA certain concentration effect was also observed such thatthe inhibitory effect increased with concentration In ad-dition as shown in Figure 8(b) the inhibitory effect of thecompounds on hemoglobin oxidation weakened with time-is was because AAPH could continuously release ROO_

over time

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

Abso

rban

ce at

535

nm

Time (h)

(a)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

Time (h)

(b)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

07

08

09

Time (h)

(c)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 1400Time (h)

01

02

03

04

05

06

07

08

09

(d)

Abso

rban

ce at

535

nm

200 400 600 800 1000 1200 140001

02

03

04

05

06

07

08

09

Time (h)

(e)

Figure 6 Variation in the absorbance of piperine derivatives in AAPH-induced oxidative damage in DNA over time 0 μM () 80 μM ()160 μM () (a) 5a (b) 5b (c) 5c (d) 5d (e) Piperine

0 360 480 1080

0

10

20

30

40

50

Time (min)

AA

PH-in

duce

d D

NA

dam

age i

nhib

ition

rate

() lowast

lowast

lowast

Figure 5 Inhibition effect of piperine derivatives on AAPH-induced oxidative damage in DNA 5a 160 μM ( ) 5a 80 μM ( ) 5b 160 μM( ) 5b 80 μM ( ) 5c 160 μM ( ) 5c 80 μM ( ) 5d 160 μM ( ) 5d 80 μM ( ) piperine 80 μM ( ) lowastindicates a significant differencefrom the piperine group (plt 005)

8 Journal of Chemistry

36 Influence of Piperine Derivatives on GSH-Px T-SOD andCATActivities in AAPH-Treated Erythrocytes GSH-Px is anantioxidant enzyme present in cells that catalyzes the re-duction of glutathione (GSH) and the removal of peroxidesin the body such as H2O2 in cells causing antilipid oxidation[30] As shown in Figure 9(a) the GSH-Px content inerythrocytes belonging to the control group was stable whilethat in erythrocytes of the AAPH group showed a statisti-cally significant gradual decrease with increasing incubationtime Following the addition of 50 μMof piperine derivativesto erythrocytes prior to the induction of oxidative damage byAAPH the piperine derivatives did not significantly influ-ence the erythrocyte GSH-Px content after 1 h of incubationcompared with the AAPH group After 2 h of incubationcompounds 5a and 5b significantly increased the GSH-Pxcontent (plt 005) after 4 h of incubation only the GSH-Pxcontent in the 5b and 5d groups was increased compared

with that in the AAPH group -ese results indicate that theamino acid derivatives of piperine can increase GSH-Pxactivity in erythrocytes Particularly compounds 5b and 5dproduced the strongest effects with the differences beingstatistically insignificant compared with the positive drugie vitamin C (VC) (pgt 005)

SOD is a key enzyme for removing free radicals withinthe body -e SOD activity level serves as an indicator of thebodyrsquos ability to remove free radicals [30 31] Figure 9(b)shows the influence of amino acid derivatives of piperine onT-SOD activity in AAPH-treated erythrocytes In the AAPHgroup with increasing incubation time T-SOD activitydecreased gradually Following the addition of 50 μM ofpiperine derivatives T-SOD activity increased after 1 h ofincubation albeit insignificantly compared with the AAPHgroup (pgt 005) After 2 h of incubation compounds 5a 5b5c and 5d increased T-SOD activity significantly (plt 005)

0

5

10

15

20

25

30

35

40

Met

Hb

()

Control AAPH 10 20Concentration (μM)

5

lowast

lowast

(a)

Time (h)

Met

Hb

()

2 3 40

10

20

30

40

50

(b)

Figure 8 (a) Protective effects of piperine derivatives on AAPH-induced hemoglobin oxidation (3 h) (b) Kinetic curve of the protectiveeffect of 20 μMpiperine derivatives on AAPH-induced hemoglobin oxidation Control ( ) AAPH ( ) AAPH+ 5a ( ) AAPH+ 5b ( )AAPH+ 5c ( ) AAPH+ 5d ( ) lowastindicates a significant difference from the control group (plt 005) indicates a significant differencefrom the control group (plt 005)

0

10

20

30

40

50

60

70

80H

emol

ysis

()

Control AAPH 10 20Concentration (μM)

5

lowast

lowast

(a)

Hem

olys

is (

)

0

20

40

60

80

100

Time (h)2 3 4

(b)

Figure 7 (a) Protective effect of piperine derivatives on AAPH-induced hemolysis of erythrocytes (3 h) (b) Kinetic curve of the protectiveeffect of piperine derivatives on AAPH-induced hemolysis of erythrocytes at 20 μM Control ( ) AAPH ( ) AAPH+ 5a ( ) AAPH+ 5b( ) AAPH+ 5c ( ) AAPH+ 5d ( ) lowastindicates a significant difference from the control group (plt 005) indicates a significantdifference from the AAPH group

Journal of Chemistry 9

while the positive control and prototype drug (piperine) hadno significant influence on erythrocyte T-SOD activity(pgt 005) After 4 h of incubation compound 5d increasedT-SOD activity significantly (pgt 005) while other com-pounds had no significant influence on T-SOD activity(pgt 005) -ese results highlight the significant enhance-ment caused by piperine derivatives of T-SOD activity inerythrocytes with AAPH-induced oxidative damage com-pared to the relatively inferior effect of the prototypecompound and of the positive drug VC

CAT an abundant enzyme in erythrocytes catalyzesH2O2 decomposition which reduces oxidative damagecaused by OHbull an oxidation product formed from H2O2under catalysis by metal ions [30 31] As shown inFigure 9(c) CAT activity in the AAPH group decreasesgradually relative to that in the control group with in-creasing incubation time Following the addition of 50 μMofpiperine derivatives compound 5c and piperine increasederythrocyte CATactivity after 1 h of incubation After 2 h ofincubation compounds 5a and 5b and piperine increasedCAT activity in erythrocytes significantly (plt 005) whilethe positive control had no significant influence on CAT

activity (pgt 005) After 4 h of incubation compound 5bincreased CAT activity significantly (pgt 005) while othercompounds had no significant influence (pgt 005) -eseresults indicate that piperine derivatives produce signifi-cant differences in CAT activity in AAPH-treated raterythrocytes

4 Conclusion

In the present study piperine-amino acid derivatives weresynthesized and the results indicate that the free radicalremoval ability and total reductive ability of the phenolichydroxyl-containing piperine-amino acid derivatives werehigher than those of the parent compound Specificallycompounds 5a and 5b have free radical removal perfor-mances comparable to that of VC-e derivatives have goodinhibitory effects on AAPH-induced oxidative DNA dam-age while certain compounds also show inhibitory effects onAAPH-induced hemolysis in rat erythrocytes Particularlycompound 5b can significantly inhibit AAPH-inducederythrocyte hemolysis and hemoglobin oxidation In addi-tion the experiments further verified that the protection

Time

GSH

-Px

(U)

1h 2h 4h0

100

200

300

400

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

(a)

Time

SOD

activ

ity (U

mgH

b)

1h 2h 4h0

5

10

15

20

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

(b)

Time1h 2h 4h

CAT

activ

ity (U

mgH

b)

0

1

2

3

4

5

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast lowast

lowast

lowast

lowast

lowast

(c)

Figure 9 Influence of piperine derivatives on the (a) GSH-Px content (b) T-SOD activity and (c) CATactivity in AAPH-treated rat erythrocyteslowastindicates a significant difference from the control group (plt 005) Control ( ) AAPH ( ) AAPH+5a ( ) AAPH+5b ( ) AAPH+5c( ) AAPH+5d ( ) AAPH+piperine ( ) AAPH+VC ( ) indicates a significant difference from the AAPH group (plt 005)

10 Journal of Chemistry

provided by piperine derivatives against AAPH-inducedoxidative damage can be achieved by preserving the activityof the antioxidant enzyme system (GSH-Px T-SOD andCAT) within cells -e protection by piperine derivativeswas also found to be superior to that by piperine and vitaminC -is paper described a structural modification methodthat could effectively improve the antioxidant ability ofpiperine-is type of compound could be a candidate for thedevelopment and research of oxidative damage-relateddiseases (hyperlipidemia diabetes cancer whitening etc)

Data Availability

-e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

-is work was financially supported by the Shaanxi Pro-vincial Department of Education serving local (industrial-ization) research project (no 15JF028) and the ScientificResearch Fund of Xirsquoan Medical University(nos2016YXXK09 2018DC-12 and 201825012)

References

[1] J P Adjimani and P Asare ldquoAntioxidant and free radicalscavenging activity of iron chelatorsrdquo Toxicology Reportsvol 2 pp 721ndash728 2015

[2] I Dalle-Donne R Rossi R Colombo D Giustarini andA Milzani ldquoBiomarkers of oxidative damage in humandiseaserdquo Clinical Chemistry vol 52 no 4 pp 601ndash623 2006

[3] L J Machlin and A Bendich ldquoFree radical tissue damageprotective role of antioxidant nutrientsrdquo Fe FASEB Journalvol 1 no 6 pp 441ndash445 1987

[4] N SatoW Li H Takemoto et al ldquoComprehensive evaluationof antioxidant effects of Japanese Kampo medicines led toidentification of Tsudosan formulation as a potent antioxidantagentrdquo Journal of Natural Medicines vol 73 no 1pp 163ndash172 2018

[5] N Bao Z Sun H Baigude G Borjihan and Z Jin ldquoLightinduced isomerization of piperlonguminine to scutifoliamideA isopiperlonguminine and hoffmannseggiamide Ardquo Phy-tochemistry Letters vol 16 pp 324ndash327 2016

[6] T Velpandian R Jasuja R K Bhardwaj J Jaiswal andS K Gupta ldquoPiperine in food interference in the pharma-cokinetics of phenytoinrdquo European Journal of Drug Meta-bolism and Pharmacokinetics vol 26 no 4 pp 241ndash247 2001

[7] A Duangjai K Ingkaninan S Praputbut andN Limpeanchob ldquoBlack pepper and piperine reduce cho-lesterol uptake and enhance translocation of cholesteroltransporter proteinsrdquo Journal of Natural Medicines vol 67no 2 pp 303ndash310 2013

[8] D E Eigenmann C Durig E A Jahne et al ldquoIn vitro blood-brain barrier permeability predictions for GABAA receptormodulating piperine analogsrdquo European Journal of Phar-maceutics and Biopharmaceutics vol 103 pp 118ndash126 2016

[9] Y A Samra H S Said N M Elsherbiny G I Liou M M El-Shishtawy and L A Eissa ldquoCepharanthine and Piperineameliorate diabetic nephropathy in rats role of NF-κB andNLRP3 inflammasomerdquo Life Sciences vol 157 pp 187ndash1992016

[10] R Mittal and R L Gupta ldquoIn vitro antioxidant activity ofpiperinerdquoMethods and Findings in Experimental and ClinicalPharmacology vol 22 no 5 pp 271ndash274 2000

[11] Z Zarai E Boujelbene N Ben Salem Y Gargouri andA Sayari ldquoAntioxidant and antimicrobial activities of varioussolvent extracts piperine and piperic acid from Piper nig-rumrdquo LWT-Food Science and Technology vol 50 no 2pp 634ndash641 2013

[12] A Yasir S Ishtiaq M Jahangir et al ldquoBiology-orientedsynthesis (BIOS) of piperine derivatives and their comparativeanalgesic and antiinflammatory activitiesrdquo MedicinalChemistry vol 14 no 3 pp 269ndash280 2018

[13] R S Vijayakumar D Surya and N Nalini ldquoAntioxidantefficacy of black pepper (Piper nigrumL) and piperine in ratswith high fat diet induced oxidative stressrdquo Redox Reportvol 9 no 2 pp 105ndash110 2004

[14] T Miyazawa K Nakagawa S H Kim et al ldquoCurcumin andpiperine supplementation of obese mice under caloric re-striction modulates body fat and interleukin-1βrdquo Nutrition ampMetabolism vol 15 no 1 p 12 2018

[15] A Khajuria N -usu U Zutshi and K L Bedi ldquoPiperinemodulation of carcinogen induced oxidative stress in intes-tinal mucosardquo Molecular and Cellular Biochemistry vol 189no 12 pp 113ndash118 1998

[16] S Inder Pal J Shreyans Kumar K Amandeep et al ldquoSyn-thesis and antileishmanial activity of piperoyl-amino acidconjugatesrdquo European Journal of Medicinal Chemistry vol 45no 8 pp 3439ndash3445 2010

[17] T Koichi M Takaki and S Yoshiaki ldquoSynthesis and bio-logical evaluation of piperic acid amides as free radicalscavengers and α-glucosidase inhibitorsrdquo Chemical amp Phar-maceutical Bulletin vol 63 no 5 pp 326ndash333 2015

[18] J M Latorres D G Rios G Saggiomo W Wasielesky andC Prentice-Hernandez ldquoFunctional and antioxidant prop-erties of protein hydrolysates obtained from white shrimp(Litopenaeus vannamei)rdquo Journal of Food Science and Tech-nology vol 55 no 2 pp 721ndash729 2018

[19] A Moayedi L Mora M C Aristoy M Safari M Hashemiand F Toldra ldquoPeptidomic analysis of antioxidant and ACE-inhibitory peptides obtained from tomato waste proteinsfermented using Bacillus subtilisrdquo Food Chemistry vol 250pp 180ndash187 2018

[20] L Zheng H Dong G Su Q Zhao and M Zhao ldquoRadicalscavenging activities of Tyr- Trp- Cys- andMet-Gly and theirprotective effects against AAPH-induced oxidative damage inhuman erythrocytesrdquo Food Chemistry vol 197 no Part App 807ndash813 2016

[21] M Miceli E Roma P Rosa et al ldquoSynthesis of benzofuran-2-one derivatives and evaluation of their antioxidant capacity bycomparing DPPH assay and cyclic voltammetryrdquo Moleculesvol 23 no 4 p 710 2018

[22] K -aipong U Boonprakob K Crosby et al ldquoComparisonof ABTS DPPH FRAP and ORAC assays for estimatingantioxidant activity from guava fruit extractsrdquo Journal of FoodComposition amp Analysis vol 19 no 6-7 pp 669ndash675 2006

[23] X-R Gong G-L Xi and Z-Q Liu ldquoActivity of coumarin-oxadiazole-appended phenol in inhibiting DNA oxidationand scavenging radicalrdquo Tetrahedron Letters vol 56 no 45pp 6257ndash6261 2015

Journal of Chemistry 11

[24] G-L Xi and Z-Q Liu ldquoCoumarin sharing the benzene ringwith quinoline for quenching radicals and inhibiting DNAoxidationrdquo European Journal of Medicinal Chemistry vol 95pp 416ndash423 2015

[25] H R Shin B R You and W H Park ldquoPX-12-induced HeLacell death is associated with oxidative stress and GSH de-pletionrdquo Oncology Letters vol 6 no 6 pp 1804ndash1810 2013

[26] S B Nimse D Pal A Mazumder et al ldquoSynthesis of cin-namanilide derivatives and their antioxidant and antimi-crobial activityrdquo Journal of Chemistry vol 2015 Article ID208910 5 pages 2015

[27] M Oyaizu ldquoStudies on products of browning reactionAntioxidative activities of products of browning reactionprepared from glucosaminerdquo Fe Japanese Journal of Nu-trition and Dietetics vol 44 no 6 pp 307ndash315 1986

[28] Y Yoshioka X Li T Zhang et al ldquoBlack soybean seed coatpolyphenols prevent AAPH-induced oxidative DNA-damagein HepG2 cellsrdquo Journal of Clinical Biochemistry and Nu-trition vol 60 no 2 pp 108ndash114 2017

[29] R C Chiste M Freitas A Z Mercadante and E FernandesldquoCarotenoids inhibit lipid peroxidation and hemoglobinoxidation but not the depletion of glutathione induced byROS in human erythrocytesrdquo Life Sciences vol 99 no 1-2pp 52ndash60 2014

[30] R Ozturk-Urek L A Bozkaya and L Tarhan ldquo-e effects ofsome antioxidant vitamin-and trace element-supplementeddiets on activities of SOD CAT GSH-Px and LPO levels inchicken tissuesrdquo Cell Biochemistry and Function vol 19 no 2pp 125ndash132 2001

[31] O Levy Y Achituv Y Z Yacobi N Stambler andZ Dubinsky ldquo-e impact of spectral composition and lightperiodicity on the activity of two antioxidant enzymes (SODand CAT) in the coral Favia favusrdquo Journal of ExperimentalMarine Biology and Ecology vol 328 no 1 pp 35ndash46 2006

12 Journal of Chemistry

(CH3)2) 13C NMR (101MHz DMSO-d6) δ 17310 1653914789 14770 13985 13812 13078 12513 12407 1226910840 10564 10123 5729 2983 1917 1810 ESI mz3181 [M+ 1]+

229 Piperine L-Leucinate Conjugate (4c C18H21NO5)-e synthesis process of compound 4c was similar to that ofcompound 4a -e product was a white solid (yield 857)1H NMR (400MHz DMSO-d6) δ 1253 (s 1H -COOH)831-829 (d 1H CHNHCO) 728 (d 1H CHCH-CO)720-714 (m 1H Ar-CH) 702-686 (m 4H Ar-H Ar-CHCH-) 619-615 (d 1H CHCH-CO) 605 (s 2H-OCH2O-) 435-430 (m 1H CONHCH) 167-161 (m 1HCH (CH3)2) 158-153 (m 2H CH2CH (CH3)2) 092-086(dd 6H CH (CH3)2) 13C NMR (101MHz DMSO-d6) δ17417 16518 14789 14771 13986 13813 13078 1251312394 12268 10840 10564 10122 5029 4000 24372282 2127 ESI mz 3321[M+ 1]+

2210 Piperine L-Methionine Conjugate (4d C17H19NO5S)-e synthesis process of compound 4d was similar to that ofcompound 4a -e product was a pale yellow solid (yield713) 1H NMR (400MHz DMSO-d6) δ 1271 (s 1H-COOH) 837-835 (d 1H CHNHCO) 729-728 (d 1HCHCH-CO) 721-715 (m 1H Ar-CH) 702-686 (m 4HAr-H Ar-CHCH-) 618-615 (d 1H CHCH-CO) 605 (s2H -OCH2O-) 444-443 (m 1H CONHCH) 206-186 (m5H SCH2 SCH3) 13C NMR (101MHz DMSO-d6) δ 1739116582 14839 14823 14049 13875 13127 12561 1243212322 10891 10613 10174 5154 3123 3022 1503 ESImz 3501[M+ 1]+

2211 ((2E4E)-5-(34-Dihydroxyphenyl)penta-24-dienoyl)Alanine (5a C14H15NO5) Compound 4a (200mg069mmol) was dispersed in 10mL of anhydrous DCM ADCM (5mL) solution of boron tribromide (2609 μL276mmol) was added dropwise to the reaction mixture inan ice-bath condition After 4 h the reaction was quenchedby adding 10mL of ice-cold saturated ammonium chloridesolution in an ice-bath condition After stirring at roomtemperature for 05 h ethyl acetate (3times15mL) was used forthe extraction -e organic phase was dried with anhydroussodium sulfate for 24 h and then filtered to obtain theconcentrated crude product -e crude product was thenpurified by column chromatography on silica gel (devel-oping agent DCM MeOH HAc 75 1 01) to afford3652mg of the product (5a) (yield 191) as an orangesolid 1H NMR (400MHz DMSO-d6) 983 (s 2H -OH)805-803 (d 1H CHNHCO) 717-711 (m 1H CHCH-CO) 695 (s 1H Ar-CH) 684-6671 (m 4H Ar-H Ar-CHCH-) 618-614 (d 1H CHCH-CO) 430-427 (m1H CONHCH) 128-126 (d 3H -CH3) ESI mz 27819[M+ 1]+

2212 ((2E4E)-5-(34-Dihydroxyphenyl)penta-24-dienoyl)Valine (5b C16H19NO5) -e synthesis process of com-pound 5b was similar to that of compound 5a -e product

was a dark orange solid (yield 123) 1H NMR (400MHzDMSO-d6) δ 976 (s 2H -OH) 791 (s 1H CHNHCO)717-711 (m 1H CHCH-CO) 696 (s 1H Ar-CH) 684-674 (m 4H Ar-H Ar-CHCH-) 629-626 (d 1HCHCH-CO) 422-419 (m 1H CONHCH) 210 (s 1HCH (CH3)2) 088-081 (m 6H CH (CH3)2) ESI mz 3201[M+ 1]+

2213 ((2E4E)-5-(34-Dihydroxyphenyl)penta-24-dienoyl)Leucine (5c C17H21NO5) -e synthesis process of com-pound 5c was similar to that of compound 5a -e productwas a dark orange solid (yield 293) 1H NMR (400MHzDMSO-d6) δ 931 (s 2H -OH) 823-821 (d 1HCHNHCO) 719-712 (m 1H CHCH-CO) 695 (d 1HAr-CH) 686-671 (m 4H Ar-H Ar-CHCH-) 614-610(d 1H CHCH-CO) 432-430 (m 1H CONHCH) 165-160 (m 1H CH (CH3)2) 156-152 (m 2H CH2CH(CH3)2) 091-085 (dd 6H CH (CH3)2) ESI mz 3202[M+ 1]+

2214 ((2E4E)-5-(34-Dihydroxyphenyl)penta-24-dienoyl)Methionine (5d C16H19NO5S) -e synthesis process ofcompound 5d was similar to that of compound 5a -eproduct was a dark green solid (yield 204) 1H NMR(400MHz DMSO-d6) δ 1269 (s 1H -COOH) 931 (s 1H-OH) 902 (s 1H -OH) 831-826 (m 1H CHNHCO) 720-714 (m 1H CHCH-CO) 695 (m 1H Ar-CH) 787-672(m 4H Ar-H Ar-CHCH-) 614-610 (d 1H CHCH-CO) 443-437 (m 1H CONHCH) 207-202 (m 5H SCH2SCH3) ESI mz 3382[M+ 1]+

23 ABTS Radical-Scavenging Properties -e ABTS radical-scavenging activities were assayed according to the methodreported by-aipong et al [22] (1) ABTS+ aqueous solution(7mmolL) preparation 30mg ABTS+ was weighed 8mL ofultrapure water was added and it was dissolved by ultra-sonication (2) K2S2O8 solution preparation 10mg K2S2O8was weighed 15mL of ultrapure water was added and it wasdissolved by ultrasonication (3) ABTS working solutionpreparation (1) and (2) were mixed at the ratio of 1 1 andkept in the dark for 12 to 16 h-e abovemixture was dilutedfour to eight times with 95 ethanol such that it had anabsorbance of ca 07 Abs at 734 nm to obtain the ABTSworking solution After keeping the 300 μL ABTS workingsolution and 100 μL ethanol solution of compounds (4andash4d5andash5d and piperine) with a certain concentration gradientin the dark for 30min their absorbances at 734 nm weremeasured -e experiment was repeated three times inparallel

24 DPPH Radical-Scavenging Properties -e DPPH radi-cal-scavenging activities were assayed according to themethod reported by Nimse et al [26] -e DPPH workingsolution was prepared by dissolving 20mg DPPH in 250mLof 95 ethanol and diluting it until its absorbance was ca 08at 517 nm After keeping 200 μL DPPHmiddot working solutionand 200 μL ethanol solution of compounds (4andash4d 5andash5d

4 Journal of Chemistry

and piperine) with a certain concentration gradient in thedark for 30min their absorbances at 517 nmwere measured-e experiment was repeated three times in parallel

25 Measurement of Total Reducing Ability Total reducingability of piperine derivatives was detected according to themethod reported by Oyaizu [27] First 100μL of PBS (02MpH 66) solution 100μL of 1 potassium ferricyanide solu-tion and 20μL of compound solution (1600μM 800μM) weremixed together -e mixed solution was then heated in a 50degCwater bath for 20min and cooled in an ice bath Subsequently10 TCA solution was added to quench the reaction -emixture was then centrifuged at 3000 rmin for 10min and200μL of the supernatant was taken to add in 200μLH2O and40μL 01 FeCl3 solution-e solutionwas allowed to stand for10min before measuring its absorbance at 700nm

26 Inhibiting DNA Oxidation -e ability of piperine de-rivatives to inhibit DNA oxidation was detected by Gonget alrsquos reported methods [23] A DNA solution (224mgmL) and an AAPH solution (400 μM) were prepared usingPBS1 (NaH2PO4 19mM Na2HPO4 81mM and EDTA10 μM) as the solvent First 804mL of DNA solution and60 μL of compound solution (24mM 12mM 0) were mixedtogether and 900 μL AAPH solution was added -e well-mixed solution was then poured into 18 tubes (400 μLtube)which were placed in a 37degC water bath for 30min 6 h and8 h respectively After incubation 200 μL of TBA solution(TBA 025 g NaOH 01 g final volume brought to 25mLusing double-distilled water) and 200 μL of TCA solution(TCA 075 g final volume brought to 25mL using double-distilled water) were added to the tubes which were wellshaken and heated in a boiling water bath for 15min -eywere then cooled in an ice bath Subsequently 300 μL of n-butanol was added and a vortex was used to extract theresulting TBARS (thiobarbituric acid reactive material)Finally the tubes were centrifuged at 1500 rmin for 3minand 100 μL of the supernatant was taken out for the ab-sorbance measurement at 535 nm

27 Erythrocyte Hemolysis Assay -e inhibition of piperinederivatives on AAPH-induced erythrocyte hemolysis wasevaluated by the method reported by Zheng et al [20] SD ratswere anesthetized by injecting 20 urethane intraperitone-ally Blood was sampled from the heart and was collected inheparinized tubes -e tubes were then centrifuged at3500 rmin for 5min to separate the erythrocytes which werewashed four times using centrifugal washing with PBS (pH74 containing 137mM NaCl 27mM KCl 81mMNa2HPO4 and 15mM KH2PO4) -e erythrocytes weredispersed with PBS solution to obtain a 10 erythrocytesuspension Subsequently 100 μL of a 10 erythrocyte sus-pension with 50 μL of compound solution or PBS was in-cubated at 37degC for 30min -en 100 μL PBS solution of200mMAAPHwas added and themixture was incubated fora certain period of time at 37degC For the control group theAAPH solution was substituted with PBS After incubation

200 μL of the reaction solution was diluted with 1mL of PBSand was centrifuged at 3500 rmin for 5min -e absorbance(APBS) of the supernatant was measured at 540 nm using amicroplate reader -e same volume of the reaction solutionwas diluted with 1mL of distilled water to obtain a completehemolysis solution -e supernatant absorbance (Awater) wasmeasured under the same condition -e hemolysis rate wascalculated according to the following equation

erythrocyte hemolysis () Apbs

Awatertimes 100 (1)

28 Measurement of Hemoglobin Oxidation -e inhibitionof piperine derivatives on hemoglobin oxidation was alsoevaluated by the method reported by Zheng et al [20] First200 μL of erythrocyte suspension was diluted with 1mL ofdistilled water and centrifuged at 3500 rmin for 5min -esupernatant absorbance was measured at 630 nm and700 nm and recorded as A630 and A700 respectively Inaddition the supernatant was treated with 10 μL of 5potassium ferricyanide and the absorbances at 630 nm and700 nm were measured which were recorded as Ahb630 andAhb700 respectively -e hemoglobin oxidation rate wascalculated according to the following equation

Hemoglobin oxidation () A630 minus A700

Ahb630 minus Ahb7001113890 1113891 times 100

(2)

29 Influence of Piperine Derivatives on the AntioxidantEnzyme System of AAPH-Treated Rat Erythrocytes -ecompound solution (50μL) was added to 100μL of a 10erythrocyte suspension and incubated at 37degC for 30min -en100μL of 200mmolL AAPH was added and the mixture wasfurther incubated at 37degC for 1ndash4h After incubation ultrapurewater precooled to 4degCwas added to lyse all the erythrocytes-eresultant mixture was centrifuged and the sediment was re-moved andwashed three times in PBS 990μL of double-distilledwater precooled to 4degC was then added to 10μL of theerythrocyte sediment and the mixture was stored in a minus80degCfreezer before use-e glutathione peroxidase (GSH-Px) contentand the total superoxide dismutase and catalase (CAT) activitieswere determined using assay kits in accordance with themanufacturerrsquos instructions

210 Statistical Analysis Each experiment was repeated atleast three times in parallel -e results were reported asaverageplusmn standard deviation (SD) SPSS version 190 (SPSSInc Chicago IL) was used for the statistical analysis -edata were analyzed using one-way ANOVA-e significanceof difference was determined by the LSD range test(plt 005)

3 Results and Discussion

31 Radical-Scavenging Activities of 4andash4d and 5andash5d De-termined inDPPHandABTSAssays -e radical-scavenging

Journal of Chemistry 5

ability of piperine derivatives (4andash4d and 5andash5d) andpiperine was evaluated using the DPPH and ABTS assays-e results are shown in Figure 2 It can be seen from thefigure that compounds 4andash4d had no significant scavengingeffect on the DPPH radicals below the concentration of160 μM and the scavenging rate was below 20 Amongthem 4a 4b and 4c showed a stronger scavenging abilitythan the prototype compound (piperine) at a high con-centration (plt 005) It can be seen that after the amino acidderivatization of piperine its DPPH radical-scavengingability improved however the effect was not obviousCompounds containing phenolic hydroxyl groups (5andash5d)were obtained by removing the acetal from compounds4andash4d It can be observed from Figure 2 that the phenolichydroxyl groups played a significant role in improving theDPPH radical-scavenging ability of the compounds At aconcentration of 160 μM the scavenging rates of DPPH ofthe four compounds namely 5a 5b 5c 5d and VC werenot significantly different at 800plusmn 107 781plusmn 036766plusmn 609 787plusmn 078 (plt 005) and 9334plusmn 142respectively -e values of IC50 for 5a 5b 5c 5d and VCwere 575 μM 653 μM 664 μM 809 μM and 5940 μMrespectively It can be seen that the DPPH radical-scav-enging effect of 5a is similar to that of VC in the concen-tration range of 10ndash160 μm

-e scavenging effect of piperine derivatives on ABTSradicals was also evaluated in this experiment It can be seenfrom Figure 3 that at concentrations of 25 to 80 μM thecompounds 4andash4d showed a slightly stronger ABTS radical-scavenging effect than the prototype compound piperinebut the improvement was not significant It can also be seenthat the ABTS radical-scavenging rates of compounds 5a5b 5c 5d and VC at 80 μM concentration were882plusmn 304 7944plusmn 275 7636plusmn 289 5426plusmn 508and 7850plusmn 549 (plt 005) respectively and the values ofIC50 were 427 μM 490 μM 518 μM 518 μM and 493 μMrespectively Among them compounds 5a and 5b showedthe most significant ABTS radical-scavenging abilitieswhich were slightly stronger than that of VC It can be seenthat the presence of phenolic hydroxyl groups in thestructure of piperine derivatives had a significant effect onimproving the DPPH and ABTS radical-scavenging abilitiesof the compounds

32 Reducing Ability of Piperine Derivatives -e reducingability is an important indicator to evaluate the antioxidantability of compounds In this experiment the method de-scribed by Oyaizu [27] was used to test the reducing ability ofthe compound -e principle of reducing ability determi-nation is that a reducing substance reacts with potassiumferricyanide (K3Fe(CN)6) to form potassium ferrocyanide(K4Fe(CN)6) which then reacts with ferric chloride to formPrunbullrsquos Blue (KFe[Fe(CN)6]) that has the maximumabsorbance at 700 nm -erefore the larger the absorbanceat 700 nm the stronger the reducing ability of the sample Itcan be seen from Figure 4 that all compounds were sig-nificantly concentration-dependent such that the reducingability at a high concentration (160 μM) was stronger than

Concentration (μM)

DPP

H-s

cave

ngin

g ac

tivity

()

0 20 40 60 80 100 120 140 160 180

0

20

40

60

80

100

lowast

Figure 2 DPPH radical-scavenging activity of 4a () 4b () 4c() 4d () 5a () 5b () 5c () 5d () piperine () and VC() lowastindicates a significant difference from the piperine group(plt 005)

Concentration (μM)

ABT

S-sc

aven

ging

activ

ity (

)

0 20 40 60 80

0

20

40

60

80

100

lowast

Figure 3 ABTS radical-scavenging activity of 4a () 4b () 4c() 4d () 5a () 5b () 5c () 5d () piperine () and VC() lowastindicates a significant difference from the piperine group(plt 005)

4a 4b 4c 4d 5a 5b 5c 5d

TBH

Q

Pipe

rine

00

02

04

06

08

10

12

Abso

rban

ce at

700

nm

lowast

lowastlowast lowast lowast

lowast lowast lowast

Figure 4 Measurement of total reducing ability 80 μM ( )160 μM ( ) lowastindicates a significant difference from the piperinegroup (plt 005) indicates a significant difference from the TBHQgroup (plt 005)

6 Journal of Chemistry

that at a low concentration (80 μM) -e experimental re-sults showed that the reductive ability of compounds 4a 4b4c and 4d was similar to that of piperine -e reducingstrength of the compound has a certain correlation with thenumber of active hydrogen atoms supplied by the com-pound Compounds 5a 5b 5c and 5d all had three activehydrogen atoms TBHQ had two active hydrogen atoms andpiperine had no active hydrogen atom From Figure 4 it canbe seen that at a concentration of 160 μM the absorbancesof 5a 5b 5c and 5d at 700 nm were 0999plusmn 00100880plusmn 0022 0866plusmn 0021 and 0860plusmn 0036 respectively-e absorbances of piperine and TBHQ were 0247plusmn 0015and 0261plusmn 0010 respectively -is showed that the re-ducing ability of piperine was comparable to that of TBHQwhich was not strong However compounds 5andash5d had arelatively strong reducing ability which was related to theirelectron-donating ability (hydrogen-donating ability)

33 Protective Effects of 5andash5d on AAPH-Induced DNAOxidation AAPH can react with oxygen in the air to produceperoxy radicals (ROO_) at a steady rate ROO_ can capture thehydrogen atom supplied by the C-4prime atom in DNA whichcould result in the DNAmolecule unwinding and generating avariety of carbonyl-containing small molecular substances[23 28] -ese substances can react with TBA to producethiobarbituric acid reactive species (TBARS λmax 535nm)under acidic conditions-erefore in this study the content ofcarbonyl-containing small-molecule compounds produced byAAPH-oxidized DNA was quantitatively calculated by testingthe absorbance of TBARS thereby tracking the extent ofAAPH-induced oxidative damage in DNA As shown in thecontrol experiment in Figure 5 in the process of DNA oxi-dation induced by AAPH the absorbance of TBARS increasedwith time indicating that as the reaction time increased morecarbonyl-containing small-molecule compounds were pro-duced as a result of AAPH-induced oxidative damage in DNAIt can be seen from Figure 6 that the addition of piperine didnot affect the increase in absorbance of TBARS in the above-mentioned system -is indicated that neither the potentialantioxidant units N-H nor the conjugated double bonds in thepiperine molecule could significantly inhibit AAPH-inducedoxidative damage in DNA However when the other fivederivatives were added to the above system the increase in theabsorbance of TBARS was significantly reduced -is showedpreliminarily that phenolic hydroxyl groups could protectDNA from AAPH-induced oxidative damage -e inhibitioneffect of compounds on AAPH-induced oxidative damage inDNA is shown in Figure 5 from which it can be seen that theinhibition effect of compounds 5a-5b onAAPH-induced DNAoxidative damage was concentration-dependent such that theinhibition rate at 160μM was higher than that at 80μM Inaddition as AAPH could generate ROO_ continuously in theoxidative damage process the inhibition rate of compounds5a-5b on DNA oxidative damage first increased and thendecreased with time and was the highest at 360min

34 Protective Effects of 5andash5d on AAPH-Induced HemolysisinErythrocytes In this study compounds 5andash5dwith strong

radical-scavenging ability were studied -e protective effectof derivatives on the AAPH-induced hemolysis of raterythrocytes was discussed It can be seen from Figure 7(a)that the erythrocyte suspension was stable and the hemolysisrate was 1445 in the PBS solution (pH 74) (control group)after incubation at 37degC for 3 h No obvious hemolysis wasobserved After adding AAPH under the same condition thehemolysis of erythrocytes increased to 6208 (AAPHgroup) -e significant increase in hemolysis showed thatAAPH could induce damage in erythrocytes thereby in-ducing hemolysis When a low concentration (20 μM) ofpiperine derivatives was added to the erythrocytes thehemolysis rates all decreased significantly (plt 005) andshowed significant concentration dependency such that thehigher the compound concentration the lower the hemo-lysis rate All four types of piperine derivatives reduced thehemolysis rate of erythrocytes to less than 50 Amongthem compound 5d showed the most prominent effect andreduced the hemolysis rate to 2590 after 3 h of incubation

-e protection time of 5a-5d on the AAPH-inducedhemolysis of erythrocytes is shown in Figure 7(b) It can beseen that the erythrocytes were stable in the PBS solutionand no significant change in the hemolysis rate was observedwith time -e hemolysis of the erythrocyte suspensioncaused by AAPH-induced oxidative damage showed a sig-nificant time effect such that the hemolysis rate increasedwith increasing damage time -e hemolysis rate increasedfrom 3664 to 7475 from 2 h to 4 h-is was because theantioxidant system of erythrocytes (GSH SOD CAT etc)could inhibit the oxidative damage from ROO_ in the initialstage However due to the consumption of antioxidants andthe increase of free radical chain reactions over time theerythrocyte membrane became damaged and the hemolysisoccurred rapidly -e addition of piperine derivatives couldeffectively suppress hemolysis As shown in the figure thehemolysis rate of the AAPH group was 3664 after 2 h ofincubation while the hemolysis rates of 5a 5b 5c and 5dgroups were reduced to 2808 3371 1899 and1492 respectively -erefore the piperine derivatives hada certain protective effect on hemolysis caused by oxidativedamage Among all groups compound 5d showed the besteffect before 3 h while compound 5a had the most signif-icant effect at 4 h

35 Effects of 5andash5d on AAPH-Induced HemoglobinOxidation in Erythrocytes Hemoglobin is the main proteinin erythrocytes Excessive free radicals can cause hemoglobinto oxidize and form methemoglobin thereby leading to thedisorder of erythrocyte function [20 29] -is study furtherdemonstrated the inhibitory effect of derivatives on oxi-dative damage in erythrocytes by examining the content ofmethemoglobin in erythrocytes -e effect of piperine de-rivatives against AAPH-induced hemoglobin oxidation inerythrocytes is shown in Figure 8 After 3 h of incubation(Figure 8(a)) the methemoglobin content in the normalcontrol group was low (174) whereas it was relatively high(2565) after oxidation by AAPH In addition as shown inFigure 8(b) the methemoglobin content in the normal

Journal of Chemistry 7

group was relatively stable while that in the AAPH groupcontinued to increase with time -us it can be seen thatAAPH could induce the continuous oxidation ofhemoglobin

It can be seen from Figure 8(a) that after 3 h of in-cubation the compounds showed some inhibitory effect onthe production of methemoglobin Among them

compounds 5c and 5d showed the most significant effectsA certain concentration effect was also observed such thatthe inhibitory effect increased with concentration In ad-dition as shown in Figure 8(b) the inhibitory effect of thecompounds on hemoglobin oxidation weakened with time-is was because AAPH could continuously release ROO_

over time

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

Abso

rban

ce at

535

nm

Time (h)

(a)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

Time (h)

(b)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

07

08

09

Time (h)

(c)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 1400Time (h)

01

02

03

04

05

06

07

08

09

(d)

Abso

rban

ce at

535

nm

200 400 600 800 1000 1200 140001

02

03

04

05

06

07

08

09

Time (h)

(e)

Figure 6 Variation in the absorbance of piperine derivatives in AAPH-induced oxidative damage in DNA over time 0 μM () 80 μM ()160 μM () (a) 5a (b) 5b (c) 5c (d) 5d (e) Piperine

0 360 480 1080

0

10

20

30

40

50

Time (min)

AA

PH-in

duce

d D

NA

dam

age i

nhib

ition

rate

() lowast

lowast

lowast

Figure 5 Inhibition effect of piperine derivatives on AAPH-induced oxidative damage in DNA 5a 160 μM ( ) 5a 80 μM ( ) 5b 160 μM( ) 5b 80 μM ( ) 5c 160 μM ( ) 5c 80 μM ( ) 5d 160 μM ( ) 5d 80 μM ( ) piperine 80 μM ( ) lowastindicates a significant differencefrom the piperine group (plt 005)

8 Journal of Chemistry

36 Influence of Piperine Derivatives on GSH-Px T-SOD andCATActivities in AAPH-Treated Erythrocytes GSH-Px is anantioxidant enzyme present in cells that catalyzes the re-duction of glutathione (GSH) and the removal of peroxidesin the body such as H2O2 in cells causing antilipid oxidation[30] As shown in Figure 9(a) the GSH-Px content inerythrocytes belonging to the control group was stable whilethat in erythrocytes of the AAPH group showed a statisti-cally significant gradual decrease with increasing incubationtime Following the addition of 50 μMof piperine derivativesto erythrocytes prior to the induction of oxidative damage byAAPH the piperine derivatives did not significantly influ-ence the erythrocyte GSH-Px content after 1 h of incubationcompared with the AAPH group After 2 h of incubationcompounds 5a and 5b significantly increased the GSH-Pxcontent (plt 005) after 4 h of incubation only the GSH-Pxcontent in the 5b and 5d groups was increased compared

with that in the AAPH group -ese results indicate that theamino acid derivatives of piperine can increase GSH-Pxactivity in erythrocytes Particularly compounds 5b and 5dproduced the strongest effects with the differences beingstatistically insignificant compared with the positive drugie vitamin C (VC) (pgt 005)

SOD is a key enzyme for removing free radicals withinthe body -e SOD activity level serves as an indicator of thebodyrsquos ability to remove free radicals [30 31] Figure 9(b)shows the influence of amino acid derivatives of piperine onT-SOD activity in AAPH-treated erythrocytes In the AAPHgroup with increasing incubation time T-SOD activitydecreased gradually Following the addition of 50 μM ofpiperine derivatives T-SOD activity increased after 1 h ofincubation albeit insignificantly compared with the AAPHgroup (pgt 005) After 2 h of incubation compounds 5a 5b5c and 5d increased T-SOD activity significantly (plt 005)

0

5

10

15

20

25

30

35

40

Met

Hb

()

Control AAPH 10 20Concentration (μM)

5

lowast

lowast

(a)

Time (h)

Met

Hb

()

2 3 40

10

20

30

40

50

(b)

Figure 8 (a) Protective effects of piperine derivatives on AAPH-induced hemoglobin oxidation (3 h) (b) Kinetic curve of the protectiveeffect of 20 μMpiperine derivatives on AAPH-induced hemoglobin oxidation Control ( ) AAPH ( ) AAPH+ 5a ( ) AAPH+ 5b ( )AAPH+ 5c ( ) AAPH+ 5d ( ) lowastindicates a significant difference from the control group (plt 005) indicates a significant differencefrom the control group (plt 005)

0

10

20

30

40

50

60

70

80H

emol

ysis

()

Control AAPH 10 20Concentration (μM)

5

lowast

lowast

(a)

Hem

olys

is (

)

0

20

40

60

80

100

Time (h)2 3 4

(b)

Figure 7 (a) Protective effect of piperine derivatives on AAPH-induced hemolysis of erythrocytes (3 h) (b) Kinetic curve of the protectiveeffect of piperine derivatives on AAPH-induced hemolysis of erythrocytes at 20 μM Control ( ) AAPH ( ) AAPH+ 5a ( ) AAPH+ 5b( ) AAPH+ 5c ( ) AAPH+ 5d ( ) lowastindicates a significant difference from the control group (plt 005) indicates a significantdifference from the AAPH group

Journal of Chemistry 9

while the positive control and prototype drug (piperine) hadno significant influence on erythrocyte T-SOD activity(pgt 005) After 4 h of incubation compound 5d increasedT-SOD activity significantly (pgt 005) while other com-pounds had no significant influence on T-SOD activity(pgt 005) -ese results highlight the significant enhance-ment caused by piperine derivatives of T-SOD activity inerythrocytes with AAPH-induced oxidative damage com-pared to the relatively inferior effect of the prototypecompound and of the positive drug VC

CAT an abundant enzyme in erythrocytes catalyzesH2O2 decomposition which reduces oxidative damagecaused by OHbull an oxidation product formed from H2O2under catalysis by metal ions [30 31] As shown inFigure 9(c) CAT activity in the AAPH group decreasesgradually relative to that in the control group with in-creasing incubation time Following the addition of 50 μMofpiperine derivatives compound 5c and piperine increasederythrocyte CATactivity after 1 h of incubation After 2 h ofincubation compounds 5a and 5b and piperine increasedCAT activity in erythrocytes significantly (plt 005) whilethe positive control had no significant influence on CAT

activity (pgt 005) After 4 h of incubation compound 5bincreased CAT activity significantly (pgt 005) while othercompounds had no significant influence (pgt 005) -eseresults indicate that piperine derivatives produce signifi-cant differences in CAT activity in AAPH-treated raterythrocytes

4 Conclusion

In the present study piperine-amino acid derivatives weresynthesized and the results indicate that the free radicalremoval ability and total reductive ability of the phenolichydroxyl-containing piperine-amino acid derivatives werehigher than those of the parent compound Specificallycompounds 5a and 5b have free radical removal perfor-mances comparable to that of VC-e derivatives have goodinhibitory effects on AAPH-induced oxidative DNA dam-age while certain compounds also show inhibitory effects onAAPH-induced hemolysis in rat erythrocytes Particularlycompound 5b can significantly inhibit AAPH-inducederythrocyte hemolysis and hemoglobin oxidation In addi-tion the experiments further verified that the protection

Time

GSH

-Px

(U)

1h 2h 4h0

100

200

300

400

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

(a)

Time

SOD

activ

ity (U

mgH

b)

1h 2h 4h0

5

10

15

20

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

(b)

Time1h 2h 4h

CAT

activ

ity (U

mgH

b)

0

1

2

3

4

5

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast lowast

lowast

lowast

lowast

lowast

(c)

Figure 9 Influence of piperine derivatives on the (a) GSH-Px content (b) T-SOD activity and (c) CATactivity in AAPH-treated rat erythrocyteslowastindicates a significant difference from the control group (plt 005) Control ( ) AAPH ( ) AAPH+5a ( ) AAPH+5b ( ) AAPH+5c( ) AAPH+5d ( ) AAPH+piperine ( ) AAPH+VC ( ) indicates a significant difference from the AAPH group (plt 005)

10 Journal of Chemistry

provided by piperine derivatives against AAPH-inducedoxidative damage can be achieved by preserving the activityof the antioxidant enzyme system (GSH-Px T-SOD andCAT) within cells -e protection by piperine derivativeswas also found to be superior to that by piperine and vitaminC -is paper described a structural modification methodthat could effectively improve the antioxidant ability ofpiperine-is type of compound could be a candidate for thedevelopment and research of oxidative damage-relateddiseases (hyperlipidemia diabetes cancer whitening etc)

Data Availability

-e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

-is work was financially supported by the Shaanxi Pro-vincial Department of Education serving local (industrial-ization) research project (no 15JF028) and the ScientificResearch Fund of Xirsquoan Medical University(nos2016YXXK09 2018DC-12 and 201825012)

References

[1] J P Adjimani and P Asare ldquoAntioxidant and free radicalscavenging activity of iron chelatorsrdquo Toxicology Reportsvol 2 pp 721ndash728 2015

[2] I Dalle-Donne R Rossi R Colombo D Giustarini andA Milzani ldquoBiomarkers of oxidative damage in humandiseaserdquo Clinical Chemistry vol 52 no 4 pp 601ndash623 2006

[3] L J Machlin and A Bendich ldquoFree radical tissue damageprotective role of antioxidant nutrientsrdquo Fe FASEB Journalvol 1 no 6 pp 441ndash445 1987

[4] N SatoW Li H Takemoto et al ldquoComprehensive evaluationof antioxidant effects of Japanese Kampo medicines led toidentification of Tsudosan formulation as a potent antioxidantagentrdquo Journal of Natural Medicines vol 73 no 1pp 163ndash172 2018

[5] N Bao Z Sun H Baigude G Borjihan and Z Jin ldquoLightinduced isomerization of piperlonguminine to scutifoliamideA isopiperlonguminine and hoffmannseggiamide Ardquo Phy-tochemistry Letters vol 16 pp 324ndash327 2016

[6] T Velpandian R Jasuja R K Bhardwaj J Jaiswal andS K Gupta ldquoPiperine in food interference in the pharma-cokinetics of phenytoinrdquo European Journal of Drug Meta-bolism and Pharmacokinetics vol 26 no 4 pp 241ndash247 2001

[7] A Duangjai K Ingkaninan S Praputbut andN Limpeanchob ldquoBlack pepper and piperine reduce cho-lesterol uptake and enhance translocation of cholesteroltransporter proteinsrdquo Journal of Natural Medicines vol 67no 2 pp 303ndash310 2013

[8] D E Eigenmann C Durig E A Jahne et al ldquoIn vitro blood-brain barrier permeability predictions for GABAA receptormodulating piperine analogsrdquo European Journal of Phar-maceutics and Biopharmaceutics vol 103 pp 118ndash126 2016

[9] Y A Samra H S Said N M Elsherbiny G I Liou M M El-Shishtawy and L A Eissa ldquoCepharanthine and Piperineameliorate diabetic nephropathy in rats role of NF-κB andNLRP3 inflammasomerdquo Life Sciences vol 157 pp 187ndash1992016

[10] R Mittal and R L Gupta ldquoIn vitro antioxidant activity ofpiperinerdquoMethods and Findings in Experimental and ClinicalPharmacology vol 22 no 5 pp 271ndash274 2000

[11] Z Zarai E Boujelbene N Ben Salem Y Gargouri andA Sayari ldquoAntioxidant and antimicrobial activities of varioussolvent extracts piperine and piperic acid from Piper nig-rumrdquo LWT-Food Science and Technology vol 50 no 2pp 634ndash641 2013

[12] A Yasir S Ishtiaq M Jahangir et al ldquoBiology-orientedsynthesis (BIOS) of piperine derivatives and their comparativeanalgesic and antiinflammatory activitiesrdquo MedicinalChemistry vol 14 no 3 pp 269ndash280 2018

[13] R S Vijayakumar D Surya and N Nalini ldquoAntioxidantefficacy of black pepper (Piper nigrumL) and piperine in ratswith high fat diet induced oxidative stressrdquo Redox Reportvol 9 no 2 pp 105ndash110 2004

[14] T Miyazawa K Nakagawa S H Kim et al ldquoCurcumin andpiperine supplementation of obese mice under caloric re-striction modulates body fat and interleukin-1βrdquo Nutrition ampMetabolism vol 15 no 1 p 12 2018

[15] A Khajuria N -usu U Zutshi and K L Bedi ldquoPiperinemodulation of carcinogen induced oxidative stress in intes-tinal mucosardquo Molecular and Cellular Biochemistry vol 189no 12 pp 113ndash118 1998

[16] S Inder Pal J Shreyans Kumar K Amandeep et al ldquoSyn-thesis and antileishmanial activity of piperoyl-amino acidconjugatesrdquo European Journal of Medicinal Chemistry vol 45no 8 pp 3439ndash3445 2010

[17] T Koichi M Takaki and S Yoshiaki ldquoSynthesis and bio-logical evaluation of piperic acid amides as free radicalscavengers and α-glucosidase inhibitorsrdquo Chemical amp Phar-maceutical Bulletin vol 63 no 5 pp 326ndash333 2015

[18] J M Latorres D G Rios G Saggiomo W Wasielesky andC Prentice-Hernandez ldquoFunctional and antioxidant prop-erties of protein hydrolysates obtained from white shrimp(Litopenaeus vannamei)rdquo Journal of Food Science and Tech-nology vol 55 no 2 pp 721ndash729 2018

[19] A Moayedi L Mora M C Aristoy M Safari M Hashemiand F Toldra ldquoPeptidomic analysis of antioxidant and ACE-inhibitory peptides obtained from tomato waste proteinsfermented using Bacillus subtilisrdquo Food Chemistry vol 250pp 180ndash187 2018

[20] L Zheng H Dong G Su Q Zhao and M Zhao ldquoRadicalscavenging activities of Tyr- Trp- Cys- andMet-Gly and theirprotective effects against AAPH-induced oxidative damage inhuman erythrocytesrdquo Food Chemistry vol 197 no Part App 807ndash813 2016

[21] M Miceli E Roma P Rosa et al ldquoSynthesis of benzofuran-2-one derivatives and evaluation of their antioxidant capacity bycomparing DPPH assay and cyclic voltammetryrdquo Moleculesvol 23 no 4 p 710 2018

[22] K -aipong U Boonprakob K Crosby et al ldquoComparisonof ABTS DPPH FRAP and ORAC assays for estimatingantioxidant activity from guava fruit extractsrdquo Journal of FoodComposition amp Analysis vol 19 no 6-7 pp 669ndash675 2006

[23] X-R Gong G-L Xi and Z-Q Liu ldquoActivity of coumarin-oxadiazole-appended phenol in inhibiting DNA oxidationand scavenging radicalrdquo Tetrahedron Letters vol 56 no 45pp 6257ndash6261 2015

Journal of Chemistry 11

[24] G-L Xi and Z-Q Liu ldquoCoumarin sharing the benzene ringwith quinoline for quenching radicals and inhibiting DNAoxidationrdquo European Journal of Medicinal Chemistry vol 95pp 416ndash423 2015

[25] H R Shin B R You and W H Park ldquoPX-12-induced HeLacell death is associated with oxidative stress and GSH de-pletionrdquo Oncology Letters vol 6 no 6 pp 1804ndash1810 2013

[26] S B Nimse D Pal A Mazumder et al ldquoSynthesis of cin-namanilide derivatives and their antioxidant and antimi-crobial activityrdquo Journal of Chemistry vol 2015 Article ID208910 5 pages 2015

[27] M Oyaizu ldquoStudies on products of browning reactionAntioxidative activities of products of browning reactionprepared from glucosaminerdquo Fe Japanese Journal of Nu-trition and Dietetics vol 44 no 6 pp 307ndash315 1986

[28] Y Yoshioka X Li T Zhang et al ldquoBlack soybean seed coatpolyphenols prevent AAPH-induced oxidative DNA-damagein HepG2 cellsrdquo Journal of Clinical Biochemistry and Nu-trition vol 60 no 2 pp 108ndash114 2017

[29] R C Chiste M Freitas A Z Mercadante and E FernandesldquoCarotenoids inhibit lipid peroxidation and hemoglobinoxidation but not the depletion of glutathione induced byROS in human erythrocytesrdquo Life Sciences vol 99 no 1-2pp 52ndash60 2014

[30] R Ozturk-Urek L A Bozkaya and L Tarhan ldquo-e effects ofsome antioxidant vitamin-and trace element-supplementeddiets on activities of SOD CAT GSH-Px and LPO levels inchicken tissuesrdquo Cell Biochemistry and Function vol 19 no 2pp 125ndash132 2001

[31] O Levy Y Achituv Y Z Yacobi N Stambler andZ Dubinsky ldquo-e impact of spectral composition and lightperiodicity on the activity of two antioxidant enzymes (SODand CAT) in the coral Favia favusrdquo Journal of ExperimentalMarine Biology and Ecology vol 328 no 1 pp 35ndash46 2006

12 Journal of Chemistry

and piperine) with a certain concentration gradient in thedark for 30min their absorbances at 517 nmwere measured-e experiment was repeated three times in parallel

25 Measurement of Total Reducing Ability Total reducingability of piperine derivatives was detected according to themethod reported by Oyaizu [27] First 100μL of PBS (02MpH 66) solution 100μL of 1 potassium ferricyanide solu-tion and 20μL of compound solution (1600μM 800μM) weremixed together -e mixed solution was then heated in a 50degCwater bath for 20min and cooled in an ice bath Subsequently10 TCA solution was added to quench the reaction -emixture was then centrifuged at 3000 rmin for 10min and200μL of the supernatant was taken to add in 200μLH2O and40μL 01 FeCl3 solution-e solutionwas allowed to stand for10min before measuring its absorbance at 700nm

26 Inhibiting DNA Oxidation -e ability of piperine de-rivatives to inhibit DNA oxidation was detected by Gonget alrsquos reported methods [23] A DNA solution (224mgmL) and an AAPH solution (400 μM) were prepared usingPBS1 (NaH2PO4 19mM Na2HPO4 81mM and EDTA10 μM) as the solvent First 804mL of DNA solution and60 μL of compound solution (24mM 12mM 0) were mixedtogether and 900 μL AAPH solution was added -e well-mixed solution was then poured into 18 tubes (400 μLtube)which were placed in a 37degC water bath for 30min 6 h and8 h respectively After incubation 200 μL of TBA solution(TBA 025 g NaOH 01 g final volume brought to 25mLusing double-distilled water) and 200 μL of TCA solution(TCA 075 g final volume brought to 25mL using double-distilled water) were added to the tubes which were wellshaken and heated in a boiling water bath for 15min -eywere then cooled in an ice bath Subsequently 300 μL of n-butanol was added and a vortex was used to extract theresulting TBARS (thiobarbituric acid reactive material)Finally the tubes were centrifuged at 1500 rmin for 3minand 100 μL of the supernatant was taken out for the ab-sorbance measurement at 535 nm

27 Erythrocyte Hemolysis Assay -e inhibition of piperinederivatives on AAPH-induced erythrocyte hemolysis wasevaluated by the method reported by Zheng et al [20] SD ratswere anesthetized by injecting 20 urethane intraperitone-ally Blood was sampled from the heart and was collected inheparinized tubes -e tubes were then centrifuged at3500 rmin for 5min to separate the erythrocytes which werewashed four times using centrifugal washing with PBS (pH74 containing 137mM NaCl 27mM KCl 81mMNa2HPO4 and 15mM KH2PO4) -e erythrocytes weredispersed with PBS solution to obtain a 10 erythrocytesuspension Subsequently 100 μL of a 10 erythrocyte sus-pension with 50 μL of compound solution or PBS was in-cubated at 37degC for 30min -en 100 μL PBS solution of200mMAAPHwas added and themixture was incubated fora certain period of time at 37degC For the control group theAAPH solution was substituted with PBS After incubation

200 μL of the reaction solution was diluted with 1mL of PBSand was centrifuged at 3500 rmin for 5min -e absorbance(APBS) of the supernatant was measured at 540 nm using amicroplate reader -e same volume of the reaction solutionwas diluted with 1mL of distilled water to obtain a completehemolysis solution -e supernatant absorbance (Awater) wasmeasured under the same condition -e hemolysis rate wascalculated according to the following equation

erythrocyte hemolysis () Apbs

Awatertimes 100 (1)

28 Measurement of Hemoglobin Oxidation -e inhibitionof piperine derivatives on hemoglobin oxidation was alsoevaluated by the method reported by Zheng et al [20] First200 μL of erythrocyte suspension was diluted with 1mL ofdistilled water and centrifuged at 3500 rmin for 5min -esupernatant absorbance was measured at 630 nm and700 nm and recorded as A630 and A700 respectively Inaddition the supernatant was treated with 10 μL of 5potassium ferricyanide and the absorbances at 630 nm and700 nm were measured which were recorded as Ahb630 andAhb700 respectively -e hemoglobin oxidation rate wascalculated according to the following equation

Hemoglobin oxidation () A630 minus A700

Ahb630 minus Ahb7001113890 1113891 times 100

(2)

29 Influence of Piperine Derivatives on the AntioxidantEnzyme System of AAPH-Treated Rat Erythrocytes -ecompound solution (50μL) was added to 100μL of a 10erythrocyte suspension and incubated at 37degC for 30min -en100μL of 200mmolL AAPH was added and the mixture wasfurther incubated at 37degC for 1ndash4h After incubation ultrapurewater precooled to 4degCwas added to lyse all the erythrocytes-eresultant mixture was centrifuged and the sediment was re-moved andwashed three times in PBS 990μL of double-distilledwater precooled to 4degC was then added to 10μL of theerythrocyte sediment and the mixture was stored in a minus80degCfreezer before use-e glutathione peroxidase (GSH-Px) contentand the total superoxide dismutase and catalase (CAT) activitieswere determined using assay kits in accordance with themanufacturerrsquos instructions

210 Statistical Analysis Each experiment was repeated atleast three times in parallel -e results were reported asaverageplusmn standard deviation (SD) SPSS version 190 (SPSSInc Chicago IL) was used for the statistical analysis -edata were analyzed using one-way ANOVA-e significanceof difference was determined by the LSD range test(plt 005)

3 Results and Discussion

31 Radical-Scavenging Activities of 4andash4d and 5andash5d De-termined inDPPHandABTSAssays -e radical-scavenging

Journal of Chemistry 5

ability of piperine derivatives (4andash4d and 5andash5d) andpiperine was evaluated using the DPPH and ABTS assays-e results are shown in Figure 2 It can be seen from thefigure that compounds 4andash4d had no significant scavengingeffect on the DPPH radicals below the concentration of160 μM and the scavenging rate was below 20 Amongthem 4a 4b and 4c showed a stronger scavenging abilitythan the prototype compound (piperine) at a high con-centration (plt 005) It can be seen that after the amino acidderivatization of piperine its DPPH radical-scavengingability improved however the effect was not obviousCompounds containing phenolic hydroxyl groups (5andash5d)were obtained by removing the acetal from compounds4andash4d It can be observed from Figure 2 that the phenolichydroxyl groups played a significant role in improving theDPPH radical-scavenging ability of the compounds At aconcentration of 160 μM the scavenging rates of DPPH ofthe four compounds namely 5a 5b 5c 5d and VC werenot significantly different at 800plusmn 107 781plusmn 036766plusmn 609 787plusmn 078 (plt 005) and 9334plusmn 142respectively -e values of IC50 for 5a 5b 5c 5d and VCwere 575 μM 653 μM 664 μM 809 μM and 5940 μMrespectively It can be seen that the DPPH radical-scav-enging effect of 5a is similar to that of VC in the concen-tration range of 10ndash160 μm

-e scavenging effect of piperine derivatives on ABTSradicals was also evaluated in this experiment It can be seenfrom Figure 3 that at concentrations of 25 to 80 μM thecompounds 4andash4d showed a slightly stronger ABTS radical-scavenging effect than the prototype compound piperinebut the improvement was not significant It can also be seenthat the ABTS radical-scavenging rates of compounds 5a5b 5c 5d and VC at 80 μM concentration were882plusmn 304 7944plusmn 275 7636plusmn 289 5426plusmn 508and 7850plusmn 549 (plt 005) respectively and the values ofIC50 were 427 μM 490 μM 518 μM 518 μM and 493 μMrespectively Among them compounds 5a and 5b showedthe most significant ABTS radical-scavenging abilitieswhich were slightly stronger than that of VC It can be seenthat the presence of phenolic hydroxyl groups in thestructure of piperine derivatives had a significant effect onimproving the DPPH and ABTS radical-scavenging abilitiesof the compounds

32 Reducing Ability of Piperine Derivatives -e reducingability is an important indicator to evaluate the antioxidantability of compounds In this experiment the method de-scribed by Oyaizu [27] was used to test the reducing ability ofthe compound -e principle of reducing ability determi-nation is that a reducing substance reacts with potassiumferricyanide (K3Fe(CN)6) to form potassium ferrocyanide(K4Fe(CN)6) which then reacts with ferric chloride to formPrunbullrsquos Blue (KFe[Fe(CN)6]) that has the maximumabsorbance at 700 nm -erefore the larger the absorbanceat 700 nm the stronger the reducing ability of the sample Itcan be seen from Figure 4 that all compounds were sig-nificantly concentration-dependent such that the reducingability at a high concentration (160 μM) was stronger than

Concentration (μM)

DPP

H-s

cave

ngin

g ac

tivity

()

0 20 40 60 80 100 120 140 160 180

0

20

40

60

80

100

lowast

Figure 2 DPPH radical-scavenging activity of 4a () 4b () 4c() 4d () 5a () 5b () 5c () 5d () piperine () and VC() lowastindicates a significant difference from the piperine group(plt 005)

Concentration (μM)

ABT

S-sc

aven

ging

activ

ity (

)

0 20 40 60 80

0

20

40

60

80

100

lowast

Figure 3 ABTS radical-scavenging activity of 4a () 4b () 4c() 4d () 5a () 5b () 5c () 5d () piperine () and VC() lowastindicates a significant difference from the piperine group(plt 005)

4a 4b 4c 4d 5a 5b 5c 5d

TBH

Q

Pipe

rine

00

02

04

06

08

10

12

Abso

rban

ce at

700

nm

lowast

lowastlowast lowast lowast

lowast lowast lowast

Figure 4 Measurement of total reducing ability 80 μM ( )160 μM ( ) lowastindicates a significant difference from the piperinegroup (plt 005) indicates a significant difference from the TBHQgroup (plt 005)

6 Journal of Chemistry

that at a low concentration (80 μM) -e experimental re-sults showed that the reductive ability of compounds 4a 4b4c and 4d was similar to that of piperine -e reducingstrength of the compound has a certain correlation with thenumber of active hydrogen atoms supplied by the com-pound Compounds 5a 5b 5c and 5d all had three activehydrogen atoms TBHQ had two active hydrogen atoms andpiperine had no active hydrogen atom From Figure 4 it canbe seen that at a concentration of 160 μM the absorbancesof 5a 5b 5c and 5d at 700 nm were 0999plusmn 00100880plusmn 0022 0866plusmn 0021 and 0860plusmn 0036 respectively-e absorbances of piperine and TBHQ were 0247plusmn 0015and 0261plusmn 0010 respectively -is showed that the re-ducing ability of piperine was comparable to that of TBHQwhich was not strong However compounds 5andash5d had arelatively strong reducing ability which was related to theirelectron-donating ability (hydrogen-donating ability)

33 Protective Effects of 5andash5d on AAPH-Induced DNAOxidation AAPH can react with oxygen in the air to produceperoxy radicals (ROO_) at a steady rate ROO_ can capture thehydrogen atom supplied by the C-4prime atom in DNA whichcould result in the DNAmolecule unwinding and generating avariety of carbonyl-containing small molecular substances[23 28] -ese substances can react with TBA to producethiobarbituric acid reactive species (TBARS λmax 535nm)under acidic conditions-erefore in this study the content ofcarbonyl-containing small-molecule compounds produced byAAPH-oxidized DNA was quantitatively calculated by testingthe absorbance of TBARS thereby tracking the extent ofAAPH-induced oxidative damage in DNA As shown in thecontrol experiment in Figure 5 in the process of DNA oxi-dation induced by AAPH the absorbance of TBARS increasedwith time indicating that as the reaction time increased morecarbonyl-containing small-molecule compounds were pro-duced as a result of AAPH-induced oxidative damage in DNAIt can be seen from Figure 6 that the addition of piperine didnot affect the increase in absorbance of TBARS in the above-mentioned system -is indicated that neither the potentialantioxidant units N-H nor the conjugated double bonds in thepiperine molecule could significantly inhibit AAPH-inducedoxidative damage in DNA However when the other fivederivatives were added to the above system the increase in theabsorbance of TBARS was significantly reduced -is showedpreliminarily that phenolic hydroxyl groups could protectDNA from AAPH-induced oxidative damage -e inhibitioneffect of compounds on AAPH-induced oxidative damage inDNA is shown in Figure 5 from which it can be seen that theinhibition effect of compounds 5a-5b onAAPH-induced DNAoxidative damage was concentration-dependent such that theinhibition rate at 160μM was higher than that at 80μM Inaddition as AAPH could generate ROO_ continuously in theoxidative damage process the inhibition rate of compounds5a-5b on DNA oxidative damage first increased and thendecreased with time and was the highest at 360min

34 Protective Effects of 5andash5d on AAPH-Induced HemolysisinErythrocytes In this study compounds 5andash5dwith strong

radical-scavenging ability were studied -e protective effectof derivatives on the AAPH-induced hemolysis of raterythrocytes was discussed It can be seen from Figure 7(a)that the erythrocyte suspension was stable and the hemolysisrate was 1445 in the PBS solution (pH 74) (control group)after incubation at 37degC for 3 h No obvious hemolysis wasobserved After adding AAPH under the same condition thehemolysis of erythrocytes increased to 6208 (AAPHgroup) -e significant increase in hemolysis showed thatAAPH could induce damage in erythrocytes thereby in-ducing hemolysis When a low concentration (20 μM) ofpiperine derivatives was added to the erythrocytes thehemolysis rates all decreased significantly (plt 005) andshowed significant concentration dependency such that thehigher the compound concentration the lower the hemo-lysis rate All four types of piperine derivatives reduced thehemolysis rate of erythrocytes to less than 50 Amongthem compound 5d showed the most prominent effect andreduced the hemolysis rate to 2590 after 3 h of incubation

-e protection time of 5a-5d on the AAPH-inducedhemolysis of erythrocytes is shown in Figure 7(b) It can beseen that the erythrocytes were stable in the PBS solutionand no significant change in the hemolysis rate was observedwith time -e hemolysis of the erythrocyte suspensioncaused by AAPH-induced oxidative damage showed a sig-nificant time effect such that the hemolysis rate increasedwith increasing damage time -e hemolysis rate increasedfrom 3664 to 7475 from 2 h to 4 h-is was because theantioxidant system of erythrocytes (GSH SOD CAT etc)could inhibit the oxidative damage from ROO_ in the initialstage However due to the consumption of antioxidants andthe increase of free radical chain reactions over time theerythrocyte membrane became damaged and the hemolysisoccurred rapidly -e addition of piperine derivatives couldeffectively suppress hemolysis As shown in the figure thehemolysis rate of the AAPH group was 3664 after 2 h ofincubation while the hemolysis rates of 5a 5b 5c and 5dgroups were reduced to 2808 3371 1899 and1492 respectively -erefore the piperine derivatives hada certain protective effect on hemolysis caused by oxidativedamage Among all groups compound 5d showed the besteffect before 3 h while compound 5a had the most signif-icant effect at 4 h

35 Effects of 5andash5d on AAPH-Induced HemoglobinOxidation in Erythrocytes Hemoglobin is the main proteinin erythrocytes Excessive free radicals can cause hemoglobinto oxidize and form methemoglobin thereby leading to thedisorder of erythrocyte function [20 29] -is study furtherdemonstrated the inhibitory effect of derivatives on oxi-dative damage in erythrocytes by examining the content ofmethemoglobin in erythrocytes -e effect of piperine de-rivatives against AAPH-induced hemoglobin oxidation inerythrocytes is shown in Figure 8 After 3 h of incubation(Figure 8(a)) the methemoglobin content in the normalcontrol group was low (174) whereas it was relatively high(2565) after oxidation by AAPH In addition as shown inFigure 8(b) the methemoglobin content in the normal

Journal of Chemistry 7

group was relatively stable while that in the AAPH groupcontinued to increase with time -us it can be seen thatAAPH could induce the continuous oxidation ofhemoglobin

It can be seen from Figure 8(a) that after 3 h of in-cubation the compounds showed some inhibitory effect onthe production of methemoglobin Among them

compounds 5c and 5d showed the most significant effectsA certain concentration effect was also observed such thatthe inhibitory effect increased with concentration In ad-dition as shown in Figure 8(b) the inhibitory effect of thecompounds on hemoglobin oxidation weakened with time-is was because AAPH could continuously release ROO_

over time

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

Abso

rban

ce at

535

nm

Time (h)

(a)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

Time (h)

(b)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

07

08

09

Time (h)

(c)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 1400Time (h)

01

02

03

04

05

06

07

08

09

(d)

Abso

rban

ce at

535

nm

200 400 600 800 1000 1200 140001

02

03

04

05

06

07

08

09

Time (h)

(e)

Figure 6 Variation in the absorbance of piperine derivatives in AAPH-induced oxidative damage in DNA over time 0 μM () 80 μM ()160 μM () (a) 5a (b) 5b (c) 5c (d) 5d (e) Piperine

0 360 480 1080

0

10

20

30

40

50

Time (min)

AA

PH-in

duce

d D

NA

dam

age i

nhib

ition

rate

() lowast

lowast

lowast

Figure 5 Inhibition effect of piperine derivatives on AAPH-induced oxidative damage in DNA 5a 160 μM ( ) 5a 80 μM ( ) 5b 160 μM( ) 5b 80 μM ( ) 5c 160 μM ( ) 5c 80 μM ( ) 5d 160 μM ( ) 5d 80 μM ( ) piperine 80 μM ( ) lowastindicates a significant differencefrom the piperine group (plt 005)

8 Journal of Chemistry

36 Influence of Piperine Derivatives on GSH-Px T-SOD andCATActivities in AAPH-Treated Erythrocytes GSH-Px is anantioxidant enzyme present in cells that catalyzes the re-duction of glutathione (GSH) and the removal of peroxidesin the body such as H2O2 in cells causing antilipid oxidation[30] As shown in Figure 9(a) the GSH-Px content inerythrocytes belonging to the control group was stable whilethat in erythrocytes of the AAPH group showed a statisti-cally significant gradual decrease with increasing incubationtime Following the addition of 50 μMof piperine derivativesto erythrocytes prior to the induction of oxidative damage byAAPH the piperine derivatives did not significantly influ-ence the erythrocyte GSH-Px content after 1 h of incubationcompared with the AAPH group After 2 h of incubationcompounds 5a and 5b significantly increased the GSH-Pxcontent (plt 005) after 4 h of incubation only the GSH-Pxcontent in the 5b and 5d groups was increased compared

with that in the AAPH group -ese results indicate that theamino acid derivatives of piperine can increase GSH-Pxactivity in erythrocytes Particularly compounds 5b and 5dproduced the strongest effects with the differences beingstatistically insignificant compared with the positive drugie vitamin C (VC) (pgt 005)

SOD is a key enzyme for removing free radicals withinthe body -e SOD activity level serves as an indicator of thebodyrsquos ability to remove free radicals [30 31] Figure 9(b)shows the influence of amino acid derivatives of piperine onT-SOD activity in AAPH-treated erythrocytes In the AAPHgroup with increasing incubation time T-SOD activitydecreased gradually Following the addition of 50 μM ofpiperine derivatives T-SOD activity increased after 1 h ofincubation albeit insignificantly compared with the AAPHgroup (pgt 005) After 2 h of incubation compounds 5a 5b5c and 5d increased T-SOD activity significantly (plt 005)

0

5

10

15

20

25

30

35

40

Met

Hb

()

Control AAPH 10 20Concentration (μM)

5

lowast

lowast

(a)

Time (h)

Met

Hb

()

2 3 40

10

20

30

40

50

(b)

Figure 8 (a) Protective effects of piperine derivatives on AAPH-induced hemoglobin oxidation (3 h) (b) Kinetic curve of the protectiveeffect of 20 μMpiperine derivatives on AAPH-induced hemoglobin oxidation Control ( ) AAPH ( ) AAPH+ 5a ( ) AAPH+ 5b ( )AAPH+ 5c ( ) AAPH+ 5d ( ) lowastindicates a significant difference from the control group (plt 005) indicates a significant differencefrom the control group (plt 005)

0

10

20

30

40

50

60

70

80H

emol

ysis

()

Control AAPH 10 20Concentration (μM)

5

lowast

lowast

(a)

Hem

olys

is (

)

0

20

40

60

80

100

Time (h)2 3 4

(b)

Figure 7 (a) Protective effect of piperine derivatives on AAPH-induced hemolysis of erythrocytes (3 h) (b) Kinetic curve of the protectiveeffect of piperine derivatives on AAPH-induced hemolysis of erythrocytes at 20 μM Control ( ) AAPH ( ) AAPH+ 5a ( ) AAPH+ 5b( ) AAPH+ 5c ( ) AAPH+ 5d ( ) lowastindicates a significant difference from the control group (plt 005) indicates a significantdifference from the AAPH group

Journal of Chemistry 9

while the positive control and prototype drug (piperine) hadno significant influence on erythrocyte T-SOD activity(pgt 005) After 4 h of incubation compound 5d increasedT-SOD activity significantly (pgt 005) while other com-pounds had no significant influence on T-SOD activity(pgt 005) -ese results highlight the significant enhance-ment caused by piperine derivatives of T-SOD activity inerythrocytes with AAPH-induced oxidative damage com-pared to the relatively inferior effect of the prototypecompound and of the positive drug VC

CAT an abundant enzyme in erythrocytes catalyzesH2O2 decomposition which reduces oxidative damagecaused by OHbull an oxidation product formed from H2O2under catalysis by metal ions [30 31] As shown inFigure 9(c) CAT activity in the AAPH group decreasesgradually relative to that in the control group with in-creasing incubation time Following the addition of 50 μMofpiperine derivatives compound 5c and piperine increasederythrocyte CATactivity after 1 h of incubation After 2 h ofincubation compounds 5a and 5b and piperine increasedCAT activity in erythrocytes significantly (plt 005) whilethe positive control had no significant influence on CAT

activity (pgt 005) After 4 h of incubation compound 5bincreased CAT activity significantly (pgt 005) while othercompounds had no significant influence (pgt 005) -eseresults indicate that piperine derivatives produce signifi-cant differences in CAT activity in AAPH-treated raterythrocytes

4 Conclusion

In the present study piperine-amino acid derivatives weresynthesized and the results indicate that the free radicalremoval ability and total reductive ability of the phenolichydroxyl-containing piperine-amino acid derivatives werehigher than those of the parent compound Specificallycompounds 5a and 5b have free radical removal perfor-mances comparable to that of VC-e derivatives have goodinhibitory effects on AAPH-induced oxidative DNA dam-age while certain compounds also show inhibitory effects onAAPH-induced hemolysis in rat erythrocytes Particularlycompound 5b can significantly inhibit AAPH-inducederythrocyte hemolysis and hemoglobin oxidation In addi-tion the experiments further verified that the protection

Time

GSH

-Px

(U)

1h 2h 4h0

100

200

300

400

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

(a)

Time

SOD

activ

ity (U

mgH

b)

1h 2h 4h0

5

10

15

20

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

(b)

Time1h 2h 4h

CAT

activ

ity (U

mgH

b)

0

1

2

3

4

5

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast lowast

lowast

lowast

lowast

lowast

(c)

Figure 9 Influence of piperine derivatives on the (a) GSH-Px content (b) T-SOD activity and (c) CATactivity in AAPH-treated rat erythrocyteslowastindicates a significant difference from the control group (plt 005) Control ( ) AAPH ( ) AAPH+5a ( ) AAPH+5b ( ) AAPH+5c( ) AAPH+5d ( ) AAPH+piperine ( ) AAPH+VC ( ) indicates a significant difference from the AAPH group (plt 005)

10 Journal of Chemistry

provided by piperine derivatives against AAPH-inducedoxidative damage can be achieved by preserving the activityof the antioxidant enzyme system (GSH-Px T-SOD andCAT) within cells -e protection by piperine derivativeswas also found to be superior to that by piperine and vitaminC -is paper described a structural modification methodthat could effectively improve the antioxidant ability ofpiperine-is type of compound could be a candidate for thedevelopment and research of oxidative damage-relateddiseases (hyperlipidemia diabetes cancer whitening etc)

Data Availability

-e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

-is work was financially supported by the Shaanxi Pro-vincial Department of Education serving local (industrial-ization) research project (no 15JF028) and the ScientificResearch Fund of Xirsquoan Medical University(nos2016YXXK09 2018DC-12 and 201825012)

References

[1] J P Adjimani and P Asare ldquoAntioxidant and free radicalscavenging activity of iron chelatorsrdquo Toxicology Reportsvol 2 pp 721ndash728 2015

[2] I Dalle-Donne R Rossi R Colombo D Giustarini andA Milzani ldquoBiomarkers of oxidative damage in humandiseaserdquo Clinical Chemistry vol 52 no 4 pp 601ndash623 2006

[3] L J Machlin and A Bendich ldquoFree radical tissue damageprotective role of antioxidant nutrientsrdquo Fe FASEB Journalvol 1 no 6 pp 441ndash445 1987

[4] N SatoW Li H Takemoto et al ldquoComprehensive evaluationof antioxidant effects of Japanese Kampo medicines led toidentification of Tsudosan formulation as a potent antioxidantagentrdquo Journal of Natural Medicines vol 73 no 1pp 163ndash172 2018

[5] N Bao Z Sun H Baigude G Borjihan and Z Jin ldquoLightinduced isomerization of piperlonguminine to scutifoliamideA isopiperlonguminine and hoffmannseggiamide Ardquo Phy-tochemistry Letters vol 16 pp 324ndash327 2016

[6] T Velpandian R Jasuja R K Bhardwaj J Jaiswal andS K Gupta ldquoPiperine in food interference in the pharma-cokinetics of phenytoinrdquo European Journal of Drug Meta-bolism and Pharmacokinetics vol 26 no 4 pp 241ndash247 2001

[7] A Duangjai K Ingkaninan S Praputbut andN Limpeanchob ldquoBlack pepper and piperine reduce cho-lesterol uptake and enhance translocation of cholesteroltransporter proteinsrdquo Journal of Natural Medicines vol 67no 2 pp 303ndash310 2013

[8] D E Eigenmann C Durig E A Jahne et al ldquoIn vitro blood-brain barrier permeability predictions for GABAA receptormodulating piperine analogsrdquo European Journal of Phar-maceutics and Biopharmaceutics vol 103 pp 118ndash126 2016

[9] Y A Samra H S Said N M Elsherbiny G I Liou M M El-Shishtawy and L A Eissa ldquoCepharanthine and Piperineameliorate diabetic nephropathy in rats role of NF-κB andNLRP3 inflammasomerdquo Life Sciences vol 157 pp 187ndash1992016

[10] R Mittal and R L Gupta ldquoIn vitro antioxidant activity ofpiperinerdquoMethods and Findings in Experimental and ClinicalPharmacology vol 22 no 5 pp 271ndash274 2000

[11] Z Zarai E Boujelbene N Ben Salem Y Gargouri andA Sayari ldquoAntioxidant and antimicrobial activities of varioussolvent extracts piperine and piperic acid from Piper nig-rumrdquo LWT-Food Science and Technology vol 50 no 2pp 634ndash641 2013

[12] A Yasir S Ishtiaq M Jahangir et al ldquoBiology-orientedsynthesis (BIOS) of piperine derivatives and their comparativeanalgesic and antiinflammatory activitiesrdquo MedicinalChemistry vol 14 no 3 pp 269ndash280 2018

[13] R S Vijayakumar D Surya and N Nalini ldquoAntioxidantefficacy of black pepper (Piper nigrumL) and piperine in ratswith high fat diet induced oxidative stressrdquo Redox Reportvol 9 no 2 pp 105ndash110 2004

[14] T Miyazawa K Nakagawa S H Kim et al ldquoCurcumin andpiperine supplementation of obese mice under caloric re-striction modulates body fat and interleukin-1βrdquo Nutrition ampMetabolism vol 15 no 1 p 12 2018

[15] A Khajuria N -usu U Zutshi and K L Bedi ldquoPiperinemodulation of carcinogen induced oxidative stress in intes-tinal mucosardquo Molecular and Cellular Biochemistry vol 189no 12 pp 113ndash118 1998

[16] S Inder Pal J Shreyans Kumar K Amandeep et al ldquoSyn-thesis and antileishmanial activity of piperoyl-amino acidconjugatesrdquo European Journal of Medicinal Chemistry vol 45no 8 pp 3439ndash3445 2010

[17] T Koichi M Takaki and S Yoshiaki ldquoSynthesis and bio-logical evaluation of piperic acid amides as free radicalscavengers and α-glucosidase inhibitorsrdquo Chemical amp Phar-maceutical Bulletin vol 63 no 5 pp 326ndash333 2015

[18] J M Latorres D G Rios G Saggiomo W Wasielesky andC Prentice-Hernandez ldquoFunctional and antioxidant prop-erties of protein hydrolysates obtained from white shrimp(Litopenaeus vannamei)rdquo Journal of Food Science and Tech-nology vol 55 no 2 pp 721ndash729 2018

[19] A Moayedi L Mora M C Aristoy M Safari M Hashemiand F Toldra ldquoPeptidomic analysis of antioxidant and ACE-inhibitory peptides obtained from tomato waste proteinsfermented using Bacillus subtilisrdquo Food Chemistry vol 250pp 180ndash187 2018

[20] L Zheng H Dong G Su Q Zhao and M Zhao ldquoRadicalscavenging activities of Tyr- Trp- Cys- andMet-Gly and theirprotective effects against AAPH-induced oxidative damage inhuman erythrocytesrdquo Food Chemistry vol 197 no Part App 807ndash813 2016

[21] M Miceli E Roma P Rosa et al ldquoSynthesis of benzofuran-2-one derivatives and evaluation of their antioxidant capacity bycomparing DPPH assay and cyclic voltammetryrdquo Moleculesvol 23 no 4 p 710 2018

[22] K -aipong U Boonprakob K Crosby et al ldquoComparisonof ABTS DPPH FRAP and ORAC assays for estimatingantioxidant activity from guava fruit extractsrdquo Journal of FoodComposition amp Analysis vol 19 no 6-7 pp 669ndash675 2006

[23] X-R Gong G-L Xi and Z-Q Liu ldquoActivity of coumarin-oxadiazole-appended phenol in inhibiting DNA oxidationand scavenging radicalrdquo Tetrahedron Letters vol 56 no 45pp 6257ndash6261 2015

Journal of Chemistry 11

[24] G-L Xi and Z-Q Liu ldquoCoumarin sharing the benzene ringwith quinoline for quenching radicals and inhibiting DNAoxidationrdquo European Journal of Medicinal Chemistry vol 95pp 416ndash423 2015

[25] H R Shin B R You and W H Park ldquoPX-12-induced HeLacell death is associated with oxidative stress and GSH de-pletionrdquo Oncology Letters vol 6 no 6 pp 1804ndash1810 2013

[26] S B Nimse D Pal A Mazumder et al ldquoSynthesis of cin-namanilide derivatives and their antioxidant and antimi-crobial activityrdquo Journal of Chemistry vol 2015 Article ID208910 5 pages 2015

[27] M Oyaizu ldquoStudies on products of browning reactionAntioxidative activities of products of browning reactionprepared from glucosaminerdquo Fe Japanese Journal of Nu-trition and Dietetics vol 44 no 6 pp 307ndash315 1986

[28] Y Yoshioka X Li T Zhang et al ldquoBlack soybean seed coatpolyphenols prevent AAPH-induced oxidative DNA-damagein HepG2 cellsrdquo Journal of Clinical Biochemistry and Nu-trition vol 60 no 2 pp 108ndash114 2017

[29] R C Chiste M Freitas A Z Mercadante and E FernandesldquoCarotenoids inhibit lipid peroxidation and hemoglobinoxidation but not the depletion of glutathione induced byROS in human erythrocytesrdquo Life Sciences vol 99 no 1-2pp 52ndash60 2014

[30] R Ozturk-Urek L A Bozkaya and L Tarhan ldquo-e effects ofsome antioxidant vitamin-and trace element-supplementeddiets on activities of SOD CAT GSH-Px and LPO levels inchicken tissuesrdquo Cell Biochemistry and Function vol 19 no 2pp 125ndash132 2001

[31] O Levy Y Achituv Y Z Yacobi N Stambler andZ Dubinsky ldquo-e impact of spectral composition and lightperiodicity on the activity of two antioxidant enzymes (SODand CAT) in the coral Favia favusrdquo Journal of ExperimentalMarine Biology and Ecology vol 328 no 1 pp 35ndash46 2006

12 Journal of Chemistry

ability of piperine derivatives (4andash4d and 5andash5d) andpiperine was evaluated using the DPPH and ABTS assays-e results are shown in Figure 2 It can be seen from thefigure that compounds 4andash4d had no significant scavengingeffect on the DPPH radicals below the concentration of160 μM and the scavenging rate was below 20 Amongthem 4a 4b and 4c showed a stronger scavenging abilitythan the prototype compound (piperine) at a high con-centration (plt 005) It can be seen that after the amino acidderivatization of piperine its DPPH radical-scavengingability improved however the effect was not obviousCompounds containing phenolic hydroxyl groups (5andash5d)were obtained by removing the acetal from compounds4andash4d It can be observed from Figure 2 that the phenolichydroxyl groups played a significant role in improving theDPPH radical-scavenging ability of the compounds At aconcentration of 160 μM the scavenging rates of DPPH ofthe four compounds namely 5a 5b 5c 5d and VC werenot significantly different at 800plusmn 107 781plusmn 036766plusmn 609 787plusmn 078 (plt 005) and 9334plusmn 142respectively -e values of IC50 for 5a 5b 5c 5d and VCwere 575 μM 653 μM 664 μM 809 μM and 5940 μMrespectively It can be seen that the DPPH radical-scav-enging effect of 5a is similar to that of VC in the concen-tration range of 10ndash160 μm

-e scavenging effect of piperine derivatives on ABTSradicals was also evaluated in this experiment It can be seenfrom Figure 3 that at concentrations of 25 to 80 μM thecompounds 4andash4d showed a slightly stronger ABTS radical-scavenging effect than the prototype compound piperinebut the improvement was not significant It can also be seenthat the ABTS radical-scavenging rates of compounds 5a5b 5c 5d and VC at 80 μM concentration were882plusmn 304 7944plusmn 275 7636plusmn 289 5426plusmn 508and 7850plusmn 549 (plt 005) respectively and the values ofIC50 were 427 μM 490 μM 518 μM 518 μM and 493 μMrespectively Among them compounds 5a and 5b showedthe most significant ABTS radical-scavenging abilitieswhich were slightly stronger than that of VC It can be seenthat the presence of phenolic hydroxyl groups in thestructure of piperine derivatives had a significant effect onimproving the DPPH and ABTS radical-scavenging abilitiesof the compounds

32 Reducing Ability of Piperine Derivatives -e reducingability is an important indicator to evaluate the antioxidantability of compounds In this experiment the method de-scribed by Oyaizu [27] was used to test the reducing ability ofthe compound -e principle of reducing ability determi-nation is that a reducing substance reacts with potassiumferricyanide (K3Fe(CN)6) to form potassium ferrocyanide(K4Fe(CN)6) which then reacts with ferric chloride to formPrunbullrsquos Blue (KFe[Fe(CN)6]) that has the maximumabsorbance at 700 nm -erefore the larger the absorbanceat 700 nm the stronger the reducing ability of the sample Itcan be seen from Figure 4 that all compounds were sig-nificantly concentration-dependent such that the reducingability at a high concentration (160 μM) was stronger than

Concentration (μM)

DPP

H-s

cave

ngin

g ac

tivity

()

0 20 40 60 80 100 120 140 160 180

0

20

40

60

80

100

lowast

Figure 2 DPPH radical-scavenging activity of 4a () 4b () 4c() 4d () 5a () 5b () 5c () 5d () piperine () and VC() lowastindicates a significant difference from the piperine group(plt 005)

Concentration (μM)

ABT

S-sc

aven

ging

activ

ity (

)

0 20 40 60 80

0

20

40

60

80

100

lowast

Figure 3 ABTS radical-scavenging activity of 4a () 4b () 4c() 4d () 5a () 5b () 5c () 5d () piperine () and VC() lowastindicates a significant difference from the piperine group(plt 005)

4a 4b 4c 4d 5a 5b 5c 5d

TBH

Q

Pipe

rine

00

02

04

06

08

10

12

Abso

rban

ce at

700

nm

lowast

lowastlowast lowast lowast

lowast lowast lowast

Figure 4 Measurement of total reducing ability 80 μM ( )160 μM ( ) lowastindicates a significant difference from the piperinegroup (plt 005) indicates a significant difference from the TBHQgroup (plt 005)

6 Journal of Chemistry

that at a low concentration (80 μM) -e experimental re-sults showed that the reductive ability of compounds 4a 4b4c and 4d was similar to that of piperine -e reducingstrength of the compound has a certain correlation with thenumber of active hydrogen atoms supplied by the com-pound Compounds 5a 5b 5c and 5d all had three activehydrogen atoms TBHQ had two active hydrogen atoms andpiperine had no active hydrogen atom From Figure 4 it canbe seen that at a concentration of 160 μM the absorbancesof 5a 5b 5c and 5d at 700 nm were 0999plusmn 00100880plusmn 0022 0866plusmn 0021 and 0860plusmn 0036 respectively-e absorbances of piperine and TBHQ were 0247plusmn 0015and 0261plusmn 0010 respectively -is showed that the re-ducing ability of piperine was comparable to that of TBHQwhich was not strong However compounds 5andash5d had arelatively strong reducing ability which was related to theirelectron-donating ability (hydrogen-donating ability)

33 Protective Effects of 5andash5d on AAPH-Induced DNAOxidation AAPH can react with oxygen in the air to produceperoxy radicals (ROO_) at a steady rate ROO_ can capture thehydrogen atom supplied by the C-4prime atom in DNA whichcould result in the DNAmolecule unwinding and generating avariety of carbonyl-containing small molecular substances[23 28] -ese substances can react with TBA to producethiobarbituric acid reactive species (TBARS λmax 535nm)under acidic conditions-erefore in this study the content ofcarbonyl-containing small-molecule compounds produced byAAPH-oxidized DNA was quantitatively calculated by testingthe absorbance of TBARS thereby tracking the extent ofAAPH-induced oxidative damage in DNA As shown in thecontrol experiment in Figure 5 in the process of DNA oxi-dation induced by AAPH the absorbance of TBARS increasedwith time indicating that as the reaction time increased morecarbonyl-containing small-molecule compounds were pro-duced as a result of AAPH-induced oxidative damage in DNAIt can be seen from Figure 6 that the addition of piperine didnot affect the increase in absorbance of TBARS in the above-mentioned system -is indicated that neither the potentialantioxidant units N-H nor the conjugated double bonds in thepiperine molecule could significantly inhibit AAPH-inducedoxidative damage in DNA However when the other fivederivatives were added to the above system the increase in theabsorbance of TBARS was significantly reduced -is showedpreliminarily that phenolic hydroxyl groups could protectDNA from AAPH-induced oxidative damage -e inhibitioneffect of compounds on AAPH-induced oxidative damage inDNA is shown in Figure 5 from which it can be seen that theinhibition effect of compounds 5a-5b onAAPH-induced DNAoxidative damage was concentration-dependent such that theinhibition rate at 160μM was higher than that at 80μM Inaddition as AAPH could generate ROO_ continuously in theoxidative damage process the inhibition rate of compounds5a-5b on DNA oxidative damage first increased and thendecreased with time and was the highest at 360min

34 Protective Effects of 5andash5d on AAPH-Induced HemolysisinErythrocytes In this study compounds 5andash5dwith strong

radical-scavenging ability were studied -e protective effectof derivatives on the AAPH-induced hemolysis of raterythrocytes was discussed It can be seen from Figure 7(a)that the erythrocyte suspension was stable and the hemolysisrate was 1445 in the PBS solution (pH 74) (control group)after incubation at 37degC for 3 h No obvious hemolysis wasobserved After adding AAPH under the same condition thehemolysis of erythrocytes increased to 6208 (AAPHgroup) -e significant increase in hemolysis showed thatAAPH could induce damage in erythrocytes thereby in-ducing hemolysis When a low concentration (20 μM) ofpiperine derivatives was added to the erythrocytes thehemolysis rates all decreased significantly (plt 005) andshowed significant concentration dependency such that thehigher the compound concentration the lower the hemo-lysis rate All four types of piperine derivatives reduced thehemolysis rate of erythrocytes to less than 50 Amongthem compound 5d showed the most prominent effect andreduced the hemolysis rate to 2590 after 3 h of incubation

-e protection time of 5a-5d on the AAPH-inducedhemolysis of erythrocytes is shown in Figure 7(b) It can beseen that the erythrocytes were stable in the PBS solutionand no significant change in the hemolysis rate was observedwith time -e hemolysis of the erythrocyte suspensioncaused by AAPH-induced oxidative damage showed a sig-nificant time effect such that the hemolysis rate increasedwith increasing damage time -e hemolysis rate increasedfrom 3664 to 7475 from 2 h to 4 h-is was because theantioxidant system of erythrocytes (GSH SOD CAT etc)could inhibit the oxidative damage from ROO_ in the initialstage However due to the consumption of antioxidants andthe increase of free radical chain reactions over time theerythrocyte membrane became damaged and the hemolysisoccurred rapidly -e addition of piperine derivatives couldeffectively suppress hemolysis As shown in the figure thehemolysis rate of the AAPH group was 3664 after 2 h ofincubation while the hemolysis rates of 5a 5b 5c and 5dgroups were reduced to 2808 3371 1899 and1492 respectively -erefore the piperine derivatives hada certain protective effect on hemolysis caused by oxidativedamage Among all groups compound 5d showed the besteffect before 3 h while compound 5a had the most signif-icant effect at 4 h

35 Effects of 5andash5d on AAPH-Induced HemoglobinOxidation in Erythrocytes Hemoglobin is the main proteinin erythrocytes Excessive free radicals can cause hemoglobinto oxidize and form methemoglobin thereby leading to thedisorder of erythrocyte function [20 29] -is study furtherdemonstrated the inhibitory effect of derivatives on oxi-dative damage in erythrocytes by examining the content ofmethemoglobin in erythrocytes -e effect of piperine de-rivatives against AAPH-induced hemoglobin oxidation inerythrocytes is shown in Figure 8 After 3 h of incubation(Figure 8(a)) the methemoglobin content in the normalcontrol group was low (174) whereas it was relatively high(2565) after oxidation by AAPH In addition as shown inFigure 8(b) the methemoglobin content in the normal

Journal of Chemistry 7

group was relatively stable while that in the AAPH groupcontinued to increase with time -us it can be seen thatAAPH could induce the continuous oxidation ofhemoglobin

It can be seen from Figure 8(a) that after 3 h of in-cubation the compounds showed some inhibitory effect onthe production of methemoglobin Among them

compounds 5c and 5d showed the most significant effectsA certain concentration effect was also observed such thatthe inhibitory effect increased with concentration In ad-dition as shown in Figure 8(b) the inhibitory effect of thecompounds on hemoglobin oxidation weakened with time-is was because AAPH could continuously release ROO_

over time

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

Abso

rban

ce at

535

nm

Time (h)

(a)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

Time (h)

(b)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

07

08

09

Time (h)

(c)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 1400Time (h)

01

02

03

04

05

06

07

08

09

(d)

Abso

rban

ce at

535

nm

200 400 600 800 1000 1200 140001

02

03

04

05

06

07

08

09

Time (h)

(e)

Figure 6 Variation in the absorbance of piperine derivatives in AAPH-induced oxidative damage in DNA over time 0 μM () 80 μM ()160 μM () (a) 5a (b) 5b (c) 5c (d) 5d (e) Piperine

0 360 480 1080

0

10

20

30

40

50

Time (min)

AA

PH-in

duce

d D

NA

dam

age i

nhib

ition

rate

() lowast

lowast

lowast

Figure 5 Inhibition effect of piperine derivatives on AAPH-induced oxidative damage in DNA 5a 160 μM ( ) 5a 80 μM ( ) 5b 160 μM( ) 5b 80 μM ( ) 5c 160 μM ( ) 5c 80 μM ( ) 5d 160 μM ( ) 5d 80 μM ( ) piperine 80 μM ( ) lowastindicates a significant differencefrom the piperine group (plt 005)

8 Journal of Chemistry

36 Influence of Piperine Derivatives on GSH-Px T-SOD andCATActivities in AAPH-Treated Erythrocytes GSH-Px is anantioxidant enzyme present in cells that catalyzes the re-duction of glutathione (GSH) and the removal of peroxidesin the body such as H2O2 in cells causing antilipid oxidation[30] As shown in Figure 9(a) the GSH-Px content inerythrocytes belonging to the control group was stable whilethat in erythrocytes of the AAPH group showed a statisti-cally significant gradual decrease with increasing incubationtime Following the addition of 50 μMof piperine derivativesto erythrocytes prior to the induction of oxidative damage byAAPH the piperine derivatives did not significantly influ-ence the erythrocyte GSH-Px content after 1 h of incubationcompared with the AAPH group After 2 h of incubationcompounds 5a and 5b significantly increased the GSH-Pxcontent (plt 005) after 4 h of incubation only the GSH-Pxcontent in the 5b and 5d groups was increased compared

with that in the AAPH group -ese results indicate that theamino acid derivatives of piperine can increase GSH-Pxactivity in erythrocytes Particularly compounds 5b and 5dproduced the strongest effects with the differences beingstatistically insignificant compared with the positive drugie vitamin C (VC) (pgt 005)

SOD is a key enzyme for removing free radicals withinthe body -e SOD activity level serves as an indicator of thebodyrsquos ability to remove free radicals [30 31] Figure 9(b)shows the influence of amino acid derivatives of piperine onT-SOD activity in AAPH-treated erythrocytes In the AAPHgroup with increasing incubation time T-SOD activitydecreased gradually Following the addition of 50 μM ofpiperine derivatives T-SOD activity increased after 1 h ofincubation albeit insignificantly compared with the AAPHgroup (pgt 005) After 2 h of incubation compounds 5a 5b5c and 5d increased T-SOD activity significantly (plt 005)

0

5

10

15

20

25

30

35

40

Met

Hb

()

Control AAPH 10 20Concentration (μM)

5

lowast

lowast

(a)

Time (h)

Met

Hb

()

2 3 40

10

20

30

40

50

(b)

Figure 8 (a) Protective effects of piperine derivatives on AAPH-induced hemoglobin oxidation (3 h) (b) Kinetic curve of the protectiveeffect of 20 μMpiperine derivatives on AAPH-induced hemoglobin oxidation Control ( ) AAPH ( ) AAPH+ 5a ( ) AAPH+ 5b ( )AAPH+ 5c ( ) AAPH+ 5d ( ) lowastindicates a significant difference from the control group (plt 005) indicates a significant differencefrom the control group (plt 005)

0

10

20

30

40

50

60

70

80H

emol

ysis

()

Control AAPH 10 20Concentration (μM)

5

lowast

lowast

(a)

Hem

olys

is (

)

0

20

40

60

80

100

Time (h)2 3 4

(b)

Figure 7 (a) Protective effect of piperine derivatives on AAPH-induced hemolysis of erythrocytes (3 h) (b) Kinetic curve of the protectiveeffect of piperine derivatives on AAPH-induced hemolysis of erythrocytes at 20 μM Control ( ) AAPH ( ) AAPH+ 5a ( ) AAPH+ 5b( ) AAPH+ 5c ( ) AAPH+ 5d ( ) lowastindicates a significant difference from the control group (plt 005) indicates a significantdifference from the AAPH group

Journal of Chemistry 9

while the positive control and prototype drug (piperine) hadno significant influence on erythrocyte T-SOD activity(pgt 005) After 4 h of incubation compound 5d increasedT-SOD activity significantly (pgt 005) while other com-pounds had no significant influence on T-SOD activity(pgt 005) -ese results highlight the significant enhance-ment caused by piperine derivatives of T-SOD activity inerythrocytes with AAPH-induced oxidative damage com-pared to the relatively inferior effect of the prototypecompound and of the positive drug VC

CAT an abundant enzyme in erythrocytes catalyzesH2O2 decomposition which reduces oxidative damagecaused by OHbull an oxidation product formed from H2O2under catalysis by metal ions [30 31] As shown inFigure 9(c) CAT activity in the AAPH group decreasesgradually relative to that in the control group with in-creasing incubation time Following the addition of 50 μMofpiperine derivatives compound 5c and piperine increasederythrocyte CATactivity after 1 h of incubation After 2 h ofincubation compounds 5a and 5b and piperine increasedCAT activity in erythrocytes significantly (plt 005) whilethe positive control had no significant influence on CAT

activity (pgt 005) After 4 h of incubation compound 5bincreased CAT activity significantly (pgt 005) while othercompounds had no significant influence (pgt 005) -eseresults indicate that piperine derivatives produce signifi-cant differences in CAT activity in AAPH-treated raterythrocytes

4 Conclusion

In the present study piperine-amino acid derivatives weresynthesized and the results indicate that the free radicalremoval ability and total reductive ability of the phenolichydroxyl-containing piperine-amino acid derivatives werehigher than those of the parent compound Specificallycompounds 5a and 5b have free radical removal perfor-mances comparable to that of VC-e derivatives have goodinhibitory effects on AAPH-induced oxidative DNA dam-age while certain compounds also show inhibitory effects onAAPH-induced hemolysis in rat erythrocytes Particularlycompound 5b can significantly inhibit AAPH-inducederythrocyte hemolysis and hemoglobin oxidation In addi-tion the experiments further verified that the protection

Time

GSH

-Px

(U)

1h 2h 4h0

100

200

300

400

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

(a)

Time

SOD

activ

ity (U

mgH

b)

1h 2h 4h0

5

10

15

20

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

(b)

Time1h 2h 4h

CAT

activ

ity (U

mgH

b)

0

1

2

3

4

5

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast lowast

lowast

lowast

lowast

lowast

(c)

Figure 9 Influence of piperine derivatives on the (a) GSH-Px content (b) T-SOD activity and (c) CATactivity in AAPH-treated rat erythrocyteslowastindicates a significant difference from the control group (plt 005) Control ( ) AAPH ( ) AAPH+5a ( ) AAPH+5b ( ) AAPH+5c( ) AAPH+5d ( ) AAPH+piperine ( ) AAPH+VC ( ) indicates a significant difference from the AAPH group (plt 005)

10 Journal of Chemistry

provided by piperine derivatives against AAPH-inducedoxidative damage can be achieved by preserving the activityof the antioxidant enzyme system (GSH-Px T-SOD andCAT) within cells -e protection by piperine derivativeswas also found to be superior to that by piperine and vitaminC -is paper described a structural modification methodthat could effectively improve the antioxidant ability ofpiperine-is type of compound could be a candidate for thedevelopment and research of oxidative damage-relateddiseases (hyperlipidemia diabetes cancer whitening etc)

Data Availability

-e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

-is work was financially supported by the Shaanxi Pro-vincial Department of Education serving local (industrial-ization) research project (no 15JF028) and the ScientificResearch Fund of Xirsquoan Medical University(nos2016YXXK09 2018DC-12 and 201825012)

References

[1] J P Adjimani and P Asare ldquoAntioxidant and free radicalscavenging activity of iron chelatorsrdquo Toxicology Reportsvol 2 pp 721ndash728 2015

[2] I Dalle-Donne R Rossi R Colombo D Giustarini andA Milzani ldquoBiomarkers of oxidative damage in humandiseaserdquo Clinical Chemistry vol 52 no 4 pp 601ndash623 2006

[3] L J Machlin and A Bendich ldquoFree radical tissue damageprotective role of antioxidant nutrientsrdquo Fe FASEB Journalvol 1 no 6 pp 441ndash445 1987

[4] N SatoW Li H Takemoto et al ldquoComprehensive evaluationof antioxidant effects of Japanese Kampo medicines led toidentification of Tsudosan formulation as a potent antioxidantagentrdquo Journal of Natural Medicines vol 73 no 1pp 163ndash172 2018

[5] N Bao Z Sun H Baigude G Borjihan and Z Jin ldquoLightinduced isomerization of piperlonguminine to scutifoliamideA isopiperlonguminine and hoffmannseggiamide Ardquo Phy-tochemistry Letters vol 16 pp 324ndash327 2016

[6] T Velpandian R Jasuja R K Bhardwaj J Jaiswal andS K Gupta ldquoPiperine in food interference in the pharma-cokinetics of phenytoinrdquo European Journal of Drug Meta-bolism and Pharmacokinetics vol 26 no 4 pp 241ndash247 2001

[7] A Duangjai K Ingkaninan S Praputbut andN Limpeanchob ldquoBlack pepper and piperine reduce cho-lesterol uptake and enhance translocation of cholesteroltransporter proteinsrdquo Journal of Natural Medicines vol 67no 2 pp 303ndash310 2013

[8] D E Eigenmann C Durig E A Jahne et al ldquoIn vitro blood-brain barrier permeability predictions for GABAA receptormodulating piperine analogsrdquo European Journal of Phar-maceutics and Biopharmaceutics vol 103 pp 118ndash126 2016

[9] Y A Samra H S Said N M Elsherbiny G I Liou M M El-Shishtawy and L A Eissa ldquoCepharanthine and Piperineameliorate diabetic nephropathy in rats role of NF-κB andNLRP3 inflammasomerdquo Life Sciences vol 157 pp 187ndash1992016

[10] R Mittal and R L Gupta ldquoIn vitro antioxidant activity ofpiperinerdquoMethods and Findings in Experimental and ClinicalPharmacology vol 22 no 5 pp 271ndash274 2000

[11] Z Zarai E Boujelbene N Ben Salem Y Gargouri andA Sayari ldquoAntioxidant and antimicrobial activities of varioussolvent extracts piperine and piperic acid from Piper nig-rumrdquo LWT-Food Science and Technology vol 50 no 2pp 634ndash641 2013

[12] A Yasir S Ishtiaq M Jahangir et al ldquoBiology-orientedsynthesis (BIOS) of piperine derivatives and their comparativeanalgesic and antiinflammatory activitiesrdquo MedicinalChemistry vol 14 no 3 pp 269ndash280 2018

[13] R S Vijayakumar D Surya and N Nalini ldquoAntioxidantefficacy of black pepper (Piper nigrumL) and piperine in ratswith high fat diet induced oxidative stressrdquo Redox Reportvol 9 no 2 pp 105ndash110 2004

[14] T Miyazawa K Nakagawa S H Kim et al ldquoCurcumin andpiperine supplementation of obese mice under caloric re-striction modulates body fat and interleukin-1βrdquo Nutrition ampMetabolism vol 15 no 1 p 12 2018

[15] A Khajuria N -usu U Zutshi and K L Bedi ldquoPiperinemodulation of carcinogen induced oxidative stress in intes-tinal mucosardquo Molecular and Cellular Biochemistry vol 189no 12 pp 113ndash118 1998

[16] S Inder Pal J Shreyans Kumar K Amandeep et al ldquoSyn-thesis and antileishmanial activity of piperoyl-amino acidconjugatesrdquo European Journal of Medicinal Chemistry vol 45no 8 pp 3439ndash3445 2010

[17] T Koichi M Takaki and S Yoshiaki ldquoSynthesis and bio-logical evaluation of piperic acid amides as free radicalscavengers and α-glucosidase inhibitorsrdquo Chemical amp Phar-maceutical Bulletin vol 63 no 5 pp 326ndash333 2015

[18] J M Latorres D G Rios G Saggiomo W Wasielesky andC Prentice-Hernandez ldquoFunctional and antioxidant prop-erties of protein hydrolysates obtained from white shrimp(Litopenaeus vannamei)rdquo Journal of Food Science and Tech-nology vol 55 no 2 pp 721ndash729 2018

[19] A Moayedi L Mora M C Aristoy M Safari M Hashemiand F Toldra ldquoPeptidomic analysis of antioxidant and ACE-inhibitory peptides obtained from tomato waste proteinsfermented using Bacillus subtilisrdquo Food Chemistry vol 250pp 180ndash187 2018

[20] L Zheng H Dong G Su Q Zhao and M Zhao ldquoRadicalscavenging activities of Tyr- Trp- Cys- andMet-Gly and theirprotective effects against AAPH-induced oxidative damage inhuman erythrocytesrdquo Food Chemistry vol 197 no Part App 807ndash813 2016

[21] M Miceli E Roma P Rosa et al ldquoSynthesis of benzofuran-2-one derivatives and evaluation of their antioxidant capacity bycomparing DPPH assay and cyclic voltammetryrdquo Moleculesvol 23 no 4 p 710 2018

[22] K -aipong U Boonprakob K Crosby et al ldquoComparisonof ABTS DPPH FRAP and ORAC assays for estimatingantioxidant activity from guava fruit extractsrdquo Journal of FoodComposition amp Analysis vol 19 no 6-7 pp 669ndash675 2006

[23] X-R Gong G-L Xi and Z-Q Liu ldquoActivity of coumarin-oxadiazole-appended phenol in inhibiting DNA oxidationand scavenging radicalrdquo Tetrahedron Letters vol 56 no 45pp 6257ndash6261 2015

Journal of Chemistry 11

[24] G-L Xi and Z-Q Liu ldquoCoumarin sharing the benzene ringwith quinoline for quenching radicals and inhibiting DNAoxidationrdquo European Journal of Medicinal Chemistry vol 95pp 416ndash423 2015

[25] H R Shin B R You and W H Park ldquoPX-12-induced HeLacell death is associated with oxidative stress and GSH de-pletionrdquo Oncology Letters vol 6 no 6 pp 1804ndash1810 2013

[26] S B Nimse D Pal A Mazumder et al ldquoSynthesis of cin-namanilide derivatives and their antioxidant and antimi-crobial activityrdquo Journal of Chemistry vol 2015 Article ID208910 5 pages 2015

[27] M Oyaizu ldquoStudies on products of browning reactionAntioxidative activities of products of browning reactionprepared from glucosaminerdquo Fe Japanese Journal of Nu-trition and Dietetics vol 44 no 6 pp 307ndash315 1986

[28] Y Yoshioka X Li T Zhang et al ldquoBlack soybean seed coatpolyphenols prevent AAPH-induced oxidative DNA-damagein HepG2 cellsrdquo Journal of Clinical Biochemistry and Nu-trition vol 60 no 2 pp 108ndash114 2017

[29] R C Chiste M Freitas A Z Mercadante and E FernandesldquoCarotenoids inhibit lipid peroxidation and hemoglobinoxidation but not the depletion of glutathione induced byROS in human erythrocytesrdquo Life Sciences vol 99 no 1-2pp 52ndash60 2014

[30] R Ozturk-Urek L A Bozkaya and L Tarhan ldquo-e effects ofsome antioxidant vitamin-and trace element-supplementeddiets on activities of SOD CAT GSH-Px and LPO levels inchicken tissuesrdquo Cell Biochemistry and Function vol 19 no 2pp 125ndash132 2001

[31] O Levy Y Achituv Y Z Yacobi N Stambler andZ Dubinsky ldquo-e impact of spectral composition and lightperiodicity on the activity of two antioxidant enzymes (SODand CAT) in the coral Favia favusrdquo Journal of ExperimentalMarine Biology and Ecology vol 328 no 1 pp 35ndash46 2006

12 Journal of Chemistry

that at a low concentration (80 μM) -e experimental re-sults showed that the reductive ability of compounds 4a 4b4c and 4d was similar to that of piperine -e reducingstrength of the compound has a certain correlation with thenumber of active hydrogen atoms supplied by the com-pound Compounds 5a 5b 5c and 5d all had three activehydrogen atoms TBHQ had two active hydrogen atoms andpiperine had no active hydrogen atom From Figure 4 it canbe seen that at a concentration of 160 μM the absorbancesof 5a 5b 5c and 5d at 700 nm were 0999plusmn 00100880plusmn 0022 0866plusmn 0021 and 0860plusmn 0036 respectively-e absorbances of piperine and TBHQ were 0247plusmn 0015and 0261plusmn 0010 respectively -is showed that the re-ducing ability of piperine was comparable to that of TBHQwhich was not strong However compounds 5andash5d had arelatively strong reducing ability which was related to theirelectron-donating ability (hydrogen-donating ability)

33 Protective Effects of 5andash5d on AAPH-Induced DNAOxidation AAPH can react with oxygen in the air to produceperoxy radicals (ROO_) at a steady rate ROO_ can capture thehydrogen atom supplied by the C-4prime atom in DNA whichcould result in the DNAmolecule unwinding and generating avariety of carbonyl-containing small molecular substances[23 28] -ese substances can react with TBA to producethiobarbituric acid reactive species (TBARS λmax 535nm)under acidic conditions-erefore in this study the content ofcarbonyl-containing small-molecule compounds produced byAAPH-oxidized DNA was quantitatively calculated by testingthe absorbance of TBARS thereby tracking the extent ofAAPH-induced oxidative damage in DNA As shown in thecontrol experiment in Figure 5 in the process of DNA oxi-dation induced by AAPH the absorbance of TBARS increasedwith time indicating that as the reaction time increased morecarbonyl-containing small-molecule compounds were pro-duced as a result of AAPH-induced oxidative damage in DNAIt can be seen from Figure 6 that the addition of piperine didnot affect the increase in absorbance of TBARS in the above-mentioned system -is indicated that neither the potentialantioxidant units N-H nor the conjugated double bonds in thepiperine molecule could significantly inhibit AAPH-inducedoxidative damage in DNA However when the other fivederivatives were added to the above system the increase in theabsorbance of TBARS was significantly reduced -is showedpreliminarily that phenolic hydroxyl groups could protectDNA from AAPH-induced oxidative damage -e inhibitioneffect of compounds on AAPH-induced oxidative damage inDNA is shown in Figure 5 from which it can be seen that theinhibition effect of compounds 5a-5b onAAPH-induced DNAoxidative damage was concentration-dependent such that theinhibition rate at 160μM was higher than that at 80μM Inaddition as AAPH could generate ROO_ continuously in theoxidative damage process the inhibition rate of compounds5a-5b on DNA oxidative damage first increased and thendecreased with time and was the highest at 360min

34 Protective Effects of 5andash5d on AAPH-Induced HemolysisinErythrocytes In this study compounds 5andash5dwith strong

radical-scavenging ability were studied -e protective effectof derivatives on the AAPH-induced hemolysis of raterythrocytes was discussed It can be seen from Figure 7(a)that the erythrocyte suspension was stable and the hemolysisrate was 1445 in the PBS solution (pH 74) (control group)after incubation at 37degC for 3 h No obvious hemolysis wasobserved After adding AAPH under the same condition thehemolysis of erythrocytes increased to 6208 (AAPHgroup) -e significant increase in hemolysis showed thatAAPH could induce damage in erythrocytes thereby in-ducing hemolysis When a low concentration (20 μM) ofpiperine derivatives was added to the erythrocytes thehemolysis rates all decreased significantly (plt 005) andshowed significant concentration dependency such that thehigher the compound concentration the lower the hemo-lysis rate All four types of piperine derivatives reduced thehemolysis rate of erythrocytes to less than 50 Amongthem compound 5d showed the most prominent effect andreduced the hemolysis rate to 2590 after 3 h of incubation

-e protection time of 5a-5d on the AAPH-inducedhemolysis of erythrocytes is shown in Figure 7(b) It can beseen that the erythrocytes were stable in the PBS solutionand no significant change in the hemolysis rate was observedwith time -e hemolysis of the erythrocyte suspensioncaused by AAPH-induced oxidative damage showed a sig-nificant time effect such that the hemolysis rate increasedwith increasing damage time -e hemolysis rate increasedfrom 3664 to 7475 from 2 h to 4 h-is was because theantioxidant system of erythrocytes (GSH SOD CAT etc)could inhibit the oxidative damage from ROO_ in the initialstage However due to the consumption of antioxidants andthe increase of free radical chain reactions over time theerythrocyte membrane became damaged and the hemolysisoccurred rapidly -e addition of piperine derivatives couldeffectively suppress hemolysis As shown in the figure thehemolysis rate of the AAPH group was 3664 after 2 h ofincubation while the hemolysis rates of 5a 5b 5c and 5dgroups were reduced to 2808 3371 1899 and1492 respectively -erefore the piperine derivatives hada certain protective effect on hemolysis caused by oxidativedamage Among all groups compound 5d showed the besteffect before 3 h while compound 5a had the most signif-icant effect at 4 h

35 Effects of 5andash5d on AAPH-Induced HemoglobinOxidation in Erythrocytes Hemoglobin is the main proteinin erythrocytes Excessive free radicals can cause hemoglobinto oxidize and form methemoglobin thereby leading to thedisorder of erythrocyte function [20 29] -is study furtherdemonstrated the inhibitory effect of derivatives on oxi-dative damage in erythrocytes by examining the content ofmethemoglobin in erythrocytes -e effect of piperine de-rivatives against AAPH-induced hemoglobin oxidation inerythrocytes is shown in Figure 8 After 3 h of incubation(Figure 8(a)) the methemoglobin content in the normalcontrol group was low (174) whereas it was relatively high(2565) after oxidation by AAPH In addition as shown inFigure 8(b) the methemoglobin content in the normal

Journal of Chemistry 7

group was relatively stable while that in the AAPH groupcontinued to increase with time -us it can be seen thatAAPH could induce the continuous oxidation ofhemoglobin

It can be seen from Figure 8(a) that after 3 h of in-cubation the compounds showed some inhibitory effect onthe production of methemoglobin Among them

compounds 5c and 5d showed the most significant effectsA certain concentration effect was also observed such thatthe inhibitory effect increased with concentration In ad-dition as shown in Figure 8(b) the inhibitory effect of thecompounds on hemoglobin oxidation weakened with time-is was because AAPH could continuously release ROO_

over time

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

Abso

rban

ce at

535

nm

Time (h)

(a)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

Time (h)

(b)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

07

08

09

Time (h)

(c)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 1400Time (h)

01

02

03

04

05

06

07

08

09

(d)

Abso

rban

ce at

535

nm

200 400 600 800 1000 1200 140001

02

03

04

05

06

07

08

09

Time (h)

(e)

Figure 6 Variation in the absorbance of piperine derivatives in AAPH-induced oxidative damage in DNA over time 0 μM () 80 μM ()160 μM () (a) 5a (b) 5b (c) 5c (d) 5d (e) Piperine

0 360 480 1080

0

10

20

30

40

50

Time (min)

AA

PH-in

duce

d D

NA

dam

age i

nhib

ition

rate

() lowast

lowast

lowast

Figure 5 Inhibition effect of piperine derivatives on AAPH-induced oxidative damage in DNA 5a 160 μM ( ) 5a 80 μM ( ) 5b 160 μM( ) 5b 80 μM ( ) 5c 160 μM ( ) 5c 80 μM ( ) 5d 160 μM ( ) 5d 80 μM ( ) piperine 80 μM ( ) lowastindicates a significant differencefrom the piperine group (plt 005)

8 Journal of Chemistry

36 Influence of Piperine Derivatives on GSH-Px T-SOD andCATActivities in AAPH-Treated Erythrocytes GSH-Px is anantioxidant enzyme present in cells that catalyzes the re-duction of glutathione (GSH) and the removal of peroxidesin the body such as H2O2 in cells causing antilipid oxidation[30] As shown in Figure 9(a) the GSH-Px content inerythrocytes belonging to the control group was stable whilethat in erythrocytes of the AAPH group showed a statisti-cally significant gradual decrease with increasing incubationtime Following the addition of 50 μMof piperine derivativesto erythrocytes prior to the induction of oxidative damage byAAPH the piperine derivatives did not significantly influ-ence the erythrocyte GSH-Px content after 1 h of incubationcompared with the AAPH group After 2 h of incubationcompounds 5a and 5b significantly increased the GSH-Pxcontent (plt 005) after 4 h of incubation only the GSH-Pxcontent in the 5b and 5d groups was increased compared

with that in the AAPH group -ese results indicate that theamino acid derivatives of piperine can increase GSH-Pxactivity in erythrocytes Particularly compounds 5b and 5dproduced the strongest effects with the differences beingstatistically insignificant compared with the positive drugie vitamin C (VC) (pgt 005)

SOD is a key enzyme for removing free radicals withinthe body -e SOD activity level serves as an indicator of thebodyrsquos ability to remove free radicals [30 31] Figure 9(b)shows the influence of amino acid derivatives of piperine onT-SOD activity in AAPH-treated erythrocytes In the AAPHgroup with increasing incubation time T-SOD activitydecreased gradually Following the addition of 50 μM ofpiperine derivatives T-SOD activity increased after 1 h ofincubation albeit insignificantly compared with the AAPHgroup (pgt 005) After 2 h of incubation compounds 5a 5b5c and 5d increased T-SOD activity significantly (plt 005)

0

5

10

15

20

25

30

35

40

Met

Hb

()

Control AAPH 10 20Concentration (μM)

5

lowast

lowast

(a)

Time (h)

Met

Hb

()

2 3 40

10

20

30

40

50

(b)

Figure 8 (a) Protective effects of piperine derivatives on AAPH-induced hemoglobin oxidation (3 h) (b) Kinetic curve of the protectiveeffect of 20 μMpiperine derivatives on AAPH-induced hemoglobin oxidation Control ( ) AAPH ( ) AAPH+ 5a ( ) AAPH+ 5b ( )AAPH+ 5c ( ) AAPH+ 5d ( ) lowastindicates a significant difference from the control group (plt 005) indicates a significant differencefrom the control group (plt 005)

0

10

20

30

40

50

60

70

80H

emol

ysis

()

Control AAPH 10 20Concentration (μM)

5

lowast

lowast

(a)

Hem

olys

is (

)

0

20

40

60

80

100

Time (h)2 3 4

(b)

Figure 7 (a) Protective effect of piperine derivatives on AAPH-induced hemolysis of erythrocytes (3 h) (b) Kinetic curve of the protectiveeffect of piperine derivatives on AAPH-induced hemolysis of erythrocytes at 20 μM Control ( ) AAPH ( ) AAPH+ 5a ( ) AAPH+ 5b( ) AAPH+ 5c ( ) AAPH+ 5d ( ) lowastindicates a significant difference from the control group (plt 005) indicates a significantdifference from the AAPH group

Journal of Chemistry 9

while the positive control and prototype drug (piperine) hadno significant influence on erythrocyte T-SOD activity(pgt 005) After 4 h of incubation compound 5d increasedT-SOD activity significantly (pgt 005) while other com-pounds had no significant influence on T-SOD activity(pgt 005) -ese results highlight the significant enhance-ment caused by piperine derivatives of T-SOD activity inerythrocytes with AAPH-induced oxidative damage com-pared to the relatively inferior effect of the prototypecompound and of the positive drug VC

CAT an abundant enzyme in erythrocytes catalyzesH2O2 decomposition which reduces oxidative damagecaused by OHbull an oxidation product formed from H2O2under catalysis by metal ions [30 31] As shown inFigure 9(c) CAT activity in the AAPH group decreasesgradually relative to that in the control group with in-creasing incubation time Following the addition of 50 μMofpiperine derivatives compound 5c and piperine increasederythrocyte CATactivity after 1 h of incubation After 2 h ofincubation compounds 5a and 5b and piperine increasedCAT activity in erythrocytes significantly (plt 005) whilethe positive control had no significant influence on CAT

activity (pgt 005) After 4 h of incubation compound 5bincreased CAT activity significantly (pgt 005) while othercompounds had no significant influence (pgt 005) -eseresults indicate that piperine derivatives produce signifi-cant differences in CAT activity in AAPH-treated raterythrocytes

4 Conclusion

In the present study piperine-amino acid derivatives weresynthesized and the results indicate that the free radicalremoval ability and total reductive ability of the phenolichydroxyl-containing piperine-amino acid derivatives werehigher than those of the parent compound Specificallycompounds 5a and 5b have free radical removal perfor-mances comparable to that of VC-e derivatives have goodinhibitory effects on AAPH-induced oxidative DNA dam-age while certain compounds also show inhibitory effects onAAPH-induced hemolysis in rat erythrocytes Particularlycompound 5b can significantly inhibit AAPH-inducederythrocyte hemolysis and hemoglobin oxidation In addi-tion the experiments further verified that the protection

Time

GSH

-Px

(U)

1h 2h 4h0

100

200

300

400

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

(a)

Time

SOD

activ

ity (U

mgH

b)

1h 2h 4h0

5

10

15

20

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

(b)

Time1h 2h 4h

CAT

activ

ity (U

mgH

b)

0

1

2

3

4

5

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast lowast

lowast

lowast

lowast

lowast

(c)

Figure 9 Influence of piperine derivatives on the (a) GSH-Px content (b) T-SOD activity and (c) CATactivity in AAPH-treated rat erythrocyteslowastindicates a significant difference from the control group (plt 005) Control ( ) AAPH ( ) AAPH+5a ( ) AAPH+5b ( ) AAPH+5c( ) AAPH+5d ( ) AAPH+piperine ( ) AAPH+VC ( ) indicates a significant difference from the AAPH group (plt 005)

10 Journal of Chemistry

provided by piperine derivatives against AAPH-inducedoxidative damage can be achieved by preserving the activityof the antioxidant enzyme system (GSH-Px T-SOD andCAT) within cells -e protection by piperine derivativeswas also found to be superior to that by piperine and vitaminC -is paper described a structural modification methodthat could effectively improve the antioxidant ability ofpiperine-is type of compound could be a candidate for thedevelopment and research of oxidative damage-relateddiseases (hyperlipidemia diabetes cancer whitening etc)

Data Availability

-e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

-is work was financially supported by the Shaanxi Pro-vincial Department of Education serving local (industrial-ization) research project (no 15JF028) and the ScientificResearch Fund of Xirsquoan Medical University(nos2016YXXK09 2018DC-12 and 201825012)

References

[1] J P Adjimani and P Asare ldquoAntioxidant and free radicalscavenging activity of iron chelatorsrdquo Toxicology Reportsvol 2 pp 721ndash728 2015

[2] I Dalle-Donne R Rossi R Colombo D Giustarini andA Milzani ldquoBiomarkers of oxidative damage in humandiseaserdquo Clinical Chemistry vol 52 no 4 pp 601ndash623 2006

[3] L J Machlin and A Bendich ldquoFree radical tissue damageprotective role of antioxidant nutrientsrdquo Fe FASEB Journalvol 1 no 6 pp 441ndash445 1987

[4] N SatoW Li H Takemoto et al ldquoComprehensive evaluationof antioxidant effects of Japanese Kampo medicines led toidentification of Tsudosan formulation as a potent antioxidantagentrdquo Journal of Natural Medicines vol 73 no 1pp 163ndash172 2018

[5] N Bao Z Sun H Baigude G Borjihan and Z Jin ldquoLightinduced isomerization of piperlonguminine to scutifoliamideA isopiperlonguminine and hoffmannseggiamide Ardquo Phy-tochemistry Letters vol 16 pp 324ndash327 2016

[6] T Velpandian R Jasuja R K Bhardwaj J Jaiswal andS K Gupta ldquoPiperine in food interference in the pharma-cokinetics of phenytoinrdquo European Journal of Drug Meta-bolism and Pharmacokinetics vol 26 no 4 pp 241ndash247 2001

[7] A Duangjai K Ingkaninan S Praputbut andN Limpeanchob ldquoBlack pepper and piperine reduce cho-lesterol uptake and enhance translocation of cholesteroltransporter proteinsrdquo Journal of Natural Medicines vol 67no 2 pp 303ndash310 2013

[8] D E Eigenmann C Durig E A Jahne et al ldquoIn vitro blood-brain barrier permeability predictions for GABAA receptormodulating piperine analogsrdquo European Journal of Phar-maceutics and Biopharmaceutics vol 103 pp 118ndash126 2016

[9] Y A Samra H S Said N M Elsherbiny G I Liou M M El-Shishtawy and L A Eissa ldquoCepharanthine and Piperineameliorate diabetic nephropathy in rats role of NF-κB andNLRP3 inflammasomerdquo Life Sciences vol 157 pp 187ndash1992016

[10] R Mittal and R L Gupta ldquoIn vitro antioxidant activity ofpiperinerdquoMethods and Findings in Experimental and ClinicalPharmacology vol 22 no 5 pp 271ndash274 2000

[11] Z Zarai E Boujelbene N Ben Salem Y Gargouri andA Sayari ldquoAntioxidant and antimicrobial activities of varioussolvent extracts piperine and piperic acid from Piper nig-rumrdquo LWT-Food Science and Technology vol 50 no 2pp 634ndash641 2013

[12] A Yasir S Ishtiaq M Jahangir et al ldquoBiology-orientedsynthesis (BIOS) of piperine derivatives and their comparativeanalgesic and antiinflammatory activitiesrdquo MedicinalChemistry vol 14 no 3 pp 269ndash280 2018

[13] R S Vijayakumar D Surya and N Nalini ldquoAntioxidantefficacy of black pepper (Piper nigrumL) and piperine in ratswith high fat diet induced oxidative stressrdquo Redox Reportvol 9 no 2 pp 105ndash110 2004

[14] T Miyazawa K Nakagawa S H Kim et al ldquoCurcumin andpiperine supplementation of obese mice under caloric re-striction modulates body fat and interleukin-1βrdquo Nutrition ampMetabolism vol 15 no 1 p 12 2018

[15] A Khajuria N -usu U Zutshi and K L Bedi ldquoPiperinemodulation of carcinogen induced oxidative stress in intes-tinal mucosardquo Molecular and Cellular Biochemistry vol 189no 12 pp 113ndash118 1998

[16] S Inder Pal J Shreyans Kumar K Amandeep et al ldquoSyn-thesis and antileishmanial activity of piperoyl-amino acidconjugatesrdquo European Journal of Medicinal Chemistry vol 45no 8 pp 3439ndash3445 2010

[17] T Koichi M Takaki and S Yoshiaki ldquoSynthesis and bio-logical evaluation of piperic acid amides as free radicalscavengers and α-glucosidase inhibitorsrdquo Chemical amp Phar-maceutical Bulletin vol 63 no 5 pp 326ndash333 2015

[18] J M Latorres D G Rios G Saggiomo W Wasielesky andC Prentice-Hernandez ldquoFunctional and antioxidant prop-erties of protein hydrolysates obtained from white shrimp(Litopenaeus vannamei)rdquo Journal of Food Science and Tech-nology vol 55 no 2 pp 721ndash729 2018

[19] A Moayedi L Mora M C Aristoy M Safari M Hashemiand F Toldra ldquoPeptidomic analysis of antioxidant and ACE-inhibitory peptides obtained from tomato waste proteinsfermented using Bacillus subtilisrdquo Food Chemistry vol 250pp 180ndash187 2018

[20] L Zheng H Dong G Su Q Zhao and M Zhao ldquoRadicalscavenging activities of Tyr- Trp- Cys- andMet-Gly and theirprotective effects against AAPH-induced oxidative damage inhuman erythrocytesrdquo Food Chemistry vol 197 no Part App 807ndash813 2016

[21] M Miceli E Roma P Rosa et al ldquoSynthesis of benzofuran-2-one derivatives and evaluation of their antioxidant capacity bycomparing DPPH assay and cyclic voltammetryrdquo Moleculesvol 23 no 4 p 710 2018

[22] K -aipong U Boonprakob K Crosby et al ldquoComparisonof ABTS DPPH FRAP and ORAC assays for estimatingantioxidant activity from guava fruit extractsrdquo Journal of FoodComposition amp Analysis vol 19 no 6-7 pp 669ndash675 2006

[23] X-R Gong G-L Xi and Z-Q Liu ldquoActivity of coumarin-oxadiazole-appended phenol in inhibiting DNA oxidationand scavenging radicalrdquo Tetrahedron Letters vol 56 no 45pp 6257ndash6261 2015

Journal of Chemistry 11

[24] G-L Xi and Z-Q Liu ldquoCoumarin sharing the benzene ringwith quinoline for quenching radicals and inhibiting DNAoxidationrdquo European Journal of Medicinal Chemistry vol 95pp 416ndash423 2015

[25] H R Shin B R You and W H Park ldquoPX-12-induced HeLacell death is associated with oxidative stress and GSH de-pletionrdquo Oncology Letters vol 6 no 6 pp 1804ndash1810 2013

[26] S B Nimse D Pal A Mazumder et al ldquoSynthesis of cin-namanilide derivatives and their antioxidant and antimi-crobial activityrdquo Journal of Chemistry vol 2015 Article ID208910 5 pages 2015

[27] M Oyaizu ldquoStudies on products of browning reactionAntioxidative activities of products of browning reactionprepared from glucosaminerdquo Fe Japanese Journal of Nu-trition and Dietetics vol 44 no 6 pp 307ndash315 1986

[28] Y Yoshioka X Li T Zhang et al ldquoBlack soybean seed coatpolyphenols prevent AAPH-induced oxidative DNA-damagein HepG2 cellsrdquo Journal of Clinical Biochemistry and Nu-trition vol 60 no 2 pp 108ndash114 2017

[29] R C Chiste M Freitas A Z Mercadante and E FernandesldquoCarotenoids inhibit lipid peroxidation and hemoglobinoxidation but not the depletion of glutathione induced byROS in human erythrocytesrdquo Life Sciences vol 99 no 1-2pp 52ndash60 2014

[30] R Ozturk-Urek L A Bozkaya and L Tarhan ldquo-e effects ofsome antioxidant vitamin-and trace element-supplementeddiets on activities of SOD CAT GSH-Px and LPO levels inchicken tissuesrdquo Cell Biochemistry and Function vol 19 no 2pp 125ndash132 2001

[31] O Levy Y Achituv Y Z Yacobi N Stambler andZ Dubinsky ldquo-e impact of spectral composition and lightperiodicity on the activity of two antioxidant enzymes (SODand CAT) in the coral Favia favusrdquo Journal of ExperimentalMarine Biology and Ecology vol 328 no 1 pp 35ndash46 2006

12 Journal of Chemistry

group was relatively stable while that in the AAPH groupcontinued to increase with time -us it can be seen thatAAPH could induce the continuous oxidation ofhemoglobin

It can be seen from Figure 8(a) that after 3 h of in-cubation the compounds showed some inhibitory effect onthe production of methemoglobin Among them

compounds 5c and 5d showed the most significant effectsA certain concentration effect was also observed such thatthe inhibitory effect increased with concentration In ad-dition as shown in Figure 8(b) the inhibitory effect of thecompounds on hemoglobin oxidation weakened with time-is was because AAPH could continuously release ROO_

over time

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

Abso

rban

ce at

535

nm

Time (h)

(a)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

Time (h)

(b)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 140001

02

03

04

05

06

07

08

09

Time (h)

(c)

Abso

rban

ce at

535

nm

0 200 400 600 800 1000 1200 1400Time (h)

01

02

03

04

05

06

07

08

09

(d)

Abso

rban

ce at

535

nm

200 400 600 800 1000 1200 140001

02

03

04

05

06

07

08

09

Time (h)

(e)

Figure 6 Variation in the absorbance of piperine derivatives in AAPH-induced oxidative damage in DNA over time 0 μM () 80 μM ()160 μM () (a) 5a (b) 5b (c) 5c (d) 5d (e) Piperine

0 360 480 1080

0

10

20

30

40

50

Time (min)

AA

PH-in

duce

d D

NA

dam

age i

nhib

ition

rate

() lowast

lowast

lowast

Figure 5 Inhibition effect of piperine derivatives on AAPH-induced oxidative damage in DNA 5a 160 μM ( ) 5a 80 μM ( ) 5b 160 μM( ) 5b 80 μM ( ) 5c 160 μM ( ) 5c 80 μM ( ) 5d 160 μM ( ) 5d 80 μM ( ) piperine 80 μM ( ) lowastindicates a significant differencefrom the piperine group (plt 005)

8 Journal of Chemistry

36 Influence of Piperine Derivatives on GSH-Px T-SOD andCATActivities in AAPH-Treated Erythrocytes GSH-Px is anantioxidant enzyme present in cells that catalyzes the re-duction of glutathione (GSH) and the removal of peroxidesin the body such as H2O2 in cells causing antilipid oxidation[30] As shown in Figure 9(a) the GSH-Px content inerythrocytes belonging to the control group was stable whilethat in erythrocytes of the AAPH group showed a statisti-cally significant gradual decrease with increasing incubationtime Following the addition of 50 μMof piperine derivativesto erythrocytes prior to the induction of oxidative damage byAAPH the piperine derivatives did not significantly influ-ence the erythrocyte GSH-Px content after 1 h of incubationcompared with the AAPH group After 2 h of incubationcompounds 5a and 5b significantly increased the GSH-Pxcontent (plt 005) after 4 h of incubation only the GSH-Pxcontent in the 5b and 5d groups was increased compared

with that in the AAPH group -ese results indicate that theamino acid derivatives of piperine can increase GSH-Pxactivity in erythrocytes Particularly compounds 5b and 5dproduced the strongest effects with the differences beingstatistically insignificant compared with the positive drugie vitamin C (VC) (pgt 005)

SOD is a key enzyme for removing free radicals withinthe body -e SOD activity level serves as an indicator of thebodyrsquos ability to remove free radicals [30 31] Figure 9(b)shows the influence of amino acid derivatives of piperine onT-SOD activity in AAPH-treated erythrocytes In the AAPHgroup with increasing incubation time T-SOD activitydecreased gradually Following the addition of 50 μM ofpiperine derivatives T-SOD activity increased after 1 h ofincubation albeit insignificantly compared with the AAPHgroup (pgt 005) After 2 h of incubation compounds 5a 5b5c and 5d increased T-SOD activity significantly (plt 005)

0

5

10

15

20

25

30

35

40

Met

Hb

()

Control AAPH 10 20Concentration (μM)

5

lowast

lowast

(a)

Time (h)

Met

Hb

()

2 3 40

10

20

30

40

50

(b)

Figure 8 (a) Protective effects of piperine derivatives on AAPH-induced hemoglobin oxidation (3 h) (b) Kinetic curve of the protectiveeffect of 20 μMpiperine derivatives on AAPH-induced hemoglobin oxidation Control ( ) AAPH ( ) AAPH+ 5a ( ) AAPH+ 5b ( )AAPH+ 5c ( ) AAPH+ 5d ( ) lowastindicates a significant difference from the control group (plt 005) indicates a significant differencefrom the control group (plt 005)

0

10

20

30

40

50

60

70

80H

emol

ysis

()

Control AAPH 10 20Concentration (μM)

5

lowast

lowast

(a)

Hem

olys

is (

)

0

20

40

60

80

100

Time (h)2 3 4

(b)

Figure 7 (a) Protective effect of piperine derivatives on AAPH-induced hemolysis of erythrocytes (3 h) (b) Kinetic curve of the protectiveeffect of piperine derivatives on AAPH-induced hemolysis of erythrocytes at 20 μM Control ( ) AAPH ( ) AAPH+ 5a ( ) AAPH+ 5b( ) AAPH+ 5c ( ) AAPH+ 5d ( ) lowastindicates a significant difference from the control group (plt 005) indicates a significantdifference from the AAPH group

Journal of Chemistry 9

while the positive control and prototype drug (piperine) hadno significant influence on erythrocyte T-SOD activity(pgt 005) After 4 h of incubation compound 5d increasedT-SOD activity significantly (pgt 005) while other com-pounds had no significant influence on T-SOD activity(pgt 005) -ese results highlight the significant enhance-ment caused by piperine derivatives of T-SOD activity inerythrocytes with AAPH-induced oxidative damage com-pared to the relatively inferior effect of the prototypecompound and of the positive drug VC

CAT an abundant enzyme in erythrocytes catalyzesH2O2 decomposition which reduces oxidative damagecaused by OHbull an oxidation product formed from H2O2under catalysis by metal ions [30 31] As shown inFigure 9(c) CAT activity in the AAPH group decreasesgradually relative to that in the control group with in-creasing incubation time Following the addition of 50 μMofpiperine derivatives compound 5c and piperine increasederythrocyte CATactivity after 1 h of incubation After 2 h ofincubation compounds 5a and 5b and piperine increasedCAT activity in erythrocytes significantly (plt 005) whilethe positive control had no significant influence on CAT

activity (pgt 005) After 4 h of incubation compound 5bincreased CAT activity significantly (pgt 005) while othercompounds had no significant influence (pgt 005) -eseresults indicate that piperine derivatives produce signifi-cant differences in CAT activity in AAPH-treated raterythrocytes

4 Conclusion

In the present study piperine-amino acid derivatives weresynthesized and the results indicate that the free radicalremoval ability and total reductive ability of the phenolichydroxyl-containing piperine-amino acid derivatives werehigher than those of the parent compound Specificallycompounds 5a and 5b have free radical removal perfor-mances comparable to that of VC-e derivatives have goodinhibitory effects on AAPH-induced oxidative DNA dam-age while certain compounds also show inhibitory effects onAAPH-induced hemolysis in rat erythrocytes Particularlycompound 5b can significantly inhibit AAPH-inducederythrocyte hemolysis and hemoglobin oxidation In addi-tion the experiments further verified that the protection

Time

GSH

-Px

(U)

1h 2h 4h0

100

200

300

400

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

(a)

Time

SOD

activ

ity (U

mgH

b)

1h 2h 4h0

5

10

15

20

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

(b)

Time1h 2h 4h

CAT

activ

ity (U

mgH

b)

0

1

2

3

4

5

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast lowast

lowast

lowast

lowast

lowast

(c)

Figure 9 Influence of piperine derivatives on the (a) GSH-Px content (b) T-SOD activity and (c) CATactivity in AAPH-treated rat erythrocyteslowastindicates a significant difference from the control group (plt 005) Control ( ) AAPH ( ) AAPH+5a ( ) AAPH+5b ( ) AAPH+5c( ) AAPH+5d ( ) AAPH+piperine ( ) AAPH+VC ( ) indicates a significant difference from the AAPH group (plt 005)

10 Journal of Chemistry

provided by piperine derivatives against AAPH-inducedoxidative damage can be achieved by preserving the activityof the antioxidant enzyme system (GSH-Px T-SOD andCAT) within cells -e protection by piperine derivativeswas also found to be superior to that by piperine and vitaminC -is paper described a structural modification methodthat could effectively improve the antioxidant ability ofpiperine-is type of compound could be a candidate for thedevelopment and research of oxidative damage-relateddiseases (hyperlipidemia diabetes cancer whitening etc)

Data Availability

-e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

-is work was financially supported by the Shaanxi Pro-vincial Department of Education serving local (industrial-ization) research project (no 15JF028) and the ScientificResearch Fund of Xirsquoan Medical University(nos2016YXXK09 2018DC-12 and 201825012)

References

[1] J P Adjimani and P Asare ldquoAntioxidant and free radicalscavenging activity of iron chelatorsrdquo Toxicology Reportsvol 2 pp 721ndash728 2015

[2] I Dalle-Donne R Rossi R Colombo D Giustarini andA Milzani ldquoBiomarkers of oxidative damage in humandiseaserdquo Clinical Chemistry vol 52 no 4 pp 601ndash623 2006

[3] L J Machlin and A Bendich ldquoFree radical tissue damageprotective role of antioxidant nutrientsrdquo Fe FASEB Journalvol 1 no 6 pp 441ndash445 1987

[4] N SatoW Li H Takemoto et al ldquoComprehensive evaluationof antioxidant effects of Japanese Kampo medicines led toidentification of Tsudosan formulation as a potent antioxidantagentrdquo Journal of Natural Medicines vol 73 no 1pp 163ndash172 2018

[5] N Bao Z Sun H Baigude G Borjihan and Z Jin ldquoLightinduced isomerization of piperlonguminine to scutifoliamideA isopiperlonguminine and hoffmannseggiamide Ardquo Phy-tochemistry Letters vol 16 pp 324ndash327 2016

[6] T Velpandian R Jasuja R K Bhardwaj J Jaiswal andS K Gupta ldquoPiperine in food interference in the pharma-cokinetics of phenytoinrdquo European Journal of Drug Meta-bolism and Pharmacokinetics vol 26 no 4 pp 241ndash247 2001

[7] A Duangjai K Ingkaninan S Praputbut andN Limpeanchob ldquoBlack pepper and piperine reduce cho-lesterol uptake and enhance translocation of cholesteroltransporter proteinsrdquo Journal of Natural Medicines vol 67no 2 pp 303ndash310 2013

[8] D E Eigenmann C Durig E A Jahne et al ldquoIn vitro blood-brain barrier permeability predictions for GABAA receptormodulating piperine analogsrdquo European Journal of Phar-maceutics and Biopharmaceutics vol 103 pp 118ndash126 2016

[9] Y A Samra H S Said N M Elsherbiny G I Liou M M El-Shishtawy and L A Eissa ldquoCepharanthine and Piperineameliorate diabetic nephropathy in rats role of NF-κB andNLRP3 inflammasomerdquo Life Sciences vol 157 pp 187ndash1992016

[10] R Mittal and R L Gupta ldquoIn vitro antioxidant activity ofpiperinerdquoMethods and Findings in Experimental and ClinicalPharmacology vol 22 no 5 pp 271ndash274 2000

[11] Z Zarai E Boujelbene N Ben Salem Y Gargouri andA Sayari ldquoAntioxidant and antimicrobial activities of varioussolvent extracts piperine and piperic acid from Piper nig-rumrdquo LWT-Food Science and Technology vol 50 no 2pp 634ndash641 2013

[12] A Yasir S Ishtiaq M Jahangir et al ldquoBiology-orientedsynthesis (BIOS) of piperine derivatives and their comparativeanalgesic and antiinflammatory activitiesrdquo MedicinalChemistry vol 14 no 3 pp 269ndash280 2018

[13] R S Vijayakumar D Surya and N Nalini ldquoAntioxidantefficacy of black pepper (Piper nigrumL) and piperine in ratswith high fat diet induced oxidative stressrdquo Redox Reportvol 9 no 2 pp 105ndash110 2004

[14] T Miyazawa K Nakagawa S H Kim et al ldquoCurcumin andpiperine supplementation of obese mice under caloric re-striction modulates body fat and interleukin-1βrdquo Nutrition ampMetabolism vol 15 no 1 p 12 2018

[15] A Khajuria N -usu U Zutshi and K L Bedi ldquoPiperinemodulation of carcinogen induced oxidative stress in intes-tinal mucosardquo Molecular and Cellular Biochemistry vol 189no 12 pp 113ndash118 1998

[16] S Inder Pal J Shreyans Kumar K Amandeep et al ldquoSyn-thesis and antileishmanial activity of piperoyl-amino acidconjugatesrdquo European Journal of Medicinal Chemistry vol 45no 8 pp 3439ndash3445 2010

[17] T Koichi M Takaki and S Yoshiaki ldquoSynthesis and bio-logical evaluation of piperic acid amides as free radicalscavengers and α-glucosidase inhibitorsrdquo Chemical amp Phar-maceutical Bulletin vol 63 no 5 pp 326ndash333 2015

[18] J M Latorres D G Rios G Saggiomo W Wasielesky andC Prentice-Hernandez ldquoFunctional and antioxidant prop-erties of protein hydrolysates obtained from white shrimp(Litopenaeus vannamei)rdquo Journal of Food Science and Tech-nology vol 55 no 2 pp 721ndash729 2018

[19] A Moayedi L Mora M C Aristoy M Safari M Hashemiand F Toldra ldquoPeptidomic analysis of antioxidant and ACE-inhibitory peptides obtained from tomato waste proteinsfermented using Bacillus subtilisrdquo Food Chemistry vol 250pp 180ndash187 2018

[20] L Zheng H Dong G Su Q Zhao and M Zhao ldquoRadicalscavenging activities of Tyr- Trp- Cys- andMet-Gly and theirprotective effects against AAPH-induced oxidative damage inhuman erythrocytesrdquo Food Chemistry vol 197 no Part App 807ndash813 2016

[21] M Miceli E Roma P Rosa et al ldquoSynthesis of benzofuran-2-one derivatives and evaluation of their antioxidant capacity bycomparing DPPH assay and cyclic voltammetryrdquo Moleculesvol 23 no 4 p 710 2018

[22] K -aipong U Boonprakob K Crosby et al ldquoComparisonof ABTS DPPH FRAP and ORAC assays for estimatingantioxidant activity from guava fruit extractsrdquo Journal of FoodComposition amp Analysis vol 19 no 6-7 pp 669ndash675 2006

[23] X-R Gong G-L Xi and Z-Q Liu ldquoActivity of coumarin-oxadiazole-appended phenol in inhibiting DNA oxidationand scavenging radicalrdquo Tetrahedron Letters vol 56 no 45pp 6257ndash6261 2015

Journal of Chemistry 11

[24] G-L Xi and Z-Q Liu ldquoCoumarin sharing the benzene ringwith quinoline for quenching radicals and inhibiting DNAoxidationrdquo European Journal of Medicinal Chemistry vol 95pp 416ndash423 2015

[25] H R Shin B R You and W H Park ldquoPX-12-induced HeLacell death is associated with oxidative stress and GSH de-pletionrdquo Oncology Letters vol 6 no 6 pp 1804ndash1810 2013

[26] S B Nimse D Pal A Mazumder et al ldquoSynthesis of cin-namanilide derivatives and their antioxidant and antimi-crobial activityrdquo Journal of Chemistry vol 2015 Article ID208910 5 pages 2015

[27] M Oyaizu ldquoStudies on products of browning reactionAntioxidative activities of products of browning reactionprepared from glucosaminerdquo Fe Japanese Journal of Nu-trition and Dietetics vol 44 no 6 pp 307ndash315 1986

[28] Y Yoshioka X Li T Zhang et al ldquoBlack soybean seed coatpolyphenols prevent AAPH-induced oxidative DNA-damagein HepG2 cellsrdquo Journal of Clinical Biochemistry and Nu-trition vol 60 no 2 pp 108ndash114 2017

[29] R C Chiste M Freitas A Z Mercadante and E FernandesldquoCarotenoids inhibit lipid peroxidation and hemoglobinoxidation but not the depletion of glutathione induced byROS in human erythrocytesrdquo Life Sciences vol 99 no 1-2pp 52ndash60 2014

[30] R Ozturk-Urek L A Bozkaya and L Tarhan ldquo-e effects ofsome antioxidant vitamin-and trace element-supplementeddiets on activities of SOD CAT GSH-Px and LPO levels inchicken tissuesrdquo Cell Biochemistry and Function vol 19 no 2pp 125ndash132 2001

[31] O Levy Y Achituv Y Z Yacobi N Stambler andZ Dubinsky ldquo-e impact of spectral composition and lightperiodicity on the activity of two antioxidant enzymes (SODand CAT) in the coral Favia favusrdquo Journal of ExperimentalMarine Biology and Ecology vol 328 no 1 pp 35ndash46 2006

12 Journal of Chemistry

36 Influence of Piperine Derivatives on GSH-Px T-SOD andCATActivities in AAPH-Treated Erythrocytes GSH-Px is anantioxidant enzyme present in cells that catalyzes the re-duction of glutathione (GSH) and the removal of peroxidesin the body such as H2O2 in cells causing antilipid oxidation[30] As shown in Figure 9(a) the GSH-Px content inerythrocytes belonging to the control group was stable whilethat in erythrocytes of the AAPH group showed a statisti-cally significant gradual decrease with increasing incubationtime Following the addition of 50 μMof piperine derivativesto erythrocytes prior to the induction of oxidative damage byAAPH the piperine derivatives did not significantly influ-ence the erythrocyte GSH-Px content after 1 h of incubationcompared with the AAPH group After 2 h of incubationcompounds 5a and 5b significantly increased the GSH-Pxcontent (plt 005) after 4 h of incubation only the GSH-Pxcontent in the 5b and 5d groups was increased compared

with that in the AAPH group -ese results indicate that theamino acid derivatives of piperine can increase GSH-Pxactivity in erythrocytes Particularly compounds 5b and 5dproduced the strongest effects with the differences beingstatistically insignificant compared with the positive drugie vitamin C (VC) (pgt 005)

SOD is a key enzyme for removing free radicals withinthe body -e SOD activity level serves as an indicator of thebodyrsquos ability to remove free radicals [30 31] Figure 9(b)shows the influence of amino acid derivatives of piperine onT-SOD activity in AAPH-treated erythrocytes In the AAPHgroup with increasing incubation time T-SOD activitydecreased gradually Following the addition of 50 μM ofpiperine derivatives T-SOD activity increased after 1 h ofincubation albeit insignificantly compared with the AAPHgroup (pgt 005) After 2 h of incubation compounds 5a 5b5c and 5d increased T-SOD activity significantly (plt 005)

0

5

10

15

20

25

30

35

40

Met

Hb

()

Control AAPH 10 20Concentration (μM)

5

lowast

lowast

(a)

Time (h)

Met

Hb

()

2 3 40

10

20

30

40

50

(b)

Figure 8 (a) Protective effects of piperine derivatives on AAPH-induced hemoglobin oxidation (3 h) (b) Kinetic curve of the protectiveeffect of 20 μMpiperine derivatives on AAPH-induced hemoglobin oxidation Control ( ) AAPH ( ) AAPH+ 5a ( ) AAPH+ 5b ( )AAPH+ 5c ( ) AAPH+ 5d ( ) lowastindicates a significant difference from the control group (plt 005) indicates a significant differencefrom the control group (plt 005)

0

10

20

30

40

50

60

70

80H

emol

ysis

()

Control AAPH 10 20Concentration (μM)

5

lowast

lowast

(a)

Hem

olys

is (

)

0

20

40

60

80

100

Time (h)2 3 4

(b)

Figure 7 (a) Protective effect of piperine derivatives on AAPH-induced hemolysis of erythrocytes (3 h) (b) Kinetic curve of the protectiveeffect of piperine derivatives on AAPH-induced hemolysis of erythrocytes at 20 μM Control ( ) AAPH ( ) AAPH+ 5a ( ) AAPH+ 5b( ) AAPH+ 5c ( ) AAPH+ 5d ( ) lowastindicates a significant difference from the control group (plt 005) indicates a significantdifference from the AAPH group

Journal of Chemistry 9

while the positive control and prototype drug (piperine) hadno significant influence on erythrocyte T-SOD activity(pgt 005) After 4 h of incubation compound 5d increasedT-SOD activity significantly (pgt 005) while other com-pounds had no significant influence on T-SOD activity(pgt 005) -ese results highlight the significant enhance-ment caused by piperine derivatives of T-SOD activity inerythrocytes with AAPH-induced oxidative damage com-pared to the relatively inferior effect of the prototypecompound and of the positive drug VC

CAT an abundant enzyme in erythrocytes catalyzesH2O2 decomposition which reduces oxidative damagecaused by OHbull an oxidation product formed from H2O2under catalysis by metal ions [30 31] As shown inFigure 9(c) CAT activity in the AAPH group decreasesgradually relative to that in the control group with in-creasing incubation time Following the addition of 50 μMofpiperine derivatives compound 5c and piperine increasederythrocyte CATactivity after 1 h of incubation After 2 h ofincubation compounds 5a and 5b and piperine increasedCAT activity in erythrocytes significantly (plt 005) whilethe positive control had no significant influence on CAT

activity (pgt 005) After 4 h of incubation compound 5bincreased CAT activity significantly (pgt 005) while othercompounds had no significant influence (pgt 005) -eseresults indicate that piperine derivatives produce signifi-cant differences in CAT activity in AAPH-treated raterythrocytes

4 Conclusion

In the present study piperine-amino acid derivatives weresynthesized and the results indicate that the free radicalremoval ability and total reductive ability of the phenolichydroxyl-containing piperine-amino acid derivatives werehigher than those of the parent compound Specificallycompounds 5a and 5b have free radical removal perfor-mances comparable to that of VC-e derivatives have goodinhibitory effects on AAPH-induced oxidative DNA dam-age while certain compounds also show inhibitory effects onAAPH-induced hemolysis in rat erythrocytes Particularlycompound 5b can significantly inhibit AAPH-inducederythrocyte hemolysis and hemoglobin oxidation In addi-tion the experiments further verified that the protection

Time

GSH

-Px

(U)

1h 2h 4h0

100

200

300

400

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

(a)

Time

SOD

activ

ity (U

mgH

b)

1h 2h 4h0

5

10

15

20

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

(b)

Time1h 2h 4h

CAT

activ

ity (U

mgH

b)

0

1

2

3

4

5

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast lowast

lowast

lowast

lowast

lowast

(c)

Figure 9 Influence of piperine derivatives on the (a) GSH-Px content (b) T-SOD activity and (c) CATactivity in AAPH-treated rat erythrocyteslowastindicates a significant difference from the control group (plt 005) Control ( ) AAPH ( ) AAPH+5a ( ) AAPH+5b ( ) AAPH+5c( ) AAPH+5d ( ) AAPH+piperine ( ) AAPH+VC ( ) indicates a significant difference from the AAPH group (plt 005)

10 Journal of Chemistry

provided by piperine derivatives against AAPH-inducedoxidative damage can be achieved by preserving the activityof the antioxidant enzyme system (GSH-Px T-SOD andCAT) within cells -e protection by piperine derivativeswas also found to be superior to that by piperine and vitaminC -is paper described a structural modification methodthat could effectively improve the antioxidant ability ofpiperine-is type of compound could be a candidate for thedevelopment and research of oxidative damage-relateddiseases (hyperlipidemia diabetes cancer whitening etc)

Data Availability

-e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

-is work was financially supported by the Shaanxi Pro-vincial Department of Education serving local (industrial-ization) research project (no 15JF028) and the ScientificResearch Fund of Xirsquoan Medical University(nos2016YXXK09 2018DC-12 and 201825012)

References

[1] J P Adjimani and P Asare ldquoAntioxidant and free radicalscavenging activity of iron chelatorsrdquo Toxicology Reportsvol 2 pp 721ndash728 2015

[2] I Dalle-Donne R Rossi R Colombo D Giustarini andA Milzani ldquoBiomarkers of oxidative damage in humandiseaserdquo Clinical Chemistry vol 52 no 4 pp 601ndash623 2006

[3] L J Machlin and A Bendich ldquoFree radical tissue damageprotective role of antioxidant nutrientsrdquo Fe FASEB Journalvol 1 no 6 pp 441ndash445 1987

[4] N SatoW Li H Takemoto et al ldquoComprehensive evaluationof antioxidant effects of Japanese Kampo medicines led toidentification of Tsudosan formulation as a potent antioxidantagentrdquo Journal of Natural Medicines vol 73 no 1pp 163ndash172 2018

[5] N Bao Z Sun H Baigude G Borjihan and Z Jin ldquoLightinduced isomerization of piperlonguminine to scutifoliamideA isopiperlonguminine and hoffmannseggiamide Ardquo Phy-tochemistry Letters vol 16 pp 324ndash327 2016

[6] T Velpandian R Jasuja R K Bhardwaj J Jaiswal andS K Gupta ldquoPiperine in food interference in the pharma-cokinetics of phenytoinrdquo European Journal of Drug Meta-bolism and Pharmacokinetics vol 26 no 4 pp 241ndash247 2001

[7] A Duangjai K Ingkaninan S Praputbut andN Limpeanchob ldquoBlack pepper and piperine reduce cho-lesterol uptake and enhance translocation of cholesteroltransporter proteinsrdquo Journal of Natural Medicines vol 67no 2 pp 303ndash310 2013

[8] D E Eigenmann C Durig E A Jahne et al ldquoIn vitro blood-brain barrier permeability predictions for GABAA receptormodulating piperine analogsrdquo European Journal of Phar-maceutics and Biopharmaceutics vol 103 pp 118ndash126 2016

[9] Y A Samra H S Said N M Elsherbiny G I Liou M M El-Shishtawy and L A Eissa ldquoCepharanthine and Piperineameliorate diabetic nephropathy in rats role of NF-κB andNLRP3 inflammasomerdquo Life Sciences vol 157 pp 187ndash1992016

[10] R Mittal and R L Gupta ldquoIn vitro antioxidant activity ofpiperinerdquoMethods and Findings in Experimental and ClinicalPharmacology vol 22 no 5 pp 271ndash274 2000

[11] Z Zarai E Boujelbene N Ben Salem Y Gargouri andA Sayari ldquoAntioxidant and antimicrobial activities of varioussolvent extracts piperine and piperic acid from Piper nig-rumrdquo LWT-Food Science and Technology vol 50 no 2pp 634ndash641 2013

[12] A Yasir S Ishtiaq M Jahangir et al ldquoBiology-orientedsynthesis (BIOS) of piperine derivatives and their comparativeanalgesic and antiinflammatory activitiesrdquo MedicinalChemistry vol 14 no 3 pp 269ndash280 2018

[13] R S Vijayakumar D Surya and N Nalini ldquoAntioxidantefficacy of black pepper (Piper nigrumL) and piperine in ratswith high fat diet induced oxidative stressrdquo Redox Reportvol 9 no 2 pp 105ndash110 2004

[14] T Miyazawa K Nakagawa S H Kim et al ldquoCurcumin andpiperine supplementation of obese mice under caloric re-striction modulates body fat and interleukin-1βrdquo Nutrition ampMetabolism vol 15 no 1 p 12 2018

[15] A Khajuria N -usu U Zutshi and K L Bedi ldquoPiperinemodulation of carcinogen induced oxidative stress in intes-tinal mucosardquo Molecular and Cellular Biochemistry vol 189no 12 pp 113ndash118 1998

[16] S Inder Pal J Shreyans Kumar K Amandeep et al ldquoSyn-thesis and antileishmanial activity of piperoyl-amino acidconjugatesrdquo European Journal of Medicinal Chemistry vol 45no 8 pp 3439ndash3445 2010

[17] T Koichi M Takaki and S Yoshiaki ldquoSynthesis and bio-logical evaluation of piperic acid amides as free radicalscavengers and α-glucosidase inhibitorsrdquo Chemical amp Phar-maceutical Bulletin vol 63 no 5 pp 326ndash333 2015

[18] J M Latorres D G Rios G Saggiomo W Wasielesky andC Prentice-Hernandez ldquoFunctional and antioxidant prop-erties of protein hydrolysates obtained from white shrimp(Litopenaeus vannamei)rdquo Journal of Food Science and Tech-nology vol 55 no 2 pp 721ndash729 2018

[19] A Moayedi L Mora M C Aristoy M Safari M Hashemiand F Toldra ldquoPeptidomic analysis of antioxidant and ACE-inhibitory peptides obtained from tomato waste proteinsfermented using Bacillus subtilisrdquo Food Chemistry vol 250pp 180ndash187 2018

[20] L Zheng H Dong G Su Q Zhao and M Zhao ldquoRadicalscavenging activities of Tyr- Trp- Cys- andMet-Gly and theirprotective effects against AAPH-induced oxidative damage inhuman erythrocytesrdquo Food Chemistry vol 197 no Part App 807ndash813 2016

[21] M Miceli E Roma P Rosa et al ldquoSynthesis of benzofuran-2-one derivatives and evaluation of their antioxidant capacity bycomparing DPPH assay and cyclic voltammetryrdquo Moleculesvol 23 no 4 p 710 2018

[22] K -aipong U Boonprakob K Crosby et al ldquoComparisonof ABTS DPPH FRAP and ORAC assays for estimatingantioxidant activity from guava fruit extractsrdquo Journal of FoodComposition amp Analysis vol 19 no 6-7 pp 669ndash675 2006

[23] X-R Gong G-L Xi and Z-Q Liu ldquoActivity of coumarin-oxadiazole-appended phenol in inhibiting DNA oxidationand scavenging radicalrdquo Tetrahedron Letters vol 56 no 45pp 6257ndash6261 2015

Journal of Chemistry 11

[24] G-L Xi and Z-Q Liu ldquoCoumarin sharing the benzene ringwith quinoline for quenching radicals and inhibiting DNAoxidationrdquo European Journal of Medicinal Chemistry vol 95pp 416ndash423 2015

[25] H R Shin B R You and W H Park ldquoPX-12-induced HeLacell death is associated with oxidative stress and GSH de-pletionrdquo Oncology Letters vol 6 no 6 pp 1804ndash1810 2013

[26] S B Nimse D Pal A Mazumder et al ldquoSynthesis of cin-namanilide derivatives and their antioxidant and antimi-crobial activityrdquo Journal of Chemistry vol 2015 Article ID208910 5 pages 2015

[27] M Oyaizu ldquoStudies on products of browning reactionAntioxidative activities of products of browning reactionprepared from glucosaminerdquo Fe Japanese Journal of Nu-trition and Dietetics vol 44 no 6 pp 307ndash315 1986

[28] Y Yoshioka X Li T Zhang et al ldquoBlack soybean seed coatpolyphenols prevent AAPH-induced oxidative DNA-damagein HepG2 cellsrdquo Journal of Clinical Biochemistry and Nu-trition vol 60 no 2 pp 108ndash114 2017

[29] R C Chiste M Freitas A Z Mercadante and E FernandesldquoCarotenoids inhibit lipid peroxidation and hemoglobinoxidation but not the depletion of glutathione induced byROS in human erythrocytesrdquo Life Sciences vol 99 no 1-2pp 52ndash60 2014

[30] R Ozturk-Urek L A Bozkaya and L Tarhan ldquo-e effects ofsome antioxidant vitamin-and trace element-supplementeddiets on activities of SOD CAT GSH-Px and LPO levels inchicken tissuesrdquo Cell Biochemistry and Function vol 19 no 2pp 125ndash132 2001

[31] O Levy Y Achituv Y Z Yacobi N Stambler andZ Dubinsky ldquo-e impact of spectral composition and lightperiodicity on the activity of two antioxidant enzymes (SODand CAT) in the coral Favia favusrdquo Journal of ExperimentalMarine Biology and Ecology vol 328 no 1 pp 35ndash46 2006

12 Journal of Chemistry

while the positive control and prototype drug (piperine) hadno significant influence on erythrocyte T-SOD activity(pgt 005) After 4 h of incubation compound 5d increasedT-SOD activity significantly (pgt 005) while other com-pounds had no significant influence on T-SOD activity(pgt 005) -ese results highlight the significant enhance-ment caused by piperine derivatives of T-SOD activity inerythrocytes with AAPH-induced oxidative damage com-pared to the relatively inferior effect of the prototypecompound and of the positive drug VC

CAT an abundant enzyme in erythrocytes catalyzesH2O2 decomposition which reduces oxidative damagecaused by OHbull an oxidation product formed from H2O2under catalysis by metal ions [30 31] As shown inFigure 9(c) CAT activity in the AAPH group decreasesgradually relative to that in the control group with in-creasing incubation time Following the addition of 50 μMofpiperine derivatives compound 5c and piperine increasederythrocyte CATactivity after 1 h of incubation After 2 h ofincubation compounds 5a and 5b and piperine increasedCAT activity in erythrocytes significantly (plt 005) whilethe positive control had no significant influence on CAT

activity (pgt 005) After 4 h of incubation compound 5bincreased CAT activity significantly (pgt 005) while othercompounds had no significant influence (pgt 005) -eseresults indicate that piperine derivatives produce signifi-cant differences in CAT activity in AAPH-treated raterythrocytes

4 Conclusion

In the present study piperine-amino acid derivatives weresynthesized and the results indicate that the free radicalremoval ability and total reductive ability of the phenolichydroxyl-containing piperine-amino acid derivatives werehigher than those of the parent compound Specificallycompounds 5a and 5b have free radical removal perfor-mances comparable to that of VC-e derivatives have goodinhibitory effects on AAPH-induced oxidative DNA dam-age while certain compounds also show inhibitory effects onAAPH-induced hemolysis in rat erythrocytes Particularlycompound 5b can significantly inhibit AAPH-inducederythrocyte hemolysis and hemoglobin oxidation In addi-tion the experiments further verified that the protection

Time

GSH

-Px

(U)

1h 2h 4h0

100

200

300

400

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

(a)

Time

SOD

activ

ity (U

mgH

b)

1h 2h 4h0

5

10

15

20

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowastlowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

(b)

Time1h 2h 4h

CAT

activ

ity (U

mgH

b)

0

1

2

3

4

5

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast lowast

lowast

lowast

lowast

lowast

(c)

Figure 9 Influence of piperine derivatives on the (a) GSH-Px content (b) T-SOD activity and (c) CATactivity in AAPH-treated rat erythrocyteslowastindicates a significant difference from the control group (plt 005) Control ( ) AAPH ( ) AAPH+5a ( ) AAPH+5b ( ) AAPH+5c( ) AAPH+5d ( ) AAPH+piperine ( ) AAPH+VC ( ) indicates a significant difference from the AAPH group (plt 005)

10 Journal of Chemistry

provided by piperine derivatives against AAPH-inducedoxidative damage can be achieved by preserving the activityof the antioxidant enzyme system (GSH-Px T-SOD andCAT) within cells -e protection by piperine derivativeswas also found to be superior to that by piperine and vitaminC -is paper described a structural modification methodthat could effectively improve the antioxidant ability ofpiperine-is type of compound could be a candidate for thedevelopment and research of oxidative damage-relateddiseases (hyperlipidemia diabetes cancer whitening etc)

Data Availability

-e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

-is work was financially supported by the Shaanxi Pro-vincial Department of Education serving local (industrial-ization) research project (no 15JF028) and the ScientificResearch Fund of Xirsquoan Medical University(nos2016YXXK09 2018DC-12 and 201825012)

References

[1] J P Adjimani and P Asare ldquoAntioxidant and free radicalscavenging activity of iron chelatorsrdquo Toxicology Reportsvol 2 pp 721ndash728 2015

[2] I Dalle-Donne R Rossi R Colombo D Giustarini andA Milzani ldquoBiomarkers of oxidative damage in humandiseaserdquo Clinical Chemistry vol 52 no 4 pp 601ndash623 2006

[3] L J Machlin and A Bendich ldquoFree radical tissue damageprotective role of antioxidant nutrientsrdquo Fe FASEB Journalvol 1 no 6 pp 441ndash445 1987

[4] N SatoW Li H Takemoto et al ldquoComprehensive evaluationof antioxidant effects of Japanese Kampo medicines led toidentification of Tsudosan formulation as a potent antioxidantagentrdquo Journal of Natural Medicines vol 73 no 1pp 163ndash172 2018

[5] N Bao Z Sun H Baigude G Borjihan and Z Jin ldquoLightinduced isomerization of piperlonguminine to scutifoliamideA isopiperlonguminine and hoffmannseggiamide Ardquo Phy-tochemistry Letters vol 16 pp 324ndash327 2016

[6] T Velpandian R Jasuja R K Bhardwaj J Jaiswal andS K Gupta ldquoPiperine in food interference in the pharma-cokinetics of phenytoinrdquo European Journal of Drug Meta-bolism and Pharmacokinetics vol 26 no 4 pp 241ndash247 2001

[7] A Duangjai K Ingkaninan S Praputbut andN Limpeanchob ldquoBlack pepper and piperine reduce cho-lesterol uptake and enhance translocation of cholesteroltransporter proteinsrdquo Journal of Natural Medicines vol 67no 2 pp 303ndash310 2013

[8] D E Eigenmann C Durig E A Jahne et al ldquoIn vitro blood-brain barrier permeability predictions for GABAA receptormodulating piperine analogsrdquo European Journal of Phar-maceutics and Biopharmaceutics vol 103 pp 118ndash126 2016

[9] Y A Samra H S Said N M Elsherbiny G I Liou M M El-Shishtawy and L A Eissa ldquoCepharanthine and Piperineameliorate diabetic nephropathy in rats role of NF-κB andNLRP3 inflammasomerdquo Life Sciences vol 157 pp 187ndash1992016

[10] R Mittal and R L Gupta ldquoIn vitro antioxidant activity ofpiperinerdquoMethods and Findings in Experimental and ClinicalPharmacology vol 22 no 5 pp 271ndash274 2000

[11] Z Zarai E Boujelbene N Ben Salem Y Gargouri andA Sayari ldquoAntioxidant and antimicrobial activities of varioussolvent extracts piperine and piperic acid from Piper nig-rumrdquo LWT-Food Science and Technology vol 50 no 2pp 634ndash641 2013

[12] A Yasir S Ishtiaq M Jahangir et al ldquoBiology-orientedsynthesis (BIOS) of piperine derivatives and their comparativeanalgesic and antiinflammatory activitiesrdquo MedicinalChemistry vol 14 no 3 pp 269ndash280 2018

[13] R S Vijayakumar D Surya and N Nalini ldquoAntioxidantefficacy of black pepper (Piper nigrumL) and piperine in ratswith high fat diet induced oxidative stressrdquo Redox Reportvol 9 no 2 pp 105ndash110 2004

[14] T Miyazawa K Nakagawa S H Kim et al ldquoCurcumin andpiperine supplementation of obese mice under caloric re-striction modulates body fat and interleukin-1βrdquo Nutrition ampMetabolism vol 15 no 1 p 12 2018

[15] A Khajuria N -usu U Zutshi and K L Bedi ldquoPiperinemodulation of carcinogen induced oxidative stress in intes-tinal mucosardquo Molecular and Cellular Biochemistry vol 189no 12 pp 113ndash118 1998

[16] S Inder Pal J Shreyans Kumar K Amandeep et al ldquoSyn-thesis and antileishmanial activity of piperoyl-amino acidconjugatesrdquo European Journal of Medicinal Chemistry vol 45no 8 pp 3439ndash3445 2010

[17] T Koichi M Takaki and S Yoshiaki ldquoSynthesis and bio-logical evaluation of piperic acid amides as free radicalscavengers and α-glucosidase inhibitorsrdquo Chemical amp Phar-maceutical Bulletin vol 63 no 5 pp 326ndash333 2015

[18] J M Latorres D G Rios G Saggiomo W Wasielesky andC Prentice-Hernandez ldquoFunctional and antioxidant prop-erties of protein hydrolysates obtained from white shrimp(Litopenaeus vannamei)rdquo Journal of Food Science and Tech-nology vol 55 no 2 pp 721ndash729 2018

[19] A Moayedi L Mora M C Aristoy M Safari M Hashemiand F Toldra ldquoPeptidomic analysis of antioxidant and ACE-inhibitory peptides obtained from tomato waste proteinsfermented using Bacillus subtilisrdquo Food Chemistry vol 250pp 180ndash187 2018

[20] L Zheng H Dong G Su Q Zhao and M Zhao ldquoRadicalscavenging activities of Tyr- Trp- Cys- andMet-Gly and theirprotective effects against AAPH-induced oxidative damage inhuman erythrocytesrdquo Food Chemistry vol 197 no Part App 807ndash813 2016

[21] M Miceli E Roma P Rosa et al ldquoSynthesis of benzofuran-2-one derivatives and evaluation of their antioxidant capacity bycomparing DPPH assay and cyclic voltammetryrdquo Moleculesvol 23 no 4 p 710 2018

[22] K -aipong U Boonprakob K Crosby et al ldquoComparisonof ABTS DPPH FRAP and ORAC assays for estimatingantioxidant activity from guava fruit extractsrdquo Journal of FoodComposition amp Analysis vol 19 no 6-7 pp 669ndash675 2006

[23] X-R Gong G-L Xi and Z-Q Liu ldquoActivity of coumarin-oxadiazole-appended phenol in inhibiting DNA oxidationand scavenging radicalrdquo Tetrahedron Letters vol 56 no 45pp 6257ndash6261 2015

Journal of Chemistry 11

[24] G-L Xi and Z-Q Liu ldquoCoumarin sharing the benzene ringwith quinoline for quenching radicals and inhibiting DNAoxidationrdquo European Journal of Medicinal Chemistry vol 95pp 416ndash423 2015

[25] H R Shin B R You and W H Park ldquoPX-12-induced HeLacell death is associated with oxidative stress and GSH de-pletionrdquo Oncology Letters vol 6 no 6 pp 1804ndash1810 2013

[26] S B Nimse D Pal A Mazumder et al ldquoSynthesis of cin-namanilide derivatives and their antioxidant and antimi-crobial activityrdquo Journal of Chemistry vol 2015 Article ID208910 5 pages 2015

[27] M Oyaizu ldquoStudies on products of browning reactionAntioxidative activities of products of browning reactionprepared from glucosaminerdquo Fe Japanese Journal of Nu-trition and Dietetics vol 44 no 6 pp 307ndash315 1986

[28] Y Yoshioka X Li T Zhang et al ldquoBlack soybean seed coatpolyphenols prevent AAPH-induced oxidative DNA-damagein HepG2 cellsrdquo Journal of Clinical Biochemistry and Nu-trition vol 60 no 2 pp 108ndash114 2017

[29] R C Chiste M Freitas A Z Mercadante and E FernandesldquoCarotenoids inhibit lipid peroxidation and hemoglobinoxidation but not the depletion of glutathione induced byROS in human erythrocytesrdquo Life Sciences vol 99 no 1-2pp 52ndash60 2014

[30] R Ozturk-Urek L A Bozkaya and L Tarhan ldquo-e effects ofsome antioxidant vitamin-and trace element-supplementeddiets on activities of SOD CAT GSH-Px and LPO levels inchicken tissuesrdquo Cell Biochemistry and Function vol 19 no 2pp 125ndash132 2001

[31] O Levy Y Achituv Y Z Yacobi N Stambler andZ Dubinsky ldquo-e impact of spectral composition and lightperiodicity on the activity of two antioxidant enzymes (SODand CAT) in the coral Favia favusrdquo Journal of ExperimentalMarine Biology and Ecology vol 328 no 1 pp 35ndash46 2006

12 Journal of Chemistry

provided by piperine derivatives against AAPH-inducedoxidative damage can be achieved by preserving the activityof the antioxidant enzyme system (GSH-Px T-SOD andCAT) within cells -e protection by piperine derivativeswas also found to be superior to that by piperine and vitaminC -is paper described a structural modification methodthat could effectively improve the antioxidant ability ofpiperine-is type of compound could be a candidate for thedevelopment and research of oxidative damage-relateddiseases (hyperlipidemia diabetes cancer whitening etc)

Data Availability

-e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

-is work was financially supported by the Shaanxi Pro-vincial Department of Education serving local (industrial-ization) research project (no 15JF028) and the ScientificResearch Fund of Xirsquoan Medical University(nos2016YXXK09 2018DC-12 and 201825012)

References

[1] J P Adjimani and P Asare ldquoAntioxidant and free radicalscavenging activity of iron chelatorsrdquo Toxicology Reportsvol 2 pp 721ndash728 2015

[2] I Dalle-Donne R Rossi R Colombo D Giustarini andA Milzani ldquoBiomarkers of oxidative damage in humandiseaserdquo Clinical Chemistry vol 52 no 4 pp 601ndash623 2006

[3] L J Machlin and A Bendich ldquoFree radical tissue damageprotective role of antioxidant nutrientsrdquo Fe FASEB Journalvol 1 no 6 pp 441ndash445 1987

[4] N SatoW Li H Takemoto et al ldquoComprehensive evaluationof antioxidant effects of Japanese Kampo medicines led toidentification of Tsudosan formulation as a potent antioxidantagentrdquo Journal of Natural Medicines vol 73 no 1pp 163ndash172 2018

[5] N Bao Z Sun H Baigude G Borjihan and Z Jin ldquoLightinduced isomerization of piperlonguminine to scutifoliamideA isopiperlonguminine and hoffmannseggiamide Ardquo Phy-tochemistry Letters vol 16 pp 324ndash327 2016

[6] T Velpandian R Jasuja R K Bhardwaj J Jaiswal andS K Gupta ldquoPiperine in food interference in the pharma-cokinetics of phenytoinrdquo European Journal of Drug Meta-bolism and Pharmacokinetics vol 26 no 4 pp 241ndash247 2001

[7] A Duangjai K Ingkaninan S Praputbut andN Limpeanchob ldquoBlack pepper and piperine reduce cho-lesterol uptake and enhance translocation of cholesteroltransporter proteinsrdquo Journal of Natural Medicines vol 67no 2 pp 303ndash310 2013

[8] D E Eigenmann C Durig E A Jahne et al ldquoIn vitro blood-brain barrier permeability predictions for GABAA receptormodulating piperine analogsrdquo European Journal of Phar-maceutics and Biopharmaceutics vol 103 pp 118ndash126 2016

[9] Y A Samra H S Said N M Elsherbiny G I Liou M M El-Shishtawy and L A Eissa ldquoCepharanthine and Piperineameliorate diabetic nephropathy in rats role of NF-κB andNLRP3 inflammasomerdquo Life Sciences vol 157 pp 187ndash1992016

[10] R Mittal and R L Gupta ldquoIn vitro antioxidant activity ofpiperinerdquoMethods and Findings in Experimental and ClinicalPharmacology vol 22 no 5 pp 271ndash274 2000

[11] Z Zarai E Boujelbene N Ben Salem Y Gargouri andA Sayari ldquoAntioxidant and antimicrobial activities of varioussolvent extracts piperine and piperic acid from Piper nig-rumrdquo LWT-Food Science and Technology vol 50 no 2pp 634ndash641 2013

[12] A Yasir S Ishtiaq M Jahangir et al ldquoBiology-orientedsynthesis (BIOS) of piperine derivatives and their comparativeanalgesic and antiinflammatory activitiesrdquo MedicinalChemistry vol 14 no 3 pp 269ndash280 2018

[13] R S Vijayakumar D Surya and N Nalini ldquoAntioxidantefficacy of black pepper (Piper nigrumL) and piperine in ratswith high fat diet induced oxidative stressrdquo Redox Reportvol 9 no 2 pp 105ndash110 2004

[14] T Miyazawa K Nakagawa S H Kim et al ldquoCurcumin andpiperine supplementation of obese mice under caloric re-striction modulates body fat and interleukin-1βrdquo Nutrition ampMetabolism vol 15 no 1 p 12 2018

[15] A Khajuria N -usu U Zutshi and K L Bedi ldquoPiperinemodulation of carcinogen induced oxidative stress in intes-tinal mucosardquo Molecular and Cellular Biochemistry vol 189no 12 pp 113ndash118 1998

[16] S Inder Pal J Shreyans Kumar K Amandeep et al ldquoSyn-thesis and antileishmanial activity of piperoyl-amino acidconjugatesrdquo European Journal of Medicinal Chemistry vol 45no 8 pp 3439ndash3445 2010

[17] T Koichi M Takaki and S Yoshiaki ldquoSynthesis and bio-logical evaluation of piperic acid amides as free radicalscavengers and α-glucosidase inhibitorsrdquo Chemical amp Phar-maceutical Bulletin vol 63 no 5 pp 326ndash333 2015

[18] J M Latorres D G Rios G Saggiomo W Wasielesky andC Prentice-Hernandez ldquoFunctional and antioxidant prop-erties of protein hydrolysates obtained from white shrimp(Litopenaeus vannamei)rdquo Journal of Food Science and Tech-nology vol 55 no 2 pp 721ndash729 2018

[19] A Moayedi L Mora M C Aristoy M Safari M Hashemiand F Toldra ldquoPeptidomic analysis of antioxidant and ACE-inhibitory peptides obtained from tomato waste proteinsfermented using Bacillus subtilisrdquo Food Chemistry vol 250pp 180ndash187 2018

[20] L Zheng H Dong G Su Q Zhao and M Zhao ldquoRadicalscavenging activities of Tyr- Trp- Cys- andMet-Gly and theirprotective effects against AAPH-induced oxidative damage inhuman erythrocytesrdquo Food Chemistry vol 197 no Part App 807ndash813 2016

[21] M Miceli E Roma P Rosa et al ldquoSynthesis of benzofuran-2-one derivatives and evaluation of their antioxidant capacity bycomparing DPPH assay and cyclic voltammetryrdquo Moleculesvol 23 no 4 p 710 2018

[22] K -aipong U Boonprakob K Crosby et al ldquoComparisonof ABTS DPPH FRAP and ORAC assays for estimatingantioxidant activity from guava fruit extractsrdquo Journal of FoodComposition amp Analysis vol 19 no 6-7 pp 669ndash675 2006

[23] X-R Gong G-L Xi and Z-Q Liu ldquoActivity of coumarin-oxadiazole-appended phenol in inhibiting DNA oxidationand scavenging radicalrdquo Tetrahedron Letters vol 56 no 45pp 6257ndash6261 2015

Journal of Chemistry 11

[24] G-L Xi and Z-Q Liu ldquoCoumarin sharing the benzene ringwith quinoline for quenching radicals and inhibiting DNAoxidationrdquo European Journal of Medicinal Chemistry vol 95pp 416ndash423 2015

[25] H R Shin B R You and W H Park ldquoPX-12-induced HeLacell death is associated with oxidative stress and GSH de-pletionrdquo Oncology Letters vol 6 no 6 pp 1804ndash1810 2013

[26] S B Nimse D Pal A Mazumder et al ldquoSynthesis of cin-namanilide derivatives and their antioxidant and antimi-crobial activityrdquo Journal of Chemistry vol 2015 Article ID208910 5 pages 2015

[27] M Oyaizu ldquoStudies on products of browning reactionAntioxidative activities of products of browning reactionprepared from glucosaminerdquo Fe Japanese Journal of Nu-trition and Dietetics vol 44 no 6 pp 307ndash315 1986

[28] Y Yoshioka X Li T Zhang et al ldquoBlack soybean seed coatpolyphenols prevent AAPH-induced oxidative DNA-damagein HepG2 cellsrdquo Journal of Clinical Biochemistry and Nu-trition vol 60 no 2 pp 108ndash114 2017

[29] R C Chiste M Freitas A Z Mercadante and E FernandesldquoCarotenoids inhibit lipid peroxidation and hemoglobinoxidation but not the depletion of glutathione induced byROS in human erythrocytesrdquo Life Sciences vol 99 no 1-2pp 52ndash60 2014

[30] R Ozturk-Urek L A Bozkaya and L Tarhan ldquo-e effects ofsome antioxidant vitamin-and trace element-supplementeddiets on activities of SOD CAT GSH-Px and LPO levels inchicken tissuesrdquo Cell Biochemistry and Function vol 19 no 2pp 125ndash132 2001

[31] O Levy Y Achituv Y Z Yacobi N Stambler andZ Dubinsky ldquo-e impact of spectral composition and lightperiodicity on the activity of two antioxidant enzymes (SODand CAT) in the coral Favia favusrdquo Journal of ExperimentalMarine Biology and Ecology vol 328 no 1 pp 35ndash46 2006

12 Journal of Chemistry

[24] G-L Xi and Z-Q Liu ldquoCoumarin sharing the benzene ringwith quinoline for quenching radicals and inhibiting DNAoxidationrdquo European Journal of Medicinal Chemistry vol 95pp 416ndash423 2015

[25] H R Shin B R You and W H Park ldquoPX-12-induced HeLacell death is associated with oxidative stress and GSH de-pletionrdquo Oncology Letters vol 6 no 6 pp 1804ndash1810 2013

[26] S B Nimse D Pal A Mazumder et al ldquoSynthesis of cin-namanilide derivatives and their antioxidant and antimi-crobial activityrdquo Journal of Chemistry vol 2015 Article ID208910 5 pages 2015

[27] M Oyaizu ldquoStudies on products of browning reactionAntioxidative activities of products of browning reactionprepared from glucosaminerdquo Fe Japanese Journal of Nu-trition and Dietetics vol 44 no 6 pp 307ndash315 1986

[28] Y Yoshioka X Li T Zhang et al ldquoBlack soybean seed coatpolyphenols prevent AAPH-induced oxidative DNA-damagein HepG2 cellsrdquo Journal of Clinical Biochemistry and Nu-trition vol 60 no 2 pp 108ndash114 2017

[29] R C Chiste M Freitas A Z Mercadante and E FernandesldquoCarotenoids inhibit lipid peroxidation and hemoglobinoxidation but not the depletion of glutathione induced byROS in human erythrocytesrdquo Life Sciences vol 99 no 1-2pp 52ndash60 2014

[30] R Ozturk-Urek L A Bozkaya and L Tarhan ldquo-e effects ofsome antioxidant vitamin-and trace element-supplementeddiets on activities of SOD CAT GSH-Px and LPO levels inchicken tissuesrdquo Cell Biochemistry and Function vol 19 no 2pp 125ndash132 2001

[31] O Levy Y Achituv Y Z Yacobi N Stambler andZ Dubinsky ldquo-e impact of spectral composition and lightperiodicity on the activity of two antioxidant enzymes (SODand CAT) in the coral Favia favusrdquo Journal of ExperimentalMarine Biology and Ecology vol 328 no 1 pp 35ndash46 2006

12 Journal of Chemistry