phytochemical studies of the phenolic substances in aster glehni extract and its sedative and...

Arch Pharm Res Vol 35, No 3, 423-430, 2012DOI 10.1007/s12272-012-0304-7

423

Phytochemical Studies of the Phenolic Substances in Aster glehni Extract and its Sedative and Anticonvulsant Activity

Agung Nugroho1, Myung-Hoe Kim2, Jongwon Choi3, Jae Sue Choi4, Won Tae Jung5, Kyung-Tae Lee6, andHee-Juhn Park2

1Department of Agro-industrial Technology, Faculty of Agriculture, Lambung Mangkurat University, Indonesia 70714,2Department of Pharmaceutical Engineering, Sangji University, Wonju 220-702, Korea, 3College of Pharmacy, KyungsungUniversity, Busan 608-736, Korea, 4College of Pharmacy, Pukyong National University, Busan 609-735, Korea, 5GlobalR&D Center, Korea United Pharm, Seoul 616-12, Korea, and 6College of Pharmacy, Kyung-Hee University, Seoul 220-701, Korea

(Received January 3, 2011/Revised July 7, 2011/Accepted July 20, 2011)

On high performance liquid chromatography, the caffeoylquinic acid (CQ) occupying the high-est proportion of the water-ethanol (7:3) extract of Aster glehni (Compositae) leaves was 3-O-p-coumaroylquinic acid (46.10 ± 4.22 mg/g of dried weight) among CQs tested. The IC50 of thewater-ethanol (7:3) extract was 4.23 ± 0.24 µg/mL in the peroxynitrite (ONOO−)-scavengingassay. Phytochemical isolation from A. glehni extract yielded three kaempferol glycosides. Thewater-ethanol (7:3) extract and both p-coumaric acid and caffeic acid, phenylpropanoid moi-eties of CQs, had sedative effects in pentobarbital-induced sleeping time in mice and anticon-vulsant effects in pentylenetetrazole-induced mice. Furthermore, the phenolic substance-richW-E (7:3) extract of A. glehni could be used to treat anxiety or convulsion partly due to its per-oxynitrite-scavenging mechanism.Key words: Aster glehni, Compositae, Caffeoylquinic acid, Sedative, Anticonvulsant, Perox-ynitrite, High performance liquid chromatography

INTRODUCTION

Convulsions are caused by an imbalance betweenthe excitatory neurotransmitter, glutamic acid andthe inhibitory neurotransmitter, γ-aminobutyric acid(GABA). Since 1980s, the development of anticonvul-sant drugs has mainly been based on this understand-ing (Crawford, 1963; Baughman and Gilbert, 1980;Croucher et al., 1982). Pentylenetetrazole (PTZ), whichis an excitatory agent in the central nervous system(CNS), increases glutamic acid but decreases GABA(Metcalf, 1979). The hippocampus is responsible forcognition, sensation, learning, and memory and it is

vulnerable to hypoxia-ischemia, seizure and sustainedstress. In addition, sustained stress causes synapsesto accumulate glutamate and thereby results insequential intracellular biochemical changes, neurode-generative disorders and even death (Dutar et al.,1995; Wang et al., 2006).

The leaves of Aster glehni Franchet et Sckmidt(Compositae) distributed in Island Ulleung-do isedible for food called chwinamul and is used to treatdiabetes mellitus, hypercholesterolemia, insomnia,and cardiovascular disease (Kim, 1996). After observ-ing the high content of phenolic substances in theextract of A. glehni leaves and the high peroxynitrite(ONOO−)-scavenging effect in our preliminary studies,we intended to develop a functional food from thismountainous vegetable. Therefore, we sought to producea phenolic substance-rich extract with high peroxyni-trite-scavenging activity using only water (W), ethanol(E) or their mixed W-E solvents because only W and/or E can be used for functional foods. The extracts ob-tained from A. glehni using various ratios of W-E as

Correspondence to: Hee-Juhn Park, Department of Pharmaceu-tical Engineering, Sangji University, Wonju 220-702, KoreaTel: 82-33-730-0564, Fax: 82-33-730-0564Email: [email protected]

Selected by Editors, See page 389

424 A. Nugroho et al.

the extracting solvent were qualitatively and quanti-tatively analyzed for phenolic substances and thentheir peroxynitrite-scavenging effects were evaluated.

The ionic and very reactive peroxynitrite initiateslipid peroxidation, breaks DNA and binds with thiolsVirag et al., 2003). This ion causes Alzheimer’s disease,Huntington’s chorea, and Parkinson’s disease (Greenacreand Ischiropoulos, 2001). Since peroxynitrite inducesprotein oxidation accompanied by reacting withmethionine, cysteine, tryptophane or tyrosine residuesand nitration on tyrosine and tryptophane residues(Virag et al., 2003).

We have reported the composition of caffeoylquinicacids (CQs) from chwinamul and the peroxynitrite-scavenging effects (Nugroho et al., 2009), antiul-cerogenic (Nugroho et al., 2010b), antihyperlipidemic(Nugroho et al., 2010a) and hepatoprotective (Choi etal., 2005) effects. In addition, CQs have peroxynitrite-scavenging (Nugroho et al., 2010c), hepatoprotective(Basnet et al., 1996), antidiabetic (Alonso-Castro etal., 2008) and antiobesity effects (Koya-Miyata et al.,2009). CQs are commonly high in the young stage ofchwinamul and may be responsible for the pharma-cological activity (Nugroho et al., 2009).

There have been many reports on the sedative oranticonvulsant actions of natural phenolic compounds.In addition, leaves are generally enriched in polyphe-nols. Therefore, in the present study, we explored thepossibility of developing a functional food from A.glehni distributed in Island Ulleung-do (Kim, 1996),as it contains high levels of CQs and shows a potentperoxynitrite-scavenging effect. For functional foods,only water (W) and ethanol (E) can be used as theextracting solvents. Therefore, we sought to create anextract from the leaves of A. glehni that containedhigh level of CQs and high peroxynitrite-scavengingactivity, extracting with W and E solvents. In addition,the W-E (7:3) extract was used in the pentobarbital-induced sleeping time test and PTZ-induced convulsiontest in mice to evaluate the sedative and anticonvul-sant activities, respectively.

MATERIALS AND METHODS

Instruments and reagentsThe Varian HPLC system consisted of a Prostar 210

pump and a Prostar 325 UV-Vis detector: the columnwas Shiseido (Chuoku) Capcell Pak C18 column (5µm, 4.6 mm × 250 mm). Methanol (J.T Baker Co.) wasused as the mobile phase. The seven standard com-pounds were from Prof. Kang Ro Lee (College of Phar-macy, SungKyunKwan University). Dihydrorhodamine123 (DHR 123) and peroxynitrite (ONOO−), which were

used for the peroxynitrite-scavenging assay, were pur-chased from Molecular Probes (Eugene) and CaymanChemical Co., respectively. PTZ, p-coumaric acid, andcaffeic acid used for the anticonvulsant assay werefrom Sigma Co.

Plant materialThe leaves of A. glehni Franchet et Sckmidt (Com-

positae), collected at Island Ulleung-do, were driedand crushed for extraction. This plant was identifiedby Jong Hee Park (College of Pharmacy, Pusan NationalUniversity). The voucher specimen (# natchem-31)was deposited at the Laboratory of Natural ProductChemistry of the Department of Pharmaceutical En-gineering, Sangji University, Wonju, Korea.

Extraction, fractionation and isolationThe plant material (500 g) was extracted with MeOH

three times under reflux. The extracted material wasfiltered, dried on a rotary evaporator under reducedpressure, and then freeze-dried to give the MeOH ex-tract (73 g). This MeOH extract was partitioned threetimes between 800 mL H2O and 800 mL CHCl3 andthe CHCl3-soluble part was concentrated to dryness togive a CHCl3 fraction (26.7 g). The residual aqueouslayer was fractionated with 800 mL BuOH threetimes to yield the BuOH fraction (10.5 g).

The BuOH fraction (10.0 g) was chromatographedover silica gel column using CHCl3-MeOH-H2O (7:3:1,lower phase) as an eluent and collected as each 60 mLfractions. The concentrated product of subfraction #1was named Fr. A. Subfractions # 2-8 were combined,concentrated and the resultant residue was named Fr.B. The concentrate of subfractions # 14-54 was namedFr. C. Here, Fr. B was chromatographed over ODScolumn with the eluting solvent MeOH-H2O (1:1) andcollected as each 20 mL subfractions. Subfractions #7-13 were concentrated and then the residue wasrecrystallized in MeOH to afford compound 1 (45 mg,yellowish powder). Similar to Fr. B, ODS column chro-matography of Fr. C was performed and 20 mL sub-fractions were collected. Concentration of aliquots # 3-4 and # 13-18 and successive recrystallization fromMeOH yielded compound 2 (110 mg, yellowish powder,kaempferol 3-O-β-D-galactopyranoside) and compound3 (150 mg, kaempferol 3-O-β-D-galactopyranoisde),respectively. Physicochemical and spectroscopic dataof the three compounds are described below, and com-pounds 1-3 were identified as kaempferol 3-O-β-D-rhamnopyranoisde (afzelin), kaempferol 3-O-β-D-gal-actopyranoside, kaempferol 3-O-β-D-glucopyranoisde(astragalin), respectively.

Phytochemical Studies of the Phenolic Substances in Aster glehni 425

Kaempferol 3-O-α-L-rhamnopyranoside, afzelin(1)Yellowish needles from MeOH-H2O (1:1), m.p. 173-178oC; UV λmax (MeOH) nm: 268, 357; IR νmax cm−1: 3400-3100 (broad, OH), 1655 (α,β-unsaturated ketone), 1605(aromatic C=C), 1355, 1170, 1100-1000 (glycosidic C-O); 1H-NMR (DMSO-d6, 500 MHz): δ 0.81 (3H, d, J =5.6 Hz, rha-CH3), 5.32 (1H, d, J = 1.47 Hz, H-1''), 6.21(1H, d, J = 2.0 Hz, H-6), 6.41 (1H, d, J = 2.0 Hz, H-8),6.92 (2H, d, J = 8.8 Hz, H-3’, 5’), 7.76 (2H, d, J = 8.8 Hz,H-3’, 5’); 13C-NMR (DMSO-d6, 125 MHz): δ kaempferol -93.8 (C-8), 98.8 (C-6), 104.2 (C-10), 115.5 (C-3’, 5’), 120.7(C-1’), 130.6 (C-2’, 6’), 134.3 (C-3), 156.6 (C-9), 157.3(C-2), 160.0 (C-4’), 161.4 (C-5), 164.3 (C-7), 177.8 (C-4),L-Rha - 101.8 (C-1’’), 74.2 (C-2’’), 76.4 (C-3’’), 69.9 (C-4’’), 77.4 (C-5’’), 60.9 (C-6’’).

Kaempferol 3-O-β-D-galactopyranoside (2)Yellowish powder, m.p. 220-224oC; IR νmax (KBr, cm−1):3457 (OH), 1659 (α,β-unsaturated ketone), 1605, 1560,1491 (aromatic C=C), 1362, 1262, 1183, 1062 (glycosidicC-O); UV λmax (MeOH) nm: 265, 356; 1H-NMR (500MHz, DMSO-d6): δ 8.06 (2H, d, J = 8.8 Hz, H-2’, 6’),6.85 (2H, d, J = 8.8 Hz, H-3’, 5’), 6.42 (1H, d, J = 1.9Hz, H-8), 6.18 (1H, d, J = 1.9 Hz, H-6), 5.39 (1H, d, J= 7.5 Hz, H-1’’); 13C-NMR (DMSO-d6, 125 MHz): δkaempferol - 93.7 (C-8), 99.4 (C-6), 104.0 (C-10), 115.1(C-3’, 5’), 120.9 (C-1’), 131.0 (C-2’, 6’), 133.3 (C-3),156.4 (C-2), 156.4 (C-9), 160.0 (C-4’), 161.2 (C-5), 164.2(C-7), 177.6 (C-4), D-gal - 60.2 (C-6’’), 67.9 (4’’), 71.2 (C-2’’), 73.1 (C-3’’), 75.6 (C-5’’), 101.7 (C-1’’).

Kaempferol 3-O-β-D-glucopyranoside, astragalin(3)Yellowish powder, m.p. 230-233oC; FeCl3, Mg-HCl, Zn-HCl, Molish tests: positive; IR νmax cm−1: 3619-3000(broad, OH), 1655 (α,β-unsaturated ketone), 1606, 1562,1506 (aromatic C=C), 1360, 1291, 1179, 1056, 1011(glycosidic C-O), 799; UV λmax (MeOH) nm: 267, 300(sh), 352; 1H-NMR (DMSO-d6, 500 MHz): δ 5.54 (1H,d, J = 7.2 Hz, H-1 of D-Glc), 6.21 (1H, d, J = 2.1 Hz, H-6), 6.43 (1H, d, J = 2.1 Hz, H-8), 6.89 (2H, d, J = 8.8Hz, H-3’, 5’), 8.04 (2H, d, J = 8.8 Hz, H-2’, 6’); 13C-NMR (DMSO-d6, 125 MHz): δ kaempferol - 156.3 (C-2), 133.2 (C-3), 177.4 (C-4), 161.2 (C-5), 98.7 (C-6),164.2 (C-7), 93.6 (C-8), 156.3 (C-9), 103.9 (C-10), 120.9(C-1), 130.7 (C-2), 115.0 (C-3), 159.9 (C-4), 115.0 (C-5),130.8 (C-6), D-Glc - 100.9 (C-1), 74.2 (C-2), 76.4 (C-3),69.9 (C-4), 77.4 (C-5), 60.8 (C-6).

Preparation of samples for HPLC analysisFinely cut leaves of A. glehni were separately ex-

tracted at 40oC with 400 mL of H2O and W-E (7:3), W-

E (5:5), W-E (3:7) and EtOH under sonication for 6 h.After filtration, the filtered solutions were concentrat-ed in vacuo, freeze-dried and used for HPLC analysis.

HPLC analysis conditionThe present HPLC analysis was performed as de-

scribed previously (Nugroho et al., 2009). In brief,seven CQs and the four extracts dissolved in 80%MeOH were filtered using a syringe filter and injectedfor HPLC analysis. Fixed wavelength of UV detectorwas 246 nm. The two mobile phases of 0.05% phos-phoric acid (solvent A) and MeOH (solvent B) wereused for gradient elution at the rate of 1.00 mL/min:0-10 min, 60% A : 40% B; 10-20 min, 50% A : 50% B;20-30 min, 40% A : 60% B; 30-35 min, 60% A : 40% B.The two flavonoids, astragalin and kaempferol, werealso used as the standard compounds for the analysis.Under these HPLC condition identical, the retentiontimes of astragalin and kaempferol were 14.4 min and24.3 min, respectively. Plotting the peak area (y, counts)vs concentration (x, µg/mL), regression equations of y= 27.10x + 50.50 (R2 = 0.998) and y = 54.85x + 287.01(R2 = 0.999) for astragalin and kaempferol, respecti-vely. Sample solutions were injected onto the HPLCsystem at 1.000 mg/mL and the contents were deter-mined from the regression equation.

Assay for peroxynitrite-scavenging activityPeroxynitrite-scavenging effect was determined by

modifying the method described by Kooy et al. (1994).This method involves monitoring the highly fluores-cent rhodamine 123, which is formed rapidly from non-fluorescent DHR 123 under peroxynitrite. Rhodaminebuffer (pH 7.4) consisted of 50 mM sodium phosphatedibasic, 50 mM sodium phosphate monobasic, 90 mMsodium chloride, 5 mM potassium chloride, and 100µM DTPA (diethylenetriaminepentaacetic acid). Thefinal DHR 123 concentration was 5 µM. The bufferused in this assay was prepared prior to use and plac-ed on ice. The extract was dissolved in 10% DMSOprior to use. The background and fluorescence inten-sities were measured 5 min after treatment with andwithout authentic peroxynitrite (10 µM) dissolved in0.3 N sodium hydroxide. The fluorescent intensity ofoxidized DHR 123 was measured at excitation andemission wavelengths of 480 nm and 530 nm, respec-tively, using microplate fluorescent reader FL 500(Bio-Tek Instruments Inc.). Peroxynitrite-scavengingactivity was calculated as the final fluorescence inten-sity minus the background fluorescence, via the detec-tion of DHR oxidation. L-Penicillamine was used as apositive control.


Animal and sample solutionsFour or five-month-old ICR male mice weighing 20 ±

2 g were purchased from Hyochang Science. Mice weremaintained under constant conditions (temperature:20 ± 2oC, humidity: 40-60%, light/dark cycle: 12 h) forone week. All animal experiments were approved bythe University of Kyungsung Animal Care and UseCommittee, and all procedures were conducted inaccordance with the “Guide for the Care and Use ofLaboratory Animals” published by the National In-stitutes of Health. The sample solutions dissolved inTween 80 [tween 80 : saline (1:4)] were administeredto the mice. Lorazepam (i.p.) and pentobarbital (p.o.)were used as positive controls.

Pentobarbital-induced sleeping timeSeven mice were included in each group for this assay.

This assay was performed by the method described byMa et al. (2009). Thirty mg/kg of pentobarbital sodiumwas intraperitoneally injected 30 min after lorazepamadministration (i.p.) and 30 min after sample admin-istration (p.o.). The time from the disappearance ofrighting reflex to its reappearance was measured.

PTZ-induced convulsionTen mice were included in each group. This assay

was performed by the method described by Vogel(2001). Samples were orally administered once a dayfor a week. And then 1 h after the final treatment ofsample, animals were intraperitoneally injected PTZ(70 mg/kg, s.c.). Then, the onset time and tonic exten-sive on convulsion and mortality were observed.

RESULTS

The extraction rates of W, W-E (7:3), W-E (3:7) andE were 17.2%, 19.4%, 17.2%, and 8.1%, respectively.

The yield was lowest with E solvent and highest withW-E (7:3) solvent. The CQs in those four extracts wereanalyzed by HPLC using seven standard compounds.After this experiment, the extraction rates were againevaluated using the W-E solvents at W-E ratios, (5:5),(6:4), (7:3), (8:2) and (9:1) to find more adequate solv-ents. The W-E solvents, (6:4), (7:3), and (8:2) producedsimilar extraction efficiency (approx. 19%) but thehighest (19.6%) in W-E (7:3) (Fig. 1).

Column chromatography of the BuOH fraction yield-ed three flavonoid glycosides that were identified askaempferol 3-O-β-D-rhamnopyranoisde (afzelin) (Parket al., 1991), kaempferol 3-O-β-D-galactopyranoisde(Hur et al., 1998), kaempferol 3-O-β-D-glucopyranoisde(astragalin) (Lee et al., 2003) by spectroscopic evidence.The CQs identified by HPLC using standard compoundswere 3-pCQ, 3,4-DQ, 5-CQ, and 3,5-DQ (Fig. 2). Amongthe identified CQs, 3,4-DQ was found only in the E ex-tract and 4,5-DQ was found in the W-E (7:3) extract;

Fig. 1. Extraction yield (%) of A. glehni with solvent vari-ance (water-ethanol system). Data represent mean ± S.D. (n= 3). Values sharing the same letter are not significantlydifferent (p < 0.05) by Duncan’s multiple range test.

Fig. 2. Structure of phenolic compounds identified in the extracts of A. glehni and of p-coumaric acid and caffeic acid usedfor animal experiment


meanwhile 3,5-DQ existed in all four extracts measur-ed. The two DQs, 3,4-DQ and 4,5-DQ, may be isomeriz-ed since 3,5-DQ was found in the W extract. As shownin Fig. 3, polar compounds are shown at the shorterretention times and less polar ones at the later timesbecause the column contains the reverse phase sta-tionary phase (ODS). In particular, the contents of 5-CQ and 3-pCQ were high in the W-E (7:3) extract. Thecompound, 3-pCQ, was highest in the W-E (7:3) ex-tract (46.10 mg/g) but very low in the E extract (6.59mg/g). In addition, the sum of the identified CQs was

38.7% of the W-E (7:3) extract weight and 20.7% of theextract weight in the E extract. The CQ proportionwas highest in the W-E (7:3) extract among the testedextracts. When the contents of the two flavonoids werequantitatively compared, astragalin was much higherthan kaempferol. This result indicates that the flavo-noid exists in the glycoside form; the contents of as-tragalin and kaempferol were 13.2 mg/g and 0.23 mg/g, respectively (Table I).

To evaluate the peroxynitrite-scavenging effect, theextracts were tested at concentrations of 2, 5, and 10mg/g and the results are shown in Table II. The threeextracts other than the E extract exhibited similarIC50 values. Comparing the IC50s of peroxynitrite-scavenging effect, the activity of the extracts were inorder of L-penicillamine (1.97 ± 0.09 µg/mL), W-E (3:7)(3.22 ± 0.19 µg/mL), W (3.92 ± 0.07 µg/mL), W-E (7:3)(4.23 ± 0.24 µg/mL), and E (5.77 ± 0.31 µg/mL). Insummary, W-E (7:3) extract contained the highestyield of extract and CQ proportion as well as peroxy-nitrite-scavenging activity level similar to that of thestrongest W-E (3:7) extract. Therefore, the W-E (7:3)extract is desirable for the preparation of functionalfood, though other W-E extracts, e.g. W-E (6:4) and W-E (8:2), are available. However, the E is not adequatefor efficient extraction of phenolic substances. W solventmay be available for extraction of phenolic compoundsbut its yield was relatively lower.

The sedative effect was evaluated by measuring thepentobarbital-induced sleeping time in mice, and theresults are shown in Table III. We used p-coumaricacid and caffeic acid for the test because the identifiedCQs possess those moieties. Lorazepam, which is atranquilizer, was employed in the experiment as apositive control. Treatment of mice with lorazepamprolonged the sleeping time induced by pentobarbitalby 5.38-fold. p-Coumaric acid and caffeic acid delayedthe sleeping time by 3.36-fold and 2.74-fold, respectively,at the 20 mg/kg dose. The W-E (7:3) extract lengthenedthe sleeping time by 1.25- and 1.57-fold at the 100 and200 mg/kg doses compared with pentobarbital alone.The effects on pentobarbital-induced convulsions areshown in Table IV. As with the data on pentobarbital-induced sleeping time, treatment with the W-E (7:3)extract (100 and 200 mg/kg, p.o.) and both p-coumaricacid and caffeic acid (10 and 20 mg/kg, p.o.) signifi-cantly inhibited tonic convulsion in terms of onsettime, tonic extensive and mortality.

DISCUSSION

In the present study, we aimed to produce a functionalfood with sedative and anticonvulsant effects from the

Fig. 3. HPLC chromatograms of the four extracts from A.glehni


leaves of A. glehni, a mountainous vegetable beneficialfor health. W-E solvent was selected because it wasmost efficient for obtaining a phenolic substance-richextract. The W-E (7:3) extract contained 38.7% CQsand 3-pCQ was 65% of the total CQ identified. Thecontent of flavonoid was relatively lower compared to

that of CQs and mainly existed in the glycoside formrather than the aglycone form. The content of astrag-alin in the W-E (3:7) extract was higher than in otherextracts.

Based on these results, CQs are abundant as pheno-lic substances compared to flavonoids and they maybe responsible for the peroxynitrite-scavenging effectof this extract. In particular, 3-pCQ was most enrich-ed in the extract among the CQs. We previously re-ported the peroxynitrite-scavenging effect of CQs iso-lated from A. princeps var. orientalis. Peroxynitritecan be formed through the reaction between super-oxide anion radical (•O2

−) and nitric oxide (NO•) (Radiet al., 1991), causing the peroxidation of lipid and pro-tein, cytotoxicity and neurotoxicity (Haenen et al.,1997). Overproduction of peroxynitrite causes hyper-cholesterolemia, atherosclerosis, obesity or diabetesmellitus (Patcher et al., 2005; Drel et al., 2007; Kordaet al., 2008).

Many phenolic substances in plants contribute tothe prevention of neurotoxicity caused by excess glu-tamate and thereby to recovery of movement, cognition,learning and sensation via antioxidant mechanism(Karczmar, 1993; Vauzour et al., 2010). It is reported

Table I. Content of caffeoylquinic acids and flavonoids in A. glehni

CompoundsSolvent for extraction

Water (W) Ethanol (E) W-E (7:3) W-E (3:7)3,4-DQ ND a11.61 ± 0.02a ND ND3,5-DmQ ND ND ND ND3,5-DQ 12.02 ± 0.11 12.17 ± 0.33 14.88 ± 0.98 14.43 ± 0.114,5-DQ ND ND 11.63 ± 0.51 ND5-CQ 19.87 ± 0.11 14.76 ± 0.17 13.34 ± 0.24 13.69 ± 0.183-CQ 12.06 ± 0.11 11.74 ± 0.11 14.93 ± 0.40 13.25 ± 0.053-pCQ 45.97 ± 0.61 16.39 ± 0.67 46.10 ± 4.22 34.06 ± 0.80Sum (mg/g) 59.92 ± 0.83 16.67 ± 0.53 70.89 ± 2.56 55.43 ± 0.11% of extract 34.94 ± 0.49 20.70 ± 0.66 38.74 ± 1.40 30.04 ± 0.59Astragalin 11.43 ± 0.19 14.08 ± 0.23 18.18 ± 0.56 113.2 ± 0.48Kaempferol 10.15 ± 0.01 10.16 ± 0.02 10.20 ± 0.01 10.23 ± 0.03

aValues represent mean ± S.D. based on three experiments. ND: not detected

Table II. Peroxynitrite-scavenging effect of the extracts from the leaves of A. glehni

TreatmentPeroxynitrite scavenging (%)

IC502 µg/mL 5 µg/mL 10 µg/mL

EtOH ext. 27.8 ± 1.36 45.0 ± 2.26 76.0 ± 0.31 5.77 ± 0.31W-E (3:7) ext. 41.2 ± 1.60 62.5 ± 0.98 85.6 ± 1.12 3.22 ± 0.19W-E (7:3) ext. 29.8 ± 57.6 57.6 ± 3.17 82.6 ± 1.39 4.23 ± 0.24H2O ext. 25.7 ± 0.63 63.0 ± 1.28 83.3 ± 0.19 3.92 ± 0.07L-Penicillamine 13.9 ± 0.98 (0.2)a 44.6 ± 0.81 (1.0) 66.6 ± 0.69 (5.0) 1.97 ± 0.09

Data represent mean ± S.D. (n = 3).aValue (unit, µg/mL) in parentheses represents the concentration of L-penicillamine (positive control).

Table III. Effect of the 30% EtOH extract of A. glehni, p-coumaric acid and caffeic acid on the sodium pentobar-bital-induced sleeping time in mice

Group Dose (mg/kg) Duration (min)Vehicle 150.0 ± 13.9g

30% EtOH extract 100 162.5 ± 18.8g

200 178.5 ± 19.5ef

p-coumaric acid 110 109.9 ± 12.3d

120 167.8 ± 23.6b

Caffeic acid 10 189.5 ± 19.4e

20 137.2 ± 19.5c

Lorazepam 15 269.0 ± 29.1a

The assay procedure was described in the experimentalmethods. Values are mean ± S.D. for seven experiments.Values followed by the same letter are not significantlydifferent (p < 0.05).


that PTZ has the convulsive mechanism due to the in-crease of cerebral glutamate (Metcalf, 1979). Therefore,it appears that W-E (7:3) extract (30% aqueous etha-nol extract) should have sedative and anticonvulsantactivity based on its antioxidant mechanism, such asperoxynitrite scavenging effect, partly due to a highlevel of phenolic substances in the extract. Vauzour etal. (2010) reported that tyrosol, p-coumaric acid andcaffeic acid have neuroprotective activities through anantioxidant mechanism.

The sedative and anticonvulsant effects of p-coumaricacid and caffeic acid have not been reported previously,although their neuroprotective effects were known(Vauzour et al., 2010). p-Coumaric acid may moreeasily traverse the blood-brain-barrier (BBB) becauseBBB allows less polar substances to pass (Vauzour etal., 2010). However, CQs are more polar than the cor-responding phenylpropanoids, p-coumaric acid andcaffeic acid. Since CQs and flavonoid glycosides haveester and glycoside bonds, respectively, their hydroly-sis in the human body may increase the bioactivity. Onthe other hand, amyloid β, which can cause Alzheimer’sdisease, results in neurotoxicity through excessiveproduction of peroxynitrite (Butterfield et al., 2007).However, a lot of natural antioxidants that can en-hance the cognitive function decreased by amyloid βhave been reported.

The p-coumaroyl and caffeoyl moieties of the CQs ofA. glehni appeared to prevent the convulsion causedby PTZ through an antioxidant mechanism. In addi-tion, p-coumaric acid and caffeic acid significantlyprolonged pentobarbital-induced sleeping time. Suchactions were also observed upon treatment with A.glehni extract, indicating that this extract has sedativeand anticonvulsant activities, probably due to its per-oxynitrite-scavenging effect. Activated microglia by

neuronal injury or inflammatory stimulation overpro-duces nitric oxide by inducible nitric oxide synthase(iNOS) and reactive oxygen species (ROS) such as su-peroxide anion, resulting in neurodegenerative dis-eases. The toxic peroxynitrite, the reaction product ofNO and superoxide anion radical, further contributesto oxidative neurotoxicity (Demchenko et al., 2003).

Therefore, the leaves of A. glehni could be used totreat anxiety, insomnia, convulsion, and stress. Theefficient extraction method for phenolic substancescould also be used to develop functional food to treathypercholesterolemia, atherosclerosis, obesity anddiabetes mellitus, since this extraction yields a highquantity of CQs. This is the first report to identify thecomposition of phenolic substances and sedative andanticonvulsant effects of the extract of A. glehni.

ACKNOWLEDGEMENTS

This work (Grants No. 2010C142201) was supportedby Business for Cooperative R&D between Industry,Academy, and Research Institute funded by the KoreaSmall and Medium Business Administration in 2010.

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Table IV. Effect of the 30% EtOH extract of A. glehni, p-coumaric acid and caffeic acid on the pentylenetetrazole-induced convulsion and mortality in mice

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phytochemical studies of the phenolic substances in aster glehni extract and its sedative and...

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