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Page 1: Name : Dr. Danish Iqbal Department: MDL, CAMS Publications .... D… · RD Nabeel Ahmad, Sharad Bhatnagar, Ritika Saxena, Danish Iqbal, A.K. Ghosh Materials Science and Engineering:

Name : Dr. Danish Iqbal Department:______MDL, CAMS__ Publications:

Indexing Number of

Publications

ISI 3

Scopus

Others

TOTAL 3

Page 2: Name : Dr. Danish Iqbal Department: MDL, CAMS Publications .... D… · RD Nabeel Ahmad, Sharad Bhatnagar, Ritika Saxena, Danish Iqbal, A.K. Ghosh Materials Science and Engineering:

TITLE CITED BY YEAR

Biosynthesis and characterization of gold nanoparticles: Kinetics, in vitro and in vivo study. RD Nabeel Ahmad, Sharad Bhatnagar, Ritika Saxena, Danish Iqbal, A.K. Ghosh Materials Science and Engineering: C. 78, 553-564

1 2017

Investigating The Role of Novel Bioactive Compound from Ficus Virens Ait on Cigarette Smoke Induced Oxidative Stress and Hyperlipidemia in Rats

D Iqbal, A Khan, I Ansari, MS Khan Iranian Journal of Pharmaceutical Research 16 (3), 1089-1103

2017

Extenuating the role of Ficus virens Ait and its novel bioactive compound on antioxidant defense system and oxidative damage in cigarette smoke exposed rats

D Iqbal, MS Khan, A Khan, S Ahmad Biomedical Research and Therapy 3 (07), 723-732

2016

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Biosynthesis and characterization of gold nanoparticles: Kinetics, in vitroand in vivo study

Nabeel Ahmad a, Sharad Bhatnagar a,b, Ritika Saxena a, Danish Iqbal a,c, A.K. Ghosh d, Rajiv Dutta e,⁎a Department of Biotechnology, School of Engineering & Technology, IFTM University, Lodhipur Rajput, Moradabad, U.P., Indiab Graduate School of Life and Environmental Sciences, University of Tsukuba, Japanc Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Majmaah University, Al-majma'ah 11952, Saudi Arabiad School of Pharmaceutical Sciences, IFTM University, Lodhipur Rajput, Moradabad, U.P., Indiae Department of Biotechnology, Sharda University, Plot No: 32 &34, Knowledge Park III, Greater Noida, 201 306, U.P., India

a b s t r a c ta r t i c l e i n f o

Article history:Received 15 December 2016Received in revised form 23 March 2017Accepted 26 March 2017Available online 05 April 2017

This study reports a facile, cost effective, nontoxic and eco-friendly method for the synthesis of gold nanoparti-cles. In this paper, leaf extract ofMentha piperitawas successfully used to reduce chloroauric acid, leading to syn-thesis of gold nanoparticles (AuNPs). The synthesized nanoparticles were further characterized by UV–visiblespectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, transmission electronmicrosco-py and field emission scanning electronmicroscopy. Kinetics studies like effect of volume of leaf extract, precur-sor, pH, temperature for the synthesis of AuNPs were studied spectrophotometrically. Synthesized AuNPs werefound to possess hexagon structure where size of nanoparticles was ~78 nm in diameter. These biologically syn-thesized AuNPs exhibited significant activity against cancerous cell lines MDA-MB-231 and A549 and was com-pared with the normal 3T3-L1 cell line. Anti-inflammatory and analgesic activities were studied on a Wistar ratmodel to gauge the impact of AuNPs for a probable role in these applications. AuNPs gave positive results for boththese activities, although the potency was less as compared to the standard drugs. These results suggested thatthe leaves extract of Mentha piperita is a very good bioreductant for the synthesis of AuNPs and have potentialfor various biomedical and pharmaceutical applications.

© 2017 Elsevier B.V. All rights reserved.

Keywords:Gold nanoparticlesGreen synthesisKineticsCancerous cell linesAnti-inflammatoryAnalgesic animal model

1. Introduction

In this new era of material science, nanotechnology is incessantlycarving its own forte as an emerging field of research. Applicability ofnanoparticles (NPs) and nanomaterials is evolving rapidly; from dirt re-sistant clothes to targeted drug delivery for tumours, thewide spectrumof nanotechnology is rapidly advancing its relevance in current technol-ogy. Since nanomaterials and nanoparticles are the cornerstones of thisinterdisciplinary field, a constant improvisation of their productiontechniques becomes imperative [1,2].

The synthesis of nanoparticles can be accomplished by either physi-cal methods or chemical methods. Physical methods include evapora-tion, condensation, laser ablation. Whereas chemical methods includeuse of chemicals for reduction such as hydrazine and sodium borohy-dride. A new approach of biologically synthesizing nanoparticles hascome into play. This technique is also referred as green synthesis andmakes use of natural reducing agents such as plants and microbes [3,4]. Production of nanoparticles by biosynthesis technique is considered

more inexpensive, ecological and appropriate for mass production as itworks at low pressure and temperature as well as requires less energy.This technique is optedmore in comparison to the physical and classicalchemical methods due to lack of noxious derivatives and subsequentstability of the product [5]. Decrease in degradation of the product, syn-thesis of NPs utilizing the biological materials has garnered attentiondue to their unusual optical, chemical, photo electrochemical and elec-tronic properties [6]. The aforementioned qualities have put the synthe-sis of metal NPs evolving from the biological systems such as microbes,fungi and several plant extractsfirmly in the spotlight [7]. In comparisontomicrobes, theNPs fashioned byplants tend to bemore stablewhereasmicrobes have immense propensity to induce rapid synthesis of NPs. Inconjunction to stability of NPs produced, employing plant sources forNPs synthesis can be beneficial and economical over other biologicalmethods due to reduction in time of maintenance of cell cultures andcomparatively simple scale up of operation [8]. Extracellular nanoparti-cle synthesis utilizing the plant leaf extracts instead of the whole plantsis deemed to be further cost effective due to uncomplicated extractionand purification. Moreover, the NPs exhibit shape and size dependentproperties, which impart them with an edge to be used in differentspheres of applications on the basis of their morphology and diameter[9]. Therefore, controlling these aspects becomes a vital task, one

Materials Science and Engineering C 78 (2017) 553–564

⁎ Corresponding author.E-mail address: [email protected] (R. Dutta).

http://dx.doi.org/10.1016/j.msec.2017.03.2820928-4931/© 2017 Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

Materials Science and Engineering C

j ourna l homepage: www.e lsev ie r .com/ locate /msec

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which can be achieved by varying reducing agents, capping agents andstabilizers. In this regards, the presence of natural reducing agents, cap-ping agents and stabilizers in plants present them with significant ben-efits over other biological synthesis techniques [10–12].

Gold nanoparticles (AuNPs) have been brought to forefront of thenanotechnology revolution owing to their ease of synthesis, surfacemodifications, larger surface area/volume ratio, biocompatibility andnumerous medical applications. Colloidal gold solutions have gathereda lot of attention because of their cytotoxic properties. These propertiescan be harnessed in the areas of pharmacology, medicine, food industry,and water purification, etc. The biocompatibility of AuNPs makes theman interesting target for potential biological applications such as drugdelivery, tumor, tissue imaging and sensing among others [13–15]. Sev-eral plants have exhibited the ability to reduce gold macromolecules tonanoparticles. For example Aloe vera [16], tamarind [17], Cinnamomumcamphora [18], Benincasa hispida [19], Rosa damascene [45] and seed ex-tract of pomegranate [20]. The presence of numerous reducing, cappingand stabilizing agents in the plant extracts give rise to NPs with uniqueproperties, such as, AuNPs synthesized using pomegranate extract werefound to have greater cytotoxicity against multi drug resistant patho-gens [21].

A major application of AuNPs could be in the field of medicine as anantimicrobial agent. As the world is struggling with the problem of an-tibiotic resistance of microbes, a few experiments involving AuNPs andother nanoparticles have exuded a glimmer of solution. AuNPs produc-tion utilizing cefaclor as a reducing agent have exhibited a strong anti-microbial activity against Staphylococcus aureus as well as Escherichiacoli covering both ends of the spectrum of gram nature. Similarly,AuNPs coated with fluconazole have demonstrated increased activityagainst Aspergillus niger, Candida albicans and Aspergillus flavus [22].Similar results have been obtained by using flower extracts of Plumeriaalba to synthesize AuNPs, clearly elucidating that different reducingagents can give rise to NPs with different properties [23]. Silver nano-particles have been studied extensively on account of antimicrobial ac-tivity but AuNPs have had limited exposure in research till now. Anotherfactor tipping the scales in favour of research is that AuNPs have beenreported to be non-toxic compared to other metallic NPs [24,25].

In the current research, a swift facile method was developed for thefabrication of AuNPs by utilizing the leaf extract of Mentha piperita (M.piperita). The synthesized gold nanoparticles were characterized by nu-merous techniques like Spectroscopic methods (UV–vis spectropho-tometer, FTIR, DLS) and microscopic methods (TEM, FESEM). Aftercharacterization, kinetic studies like effect of volume of leaf extract, pre-cursor, pH, and temperature were analyzed for the synthesis of goldnanoparticles. In vitro studies on various cancerous cell lines and Invivo studies like anti-inflammatory activity and analgesic activity wereperformed and described in this manuscript. The preparation and appli-cation procedure is illustrated in Fig. 1.

2. Experimental section

2.1. Biogenesis of gold nanoparticles from M. piperita

2.1.1. Chemicals and reagentsAll the chemicals and reagents used in this study were of high ana-

lytical grade and were procured from Sigma-Aldrich Co. (St Louis, MO,USA). Deionized water was employed in all the experiments, until stat-ed otherwise. Fresh leaves of Mentha piperita were harvested and col-lected from Local Village of Pakbara Moradabad, Uttar Pradesh, INDIA.

2.1.2. Preparation of aqueous extract of Mentha piperita (M. piperita)In order to remove the dust particles leaves of the plant Mentha

piperita (M. piperita) were washed properly with deionized water. Sub-sequently, the leaves were air dried to wipe out the moisture and werelater chopped into small pieces. 25 g of chopped leaves were boiled for15min in 100ml distilled water at 100 °Cwith continuous stirring. Thismixture was kept for certain time in order to bring it at room tempera-ture, followed by filtering. This filtered leaf extract was stored in refrig-erator for further use and has been used as obtained in all theexperiments unless stated otherwise.

2.1.3. Biosynthesis of gold nanoparticlesFor the reduction of Au3+ to Au0, protocol of Mubarak Ali et al. was

usedwithmodification [26]. Typically 5ml ofM. piperita leaf extractwas

Fig. 1. Schematic illustration of the preparation and application of gold nanoparticles.

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added to 45 ml of 1 mM aqueous HAuCl4 (Chloroauric acid) solution indark. This blendwas heated to 40 °C under vigorous stirring bymagnet-ic stirrer for fewminutes. After a fewminutes, initiation of synthesis canbe clearly observed from the rapid change in the color of mixture fromlight yellow to ruby red. SynthesizedAuNPswere kept under gentle stir-ring conditions for 24 h. This was followed by centrifugation at10,000 rpm for 15 min, ultimately resulting in dispersion of the pelletin deionized water.

2.2. Characterization of gold nanoparticles

UV–visible spectrophotometry, Fourier transform infrared spectro-photometry (FTIR), transmission electron microscopy (TEM), dynamiclight scattering (DLS) and field emission scanning electron microscopy(FESEM) were used for characterization of AuNPs and leaf extract.

2.2.1. UV–vis spectrophotometric analysisThe AuNPs samples were analyzed in the scanning range of 300–

800 nmwith resolution being 1 nm, using Perkin-Elmer UV–vis doublebeam spectrophotometer.

2.2.2. FTIR spectroscopyAuNPs and leaf extract samples were analyzed using potassium bro-

mide pellet method. In this method, samples were distributed into KBrmatrix, mixed thoroughly and were pelletized. These pellets werethen exposed to IR waves in the range of 400–4000 cm−1 to obtainthe spectra. Perkin Elmer Spectrum BX, FT-IR (USA) was used to recordFourier transform infrared spectrum at room temperature. FTIR analysiswas aimed at the recognition of bio-functional groups attached to thenanoparticles.

2.2.3. Transmission electron microscope analysisTransmission electron microscopy (TEM) was used to study the

morphology and particle size of synthesized AuNPs. For this, HitachiModel H-7500 was used with an acceleration voltage of 200 kV.

2.2.4. Dynamic light scattering spectroscopyDynamic light scattering (DLS) spectroscopy was used to study the

size distribution of nanoparticles. DynaPro-TC-04 spectrophotometerequipped with a temperature-controlled microsampler was used DLSmeasurements. Stokes-Einstein equation was used to calculate themean hydrodynamic radii (Rh):

Rh ¼ kT=6πηD;

where Rh is the hydrodynamic radius, k is Boltzmann's constant, T is ab-solute temperature, η is the viscosity of solvent, and D is translationaldiffusion coefficient.

2.2.5. Field emission scanning electron microscopyField emission scanning electron microscopy JEOL JSM 6700F (JEOL,

Tokyo, Japan) was used to determine the surfacemorphology of AuNPs.

2.3. Kinetics of biosynthesized gold nanoparticles

Various aspects governing the synthesis of AuNPs like effect of vol-ume of leaf extract, pH, and temperature were studied to determinethe significance of these factors over the synthesis.

2.3.1. Effect of volume of leaf extract on synthesis of gold nanoparticlesIn order to determine the impact of volume of leaf extract over the

AuNPs synthesis, the volume of M. piperita extract was varied from 5to 25 ml in 100 ml of 1 mM HAuCl4 solution. UV–vis spectroscopy wasused to monitor the synthesis process and record the observations.

2.3.2. Effect of concentration of precursor on synthesis of gold nanoparticlesThe amount of precursor present in the synthesis mixture potential-

ly impacts the synthesis. To study this, various concentrations ofHAuCl4, in the range 1 mM–5 mM, were tested during the synthesis.UV–vis spectroscopy was used to observe the results.

2.3.3. Effect of pH on synthesis of gold nanoparticlespH is an important variable for synthesis of AuNPs. The effect of pH

was studied in the range of 4 to 9 which covered acidic and alkalinerange. Other parameters were kept steady and monitoring of reactionwas accomplished by UV–vis spectroscopy.

2.3.4. Effect of temperature on synthesis of gold nanoparticlesTemperature effect on the reaction ratewas gauged by observing the

synthesis of AuNPs using 1 mM Chloroauric acid in the range of 10 °C–50 °C for 6 h. Other parameters were kept constant for the determina-tion of the impact of this single factor over the rate of reaction. Therate of production was observed spectrophotometrically.

2.4. In vitro cytotoxicity assay of biosynthesized gold nanoparticles

2.4.1. Cell cultureMouse embryonic fibroblasts cells (3T3-L1 cell line), Human breast

cancer cells (MDA-MB-231 cell line) and lung adeno carcinoma cells(A549 cell line) were procured from National Centre for Cell Science,Pune, India. DMEM media containing L-glutamine and 4.5 g/l glucosewas supplementedwith 10% FBS (Foetal bovine serum), penicillin/strep-tomycin (250 U/ml), gentamycin (100 μg/ml) and amphotericin B(1mg/ml)was used to grow the cell lines as amonolayer. The incubationconditions included a temperature of 37 °C in a humidified atmosphereof 5% CO2. Confluency of the cells was established 24 h before growth.

2.4.2. MTT assayCell viability study was performed to verify the cytotoxic effect of

nanoparticles by using the conventional MTT-reduction assay withfew modifications [27]. Briefly, the control 3T3-L1 cells, the test MDA-MB-231 cells and A549 cells were seeded independently in a 96-wellplate at the density of 5 × 103 cells/well. 200 μl of DMEM with 10%FBS used as a growth media and the cells were allowed to grow andgain confluence for 24 h. Following this period, media was replacedwith varying concentrations of AuNPs. The concentration range wasfrom 18.75 μg/ml to 300 μg/ml and at least three wells were incubatedwith each concentration for 48 h to determine the average effect ofthe concentration. The results in the form of formazan products weremeasured at 540 nm using a scanning multi well spectrophotometer.The outcome of the experiment was calculated by using the mean oftriplicates. Growth inhibition by 50% or more was considered to behighly significant. Dose-response curves were used to evaluate the per-centage of growth inhibition by the following formula:

%growth inhibition¼ 100− Mean OD of test group=Mean OD of control group½ � � 100

The effect of inhibitionwas calculated by using the following formula:

%inhibition ¼ Absorbance of the control−Absorbance of sample=Absorbance of control� 100

2.4.3. Measurement of cyto-morphological changes in MDA-MB-231 andA549 cells

Pre-treatment of the cells was carried out by exposure to varyingconcentrations of AuNPs followed by 24 h incubation period at 37 °Cin 5% CO2 atmosphere. The gross cytomorphological changes were ob-served under an inverted phase contrast microscope (Nikon EclipseTi-S, Nikon Corporation Japan).

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2.5. Investigation of in vivo approach of biosynthesized gold nanoparticles

2.5.1. In vivo study experimental animals and conditionsWistar albino rats having 150–200 g weight were chosen for the in

vivo activity. The rats were kept in polypropylene cages, in air condi-tioned area at 23 ± 2 °C, 50–60% relative humidity with 10 h/14 hlight/dark cycle under hygienic conditions to ensure their comfort.Food was given in form of slandered dried pellet diet and consumptionof water was ad libitum. The rats were given 14 days for acclimatizationprior to the conduction of tests. Food was withdrawn 12 h prior to thedrug administration study. This protocol was approved by the Institu-tional Animal Ethical Committee (IAEC) IFTM University, Moradabad,Uttar Pradesh, India (Protocol approval no. 2012/837/ac/PhD/05).

2.5.2. Carrageenan induced paw edema methodSix animals each was categorized as one group and were treated as

following for carrageenan induced paw edema:

(i) Group 1: acted as control.(ii) Group 2: treated with oral dosage of standard indomethacin at

10 mg/kg.(iii) Group 3: treated with oral dosage of AuNPs colloidal suspension

at 0.3 mg/kg.

2.5.2.1. Procedure. Three groups (control, standard and test) of six ratseach were selected for the anti-inflammatory activity. Edema was in-duced by injecting 0.1 ml of 1% carrageenan sterile saline solution intothe hind paw of the animal under the plantar aponeurosis [28]. 0.1 mlof saline solution was injected into the other hind paw so as tomeasurethe effects on the contralateral paw. Four days prior to the edema in-ducement, colloidal AuNPs suspension was administered daily in theform of oral dosage. Digital plethysmograph was used to measure thevolume of the paw and edema formation was followed over 3 h period.The control group was treated with 1% Carboxymethylcellulose (CMC),the standard groupwas treatedwith 10mg/kg of indomethacin and thetest group was treated with 0.3 mg/kg of AuNPs suspension(0.2 mg/ml). All the groups were treated 30 min preceding thecarageenan injection. The dosage was decided based on the previouspublished literature [29]. The increase in the paw volume after inflam-mation was calculated by deducting the volume of hind paw injectedwith saline from contralateral paw. Degree of edema inhibition was cal-culated to determine the anti-inflammatory effects of the synthesizedAuNPs [30].

2.5.2.2. Evaluation parameters. % inhibition in edema

%I ¼ Icontrol−Iedma

Icontrol� 100

where Icontrol and Iedema are the mean inflammation values observedin control and test groups. Inhibition of the inflammation is representedby the decrease in edema formation.

2.5.3. Test for analgesic activity on rats: radiant heat tail-flick modelRadiant tail flick method was used to determine the analgesic activ-

ity of AuNPs as described by D'Amour and Smith [31]. In this method,prescreened rats were studied and change in their sensitivity towardsstimuli was measured. The intensity of light being able to induce heatstress was defined experimentally as the intensity at which animals

withdrew tails in 2–4 s. Nichrome wire acted as a heat source, with5 A current passing through it maintaining a constant amount. Thedrugs were administered orally to the animals, and thereafter the heatstress was applied. The standard group was treated with 30 mg/kg ofpentazocine and the test group was treated with 0.025 mg/ml solutionof AuNPs. The latency of the tail flick was measured using ananalgesiometer. Heat was applied to the site within 2 cm from tail'sroot and the distance between heat source and tail was 1.5 cm. Toelude any tissue injury, the cut off time of the reaction was kept around+10 s. The measurement of the tail flick was taken after 30min of test/standard administration.

2.6. Evaluation

The time taken by the rats to withdraw their tails was taken as thereaction time. The measurements were taken from 30 min to 180 minwith a 30 min interval in between.

2.7. Statistical analysis

All datawere expressed asMean± SEM (n=6) and analyzed usingoneway ANOVA followed by Dunnet's “t-test”. *p b 0.05, **p b 0.01, ***pb 0.001 when control group compared with other treated groups.

Table 1Color change during synthesis of nanoparticles.

S. no. AqueousHAuCl4 Color

Gold nanoparticlescolor

Color changein 15 min

Color change after24 h

1. Pale Yellow Red Ruby red No change in color

Fig. 2. Synthesis of AuNPs.

Fig. 3. UV Vis Spectra of AuNPs.

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3. Results and discussion

3.1. Biosynthesis of gold nanoparticles from Mentha piperita (M. piperita)

As the synthesis of gold nanoparticles begins, the aqueous pale yel-low colored solution changes to ruby red color due to excitation of Sur-face Plasmon Resonance (SPR), which is a unique trait of AuNPs [32].When the leaf extract ofM. piperita was added to the aqueous solutionof chloroauric acid with continuous stirring at room temperature, thecolor turns from pale yellow to ruby red. This change in color representsthe primary confirmation of creation of AuNPs. The results are shown in

Table 1. The bioreduction of Au + confirms the synthesis of gold nano-particles as shown in Fig. 2(a, b).

3.2. Characterization of metallic nanoparticles

3.2.1. UV–vis spectrophotometric analysisFor characterizing the gold nanoparticles wavelength ranges of 500–

550nmwasmeasured. TheUV–vis spectrumofmetallic nanoparticles isshown below in Fig. 3. The distinct peak was observed at 540 nm forAuNPs which corresponds to Surface Plasmon Resonance of the

Fig. 4. (a) FTIR Spectrum for Mentha piperita extract. (b) FTIR Spectrum for the synthesized AuNPs.

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formation of gold nanoparticles. The presence of ruby red color and thepeak at 520 nm verify the production of AuNPs [33,34].

3.2.2. FTIR analysisThe FTIR spectrum of the gold nanoparticles of M. piperita, is shown

in Fig. 4(a, b). Six bands were present at about 3390.3, 2138.17, 1645.8,1079.19, 1404.16 and 1079.19 cm−1 for plant extract ofM. piperita andat 3399.3, 2135.20, 1645.10, 1076.21 and 721.15 cm−1 for gold nano-particles. The characteristic bands observed in the spectra at3390.3 cm−1 and 3399.3 cm−1 region refer to the two N\\H stretchingbands namely primary amine and primary amide linkage spectra of pro-teins. Among all these, the peaks at 2138.17, 1645.8, 1404.16, 1079.19,757.13 and 720.13 cm−1 appeared similar in the spectrum of AuNPs.The bands at 2138.17 cm−1 refers to the region of alkynes whereasthe 2135.20 cm−1 band observed in AuNPs correspond to triple bondsC`C and C`Nwhich shows weak stretching band of alkynes moleculeassociated with the methyl group present in leaf extracts of M. piperita.The bands at 1645 cm−1 are due to amide II bond conjugated at C_Ofromproteins. 1404.16 cm−1 region correspond to the C\\Hbending vi-bration which is arising due to the alkenes whereas this peak disappearsin AuNPs.Weak bands appeared at 1079.19 cm−1 and 1076.21 cm−1 dueto C\\C stretches caused by alcohols, esters and ethers present in leaf ex-tract and gold nanoparticles. The peaks 757.13 cm−1 and 720.13 cm−1 inthe leaf extract, similar peak 721.15 cm−1 in the AuNPs were observeddue to the aromatic C\\H stretch. Some workers observed modificationsof these bands after gold recovery with S. cerevisae [35]. Furthermore,Kuyucak and Volesky determined by FTIR that carboxyl groups were in-volved in the gold recovery with the brown alga Sargassum and proposedthe formation of oxygen bridges between gold and these groups [36].Other functional groups have also been associated to gold recovery, butreduction has not been reported. Some workers using FTIR mention thatacetyl groups could also be involved in metal sorption of trivalent metalswith brown algae [37]. Other workers modified chitosan with lysine, richin amino groups, as a chelating ligand for the recovery of gold and otherprecious metals [38].

3.2.3. Transmission electron microscope analysisTransmission electron microscopy (TEM) was used to envisage the

size along with shape of synthesized gold nanoparticles. It is evidentfrom the Fig. 5 that maximum synthesized particles were poly-dis-persed, hexagonal in shape and the average size estimated was~78 nm. The size of synthesized individual gold nanoparticles rangedbetween 40 and 60 nm, with 40.7 nm being minimum and 57.6 nmbeing maximum.

3.2.4. DLS measurements analysisThe aim of dynamic light scattering measurements was carried out

to determine the size distribution and polydispersity of producedAuNPs. Rh of AuNPs ranged from 40 nm to 80 nm (Fig. 6). The majorityof particles were in range of 70 nm, with 60 nm and 80 nm size werealso prominent. This was clearly elucidated by the intensity obtainedduring the DLS measurements.

3.2.5. Field emission scanning electron microscope (FESEM) analysisThe micrograph observation (Fig. 7 displayed synthesized AuNPs

having rough surfaces which may be spherical, triangular, decahedraland uneven beads shaped with varying dimensions. It can be inferredfrom the rough contoured surface that bioreduction is taking place atthe surface level.

3.3. Kinetics of biosynthesized gold nanoparticles

3.3.1. Effect of volume of leaf extract on AuNPs synthesisConsequences of varying the volume of leaf extract upon the AuNPs

production were featured by UV–vis spectroscopy. It was found thatwith increase in volume of leaf extract, the intensity of the band ofAuNPs (520 nm) also increases as shown in Fig. 8. As elucidated fromthe Figure, the spectra for 5 ml and 10 ml for AuNPs exhibit broadpeaks which correspond to production of larger sized particles.

The peak starts becoming narrower and narrower with further in-crease in volume of leaf extract. The highest peak was obtained whenthe volume of leaf extract was increased to 25 ml, but at 20 ml and25 ml leaf extract volume, a clear shift in peak was observed [39,40].Hence, the optimum volume of leaf extract was found to be 20 ml ofM. piperita leaf extract for 100 ml of HAuCl4 (0.001 M).

3.3.2. Effect of concentration of precursors on synthesis of goldnanoparticles

UV–vis spectrum studies were performed by varying the concentra-tion of the precursor's chloroauric acid to understand the effect of pre-cursor concentration over the production of AuNPs. The UV–visspectrum (Fig. 9) shows effect of chloroauric acid concentration onthe synthesis of gold nanoparticles.

During initial synthesis by utilizing 1 mM chloroauric acid, surfaceplasmon absorption bandwas observed at 520 nm, which characterizesAuNPs synthesis. At the initial concentration of 1 mM a narrow bandwith a slight increase in absorbancewas obtained and, as the concentra-tion of precursor increased, so did the absorbance value. The peak forAuNPs was reached at 520 nm. The absorption increased steadily asthe metal ion concentration increased in the reaction mixture from

Fig. 5. TEM micrograph shows the size of synthesized AuNPs.

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1 mM to 5 mM. A clear red shift occurred in AuNPs synthesis when theconcentration was raised above 3 mM. Thus, the optimization studyconcluded that the increasing the concentration of available ions

increases the concentration of synthesized nanoparticles. 3 mM wasconsidered to be optimum precursor concentration for chloroauricacid, but at 1mMconcentration, the nanoparticles synthesis and size re-duction was rapid because of availability of larger number of functionalgroups. While increasing the substrate concentration the large size andaggregation of nanoparticles was occurred due to the occurrence ofcompetition between gold ions and functional groups of 10 ml leaf ex-tract. Similar to our study, other research group also optimized nano-particles formation using different concentrations of HAuCl4 and1 mM concentration was found to be suitable for the synthesis [41].

3.3.3. Effect of pH on synthesis of nanoparticlesThe consequences of changing pH during synthesis on the creation

of nanoparticles were studied by studying the UV–vis spectra of the re-action mixtures at different pH values. The results shown in Fig. 10clearly elucidates that pH is one of the primary factors responsible forAuNPs synthesis.

The rate of production of AuNPswas found to increase from acidic toneutral environment, i.e., as the pH increased from 4 to 7, evident by thesteady increase in the absorbance value. The absorption reduces as thepH is increased further and in highly alkaline environment, at pH 9,the synthesis decreased markedly. At highly acidic and alkaline condi-tions, i.e. at pH 4 and pH 9, clear blue shift and red shift were observedand the wider distribution indicates formation of large aggregates. Theformation of gold nanoparticles takes place swiftly in neutral and slight-ly basic pH. The ionization of phenolic groups in these pH rangesmay beheld responsible for this rapid synthesis. On the other hand, anionic

Fig. 6. DLS analysis of synthesized AuNPs.

Fig. 7. FESEM imaging of AuNPs.

Fig. 8. Effect of volume of leaf extract on AuNPs.

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electrostatic repulsion of groups in the reaction mixture in the acidicranges may lead to decreased formation rate and aggregation ofAuNPs in acidic pH. At a very high pH value, there is a likelihood ofAuOH precipitation. Therefore, it was concluded that the best pH forsynthesis is indeed a neutral one.

3.3.4. Effect of temperature on synthesis of gold nanoparticlesReaction rate was found to be slower at the lower temperatures.

Therewas no significant increase from 10 °C to 30 °C, but as the temper-ature reached 40 °C, the reaction rate increased rapidly. Maximum re-duction of Au ions was obtained after 5 h. The highest rate of reactionand bioreduction was observed at 50 °C and considered as the optimaltemperature for synthesis of AuNPs, even though the absorbance valuesat 40 °C and 50 °C were quite similar. This can be clearly seen in Fig. 11elucidating the synthesis of AuNPs at different temperatures.

3.4. Investigation of in vitro cytotoxicity assay of gold nanoparticles

3.4.1. Cytotoxic activity of biosynthesized gold nanoparticles against 3T3-L1, MDA-MB-231 and A549 cells

Toxicological analysis is one of the cornerstones for the applicationof metallic nanoparticles in medicinal applications. In order to explore

the role of biogenic synthesized gold nanoparticles in potential treat-ment of human lung andbreast carcinomas, the effect of the these nano-particles was examined taking cell proliferation and cell death in 3T3-L1, MDA-MB-231 and A549 cell lines in account. The cell viability wasfound to be decreasing almost linearly with increasing concentrationsof AuNPs in the case of MDA-MB-231 and A549 [42]. It is clear fromthe results shown in Figs. 12 and 13 that the treatment with 18.75,37.5, 75, 150 and 300 μg/ml of gold nanoparticles leads to a dose-depen-dent increase in cell death in MDA-MB-231 and A549 cell lines. The ef-fect of gold nanoparticles was not cytotoxic even in the highest rangesof tested concentrations for 3T3 L1 cell line (Fig. 14). The cell viabilityindex remained high even after being subjected to highest concentra-tions of the nanoparticles because cellular uptake of nanoparticles togeneration of reactive oxygen species which ultimately give rise to oxi-dative stress. Moreover nanoparticles easily cross the nuclear mem-brane and they can therefore interact with DNA, directly or indirectly,although the exact mechanism for this interaction is not yet known. Ithas been shown that synthesized AuNPs induces cell damage throughthe loss of cell membrane, oxidative stress and apoptosis. Since the in-teraction of nanoparticleswith the cell is dependent on cellmembrane'sstructure, integrity, cellmorphology and cellularmechanisms to combatreactive oxygen species, it can be hypothesized that maybe cancerouscell lines have structural differences as well as have deficient functionsto combat ROS as compared to normal cells, due to which the resultsof interaction with nanoparticles are different.

3.4.2. Effect of gold nanoparticles on cell morphologyTo further verify the results obtained in the treatment of cancerous

cell lines with metallic nanoparticles, the changes in cell morphologyafter treatment was studied. The cells were incubated with AuNPs, atthe same concentrations at which cell viability testing was done. Inthe concentration ranges of 37.5–300 μg/ml, the AuNPs induced signifi-cantly noticeable morphological alterations in MDA-MB-231 cell line,which increased relentlessly with increasing concentrations of AuNPs(Fig. 15). The cells exhibited remarkably shrunken cell membranesamong other debris for the higher concentrations of 150 and 300 μg/ml for 24 h, indicating severe cell damage and death. Cells were foundto sensitive to AuNPs increasing concentration and changes in cell mor-phology along with a decrease in cell number corroborates the claim ofcancerous cell death by metallic nanoparticles. Comparison to the con-trol cells suggested that the synthesized gold nanoparticles were veryFig. 10. Effect of pH on AuNPs.

Fig. 11. Effect of temperature on synthesis of Au.

Fig. 9. Effect of concentration of precursors on synthesis of AuNPs.

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sensitive. Previously, synthesized gold nanoparticles inducing cytotox-icity were discussed by Mukherjee et al. [43]. Similar results as shownin Fig. 16 were obtained for A-549 cell line with induced morphologicalchanges and decrease in cell number being the key features. At higherconcentrations of gold nanoparticles, the decrease in cell number cor-roborates with cell viability assay. Thus, this study clearly demonstratesthatmetallic nanoparticles provoke cell death inMDA-MB-231 cells andA549 cells and could become an important weapon in the armory totreat cancer.

3.5. In vivo approach of biosynthesized gold nanoparticles

3.5.1. Anti-inflammatory action of gold nanoparticles on ratsA considerable anti-inflammatory activity was produced by gold

nanoparticles in the rat model. For the first hour, maximum activity ofAuNPs was observed, as evident by the reduction in paw volume be-tween 30 min and 1 h mark. A decline in the activity was noticed bythe 2 hmark, but the effectiveness prevailedwas still significant. The ef-fectiveness started to tail off in the third hour. A comparison was done

Fig. 12. Cytotoxic activity 3T3-L1 cell line.

Fig. 13. Cytotoxic activity A549 cell lines.

Fig. 14. Cytotoxic activity MDA-MB-231 lines.

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with standard indomethacin drug and whose activity was significanteven after 3 h, as shown in the Fig. 17. This result shows that gold nano-particles are not as effective as the standard drugs but theymay offer aninteresting possibility when used in conjunction with standard drugs.

The Table 2 given below represents the results of the experiments.Results are expressed as Mean ± SEM (n = 6) analyzed by one wayANOVA followed by Dunnet's test. *p b 0.05, **p b 0.01, ***p b 0.001when control group compared with other treated groups.

3.5.2. Test for analgesic activity on ratsThe decrease in tail withdrawal time after administration of AuNPs

hints at a potent analgesic activity (Fig. 18). The inhibition percentagewas 8% for AuNPs as compared to the standard drug pentazocine(44.70%).This study suggests that AuNPs might be used in combinationwith the standard drugs to enhance their efficiency. The results concurwith previously published studies stating that metallic NPs do exhibitanalgesic activities [44]. The data has been presented in Table 3 and isrepresented graphically in Fig. 18.

Fig. 15. Phase contrast microscopic images A-549.

Fig. 16. Phase contrast microscopic images MDA-MB-231.

Fig. 17. Anti-inflammatory properties of gold nanoparticles vs standard indomethacin.

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Results are expressed as Mean ± SEM (n= 6) analyzed by one wayANOVA followed by Dunnet's test. *p b 0.05, **p b 0.01, ***p b 0.001when control group compared with other treated groups.

4. Conclusion

Chemical synthesis requires specific catalyst and regulated reactionenvironment at fixed conditions like temperature and pressure. Thusspecific requirements and toxicity generated by these methods makesthem less favorable choice. The drawback of this method is over passedby the ecofriendlymethods, as they utilize less toxic reactants and addi-tives and could occur at room temperature conditions. Hence, greensynthesis is a more preferential approach. The synthesis of AuNPsusingM. piperita leaf extract represents thepotency of biological sourcesin creation of nanosized materials. The AuNPs were polydispersed,~78 nm sizes on an average, having spherical or decahedral shape con-sistent with in the surface reduction. The presence of phenolic com-pounds, as evident by FTIR analysis, in the plant extract plays a majorrole in reduction. The kinetics of formation was studied in order to de-termine the optimal conditions for the synthesis of the AuNPs. 3mMso-lution of chloroauric acid and 20 ml of the leaf extract was found to bebest conditions for synthesis. Effect of pH and temperature on synthesisof AuNPs clearly indicates that neutral pH and higher temperature rath-er than ambient temperature favors the synthesis. The AuNPswere test-ed against the cancerous cell lines as well as normal cells to determinethe cytotoxicity in order to explore a possible role in cancer treatment.

The adipocyte cell line 3T3-L1 was used as a control whereas humanbreast cancer cell line MDA MB-231 and lung cancer cell line A549were used as test subjects. The treatment with AuNPs exhibited a dosedependent response against cancerous cell line and was quite compel-ling at higher dosages of around 300 μg/ml. The changes in the cell mor-phology were evident when viewed under phase contrast microscopy.More importantly, AuNPs showed a high degree of selectivity againstcancerous cell line which was underlined by the fact that even thehigh dosage of AuNPs did not affect the 3T3-L1 cell line adversely,which was apparent from the results of cell viability assay and steadycell morphology. AuNPs were also tested for possible analgesic andanti-inflammatory activity. The reduction in tail flick amounted to 8%after application of AuNPs compared to 44.70% of standard pentazocine,confirming the presence of veryweak analgesic action. The anti-inflam-matory action of AuNPs was quite significant up to 1 h, after which agradual decline in potency was observed, whereas the action of stan-dard indomethacin remained significant even after 2 h. These results in-dicate that AuNPs possess an anti-inflammatory and analgesic action,albeit in a small quantity. Nevertheless, these properties of AuNPs canbe harnessed to enhance the potency of standard drugs in conjunction.

Acknowledgement

The authors wish to acknowledge Prof RMDubey, Vice Chancellor ofthe IFTM University, Moradabad; Prof Sanjay Mishra and Km. SartajKhan, Department of Biotechnology; Mr. Phool Chand and Mr. SayedSalman Ali, Assistant Professor, School of Pharmaceutical Sciences andResearch, IFTM University, Moradabad for providing all the necessaryfacilities, support, and encouragement to the authors to complete thistask.Wewould also like to extend our gratitude to the SAIF Chandigarh,India for characterization of samples.

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(30 mg/kg)5.86 ± 0.149 8.48 ± 0.382*** 44.70%

3 AuNPs 6.00 ± 0.165 6.48 ± 0.212* 08.00%

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Original Article

Investigating The Role of Novel Bioactive Compound from Ficus Virens Ait on Cigarette Smoke Induced Oxidative Stress and Hyperlipidemia in

Rats

Danish Iqbala, b, Amir Khanc, Irfan A Ansaria and M. Salman Khana*

aClinical Biochemistry and Natural Product Research Laboratory, Department of Biosciences, Integral University Lucknow-226026, India. bDepartment of Medical Laboratory Sciences, College of Applied medical Sciences, Majmaah University, Al-majma’ah-11952, Saudi Arabia. cDepartment of Maxillofacial Surgery (Biochemistry), College of Dentistry, Taif University, KSA.

Abstract

The present study is premeditated to extenuate the role of Ficus virens extract and its bioactive compound on cigarette smoke, an important risk factor for CVD, induced oxidative stress and hyperlipidemia. Cigarette smoke (CS) exposure to rats results in significant loss of body weight and increases blood carbon monoxide saturation (carboxyhemoglobin), nicotine, plasma TC, TG, and LDL-C levels but reduced level of antiatherogenic HDL-C. Moreover, owing to substantial oxidative stress generated in rats due to cigarette smoke a significant increase in plasma and erythrocytes lipid peroxidation products were observed which was well correlated with increase in ex-vivo BDC (48%) and MDA (53%) level (p < 0.001). Simultaneous administration of FVBM extract at higher dose (100 mg/rat) and F18 (n-Octadecanyl-O-α-D-glucopyranosyl(6’→1’’)-O-α-D-glucopyranoside) compound to CS-exposed rats effectively blocked the increase in plasma lipid and lipoprotein levels (p < 0.001) which was due to the marked suppression in the hepatic HMG-CoA reductase activity (p < 0.001) and significantly inhibit the lipid peroxidation process thus preventing the membrane damage, LDL oxidation, and in turn subsequent atherosclerosis. Thus, the results clearly demonstrated the protective role of FVBM extract and F18 compound in risk factor induced cardiovascular disease.

Keywords: Cigarette smoke; Oxidative stress; Hyperlipidemia; Ficus virens; Bioactive compound.

Copyright © 2017 by School of PharmacyShaheed Beheshti University of Medical Sciences and Health Services

Iranian Journal of Pharmaceutical Research (2017), 16 (3): 1089-1103Received: March 2016Accepted: November 2016

* Corresponding author: E-mail: [email protected]

Introduction

Cardiovascular diseases (CVD) contribute around 17.3 million deaths globally (1). Cigarette smoking is one of the major risk factor for CVD and is the leading cause of preventable death and a major public health concern (1). Majority of compounds in cigarette smoke such as nicotine

and carbon monoxide (CO) have been reported to further increase the risk of CVD in chronic smokers (2). Smoking can raise the cholesterol and free fatty acid concentrations in blood by increasing plasma total cholesterol (TC), triglycerides (TG), and low density lipoprotein-cholesterol (LDL-C) including Apo-B and decreasing the cholesterol and ApoA-1 level of high density lipoprotein (HDL) (3). Significant generation of free radicals and subsequent oxidative stress during smoking causes lipid

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Iqbal D et al. / IJPR (2017), 16 (3): 1089-1103

1090

peroxidation, LDL oxidation and decreased levels of antioxidants in the plasma of smokers (4) that ultimately results in CVD (3). Cigarette smokers encounter a sustained free radical load that can contribute to the oxidation of LDL in-vitro (5, 6), although data are conflicting (7). Moreover, erythrocytes from CS induced-hypercholesterolemia or oxidative stress may result in accelerated peroxidation reactions, cellular aberration, and alterations in lipid and protein structure (8). Thus, natural compounds with antioxidant properties contribute the protection of cell and tissue against deleterious effects caused by CS generated reactive oxygen species (ROS). Several natural antioxidants have been experimentally proved as protective agents against smoke induced-oxidative stress and hyperlipidemia (9-11). Ficus species due to their strong antioxidant and biological properties are known previously to diffuse the toxic free radical and can be used as a possible protective agent for treatment of oxidative stress related disorders (12-14). We previously described and documented that Ficus virens bark methanolic (FVBM) extract contained large amount of antioxidant with significant hypolipidemic property (15, 16), the work presented in this manuscript extenuate the protective role of FVBM extract and its principal bioactive compound, n-Octadecanyl-O-α-D-glucopyranosyl(6’→1’’)-O-α-D-glucopyranoside in CS-induced oxidative stress and hyperlipidemia.

Experimental

ChemicalsBradford dye (Sigma Aldrich, India),

hydrogen peroxide (H2O2), isopropeol, glacial acetic acid (Merck Pvt Ltd, India), hemoglobin assay kit, total cholesterol (TC) and triglycerides (TG) kits were procured from Span Diagnostics Ltd. (India). Rodent Chow (Ashirwad pellets), capston cigarette (Capston, India) and all other chemicals were procured from Himedia Laboratories, Mumbai, India. All other chemicals and solvents used in this study were of analytical grade.

Isolation of Bioactive compoundBioactive compound; n-Octadecanyl-

O-α-D-glucopyranosyl (6 ′→1″)-O-α-D-glucopyranoside (F18) from FVBM extract has been isolated according to Iqbal et al. (16).

AnimalsMale Sprague-Dawley (SD) rats weighed

around 100-150 gm were procured from Indian Institute of Toxicology Research Center, Lucknow. The study protocol was approved by Institutional Animal Ethics Committee (IAEC) (registration number: IU/Biotech/project/CPCSEA/13/11). The rats have been housed 5 per cage for one week in the animal house for acclimatization at a temperature of 21-22 ˚C with 12 h light and dark cycle. The rats were given standard diet and water ad libitum.

Dose preparationSequentially extracted FVBM extract, its

bioactive fraction (F18) and reference drug atorvastatin were dissolved in 10% dimethyl sulfoxide (DMSO) at different concentrations and were homogenized with saline. The doses of the extracts were selected on the basis of previously published reports (16-18).

Diet/exposure to cigarette smokeFVBM extract, its bioactive fraction (F18)

and atorvastatin suspension was administrated through gastric intubation in two divided doses (morning and evening) of 0.5 mL each/rat/day. Rats in smoking control group received 0.5 mL of saline containing 10% DMSO (vehicle) twice daily while rats in normal control group received 0.5 mL of saline containing 10% DMSO twice daily. The rats were divided randomly and equally (5 rats in each group) in groups as illustrated in Table 1. Rats were exposed to cigarette smoke in morning by keeping two rats in bottomless metallic container (10 ×11 × 16 inch), having two holes of 3 and 1.5 cm diameter, one on the either side. A burning cigarette was introduced through one hole (3 cm) and the other hole (1.5 cm) was used for ventilation. Animals were exposed to CS for 30 minutes, daily for 4 weeks with interval of 10 min between each 10 min exposure, using 3 cigarettes/day/2 rats in each group (11).

Collection of blood, plasma and packed

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Effect of Ficus Virens Ait on Cigarette Smoke Induced Oxidative Stress and Hyperlipidemia in Rats

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erythrocytesAt the end of the experiment, all the rats

were anaesthetized and blood was collected in heparinized tubes by cardiac puncture. Plasma was collected from blood by centrifugation at 2,500 rpm for 30 min, aliquoted and stored at either 4 or −20 ˚C for future use.

Packed erythrocytes hemolysate was prepared as described by Lakshmi and Rajagopal (19). The packed erythrocytes obtained after the separation of plasma and buffy coat, were washed thrice with normal saline and a portion of washed erythrocytes was lysed in hypotonic (10 mM) sodium phosphate buffer, pH 7.4. A portion of the washed packed erythrocytes was stored at 4 ˚C for further use.

Collection of organ and preparation of homogenate

After the experiment, liver from the rats was promptly excised and chilled in ice-cold saline. After washing with saline, it was blotted and weighed. One g of wet tissues were cut into pieces and homogenized with 9 mL of chilled 0.1 M sodium phosphate buffer, pH 7.4 (containing 1.17% KCl) in a waring blender. The homogenate was centrifuged at 1,000 rpm for 10 min at 4 ˚C and finally was aliquoted and stored at −20 ˚C.

Determination of hemoglobin (Hb) in bloodHemoglobin level was estimated by

cyanmethemoglobin method of Drabkin and Austin [20] according to the procedure described in the instruction sheet enclosed with the reagent kit supplied by Span diagnostic. The percent blood hemoglobin was determined by measuring

the absorbance of cyanomethemoglobin at 540 nm in eppendorf spectrophotometer using a hemoglobin standard.

Determination of nicotine content in bloodNicotine content in blood samples was

determined by the method of Varley et al. (21). Nicotine standard was treated in the similar manner and used in the calculation of nicotine present in the blood samples.

Determination of carbon monoxide saturation in a mixture containing hemoglobin and carboxy hemoglobin

Carbon monoxide saturation (SCO) in blood samples was determined by the method of Varley et al. (21). The carbon monoxide saturation was calculated from the equation.

SCO% = (2.44 D538/ D578-2.68) 100

The constants 2.44 and 2.68 have been calculated from a series of 30 measurements of D538/ D578 to 0% COHb (100% Hb) and 100% COHb. The isobestic point was established at λ = 578 ± 0.5 nm (n = 100).

Isolation of Plasma LDL and HDLThe precipitation method described by

Wieland and Seidel (22) was used for the isolation of plasma LDL and the method of Patsch et al. (23) was used for the isolation of HDL.

Measurement of ex-vivo and in-vitro Cu++-mediated susceptibility of LDL to oxidation

The ex-vivo and in-vitro Cu++-mediated susceptibility of isolated LDL to oxidation

Table 1. Protocol for the treatment of cigarette smoke induce hyperlipidemia in rats.

Group Treatment

N-C Normal control

S-C Cigarette smoking control + vehicle

FVT-1 Smoke-exposed + plant extract (FVBM) (50 mg/rat/day)

FVT-2 Smoke-exposed + plant extract (FVBM) (100 mg/rat/day)

CT Smoke-exposed + bioactive compound (F18) (1 mg/rat/day)

AT Smoke-exposed + standard (Atorvastatin) (1 mg/rat/day)

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was assessed by determining the lag phase of conjugated diene (CD) formation using the method of Esterbauer et al. (24, 25). Conjugated diene was calculated by using an extinction coefficient of 2.52×104 M-1 cm-1 and expressed as nmole malondialdehyde (MDA) equivalent per mg LDL protein. The MDA content in LDL was assayed by the method of Niehaus et al. (26). The MDA concentration of the samples was calculated by using an extinction coefficient (1.56×105 M-1cm-1).

Measurement of plasma “total antioxidant power” (FRAP)

The method of Benzie and Strain (27) was used for measuring the ferric reducing ability of plasma (FRAP assay) which estimates the “total antioxidant power”, with minor modification.

Measurement of MDA release from intact erythrocytes

The procedure of Cynamon et al. (28) was employed for the determination of MDA release from erythrocytes.

Determination of MDA content in erythrocytesThe determination of MDA in erythrocytes

was carried out by adopting standardized protocol of Stocks and Dormandy (29).

Determination of plasma triglycerides and very low density lipoprotein-cholesterol

Plasma TG was determined by using enzymatic kit (Merck, India) based on glycerol-3-phosphate oxidase peroxides (GPO-POD) method (30). The very low density lipoprotein-cholesterol (VLDL-C) in plasma was calculated by dividing plasma TG values (mg/dL) by a factor of 5 as described by Friedewald et al. (31).

Determination of total cholesterol in plasma and lipoprotein

Plasma TC, LDL-C and HDL-C were determined by using cholesterol enzymatic kit (Merck, India) based on cholesterol oxidase phenol aminophenazone (CHOD-PAP) method and the results were expressed as mg/dL.

Assay of HMG-CoA reductase activity in liver homogenate

HMG-CoA reductase enzyme activity in liver homogenate was estimated indirectly by method of Rao and Ramakrishnan (32).

Protein estimationThe protein concentration of plasma, LDL and

liver homogenate was analysed by the method of Bradford (33), using bovine serum albumin as standard. Aliquots were first precipitated with 10% TCA followed by centrifugation at 1500 rpm for 10 min. The pellets containing protein were dissolved in 0.5 N NaOH and suitable aliquots were used for protein determination.

Data analysisFor all assays, samples were analyzed in

triplicate and the results were expressed as mean ± SD and the results were evaluated using one-way analysis of variance (ANOVA) and two tailed Students t-test. Statistical significance were expressed as *p < 0.05, **p < 0.01 and ***P < 0.001.

Results

Average body weight of rats in each group before and after 4 weeks of treatment

As shown in Table 2, there was a significant decrease in body weight of smoke-exposed rats from 130.85 gm ± 5.56 in N-C to 80.56 gm ± 3.14 gm in S-C (p < 0.001) rats after 4 weeks of exposure to CS. Whereas, the average body weight of smoke-exposed rats treated with different doses of F. virens methanolic extract (FVBM-50 and FVBM-100), bioactive compound (F18), and atorvastatin was 90.28 ± 3.14, 115.57 ± 4.45, 125.28 ± 5.14, and 120.57 ± 5.45 (gm) (p < 0.001), respectively, which indicates a significant regain of average body weight when compared to S-C rats.

Impact on hemoglobin (Hb), blood carbon monoxide saturation and blood nicotine in smoke-exposed rats treated for 4 weeks

The results presented in Table 3, indicated the Hb, blood carbon monoxide saturation (carboxyhemoglobin) and blood nicotine level in N-C, S-C, and plant extract bioactive compound treated rats. Hemoglobin level was significantly reduced by 22% (p < 0.01) in smoke-exposed

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(S-C) rats, when compared to N-C value. However, treatment with higher dose of plant extract (100 mg/rat), purified compound (F18) and atorvastatin showed a highly significant increase in Hb concentration of 26%, 27% and 21% (p < 0.01) respectively, when compared to S-C rats. Both FVBM extract and F18 bioactive compound administration to smoke-exposed rats restored the Hb levels close to normal

value. Furthermore, in S-C rats blood carbon monoxide saturation and blood nicotine levels were increased from 3.26 (SCO%) and 0.998 µg/mL in N-C to 5.78 (77%) and 2.16 µg/mL (116%) (p < 0.001) respectively. After 4 weeks of treatment blood carbon monoxide saturation and blood nicotine levels showed reduction of 11% (p < 0.001) and 2% (p < 0.05) in FVT-1; 30% (p < 0.001) and 32% (p < 0.001) in FVT-2,

Table 2. Average body weight of rats in each group before and after 4 weeks of FVBM extract, F18 bioactive compound and atorvastatin treatment.Group* Before treatment After treatment

N-C 125.14 ± 2.67 130.85 ± 5.63

S-C 135.85 ± 3.93 80.56 ± 3.14 a

FVT-1 125.28 ± 7.86 90.28 ± 3.14 c

FVT-2 133.46 ± 6.07 115.57 ± 4.45 a

C-T 122.24 ± 7.86 125.28 ± 5.14 a

A-T 126.78 ± 6.07 120.57 ± 5.45 a

*Values are mean (grams) ± SD from 5 rats in each group.N-C: normal control, S-C: smoke-exposed control, FVT-1: fed 50 mg FVBM extract/rat/day, FVT-2: fed 100 mg FVBM extract/rat/day, C-T: fed 1 mg F18 bioactive compound/rat/day and A-T: given 1 mg atorvastatin/rat/day for 4 weeks.Significantly different from N-C at ap < 0.001.Significantly different from S-C at ap < 0.001.Significantly different from S-C at cp < 0.05.

Table 3. Impact of FVBM extract, F18 bioactive compound and atorvastatin on blood haemoglobin, carbon monoxide saturation and nicotine in cigarette smoke-exposed rats after 4 weeks of treatment.

Group* Haemoglobin (g/dL) Carbon monoxidesaturation (SCO%) Nicotine (µg/mL)

N-C 14.86 ± 0.764 3.26 ± 0.089 0.998 ± 0.012

S-C 11.60 ± 0.515(-21.9%)b

5.78 ± 0.095(+77.3%)a

2.16 ± 0.033(+116.4%)a

FVT-1 12.35 ± 0.617(+6.4%)c

5.16 ± 0.080(-10.7%)a

2.11 ± 0.024(-2.3%)d

FVT-2 14.62 ± 0.721(+26.1%)b

4.07 ± 0.091(-29.6%)a

1.48 ± 0.042(-31.5%)a

C-T 14.74 ± 0.732(+27.1%)b

4.76 ± 0.078(-17.6%)a

1.59 ± 0.035(-26.4%)a

A-T 13.99 ± 0.677(+20.6%)b

3.97 ± 0.043(-31.3%)a

1.98 ± 0.022(-8.3%)b

*Values are mean ± SD from blood of 5 rats in each group.Significantly different from N-C at bp < 0.01.Significantly different from S-C at ap < 0.001Significantly different from S-C at bp < 0.01.Significantly different from S-C at cp < 0.01.Non-significantly different from S-C at dp > 0.05.

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18% (p < 0.001) and 26% (p < 0.001) in C-T and 31% (p < 0.001) and 8% (p < 0.01) in A-T rats, respectively, in comparison to values in S-C rats.

Effect on plasma lipids and lipoprotein levelsThe results illustrated in Figure 1 showed

that all the plasma lipids parameters, TC, TG, and non-HDL-C were significantly increased from 85.42, 64.05, and 55.53 mg/dL in N-C rats to 136.87, 171.05, and 125.93 mg/dL (p < 0.001) respectively in S-C rats. After 4 weeks of treatment with FVBM extract (50 and 100 mg/rat/day), levels of TC, TG and non-HDL-C were significantly decreased by 20%, 30%, 29% and 30%, 50%, 47% (p < 0.001) respectively, when compared to corresponding S-C values. Whereas, marked reduction of 33%, 55%, 56% and 36%, 61%, 54% (p < 0.001) was observed in TC, TG and non-HDL-C level of F18 and atorvastatin treated rats, when compared to corresponding values in S-C group.

Moreover, plasma LDL-C and VLDL-C levels were significantly increased from 42.7 and 12.8 mg/dL in N-C to 91.7 mg/dL (115%) and 34.2 mg/dL (167%) (p < 0.001) respectively, in S-C rats. After 4 weeks of treatment with FVBM extract (at higher dose), both LDL-C and VLDL-C levels showed a significant reduction of 46% and 50%, (p < 0.001) respectively, whereas, FVT-1 group exhibited much less reduction. Furthermore, LDL-C and VLDL-C level in C-T group were significantly reduced by 50% and

55% (p < 0.001) respectively, in comparison to corresponding values in S-C rats

which was almost equivalent to the reduction observed in atorvastatin treated rats. Plasma HDL-C level were decreased from 30 mg/dL in N-C to 11 mg/dL (63%) (p < 0.001), in S-C values which was subsequently attenuated after the treatment with FVBM extract, bioactive compound and standard. Further, our result also depicted a significant decrease in HDL-C/LDL-C (5.8 fold) and HDL-C/TC (4.4 fold) ratio and a increase in TC/HDL-C (4.4 fold) and LDL-C/HDL-C (5.9 fold) ratios in CS-exposed hyperlipidemic rats (p < 0.001). The atorvastatin and bioactive compound treated rats exhibited marked increase of 4.1, 5.3 and 4.4, 5.8 fold (p < 0.001) in HDL-C/TC and HDL-C/LDL-C ratio (Table 4).

Regulation of enzymatic activity of hepatic HMG-CoA reductase

The result also exhibited a significant increase of 2.31 fold (p < 0.001) in hepatic HMG-CoA reductase activity; the rate limiting enzyme in the biosynthetic pathway of cholesterol when compared to N-C value (Figure 2). Among all the treated groups FVT-2 and C-T exhibited marked decline of 1.99 and 2.21 fold (p < 0.001) in HMG-CoA reductase activity, respectively (Figure 2), which was better or equivalent to the decline observed in atorvastatin treated rats.

Figure 1. Effect of FVBM extract, F18 bioactive compound and atorvastatin on plasma triglycerides, total cholesterol, non-HDL-cholesterol, LDL-C, HDL-C and VLDL-C in cigarette smoke-exposed rats after 4 weeks of treatment. †For the calculation of non-HDL-C, data is taken from Figure 1. *Values are mean (mg/dL) ± SD from plasma, LDL-C, HDL-C and VLDL-C of 5 rats in each group. Significantly different from N-C at ap < 0.001. Significantly different from S-C at ap < 0.001.

level of F18 and atorvastatin treated rats, when compared to corresponding values in

S-C group.

Figure 1. Effect of FVBM extract, F18 bioactive compound and atorvastatin on plasma triglycerides, total cholesterol, non-HDL-cholesterol, LDL-C, HDL-C and VLDL-C in cigarette smoke-exposed rats after 4 weeks of treatment. †For the calculation of non-HDL-C, data is taken from Figure 1. *Values are mean (mg/dL) ± SD from plasma, LDL-C, HDL-Cand VLDL-C of 5 rats in each group. Significantly different from N-C at ap < 0.001. Significantly different from S-C at ap < 0.001.

Moreover, plasma LDL-C and VLDL-C levels were significantly increased

from 42.7 and 12.8 mg/dL in N-C to 91.7 mg/dL (115%) and 34.2 mg/dL (167%) (p <

0.001) respectively, in S-C rats. After 4 weeks of treatment with FVBM extract (at

higher dose), both LDL-C and VLDL-C levels showed a significant reduction of 46%

and 50%, (p < 0.001) respectively, whereas, FVT-1 group exhibited much less

reduction. Furthermore, LDL-C and VLDL-C level in C-T group were significantly

reduced by 50% and 55% (p < 0.001) respectively, in comparison to corresponding

values in S-C rats

which was almost equivalent to the reduction observed in atorvastatin treated

rats. Plasma HDL-C level were decreased from 30 mg/dL in N-C to 11 mg/dL (63%)

(p < 0.001), in S-C values which was subsequently attenuated after the treatment with

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Impact on plasma total antioxidants and lipid peroxidation products

Data in Table 5 demonstrate the antioxidant efficacy of FVBM extract, F18 bioactive compound and atorvastatin on ex-vivo plasma concentrations of total antioxidants, CD, LOOH, and MDA in CS-exposed rats. Data illustrated that cigarette smoke causes substantial decrease in plasma total antioxidants level and was reduced by 48% (p < 0.001) while CD, LOOH, and MDA levels were increased by 163%, 182%, and 116% (p < 0.001) respectively. Administration of FVBM extract (50 and 100 mg/rat/day), F18 and atorvastatin to smoke-exposed rats significantly increased the total antioxidants levels by 28%, 84%, 79% and 17% (p < 0.001), respectively, when compared to S-C value. On the other hand, CD, LOOH and MDA levels were significantly decreased by 30% (p < 0.001), 32% (p < 0.001) and 27% (p < 0.01) in FVT-1; 57%, 55% and 45% (p < 0.001) in FVT-2; 61%, 63% and 52% (p < 0.001) in C-T and 24% (p < 0.01), 21% (p < 0.001) and 20% (p < 0.05) in A-T rats, when compared to corresponding values in smoke-exposed rats.

Effect on membrane lipid peroxidation in

erythrocytesAs seen in Table 6, erythrocytes from smoke-

exposed rats (S-C) group showed a greater susceptibility to hydrogen peroxide- induced lipid peroxidation than those from N-C group. The MDA level was substantially increased by 125% (p < 0.001) in S-C rats, when compared to N-C value. Formation of MDA was markedly decreased by 23%, 46%, 54%, and 25% (p < 0.001) after the administration of FVBM extract, F18 and atorvastatin, respectively, when compared to the corresponding values in S-C. Similarly, H2O2-mediated release of MDA erythrocytes was increased from 22.45 in N-C to 46.6 nmol/gHb (108%) (p < 0.001) in S-C rats. A highly significant decrease of 27%, 36%, 50% and 21% (p < 0.001) in MDA release was seen in smoke-exposed rats treated with FVBM extract, F18, and atorvastatin, respectively, when compared to corresponding values in S-C rats.

Antioxidant effect on basal and maximal level of CD formation and MDA content in LDL

As depicted in Table 7, the ex-vivo basal CD level of LDL in smoke-exposed rats was increased by 48%, in comparison to the corresponding N-C values. Administration of

Table 4. Effect of FVBM Extract, F18 bioactive compound and atorvastatin on the ratios of plasma HDL-C/TC, HDL-C/LDL-C, TC/HDL-C and LDL-C/HDL-C in cigarette smoke-exposed rats after 4 weeks of treatment.

Group*/Ratio† HDL-C/TC HDL-C/LDL-C TC/HDL-C LDL-C/ HDL-C

N-C 0.35 ± .015 0.70 ± 0.031 2.85 ± 0.105 1.43 ± 0.057

S-C0.08 ± 0.003 0.12 ± 0.006 12.51 ± 0.46 8.38 ± 0.35

(-4.4 f)a (-5.8 f)a (+4.4 f)a (+5.9 f)a

FVT-10.18 ± 0.008 0.30 ± 0.014 5.55 ± 0.24 3.33 ± 0.186

(+2.3 f)a (+2.1 f)a (-2.3 f)a (-2.5 f)a

FVT-20.30 ± 0.013 0.58 ± 0.026 3.33 ± 0.135 1.73 ± 0.079

(+3.8 f)a (+4.8 f)a (-3.8 f)a (-4.8 f)a

C-T0.33 ± 0.016 0.65 ± 0.029 3.04 ± 0.091 1.53 ± 0.061

(+4.4 f)a (+5.8 f)a (-4.4 f)a (-5.9 f)a

A-T0.33 ± 0.015 0.64 ± 0.028 3.03 ± 0.129 1.56 ± 0.064

(+4.1 f)a (+5.3 f)a (-4.1 f)a (-5.4 f)a

N-C: normal control, S-C: smoke-exposed control, FVT-1: fed 50 mg FVBM extract/rat/day, FVT-2: fed 100 mg FVBM extract/rat/day, C-T: fed 1 mg F18 bioactive compound/rat/day and A-T: given 1 mg atorvastatin/rat/day for 4 weeks.†For the calculation of ratios, data is taken from Figure 1.*Values are mean (ratio) ± SD from plasma of 5 rats in each group.Significantly different from N-C at ap < 0.001.Significantly different from S-C at ap < 0.001.

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FVBM-50, FVBM-100, F18 and atorvastatin to these smoke-exposed stressed rats partially blocked their in-vivo LDL oxidation and reduced their basal CD levels by 11%, 27%, 29% and 8%, respectively, in comparison to the corresponding S-C values. The maximal CD value of LDL in N-C was substantially increased by 430% in comparison to corresponding basal CD value in N-C. When compared to corresponding maximal CD value in N-C rats, LDL associated CD formation of S-C rats was increased by 43%.

Being a potent antioxidant, FVBM-100 and F18 significantly blocked the maximal CD concentration and reduced them by 22% and 27%, respectively, in comparison to corresponding maximal values in S-C rats. As expected, the lag phase time of LDL oxidation was reduced from 88 min in N-C to 68 min in S-C. Treatment of smoke-exposed rats with FVBM-50, FVBM-100, F18 and ATR, restored the lag phase time of LDL oxidation to 75 min, 85 min, 87 min, and 73 min, respectively. Similar to ex-vivo basal and in-vitro Cu++ catalyzed maximal CD values, the ex-vivo basal MDA content in LDL was significantly increased by 53% (p < 0.01) in S-C rats, when compared to corresponding values in N-C rats. FVBM-50, FVBM-100, F18 and atorvastatin treatment to smoke-exposed rats significantly blocked the ex-vivo increase in LDL MDA formation in S-C rats and reduced their levels by 14% (p < 0.01), 28% (p < 0.001), 32% (p < 0.001) and 13% (p < 0.05) respectively. An

almost similar pattern was observed in maximal MDA content of LDL.

Discussion

Cigarette smoking (CS) is the foremost cause of morbidity and mortality worldwide (34, 35) and is considered to be the preventable risk factor for CVD (3). The current manuscript basically illustrates the use of FVBM extract and F18 compound in prevention and protection of CS-induced oxidative stress, hypercholesterolemia, and subsequent atherosclerosis. As shown in Table 2, there was significant decrease in body weight of smoke-exposed rats that may be due to reduced food intake or gastrointestinal irritation. Other reports also stated that smoker›s weight on average is about 4 kg less than non-smokers, mainly because of reduced food intake (36). Smoke-exposed rats treated with different doses of Ficus virens methanolic extract (FVBM-50 & FVBM-100), bioactive compound (F18) and atorvastatin indicates a significant regain of average body weight when compared to S-C rats.

The gas phase and tar of cigarettes contain free radicals, responsible for oxidative stress, has been hypothesized to be involved in the pathogenesis of smoking-related atherosclerosis (6). Moreover, cigarette smoking generates many toxic and carcinogenic compounds harmful to the health and was an unlikely cause for atherosclerosis, such as nicotine, nitrogen

Figure 2. In-vivo regulation of hepatic HMG-CoA reductase activity in cigarette smoke-exposed rats treated with FVBM extract, F18 bioactive compound and atorvastatin for 4 weeks of treatment. †Expressed as ratio of HMG-CoA to Mevalonate; lower the ratio higher the enzyme activity. *Values are mean ± SD from liver homogenate of 5 rats in each group. Significantly different from N-C at ap < 0.001. Significantly different from S-C at ap < 0.001.

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oxides, carbon monoxide (CO), hydrogen cyanide and free radicals (37-40 and 2). Similar to these reports, our results presented in Table 3, indicates blood carbon monoxide saturation and blood nicotine levels in smoke exposed rats were significantly increased and after 4 weeks a significant decrease was observed, in comparison to values in S-C rats. Moreover, both FVBM extract and F18 bioactive compound administration to smoke-exposed rats restored the Hb levels close to normal value. These results specify a strong protective effect of FVBM extract and F18 compound, which may help in lowering the menace of myocardial infarction in smokers.

Numerous smoking consequences have been described as being atherogenic, like direct vascular actions, oxidative stress, thrombogenic factors and secondary dyslipidemia (4, 40 and 41). In this context, our results illustrated that, all the plasma lipids parameters, TC, TG, non-HDL-C, LDL-C and VLDL-C levels were significantly increased (Figure 1). This increase in cholesterol and TG level of CS-exposed rats is in consensus with previously published reports

(3, 42 and 43). Whereas, HDL-C level was significantly decreased by 63.4% (p < 0.001) in S-C rats when compared to N-C value (Figure 1). The increase in plasma TC level in S-C rats is apparently due to increased cholesterol synthesis (42-44), through the induction of hepatic HMG-CoA reductase activity-the rate limiting enzyme in the biosynthetic pathway of cholesterol. Chen and Loo (7) earlier reported that the increase in the cholesterol content of TG rich VLDL might be due to CS-induced reduction of lipoprotein lipase, which is responsible for the removal of TG from VLDL particles. Thus, the increased level of VLDL-C in S-C rats is also responsible for high concentrations of atherogenic cholesterol rich circulating LDL. The observed high level of VLDL-C in CS-exposed rats may responsible for reduced level of antiatherogenic HDL-C because of decreased availability of phospholipid remnants needed for the configuration of HDL from VLDL and a concomitant decline in lecithin cholesterol: acyltransferase (LCAT) activity.

Administration of FVBM extract at higher dose (100 mg/rat) and F18 bioactive compound to CS-exposed rats effectively blocked the

Table 5. Antioxidant impact of FVBM, F18 bioactive compound and atorvastatin on plasma total antioxidants, CD, LOOH and MDA contents in cigarette smoke-exposed rats after 4 weeks of treatment.

Group* CD LOOH MDA Total antioxidants

N-C 2.86 ± 0.141 1.02 ± 0.022 1.24 ± 0.015 85.47 ± 3.46

S-C7.52 ± 0.325 2.88 ± 0.104 2.68 ± 0.125 44.21 ± 2.04

(+162.9%)a (+182.4%)a (+116.1%)a (-48.27%)a

FVT-15.28 ± 0.216 1.96 ± 0.083 1.95 ± 0.098 56.42 ± 2.79

(-29.8%)a (-31.9%)a (-27.2%)b (+27.6%)a

FVT-23.21 ± 0.110 1.29 ± 0.034 1.46 ± 0.062 81.53 ± 3.16

(-57.3%)a (-55.2%)a (-45.5%)a (+84.4%)a

C-T2.91 ± 0.108 1.08 ± 0.028 1.28 ± 0.042 79.18 ± 3.73

(-61.3%)a (-62.5%)a (-52.2%)a (+79.1%)a

A-T5.74 ± 0.209 2.28 ± 0.026 2.14 ± 0.044 51.79 ± 2.92

(-23.7%)b (-20.8%)a (-20.1%)c (+17.1%)a

N-C: normal control, S-C: smoke-exposed control, FVT-1: fed 50 mg FVBM extract/rat/day, FVT-2: fed 100 mg FVBM extract/rat/day, C-T: fed 1 mg F18 bioactive compound/rat/day and A-T: given 1 mg atorvastatin/rat/day for 4 weeks.CD: Conjugated diene, LOOH: Lipid hydroperoxide, MDA: Malondialdehyde.*Values are mean (µmole/dL) ± SD from plasma of 5 rats in each group.Significantly different from N-C at ap < 0.001.Significantly different from S-C at ap < 0.001.Significantly different from S-C at bp < 0.01.Significantly different from S-C at cp < 0.05.

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increase in plasma lipid and lipoprotein level and reversed them to a level close to their normal control values, which is almost comparable to amelioration exhibited by standard drug atorvastation in cigarette smoke-exposed rats. Moreover, the present study observed diminished level of HDL-C in cigarette smoke-exposed rats when compared to normal rats which were in agreement with earlier reports (44, 45). Simultaneous administration of FVBM extract and F18 compound to CS-exposed rats significantly increases the HDL-C level which might be due to its profound antioxidant activity which in turn offers better protection to LDL as well as HDL from oxidative stress through its associated antioxidant enzyme paraoxonase (PON) (46). Consistent with earlier reports (47, 48) that established lipoprotein ratios, LDL-C to HDL-C and HDL-C to TC, are good forecaster for the existence and severity of CAD, our results depicted a significant decrease in HDL-C/LDL-C and HDL-C/TC ratio and a increase in TC/HDL-C and LDL-C/HDL-C ratios in CS-exposed hyperlipidemic rats. The atorvastatin and bioactive compound treated rats exhibited

marked increase in HDL-C/TC and HDL-C/LDL-C ratio. An opposite pattern was observed in TC/HDL-C and LDL-C/HDL-C ratios. The results indicated a significant restoration of these ratios close to the normal values, which in turn implify the normalization of lipoprotein associated cholesterol level in CS-exposed rats treated with FVBM extract, F18 and atorvastatin.

Enhanced plasma TC level in cigarette smoke-exposed rats is apparently due to significant amplification in hepatic HMG-CoA reductase activity-the rate limiting enzyme in the biosynthetic pathway of cholesterol (Figure 2). Moreover, the significant amelioration in plasma and lipoprotein lipid level exerted by these fractions was due to the marked suppression in the hepatic HMG-CoA reductase activity. Among all the treated groups FVT-2 and C-T exhibited marked decline in HMG-CoA reductase activity, respectively (Figure 2), which was better or equivalent to the decline observed in atorvastatin treated rats. These data suggest that FVBM extract/compound may exert their cholesterol lowering effect in CS-exposed hyperlipidemic rats via suppression of hepatic

Table 6. Basal MDA contents and its H2O2-induced MDA release in intact erythrocytes of cigarette smoke-exposed rats after 4 weeks of FVBM extract, F18 bioactive compound and atorvastatin treatment.

Group* MDA content (nmole/g Hb) MDA release (percent)

N-C 5.67 ± 0.24 22.45 ± 0.92

S-C12.74 ± 0.64 46.6 ± 2.13

(+124.7%)a (+107.6%)a

FVT-19.78 ± 0.46 34.26 ± 1.92

(-23.2%)a (-26.5%)a

FVT-26.82 ± 0.31 29.87 ± 1.12

(-46.4%)a (-35.9%)a

C-T5.82 ± 0.23 23.68 ± 1.02

(-54.3%)a (-49.2%)a

A-T9.54 ± 0.32 36.81 ± 1.24

(-25.1%)a (-21.0%)a

N-C: normal control, S-C: smoke-exposed control, FVT-1: fed 50 mg FVBM extract/rat/day, FVT-2: fed 100 mg FVBM extract/rat/day, C-T: fed 1 mg F18 bioactive compound/rat/day and A-T: given 1 mg atorvastatin/rat/day for 4 weeks.CD: Conjugated diene, LOOH: Lipid hydroperoxide, MDA: Malondialdehyde.*Values are mean (µmole/dL) ± SD from plasma of 5 rats in each group.Significantly different from N-C at ap < 0.001.Significantly different from S-C at ap < 0.001.Significantly different from S-C at bp < 0.01.Significantly different from S-C at cp < 0.05.

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HMG-CoA reductase m-RNA expression which in turn cause inhibition of cholesterol synthesis (49). These data are in agreement with previous published reports that revealed hypolipidemic property of natural bioactive compound might be due to decrease in the efficiency of HMG-CoA reductase m-RNA translation and increasing the degradation of reductase protein post translationally (50-52). Our results represent an initial demonstration and exhibit strong rationale in support of the use of FVBM extract/bioactive compound, preferably F18, as a functional food in the prevention and treatment of tobacco induced dyslipidemia/hyperlipidemia and atherosclerosis.

It is well known that CS is associated with substantial increase in oxidative stress which is mainly due to increased lipid peroxidation

or reduced antioxidants (3, 4). Throughout the course of CS-induced oxidative stress, oxygen derived free radicals like superoxide (O¨), hydrogen peroxide (H2O2), hydroxyl (•HO), peroxyl (ROO), alkoxy (RO) and nitric oxide (NO) are known to be generated in the cell, which can lead to oxidative modification of lipids and cause membrane damage, resulting in cell death (53, 4). It was previously found that serum MDA concentrations were higher in smokers and rats exposed to CS (54, 4). Similar to plasma, erythrocytes are also susceptible to damage by CS-induced oxidative stress because they are constantly exposed to both extracellular and intracellular sources of ROS and cause profound alteration in the structure and function of cell membrane that lead to cell death (8). Cigarette smoke is highly responsible for

Table 7. Ex-vivo and Cu++-catalyzed in-vitro oxidation of LDL, from plasma of cigarette smoke-exposed rats after 4 weeks of FVBM extract, F18 bioactive compound and atorvastatin treatment.

Group*

LDL oxidation+

Conjugated diene formation* MDA content#

Basal Maximal Lag Phase** Basal Maximal¥

N-C 178.49 945.86 88 5.43±0.212 15.26

(+430%)

S-C264.36 1348.56 68 8.64±0.364a 23.65

(+48.3%)† (+42.6%)€ (-22.7%)¶ (+53.2%)† (+54.9%)€

FVT-1234.72 1264.35 75 7.43±0.202 b 20.26

(-11.4%)†† (-6.2%)α (+10.3%)§ (-14.0%)†† (-14.3%) ♯

FVT-2192.41 1048.95 85 6.24±0.244 a 18.45

(-27.3%)†† (-22.2%) α (+25.0%)§ (-27.8%)†† (-21.9%) ♯

C-T187.32 989.35 87 5.87±0.231 a 16.6

(-29.2%)†† (-26.6%) α (+27.9%)§ (-32.1%)†† (-29.8%) ♯

A-T244.54 1294.28 73 7.56±0.292 c 20.43

(-7.5%)†† (-4.0%) α (+7.4%)§ (-12.5%)†† (-13.6%) ♯

N-C: normal control, S-C: smoke-exposed control, FVT-1: fed 50 mg FVBM extract/rat/day; FVT-2: fed 100 mg FVBM extract/rat/day, C-T, fed 1 mg F18 bioactive compound/rat/day and A-T: given 1 mg atorvastatin/rat/day for 4 weeks.MDA: Malondialdehyde.*The CD values are expressed as nmole MDA equivalents/mg protein. Basal conjugated diene represent the in-vivo status of oxidized LDL. **The lag phase is defined as the interval between the intercept of the tangent of the slope of the curve with the time expressed in minutes. ¥The maximum in-vitro oxidation of LDL was achieved after 12 h of incubation with CuSo4 in each group, †Percent increase with respect to basal value in N-C, ††Percent decrease with respect to basal value in S-C, ¶Percent decrease with respect to lag phase value in N-C, §Percent increase with respect to lag phase value in S-C, €Percent increase with respect to maximal value in N-C, ♯Percent decrease with respect to maximal value in S-C, Significantly different from N-C at aP < 0.001. +Values are obtained from LDL subpopulation, isolated from plasma of 5 rats in each group.Significantly different from N-C at ap < 0.001, significantly different from S-C at ap < 0.001, significantly different from S-C at bp < 0.01, significantly different from S-C at cp < 0.05.

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permanent inflammation and leads to imbalance in the profile of lipid peroxidation products (55), which can cause number of membrane changes including lipid peroxidation in CS-exposed erythrocytes of rats (8). The studies also showed that occupationally exposed human subjects indicate enhance lipid peroxidation and alter antioxidant systems in erythrocytes (56).

Consistent with these reports, our results demonstrated a significant increase in lipid peroxidation products in plasma and erythrocytes of subchronic cigarette smoke-exposed rats. The increase in plasma lipid peroxidation product is well correlated with a significant decline in plasma total antioxidant content which in turn is consistent with the prooxidant effect of CS in rats. These results are in consensus with previous finding (57), where an increase correlation between plasma MDA level and antioxidants has been reported in CS-exposed animals.

However, protective role of lipid lowering agent with potent antioxidant property, such as FVBM extract and F18 compound, on the formation of lipid peroxidation products in plasma and erythrocytes of CS-exposed rats has not been reported. Our results showed that supplementation of FVBM extract and F18 bioactive compound to CS-exposed rats caused a significant decrease in plasma and erythrocytes lipid peroxidation products with a concomitant and significant increase in plasma total antioxidants and restored their levels close to corresponding control values in N-C (Table 6). These results are in concordant with our previously published in-vitro data that demonstrated the profound antioxidant property of FVBM extract and F18 bioactive compound (14, 16). Here it is interesting to mention that F18 compound, at a dose of 1 mg showed significant amelioration in reducing CS-induced oxidative stress parameters which were almost comparable to the FVBM extract at higher dose. The above data indicate that FVBM extract at higher dose and F18 compound strongly inhibit the lipid peroxidation process initiated by free radicals, thus preventing the membrane damage.

Several lines of research have established that lipoproteins play a pivotal role in atherogenesis (58, 59). Exposure of CS produces enhanced free radical that leads to the oxidative modification

of lipoprotein (6). During oxidative modification native LDL is converted to Ox-LDL which is a key mediator of atherosclerosis and plasma concentrations has been found to be elevated in a range of cardiovascular diseases (60).

Previous studies showed that elevated level of Ox-LDL concentrations in plasma have been found in smoke-exposed rats (61). The ex-vivo BDC levels and MDA have been suggested to reflect the in-vivo oxidation of LDL (62). The result demonstrated that LDL in the CS-exposed dyslipidemic rats had an increased susceptibility to oxidation when compared to LDL of normal rats. Moreover, our result showed that the ex-vivo BDC and MDA levels of LDL in CS-exposed hyperlipidemic rats were significantly increased. This increase susceptibility may result from increased oxidative stress, decrease total antioxidant and increase LDL cholesterol content. To the best of our knowledge no one to date has demonstrated the potent protective effect of F. virens extract or the isolated bioactive compound on the ex-vivo and in-vitro Cu++-catalyzed oxisability of plasma LDL of CS-exposed dyslipideamic rats. Simultaneous supplementation of FVBM extract and bioactive compound to CS-exposed rats blocked the in-vivo and in-vitro oxidation of LDL. As expected, the lag phase time of LDL oxidation was reduced and restored significantly after treatment. Similarly, ex-vivo basal and Cu++

induced maximal formation of MDA in LDL was significantly decreased in CS-exposed rats treated with plant extract and the bioactive compound. The data summarises that FVBM extract at a lower dose was least effective as an antioxidant and was only able to partial restoration of the above lipid peroxidation parameters. The potent hypolipidemic property of FVBM extract (at higher dose) and the bioactive compound, as discussed above, is consistent with the excellent antioxidant property of these fractions.

The data summarises that FVBM extract at a lower dose was least effective as an antioxidant and was only able to partial restoration of the above lipid peroxidation parameters. The potent hypolipidemic property of FVBM extract (at higher dose) and the bioactive compound, as discussed above, is consistent with the excellent antioxidant property of these fractions.

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Conclusions

In conclusion our combined results clearly demonstrated the protective role of FVBM extract and F18 compound in risk factor induced cardiovascular disease. Moreover, the test fractions/pure compound exhibited significant protection against CS-induced severe oxidative stress and hyperlipidemia, which indicates that F18/FVBM extract are highly promising natural antioxidant and also can be used as an antiperoxidative, hypolipidemic, and antiatherogenic agent. However, further large scale clinical trials in hyperlipidemic smokers with and without coronary heart disease are required to substantiate their antioxidative, hypolipidemic, and atheroprotective properties.

Acknowledgements

The author would like to appreciate Prof. S.W. Akhtar, vice chancellor, for providing state-of-the-art research laboratory and animal house for smooth succession of this work. The author would also like to thank Deanship of Scientific Research at Majmaah University for their support and contribution to this study.

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Biomedical Research and Therapy 2016, 3(7): 723-732 ISSN 2198-4093 www.bmrat.org

723 The role of Ficus virens Ait and its novel bioactive compound

ORIGINAL RESEARCH

Extenuating the role of Ficus virens Ait and its novel bioactive compound on antioxidant defense system and oxidative damage in cigarette smoke exposed rats

Danish Iqbal1,2, M. Salman Khan1*, Amir Khan3, Saheem Ahmad1

1Clinical Biochemistry and Natural Product Research Laboratory, Department of Biosciences, Integral University Lucknow-226026, India 2Department of Medical Laboratory Sciences, College of Applied medical Sciences, Majmaah University, Al-majma’ah-11952, Saudi Arabia 3Department of Maxillofacial Surgery (Biochemistry), College of Dentistry, Taif University, KSA

*Corresponding author: [email protected]

Received: 21 June 2016 / Accepted: 13 July 2016/ Published online: 26 July 2016 ©The Author(s) 2016. This article is published with open access by BioMedPress (BMP)

Abstract— Introduction: Production of free radicals is associated with cigarette smoke (CS) which in turn generates oxidative stress, could be responsible for alterations in the activities of enzymatic and non-enzymatic antioxidants that links with atherosclerosis. Methods: Therefore, the putative preventive effects of F. virens extract and its bioactive compound (F18), n-Octadecanyl-O-α-D-glucopyranosyl(6’→1’’)-O-α-D-glucopyranoside were investigated on overall enzyme and non-enzymatic defense system and in oxidative stress CS-exposed rats. Results: The enzymatic activities of hepatic and lung CAT, SOD, Gred and GST in CS exposed rats were significantly decreased, while Gpx activity in CS exposed rats was increased. Similarly, hepatic and lung GSH content was reduced when compared to value of normal control group. Simultaneous administration of FVBM extract (50 and 100 mg/rat) and F18 bioactive compound (1 mg/rat) significantly increases hepatic and lung CAT, SOD, Gred and GST activity as well GSH concentration coupled with decrease in Gpx level in CS-exposed stress rats. Moreover, our histological observations concludes the pulmonary congestion, thickening of interalveolar septa and foci of collapsed alveoli with subsequent dilation of the adjoining alveolar spaces as well as development of large irregular spaces in rats lung exposed to cigarette smoke. Similarly, the liver also showed morphological alterations with congestion in central vein, portal inflammation and necrosis in CS-exposed rats. These morphological changes reversed significantly after treatment with FVBM extract and F18 compound. Conclusion: Thus biochemical and histopathological studies suggested that, FVBM extract and F18 showed its protective nature against CS-exposed rats.

Keywords: Cigarette smoke, Oxidative stress, Antioxidant defense system, Histopathology, Ficus virens

INTRODUCTION

Oxidative stress is one of the major symptoms

accompanying physiological functions and many

pathological conditions such as cancer, diabetes,

chronic obstructive pulmonary disease, cardiovascular

and neurodegenerative diseases and also in the aging

process itself (Aruoma, 1998; Santilli et al., 2015; Sen et

al., 2010). Cigarette smoking (CS) contributes a

considerable amount of free radicals, estimated as 1014

and 1015 free radicals/puff in the tar and gas phases

(Church and Pryor, 1985) and is the major risk factor

as well as leading cause cardiovascular disease (CVD)

(Messner and Bernhard, 2014; WHO, 2015).

Moreover, CS generate free radicals that could be

responsible for alterations in the activities of

enzymatic viz., superoxide dismutase (SOD), catalase

(CAT), glutathione peroxidase (Gpx), glutathione

reductase (Gred), glutathione-s-transferase (GST) and

non-enzymatic, namely GSH, antioxidants (Ramesh et

al., 2007; Ramesh et al., 2015; Thirumalai et al., 2011).

DOI 10.7603/s40730-016-0033-5

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724 The role of Ficus virens Ait and its novel bioactive compound

Various studies suggested that medicinal plants are

source for the prevention of numerous oxidative stress

related diseases (Akhter et al., 2013; Alvi et al., 2015;

Khan et al., 2013; Salvamani et al., 2014). Thus, natural

compounds with antioxidant properties could

contribute to the protection of cell and tissue against

deleterious effects caused by CS generated reactive

oxygen species (ROS). Ficus species exhibits strong

antioxidant and biological properties, known to

diffuse the toxic free radical and can be used as a

possible protective agent for treatment of oxidative

stress related disorders (Iqbal et al., 2014a; Sirisha et

al., 2010; T S et al., 2013). We have previously shown

that Ficus virens bark methanolic (FVBM) extract

contained large amount of antioxidant with significant

hypolipidemic property (Iqbal et al., 2014b; Iqbal et al.,

2015). The current investigation demonstrates the

protective role of FVBM extract and its principal

bioactive compound, n-Octadecanyl-O-α-D-

glucopyranosyl(6’→1’’)-O-α-D-glucopyranoside in

cigarette smoke-induced oxidative stress.

MATERIALS AND METHODS

Chemical Reagents

Bradford dye was purchased from Sigma Aldrich, In-

dia, potassium dichromate, hydrogen peroxide (H2O2),

glacial acetic acid was procured from Merck Pvt Ltd,

India; capston cigarette from Capston, India ltd. All

other chemicals were procured either from Himedia

Laboratories, Mumbai, India or of analytical grade.

Isolation of Bioactive compound

Bioactive compound; n-Octadecanyl-O-α-D-

glucopyranosyl (6→1″)-O-α-D-glucopyranoside (F18)

from FVBM extract was isolated by following the pro-

tocol (Iqbal et al., 2015).

Animals

Male Sprague-Dawley (SD) rats weighed around 100-

150 gm were procured from Indian Institute of

Toxicology Research Center, Lucknow. The proposed

study was approved by Institutional Animal Ethics

Committee (IAEC) (registration number:

IU/Biotech/project/CPCSEA/13/11). The rats were

housed 5 per cage for one week in the animal house

for acclimatization at a temperature of 21-22˚C with 12

hours light and dark cycle. The rats were given

standard diet and water ad libitum.

Dose preparation

Sequentially extracted FVBM extract, its bioactive frac-

tion (F18) and reference drug atorvastatin were dis-

solved in 10 % dimethyl sulfoxide (DMSO) at different

concentrations and were homogenized with saline.

The doses of the extracts were selected on the basis of

previously published reports (Iqbal et al., 2015; T S et

al., 2013).

Diet/exposure to cigarette smoke

FVBM extract, its bioactive fraction (F18) and

atorvastatin suspension was administered through

gastric intubation in two divided doses (morning and

evening) of 0.5 ml each/rat/day. Rats in smoking

control group received 0.5 ml of saline containing 10

% DMSO (vehicle) twice daily while rats in normal

control group received 0.5 ml of saline containing 10

% DMSO twice daily. The rats were divided randomly

and equally (5 rats in each group) in groups as

illustrated in Table 1.

Table 1. Protocol for the treatment of cigarette smoke-

induced oxidative stress in rats

Group Treatment

N-C Normal control

S-C Cigarette smoking control + vehicle

FVT-1 Smoke-exposed + plant extract (FVBM) (50

mg/rat/day)

FVT-2 Smoke-exposed + plant extract (FVBM) (100

mg/ rat/day)

CT Smoke-exposed + bioactive compound (F18) (1

mg/ rat/day)

AT Smoke-exposed + standard (Atorvastatin) (1

mg/ rat/day)

Rats were exposed to cigarette smoke in the morning

by keeping two rats in bottomless metallic container

(10 x 11 x 16 inch), having two holes of 3 and 1.5 cm

diameter, one on the either side. A burning cigarette

was introduced through one hole (3 cm) and the other

hole (1.5 cm) was used for ventilation. Animals were

exposed to CS for 30 minutes, daily for 4 weeks with

interval of 10 min between each 10 min exposure,

using 3 cigarettes/day/2 rats in each group (Anbarasi

et al., 2006).

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725 The role of Ficus virens Ait and its novel bioactive compound

Collection of different organs

Liver and lung were excised and kept in ice-cold sa-

line. A portion of liver and lung were immediately

fixed in 10 % neutral formalin for histopathological

studies.

Preparation of homogenate and post mitochondrial

supernatant

At the end of the experiment, liver and lung from the

rats were promptly excised and chilled in ice-cold sa-

line. After washing with saline, it was blotted and

weighed. One gram of wet tissues was cut into pieces

and homogenized with 9 ml of chilled 0.1 M sodium

phosphate buffer, pH 7.4 (containing 1.17 % KCl) in a

waring blender. The homogenate was centrifuged at

1,000 rpm for 10 min at 4˚C, aliquoted and stored at

−20˚C. The remaining portion of the liver and lung

homogenates was centrifuged at 12, 000 rpm for 20

min at 4˚C. The post mitochondrial supernatant (PMS)

thus obtained was also aliquoted and stored at –20˚C

for future use.

Activities of antioxidant enzymes

The enzymatic activity of catalase in PMS of liver and

lung was measured by adopting the procedure of

Sinha (1972). Enzymatic activity of SOD in PMS

fraction of liver and lung was determined by the

method as described by Kakkar et al. (1984) based on

the 50 % inhibition of the formation of nicotinamide

adenine dinucleotide (NADH)-phenazine methosulphate-

nitroblue tetrazolium formazan at 560 nm. Glutathione

peroxidase activity in liver and lung homogenate was

assayed by modifying of the previous protocols

(Hafeman et al., 1974; Mills, 1959). The enzymatic

activity of Gred in liver and lung was determined

according to the method of Carlberg and Mannervik

(Eriksson et al., 1975). Method of Habig et al. (1974)

was used to measure the GST activity in PMS fraction

of liver and lung.

Activity of non-enzymatic antioxidant

For the determination of GSH content in liver or lung

homogenate, the previous methods were followed

(Ellman, 1959; Sedlak and Lindsay, 1968).

Histopathological studies of liver and lung

For histopathological study, a portion of liver and

lung were used. For microscopic preparation of the

above tissues, method of Disbrey and Rach (1970) was

used. Two formalin fixed samples from each tissue

were embedded in paraffin and sectioned after block

preparation. The paraffin block was sectioned in 4~5

μm thickness by using a microtome to make a slide

and then it was dyed with hematoxylin-eosin (H&E)

and observed under a light microscope and

photographs were taken (JA, 2001) .

Protein estimation

The protein concentration of liver homogenate and

PMS was analyzed by the method of Bradford (1976)

using bovine serum albumin as standard. Aliquots of

liver homogenates and PMS were first precipitated

with 10 % TCA followed by centrifugation at 1500

rpm for 10 min. The protein pellets were dissolved in

0.5 N NaOH and used for protein determination.

Data analysis

For all assays, samples were analysed in triplicate and

the results were expressed as mean ± SD. The results

were evaluated using one-way analysis of variance

(ANOVA) and two tailed Students T-test. Statistical

significance were expressed as *p<0.05, **p<0.01 and

***p <0.001.

RESULTS

Regulatory effects of FVBM extract, F18 bioactive

compound and atorvastatin on antioxidant defense

system in smoke-exposed rats after 4 weeks of

treatment

Free radicals released by cigarette smoke (CS)

generate oxidative stress. This process could be

responsible for alterations in the activities of

enzymatic and non-enzymatic antioxidants and

development of atherosclerosis. Since, protection

against oxidative stress/ROS is provided by enzymatic

and non-enzymatic antioxidants, therefore, the status

of antioxidant enzymes, such as CAT, SOD, Gpx, GST

and Gred including GSH concentrations in liver and

lung of experimental smoke-exposed rats are

important.

Impact on the regulation of hepatic and lung cata-

lase, superoxide dismutase, glutathione peroxidase,

glutathione reductase, glutathione-s-transferase ac-

tivities and reduced glutathione content

The enzymatic activities of hepatic CAT, SOD, Gred

and GST in S-C rats were significantly decreased from

N-C values of 62.28, 7.65, 18.29 and 132.8. U/mg

protein to 32.45, 4.8, 11.28 and 68.12 U/mg protein,

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726 The role of Ficus virens Ait and its novel bioactive compound

respectively, while Gpx activity in S-C rats was

increased from N-C values of 44.2 to 57.28 U/mg

protein (Fig. 1). The restoration in above enzymatic

activities of hepatic CAT, SOD, Gred and GST in FVT-

2 treated rat was 24, 32, 48 and 36 % of respective

normal control values. The values of these enzymes in

C-T group, in comparison to S-C values, were

significantly increased by 35, 53, 60 and 43 %

respectively. In contrast the hepatic Gpx activity was

significantly decreased by 14, 18, 20 and 10 % in FVT-

1, FVT-2, C-T and A-T rats. As depicted in fig. 3, the

hepatic GSH content were reduced by 43 % when

compared to value of N-C group. Administration of

FVBM extract, F18 bioactive compound and

atorvastatin to CS-exposed rats resulted in a

significant increase in GSH content by 22, 32, 45 and

18 % respectively.

Figure 1. Impact of FVBM extract,F18 bioactive compound andatorvastatin on Liver CAT, SOD, Gpx, Gred and GST activities in

cigarette smoke-exposed rats after 4 weeks of treatment. One unit (U/ mg protein) of Gpx activity is defined as nmole oxidized glutathione

formed/min/mg homogenate protein. One unit (U/ mg protein) of Gred activity is defined as nmole NADPH oxidized/min/mg PMS protein. One

unit (U/ mg protein) of GST activity is defined as the nmole of 1-chloro 2,4-dinitrobenzene (CDNB) conjugate formed/min/mg PMS protein. One unit

(U/mg protein) of CAT activity is defined as the μmoles of H2O2 decomposed/min/mg protein. One unit (U/mg protein) of SOD activity is defined as

the amount of enzyme required to inhibit O.D. at 560 nm of chromogen production by 50 % in one minute. Values are mean SD from

homogenate/PMS fraction of liver of 5 rats in each group. Significantly different from N-C at ap<0.001, significantly different from S-C at ap<0.001, significantly

different from S-C at bp<0.01, significantly different from S-C at cp<0.05.

Figure 2. Impact of FVBM extract, F18 bioactive compound andatorvastatin on lung CAT, SOD, Gpx, Gred and GST activities in

cigarette smoke-exposed rats after 4 weeks of treatment. One unit (U/ mg protein) of Gpx activity is defined as nmole oxidized glutathione

formed/min/mg homogenate protein. One unit (U/ mg protein) of Gred activity is defined as nmole NADPH oxidized/min/mg PMS protein. One

unit (U/ mg protein) of GST activity is defined as the nmole of 1-chloro 2,4-dinitrobenzene (CDNB) conjugate formed/min/mg PMS protein. One unit

(U/mg protein) of CAT activity is defined as the μmoles of H2O2 decomposed/min/mg protein. One unit (U/mg protein) of SOD activity is defined as

the amount of enzyme required to inhibit O.D. at 560 nm of chromogen production by 50 % in one minute. Values are mean SD from homoge-

nate/PMS fraction of Lung of 5 rats in each group. Significantly different from N-C at ap<0.001. Significantly different from N-C at bp<0.01. Significantly different

from S-C at ap<0.001. Significantly different from S-C at bp<0.01. Significantly different from S-C at cp<0.05.

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727 The role of Ficus virens Ait and its novel bioactive compound

Moreover, similar pattern was observed in lung en-

zymatic and non-enzymatic antioxidant defense sys-

tem. As depicted in Fig. 2 the enzymatic activities of

lung CAT, SOD, Gred and GST in S-C rats were signif-

icantly decreased from N-C value of 32.5, 14.7, 24.3

and 127.7 U/mg protein to 21.86, 9.56, 11.78 and 102.47

U/mg protein (Fig. 2). Whereas, lung Gpx activity in S-

C rats was significantly increased from N-C values of

59.68 to 82.73 U/mg protein. The restoration in above

enzymatic activities of lung CAT, SOD, Gred and GST

in FVT-2 treated rat was 34, 40, 41 and 20 % of respec-

tive N-C values. The values of these enzymes in C-T

group, in comparison to N-C values were significantly

increased by 42, 50, 47 and 24 % respectively. In con-

trast the lung Gpx activity was significantly decreased

by 11, 17, 26 and 16 % in FVT-1, FVT-2, C-T and A-T

treated rats. Similarly, lung GSH content which was

significantly reduced by 33 % in S-C rats as signifi-

cantly increased by 20, 37, 46 and 28 % after adminis-

tration of FVBM extract, F18 bioactive compound and

atorvastatin (Fig. 3). Here it is interesting to mention

that atorvastatin also exhibited significant ameliora-

tion in all the enzymatic and non-enzymatic activities

but is considered to be less effective than higher dose

of FVBM extract and F18 bioactive compound.

In summary, hepatic and lung CAT, SOD, Gred, GST

enzymes and non-enzymatic GSH, which constitute a

mutually supportive team of defense against ROS, are

significantly ameliorated after feeding of FVBM

extract and F18 bioactive compound as well as

substantially quenches the free radicals, thus

positively normalizing the above enzyme levels close

to normal values.

Figure 3. Effect of FVBM extract,F18 bioactive compound andatorvastatin on liver and lung GSH content in cigarette smoke-

exposed rats after 4 weeks of treatment. Values are mean (nmole SH group/mg protein) SD from homogenate of liver or lung of 5 rats in

each group. Significantly different from N-C at ap<0.001. Significantly different from S-C at ap<0.001. Significantly different from S-C at bp<0.01. Significantly different from S-C at cp<0.05.

Histopathological studies of liver and lung

It is apparent from Fig. 4A that histopathology of the

liver of the control rats showed normal looking uni-

form hepatocytes with small vesicular nuclei and ab-

undant eosinophilic granular cytoplasm with distinct

cell boundaries. Architecture of liver is well main-

tained. Normal Interstitial cells with vascularity. CS-

exposed rats showed smaller hepatocytes with small

vesicular or pyknotic nuclei and reduced granularity

of cytoplasm. Cytoplasmic boundaries are relatively

indistinct. Architecture not well maintained. Intersti-

tial cells increased and vascularity is reduced (Fig.

4B). Administration of FVBM extract at lower dose

has shown smaller hepatocytes with smaller vesicular

nuclei and eosinophilic cytoplasm with indistinct cell

boundaries showing proliferation. Architecture not

well maintained but vascularity has relatively in-

creased (Fig. 4C). Moreover, liver section of FVT-2 rats

(higher dose) (Fig. 4D) showed proliferated normal

hepatocytes with normal vesicular nuclei and abun-

dant eosinophilic cytoplasm with distinct cell bounda-

ries. Architecture well maintained with normal inters-

titial cell and vascularity. Similarly, liver section of C-

T rats showed proliferative hepatocytes with normal

morphology as well as architecture. Interstitial cells

normal with normal vascularity, also no toxic effects

noted (Fig. 4E). Furthermore, liver section of A-T rats

showed normal hepatocytes with normal vesicular

nuclei and abundant eosinophilic cytoplasm alongwith

well maintained normal architecture and vascularity (Fig. 4F).

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728 The role of Ficus virens Ait and its novel bioactive compound

Figure 4. Histology of liver section. A. Microphotograph of liver section from normal control rats, B. Microphotograph of liver

section from cigarette smoke-exposed rats after 4 weeks of cigarette smoke exposure, C. Microphotograph of liver section from

cigarette smoke-exposed rats after 4 weeks of FVBM-50 treatment, D. Microphotograph of liver section from cigarette smoke-

exposed rats after 4 weeks of FVBM-100 treatment, E. Microphotograph of liver section from cigarette smoke-exposed rats after 4

weeks of F18 bioactive compound treatment, F. Microphotograph of liver section from cigarette smoke-exposed rats after 4 weeks of

atorvastatin treatment. Figures were captured at 100X.

Histopathology of lung in N-C group shows normal

lung stroma and characteristic spongy appearance of

the lung with normal looking numerous alveolar

spaces, had normal blood vessels and bronchi. The

bronchoalveolar unit parenchyma in the normal lung

was within the limits (Fig. 5A). The lung section of CS-

exposed rats showed the increased volume of stroma

with presence of few collections of stromal cells with

reduced alveolar spaces, reduced vascularity and con-

stricted bronchi (Fig. 5B). There was obliteration of

most alveoli and subsequently compensatory dilata-

tion and expansion of the contiguous ones with de-

struction of alveolar wall. On treatment with lower

dose of FVBM extract, section of lung shows reduced

stroma, increased alveolar spaces, vascularity with

patent blood vessels and dilated bronchi (Fig. 5C). On

the other hand, treatment with higher dose, section of

lung showed changes as described above but bronchi

are relatively constricted (Fig. 5D). In addition, in lung

section of C-T rat observed that stroma is further re-

duced with presence of large alveolar spaces. Blood

vessels were normal but smaller bronchi were further

constricted (Fig. 5E). Furthermore, lung slice of ator-

vastatin treated group showed the alveolar spaces

comparatively less than normal with corresponding

increase in stromal elements. Blood vessels and bron-

chi were normal in the presence of few constricted

smaller bronchi (Fig. 5F).

DISCUSSION

Cigarette smoking (CS) is the foremost cause of

morbidity and mortality worldwide (Alam et al., 2013;

Jha et al., 2008) and is considered to be the preventable

risk factor for CVD (Ambrose and Barua, 2004).

Oxidative stress caused by CS are responsible for

enhanced lipid peroxidation, protein oxidation, DNA

damage and endothelial damage that could results in

various diseases such as CVD, stroke,different types

of cancer and diabetes (Messner and Bernhard, 2014;

Mohod et al., 2014; Valavanidis et al., 2009; Willi et al.,

2007).

Cigarette smoke is a complex situation possessing an

array of free radicals and ROS (Pryor and Stone, 1993).

These, free radicals are highly reactive molecules

occurs in normal consequence of a variety of

biochemical reactions (S, 2011). And their overload

from exogenous sources like, smoking, alcohol abuse,

UV radiations and air pollution added to the

endogenous production of free radicals that results in

oxidative stress and oxidative damage to the tissues

(Ghobashy et al., 2010; Mohod et al., 2014; Wu and

Cederbaum, 2003) as well as to DNA, proteins and

membrane lipids (Cosmas Achudume and Aina, 2012;

Wu and Cederbaum, 2003).

Under oxidative stress conditions, the antioxidant

enzyme levels are altered, in order to cope with the

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729 The role of Ficus virens Ait and its novel bioactive compound

tremendous increase in the production of ROS

(Anbarasi et al., 2006; McCord, 1993; Mohod et al.,

2014). However, after prolonged exposure, the toxic

effects of cigarette smoke emerge to override the

adaptive mechanism of the body tissues, as indicated

by a decrement in the levels of these enzymes (Hulea

et al., 1994). It is clear from the above discussion CS-

induces paramount oxidative stress and is consistent

with our results that demonstrate significant decrease

in liver and lung enzymatic activity of SOD, CAT,

Gred, GST including the GSH level, while Gpx activity

was significantly increased in CS-exposed rats.

In antioxidant defense system SOD is known to be the

first enzyme, responsible for scavenging the

superoxide radicals to form H2O2 (Andersen et al.,

1997). Furthermore, H2O2 is scavenged by

catalase/GSH (glutathione peroxidase) or it facilitate

in the development of highly reactive oxygen species

(Inoue et al., 2013; Mohod et al., 2014). The tar phase

of cigarette smoke contains quinone–semiquinone

radicals which are capable of reducing molecular

oxygen to superoxide radicals whose extreme

generation inactivates this enzyme (Church and Pryor,

1985; Durak et al., 2002). The proposed decrease in

SOD activity in CS-exposed rats could have resulted

from its inactivation by tar phase oxidants. The

presence and production of the free radicals from

smoke lower enzymatic activity of CAT, leading to

accumulation of H2O2 and lipid hydroperoxides which

further deteriorate the tissue damage (Pryor and

Stone, 1993). Inhibition of CAT activity in rat liver and

lung by cigarette smoke was observed during the

present study which is in conjunction of previous

reports (Luchese et al., 2009; Ramesh et al., 2007;

Ramesh et al., 2010).

Figure 5. Histology of lung section. A. Microphotograph of lung section from normal control rats, B. Microphotograph of lung

section from cigarette smoke-exposed rats after 4 weeks of cigarette smoke exposure, C. Microphotograph of lung section from

cigarette smoke-exposed rats after 4 weeks of FVBM-50 treatment, D. Microphotograph of lung section from cigarette smoke-

exposed rats after 4 weeks of FVBM-100 treatment, E. Microphotograph of lung section from cigarette smoke-exposed rats after 4

weeks of F18 bioactive compound treatment, F. Microphotograph of lung section from cigarette smoke-exposed rats after 4 weeks of

atorvastatin treatment. Figures were captured at 100X.

However, a decrease in the activity of CAT in the

present study suggests the in-vivo decrease in

antioxidant level which in turn unable to defend the

oxidative stress generated via cigarette smoke.

Treatment with FVBM extract and F18 bioactive

compound to smoke-exposed rats resulted in a

significant increase in both liver and lung SOD and

CAT activity which further strengthen the potent free

radical scavenging property of plant extract and

bioactive compound. Reduced glutathione play an

important role in glutathione-dependent antioxidant

system that most probably act as free radicals

scavenger or a substrate for Gpx and GST during the

detoxification of H2O2 (Masella et al., 2005).

Glutathione is maintained in body from its oxidized

form by the enzyme Gred, which requires NADPH as

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730 The role of Ficus virens Ait and its novel bioactive compound

a cofactor (Carmel-Harel and Storz, 2000). Previously

it has been reported that GSH was depleted during

CS-exposure in various tissues. Meanwhile, the

decrease in tissue GSH levels in CS-exposed rats may

be because of declined Gred activity and probably

reduced NADPH supply (Masella et al., 2005). GST is

involved in the detoxification of ROS and toxic

compounds from cigarette smoke, by conjugating

them to GSH. This conjugation reaction results in

depletion of the intracellular GSH that may further

enhances oxidant injury probably due to non-

availability of GSH for antioxidant enzymes such as

Gpx. Recent experimental data support the

assumption that CS exposure increases oxidative

stress and act as a potential mechanism for initiating

cardiovascular dysfunction (Ambrose and Barua,

2004).

Our data is well in agreement with above discussed

reports and observed the significant decline in GSH,

Gred and GST level in contrast the Gpx activity in

liver and lung of CS-exposed rats was significantly

increased which is due to inability of CAT to cope

with the oxidative stress. Helen and Vijayammal

(1997) also observed a similar decrease in the activity

of SOD, CAT, Gred and an increase in Gpx in rats

exposed to cigarette smoke. Gpx is also a scavenging

enzyme, but an increase in its activity in tissues of CS-

exposed rats may further reduce the GSH content. In

addition, an increased Gpx activity represents a

compensatory mechanism to degrade H2O2.

Simultaneous administration of FVBM extract (50 and

100 mg/rat) and F18 bioactive compound (1 mg/rat)

significantly increases CAT, SOD, Gred and GST

activity as well GSH concentration coupled with

decrease in Gpx level in CS-exposed stress rats. Thus,

it has been concluded that administration of FVBM

extract at higher dose and F18 bioactive compound to

CS-exposed rats restore the enzymatic (SOD, CAT,

Gred, GST and Gpx) and non-enzymatic (GSH)

antioxidant defense system and thus protect liver and

lung cells against oxidative stress-induced damage by

directly counteracting ROS/free radicals and by

activating the overall antioxidant defense systems.

Histological observations noticed the pulmonary con-

gestion and thickening of interalveolar septa in rats

exposed to cigarette smoke. The present study showed

devastation of some alveolar walls and foci of col-

lapsed alveoli with subsequent dilation of the adjoin-

ing alveolar spaces and development of large irregular

spaces (emphysematous changes). Cigarette smoking

causes endothelial dysfunction due to increased oxi-

dative stress (Messner and Bernhard, 2014). Our re-

sults coincided with those who interrelated the thick-

ening of the alveolar septa to modification in the vas-

cular bed resulting in inflammatory infiltration and

oedema (Hora et al., 2003). Chronic cigarette smoke

exposure causes lung tissue destruction might be due

to increased production of metalloproteinases (MMP)

proteolytic enzymes by macrophages (Barnes et al.,

2003; Taraseviciene-Stewart and Voelkel, 2008). The

liver showed congestion in central vein, portal in-

flammation and necrosis in CS-exposed rats. These

morphological changes were significantly reversed

after treatment with FVBM extract and F18 com-

pound. Thus, biochemical and histopathological stud-

ies suggested that, FVBM extract and F18 showed its

protective nature against CS-exposed rats.

CONCLUSION

In conclusion, our combined results clearly demon-

strated the protective role of FVBM extract and F18

compound in CS-induced severe oxidative stress. The

results are well supported by histopathological obser-

vations and indicates that F18/FVBM extract are

highly promising natural antioxidant as well as can be

used as an antioxidant and antiatherogenic agent.

However, further large-scale clinical trials in smokers

with and without coronary heart disease are required

to substantiate their antioxidative, atheroprotective

properties.

Acknowledgements

The Authors like to thank Prof. S.W. Akhtar, vice

chancellor, for providing state-of-the-art research

laboratory and animal house for smooth succession of

this work. The author would also like to thank

Deanship of Scientific Research at Majmaah

University for their support and contribution to this

study.

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Iqbal et al., 2016                                                                                                                 Biomed Res Ther 2016, 3(7): 723-732

731 The role of Ficus virens Ait and its novel bioactive compound

Competing Interests

The authors declare they have no competing interests.

Open Access

This article is distributed under the terms of the Creative

Commons Attribution License (CC-BY 4.0) which permits

any use, distribution, and reproduction in any medium,

provided the original author(s) and the source are credited.

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Cite this article as:

Iqbal, D., Khan, M., Khan, A., & Ahmad, S. (2016).

Extenuating the role of Ficus virens Ait and its novel

bioactive compound on antioxidant defense system

and oxidative damage in cigarette smoke exposed rats.

Biomedical Research and Therapy, 3(7), 723-732.