Screening of in-vitro antioxidant activity of Hippophae salicifolia berries extract

In-vitro antioxidant activity of Hippophae salicifolia ISSN: 2394-0026 (P)Original Research Article ISSN: 2394-0034 (O)Screening of in-vitro antioxidant activity ofHippophae salicifolia berries extractSanthrani Thakur*, Pradeepthi Chilikuri, Bindu Pulugurtha, Lavanya YaidikarDivision of Pharmacology, Institute of Pharmaceutical Technology, Sri Padmavati Mahila Visvavidyalayam (Women’s University), Tirupati-517502, Andhra Pradesh, India.AbstractThe present study aimed to study the preliminary phytochemical analysis, standardisation and in-vitro antioxidant evaluation of Hippophae salicifolia (HS) berries extracts. Briefly, the berries weresubjected to alcoholic and aqueous extraction. The preliminary phytochemical analysis wasperformed for aqueous and alcoholic extracts. The aqueous and alcoholic extracts are standardisedfor total phenolic, total tannin and total flavonoid content. The extracts were evaluated for its in-vitro free radical scavenging activities against, 2’-diphenyl-1- picrylhydrazyl (DPPH) radicals, hydroxylradicals, nitric oxide radicals and total antioxidant activity. We found that alcoholic extract showedthe presence of alkaloids, phenols, tannins, saponins, flavonoids and aqueous extract showed thepresence of flavonoids, tannins, carbohydrates, and phenols. The extracts of HS berries werestandardized for their total phenolic, tannin, flavonoid and vitamin-C content. The in vitroantioxidant activity revealed that both the extracts showed significant scavenging activities againstfree radicals. It can be concluded that both the extracts are effective in scavenging the free radicals.The flavonoids, tannins, vitamin-C and saponins may be attributed to free radical quenching activityof Hippophae salicifolia.Key wordsFree radical scavenging, Antioxidant, Hippophae salicifolia, Berries, Extract. Introduction*Corresponding Author: Santhrani Thakur Free radicals are produced as a by productE mail: [email protected] during metabolism in human beings. TheseReceived on: 22-09-2014 How to cite this article: Santhrani Thakur, Pradeepthi Chilikuri, BinduAccepted on: 29-09-2014 Pulugurtha, Lavanya Yaidikar. Screening of in-vitro antioxidant activity of Hippophae salicifolia berries extract. IAIM, 2014; 1(2): 1-8. Available online at www.iaimjournal.comInternational Archives of Integrated Medicine, Vol. 1, Issue. 2, October, 2014. Page 1Copy right © 2014, IAIM, All Rights Reserved.

In-vitro antioxidant activity of Hippophae salicifolia ISSN: 2394-0026 (P) ISSN: 2394-0034 (O)liberated free radicals cause damage to cellular percentage yield was found to be 7.63% w/w.bio-molecules such as lipids, proteins and DNA The extracts were subjected to various chemical[1]. Our body defensive mechanism in the form tests in order to detect the presence of differentof antioxidants against the free radicals, phytoconstituents [6, 7].maintains equilibrium between generated freeradicals and antioxidants. If any riot in this Standardization of HSequilibrium leads to oxidative stress [2]. It is The total phenolic content of the extract wasresponsible for a number of pathological determined with the Folin-Ciocalteau assay anddiseases such as aging, cancer, diabetes, expressed as milligrams of gallic acid equivalentsatherosclerosis and other neurodegenerative (GAE) per 100 grams dry mass (mg GAE/100g)disorders [3]. An inequity between free radicals [8]. The total flavonoid content was measuredand the innate antioxidant capacity of the body, with an aluminium chloride colorimetric assayintended for the use of antioxidants [4]. Natural and expressed as milligrams of (+) Quercetinantioxidants have better therapeutic efficacy in equivalents (QE) per 100 gram dry mass (mgprotecting cells from the injury of free radical QE/100 g) [9]. The total tannin content wasover synthetic one. So, immense interest is determined by the Folin-Denis assay methodfocused on the development of antioxidants and expressed as the equivalents of tannic acidfrom traditional herbal plants. (TE) (mg TE/100g extract) [10].Hippophae salicifolia of Elaeagnaceae family is In vitro antioxidant activityone such plant species distributed in Northern The free radical scavenging activity of theHimalayas. The plant is rich in tocopherols, extracts of Hippophae salicifolia berries wascarotenoids, vitamin-C, omega-3 fatty acid and determined by using various in vitro assays suchvitamin-K. Tocopherol, vitamin-C, omega-3 fatty as DPPH radical scavenging assay, hydrogenacids are shown to have potent antioxidants [5]. peroxide radical scavenging assay, nitric oxideTherefore to have the scientific validation on radical scavenging assay, total antioxidantantioxidant activity, the berries were taken for capacity, reducing power assay.the present study. The aqueous and alcoholicextracts of HS berries were used to investigate DPPH radical scavenging assaythe antioxidant activity using in-vitro methods. DPPH scavenging activity was measured with Spectrophotometric Method [11]. To the extractMaterial and Methods solution of concentration ranging from 40 microgram to 200 microgram, 4 ml of DPPH wasHippophae salicifolia berries added and was made up to 5 ml with ethanol,Hippophae salicifolia berries powder was incubated for 30 minutes at room temperature.purchased from Changsha Organic Herb Inc., The absorbance was measured at 517 nmChina. The alcoholic extract was prepared by against blank. The percentage of inhibition ofsubjecting the berry powder to extraction with DPPH was calculated as follows. Ascorbic acidalcohol, refluxed for about 6-8 hours, filtered was used as standard and the scavenging effectand evaporated to dryness by rotary film of DPPH was expressed in terms of ascorbic acidevaporator at low temperature under reduced equivalents.pressure. The percentage yields were found tobe 4.27% w/w. The aqueous extract was Hydrogen peroxide radical scavenging assayprepared by heat distillation method. TheInternational Archives of Integrated Medicine, Vol. 1, Issue. 2, October, 2014. Page 2Copy right © 2014, IAIM, All Rights Reserved.

In-vitro antioxidant activity of Hippophae salicifolia ISSN: 2394-0026 (P) ISSN: 2394-0034 (O)The scavenging effect of hydrogen peroxide was The alcoholic and aqueous extracts ofdetermined according to the method of Ruch, et Hippophae salicifolia berries were tested foral. [12]. 1 ml of extract solution was treated with different phytoconsituents like alkaloids,0.6 ml of hyrogen peroxide for 10 minutes; the glycosides, saponins, tannins, terpinoids, sugars,absorbance was read at 230 nm against blank. phenolic compounds, flavanoids, protein,Ascorbic acid was used as standard and the carbohydrates and volatile oils using standardscavenging effect of hydrogen peroxide was procedures and the results are given in Table - 1.expressed in terms of ascorbic acid equivalents. The total phenolic, tannin, and flavonoid content of alcoholic and aqueous extracts of HS wereNitric oxide scavenging assay quantified and results were given in Table - 2.Nitric oxide was generated from sodiumnitroprusside and its scavenging effect was Table - 1: Preliminary Phytochemical analysis ofdetermined [13, 14]. Different concentration of alcoholic and aqueous extracts of Hippophaeextract solution in phosphate buffer was salicifolia berries.incubated with sodium nitroprusside for 5 hoursat 25 °C. Control experiments were performed Test for Alcoholic Aqueouswith equal amount of buffer instead of extract Extracts of Extracts ofsolution. After 5 hours of incubation, 0.5 ml of HS HSsupernatant liquid was removed and 0.5 ml of Alkaloids -ve -veGriess reagent was added. The absorbance of Tannins +ve +vethe chromphpore formed during diazotization Phenolic +ve +vewith sulphanilamide and its subsequent coupling compoundswas read at 546 nm. Ascorbic acid was used as Glycosides -ve -vestandard and the nitric oxide scavenging was Proteins -ve -veexpressed in terms of ascorbic acid equivalents. Flavonoids +ve +veTotal antioxidant capacity Sterols +ve -veThe total antioxidant capacity was determined Fixed oils -ve -veby Spectrophotometric method [15]. Extract Volatile oils -ve -vetest solution of concentration ranging from 40 to Triterpenoids -ve +ve200 µg was taken in eppendroff tube and 1 ml of Saponins -ve -vereagent containing 0.6 mM sulphuric acid, 28 Sugars +ve +vemM sodium phosphate and 4 mM ammominum Carbohydrates -ve +vemolybadate were added. The tubes were DPPH radical scavenging activityincubated at 95 °C for 90 minutes and werecooled to room temperature; the absorbance DPPH radical scavenging activity of alcoholic andwas read at 695 nm. Ascorbic acid was used as aqueous extracts of HS was measured andstandard and the total antioxidant capacity was compared with Ascorbic acid. The percentageexpressed in terms of ascorbic acid equivalents. inhibition of DPPH radical by the extracts wasResults found to increase with increasing concentration of extracts as well as standard ascorbic acid. The percentage inhibition and IC50 concentrations ofPreliminary Phytochemical analysis and extracts and ascorbic acid are given in Table - 3.Standardization of Hippophae salicifoliaInternational Archives of Integrated Medicine, Vol. 1, Issue. 2, October, 2014. Page 3Copy right © 2014, IAIM, All Rights Reserved.

In-vitro antioxidant activity of Hippophae salicifolia ISSN: 2394-0026 (P) ISSN: 2394-0034 (O)Table - 2: Standardization of extracts of HS total antioxidant capacity of the extracts wasberries. found to increase with increasing concentration Alcoholic Aqueous of in extracts as well as standard ascorbic acid. Extracts of HS Extracts of The percentage of inhibition and IC50 HS concentrations of extracts and ascorbic acid are 14.36 ± 1.23 65.89 ± 1.11 given in Table - 6.Totalphenols Discussion(mg GAE/100g extract) 22.78 ± 1.06 23.55 ± 1.57 The Hippophae salicifolia berries were extractedTotal tannins 67.42 ± 1.43 54.68 ± 0.96 with alcohol and water. The extracts were(mg TAE/ tested for different phytoconsituents like100g extract) alkaloids, glycosides, saponinins, tannins,Total terpinoids, reducing sugars, phenolicflavonoids compounds, flavanoids, proteins, carbohydrates(mg QE/ and volatile oils. The information on the100g extract) phytochemical composition of a plant will giveHydrogen peroxide scavenging activity insight to screen for its biological properties [16, 17, 18]. The extracts are standardized for itsHydrogen peroxide scavenging activity of total phenolic, tannin and flavonoid content.alcoholic and aqueous extracts of HS was Now-a-days, plant materials rich in phenolicmeasured and compared with Ascorbic acid. compounds are used widely in industries toThe percentage inhibition of hydrogen peroxide improve the quality and nutrition of the foodradical by the extracts was found to increase [19]. Phenolic compounds are considered aswith increasing concentration of extracts as well secondary metabolites. Flavonoids areas standard ascorbic acid. The percentage recognized as polyphenolic compounds.inhibition and IC50 concentrations of extracts Phenolics and Flavonoids are entangled asand ascorbic acid are given in Table - 4. potent free radical scavengers and exert its antioxidant activity due to the presence ofNitric oxide scavenging activity hydroxyl group [20, 21]. They are shown to haveNitric oxide scavenging activity of alcoholic and biological properties such as anti-inflammatory,aqueous extracts of HS was measured and anti-hepatotoxic, anti-ulcer, anti-allergic, anti-compared with Ascorbic acid. The percentage viral and anti-cancer activities [22].inhibition of nitric oxide by the extracts was An in vitro antioxidant studies are widely used tofound to increase with increasing concentration screen various plants containing phenolic andof extracts as well as standard ascorbic acid. The flavanoid constituents. Free radicals are knownpercentage inhibition and IC50 concentrations of to play a unambiguous role in a wide variety ofextracts and ascorbic acid are given in Table - 5. pathological manifestations. Antioxidants fightTotal antioxidant activity against free radicals and protect us from variousTotal antioxidant activity of alcoholic and diseases. They exert their action either byaqueous extracts of HS was measured and scavenging the reactive oxygen species orcompared with Ascorbic acid. The percentage of strengthening the antioxidant defense mechanisms. DPPH radical, a free radical widelyInternational Archives of Integrated Medicine, Vol. 1, Issue. 2, October, 2014. Page 4Copy right © 2014, IAIM, All Rights Reserved.

In-vitro antioxidant activity of Hippophae salicifolia ISSN: 2394-0026 (P) ISSN: 2394-0034 (O)used for evaluating radical scavenging activity of Conclusionthe plant extract [23]. Scavenging of DPPHradical is related to the inhibition of lipid It can be concluded that HS berries extractsperoxidation [24]. Antioxidants either transfer showed potent anti-oxidant activities as evidentan electron or a hydrogen atom to DPPH radical, from scavenging against different radicals. Thethus neutralizing its free radical character [25]. aqueous extract showed highest antioxidantIn the present study, the percentage of activity than alcoholic extract of HS berries. Thescavenging effect on the DPPH radical was content of total phenols, total flavonoids may beconcomitantly increased with the increase in the attributed to its antioxidant activity.concentration of both the extracts. From theresults it is observed that HS berries possess Acknowledgementshydrogen donating capabilities, through whichscavenges the free radicals. The authors would like to thank UniversityHydrogen peroxide, an oxidative metabolite turn Grants Commission (UGC, Grant No. 39-174 (SR)to generate hydroxyl radicals (OH) resulting in - 2010), New Delhi for their financial support toinitiation and propagation of lipid peroxidation. carry out this research work.The hydrogen peroxide scavenging activity ofalcoholic and aqueous extract of HS was Referencesmeasured and compared with Ascorbic acid. Theextracts showed good hydrogen peroxide 1. Huang DJ, Chen HJ, Lin CD, Lin YH.scavenging activity. The ability of the extracts to Antioxidant and anti-proliferativequench OH radical seems to be directly related activities of water spinach (Ipomeato the prevention of the lipid peroxidation [26]. aquatica Frosk.) constituents. Bot Bull Acad Sin., 2005; 406: 99-106.Peroxy nitrite is a potent pleiotropic inhibitor of 2. Youwei Z, Jinlian Z, Yonghong P. Aphysiological processes such as smooth muscle comparative study on the free radicalrelaxation, neuronal signaling, inhibition of scavenging activities of some freshplatelet aggregation and regulation of cell flowers in Southern China. LWT-Foodmediated toxicity. Nitric oxide (NO) is a Sci. Technol., 2008; 41: 1586-1591.diffusible free radical that plays many roles as an 3. Scalbert A, Manach C, Remesy C,effectors molecule in diverse biological systems Morand C. Dietary polyphenols and theincluding neuronal messenger, vasodilatation prevention of diseases critical. Reviewsand antimicrobial and antitumor activities [27]. in Food Sciences and Nutrition. 2005;The Nitric oxide scavenging activity of alcoholic 45: 287-306.and aqueous extract of HS was measured and 4. Gulcin I, Berashvili D, Gepdiremen A.compared with Ascorbic acid. The total Antiradical and antioxidant activity ofantioxidant capacity of the extracts was total anthocyanins from Perillameasured spectrophotometrically through pankinensis decne. J Ethnopharmacol,ammonium molybdate method. The present 2005; 101: 287–293.study demonstrated that both the extracts 5. Rosch D, Bergmann M, Knorr D, Krohexhibited highest antioxidant capacity for LW. Structure-antioxidant efficiencyammonium molybdate reduction. relationships of phenolic compounds and their contribution to the antioxidantInternational Archives of Integrated Medicine, Vol. 1, Issue. 2, October, 2014. Page 5Copy right © 2014, IAIM, All Rights Reserved.

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In-vitro antioxidant activity of Hippophae salicifolia ISSN: 2394-0026 (P) ISSN: 2394-0034 (O)Traditional, complementary and (Dimocarpuslongum Lour.) peel. FoodAlternative Medicines, 2008; 5(1): 61- Chemistry, 2008; 106: 1264-1270.73. 26. Babu BH, Shylesh BS, Padikkala J.23. Bhuiyan MAR, Hoque MZ, Hossain SJ. Antioxidant and hepatoprotective effectFree Radical Scavenging Activities of of Alanthus icicifocus. Fitoterapia 2001,Zizyphus mauritiana. World J. Agr. Sci., 72:272–277.2009; 5: 318-322. 27. Karuppagounder SS, Madathil SK,24. Rekka E., Kourounakis PN. Effect of Pandey M, Haobam R, Rajamma U,hydroxyethyl rutenosides and related Mohanakumar KP. Quercetin up-compounds on lipid peroxidation and regulates mitochondrial complex-Ifree radical scavenging activity-some activity to protect against programmedstructural aspects. J. Pharm Pharmacol., cell death in rotenone model of1991; 43: 486-491. Parkinson's disease in rats.25. Pan Y, Wang K, Huang S, Wang H, Mu X, Neuroscience. 2013; 16(236):136-148.He C. et al. Antioxidant activity ofmicrowave-assisted extract of longan Conflict of interest: None declared.Table - 3: DPPH radical scavenging activity of extracts of HS.Concentration of Ascorbic acid, Percentage of inhibitionAlcoholic Extracts of HS and AqueousExtracts of HS (µg/mL) Ascorbic acid Alcoholic Aqueous Extracts40 of HS80 Extracts of HS 37.44 ± 1.58120 49.77 ± 1.37160 90.16 ± 0.91 42.65 ± 0.61 51.98 ± 1.78200 62.67 ± 1.85IC50 92.44 ± 1.06 59.66 ± 0.45 72.56 ± 1.99 119.34 μg/mL 94.17 ± 1.21 63.78 ± 0.37 95.33 ± 1.54 74.67 ± 1.02 97.11 ± 1.91 78.12 ± 1.11 20.36 μg/mL 48.22 μg/mLValues are expressed as Mean ± SEM (N=3 Readings).Table - 4: Hydrogen peroxide scavenging activity of extracts of HS.Concentration of Ascorbic acid, Percentage of inhibition Aqueous ExtractsAlcoholic Extracts of HS and Aqueous Ascorbic acid Alcoholic of HSExtracts of HS (µg/mL) 64.51 ± 0.2240 Extracts of HS 66.36 ± 0.2580 26.40 ± 3.46 49.59 ± 0.23 68.92 ± 0.31120 35.91 ± 1.25 61.80 ± 0.32 73.68 ± 0.28160 42.17 ± 3.59 64.64 ± 0.31 76.84 ± 0.15200 69.58 ± 2.28 66.01 ± 0.15 30.76 μg/mLIC50 87.23 ± 3.31 76.35 ± 0.28 142.82 μg/mL 40.38 μg/mLValues are expressed as Mean ± SEM (N=3 Readings). Page 7International Archives of Integrated Medicine, Vol. 1, Issue. 2, October, 2014.Copy right © 2014, IAIM, All Rights Reserved.

In-vitro antioxidant activity of Hippophae salicifolia ISSN: 2394-0026 (P) ISSN: 2394-0034 (O)Table - 5: Nitric Oxide scavenging activity of extracts of HS.Concentration of Ascorbic acid, Percentage of inhibition Aqueous ExtractsAlcoholic Extracts of HS and Aqueous Ascorbic acid Alcoholic of HSExtracts of HS (µg/mL) 45.34 ± 0.7440 Extracts of HS 48.56 ± 0.2280 26.40 ± 3.46 47.42 ± 1.21 52.98 ± 0.64120 35.91 ± 1.25 48.55 ± 2.01 58.77 ± 0.01160 42.17 ± 3.59 51.98 ± 0.51 68.99 ± 0.01200 69.58 ± 2.28 54.22 ± 0.15 115.23 μg/mLIC50 87.23 ± 3.31 64.10 ± 0.01 142.82 μg/mL 119.5 μg/mLValues are expressed as Mean ± SEM (N=3 Readings).Table - 6: Total antioxidant capacity of extracts of HS.Concentration of Ascorbic acid, Percentage of inhibition Aqueous ExtractsAlcoholic Extracts of HS and Ascorbic acid Alcoholic Extracts of of HSAqueous Extracts of HS (µg/mL) 55.94 ± 0.7840 HS 62.79 ± 0.8480 57.69 ± 1.27 46.47 ± 0.54 70.24 ± 0.37120 68.57 ± 2.98 51.93 ± 0.89 75.58 ± 0.65160 75.75 ± 1.92 62.86 ± 0.78 87.96 ± 0.66200 85.14 ± 2.91 83.26 ± 0.96 36.38 µg/mLIC50 96.67 ± 1.91 89.97 ± 0.078 36.21 µg/mL 77.67 µg/mLValues are expressed as Mean ± SEM (N=3 Readings).International Archives of Integrated Medicine, Vol. 1, Issue. 2, October, 2014. Page 8Copy right © 2014, IAIM, All Rights Reserved.