Patcharawan Tanamatayarat

School of Pharmaceutical Sciences,University of Phayao,Phayao 56000,Thailand

Antityrosinase,antioxidative activities,and brine shrimp lethality of ethanolic extracts from Protium serratum(Wall.ex Colebr.)Engl.

Patcharawan Tanamatayarat*

School of Pharmaceutical Sciences,University of Phayao,Phayao 56000,Thailand

ARTICLE INFO

Article history:

Received 3 Feb 2016

Receivedinrevisedform22Feb2016 Accepted 11 Aug 2016

Available online 15 Oct 2016

Antityrosinase

Protium serratum

Antioxidant

Phytochemistry

Free radical

Objective:To evaluate all parts of Protium serratum (Wall.ex Colebr.)Engl. (P.serratum)for their phytochemistry and biological activities including antityrosinase, antioxidant,and brine shrimp lethality.

Methods:Nine ethanolic extracts from different P.serratum parts such as twig,whole fruit,pericarp,and root were investigated for their phytochemical screening and biological activities in terms of tyrosinase inhibition,antioxidant against 1,1-diphenyl-2-picrylhydrazyl and hydroxyl radicals,and lethality to brine shrimp larvae.

Results:Phytochemical screening also revealed the presence of flavonoids,condensed tannins,alkaloids,triterpenoids,steroids,andsugarsinP.serratum.Therootextractwasthe most effective for antityrosinase activity with IC50of(21.63±0.31)μg/mL,and the leaf extract exhibited the highest antioxidation activity using 1,1-diphenyl-2-picrylhydrazyl and hydroxyl radical scavenging methods,with IC50of(4.34±0.09)and(119.80±1.01)μg/ mL,respectively.The essential oil extracted from the whole fruit displayed the highest toxicity against brine shrimp,with LC50of(3.57±1.82)μg/mL.

Conclusions:This study indicates that ethanolic extracts from each P.serratum part have differences in phytochemistry and biological activities(antityrosinase,antioxidant, and brine shrimp lethality).Some parts of the plant should be considered in the further study.

1.Introduction

There has been growing interest in use of traditional medicines including plants,animals,and mineral for new drug development, as the medicinal properties of natural materials have been proved for effectiveness against disease.Natural products play a key role in medicinal therapysuch as artemisinin fromArtemisia annuafor malaria[1],reserpine from Rauvolfia serpentina for hypertension [2],and taxol from Taxus brevifolia for cancer[3].Thus,natural materials are studied to protect the human body from diseases through their anticancer,antioxidant,and antityrosinase inducing abilities.

In Thailand,there are a large number of biodiversity,with approximately 12000 recorded species of vascular plants.Some flora are indigenous,some are rare,and many may have as yet unknown medicinal properties[4].It is interesting to study these plants for foods and medicines.Burseraceae is a family of trees and shrubs.There are 16 genera and 550 species in the world[5]. Several biological activities of the plants in this family were examined such as anti-inflammatory,antioxidation,antimalaria, and neuroprotective.Previous studies indicated that methyl-3,4,5-trihydroxybenzoate from the hexane extract of Dacryodes edulis stem bark inhibited 3D7(chloroquine-susceptible)and Dd2(multidrug-resistant)strains of Plasmodium falciparum[6]. Moreover,sesquiterpenes such as myrrhterpenoids K and N from the resin of Commiphora myrrha showed neuroprotective effects against 1-methyl-4-phenyl-pyridinium-induced neuronal cell death in dopaminergicneuroblastoma SH-SY5Y cells[7]. Additionally,triterpenoid compounds such as boscartene A-K from Boswellia carterii gum resin exhibited different hepatoprotective activities againstD-galactosamine-induced human hepatic(HL-7702)cell injury[8].Sesquiterpenoid compound such as guaia-4β,7β,10α-trihydroxy-5 ene from the resinous exudates of Commiphora opobalsamum showed cytotoxicity against human cervical epithelioid carcinoma(HeLa)and liverhepatocellular(HepG2)celllineswithIC50of15.4and8.7μmol/L, respectively[9].Moreover,two new prenylated flavonoids such as 6-(3,3-dimethylallyl)-2,3-dihydrokaempferol-3-β-O-glucoside and 6-(3,3-dimethylallyl)-naringenin-7-β-O-glucoside were isolated from stem wood of Commiphora opobalsamum [10]. Ethanolic extract from Protium serratum(Wall.ex Colebr.) Engl.(P.serratum)leaf exhibited over 50%inhibition of plaque formation in herpes simplex virus type 1 and poliovirus[11]. Methanolic extract from P.serratum leaf showed inhibition of protein denaturation at 1000μg/mL and thrombolytic activity[12].

*Correspondingauthor:Dr.Patcharawan Tanamatayarat,Schoolof Pharmaceutical Sciences,University of Phayao,Phayao 56000,Thailand.

Tel:+66 54 466 66

Fax:+66 54 466 690

E-mail:patcharawan_070@outlook.co.th

Peer review under responsibility of Hainan Medical University.The journal implements double-blind peer review practiced by specially invited international editorial board members.

2221-1691/Copyright©2016 Hainan Medical University.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Inaddition,thereare5generaand20speciesoftheBurseraceae family in Thailand.These genera comprise Canarium,Santiria, Dacryodes,Garuga,and Protium[13].P.serratum was found in evergreen forest,1500 m above sea level in the north and the northeast of Thailand[13].This fruit of this plant tastes slightly sweet and sour and is used as a food source.Also,the roots are used for their detoxicity and antipyretic activity[14].However, little research has previously been conducted on the biological activity and chemical constituents of this plant.Therefore,this study investigated the antityrosinase,antioxidative properties,the brine shrimp lethality and screening of the phytochemicals of

P.serratum.

2.Materials and methods

2.1.Chemicals

1,1-Diphenyl-2-picrylhydrazyl(DPPH),tyrosinase enzyme, 2-deoxyribose,and kojic acid wereobtained fromSigma-Aldrich, USA.Ascorbic acid was purchased from Ajax Finechem, Australia.EthanolfromLabscanwascommercialgradewhichwas redistilled before use.

2.2.Plant sample

P.serratumwascollectedfromChiangRaiProvinceonAugust 15,2014.This plant was identified by J.F.Maxwell at Biological Herbarium,Department of Biology,Faculty of Science,Chiang Mai University in April,2015.The voucher specimen is No.26.

2.3.Plant extraction

AllfreshpartsofP.serratumwerewashed,dried,andchopped at room temperature.Five grams of dried plant material were maceratedwithethanolthreetimes.Thefiltratewasthencollected, andthesolventevaporatedusingarotaryevaporator.Fivegramsof fruit pericarp(rind)were macerated with petroleum ether three times,and the macerated solution was filtered and dried.Essential oilfromthefruit(wholefruit)wasextractedwithsteamdistillation.

2.4.Preliminary phytochemical screening

The phytochemical tests were evaluated using the methods of Kar[15]and Evans[16].An ethanolic solution of each sample was prepared for the testing of flavonoids,sugars,and phenolic compounds.To test for tannins,the samples were dissolved in distilled water.The dried samples were investigated for alkaloids, triterpenoids,and steroids.

2.5.Biological activities

2.5.1.Tyrosinase inhibitory assay

The assay technique for dopachrome formation with antityrosinaseactivityfollowedthemethodsofMapunyaetal.[17]and Potduang et al.[18]with some modifications.Twelve millimoles per liter ofL-dopa were used as the substrate.Ethanolic extracts were prepared with 100,50,10,5,and 1μg/mL in triplicate. Mushroom tyrosinase enzyme was assayed as 333 unit/mL.The testing was divided into four groups A,B,C,and D using a 96-well plate.Group A included 30μL of mushroom tyrosinase enzyme and 70μL of phosphate buffer saline(PBS;pH 6.5). Group B contained 100μL of PBS.GroupC consisted of30μL of tyrosinase enzyme and 70μL of sample.Group D was composed of 30μL of PBS and 70μL of sample.After 10 min incubation, 110μL ofL-dopa was added to each group.The absorbance was measured at a wavelength of 492 nm using microplate reader (BioTek Synergy H1,USA).The percentage inhibition was calculated to evaluate the activity using positive control of kojic acid.Fifty percent of the inhibitory concentration was evaluated using probit analysis.

2.5.2.Antioxidative activities

2.5.2.1.DPPH free radical scavenging activity

TheDPPHassayfollowed themethodsofChungetal.[19]and Molyneux[20]with slight modifications.DPPH was prepared in ethanol at 75μg/mL.The ethanolic extracts were dissolved in 1%dimethyl sulfoxide(DMSO)and serial dilutions were 100, 50,10,5,and 1μg/mL in ethanol.All concentrations were performed in triplicate.The ratio of ethanolic extract and DPPH solution foreach concentration was1:1.After60 min incubation,absorbance was read by UV-vis spectrophotometer (JASCO V-630 PC,Japan)at a wavelength of 518 nm. Ascorbic acid was used as the positive control.Negative controls were ethanolic extracts without DPPH or ascorbic acid without DPPH.The scavenging activity,calculated by the following equation,was analyzed as 50%of the inhibitory concentration.

where ADPPHis the absorbance ofDPPH,Asample+DPPHis the absorbance of the sample and DPPH solution,and Asampleis the absorbance of the sample solution without DPPH solution.

2.5.2.2.Hydroxyl radical assay

The method was adapted from Awah and Verla[21].All reagents were prepared in phosphate buffer(pH 7.4)such as 10 mmol/L ferrous sulfate,10 mmol/L ethylenediaminetetraacetic acid,and 10 mmol/L 2-deoxyribose.The extracts dissolved in 1%DMSO and ascorbic acid used as the positive control,were assayedas1000,500,100,50,and10μg/mLinphosphatebuffer. Each concentration of samples and positive control were tested in triplicate.The mixture solution contained 100μL of 10 mmol/L ferrous sulfate,100μL of 10 mmol/L ethylenediaminetetraaceticacid,200μL of 10 mmol/L 2-deoxyribose,20μL of extract, 1.38mLof50mmol/L phosphate buffer,and200μLof10 mmol/ L H2O2,respectively.After incubation for 60 min at room temperature,the reaction was stopped with 1 mL of 2.8%trichloroacetic acid and 1 mL of 1%thiobarbituric acid.The mixture solutionwasthenboiledat100°Cfor15min,beforecoolinginan ice bath.Absorbance was monitored using UV-vis spectrophotometer at a wavelength of 532 nm(JASCO V-630 PC,Japan). Fifty percent of the inhibitory concentration was calculated from the percentage of scavenging activity.The positive control was ascorbic acid,and the negative control was sample without 2-deoxyribose and ascorbic acid without 2-deoxyribose.All concentrations were carried out in triplicate.The hydroxyl scavenging activity was calculated following the equation:

3.Results

3.1.Phytochemical screening

All partsofP.serratum were screened and analyzed;the results showed different chemical groupings.The roots,the twigs,and the pericarps(rind)were tested positively for alkaloids.Triterpenoids were found in petroleum ether extracted from the pericarps,the roots,the pericarps(rind),and the whole fruits.However,only the leaves gave a positive test for steroids.Positive tests for tannins were detected in the pericarps(rind),roots,twigs,and leaves.The petroleumetherextractedfromtheroots,twigs,thepericarps(rind), and leaves exhibited a positive test for flavonoids.Moreover,all parts of the plant were tested positively for sugars(Table 1).

Table 1 Phytochemical tests of ethanolic extracts from P.serratum.

where Acontrolis the 2-deoxyribose oxidation without sample extracts or ascorbic acid,Asampleis the absorbance of the sample or ascorbic acid,and Asamplewithout2-deoxyriboseis the sample without 2-deoxyribose or ascorbic acid without 2-deoxyribose.

2.6.Brine shrimp lethality

The general toxicity test was adapted from the test of Mclaughlin et al.[22]with some modifications.Artificial sea water(Jor Charoen aquarium;Thailand)was prepared at 38 g/ L in a small tank.Artemia salina L.eggs(S.K.Trading; China)were then added to the tank.After 48 h,ten brine shrimp larvae were added into each vial of plant extract solution(dissolved in 1%of DMSO)and prepared at 1000, 100,10 and 1μg/mL in triplicate.After incubation for 24 h, the brine shrimp larvae were counted and the survivors were recorded.The percentage mortality was analyzed to be LC50.

3.2.Tyrosinase inhibitory activity

The results of antityrosinase activity were classified into four groups:highly active(IC50≤15.00μg/mL),moderately active (IC50:15.01-50.00μg/mL),weaklyactive(IC50:50.01-100.00μg/ mL),and inactive(IC50＞100.00μg/mL).Roots,twigs,and leaves showed moderate activity against mushroom tyrosinase.Kojic acid,a positive control,showed high activity against tyrosinase enzyme with IC50of(12.21±0.62)μg/mL(Table 2).

3.3.Antioxidative activities

3.3.1.DPPH free radical scavenging activity

The DPPH scavenging activity of all ethanolic extracts from P.serratum was divided into four groups:highly active (IC50≤5.00μg/mL),moderately active(IC50:5.01-50.99μg/ mL),weakly active(IC50:51.00-100.00μg/mL),and inactive (IC50＞100.00μg/mL).The ethanolic extracts from leaves showed high activity.Moderate radical scavenging activity was found in roots,pericarps(rind),and twigs(Table 2).Ascorbic acid showed high activity with IC50of(3.00±0.14)μg/mL, however,the pulp fruits and essential oil from the fruits were inactive.

Table 2 IC50values(μg/mL)and classified activities of ethanolic extracts from parts of P.serratum.

3.3.2.Hydroxyl radical activity

The detection of hydroxyl radical scavenging activity was classified into four groups:highly active(IC50≤50.00μg/mL), moderately active(IC50:50.01-100.99μg/mL),weakly active (IC50:101.00-500.00μg/mL),and inactive(IC50＞500.00μg/ mL).Ethanolic extracts from all parts of P.serratum showed weak activity against hydroxyl radicals with IC50of (113.48±0.67)-(350.37±0.50)μg/mL.However,ascorbic acid exhibited moderate activity(Table 2).

3.4.Brine shrimp lethality

Thelevel oftoxicityagainst brine shrimp was classified asfour groups:highly toxic(LC50＜10.00μg/mL),moderately toxic (LC50:10.00-100.00μg/mL),weakly toxic(LC50:100.00-1000.00μg/mL),andinactive(LC50＞1000.00μg/mL).Onlythe essential oil exhibited high toxicity with LC50of(3.57±1.82)μg/ mL.Roots,petroleum ether extract from the pericarps,and twigs showed moderate toxicity,with LC50of(14.14 ± 1.76) -(27.10±2.39)μg/mL.Wholefruitsandleaveswereweaklytoxic (Table 2).Nevertheless,the pulp fruits,pericarps(rind),and seeds were not determined.

4.Discussion

All parts of P.serratum are utilized for fruit,food,and traditionalmedicine.Secondarymetabolitecompoundsinthisplantare important for new drug discovery processes.Preliminary phytochemical screening can determine the biological activities;and results showed that each part of this plant contained different chemical compounds including flavonoids,alkaloids,triterpenoids,steroids,tannins,and sugars.The roots,twigs and pericarps (rind)all contained alkaloids which have been used as anticancer, antimalaria,anti-tussive,analgesic,and antispasmodic agents[23]. Condensedtanninswerefoundinthepericarps(rind),roots,fruits, twigs,andleaves.Tanninsarepolyphenoliccompoundswhichcan be divided into two groups including true and pseudo tannins [23,24].True tannins comprise condensed and hydrolyzable tannins.In terms of biological activities,tannins are used as antitussive,astringent,antitumor,and antioxidative agents[23,24]. Flavonoids,comprising benzo-γ-pyrone,were found in petroleum ether of pericarps,roots,twigs,the pericarps(rind),and leaves.It was found that flavonoids have been reported to have biologicalactivitiessuch asantioxidative,antiulcer,antiinflammatory,and anti-viral agents[23,25].Deoxy sugars with chemical structures of flavonoids,triterpenoids,and alkaloids were detected in all parts of the plant[24].Steroids were mainly found in the leaf.Previous research indicated that the biological activities of steroids were anti-inflammatory,hormonal,and antifungal[24].Triterpenoids were presented in P.serratum in the pericarps,whole fruits,and petroleum ether extract from pericarps.Triterpenoids exhibit biological activities such as antiseptic,anthelmintic,and antimicrobial agents.Coumarin and steroids(scopoletin,β-sitostenone,andβ-amyrin)were isolated from the stem barks of P.serratum[26].

Skin color has an influence on both males and females,especially irregular dark skin.There are many causes of hyperpigmentation,suchasinflammationandUV[27,28].Thecausesofdark pigmentation are associated with abnormal melanin synthesis after acneandskininflammation.Inthegeneralmelaninprocess,L-dopa is an intermediate substance which is changed into dopaquinone and dopachrome by the tyrosinase enzyme[29].Thus,this research may discover a new natural product to decrease hyperpigmentation,using tyrosinase inhibition.The tyrosinase inhibitory activities of the roots,twigs,and leaves extract from P.serratum were found to be weak compared with kojic acid. However,the ethanolic root extract showed the highest activity against the tyrosinase enzyme compared with other parts.Results indicated that tyrosinase inhibition of ethanolic extract from P.serratum roots and twigs could be associated with their chemical substances in phytochemical screening.

Oxidative stress relates to reactive oxygen species and other species which lead to macromolecular damages,and cardiovascular complaints,Alzheimer,and cancer as a result[30]. Antioxidants are agents that protect the human body[31].DPPH assay for antioxidative agent screening is widely used because it is simple and rapid.The antioxidant gives the electron or proton to a stable nitrogen in DPPH radical.The method indicates ability of antioxidative agents[32].The antioxidative activities of P.serratum extracts could result in the formation of new compounds to decrease free radical-induced oxidative stress. Fourninthsoftheplantextractsexhibitedinactivity against DPPHradicals.The ethanolic extract from leaves showed the highest activity against DPPH radicals,as compared with the other parts. However,the leaves extract was weak,as compared to ascorbic acid,a positive control.Previous study in India examining fruit and leaves extracts from P.serratum exhibited DPPH radical scavenging activity with IC50of 29 and 52μg/mL,respectively [33].Reports of other species also showed that the methanolic extract from Protium neglectum leaves was composed of polyphenol,flavonoid,and tannin compounds.The extract exhibited 85.44%against DPPH radicals[34].

Moreover,Fenton's reaction which is caused by hydroxyl radicals related to biological activities such as anti-inflammatory,antiviral,and anti-arthritic activities[12].In this study,the essential oil from fruit exhibited the highest activity against the hydroxyl radical.However,the scavenging ability of the hydroxyl radical of the essential oil from the fruit was weak,as compared to ascorbic acid.Previous research demonstrated that methanolic extracts of fruits and leaves from P.serratum exhibited hydroxyl radical scavenging activity with IC50of 920 and 3460μg/mL, respectively[33].Other species have also shown hydroxyl radical scavenging activity.The ether extract from Protium kleinii orαamyrin pentacyclic triterpene can inhibit 12-O-tetradecanoylphorbol-13-acetate which induced edema in mice ears[35]. Moreover,α-amyrin andβ-amyrin in resin from Protium heptaphyllum at a dose of 400 mg/kg can decrease vascular permeability induced by acetic acid in mice[36].These results demonstrated that effect of hydroxyl radicals on the human body was higher than that of the DPPH radicals.Therefore,scavenging activity of hydroxyl radical of this plant may predict antioxidative activity in in vivo,as compared with DPPH radicals.

The brine shrimp lethality assay was tested to predict the preliminary toxicity[23].Results revealed that essential oil from the fruit has the highest toxicity.The other extracts showed various toxicities ranging from moderate to weak and inactive. Previous research indicated that the methanolic extract from P.serratum leaves,and the dichloromethane extract from P.serratum stem barks showed toxicity to brine shrimp with LC50of 22.91 and 9.64μg/mL,respectively[37].This level of toxicity suggested that some parts of the plant should be further studied for antitumor activity.

The chemical components of all parts of P.serratum consisted of flavonoids,condensed tannins,alkaloids,triterpenoids, steroids,and deoxy sugars.The results suggested that all parts of the plant showed different biological activities.The biological activities determined in this experiment may suggest the usage of P.serratum roots in traditional medicine.Additionally,the leaves,twigs and roots of P.serratum are interesting for the isolation of bioactive compounds,and therefore worth further investigation.

Conflict of interest statement

I declare that I have no conflict of interest.

Acknowledgments

I am grateful to School of Pharmaceutical Sciences,University of Phayao for facilities in this research.I wish to thank Napaporn Aeamla-Or who assisted in the proofreading of the manuscript.

[1]Kong LY,Tan RX.Artemisinin,a miracle of traditional Chinese medicine.Nat Prod Rep 2015;32:1617-21.

[2]Bindu S,Rameshkumar KB,Kumar B,Singh A,Anilkumar C. Distribution of reserpine in Rauvolfia species from India-HPTLC and LC-MS studies.Ind Crops Prod 2014;62:430-6.

[3]Guerra-Bubb J,Croteau R,Williams RM.The early stages of taxol biosynthesis:an interim report on the synthesis and identification of early pathway metabolites.Nat Prod Rep 2012;29: 683-96.

[4]Office of Natural Resource and Environmental Policy and Planning.Thailand:national report on the implementation of convention on biological diversity.Bangkok:Office of Natural Resource and Environmental Policy and Planning;2009.[Online]Available from:www.cbd.int/doc/world/th/th-nr-04-en.pdf[Accessed on 10th February,2016].

[5]Daly DCB,Fine PVA,Martinez-Habibe MC.Burseraceae:a model for studying the Amazon flora.Rodriguesia 2012;63(1): 21-30.

[6]Zofou D,Tematio EL,Ntie-Kang F,Tene M,Ngemenya MN, Tane P,et al.New antimalarial hits from Dacryodes edulis(Burseraceae)-part I:isolation,in vitro activity,in silico“drug-likeness”and pharmacokinetic profiles.PLoS One 2013;8:e79544.

[7]Xu J,Guo Y,Zhao P,Guo P,Ma Y,Xie C,et al.Four new sesquiterpenes from Commiphora myrrha and their neuroprotective effects.Fitoterapia 2012;83:801-5.

[8]Wang YG,Ma QG,Tian J,Ren J,Wang AG,Ji TF,et al.Hepatoprotective triterpenes from the gum resin of Boswellia carterii. Fitoterapia 2016;109:266-73.

[9]Yang JL,Shi YP.Cyclartane-type triterpenoids and sesquiterpenoids from the resinous exudates of Commiphora opobalsamum. Phytochemistry 2012;76:124-32.

[10]Suleiman MHA.Prenylted flavonoids from the stem wood of Commiphora opobalsamum(L.)Engl.(Burseraceae).J King Saud Univ Sci 2015;27:71-5.

[11]LipipunV,KurokawaM,SuttisriR,TaweechotipatrP,PramyathinP, Hattori M,et al.Efficacy of Thai medicinal plant extracts against herpes simplex virus type 1 infection in vitro and in vivo.Antiviral Res 2003;60:175-80.

[12]SayeedMA,RashidMM,KabirMF,AlamR,IslamMS,DharR,etal. Invitroanti-arthritic andthrombolyticactivitiesofmethanolic extract of Protium serratum leaves.J Med Plants Res 2014;8(16):615-8.

[13]Pooma R.A preliminary account of Burseraceae in Thailand.Thai For Bull Bot 1999;27:53-82.

[14]Gardner S,Sidisunthorn P,Anusarnsunthorn V.A field guide to forest trees of Northern Thailand.Bangkok:Kobfai Publishing Project;2007.

[15]Kar A.Pharmacognosy and pharmacobiotechnology.2nd ed.New Delhi:New Age International(P)Ltd.;2007.

[16]Evans WC.Trease and Evans'pharmacognosy.16th ed.New York:Elsevier Limited;2009.

[17]Mapunya MB,Hussein AA,Rodriguez B,Lall N.Tyrosinase activity of Greyia flanaganii(Bolus)constituents.Phytomedicine 2011;18:1006-12.

[18]PotduangB,ChongsiriroegC,BenmartY,GiwanonR,SupatanakulW, Tanpanich S.Biologicalactivitiesof Scheffleraleucantha.Afr J Tradit Complement Altern Med 2006;4:157-64.

[19]Chung SK,Osawa T,Kawakishi S.Hydroxy radical-scavenging effects of spices and scavengers from brown mustard(Brassica nigra).Biosci Biotech Biochem 1997;61:188-223.

[20]Molyneux P.The use of the stable free radical diphenylpicrylhydrazyl(DPPH)for estimating antioxidant activity.Songklanakarin J Sci Technol 2004;26:211-9.

[21]Awah FM,Verla AW.Antioxidant activity,nitric oxide scanvenging activity and phenolic contents of Ocinum gratissinum leaf extract.J Med Plants Res 2010;4:2479-87.

[22]Mclaughlin JL,Roger LL,Anderson JE.The use of biological assays to evaluate botanicals.Drug Inf J 1998;32:513-24.

[23]Heinrich M,Barnes J,Gibbons S,Williamson E.Fundamentals of pharmacognosy and phytotherapy.2nd ed.London:Elsevier;2012.

[24]Okuda T,Ito H.Tannins of constant structure in medicinal and food plants-hydrolyzable tannins and polyphenols related to tannins.Molecules 2011;16:2191-217.

[25]Kumar S,Pandey AK.Chemistry and biological activities of flavonoids:an overview.ScientificWorldJournal 2013;2013:162750.

[26]Ara K,Rahman MS,Rahman AHM,Hasan CM,Rashaid MA. Terpenoids and coumarin from Bursera serrata Wall.Dhaka Univ J Pharm Sci 2009;8:107-10.

[27]Gendreau I,Angers L,Jean J,Pouliot R.Pigmented skin models: understand the mechanisms of melanocytes.In:Andrades JA,editor. Regenerative medicine and tissue engineering.Rijeka:InTech;2013.

[28]InoueY,HasegawaS,YamadaT,DateY,MizutaniH,NakataS,etal. Analysis of the effects of hydroquinone and arbutin on the differentiation of melanocytes.Biol Pharm Bull 2013;36(11):1722-30.

[29]Chang TS.An updated review of tyrosinase inhibitors.Int J Mol Sci 2009;10:2440-75.

[30]Rahman T,Hosen I,Islam MMT,Shekhar HU.Oxidative stress and human health.Adv Biosci Biotechnol 2012;3:997-1019.

[31]BirbenE,SahinerUM,SackesenC,ErzurumS,KalayciO.Oxidative stress and antioxidant defense.World Allergy Organ J 2012;5:9-19.

[32]Prior RL,Wu X,Schaich K.Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements.J Agric Food Chem 2005;53:4290-302.

[33]Prakash D,Upadhyay G,Gupta C.Total phenol and antioxidant activity of some fruits and their under-utilized parts.Int Food Res J 2013;20:1717-24.

[34]Padilla FC,Rincon M,Rached LB,Suarez A.Protium neglectum, Podocalyx loranthoides,and Brosimum utile source of flavonoids and other phenolic compounds with antioxidant activity.Rev Fac Farm 2008;71:8-14.

[35]Otuki MF,Vieira-Lima F,Malheiros A,Yunes RA,Calixto JB. Topical antiinflammatory effects of the ether extract from Protium kleinii andαalpha-amyrin pentacyclic triterpene.Eur J Pharmacol 2005;507:253-9.

[36]OliveiraFA,Vieira-JúniorGM,ChavesMH,AlmeidaFR, Florˆencio MG,Lima RC Jr,et al.Gastroprotective and antiinflammatory effects of resin from Protium heptaphyllum in mice and rats.Pharmacol Res 2004;49:105-11.

[37]Islam MR,Kabir MF,Alam R,Dhar R,Rana MN,Islam ME,et al. Sedative,membrane stability,cytotoxic and antioxidant properties of methanol extract of leaves of Protium serratum Wall.Asian Pac J Trop Dis 2014;4:S928-33.

Original article http://dx.doi.org/10.1016/j.apjtb.2016.10.001