Phytochemical Screening, In vitro Antifungal and Antioxidant Activity of Essential Oil from roots of Rheum webbianum Royle from Himalayan Region

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1 ORIGINAL ARTICLE Phytochemicl Screening, In vitro Antifungl nd Antioxidnt Activity of Essentil Oil from roots of Rheum webbinum Royle from Himlyn Region Snjy Kumr 1, Mohmmd S. Jved 1, Schin Gupt 2, Rishendr Kumr 3 1 Deprtment of Plnt Pthology, Kumun University, D.S.B.Cmpus, Ninitl , Uttrkhnd, Indi, 2 Deprtment of Pthology, Sher-e-Kshmir University of Agriculturl Sciences nd Technology, Jmmu , Jmmu nd Kshmir, Indi, 3 Deprtment of Biotechnology, Kumun University, Ninitl , Uttrkhnd, Indi Abstrct Aim: The im of our study is to evlute the phytogenic chemicl compounds nd ssess their ntifungl nd ntioxidnt ctivity of essentil oil of Rheum webbinum Royle growing in greter Himlyn region. Mterils nd Methods: In the present study, the phytochemicl constituents of essentil oil were isolted by stem distilltion nd screened by gs chromtogrphy (GC) nd GC-mss spectrometry nlysis in reltion with their Kovts indices from R. webbinum is rich in oxygented monoterpenoids nd sesquiterpenoids. The essentil oil ws further evluted for their ntifungl ctivity by well diffusion method nd ntioxidnt ctivity by 1, 1-diphenyl- 2-picrylhydrzyl scvenging (DPPH) scvenging ssy t vrious concentrtions. Results: The mjor chemicl constituent s were undecnone (19.72%), mliol (13.03%), 4-vinyl guicol (10.40%), 2E-undecnl (7.81%), vlerinol (6.87%), viridiflorol (4.7%), euclyptol (2.87%), nd eugenol (2.98%) s the mjor constituents. The oil from the eril prts of R. webbinum hs shown moderte ntifungl nd ctivity with 100% myceli growth inhibition ginst Alternri lternt t concentrtion of 2400 µg/ml nd Bipolris mydis nd Rhizoctoni solni t concentrtion of 2800 µg/ml. However, Fusrium oxysporum ws found less susceptible for this oil. The IC 50 showed rnge from µg/ml to µg/ml s compred with stndrd fungicides with IC 50 vlues rnging from 37.8 µg/ml to 88.6 µg/ml. The free rdicl scvenging ctivity of R. webbinum oil employed by in vitro ssy methods like DPPH t concentrtion of 600 µg/ml ws 83%, respectively. Conclusion: Our study showed tht mliol followed by ρ-vinylguicol s the mjor components in this oil which ws bsent in previous findings. The essentil oil hd potent ntifungl nd ntioxidnt ctivity, respectively. Key words: Antifungl, ntioxidnt, essentil oil, free rdicl, phytochemicls, Rheum webbinum INTRODUCTION The vlley Jmmu nd Kshmir is regrded s hub for the medicinl plnts. The people there were using these medicinl plnts for their cure nd preventing vrious diseses since ncient times. There re bout totl of 937 plnt species belonging to 129 fmilies hve been reported from Jmmu nd Kshmir hving trditionl medicinl uses. [1] The genus Rheum belonging to fmily Polygoncee represented by 48 gener nd 1200 species with 12 gener in Indi distributed in Himlyn region between 2800 nd 3800 m. The genus Rheum contins 50 species, of which 12 re present in Indi. [2] The Rheum webbinum is perennil herbceous plnt which cquired the sttus of endngered ctegory. It is used for fever, cough, nd dirrhe, nd menstrul nd liver disorders. [3] It is lso used to cure inflmmtory diseses nd oxidtive stress relted to injuries. [4] Rheum emodi hs constituents such s nthrquinone nd stilbene which confer nticncer, nti-inflmmtory, ntimicrobil, ntiulcer, nd heptoprotective ctivities. [5] The methnolic nd queous extrct of R. emodi possessed ntimicrobil Address for correspondence: Mohmmd S. Jved, Deprtment of Chemistry, Kumun University, D.S.B.Cmpus, Ninitl , Uttrkhnd, Indi. E-mil: msuhil12003@yhoo.co.in Received: Revised: Accepted: Interntionl Journl of Green Phrmcy LPU Conference Apr-Jun 2018 Specil Issue 95

2 ctivity ginst bcteri Pseudomons eruginos nd Bcillus megterium nd fungi Fusrium solni nd Aspergillus flvous with zone of inhibition rnging between 0.9 nd 1.8 cm. [6] Methnolic nd queous extrcts of Rheum ribes Linn. Leves possess nti-ulcer ctivity using pylorus ligtion method t concentrtion 200 mg/kg produces protective effect on ulcer-induced models. [7] The root extrcts of R. emodi exhibit ntifungl ctivity ginst Aspergillus fumigtus, Cndid lbicns, Cryptococcus neoformns, nd Trichophyton mentgrophytes (MIC = µg/ml). [8] Root extrct of R. rhbrbum hs ntibcteril, ntivirl, heptoprotective, nti-ulcer, [9] nd loe emodin of root extrct hs ntiinflmmtory responses. [10] Rhein possesses ntibcteril ctivity ginst Escherichi coli K12 with MIC vlue of 128 µg/ml. [11] Anthrquinones C-glycosides occur in the form of 10-hydroxy-cscroside C, 10-hydroxy-cscroside D, 10R-chrysloin-1-O-β-D-glycoside, cscroside C, cscroside D, nd cssiloin. Other compounds extrcted ws rutin, rheinl, rhein-11-o-β-d-glucopyrnoside, torchrysone- 8-β-D-glucoside, epictechin, nd uronols (corpusin nd mesopsin). [12] The root extrcts of R. ribes contin loe emodin, chrysophnol, loe emodin-8-β-o-glucoside, sennoside A, nd rhponticin hs ntimicrobil properties, wheres shoots of R. ribes contin quercetin, 5-deoxy-quercetin, quercetin- 3-O-rhmnoside, quercetin-3-o-glctoside, uercetin-3- O-rutinoside, nd quercetin-3-o-rutinoside. [13] Sulfemodin- 8-O-β-D-glucoside, revndchinone-1, revndchinone-2, revndchinone-3, revndchinone-4, 6-methyl-rhein, nd 6-methyl-loe-emodin. [14] The chloroform nd methnolic extrcts from the roots nd stems of R. ribes hve ntioxidnt ctivity, in 1,1-diphenyl-2-picrylhydrzyl scvenging (DPPH) scvenging, superoxide nion rdicl scvenging, ferric reducing power, nd cupric reducing power. [15] Our present work reveled with phytochemicl investigtion nd their bioctivity of R. webbinum plnt. Plnt mteril MATERIAL AND METHODS The fresh plnt mteril ws collected from high ltitudes of Bhless (Dod), Jmmu nd Kshmir (Indi) t n elevted ltitude of 3000 m in the month of August The roots were wshed with cold wter nd their ded skin ws skimmed off nd ws used for the extrction of oil. The preliminry plnt identifiction ws done by Prof. P. C. Pndey, Deprtment of Botny, Kumun University, Ninitl. The plnt ws further confirmed by Botnicl Survey of Indi, Dehrdun, Voucher specimen R. webbinum Royle, Acc. No where herbrium of plnt specimens hs been deposited. glss condenser. The distillte ws sturted with NCl nd extrcted with hexne. The hexne extrct is dried with sodium sulfte (N 2 SO 4 ) nd the solvent removed with Rotovp t moderte pressure nd 38 C temperture nd stored t 4 C for further nlysis. All chemicl nd regents of nlyticl grde nd were obtined from Merk, Mumbi, Indi. Chemicl nd regents All chemicls nd regents used were of nlyticl grde. Hexne, nhydrous N 2 SO 4, dimethyl sulfoxide (DMSO), ether, ethnol, nd sodium hypochlorite were obtined from Merk, Mumbi, Indi, wheres potto dextrose gr (PDA), potto dextrose broth (PDB), 2,2-diphenyl-1-picrylhydrzyl (DPPH), scorbic cid, α-tocopherol, nd dextrose (D-glucose) were obtined from HiMedi Pvt., Ltd., Mumbi Indi Anlysis of essentil oil by gs chromtogrphy (GC) nd GC-mss spectrometry (MS) nlysis The nlysis of the oil ws done using gs chromtogrph (Shimdzu GC QP-2010) equipped with RTx-5 MS cpillry column, (30.0 m 0.25 mm, film thickness: 0.25 µm). The oven temperture (50 C 280 C) ws progrmmed t 50 C for first 2 min, then 3 C/min to 200 C, nd then 10 C/min to 280 C, fter which it ws mintined isothermlly t 280 C for 8 min. N 2 ws used s the crrier gs t ml/min. The injector temperture ws 250 C, detector temperture 260 C, nd the injection volume 0.5 µl using 10% solution of the oil in n-hexne. The GC-MS nlysis ws crried out with GC-MS QP 2010 (Shimdzu) fitted with RTx-5 MS cpillry column, (30.0 m 0.25 mm, film thickness: 0.25 µm). The oven temperture (50 C 280 C) ws progrmmed t 50 C for first 2 min, then 3 C/min to 200 C, nd then 10 C/min to 280 C. After which, it ws mintined isothermlly t 280 C for 8 min. N 2 ws used s the crrier gs. The injection volume 0.5 µl nd split rtio were 1:90. The mss spectr were tken t 70eV. The percentge by pek re normliztion ws tken to express the reltive percentge of the oil constituents. Identifiction of compounds Identifiction of different chemicl constituents of the essentil oil ws done by compring their Retention indices/ Kovts indices in reltion to series of n-lknes (C 6 -C 33 ) indices on the RTx-5 MS cpillry column, either with those of published dt [16] or with uthentic smples which were further supported by NIST nd WILEY mss spectrl librry serches. The results re presented in Tble 1. Isoltion of essentil oil The essentil oil ws obtined by stem distilltion of fresh plnt mteril (8 kg roots) using copper still fitted with spirl Plnt pthogenic fungi nd their culture The folige born fungi were obtined Division of Plnt of Pthology, FOA, Chth, Sher-e-Kshmir University of Interntionl Journl of Green Phrmcy LPU Conference Apr-Jun 2018 Specil Issue 96

3 Tble 1: Phytochemicl composition of roots essentil oil from R. webbinum Compound % in the oil Method of identifiction R.I. Others p cymene b, c, d limonene b, c, d 1,8 cineole c, d Z β ocimene b, c, d β epoxystyrene c, d undecnone b, c, d, linlool b, c, d n nonenl c, d cis thujone c, d terpinen 4 ol b, c, d α terpineol c, d linlool cette c, d 2 decnl c, d 4 ethylguicol c, d bornyl cette b, c, d ρ vinyl guicol c, d eugenol b, c, d 2E undecenl b, c, d α copene c, d E cryophyllene b, c, d trns β bergmotene c, d 9 epi E cryophyllene b, c, d β ptchoulene c, d α bulnesene c, d β selinene c, d kessne c, d mliol c, d cryophyllene oxide b, c, d Diethyl phthlte c, d viridiflorol b, c, d β eudesmol b, c, d vlerinol c, d longifolol b, c, d neocinidilide c, d Z nuciferol c, d isofenchol c, d phytone c, d isobutyl phthlte c, d elol c, d eicosne c, d n henicosne c, d n tetrcosne c, d Compound Agriculturl Sciences & Technology, Jmmu (SKUAST-J). The pure culture of fungl nd species ws mintined in PDA nd stored below 4 C. The pthogenic fungi, nmely Bipolris mydis, Rhizoctoin solni, Alternri lternt, nd Fusrium oxysporum, were cultured on PDA medium in sterilized Petri dishes. In vitro ntifungl ssy % in the oil Sesquiterpenoids 9.56 Oxygented sesquiterpenoids Monoterpenoids 3.67 Oxygented monoterpenoids Totl identified Yield Tble 1: (Contd) 6.6 g (4.05% by weight) Poisoned food technique [17] using PDA s medium ws used to check the ntifungl growth of oil ginst test fungi. The different concentrtion of essentil oil ws prepred by dissolving pproprite mount of oil in 10% DMSO nd double-distilled wter into 20 ml of PDA to obtin the desired concentrtion. [18] Mycelil plugs from edges of ech culture were plced in the center of ech PDA plte. The prepred pltes were inoculted septiclly with ssy discs test fungus ws inoculted t 25 ± 2 C for 3 8 dys until growth reched t periphery of the plte. Growth inhibition of ech fungl strin ws clculted s percentge inhibition of rdil growth reltive to control using the formul s C-T % myceli inhibition = 100 C Where C is the concentrtion of control plte nd T is the rdil growth of test plte. The pltes were used in triplicte for ech tretment. IC 50 vlues were grphiclly obtined from dosge response curves bsed on mesurement t different concentrtions. Determintion of minimum inhibitory concentrtion (MIC) Method of identifiction R.I. Others Retention index (RI) reltive to homologous series of n lkne (C 6 C 32 ) on RTx 5MS Cpillry Column. b Compound checked by uthentic stndrds compounds. c Retention index (RI). d MS, NIST08.LIB, nd WILEY8.LIB librries spectr nd the literture. R. webbinum: Rheum webbinum The MIC of oil ws determined by gr diffusion method. [19] The oil ws dissolved on 10% DMSO. A 10 µl of Interntionl Journl of Green Phrmcy LPU Conference Apr-Jun 2018 Specil Issue 97

4 spore suspension of ech fungl strin ws inoculted in test tube in PDB medium nd inoculted for 3 8 dys. The control tubes contining PDB medium were inoculted only with fungl suspension. The MIC is the minimum concentrtion of oil in µg/ml where no visible growth is observed. In vitro ntioxidnt ctivity The DPPH ssy ws done ccording to the method described by Brnd-Willims et l [20] The DPPH g ws dissolved in ethnol nd mde up to 100 ml with doubledistilled wter. The ethnol (20%) 20 ml nd 80 ml doubledistilled wter ws prepred. The 100 µm DPPH (50 µl) ws dded to equl volume of 20% ethnol to generte 400 µl DPPH. The oil smples of different concentrtions were tken in different test tubes nd dded DPPH 400 µl nd mke volume up to 100 µl with double-distilled wter. Then, it ws shken vigorously nd tken in the drk for 20 min t room temperture. The reduction in bsorbnce ws recorded t 520 nm in ultrviolet (UV)-visible spectrometer. Ascorbic cid nd α-tocopherol ws used s stndrd nd controlled bsorbnce of DPPH ws tken without dding oil smple nd ll the ssys ws crried out in triplicte. Scvenging effect (%) of free rdicl DPPH ws clculted s: Scvenging effect %ge = Absorbnce of control-absorbnce of oil smple 100 Absorbnce of control IC 50 ssy ws clculted grphiclly using curve by plotting ntioxidnt cpcity or percentge inhibition versus corresponding smple concentrtions. RESULTS AND DISCUSSION Figure 1: Effect of essentil oil on myceli growth of test fungi t different concentrtions. (B. m. - Bipolris mydis, R. s. - Rhizoctoni solni, A.. - Alternri lternte, nd F. o. - Fusrium oxysporum The stem distilltion of fresh plnt mteril (8 kg) gives 6.5 g of oil yield with drk yellow in color nd unplesnt smell. This oil ws dominted by oxygented sesquiterpenoids nd oxygented monoterpenoids wheres monoterpenoids nd sesquiterpenoids constituted s minor components. Among oxygented sesquiterpenoids mliol (13.03%), ρ-vinylguicol (10.40%), 2E-undecnl (7.81%), vlerinol (6.87%), viridiflorol (4.7%), Z-nuciferol (4.21%), nd β-eudesmol (2.73%) were the mjor compounds wheres Tble 2: % myceli growth inhibition by essentil oil from roots of R. webbinum Pthogenic fungi (µg/ml) B. mydis 00.00± ± ± ± ± ±0.00 R. solni 03.05± ± ± ± ± ±0.00 A. lternt 08.22± ± ± ± ± ±0.00 F. oxysporum 03.34± ± ± ± ± ±0.80 vlues within column re given s men SD of three experiments. R. webbinum: Rheum webbinum. B. mydis: Bipolris mydis, R. solni: Rhizoctoni solni, A. lternte: Alternri lternt, F. oxysporum: Fusrium oxysporum Tble 3: IC50 nd MIC vlues of essentil oil nd fungicides (positive control) ginst test pthogens Pthogenic fungi Essentil oil Amphotericin Fungicide (positive control) Clotrimzole IC 50 MIC b IC 50 MIC b IC 50 MIC b B. mydis NA 600 NA 1200 R. solni NA 42.2 NA A. lternt NA NA F. oxysporum NA NA 600 NA: Not ppered. Concentrtion tht produces 50% inhibitory effect on myceli growth. b Minimum inhibitory concentrtion (g/ml). B. mydis: Bipolris mydis, R. solni: Rhizoctoni solni, A. lternte: Alternri lternt, F. oxysporum: Fusrium oxysporum Interntionl Journl of Green Phrmcy LPU Conference Apr-Jun 2018 Specil Issue 98

5 b undecnone (19.72%), 1,8-cineole (1.83%), terpinen- 4-ol (1.57%), nd α-terpineol (1.23%) were mjor mong oxygented monoterpenoids [Tble 1]. The mliol nd ρ-vinylguicol were the mjor constituents which were bsent in previous findings. This chnge in composition is due to environmentl dpttion, climtic conditions, nd ltitude fctor of the plnts. c Figure 2: (-c) Effect of different concentrtions of essentil oil nd controls on 1, 1-diphenyl-2-picrylhydrzyl scvenging ssy The oil inhibits the growth of myceli strins in dosedependent mnner. The essentil oil show vried effect t different concentrtions. The oil ws found to be effective for ll the pthogenic test fungi. The inhibitory effect of oil vries from 8.71% to %. The oil of R. webbinum completely inhibits the myceli growth of A. lternt t 2400 µg/ml nd B. mydis nd R. solni t 2800µg/ml wheres F. oxysporum is to be less susceptible for this oil [Tble 2 nd Figure 2]. The essentil oil of this plnt is endowed with ntibcteril, ntivirl, ntiulcer, nd ntiinflmmtory gent. The nturl product cts s potent biofungicides nd ntibcteril gents. The reports on ntimicrobil ctivity of R. emodi ginst Bcillus subtilis nd P. eruginos showed potent ZOI rnges from 0.7 to 1.4 cm, [8] nd rhizome extrct hs ntifungl ctivity ginst Aspergillus niger nd C. lbicns. [21] The investigtions on ntimicrobil ctivity of different plnt extrct ginst plnt pthogens hve been performed worldwide, [22,23] hence our result hve importnt becuse it provides informtion bout the essentil oil. Our work deled with ntifungl ctivity of essentil oil from roots of R. webbinum. The oil hd shown potent effect ginst A. lternt, R. solni, nd Bipolris mydis t concentrtions of 2400 µg/ml nd 2800 µg/ ml, respectively [Tble 2 nd Figure 1]. The result of our studied reserch work showed tht oil is sfe potent nd effective biofungicide. Tble 4: Absorbnce shown by the essentil oil nd control (Ascorbic cid nd Vitmin E) t different concentrtions Concentrtion (µg/ml) b Figure 3: ( nd b) %ge inhibition of 1, 1-diphenyl-2- picrylhydrzyl scvenging ssy shown by Pie-chrt nd Histogrm t vrious concentrtions. (%ge inhibition= essentil oil, control 1= scorbic cid, control 2= α-tocopherol) Essentil oil Absorbnce Ascorbic cid α tocopherol 100 µg/ml 0.201± ± ± µg/ml 0.437± ± ± µg/ml 0.563± ± ± µg/ml 0.736± ± ± µg/ml 1.193± ± ± µg/ml 1.536± ± ±0.042 Vlues in the column re men of three bsorbnce results±sd This study ffirms tht in vitro ntioxidnt ctivity of essentil oil from roots of R. webbinum ws comprble to Interntionl Journl of Green Phrmcy LPU Conference Apr-Jun 2018 Specil Issue 99

6 Conc. (µg/ml) Tble 5: Percentge scvenging ctivity of different concentrtion of essentil oil nd control %ge of DPPH scvenged by Essentil oil %ge of DPPH scvenged by Ascorbic cid %ge of DPPH scvenged by α Tocopherol DPPH: 1, 1 diphenyl 2 picrylhydrzyl scvenging those of stndrd scorbic cid nd Vitmin E [Tble 5]. [15,24] Higher bsorbnce indicted higher reducing power. The present study indicted tht the oil t concentrtion of 600 µg/ml could ply n importnt role in the mngement of oxidtive stress. Thus, it ws considered tht the essentil oil hd ntioxidnt ctivity ginst DPPH rdicl [Tbles 3, 4 nd Figure 3]. Sttisticl nlysis For ll the tests, the men vlues nd stndrd devitions were clculted nd dt were nlyzed using SPSS 16.0 sttisticl softwre. The one-wy nlysis of vrince ws pplied for clculting the results. The mens ws compred by Duncn tests t level of significnce of P CONCLUSIONS This study showed tht mlliol followed by ρ-vinyl-guicol s the mjor components in this oil which ws bsent in previous findings. The essentil oil from the roots hs been responsible for its ntifungl ctivity. However, the synergic effect is found to be responsible for its bioctivity. As result of its ntifungl ctivity, it cn be used s biofungicides nd which is more sfe nd eco-friendly s compred with synthetic chemicl fungicides. This oil hs therpeutic potentil s nturl ntioxidnt in reducing free rdicl scvenging ction. ACKNOWLEDGMENTS We re highly thnkful to Prof. P. C. Pndey, Kumun University, Ninitl nd Kumr Ambrish, Botnicl Survey of Indi, Dehrdun, Indi, for plnt identifiction. We re lso thnkful to Prof. A. B. Melkni, HOD, Chemistry, nd Prof. S. P. S. Meht, D. S. B. Cmpus, KU, Ninitl, nd Dr. Schin Gupt, Sher-e-Kshmir University of Agriculturl sciences nd Technology, Jmmu for providing necessry fcilities. Furthermore, we re thnkful to Dr. Rishendr Kumr, Deprtment of Biotechnology, Bhimtl Cmpus, Ninitl (Indi), for their vluble necessrily guidnce. REFERENCES 1. Girol S, Shrm J, Bedi YS. Across-culturl nlysis of Jmmu, Kshmir nd Ldkh (Indi) medicinl plnts use. J Ethnophrmcol 2014;155: Cristenhuz MJ, Dyng JN. The number of known plnt species in world nd its nnul increse. Phytotx 2016;261: Rehmn H, Begum W, Anjum F, Tbsum H. Rheum emodi (Rhubrb): A fscinting herb. J Phrmcogn Phytochem 2014;3: Chi YY, Wng F, Li YL, Liu K, Xu H. Antioxidnt Activities of stilbenoids from Rheum emodi Wll. Evid Bsed Complement Altern Med 2012;2012: Nzir S, Shrm S, Sxen M, Abrr M, Ajz M. Rheum emodi: Phytochemistry, bioctive compound nd their biologicl ctivity. Int J Phytophrmocol 2013;4: Ahmed T, Slm MD. Antimicrobil ctivity of methnolic extrct nd queous extrct of Rheum emodi. Int J Phrm Sci Rev Res 2015;30: Sindhu RK, Kumr P, Kumr J, Kumr A, Aror S. Investigtion into the nti-ulcer ctivity of Rheum emodi Linn. Leves extrcts. Int J Phrm Phrm Sci 2010;2: Agrwl SK, Singh SS, Verm S, Kumr S. Antifungl ctivity of nthroquinone derivtives from Rheum emodi. J Ethnophrmcol 2000;72: Prk MY, Kwon HJ, Sung MK. Evlution of loin nd loe-emodin s nti-inflmmtory gents by using murine mcrophges. Biosci Biotechnol Biochem 2009;73: Hu B, Zng H, Meng X, Wng F, Wng P. Aleo-emodin from Rheum rhbrbrum inhibits lippolyscchrideinduced inflmmtory responses in RAW264.7 mcrophges. J Ethn Phrmcol 2014;155: Htno T, Uebyshi H, Ito H, Shiot S, Tsuchiy T, Yoshid T. Phenolic constituents of cssi seeds nd ntibcteril effect of some nphthlene s nd nthroquinones on methicillin-resistnt Stphylococcus ureus. Chem Phrm Bull 1999;47: Krenn L, Prdhn R, Reznicek G, Koop B. Anthrone C glucosides from Rheum emodi. Chem Phrm Bull 2004;52: Tosum F, Akyuz-Kizily C. Anthrquinones nd Interntionl Journl of Green Phrmcy LPU Conference Apr-Jun 2018 Specil Issue 100

7 flvonoids from Rheum ribes. J Fc Phrm Ankr 2003;32: Zrger B, Msoodi MH, Ahmed B, Gnie SA. Phytoconstituents nd therpeutic uses of Rheum emodi wll. Ex. Meissn. Food Chem 2011;128: Ozturk M, Ozturk FA, Doru ME, Topcu G. Antioxidnt ctivity of roots nd stem extrcts of Rheum ribes. Food Chem 2007;103: Adm RP. Identifiction of Essentil Oils Components by Gs Chromtogrphy/Mss Spectrometry. Crol Strem IL: Allured Publishing Corportion; Groover RK, Moore JD. Txicometric studies of fungicides ginst brown rot orgnism rot orgnisms Sclerotini fructicol nd S. lx. Phytopthology 1962;52: Feng W, Zheng X. Essentil oil to control Alternri lternt in-vivo nd in-vitro. Food Control 2007;18: Leelsuphkul W, Himmne P, Chuentchitt S. Growth inhibitory properties of Bcillus subtilis strins nd their metbolites ginst the green model pthogen (Penicellium digittum scc.) of citrus fruit. Posthrvest Biol Technol 2008;48: Brnd-Willims W, Cuvelier ME, Berset C. Use of free rdicl method to evlute ntioxidnt ctivity. Lebensem Wiss U Technol 1995;28: Srivstv S, Singh RP. Antifungl ctivity of essentil oil of Murry koenigii (L) Spreng. Indin Perfumer 2001;45: Wni SA, Shh KW, Ahmed MA. Antifungl ctivities of methnolic extrcts of Podophyllum hexndrum nd Rheum emodi Wll ginst humn pthogenic fungl strins. J Phermceuticl Sci Revised Res 2013;9: Dormn HJ, Dens SG. Antimicrobil gents from plnts: Antimicrobil ctivity of plnt voltile oils. J Appl Microbiol 1999;88: Brc A, Sortino C, Politi M. Antioxidnt ctivity of flvonoids from Licni licnieflor. J Ethnophrmcol 2007;79: Source of Support: Nil. Conflict of Interest: None declred. Interntionl Journl of Green Phrmcy LPU Conference Apr-Jun 2018 Specil Issue 101