Int.J.urr.Microbiol.pp.Sci (2017) 6(10): 302-313 Interntionl Journl of urrent Microbiology nd pplied Sciences ISSN: 2319-7706 Volume 6 Number 10 (2017) pp. 302-313 Journl homepge: http://www.ijcms.com Originl Reserch rticle https://doi.org/10.20546/ijcms.2017.610.037 ssessment of Metl helting, Bioutogrphy nd Spot Screening by TL of Green Te (melli sinensis) rti Ghbru 1* nd R.G. Sud 2 1 Deprtment of Bsic Sciences, ollege of Forestry, UHF, Nuni, Soln, HP, Indi 2 Deprtment of hemistry nd Biochemistry, OBS, SKHPV, Plmpur, HP, Indi *orresponding uthor B S T R T K e y w o r d s Green te, Metl helting ions, ntibcteril ssy, ntioxidnt ctivity, Phytochemicl screening, melli sinensis. rticle Info ccepted: 04 September 2017 vilble Online: 10 October 2017 Introduction Te is receiving incresed interest from scientists due to its biologicl properties nd the presence of bioctive substnces like polyphenols which give nutrceuticl vlues to well brew cup of te. The study ws crried out s prerequisite to evlute the therpeutic potentil of green te. The ntioxidnt potentil of Kngr te in terms of metl ions cheltion ws determined. Ferrous ions cheltion potentil of te powders (in terms of I 50 (Fe ) vlues) ws in between the I 50 (Fe ) vlues of EDT nd citric cid. The I 50 (Fe ) exhibited correltion with T nd flvnols. comprtive TL (Thin lyer chromtogrphy) bioutogrph study between queous solutions of stndrd ctechins (0.5 mg ml -1 ) nd te powder (1 mg ml -1 ) indicted tht EGG ws one of the ctive components present in te solution which might be responsible for ntibcteril ctivity of Kngr te. Growth inhibition of bcteril pthogens (B. cereus nd S. ureus) by queous solution of te powder ws found to be influenced by the ddition of ferrous ions which suggest tht the ntibcteril ctivity of te ctechins could be due to their bility to chelte ferrous ions, indicting tht this ctivity is bcteriosttic rther thn bctericidl in nture. For more thn 1000 yers green te hs been used in hin nd Jpn s medicl herb nd helthful beverge. The trditionl hinese medicine hs recommended this plnt for hedches, body ches nd pins, digestion, depression, detoxifiction, s n energizer nd, in generl, to prolong life. Te is mde from young shoots nd leves of the plnt melli sinensis (Willson nd lifford 1992; brer et l., 2006; Shrm et l., 2007). Resons for the vst use of medicinl plnts re high cost of llopthic drugs nd their side effects (Mrwt et l., 2008). Mjority of the humn beings died mostly due to infectious bcteril diseses in the developing countries (Nthn 2004). The synthetic ntibiotics tht re being used lso becme ineffective becuse the pthogens got resistnce ginst these ntibiotics (Wlsh nd myes, 2004). Vrious strtegies hve been developed during the pst decde e.g., biologicl screening, isoltion s well s clinicl trils for vriety of plnts to explore the secret of their therpeutic ctions. Now dy, bioctive plnt extrcts re considered s 302
Int.J.urr.Microbiol.pp.Sci (2017) 6(10): 302-313 promising source of biologicl friendly ntibcteril gents (Koehn nd rter 2005). Fresh te leves contin cffeine, te polyphenols, te polyscchrides, nd necessry nutrients, such s protein, mino cids, lipids, nd vitmins. Generlly, four chemicl components free mino cids, totl te polyphenols, soluble sugrs, nd cffeine re considered indictors of te qulity (Ymmoto et l., 1997; Bshir et l., 2014; Gogoi nd Bru 2017). It is of the interest to note tht the polyphenols found in fresh green te shoots re different from other polyphenols of plnt origin s most of them re found only in leves of te plnt. Te ctechins hve been used s ntioxidnt compounds in mny food mtrices such s mets, poultries, fishes nd vegetble oils (Yilmz 2006). Since te hs n ntimicrobil ctivity ginst lrge spectrum of pthogenic bcteri (hou et l., 1999, Grmz et l., 2005; Friedmn et l., 2006; Freidmn 2007). Its low ph vlue, of pproximtely 4.2, mkes te comptible with mny food products in term of cidity. Epidemiologicl studies during the recent yers hve suggested tht green te reduces risks of severl chronic diseses including cncer nd crdiovsculr disorders by incresing plsm ntioxidnt cpcity in humns (Nkgw et l., 1999; Duthie et l., 2000). In the present study, we hve investigted the bioctivity of green te, cultivted t Wh te estte, Rjpur, HP. Mterils nd Methods Preprtion of te powders trnsferred into glss tubes fitted with irtight stoppers which were stored in vcuum desicctor. Estimtion of totl ctechins Totl ctechins were lwys estimted in freshly prepred te extrcts by the method of Sun et l., (1998). Thin- lyer chromtogrphy Thin lyer chromtogrphy ws done on glss plte coted with silic gel G by the method of Sthl (1969). Stndrd solutions were prepred (0.5 mg ml -1 ). (+)-ctechin, ( )- epictechin, ( )-epiglloctechin gllte, ( )- epictechin gllte nd ( )-epiglloctechin were procured from (Sigm, US). Solvent system ws used: hloroform: Ethyl cette: cetic cid:: 25:75:0.5 (By volume) Metl ions cheltion ssy The binding potency of metl ions by te ctechins ws evluted using method of Tng et l., (2002). The technique for the evlution of metl ions cheltion by the te ctechins method ws stndrdized using stndrd chelting gents nmely EDT (1 mm) nd citric cid (25 mm). The vlue of slope nd y-intercept were evluted from plot (Figures 1) between % chelting ctivity nd concentrtion of EDT nd itric cid. I 50 (Fe ) vlue (the mount of metl cheltor necessry to decrese the initil ferrous ions concentrtion by 50%) for EDT ws evluted using the following eqution: I 50 (Fe ) Vlue = (50 y-intercept)/ slope Te powders were prepred by lyophilizing queous extrcts of dried green te with the help of Edwrds EF 4 Modulyo freeze dryer nd Heto Power Dry LL 3000 freeze dryer. The powders so obtined were immeditely 303 Evlution of te smples The metl ions cheltion ctivity of te ctechins ws evluted in queous solutions of te powders by following protocol:
Int.J.urr.Microbiol.pp.Sci (2017) 6(10): 302-313 The smple solution hve ple yellow color nd smple volumes used for evluting metl ions chelting ctivity were quite lrge, it ws noted tht smple form blue color with ferrous ions. To correct this occurrence, smple blnks contining the pproprite dilution of ech volume of smple with 2 mm Fel 2.7H 2 O solution (0.1 ml) were used. The bsorbnce of ech smple blnk ws subtrcted from the bsorbnce of smple, respectively. The finl bsorbnce so obtined ws used for further clcultions. ntibcteril ctivity Growth inhibitory ctivity of te powder of fresh green te shoots ws evluted ginst selected pthogenic bcteri i.e. Bcillus cereus (MT-1272) nd Stphylococcus ureus (MT-96) procured from the Institute of Microbil Technology (IMTEH), hndigrh. The bcteril cultures were mintined on freshly prepred nutrient gr (N) medium nd were stored t 4±0.5 for further use. gr-well diffusion method for ntibcteril ctivity ntibcteril ctivities of te powder prepred from green te shoots were evluted by the method of Tod et l., (1989). The ntibcteril ctivity ws lwys determined in freshly prepred extrcts/solutions. Bioutogrphy TL bioutogrphic method s described by Kline nd Golb (1965) ws used to detect ctive ntibcteril components present in te powder. For this 50 µl of queous solution of te powder (1 mg ml -1 ) ws pplied on silic gel G TL plte which ws developed by using chloroform: ethyl cette: cetic cid (25:75:0.5) s the solvent system nd then dried for complete removl of solvent. Then TL plte ws plced in the Petri dish nd 0.1 ml of inoculum of Bcillus cereus (MT- 1272) grown in nutrient broth ws dded to gr medium nd this medium ws distributed over the entire TL plte. The plte ws incubted t 37 for 24 h nd the inhibition zone ws visulized with 0.1% of triphenyl tetrzolium chloride solution. Inhibition zone ws observed s cler re ginst red colored bckground. Influence of metl ions on inhibitory properties of queous solution of te powder Influence of metl ions on inhibitory properties of queous solution of te powder ws evluted using gr-well diffusion method s described by West et l., (2001). The growth inhibition of queous solution of te powder ws determined ginst B. cereus nd S. ureus using gr-well diffusion method s described bove. Zones of inhibition were observed, then 0.1 ml of ferrous sulphte solution ws dded to one well wheres sterilized double distilled wter ws dded to other well s control. Plte ws kept t 4 for 1 h to llow diffusion of the solution into the medium. The plte ws then incubted t 37 for 24 h then ppernce of growth ws observed in inhibition zone. Results nd Discussion Men totl ctechins (T) content of fresh green te shoots vried significntly in the rnge of 143.242 to 120.930 g kg -1. Te powders obtined by lyophilizing queous extrcts of green te shoot smples. The men T content in te powders vried significntly in the rnge 271.392 to 140.831 g kg -1. 304
Int.J.urr.Microbiol.pp.Sci (2017) 6(10): 302-313 Thin lyer chromtogrphy of te powder Thin lyer chromtogrphs (TL) of stndrd flvn-3-ols [(+)-ctechin (), ( )-epictechin (E), ( )-epiglloctechin (EG), ( )- epiglloctechin gllte (EGG) nd ( )- epictechin gllte (EG)] nd te powder were presented in Plte I. Metl ions cheltion The men I 50(Fe ) vlues corresponding to cheltion of Fe ions by EDT nd citric cid (verstile metl cheltors) were estimted to be 1.589 µg ml -1 nd 1455.895 µg ml -1, respectively (Tble 1). In tble 3 is given the men monthly I 50(Fe ) vlues (inhibitory concentrtion necessry to reduce the metl ions concentrtion by 50 per cent) of queous solutions (concentrtion: 5 mg ml -1 ) of te powders obtined by lyophilizing queous extrcts of smples of fresh green te shoots long with D (t 5%) nd per cent V. perusl of tble 3 indictes tht the men monthly I 50 (Fe ) vlues vried significntly in the rnge: 1243.002 to 724.953 µg ml -1 for te powders. The te powders obtined by lyophilizing queous extrcts of fresh green te shoots hrvested during ugust hd lower men monthly I 50(Fe ) vlue. lthough these vlues re comprble with the I 50 (Fe ) vlue estimted for citric cid but, fr higher in comprison to EDT. The metl ions chelting potentil in terms of I 50 (Fe ) vlues of te powders exhibited significnt negtive correltion with T, EGG, EG nd EGG+EG (Tble 4). ntibcteril ctivity In order to evlute green te for its biologicl ctivity, ntibcteril potentil of queous extrcts of smples of fresh green te shoots 305 were studied ginst stndrd bcteril pthogens i.e. Bcillus cereus (MT-1272) nd Stphylococcus ureus (MT-96) Sensitivity of selected bcteril pthogens for stndrd ctechins Before evluting the te smples for ntibcteril ctivity, the individul ctechins i.e., E, EG, EGG nd EG were tested for their ntibcteril effect on the selected pthogens (Plte II). Minimum inhibitory concentrtions (MI) of EG, EGG nd EG for B. cereus were 375, 50 nd 250 µg ml -1, respectively, wheres for S. ureus the MI of EG, EGG nd EG were 500, 250 nd 375 µg ml -1, respectively tble 5 indictes tht the MI of queous extrcts of smples of fresh green te shoots vries significntly in the rnge 724.953 to 1243.002 µg ml -1 Bioutogrphy In Plte IV the TL bioutogrphic result of queous solution of te powder ginst B. cereus is given. The component tht corresponded to the zone of inhibition ginst red bckground ws t the bottom of the TL plte where the te solution ws spotted Influence of metl ions on inhibition properties of te ctechins Growth inhibition of the queous solution of te powders on B. cereus nd S. ureus ws influenced by the ddition of ferrous ions (Plte V). Helth benefits of te drinking hve been ttributed to te ctechins. The present investigtions: ssessment of metl chelting, bioutogrphy nd spot screening of TL of green te (melli Sinensis) were crried out to elucidted biologicl ctivity of green te.
Int.J.urr.Microbiol.pp.Sci (2017) 6(10): 302-313 Tube no. Tble.1 Protocol of metl ions chelting ctivity Vol. of stock smple solution (ml) Vol. of double distilled wter (ml) Vol. of Fel 2 (ml) T 1 (ontrol) 0.000 2.700 0.100 0.200 T 2 0.240 2.460 0.100 0.200 T 3 0.360 2.340 0.100 0.200 T 4 0.480 2.220 0.100 0.200 T 5 0.600 2.100 0.100 0.200 T 6 0.720 1.980 0.100 0.200 T 7 0.840 1.860 0.100 0.200 Vol. of ferrozine (ml) Tble.2 Men totl ctechins (g kg 1 ) contents in te powders prepred from Fresh green te shoots Months Fresh green shoots Totl ctechins pril 120.930 e 140.831 f My 137.730 b 197.627 d June 136.242 b 226.024 c July 132.429 c 249.689 b ugust 142.644 271.392 September 135.609 b 251.041 b October 127.097 d 185.190 e Men 132.954 217.399 D (5%) 4.86 10.75 V (%) 2 2.82 Rnkings mens within ech column followed by the sme letter re not significntly different t p< 0.05. Tble.3 Men I 50 (Fe ) vlues (µg ml 1 ) of te powders from fresh green te shoots (FGTS) Months FGTS Men I 50(Fe ) vlues pril 1243.002 My 1160.127 b June 884.435 d July 971.934 c ugust 724.953 e September 890.731 d October 985.467 c Men 980.093 D (5%) 47.66 V (%) 2.78 Rnkings mens within ech column followed by the sme letter re not significntly different t p < 0.05. 306
Int.J.urr.Microbiol.pp.Sci (2017) 6(10): 302-313 Tble.4 orreltion coefficient mong TP, T,, E, EG, EGG nd EG nd men I 50 (Fe ) vlues of te powders from fresh green te shoots (FGTS) TP T E EG EGG EG EGG+EG TP T Men I 50(Fe ) vlues FGTS 0.77 0.87 NS NS NS 0.92 0.80 0.88 0.77 0.87 Significnt t p < 0.05; NS Not significnt. TP - totl polyphenols; T - totl ctechins; ctechin; E epictechin; EG- epiglloctechin; EGG - epiglloctechin gllte nd EG - epictechin gllte. Tble.5 Men minimum inhibitory concentrtions (mg ml 1 ) of queous extrcts of smples of Fresh green te shoots ginst selected bcteril pthogens Pthogens Months B. cereus S. ureus Men minimum inhibitory concentrtions* pril 1.541 f 2.042 b My 1.595 e 1.595 e June 1.770 c 1.770 c July 2.025 b 2.025 b ugust 1.073 g 1.541 f September 1.723 d 1.723 d October 2.253 2.253 Men 1.712 1.850 D (5%) 0.036 0.036 V (%) 1.20 1.20 Rnkings mens within ech column followed by the sme letter re not significntly different t p < 0.05; NZ No inhibition zone detected. *The minimum inhibitory concentrtions re bsed on totl ctechins content (mg ml 1 ) of queous extrcts. 307
% h e l tin g c tiv ity % h e l tin g c tiv ity Int.J.urr.Microbiol.pp.Sci (2017) 6(10): 302-313 Fig.1 - Grph between EDT nd per cent chelting ctivity; B- Grph between citric cid nd per cent chelting ctivity 90 80 60 70 50 60 50 40 40 30 30 20 10 20 10 0 2 3 4 5 6 0 0.0 2.5 5.0 7.5 10.0 onc e ntr tion of E D T ( M) concentrtion of citric cid (m M ) -Slope = 23.56; y-intercept = ( ) 50.62; r² = 0.9857; B- Slope = 0.02290; y-intercept =16.66; r² = 0.9424 Plte.1 Thin lyer chromtogrm of te powders contining significnt mounts of flvnols long with stndrd ctechins mixture Solvent system: hloroform: Ethyl cette: cetic cid:: 75:25:0.5 B Std Mix 18 19 20 21 22 23 24 25 26 27 28 E EG EG EG Plte 4.5 Thin lyer chromtogrm of te powder of pril 2009 column frctions contining significnt mounts of flvn-3-ols long with stndrd ctechins mixture 308
EGG E EG EG EGG EGG E EG Int.J.urr.Microbiol.pp.Sci (2017) 6(10): 302-313 Plte.2 Sensitivity of B. cereus nd S. ureus ginst stndrd ctechin nd its derivtives Bcillus cereus (MT-1272)Listeri monocytogenes (MT-839) E EG Streptococcus mutns(mt-890) E EG EG E EGG EG EG EG B Stphylococcus D ureus (MT-96) B EG EG EGG EGG E E F E EGG G D E Pseudomons eruginos B (MT-741) Bcillus cereus (MT-1272)Listeri monocytogenes D Plte.3 Sensitivity of B. cereus nd S. ureus for queous extrcts of fresh green te shoots E Bcillus cereus (MT-1272) Listeri monocytogenes (MT-839) Streptococcus mutns(mt-890) EGG EG B EG EG B B D Stphylococcus D ureus (MT-96) P D D B Stphylococcus ureus (MT-96) Bcillus Pseudomons cereus (MT-1272) eruginos Listeri (MT-741) monocytogenes (MT- Plte.4 TL bioutogrphy of queous solution of te powder of green te shoots ginst B. cereus long with TL of te solution nd stndrd ctechins. Solvent system: B hloroform: Ethyl cette: cetic cid:: 75:25:0.5 E EG EG EGG Mixture D D Stphylococcus ureus (MT-96) Pseudomon TL bioutogrph of te solution TL of te solution TL of stndrd ctechins 309
Int.J.urr.Microbiol.pp.Sci (2017) 6(10): 302-313 Plte.5 Growth inhibition of B. cereus nd S. ureus by queous solution of te powder () in the bsence, nd (b) showing restortion of growth in the presence of ferrous ions b Stphylococcus ureus (MT-96) b Bcillus cereus (MT-1272) Totl ctechin content Significnt men vritions in the levels of T in te powders were lmost similr to those of in fresh green te shoots (Tble 2). Misr et l., (2008) reported tht the fresh te shoots were extremely rich in phenolic compounds which cn constitute up to 300 mg/g of dry leves. Thin lyer chromtogrphy of te powder The chromtogrm indictes tht the te powders contin ll the five flvnols of interest. Bshir et l., (2014) reported tht spot screening of TL-developed pltes indicted tht the presence of ctive biologicl compounds such s flvonoids, phenols, lkloids nd glycosides. Metl ions cheltion The bility of flvnols to bind ctions of metls hving vrible vlency will restrict the metl ions from undergoing oxidtion which in turn terminte the genertion of free electrons/rdicls in biologicl system. Such rection my contribute, indirectly, to the ntioxidnt potentil of flvnols. The metl cheltion potentil of the te powders ws better thn tht of citric cid. This could be due to the presence of flvnols in te powders. Te ctechins hve been reported to exhibit significntly (p<0.05) weker chelting ctivity rnging from EGG (38%), E (30%), EG (19%) on Fe ions compred to citric cid (54%) or EDT (100%) t concentrtion of 400 ppm (Tng et l., 2002). 310
Int.J.urr.Microbiol.pp.Sci (2017) 6(10): 302-313 From significnt negtive correltion with T, EGG, EG nd EGG+EG, it is resonble to infer tht the flvnols with gllte moiety (EGG nd EG) might be responsible for the metl ions cheltion ctivity mong te ctechins. ompounds with gllte groups in their molecules, gllte of ctechins nd their polymers hve been reported to exhibit metl ions chelting ctivity (Miller et l., 1996). ntibcteril ctivity Sensitivity of selected bcteril pthogens for stndrd ctechins The lowest men MI mong the stndrd ctechins ws observed with EGG ginst B. cereus. The selected bcteril pthogens were resistnt ginst ctechin nd epictechin. These results re in greement with Tguri et l., (2004) who reported higher ntibcteril effect of EGG ginst ll bcteril groups: Stphylococcus ureus (20 strins), Slmonell (26 strins), E. coli (23 strins) nd genus Vibrio (27 strins) s compred to EG suggesting tht this could probbly be due to the presence of glloyl group in EGG which increses its ntibcteril ctivity. Tble 5 indictes tht the queous extrcts of smples of fresh green te shoots hrvested during the month of ugust hd lower MI nd hence more potent ntibcteril s compred to rest of the months (Plte III). However, hou et l., (1999) reported tht the extrct of oolong te prepred from smples of te shoots hrvested in summer flush sesons exhibited the strongest ntimicrobil ctivity followed by those prepred from smples of spring, winter nd fll. The high ntibcteril ctivity of smples ugust could be due to the presence of high levels of totl ctechins in the smple s evident from the results given in tble 2. These results corroborte with the erlier studies crried by hou nd Lin (1987) nd Ym et l., (1997) who frctionted te extrct nd reported tht the frctions rich in EG, EGG nd EG exhibit strong ntimicrobil ctivity. Bioutogrphy comprison with stndrd ctechins TL chromtogrm illustrte tht EGG ws present t bottom nd hence, EGG ws the ctive component of te smple which probbly responsible for the ntibcteril ctivity of Kngr te. These results re in greement with Freidmn et l., (2006) who reported tht gllte of ctechins i.e. ( )- glloctechin-3-gllte, ( )-epiglloctechin- 3-gllte, ( )-ctechin-3-gllte, ( )- epictechin-3-gllte, theflvin-3,3 - digllte, theflvin-3'-gllte nd theflvin- 3-gllte showed ntimicrobil ctivities t nnomolr levels. Bshir et l., (2014) reported tht TL bioutogrphy of methnol, ethnol nd DMSO extrcts of melli vrieties indicted the significnt inhibition of S. ureus, S. epidermidis nd S. mrcescens, respectively. Similr results were recorded by spot screening of TL developed pltes ginst ll clinicl bcteril pthogens. The zone of inhibition ws recorded in rnge from 10.00±0.0 mm to 28.00±0.00 mm. Influence of metl ions on inhibition properties of te ctechins It ws evident from the plte tht ddition of ferrous ions restored the growth of orgnism round the well indicting tht the ntibcteril ctivity of te ctechins is probbly due to their bility to chelte Fe ions nd mking them non-vilble for microbil growth. These observtions re in ccordnce with the erlier studies of West et l., (2001). These results lso suggest tht the growth inhibition by te ctechins is of bcteriosttic in nture. Erlier studies by Kono et l., (1994) suggested tht EGG hs 311
Int.J.urr.Microbiol.pp.Sci (2017) 6(10): 302-313 concentrtion dependnt bctericidl ctivity i.e. t concentrtion of 1 to 2 times of MI, the number of bcteri decresed to 1/10 th of originl lod, suggesting bcteriosttic ctivity, but if concentrtion ws incresed to 6 fold greter thn MI, then EGG showed bctericidl ctivity. However, Freidmn et l., (2006) reported bctericidl ctivities of the queous extrct of green te ctechins. The ferrous ions cheltion nd ntibcteril potency of green te were directly proportionl to totl ctechins contents of te in generl nd ( )-epiglloctechin gllte nd ( )-epictechin gllte contents in prticulr. The ntibcteril effect of green te ws bcteriosttic insted of bctericidl; however, more work is required to be crried out to estblish this property. The flvnols with glloyl moiety exhibit potentil biologicl ctivity. References Bshir, S., Khn BM, Bbr M, ndleeb S, Hfeez M, li S nd Khn MF. 2014. ssessment of bioutogrphy nd spot screening of TL Of green te (melli) plnt extrcts s ntibcteril nd ntioxidnt gents. Indin Journl of Phrmceuticl Sciences. 364-370 brer,., rtcho R nd Gimenez R. 2006. Beneficil effects of green te review. Journl of the mericn ollege of Nutrition 25(2): 79 99. hou,., Lin L nd hung K. 1999. ntimicrobil ctivity of te s ffected by the degree of fermenttion nd mnufcturing seson. Interntionl Journl of Food Microbiology 48(6): 125-130. hou,.., nd Lin L. 1987. ntimicrobil ctivity of te flush. In: Proceeding of 2 nd World ongress of Food Technology, Brcelon, Spin pp 287-296. Duthie, G.G., Duthie SJ nd Kyle JM. 2000. Plnt polyphenols in cncer nd hert disese: Implictions s nutritionl ntioxidnts. Nutrition Reserch Review 13(1): 79-106. Freidmn, M., 2007. Overview of ntibcteril, ntitoxin, ntivirl, nd ntifungl ctivities of te flvonoids nd tes. Moleculr Nutrition nd Food Reserch 51(1): 116 134. Friedmn, M., Henik PR, Levin E, Mndrell RE nd Kozukue N. 2006. ntimicrobil ctivities of te ctechins nd the flvins nd te extrcts ginst Bcillus cereus. Journl of Food Protection 69(2): 354 361. Gogoi,.S., nd Boru P K. 2017. Profiling of totl polyphenols nd pigments in te (cmelli Sinensis (L.) O. Kuntze) in vrious sesons for mnufcturing blck te nd green te. Interntionl Journl of Food Nutrition nd Science. 6, No. 2: 56-67. Grmz,., Korczk J nd mrowicz R. 2005. Te polyphenols their ntioxidnt properties nd biologicl ctivity review. Polish Journl of Food nd Nutrition Sciences 14(3): 219 235. Kline, R.M., nd Golb T. 1965. simple technique in developing thin-lyer bioutogrphs. Journl of hromtogrphy 18:409-411. Koehn, F.E., rter GT. 2005.The evolving role of nturl products in drug discovery. Nt Rev Drug Discov; 4:206-20. Kono, K., Ttr I, Tked S, rkw K nd Hr Y. 1994. ntibcteril ctivity of epiglloctechin gllte ginst methicillin-resistnt Stphylococcus ureus. Knsenshogku Zsshi 68(12):1518-1522. Mrwt, S.K., Khn M, hmd M, Zfr M, Rehmn F. 2008.Ethnomedicines for 312
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