B Y T TRNG I HC DC H NI PHM TH LAN NH TNH SAPONINTRONG GIO C LAM BNG SC K LP MNG KHA LUN TT NGHIP DC S Ngi hng dn ThS. Phm Tun Anh Ni thc hin B mn Dc LiuTrng i hc Dc H Ni H NI -2013 B Y T TRNG I HC DC H NI PHM TH LAN NH TNH SAPONINTRONG GIO C LAM BNG SC K LP MNG KHA LUN TT NGHIP DC S Ngi hng dn ThS. Phm Tun Anh Ni thc hin B mn Dc liuTrng i hc Dc H Ni H NI 2013 LI CM N hon thnh kha lunny,trc ht ti xin by t s knh trng vlng bit n su sc ti ThS. Phm Tun Anh, ngi thy tn tnh hng dnvtruyntchotinhngkinthccngnhnhngkinhnghim nghin cu khoa hc qu bu.Ticng xintrn trng gi li cmnti s gip ca ThS. L Thanh Bnh, ngi cho ti s h tr kp thi trong qu trnh thc hin kha lun.Ti xin chn thnh cmnccthy c, cc anh ch k thutvinang cng tc ti b mnDc Liu Trngi hcDc H Nitoiu kin ti hon thnh kha lun ng hn. Cui cng ti xin gi li cm n su sc tigianh,bn b ti, nhngngilun tintng, c v,ng vin ti trong hc tp v trong cuc sng. H Ni, ngy 21 thng 5 nm 2013 Sinh vin Phm Th Lan MC LC LI CM N T VN .................................................................................................. 1 CHNG 1.TNG QUAN .......................................................................... 2 1.1. TNG QUAN V GIO C LAM ....................................................... 2 1.1.1. c im chi Gynostemma Blume ....................................................... 2 1.1.1.1. V tr phn loi chi Gynostemma Blume ........................................... 2 1.1.1.2. c im thc vt v phn b ca chi Gynostemma Blume ............ 2 1.1.1.3. Cc loi trong chi Gynostemma Blume ti Vit Nam ....................... 3 1.1.2. Thnh phn ha hc ca cc loi Gio c lam .................................... 5 1.1.2.1. Saponin trong Gio c lam ............................................................... 5 1.1.2.2. Nghin cu v flavonoid ................................................................. 10 1.1.2.3. Cc cht khc .................................................................................. 11 1.2. TNG QUAN V H THNG SC K LP MNG HIU NNG CAO, CAMAG REPROSTAR 3, LINOMAT 5.0 ...................................... 12 1.3.1. H thng sc k lp mng hiu nng cao (high performance thin layer chromatography: HPTLC) ........................................................................... 12 1.3.2.Camag Reprostar 3 ............................................................................ 138 1.3.3.Linomat 5.0 ......................................................................................... 14 CHNG2.NGUYNLIU,THITB,NIDUNGVPHNG PHP NGHIN CU ................................................................................... 16 2.1. Nguyn liu ............................................................................................. 16 2.2. Phng tin nghin cu ......................................................................... 16 2.2.1. Thuc th, dung mi, cht chun, dch chit so snh ........................ 16 2.2.2. My mc, thit b, dng c ................................................................ 17 2.3. Ni dung v phng php nghin cu ................................................. 18 2.3.1. Kho st cc iu kin tin hnh sc k lp mng........................... 18 2.3.2. nh tnh saponin trong Gio c lam bng sc k lp mng .......... 193 CHNG 3. THC NGHIM, KT QU V BN LUN .................. 21 3.1. Thc nghim v kt qu ........................................................................ 21 3.1.1. Kho st cc iu kin tin hnh sc k lp mng ............................ 21 3.1.1.1.Chun b dch chit .......................................................................... 21 3.1.1.2. iu kin sc k .............................................................................. 21 3.1.2. nh tnh saponin trong dc liu Gio c lam ................................. 24 3.1.2.1.nh tnh cc mu thuc loi Gynostemmapentaphyllum (Thunb.) Makino ......................................................................................................... 24 3.1.2.2. nh tnh cc loi Gio c lam Vit Nam ..................................... 303 3.2. Bn Lun ............................................................................................. 3539 KT LUN V XUT ........................................................................ 404 TI LIU THAM KHO DANH MC CH VIT TT BuOH Buthanol EtOHEthanol GGynostemma GCLGio c lamNXBNh xut bn SKLMSc k lp mng DANH MC CC HNH STTTn hnh Trang 1 Hnh 1.1. Cu trc Dammaran thuc nhm Saponin triterpen tetracyclic 5 2 Hnh1.2.KhungcutrcsaponintrongG.pentaphyllum (Thunb.) Makino 6 3 Hnh1.3.CutrcepoxydammarantG.pentaphyllum (Thunb.) Makino 8 4Hnh 1.4. Cng thc cu to ca Gylongosid A9 5Hnh 1.5. Cng thc cu to ca Gylongosid B9 6Hnh 1.6 Cng thc cu to ca vinagynostesid A10 7Hnh 1.7. Camag reprostar 313 8Hnh 1.8. Linomat 5.015 9Hnh 2.1. S qu trnh chit bng n BuOH bo ha nc18 10 Hnh 3.1. Hnh nh sc k dch chit loi G. pentaphyllum (Thunb.)MakinotrongnBuOHbohanc,quanst dinhsngtngoibc sng 254 nm, 366 nm v phun hai loi thuc th hin mu 22 11 Hnh 3.2. Hnh nh sc k dch chit loi G. pentaphyllum (Thunb.) Makino c Nng trongn BuOH bo ha nc viccthtchchmlnltt1ln5lsaukhikhai trin vi h dung mi (I), quan st di nh sng t ngoi v phun thuc th hin mu 23 12 Hnh 3.3. Hnh nhsc k ca dch chit 13 mu thuc loi G. pentaphyllum (Thunb.) Makino khi quan st nh sng t ngoi bc sng 254 nm 25 13 Hnh3.4.Hnhnhsckcadchchit13muloiG. pentaphyllum(Thunb.)Makinokhiquanstnhsngt ngoibc sng 366 nm 27 14 Hnh3.5.Hnhnhsckcadchchit13muG. pentaphyllum (Thunb.)Makinosaukhiphunthucthhin mu28 15 Hnh 3.6. Hnh nh sc k ca dch chit mt s loi Gio c lam thuc chiGynostemma Blume khi quan st di nh sng t ngoi bc sng 254 nm 31 16 Hnh 3.7. Hnh nh sc k ca dch chit mt s loi Gio c lam thuc chiGynostemma Blume Vit Nam khi quan st di nh sng t ngoi c bc sng 366 nm 32 17 Hnh 3.8. Hnh nh sc k ca dch chit mt s loi Gio c lam Gynostemma Blume sau khi hin mu bng thuc th vanillin acid sulfuric 33 DANH MC CC BNG STTTn bngTrang 1Bng 1.1. Saponin thng gp trong G.pentaphyllum (Thunb.) Makino 7 2Bng 1.2. So snh gia HPTLC v TLC12 3Bng 3.1.Gi tr Rf ca cc vtquanstc trn sck ca cc mu G.pentaphyllum (Thunb.) Makino nh sng t ngoi bc sng 254 nm 25 4Bng 3.2. Gi tr Rf v mu sc ca cc vt trn sc k ca cc mu G. pentaphyllum (Thunb.) Makino sau khi hin mu 28 5Bng 3.3. Gi tr Rf ca cc vt trn sck ca dch chit mt s loi Gio c lam thuc chiGynostemma Blume khi quan st di nh sng t ngoi bc sng 254 nm 31 6Bng 3.4. Gi tr Rf v mu sc ca cc vt trn sc k ca mt s loi Gio c lam thuc chi Gynostemma Blume sau khi hin mu bng thuc th vanillin acid sulfuric 34 1 T VN Gio c lam l mt dc liu qu, c pht hin v s dng ln u Nht Bn vi tn gi cy trng sinh. Nm 1997, GS. TS. Phm Thanh K pht hin ra Gio c lam ti Lo Cai Vit Nam. Trong nhng nm gn y, Gio c lam c trng trn din rng cc a phng, c ch bin thnh cc sn phm ch Gio c lamadngvcluthngrng ri trn th trngnhGio c lam Tu Linh, Gio c lam Ba Tri, Gio c lam Tam o Do ngun gc Gio c lam phc tp, khng r rng li c s dng vi s lng ln nn rt kh kim sot dn n tnh trng dc liu gi mo, dc liu km cht lng vn lu thng t do trn th trng nh hng n sc khe ca nhn dn. V vy, vic phn tch so snh cc mu Gio c lam v la chndc liu s dng m bo chtlng thuc l ht sc cn thit. Nhng nghin cu a ra cc tiu chun truyn thng v Gio c lam trc y nh cm quan v hnh thi, c im vi phu, ch th ha hc rt kh nhgichnhxcchtlng Gio c lam. V vy, vic phn tch thnh phn ha hc c bit l thnh phn saponin bc u xy dng tiu chun vn tay ha hc ca Gio c lam l rt quan trng v v c ngha thc tin trong kim sot ngun gc v cht lng ca dc liu ny. Xut pht t yu cu thc t trn, kha lun nh tnh saponin trong Gio c lam bng sc k lp mng c thc hin vi 2 mc tiu: -nhtnhthnhphnsaponincaloiGynostemmapentaphyllum (Thunb.) Makino. -SosnhthnhphnsaponingiaccloiGCLthucchi Gynostemma Vit Nam. 2 CHNG 1.TNG QUAN 1.1. TNG QUAN V GIO C LAM 1.1.1. c im chi Gynostemma Blume 1.1.1.1. V tr phn loi chi Gynostemma Blume Theo cc ti liu Thc vt dc v phn loi thc vt [15], Cy c Vit Nam [10], chi GynostemmaBlume c xp vo h Cucurbitaceae (h Bu b). V tr ca chi Gynostemma Blume trong h thng phn loi thc vt dc nh sau: Ngnh Ngc lan Magnoliophyta Lp Ngc lan Magnoliopsida Phn lp S Dilleniidae Lin b Hoa tm Violanae B B Cucurbitales H Bu b Cucurbitaceae Chi Gynostemma. Hinnay,nhn dng 21 loi ca chi Gynostemma Blume. Trung Quc c ghi nhn 14 loi [22]. Vit Nam, chi Gynostemma Blume c 5 loi [23]. 1.1.1.2. c im thc vt v phn b ca chiGynostemma Blume Chi GynostemmaBlume c m t u tin biBlumevonm1825 da trn c im hnh thi ca loi G. simplicifolium [22]. T n nay, c thm nhiuloic m t. Cc loi thuc chi Gynostemmacccc im chung nh sau [10], [45]: Cy tho, mnh, thn leo, sng lu nm. L kp, t khi l l n, l kha rng ca. Tua cun ch i, i khi c tua cun n. Cm hoa khc gc, dng chy mnh, di, nht l i vi hoa c. Hoa nh, mu trng hoc lc nht, c 3 l bc con; cung hoa c t. i hoa hnh bnh xe, chia 5 thy, ngn. Trng hnh bnh xe, hi hn lin phn gc trng, c u nhn. Nh 5, phn gc ch nh hn lin thnh ct. Bao phn1,nhngnhncv nh2.Nhy: bu hnh cu nh, 2 3 ngn, 2 3 vi nhy vi u nhy chia 2 3 u nhn. Qu hnh cu ln hn ht u, khng m, 2 3 ht hnh trng hi dt 2 bn hoc c 3 gc. Ht sn si. Cc loi ca chi Gynostemma Blume phn b ch yu vng nhit i chuvngNamtHymalayatiNhtBn,MalaysiavNew Guinea. Loi Gynostemmapentaphyllum(Thunb.) Makino l loi ph bin nht, n phn b n, Nepal, Bangladesh, Srilanca, Lo, Myama, Hn Quc, Nht Bn v Vit Nam [45]. 1.1.1.3. Cc loi trong chi Gynostemma ti Vit Nam Hin nay c 3 loi thuc chi Gynostemma c cng b ti Vit Nam. a. Gynostemma pentaphyllum (Thunb.) Makino - c im thc vt: Cy tho, mc leo, thn cnh mnh, c gc cnh, khng lng hoc lng tha tht mu. L kp chn vt bn p, 5 7 l cht di 3 9cm, rng 1,5 3cm, mp l c rng ca.Tua cun mnh, x i nh [6]. Cm hoa c dng chm kp. Hoa c cung mnh c 1 4 mm; ng i rt ngn,thyihnhtamgic,dikhong0,7mm,nh nhn, thy trng hnh bu dc hoc mi mc, nh nhn c mt gn, nh 5. Cmhoacidngchmngnhnhoac.Hoacicivtrng ging hoa c, bu hnh cu 2 3 , vi nhy 3, nh lp 5, ngn. Qu khng t m, hnh cu,ng knh 5 6mm,khichnmuen. Ht hnh trng hoc hnh tim, ng knh 4 mm, mu nu, nh t, gc hnh tim dt. Ma hoa thng 3 10, qu thng 4 12 [5], [6], [7]. 4 Cy mc trn vi, hoa cng v t ni la, trong rng tha, lm bi t vng ng bng n vng ni cao 2000m [6]. - Phn b: ti Vit Nam, cy mc nhiu ni nh Lo Cai, Lng Sn, Qung Ninh, Ha Bnh, Tha Thin Hu, Kon Tum, ng Nai [5], [6]. b. Gynostemma longipes C.Y.W - c im thc vt: Cy dy leo. Thn cnh mnh, c 5 gc hnh ng gic. L kp chn vt. Mp l rng ca to. Ngn l nhn di. Mt trn c lng cng ri rc, mt di nhn, gn bn 9 cphnhlngchim,clngttha.Lbnnh dn. Tua cun mnh, r i mun. Hoa n tnh khc gc. Cm hoa c kp 3 ln chm, mnh. Hoa c rt nh, mu trng. i 5 ri, hnh tam gic. Trng 5, hnh tam gic, ri. Nh 5, ch nh dnh thnh 1 ct trung tm, hnh sao; bao phn 2 . Cm hoa ci kp 3 ln chm. Hoa ci, cung di 1,8 2,0mm.iv trng ging nh hoa c, b nhy cu to thng bi 3 l non hn lin, 2 3 vi nhy mp, ri; nm nhy chia 2 3; bu gia, 3 , mi c 1 ht. Qu mng, khi chn mu xanh,ng knh 6 7 mm, cung qu di 8 15 mm. Ht hnh tim, rng 3 4 mm, mu xm nht, 2 mt c hoa vn dng cc, vin ht c rng ca. - Ma hoa vo thng 8 10, ma qu vo thng 11 12. - Phn b: Ha Bnh, Lo Cai, H Giang, Cao Bng [2]. c. Gynostemma laxum (Wall.) Cogn. - Tn khc: C ym l bng, Th trng tha [5]. - c im thc vt: Cy dy leo mnh, phn nhnh nhiu, nhn hocclngtrttha, ging di 10 20 cm. L mng, l kp hnh chn vt, 3 l cht, cung di 1,5 4 cm, nhn, gn ph 5 7 cp. L cht kch c 4 10 2 3 cm; mp l 5 hnh sng rnh. Hoa khc gc. Cmhoac hnh chy, mc ngn hoc nch l, di 10 30cm,clngtmn. Trng hoa 5, ri, mu vng xanh. Kch thc 1,5 0,5 mm. Nh 5, hn lin ch nh v bao phn. Cm hoa ci ging cm hoa c, bu hnh cu, ng knh 1 mm. Vi nhy 3, ri, x i nh. Qu hnh cu, to 6 8 cm, mu vng xanh, nhn, khng t m, hi dt. Ht 2 3, hnh trng rng, ng knh 4 mm [5], [10]. Ma hoa vo thng 5. - Phn b: Cy mc cao 1200 m, trong cc khu rng, dc theo cc con sui [25], [27]. Cy mc leo cc khu rngtha,savanc,trnt st hoc cc rng cy bitrnnivi. Vit Nam, cy mc leo rngthacctnh: Lo Cai, Ha Bnh, Ninh Bnh, Qung Tr [5], [10]. 1.1.2. Thnh phn ha hc ca cc loi Gio c lam 1.1.2.1. Saponin trong Gio c lam 123456789101112131415161718192021 22 24 262723 2528 2930 Hnh 1.1.Cu trc Dammaran thuc nhm Saponin triterpen tetracyclic Cc nghin cu u tin pht hin saponin c mt trong Gio c lam thuc nhm dammaran (hnh 1.1). Dammaran l nhm saponin triterpenic c cutrc4vng(triterpenoidtetracyclic).Trongcngthcphntc30 carbon v do 6 nhm hemiterpen ghp li theo qui tc u ui. Cc saponin thucnhmnyxuthinnhiutrongcccythucchiPanaxL.,h Araliaceae.cbitccsaponintrongNhnsm(PanaxginsengC.A. 6 Mayer) cho thy nhiu tc dng qu c nhiu nh khoa hc trn th gii nghin cu. a. Loi Gynostemma pentaphyllum (Thunb.) Makino: ctrn100saponintrongthnhphnGynostemmapentaphyllum (Thunb.) Makino c phn lp v nhn dng cu trc, trong c 8 saponin gingnhloiprotoPanaxadioltrongginsenosidcaPanaxginsengC.A. Mayer l Rb1 (Gypenosid III) [31], [42], Rc [33], Rb3 (Gypenosid IV), Rd (GypenosidVIII),F2[33],Rg3[35],malonyl-Rb1vmalonyl-Rd[38]. Ngoiracngphthin Rf l 1 protoPanaxatriol [33]. Nhngginsenosidchim khong 25% tng gypenosid ton phn trong cy v l minh chng u tin ca nhm saponin nhn sm c tm thy ngoi h Araliaceae [32]. Mt s GypenosidXXVIII,XXXVII,LV,LXII,LXIIIcngtmthy trong loi Gynostemma sylvestre (Retz) Schult [48]. Cc saponin cn li chim phn ln ccgypenosidcphthinlnuloiG.pentaphyllum(Thunb.) Makino. R7R5R61 918203028 2917R3R4OR2R1 CH2OH OHOHOOHOOCH3CH2OOHGluORhaabcdefghi

Hnh 1.2a Cu trc saponin trong G. pentaphyllum (Thunb.) Makino Hnh 1.2b Cc dng cu trc R7 Hnh 1.2. Khung cu trc saponin trong G. pentaphyllum (Thunb.) Makino 7 Bng 1.1. Saponin thng gp trong G. pentaphyllum (Thunb.) Makino Cc nhm thV d R1ng glu, rham, xyl, c th 1 hoc 2, 3 ng kt hp vi nhau R2- H - OH Gypenosid I, Rb1 Gynos TN1, Gynos TN2 R3- CH3 - CH2OH - CHO Gypenosid I, Rb1 Gylongiposid I R4- OH - H =O Rb1 Gylongiposid I Gypentonosid A R5- OH - ng i; thng l glu kt hp vi rha hoc xyl Rg3, Rf Rb1, gymnemasid II R6- CH3 - CH2OH - CH2O -glu hoc CH2O xyl Rb1 R7C th l a, b, c, d, e, f, g, h hoc iCc ginsenoid u c cu trc a Loing chnh trong saponin ca G.pentaphyllum (Thunb.) Makino (hu ht thuc dng pyranose) l -D-glucose, -D-xylose, -L-rhamnose, -L-arabinose ni v tr C-3()vC-20. Cc nhm chc tiu biu l -OH, -CH3, -CHO, cc alcol v t ph bin hn c l nhm chc ceton v tr C-19 (R3). Nhm OH cng c v tr C-2() v C-12() [31], [32]. 8 R3OOR2R1OOH28 29301224262718 Hnh 1.3. Cu trc epoxy dammaran t G.pentaphyllum (Thunb.) Makino Cc saponin dng ocotillon c cu ni epoxy ti v tr C-17cngc phthinvicutrc3,12,23S,24R-tetrahydroxy-20S,25-epoxydammaran v (20S, 24S)-20,24-epoxy-dammaran-3, 12, 25-triol [34]. Ccsaponintrong G.pentaphyllum(Thunb.)Makinoasdng bt v nh hnh, ch c mt s t dng tinh th l gypenosid A [31], Gynogenin II [33] v Gynosaponin TN1 [31]. b. Loi Gynostemma longipes C.Y. Wu Nm 1993, Sun v cng s pht hin 2 saponin l Gylongosid A: 18-Norfurostan-19-al,23-hydroxy-4,4,8,14,24-pentamethyl-3-[(2-O--D-xylopyranosyl--D-xylopyranosyl)oxy]-(3,16,21)-(9Cl)vGylongosid B: 18-Norfurostan-19-al, 3-[(O-6-deoxy--L-mannopyranosyl(12)-O--D-xylopyranosyl-(12)--D-xylopyranosyl)oxy]-23-hydroxy-4,4,8,14,24-pentamethyl-,(3,16,21)- (9Cl) [40]. Nm1997,Guovcng s phn lpc3saponinnhndammarantrongc1saponinmil Gylongiposid1:19-oxo-3,20(S),21-trihydroxydammar-24-ene-3-O-{[-L-rhamnopyranosyl(1-2)][-D-xylopyranosyl(1-3)]}-L-arabinopyranoside, cn 2 cht cn li l gypenosid XLIX v ginsenosid Rb1 [24]. 9 Hnh 1.4. Cng thc cu to ca Gylongosid A Hnh 1.5. Cng thc cu to ca Gylongosid B Nm2010,PhanTh Thotin hnh nghin cu loi Gynostemma longipes VitNamvphnlpc 1 saponin l GCL1, da vo ph khi ESI MS, 1H NMR, 13C NMR, DEPT, HMBC, HSQC, 1H - 1H COSYxcnhccutrccaGCL1l(20S,24R)-3,12,25-trihydroxy-20,24-epoxydammaran3-O-{[-L-rhamnopyranosy(12)][-L-rhamnopyranosy(13)][-D-xylopyranosy(16)]}-D-glucopyranoside, y l mt saponin mi nn c t tn l vinagynosteside A [17]. 10 Hnh 1.6. Cng thc cu to ca vinagynostesid A c. Mt s loi khc: -phnlpcsaponin1-8tphntrnmttcaloi Gynostemma pubescens (Gagnep.) C.Y.Wu. Trong s chng, hp cht 1-4, c 8 nhm carboxyl c 2 v tr C21 v C29, 5 c cha mt nhm carboxylic C-21 v mt nhm chc aldehyd C-29, 6 v 7 cha nhm carboxylic C-21 v1nhmhydroxymethylenC-29[46].nghincuphnlp6 dammaranglycosidmitdchchitethanolcaphntrnmttloi GynostemmacardiospermumCogniaux ex Oliver. Phn aglycol triterpenoid cnhmcarbonylcC21vC28,lnutinctmthytrongh Cucurbitaceae [48]. 1.1.2.2. Nghin cu v flavonoid Flavonoid cng l mt trong nhng nhm cht chnh trong cc loi thuc chi Gynostemma Blume nhng t c nghin cu.- T Gynostemmapentaphyllum (Thunb.) Makino pht hin mt s flavonoid lombuin,ombuoside,rutin[36],quercetin-di-(rhamno)-hexosid, 11 quercetin-rhamno-hexosid,kaempferol-rhamno-hexosidvkaempferol-3-O-rutinosid [38]. - Nm2006,Yinv cc cng s phn lp c 3 flavonoid l rutin, kaempferolvquercetintloiGynostemmacardiospermumCogniauxex Oliver [48]. - Mt nghin cu ca cc tc gi ti i hc Y Qung Ty (Trung Quc)so snh hm lng ca flavonoid trong 6 loi Gynostemma thu hi ti Qung Tybngphngphpoquangphtibcsng510nm,sosnhvi chunnirutinchoktquhmlngflavonoidtonphntrongloi Gynostemma guangxiense X.X.Chen&D.H.Qin l cao nht [28]. 1.1.2.3. Cc cht khc - Sterol: chim1lng nh (khong 0.0001%) bao gm cc loi ergostanol, sitosterol v stigmasterol [34]. -NghincuphngphpxcnhchlorophyllvdnchttrongG. pentaphyllum (Thunb.) Makino bng HPLC-MS. tch v xc nh c trongG.pentaphyllum(Thunb.)Makinoc15chlorophyllvdncht: Pheophytina, pheophytin a, chlorophyll a, chlorophyll a, hydroxypheophytin a,hydroxypheophytina,pheophytinb,pheophytinb,chlorophyllb, chlorophyllb,hydroxychlorophyllb,hydroxypheophytinbv hydroxypheophytin b [26]. - Bng phng php phn tch in di mao qun pht hin trong dch chit nc G. pentaphyllum (Thunb.) Makino c 1 loi hetero polysaccharid khng tinhbtcthnhphnchnhlmonosaccharid,galactose,arabinose, rhamnose, galacturonic acid, xylose, manosev acid glucuronic [46]. - Nhm alcaloid c bo co l khng c trong G.pentaphyllum (Thunb.) Makino [19]. 12 1.2. TNG QUAN V H THNG SC K LP MNG HIU NNG CAO, CAMAG REPROSTAR 3, LINOMAT 5.0 1.3.1.Hthngscklpmnghiunngcao(highperformancethin layer chromatography: HPTLC) H thng thit b tinhvi,ciu khin bi mt nn tng phn mm tch hp m bo n mc cao nht tnh hu ch, tin cy, v lp li ca cc kt qu [20], [38]. Cc dng c: - Thit b chia mu. - Thit b trin khai sc k. - Trin khai sc k theo chiu dc. - Trin khai sc k theo chiu ngang. - Trin khai sc k t ng. - Trin khai nhiu sc k . - Thit b densitometer. - My qut. - My lu kt qu: lu hnh nh sc k cc bc sng t ngoi v sau khi phun thuc th [38]. Bng 1.2. So snh gia HPTLC v TLC [38], [37] c imHPTLCTLC K thutT ngTh cng Kch thc ht hp ph 5 6m10 12m dy bn mng100m250m Chiu cao a12m30m Hiu quCao do kch thc ht nh hn Thp hn 13 Phn tch 3 5 cm10 15 cm Thi gian phn tch Khong cch di chuyn ngn hn nn thi gian phn tch gim Lu hn Pha tnhNhiu s la chn nh silicagel trong trng hp thng thng v C8, C18 cho pha o Silicagel, kielsulguhr, alumina Bnh khai trinKiu mi do yu cu lng nh hn ca pha ng Yu cu lng ln hn Ly muT ngTh cng Th tch chm0,1 0,5 l1 5 l S vt 36 10 Qut hnh nhS dng UV / my qut hunh quang qut ton b sc k v my qut l hnh thc cao densitometer Khng Thi gian tch3 20 pht20 200 pht Gii hn pht hin ( hp ph ) 100 500 pg1 -5 ng Gii hn pht hin hunh quang 5 10 pg50 100 pg 1.3.2. Camag Reprostar 3 14 Hnh 1.7. Camag Reprostar 3 Camag reprostar 3, l b phn chp hnh nh sck, gm mt my nh ng v cc thit b p ng yu cu to ra mt bc sng nh sng ph hp. Cc loi bc sng nh sng: - 254 nm, UV bc sng ngn nh sng thng. - 366nm, UV bc sng di nh sng thng. - nh sng trng 400 750 nm nh sng thng, nh sng truyn qua, nh sng thng kt hp nh sng truyn qua. My nh trong cc reprostar c th l my nhthngthng, my nh ly nh ngay, tuy nhin tt nht l nn s dng my nh k thut s, hoc mt camera hin i. m bo hnh nh sc nt v kh nng lp li gia cc ln chp, trong camag reprostar 3 cn: - Mt ng knh c tiu c c nh. - B qua chcnngzoom m bo hnh nh ca cng mtanh dng lun c cng kch thc. - Thi gian bn mng tip xc vi nh sng c tnh ton t ng bng winCATS vng sng ca hnh nh c ti u. V t m bo loi tr hon ton nh sng ca mi trng xung quanh, docammag reprostar 3 thc hinc vic chp nhdi cc loi nh sng trong mt phng khng c lm ti [51]. 1.3.3. Linomat 5.0 15 Hnh 1.8. Linomat 5.0 Cc Linomat 5.0 l thit b c lp v l cng c l tng cho vic a mu ln bn mng trin khai sc k lp mng.Vi phin bnciu khin bng phn mm cho php HPTLC c ng dng rng ri. Linomat 5.0 l mt thit b bn t ng c th thay th cho thit b t ng ATS 4 m khng nh hng n cht lng ca h thng. Thit b ny c dng trong phntchnhtnhvnhlng, ph hp vilng mu trung bnh. Tri ngc vi ATS 4, vic thay i mu i vi Linomat 5.0 c thc hin th cng. Cc tnh nng chnh:- Hot ng theo ch c lp hoc theo winCATS. - a mu thnh di hp bng cch phun k thut. - p dng cc gii php trn sc k phng. - Hotngbn t ng, victhayi mu (lm sch,lmy v thay th ng tim) c thc hin bng tay [20]. 16 CHNG 2. NGUYN LIU, THIT B, NI DUNG V PHNG PHP NGHIN CU 2.1. Nguyn liu Nguyn liu l phn trn mt t ca 4 loi Gio c lam vi 16 mu khc nhau bao gm: - Loi Gynostemmapentaphyllum(Thunb.) Makino thu hi ti cca phngkhcnhaugm:Tamo (5 mu),Lt (4 mu),c Nng (1 mu), Ha Bnh (3 mu). Trong 5 muTamo, c 3 mu c cch trng khc nhau (trng ngoi nng, trng gim hom trong nh li, trng xen k vi cy rngvcynqu), 2 mu thu hi ti thiim khc nhau (3 thng, 4 thng). - Loi Gynostemma longipes C.Y.W - Loi Gynostemma laxum (Wall).Cogn. - Loi Gynostemma pubescen (Gagn.) C.Y.W. Dc liu sau khi thuhicphi nithongmt,sy nhit600C (t sy Shellab c qut thng gi),sauc nghin thnh bt v c bo qun trong ti nilon ni kh ro, thong mt. Nguyn liu thnh phm: ch Gio c lam BaTri(c cung cp bi cng ty TNHH Hong Tng), Gio c lam Tu Linh (c cung cp bi cng ty TNHH Tu Linh).2.2. Phng tin nghin cu 2.2.1. Thuc th, dung mi, cht chun, dch chit so snh a. Cc thuc th: - Dung dch H2SO4 10%/ EtOH. - Dung dch vanillin - acid sulfuric c to bi 2 dung dch: Dung dch 1: vanillin 1%/EtOH. 17 Dung dch 2: H2SO4 10%/EtOH. Khi s dng th trnnglng dung dch 1 v dung dch 2, phun ln bn mng v sy nhit 110oC trong 5 - 10 pht hin mu. b. Dung mi - EtOH, n BuOHt tiu chun chit xut, c cung cp bi cng ty sinh ha Xilong Trung Quc. - CHCl3, MeOH tinh khitt tiu chunphntchdng trin khai sc k c cung cp bi cng ty ha dc phm Daejung Hn Quc. c. Cht chun Rb1, Rg1 - Ginsenoside Rb1 phn lp t Panax.ginseng C.A.Mey,c cung cp bi cng ty Shanghai tautobiotech, tinh khit t 97%. - Ginsenoside Rg1 phn lp t Panax.ginseng C.A.Mey,c cung cp bi cng ty Shanghai tautobiotech, tinh khit t 98%. d. Dch chit so snh: dch chit Gynostemmacha 98% Gypenosid, c cung cp bi cng ty Changsha. 2.2.2. My mc, thit b, dng c - Bn mng trng sn Silicagel 60 GF254 caMERCK(c) c hot ho trongt sy BINDER nhit 110oC trong 60 pht. - Cn k thut Precisa (Switzerland). - T sy SHELLAB (c). - My ct chn khng BCHi ROTAVAPOR R-200. - My chm sc k Linomat 5.0 (CAMAG, MuTTenz Switzerland). - H thng my CAMAG REPROSTAR 3 cng phn mm winCATS. - Ni un cch thy, bnh chit v b dng c chit hi lu. - Bnhnh mc, pipet, ngong v cc dng c phn tch cn thit khc. 18 2.3. Ni dung v phng php nghin cu 2.3.1. Kho st cc iu kin tin hnh sc k lp mng - Phng php chun b dch chit: Theomc1.1.2.1saponintrongGioclamthucnhmdammaran, xut hin nhiu trong cc cy thuc chi Panax L., h Araliaceae. Saponin l hp cht phn cc mnh, tan tt trong n BuOH. Cc ti liu [25], [30], [41], [43]nghincu v thnh phn saponin trong mt s loi Nhn sm, kt qu cho thy n BuOH bo ha nc chit xut c phn ln saponin trong dc liu. V vy, kha lun la chn phng php chit bng n BuOH bo ha nc. Qu trnh chitnhsau: Cn 2 gam btdc liu, chit hilucch thy bng 15 ml EtOH 70% trong 10 pht. Lc ly dch chit EtOH 70%, c cch thy n 5 ml, cho vo bnh gn lc vi 5 ml n BuOH bo ha nc. Dch chit n BuOH c c cch thy n 1 ml em chm sc k. Hnh 2.1. S qu trnh chit bng n BuOH bo ha nc 2 gam dc liu Dch chit EtOH 70% 5 ml dch chitEtOH 70% Dch chit n - BuOH Chit hi lu cch thy bng 15 ml EtOH 70% Lc, c cch thy Lc vi 5 ml n BuOH bo ha nc C cch thy 1ml dch chit n BuOH em chm sc k 19 - La chn h dung mi: Theo mt s ti liu tham kho:Dcin Vit Nam [16],Dcin TrungQuc[21],iviSKLMcasaponindammaranchdungmi CHCl3 MeOH H2O vi hai t l (7 : 3 : 1) v (13 : 7 : 2) cho kh nng phn tch tt. V th, chng ti la chn h dungmitrn trin khai sc k, ng thi iu chnh t l dung mi kt qu phn tch ca cc vt l tt nht. - La chn thuc th hin mu: +Dung dch H2SO4 10%/EtOH sau khi phun ln bn mng sc k v sy nng s cho mu tm hoa c vi cc hp cht terpenoid. +Dung dch vanillin- acidsulfuric sau khi phun ln bn mng v sy nng s hin mu khc nhau vi cc hp cht khc nhau. - Kho st th tch chm sc k: DingShuli,ZhuZhaoyinghincuhmlng saponin trong 7 loi thuc chi Gynostemma Blume trong c cc loi G. pentaphyllum (Thunb.) Makino, G.laxum(Wall.) Cogn., G. longipesC.Y.W. Kt qu l v thn ca cc loi ny u cha 2,37 6,98% saponin (so vi trng lng kh) [50]. m bo hnh nh sc k cho cc vt phn tch r rng thun li cho phn tch kt qu, chng ti tin hnh kho st trn dch chit 2 : 1 (chit t 2 gam dc liu v c dch chit trong n BuOHn 1 ml) vi cc th tch chm sc k sau: 1l; 2l; 3l; 4l; 5l. 2.3.2.nh tnh saponin trong Gio c lam bng sc k lp mng Cc mu dch chit c chm trn bn mng silicagel GF254 bng my Linomat 5.0, chp nh sc k bng h thng my CAMAG REPROSTAR 3 cng phn mm winCATS. 20 -Thnghim1:nhtnhsaponintrong13mucaloiG. pentaphyllum (Thunb.) Makino, tm cc vt c trng cho loi, t chn ra cc mu i din cho loi G. pentaphyllum (Thunb.) Makino. - Th nghim 2: So snh thnh phn saponin gia mt s loi trong chi Gynostemma Blume trng v thu hi ti Vit Nam, ng thi so snh vi mt s ch phm v cht chun. 21 CHNG 3. THC NGHIM, KT QU V BN LUN 3.1. Thc nghim v kt qu 3.1.1. Kho st cc iu kin tin hnh sc k lp mng 3.1.1.1. Chun b dch chit S dng phng php chit xut nh mc 2.3.1 vi mt mu ca loi G. pentaphyllum (Thunb.) Makino thu hi ti c Nng.3.1.1.2. iu kin sc k - La chn h dung mi:Nhiu nghin cu chng minh thnh phn saponin trong Gio c lam c nhiuimtngng vi thnh phn saponin trong cc cy thuc chi PanaxL.nh tnh saponin trong Nhn sm bng SKLM, s dng h dung mi CHCl3: MeOH: H2O vi nhiu t l khcnhaunhsau(7: 3: 1); (7: 3: 0,8); (7: 3: 0,5); (7: 3 : 0,25) cho thy kh nngphntchtt [41], [44]. V vy, kha luns dng h dung mi CHCl3: MeOH: H2Oviu chnh thnh t l (7: 3: 0,4) (I) khai trin sc k, kt qucho thy cc vt phn tch tt, thun li cho phn tch kt qu. - La chn thuc th hin mu: Tin hnhtrn 2 bn mng sc k c chm cng mt dch chit dc liu v khai trin vi cng mt h dung mi (I). Sau khi khai trin, quan st bn mng di nh sng t ngoi bc sng 254 nm v 366 nm ri phun thuc th hin mu. Vi bn mng 1 phun thuc th H2SO4 10%/EtOH, bn mng 2 phun thuc th vanillin acid sulfuric, sy c 2 bn mng trong 5 10 pht. Kt qu thu c sc k nh trong hnh 3.1. 22

Hnh 3.1. Hnh nh sc k dch chit loi G. pentaphyllum (Thunb.) Makino trong n BuOH bo ha nc, quan st di nh sng t ngoi bc sng 254 nm, 366 nm v phun hai loi thuc th hin mu Nhn xt: Trn hnh nh sc k , sau khi phun thuc th vanillin acid sulfuricquan st c 8 vt trong c 7 vt tng ng vi kt qu sc k sau khi phun thuc th H2SO4 10%/EtOH. Hn na, thuc th vanillin acid sulfuric chomurhnvbnhnthuc th H2SO4 10%/EtOH.Doccphp nh tnh trong kha lun u s dng thuc th vanillin acidsulfuric.- La chn th tch chm sc k: Sau khi la chn c phng php chun b dch chit, h dung mi v thuc th hin mu, dch chit t loi G. pentaphyllum (Thunb.) Makino c chm ln bn mng sc k vi cc th tch 1l, 2l, 3l, 4l, 5l la chn c th tch chm ph hp nht. Sau khi khai trin sc k, kt qu c th hin hnh 3.2. 23 Hnh 3.2.Hnh nh sc k dch chit loi G. pentaphyllum (Thunb.) Makino c Nng trongn BuOH bo ha nc vi cc th tch chm ln lt t 1l n 5l sau khi khai trin vi h dung mi (I), quan st di nh sng t ngoi v phun thuc th hin mu 24 Nhn xt: Theo hnh nh sck th vi th tch chm 3l cc vt tch nhau r rng v m ca cc vt va quan st c di nh sng t ngoiv sau khi phun thuc th hin mu. Vi ccth tch chm 1l; 2l, cc vt tch nhau r nhng nht, gy kh khn cho phn tch kt qu. Vi cc th tch chm 4l; 5l, cc vt m cha tch c khi nhau hon ton. V vy, cc th nghim trong kha lun dng th tch chm sc k l 3l. Kt lun: T kt qu kho st trn, nhng th nghimnh tnh bngphng php sc k lp mng trong kha lunc tin hnh theo nhngiu kin nh sau: - Dch chm sc k c chun b theo phng php chit xut bng n BuOH bo ha nc. - Th tch dch chit chm ln bn mng: 3l. - H dung mi khai trin: CHCl3 : MeOH : H2O (7 : 3 : 0,4). - Sau khi khai trin, sc k c quan st di nh sng t ngoi c bc sng 254nm, 366nm v hin mu bng thuc th vanillin acid sulfuric. 3.1.2. nh tnh saponin trong dc liu Gio c lam 3.1.2.1. nh tnh cc mu thuc loi Gynostemma pentaphyllum (Thunb.) Makino p dng cc iu kin sc k la chn tin hnh nh tnh saponin trong 13 mu ca loi G. pentaphyllum (Thunb.) Makinovi cc k hiu l: T1, T2, T3: cc mu Tam o c trng theo cc cch khc nhau. B3, B4: cc mu Tam o c thu hi ti cc thi im khc nhau (3 thng, 4 thng sau khi trng). 1, 2, 3, 4: cc mu Lt c trng theo cc cch khc nhau. N: mu mc t nhin thu hi c Nng. TR: mu Ha Bnh c trng trong rm. 25 NN: mu Ha Bnh c trng ngoi nng. GN: mu Gio c lam ngt thu hi Ha Bnh. Kt qu:- Khi quan st bc sng t ngoi 254 nm: Hnh nh sc k v kt qu phn tch sc k c th hin hnh 3.3 v bng 3.1. Hnh 3.3. Hnh nh sc k ca dch chit 13 mu thuc loi G. pentaphyllum (Thunb.) Makino khi quan st nh sng t ngoi bc sng 254 nm Phn tch kt qu Bng 3.1.Gi tr Rf ca cc vt quan st c trn sc k ca cc mu G. pentaphyllum (Thunb.) Makino nh sng t ngoi bc sng 254 nm STTRfCc mu th T1T2T3B3B41234NTRNNGN 10,78+ + + + + +++ +++ ++ +20,71+ +30,66+40,61+ ++ ++ + + + + ++ + ++ ++ + +50,43+ + + + + + ++ + ++ ++ ++ ++60,38+ + + +70,32+ + + + ++ + ++ ++80,24+ + ++ + + + ++ + + +90,14++ ++ ++ ++ ++ +Ghi ch: (+): c vt nht; (++): vt m; (+++): vt rt m 26 Nhn xt: +Nhm cc mu G. pentaphyllum (Thunb.) Makino trng Tamo (T1, T2, T3, B3, B4): sc k nm mu ny tng t nhau khi quan st di nh sng t ngoibc sng 254 nm. Trn sck c nmmuu xut hin 4 vt c Rf ln lt l: 0,61; 0,43; 0,24; 0,14 vi m nht khc nhau cc mu; trong 2 vt c Rf bng 0,61; 0,14 l m nht. Ngoi ra, bn muT1, T2, T3, B3 cn xut hin vt c Rf bng 0,38; hai mu T2, T3 xut hin vt c Rf bng 0,78. +Nhm nm mu G. pentaphyllum (Thunb.) Makino trng Lt v mu thu hi c Nng (1, 2, 3, 4, N): Trn sc k ca nm mu u xut hin 2 vt c gi tr Rf bng 0,61 v 0,43. c bit hai mu 3 v Ncxut hin 1 vtm Rf bng 0,32; vtnycngxut hin 4 nhngnhthn.Ngoira 4vNcnxut hin vt c Rf bng 0,24. Ring muNxut hin mt vt Rf bng0,66.Nhvytrongnmmu ny, mu t nhin thu hi c Nng (N) cho nhiu vt nht trn sc k , cc vt xut hin cng m nht. +Nhm cc mu G. pentaphyllum(Thunb.) Makino trng Ha Bnh (TR, NN): Sck ca hai mu nytngt nhauvi 4 vt c gi tr Rf bng0,78; 0,61; 0,43; 0,24. Ring mu trng trong rm (TR) xut hin thm 2 vt Rf bng 0,71 v 0,32. +Mu Gio c lam ngt thu hi Ha Bnh (GN): Trn sck ca mu ny, cc vt xut hin nht hn so vi 12 mu khc. mu ny xut hin 5 vt nht trn sc k vi gi tr Rf bng 0,78; 0,71; 0,61; 0,24 v 0,14. Nh vy, khi quan st sc k nh sng t ngoi c bc sng 254nm, tt c 13 mu G. pentaphyllum (Thunb.) Makino u xut hin 1 vt m c Rf bng 0,61. 27 - Khi quan st bc sng 366 nm: Hnh nh sck 13 mu G. pentaphyllum (Thunb.) Makino khi quan st nh sng t ngoi c bc sng 366 nm c th hin hnh 3.4. Hnh 3.4. Hnh nh sc k ca dch chit 13 mu loi G. pentaphyllum (Thunb.) Makino khi quan st nh sng t ngoi bc sng 366 nm Nhn xt: Trn hnh nh sc k ca 13 mu G. pentaphyllum (Thunb.) Makino, khi quan st di nh sng t ngoi c bc sng 366 nm, vt hunh quang xut hin tt c cc mu. Cc vt cn li c hunh quang rt nht. Mt khc, mc tiu ca ti t ra l nh tnh saponin trong dc liu GCL, khi tham kho ti liu nghin cu v thnh phn ha hc ca GCL th saponin trong dc liu ny thuc nhm triterpenoid, trong cu trc hu nh khng c cc lin ktilinhp, nn cc hp cht ny khng pht hunh quang bc sng 366 nm. Kt hp vi hnh nh sck ca 13 mu GCL (hnh 3.4), chng ti nhn thy hnh nh cc vt ny khng c gi tr c trng cho cc hp chtsaponintrongGCL.Do,chngtikhngphntchsuhn v cc vt trn sc k ca 13 mu G.pentaphyllum (Thunb.) Makino khi quan st di nh sng t ngoi c bc sng 366 nm.28 - Sau khi hin mu vi thuc th: Hnh nh sck v kt qu phn tch cc vt trn sc k c th hin hnh 3.5 v bng 3.2. Hnh 3.5. Hnh nh sc k ca dch chit 13 mu G. pentaphyllum (Thunb.) Makino sau khi phun thuc th hin muBng 3.2. Gi tr Rf v mu sc ca cc vt trn sc k ca cc mu G. pentaphyllum (Thunb.) Makino sau khi hin mu STT Rf Cc mu th T1T2T3B3B41234NTRNNGN 10,82 t ++t +++t +++t ++t +++t +t +t ++t +t +t ++t +t ++20,78 vn +vn ++vn ++vn ++ 30,74 n ++ 40,65 t ++t ++t ++t ++t ++t ++t ++t +t +t ++t ++t ++t ++50,62 x +x +x +x ++x +x +x +60,50 x ++x ++x ++x ++x ++x +x +x +t +70,46 vn +vn +vn +vn +vn +vn +vn +vn +vn +vn +t +80,37 vn +vn +vn +x ++x +++x +++x +x +++x +++x ++t +90,31 x +x +x +x +x +x + 100,26 x (+) x +x +x +x +x +t +110,15 vn +vn +vn +vn + 120,10 x +x ++x ++x +x + Ghi ch: vn: vng nu; x: xanh; t: tm 29 (+): c vt nht; (++): vt m; (+++): vt rt m Nhn xt: Sau khi hin mu bng thuc th vanillin acid sulfuric, quan st trn sc k ca 13 mu loi G. pentaphyllum (Thunb.) Makino nhn thy c 2 vtmutmcRfbng0,82v0,65xuthinc13muloiG. pentaphyllum (Thunb.) Makino. Cc vt cn li trn sc k xut hin theo tng nhm mu dc liu nh sau: +Nhm cc mu G. pentaphyllum (Thunb.) Makino trng Tam o: sc k ca T1, T2, T3, B3, B4 tng i ging nhau v c s lng; mu sc v gi tr Rf ca cc vt. Trong c bit c 1 vt m mu xanh Rf l 0,5. +Nhm cc mu G. pentaphyllum(Thunb.) Makino trng Lt, v mu thu hi c Nng: Nu nh 5 mu G. pentaphyllum (Thunb.) Makino trng Tamocctrngbi 1 vtm mu xanh Rf bng 0,5 th cc mu thu hi Lt, c Nng (N) v Ha Bnh (TR, NN) c c trngbi 1 vtm mu xanh Rf bng 0,37. Trong nhm mu ny, mu dc liu thu hi c Nng v mu trng trong rm Ha Bnh xut hin nhiu vt v cc vt cng m hn trn sc k .+Ring sck ca mu GCL ngt khc bit nhiu so vi 12 mu thucloiG.pentaphyllum(Thunb.)Makinokhiccvtxuthinnhiu nhng rt nht.T nhng kt qu nghin cu trn, chng ti la chn ra nm mu i din cho tng nhm mu ca loi G.pentaphyllum (Thunb.)Makino so snh vi cc loi G. laxum (Wall.) Cogn, G. longipes C .Y. W,G. pubescens (Gagn.) C. Y. W v hai mu ch phm ch Gio c lam Ba Tri, ch Gio c lam Tu Linh. Nm mu c la chn l T3, 2, N, TR, GN. 30 3.1.2.2. nh tnh cc loi Gio c lam Vit Nam Th nghim nh tnh ny c tin hnh vi iu kin sc k lp mng la chn trn cc mu nh sau: Rb1, Rg1: Hai saponin c cng b c trong thnh phn ha hc loi G. pentaphyllum (Thunb.) Makino [40]. TQ: Gypenosid Trung Quc. T3,2,N:ccmu G.pentaphyllum (Thunb.)Makinoc trng Tam o, Lt, c Nng. TR:muG.pentaphyllum(Thunb.)MakinoHaBnhctrng trong rm. GN: mu Gio c lam ngt thu hi Ha Bnh. G. lax: Gynostemma laxum (Wall.) Cogn. G. log: Gynostemma longipes C.Y.W. G. pub: Gynostemma pubescens (Gagn.) C.Y.W. Ch: ch Gio c lam Ba Tri. TL: ch Gio c lam Tu Linh. Ring cc mu Rb1, Rg1, v Gypenosid Trung Qucc chun b chm sc k nh sau: -MuGypenosidTrungQuc:ly1gammuhatantrong1mln BuOHbo ha nc, lc qua giy lc em chm sc k, th tch chm sc k l 8l. -CcchtchunRb1,Rg1:cphathnhdungdchcnng 1mg/ml v c chm sc k vi th tch 3l. Kt qu: - Hnh nh sc k v kt qu phn tch sc k cc mu khi quan st di nh sng t ngoicbc sng 254 nmc th hin hnh 3.6 v bng 3.3. 31 Hnh 3.6. Hnh nh sc k ca dch chit mt s loi Gio c lam thuc chiGynostemma Blume khi quan st di nh sng t ngoi bc sng 254 nm Phn tch kt qu Bng 3.3. Gi tr Rf ca cc vt trn sc k ca dch chit mt s loi Gio c lam thuc chi Gynostemma Blume khi quan st di nh sng t ngoi bc sng 254 nm STTRfCc mu th Rb1Rg1TQT32NTRGNG.laxG.logG.pubChTL 10,78+ + +++ +++ + +++ + + ++20,66+ + + ++30,61+ + + ++ + + +++ + + + ++40,47+50,43+ + ++ ++ + + + ++60,35++ + +70,32+ + + + ++ + +80,24+ + + + + + + ++90,14++ Ghi ch: (+): c vt nht; (++): vt m; (+++): vt rt m Nhn xt: Khi quan st sck di nh sng t ngoicbc sng 254 nm, hai cht chun Rb1, Rg1 khng xut hin vt. Trong 11 mu cn li (gm 1 mu Gypenosid Trung Quc v 10 mu GCL Vit Nam), u xut hin mt 32 vt chung c gi tr Rf bng 0,61. Da vo hnh nh sc k v gi tr Rf ca cc vt c th nhn thy cc mudc liu loi G.pentaphyllum (Thunb.) Makino trng Lt (2), mu thu hi c Nng (N) v mu trng Ha Bnh tng i ging mu ch GCL Ba Tri, v mu ch GCL Tu Linh. - Hnh nh sc k dch chit mt s loi GCL thuc chi Gynostemma Blume khi quan st nh sng t ngoi c bc sng 366 nm c th hin hnh 3.7. Hnh 3.7. Hnh nh sc k ca dch chit mt s loi Gio c lam thuc chiGynostemma Blume Vit Nam khi quan st di nh sng t ngoic bc sng 366 nm Tngt nhhnhnh sc k 13 muG.pentaphyllum (Thunb.) Makino (Hnh 3.4) khi quan st di nh sng t ngoi c bc sng 366 nm, cc vtquanstckhngctrngchocchp cht saponin. Do vy, chng ti khng phn tch su vo sc k v gi tr Rf ca cc vt cht trn sc k ny. - Hnh nh sck v kt qu phn tch sck dch chit mt s loiGCLthucchiGynostemmaBlumesaukhihinmubngthucth vanillin acid sulfuric c th hin hnh 3.8 v bng 3.4. 33 Hnh 3.8.Hnh nh sc k ca dch chit mt s loi Gio c lam Gynostemma Blume sau khi hin mu bng thuc th vanillin acid sulfuric Nhn xt:- Sau khi hin mu bng thuc th vanillin acid sulfuric, trn sc k ca10 mu Gio c lam thuc chi Gynostemma Blume trng v thu hi ti Vit Nam cho 2 vt mu tm c trng vi Rf l 0,82 v 0,65. - Hai cht chun Rb1, Rg1 c mu tm hoa c vi Rf c gi tr ln lt l 0,13; 0,43. - Trn sc k ca ba mu GCL loi G. pentaphyllum (Thunb.) Makino trng ti Lt (2), thu hi ti c Nng (N) v trng ti Ha Bnh (TR) xut hin cc vt c gi tr Rf v mu sc tng t cc vt xut hin hai mu ch phm ch GCL Ba Tri v GCL Tu Linh.- Hnh nh sck ca mu G.pentaphyllum (Thunb.) Makino trng tiTamo(T3)tngt hnh nh sck ca mudc liu loi G. longipes C.Y.W v G. pubescens (Gagn.) C.Y.W (G. pub). 34 - Mu dc liu GCL ngt thu hi Ha Bnh (GN) v GCL thuc loi G.laxum (Wall.) Cogn c hnh nh sc k khc nhau v khc tt c cc mu cn li. - Mu Gypenosid ca Trung Quc xut hin 4 vtm trn sck, tuy nhin 4 vt ny khng xut hin hoc xut hin rt nht cc mu GCL trong th nghim. Bng 3.4. Gi tr Rf v mu sc ca cc vt trn sc k ca mt s loi Gio c lam thuc chi Gynostemma Blume sau khi hin mu bng thuc thvanillin acid sulfuric STT RfCc mu th Rb1Rg1TQT32NTRGNG.lax G.logG.pubChTL 10,82t ++t +t +t +++t +t +t ++t +t +t ++20,78v ++v ++v ++v +v ++30,74t ++t +t + 40,68v ++ 50,65t ++t ++t ++t ++t ++t +t ++t ++t +t ++60,60vn +vn +t +t +t +x ++70,54xn ++ 80,49t ++t +t ++t ++t +90,43t ++vn +vn +++vn +vn +vn +t +vn +vn +vn +vn +vn ++100,36t ++t +t ++t ++t ++t +t +t +t ++110,30n ++n +n +n +n + 120,24n +n +n +n +n +n +130,17n ++n +n + 140,13t ++n +n +n + 150,10n + n +n + Ghi ch: n: nu; t: tm; v: vng; vn: vng nu; xn: xanh nu (+): c vt nht; (++): vt m; (+++): vt rt m 35 3.2. Bn Lun Sc k lp mng l mt phng php c WHO v dc in nhiu nc p dng trong kim tra chtlngdc liu.ylphngphpn gin, chi ph thp, c th p dng vi hu ht cc hp cht. V vy, SKLM c s dngnhlmtphngtin ph bintrongphntchdc liu t nghin cu sng lc ban u n bn nh lng v nh lng. nh tnh v so snh c cc mu GCL nghin cu, cc iu kin tinhnhSKLMc kho st gm c: chun b dch chm sc k, h dung mi khai trin v thuc th hin mu. Trong dch chit c chun b bng cch chit xutdc liu vi EtOH 70% l dung mi c kh nngha tan nhiu hp cht t dc liu, sau dch chit c bc hi dung mi v chit vi n BuOH bo ha nc. n BuOH bo ha nc l dung mi kh phn cc nn ph hp chit xut cc hp cht nhm saponin v loi bt mt s tp cht trong dch chit. Nhng iu kin cn li tin hnh SKLM nh th tch chm dch chit ln bn mng, h dung mi khai trin, thuc th hin mu u c kho st la chn ra iu kin ph hp nht cc vt hin r rng v kh nng tch ca cc vt tt. VinhngiukinSKLMlachn,13muG.pentaphyllum (Thunb.) Makino c nh tnh. Sau khi quan st hnh nh sc k di nh sng t ngoi v hin mu vi thuc th vanillin acid sulfuric, tt c 13 mu G. pentapyllum (Thunb.) Makino u xut hin 1 vt m di nh sng t ngoi c bc sng 254 nm vi Rf bng 0,61 v hai vt m mu tm c Rf bng 0,82 v 0,65 sau khi hin mu. Da vo gi tr Rf ca cc vt cn li trn sck nhn thy 13 mu G.pentaphyllum (Thunb.) Makino khi trng ti cc a im khc nhau th s c cc vt c trng khc nhau. C th 13 mu ny c th chia ra thnh ba nhm c hnh nh sck tngt nhau l: nhm nm dc liu trng ti Tam o (T1, T2, T3, B3, B4), nhm by dc 36 liu trng ti Lt (1, 2, 3, 4), c Nng (N), Ha Bnh (TR, NN) v nhm dc liu GCL ngt trng ti Ha Bnh (GN).Nhm nm mu trng ti Tam o c c trng bi mt vt m Rf bng 0,14 khi quan st di nh sng t ngoi bc sng 254 nm v mt vt m mu xanh c Rf bng 0,50 sau khi hin mu. Cc vt cn li xut hin tng t nhau trn c nm muv gi tr Rf v mu sc, ch khc nhau mc m nht. Nh vy, nm mu GCL trng ti Tam o mc d cch trng khc nhau nhngthnhphn ha hctngt nhau ch khc nhau v hm lng cc cht. c bit khi so snh hai mu B3, B4 l hai mu thu hoch sau khi trng c 3 thng v 4 thng th cc vt trn sc k ca B3 m hn muB4.ylmt gi v thiimthuhidc liu vathuc dc liu c hm lng hot cht cao va tit kim c chi ph khi thi gian trng c rt ngn. Nhm by mu GCL trng ti Lt, c Nng v Ha Bnh c c trngbi mt vtm c gi tr Rf bng 0,43 khi quanstdi nh sng t ngoi c bc sng 254 nm v mt vt m mu xanh c gi tr Rf bng 0,37 sau khi hin mu. Trong cc mu ny th mu thu hi ti c Nng xut hin cc vtmhnsovi cc mu cn li. Ring hai mu trng ti Ha Bnh (TR, NN) khc nhau v cch trng: TR l mu trong rm, NN l mu ngoi nng th mu TR c hm lng cc cht cao hn so vi mu NN. y l iu ng lu trong qu trnh trng trt dc liu GCL thu c dc liu c hm lng hot cht cao. Ring mu GCL ngt (GN) thu hi ti Ha Bnh cho hnh nh khc bit so vi 12 mu trn vi s lng cc vt xut hin rt nhiu nhng nht mu. Vi ba nhm dc liu chia theo hnh nh sc k nh trn hon ton ph hp vi v cabanhmdc liuny.Nhmnmmu GCL trng ti Tam o c v rt ng, nhm bydc liu trng tiLt,c Nng v Ha 37 Bnh c v ng nh, sau v ng l v ngt; cn mu GCL ngt th c v ngt nh ng tn gi ca n. Khi tm hiu qua cc ti liu, chng ti thy rng: dc liu GCL thuc loi G. pentaphyllum (Thunb.) Makino u tin c s dng Nht Bn lm cht to v ngt [50]. Sau ny ngi ta tm ra nhng tc dng mi ca dc liu ny nh l mt v thuc b v mi hn l tc dng lm gim lipid mu, h cholesterol, chng oxy ha v c hot tnh chngungth[7], [8], [11],[13].DovyrtnhiunghincuctinhnhtrnloiG. pentaphyllum (Thunb.) Makino, nhiu ch phm t loi ny c sn xut v a ra th trng. Lc ny, bn cnh GCL c v ngt cn xut hin dc liu c v ng cng thuc loi G.pentaphyllum (Thunb.) Makino. Do vy vic phn bit c cc dc liu GCL c v ng v v ngt bng SKLM rt c ngha gp phn tiu chun ha ngun nguyn liu ny. Saukhinh tnh 13 mudc liu G.pentaphyllum (Thunb.) Makino, nmmui dinchoccnhmc la chn tip tcnh tnh so snhvimtschtchun(Rb1,Rg1),GypenosidTrungQuc,haich phm ch trn th trng (ch Gio c lam Ba Tri v Gio c lam Tu Linh) v ba loi khc cng chi GynostemmaBlume (G.laxum (Wall.) Cogn., G. longipesC. Y. W v G.pubescens (Gagn.)C.Y.W).Nmmuc la chn gm c T3, 2, N, TR v GN. Kt qu l trn hnh nh sc k , sau khi hin mu, hai vt mu tm c Rf bng 0,82 v 0,65 xut hin trn tt c ccmuGCLtrngvthuhiVitNam,nhngkhngxuthin Gypenosid Trung Quc.Nhvy, c th kt lun hai vtnyctrngcho dc liu GCL thuc chi Gynostemma Blume trng v thu hi Vit Nam. Da vo gi tr Rf v mu sc cc vt cn li trn sc k , d dng nhn thy hai mu ch phm (Ch, TL) xut hin cc vt tng t cc vt ca nhmdc liu loi G.pentaphyllum (Thunb.) Makino thu hi tiLt, 38 c Nng v Ha Bnh. Nh vy c th kt lun nguyn liu sn xut hai ch phm ny t loi G. pentaphyllum (Thunb.) Makino. Sosnhtrctipdatrnhnhnhsck,muG.pentaphyllum (Thunb.) Makino trng ti Tam o (T3) tng t hai mu dc liu loi G. longipes C. Y. W. v G. pubescens (Gagn.) C. Y. W. S ging nhau ny ph hp vi s ging nhau v v ng ca cc mu dc liu ny. Trong ba mu trn, s ging nhau v hnh nh sc k ca hai loi G. longipes C. Y. W. v G. pubescens (Gagn.) C. Y. W. c th l gii do c im hnh thi hai loi nykhgingnhau,thmchtrongmtskhaphnloi,G.pubescens (Gagn.)C.Y.W.khngc xp thnh loi ring [14], [24]. Vit Nam hin nay cng c nhng kin khc nhau v vic phn loi gia hai loi ny. Ring loi G.pentaphyllum (Thunb.) Makino c nhngcim khc bit vihailoicnli.HnnatrnhnhnhsckccmuloiG. pentaphyllum (Thunb.) Makino trng v thu hi tiLt,HaBnh,c Nng u c hnh nh sc k khc bit r rng so vi hai loi G. longipes C. Y. W. v G. pubescens (Gagn.) C. Y. W. Do vy cn c nhng nghin cu thm v phn loi thc vt, vthnh phn ha hc v gen xc nh li chnh xc tn khoa hc ca cc mu dc liu trng ti Tam o. Mu GCL ngt v mu G. laxum (Wall.) Cogn. u c c trng bi hnh nh sc k ring, khng trng vi sc k ca cc mu khc nghin cu trong kha lun. KhisosnhccmuGCLtrngvthuhitiVitNamvimu Gypenosid trung Quc nhn thy hnh nh sc k khc nhau, do d c th kt lun mu Gypenosid Trung Quc c thnh phn ha hc khc vi cc mu GCL trng v thu hi tiVit Nam. Trong th nghim ny, hai cht chun Rb1 v Rg1 l hai saponin c cng b l c trong thnh phn ca cc loi thuc chi Gynostemma Blume nn 39 c chm trn cng mt bn mng v khai trin trong cng iu kin vi cc mu GCL trng v thu hi ti Vit Nam. Tuy nhin trn sc k ca cc mu GCL u khng xut hin vt c gi tr Rf v mu sc tng ng Rb1, Rg1. Nh vy c th kt lun cc mu GCL nghin cu khng c Rb1 v Rg1 hoc c th c nhng khng nm trong kh nng pht hin ca phng php SKLM tin hnh trong kha lun ny. 40 KT LUN V XUT 1. Kt lun: Sau mt thi gian tin hnh thc nghim, kha lun thu c mt s kt qu sau: khostc nhngiu kin ph hp tin hnh sc k lp mng nh tnh dc liu GCL vi nhm hot cht chnh l saponin. nh tnh c 13 mu GCL thuc loi G. pentaphyllum (Thunb.) Makino: - Trn hnh nh sck quan st nh sng t ngoicbc sng 254nm, 13 munyu xut hin mt vt c gi tr bng0,61.Nmmu Gio c lam trng ti Tam o (T1, T2, T3, B3, B4) c c trng bi vt c gi tr Rf bng 0,14. By mu GCL trng tiLt,c Nng v Ha Bnh (1, 2, 3, 4, N, TR, NN) c c trng bi vt c gi tr Rf bng 0,43. Mu GCL ngt cho hnh nh sc k vi cc vt rt nht. - Trn hnh nh sc k quan st nh sng thng sau khi hin mu bng thuc th vanillin acid sulfuric c 2 vt mu tm vi Rf bng 0,82 v 0,65 xut hin c 13 mu. Tng t khi quan st di nh sng t ngoi bc sng 254 nm, nm mu G. pentaphyllum (Thunb.) Makino trng ti Tam ou xut hin mt vt mu xanh c gi tr Rf bng 0,50; trong khi by mu ca loi ny trng Lt, c Nng v Ha Bnh u xut hin mt vt mu xanh c gi tr Rf bng 0,37; mu GCL ngt xut hin nhiu vt mu tm nhng rt nht. La chn c cc mu c trng cho loi G. pentaphyllum (Thunb.) Makino so snh vi cc mu G. laxum (Wall) Cogn., G. longipes C. Y. W, G. pubescens (Gagn) C. Y. W, Rb1, Rg1, Gypenosid Trung Quc v hai ch phm ch GCL Ba Tri v GCL Tu Linh. 41 -Trnhnhnhsckquanstnhsngtngoibcsng 254nm, tt c cc mu GCL v mu Gypenosid Trung Quc xut hin mt vt c gi tr Rf bng 0,61. Da vo gi tr Rf ca cc vt cn li, nhn thy hai mu ch phm ch GCL Ba Tri v Tu Linh c hnh nh sc k tng t cc mu GCL G. pentaphyllum (Thunb.) Makino trng ti Lt, c Nng, Ha Bnh. - Sau khi hin mu bng thuc th vanillin acid sulfuric, trn hnh nh sck ca tt c cc mu GCL thuc cc loi khc nhau thu hi ti Vit Nam u xut hin hai vt mu tm c gi tr Rf bng 0,82; 0,65. Hai vt ny khng trng vi Rb1, Rg1 v Gypenosid Trung Quc.Nhvyy l 2 vt c trng cho cc loi GCL thu hi Vit Nam. Hai ch phm ch GCL Ba Tri v Tu Linh c hnh nh sc k tng t hnh nh sc k ca loi G. pentaphyllum (Thunb.) Makino thu hi Lt, c Nng v Ha Bnh. Cc mu G.pentaphyllum (Thunb.) Makino trng Tamo xut hin cc vt tngt mu G.longipes C. Y. W v G.pubescens (Gagn.) C. Y. W. Hnh nh sc k ca mu GCL ngt v mu G. laxum (Wall.) Cogn khng ging cc mu khc. Tt c cc mu GCL thu hi Vit Nam khng xut hin vt Rb1, Rg1 v khng ging mu Gypenosid Trung Quc. 2. xut:Trong khun kh kha lun, loi G.pentaphyllum (Thunb.)Makino c nh tnh trn 13 mu nhng cc loi GCL khc mi c nh tnh trn mt mudc liu. Do vy, tngtnhthuyt phc cho nghin cu ny, chng ti xin a ra 3 xut nh sau: - Thu hi thm mu dc liu ca cc loi Gynostemma Blume ti cc a im khc nhau vo cc thi im khc nhau nh tnh bng sc k lp mng. 42 - Trin khai trn bn mng sc k hiu nng cao cc vt tch nhau r rng hn. - Nghin cu thm v c im thc vt, thnh phn ha hc v gen xc nh chnh xc tn loi GCL trng ti Tam o. 43 1 TI LIU THAM KHO Ti liu ting Vit 1. Trn T An (2006), Trngi hcDc H Ni, Haphntchtp2, trang 205 222. 2.AndrewchevallierFnimh(2006),Dcthotonth,NXBtnghp thnh ph H Ch Minh. 3. Phm Tun Anh (2008), Nghincuthnhphnhahcvmtstc dng sinh hc ca cy Gio c lam thu hi ti Sapa, Lun vn thc s Dc hc, Trng i hc Dc H Ni. 4. Vc Cnh (1999), Ngincuthnhphnhahcvmtstcdng sinh hc ca cy Gynostemma pentaphyllum(Thunb.) Makino, Cucurbitaceae, Kha lun tt nghip Dc s i hc, Trng i hc Dc H Ni. 5. V Vn Chi (1997), T in cy thuc Vit Nam, tp 2, tr 308 309. 6. V Vn Chi (1997), T in thc vt thng dng, tp 2, tr 1322, 1323. 7. Nguyn Tin Dn, Nghincuthnhphnhahcvtcdngsinhhc ca cy tht dip m, Lun vn thc s Dc hc, Trng i hc Dc H Ni. 8. Nguyn Th Thanh Duyn (2000), Tip tc nghin cu thnh phn ha hc vtcdngsinhhccacythtdipm, Kha lun tt nghipDcs i hc, Trng i hc Dc H Ni. 9. Nguyn Vn n, Nguyn Vit Tu (1985), Phng php nghin cu ha hc cy thuc, NXB Y hc, tr 243 289, 327 347, 42 57. 10. Phm Hong H (1999), CycVitNam, NXB Tr, quyn I, tr 563 575. 11. Phm Thanh Hng (2003), Nghin cu thnh phn ha hc v tc dng sinh hc ca cy Gio c lam, Kha lun tt nghip Dc s i hc, Trng i hc Dc H Ni. 2 12. Kurt Randerath, Nguyn Hu By, Trn Trung Nam (dch), (1974), Sc k lp mng, NXB Y hc, tr 19, 22 45, 59 126. 13. Phm Thanh K, Phan Th Phi Phi, Phm Th Thu Anh (2007), Nghin cutcdngtngpngmindchcadcliuGioclam, (Gynostemma pentaphyllum (Thunb.) Makino), Tp ch thng tin Y dc (5). 14. Phm Thanh K (2006), Thc tp Dc liu, Kim nghim dc liu bng phng php ha hc, Trng i hc Dc H Ni, tr 14 15. 15. Trn Vn n (2004), Thc vt dc v phn loi thc vt, B mn Thc vt, Trng i hc Dc H Ni. 16. Trnh Vn Qu, Nguyn Vn Tu (2009), Ch tch Hi ng Dc in Vit Nam, Dc in Vit Nam IV, mc 5.4, PL 130, PL 13, chuyn lun dc liu, v thuc nhn sm, tam tht. 17. Phan Th Tho (2010), Nghincucimvihcvthnhphnha hc ca cy Gio c lam thu hi ti Ha Bnh, Kha lun tt nghip Dc s i hc, Trng i hc Dc H Ni.18. NgVnThu(2006), BigingDcliutpI. B mnDc Liu, Trng i hc Dc H Ni. Ti liu ting Anh 19.Arbain,D.,etal.(1989),SurveyofsomeWestSumatranplantsfor alkaloids. Econ. Bot. 43 (1): p. 73 - 78. 20. Arshad Hala, HPTLC Instrumentation: An overview, Seth GlBihani SD college of technical education, Institute of pharmaceutical Science and drug research gagan path, Sri ganganagar, Rajasthan 335.001, India. 21. Chinese pharmacopoeia commission, Beijing 100061, China, TheChinese Pharmacopoeia 2010. 22. Elmer Drew, Merill (1904 1992), Note worthy Pilippine plants, Bureau of Public Crinting, Manila. 3 23.TheodoreAlbertGeissman(1962),Chromatographicmethod,The chemistry of flavonoid compounds, Macmillan, pp 32, 35 45. 24. Guo, X. L., T. J . Wang, et al. (1997). Studies on the chemical constituents of Gynostemma longipes C.Y. Wu. Yao Xue Xue Bao32(7): 524-529. 25. Home Nanzhu (2001), Dictionary of seed plants name, Latin Chinese English, Science press China. 26.Huang,S.C.,etal.(2008),Determinationofchlorophyllsandtheir derivatives in Gynostemma pentaphyllum Makino by liquid chromatography-mass spectrometry. JPharm Biomed Anal. 48(1): p. 10. 27.Instituteofscienceandtechnology(1922),ThePhilippinefournalof Science vol. 20, Bureau of printing, Manila. 28. J iang, W., Zhou, Y.; Li, J . (2006), Assaying total flavonoids in 6 kinds ofGynostemmamade in Guangxi. Zhongguo Yaofang. 17(1): p. 74-75.5-12. 29.Kao,T.H.,etal.(2008),Determinationofflavonoids andsaponinsin Gynostemma pentaphyllum (Thunb.) Makino by liquid chromatography-mass spectrometry. Anal Chim Acta. 626(2): p. 200-11. 30. J ung Hea Kim, Eun J ung Chang and Hoon-Il Oh*, et al. (2005).Saponin production in submerged adventitious root culture of Panaxginseng as affected by culture conditions and elicitors , Asiapacific journal of molecular biology and biotechnology, 2005. Vol. 13 (2): 87 91. 31.Kuwahara,M.,F.Kawanishi,etal.(1989).Dammaranesaponinsof GynostemmapentaphyllumMakinoandisolationofmalonylginsenosides-Rb1,-Rd,andmalonylgypenosideV.Chem.Pharmaceut.Bull.37(1):135139. 32.Liu,X.,W.Ye,etal.(2004).Fivenewocotillone-typesaponinsfrom Gynostemma pentaphyllum. J . Nat. Prod67: 11471151. 4 33. Mackay, M., J . Wei, et al. (1991). Structure of a new dammarane-type triterpenefromGynostemmapentaphyllum(Thunb.)Makino.Acta Crystallogr. Sec. C: Crystal Struct. Commun.47: 790793. 34. Marino, A., Elberti, M. G.; Cataldo, A. (1989).Sterols from Gynostemma pentafillum. Boll Soc Ital Biol Sper. 65(4): p. 317-9. 35.Qin,Z.,L.Zhao,etal.(1992).Saponinconstituentsandresourceof Gynostemma pentaphyllum. Tianran Chanwu Yanjiu Yu Kaifa4(1): 83-98. 36. Razmovski-Naumovski, V., R. Duke, et al. (2005b). (20S), 2a; 3b; 12b- Trihydroxydammar-24-ene 20-O-b-D-glucopyranoside (GynosaponinTN1) as the 2,5-methanol solvate.Acta Crystallogr. Sec. E61(5): o1239o1241. 37.PiiSalo(2007),Thinlayerchromatographywithultravioletandmas spectrometric detection from preparative layer to miniaturied ultra thin layer technique , page 16 18, 26 28, 35 39. 38.ManmohanSrivastava(2011),Highperformancethinlayer chromatography, chapter 3, part 2, pp 41 54. 39. Mary F. Striegel, J o Hill (1996), Thinlayerchromatographyforbinding media analysis, chapter 2 pp 19 24, chapter 3 pp 25 35. 40.Sun,W.,Z.Sha,etal.(1993).Saponinconstituentsof Changgengjiaogulan (Gynostemma longipes). Zhongcaoyao24(12): 619-622. 41. Surendar. M*1 , Sai Kumar.P1 , Shyam Prasad.T1 , Madhava Reddy. A1 , Nagulu.M2,HariPrasad.P2,ShaikShabber2,VamshiSharath.K2,etal. (2011).Extraction, isolation and purification of saponins from herbal plants . Herbal tech industry / September 2011, page 121; 131; 141; 151; 161; 171. 42. Takemoto, T., S. Arihara, et al. (1983a). Studies on the constituents of GynostemmapentaphyllumMakino.I.StructuresofGypenosideI-XIV. Yakugaku Zasshi103(2): 173-185. 5 43. Samu Tanaka, *,a Eyong Chae Han,b Hiroyuki Yamaguchi,c Hiromichi Matsuura,cToshiyukiMurakami,dToshioTaniyama,dandMasayuki Yoshikawad (2000), Saponins of plants of Panax species collected in central Nepan, and their chemotaxonomical significane. III. 1). Chem.Pharm.Bull. 48(6) 889 892 (2000). 44. J ianyong Wu*, Lidong Lin, Foo tim Chau, et al. (2000).Ultrasound assistedextractionofginsengsaponinsfromginsengrootsandcultured ginseng cell , ultrasonics sonochemistry 8 (2001): 347 352. 45. Wu zhengYi, Peter H. Raven, Hong Deynan (2008), Flora of China, vol. 19, Science Press China. 46.Yang,X.,etal.(2008),Isolationandcharacterizationof immunostimulatorypolysaccharidefromanherbtea,Gynostemma pentaphyllum(Thunb.) Makino. JAgric Food Chem. 56(16): p. 6905-9. 47.Yang,Z.,Q.Chen,etal.(2007),Dammarane-typeglycosidesfrom Gynostemma pubescens. Phytochemistry68(13): 1752-1761.48. Yin, F., Y.Zhang, et al. (2006),Triterpene saponins fromGynostemma cardiospermum, JNat Prod69(10): 1394-1398.Yoshikawa, K., S. Arihara,et al. (1991). Dammarane saponins from Gynostemma sylvestre. Ti liu ting Trung Quc 49. Blumert M and Liu J ialiu (1999), Jiaogulan: China'sImmortalityHerb, Torchlight Pub., Badger, CA. 50.DingShuli,ZhuZhaoyi(1992),ResourcesofgenusGynostemmaand determination of their total saponins contents, Zhongcaoyao (Chineseherbal medicine), 23(12), 627 9.Ti liu t internet 51. http://www.kobis.si/material/DigiStore2brochure.pdf.