HOI NGH! KHOA HQC C(!)NG N G H f SiNH HQC T O / " ' ^'
NGHIDN CU'U CHIET XUAT VA BU'CITC DAU KHAO SAT HOAT TINH KICH THICH MIEN DICH VA DOC TINH CAP COA MOT SO DICH CHitT TCf L^ e u eCi « {Carica papaya L.)
05 Thj Hoa Vi6n
Vidn Cong ngh$ Sinh htfc va Cong ngh$ Thirc phim, T/uting Dai hqc Bach t^oa HS Not
T 6 M TAT
Quy trinh cong nghe vci cac tbong s6 thicb hgp dc chifit xuat cic chit phin c\rc, flavonoid, s^wnin vi alkaloid tii Ii du d i {Carica papaya) da Avqc lya chon. Him lugng cac nhom chit trong li du du (tinh theo % nguyen Ii§u kho) giam din tii cac chit phin c(rc, alkaloid, flavonoid va s^Kniin; ham lugng uong iing cua cac nhom chat trSn la 6,02%; 3,40%; 1,67% vi 0.11%. Trong bon nhfim chit (cic chit phan cyc, alkaloid, flavonoid vi saponin) da thii ng^l^m hojt dnh kich thich mien dich thong qua khi nang Idch thich su phit tii&i cua tc tiio lynyhocyle lich chufit, chi cfi nhfim cic chit phSn c\rc va saponin li cfi hoat tinh. SD50 (lieu kich diich 50%
sy phit tri^n ciia tfi bio lynqihocyte) ciia cac chit phan cyc vi saponin tirong ung li 287,87 vi 192,99 pg/ml. Cic chat phan cite vi cic alkaloid chiat xuit tir li du du khfl khong co dgc tinh cip v6i chugt 6 lieu da thii nghiem (10000 mg ho?t chat/kg thfi trgng).
7i> khoa: Alkaloid, cac chit pban cyc, flavonoid, saponin, dgc tinh cip, ho?t tinh kich thich miSn dich.
MdOAU
Du dO cd t6n khoa hgc la Carrca papaya, thupc hp du du Papayaceae, c6 ngu6n goc h> cdc nirdc Taing Wj, vk sau d6 duo'c hfing ph6 biSn * c4c nmSfc nhi#t ddi vk c$n nhi^t d6i. Chiing c6n din?c gpi Ui "Paw Paw" * Australia v^ New Zealand, vk khflng c6 lign quan gi voi cSy Paw Paw it Bkc My - c6 t6n khoa hpc Id Asimina tribola.Nkm 1962, bai thu6c ch&a b$nh ung thu tir Id du du 6k dirge ong Stan Sheldon dr viing Gold Cost. Australia si> dgng d l chOa lanh b^nh ung thir phdi. Sau dd, dng Stan Sheldon da phd bi^n cho mOt s6 b$nh nhkn ung thu- khdc vd 16 ngudi nira da dup'c chira khdi b^nh. Din ndm 1978, dng Stan Sheldon Ok tilt 1$ cho bdo chi bill, vd cSu chuy$n cCing bdi thu6c dCing Id du dO chOa Idnh b6nh ung thu ciia dng da dup'c ddng trdn td t^p chi Gold Cost Bulletin vdi nhan d l 'PawPaw Cancer Plea Bears Fmit", vd cung dd dirge de c|p trong quyln sdch "Papaya The Medicine Tree* (Harald W. Tietze, 1997). Sau dd, cdc bdc sT Australia dd thir nghl^m vd cdng nhan cdng hi$u cOa la du dii trong dilu tri ung thu.trong s6 dd (x> dng Betty Ellingsworth, gidm d6c b$nh vi|n Nerang Nursey vd dng Vem Forest cCing mOt sd chuydn gia ciia b$nh vien Labrator (Hulda Clark. 2010). Ngodi cdc t h i ddn Australia, cdc th6 dan Mehico cQng Ok sir dgng la du du d l dilu tri ung thu. G^n ddy, mOt s6 nhd khoa hpc d Trung tdm Ung thu, Dai hpc Florida, My vd D^i hpc Tokyo. Nhdt Bdn da hp'p tdc vdl nhau d l nghidn ci>u v l khd ndng phdng ching ung thu cOa Id du OQ. Kit qud cdng b l ciia hp da chl ra rang djch chilt nude cua Id du dCi cd khd ndng i>c c h l su phdt triin cua 10 ddng t l bdo ung thu thip nghipm. Khi cho 10 loai t l bdo ung thir khdc nhau tilp xOc vdi djch d i i l t ben, 24 gid sau cd t h i thdy tic dd phdt triin cua t l bdo chdm hin Igi, vd neu ning dO chit chilt xuat cdng cao. h i ^ qud khdng vd di§t t l bdo ung thucdng rd r#t. Trong mpt thi nghi§m tilp theo, cdc nhi khoa hpc cdn phdt hl^n ra cdc hogt chit chiet xuit tir Id du dCi cd khd ndng thiic diy sg sdn sinh ra te bdo lympho Thi - t l bdo ddng vai trd quan trpng trong h^ miln djch ciJa nqudi(Nonho Otsuki, Nam H. Dang et al., 2010). CGng trong nghidn cdu ndy, cdc tdc gid Ok cdng b l ring cdc hogt chat chilt xuit tir Id du dii cd tac dgng khdng ung thu, nhung khdng gdy ddc d l i vdi cdc t l bdo Idnh tinh khdc.
Ld du Oil cd chi>a cdc alkaloid nhu carpaine, pseudocaqjaine. dehydrocarpaine vd choline. Ngodi ra, trang Id du dD cfin cd saponin, carposide, myrosin. rutin, resin, tannins, flavonols, benzylglucosinolate, linalool, malic acid, methyl salicylate, enzym papain vd chymopapain, caici, sit. magne, mangan, photpho, kali. Kem, beta-caroten, cdc vitamin nhdm B, vitamin A, vitamin E vd vitamin C (bt)ng Id cao hon trong qua).Ndm 2007, mdt nhdm nhd khoa hpc d Italia da cdng b6 ring trong djch chilt methanol cua Id du dO cd chii-a cdc phenolic acid nhu caffeic add (0,25mg/g Id khd). coumaric acid (0,33 mg/g), protocatechuic acid (0,11 mg/g), kaempfenal (0,03 mg/g), quere^etln (0,04 mg/g). 5,7-dimeUioxycoumarin (0,14 m^/g)(/V.U. Ogan, 1971).0' Vi§t Nam. trong cdc bdi thulc chi>a ung ttiu luu tnjyln ddn gian, cd bdi thulc u6ng nude sac td du dil de hB trp dilu trj ung thu, dgc bi§t la ung thu phii vd ung thu vO. Nam 1998. bd Ld Thj Ddng, bdc si Bd Cdng An, d thdnh phi H6 Chl Minh da si> dyng bdi thulc Id du dCi md dng Stan Sheldon - ngudi Oc Oa tir chCra khfii b§nh ung thu phoi thdng qua tdi li#u cOa Oc md bd thu thgp dupe d l dilu frj cho chong bd Id dng BCii Hoan, 80 tuii, b|
ung thu ludi piai dogn cudi. Sau mdt thdi gian dieu trj, dng Hoan da khoi bgnh ung thu, si>c khde hii phyc, vd dng da sing dugc den 89 tuii. Tir ndm 2006, trdn bao Si>c khde vd Ddi sing, cd mpt sd bdi viet cila Gido su Nguyin Xudn Hiln, nguydn Tnrdng Khoa Da lilu, B§nh vi$n Qudn y 108 dd ndi v l tdc dgng cue Id du du d l i vdi b§nh ung thu. GS.
Hien cQng dd cd dugc bdi thulc do bd Le Thj Ddng cung cap. GS. dd khuyen cdo vd pho cdp cho nhilu bgnh nhdn dang mac b§nh ung thu(Ld ttij Ddng, 1998, Nguyin Xudn Hiln, 2006).
Nghien ci>u ciia chOng tli nhim myc tigu gdp phin ldm sang td ca sd khoa hoc ciia viec su dyng Id du Oil trang h8 trp dieu tli ung thu(Do Thi Hoa Vien, Phung Thi Thuy, 2012; Do Thi Hoa Vien, Ho Thi Ha, 2013). Trong phgm vi bdi bdo nSv, chOng tdi da su dgng mdt so dung mdi ddc higu d l chilt xuit 4 nhdm hogt chit chinh trong id du du Id nhdm cdc chat phdn cgc, flavonoid. saponin vd alkaloid; ding thdi budc diukhao sdt in vitro khd nang dilu hda miln djch vd Ihi) nghidm in vivo ddc tinh cap cua cdc nhdm hogt chit tren.
NGUYEN LIEU vA PHU-ONG P H A P Nguydn ligu, hda chit
La du dCi dugc ttiu hdi d xa Ddng Hdi, huygn Ddng fivnh. Hd Ndi; sau do duge say khd vd xay nhd.
Hda chit nghidn cuu: Hexane, dichloromethane, ethanol, methanol vd cac hda chit khdc diing trong cdc phdp thi*
_T.,.-.; — r ; ; ;QC C O N G N G H f SINH HOC T O A N Q U O C 2013
nghigm hogt tinh kfch thich miln djch, ddc tinh cap do hang Sigma cung d p . Chupt BALB/C.
Phiromg phdp nghiin ci>u Chiet xuat cic chit phan c(fc
Chilt logi cdc hgp chit khdng phan cgc bang CH2CIZ100%, t^ le nguydn lieu/dung mdi = 1/3 (g/ml). Chiet 3 lan trdn may lac. mli l l n 24 gid d nhigt dd phdng, Ipc chdn khdng Ihu djch chilt, cd quay chdn khdng de ttiu hii dung mdi. Phan ba chilt d l hting tu hut cho tiay hoi hit dk;hk)romethane, roi tiep tyc ehilt bdng metanol 100% a nhidt dd phdng. ty lg nguydn li^u/dung mdi = 1/3 (g/ml). Chilt 3 lln trin mdy lac, mli lln 24 gid. Lpc chdn khdng thu djdi ehilt, cd quay chan ktidng den hit dung mdi. Sau dd tiln hdnh sly ddng khd de thu bpt ehilt cdc chat phdn cgc(K. Hostedmann et al., 2000).
Chiet xuat cic hij/p chat flavonoid
Sir dyng bpt ehilt cdc hpp chit phdn cgc tir la du dd d i chilt cde hgp chat flavonoid theo quy frinh cdng nghe nhu sau:
hda tan bdt chilt trong nude ndng 40°C, sau dd chiet vdi ettiyl acetate, thu djch chilt euiyl acetate roi ed quay chdn khdng d l ttiu hoi dung mli. Tiln hdnh sly ddng khd djch chilt dd bay hoi het dung mdi d l thu bOt chilt flavonoid thd todn phln(Lgi Thj Kim Dung, 2004).
Chiet xuat cic hop chit saponin
Sir dung bdt chilt cdc chit ptidn cgc tir id du dij de tiep h,ic chiet xuat cdc hgp chit saponin ting s i . Quy frinh chilt xuit dugc thgc hign nhu sau: hda tan bpt chilt cde hgp chat phdn cue vdi nude elt, sau dd d i i l t Idng Idng vdi n-butanol, djch ehilt n-butanol dugc cd quay d l thu hii dung mdi vd ttiu egn ehilL Tiln hdnh sly ddng khd cdn chilt de Uiu nh|n saponin ttid todn phln(K. Hostettmann et al.. 2000).
Chiet xuit cic /ipp chat alltaloid
Chilt xuit edc alkaloid nhu sau: Id du dii khd xay nhd, chilt bing ethanol, sau dd boc hoi dung mdi bing cd quay chdn khdng d l thu can chilt kilm hda egn chilt bang K^COa din pH 8.5; rlj chilt tiep bang dichloromethane, Idm khan nude djch ehilt dichloramethane bing NazS04. Cd quay chan khdng d i thu hii dung mdi vd thu egn chilt chira edc hgp chat alkaloid, sau dd sly ddng khd de ttiu bdt chilt cdc hgp chit alkalokJ thd(Nguyen Tudng Vdn et al., 1983).
Xie Olnh h09t Hnh kich Otich miin tfjch ciia c^c nhdm tm^t chit
Nguyen tae eua phuong phdp: Ddnh gid hogt tinh kleh thich miSn dich (khd ndng kich thich su phat trien ciia t l bao lympho Idch chudt) ciia cdc hogt chit Uieo phuong phdp MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphen^tetrazollum bromide)(Mosmann T., 1983).
Xie 6lnh d^ tfnh cip cua dc nhdm ho^t chit
Nguyen tac ciia phuong phdp: Xdc d|nh dp dpc d p eua d e hogt chit ttidng qua lilu gdy chet 50% dpng vdt ttii nghidm LD50 (Lettial Dose). Ddng v$t ttil nghiem Id chudt B/U.B/c.Cdng thue tinh LD50 nhu sau:
LOurXk-d/Z-d/nxZm,
Trong OOLDsd^ liiu gky chet 50%
ddng v$t thi nghidm; n: s6 ddng v$t su di/ng trong timg 16 thi nghi$m;k:
s6 Id d0ng v$t; mf s6 dgng v$t chit dim theo timg 16 trong 72 giir, d:
khoing cdch giiia cdc mdc liiu thudc, Xu: liiu thuic thap nhat giy cMt 100% d6ng v$t thi nghidm Can dr theo tidu chuin eua to chuc OECD, niu hogt chit Uii> nghidm ed LOso Idn hon 2000 mg/kgP thi dirge xem Id an toan va khdng cd dd ddc d p ttnh.
K^T QUA V A T H A O LUAN K i t qud chilt x u i t
Chiet xuit cic hpp chit phan C(re; Khdo sat d c yeu to dnh hudrng din hi$u qua chilt xuat nhu kich ttiudc nguydn lidu. then gian ehilt, t^ Id nguydn li$u/dunc| mdi bdng phuong phdp t l i uu co diln
Chiet bang dichloromethane (3 Sn x 24 h/1 lan, NL/DM = 1/3. r = 30°C|
i
Dich chiet dichloromethane
Chift bine methanol^ l3n x 24 h/1 lan, NL/DM = 1/3. T' = 30°cl
3 :
tflnh 1. Quy trtnh dilet xuat cic h^p chat phan ci^c
HOI NGH! KHOA HQC C O N G NGH? SINH HQC T O A M ?
dga vao lugng chat chiet Uiu dugc. Ket qua khdo sdt eho ttily: cdc ttidng s i ky thudt ttilch hgp eho qud trinh chilt edc chat phdn cgc tif cuing td du dii khd Id d hai cdng dogt chiet bdng dichkiromethane vd methanol diu dugc ttigc hi$n trdn mdy ldc, d nhidt dp phdng, nguydn lidu dugc xay nhd din kich thudc 1,5 mm; chiet 3 tin (24 gid/1 lan), ty lg NL^M d a mli l l n chilt Id 1/3.Quy frinh ehilt cdc hogt chit phan cgc tir Id du dCi duge md td nhu trong Hinh 1.Chilt cdc chit phdn circ theo quy trinh cong ngh§
vdi d c ttidng s i ky ttiudt ttiieh hgp nhu tren. Tlln hdnh ehilt 3 lan,jly kit qud tmng binh. Lugng b6t chiet cic /lo^f chit fOian cgc tfiu 6it^ la 6,02 % so vdi khli lugng nguydn lidu khd ban dau.
Bdt ehilt d c chit phdn cgc dugc bdo qudn ttong Ip nOt nhdm mdu ndu, d nhi#t dp -20''C; vd dugc tilp tyc so dgng d l chilt cdc hogt chit flavonokl, saponin vd Uiu nghigm cde hogt tinh sinh hpc.
H6a u n bdt chift trong nirdc n6ng 40°CW '$ NL/HjO = 1/10 g/ml)
i
Chiet bing ethyl acetate(2 iSn chiet, ty 1$ didi chi^t ninic/DM = 1/1)
Hlnh 2. Quy trinh chi^t xuSt dc hpp ch£t flavonoid
Chiit xuat cic hgp chit navonofd:Sir dyng bit chilt edc hgp chit phan cgc d l chiet cdc hgp chit flavonoid theo qiw frinh d n g nghg neu d phan phuong phdp nghidn ciiu. Tlln hdnh khdo sdt mOt s6 thdng s i ky ttiugt dnh hudng din kit qud chilt xuit nhu: ty 1§ giua bpt chilt cdc hpp chit phdn cgc vdi nude ndng 40°C, ty 1$ giira djch chilt nude vdi dung mli ettiyl acetate, vd s i lln chilt idng Idng bdng ethyl acetate. Tidu ehl ddnh gia la lugng chit ehilt ttiu dugc. Qjjy trinh ehilt xuit d c hgp chit flavonoid dugc md td nhu ttong hlnh 2.Ap dgng quy ttlnh chilt xuit nhu d Hlnh 2, Uen hdnh chiit xuat 3 lln vd liy kit qud frung binh, ttiu dugc b^t chiit flavonoid th6 v6f him Iwgng la 1.67 %Jfio vdi nguyfin ligu khd). Bit chilt cde hogt chat flavonoid dupe bdo qudn trang Ip nirt nhdm mdu ndu, d nhigt dd -20''C; vd dugc sir dyng d i thu nghiSm d c hogt tinh sinh hpc.
Chiet xuit cic hgp chat a/fta/oW:Chilt xuit d c alkaloid thd dugc thgc hign nhu fla md td d phin phuong phdp nghidn ci>u. Vide chilt xuit bing cdc dung mli ethanol vd dichloromettiane dugc thgc hign ttdn mdy lie, d nhi|t d0 SO'C. —
d ^ ^ ^ ^ l S flu ail xav nho (kich Ihi/dc 1,5 mm) ^ ^ ^ J ! ! ?
Oiiet bSne ethanol 80 iUH IS mJH,0 = 1/10 e/ml. chiet 3 \ln x 24 h/1 ISn)
Boc hcfi dung moi thu c^n chi£t ethanol
Kiem hoa bine dune djch K,COi 10 % a4n pH 8.5 Chiet bang dichloromethane!ty Ig NL/OM = 1/2 mg/ml, chiet 3 lan)
o djch chiet 6i I091 cac thanh ph^n tan trong nu'd'c(3 lan, ty l | dich chiet/H]0 = 1/3)
DjCh chiet dichloramethane
Lam khan nird'c bane NajSOj
Sav done kho
••=dl|^___ Alkaloid tha toan ph3n __,JZ—^^^"
Hmh 3. Qi-y trinh chiet xuat cac h(rp chat alkaloid
IQC C O N G NGHE SINH HQC TOAN QUOC 2013
Tiin hdnh khao sdt d l lua dipn edc dilu kidn ehilt xuat thich hgpbing phuong phdp t& uu d diln (ttiay d l i ttidng so d n khdo sdt vd d d|nh cdc ttidng so khdc). O l i vdi eac cdng dogn ttiu 4 (kilm hda e^n chiet ettianol) vd edng dogn Uiu 6 (rua djch cNit dichloromethane bing nude dt):ehpn d c thdng so k^ thu$t theo nhu tdi ligu tham khao.
Vdi d c thing s i ky thudt dd dugc lua chpn, quy ttinh chiet xull cde alkaloid tit Id du dd khd dugc tdm tat nhu bxing htnh 3.Ap dung quy trinh chiet xuat nhu trong Hlnh 3, tiln hdnh chilt xuit 3 lln vd tinh kit qua trung binh, thu dugc b^t chiit cac hgp chat alltaloid tir li du (ft) vcri ham ftegng 3,40 % (so viri nguyen li$u kho). Theo d c tai ligu da cdng b l . nhdm alkaloid cd trong Id du du gom d c hogt chat carpaine vd cac ddn xuit methyl carpaine, ettiyl carpaine, carpaine chloroaurate, carpaine hydrochloride, nitro- carpaine; pseudocarpaine; dehydrocarpaine I vd II; choline vd earposide(A.U. Ogan, 1971;
Nguyin Tudng Vdn et al, 1983). Ket qud
Bgt diiet cac h<n) chat phan cvc
Hoa tan bqt chiet trong nu^c c£t(ty )# NI/H2O = 1/10 g/ml)
- butanoi{2 lan chiet, ty l# djdi chiet nuac/DM = 1/1)
1
Hlnh 4. Quy trinh chi^t xu£t cac hop diKl saponin
ehilt xuat eho thiy cdc hgp chat alkaloid thfi todn phan d mgt trong Id du du vdi hdm lugng khd cao. va phii hgp vdi d c kit qud dd cdng bo.Bdt alcaloid dugc bao quan trong Ip niit nhdm mdu ndu, 0 nhigt dd -20°C.
Chiit xuit cac hgp chat saponin: Sd dyng bdt chiet cdc ctiat ptidn cgc tu Id du du de tilp tje chiit xuit cac hgp chit saponin ting s i . Quy trinh chilt xuit dugc thgc hi$n nhu dd md td d fren. Tiln hdnh khdo sdt mpt sd ttifing s i k^ thudt dnh hudng din kit qua chiit xuit saponin nhu:ty tg giOa bdt chiit cdc hgp chit phdn cgc vdi nude d t , ty lg giua didi ehilt nude vdi dung mdi n - butanol, vd s i lln chilt long idng bdng n - butanol. Tidu chi ddnh gia Id hdm lupng bdt chilt thu duge.Quy frinh ehilt xuat d e hgp chit saponin duge md ta nhu frong hinh 4.Ap dung quy trinh chiet xuat nhu trong hlnh 4, tiin hdnh chiit xuit 3 lln vd tinh k i t qua tmng binh, thu dugc bgt chiet cac hgp c/iat saponin tir 14 du dH v&i him lugng 0,11 % (so vdi nguyen ligu khd). Kit qud cho thdy cdc hgp chit saponin d mdt trong Id du dii vdi ham lugng thip, dilu ndy cung phii hgp vdi d e tdi lidu dd edng bo(A.U. Ogan. 1971; Nguyen Tudng Vdn et al, 1983). Bdt chilt cdc hogt chit saponin dugc bdo quan trong Ip niit nhdm mdu ndu, d nhigt dd -20°C; vd dugc sir dyng de thu nghidm d c hogt tinh sinh hoc.
Kit qud chilt xuit cdc nhdm chit phdn cge, flavonoid, saponin vd alkaloid duge tdm tat trong Bdng 1.
Bang 1. K4t qud chiet x u i t cdc nhom chat phan cgc, flavonoid, saponin vi alkaloid t u cuong vd ta du diJ kho Nh6m chat
Ham lirpngl* so won NL khfl)
Phancgc 6,02
Alkakiid 3,40
Flavonoid 1,67
Saponin 0.11 K i t qud thi> nghigm hogt tinh kich thich miln djch
Cde mSu thir nghidm: d e chit phdn cgc, d c alkaloid, d c flavonoid va d c saponin dugc thir nghigm tren t l bao lympho tach tu Idch chudt d i xdc dinh kha ndng kich thich miln dich.ChIt thd ndo d SI > 1,5 se dugc xem Id d kha ndng kleh thich mien djeh, Nfing dp kfch thich 50% sg phdt ttiin eiia te bao (SD - stimulate dose) se duge xdc ffinh nhd vdo phin mim mdy tinh TableCurve.Kit qua thir nghiem nhu trong t>ang 2.
Bdng 2. Khd nang kich thich te b&o miSn djch cua cac nhom chat t h u n g h i f m
N&ngd$ chilt t h i « « / m l )
lOOO zoo 40 8 SO«^Hg/ml)
SI (chl so kich thich t^ t>ao phat trien so vdi doi chumg am)
Phincire
1.835 1.472 0,994 0,932 287,87
Alkaloid
3,190 (mdu CUO chat nghiin cuu do bi l6ng]
1,275 1,063 0,953
Flavonoid
1,320 1,174 0,924 0.906
>1000 Saponin
2,127 1,481 1,187 0,965 192,99
Djch tot (d/c duumg)
1.492 ft none dp thir nghidm 10 mg/ml
HOI NGHI KHOA HQC C O N G NGHE SINH HQC T*^
Trong so 4 miu ttiu nghidm nhim kiem tra hogt tinh kich Ihfcti miln djch ttidng qua kha ndng kich hogi te bdo hmphfl Idch phdt ttiln in vitro tiii: djch chiit tir la du du hroi vd d e ehit chilt flavonoid khdng cd khd nang kich Uiieh mien^tdi d ning dd da ttiu nghidm; cde chat chiit phdn cge vd cac chit chiit saponin cd Idid ndng kich ttiieh t l bdo lympho p h ^ ttiln, ning dd kich Uifch 50% sg phdt triin d a t l bdo lympho cOa d c chit chiit phdn cgc vd d e chit chiit saponm I M lugt Id ^7,87 vd 192,99 pg/ml; d c ehit ehilt alkaloid d hogt ttnh rit y l u ndn ed ttii xem Id khdng cd hogt tinh. | K i t qud thir nghifm d^c tinh c i p
Kit qud d Bdng 1 cho Uily cdc d i l t phdn cge vd alkaloid la 2 nhdm chat cd hdm lugng ddng k i frong Id du dii khd; hon ttii nira kit qua ttiu nghifm d Bdng 2 cho ttily cac ehit phdn egc_d hogt Unh kich Bifch miln djeh, kit qud ttii> nghifm, hogt tinh gay ddc tt*n mft s6 ddng t i bdo ung Uiu cOng cho Uily ring nhdm ehit allralojd cd hogt tinh di$t vd 0^ chlcfc^
ddng t l bdo ung ttiu da Uii> nghifm Id t i t nhit ttong 4 nhdm chit chilt [19]; vl v$y nhdm d c chit phdn cgc vd nhIra,:
alkak>k] duge chpn d i ttiii- ngtuf m dpc tinh d p .
€)dc ttnh d p d a d c hogt dial phdn cge vd d e alkaloid dugc tien hdnh ttieo phuong phdp cda Abrham (1978) nhu da md td ttong phin phuong phdp. Sau khi dio uing d c hoat chit, chuft dugc ttieo ddi lidn tyc frong vdng 24 gid Oli vM d e biiu hifn chirc ndng, ding ttidi d i m s i lugng chudt ehit d h>ng Id ttong vdng 72 gid. K i t qud thu dugc v l tinh dfc d p cOa cdc hogt ehit phdn cgc dugc trinh bdy d Bdng 3 vd Bdng 4.
Bdng 3. D$c tinh c^p cda cdc boat chat phin cgc trSn chuot BALB/c
Ld
1 2 3 4 5
Lupng cdc hoat c h i t phdn ci/c (mg/kgp/lln uIng duy nhdt)
1000 2500 5000 7500 10000
S6 chu$t chet/sd chuQt s i n g sau 72 gid
0/6 0/6 0/6 0/6 0/6
B i l u h i f n chirc ndng trong v6ng 24 gid Chupt khoe manh, di c h u y i n , dn u l n g binh ttiirfrng,
phdn xg dnh s d r ^ vd dm ttianh t i t nt nt nt nt
Kit qud d bdng 3 cho ttily:tlt d d e Id diu khdng d ehuft bj chit dd uIng hogt chat d ning dp cao nhit 1OOO0 mg/kgP/lIn duy nhit. B i ^ hifn chi>c ndng ciia chudt thF nghifm eho thiy ring chupt d cdc Id thi nghifm uIng d !Ilu cao nhit 10000 mg/kgP v i n khde mgnh, di chuyin, dn uong binh thudng, phdn xa dnh sdng, dm thanh t i t vd kh&ig ob chu|t bj chit trong vdng 72 gid. Nhu vfy, d c hogt chit phdn cue ehiit xuat b> la du dti khdng t h i hifn dd dfe d p tSnti d cdc lieu thu nghifm ttt>ng ttil nghifm ndy vd khfing xac djnh duge LDso (lilu gdy d i i t 50% dfng vft thi nghifm).
Bdng 4. D$c tinh d p ciia cdc alkaloid trfin chufit B/U.B/c Lfl
1
2 3 4 5
Lupng aikalokj (mg/kgp/lln ulng duy nhit)
1000
2500 5000 7500 10000
56 chu^t chet/so chu$t s i n g sau 72 glcr
0/6
0/6 0/6 0/6 0/6
B i l u hien chi>c nang trang vdng 24 gid
Chu^t khod mgnh, di chuyin, dn u6ng binh thudng.
phdn xg dnh sdng vd dm thanh t6t nt nt nt
Chu^t khod mgnh, di chuyin, an uIng binh ttiirdng, phdn xa dnh sdng vd dm ttianht6t (ISng hcri xO) Kit qud ttiLP nghifm d bdng 4 eho ttily: t i t ca cdc Id diu khdng d chupt bj chit dii uIng hogt chit d nong df cao nlA 10000 mg/kgp/lln duy n h i t Bilu hifn chirc ndng cOa chupt ttii nghifmcho ttily: chupt d d c Id ttil nghifm uIng d liftu cao nhit 10000 mgftgP vSn khde mgnh, Idng hoi xii, nhung v i n di ehuyin, an uIng binh ttiudng. phdn xa dnh sdng,dm ttianh tot vd khfing ed chupt bj chit ttong vdng 72 gid. Nhu vdy, d c hogt d i i t alkaloid chill xuit tir Id du Oil khfing ttii hifn df dfe d p tfnh d cae lieu ttid nghifm trong thi nghiem ndy vd khfing xae djnh dugc LDso.
K^T LUAN
Hdm lugng d c nhdm chit ttong Id du du (% so vdi nguydn lieu khd) gidm din tt> phdn cgc, alkaloid, flavonokl vd cuS ciing Id saponin; d c gid &i hdm lugng tuong dng vdi d c nhdm chit frdn Id 6,02 %; 3,40 %; 1,67 % vd 0,11 %. Trongi mdu Uiu nghifm (ede hogt chit phdn cgc, d e alkaloid, d c flavonoid, d e saponin) nhdm kilm tra hogt tinh k[chlhj|
miin djch ttidng qua khd ndng kieh hogt t i bdo lympho lach phdt friln in vitro thi dil d nhdm d e hogt chit phdn c m d e hogt chat saponin cfi kha ndng kich ttilch t l bao lympho phdt ttiin. Ning d f kich thfch 50% su phdt triin d a t i H j lympho cfla cdc hogt chat phdn egc vd cdc hogt chit saponin lin lugt Id 287,87 vd 192,99 pg/ml. Cdc hogt c h i m cgc vd d c hoat chit alkaloid chilt xuit tu Id du du khd diu khfing gdy ddc d p tfnh doi vdi chuft d liiu dd Uii> nghifm I
C C O N G N G H $ SINH HQC TOAN Q U O C 2013
TAI U $ U THAM KHAO
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RESEARCH ON EXTRACTION AND PRELIMINARYSURVEY ON
IMMUNOLOGICAL STIMULATING ACTIVITY AND ACUTE TOXICITY OF SOME EXTRACTS FROMCARICA PAPAYAL. LEAVES
D o T h i H o a V i e n
School of Biotechnology and Food Technology, Hanoi University of Science and Technology
SUMMARY
Technological process and suitable fectors for extraction of polar conqioimds, tlavotioids, saponins and alkaloids from Carica papaya leaves were selected. The content of conqxiund groups in papaya leaves (% of dry material) decreased from polar conqiounds, alkaloids, flavonoids and saponins; the content ofthe atiove groups was 6.02 % , 3.40%, 1.67% and O.Il%Te$pectiveIy.
In four groups (polar compounds, alkaloids, flavonoids, saponins) of tested immunological stimulating activity through activating ce^Hibility of growtti oflynqihocytes of mouse spleen, only polar conqiounds and saponins were active.TheSD]o(stiinulating dose of S0% of development of lymphocyte) of polar confounds and sapoiiinswere287.87 and 192.99 pg/mlrespectively. The polar compounds and alkaloids extracted from dried papaya leaves were not toxic wilh tested mice in acute toxicity tesu'n vrwowith tested dose (10000 mg of con^unds/kgP).
Key words. Acute toxicity, alkaloid, immunological stimulating activity, flavonoids, polar compounds, saponins.
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