• Nghien CLFU - Ky thuat
n§ng nhung chi s i gan khong tSng vi k6m theo sy huy hoai gan Icim mit di nhieu th^nh phan trong gan. Lo dung thuic ton thuang nhe han, chu yiu la qua trinh viem ISm cho chi s i gan tang len. So sanh giu'a 2 16 dung thuic cho thiy 16 dung liiu cao chT s i gan giam so vo'i 16 dung liiu thip, chung to qua trinh viem 6' 16 nay diin ra nhe han so vo'i 16 dung liiu thip. Mat khac, khi dung FLPT eung cai thien dang k i hinh anh dai the, hinh anh vi t h i gan so vgi nhom gay dpc kh6ng dung thuic.
Kit qua nghien
CLPUeua chung toi phu hgp VO'I kit qua cua Yuan L.P., va CS '^' v i tac dung bao ve gan cua flavonoid toan phin cay dan kim.
K§t luan
Flavonoid toan phin cay dan kim dung liiu 60 mg/kg va 120 mg/kg co tac dung lam giam hoat dp cac enzym AST va ALT, lam tang chi s i gan va cai thien hinh anh dai thi va vi thi t i bao gan tren m6 hinh gay dpc bing CCU cc chupt thi nghiem.
Summary
The hepatoprotectivity of the total flavonoids of Bidens pilosa L. (a traditional medicinal plant of common and wide occurence in Viet Nam) was evaluated in carbon tetrachloride-induced liver injuries in rats. The total flavonoids
exhibited evident hepatoprotective effects at the dose range of 60 mg/kg and 120 mg/kg.
Keywords: Bidens pilosa, total flavonoid, hepatoprotective. • •
Tai lieu tham khao
1. Do Tat Lgi, Nhiing cay thu6c va vi thuoc Viet Nam, Nxb y/7pc,(2005)tr 120-121.
2. Vien Dugc lieu, Cay thuoc va dong vat la thuoc a Viet Nam, tap I, 816-818. Nxb KHKT, Ha Noi, (2004).
3. Vien Dugc lieu, PhLPong phap nghien CLPU tac dung dugc ly cua thuoc tCf thao dugc, Nxb KHKT, (2006), Ha NQL
4. Pham Van Vugng, Hoang Van Luong, Nguyin Van Long, Pham Thanh Ky, Nghien cuu thanh phan hoa hpc cay dan kim {Bidens pilosa .L, Astearaceae), Tap chi Y-DwQv hoc Quan Sw, Vol 34, N°9/2009, trang 94-98.
5. Pham Van Vugng, Pham Thanh Ky, Hoang Van Luong, Phan Van Kiem, Cac hgp chat flavonoid chiet xu§t t u cay den kim (Bidens pilosa L.), Tap chi Dwac hoc, so 409, thang 05/2010, trang 48-53.
6. Kathleen A.D, Hepatic principles. Tocxicologic emergencies, Jeanman M.Roche, (1998), pp 213 - 225.
7. Yuan LP., Chen F.H., Ling L, Dou P.F., Bo H., Zhong M.M., Xia L.J., Protective effects of total flavonoids of Bidens pilosa L. (TFB) on animal liver injury and liver fibrosis. Journal of Ethnopharmacology 116:(2008),p 539-546.
Nghien ciru dinh tinh, djnh lu'O'ng kaempferol trong la trinh nir hoang cung
bang Phu'O'ng phap HPLC
Dat van d§
San pham crila tCp cay trinh nu hoang cung (Crinum latifolium L, hp Thuy tien Amaryllidaeeae) (TNHC) da duge su dung trong diiu tri phi dai lanh tinh tuyin tiin liet va u xa tu eung'^'^'
Nguyen Nhat Thanh', Le Thi Minh Thao' Hoang Minh Chau^, Nguyen Thi Ngoc Tram^
'Trung tam Kiem nghiem T-MP-TP TP.HCM
^Khoa Duac, Dai hoc YDuac TP.HCM
^Cong ty TNHH Thien Duac
Nhim chuan hoa dugc lieu de gop phan nang cao t^c dung diiu trj eua san phim eo nguin gie tii TNHC chung toi da tiin hanh nghien
CLPUdjnh tinh, djnh lugng eae flavonoid trong la TNHC va chpn kaempferol la chit dac trung de dinh lugng
(2,3118 TAP CHI DUOC HOC- 11/2010 (SO 415 NAM 50)
Nghien CLPU - Ky thuat
D6i tu'O'ng va phu^ang phap nghien
CLPU
Ddi tuang nghien cu'u
M i u nghien cuu: La kh6 TNHC do C6ng ty TNHH Thien Dugc eung c i p duge tring 6" trang trai Long Thanh-Dong Nai
C h i t d i i chiiu: kaempferol cua hang Sigma- Aldrich, Thuy ST, LOT 1325550, dp tinh khiit 99,5 %, dp i m 5,83 %. May s i c ky long hieu nang cao SHIMADZU, detector PDA
Phu'O'ng phap nghien ciru Chuan bj mau
Mau thw: Can chinh xac 10 g bpt la TNHC, lam i m bing h i n hgp dung moi methanol - nuac (9:1) day kin, d i yen 1 gio'. Them 100 ml h i n hgp dung m6i methanol - nuac (3:1), l i e sieu am 30 phut, ngam 30 phut, lam 3 l i n , Ipc qua giiy Ipc. Ba dugc chiit 4 l i n nhu tren cho d i n kiet flavonoid. Djch chiit, eo each thuy d i n con khoang 50 ml, chiit loai tap vo'i n-hexan cho d i n h i t m i u . Dich da loai tap eo each thuy d i n eon khoang 25 ml, them vao djch chiit 25 ml acid hydrochloric 10 %, dun each thuy h i i luu 45 phut d i thuy phan flavonoid, c h i i t flavonoid aglycon bing ethyl acetat cho d i n kiet. Gop djch chiit Ipe qua natri sulphat khan, djch Ipe dugc e6 each thuy to'i c i n kh6. Hoa tan va chuyin toan bp c i n vao binh dinh muc 10 ml bing methanol HPLC, them methanol d i n vach, l i e d i u , Ipc qua giiy ipc, r i i ipe t i i p qua mang Ipc 0,45pm ( dung djch thCr).
Miu chuin: kaempferol pha trong methanol loai HPLC, CO ning dp chinh xac khoang lOOpg/ml.
Dieu kien sac ky
C6t RP18 (250 X 4,6mm; 5Mm), Detector PDA - 320 nm, Pha dpng: Acetonitril - Dem kali dihydrophosphat 100 mM, pH 3 vo'i chuang trinh rua giai nhu sau:
Bang 1: Chwang trinh pha dpng Thai gian
(phut) 0^20 20^30 30^55
B, J. .. -. % f^ei' phosphat, pH
% acetonitnl •>
20 80 25 75 30 70 Toe dp dong: 1,5 ml/phut. The tich tiem: 20 pi
Xay dwng va danh gia phwang phap djnh tinh kaempferol
Tien h^nh s i c ky theo d i i u kien da chpn, so s^nh s i c ky d i vS p h i h i p thu cua m i u thu v^
m i u chuin dya tr6n h$ so SI.
Danh gia phwang phap dinh Iwang Tinh thich hgp cua he thing sac ky, khoang tuyin tinh cua phugng phap djnh lugng, dp lap lai vS dp dung cua phuang phap, x^e djnh gi6'i han djnh lugng.
:i !~
Hinh 1: Sac ky dd cua chuin kaempferol
Hinh 2: Sac ky dd cua miu li TNHC
i'/iaiicisw :>fi-i'?.
TAP CHI DUOC HOC - 11/2010 (SO 415 NAM 50)
~ j,.»^ai-V^i4 ^ . . s-n-.-'.. iira^y^y-srs ;^«vEfv^-s.i, . . . . •. . . . Hem*.: Cbaaji kwiTifise-S
A -" i.tir. ^7f ^& i-5 l-^'J '
Hinh 3: Dwang biiu diin chi sd twang tw SI cua phd UV giwa chuin kaempferol va miu thw li TNHC
K e t q u a v a b a n l u a n
Xay diFng va danh gia phu'ang phap dinh tinh kaempferol
T i i n hanh s i c ky theo cac thong s i da lya chpn, k i t qua cho thiy e^e pie tach ro rang, nhiiu n i n t h i p , s i c ky d i cua m i u thu la TNHC cho pic CO eung tho'i gian luu (tp ~ 43 phut) vo'i pic eua kaempferol trong s i c ky d i eua m i u chuin. Tai tho'i gian luu cua pic kaempferol tren s i c ky d i thu va chuin cho p h i trung khft len nhau VO'I he s i SI la 0,9998.
K i t qua cho thiy pic tren s i c ky d i cua m i u thu id tinh khiit. Cac thanh phin khae trong m i u thu kh6ng anh huang d i n qua trinh phan tich kaempferol a cac diiu kien s i c ky da lya chpn.
19
Nghien CLFU - Ky thuat
Danh gia phu'ang phap Tinh thich hap cua he thong
Tiem 6 Ian dung djch kaempferol ning d6 100 pg/ml. Kit qua trinh bay 6' bang 2. He thing sic ky dat tinh tuang thich he thing, cac diiu kien sic ky va he thing sic ky phu hgp va dam bao sy on djnh eua phep phan tich djnh lugng kaempferol.
Bang 2: Kit qua twang thich he thing cua kaempferol
1 0 0 0 0 0 0 0 , o 8 0 0 0 0 0 0 -!
Q .
.g 6 0 0 0 0 0 0 c dOOOOOO
< ( U .
' Q 2 0 0 0 0 0 0 0
0
y = = 32805*x + 9361 R2 = 0^999
. •:• ^ • ^ • ' ' ' ^ ^
1 0 0 2 0 0
Nong do ( pg/ml)
3 0 0
Thai gian Iwu Dien tich pic So dia ly thuyet
He so bat doi
x"
43,64 3361993
13232 1,0161
RSD (%) 1,54 1,64 1,19 1,02
Khoang tuyen tinh cua phwang phap Kit qua trinh bay 6' bang 3 vS hinh 4, cho thiy CO sy tuang quan tuyin tinh giO'a ning d6 kaempferol va dien tich pie chuin kaempferol.
Khoang tuyin tinh nay phu hgp d i djnh lugng kaempferol.
Bang 3: Twang quan giwa ndng do vi dien tich pic
Hinh 4: Dwang bieu dien sw twang quan giO'a ndng dp kaempferol va tile dien tich pic thu dwac
Kit qua khao s^t cho thiy vo'i khoang ning d6 eua kaempferol tCp 25pg/ml din 250 pg/ml e6 sy tugng quan tuyin tinh giu'a ning dp va dien tich pie tugng ung.
Dp lap cua phu'ang phap
Dp lap.cua phugng phap dugc xac djnh bing each tiin hanh 6 thi nghiem rieng biet tren miu thu. Kit qua duge ghi tren bang 4.
Bang 4: Kit qua khao sit dp lap cua phwang phap
STT 1 2 3 4 5 6
Nong dp (pg/ml) 25 50 100 150 200 250
Dien tich pic 852505 1645150 3268333 4917436 6575317 8221831
Ten mau
S6 mau
Ham Iwang kaempferol
tim thay
Cac so lieu thong ke
La TNHC 225,98+11,34 pg/g
S=9,13;RSD:
4,0 %
Do dung cua phu'ang phap
Dp dung cua phugng phap dugc x^c djnh bing phuang phap them chuin, kit qua thu dugc ghi a bang 5.
Bang 5: Kit qua khao sat dp dung cua phwang phip
Ten mauMiu la TNHC
So miu lap Lwang them vao (pg) Lwang thu hoi (pg)
1 100 96,66 2 100 93,08 3 100 97,84
Ti le thu hoi (%)
95,86 ±6,15
So lieu thong ke n = 3 SD = 2,84 RSD=2,58
Kit qua khao sat cho thay, kha nang thu h6i lai cua phuang phap HPLC eo thi chip nhan duge vo'i phugng phap djnh lugng hoat chit eo ham lugng thip va eo thanh phin phuc tap nhu la trinh nO' hoang cung.
Xac djnh giai han djnh tu'ang
Tu' miu phan tich la TNHC, lin lugt pha loang miu thCp tung buo'c. Miu pha loang ti le 1/500 cho tin hieu dap Cpng e6 thi xac djnh duge so vo'i miu tring la methanol. Can eu tren dp lenh chuin eua miu tring va he s i goe eua duo'ng hii quy ning dp pha loang. Gio'i han
20
phat hien eua kaempferol trong miu phan tich dugc tinh theo c6ng thCpc''*':
LOD = F*SD/b Trong do:
F: he sd dwac tinh li : 3.3 cho LOD va 10 cho LOO
SD: dp lech chuin cua miu tring b: He sd gdc cua dwang hdi quy
Kit qua xac djnh dugc gio'i han phat hien kaempferol trong la TNHC (LOD) la 50,9 ng/g va gio'i han djnh lugng kaempferol trong \A TNHC (LOQ) la 169,8 ng/g. •:,.,:.:.-. ^.^ ;.;.
TAP CHI DUOC H O C - 11/2010 (SO 415 NAM 50)
Nghien CLPU - Ky thuat
Ket luan
K i t qua nghifen cuu d§ khao s^t xSc djnh c^c dieu ki0n phSn tfeh HPLC d i djnh tinh vd djnh lugng kaempferol trong la TNHC. Phuang phSp phan tich da dugc t h i m djnh dat yeu e i u v i tfnh dae hieu v^ d6 chinh xac, co t h i dung de xac djnh kaempferol trong 1^ TNHC.
Summary
A method for assay of kaempferol in Folium Crinum latifolium by HPLC was introduced. The chromatographic conditions were: Column - RP18 (5 pm; 250 x 4,6mm), detector - PDA at 320 nm. Mobile phase - Mixture of Aeetonitrile - 100 mM potassium dihydrogen phosphate buffer pH = 3.0. Elution was programmed in three steps (I, II and III) Aeetonitrile (A%) and Buffer (B%) and the respective eluent time (T minutes) as follows: I (A - 20% and B - 80%, T
= 0^20; II (A - 25% and B - 75%, T = 20-^30 and III (A - 30% and B - 70%; T = 30-^55);
Flow rate: 1.5 ml/min; Injection volume : 20 pi.
The proposed method was of satisfactory
selectivity and precision (recoveries = 95.86 %, repeatability » 4.0 %).
Tai lieu tham khao
1. Mai Dinh Tn, Nguyen Cong Mac.
Phenylpropanoid va flavonol glycosid dugc co lap tu la tuci cay trinh nu hoang cung (Crinum latifolium.L).
T^p chi Hoa hQC, (2005) 43(2), tr 162-164
2. Mai Dinh Trj, Nguyen Cong Mac, Le Viet Tien.
Mo* s6 hgp chat mai dugc co Igp tu la tuai cay trinh nif licang cung (Crinum latifolium.L). Hoa hoc the ky XXI vi sw phat trien ben vCmg - Tuyen t$p cic session: t$p III 2003/s6 6,tr 120-123
3. Nguyen Hai Nam, Nguyin Tien Vung. Phan lap va xac dinh cau true cua 2 flavonoid tu Crinum latifolium. L. Tap chi Dwg/c hpc 1/2006, tr 7-9
4. J. Ermer and J H.McB. Miler. Method validation in Pharmaceutical analysis, l/V/LEy-\/C/-/,2005, pages 101-119
5. Nam NH, Kim Y, You YJ, Hong DM, Kim MM, Ahn BZ. New constituents from Crinum latifolium with inhibitory effects against tube-like formation of human umbilical venous endothelial cells. Nat Prod Res, 2004 dec; 18(6), pages 485-491.
Hieu qua cua propyl galat trong vif c
• nu A
gift on dinh mot so hoat chat
CO tinh chat sinh hoc trong dau gac
Nguyen Tuang Vy, Tran Nguyen Ha
Truang dai hoc Duac Ha NoiDat van de
Hien nay dau g i e dugc s u dung r i t rpng rai tren thj trub'ng, do trong d i u g i e e6 nhiiu thanh phin hoS hpc nhu carotenoids, acid b6o kh6ng no v^ r i t nhieu cac nguyen t i vi lugng khac e6 tac dung hieu qua nhu 1^ thuic ph6ng Vci chu'a benh. thyc p h i m ehu'c nang bo duo'ng.
Tuy nhien, thanh phin hoa hpc quan trpng trong d i u g i e nhu carotenoids r i t d i bj oxy hoa, ham lugng se giam r i t nhanh sau mot tho'i gian n i u kh6ng duge bao quan t i t '^' "*'. Buo'c d i u cho t h i y bao quan d i u g i e trong d i i u
kien moi trub'ng sue khi tra nita v^ 6' nhiet d6 lanh 5 ± 3°C thi h^m lugng carotenoids giam ft han so vai e^e d i i u kien nghien cuu khcic'^'.
Propyl galat (E310) IS c h i t c h i n g oxy hoS, hay dugc dua vSo thyc p h i m c6 chua d i u , chat beo de ngan can quS trinh oxy hoS lam giam hSm lugng cac c h i t c6 Igi d i bj oxy hoci.
Vi vay c6ng trinh nay chung t6i th6ng bSo k i t qua su dung phuang phap s i c ky long hieu nang cao d i nghien CLPU dp i n djnh cua dau g i e khi dugc them c h i t bao quan propyl galat vS bao quan 6' cSe dieu kien khac nhau.