• N g h i e n
CLPU- K y t h u a t
r
Nghien cuu tac dung chong oxi hoa cua
A r r
flavonoid toan phan chiet xuat tir cay don kim (Bidens pilosa L, Asteraceae^ tren thirc nghiem
Pham Van VirongS Pham Thanh Ky^
Hoang Van Lu-ong' Hoc vien Qudn Y, Tru&ng dgi hoc Dugc Hd Noi
khi thi nghiem eho dn djnh vd trong sudt qud trinh nghien cuu. Chupt an thuc theo tieu ehuin, nude (dun soi de nguoi) udng t u do, Dpng vat nghien euu duge nudi trong chudng rieng d i trdnh lay cheo eo t h i xay ra theo dudng h6 h i p va t i i p xiic. Hang ngdy theo doi ghi chep d i i n b i i n k i t qua thi nghiem.
Hoa chat
Carbon tetrachlorid (Trung Qude), ede hda c h i t khde nhu tricloacetic acid (TCA), thiobarbituric acid (TBA)... dat tieu c h u i n mua tu hdng Merck {Que) vd Sigma (Hoa K y ) .
Phu'O'ng phap nghien ciru Chiet xuat flavonoid toan phan
Phan tren mat d i t edy dan kim duge thu hdi trong ngdy, ru'a saeh, thai nhd, say khd d nhiet dp khdng qud 70°C vd nghiin nhd d kfch thude thfch hgp. L i y 1kg bpt dugc ngdm ehiit 3 l i n , l i n d i u 48h, hai l i n sau 24h vdi methanol 80%
(lin d i u 5 lit, hai l i n sau mdi lan 3 lit), Gop cdc djch chiit, c i t thu hdi'dung mdi dudi dp s u i t giam dugc 130 g cao dam dac. Phdn tdn cao dam ddc vdo 1 lit nude c i t 2 lan, loai tap bdng n-hexan, ehiit kiet flavonoid bdng ethyl acetat (8 l i n , mdi l i n 300ml). Gop ede chiit ethyl acetat, c i t thu hdi dung mdi dudi dp s u i t giam duge 6,5g flavonoid todn p h i n (FLTP). FLTP edy dan kim dugc hoa tan trong nude (co 3% ethanol) de tao thdnh hdn djch cho chupt udng.
Nghien CLPU tac dung chong oxi hoa Phuang phdp nghien cuu tdc dung chdng oxi hoa dua tren xdc djnh ndng dp MDA/gan va GSH gan. Xdc djnh hdm lugng MDA trong dich ddng t h i gan theo phuang phap cua I.U.A. Vladymyrop vd cpng su, 1972, con hdm lugng GSH trong gan duge xae djnh theo phuang phap dugc ciia G.A.Hazenton vd C.A.Lang, 1980 '^'.
Dat van de
Cdy dan kim cd ten khoa hpc Bidens pilosa L, la cay mpc hoang nhiiu d nude ta tir miin nui, trung du, ddng bdng vd nhiiu nude tren t h i gidi nhu: Trung Qude, Han Qudc, Nhat Ban, Brazil,,,.vv, Ddy Id lodi cay mpc quanh nam, sue sinh san nhanh vd dugc coi nhu lodi "cd dai", Trong ddn gian, edy don duge sir dung n i u nude tdm d i diiu trj mun nhpt, dj ung, m i n ngua vd Idm nude udng d i phdng tieu chay vdo mua he '^'^l
Nghien euu v i tac dung sinh hpc, ehiing tdi da thdng bdo tde dung khdng khuin khdng n i m ciia tinh d i u edy dan kim '^', tde dung giam dau, chdng viem '"'. Bdi bdo ndy xin thdng bdo k i t qua nghien euu v i tde dung chdng oxi boa eiia flavonoid todn phin chiet x u i t tu cdy don kim.
Nguyen vat lieu va Phu'O'ng phap nghien CLPU
Nguyen lieu nghien CLFU
Phin tren mat d i t edy don kim duge thu hdi vao thdng 04 ndm 2009 tai khu vuc Ha Dong - Ha Npi, M i u tieu ban dugc PGS,TS,Vu Xudn Phuang vd ThS, Nguyin T h i Cudng (Phong thuc vat - Vien Sinh thdi vd Tdi nguyen sinh vat) xde djnh ten khoa hpc: Bidens pilosa L., thupe hp cue - Asteraceae.
Dong vat va hoa chit Dong vat
Chudt nhdt trang dong Swiss trudng thdnh, khdng phdn biet gidng, dat tieu ehuin thi nghiem, trpng lugng 20,0 ± 2,0 g, do Ban eung c i p dpng vdt thi nghiem - HVQY cung c i p .
Chupt dugc nuoi trong d i i u kien phong thi nghiem ciia Trung tam nghien euu Sinh - Y - Duge, Hpc Vien Qudn Y (nhiet dp 25-30°C, dp i m 60% vd chiiu sang 12h/ngdy) 10 ngdy trude
TAP CHi DUOC HOC - 8/2010 (SO 412 NAM 50) 51
Nghien
CLFU- Ky thuat
Chupt nhdt trdng du tieu chuin thi nghiem duge chia ngiu nhien thdnh 4 16, moi 16 7 eon:
- Lo 1 ( ehung sinh ly)udng nuoc dun soi d i nguoi,
- L6 2 (chung benh ly) udng nuoc dun sol de nguoi.
- L6 3 udng flavonoid toan phin liiu 60mg/kg (liiu 1).
- L6 4 udng flavonoid todn phin liiu 120mg/kg (liiu 2).
Chupt dugc udng thudc 7 ngdy trude khi lam thi nghiem, sau do gdy dpc ede 16 tu 2-4 bdng e c u 10% (liiu 5ml/kg TLCT) 2 gid sau khi udng thude lin cudi, Thdi gian gdy dpc 16 gid trudc khi liy gan Idm thue nghiem,
Xic djnh MDA/gan theo quy trinh:
Giit chupt bdng phuang phdp keo gian ept sdng, md nhanh liy gan, nua saeh mdu bdng nude mudi sinh ly lanh, thim kh6 bang giiy lpe, d i len dfa petri lanh, Cdn mdi miu lOOmg gan, Nghiin kho vdi tde dp 1,000 vong/phut trong 3 phut sau dd them 5ml dd Tris, nghiin vdi tdc dp 1,000 vong/phiit trong 2 phiit IJ i m 37°C trong 45 phiit Them 1ml dd TCA 30%, lie, Ipe bd tua (Bd mdt phin djeh lpe diu, niu ein). Liy 2ml djch trong, them 2ml dd TBA 0,25%, dun sdi 100°C trong 20 phut, d i nguoi va do quang d bude song 532nm.
Ham lugng MDA duge tinh theo edng thue:
D,2.V,1000 C= e,v,l,m
Trong do:
C: Hdm lugng MDA (mmol/g td chuc).
D: Matdd quang I: Chiiu ddy cuvet
e: He sd tit moi (e = 1,56 x 10^M"^ em"^) v: Thi tfch djeh do
V: Tdng thi tfch djeh ddng the vd acid trieloaeetie (ml).
m: sd mg td chue su dung,
Thay ede gid trj eu t h i trong diiu kien dd tiin hdnh thi nghiem, ta co:
C = D . 76,92
Xic djnh GSH/gan theo quy trinh:
Giit chupt bdng phuong phdp keo gidn cpt sdng, md nhanh liy gan, rua sach mdu bdng nude mudi sinh ly lanh, thim kh6 bdng giay lpc, de len dTa petri lanh. Cdn mdi miu 270 mg gan.
Them 3ml dd acid metaphosphoric 5%, nghien vdi tdc dp 3.000 vdng/phOt trong 5 phut, sau dd them 1ml dd TCA 30%, Ipc. Liy 0,5ml djch trong, them vdo 4,5 ml dd TT Ellman, u d 25°C trong 5 phiit vd do quang d bude sdng 412nm,
Tit ea cdc giai doan che boa miu tiin hdnh trong diiu kien dd dang tan (nhiet dp tu 0°C den 4°C).
Ndng dp GSH gan dugc tinh theo edng thu'c:
C x 5 x V 0,5 x m Trong dd:
X: Hdm lugng GSH (mg/g td chuc).
V: Tdng the tich djch ddng the vd acid trlchloacetie (ml).
C: Ndng dp GSH trong 1ml dung djch do (iig/ml).
m: Khdi lugng to chirc (mg).
Viee xdc djnh ndng dp GSH trong dung djch do dugc tien hdnh dua vdo phuang phdp them chuan vdi chit ehuin glutathion ciia hdng Sigma, My.
Thay cdc gid trj cu the trong diiu kien da tiin hdnh thf nghiem, ta dugc cdch tfnh ndng dp GSH trong gan theo c6ng thue:
X = 2,74 . D (mg/g td chue).
Trong dd D Id gid trj mat dp quang do duge eua miu do.
Xw ly so lieu:
Tit ca cdc sd lieu thu dugc diu dugc xii" ly thdng ke theo phuong phdp dung trong y - sinh - dugc hpc, sir dung phan mem Microsoft Excel 2003. So lieu dugc viit dudi dang trung binh ± SD vd su khac biet c6 y nghTa thdng ke vdi p<
0,01 hoac p< 0,05.
K§t qua nghien CCFU
Anh hU'O'ng ciia FLTP cay dan kim len ham lu'O'ng MDA/gan chuot thi nghiem
Su thay ddi ndng dp MDA trong gan ciia chupt nhdt trdng dugc trinh bdy d bang 1.
Bang 1: Ham lugng MDA/gan a cac Id chudt nghien ciuu
Ld nghien euu
Lo ehung sinh ly (1) Lo chung benh ly (2) Lo udng FLTP lieu 1 (3) Lo uong FLTP lieu 2 (4)
P
n
7 7 7 7
Mat do quang
0,1950 ±0,0138 0,3235 ± 0,0477 0,2536 ± 0,0446 0,2425 ± 0,0363
MDA(mmol/g to chuc) 15,00 ±1,06 24,89 ± 3,67 19,51 ±3,43 18,65 ±2,80
%sov6i(1)
100,00 165,93 130,07 124,33
% so vdi (2)
60,27 100,00
78,38 74,93 Pl.2 < 0,01 ; P4-2 < 0,01, P3-2< 0,05, P3-4>0,05, P3,4-1< 0,05
52 TAP CHI DUQC HQC - 8/2010 (SO 412 NAM 50)
Nghien
CLFU- Ky thuat
Kit qua d bang 1 cho thiy:
- Ndng do MDA trong gan chupt d 16 chirng benh ly tdng cao, phin trdm so vdi 16 chung sinh ly Id 165,93%. Su khde biet giua 2 16 ed y nghTa thdng ke vdi p < 0,01.
- So vdi 16 ehung benh ly, ndng dp MDA trong gan chupt d ede 16 gdy dpc sau dd dung FLTP
Kit qua xae djnh ham lu'O'ng GSH/gan
Bang 2: Ndng dd GSH/gan a cic Id nghien cu'u
liiu 1(60mg/kg) vd liiu 2 (120 mg/kg) diu giam cd y nghTa thdng ke, phin trdm so vdi 16 chung benh ly tuang ung Id 78,38%) vd 74,93%. Tuy nhien so vdi nhdm chung sinh ly vin cao ban.
Su khde biet ed y nghTa thdng ke vdi p < 0,05.
- So sdnh giua 16 dung FLTP liiu 1 vd liiu 2 khdng thiy c6 su khac biet (p > 0,05).
Lo nghien cwu
L6 chung sinh ly (1) Lo ehung benh ly (2) Lo udng FLTP lieu 1 (3) L6 udng FLTP liiu 2 (4)
P
n 7 7 7 7
Mat do quang 0,3694 ±0,0123 0,2374 ± 0,0559 0,3189 ±0,0650 0,3392 ± 0,0274
GSH (mg/g to ChOc) 1,012 ±0,034 0,650 ±0,150 0,8739 ±0,18 0,929 ±0,075
%sovai(1) 100,00
64,26 86,33 91,81
% so voi (2) 155,62 100,00 134,35 142,88 Pi.2 < 0,01 ; P4.2 < 0,01, p3.2< 0,05; p3_4 > 0,05, P3,4.i< 0,05 Kit qua d bang 2 eho thiy:
- Ndng dp GSH trong gan chupt d 16 ehung benh ly giam manh, phin trdm so vdi 16 ehung sinh ly 64,26%. Su khde biet giua 2 16 ed y nghTa thdng ke vdi p < 0,01.
- So vdi 16 chung benh ly, ndng dp GSH trong gan chupt d cdc 16 gdy dpc sau do diing FLTP liiu 1(60mg/kg) vd liiu 2 (120 mg/kg) diu tdng, phin trdm so vdi 16 ehung benh ly tuang ung la 134,35% vd 142,88. Tuy nhien so vdi 16 ehung sinh ly thip ban, Su khde biet ed y nghTa thdng ke vdi p < 0,05.
- So sdnh giua 16 dung FLTP liiu 1 vd liiu 2 khong thiy ed su khde biet (p > 0,05)
Ban luan
Tde dung ehdng oxi hda cua FLTP cdy don kim duge ddnh gid tren md hinh gdy dpc bdng CCI4. Ddy Id m6 hinh kinh diin dd dugc nhiiu nghien euu trong nuoc vd tren t h i gidi sir dung gdy tdn thuang gan thuc nghiem de nghien euu thude ed tdc dung chdng oxi hda, bao ve gan.
Co chi gay dpc cua CCI4 Id sau khi vdo ea the, nd bi ehuyen hda qua cytoehrom P450 thanh gdc tu do CCI 3 vd gdy peroxy hda mdng t i bdo, din tdi buy hoai t i bdo gan. Noi edeh khde, co ehe gdy dpc gan eua CCI4 Id co ehi gdy tdn thuong gan bdng gdc tu do. Vi vay chpn md hinh gdy dpc bdng CCI4 co thi giup xdc djnh true tiip tdc dung chdng oxy hda, ehdng gdc tu do ciia ehi phim thudc mudn nghien euu '^•'^l
'' Khi gdy dpc bang CCI4, ndng dp MDA trong gan tdng cao do gan bj doe Idm tdng qud trinh peroxid hda lipid mdng t i bdo. FLTP cay don kim khi sir dung d liiu 60 mg/kg hoac liiu 120 TAP CHi DUOC HQC - 8/2010 (SO 412 NAM 50)
mg/kg ldm giam MDA ro ret so vdi 16 gdy dpc khdng dung thudc, chung td FLTP eo tde dung ehdng oxy hoa, bao ve t i bdo gan, Idm giam qud trinh peroxid hda lipid d mdng t i bdo gan.
Ndng dp GSH trong gan giam khi gdy dpc bdng CCI4 vdi liiu 0,5ml/kg, chung td GSH co sin trong gan eung nhu dugc eg t h i huy dpng them da duge sir dung vdo chdng lai tdn thuang oxy hda do ehit ddc gay ra. Khi dung FLTP cay dan kim d liiu 60 mg/kg hoac lieu 120 mg/kg, ndng dp GSH trong gan tdng len ro khi so sdnh vdi nhdm gay dpc khdng diing thude. Nhu vdy FLTP edy don kim dd the hien vai trd hd trg ea thi trong viec chdng oxy hoa gdy ra bdi ehit dpc.
Kit qua nghien euu eiia ehiing toi cung phii hgp vdi ket qua cua Yuan L.P,, va CS '^' v i tdc dung ehdng oxi hda cua flavonoid todn phin edy dan kim.
K§t luan
Flavonoid todn phin cdy don kim dung liiu 60 mg/kg vd 120 mg/kg ed tac dung chdng oxi hda tren md hinh gdy dpc bang CCI4 the hien ldm giam ndng dp MDA vd lam tdng ndng dp GSH trong gan chupt thi nghiem.
Summary
The antioxidant effect of total flavonoids of Bidens pilosa L, a traditional medicinal plant widely distributed in Viet Nam were evaluated in cartoon tetrachloride (CCI4) - induced liver injury in rats. The results showed that total flavonoids at doge of 60 mg/kg and 120 mg/kg had antioxidant effect.
Keysword: Flavonoid, Bidens pilosa, antioxidant (Xem tiep trang 10)
53
• Nghien
CLFU- Ky thuat
France, Germany and Italy supplied the Vietnam market with both groups of specialized drugs and even those the domestic pharmaceutical industry was focusing on.
Whereas, from the developing countries, such as Korea and India, the supply was mainly the groups that overlapped the domestic products.
The impod of finished pharmaceutical products from developed pharmaceutical industry countries basically met the import purpose, supplementing those essential drugs the domestic pharmaceutical industry were unable to meet the national healthcare need.
Tai lieu tham khao
1. Bp Y t i , Phat triin cdng nghiep duge Viet Nam vd eae vin d i qudn binh eung elu de binh dn thj trudng duge phim, Hd Npi, (2008)
2. Cue Quan ly dugc, Tong quan nganh duge Viet Nam nam 2008. Hoat dpng eua doanh nghiep nude ngodi. Mpt sd djnh hudng quan ly nhd nude v i duge trong nam 2009, Ha Npi, (2008)
3. Chu Qudc Thjnh, Phan tich ea eiu thudc thanh phim nhap khiu giai doan 2006 - 2008, Luan van Thac sy dugc hoc, Trudng dai hpe duge Ha Npi, Ha Npi, (2009) ;.:
H o a t t i n h s i n h h p c . . . (Tiep theo trang SO)
3. T r i n Thi Da, Nguyen Thi Thanh Chi, Bill Dii'c T h u i n , Nguyen HCru DTnh, Tong hp'p, c l u triic, tinh c h i t cua mot s6 phirc c h i t platin(ll) chira piperidin va amin thorn, Tap chi hda hoc (2005), s6 2, tr 169-173.
4. Raaphorst G. P., Yang H., Wilkins D, E, Cisplatin hyperthermia and radiation treatment in human cisplatin - sensitive and resitant ligoma cell line. Int J. Hyperthenvia (1996), p.801-80.
5. Nguyen Thi Thanh Chi, T r i n Thi Da, Vu Quang Nhu, Nguyin Hiru DTnh, Tong hp'p va c l u triic cua mot s6 phirc c h i t trans - dicloro(safrol)(amintho'm) platin(ll); Tap chi hoa hoc (2007), 42 (2), 142-146.
6. Nguyin Thi Thanh Chi, T r i n Thj Da, Tong hop, c l u true va hoat tinh sinh hpc ciia phirc c h i t trans -
[PtCl2(safrol)(quinolin)] va trans -[PtCl2(safrol) (pyridin)]. Tap chi duo'c hoc (2009), (11), 32-36.
7. Nguyin Thj Thanh Chi, T r i n Thi Da, Cao Van Xuong, Tdng hgp va c l u triic ciia mot s6 phirc chat trans-dicloro(safrol)(aminbeo) platin(ll). Tap chi hoa hoc (2009), so 6.
8. Nguyen Thi Thanh Chi, Tran Thj Da, Nguyin HO'U DTnh, Tuong tac ciia kali tricloro (safrol)platinat(ll) voi amoniac, dimetylamin va dietylamin. Tap chi hoa hoc (da nhan dang)
9. Nguyin Thj Thanh Chi, T r i n Thj Da, Tong hp'p, c l u triic va hoat tinh sinh hpc mpt vai phirc chit cis - dicloro(safrol) (amin) platin(ll). Tap chi hoa hoc (2007), 45 (6), 715-721.
N g h i e n CLFU t a c d u n g . . . (Tiep theo trang 53)
Tai lieu tham khao
1. D6 T i t LD'I ., Nhung cay thuoe va vi thudc Viet Nam , Nxb Yhoc, (2005) tr 120-121.
2. Vien Dugc lieu: Cay thudc va dpng vat la thuoe a Viet Nam, Nha xuat ban khoa hoc ky thuat - Ha Noi (2003)
3. Pham Van Vupng, Pham Thanh Ky (2010), Nghien ciru thanh p h i n hoa hpc va tac dung khang khuan, khang n i m cua tinh d i u ehiit x u l t tir cay don kim (Bidens pilosa L.). Tap chi Duac lieu s6 1, tap 15, so 1/2010, trang 32-37.
4. Pham Van Vup'ng, Vu Manh Hiing, Nguyin Hoang Ngan (2007), Nghien ciru tac dung giam dau, chdng viem tren thuc nghiem ciia BP1 chiit x u l t tir cay Dan kim {Bidens pilosa L.), tap chi duoc hoc so 379. thang 11/2007, trang 9-13.
5. Vien DUO'C lieu , Phuong phap nghien ciru tac dung duo'c ly ciia thuoe tir thao duo'c, Nxb KHKT, Ha Noi (2006)
6. Kathleen A.D , Hepatic principles. Tocxicologic emergencies, Jeanman M.Roche, (1998) pp 213- 225.
7. Park E.J., Jeon C.H., Ko J., Kim J., Sohn D.H, Protective effects of curcumin in rat liver injury induced bac carbon tetrachloride. J Pharm Phanvacol (2000) 52 (4), pp 437-440.
8. Yuan L.P., Chen F.H., Ling L., Dou P.P., Bo H., Zheng M.M., Xia L.J., Protective effects of total flavonoids of Bidens pilosa L. (TFB) on animal liver injury and liver fibrosis- Joumal of Ethnophamiacology (2008) 116: p 539-546.
10 TAP CHI DUOC HQC - 8/2010 (SO 412 NAM 50)
