• Nghien ci>u - Ky thuat
3. Fonseca V. A (2009), "Defining and Characterizing the Progression of Type 2 Diabetes", American Diabetes Association, pp. S151- S155.
4. OECD guidelines for testing of chemicals. Acute
oral toxicity - Fixed dose procedure OECD 420, 2001.
5, Vogel H. G (2007), "Drug Discovery and Evaluation: Pharmacological Assays", third edition, Springer, pp. 1327-1355, 1572-1573.
{Ngiy nhin bii: 28/04/2014 - Ngiy duyit ding: 09/06/2014)
Nghien cmi doc tinh cap va doc tinh ban trirong diin cua chi pham OS35 chilt xuk tir qua xa sang (Cnidium monnieri (L.) Cuss.)
tren dong vat thuc nghiem
Dau Thuy Dirffng'*,Nguyln Tr3n Thi Giang Hirffng^
D S Thi Nguyet Que^ Nguyen Thu HangS Ngo Thi Phuong^
Nguyen Thi Hai Ygn^ Lg Minh Ha^
' Trudng Dgt hoc Y Hd Npi, - Tnrong Dgi hoc Dircrc Hd Noi
^ Vien Hoa hoc cdc Hap chdt thien nhien, Vien Hdn 1dm KH-CN Viet Nam 'E-mail: [email protected]
Summary
Acute and subchronic toxicity of OS35 (extracted from Cnidium monnieri (L.) Cuss.) was evaluated in experimental animals. The LD50 of OS35 given to mice per oral was 4.5(2.8- 7.1) (g/kg body weight), p
= 0.05. To evaluate the subchronic toxicity, OS35 was administered by oral route to rats at daily doses of 150 and 450 mg/kg body weight/day for 4 successive weeks. All the animals sun/ived with no significant changes in general signs, growth, hematological examinations (red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, white blood cell count, leukocyte formula and platelet count) (p > 0.05). Besides, no significant difference was revealed in the plasma activity of AST, ALT, plasma concentrations of protein, cholesterol and creatinine between the experimental groups of rats and the control (p > 0.05). In addition, OS35 exerted no effect on the constitution, growth, hematopoietic, hepatic and renal function in rats after such an oral administration.
Keywords: OS35, Cnidium monnieri, Acute toxicity, subchronic toxicity.
Dat van de
Suy sinh due d nam gidi la mgt hdi chirng gap vdi ti Ig n g i y c i n g eao, l i m giam dang ke e h i t l u g n g cudc s i n g v i anh h u d n g b i t lgi len chirc nang cua n h i i u he c a quan khac nhau'^'.
B i n egnh cac bien phap sir dyng thuoe theo y hgc hi^n dai, mdt xu h u d n g rat pho b i i n d Viet Nam cung n h u tren t h i gidi la p h i t hien va nghien c u u e i e thuoc dieu tn cd n g u i n goc tir d u g c ligu. Theo y hgc co t r u y i n ed nhieu d u g e
lieu d u g e sii' dyng de d i i u trj suy sinh due nam;
trong dd ed qua xa sang (ten khoa hge la Cnidium monnieri (L.) Cuss.) ed thanh phan ehinh la hoat e h i t osthol. Theo D d T i t Lai va cac n h i khoa hgc, hgp e h i t osthol ehiit xuat tir q u i xa sang ed t i e dyng tang cudng chue nang sinh ly d nam gidi '^' ^1. Tuy nhien, d nude ta v i tren t h i gidi, cho d i n nay v i n ehua ed nhieu cac nghien ciru ve ddc tinh cua cac c h i pham tir qua xa sang va hgp c h i t osthol. G i n day tir qua xa sang
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nhdm nghien euu da e h i i t x u i t va bao ehe t h i n h cdng ehe p h i m OS35 ed t i c dung tang cudng smh ly kha tot. Vi vay, d i eung e i p b i n g ehung khoa hge v i tinh an toan ciia c h i pham OS35 trong d i i u trj suy giam sinh ly nam gidi, trong bai b i o n i y chiing tdi trinh b i y c i c k i t qua nghien cuu ve ddc tinh cap va ddc tinh ban trudng dien cua c h i p h i m OS35 tren ddng vat thye n g h i i m .
Nguyen lieu, doi tu'O'ng va phu'ang phap nghien ciru
Nguyen lieu nghien ciipu
C h i p h i m OS35 la cao c h i i t con ciia qua xa sang, trong dd osthol chiem 35% vdi hieu s u i t ehiit la 3%; do Vien Hda hge eac hgp c h i t thien n h i i n , V i i n Han l i m khoa hgc edng nghe Viet Nam cung cap. Thuoe t h u d u g e pha trong dung mdi la dung djch CMC 0,5% trudc khi eho dgng vat thi nghigm udng.
Doi t u ^ n g nghien ciru
- Chudt c i n g t r i n g chiing Wistar. trudng thanh, khde manh, trgng lugng 180 ± 20 g do Hoc vien Quan y cung c i p .
- Chudt nhat trang chiing Swiss, trudng thanh, khde manh, trgng lugng 20 ± 2 g do Vien Ve sinh dich t i Trung u a n g cung c i p .
Ddng v i t thi n g h i i m d u g c nudi 5 ngay t r u d c khi t i i n h i n h nghien cuu de thieh nghi vdi mdi trudng v i d i i u kien chan nudi ciia phdng thi n g h i i m . Trudc va trong s u i t qua trinh nghien euu, dgng v i t thi nghiem d u g e nudi b i n g thuc an ehuan do Vien Vg sinh djch te Trung u a n g cung e i p , uong t h u i c t y do.
Phu'O'ng phap nghien ciru
Nghiin ciru dgc tinh cap cua ehe phim 0335 theo dwong uong tren chugt nhat trang (Theo phuvng phap Litchfield- Wilcoxon i*'^') Chudt n h i t trang trgng lugng 20 ± 2 g d u g c chia thanh tirng Id, moi Id 10 con. Cho tCrng Id ehudt uong e h i pham OS35 vdi lieu tir l i i u cao n h i t khdng g i y e h i t tdi l i i u thap n h i t gay c h i t 100% ehudt. Chugt d u g c nhjn an 12 gid t r u d c khi u i n g thuoe, v i n u i n g n u d c day du. Theo ddi so ehudt c h i t trong 72 g i d d i u va tinh trang ehung eiia chugt trong 7 ngay sau khi u i n g thuoe (an udng, hoat dgng t h i n
kinh, di lgi, b i i tiit...). N i u chugt c h i t , m i chugt de danh g i i dgi the cae ton t h u a n g eiia cac ca quan. X i e djnh l i i u c h i t 50% (LD^^) theo ty 1^
chugt c h i t trong vdng 72 gid d i u .
Nghien cu'u dgc tinh bin truvmg dien cUa chi phim OS35 theo duxmg uong tren chudt cong trang (theo huvmg dan cua WHO ''i)
Chugt cdng d u g c chia n g i u nhien l i m 3 Id, moi Id 10 con nhu sau;
- Ld ehung: uong dung mdi pha t h u i c 1 ml/
kg t h i trgng/ngay
- Ld trj 1: uong c h i p h i m OS35 l i i u 150 mg/
kg the trgng/ngay.
- Ld trj 2: uong c h i pham OS35 l i i u 450 mg/
kg t h i trgng/ngiy ( g i p 3 l i n Id trj 1).
Chugt cong t r i n g d u g c u i n g n u d c hoae thudc t h u trong 4 t u i n l i i n , mdi ngay mdt lan v i o buoi sang.
Cac chi tieu theo doi truuc va trong qua trinh nghien ciru (tru&c, sau 2 tuin va sau 4 tuin udng thuic)
- Tinh trang ehung, the trgng eiia chugt eong t r i n g .
O i n h gia chue phgn tao mau thdng qua so lugng hong c i u , the tich trung binh hong c i u , h i m lugng h u y i t s i c to, hematocrit, nong do h u y i t sac td trung binh hong c i u , ham l u g n g h u y i t s i c t i t r u n g b i n h hong c i u , so lugng bgch c i u , edng thuc bach e i u va s i luong t i i u eau.
D i n h g i i ehuc nang gan, th§n thdng qua dinh lugng ALT, AST, protein v i cholesterol, creatinin trong h u y i t thanh.
Md benh hgc: Sau 4 t u i n uong t h u i c thu, ehudt c i n g trang d u g e mo de quan sat d^i the toan bd c i c ea quan. K i i m tra ngau nhien c i u triic vi the gan, than ciia 3 0 % sd chupt eong trang d moi Id.
Xu> ly so lieu
Cae so lieu thu d u g c xir ly theo phan m i m Microsoft Excel 2007, sir dung t h u i t t o i n thong ke T-test Student. So Hgu d u g e trinh b i y dudi dgng MEAN ± SE. S y khac bigt cd y nghia thong ke khi p < 0,05.
TAP CHI DirOC HQC - 6/2014 (S6 458 NAM 54)
Nghien CIFU - Ky thuat
K e t q u a n g h i e n c u ' u
Dgc tinh c i p cua ehe pham OS35 theo dudng uong tren chudt n h i t t r i n g
& nhirng Id ehudt uong e h i pham OS35 lieu fCr 1,5 g/kg the trgng chugt t r d Idn, sau khi uong, chugt d tinh trang eham chgp, ire ehe, nam im, khdng an uong g i . Sau 30 phut, chudt c h u y i n sang trgng thai kich thich, va bgt mep, khd t h d
Bang 1 : Tyl4 chugt chit a cic 16 chudt uong chi
roi d i n den suy hd hap, tim vung d i u v i c i , d i n g thdi, ehudt cd hien t u g n g co giat r i i c h i t . Cac chugt e h i t duge m i dk quan sat dgi t h i thi quan sat thay tim, phoi, gan ciia chugt sung h u y i t . Cac ea quan khac chua quan sat thay gi bat thudng d c i c Id cd ehudt c h i t , cac chudt c h i t trong vdng 24 gid ke tCr khi d u g e eho udng thuoc t h u . Ti le chugt c h i l d u g c ghi lgi d bang 1.
pham OS35 STT
1 2 3 4 5 6 7
LiSu uong ehe pham OS35 (g/kg) 0,625
1,25 2,5 5,0 7,5 11,25
15,0
So chuot chU 0/10 1/10 3/10 5/10 7/10 8/10 10/10
% chuot chet 0 10 30 50 70 80 100 Ttr k i t qua d bang 1, tinh d u g c LDg^ ciia c h i
p h i m OS35 tren chudt nhat trang theo dudng u i n g la:
LDgo = 4,5 (2,8 - 7,1) g/kg the trgng vdi p = 0,05.
Nghien ciru dgc tinh ban tru'dng dien cua ehe pham 0 3 3 5 tren chugt cong trang
Tinh trang chung vi can nang cua chugt cong trang
Trong thdi gian thi nghiem. chugt d ea 3 Id hogt dOng binh thudng, an uong tot, p h i n khd.
Khdng t h i y b i i u hign gi b i t t h u d n g d ea 3 Id chugt trong s u i t thdi gian nghien ciru.
Sau 2 t u i n va 4 tuan u i n g t h u i c , can nang CLia chugt c i n g d ca 3 Id deu tang so vdi t r u d c khi nghien ciru (p < 0,05). Khdng ed s y khac biet ve can ngng ehudt cong gitra Id ehung v i c i c Id diing thuoc (p > 0,05).
Anh hwong cua chi phim OS35 den chuv nang tao mau
Chugt cong trang udng cac lieu 150 mg/kg the trgng v i 450 mg/kg the trgng hang ngay trong 4 tuan lien tye khdng lam thay d i i c i c chl s i danh g i i ehuc nang tao mau (bao g i m s i lugng hong cau, ham l u g n g h u y i t s i c to, hematocrit, t h i tich trung binh hong c i u , h i m lugng h u y i t sae t i trung binh hong cau, n i n g dp h u y i t s i c to trung binh hong cau, so lugng bach c i u , edng thirc bach c i u v i so l u g n g tieu cau) tren chugt c i n g trang so vdi Id ehdng va so vdi tnj-de khi uong
thuoc tai c i c thdi diem sau 2 t u i n v i sau 4 t u i n u i n g t h u i c .
Anh huwig cua chi pham OS35 tren chuv nang gan, th$n tren chugt cong tring
Chudt c i n g t r i n g u i n g cac lieu 150 mg/kg t h i trgng va 450 mg/kg the trgng hang n g i y trong 4 tuan lien tyc khdng lam thay doi c i c chi so d i n h gia ehuc nang gan (bao gom hoat dd AST, ALT va nong do protein, cholesterol trong mau), chue nang t h i n ( n i n g do creatinin trong mau) so vdi Id chirng va so vdi t r u d c khi uong thuoe tgi e i e thdi d i i m sau 2 tuan va sau 4 t u i n uong thuoc.
Anh hw&ng cua ehe pham OS35 den giai phau binh gan, than chugt cong trang thirc nghiim
vi mat dai thi cua gan, than
Tren t i t ca ehudt eong trang thuc n g h i i m ( d ca Id Chung va 2 Id uong OS35 l i i u 150 mg/kg the trgng v i 450 mg/kg t h i trgng h i n g ngay trong 4 tuan lien tuc), khdng quan s i t t h i y cd thay doi b i n h ly nao v i mat dai t h i eiia gan, than.
Hinh thii vi thi gan, thin chudt cing tring - Tren c i u triic vi the gan, c i u true gan khdng bj dao Ign.Tit ca vting tTnh mgch trung t i m lieu thuy gan va vung khoang eua khdng x a hda, khdng x i m nhap viem, khdng tang sinh ong mat. Mdt so t i bao gan thoai hda nhe. Tuy n h i i n khdng cd s y khac biet v i cau true vi the gan giu'a 2 Id diing thuoc thu' OS35 vdi Id ehirng.
TAP CHi DU'OC HQC - 6/2014 (SO 458 NAM 54) 45
Nghien CIFU -Ky thuat
- Tren eau triic vi t h i than: cac cau thgn cd hinh thai, eau triie trong gidi han binh thudng, khdng xo hoa, khdng tang sinh t i bao. Tuy nhien,
d cac Id ehudt u i n g t h u i e thir OS35. trong md dem cd eae o x i m nhgp t i bao viem mdt nhan (ehu y i u l i lympho bao), tang sinh x a .
Ld chirng Ld tri 1 Ld tri 2 Hinh 1 : Hinh anh vi thi than chudt cong tring sau 4 tuin uong thudc (HE x 400)
C:i ehudt Id ehung, nhu md than khdng viem, khdng hoai t u , e i u t h i n khdng ton thuang. Cj ehudt 16 trj 1 ( u i n g OS35 lieu 150 mg/kg t h i trgng), trong md dem ed cac o xam nhap t i bao viem va tang sinh xa. & chudt Id tri 2 (uong OS35 lieu 450 mg/kg t h i trgng), trong md dem cd cac o xam nhap viem mgn tinh.
Ban luan
Dgc t i n h cap ciia ehe p h a m OS35 tren chugt c o n g trang theo d u d n g u o n g
G i i trj LD^^ ciia c h i p h i m OS35 tren chudt n h i t t r i n g theo dudng uong d u g c xac dinh la:
LDjp = 4,5 (2,8 - 7,1) g/kg t h i trgng vdi p = 0,05.
Cae d i u higu ngd dge ehii y i u ciia ehudt la:
sau 30 phiit, ehudt ehuyen sang trang thai kich thich, va bgt mep, khd thd rdi d i n d i n suy hd h i p , tim vung d i u va co, dong thdi, chudt cd hien tugng co g i i t roi ehit. Cae chugt e h i t trong vdng 24 gid d i u sau khi d u g e eho udng thuoe t h u ; s i ehudt cdn song d u g c theo ddi t i i p trong vdng 7 ngay khdng t h i y ed b i i u hien gi dae biet, chudt v i n an u i n g , di lgi va bai tiet binh t h u d n g .
A n h h u d n g cua c h i p h a m OS35 den t i n h t r a n g c h u n g va s i r t h a y d o i can n a n g cua c h u g t c o n g t r a n g
T u k i t qua nghien euu cho t h i y c h i p h i m OS35 l i i u 150 mg/kg t h i trgng/ngay va 450 mg/
kg the trgng/ngay udng trong 4 tuan lien tue khdng lam anh hudng x i u d i n tinh trang ehung
va mue dd tang trudng eua chugt eong trang khi u i n g thuoe lien tye trong 4 t u i n .
A n h hu'O'ng cua ehe pham OS35 den chu'c n a n g tao m a u
C i c chi s i trong xet nghiem t i b i o m i u ngogi vi ed gia trj ldn trong viee danh gia chue nang tgo mau''^. Vdi lieu 150 mg/kg t h i trgng/ngay va 450 mg/kg t h i trgng/ngay, diing trong 4 t u i n lien tuc, ehe pham OS35 khdng gay anh h u d n g d i n chue nang tao mau eua ehudt eong trang.
A n h h u d n g c u a ehe p h a m 0 8 3 5 den chipc nang gan
Nghien euu anh h u d n g eua t h u i c d i i vdi ehuc nang gan la r i t c i n t h i i t khi nghien cuu dp an toan ciia mgt thuoc ''•^l
Mdt trong nhirng p h u a n g phap d i n h g i i mue do ton t h u a n g t i b i o gan la dinh lugng nong dp eae enzym ed nguon goe tai gan ed trong huyet thanh i'-^'. Trong nghien euu nay, chiing tdi djnh lugng hoat do eac enzym ALT v i AST trong huyit thanh tren ehudt e i n g t r i n g thuc nghiem ^-^^ K i t qua nghien c u u eua ehiing tdi cho t h i y hogt dg ALT va AST trong mau chugt eong trang d e i e 16 udng thuoe thiJ OS35 lieu 150 mg/kg the trgng/
ngay va 450 mg/kg the trgng/ngay tai c i c thdi d i i m sau 2 t u i n va 4 t u i n u i n g thuoc khdng tang so vdi trudc khi uong thuoc va so vdi Id chung (p
> 0,05). N h u vay, cd t h i ndi thuoc khdng gay ton thuang te bao gan chugt cong trang.
TAP CHI DlTOC HQC - 6/2014 (SO 458 NAM 54)
• Nghien CIFU - Ky thuat
Gan la ea quan ed nhieu ehuc nang doi vdi qua trinh chuyen h o i cac e h i t , trong dd cd c h u y i n h o i protid, lipid... '^'^l Theo k i t q u i nghien euu cua chung tdi, nong do protein v i cholesterol trong h u y i t thanh chupt c i n g trang thuc nghiem d 2 Id uong e h i p h i m OS35 khdng thay d i i tgi cae thdi d i i m sau 2 tuan, sau 4 t u i n uong t h u i c l i i n tye so vdi trudc khi u i n g t h u i c va so vdi 16 ehdng. T u dd cd t h i k i t luan r i n g c h i pham OS35 l i i u 150 mg/kg the trgng/ngay va 450 mg/kg t h i trgng/ngay dimg tren chudt c i n g t r i n g theo d u d n g uong trong 4 t u i n Hin tue khdng lam anh hudng d i n ehuc nang c h u y i n hoa protid va lipid cua gan.
Anh hu'd'ng cua ehe pham OS35 den chirc nang than
T h i n l i c a quan t i i t nleu, cd vai trd quan trgng b i c n h i t d i dam b i o s y hang djnh ndi mdi.
Khi dua t h u i c v i o ea the ed t h i g i y ton t h u a n g thgn, anh h u d n g d i n ehirc n i n g than. Creatinin la chat e h u y i n hoa euoi eting, san pham phan huy cua creatin phosphat, nong do creatinin khdng phu thuge vao c h i dg an. Khi cd ton thuang t h i n , creatinin tang sdm han v i tin eay han ure nen hien nay day l i mdt chf s i t h u d n g dung de danh gia va theo ddi chue nang thini^^'.
K i t qua nghien ciru ciia chiing tdi eho thay sau 2 tuan, 4 t u i n u i n g thuoc thir lien tuc, nong dg creatinin trong h u y i t thanh chugt eong trang d 2 16 tri kh6ng thay dol so vdi t r u d c khi uong thuoc v i so vdi 16 ehung. D i i u n i y chirng td c h i pham OS35 khdng anh h u d n g d i n chirc nang lgc ciia c i u than.
Anh hud'ng cua ehe pham 0 8 3 5 den g i i i phau benh gan, than chugt e i n g trang thuc nghiem
K i t qua xet nghiem md bgnh hge gan, than eua ehudt c i n g trang sau 4 tuan uong thuoc cho thay:
- Cau true vi the gan eua chugt cong trang d cac lo uong c h i pham OS35 sau 4 tuan uong thuoc khdng cd s y khae biet so vdi 16 chirng.
- Cau triie vi t h i than cua chugt eong t r i n g d Id uong c h i p h i m OS35 sau 4 tuan u i n g thuoc
cd eac o viem man tinh trong md d i m . Tuy nhien, eac c i u t h i n ed hinh t h i i , eau true trong gidi hgn binh t h u d n g v i xet nghigm creatinin danh gia chu'c n i n g than v i n trong gidi ban binh thudng.
Ket luan
C i c nghien ciru ciia chiing tdi da xac djnh d u g c :
- Gia tri LD^^ ciia ehe pham 0 8 3 5 tren chudt n h i t t r i n g theo dudng uong l i :
LDgo = 4,5 (2,8 - 7,1) g/kg t h i trgng vdi p = 0,05.
- Che pham OS35 l i i u 150 mg/kg t h i trgng/
ngay va 450 mg/kg t h i trgng/ngay udng lien tuc trong 4 tuan khdng anh hudng den tinh trgng ehung, e i n nang, ehuc nang tgo mau va ehuc nang gan v i than tren chugt e i n g t r i n g thye nghiem. Tuy nhien, c i n theo ddi anh hudng eua thuoe len hinh thai, eau true eiia than.
Ldi earn a n : Cdng trinh di/oc hoan thanh v&i SW tai trg kinh phi cua Chuang trinh Hda Dwgc - Bi Cdng Thwang, mi sd de tii CNHD.
DT.049/13-15.
Tai lieu tham khao 1. E. Wespes, I. Eardley, F. Giuliano, D.
Hatzichristou, K, Hatzimouratidis, I. Moncada, A Salonia, Y. Vardi (2013), "Guidelines on Male Sexual Dysfunction", European Association of Urology.
2. Bp mon Y hpc co truyen, Trudng Dai hgc V Ha Noi (2006), y hgc co truyin (Dong Y), Nha xuat ban Y hoc.
3. D 6 Tat Lgi (2004), Nhirng cay thuoc va vj thuic Viet Nam, Nha xuit ban Y hpc, trang 82.
4. Litfield, Wilcoxon (1948), "A simplified method of evaluating dose-effect experiments", J. Pharmacol.
Exp. Ther, pp. 9 9 - 1 1 3 .
5. D 8 Trung Dam (1996), Phuong phap xac dmh doc tinh cap cua thudc, Nha xuat ban Y hpc
6. World Health Organisation (2000), "General Guidelines for Methodologies on Research and Evaluation of Traditional Medicine".
7. Nguyin The Khanh, Pham Tir Duong (2001), Xet nghi$m su dung trong lam sang, Nha xuat ban Y hgc.
8 Vu Dinh Vinh (2001), Hudng dan sO dung cac xet nghi$m sinh hoa, N h i xuat ban Y hpc.
{Ngiy nhin bai: 16/04/2014 - Ngay duyet dang: 06/09/2014) TAP CHi DlTQC HOC - 6/2014 (SO 458 NAM 54)
