N g h i e n c u ? u - K y t h u a t cordata Thunb.) Vidt Nam vai hai loai dung mdi nude va dung mdi c6n. Ket qua la dd thu duac hai miu cao giau flavonoid la DLDC-N-TH, DLDC-E- TH. Cac cao chiit nay dd duac nghidn cuu tac dung duac ly trdn ca tran va ddng cim mau. Kit qua khao sat cho thiy cao gidu flavonoid chiet bing dung mdi cdn DLDC-E-TH cd tdc dyng trdn ca tran vd ddng cim mdu tdt han cao chiit bdng dung mdi nude DLDC-N-TH.
Nhu vdy budc diu sdng Ipc chiing tdi thiy nguydn lidu d i san xuit thudc dieu trj trT tif cdy diip cd Vidt Nam se duac chpn Id cao chiit bdng dung mdi cdn vd ddy Id ca sd khoa hpc d i tidp tuc nghidn cuu tdch cac phdn doan tir cao chiit cdn tien tdi nghidn cuu tdc dyng duac ly cua cdc phdn doan flavonoid.
S u m m a r y
The effects of the total flavonoids extracted with ethanol (DLDC-E-TH) and water (DLDC-N- TH) from Vietnamese Houttuynia cordata Thunb. on the smooth muscle contraction and blood coagulation-hemostasis were screened for treatment of hemorrhoid. Experimental studies
were carried out on in situ and isolated organs of mouse and rabbit. The ethanolic extracts were better effective than aqueous extracts on both contraction of smooth muscle and the blood coagulation-hemostasis.
Tai lieu t h a m k h a o
1. Nguyen Dinh Hdi, Didu trj benh trT bing laser C02, Y hoc thuc hanh, (1994), 4, tr.15-19.
2. Nguyen Mgnh Nhdm, Nguyen Dinh Chi, Tinh hinh b#nh trT 0 m0t nhd mdy, Ngoai khoa, Tdng hQi Y Dirov hoc Viit Nam, (1999), 4, tr. 15-21.
3. Dd Tit Lgi, Nhirng cdy thudc va vj thudc Viet Nam, NXB KHKT, (2003), tr.40.
4. V6 Vdn Chi, Tu dien cdy thudc Viet Nam, NXB Y Hoc, Hi NOi, (1997), tr.406.
5. NguySn Vdn Ddn, NguySn Viet Tuu, Phuong phap nghien cuu hda hpc cac cdy thudc, NXB Y hoc, (1985).
6. Hodng Thanh Huang, Tran Quynh Hoa va cpng sy , gop phin nghidn ciru thdnh phin flavonoid chiet xuit tu Id cdy diep cd (Houttuynia cordata Thunb) ciia Vidt Nam, Tap chi Duoc hoc, (2002), 9, tr.13.
7. Hodng Thanh Huong, Hd Viet Bao vd c$ng sir thdm dd kha ndng chdng ung thu cua thdnh phin flavonoid chiit xuit tir Id diip ca (Houttuynia cordata Tnunb) ciia Vidt Nam, Tap chi Duoc hoc, (2003), 10, tr.9.
r
D a n h g i a a n h h i r f r n g c u a d i c h c h i e t m e t h a n o l t o a n p h a n v a d i c h c h i e t p h a n d o a n h e x a n
r e c a y c h o c m a u N a m B o ( R a d i x S a l a c i a c o c h i n c h i n e s i s L o u r . ) t r e n s u h a p t h u g l u c i d
Dat van de
Odi thdo dudng (OTO) Id bdnh rdi loan chuyen hod ddn den tdng glucose huyit vd rit nhiiu biin chirng. Theo Hidp hdi Odi thdo dudng My 2009, OTO duac chia thdnh 4 loai trong dd chu yiu Id OTO type 1 vd OTO type 2P l.
Do Thj Nguyft Que Phi T h j XuyeV, Phvong T h i f n Thiromg2, Nguyen Duy Thuan3, Nguyen Tran T h j Giang Hiroug4 'Dgi hqc Dugc Hd Nqi, , 2 Vien Dugc liiu,
3Hoc vi^n Yhoc cd truyen, 4Dgi hoc Y Hd Nqi Ciing vdi sy gia tdng nhanh chdng cua bdnh
OTO trdn todn cau vd cdc hdu qua n$ng n i do cdn bdnh ndy gdy ndn Id sy cd gdng nd lyc cua cdc nhd khoa hpc nhdm Idm rd nguydn gdy bdnh, tim ra cdc thudc didu trj, phdng ngira...
Hidn dd cd mdt sd thudc diiu trj OTO nhung
24 TAP CHI Dl/OC HOC - 4/2011 rs6 420 NAM SI 1
• N g h i e n C U M J - K y t h u a t hidu qui c6n nhiiu han chi (cac thuoc hiu nhu chua c6 tac dyng ha glucose huyet lau dai k i ca khi dd dung phoi hgp, hidu qua ngdn ngua biin chung han chi... (2- 4 1), nhiiu tac dyng khdng mong mudn lidn quan din chinh co chi tac dung cua chung, da sd cac thudc cdn gdy tdng cdn khi diiu trj...
Glucid vd cdc polysaccharid khdc, khi dua vdo dudng tidu hda, d i cd the dugc hip thu trudc hit phai nhd enzym amylase chuyin thdnh cdc oligosaccharid sau dd nhd a- glucosidase (nhu glucoamylase, dextrinase isomaltase, maltase, sucrase) chuyen thdnh monosaccharid vd co t h i chi hip thu dugc cdc monosarcarid. Cdc chit uc che a-glucosidase Idm cdc oligosaccharid khdng the bj thuy phdn thdnh cdc monosaccharid do dd cd tdc dung uc chi tdng glucose huyet sau khi dn trdn in vivo.
Kit qua ddnh gid tdc dyng ha glucose huyit cua cdn dich chiet methanol todn phan vd cin djch chiit cdc phdn doan n-hexan, cua rd cdy chdc mdu Nam Bd (Salacia cochinchinesis LourJ thu hdi tai Vidt Nam cho thiy, cdn djch chiit methanol todn phin, phdn doan n-hexan vd phdn dogn ethylacetat diu cd tdc dung hg glucose huyet rd rdt (p<0,05) trdn chudt nhdt gdy ddi thdo dudng bdng streptozocin, trong dd muc dd ha glucose huyit cua cdn djch chiit phdn doan n-hexan ldn hon rd rdt so vdi phdn doan ethylacetat m. VI vdy, trong bdi bdo ndy chiing tdi tiin hdnh ddnh gid tdc dyng cua cdn djch chiit phdn doan n-hexan rd cdy chdc mdu Nam Bd trdn a-glucosidase (in vitro) vd trdn kha ndng dung nap sucrose d chudt cdng tring.
Ddng thdi song song ddnh gid tdc dyng cua cin djch chiit methanol "todn phin de so sdnh.
N g u y e n lieu va p h u ' o n g phap n g h i e n CLPU
Nguydn lieu
Rd cdy chdc mdu Nam Bd (Salacia cochinchinesis Lour.) dugc thu hdi tai vudn qudc gia Bach Md. Sau khi thu hdi, rd cdy dugc dem rua sach, siy khd d 60°-70°C, tdn nhd.
Chuin bj miu nghidn cuu: Dugc lidu dd Idm nhd dugc chiit bdng methanol, cit thu hdi dung mdi d dp suit giam thu dugc cdn djch chiit todn phin (DCTP). Hda cdn DCTP vdo nude, dem ldc vdi n-hexan. Cit thu hdi dung mdi d i thu liy cdn djch chiit phdn doan hexan (DCPO/hexan).
Ddng vat dung trong nghien cuu
- Chudt cdng trdng ca hai gidng, khde mgnh, trpng lugng trung binh 180 - 200g, do Hpc vidn Qudn Y cung cip. Chudt dugc nudi 5 ngdy trudc khi Idm thi nghidm tai phdng thi nghidm Bd mdn Dugc ly, Trudng Dgi hpc Dugc Hd N0L Chupt dugc cho dn thuc dn chuin do Vidn Vd sinh Dich td Trung uong cung cip vd udng nude tg do.
Hda chit, dung cu thi nghiem
a-Glucosidase, p-nitrophenyl a-D-glucoside, cdc hda chit thi nghidm khdc (KH2P04, NaOH, NaHC03...) do cdng ty Sigma cung cip
Phuang phdp nghien cuu
Danh gia kha nang (re che x- glucosidase tren in vitro cua DCTP va phin doan nghien ciru
Nguyin tic: Dya vdo phan ung thuy phdn p- nitrophenyl a-D-glucosid dudi xuc tdc cua a- glucosidase tgo thdnh p-nitrophenol. Djnh lugng p-nitrophenol tgo thdnh bdng phuang phdp do quang d budc sdng 405nmI5l
a-glucosidase
p-nitrophenyl a-D-gluoosid -> a-D-glucose + p-nitrophend Cich tiin hinh
0 thudc thu (cdc ndng dd thudc thu khdc nhau) vdi a-glucosidase (dd pha trong ddm phosphat) vd hdn djch p-nitrophenol a- D - glucopyranosid pha trong ddm phosphat (pH=6,8) d 37° C trong khoang 15 phut de phdn ung cd the xay ra. Dftng phan yng bdng dung djch natri carbonat 100mM. Djnh luang p- nitrophenol duac giai phdng ra bdng phuang phdp do quang d budc sdng 405nm.
Thi nghidm trdn duac Idp lgi 2 lin vdi cung mpt diiu kidn
Cich tinh kit qui
Ty Id phin trdm uc chi hogt ddng cua enzym duac tinh theo cdng thuc:
(Aching-Atho)X 100 - —
"chirng Trong dd:
X: Ty Id phin trdm uc chi hogt ddng cua enzym Acncng: Mdt dd quang cua Id chung
A,ni>: Mdt dd quang cua Id thudc thu
Ndng dd uc chi 50% (IC50) dugc tinh dya trdn dd thj bdn log (semilog) biiu didn sy phy thudc % uc chi enzym vdo log (ndng dd thudc
TAP CHI DUQC HQC - 4/2011 (S6 420 NAM 51) 25
• N g h i e n c m j - K y t h u a t thu-) - sir dung phan mem Sigmaplot 11 (SPCC Inc., Chicago, IL).
Danh gia tac dung cua thuoc tren kha nang dung nap sucrose cua chuot cong trang
ChuOt cong tring dVgc lira chon ngiu nhien chia thanh cac Id nhir sau
Lo 1 (16 chirng tring): uing dung moi pha thuoc.
L6 2 (Id DCTP): uong cin DCTP vdi lieu 0,70g/kg chudt*
Lo 3 (lo DCPO hexan): uong cin DCPO/hexan vdri lieu vdri lieu 0,006g/kg chupt*.
Lo 4 (16 chirng dirang): uong acarbose vcri lieu 7mg/kg chudt.
(*): tirang duong 2,9 g dirac lieu/kg chupt De chudt nhin <36i 15h trudc khi bit diu cho uing thudc. Sau khi cho chupt uing thuoc (hodc dung m6i pha thuoc) 4h, cho uong sucrose vdi lieu 2g/kg. Tiin hanh dinh luang glucose huyit tai thdi diem 0, 30 phut, 60 phut, 120 phut sau khi uong sucrose.
Ti Id % tang glucose huyet sau khi uong sucrose so vdi trade khi uong sucrose dirac tinh theo cdng thirc:
Y=100x (G'-Go)
G0
Trong dd:
Y: ti Id % tdng glucose huyet sau khi uong sucrose so vdi trirdc khi uong sucrose
Gt: nong dd glucose huyet sau khi uong sucrose t gid
G0: nong dd glucose huyet ngay trade khi uong sucrose
So lidu duoc xir ly thong kd theo phin mem Microsoft Excel 2003. Sir khdc nhau cd y nghTa thong kd khi p< 0,05. Kit qua thi nghidm dugc bieu thj bing trj so trung binh cdng/trir sai so chuan (M ± SE).
Ket qua va ban luan
Anh hudng cua djch chiit toan phin va djch chiit phan doan hexan tren o>
glucosidase {in vitro)
Can djch chiet toan phan (DCTP)
Ty Id phin trdm ire chi enzym cua cac Id thu thuoc so vdi Id chirng nhir sau:
Bang 1: Tac dung tren a-glucosidase cua DCTP
Nong dgng
thuoc thir (fjg/ml) So
mau thir % ire che enzym
4,00 8 93,24 ± 0,48
3,20 9 88,95 ±0,76
2,56 8 84,74 ± 0,56
2,05 9 70,08 ± 0,74
1,64 9 53,74 ±3,12
1,31 9 30,91 ± 1,60
|(J[I J — ! ~ I ^-cr °"
so -j ^0****^^
E i /
£ eo J S
" 0 i 40 \ /
TO J / i r t 2 3 r I /
NOIKJ do thuoc- thu Hinh 1: Do&ng cong biiu dien so phu thudc
% ire chi a-glucosidase vao ndng do DCTP Nong dd ire che 50% hoat tinh enzym ciia DCTP (tinh bing phin mem SigmaPlot 11) Id:
1,40 ± 0,29(ug/ml).
Can dich chiet phan doan hexan (DCPD/hexan)
Ty Id phin trSm ire che enzym cua cac Id thuoc so vdi Id chirng nha sau:
Bang 2: Tic dung trdn a-glucosidase cua DCPD/hexan
Nong do
thuoc thir (ug/ml) S6
mau thir % ire che enzym
4,00 9 83,32 ±1,24
3,20 8 75,61 ±1,70
2,56 9 62,41 ± 3,28
2,05 9 40,04 ±2,71
1,64 8 27,96 ± 2,40
1,31 9 16,00 ±2,10
' •' ; • i . . tti..
Hinh 2: Dudng cong biiu diin so phu thuQc % Crc chi a-glucosidase vio ndng dd DCPD/hexan
26 TAP CHI DUOC HOC - 4/2011 (S6 420 NAM SO
• N g h i e n C L F U - K y t h u a t
Nong dd ire che 50% hoat ti'nh a-glucosidase sucrose cua chudt cdng tring
cua cin DCPO/hexan (tinh bing phin mim Ty le phin tram tdng glucose huyet sau SigmaPlot 11) la: 2,23 ± 0,09 (ug/ml). cho uong sucrose so vdi thdi diem ngay trudc
Kit qua thu> tac dung cua DCTP va khi cho uing sucrose dugc ghi lai trong bang DCPD/hexan tren kha nang dung nap sau:
Bang 3: Anh huong thuoc trin kha ning dung nap sucrose cua chudt cdng tring
Lo Ti /# % tang GH
Lo 30 phut 60 phut 120 phiit
Lo 1: Chung trang (n = 7) 121,72 ±17,70 117,49 ± 10,90 94,78 ±17,14 Lo 2: uong DCTP (n = 8) 93,29 ± 12,26; p2/i >0,05 97,43 ± 10,05; p2/i >0,05 74,50 ± 11,60; D2/1 >0,05 Lo 3: uong DCPD/hexan 63,57 ± 9,90 71,91 ±9,62 65,45 ± 9,89
(n = 9) p3/i <0,05*; p3M >0,05 Pa/i <0,05*; P3/4 >0,05 P3/1 >0,05; P3/4 >0,05 Lo 4: u6ng Acarbose (n = 8) 59,26 ± 8,47; p4,i <0,01 69,04 ± 5,40; pm <0,01 58,63 ± 6,92; p4/i = >0,05
Px/yi so sanh 16 x v&i 16 yd cung mot thoi diem.
Kit qua nghidn cuu cho thiy:
Tai th&i diem 30 phut vi 60 phut sau khi uong sucrose
Muc tdng glucose huyit cua Id uong DCPD/hexan (tuong ung Id 63,57% & 71,91%), cua Id uong acarbose (tuong ung Id 59,26 % &
69,04%), deu thip hon rd ret so vdi muc tdng glucose huyet cua Id chung (121,72% & 117,49%).
Ld uong DCTP muc tdng glucose huyit thip hon khdng rd rdt so vdi Id chung (p>0,05).
Tai th&i diem 120 phut sau khi uong sucrose
% tdng glucose huyet cua ca 3 Id uong thudc (DCTP, DCPO/hexan vd acarbose) diu khdng khdc bidt cd y nghTa thdng kd so vdi Id chung trdng (p>0,05).
Ban luan
Ddnh gid tdc dyng cua cdc miu thu trdn a- glucosidase (in vitro), kit qua thi nghidm trdn cho thiy, ca cin dich chiet methanol todn phin vd cin djch chiet phdn doan hexan cua rd cdy chdc mdu Nam Bd deu cd tdc dung uc che a-glucosidase vdi IC50 tuong ung Id 1,40 vd 2,23 (u.g/ml).
Oe ddnh gid anh hudng cua cdc mdu nghidn cuu din kha ndng hip thu polysaccharid trdn in vivo chiing tdi tiin hdnh danh gid anh hudng cua cdc miu nghien cuu trdn kha ndng dung nap sucrose d chudt cdng chudt cdng. Theo kit qua thu dugc tai bang 3, chi cd cdn dich chiet phdn doan hexan (DCPO/hexan) cd tdc dung uc che tdng glucose huyet so vdi Id chung d thdi diem 30 phut vd 60 phut sau khi udng sucrose.
Cdn djch chiit todn phin (DCTP) khdng t h i hidn tdc dyng ndy.
Nhu vdy, trdn in vitro, ca DCTP vd DCPO/hexan deu the hidn tdc dung uc che a- glucosidase. Ndng dd uc che 50% (IC50) ddi vdi a-glucosidase cua DCTP (1,40 ± 0,29 ug/ml) thip hon cua djch chiit phdn doan hexan (2,23 ± 0,09 ug/ml). Dieu ndy cho thiy trong DCTP cd thi cd nhieu chit ire chi a- glucosidase hon trong DCPO/hexan. Tuy nhidn, trdn invivo, d muc lieu dd thu, chi cd DCPO/hexan t h i hidn tdc dung uc chi dung nap sucrose mdt cdch rd rdt (p<0,05), DCTP khdng the hidn tdc dyng ndy. Su khdc nhau v l tdc dung trdn in vivo cua 2 miu nghidn cuu cd the Id do trong co the qud trinh hip thu cdc polysaccharid ngodi su tham gia cua a- glucosidase cdn cd vai trd cua cdc enzym khdc (vi du nhu amylase). Trong thi nghidm ndy chiing tdi mdi chi ddnh gid dugc tdc dung cua DCTP vd DCPD/hexan ciia rd cdy chdc mdu nam bd trdn a-glucosidase.
K i t luan
Kit qua nghidn ciru cho thiy:
Ndng dd ire chi 50% (IC50) ddi vdi a- glucosidase cua:
Djch chiet methanol todn phin cua rd cdy chdc mdu Nam Bd Id: 1,40 ± 0,29 (ug/ml).
Djch chiet phdn doan hexan cua rd cdy chdc mdu Nam Bd Id: 2,23 ± 0,09 (ug/ml).
Cdn djch chiet trong phdn doan hexan (vdi lieu 0,006g/kg) cua rd cdy chdc mdu Nam BO cd tdc dyng uc chi tdng glucose huyit so vdi Id chung sau khi cho chudt udng sucrose 30 phut vd 60 phut.
Cdn djch chiit methanol todn phin (vdi liiu 0,70g/kg cdn ndng) khdng thi hidn tdc dyng ndy.
TAD r u i niTrtr- u o r - 4/1011 (SO 420 NAM 51) 27
N g h i e n C L P U - K y t h u a t S u m m a r y
Effects of the methanol extracts and those from hexane fractions of the roots of Salacia chochinchinesis on a-glucosidase (in vitro) and on sucrose tolerance were tested in rat. The methanol and hexane extracts inhibited a-glucosidase in vitro with IC^ was 1.40±0.29(pg/ml) of the methanol extracts and 2.23±0.09(/jg/ml) for the hexane extracts, respectively. The extracts from n- hexane fractions reduced sucrose tolerance in rat compared with the control at 30 minutes and 60 minutes (p<0.05), whereas the methanol extracts exhibited no effects at all (p>0.05).
Tai lieu t h a m k h a o
1. D6 Thj Nguyet Que, Nguyen Tran Thi Giang
Huang, Nguyen Duy Thuin, Budc dau danh gia tac dgng ha glucose huyet cua re cay chdc mau (Salacia chochinchinesis) tren chupt nhit gay tdng glucose huyet bing streptozocin, Tap chi Dirov hoc, 7-2009, tr 28-32.
2. DeFronzo RA, Overview of Newer Agents:
Where Treatment Is Going, The American Journal of Medicine 123, (2010) p S38-S48
3. Standards of Medical Care in Diabetes - 2009, Diabetes Care January 2009 vol. 32 no. Supplement 1 S13-S61, p1-9, 44-73.
4. Tadayyon M., Smith S.A., Insulin sensitisation in the treament of type 2 diabetes, Expert Opin Investig Drugs, (2003), 12(3), p 307- 324.
5. Vogel H. G, Drug Discovery and Evaluation:
Pharmacological Assays, Springer, (2008), p1572.
IV
D i n h l u x r n g g i n g e r o l t r o n g c a c m a u g i r n g t u o i
( Z i n g i b e r o f f i c i n a l e R o s c o e ) v a c a o g i r n g
b a n g p h u o n g p h a p H P L C
Dat van de
6 - gingerol Id chit cay cd mui tham ddc trang cua gang. Trong chuydn ludn ve gang Duac dien Vidt Nam IV dd xay dang tidu chuin djnh luang hdm luang tinh diu, cdc chit chiet duac trong nude vd trong ethanol 90%, nhung chua de cdp den quy trinh djnh luang cdc chit gingerol- mdt trong nhung thdnh phdn chu yeu tao ndn gid trj thuc su cua gung111. Quy trinh trong bdi bdo ndy chung tdi trinh bdy quy trinh djnh luang 6-gingerol trong cdc miu gung tuai (Zingiberaceae) va cao gung bdng phuang phdp HPLC de gdp phin tidu chuin hda loai duac lidu ndy.
N g u y e n vat lieu, p h i F o n g p h a p Oe tdi duac thuc hidn trdn gung tuai thu hdi tai Long An, Od Lat vd gung Trung Quoc (mua tai cha Binh Oiin, qudn Binh Chdnh TP.HCM vdo thdng 4 ndm 2010) vd ba loai cao gCrng: cao
Nguyen Ngoc Khdi, Nguyin Thi Hong Huong Khoa Dugc, Dgi hgc YDugc TP. Ho CM Minh gu-ng ^ (1-2009), cao gung K2 (12-2009) - Cao
gung Ki vd K2 do TS. Nguydn Nggc Khdi (bd mdn Duac Ldm sang, Khoa Duac) cung cip.
Cao gung N dieu che tu gung tuai Long An (4- 2010).
Chit chuin 6 - gingerol, dd tinh khiet 95%, Cat 345868, LOT D00079448 do cdng ty CALBIOCHEM, Germany cung cip. Acetonitril, methanol ddng cho HPLC (Merck), nude cit 2 lan dat tidu chuan dung cho HPLC. Silicagel Lichroprep RP 18 (Merck); Ban mong silicagel
60 F2 5 4 20 x 20 cm (Merck); Cdc hda chit khac
nhu methanol, chloroform, ethanol, aceton, petroleum ether, vanillin dat tidu chuin PA.
Hai hd thing HPLC Waters 2695, PDA Waters 2996 (ph'in mem Empower), detector MS MICROMASS® QUATTRO MICRP ™ API.
Cdt sdc ky Phenomenex C1 8 Gemini (150 x 4,6 mm, 5um). Mdy cat nude 2 lin Aquatron A 4000 D cua hdng Bibby (Germany).
28
