iu tra, nghin cu mt s thc vt Vit Nam c

tc dng h tr iu ha lng ng trong mu

ng dng cho bnh nhn i tho ng type 2
H Th Bch Ngc
Trng i hc Khoa hc T nhin
Khoa Sinh hc
Lun n Tin s ngnh: Ha sinh hc; M s: 62 42 30 15
Ngi hng dn: PGS.TS. Nguyn Vn Mi, TS. Phm Th Hng Minh
Nm bo v: 2012

Abstract. Thu thp 24 mu thc vt Vit Nam c tham kho l c kh nng trong h
tr iu tr bnh i tho ng (T). Tch chit thu cao nc nng v cao cn, th
nghim kh nng h ng huyt trn chut nht T type 2. Chit phn on cao nc
nng l vi v l ch ng bng cc dung mi c phn cc tng dn v nghin cu kh
nng h ng huyt ca cc phn on trn chut nht T type 2. Xc nh thnh
phn ha hc ca cao chit phn on c tc dng h ng huyt tt nht. Th nghim
kh nng c ch enzym -glucosidase ca cao chit phn on v cc cht tinh sch c.
Nghin cu bo ch ch phm, kh nng h ng huyt, xc nh an ton v ch tiu vi
sinh vt, c tnh cp cng nh hot tnh c ch enzym -glucosidase ca ch phm sinh
hc.

Keywords. Ha sinh hc; Thuc cha bnh; i tho ng; Bnh nhn; Thc vt

Content

M U
1. Tnh cp thit ca ti
t lu con ngi bit rng i tho ng (T) l mt bnh ri lon chuyn ha, nhng
nhng bin chng m n gy ra th gn y mi c lm sng t. T nhng nm 90 ca th k 20,
cc chuyn gia y t d bo Th k 21 l th k ca cc bnh ri lon chuyn ho. Bnh T
c th gy ra nhiu bin chng nguy him v l nguyn nhn hng u gy nhi mu c tim, t
qu, suy thn giai on cui v m la lm tng t l t vong ln gp 2-4 ln so vi ngi khng
b T.
Vic s dng cc thc phm b sung c ngun gc t thc vt cho cc bnh nhn T s c tc
dng phi hp vi vic iu tr bng thuc. Vit Nam ta s hu ngun ti nguyn thc vt ni chung, ti
nguyn thc vt cha cc hp cht t nhin c hot tnh sinh hc ni ring rt a dng. y l ngun
dc liu qu, tim nng, c gi tr kinh t v x hi rt ln lao. Ngy nay, vi s pht trin ca cc
ngnh sinh hc, ha hc cc hp cht t nhin v dc hc, hng chc ngn hot cht c trong cy c
c pht hin, nghin cu, ch bin v lm thuc cha bnh. Do vic iu tra, nghin cu
pht hin, khai thc, s dng c hiu qu cng nh bo tn, pht trin bn vng ngun ti nguyn
thc vt cha cc hp cht c hot tnh sinh hc l nhim v ln v ang t ra trc chng ta.
Xut pht t nhng yu cu v thc t trn y, chng ti tin hnh nghin cu ti: iu
tra, nghin cu mt s thc vt Vit Nam c tc dng h tr iu ho lng ng trong mu
ng dng cho bnh nhn i tho ng type 2.
2. Mc ch nghin cu
- iu tra, sng lc mt s thc vt Vit Nam c tc dng h ng huyt trn chut nht i tho
ng type 2.
- Xc nh cc cao chit phn on ca mt s thc vt c tc dng h ng huyt tt nht.
- Xc nh thnh phn ha hc ca cc phn on c tc dng h ng huyt, tinh sch cc cht v
lm sng t c ch h ng huyt ca chng.
- Bo ch ch phm c ngun gc t cc thc vt c sng lc, nghin cu kh nng v c ch h
ng huyt, xc nh c tnh cp ca ch phm.
3. Ni dung nghin cu
- Thu thp 24 mu thc vt Vit Nam c tham kho l c kh nng trong h tr iu tr bnh
T. Tch chit thu cao nc nng v cao cn, th nghim kh nng h ng huyt trn chut nht
T type 2.
- Chit phn on cao nc nng l vi v l ch ng bng cc dung mi c phn cc tng dn v
nghin cu kh nng h ng huyt ca cc phn on trn chut nht T type 2.
- Xc nh thnh phn ha hc ca cao chit phn on c tc dng h ng huyt tt nht.
- Th nghim kh nng c ch enzym -glucosidase ca cao chit phn on v cc cht tinh sch
c. Nghin cu bo ch ch phm, kh nng h ng huyt, xc nh an ton v ch tiu vi sinh
vt, c tnh cp cng nh hot tnh c ch enzym -glucosidase ca ch phm sinh hc.
4. Nhng ng gp mi ca lun n
c ng gp mi trong nghin cu tc dng h ng huyt ca l cy vi Cleistocalyx
operculatus (Roxb.) Merr.& Perry, trc y cc nghin cu ch quan tm n n vi. L kt qu
u tin ti Vit Nam xc nh c kh nng h ng huyt ca cy ch rng ca Phyllanthus
urinaria L., tm gi trn cy mt Macrosolen cochinchinensis (Lour.) Blume, c chui ht Musa
balbisiana Colla, v thn i Psidium guajava L., l ch ng Ilex kaushue S.Y.Hu trn chut nht
T type 2.
xc nh c kh nng c ch enzym -glucosidase ca 4/7 hp cht tinh sch c t l vi
gm: 2,4-dihydroxy-6-methoxy-3,5-dimethylchalcon (H6), 3-hydroxy-lup-20(29)-en-28-oic
acid (C3), triterpenoid khung olean 3-hydroxy-olean-12(13)-en-28-oic acid (LVE2), 2,3,23-
trihydroxy-urs-12en-28-oic acid (LVE4) v 01 hp cht tinh sch t l ch ng l 24 methyl (3-
hydroxy-lup-20(29)-en-24-oic acid) ester (H4). Kh nng c ch enzym -glucosidase c xc
nh l mt trong nhng c ch gy h ng huyt nhm ng dng bo ch thuc iu tr T.
bo ch ch phm Thivoda vi thnh phn t l vi, l ch ng, n vi, ch rng ca, dy
tha canh. Ch phm c xc nh c kh nng h ng huyt trn chut nht T type 2, m
bo ch tiu vi sinh vt, khng xc nh c liu gy c, c hot tnh c ch -glucosidase. L c
s tin ti nghin cu s dng cc nguyn liu thc vt cng nh ch phm Thivoda h tr cho
bnh nhn T type 2.
5. B cc lun n
 Lun n bao gm 125 trang, trong c 26 bng v 36 hnh. M u: 3 trang. Chng 1-Tng
quan ti liu: 31 trang. Chng 2- Nguyn liu v phng php nghin cu: 22 trang. Chng 3-
Kt qu v tho lun: 54 trang. Kt lun v kin ngh: 3 trang. Cc cng trnh khoa hc ca tc gi
lin quan n lun n (5 cng trnh): 1 trang. Ti liu tham kho: 11 trang, 36 ti liu ting Vit, 90
ti liu ting Anh. Ph lc: 110 trang gm 13 ph lc chi tit.
Chng 1.TNG QUAN TI LIU
1.1. BNH I THO NG
Khi nim bnh T. .. Phn loi bnh T: gm T type 1, T type 2, T thai k
v cc tnh trng tng ng huyt c bit khc.
Bnh T type 2 v tnh khng insulin: nu c c ch tc dng ca insulin v tnh khng
insulin.
Bin chng bnh i tho ng: nu c hai loi bin chng: bin chng cp tnh hn m
tng ng mu v bin chng mn tnh.
Tnh hnh bnh i tho ng trn th gii v ti Vit Nam.
1.2. S DNG THUC V THO DC TRONG IU TR I THO NG
Tng quan v s dng thuc trong iu tr T.
Nghin cu iu tr T bng tho dc trn th gii
Nghin cu iu tr T t ngun thc vt ti Vit Nam.
1.3. HOT CHT CHIT XUT T THC VT C KH NNG H NG HUYT DO
C CH ENZYM -GLUCOSIDASE
Gii thiu v c ch tc dng ca cht gy c ch enzym -glucosidase v tng quan cc dch
chit thc vt, hot cht sinh hc c kh nng c ch -glucosidase.

Chng 2. NGUYN LIU V PHNG PHP NGHIN CU

2.1. NGUYN LIU
2.1.1. Nguyn liu v i tng nghin cu
- Nguyn liu: s dng 24 i tng thc vt c thu hi theo ma, tng ng vi tng b
phn thu hi m thi im thu hi khc nhau, lc ra hoa, kt tri, pht trin sinh dng nh l,
thn , mt s mu c mua ti cc a phng.
- i tng ng vt: chut nht trng c dng Swiss, trng lng t 1822g, c cung cp
bi Vin V sinh dch t Trung ng.
2.1.2. Ha cht v thit b th nghim
- Ha cht: Tt c cc ha cht u m bo tin cy cho th nghim, ca cc hng Merck,
Sigma, Wako, Crystal Chem Inc
- Thit b: my ct quay chn khng, bnh chit phn on, h thng phn tch ELISA, my o
quang ph, cn phn tch, my li tm lnh, my o im chy, ct sc k, my o ph cng hng
t ht nhn, my o pH, my o ng huyt, t lnh su, t sy
2.2. PHNG PHP NGHIN CU
2.2.1. Phng php chit xut
2.2.1.1. X l mu
2.2.1.2. Chit mu bng nc nng
2.2.1.3. Chit mu bng cn 600
2.2.1.4. Chit thu phn on trong cc dung mi c phn cc tng dn
 T cao nc tng s (CNN) chit bng cc dung mi c phn cc tng dn: n-hexane,
ethylacetate, n-buthanol, dch chit tng ng c c quay thu hi dung mi v cao chit phn on:
cao chit n-hexane-CHe, ethylacetate-CEtA, n-buthanol-CBuOH, nc cui cn li: CNC.
2.2.2. Phng php gy chut nht T type 2
2.2.2.1. Nui chut nht bo bng ch n giu cht bo
2.2.2.2. Gy chut nht T type 2 thc nghim
2.2.2.3. nh lng ng huyt
2.2.2.4. Nghim php dung np glucose
2.2.2.5. nh lng insulin mu chut nht bng k thut ELISA
2.2.3. Nghin cu tc dng h ng huyt trn chut nht T type 2 ca: cao th 24 mu
thc vt, cao chit phn on l vi, l ch ng v ch phm Thivoda.
2.2.4. Xc nh ch s ha sinh: GOT, GPT, cholesterol, triglyceride
2.2.5. Xc nh kh nng c ch enzym -glucosidase
2.2.6. Xc nh thnh phn ha hc mt s mu thc vt
2.2.6.1. Kho st s b thnh phn ha hc trong mt s cao th thc vt
2.2.6.2. Phng php phn lp cc cht
2.2.6.3. Phng php xc nh cu trc ha hc
2.2.7. Bo ch ch phm Thivoda
2.2.8. Xc nh c tnh cp ca ch phm Thivoda
2.2.9. Phng php x l s liu

Chng 3. KT QU V THO LUN

3.1. CHIT XUT BNG NC NNG V CN 600 CC MU THC VT
Qua tham kho cc ti liu, kinh nghim dn gian v thc t ca cc bnh nhn T type 2,
chng ti la chn v thu thp 24 mu thc vt c d on c kh nng h ng huyt. iu
tra cc thc vt vi tnh k tha v pht huy, chng ti mong mun lm phong ph thm v kh
nng h ng huyt ca cc thc vt v xc nh c ch tc dng ca cc hot cht, t sng lc
cc thc vt c tc dng h ng huyt nhm h tr cho bnh nhn T.
Sau khi x l mu, hiu sut thu mu kh dao ng t 9% n 16,25%. Sau chit xut mu
kh bng nc nng v cn 600, phn trm tch chit thu cao th bng nc nng cao hn so vi
cn. Mt s cao nc c hiu sut tch chit trn 30% nh: l ch ng (31,4%), c chui ht
(36,2%), thn v l c nh ni (32,9%), l dy tha canh (40%), thn v l lc vng (32,4%), thn
v l m (37,6%), qu nhu (34%), l ta t (32,9%), l tm gi trn cy mt (30,8%). Cc cao
cn c phn trm tch chit kh cao, bao gm: c chui ht (18,9%), hoa actis (16%), l dy tha
canh (30%), l tm gi trn cy mt (14,6%)
3.2. NGHIN CU KH NNG IU HA NG HUYT CA DCH CHIT THC
VT TRN CHUT NHT T TYPE 2
3.2.1.Gy chut nht T type 2
3.2.1.1. Kt qu nui chut nht bo
Chut nhm I cho n thc n tiu chun (ND) v nhm II cho n thc n giu cht bo (HFD).
Nhn thy, chut nhm HFD tun th 8 c trng lng trung bnh l 60,284,06g tng ln gp
gn 4 ln so vi thi im trc khi cho n bo (15,450,57g) v tng gn gp i so vi nhm ND
cng thi im (35,013,03g). Trong khi nhm ND, tun th 8 trng lng trung bnh chut l
 35,013,03g, tng gn 2 ln so vi thi im ban u. Hnh 3.2 l nh hai con chut nui bo
(HFD) v nui bnh thng (ND).

Hnh 3.2. Chut nui HFD (bn tri) v chut nui ND (bn phi)

Xc nh cc ch s m mu: cholesterol ton phn, triglyceride, HDLc, LDLc. Ch s

triglyceride 5 c th nhm HFD tng ng k (tng 139,31%) v c ngha so vi 5 c th nhm
ND (p<0,001). Tng t nh vy, ch s cholesterol 5 c th thuc nhm HFD cao hn so vi
nhm ND (hn 30,3%) v s khc bit c ngha thng k, p<0,05. Hai ch s HDLc v LDLc tuy
c s khc bit nhng l hon ton ngu nhin, khng c ngha thng k.
3.2.1.2. nh lng ng huyt v insulin ca chut nht bo sau tim mng bng STZ
Chng ti ng dng m hnh ca Sawant v tp th, nhm chut HFD tim mng bng (tmb)
STZ liu 120mg/kg (HFD+STZ) c so snh vi ba nhm HFD tmb m (HFD+m), ND tmb
STZ (ND+STZ), ND tmb m (ND+m). Kt qu nh lng ng huyt ca cc nhm chut th
hin trn Hnh 3.3. Nng ng huyt nhm ND+STZ tng dn n 18,890,04 mmol/l ti 72
gi, sau li gim dn sau 7 ngy, 10 ngy, 14 ngy ln lt l:13,590,59, 8,650,57, 8,840,4
mmol/l. Vi nhm HFD+STZ nng ng huyt ti cc thi im ny rt cao, tng ng l:
22,531,12, 23,242,29, 22,962,47, 23,21,86 mmol/l, s tng ng huyt nhm ny c duy
tr kh n nh, (p<0,001).
Cc con chut HFD+STZ c n ng ng huy t cao (trn 18 mmol/l) c nh lng
insulin (Hnh 3.5). Vi nhng con c n ng insulin n m trong khoang 0,5 - 2 ng/ml l chut T
type 2. Kt qu chng ti gyc 17/20 con chu t T type2, hi u sut a t 85%.
HFD+STZ
25 2.00
HFD+ m 1.80
20 ND+STZ
nng insulin (ng/ml)
Nng ng huyt

1.50
ND+m 1.42 1.40 1.38
15 1.22
(mmol/l)

1.10
1.00 1.00 1.00 1.00
10 0.78
0.62 0.57 0.60 0.57 0.60 0.58
0.50 0.52
5
0.20 0.15 0.20
0 0.00
0h 48h 72h 7 ngy 10 ngy 14 ngy Thi gian 0 2 4 6 8 10 12 14 16 18 20
con chut

Hnh 3.3. Nng ng huyt ca cc l Hnh 3.5. nh lng nng insulin mu

chut th nghim chut
ti cc thi im HFD + STZ
3.2.1.4. Nghim php dung np glucose
Ti thi im 1 gi sau khi ung dung dch ng, ng huyt ca cc chut u tng ln,
tng nhiu nht l nhm HFD+STZ, tng ln gn 30%. Ti thi im 2 gi, trong khi ba nhm
chut i chng ng huyt gim dn v gn mc bnh thng, nhm chut HFD+STZ li c
xu hng tng ng huyt. Kh nng dung np glucose ca nhm chut HFD+STZ gim i,
khng nh chut b T type 2. Kt qu trnh by trn Bng 3.4.
Bng 3.4. Kh nng dung np glucose ca cc nhm chut
0 gi 1 gi 2 gi
(mmol/l) (mmol/l) (mmol/l)

HFD + STZ 23,201,86 30,041,23I*, II** 29,951,44I*,II**

HFD + m 8,810,34 11,201,20 9,251,1

ND + STZ 8,840,40 12,820,87 9,80,82

ND + m 6,090,46 6,331,30 6,041,12

(n=5, I so vi chut cng nhm thi im 0 gi, II so vi chut cc nhm i chng cng
thi im, *p<0,05, **p<0,001 )
3.2.2. Sng lc cc mu thc vt c kh nng h ng huyt
Cho chut nht T type 2 ung CNN v CC cc mu thc vt vi liu 500 mg/kg/ngy. C 4
t th nghim, mi t iu tra hot tnh h ng huyt ca 6 mu thc vt. Cc Hnh 3.6, 3.7,
3.8, 3.9 th hin kt qu nh lng nng ng huyt ca chut ti thi im 0 gi v 20 ngy
qua cc t th nghim.

Hnh 3.6. ng huyt ca chut T type 2

cho ung cao chit thc vt t I

Ti t I, trong s 6 mu thc vt c th nghim kh nng h ng huyt ch c duy nht

mu ch ng th hin hot tnh, c th: chut ung CNN ng huyt h 65%, chut ung CC h
64% (p<0,001) so vi thi im 0 gi. C th nng ng huyt ca nhm chut T type 2 cho
ung CNN l ch ng ti ngy th 20 l 8,30,5 mmol/l, nhm ung CC l ch ng l 8,70,8 mmol/l.
 Hnh 3.7. ng huyt ca chut T type 2 cho ung cao chit thc vt
t II

Ti t II, pht hin ra kh nng h ng huyt ca mu thn, l ch rng ca, chut ung CNN h
63%, chut ung CC h 44% (p<0,001) v mu c chui ht, chut ung CNN h 58%, chut ung CC h
55% (p<0,001). Cc con chut sau khi cho ung cao chit hon ton khe mnh v khng xut hin cc bin
chng ca bnh T.

Hnh 3.8. ng huyt ca chut T type 2 cho ung cao chit thc
vt t III

Nhn thy trong t III ch c duy nht mu l dy tha canh th hin hot tnh h ng huyt.
Chut ung CNN ng huyt h 60%, chut ung CC h 53% (p<0,001). Kt qu nghin cu ca
chng ti v hot tnh h ng huyt ca dy tha canh Gymnema sylvestre (Retz.) R.Br.ex Schult
hon ton ph hp vi kt lun ca mt s tc gi trong v ngoi nc. Khc vi nhm tc gi Trn
Vn n cho chut bnh thng v chut tng ng huyt bi STZ ung dch chit dy tha canh
trong mt thi gian ngn (tnh theo gi), chng ti cho chut nht T type 2 ung dch chit
trong 20 ngy, thy rng cao chit l dy tha canh c tc dng h ng huyt n nh trong mt thi
gian di.

Hnh 3.9. ng huyt ca chut T type 2 cho ung cao chit thc
vt t IV

Vi t iu tra cui cng, chng ti thu c 4 mu c tc dng h ng huyt. Mu l tm

gi trn cy mt: chut ung CNN h 60%, chut ung CC h 64% (p<0,001). Mu n vi: chut
ung CNN h 60%, chut ung CC h 56% (p<0,001). Mu l vi: chut ung CNN h 67%, chut
ung CC h 63% (p<0,001). Mu v thn i: chut ung CNN h 52%, chut ung CC h 48%
(p<0,001).
c nhiu cng b v tc dng h ng huyt ca l v qu i Psidium gajava Linn. i vi
v thn i, nhng cng b nghin cu tc dng h ng huyt trn th gii cn rt t v hin cha c ti
Vit Nam. Hot tnh h ng huyt ca 3 mu l tm gi trn cy mt, l vi, n vi gn tng
ng nhau. Chng ti iu tra n vi da trn nhng kt qu cng b trong v ngoi nc, y l mt
ngun thc vt v cng di do ti nc ta. Nhm lm phong ph thm v tc dng ca cy vi, chng ti
la chn v tp trung nghin cu l vi, v so vi n vi, l vi c thu hi d dng hn, hot tnh h
ng huyt th hin tng ng n vi trong khi cc cng b v tc dng v c ch h ng huyt ca
chng cn kh t i.
Sau khi iu tra 24 mu thc vt, xc nh c 8 mu c tc dng h ng huyt trn
chut T type 2: l ch ng, thn v l ch rng ca, dy tha canh, n vi, c chui ht, l
tm gi trn cy mt, l vi, v thn i. Nhn thy tm gi trn cy mt l i tng nghin cu hon
ton mi, y l cng b u tin v tc dng h ng huyt ca Macrosolen cochinchinensis (Lour.)
Blume trn th gii.
3.3. NGHIN CU DCH CHIT L VI V L CH NG
3.3.3. nh hng ca cao nc l vi, l ch ng ln gan chut
3.3.3.1. Cc ch s GOT, GPT mu chut
 Bn cnh nghin cu xc nh hot tnh h ng huyt ca cc cao chit phn on chng ti
theo di v xc nh cc ch s men gan, to tiu bn c ct gan chut. Kt qu th hin trn
Hnh 3.10 v 3.11
120

1006.8

Nng (U/l) 90

595.2
60
42.45.1
31.64.3 372.7 353.7
29.42.8
30
201.5

Hnh 3.10. Gi tr cc ch s GOT, GPT trong mu chut

0
GOT GPT
Chut T type 2 Chut b nh thng
Chut T type 2+cao nc l vi Chut T type 2+cao nc l ch ng

Chut T type 2 khng c cho ung cao chit l vi, l ch ng c biu hin gan b
tn thng, ch s men gan kh cao, ch s GOT trung bnh l 595,2U/l, GPT trung bnh l
1006,8U/l. Trong khi cc con chut T type 2 cho ung cao nc l vi v l ch ng cc
ch s men gan h thp hn so vi nhm chut i chng, tuy vn cao hn nhm chut bnh
thng. Nhm chut T type 2 cho ung cao nc l vi c GOT, GPT trung bnh tng ng l
31,64,3 v 42,45,1U/l, nhm chut T type 2 cho ung cao nc l ch ng c GOT, GPT
trung bnh tng ng l 29,42,8 v 353,7 U/l, iu ny chng t gan c du hiu c phc hi.
3.3.3.2. nh hng ln cu trc m gan
T nhng hnh nh quan st tiu bn gan chut T type 2 khng cho ung cao chit thc
vt, cc t bo gan mo m, ri rc, hnh dng t bo khng r rt, thiu s lin kt vi nhau, xut
hin mt s t bo b teo nhn, kch thc nh hn so vi cc t bo khc. Nhn t bo bt mu rt
m chng t t bo b cht. tiu bn gan chut T type 2 cho ung cao nc l vi v l
ch ng, hnh nh t bo quan st nt hn, dng nh khng nhn ra s ph hy ca t bo, nhn
t bo cng nh cc t bo c kch thc bnh thng v kh ng u, nhn t bo bt mu hon
ton bnh thng, ng thi cc t bo lin kt vi nhau, tng ng vi tiu bn gan chut nht
bnh thng.
 Gan chut T

type 2 cho ung cao nc l vi

Gan chut T

type 2 cho ung cao nc l ch

ng

Gan chut T type 2

Gan chut bnh thng

Hnh 3.11. Hnh nh quan st tiu bn c ct gan chut

Bn tri: chp nh phng i 400 ln, bn phi: phng i 1600 ln

3.3.2. Cao chit phn on l vi

3.3.2.1.Khi lng cao chit phn on l vi
Chng ti s dng 3kg bt kh l vi, chit bng nc nng thu CNN, sau tch chit phn
on qua cc dung mi. CNC chim t l ln nht (31%), sau l CBuOH (26,6%), CEtA (22%)
v thp nht l CHe (13,3%). Cc cht khng phn cc chim t l kh t so vi nhm cht phn
cc trung bnh v phn cc mnh.
3.3.2.2. Nghin cu tc dng h ng huyt ca cc phn on dch chit l vi
Hnh 3.12 l kt qu tng hp nng ng huyt sau 20 ngy cho chut ung cc cao chit
phn on l vi liu 500mg/kg.
 0h

Nng ng huyt (mmol/l)

40
3 ngy
7 ngy
30
10 ngy
20 ngy
20

10

0
L c L ung L ung L ung L ung
CHe CEtA CBuOH CNC

Hnh 3.12. Tc dng h ng huyt ca cao chit phn

on l vi

Trc thi im 7 ngy, nng ng huyt ca cc l chut c iu tr bng cc cao chit

phn on l vi tuy c s khc bit nhng khng c ngha thng k (p>0,05). Sau 7 ngy, mt s
cao chit l vi th hin hot tnh h ng huyt kh r nh CHe, CEtA v CBuOH (p<0,05).
Hot tnh ny th hin cng r ti thi im 20 ngy khi p<0,01. S d chng ti ko di thi gian
th nghim khi cho chut nht iu tr trong 20 ngy xc nh hot tnh h ng huyt ca ch
phm c n nh hay khng. CHe tuy chim t l thp nht nhng th hin hot tnh h ng
huyt, nng ng huyt trung bnh ca chut sau 20 ngy iu tr bng 6,10,7 mmol/l, tip
theo l CEtA (6,81,1mmol/l) v CBuOH (9,31,7mmol/l). Phn on CNC cng c tc dng h
ng huyt tuy rng cha tht s hiu qu (nng ng huyt sau 20 ngy iu tr bng
15,21,3 mmol/l). La chn CHe, CEtA, CBuOH xc nh thnh phn ha hc.
3.3.2.3. Hot tnh c ch enzym -glucosidase ca cc cao phn on
CHe c kh nng c ch enzym -glucosidase vi IC50 l 5,0370,6 g/ml, ng ngha vi
lng ng glucose b hn ch to ra nhiu nht, do nng ng huyt gim nhiu nht. CEtA
cng c kh nng c ch -glucosidase gn nh tng ng vi CHe, IC50 l 5,766 0,3 g/ml.
Cui cng l CBuOH c IC50 cao hn, 8,011 0,7 g/ml.
3.3.2.3. Phn lp v xc nh cu trc cc hp cht ca l vi
a. - Sitosterol hay stigmast-5-en-24R-3-ol
a ln ct 15 g CHe. Ra gii ct bng h dung mi n-hexane: ethylacetate (20:1), thu c
khi cht rn v nh hnh, tch lp li trn ct silicagel v kt tinh li trong n-hexane thu c
nhng tinh th hnh kim, khng mu, c khi lng 23mg, Rf=0,5, nng chy 135-136C. Gi l
H1. Cc d liu v ph NMR u ph hp vi -sitosterol:
26
21 21 23
25
18 20 22 24 27
12 17 28
19 29
14 15
10 8
3
HO 5

Hnh 3.13. Cu trc hp cht -sitosterol

 b. -sitosterol-glucopyranoside
Tip tc ra gii ct bng h dung mi n-hexane:ethylacetate (20:1), thu c khi cht rn
v nh hnh, c khi lng 21mg, gi l H2. Tin hnh chy sc k bn mng vi cht chun -
sitosterol-glucopyranoside nhn thy hai bng cht hon ton trng nhau c v v tr v bt mu vi
cht nhum. Cht ny l -sitosterol-glucopyranoside :
26
27
25
21 22 24

18 20 23
12 28 29
17
19
14 15
10 8
3
RO 5
R-glucopyranoside

Hnh 3.14. Cu trc hp cht H2

(-sitosterol-glucopyranoside)

c. 2,4-dihydroxy-6-methoxy-3,5-dimethylchalcone
Thay i h dung mi ra gii ct theo t l n-hexane:ethylacetate (10:1), thu c khi cht
rn v nh hnh, tch lp li trn ct silicagel v kt tinh li trong n-hexane thu c nhng tinh
th hnh kim, mu vng, c khi lng 25mg, Rf=0,7, nng chy 123-125C. Phn tch cc s liu
ph cho php khng nh H6 l mt flavonoid tn 2,4-dihydroxy-6-methoxy-3,5-
dimethylchalcon.

2 4

5' 1
HO OCH3 5

4' 6' 6
1'

3'
2'

OH O
Hnh 3.15. Cht H6 tinh sch v sc k bn Hnh 3.16. Cu trc H6 (2,4-dihydroxy-6-methoxy-
mng cht H6 3,5- dimethylchalcon)

d. 3-hydroxy-lup-20(29)-en-28-oic acid
a ln ct 15 g CEtA, ra gii ct bng dung mi chloroform thu c hp cht C3 c khi
lng 41,9 mg l cht rn v nh hnh, mu trng, c Rf = 0,76, im nng chy 282 -2840C. Kt
hp cc s liu ph v ti liu tham kho cho php xc nh C3 l 3-hydroxy-lup-20(29)-en-28-oic
acid (bentulinic acid).

Hnh 3.17. Cht tinh sch (phi) v xc nh cu trc C3 (3-hydroxy-lup-

20(29)-en-28-oic acid) (tri)

e. Triterpenoid khung olean (3-hydroxy-olean-12(13)en-28-oic acid) Thay i h dung mi

ra gii ct chloroform:methanol theo t l 9:1 thu c khi cht rn khng mu, c khi lng
27,2mg, Rf= 0,56, nng chy 262-263C. Gi cht ny l LVE2. Tt c cc d liu ph thu c
cho php khng nh LVE2 l triterpen kiu khung olean vi tn gi 3-hydroxy-olean-12(13)-
en-28-oic acid.

Hnh 3.18. Sc k bn mng (phi) v cu trc ca LVE2

3-hydroxy-olean-12(13)-en- 28-oic acid (tri)

f. 2,3,23-trihydroxy-urs-12en-28-oic acid
Thay i h dung mi ra gii ct bng h chloroform:methanol theo t l 80:20 thu c khi
cht rn v nh hnh LVE4 c khi lng 98,8mg, Rf = 0,75, nng chy 295-2960C. Cc s liu
v ph ca LVE4 cho thy hon ton ph hp vi ph ca asiatic acid hay 2,3,23-trihydroxy-urs-
12en-28-oic acid
 30

29
20 21
19

12 18
17 22
11 OH
13
25 26
28
1 14 16
HO
9 O
2 10 8 15
27
3 5 7
HO 4 6

HO 23 24

Hnh 3.19. Sc k bn mng (phi) v cu trc ca LVE2 (3-hydroxy-olean- 12(13)-

en- 28-oic acid) (tri)

g. Hp cht C7 l mt flavonoid (quercetin)

a ln ct 15 g CBuOH, ra gii bng h dung mi chloroform: methanol t l 95:5, thu c
cht kt tinh hnh kim mu vng rm c khi lng 15mg, gi l C7. Hp cht C7 l mt flavonoid
khi so snh cc d kin ph vi cc ti liu thu c cho php kt lun y l hp cht quercetin
vi cng thc phn t C15H10O7.

Hnh 3.20. Sc k bn mng (phi) v cu trc ha hc ca quercetin (tri)

3.3.2.4. Thnh phn cc hot cht phn lp c

Trong s 7 hot cht phn lp c 2,3,23-trihydroxy-urs-12en-28-oic acid chim t l ln nht
trong cao phn on (0,66%), tip theo l 3-hydroxy-lup-20(29)-en-28-oic acid (0,28%) v 3-
hydroxy-olean-12(13)-en-28-oic acid (0,185%). Cc thnh phn cn li chim t l trong cao phn on
gn nh tng ng nhau, xp x 0,1%.
3.3.3. Phn on dch chit l ch ng
3.3.3.1. Khi lng cao chit phn on l ch ng
 CHe l ch ng chim t l thp nht (12,1%), tip n l CEtA (21,4%) v CBuOH (24%).
Chim t l cao nht l CNC (39,1%).
3.3.3.2. Nghin cu tc dng h ng huyt ca phn on dch chit l ch ng
Chng ti tp trung phn tch mu l ch ng, xc nh phn on c hot tnh h ng
huyt v nhm hot cht chnh c trong phn on cng nh tm hiu v c ch tc dng ca
chng. Di y l kt qu sau khi cho chut T type 2 iu tr bng cao chit phn on l ch
ng, th hin trn Hnh 3.21.

0h
Nng ng huyt (mmol/l)

40
3 ngy
7 ngy
30
10 ngy
20 ngy
20

10

0
L c L ung L ung L ung L ung
CHe CEtA CBuOH CNC

Hnh 3.21. Nng ng huyt chut cho ung cao chit phn on l ch ng

Cc phn on dch chit l ch ng th hin hot tnh h ng huyt ti thi im 7 ngy

(p<0,05), mnh hn ti thi im 10 ngy v 20 ngy (p<0,01). Chut cho ung CHe c ng
huyt gim t 23,71,1mmol/l v 7,21,4mmol/l. Cc phn on khc cng th hin hot tnh h
ng huyt tuy nhin cha tht s tt. Ring phn on cao nc cui li khng h c tc dng h
ng huyt. T kt qu ny chng ti quyt nh chn phn on CHe ca l ch ng nghin
cu v thnh phn ha hc v xc nh hot tnh c ch enzym -glucosidase ca cc cht tinh sch
c.
3.3.3.3. Thnh phn ha hc ca phn on n-hexane l ch ng
Ra gii ct chy CHe l ch ng bng h dung mi n-hexane:ethylacetate theo t l 95:5 thu
c khi cht rn v nh hnh. Khi cht ny c kt tinh li trn ct silicagel c ht 0,040-
0,063mm, h dung mi ra gii ct n-hexane:ethylacetate theo t l 19:1, kt tinh li trong methanol
thu c cht v nh hnh c khi lng 19,3mg, Rf= 0,8, nhit nng chy 222- 2230C gi l
H4. Kt hp cc d liu ph cho php khng nh cu trc H4 l 24-methyl (3-hydroxy-lup-
20(29)-en-24-oic acid) ester, th hin trn Hnh 3.22.
 29

20
30
21
19
12 22
18
11 13 17
25 26
28
1 14 16
9
2 10 8 15
27
3
4 5 7
HO
6
24
23 O
O

Hnh 3.22. Cu trc phn t ca H4 (24 methyl (3-hydroxy-lup-20(29)-en-24-oic acid) ester

3.3.4. Xc nh hot tnh c ch enzym -glucosidase

Xc nh hot tnh c ch enzym -glucosidase ca cc cht tinh sch t l vi v l ch ng
nhm xc nh c ch h ng huyt ca chng.
Bng 3.14. Hot tnh c ch enzym -glucosidase
Stt Tn cht CTPT IC50 (g/ml ) IC50 (mol/l)
24-methyl (3-hydroxy-lup-20(29)-en-24-oic
1 C31H50O3 3,4 0,7 71x10-8
acid) ester (H4)

2 3-hydroxy-lup 20(29)-en-28-oic acid (C3) C30H44O3 3,60,5 79,3x10-8

2,4-dihydroxy-6-methoxy-3,5-
3 C18H18O3 4,30,2 153x10-7
dimethylchalcon (H6)
2,3,23-trihydroxy-urs-12en-28-oic acid
4 C30H48O5 5,70,5 117x10-7
(LVE4)

5 3-hydroxy-olean 12(13) en-28-oic acid (LVE2) C30H48O3 6,10,3 133x 10-7

Trn y l th t 05 cht theo hot tnh c ch enzym -glucosidase gim dn (IC50 tng dn).
Hot tnh c ch enzym -glucosidase ca i chng l thuc Acarbose c xc nh vi IC50 l 0,12
mg/ml. Bc u khng nh kh nng h ng huyt ca cc cao chit phn on v cao tng nc
mt phn c quyt nh bi cc thnh phn ha hc ny. Cc cht tinh sch ny khng phi l cht
mi tuy nhin y l cng b u tin v hot tnh c ch enzym -glucosidase ca chng.
3.4. NGHIN CU BO CH V TC DNG H NG HUYT CA CH PHM
THIVODA
3.4.1. Chn lc ch phm ngun gc t thc vt c kh nng h ng huyt
Cc thc vt c chn lm thnh phn ch phm Thivoda gm: Ch rng ca
Phyllanthus urinaria L. thuc h Thu du (Euphorbiaceae); Ch ngIlex kaushue S.Y.Hu thuc
h Trm Bi (Aquifoliaceae); ViCleistocalyx operculatus (Roxb.) Merr.& Perry thuc h Sim
(Myrtaceae); Dy tha canhGymnema sylvestre (Retz.) R.Br.ex Schult thuc h Thin l
(Ascleipiadaceae)
Cc mu thc vt nghin cu c nh tn khoa hc bi PGS.TS V Xun Phng v
ThS.Nguyn Th Cng (Phng thc vt-Vin Sinh thi v Ti nguyn Sinh vt).
3.4.1.1. nh tnh thnh phn ha hc c trong 5 mu thc vt
Hot cht sinh hc c trong cc mu thc vt ny ht sc phong ph, cha mt s thnh phn
trong s cc nhm hp cht: akaloid, flavonoid, saponin, tannin, steroid, glycoside.
3.4.1.2. Tc dng h ng huyt ca cao tng nc v cn
Tin hnh chit bng nc v cn, trn 5 loi cao nc thnh phn, thu cao tng nc, trn 5
loi cao cn thnh phn thu cao tng cn. Nghin cu kh nng h ng huyt ca cao tng nc
v cao tng cn trn chut nht T type 2. Kt qu c trnh by trn Hnh 3.25.

Hnh 3.25. Nghin cu kh nng iu ha ng

huyt ca cao tng s (p*<0,05, p**<0,001)

Cao tng nc v tng cn th hin hot tnh iu ha ng huyt, c th gim tng ng

nhau, ng huyt v mc kh n nh, so vi nhm i chng chut T type 2 khng c
ung cao tng, ng huyt gim c hn 68% (p<0,001).
nghin cu tc dng phc hi cc tn thng trn gan v iu ha tnh trng ri lon lipid
mu, sau 20 ngy th nghim, tin hnh ly mu xt nghim mt s ch s ha sinh: GOT, GPT,
cholesterol, triglyceride.

Cao cn Cao cn
120 6
Cao nc 5.21.1 Cao nc
102.1 16
C 5 C
89.7 17
90
Nng mmol/l

4
Nng (U/l)

60 3
1.77 0.22* 2.440.7
25.3 6.6** 2 1.8 0.47**
18.6 5.2** 22.3 4.5** 1.32 0.18**
30 1.05 0.11**
17.3 4.3** 1

0 0
GOT GPT Cholesterol Triglycerid

Hnh 3.26 Ch s men gan (tri) v cc ch s m mu (phi)

*p<0,05, **p <0,001 (p so vi nhm i chng)
 Ch s GOT v GPT nhm ung cao nc v cao cn gim so vi l i chng, s khc nhau ny
hon ton c ngha thng k. l chut c ung cao tng nc c cc nng cholesterol v
triglyceride thp nht.
3.4.2. Nghin cu v bo ch ch phm Thivoda c tc dng iu ha ng huyt trn chut
nht m phng T type 2
3.4.2.1. Bo ch ch phm Thivoda
Nc c dng chit xut cc thc vt bo ch ch phm Thivoda.
Bng 3.16. Thnh phn ch phm Thivoda
Khi lng m mu kh m cao nc m thnh phn Thnh phn
Mu (kg) (kg) cm (kg) vin nang (mg)

N vi 4,3 1,0 0,55 70

L vi 6,0 1,0 0,57 70
Ch rng ca 5,3 1,0 0,48 70
Dy tha canh 3,5 1,0 0,51 70
Ch ng 4,8 1,0 0,55 70
T dc ----- ----- 1 150
Tng s 23,9 5,0 3,5 500

3.4.2.2. Nghin cu kh nng h ng huyt ca ch phm vin nang Thivoda trn chut nht
m phng T type 2
Trong qu trnh bo ch ch phm chng ti phi x l mu vi cc thao tc u lin
quan n x l nhit. Do nhm xc nh kh nng h ng huyt ca ch phm Thivoda trn chut,
chng ti b tr th nghim cho chut nht T type 2 ung ch phm Thivoda vi liu 500mg/kg/ngy
v so snh vi chut T type 2 iu tr bng Metformin liu 500mg/kg/ngy. Kt qu th hin trn Hnh
3.28.

0h
35
2h
Nng ng huyt (mmol/l)

30 4h
8h
25
* * 3 ngy
20 *
* * 7 ngy
* * *
* 10 ngy
15
**
20 ngy
10 **

0
Thivoda
Thivoda Acarbose
Metformin

Hnh 3.28. Kh nng h ng huyt ca ch phm Thivoda v Metformin

(*p<0,05, **p<0,01, p so vi thi im 0 gi cng nhm)
 Ti thi im 0 gi, 2 gi, 4 gi v sau 3 ngy, ng huyt ca chut cho ung ch phm
Thivoda vn mc tng i cao. Tuy nhin sau khi iu tr n ngy th 7 tr v sau ng huyt
ca chut gim dn (16,21,8 tng ng mc gim 43% ), n ngy th 20 th ng huyt ca
chut tr v mc bnh thng v n nh (7,51,4 mmol/l tng ng mc gim 71% v c
ngha thng k vi p<0,001). Nhn thy thuc Metformin c tc dng h ng huyt nhanh chng
trong vng 2 gi v 4 gi t thi im bt u s dng thuc (gim t 27,62,1 xung cn 20,11,4
v 16,62,5 mmol/l) tuy nhin khi s dng thuc trong mt thi gian di th ng huyt ca chut
khng nhng khng gim m cn c du hiu tng nh.
3.4.2.3. Kh nng c ch enzym -glucosidase
Kt qu c ch enzym -glucosidase ca ch phm Thivoda: c ch 60,6 % hot tnh -
glucosidase ti nng 6,7 0,4 g/ml (tng ng theo tnh ton IC50 l 5,510.4g/ml). Ch
phm Thivoda th hin hot tnh c ch -glucosidase gn nh tng ng vi cc phn on l
vi v l ch ng.
3.4.2.4. Nghin cu c tnh cp ca ch phm Thivoda
Khng xc nh c LD50 do mc liu ti a c th cho chut ung ch c 30% chut
cht. xc nh c mc liu di liu cht (LD0) l 41,7 g mu th/kg chut. Ngoi ra ch
phm Thivoda c xc nh v ch tiu gii hn cc vi sinh vt t yu cu qui nh ca Dc in
Vit Nam IV.

KT LUN

1. Xc nh 8/24 mu thc vt c iu tra c tc dng h ng huyt trn chut nht

T type 2 gm l vi, n vi, dy tha canh, ch rng ca, ch ng, v thn i, l tm
gi trn cy mt, c chui ht. Trong cc mu l vi, thn v l ch rng ca, v thn
i, l tm gi trn cy mt, c chui ht l pht hin u tin Vit Nam v hot tnh h
ng huyt.
2. i vi mu l vi:
- xc nh c 03 cao chit phn on c kh nng h ng huyt tt trn chut T
type 2 l: CHe, CEtA, CBuOH.
- phn lp v xc nh c 07 hp cht trong l vi l -sitosterol (H1); -sitosterol
glucopyranoside (H2); 2,4-dihydroxy-6-methoxy-3,5-dimethylchalcon (H6); 3-hydroxy-lup-
20(29)-en-28-oic acid (C3); 3-hydroxy-olean-12(13)-en-28-oic acid) (LVE2) ; 2,3,23-
trihydroxy-urs-12en-28-oic acid (LVE 4); quercetin (C7).
- Cng b u tin v hot tnh c ch enzym -glucosidaseca cc cao phn on l vi v
ca cc cht tinh sch. IC50 ca cc phn on CHe, CEtA, CBuOH ln lt l: 5,0370,6;
5,7660,3; 8,0110,7 g/ml; IC50 cc hot cht H6, C3, LVE2, LVE4 tng ng l 4,30,2;
3,60,5; 6,10,3; 5,70,5 g/ml.
3. i vi mu l ch ng:
CHe l ch ng c tc dng h ng huyt trn chut T type 2 v c ch 55% hot
tnh ca -glucosidase ti nng 7,84g/ml. Hp cht H4 phn lp t CHe c xc nh l
24-methyl (3-hydroxy-lup-20(29)-en-24-oic acid) ester gy c ch 59,5% hot tnh ca enzym
-glucosidase ti nng 4 g/ml. y l pht hin mi ca chng ti v hot tnh c ch
enzym -glucosidase ca hp cht H4 v cao phn on CHe t l ch ng.
 4. Cao nc l vi v l ch ng ngoi tc dng h ng huyt cn c tc dng phc hi
gan chut T type 2 b tn thng.
5. Ch phm Thivoda c tc dng h ng huyt:
- Thnh phn ch phm Thivoda gm: l vi, n vi, l dy tha canh, thn v l ch rng
ca v l ch ng.
- Ch phm Thivoda c kh nng h ng huyt trn chut T type 2 mt cch n nh,
ti ngy th 20 v mc 7,51,4 mmol/l tng ng mc gim 71% (p<0,001).
- Ch phm Thivoda khng gy h ng huyt chut nht bnh thng.
- Ch phm c hot tnh c ch enzym -glucosidase vi IC50 l 5,510,4 g/ml.
- Khng xc nh c c tnh cp ca ch phm Thivoda, ch tiu gii hn vi sinh vt t
yu cu qui nh ca Dc in Vit Nam.

NGH
Qua nhng kt qu nghin cu thu c chng ti thy rng ngun ti nguyn thc vt ca
Vit Nam rt qu gi v tim nng. Ch phm Thivoda c ngun gc t nhng thc vt quen thuc,
ging vi cc tho dc dng trong ng y, do kh nng ch phm gy c l rt t. Lun n hy
vng s gp phn tham gia vo nng cao sc khe cng ng. t c mc tiu chng ti
xin a ra ba ngh sau:
- Nghin cu mt s c ch tc dng gy h ng huyt khc ngoi kh nng c ch enzym -
glucosidase ca ch phm Thivoda v ca cc hot cht phn lp c.
- Nghin cu v thnh phn ha hc cng nh tc dng h ng huyt ca cc hot cht tinh
sch t thn v l ch rng ca Phyllanthus urinaria L.
- Xc nh c cc tiu chun c s ca ch phm Thivoda nh ch tiu cm quan, l ha, vi
sinh vt, hm lng kim loi nng, hm lng cc cht c hi khng mong mun, c tnh
bn trng din, t xut thm d lm sng trn ngi qua cc giai on.

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