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
N g h i e n C U P U - K y t h u a t Phirang phap nghidn ciru
Tu kdt qua thyc nghidm da xdc djnh dieu kidn tien hdnh nhu sau: cdt Phenomenex C1 8
Gemini (150 x 4,6 mm, 5um), pha ddng gradient tu 0 - 25 phut acetonitril - H20 (35:65), 26 - 33 phut acetonitril - H20 (10:90), 34 - 38 phut acetonitril - H20 (35:65), phdt hidn vdi detector PDA tai 280 nm, tdc dd ddng 1 ml/
phut, nhidt dd cdt 35°C.
Xdc dinh cdc thdng sd chiet sidu dm mdu:
thdi gian sidu dm 20 phut, mdi miu chiet 4 lin.
Thdm dd thdi gian vd sd lin chiet bdng kiem tra djch chiet vdi HPLC.
Thim dinh quy trinh phdn tich 6 - gingerol v i tinh chon loc, tinh phii hap hd thdng, tinh tuyen tinh, dd Idp lai, dd dung, gidi han phdt hidn LOD vd gidi han dinh luang LOQ. Cdc thdng sd thim djnh duac khao sdt trdn cdc miu 6-gingerol chuin, gung Dd Lat vd cao gung N. Cdc kit qua duac xu ly thdng kd vdi Excel 5.0.
Djnh luang 6-gingerol trong 3 mdu cao gung vd trong 3 miu gung tuai dd lidt kd d trdn.
K i t qua n g h i e n cuoi
Xdc djnh dd i m cdc mau gung bdng phuang phdp thi tich (chung cit vdi dung mdi) theo Phy lyc 12.13 DOVN IV.
Quy trinh chuin bj m i u 6u77g tuvi
Cdn chinh xdc khoang 25 g gung tuai dd xay nhuyen, thdm 20 ml ethanol tuydt ddi vd
sidu dm 20 phut. Gan liy djch chiit, bd duac chiit tiip tyc 3 lin n&a. Gdp djch chiit vd thdm ethanol vira du 100 ml.
Cao gung
Cdn chinh xdc khoang 0,05g - 0,2g cao girng cho vdo becher 50 ml, cho vdo khoang 8 ml ethanol tuydt ddi, sidu dm trong 30 phut, loc cho vdo binh djnh mire 10 ml, dien ethanol tuydt ddi din vach.
Chuin doi chieu
Cdn chinh xdc khoang 2,1 mg chuan 6 - gingerol (hdm luang 95%) hda tan bdng methanol trong binh djnh mire 1 ml, thu duac dung djch cd ndng dd 1,995 mg/ml. Tir dung djch ndy pha lodng tiip bdng methanol de duac cdc dung djch cd ndng dd lin luat Id 0,9975;
0,4988; 0,1995 vd 0,0998 mg/ml.
Djnh tinh 6 -gingerol bdng LC-MS
Cdc miu girng vd cao girng trudc khi djnh luang duac djnh tfnh bdng LC-MS. Cdc diiu kidn thyc hidn LC nhu md td phin phuang phdp nghidn cyu vd diiu kidn thyc hidn MS:
ion hda miu bdng ESI kiiu positive, nhidt dd budng ion hda 350°C, phun miu vdi khi nitrogen 30 L/phiit, bay hoi dung mdi vdi khi nitrogen khd 10 L/phut, khoang qudt tu 100 - 1200 m/z. Sdc ky dd PDA vd MS thu duyc cho thiy cd 2 pic d 15 phOt vd 18,6 phut cd ciing phd MS vdi [M+H]+ 295,4 m/z trung vdi phd MS cua 6-gingerol'2'.
4)31 --
4 0
i»v .:Ht*'
m
1- V '"''if tf
•* f u »• •*.-
•iff
•: »•. v r •
a cc :: cc
srjn - « . : > • ' \ x ::
• v - :* cc :: cc 3£ J: *:.»
TC
I'M . "-" \^
' «.'::• ' •:'» I'X ' :i cc cc
Hinh 1: SSc ky dd LC-MS va phi MS cua 6 - gingerol trong gCrng Da Lat
TAP CHI DU'(?C HQC - 4/2011 (S6 420 NAM 51) 29
• N g h i e n C U M J - K y t h u a t Tham djnh phirang phap phan tich
a 1 1
\ I ii
J';1 .~. . & 1 \
.UJL^^.^. I, K _ Hinh 2: Sdc W dd HPLC cua cao gi>ng (a), 6-gingerol chuin (b);
(*) IR cua pic 6- gingerol trong LC MS (djnh tinh) va HPLC (PDA) co su- khic biet do tiin hanh trin hai h$
thdng sac ky khac nhau.
Tinh chon loc
Sac ky dung mdi MeOH trong cac dieu kien sic ky neu tren, sac ky dd (hinh 2) khdng cd pic nao ngoai tra pic MeOH vdi k 2,0 phut. Vdi cung dieu kien sic ky cac miu gang taoi Da Lat va cao gang cho pic cua 6- gingerol tach hoan toan, nhon va can ddi cd thdi gian lau tp, la 19,668 phut va 19,045 phut tuong ung (hinh 2) vdi tp, cua pic 6- gingerol chuin 19,009 phut (*). Nhu vdy quy trinh HPLC dat tinh dac hidu vdi 6- gingerol.
Bang 3: Cic thing sd cua qui trinh HPLC tich 6 - gingerol cic miu
Tinh phu hqp he thong
Tinh phu hap hd thdng cua qua trinh djnh luang HPLC duac ddnh gid trdn ca 3 miu 6- gingerol chuin, gung tuoi va cao gung. Mdi mdu sdc ky 6 lan. Cdc thdng sd cua qud trinh tdch HPLC thu duoc the hidn trong bang 3. Cdc RSD cua cdc thdng sd sdc ky cua 6 - gingerol trong ca 3 mdu khao sdt deu < 2 % (bang 3). Do dd hd thdng sdc ky djnh luang 6 - gingerol dgt tinh tuang thich cao.
Mau t/? Sdinh k' a Rs r N
TB 19,309 1031792 12,8 - - 1,045 11869
6- gingerol chuan SD 0,098 8552 0,075 - - 0,005 191
RSD% 0,51 0,83 0,59 - - 0,46 1,61
TB 19,553 1999322 12,97 1,086 2,129 1,033 11952 Gung Da Lat SD 0,072 3852 0,05 0,001 0,024 0,003 98
RSD% 0,37 0,19 0,41 0,07 1,11 0,3 0,82
TB 18,894 1624544 12,48 1,082 1,855 1,027 11744 Cao gung N SD 0,053 25010 0,041 0,002 0,04 0,005 118
RSD% 0,28 1,54 0,32 0,14 1,94 0,5 1,01
Tinh tuyen tinh
Tien hdnh sdc ky nhu dieu kidn da ndu d tren dung djch 6- gingerol chuin vdi cdc ndng do khdc nhau. Kit qua ghi d bang 4 cho thiy 6- gingerol vdi
khoang ndng dd tu 99,8 - 1995 ug/ml cd sy tuang quan tuyen tinh chdt che giua ndng dd vd didn tich pic the hidn qua dudng thing hdi quy vd h$ sd tuang quan R = 0,9941 (Hinh 3).
30 TAP CHI Dl/QC HQC - 4/2011 (SO 420 NAM 51)
• N g h i e n c i m - K y t h u a t
Bang 4: Sir toong quan gioa ndng dp va didn tich pic cua 6- gingerol chuin
Nong do (ug/ml) 99,75 199,5 498,75 997,5 1995
Didn tich dinh 437755 1016895 2497938 5153585 12166173
•a u
•
14000000 12000000 10000000 8000000 6000000 4000000 2000000 0
6157.6X - 413630 R2 = 0.9941
500 1000 1500 2000 2500
Ndng do (ug/ml) Hinh 3: Duong tuyin tinh gioa ndng do va dien tich pic cua 6- gingerol chuin Kiem tra tinh thich hp-p cua phirang trinh hoi
quy (trie nghidm F) vd y nghTa cua cdc hd so trong phuang trinh hdi qui (trie nghidm t): hd sd B, cd y nghTa, hd sd B2 khdng cd y nghTa, phuang trinh hdi quy cd tinh tuang thich. Vdy phuang trinh hdi quy cd dang y = 6157,6 x.
Xac dinh gioi han phat hien LOD va gioi han dinh Iwong LOQ
Tidn hdnh pha lodng mdu 6-gingerol chuan dd duac pic sdc ky cd chieu cao gap 3 lan dd nhidu dudng ndn, d dd nhay tdi da cd thd duac Bdng 5: Kit qui di chinh xic trdn cic miu
khi van hanh. Ket qua quy trinh phdn tfch HPLC 6 - gingerol da xdc djnh cd LOD = 0,65 ug/ml vd LOQ = 3,33 x LOD = 2,2 ug/ml.
Dd chinh xac
Trdn mdu gung tuai DL tien hdnh dinh luang hdm lugng 6 - gingerol trong cdc miu girng vdi 6 lin bam lidn tiep. Hdm luang 6 - gingerol trung binh (mg/g) trong girng tuai DL vd cao N lin lugt Id 16,715 vd 85,542 vdi RSD
= 1,81 % vd 1,49 %. Do dd phuang phdp xdy dung dat dp Idp lai.
Girng DL Cao girng N
X = 16,715 (mg/g); SD = 0,302; RSD =1,81 %; X = 85,542 (mg/g); SD = 1,28; RSD = 1,49 %;
e = ± 0,34 (P = 0,95, n=6); u = 16,715 ± 0,34 (mg/g) e = ± 1,45 % (P = 0,95, n=6); u = 85,542 ± 1,45 % (mg/g) Do diing
Thdm vdo miu thu mdt luang chinh xdc 6- gingerol. Tien hdnh chiet mdu thir cd thdm chuin theo quy trinh dd md td (bang 6). Hdm luang trung binh cua 6 - gingerol trong mdu gung tuai DL Id 16,715 mg/g dugc lidu khd ndn ty Id phuc hdi cho phdp Id 9 7 - 103 %. Theo kit qua bang trdn, ty Id phuc hdi cua miu ndm trong gidi hgn quy djnh
Bang 6: Khio sit di diing cua 6 - gingerol trong miu gdng Di Lat
Lwong chuan Di$n Lwovg chuan Ty 1$
thim vio tich tim thay phuc hoi (pg) dinh (ug) (%)
Djnh lugng 6 - gingerol trong cdc mdu girng tiroi va cao girng
Chuin bj miu girng tuoi vd cao gung nhu phin xdy dung phuang phdp. Tiin hdnh djnh lugng cdc miu trdn bdng phuang phdp HPLC, kit qua thu dugc thi hidn trong bang 7.
Bang 7: Him luong 6 - gingerol trong cic miu khio sit (mg/g)
227,2 2165589 228,6 100,62
Mau 6-gingerol Mau 6-gingerol Girng
Long An 15,88 Cao girng A 33,98 Girng
Da LaX 16,68 Cao girng B 18,81 Girng
Trung Quoc 13,81 Cao girng C 96,25 454,4 3903749 484,55 106,65
Ty Id phyc hdi trung binh: 103,64%
Ban luan
Trong sdc ky dd LC-MS vd HPLC (PDA detector -280 nm) cua cdc miu khao sdt cho cd
TAP CHl DUQC HQC - 4/2011 (SO 420 NAM 51) 31
N g h i e n c u m - K y t h u a t 2 pic tai 15,17 va 18,6 phut c6 ciing pho MS (c6 m/z 295,4 tuang irng vdi [M+H]+ cua 6 - gingerol) va tai 17,702 va 19,045 phut c6 ciing pho UV. Dieu nay phii hop vdi cac ket qua da cdng bo, 6 - gingerol cd 2 dang dong phan R va S (trong ty nhidn chu yeu ton tai d dang ddng phdn S)[3'. Nhu- vdy trong girng tuai tdn tai ca 2 dang R vd S cua 6 - gingerol.
Trong 3 mdu girng tiroi, 6 - gingerol cd ty Id cao nhat trong girng Od Lat vd thap nhdt trong girng Trung Qudc. Hdm lugng 6 - gingerol trong cdc mdu cao girng deu ldn hon so vdi cdc mdu girng tiroi. Giua cdc mau cao girng hdm luong 6 - gingerol khdc nhau nhieu cd the do dugc che bien tir cdc mdu girng tuoi khdc nhau, cdch che bien khdc nhau vd thdi gian bao quan khac nhau (mdu cao gung N che bien thdng 04-2010, miu cao girng K2 che bien thdng 1-2009, mdu cao gung chd bien thdng 12-2009).
K i t luan
Phuong phdp djnh lugng 6 - gingerol trong gung tuoi bang HPLC dd xdy dung don gian dd dp dyng vd dat ydu cau ve tinh chpn Ipc, tinh phii hgp hd thdng, dd chfnh xdc, dd diing. Vdi ket qua nhu vdy, phuong phdp ndy cd the dua vdo chuydn ludn ve gung trong Dugc dien Vidt Nam de gdp phdn ndng cao chat lugng cua
gung vd cdc che pham cua dugc lidu ndy.
S u m m a r y
Ginger is the rhizome of Zingiber officinale (Zingiberaceae), a plant cultivated in many tropical and subtropical countries. The constituents responsible for the pungent taste of ginger are a homologous series of phenolic ketones, known as 4-, 6-, 8-, 10- and 12- gingerols. In this study, a gradient elution reversed-phase HPLC separation of ginger extract has been developed. This method shown to be suitable for the determination 6-gingerol from fresh ginger or from ginger extracts. The HPLC analysis displayed a simple, good linearity, precision and accuracy within a wide concentration range. The results from our study could be apply in quantitative control for preparation from ginger.
Tai lieu t h a m k h a o
1. BO Y te - Dugc dien Viet Nam IV (2010), tr. 771.
2. Hongliang Jiang, Aniko M. Solyom, Barbara N.
Timmermann and David M. Gang , Characterization of Gingerol related compounds in Ginger Rhizome (Zingiber officinal Rocs) by HPLC-MS electrospray ionization, Rapid Commun.,(2005), 19, pp 2957-2964.
3. Quality Control Department of Nature Remedies Rechearch Centre, Bangalore Zingiber officinale, (1999) p. 10-20.
r ** r X a c d i n h d o t i n h k h i e t q u a n g h o c c u a d a n c h a t
o s - N - b e n z y l v a d s - N - h e p t y l p h t h a l a z i n o n b a n g
p h u o n g p h a p H P L C s u d u n g p h a t i n h b a t d o i Chirong Ngoc Nai1, Tran T h j Nguyen Dang2,
Dang Van Tinh2, Nguyen Dire Tuan2' ' Vien Kiem nghiem thuoc Thanh phd H6 Chi Minh
2Khoa Duoc, Dai hqc Y Duoc Thanh pho Ho Chi Minh Dat van de
Nhidu cdng trinh nghidn ciru trdn the gidi cho thay cdc din chit c/s-A/-alkyl phthalazinon cd tdc dung uc che chpn Ipc phosphodiesterase IV manh hon ddng phdn frans ddng thdi cdc ddng phdn cis(+) cd tdc dung mgnh hon cdc c/s(-) vd
dgng racemic | 1"2l Hidn nay, Khoa Dugc, Dgi hpc Y Dugc thdnh phd Hd Chi Minh dd tdng hgp thdnh cdng cdc din chat quang hogt cis-N- benzyl vd c/s-/V-heptyl phthalazinon bdng con dudng tdng hgp tgo mudi dia. Do tdc dyng sinh hpc ciia chit ddi quang chi t h i hidn d mdt trong
