• Nghien ciru - Ky thuat
bu*c sfing phan tich" 364 nm; toe dfl dfing:
1 ml/phut, nhiet dfi cot 28°C. the tfch fi§m: 20 pl;
n>a giai theo chutD'ng tiinh gradient vdi pha dfing gom hai dung mfii la acetonitnl va dung djch acid forniic 0,1%. Phu'cng phap djnh lu^ng duw; xSy dyng cfi sy ^rang quan hjyen tfnh ch#t che gi&a dien tfch pfc vd nong dp quemretin trang khoang nong dfl khao sm vfi-i R^ = 0,9999, cfi 6Q ISp lgi (RSD = 2,57%) ^va dp diing (tip 96,42-102,71%.
RSD = 2,27%) tot Bang phyang phap xdy dyng dyo-c, da xac i^nh ham lypng quercetin trong qua teo meo thu hai & huy§n MCi Cang Chai, flnh Y6n Bar vao thang 10/2015 la 0,00434%.
Ndi dung cua nghien ciru ndy thudc de tdi ma s6 KHCN-TB.04C/13-18. D§ tdi ndy dLfoc tai tru bdi Chumg trinh °Khoa hoc vd Cdng nghd phuc vu phdt tridn bin vung vung TSy B4C° md s6 KHCN-
TB/13-18 do Dai hpc Quoc gia Hd Npi Id Co quan chO tri.
Tki li$u tham khao
1. B6 Tat Lgi (2004), NhQng cSy thudc vd vi thuSc Vi$t Nam. Nxb. Y hpc, Hd NQI, trang 355-357.
2 V[^nDu!(?c\i^(20M). CSy thudc vadOngv0t Idm thudc & VietNam. Nxb. Khoa hpc vd K? thu^t, Ha NOi, trang 785-787.
3. FSbio R. F. Seiva. Luiz Gustavo A. Chuffa, Camila Pereira Braga et al (2012), "Quercetin ameliorates glucose and lipid metabolism and improves antioxidant status in pos^atally monosodium glutamate-tnduced metabolic alterations", Food and Chemical Toxicology.
vol 50, pp. 3556-3561.
4. ICH Guidelines (1996). Q2 (Rl) validation of analyUcal procedures: text and methodology.
5. Jennifer Swick (2011), EfTect of ffie ffavonad quercetin on adipocytes. The master thesis. University of Massachusetts Amherst.
6. Yu He, Zhimin He, Feng He, Haitong VJan (2012), "IDetemiination of quercetin, plumbagin and total flavonoids in Drosera pellata Smith, var glabrata Y. Z.
Ruan", Pharmacognosy Magazine, vol 8(2), pp, 263-267 {Ngdy nh$n bdi: 13A)5/2016 - Ngdy duydt mng: 05/07/2016) ^ ^ H
Nghien cihi chiet xuat, phan lap hop chat ^ diterpenoid chinh tir cay Idio sam cho la
{Croton tonkinensis Gagnep.)
Tran Thj Thu Hien'*, Nguyin Duy Thu^n' Nguyin Van ThoanS Phffffng Thi^n Thmmg", ly^n Phuffng ThSo' 'Hgc vi4n IT Dupc hgc c6 tniyin V141 Nam
^Vi§n Dugc lieu ' Trudng Dai hgc Dugc Ha Ngi 'E-mail: [email protected] Suinmary
Keyviords: Craton tonkinensis Gagnep.. diteipenaid.
san ptiim niy i i m ctiSt d6i ctiilu d4 dinii tinti mgt Sat vln dd
C i y klio Sim cho i i (Cmton tonkinensis Gagnep.) dm?c sft dgng lim thuoc chgng vigm logt da diy, m i n ngfta, ghe Ift Mi, ngoii ra cbn u^ che SU' Sftng viSm M , thinh phin hoat chit chinh trong la kho s i m l i cic diterpenoid.'Trong bil bio niy, chUng tgi cflng b6 quy trinh chilt xult phin igp, xic dmh cgng thftc hga hoc cua chit diterpenoid chinh tft l i ciy kho s i m ctio l i v i dftng
so m l u l i v i oinh kti6 s i m cho l i
Nguyen lidu vi p h u w n g phap nghiSn c i i u Nguygn li^u
M i u nghign cftu l i l i c i y kh6 s i m cho l i thu h i i ft Hirong Sen, huygn Mf Bfte, H i Ngi thino 1 nim 2013: d i duac x i c dmh t i n khoa hoc l i Croton tonkinensis Gagnep., m i tigu b i n s6
TAP CHl D i r o c HOC - 7/2016 (S6 483 ^ 156)
• Nghien CIFU - Ky thuat
HNip;i 7851/13. IMiu nghign cftu lifu tai Ktioa Hfta phin Hch - Tliu chuin, Vign Duac iigu. U kti6 Sim sau ktii thu h i i dug'c phcri, say khg, tan thinh bdtthgdungdechietxuat
Phuwng phap nghien cipu Phucmg phip chiit xuat, phin lip Chilt xuat dftftc liiu bang phftcng phip chiet ft nhigt dg phbng v i chiet nftng vfti methanol ethanol 4 c i c dg c6n khic nhau d l chilt nhgni chat nghign cftu
Phin l i p chit bing phu'cng phip s i c ky cgt vfti chat hipphgiisilicageieo (cft hat 40-63|im Merck) pha thftftng v i rfta giii vfti hg dung mbl thich hop
Phifcmg phdp xac dinh cau trdc hda hgc Xic dinh d u tnjc ho'p chit phin i i p du'oc dira trSn c i o tinh chit hba Ki vi cic phu'ong phip pho bao gbm: pho h6ng ngoai (/R). khli p h i (MS), phi cgng hftftng tft hat nhgn ('H-NMR v i "ONMR) vi so sinh vfti dft ligu trong cic t i l ligu tham khao Nhigt dg ngng chay (mp) duac do bing miy Gallenkamp (Sanyo-Nhit Ban). P h i cgng hu'ong tft ghi bSng miy Bniker AM500-FT-NiVlR Spectrometer vfti chat chuin ngi l i TMS. Ph6 klioi ESI-US Suae ghi trin miy AGILENT 1100LC- MSD Trap. P h i IR duac ghi trgn miy GX PeiWn Elmer (M?). P h i UV-VIS dm?c ghi trgn miy LIV- 1800 Spectrometer (Shlmadzu-Nhgt Ban).
Phuvng phip dinh dnh S|/ cd m^t ent-kauran diterpenoid chlnh vi kiim tra dd dnh khiit
^ Kilm tra dg tinh kliilt v i dinh tinh sg' eg mgt cua ent-kauran diterpenoid chinh ft i i vg cinh cgy khg Sim cho l i bSng s i c kj Iftp mong (SKLM) v i sic k> long higu ning cao (HPLC). TLC trgn ban mong tring sin pha thftftng silica gel 60 F (Merck), quan s i t a duOi d i n tft ngoai budc sgng 254 v i 365 nm, thuoc thft hign m i u i i H SO 10%
trong ethanol, sly ban mong ft 120°C d i n khi hign vgl HPLC duoc thg'c hign trin hg thing HPLC - LC 10A (Shimadzu-Nhgt Ban). Dilu kign HPLC' a5tBondapak™ CIS (3,9 mm IDx300 mm, 5 pm) Hing Miliipore, pha dgng: Acetonittii (A): Nu'ftc d t hai lin (B) (65-35): tic dg dgng 0,4 ml/phuf the tich tl§m 10 pi: detector UV 233 nm: thfti gian s i c ky: 45 phftt
Chiit xuit, phin Ifp chit
. , ^ f , ' , ' ! ' ? , * . ^ " ' ' " '*3) " " ^ ^ "S^"' * i l t vfti MeOH (30 lit) ft nhlgt dg phbng (2 lin x 72 gift/iln)- chilt nflng tilp vfti MeOH (1 iln x 25 lit): cb thu hii dung mfli duo-c d n MeOH (960 g): d n duvc chilt vfti dicloromethan vg d t loai dung mgi thu duftc
T » P C H l D i r < y : H p c - 7 / 2 0 1 6 ( s 6 4 8 ? i a 7 ^
can didonamethan (446 g). C i n didoramethan dftftc phin tich tren cbt silica gel 60 (Merok) vfti dung mbl n>a giai l i n-Hex - EtOAc (4-1)- C i c phin doan dup'c kiem tra bing SKLM, thu 'duvc phan doan F2: Ket tinh nhilu i l n phin doan niy thu dfto'Cchit CT-1 (3,529 g). Tiln hinh 3 ian chilt xuit v i phin lgp thu duvc 10,67 g chit CT-1.
K§t qua va ban luan Xac djnh c l u tnjc
Chat nin CT-1 thu duyc dufti dang tinh the hmh kim khbng miu, mp. 148-150''C. Tan t i t trong IMeOH, EtOAc, k i m tan trong ether diu, benzen Pho UV-ViS (MeOH) X„^ nm : 233. P h i IR (KBr) v „ c m ' : 1126, 1238 (C-O), 1642 (C=C), 1743 1716 (C=0), 2944 (C-H), 3468 (OH) P h i ESI^
MS : mte 383 [M + Na]*, M = 360 ( C ^ H „ 0 ) P h i
•H-NMR (CDCI,, 500 MHz). 6 (ppm)' 1 80 (1H s H J : 0,74 (IH, td. J = 10,6: 4,0 Hz: H J , 1,65 ( I H ' m, H J : 1,51 (IH, m, H J ; 1,38 (IH, b | J = 4,0 Hz' H J ; 1,35 (IH, m, H J ; 1,28 (IH, dd, J = 1,6: 2 6 Hz, H J ; 1,45 (1H, d, J = 6,2 Hz, H J ; 1,72 (1H m. H ^ ; 4,04 (IH, d, J = 15,0 Hz, H J ; 1,23 ( 1 H ' bd, J = 9,0 Hz. H J ; 1.77 (IH, s, H„) ?,48 (IH. m, H J : 1,96 (IH, m, H , J : 1,70 (IH, m, H , J ; 3,11 m. s, H J : 2,06 (IH, bd, J = 2,5 Hz, H,!); 2 08 (1H, s, H ' J : 5,29 (IH, s, H , J ; 6,97 (IH, s. H , )•
3,66 (IH, d, J = 11 Hz; HjXe7 (1H, dd, J = s'o 16,0 Hz: H, J ; 0,86 (3H, s); 1,14 (3H, s); 2,09 ( 3 H ' s). "ONMff (COa,, 125 MHz), 5 (ppm): 38,5 (C- 1); 17,5 (C-2); 35,4 (C-3); 36,4 (C^); 46,3 (C-6)- 27,7 (&6): 70,8 (C-7): 58,4 (C-6); 61,8 (C-9); 39 6 (C-10); 18,2 (C-11); 32,8 (C-12): 37,6 (C-13): 27 9 (C-14): 209,8 (C-15): 149,2 (C-16); 114 9 (C-17)- 72,3 (C-18): 17,5 (C-19): 18,0 (C-20): 171 2 ( C - n 21,1 (C-2'). ' \ /.
P h i IR cho d c pic dgc tn^ng 1126, 1238 (C- O), 1642 (C=C), 1743, 1716 (C=0), 2944 (C-H) vg 3468 (OH). Nhu viy, trong cbng thftc cua hvp chit CT-1 cfl nhflm hydroxy (-OH), nhflm oxo (RC(R')=0) v i lign kit dbi C=C Phi ESt-US cho pio ion tai rn* = 383 [M -i- Na]' phd hyp vfti cbng thu'c phan^tft cua hp'p chit i i CJi^p (M = 360 d.v.C). Pho 'H-NMR chi ra tin higu dgc trung cOa 2 proton olefin tai 5„ 5,29; 5,97 ppm, 1 proton cija nhflm carbinol tai 5„ 4,04 (IH, d, J = 15 0 Hz H )- 3 nhbm methyl tai 8„ 0,85; 1,14; 2,09 ppm C i c hgng s6 tuvng t i c ciia H, cho phgp x i c nhgn d u hlnh p cua proton n i y P h i "C-NMR cho thiy hi?p chit CT-1 cfl 21 carbon, trong db cb 1 nhflm oxo tai 5^209,8 ppm (C-15); 1 nhbm carbcnyl tai 6 171,2 ppm (C-1'), 2 carbon n l i dgi tgi 5^ 1492 (C^
16), 114,9 (C-17), mgt carbon bgc bin gin oxy tai
• Nghien ciru - Ky thuat
5c 'O'S ppm. Tft k i t qua phin tich tren k i t hop so sanh vfti t i i iigu PI cb the khing dinh hop chat CT-1 chinh l i ent-7|&4iydroxy-15-oxo kaur-1&*n-18-yl acetat (hinhl).
K i t qua gfti y: 06 t h l dung CT-1 phgn l i p duvc i i m chit d l i cbilu d^ nghign cftu x i y dftng d c phuvng phip dinh tinh, dmh tuvng duvc iigu k h i s i m cho l i nhim x i c dinh h i m luvng cua nfl trong c i c m i u duvc ligu v i phgc vg cflng t i c tigu chuin hfla v| thulc niy
Hinh 1: Cdng thdc du tao chit CT-1 Kiem tra dp tinh khigt cua CT-1 Kiem tra db tinh khiet cua CT-1 bing SKLM, khai triln vfti c i c hg dung mbi khic nhau: Hex : EtOAc (3:1); l3icloromethan: MeOH (50:1), Benzen : EtOAc (5.2) chat r i n CT-1 d i u cho mgt v i t tibn vfti g i i tri R, tuvng ftng bing 0,42:0,66; 0,59. Hign miu trgn SKLM vfti thuoc thft H,SO, 10% /EtOH cho phit quang mau ving sing dufti i n h sing UV 366 nm v i cb m i u tim duVi i n h sing thu'ftng.
Kilni tra bing HPLC cho thiy chit CT-1 cfl dg tinh khiit 96% (tinh theo phan t i i m dign tich pic) (hinh 2).
Hinh 2:Siakfdd HPLC cda chit CT-1 Djnh tinh sip co m j t c h i t CT-1 ft l i v i canh c i y k h i sam cho la
Binh tinh tling SKLM Miu thit: chilt sigu i m bbt duvc iigu trong MeOH (30 phot), i p c K cfln 3 ml. Mlu ddi chiiu: Dung dich chit C T - 1 "
0,5 mg/ml trong MeOH; Hg dung mdi tnin khal- Hex: EtOAc = 2:1 (v:v). K i t qua t h l hign ft hinh 3 V366nmliu*cpluinTT tAr-254nmlii«ii: khi phun TT " ^ 366 nm sau phun TTH,SO Anh sing thuOng sau phun TT
'""'EtOH H,SO,10%/EtOH
Hinh 3; Sic ky dd chit CT-1.
Shi cliu: C-Canh khi Sim cho li: a:
i-J^y^'l^ ' ' " ' " ' " " " ^ ^'*' " n " - i w c iigu khg Sim cho l i theo duvc ligu chuin vfti hg dunq mgi benzen : EtOAc (ty ig 95.5), c i c d i u higu d l dinh tinh chi hufl'ng din thinh phin alkaloid v i tiavonoid m i chua giup nhgn bilt su cfl mat ciia ent-kauranH. Nghign cftu niy dung chit d l i chilu CT-1 1^ hg dung mbl Hex EtOAc (2:1) d i cho k l ^ qua dinh tinh t i t ng ring. So vfti tigu chuin 13DVN iV su dung dung mbi bdt dbc hai v i kinh te hon (thay benzen bing n-hexan). v i xic dinh
iividnh ciy khi sim cho ii CHl ail cUtu CT-1: LU khi Sim cho li.
rfi ring hoat chit chlnh ciia K h i s i m cho l i d i e trung cho duvc iigu n i y K i t qua nghign cftu niy cb t h l duftc sft dung che cflng t i c tigu chuin hba du'ftc ligu vg sau.
Binh dnh chil CT-I bi„g HPLC Vfti digu kign s i c ky v i phuvng phip xft |y m i u d i Iga chpn, s i c ky d l thu duvc cho i c oic tich tgong d l i r« ring, t h l hign qua s i c ky d i
(Xem hip trang 67) TAP CHl pu^tjic Hpc - 7/2016 (sd^^gi^T^
