Tap chi Khac hoc 2012:2lo 134-138 ^"^ ^ * " C"" Th«
NGHIEN Ctru THANH PHAN HOA HOC TRONG CAO ETHYL ACETATE CUA RE CAU (ARECA CATECHU!^)
Le Thanh Phudc vd Bdnh Nguyin Anh Hdo'
ABSTRACT
This paper presents the results on separating identifying the chemical composition in Areca catechu L. roots in Phong Dien district. Can Tho city. The results showed that two compounds: lamotrigine (C9H-K^l2) and a,a-trehalose (C,2H220,d ^ere isolated from the ethyl acetate extracts. The stiuctures of these compounds have been elucidated by modern spectroscopic methods. ESI-MS 'H-NMR. "C-NMR, HSQC, COSY and HMBC.
Keywords: Areca catechu L., conq/onents, lamotrigine, a,cc-trehalose, root Title: Study on the chemical components in ethyl acetate extract from Areca catechu L.
root
T 6 M TAT
Bdi bdo trinh bay cdc kit qud nghien cuu vi chiit, tdch. dinh danh thanh phdn hoa hoc tiong ri Cau trdng tgi huyen Phong Diin. thanh phd Cdn Tha. Kit qud: hai hop chdt:
lamotrigine (C9H-N5CI2) vd a,a-trehalose (C12H22OU) duoc co Igp tit cao ethyl acetate. Cdu true hoa hgc cua cdc chdt ndy dd dugc xdc dinh bdng cdc logi pho ESI-MS, 'H-NMR, '^C-NMR. HSQC. COSY vd HMBC.
Tu khoa: Areca catechu L., lamotrigine (CgHrNsClz) vd a,a-trehalose (C12H22OJ1).
1 D,4.TVANDE
d Viet Nam. Cau la cay Udng liu ddi rat quen thupc d khap noi, nhat la d vimg tnmg du v i ddng bing. Ciy Cau cd ten khoa hpc la Areca catechu L., thudc hp Cau (Arecaceae) (Dd Tit Loi. 1995). Cic bd phan cua ciy Cau dupe diing nhieu Uong y hpc cd truyin de chiia nhieu benh nhu: hat Cau dimg chiia benh san xa mit.
sin li, chiia viem rudt.... v6 qui Cau dimg tri thuy thung. bung diy trudng, bi tieu tien; rl Cau noi cd tic dung bd duong. chOa dii nhit (Dd Huy Bich, 2004). Tren t h i gidi, Cau d i dupe ket hop mdt sd nguyen lieu thien nhien khac tao chit khing oxi hda dimg lam my phim, lam thudc chdng benh trim cam, benh cao huylt i p (P. Wetwitayaklunga et ah, 2006).
Thinh phan hda hoc Uong cic phin khic nhau cua ciy Cau: than, la, trai,... da dupe nhieu tic gia trong v i ngoii nude nghien ciiu. Tuy nhien, viec nghien ciiu thanh phin hda hpc ciia re ciy Cau cd rit it bii b i o dupe cdng bd (Dao Himg Cudng, 2010). Trong cac nghien ciiu trudc diy chiing tdi da cd lap v i dinh danh dupe hai hpp chat: lupeol va lupeol acetate cd trong dich chiet petroleum ether (Le Thanh Phudc et al. 2011). De tiep tuc gdp phin nghien ciiu thanh phin hda hpc ciia re Cau v i lam sing td them ca sd khoa hpc cho c i c bii thudc d i n gian, chiing tdi phin lip v i xic dinh ciu tnic mpt sd hpp chit tir cao ethyl acetate cua re Cau (Areca catechu L.) d huyen Phong Dien, thaiih phd C i n Tha.
' Khoa Khoa hpc Ty nhien. Trudng Dgi h(jc Cin Ther
Tgp chi Khoa hgc 2012:21a 134-138 Tnrong Dgi hgc Cdn Tha
2 PHlTONG TifiN VA PHU'ONG PHAP 2.1 Nguyen lieu
Re Cau non thu hai tgi huyen Phong Dien, thinh phd Can Tha. Re Cau dupe nia sgch, loai bd phin siu, siy khd d nhiet dp 55°C ddn khoi lupng khdng ddi v i xay nhd trudc khi su dung. Dp i m tnmg binh ciia mau rl Cau la 8.8%.
Re Cau duac dinh danh khoa hoc la re cua Areca catechu L. bdi Ths. Ngd Thanh Phong, Bd Mdn Sinh, Khoa Khoa hpc Tu Nhien, Dgi Hpc Cin Tha.
2.2 P h u a n g p h a p
Chilt hoat chit: bpt re Cau (3500 g) dupe chilt ngim kiet vdi ethanol 96° (EtOH) Uong 7 ngay, tich liy phin long dem cd quay dudi ap suit kem thu cao EtOH thd (305 g). Cho phin cao thd hda tan Uong mdt lupng nude cit nhat dinh, sau do chiet Idng Idng lin lupt vdi cic dung mdi peuoleum ether (PE), ethyl acetate (EtOAc), w-butanol (BuOH). Thu gom cic dich trich va sau khi logi dung mdi dudi i p suit kem thu dupe cac cao PE (59 gj, cao EtOAc (49 g) va cao BuOH (56.5 g), tuong irng.
Phin lip cic chat tir cao ethyl acetate: thyc hi?n sac ky cpt, chit hip phu l i silica gel, sir d\mg nhiing he dung mdi giai ly gdm PE, EtOAc v i methanol (MeOH) cd dp phin cue ting din. Theo ddi qui trinh sac ky cdt bang sac ky bin mdng vdi he dung mdi giai ly la chloroform:methanol (CHCl3:]VIeOH), thudc thii hien vet la dung dich acid suLftiric 10% Uong ethanol hoac dung dich KMn04. Cac phin dogn the hien R/ gidng nhau tren sic ky ban mdng dupe gdp Igi. Tien hinh sic ky cdt lin 2 vdi cac phan dogn gidng nhau d l cd lip dupe chit sgch.
Xac dinh cau Uiic ciia cac chat da cd lip dupe bing each sir dung cac phuang phap phd nghiem: MS, ' H - N M R , ' ^ C - N M R , COSY, HMBC. Pho NMR dupe do Uen may Bruker Advance 500 MHz (Vien Cdng Nghe, 18 Hoang Qudc Viet, C i u Giiy, Ha Ndi).
Silica gel 60 F254 (0.04-0.05 mm), ban mdng TLC cua hang Merck, Due. Cac hoa chit tinh khilt khic cd xuit xii tir Tnmg Qudc.
3 K E T QUA VA THAO L U ^ 3.1 Ket qua s i c \^ cot
Tir cao EtOAc (9.023 g), tiln hinh sic ky cpt thudng vdi h? dung mdi giii ly PE:EtOAc va EtOAc:MeOH cd dp phin cue ting din. Kiem tra cic phan doan bing TLC. Cic phin dogn co % gidng nhau dupe gom chung. KSt qui d phan dogn EtOAciMeOH = 95:5 thu dupe 50 mg chit sgch dudi dang tinh the mau tring, TLC giii ly bing he CHCl3:MeOH:NH40H = 85:10:5 hi?n hinh bing thudc thii KMn04 cho vit Udn mau tring cd R/^ = 0.63 ky hieu tinh the nay la Phuoc- Hao4H.
0 phin dogn EtOAc:MeOH = 1:2 thu duac 25 mg chit sach dudi dgng tinh thi m i u u i n g , TLC giai ly bing he CHCl3:MeOH= 4:6 hien hmh bing thudc thu
Tap chi Khoa hgc 2012:21a 134-138 Truong Dgi hgc Cdn Tha
H2SO4 10% uong EtOH cho vit udn mau dd cd R/ = 0.48 ky hieu tinh t h i niy la Phuoc-Hao6.
3.2 Ket qua dir lieu pho Hop chat Phuoc-Hao4H
Phd 'H-NMR (500 Mz, DMSO, 6 ppm, J= Hz): trong phan tir Phuoc-Hao4H cd 7 tin hi?u proton, trong dd cd 3 tin hieu ciia cic proton gin Uen vdng benzen d cac vi tri 7.71 (dd,J= 8; 1.5, Ha), 7.44 it,J= 8; 7.5, Ht), 7.36 (_dd, J= 7.5; 1.5, H,). Tin hieu cpng hudng d 7.44 ppm dgng triplet nhung lai cd hai gii Uj J=% va 7.5 Hz la vi hai proton Ha va He khdng tuang duong v l mat hda hpc. Tir hing so ghep cap J, ta thiy Ha va Kb d vi tri ortho vdi nhau, Hb v i H^ d vi \ri ortho vdi nhau, Hg v i H^
d vj tri meta vdi nhau. Bdn tin hieu proton d 6.90 (2H, br s, -NHi), 6.42 (2H, br s, -NH2).
Phd '*C-NMR (125.8 MHz, DMSO, 5 ppm) kit hpp DEPT cho thiy Uong phan tir Phuoc-Hao4H cd 9 carbon trong dd cd 6 carbon tu cip.
Phd HMBC cho thiy su tuang quan giira proton Ha vdi tin hieu Sc 131.58, proton Hb vdi tin hi^u 6c 136.76, 131.99, proton H^ vdi tin hieu 5c 138.29, 131.58.
Hinh I: Tinmg quan HMBC cua h o p chat Phuoc-Hao4H
Phd ESI-MS: cho bilt [M+H]" = 257.1 amu tuang iing vdi phin tir cd khdi lupng phin tir la 256.1 (C9H7N5CI2).
Tu nhutig dii ki?n tren Phuoc-Hao4H duac nhin danh la lamotrigine hay cdn gpi la 6-(2.3-dichlorophenyl)-l,2,4-triazme-3,5-diamine. Kit q^ua niy phu hpp vdi kit qua ciia Guntoori et al 2003. Kit qua do GC-MS dich chiet alkaloid tir re Cau ciia PGS.TS. Dio Hung Cudng, Dai Hpc Da N i n g (2010) cung cho thiy Uong r l Cau cd lamotrigine.
Lamotrigine cd tic dung tri ddng kinh, bfnh parkmson va tic dung len kenh sodium (Na*) (N. R. Mirza et al, 2005).
Hinh 2: CSng thirc cau tao hoa hoc ciia lamotrigine
Tgp chi Khoa hgc 2012.21a 134-138 Truong Dgi hgc Cdn Tho
Hgrp c h i t Phuoc-Hao6
Phd ESI-MS: cho bilt [M+H]"^ ^ 3 6 1 . 3 amu tuong ihig vdi phin tir cd khdi lupng p h i n tu la 360.3 (Ci2H220,i.H20).
Phd ' H - N M R (500 MHz, DMSO, 5 ppm, J = Hz): xuit hien tin hieu proton anomer d 4.87 (IH, d,J= 3.5, H] v i H i ) , va 5 tin hieu proton g i n U8n vdng dudng d 3 . 2 3 (IH, m, Hj va H2), 3.54 (IH, m, H3 v i H3), 3.13 (IH, m, H4va H4-), 3.64 ( I H , m. H5 v i H5), 3.46 (IH, m. Hga v i H f a ) , 3-54 (IH, m, Heb v i Hsb-)- Phd ' ^ C - N M R (125.8 MHz, DMSO, 5 ppm) k i t hpp vdi pho DEPT: cho 1 tin hieu carbon acetal 93.08 (Ci v i C i ) , bdn miii tin hieu carbon m e t h i n k l O d c i c vi tri 70.12 (C4 va C4.), 71.6 (C2 v i C^), 72.44 (C5 va C j ) , 72.87 (C3 va C^-) va mpt carbon metylen k l O (-CH2-O) d 60.76 (Ce va Ce).
Phd HSQC cho thiy carbon - C H i - tai 5c= 60.76 (Ce va CeO tuan^ quan vdi hai proton tgi SH = 3.46 va 5H = 3.54 ppm. Chung td hai proton nay lin lupt l i Hea va H6b. Dua vao phd HSQC, l i n lupt xac dinh dupe dp dich chuyen hda hpc H3, H4, Hs.
Phd hai chilu HMBC (DMSO, 5 ppm): cho thiy proton tgi 3.13 (IH, ni, H4) Urong tac vdi carbon tgi 6c = 72.44 (C5), 6c = 72.87 (C3), 5c = 60.76 (Cg); proton tgi 3.23 (IH, m, H2) tuong tic vdi carbon tai 5c = 72.44 (C5); proton tai 4.87 (IH, d, J=
3.5, H,) mang tac vdi carbon tai 5c = 72.44 (Cj), 5c = 72.87 (C3).
Tin Weu 6 H = 4.87 ppm (IH, (^7= 3.5, Hi v i Hi) tren p h d ' H - N M R vi 5,;= 93.08 ppm tren phd '^C-NMR khing dinh hpp chit Phuoc-Hao6 co ciu Wnh a-pyranoside. Proton ghep c i p true -xich dgo vdi Hi phai la H2 5H= 3.23 (IH, m, Hi).
Tir dii' ligu phd Uen kit hpp vdi tii lieu tham khio (Ivanka Jeri et al. 2006), chdng tdi xac dinh Phuoc-Hao6 l i [a-D-glucose-(l-)-l)-a-D-gIucose] hay a,a-D-trehaIose.
a,a-D-Trehalose cd tic dung ire che men a-glucosidase nen dupe ung dyng rong trong dilu tri benh dai thao dudng (Ivanova et al, 2009).
Hinh 3: C6ng thirc cSu tao Phuoc-Hao6
4 K E T L U i 4 J V
D i phin lip va xac dinh ciu tnic cua hai hpp chat: lamoUigine (C9H7N5CI2) v i a,a-Uehalose (C12H22O1 j) tir r l Cau. Diy la nhutig chit diu cd hogt tinh sinh hpc
T<^ chi Khoa hgc 2012:21a 134-138 Trudng Dgi hgc Cdn TTur
nen d i giai thich ducrc ly do tgi sao r l Cau dupe dung chua mpt sd benh trong y hpc din gian.
TAI LEEU THAM K H A O
Dao Himg Cuong, Bui Ngoc Phuong Chau, 2010. Nghien cuu chiet tdch. xdc dfnh thdnhphdn hgp chdt hoa hgc trong hgi vd ri cdy Cau, Tuyen tap bao cao hoi nghj Sinh vien n ^ S n cuu khoa hpc lan £bii 7. trang 4 6 4 - ^ 8 .
Do Huy Bich, 2004. Cay thudc vd dgng vgt lam thudc d Viet Nam, NXB Khoa hoc va Ky thuat, Hanoi, (I), tr. 350-353.
D S Tit Loi, 1995. Nhiing cdy thudc va vi thudc Viit Nam, NXB Khoa hoc va Ky AuSt, tr. 172-174.
Guntoori et al., 2003. Efficient process for the preparation of lamotrigine andrelaled 3.5-diamino-6-substituted-I,2,4-tnccines, United Slates Patent, US 6586593B1.
Ivanka Jeric et al., 2006. Syrahesis ofTrehalose-cenieredDipeptide Esters, Croatica Chemica Acta, 79 (2) 261-272.
Ivanova et al., 2009. Isolation and identification of alpha,alpha-trehalose and glycerol from an m-ctic psychrololerant Streplomyces sp. SB9 and their possible role in the strain's survival. Prep Biochem Biotechnol 39{l):46-56.
Le Thanh Phudc, Banh Nguyen Anh Hao, 2011, Gop phdn khdo sdt thdnhphdn hoa hoc cua re Cau (Areca catechu L.>. Tap chi Khoa hoc Dai hoc Can TTia, 19b, 80-84.
N . R. Mirza et al., 2005. Lamotrigine has an anxiolytic like profile in the rat conditioned emotional response test of anxiety: a potential role for sodium channels, P s y c h o p h a r m a c o l o g y 180: 1 5 9 - 1 6 8 .
