Tap chi Khoa hoc Tnrang Dai hoc Cdn Tha PhAn A: Khoa hoc Tv nhiin. Cong nghi va Moi mrang 30(2014,: 15-19
Tap chl Khoa hpc Tru'dng Dai hoc Can Thd
website: sj.ctu.edu.vn 1
B A H O P C H A T TU* C A O E T H Y L A C E T A T E C U A T R A I 6 M O I {CASSM GRANDIS L-F)
Ngo Qu6c Luan', Le Van Rii', D6 Hoang Vinh', Ngo Khdc Khong Minh^ va Nguyen Ngoc Hanh^
' Khoa Suphgm, Tnrong Dgi hgc Cdn Tho
^ Truong Dgi hgc Bdch Khoa, Dgi hoc Quoc gia Thdnh pho Ho Chi Minh
^ Viin Cong nghe Hog hgc, Thdnh pho Ho Chi Minh Thong tin chung:
Ngdy nhgn: 01/07/2013 Ngdy chdp nhgn: 25/02/2014 TUle:
Three natural compounds fi-om ethyl acetate extracts of fruit of Cassia grandis Lf
Tie khda:
Trdi O moi. cinnamic acid Keywords:
Fruit of Cassia prandis Lf. cinnamic acid, beta-sitosterol
ABSTRACT
From the ethyl acetate extracts of the fruit of Cassia srandis Li from An Giang Province, three compounds (apigenin-7-O-^D- glucopyranoside, cinnamic acid and beta-sitosterol) were isolated. Their structures were interpreted by spectra including 'H-NMR, '^C-NMR, HSQC, HMBC. MS and based on published data.
TOM TAT
Tif dich chiit ethyl acetate cita trdi O moi a Tinh An Giang, ba hap chdt dd duac co lap vd nhdn danh la apigenin-7-O-p-D- glucopyrgnoside, cinngmic acid vd beta-sitosterol. Cdu true cdc chdt ndy duac xdc dinh bdng cdc phuang phdp pho Men dgi nhu 'H-NMR, '/C-NMR. HSQC. HMBC. MS vd so sdnh vai tdi lieu da cong bo.
1 G i a i T H q : u
Cay O mdi da dugc gidi thieu trong cdc bdi bao trudc day''- *], nhdm nghign ciiu bdt ddu nghien cuu cdy nay tir nam 2011 va dd cdng bd phdt hien dugc II hgp chdt trong la 6 mdi''--'-'*l Bai bdo ndy tilp tuc cdng bd them 3 hgp ehdt nira, trong dd cd 2 hgp chdt mdi phat hien tir trdi 6 mdi.
2 THV'CNGHI?M 2.1 Nguyen heu
Nguyen lieu tir cac bg phdn cua cdy 6 mdi dugc ThS. Ddng Minh Qudn-Trudng Dai hoc Cdn Tho gidm dinh. Trai 6 mdi chin thu hai vdo thdng 10/2012 tgi huyen Phii Tan, tinh An Giang. Sau khi riia sach. loai bd phdn sdu mgt, sdy khd d SO^C, taeh Idy hgt de rieng, cac phan eon lai xay thanh bgt Idm nguygn lieu.
2.2 Phuong phap ngbien clhi
22.7 Phircmgphdp chief xudt, co ldp cdc hop chdt Chiet rdn-ldng, Idng-ldng, soxhiet bdng cac dung mdi edn 96°, n-hexane, ethyl acetate, methanol. Cd loai dung mdi bang mdy cd quay dudi dp suit kem Buchi R-210.
- Sac ky Idp mdng (SKLM) sir dung ban nhdm silica gel 6OF2M (Merck) ttang sdn dd day 0,2 mm. Phdt hign vet bang den tii ngoai hai budc sdng 254 va 365 nm, hode phun dung djch H2SO4 10% ttong ethanol va ha ndng tren bip dien.
- Sdc ky cdt pha thuong (SKCPT), nhilu kich cd cdt tuy lugng mdu, chdt hdp phu la silica gel ed hgt 0,040-0,063 mm (230-400 mesh) cua cac hang Himedia, Scbarlau, Merck.
Lam sach chat bang each kit tinh Iai nhieu ldn ttong cae dung mdi tmh khilt khdc nhau.
Tap chi Khoa hgc Tnr&ng Dai hpc Cdn Tha Phdn A: Khoa hoc Tic nhien. Cong ngh? va Mdi n-itang 30 (2014): 15-19 2.2.2 Phucmg phdp xdc djnh cdu trOc vg nhAi dtaih
Diem tan c h ^ (mp.) dugc do tren may Electrothennal 9100 (U.K), diing mao qudn khdng higu chinh. Cac pho 'H-NMR, '^C-NMR, HSQC, HMBC duge ghi tren may Bruker AM500 FT-NMR Speettometer tgi Vien Hda hgc-Vien Han ldm Khoa hgc vd Cdng nghe Viet Nam. Phd khdi lugng dugc do ttgn may HP 1100 Series, LC/MSD Trap, Agilent tai Vien Cdng nghg Hoa hgc-Vien Han lam Khoa hoe vd Cdng nghe Viet Nam.
2.3 Chiet xuat va phan I|p cac bgp chat Bgt khd nguyen ligu trdi 6 mdi (4,0 kg) dugc tgn trich vdi cdn 96°, lge bd ba, sau do cd dac vd say d 50°C duge 380 g cao F. Phdn tan cao F ttong nudc-cdn am vd chiet ki^t vdi n-hexane, ed loai dung mdi thu dugc 15 g cao FH. Phdn khdng tan trong n-hexane tigp tuc dugc chiet kiet vdi ethyl acetate vd cd logi dung mdi thu dugc 65 g eao FE.
Tii 60 g cao FE tiln hdnh SKCPT vdi cac hg dung mdi n-hexane:ethyl acetate (H:E) ty le thay ddi theo hudng tang ddn dd phdn cyc. Cdc phdn dogn con gidng nhau duge gom lgi, dugc 12 phdn dogn cd cdc vet chdt khae nhau (FEOl, FE02...
FEI2). Tgi phdn doan FE05 (H:E = 5:5) thu duge cdn chiia kgt tinh mau trdng, ket tinh Igi nhigu Idn trong n-hexane thu dugc 50 mg chdt sgch ddt tgn la CGFE04.
Tai phdn dogn FEIO (ethyl acetate 100%) thu dugc 0,54 g can chira vet chinh mau vdng. Tigp tuc tiln hdnh SKCPT can FEIO Idn 2 vdi hg dung mdi CHCl3:CH30H (C:M) = 85:15. Tdng s6 phan doan gom dugc la 7 (FElO-1, FElO-2... FElO-7). CJ phan doan FElO-2 thu dugc chdt bdt kgt tinh mdu vdng. ket tinh Igi trong CHCI3 va kilm tta bdng SKLM thu dugc 10 mg chat sach dat ten Id CGFE03.
Tai phdn doan FE02 (H:E = 8:2) dm duge 0,32 g cdn chiia vet ehinh mdu tim. Tigp tue tien hanh SKCPT cdn FE02 ldn 2 vdi he dung mdi H:E = 8:2.
Tong sd phdn doan gom dugc la 15 (FE02-1, FE02-2... FE02-15). 6 phan doan FE02-6 thu dugc chdt kgt tinh mau trdng kha sach, kgt tinh lai trong n-hexane va kiem tra bang SKLM thu dugc 16 mg chdt sach dat ten la CGFE05.
Tiln hanh do digm chay, phdn tich phd MS vd NMR cac mdu chat phan lap.
3 KET QUA VA THAO LUAN 3.1 Hgp chit CGFE03
Hgp ehdt CGFE03 kit tinh hinh kim mau vang trong MeOH, mp. 236''C. SKLM giai ly bdng
he dung mdi CJv) = 8:2, hign vgt bdng dung dich H7SO4 10% ttong ethanol vd ho ndng eho vgt mau vang, R/= 0,18. Ngoai ra edn cho phan ung duong tinh vdi thudc thii FeCb/ethanol chiing td day la ddn xudt phenolic.
Phd ESAPI-MS Negative cho pie ion phdn tii m/z [M-H]" = 431 vd edc manh khae phii hgp vdi cdng thiic phdn tir C21H20O10 (M = 432 dvC).
Phd 'H-NMR (DMSO-rftf, 500 MHz, SH ppm, J Hz) cho tin higu dgc ttung cua 7 proton vdng benzene (5H ttong khoang 6,45-7,97), ttong dd cd 2 cap proton tuong duang; 2 proton ciia 2 nhdm -OH gan tryc tigp tren vdng benzene (SH 10,40 va 12,96). Ngoai ra cdn cd tin hieu ciia 11 proton ciia 1 dan vi dudng hexose (5H ttong khodng 3,16-5,40), trong dd cd 1 tin higu proton anomer 5H 5,06.
Phd '^C-NMR (DMSO-t/fi, 125 MHz. 6c ppm) ket hgp vdi ky thudt DEPT cho thdy U'n hieu dgc trung eiia khung sudn flavone 15 carbon.
Trong do cd 14 carbon vdng benzene (5c ttong khoang 99,48-164,21) gdm 7 carbon methin (cd 2 cap carbon tuong duang), 7 carbon tii cap ma ttong dd 5 carbon ed 5c d vimg trudng thdp ban do cd gan vdi nhdm chiia nguygn td cd do dm dign ldn, cd thi la oxygen va 1 carbon carbonyl ed 5c 181,93. Ngoai ra cdn cd tin hieu cua 6 carbon vdng dudng hexose (khong chiia carbon methyl), ttong dd cd 5 tin hieu dang hydroxy methine, 1 hydroxy methylene, tin hieu d 5c 99,89 Id carbon anomer.
Cac phd HSQC va HMBC cho thdng tin vl cdc mdi tuong quan giup qui ngp cdc gid tri 5H vd 5c phil bcrp vdo khung flavone va vdi^ dudng hexose: Trong cum proton ciia vdng duang, ngodi cac proton khdng gdn ttye "tigp vdi carbon thi proton anomer cd do ddi hda hge ldn nhdt (5H 5,06;
d; J= 7,5), gan trye tiep vdi carbon anomer (cd do ddi hda bgc ldn nhat so vdi cdc carbon cdn lgi ciia vdng dudng, 5c 99,89) va cd tuong quan vdi carbon 5c 162,91 chung td dan vi dudng gdn vdi khung flavone tgi vi tti C-7 (xdc dinh C-7 nhd tuong quan vdi H-6 va H-8). Vdi do ddi bda hge cua cac carbon vdn| dudng C-1">C-5">C-3">C- 2'>C-4">C6" cho bigt day la dudng glucose vd day la dudng dgng p vi proton anomer cd J- 7,5.
Tir cdc dgc digm tten eho phep dy doan CGFE03 thugc loai flavonoid cd gdn I dan vi dudng glucose, tta ciiu tai lieu thdy trijng khdp vdi apigenin-7-O-^D-glueopyranoside. Dii lieu phd NMR sau khi phan tich vd so sdnh vdi tdi lieu tham khdo nhu sau:
Tap chi KIwa l,oc TmangDti Im Cin Tha Phin A: Khoa Im Ttr nhiin. Cong nghi va Moi mr&ng: 30 (2014): 15-19 Bang 1: So sanh dir lieu ph6 NMR cua CGFEOS va apigenin-7-0-/;-D.gliicopyranosl(ie
VltriC/H CGFE03
"C-N1*«,H.NMR ' „ . . . " , ' : (DMSO-d6,500 MHz)
125 MHz)
Apigenin-7-0:j?-D-glncQpvranoside'^l
"C-NMR (CD3OD, 125 MHz)
'H-NMR (CDJOD, 500 MHz) 2
3 4 5 6 7 8 9 10 2', 6'
r
3', 5' 4' 5-OH 4'-OH 1"
2"
3"
4"
5"
6"
2"-0H 3"-OH 4"-0H 6"-0H
164,21 103,07 6,87 (lH,i) 181,93 156.89
99,48 6,45 CIH,i/,J= 2,0) 162.91
94,80 6,83 (lH,<i,y= 2,5) 161,06
105,29 120,99
128.54 7,96 (2H,rf, .7=9,0) 115,94 6,94 (2H,rf,J= 9,0) 161.30
12,96 (IH.s) 10,40 (IH.s) 99,89 5,06(lH,rf,y= 7,5) 73,06 3.16-3,46 (4H,m) 76,40
69,53 77,13
3,49 (1H, dd. J=6flviJ= 12,0)
" ' ' 3,71 (lH,rfrf,y= 5,5 v a J = 10,5) 5,40 (lH,rf,y= 5,0) 5,13(lH,rf,y=4,5) 5,07 (IH, <;,,/= 2,5) 4.61 (IH. (.7=5.0)
164,41(«) 106,57 6,85 (lH,s) 180,45 166,25(*)
104,83 6,83(lH,rf,J=2,l)(») 160,16(*)
99,34 6 , 7 1 ( l H , i J = 2 , l ) ( » ) 160,72(»)
109,39 123,32
129,51 7,83 (2H, i ./= 8,8) 117,10 6,92 (2H, rf, ./= 8,8) 162,60(«)
105,11 4,90 (lH,rf,y= 7,6) 74,83 3,25-3,50 (4H, m) 77,58
71,80 78,66
„ , , 3,73(lH,rfrf,/=ll,9vay=5,4) ''^•'" 3,87(lH,rfrf,./=ll,9vaJ=2,2)
Chil Ihich: (*) DH lieu phii hap nhttng gdn chua khap vi tri so vdi v&i CGFE03 Tir cac dir kien tren ket hgp so sanh vdi tai
liguf*^ cdu ttiic ciia CGFE03 dugc dl nghj nhu Hinh 1 va dinh danh la apigenin"7-0-y9-D- glucopyranoside (1).
Hinb 1; C^u triic hgp chat CGFE03 3.2 Hpp ch^t CGFE04
Hgp chdt CGFE04 kit tinh ttong n-hexane hinh kim to, ttdng vd khd ttong, mp. 134-135°C. SKLM gidi ly bdng he dung mdi C:M = 98:2. khdng hign vlt bdng dung dich H2SO4 10% ttong ethanol, ho ndng thi bay hai. soi vet dudi den UV vd ddnh ddu do dugc R, = 0,31.
- Phd 'H-NMR (CDCI3, 500MHz, 5H ppm, J Hz) cho 4 tin hieu ciia 7 proton, ttong do cd 2 cap proton tuong ducmg tuong uiig vdi 2 cap carbon methine tuong duang tten phd '^C-NMR dgc ttung cho vdng benzene. Ha: proton olefin 5H 6,46 (J = 15,5) vd 7,80 (J= 16,0) dvj tti ttans vi cd Jldn.
Phd '^C-NMR (CDCI3, 125MHz, 5c ppm) kit hgp ky thudt DEPT cho 7 tin hieu ciia 9 carbon.
Trong dd cd 7 carbon methine (ed 2 cap carbon methine tuang duang dde tnmg cho vdng benzene), 1 carbon bdc 4 vd 1 carbon carbonyl (5c 172,45).
TLT cdc dac diem ttdn eho phep dy doan COBE04 la ddn xudt phenyl ciia carboxyiic acid khdng no, tra ciiu tdi ligu thdy trung khdp viM cinnamic acid. Dii lieu phd NMR sau khi phdn tieh, kilm tra lgi ttgn cdc pho HSQC, HMBC vd so sdnh vdi tdi lieu tham khao dugc trinh bdy ttong Bdng 2.
Tap chi Khoa hpc Truang Dai hoc Cin Tha Phdn.4 Khoa hoc Ta nhiin. Cong nglii ra .Moi tnrong- 30(2014)- J 5-19 Bang 2: So sanh dir lieu pbd NMR cua CGFE04 va cinnamic acid
CGFE04 Cinnamic acid'"'
"C-NMR (CDCb, 125 MHz)
'H-NMR (CDCb, 500 MHz)
"C-NMR (CDCb, 120 MHz)
'H-NMR (CDCb, 400 MHz) 1
2 3 1' 2', 6' 3', 5' 4'
172,45 117,34 147,09 134,07 128,38 128,97 130,75
6,46 (lH,rf, .7=15,5) 7,80 (lH,rf,y= 16,0) 7,55-7,57 (2H, m) 7,39-7,42 (3H, m) 7,39-7,42 (3H, m)
172,06
6,47 (IH, J, 7=16,0) 147,11 7,8UlH,rf,y=16,0) 133,99
128,37 7,57 (2H,m) 128,96 7,42 (3H,m) 130,77 7.42 (3H,m) Tir cac dir kien tren ket hop so sanh voi tai
lieu'*', cSu true cOa CGFE04 dugc dl nghi nhu Hinh 2 va dinh danh la cinnamic acid (2).
Hlnh 2: Cin true hgf chat CGFG04 3.3 Hop ch^t CGFE05
COFE05 kSt tinh trong CHCb 4 djng hot min, mau tring, mp. 136-137''C. SKLM cho mot vet mau tim khi phun dung dich H2SO4 10% trong ethanol vi ha nong, co R = 0,26 (CHCljiMeOH = 95:5).
Pho khoi lugng cho pic ion phSn tu m/z [M+Hf*" = 415 tuong ung voi cong thiic phan tir Ci9HsoO=414dvC.
- Ph6 'H-NMR (CDCI3, 500MHz, 5H ppm, J Hz) cho 6 tin hieu proton methyl dac trimg cua hgp chSt sterol trong khoang SH 0,68-1,01; 1 tin hiSu proton n6i doi a 5H 5,35-5,36 (m); 1 tin hieu proton cua carbon gan voi nhom OH o 5H 3,50-3,54 (m).
- Ph6 "C-NMR (CDCb, 125MHz, 5c ppm) kit hgp ky thuat DEPT cho 28 tin hieu cua tong so 29 carbon. Trong do c6 6 carbon methyl, 11 carbon methylene, 9 carbon methine va 3 carbon tix cSp.
Tir cac dac dilm tren cho phep dit doan CGFE05 thugc nhom sterol, tra cuu tai lieu thSy trirng kliop vdi 6ero-sitosterol. Du iieu pho NMR sau khi phait tich, so sanh voi tai li^u tham khao'^' nhu sau:
Phd 'H-NMR (CDCb, 500 MHz. 6H ppm, J Hz)- 0.70 (3H, s. H-18); 0,84 (3H, s, H-29); 0.92 (6H. s. H-26. 27); 0.95 (3H, s. H-21); 1.03 (3H, s.
H-19);... 3.55 (IH, d, J= 5,0; H-3) va 5,37 (IH, i , y = 4.5; H-6). Ph6 '^C-NMR (CDCI3, 125 MHz, 5c ppm): 12.65 (C-18); 19,19 (C-29); 19,45 (C-19);
19,80 (C-21): 20,22 (C-26); 21,50 (C-27); 23.49 (C-U); 24,71 (C-28); 26,51 (C-15); 28,65 (C-23);
29,58 (C-16); 31,31 (C-25); 32,06 (C-8); 32,32 (C- 2); 32,32 (C-7); 34,37 (C-22); 36,55 (C-20); 36,91 (C-10); 37,67 (C-1); 40,19 (C-12); 42,70 (C-4);
42,73 (C-13); 46,25 (C-24); 50,55 (C-9); 56,48 (C- 17); 57,18 (C-14); 73,20 (C-3); 122,11 (C-6) va 141,17 (C-5).
Tir cac diJ lieu pho tren day ket hgp so sanh vol tai lieu''- '1 CO the nh^in dmh CGFE05 la bela- sitosterol (3) (Hinh 3).
4 6
Hinh 3: Can true hgp chat CGFE05 ( K E T L H . ^
Tir trai cay 6 m6i a Tinh An Giang d2 co lap dugc 3 hgp chat la apigenin-7-t>.^-D- glucopyranoside (1), cinnamic acid (2), bela- sitosterol (3). Trong do, cac hgp chSt (1) va (2), ISn diu ti^n dugc co lap tii' loai cay nay. Nho pho 2D- NMR CO the xac dinh chinh xac vi tri cac proton va carbon trong cau true cua hgp chat (1).
TAl LlEU THAM KHAO
1. Le Tiln DOng va ctv (2011). Khao sat thanh phin hoa hgc phan doan phan cue cua la cay 6 moi-Cassia grandis L.. Tap chi Hoa hgc T.49(6A-2011), trang 403-407.
Tap chi Khoa hoc TrudngDai hoc Cdn Tha Phin A: Khoa hoc Tir nhiin. CSng nghi va Moi Inidng: 30 /2014/ 15-19 2. LeTienDiingvactv(2012).Hgpc_hat
flavonoit va antraquinon tir la cay O m6i. Tap chi Hoa hgc T.50(5A-2012). ttang 200-202.
3. Ng6 Quoc Luan va ctv (2012). Hgp chat flavonoid til la O moi Cassia grandis L..
T^p chi Khoa hgc va Cong nghe. Tap 50, So 3A, 2012. ttang 296-301.
4. Ngo Quoc Luan va ctv (2013). Phan lap va nhan danh hai hgp chat tir dich chilt ethyl acetate ciia ttai 6 moi Cassia grandis L.f.
Tap chl Khoa hgc, Trudng Dai hgc Cin Tho, S6 26(Phan A)-2013. ttang 30-34.
5. Nguyin Quyet Tiln, Pham Thi H6ng Minh, Nguyen (juang An, Tnrong Tiii Thanh Nga, Nguyin Nggc Tuin, Doan V5n Tuin, Pham HlhjDiln(2011).Mgtsdkltquanghien ciju ban dau ve thanh phin hda hgc ciia cay Bang Lang nuoc. T^p chi Khoa hgc va Cong nghe, Dai hgc Thai Nguyen, 83(07), 15-18.
6. Renmin Uu, Aifeng Li, Ailing Sun (2004).
Preparative isolation and purification of hydroxyanthiaqiuBones and cinnamic acid from the Chinse medicinal herb Rheum officinajle Baill. By k^i-speed counter- current chn>mati)gta|%. Joumal of Chromatography A, 1052(2004): 217-221.
7. TayfunEt5-0z,"U.SebnemHarput,Iclal Saracoglu, Ihsan Calls. Phenolic Compounds from Scutellaria pontjca. Turk.
J. Chem. 26 (2002), pp. 581-588.
8. Ton N& Lien Huong va ctv (2011). Nghien ciru thanh phin hoa hgc ciia than cay 06 xuoc (Achyranflies aspera L.) a Tra Vinh.
Tap chi Khoa hgc, Truong Dai hgc Cin Thg, S619b-2011,ttang56-61.
