KHO ST THNH PHN HO HC CY MP GAI (LASIA SPINOSA L.) INVESTIGATION OF CHEMICAL CONSTITUENTS OF LASIA SPINOSA L. Nguyn Minh ng1, Nguyn Tn Pht2, Phng Vn Trung2, Nguyn Ngc Hnh2 1 i hc Cn Th, 2Vin Cng Ngh Ho Hc-Vin Khoa Hc v Cng Ngh Vit Nam t vn Cy Mp gai (Lasia spinosa L.) cn c gi l ry gai, sn thc gai, thuc h ry (Araceae). Cy thuc loi cy tho, cao 0,4-0,7 m, thn r v cung l u c gai. nc ta, cy phn b ri rc khp cc a phng vng ng bng, trung du v ni thp. B phn thng dng l thn v r. Cy c tnh mt, c tc dng tiu vim, gii c, thanh nhit tr thp. Theo kinh nghim dn gian v y hc c truyn mp gai l ngun dc liu quan trng dng cha ho, au hng, ph thng suy gan, di chng do st rt[1,2]. Trong nhng nm gn y, c nhiu nghin cu v thnh phn ha hc v hot tnh sinh hc cy mp gai Vit Nam cng nh trn th gii[3,4,5,6,7]. Trong bi bo ny, chng ti trnh by vic phn lp v xc nh cu trc ca cc cht t cao ethyl acetate v cao butanol ca cy mp gai. Nguyn liu v phng php Nguyn liu: Cy mp gai (Lasia spinosa L.) c cung cp bi Cng ty dc Domesco, trng ti x An C, huyn Tnh Bin, tnh An Giang, thu hi phn trn mt t vo thng 12 nm 2011, cy c 3 nm tui. Phng phpxc nh cu trc v chit xut: im nng chy c o trn my ELECTROTHERMAL 9100 (UK), mao qun khng hiu chnh. Cc ph cng hng t ht nhn: 1H-NMR, 13C-NMR, DEPT, COSY, HSQC, HMBC c ghi trn my BRUCKER AVANCE (500 MHz) dch chuyn ho hc tnh theo (ppm), hng s tng tc (J) tnh bng Hz. Ph khi lng c o trn my AGILENT TECHNOLOGIES 6120 (Quadrupole LC/MS). Sc k lp mng (TLC) c thc hin trn bn nhm silica gel Merck-GF60F254 trng sn, kch thc 20 20 cm, dy lp hp ph 0,2 mm ca hng Merck, Germany. Sc k ct trung p dng silica gel 60, Merck, ng knh ht 0,040-0,063 mm. Thn l cy mp gai kh (1,5 kg) c trch kit bng chit siu m vi ethanol 96% o . 60 C Sau khi thu hi dung mi, c cao ethanol (117 g), cao ny c chit lng-lng ln lt vi n-hexane, ethyl acetate v butanol thu c cc cao tng ng l cao LSH 27,5 g (chim 23,5%); cao LSE 4,5 g (chim 3,8%) v cao LSB 11 g (chim 9,4%). T cao LSE (4,5 g) tin hnh sc k ct trung p vi h dung mi c phn cc tng dn n-hexane, ethyl acetate v methanol; ti phn on 5 tin hnh hnh sc k ct trung p vi h dung mi CHCl3 MeOH (93 : 7), thu c hp cht (1) (5 mg); ti phn on 6 tin hnh hnh sc k ct trung p vi h dung mi CHCl3 MeOH 20 (8 : 2 : 0,2), H thu c hp cht (2) (27 mg). T cao LSB (11 g) tin hnh sc k ct trung p vi h dung mi c phn cc tng dn n-hexane, ethyl acetate, methanol v nc. Ti phn on 7, thu c hp cht (3) (30mg). Kt qu v tho lun

Hp cht (1) thu c di dng bt, mu trng; im nng chy: 298-300C; sc k lp mng (TLC) trin khai bng hn hp dung mi CHCl3 MeOH (9 : 1) hin mu bng dung dch H2SO4 10% trong EtOH, cho vt trn xanh tm c Rf = 0,25 khi h nng. Ph 1H-NMR (500 MHz, DMSO, ppm) cho tn hiu ca 3 proton olefin H 5,33 (1H, d, J = 5, H6), 5,03 (1H, dd, J = 9 v 15, H23) v 5,20 (1H, dd, J = 8 v 14,5, H22); 5 proton ca nhm methin k oxygen H 3,46 (1H, m, H3), 2,90 (1H, td, J = 5 v 9, H2), 3,14 (1H, td, J = 4 v 8,5, H3), 3,02 (1H, td, J = 4 v 9, H4) v 3,07 (1H, dd, J = 4 v 13,5, H5); 2 proton ca nhm methylen k oxygen H 3,65 (1H, ddd, J = 11, 6 v 2, H6a) v 3,40 (1H, m, H6b); 1 proton anomer H 4,21 (1H, d, J = 8, H1); proton ca 6 nhm methyl H 0,671,01. Ph 13C-NMR (125 MHz, DMSO, ppm) kt hp DEPT cho tn hiu ca 35 carbon; trong c 1 carbon t cp ni i C 140,4 (C5); 3 carbon olefin C 136,7 (C22), 128,8 (C23) v 121,0 (C6); 1 carbon acetal C 100,8 (C1); 5 carbon methin k oxygen C 76,9 (C3), 73,5 (C2), 76,8 (C3), 70,2 (C4) v 76,7 (C5); 1 carbon methylen k oxygen C 61,1 (C6); 6 carbon methyl; 2 carbon t cp; 9 carbon methylen; 7 carbon methin. Chng t (1) c khung aglycon l stigmasterol 29 carbon, mang ni i v tr 5 v v tr 22 vi phn ng 6 carbon. Ph COSY v HSQC, xc nhn y l ng Dglucose. Proton anomer H 4,21 (1H, d, J = 8, H1) chng t l ng . Ngoi ra, proton anomer H 4,21 (1H, d, J = 8, H1) cn tng tc vi carbon C 76,9 (C3), chng t phn ng gn vo khung aglycon v tr C3. T d liu ph 1H-NMR, 13C-NMR, kt hp vi ph DEPT, COSY, HSQC, HMBC; cc c trng vt l v so snh vi cc ti liu cng b[8], chng ti nhn danh hp cht (1) l: 3-O-[-D-glucopyranosyl]-stigmasta-5,22-dien. Hp cht (2) thu c di dng bt, mu vng; im nng chy: 269-270C; sc k lp mng (TLC) trin khai bng hn hp dung mi CHCl3 MeOH 20 (7 : 3 : 0,5) hin H mu bng dung dch H2SO4 10% trong EtOH, cho vt trn mu vng c Rf = 0,5 khi h nng. Ph khi lng MS cho mi ion gi phn t vi m/z [M-H]- = 431 tng ng vi cng thc phn t C21H20O10. Ph 1H-NMR (500 MHz, DMSO, ppm) (Bng 1) cho tn hiu ca cc proton vng thm H 6,27-8,01. Trong 1 proton ca vng thm b th 5 ln H 6,27 (1H, s, H6) v 4 proton ca vng thm th i xng 1,4 H 6,88 (1H, d, J = 8,5, H2, H6) v H 8,01 (1H, d, J = 8,5, H3, H5); 1 proton ca nhm hydroxyl kim ni H 13,17 (1H, s, OH5). Ngoi ra, cn c proton anomer H 4,68 (1H, d, J = 9,5, H1) v proton methin k oxygen H 3,22-3,84. Ph 13C-NMR (125 MHz, DMSO, ppm) kt hp vi DEPT cho tn hiu ca 21 carbon; trong c 5 carbon t cp vng thm k oxygen C 163,9 (C2), 160,3 (C5), 162,6 (C7), 156,0 (C9) v 161,1(C4); 3 carbon t cp vng thm C 104,6 (C8), 104,0 (C10) v 121,6 (C1); 1 carbon carbonyl C 182,0 (C4); 6 carbon methin vng thm C 102,4 (C3), 98,2 (C6), 128,9 (C2, C6) v 115,8 (C3, C5); 5 carbon methin k oxygen v 1 nhm methyl k oxygen C 61,3 (C6) cho thy hp cht (2) l flavonoid glucoside vi phn ng 6 carbon. Ph COSY v HSQC xc nhn n v ng l D-glucose. Hng s ghp cp J ca proton anomer H 4,68 (1H, d, J = 9,5, H1), cho bit y l ng -Dglucose. Ph HMBC cho thy proton anomer H 4,68 (1H, d, J = 9,5, H1) tng tc vi

carbon t cp vng thm C 104,6 (C8), vy phn ng phi gn vo khung aglycon v tr C8; ngoi ra H1 cng cho tng tc vi carbon t cp vng thm C 162,6 (C7) v 156,0 (C9) do proton duy nht ca vng A H 6,27 (1H, s, H6). iu ny cng c chng minh, proton H 6,27 (1H, s, H6) tng tc vi carbon t cp vng thm C 104 (C10); 104,6 (C8); 160,3 (C5) v 162,6 (C7). Ngoi ra, trn ph HMBC cng cho thy proton H 6,78 (1H, s) tng tc vi carbon carbonyl C 182,0 (C4) v carbon t cp vng thm k oxygen C 163,9 (C2), vy proton ny l H3. T d liu ph 1H-NMR, 13C-NMR, kt hp vi ph DEPT, COSY, HSQC, HMBC; cc c trng vt l v so snh vi cc ti liu cng b[9], chng ti nhn danh hp cht (2) l: Vitexin. Hp cht (3) thu c di dng bt, mu vng sng; im nng chy: 213-215C; sc k lp mng (TLC) trin khai bng hn hp dung mi CHCl3 MeOH 20 (7 : 3 : 0,5) H hin mu bng dung dch H2SO4 10% trong EtOH, cho vt trn mu vng c Rf = 0,25 khi h nng. Ph khi lng MS cho mi ion gi phn t vi m/z [M-H]- = 593 tng ng vi cng thc phn t C27H30O15. Tng t cht (3) cng c d liu ph gn nh cht (2) (Bng 1), gm 1 khung flavonoid v 2 n v ng. Ph COSY v HSQC xc nhn n v ng th hai l D-glucose. Hng s ghp cp J ca proton anomer H 3,91 (1H, d, J = 7,5, H1), cho bit y l ng . Ph HMBC cho thy s tng tc gia proton anomer ca ng th hai H 3,91 (1H, d, J = 7,5 Hz, H1) tng tc vi C 81,1 (C2), vy ng th 2 gn vo phn ng th nht ti v tr C2. T d liu ph 1H-NMR, 13C-NMR, kt hp vi ph DEPT, COSY, HSQC, HMBC; cc c trng vt l v so snh vi cc ti liu cng b[9], chng ti nhn danh hp cht (3) l: Vitexin-2-O--D-glucopyranoside.29 21 12 19 OH 4' HO HO 3' 6' 5' 2' O O OH 1' H 2 3 4 1 10 5 6 11 9 8 7 18 13 14 20 17 16 15 28 22 23 24 25 26

HO OH 3'' 2'' HO HO 6 7 1'' 8

HO OH

4'' 5'' O

6''

3'''

4''' 6'''

OH 3' 2' 2 3 1' 4' 5' 6' OH

HO 1'''

2'''

27

9 O 4 O

OH HO OH 4'' 6'' 3'' OH 5'' 2'' O O 1'' 2' O 8 9 O 4 O 2 3 1'

5'''

3' 4' 5' 6'

OH

HO 7 6

5 OH

10

5 OH

10

3-O-[-D-glucopyranosyl]stigmasta-5,22-dien (1)

Vitexin (2)

Vitexin-2-O--Dglucopyranoside (3)

Bng 1: D liu ph 1H, 13C-NMR v HMBC ca cht (2) v (3)

V tr 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5

C NMR ppm (2) (3) 163,9 163,7 102,4 102,6 182,0 182,0 160,3 160,6 98,2 98,1 162,6 162,6 104,6 103,6 156,0 156,2 104,0 103,8 121,6 121,7 128,9 128,8 115,8 115,8 161,1 161,0 115,8 115,8 128,9 128,8 73,3 71,5 70,8 78,6 81,7 70,6 81,1 78,4 70,1 81,7

13

H NMR ppm, J = Hz (2) 6,78 (1H, s) OH5 13,17 (1H, s) 6,27 (1H, s) (3) 6,74 (1H, s) OH5 13,15 (1H, s) 6,23 (1H, s) (2)

1

1

HMBC H13C (3)

C1,C2,C4,C10 C1,C2,C10 C5,C7,C8,C10 C8,C10

8,01 (1H, d, J = 9) 6,88 (1H, d, J = 8,5) 6,88 (1H, d, J = 8,5) 8,01 (1H, d, J = 9) 4,68 (1H, d, J = 9,5)

7,99 (1H, d, J = 8,5) 6,89 (1H, d, J = 8,5) 6,89 (1H, d, J = 8,5) 7,99 (1H, d, J = 8,5) 4,79 (1H, d, J = 9,5)

C2,C4,C6 C1,C4,C5 C1,C3,C4 C2,C2,C4 C7, C8, C9, C2,C3,C4 C8,C1,C3 C5 C5 C3,C4

C2,C4,C6 C1,C4,C5 C1,C3,C4 C2,C2,C4 C7, C8, C9,C2,C3 C1,C3 C2,C4 C3

3,84 (1H, t, J = 9) 4,07 (1H, t, J = 9,5) 3,27 (2H, m) 3,41-3,46 (3H, m) 3,27 (2H, m) 3,41-3,46 (3H, m) 3,40 (1H, m) 3,24-3,27 (2H, m) H6a 3,76 (1H, dd, J = 4,5 v H6a 3,74-3,77 (1H, dd, J 61,3 60,9 10,5) = 5,5 v 10,5) H6b 3,52 (1H, m) H6b 3,41-3,46 (3H, m) 105,1 3,91 (1H, d, J = 7,5) 74,3 2,77 (1H, t, J = 8) 76,2 2,88-2,94 (2H, m) 75,9 2,46 (1H, m) 2,88-2,94 (2H, m) 69,5 H6a 3,16 (1H, dd, J = 3 v 11) H6b 3,05 (1H, dd, J = 2,5 v 11)

C2,C3 C3 C4,C5

6

60,4

C5,C4

Kt lun T phn trn mt t ca cy mp gai c 3 nm tui trng ti An Giang, chng ti phn lp v nhn danh cu trc ba hp cht l: 3-O-[-D-glucopyranosyl]-stigmasta5,25(27)-dien (1), Vitexin (2) v Vitexin-2-O--D-glucopyranoside (3).

Summary

From the ethyl acetate extract of Lasia spinosa L. cultivating in An Giang province, 3-O-[-D-glucopyranosyl]-stigmasta-5,22-dien (1), Vitexin (2) and from the butanol extract, Vitexin-2-O--D-glucopyranoside (3) were isolated. Their structures were elucidated by NMR (1D and 2D NMR) and comparison with published data. Keywords: Lasia spinosa L., 3-O-[-D-glucopyranosyl]-stigmasta-5,22-dien (1), Vitexin (2) and Vitexin-2-O--D-glucopyranoside (3). Ti liu tham kho 1. Cy thuc v ng vt lm thuc Vit Nam (Tp 2), NXB Khoa hc v K thut (2003). 2. Tt Li, Nhng cy thuc v v thuc Vit Nam, NXB Y hc (2004). 3. Nguyn Th Hng Vn, Chu Vn Minh, Phm Quc Long, Braca Alessandra, Morelli Ivano, Nguyn Th Dng, Bc u nghin cu thnh phn ha hc ca cy ry gai, Tp ch Dc liu, tp 10 s 2, trang 45-49 (2005). 4. Nguyn Th Hng Vn, Chu Vn Minh, Phm Quc Long, Braca Alessandra, Morelli Ivano, Nguyn Th Dng, three flavonoid glucoside and two phenylpropane derivatives of lasia spinosa L., Biochemiscal Systematics and Ecology 34, pp. 882884 (2006). 5. W. Suthikrai, R. Jintana, S. Sophon, R. Hengtakulsin, V. Usawang, M. Kamonpatana, Effects of Lasia spinosa Thw. on growth rate and reproductive hormone of weaned Swamp buffalo and Murrah X Swamp buffalo calves, Italian Journal of Animal Science, vol. 6, pp. 532-535 (2007). 6. Fakrul Alam, Mozammel Haque, Md. Hossain Sohrab, Mohammad A. Monsur, Choudhury M. Hasan v Nuruddin Ahmed, Antimicrobial and Cytotoxic activity from Lasia spinosa and isolated Lignan, Latin American Journal of Pharmacy, pp 550-553 (2011). 7. Temjenmongla and Arun K. Yadav, Anticestodal efficacy of Lasia spinosa extract agaisnt experimental hymenolepis diminuta infections in rats, Pharmaceutical Biology, vol. 44, No. 7, pp. 499-502 (2006). 8. M. Sarwar Alam, Neeraj Chopra, Mohammed Ali, Masatake Niwa, Oleanen and stigmasterol derivatives from Ambroma Augusta, Phytochemistry, Vol. 41, No. 4, pp. 1197-1200 (1996). 9. Erika Vzquez, Estaban M.Martnez, Juan Antonio Cogordn, Guillermo Delgado, Triterpenes, phenols, and other constituents from the leaves of Ochroma pyramidale (Balsa wood, Bombacaceae). Preferred conformations of 8-C--D-glucopyranosylapigenin (vitexin), Journal of the Mexican chemical Society, julio-septiembre, ano/vol. 46, nmero 003 Sociedad Qumica de Mxico Mexico Mxico, pp. 254-258 (2002).