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NGHIN CU THNH PHN HA HC, HOT TNH SINH HC CC CHT TRONG CAO PETROLEUM ETHER V THNH PHN DINH DNG CA L CY MM I (AVICENNIA MARINA)
L Thanh Phc1 v L Hng Nhi1
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Khoa Khoa hc T nhin, Trng i hc Cn Th

Thng tin chung: Ngy nhn: 14/09/2012 Ngy chp nhn: 25/03/2013 Title: Study on chemical components, biological activities from the petroleum ether extract and nutrition components of the leaves of Avicennia marina T kha: L Mm i Avicennia marina, thnh phn ha hc, hot tnh sinh hc, thnh phn dinh dng, lupeol, betulin Keywords: Avicennia marina, chemical components, biological activities, nutrition components, lupeol, betulin

ABSTRACT
Study on the chemical ingredients from the leaves of Avicennia marina, collected in Dong Hai district, Bac Lieu province, we have isolated and identified two compounds: lupeol (C30H50O) and betulin (C30H50O2) from the petroleum ether extract. Structures of these compounds had been elucidated by modern spectroscopic methods: 1H-NMR, 13C-NMR, DEPT NMR and compared with published data. In this study, we tested and determined some biological activities of lupeol and betulin. As result, lupeol showed significant activity against human Hepatocellular carcinoma cells with an IC50 value of 93,53 g/mL. Betulin was against human Lung cancer cells with an IC50 value of 25,84 g/mL. Furthermore, when we tested nutrition of the leaves of Avicennia marina, we found out a lot of amino acids with high content, this explained why shrimp farmers have been using the leaves of Avicennia marina as natural food for shrimp. The study has been continued.

TM TT
Kho st thnh phn ho hc l cy Mm i c thu hi ti huyn ng Hi, tnh Bc Liu, chng ti c lp v nh danh c hai cht: lupeol v betulin t dch chit petroleum ether. Cu trc ha hc cc cht ny c lm sng t da vo nhng phng php ph hin i 1HNMR, 13C-NMR, DEPT NMR v so snh vi ti liu cng b. Trong , chng ti kho st v tm ra nhng hot tnh sinh hc ca lupeol v betulin. Kt qu l, lupeol c kh nng khng t bo ung th gan vi IC50 c gi tr l 93,53 g/mL. Betulin cng c kh nng chng li t bo ung th phi vi IC50 c gi tr l 25,84 g/mL. Hn th na, khi tin hnh nghin cu thnh phn dinh dng ca l Mm, chng ti nhn thy l Mm c nhiu amino acid vi hm lng cao, iu ny l gii ti sao ngi nng dn nui tm li s dng l Mm lm ngun thc n t nhin cho tm. Nghin cu vn ang c tip tc.

1 T VN Cy Mm i c tn khoa hc l Avicennia marina, thuc h Mm (Verbenaceae) (Phm

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Hong H, 2000). Cy Mm c th nhanh chng sinh sn, ln ln v pht trin tt vng gip ranh gia t v nc mn. Vai tr ln nht ca loi Mm l c nh t, do b r c

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cu trc vng chc n su xung t, n c sc chu ng c sng v gi, chu c nc mn ngp quanh nm. Theo mt s ti liu dn gian trn th gii, l ca cy Mm tr bnh au d dy, bnh nm ph n, tr cc loi ung nht (P. Thirunavukkarasu et al., 2010). V ca cy Mm dng lm thuc tr gh v cha bnh phong, cha vt thng hoi th, dit chy rn, dit giun sn v c tc dng nga thai (Phm Hong H, 2000). N cn c kh nng cha bnh ung th. V vy, cy Mm khng phi l ch l loi cy gip chng l t, cung cp g tp m cn l cy thuc qu cn c bo v v nghin cu su hn. Trn th gii c mt s cng trnh nghin cu v thnh phn ha hc v hot tnh sinh hc trn l Mm i. nc ta, ngi dn thng c thi quen s dng l Mm tr bnh theo kinh nghim dn gian tuy nhin vn cha c nhng nghin cu v thnh phn ha hc ca l Mm. Cho nn vic nghin cu v thnh phn ha hc ca l Mm i (Avicennia marina) trong iu kin hin nay l cn thit i vi ha hc v i sng. 2 PHNG PHP NGHIN CU 2.1 Phng php nghin cu

tinh ch cc cht c lp thu c cc cht sch. Xc nh cu trc ca cht tinh khit phn lp c: s dng cc phng php ph nghim: 1H-NMR, 13C-NMR, DEPT NMR v cc ti liu lin quan xc nh cu trc cc cht phn lp c. Ph NMR c o trn my Bruker Advance 500 MHz (Vin Ha hc, Vin Khoa hc v Cng ngh Vit Nam, s 18 Hong Quc Vit, Cu Giy, H Ni). Silica gel dng cho sc k ct pha thng c ht 0,040 - 0,063 mm. Sc k lp mng c thc hin trn bn mng trng sn silica gel KG 60 F254. Cc ha cht tinh khit khc c xut x t Trung Quc. 2.2 Hot tnh khng ung th Php th c thc hin ti Phng Sinh hc thc nghim, Vin Ha hc, Vin Khoa hc v Cng ngh Vit Nam. Xc nh hot tnh khng ung th i vi 3 dng t bo Hep-G2 (Hepatocellular carcinoma - ung th gan), Lu (Lung cancer - ung th phi) v RD (Rhabdo sarcoma - ung th mng tim) c cung cp bi vin v sinh dch t trung ng. Phng php xc nh: Theo phng php ca Skehan v ctv. (1990) v Likhiwitayawuid v ctv. (1993), cht chun chng dng tnh: Dng cht chun c kh nng dit t bo: Ellipiticin, Vinblastine hoc Taxol pha trong dimethyl sulfoxide (DMSO), c trn my ELISA bc sng 495- 515 nm, xc nh CS% (% t bo sng st) v IC50. 2.3 Thnh phn dinh dng Vic xc nh thnh phn dinh dng trong l cy Mm i c thc hin ti Phng th nghim chuyn su - i hc Cn Th. Thnh phn dinh dng c xc nh l hm lng amino acid c trong l Mm. Phng php xc nh: s dng phng php Faast Amino Acid Analysis Hydrolysates bng liquid chromatography mass spectrometry (LCMS) vi qui trnh v b kit do Phenomenex ZE: faastTM, Hoa K cung cp. Cc thnh phn amino acid mi gm c arginine, histidine,
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Nguyn liu: L cy Mm i c thu hi

ti x nh Thnh, huyn ng Hi, tnh Bc Liu, chn nhng l ti trng thnh, sau ra sch, ct nh, phi kh. Phng php: Chit hot cht: L Mm i c ngm trong cn 96, phn dch chit c quay loi dung mi thu c cao cn. Sau ly cao cn chit vi dung mi petroleum ether (PE) c quay loi dung mi thu c cao PE. Phn lp cht t cao PE: thc hin qu trnh sc k ct, cht hp ph l silica gel, theo di qu trnh sc k ct bng sc k bn mng (thin layer chromatography TLC), gii ly ct bt u t PE sau tng phn cc bng dung dch PE vi ethyl acetate (EtOAc) theo t l thch hp. Thuc th hin vt l dung dch sulfuric acid (H2SO4) 10% trong methanol (MeOH) v sy bn mng 110C. Cc phn on th hin Rf ging nhau trn TLC c gom li. Tin hnh sc k ct tip tc i vi cc phn on c vt c trng v khi lng ng k, sau

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isoleucine, leucine, lysine, hydroxylysine, methionine, phenylalanine, tryptophan, valine, alanine, aspartic acid, glutamic acid, glutamine, glycine, serine, proline, tyrosine, cysteine v threonine. 3 KT QU V THO LUN 3.1 Kt qu sc k ct T 10,0 g cao petroleum ether tin hnh sc k ct thng vi h dung mi gii ly PE:EtOAc v EtOAc:MeOH c phn cc tng dn. Kt qu phn on PE:EtOAc = 100:0 thu c 1,007 g mt hp cht tinh khit c tinh th hnh kim mu trng c, hin vt trn TLC cho mt vt trn mu hng tm c Rf = 0,43 (PE:EtOAc = 75:25) khi dng thuc th l H2SO4 10% trong MeOH. K hiu hp cht ny l PHUOC-NHI-01. phn on PE:EtOAc = 95:5 kt qu thu c 0,278 g mt hp cht tinh khit, c dng tinh th mu trng, hin trn TLC mt vt trn mu nu xm c Rf = 0,47 (CH2Cl2:EtOAc = 90:10). K hiu hp cht ny l PHUOC-NHI-02. 3.2 Kt qu d liu ph Hp cht PHUOC-NHI-01 Ph 1H-NMR (500 MHz, CDCl3), (ppm): 0,68 (d, 1H, J = 9,5 Hz, H-5); 0,76 (s, 3H, CH324); 0,79 (s, 3H, CH3-28); 0,83 (s, 3H, CH3-25); 0,94 (s, 3H, CH3-27); 0,97 (s, 3H, CH3-23); 1,03 (s, 3H, CH3-26); 1,68 (s, 3H, CH3-30); 1,87-1,96 (m, H-21); 2,37 (m, 1H, J = 6,0 v 5,5 Hz, H-19); 3,19 (dd, 1H, J = 5,0 v 4,5 Hz, H-3); 4,57 (br s, 1H, H-29); 4,69 (br s, 1H, H-29). So snh ph 1H-NMR ca hp cht PHUOC-NHI-01 vi ph 1H-NMR ca hp cht PHUOC-TR-01 (lupeol) ca L Thanh Phc v Phm Th Thy Trang, 2010 v kt hp vi nhng d kin trn th PHUOC-NHI01 c nhn danh l lupeol (Hnh 1). Kt qu ny cng ph hp vi kt qu ca M.S. Ali et al., 2007.
HO

Hnh 1: Cng thc cu to ca lupeol

Lupeol c th tiu dit t bo ung th u, c v ngn chn s lan truyn ca chng mt cch hiu qu. Hp cht lupeol c hot tnh gy c t bo vi dng t bo ung th gan (HepG2), A-431, H-4IIE (El Deed K.S. et al., 2003). Th nghim trn chut do i hc Hong Kong thc hin cho thy hp cht trn chng t tc dng hu hiu nht khi c p dng km theo phng php ha tr v hu nh khng gy tc dng ph. Thm ch, lupeol c th gim kch thc khi u mt cch nhanh chng v hiu qu hn c nhng loi thuc ang c s dng tr ung th nh cisplatin. Theo cc nh khoa hc, lupeol ngn chn protein NFkB, vn gip t bo ung th hi phc v tng trng. Bn cnh , lupeol gip ngi bnh gi nguyn trng lng, khng gy st cn trm trng nh khi s dng phng php ha tr. Ngoi ra lupeol cn l cht chng oxy ho v khng vim (Fernandez M.A. et al., 2001). Theo kt qu nghin cu ca L Thanh Phc v Phm Th Thy Trang, hm lng lupeol trong r cy Mm i kh cao (khong 0,21%), trong khi hm lng lupeol trong l cy Mm i chim khong 0,23%. Do , chng ta c th tch c cht lupeol vi lng ln nhm mc ch cha bnh v cy Mm c rt nhiu, d thu hi v x l. Hp cht PHUOC-NHI-02 Ph 1H-NMR (500 MHz, CDCl3), (ppm): 0,68 (t, 1H, H-5); 0,76 (s, 3H, CH3-24); 0,82 (s, 3H, CH3-26); 0,98 (s, 3H, CH3-27); 0,97 (s, 3H, CH3-23); 1,02 (s, 3H, CH3-25); 1,68 (s, 3H, CH3-30); 2,38 (dt, 1H, J = 6,0 Hz, H-19); 3,18 (d, 1H, J = 10,5 Hz, H-3); 3,33 (d, 1H, J = 11,0 Hz, H-28); 3,80 (dd, 1H, J = 11,0 v 1,5 Hz, H24

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28); 4,58 (t, 1H, J = 1,5 Hz, H-29); 4,68 (d, 1H, J = 1,5 Hz, H-29) (ppm): 14,8 (C-27); 15,4 (C-24); 16,0 (C-26); 16,1 (C-25); 18,3 (C-6); 19,1 (C-30); 20,9 (C11); 25,2 (C-12); 27,1 (C-15); 27,4 (C-2); 28,0 (C-23); 29,2 (C-16); 29,8 (C-21); 34,0 (C-22); 34,3 (C-7); 37,2 (C-10); 37,3 (C-13); 38,7 (C1); 38,9 (C-4); 40,9 (C-8); 42,7 (C-14); 47,8 (C17, C-19); 48,7 (C-18); 50,4 (C-9); 55,3 (C-5); 60,6 (C-28); 79,0 (C-3); 109,7 (C-29); 150,5 (C-20). T nhng d kin trn th hp cht k hiu l PHUOC-NHI-02 c nhn danh l betulin (Hnh 2). Kt qu ny cng ph hp vi kt qu ca Seyed Abdolmajid Ayatollahi et al., 2009.
29 20 30 12 11 25 1 2 3 24 4 10 5 23 9 8 27 7 6 26 14 15 13 18 17 16 19 21 22

3.3 Kt qu kho st hot tnh khng ung th

Ph 13C-NMR (125,8 MHz, CDCl3),

Theo kt qu trn th hp cht lupeol va c lp c (k hiu PHUOC-NHI-01) c hot tnh khng ung th gan (Hep-G2) vi gi tr IC50 l 93,53 g/mL v hp cht betulin (k hiu l PHUOC-NHI-02) c hot tnh khng ung th phi vi gi tr IC50 l 25,84 g/mL (Lu). Nh vy, hot tnh i vi dng Hep-G2 ca hp cht lupeol (k hiu PHUOC-NHI-01) v hp cht betulin (k hiu PHUOC-NHI-02) l yu.
Bng 1: Kt qu th hot tnh kh nng khng ung th ca cc cht Kt qu: Gi tr IC50 (g/mL) ca mu th trn dng t bo Lu Hep-G2 Khng th 93,53 Khng th 25,84

STT

Tn mu

1 PHUOC-NHI-01 2 PHUOC-NHI-02

3.4 Kt qu kho st thnh phn dinh dng Protein l thnh phn cht hu c chnh ca c th cc loi thy sn v cc amino acid l thnh phn cu to nn protein. C mt s amino acid thit yu ng vt thy sn khng th tng hp c m chng phi ly t thc n nh: arginin, histidin, isoleucine, leucine, lysine, methionine, phenyllalanine, threonine, tryptophan v valine. L Mm c cha y cc loi amino acid thit yu trn vi hm lng mi loi kh cao. Thc t, mt s loi rau thng s dng trong chn nui hin nay nh rau mung c hm lng amino acid thp hn so vi l Mm, rau khoai lang khng cha cc amino acid thit yu ging l Mm nh arginin, histidin, isoleucine, leucine, phenyllalanine, threonine v valine (Nguyn Cng Khn et al., 2007). Chnh v vy, c mt s nghin cu a l Mm vo lm thc n cho tm vi kt qu rt kh quan (trng lng tm tng ln n 12,3% so vi nui tm bng cc loi thc n t nhin khc) v l Mm c y cc loi amino acid thit yu cn thit cho ng vt thy sn (S. Athithan and V. Ramadhas, 2000).

CH2OH
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HO

Hnh 2: Cng thc cu to ca betulin

Betulin l mt hp cht c tm thy ph bin thc vt, c tnh khng khun, khng vim, khng st rt v khng ung th. Betulin th hin hot tnh gy c t bo trn hai dng HeLa v Hep-G2 vi cng gi tr IC50 l 40 g/mL. Betulin cng th hin hot tnh chng HIV vi gi tr IC50 l 6,1 g/mL (El Deed K.S et al., 2003). Cc nghin cu ca Miura cn cho thy betulin c tc dng bo v gan v lm gim kh nng gy c ca CdCl2 nng thp 0,1 g/mL. Tc dng bo v ca betulin c bit r rng khi thm vo mi trng nui cy t bo Hep-G2 trc khi cho CdCl2. Hn na, khi t bo Hep-G2 c vi betulin trc khi cho CdCl2 th tc dng c hi ca cadmium c gim, c ch c th l do betulin thc y s tng hp cc protein c tc dng bo v cc t bo Hep-G2 khi nh hng ca CdCl2 (N. Miura et al., 1999).

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Bng 2: Kt qu kho st thnh phn amino acid trong l Mm Kt qu th nghim Phng STT Tn ch tiu php th % trong l Mm 01 Arginine EZ: faast 0,5259 Amino Acid 02 Serine 0,2479 Analysis of 03 Glycine 0,3988 protein 04 Threonine 0,2753 Hydrolysates 05 Alanine 0,4858 by LCMS 06 Methionine 0,0454 (Phn tch 07 Proline 0,3939 Amino Acid 08 Lysine 0,4424 09 Aspartic acid ca protein 0,7968 thy phn 10 Histidine 0,3589 bng LCMS) 11 Valine 0,9970 12 Glutamic acid 0,9119 13 Isoleucine 0,5679 14 Leucine 0,3719 15 Phenylalanine 0,3413 16 Cystine 0,0311 17 Tyrosine 0,2547

tc nghin cu su sc hn v thnh phn ha hc, thnh phn dinh dng v hot tnh sinh hc ca chng. TI LIU THAM KHO
El Deed K.S., Al-Haidari R.A., Mossa J.S., Abdel Monem A.A., 2003. Phytochemcal and pharmacological studies of Maytenus forsskaoliana, Journal Saudi Pharmaceutical. 11 (4), 184-191. 2. Fernandez M.A., De las Heras B., Garcia M.D., Saenz M.T., Villar A., 2001. New insights into the mechanism of action of the anti-inflammatory triterpen lupeol, J. Pharm. Pharmcol. 53 (11), 1533-1539. 3. L Thanh Phc v Phm Th Thy Trang, 2010. Kho st thnh phn ha hc ca r cy Mm (Avicennia marina), Tp ch Khoa hc Trng i hc Cn Th, S 15(b), 9-14. 4. M.S. Ali and S. Waseemuddin Ahmed Shehla Imam, Iqbal Azhar, M. Mohtasheemul Hasan, 2007. Two triterpenes lupanone and lupeol isolated and identified from Tamarindus Indica Linn, Pak., J. Pharm. Sci. Vol 20 (2), 125-127. 5. Nguyn Cng Khn, H Th Anh o, L Hng Dng v Nguyn Th Lm, 2007. Bng thnh phn thc phm Vit Nam, NXB Y Hc, 158-165. 6. N. Miura, Y. Matsumoto, S. Miyairi, S. Nishiyama, A. Naganuma, 1999. Protective effects of triterpene compounds against the cytotoxicity of cadmium in HepG2 cells, Molecular Pharmacology 56 (6), 1324-1328. 7. Phm Hong H, 2000. Cy c Vit Nam, NXB Tr, TP H Ch Minh (II), 844-845. 8. P. Thirunavukkarasu, T. Ramanathan, L. Ramkumar and R. Shanmugapriya, 2010. Anti ulcer effect of Avicennia officinalis leaves in Albino Rats, World Applied Sciences Journal 9 (1), 55-58. 9. S. Athithan and V. Ramadhas, 2000. Bioconversion Efficiency and Growth in the White Shrimp, Penaeus indicus (Milne Edwards), Fed with Decomposed Mangrove Leaves, The ICLARM Quarterly Vol 23(1), 17-18. 10. Seyed Abdolmajid Ayatollahi, Asie Shojaii, Farzad Kobarfard, Mitra Nori, Mohamma Fathi and Mohammad Iqbal Choudhari, 2009. Terpens from aerial parts of Euphorbia splendida, Journal of Medicinal Plants Research Vol. 3(9), 660-665. 1.

4 KT LUN Trong qu trnh kho st thnh phn ha hc trong cao petroleum ether ca l cy Mm i (thu hi ti huyn ng Hi, tnh Bc Liu), bc u chng ti c lp v nh danh c hai cht lupeol v betulin. Kho st hot tnh khng ung th th lupeol c hot tnh khng ung th gan (Hep-G2) v betulin c hot tnh khng ung th phi (Lu). Kho st thnh phn dinh dng trong l Mm th cc thnh phn amino acid thit yu u c trong l Mm vi hm lng kh cao. Nh c hm lng amino acid cao hn nhiu so vi cc loi thc n khc cho ng vt thy sn nh: rau mung, rau khoai lang v cc loi l rng khc nn l Mm ngy cng c cc h nng dn nui tm a vo lm thc n cho tm vi hiu qu kinh t rt cao. Tuy nhin, nu th l Mm vi mt qu dy s lm cho lng oxy ha tan trong nc s gim i v mi trng nui tm s b nhim. V vy, mt th th no v bao nhiu l thch hp, th cn c cng trnh nghin cu rng v su hn na. Chnh v nhng l do trn m cy Mm i ni ring v cc cy Mm ni chung ngy cng c cc nh ha hc v hp cht t nhin tip
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