Tgp ehi Khoa hge Tru&ng Dgi hgc Cdn Tha Phdn A: Khoa hoc Tu nhien. Cong ngh? vo Moi Iru&ng: 25 (2013): 22-26
Tap chi Khoa hpc - Dai hpc Can Thd website: sj.ctu.edu.vn
NGHIEN c t r u THANH P H A N HOA HOC, HOAT TINH SINH HQC
CAC CHAT TRONG CAO PETROLEUM ETHER VA THANH PHAN DINH DITGNG CUA LA CAY M A M 6 I {A VICENNIA MARINA)
Le Thanh Phudc' va Le Huang Nhi' Khoa Khoa hgc Ty nhien, Trucmg Dgi hgc Cdn Tha
Thong tin chung:
Ngay nlian-14/09/2012 Ngay chdp nhgn • 25/03/2013 Title:
Study on chemical components, biological activities from the petroleum ether extract and nutrition components of the leaves ofAvicennia marina Tirkhda:
Ld Mdm oi Avicennia marina, thanh phdn hoa hgc. hogt tinh sinh hgc, thdnhphdn dinh dir&ng, lupeol. betulin Keywords:
Avicennia marina, chemical components, biological activities, nutrilion components, lupeol. betulin
ABSTRACT
Study on the chemical ingredients from the leaves ofAvicennia marina, collected in Dong Hai district, Bac Lieu province, we have isolated and identified two compounds: liq^eol (CSQHSOO) and betulin (CsoHst^j) from the petroleum ether extract. Structures of these compounds had been elucidated by modem spectroscopic methods: 'H-NMR, '^C-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 ICso value of 93,53 jjg/mL. Betulin was against human Lung cancer cells with an IC^o value of 25,84 pg/mL.
Furthermore, when we tested nutrition of the leaves ofAvicennia marina, we found out a lot of amino acids with high content, this explained why shrimp farmers have been using the leaves ofAvicennia marina as natural food for shrimp. The study has been continued.
TOM T A T
Khao sdt thanh phdn hod hgc Id cdy Mdm oi dugc thu hdi tgi huyen Dong Hdi, linh Bac Lieu, chimg toi da co Igp vd 'dfnh danh dugc hai chdt.
lupeol va betulin tir dich chiet petroleum ether. Cdu true hoa hgc cdc chdt nay da dirge ldm sdng to dua vao nhOng phuang phdp pho hien dgi H- NMR, '^C-NMR. DEPT NMR vd so sdnh vai tdi lieu da cong bo. Trong do, chung toi dd khdo sdt vd tim ra nhiing hogt tinh sinh hgc ciia lupeol vd betulin. Kit qud Id, lupeol co khd ndng khdng te bdo ung thu gan vai ICso CO gid trf la 93.53 pg/mL. Betulin cung co khd nang chong lgi te bdo ung thu phoi vol ICso co gid Iri Id 25,84 jjg/mL Han the niia, khi lien hdnh nghien diu ihdnh phdn dinh duang cua Id Mdm, chiing toi nhdn thdy la Mam co nhieu amino acid voi ham lugng cao, dieu ndy ly gidi tgi sao ngirai nong ddn nuoi torn lgi sir dung la Mam ldm nguon thuc dn tu nhien cho torn. Nghien cuu vdn dang dugc tiep tuc.
1 D A T V A N D E
Cay Mdm di cd ten khoa hgc la Avicennia marina, thudc hg Mam (Verbenaceae) (Pham
Hoang Hd, 2000). Cay Mdm cd thi nhanh chdng sinh sdn, ldn len va phat trien tdt d viing gidp ranh giiJa ddt vd nude man. Vai trd ldn nhat cua loai Mam Id cd dinh dat, do bd rl duac
Tap chi Khoa hge Tru&ng Dgi hoc Cdn Tha Phdn A • Khoa hgc Tu nhien. Cong ngh? vd Moi truong: 25 (2013): 22-^
cdu tnic vihig chdc dn sdu xudng dat, nd cd site chiu dung dugc sdng va gid, chiu dugc nude man ngap quanh nam. Theo mdt sd tai lieu ddn gian tren thi gidi, la cua cdy Mdm tri benh dau da day, benh nam d phu nii, tri cdc lo^i ung nhgt (P. Thirunavukkarasu et ai, 2010). Vd cua cay Mdm diing d l lam thudc tri ghe va chiia benh phong, chiia vit thuong hoai thu, diet chdy ran, diet giun san va cd tac dung ngira thai (Ph?m Hoang Ho, 2000). Nd cdn cd khd ndng chvta benh ung thu. Vi vdy, cay Mdm khdng phdi la chi la loai cay giup chdng Id ddt, cung cdp go tap ma cdn la cdy thudc quy cdn dugc bao ve vd nghien cuu sdu han. Tren the gidi dd cd mgt sd cdng trinh nghien cuu ve thanh phdn hda hpc vd hoat tinh sinh hgc tren la Mdm di. Ci nude ta, ngudi dan thudng cd thdi quen sir dung Id Mdm de tri b?nh theo kinh nghiem ddn gian tuy nhidn vdn chua cd nhiing nghien cuu ve thdnh phan hda hgc ciia Id Mdm. Cho nen viec nghien cuu ve thanh phdn hda hgc cua la Mdm oi (Avicennia marina) trong dieu kien hien nay la cJin thilt ddi vdi hda hgc vd ddi song.
2 PHU'DNG P H A P NGHIEN ClTU 2.1 Phirang phdp nghien ciru
N g u y e n lieu: La cay Mdm di dugc thu hai tai xd Dinh Thanh, huy?n Ddng Hdi, tinh Bac Li6u, chgn nhung la tuai da trudng thanh, sau dd nia s^ch, cdt nhd, phai kho.
Phuang phap: Chiet hoat chat: La Mam di dugc ngam trong cdn 96°, phan dich chilt cd quay loai dung mdi thu dugc cao cdn. Sau dd ldy cao con chiet vdi dung mdi petroleum ether (PE) CO quay loai dung mdi thu dugc cao PE.
Phdn lap chat tir cao PE: thuc hien qua trinh sdc ky cgt, chdt hdp phy Id silica gel, theo ddi qua trinh sdc ky cdt bdng sac ky ban mdng (thin layer chromatography TLC), gidi ly cdt bat ddu tir PE sau dd tang dp phan cgc bdng dung djch PE vdi ethyl acetate (EtOAc) theo ty le thich hop. Thudc thu hien vet la dung dich sulfuric acid (H3SO4) 10% trong methanol (MeOH) va say ban mdng d 110°C. Cdc phdn do?n thi hien R/ gidng nhau tren TLC dugc gom lai. Tiln hanh sdc ky cot liep tuc ddi vdi cdc phdn doan cd vet ddc trung va khdi lugng ddng kl, sau
dd tinh che cac chdt da cd lap thu dugc cac chat sach.
Xac dinh cau tnic ciia chdt tinh khilt dS phan Idp duac: su dung cdc phuang phap phi nghiem: ^H-NMR, ''C-NMR, DEPT NMR vd cac tai lieu lien quan de xdc dinh cdu tnic cdc chat phdn lap dugc. Phd NMR dugc do tren may Bruker Advance 500 MHz (Vien Hoa hgc, Vi|n Khoa hgc va Cdng n ^ h | Viet Nam, sd 18 Hoang Quoc Viet, Cdu Gidy, Ha Ndi).
Silica gel diJng cho sdc ky cdt pha thudng cd hat 0,040 - 0,063 mm. Sdc ky ldp jndng dugc thuc hiSn tren bdn mdng trdng sEn silica gel KG 60 F254. Cac hda chat tinh khilt khac cd xudt xd tiJ Trung Qudc.
2.2 Hoat tinh khang ung thir
Phdp thur dugc thuc hien tai Phdng Sinh hoc thuc nghiem, Vien Hda hgc, Vi6n Khoa hpc va Cdng nghe ViSt Nam.
Xdc dinh hoat tinh khang ung thu ddi vdi 3 ddng t l bao Hep-G2 (Hepatocellular carcinoma - ung thu gan), Lu (Lung cancer ung thu phdi) vd RD (Rhabdo sarcoma - ung thu mans tim) dugc cung cdp bdi vien ve sinh dich te trung uang.
Phuang phap xac dinh: Theo phucmg phdp cila Skehan va ctv. (1990) v^
Likhiwitayawuid va ctv. (1993), chdt chuan chung duang ti'nh: Diing chdt chuan cd kh4 nang diet t l bao: Ellipiticin, Vinblastine hodc Taxol pha trong dimethyl sulfoxide (DMSO), dgc tren may ELISA d budc sdng 495- 515 nm, xdc dinh CS% (% t l bao sdng sdt) va IC50.
2.3 Thdnh phan dinh dirdng
Viec xac djnh thanh phdn dinh dudng trong Id cdy Mam di dugc thuc hien tai Phdng thi nghiem chuyen sdu - Dai hgc Cdn Tha. Thdnh phdn dinh dudng dugc xac dinh la ham lugng amino acid cd trong Id Mdm.
Phuang phap xac dinh: sir dung phuang phdp Faast Amino Acid Analysis Hydrolysates bang liquid chromatography mass spectrometry (LCMS) vdi qui trinh vd bg kit do Phenomenex ZE: faast^", Hoa K^ cung cdp. Cac thanh phan amino acid mdi gdm cd arginine, histidine,
Tgp chi Khoa hgc Tnrdng Dai hgc Cdn Tho Phdn .4: Khoa hgc Tu nhien. Cong nghe va Moi Irudng: 25 (2013):
isoleucine, leucine, lysine, hydroxylysine, methionine, phenylalanine, tryptophan, valine, alanine, aspattic acid, glutamic acid, glutamine, glycine, serine, proline, tyrosine, cysteine vd threonine.
3 K E T QUA VA T H A O LUAN 3.1 Ket qua sac ky cgt
Tu 10,0 g cao petroleum ether lien hanh sac ky cgt thudng vdi he dung mdi gidi Iy PEiEtOAc vd EtOAc:MeOH cd do phdn cue tang dan. Ket qud d phan doan PE:EtOAc = 100:0 thu dugc 1,007 g mdt hgp chdt tinh khilt cd tinh the hinh kim mau trdng due, hien vet tren TLC cho mgt vet trdn mau hdng tim cd R/ = 0,43 (PErEtOAc - 75:25) khi dimg thudc thu la H2SO4 10% trong MeOH. Ky hieu hgp chdt ndy Id PHUOC-NHI-OI. 6 phdn doan PE:EtOAc = 95:5 kit qua thu dugc 0^78 g mgt hgp chdt tinh khilt, cd dang tinh thi mau trdng.
hien tren TLC mdt vet trdn mdu ndu xam cd Rf = 0,47 (CHzCUtEtOAc = 90:10). Ky hieu hgp chdt nay la PHUOC-NHI-02.
3.2 Ket qua dir lieu phd Hap chdt PHUOC-NHI-01
Phd 'H-NMR (500 MHz. CDCI3), 5 (ppm):
0,68 (d, \H,J= 9,5 Hz, H-5); 0,76 (s, 3H, CH3- 24); 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, CH3-3O);
1,87-1,96 (m, H-21): 2,37 (m, IH, 7 = 6,0 va 5,5 Hz, H-19); 3,19 (dd, I H , y = 5 , 0 vd4,5 Hz, H-3); 4,57 (br s. IH, H-29); 4,69 (br s. IH, H-29).
So sdnh phd ^ H-NMR ciia hgp chdt PHUOC-NHI-OI vdi phd ^H-NMR cua hgp chat PHUOC-TR-OI (lupeol) cua Le Thanh Phudc va Pham Thi Thuy Trang, 2010 va kit hgp vdi nhiing dii kien tren thi PHUOC-NHI- OI dugc nhdn danh Id lupeol (Hinh 1). Kit qua nay cung phii hgp vdi kit qua cua M.S. Ali et al.. 2007.
-3 Z4
Huah 1: C o n g thiirc c a n t a o c a a lupeol Lupeol cd thi tieu diet t l bao ung thu ddu, cd va ngdn chan su lan truyen cua chiing mdt cdch hieu qud. Hgp chat lupeol cd hoat tinh gay ddc te bao vdi ddng te bdo ung tfiu gan (Hep- G2), A-431, H-4IIE (El Deed K.S. et al. 2003).
Thi nghiem tren chudt do Dai hgc Hong Kong thuc hien cho thay hgp chat tren chung td tac dung hiru hieu nhdt khi dugc dp dung kem theo phuang phap hda tri va hdu nhu khdng gay tac dung phu. Thdm chi, lupeol cd the gidm kich thudc khoi u mdt each nhanh chdng va hieu qua han cd nhung loai thudc dang dugc sir dung de tri ung thu nhu cisplatin. Theo cac nha khoa hgc, lupeol da ngan chan protein NFkB, vdn giup te bao ung thu hdi phuc va tang trudng.
Ben cwih do, lupeol giup ngudi benh giu nguyen trgng Iugng, khdng gay sut can tram trpng nhu khi sii dung phuang phdp hda tri.
Ngoai ra lupeol cdn la chat chdng oxy hoa va khdng viem (Fernandez M.A. et ai, 2001).
Theo kit qua nghien ciru ciia Le Thanh Phudc vd Pham Thi Thiiy Trang, ham lugng lupeol trong re cay Mam di khd cao (khoang 0,21%), trong khi dd ham lugng lupeol trong la ca\ Mdm di chiem khodng 0,23%. Do dd, chiing ta cd the tdch dugc chdt lupeol vdi lugng ldn nhdm muc dich chiia benh vi ca\- Mam cd rat nhieu, de thu hdi va xir ly.
Hap chdt PHU(X:-NHJ-02
Phd 'H-NMR (500 MHz, CDCI3), 6 (ppm):
0.68 (t, IH, H-5); 0,76 (s, 3H. CH3-24); 0.82 (s.
3H. CH3-26); 0,98 (s, 3H. CH3-27): 0,97 (s, 3 a CH3-23); 1,02 (s. 3H, CH3-25); 1.68 (s. 3H.
CH3-3O); 2,38 (dt, IH, J= 6,0 Hz, H-19); 3.18 (d, IH, J = 10.5 Hz. H-3); 3.33 (d. IH, J = 11,0 Hz. H-28); 3.80 (dd. IH. J = 11.0 vd 1,5 Hz, H-
Tgp chi Khoa hoe Trudng Dai hoc Cdn Tha Phdn A • Khoa hgc Tv nhien. Cong nghe vd Moi Iru&ng 25 (2013): 22-^
28); 4,58 (t, IH, J- 1,5 Hz, H-29); 4,68 (d, IH, / = 1,5 Hz, H-29)
Phd 13C-NMR (125,8 MHz, CDCb), 5 (ppm): 5 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 (C- II); 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 (C- 1); 38,9 (C-4); 40,9 (C-8); 42,7 (C-14); 47,8 (C- 17, 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).
Tir nhiing dtr kien tren thi hgp chdt ky hieu la PHUOC-NHI-02 dugc nhdn danh la betulin (Hinh 2). Kit qud nay cung phii hgp vdi kit qua ciia Seyed Abdolmajid Ayatollahi etai, 2009.
Hinh 2: Cong thirc cau tao ciia betulin Betulin la mdt hgp chat dugc tim thdy phd biln d thuc vdt, cd tinh khdng khudn, khdng viem, khang sot ret vd khang ung thu. Betulin thi hi^n hoat tinh gay ddc te bdo tren hai ddng HeLa va Hep-G2 vdi cung gid tri IC50 la 40 pg/mL. Betulin cung thi hien hoat tinh chong HIV vdi gid tri IC50 la 6,1 pg/mL (El Deed K.S et ai, 2003). Cdc nghien cuu cua Miura cdn cho thdy betulin cd tac dung bdo ve gan vd lam giam kha ndng gdy ddc cua CdCL d ndng do thdp 0,1 pg/mL. Tac dung bao ve cua betulin dac bi^t ro rang khi them vdo mdi trudng nudi cdy te bao Hep-G2 trudc khi cho CdCU. Hon nira, khi te bao Hep-G2 dugc u vdi betulin trudc khi cho CdCL thi tdc dung ddc hai ciia cadmium dugc giam, co che cd thS Id do betulin thiic ddy su tdng hgp cac protein cd tdc dung bdo ve cac te bao Hep-G2 khdi dnh hudng cua CdCb (N. Miura et ai. 1999).
3.3 KSt qud khao sdt hoat tinh khang ung thu Theo kit qua tren thi hgp chdt lupeol viia co lap dugc (ki hieu PHUOC-NHI-01) cd hoat tinh khang ung thu gan (Hep-G2) vdi gid tri IC50 lik 93,53 pg/mL va hgp chdt betulin (ki hieu ]k PHUOC-NHI-02) cd hoat tinh khang ung thu phoi vdi gid tri IC50 la 25,84 pg/mL (Lu). Nhu vay, hoat tinh ddi vdi ddng Hep-G2 cua hgp chdt lupeol (kl hieu PHUOC-NHI-01) vd hgp chdt betulin (ki hieu PHUOC-NHI-02) la ylu.
Bang 1: Ket qua thu boat tinh kha nang khang ung thir ciia cdc chat
" Ket qua: Gid tri IC50 (pg/mL) cua man thir STT Ten mau tren ddng te bdo
Hep-G2 1 PHUOC-NHI-01 Khong thii 93,53 2 PHUOC-NHI-02 25,84 Khdng thii 3.4 K i t qua khao sat thanh phan dinh downg
Protein la thanh phdn chat hiiu ca chmh ciia CO thi cac lodi thuy sdn va cdc amino acid l^
thanh phdn cdu tao nen protein. Cd mdt so amino acid thilt ylu ddng v^t thuy sdn khong thi tong hgp dugc ma chiing phai ldy tir thiic an nhu: arginin, histidin, isoleucine, leucine, lysine, methionine, phenyllalanine, threonine^
tryptophan va valine. La Mdm cd chua ddy mi cac loai amino acid thilt yeu tren vdi ham lugng mdi loai khd cao. Thuc t l , mdt sd loai rau thudng sii dung trong chan nudi hien nay nhu rau mudng cd ham lugng amino acid thap hem so vdi la Mdm, rau khoai lang khdng chda cac amino acid thiet ylu gidng la Mdm nhu arginin, histidin, isoleucine, leucine, phenyllalanine, threonine va valine (Nguyin Cdng Khan et al, 2007). Chinh vi vdy, da co mgt sd nghiSn citu dua Id Mam vao lam thuc dn cho tdm vdi ket qua rdt kha quan (trgng Iugng tdm tang Ien den 12,3% so vdi nudi tdm bdng cac loai thiic an ty nhiSn khac) vi la Mam cd ddy du cac loai amino acid thiet yeu cdn thiet cho ddng vat thiiy sSii (S. Athithan and V. Ramadhas, 2000).
Tap chi Khoa hge Tru&ng Dgi hgc Cdn Tho Phdn A. Khoa hgc Tu nhien. Cong nghe vd Moi trudng: 25 (2013). 2. 2-26 Bang 2: Ket qua khao sat tbanh phdn amino acid
trong la M^m STT
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17
Ten chi tieu Arginine Serine Glycine Threonine Alanine Methionine Proline Lysine Aspartic acid Histidine Valine Glutamic acid Isoleucine Leucine Phenylalanine Cystine Tyrosine
Phiromg phap thir EZ: faast Amino Acid Analysis of protein Hydrolysates by LCMS (Phan tich Amino Acid ciia protein thiiy phSn bang LCMS)
Ket qaa thir nghiem
% trong la Mam 0,5259 0.2479 0,3988 0,2753 0,4858 0,0454 0,3939 0,4424 0,7968 0.3589 0,9970 0,9119 0,5679 0,3719 0,3413 0,0311 0,2547 4 K E T L U i ^ N
Trong qua trinh khdo sat thdnh phdn hda hgc trong cao petroleum ether cua la cdy Mdm di (thu hai tai huyen Ddng Hdi, tinh Bac Lieu), budc ddu chiing tdi d3 cd lap va dinh danh dugc hai chdt lupeol vd betulm. Khdo sat hoat tinh khdng ung thu thi lupeol cd hoat tinh khdng ung thu gan (Hep-G2) vd betulin cd hoat tinh khdng ung thu phdi (Lu). Khao sat thdnh phdn dinh duang trong Id Mam thi cdc thdnh phdn amino acid thilt yeu deu cd trong la Mdm vdi hdm lugng kha cao. Nhd cd ham lugng amino acid cao han nhilu so vdi cac loai thiic an khac cho ddng vat thiiy sdn nhu: rau mudng, rau khoai lang va cdc loai la rimg khac nSn Id Mam ngay cang dugc cdc hd ndng dan nudi tdm dua vao lam thurc an cho tdm vdi hieu qua kinh te rdt cao. Tuy nhidn, neu thd la Mam vdi mdt do qud day se lam cho tugng oxy hda tan trong nude se gidm di va mdi trudng nudi tdm se bi d nhiem.
Vi vdy, mat do tha the nao vd bao nhieu la thich hgp, thi can cd cdng trinh nghien curu rgng va sau hon niia.
Chinh vi nhiing li do tren ma cdy Mdm di ndi rieng va cac cay Mdm ndi chung ngay cang dugc cac nhd hda hgc ve hgp chdt tu nhien tilp
tuc nghien cuu sdu sac han ve thanh phan hda hgc, thdnh phdn dinh duang va hoat ttnh sinh hgc cua chung.
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anti-mflaramatory triterpen lupeol, J. Pharm.
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Ramkumar and R. Shanmugapriya, 2010. Anti ulcer effect ofAvicennia officinalis leaves in Albino Rats, World Applied Sciences Joumai 9 (1), 55-58.
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