V ^ DAI HOC QUOC GIA HA NOI TRirdNG DAI HOC KHOA HOC Tl/ NHlfiN 4e4G*4c4c4e4c4e4( : NGHlfiN ctru CAC HOP CHAT CO HOAT TINH SINH HOC TlT CAC CAY THUdc CUA V I £ T NAM T £ N D £ TAI MA SO: QT - 06 -19 CHU TRI f)t TAX: TS PHAN MINH GIANG ;.Al HGC Q U O C G l A HA N p i DT / oJ N- A HA NOI - 2006 | Bao cao torn tit a Ten de tai NghiSn curu cac hop ch^t co hoat tmh sinh hoc tur cac c^y thuoc cua Viet Nam Ma s6': QT-06-19 b Chu tri de tai TS Phan Minh Giang c Cac sinli vien tham gia NCS Le Huyen Tram ThS Dao Quoc Himg CN Nguyen Thi Tu6 d Muc tieu va noi dung nghien curu • Sang lgc lira chon mgt so c^y thuoc co gia tri su dung cao Y hoc d^n toe cua Vi6t Nam, giam dinh thuc vat, thu hai nguyen lidu thuc vat dung cho nghien cihi • Xtr ly mSu thuc vat, nghien cihi qui trinh chiet va phan tach d^ dieu che cac phdn chiet va phan doan giau hoat tinh sinh hoc • Danh gia hoat tinh sinh hgc cua cac phdn chiet va phan doan Dinh hucfng phan tach theo hoat tinh sinh hoc • Phan tach theo dinh hucfng hoat tfnh sinh hoc Nghien cihi qui trinh phan lap cac hop cha^t co hoat tinh sinh hgc bang cac phuong phap phan tfch va phan tach hien dai • Xac dinh ca'u true hoa hgc cua cac hop chat ducfc phan lap bang cac phuang phap vat ly hien dai • Danh gia hoat tfnh sinh hgc (chong oxi hoa, khang khuan va khang nam) cua cac hop chS^t dugc phan lap • Khao sat ham lugng cac hop cha't co hoat tfnh sinh hoc mgl sd cay thuoc ducfc nghien cihi e Cac ket qua dat dirge Da tien hanh sang lgc lua chgn cay Nghe xanh {Curcuma aff aeruginosa Roxb., Zingiberaceae), mgt cay thuoc mdi dugc phat hien, dang duac dai tra, va ducfc sii diing Y hoc dan tgc de dieu tri cac benh nhu dau biing va tang cudng tieu hoa lam doi tugng nghien ciiu cua de tai Da lien hanh giam dinh thuc vat va thu thap nguyen lieu thuc vat phue vu cho nghien ciiu Da xay dung dugc cac qui tnnh chiet va phan tach chgn lgc de di^u che cac phdn chiS't c6 hoat tfnh sinh hgc, //-hexan (hieu suat 3,14% so vai luong nguyen lieu kho), etyl axetat (0,6%) va ii-butanol (0,14%) Mgt phuong phap phan lap sac ky thfch hop da ducfc phat trien de nghien curu mgt each he thong ve hoa hgc va hoat tfnh sinh hgc phan chiet nhexan iix than re Nghe xanh Bang phuong phap cac hop cha't phdn chfnh curdion va zederon da ducfc phan lap v6i cac hieu sua't phan lap tuong iing la 0,1% va 0,05% so vdi lugfng nguyen lieu kho ban ddu Phuong phap ciing thfch hop d^ phat tri^n phuong phap phan lap lucfng Idfn cac hop chdt co hoat tfnh sinh hgc la curdion va zederon, cung nhu cac che phdm dugfc chiia cac nhom sesquiterpenoid khac Cac kS't qua nghien cthi cac hop chdt de bay hoi cua Nghe xanh da xac dinh dugc tdng so 23 hgp chdt (chii yeu la cac sesquiterpenoid) vdi P-elemen - mgt hgp chdt co hoat tfnh chdng ung thu la phdn chfnh cua cac sesquiterpen hydrocarbon, va curzerenon, curcumol, germacron va curdion la cac phdn chfnh ciia cac sesquiterpenoid chiia oxy, so curzerenon da dugc sit dung lam thuoc lam sang cho ung thu da va c6 tur cung Cac ket qua nghien ciiu cac hgp chdt phan cue hon da ddn den viec phan lap mirdi bdn hgp chS't tinh kiiiet tii than re Nghe xanh, curdion (1), axit palmitic (2), zederon (3), y0-sitosterol (4), stigmasterol (5), 13-hydroxygermacron (6), aeruginolacton (7), guaianediol (8), isoprocurcumenol (9), procurcumenol (10), aerugidiol (11), aeruginofuran (12), zedoarofuran (13) va aeruginon (14) Nhu vay cac hgp chat dugc phan lap CO cdu tnic ciia cac sesquiterpenoid, cac steroid va axit beo Cac sesquiterpenoid aeruginolacton (7) va aeruginofuran (12) la cac hgp chat huru ca thien nhien mdi, va aeruginon (14) la hgp chat lan dau tien du*gc phSn lap tiT thien nhien Cdu tnic hoa hgc ciia cac hgp chdt 1-14 da dugc xac dinh bang cac phuong phap ph6 hi6n dai (UV, IR, MS, ID NMR va 2D NMR) Da sir dung cac phan tfch ph6 NOED, NOESY va CD xac dinh cac cdu tnic lap thi tuyet ddi cua cac hgp chdt dugc phan lap Trong cac thu nghiem theo hucfng khang khudn va khang ndm cac phdn chie't «-hexan, etyl axetat va /i-butanol da dugc danh gia la co hoat tfnh khang ndm Chiing deu ihi hien tac dung khang Aspergillus niger, Candida albicans va Fusarium oxysporum cf miic MIC tir 25-200 )ag/ml Cac phan tach co dinh hudfng khang khudn va khang ndm da ddn den viec phan lap cac hoat chdt la curdion va zederon - Curdion va zederon (cac MIC 25 |ig/ml) co th^ dugc phat tridn cac tac nhan chdng benh thdi re thuc vat gay bdi ndm mdc Fusarium oxysporum - Curdion va zederon da dugc chiing to la cac hgp chdt khang cac vi nam gay benh cho ngucfi tuong iing la Aspergillus niger (MIC 50 |ag/ml) va Candida albicans (MIC 25 )ig/ml) - Curdion (MIC 50 ^g/ml), phdn chiet etyl axetat (MIC 200 (ag/ml) va phdn chiet ;j-butanol (MIC 50 ]ig/ml) co hoat tfnh khang vi khuan Staphylococcus aureus, Chiing co ihi la nhung ddi tugng nghien ciiu thu vi cho hudfng tim kidm cac hgp chdt co hoat tfnh khang Staphylococcus aureus khang methicillin (MRSA) va vancomycin (VRSA) Da phan lap va xac dinh dugc cac hgp chdt co hoat tfnh bao \t gan than rS Nghe xanh nhu furanodien, germacron, curdion, 13- hydroxygermacron va aerugidiol Furanodien va 13-hydroxygermacron cung la cac hgp chdt da dugc chiing td la co tac dung lie che' sir san sinh cua nito oxit (NO) Trong cac thur nghiem v^ hoat tfnh chdng oxy hoa (dgn gdc tu do) va gay dgc te' bao ddi vdi ba ddng te' bao ung thu ngudi la Hep-Gj, Fl va RD, ca curdion va zederon diu khong (hi hien cac hoat tfnh dang chu y Cac ke't qua ciia De tai da ddn den sir chuan bi cdng bd hai bai bao tren cac tap chf ciia Viet Nam Duac hpc va qudc te' Chemical and Pharmaceutical Bulletin Mgt sinh vien da tdt nghiep va ba sinh vien nam thur da hoan nghien ciiu khoa hgc khudn khd ciia de tai f Tinh hinh kinh phi cua de tai Tdng kinh phf dugc cdp: 15.000.000 ddng Cac khoan chi cila de tai da dugc phan bd nhu sau: Muc Npi dung chi So tien (dSng) Tyle (%) 109 110 114 119 Thanh toan djch vu cong cong Vat tu van phong Thue thiet bi cac loai (thue thie't bi ghi phd, thir hoat tfnh sinh hoc) Chi phi nghiep vu chuyen mon (Nguyen lidu, hoa ch^t, dung cu) Quan ly phi Tdng c6ng 600.000 873.000 6.310.000 4,0 5,8 42,1 6.617.000 44,1 600.000 4,0 15.000.000 100.0 CHU TRI Dt TAI (Ky va ghi ro ho ten) KHOA Q U A N L t (Ky va ghi ro ho ten) il 'iim Phd Chu nhiem Khoa Hoa hoc PGS TS Trdn Thi Nhir Mai TS Phan Minh Giang TRlTCfNG DAI HOC KHOA HOC Tl/NHlfiN PHOHI^UTRUONU PGSJS.^Aw^iu^am Outline Report a Project title Study on biologically active compounds from Vietnamese medicinal plants Code No.: Q T - - b Head of the project Dr Phan Minh Giang c Participating researchers PhD student Le Huyen Tram MSc Dao Qudc Hung BSc Nguyen Thi Tue d Objectives and main contents of the project • To carry out selection, taxonomic identification and collection of some interesting medicinal plants of Vietnam for the research • To perform stabilization of the plant materials, extraction and fractionation procedures for the preparation of biologically active extracts and fractions • To evaluate biological activities of the extracts and fractions To perform activity-guided fractionation of the extracts and fractions • To study isolation procedures of biologically active compounds using modem analytical and separation methods • To determine structures of the isolated compounds on the basis of modem physical methods • To evaluate biological (antioxidant, antibacterial, antifungal, and cytotoxic) activities of the isolated compounds • To examine contents of the biologically active compounds in the medicinal plants studied e Study results Curcuma aff aeruginosa Roxb., Zingiberaceae (Vietnamese name: Nghe xanh), a medicinal plant, which was recently discovered and widely cultivated for the use in the treatment of stomach ache and in the enhancement of digestion, was selected for the study The plant was taxonomically identified and collected for the study A selective extraction and fractionation procedure was established for the preparation of the biologically active n-hexan (3.14% yield on the basis of the dry material), ethyl acetate (0.6%) and /i-butanol (0.14%) extracts A suitable chromatographic isolation method was developed for the systematic chemical and biological study of the /z-hexan extract of the rhizomes of C aff aeruginosa By using this method the main constituents curdion and zederon were isolated in the yields of 0.1% and 0.05%, respectively, of the dry material This method will be suitable for the development of large-scale isolation of the biologically active curdion and zederon, as well as pharmaceutical preperations containing different groups of sesquiterpenoids The study of the volatile compounds of C aff aeruginosa determined in total 23 compounds (mainly sesquiterpenoids), of which P-elemene - an anticancer compound - was the main constituent of the sesquiterpene hydrocarbons, and curzerenone, curcumol, germacrone, and curdione were the main components of the oxygenated sesquiterpenoids, of which curzerenone was used as a clinical drug for cervix and skin carcinomas The study of the more polar compounds led to the isolation of fourteen pure compounds from the rhizomes of C aff aeruginosa, curdione (1), palmitic acid (2), zederone (3), y3-sitosterol (4), stigmasterol (5), 13-hydroxygermacrone (6), aeruginolactone (7), guaianediol (8), isoprocurcumenol (9), procurcumenol (10), aerugidiol (11), aeruginofuran (12), zedoarofuran (13), and aeruginone (14) The isolated compounds possess the structures of sesquiterpenoids, steroids, and fatty acid The sesquiterpenoids aeruginolactone (7), aeruginofuran (12) are new organic natural compounds, and aeruginone (14) was isolated from Nature for the first time The chemical structures of compounds 1-14 were determined by using modem spectroscopic methods (UV, IR, MS, ID NMR, and 2D NMR) NOED, NOESY, and CD spectra were used in the determination of the absolute stereostructures of the compounds isolated In the antibacterial and antifungal assays n-hexan, ethyl acetate, and n-butanol extracts showed inhibitory activity against Aspergillus niger, Candida albicans and Fusarium oxysporum at MICs of 25-200 (ag/ml Activity-guided fractionation led to the isolation of the bioactive compounds curdione and zederone - Curdione and zederone (MICs of 25 |ag/ml) can be developed into agents which were active against the blockage and breakdown of xylem of plants caused by Fusarium oxysporum - Curdione and zederone were demonstrated to be active against the human pathogenic fungi Aspergillus niger (MIC of 50 ^g/ml) and Candida albicans (MIC of 25 |ig/ml), respectively - Curdione (MIC of 50 ^g/ml), ethyl acetate (MIC of 200 ^ig/ml), and n-butanol (MIC of 50 fig/ml) extracts showed inhibitory effect against Staphylococcus aureus They can be selected for the study toward the discovery of active compounds against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant (VRSA) activities The hepatoprotective compounds were isolated and identified in the rhizomes of C aff aeruginosa such as furanodiene, germacrone, curdione, 13-hydroxygermacrone, and aerugidiol Furanodiene and 13-hydroxygermacrone were demonstrated to inhibit the nitric oxide (NO) production In the antioxidant (radical scavenging activity) and cytotoxic activity against the human cancer cell lines Hep-Gj, Fl, and RD bioassays curdione and zederone did not show noticeable effects The project resulted in the preparation of two publications in the national Vietnam Pharmaceutical Journal and the intemational Chemical and Pharmaceutical Bulletin One BSc student graduated and three 3'** year students completed the Research training in the framework of the project Muc luc Trang Muc luc cac chii vie't tat 1 Ldi md ddu 2 Ndi dung chfnh 2.1 Phuong phap nghien cihi 2.2 Phuong phap va thiet bi 2.3 Nguyen lieu thuc vat 2.4 Di^u che' cac phdn chiet tir than re Nghe xanh (C aff aeruginosa) 2.5 Phan tach phdn chiet n-hexan va phan lap cac sesquiterpenoid 2.6 Phuong phap thii hoat tinh sinh hoc 2.6.1 TTiir hoat tfnh khang khudn va khang ndm in vitro 2.6.2 Thijf hoat tfnh chdng oxy hoa in vitro 2.6.3 Hiir hoat tfnh gay ddc te' bao in vitro 2.7 Hdng sd vat ly va du kien phd cua cac hgp chdt dugc phan lap 2.8 Ket qua va Thao luan 11 2.8.1 Phan lap va xac dinh cdu tnic cac sesquiterpenoid tir Nghe xanh 11 (C aff aeruginosa, Zingiberaceae) 2.8.2 Danh gia cac hoat tfnh sinh hoc cua Nghe xanh 22 (C aff aeruginosa, Zingiberaceae) 3.2.1 Hoat tfnh khang khudn va khang ndm in vitro 22 3.2.2 Hoat tfnh chdng oxy hoa/n v/rr