bản tóm tắt tiếng anh nghiên cứu thành phần hóa học và hoạt tính sinh học của ba loài thuộc chi bách bộ (stemona) mọc ở lào

1 INTRODUCTION 1. Proposal So far, we do not have medicine for cancer and AIDS as well as bacteria which have good resistance to antimicrobial agents. Only few effective drugs are available in the market, but they are too expensive. In addition, some of them are also harmful to normal cells. Therefore, many scientists have been carrying out their research to looking for new drugs. Some natural products, which have strong antimicrobial, anticancer, antiimflammatory activity, are good sources for developing new drugs. The Stemonaceae family widely grows in Southeast Asian countries such as Vietnam, Laos, Thailand Stemona plants have been used for anticough, anti- helminths, anti cancer remedies in the folk medicines. So far, chemical constituents of Laos Stemona sp. are unknown. Only some studies have been done on their classification and their biological activity. Thus, I select the title for my PhD thesis as “Studies on the chemical constituents and the biological activity of three Stemona species in Laos”. 2. Subjects of the research: Studying on chemical constituents and biological activity of the roots of Stemona plants, collected in Laos, including S. cochichinensis, S. pierrei and S. tuberosa plants. 3. New results from the research 1. In the first time from the roots of S.cochinchinensis plants, collected in Savannakhet province (Laos) were isolated and structurally identified 12 metabolites, including one new: 1-(3’-hydroxy-2’-methoxyphenyl)-2-(3’’- hydroxy-5-methoxy-2,4-dimethylphenyl)ethane (K8) and 11 known compounds: β-sitosterol (K1), Sesamin (K2), Methyl cis-4-hydroxycinnamate (K3), Methyl trans-4-hydroxycinnamate (K4), 5’-methoxy-4’-hydroxy-1’-(1-methoxyprop-2- enyl)benzen (K5), Methyl 4’-hydroxy-3’-methoxycinnamate (K6), Stemanthrene C (K7), Stemanthrene A (K9), Pinoresinol (K10), Maistemonine (K11) and Isomaistemonine (K12). 2. From the roots of S. pierrei plants, seven compounds have been isolated and structurall identified as Sesamin (K2), Stemobenzofuran A (K13, new), Stemanthrene B (K14), Syrigaresinol (K15), Benzoic acid (K16), p-Anisic acid (K17), methyl 1’-hydroxy-1’-(4-hydroxy-3-methoxyphenyl)propylate (K18, new). 3. From the roots of S. tuberosa plants, six compounds have been isolated and structurall identified as: Stemophenanthren A-D (K19-K22), Stemostemanthren E (K23) and Stemobenzofuran B (K24). All six compounds are new ones. 4. Eight compounds were tested on a cytotoxicity towards for cancer lines, such as KB, Hep-G2, Lu, MCF7. The results showed that K21, K22 and K23 obtained good cytotoxicity towards 4 cancer cell lines with IC 50 values from 4.49 to 63.65 µg/ml. Five compounds ( K7, K8, K9, K19 and K20) have a middle cytotoxicity towards KB cell line (IC 50 values from 20.67 to 82.30 µg/ml). 4. Structure of PhD thesis PhD thesis contained 140 pages with 31 tables, 71 pictures, 101 references, including Introduction (2 pages), Chapter 1: Overview of reseach (25 pages), Chapter 2 2: Materials and Methods (6 pages), Chapter 3: Experiments (20 pages), Chapter 4: Results and discussion (77 pages), Conclusion (2 page). CHAPTER 1: OVERVIEW OF RESEARCH 1.1. INTRODUCTION FOR STEMONACEAE FAMILY 1.1.1. Classification of Stemonaceae Stemonaceae is belonging to Pandanales order. It is a family of monocotyledonous flowering plants (Monocosts) and Angiosperms. It is devided into four genera, including Croomia.Torr, Stemona. Lour, Stichoneuron Hook. F and Pentastemona Steenis. The genus Stemona is biggest with about 30 species. These plants grow everywhere, but concentrated in China, Vietnam, Laos, Cambodia and Thailand. Below are the classification of Stemonaceae. Table 1.1: Classification of the genus Croomia based on WCSP (Word Checklist of Selected Plant family) up to 1/1/2011 1. Croomia heterosepala (Baker) Okuyama 2. Croomia japonica Miq. 3. Croomia pauciflora (Nutt.) Torr. Table 1.2: Classification of the genus Stemona Lour based on WCSP (Word Checklist of Selected Plant family) up to 1/1/2011 1. Stemona angusta I.R.H.Telford 2. Stemona aphylla Craib 3. Stemona australiana (Benth.) C.H.Wright 4. Stemona burkillii Prain 5. Stemona cochinchinensis Gagnep. 6. Stemona collinsiae Craib 7. Stemona curtisii Hook.f. 8. Stemona griffithiana Kurz 9. Stemona hutanguriana Chuakul 10. Stemona japonica (blume) Miq. 11. Stemona javanica (Kunth) Engl. 12. Stemona kerrii Prain 13. Stemona kurzii Prain 14. Stemona lucida (R.Br.) Duyfjes 15. Stemona mairei (H.Lév.) K.Krause 16. Stemona parviflora C.H.Wright 17. Stemona phyllantha Gagnep 18. Stemona pierrei Gagnep 19. Stemona prostrata I.R.H.Telford 20. Stemona sessilifolia (Miq.) Miq. 21. Stemona squamigera Gagnep 22. Stemona tuberosa Lour. 23. Stemona tuberosa var. moluccana (Blume) ined. 24. Stemona tuberosa var. tuberosa Table 1.3: Classification of the genus Stichoneuron Hook. F based on WCSP (Word Checklist of Selected Plant family) up to 1/1/2011 1. Stichoneuron caudatum Ridl. 2. Stichoneuron membranaceum Hook.f. Table 1.4: Classification of the genus Pentastemona based on WCSP (Word Checklist of Selected Plant family) up to 1/1/2011 1. Pentastemona egregia (Schott) Steenis 3 2. Pentastemona sumatrana Steenis 1.1.2. Introduction to the genus Stemona The genus Stemona has about 30 species. In Vietnam, six species belonging to this genus have been collected and identified. They are Stemona cochinchinensis Gagnep, Stemona collinsiae Craib, Stemona pierrei Gagnep, Stemona phyllantha Gagnep, Stemona saxorum Gagnep and Stemona tuberosa. Lour. Laos has 11 species of this genus. They are S.tuberosa., S. phyllantha Gagnep., S. squamigera Gagnep., S. cochinchinensis Gagnep., S. pierrei Gagnep., S. saxorum Gagnep., S. collinsae Craib., S. aphylla Craib., S. burkillii Prain., S. griffithiana Kurz and S. kerrii Craib. They distribute in the mountains in the north, delta in the middle and the south of Laos. Stemona genus has several trivial names such Samzip, Mưnang, Ìnhạng (Lao and Thai). They have stems or clim in moist area, such as in south east Asia. Each species has typical characters as below:  Stemona sp have stems, mostly grow in moist area (near rivers, streams or lacks). Normally, they are in delta.  Stemona sp as climb plants mainly are available in forests. They are about 1.4 m length. 1.1.3. Usage Based on traditional medicines, Stemona sp is sweet, pungent. They have been used as anticough and anti-helminths remedies. People in Laos use all plant parts to make nematocidal medicines, insecticide. 1.2. CHEMICAL CONSTITUENTS OF STEMONA SP. Stemona sp. is a rich source of secondary metabolites such as alkaloids, stilbenoids, stemanthrenes, stemofurans. Alkaloids isolated from Stemona sp have various carbon skeletons. However, they have the same pehydroazaazulen core. So far, more than 90 alkaloids have been purified from Stemona sp. They are devided into five skeleton as following: N O O N N O N O O O N O O N O O V IV III II I Figure 1.1. Five alkaloid skeletons from Stemona sp CHAPTER 2: MATERIALS AND METHODS 4 2.1. MATERIALS 2.1.1. Plant materials Stemona cochinchinensis was collected in Savannakhet province, Laos in October 2010. It was identified by MSc. Nguyen The Anh, Institute of Chemistry, Vietnamese Academy of Science and Technology. A voucher specimen (KPN- 01.2010) was deposited at the Hebarium of Faculty of Chemistry, Hanoi National University of Education, Hanoi Figure 2.1: Stemona cochinchinensis S. peirrei were collected in Savannakhet Province, Southern Laos, in June 2011 by Mr. Khamko V. A. and identified by MSc. Nguyen The Anh, Institute of Chemistry, Vietnamese Academy of Science and Technology. A voucher specimen (KPN-02.2011) was deposited at the Hebarium of Faculty of Chemistry, Hanoi National University of Education, Hanoi. Figure 2.2: Stemona peirrei Stemona tuberosa was collected in Attapu Province, Southern Laos, in July 2011 by one of the authors (Mr. Khamko V.A) and identified by Dr.Nguyen Thi Lien (Faculty of Biology, HNUE, Hanoi) as Stemona tuberosa Lour. A voucher specimen (KPN-03.2011) was deposited at the Hebarium of Faculty of Chemistry, HNUE, Hanoi, Vietnam. Figure 2.3: Stemona tuberosa 5 2.1.2. Chemicals: In this PhD thesis all chemicals (MeOH, n-hexane, ethyl acetate…) are pure for analytic purpose. 2.2. METHODS 2.2.1. Extraction and Isolation Selected plants were treated by the same methods for researching natural products. Compounds were isolated by several chromatographic methods such as thin layer chromatography (TLC), column chromatograpgy (CC), reversed phase column chromatography (RP-18), preparative HPLC (prep. HPLC). 2.2.2. Structural elucidation Their structures will be elucidated by spectroscopic analysis such as Infrared spectrum (IR), Ultra violet spectrum (UV-Vis), Mass spectrum (MS) and Nuclear Magnetic Resonance (NMR) including 2D NMR. In particular cases, chemical reactions might be employed to get crystals for X-ray crystallographic analysis. IR spectrum was recorded on a JASCO FT/IR-5300, at Department of physical chemistry, Faculty of Chemistry, Hanoi University of Education. Uv-vis spectrum was measured on a Shimadzu UV-1650PC instrument in MeOH at Department of physical chemistry, Faculty of Chemistry, Hanoi University of Education. FT-ICR-MS spectrum was recoded on a Variance 320-MS instrument. 1D and 2D NMR spectra were recorded on a Brucker Avance 500 MHz with TMS (trimethylsilan) as standard at Institute of Chemistry, VAST. Some compounds were measured their MS on Brucker Apex III (FT-ICR-MS) at Leibniz Institute of Plant Biochemistry in Germany. CHAPTER 3: EXPERIMENTS 3.1. Roots of S. cochinchinensis The powder of dried roots of Stemona cochinchinensis (3.0 kg) was extracted as shown in scheme 3.1. Scheme 3.1. Isolation process of Stemona cochinchinensis After running TLC with different solvent systems, we found a suitable solvent systems for column chromatography. The crude extract was purified as follow: Scheme 3.2. Separation of compounds from n-hexane extract of S. cochinchinensis Dried sample MeOH:H 2 O (80:20) Extract EtOAc extract n-hexane extract n-buthanol extract n-hexane, EtOAc and n-buthanol 6 Scheme 3.3. Separation of compounds from EtOAc extract of S. cochinchinensis 3.2. Roots of S. pierrei Scheme 3.4. Separation of compounds from n-hexane extract of S. pierrei SKC / n:E /8:1 n-Hexan extract (CH: 5,76g) CH1(K1) 1,05g CH2B3(K4) 7mg CH2B2(K3) 4mg CH2B1(K2) 9mg CH2 1,3g SKC / n: E/4:1 CH2B 78mg CH2C 63mg CH2C2(K5) 9mg HPLC/ n:E /1:1 HPLC/ n:E /2:1 CE2 165mg SKC / n: E/1:1 CE2B 77mg CE2B1 (K10) 7mg CE2B2 (K11) 9mg CE2B3 (K12) 5mg HPLC / n: E/3:7 EtOAc extract (CE:7.67g) SKC/n:E/4:1 CE1 0,61g CE1D1 (K9) 17mg CE1C1 (K8) 4mg SPD /C:M/1:1 HPLC/ n: E/3:2:1 SKC / n: E/ 3:1 CE1A (K6) 88mg CE1B (K7) 83mg CE1C 181mg CE1D 72mg SKC/ n: E/4:1 SKC/ n:E /8:1 n-Hexane extract PH (3.17g) PH3C 66mg SKC/ n: E/3:1 PH3 322mg PH3C1 (K13) 4mg HPLC n: E/3:2 PH1 414mg PH1B1 (K2) 5mg PH1B 56mg HPLC/ n: E/3:1 7 Scheme 3.5. Separation of compounds from EtOAc extract of S. pierrei 3.3. Roots of S. tuberosa Scheme 2.6. Separation of compounds from n-hexane extract of S. tuberosa 3.4. Bioassays Some isolated compounds were tested their antimicrobial, antifungal, cytotoxicity and their results are described in tables 4.18. CHAPTER 4: RESULTS AND DISCUSSION 4.1. DETERMINATION OF STRUCTURES OF ISOLATED COMPOUNDS FROM S. COCHICHINENSIS PE1 657mg PE3 325mg PE4 233mg SKC n:E/5:1 SKC/n:E/3:1 SKC/n:E/2:1 HPLC/n:E/1:1 HPLC/n:E/1:1 HPLC/n:E/2:1 HPLC/n:E/3:2 PE4A 43mg PE3B 53mg PE1B 83mg PE1D 69mg PE4A2 (K18) 5mg PE3B2 (K17) 5mg PE3B1 (K16) 3mg PE1D2 (K12) 6mg PE1D3 (K15) 7mg PE1D1 (K11) 7mg PE1B1 (K14) 7mg PE1B2 (K7) 5mg EtOAc ext. (PE: 6.67g) SKC n:E/ 6:1 TH5 0.78g SKC/n:E/3:2 HPLC/n:E/1:1 TH5B 57mg TH5B4 (K24) 9mg TH5B1 3mg TH2 1.27g SKC/ n:E/2:1 HPLC/n:E/3:2 TH2C1 (K19) 4mg TH2C2 (K20) 8mg TH2C 97mg TH2C4 (K22) 12mg TH2C5 (K23) 5mg TH2C3 (K21) 6mg n-Hexane ext. (TH:14.76g) SKC/ n:E/ 10:1 8 4.1.1. Chemical constituents of S. cochichinensis Crude extract of S. Cochichinensis were separated as scheme 3.1 and followed by schemes 3.2, 3.3 to give 12 compounds.  K1 (β-sitosterol): This compound K1 was purified by recrystallization as a white crystals, well- dissolved in chloroform and n-hexane. It was determined as β-sitosterol, one of the most popular sterols in plants. Figure 4.1: 1 H NMR spectrum of K1 in CDCl 3  K2(Sesamin): NMR spectra and data of K2 are described in figure 4.2 and table 4.1. Figure 4.2: 1 H and 13 C NMR spectra of K2 in CDCl 3 K2 is determined to be Sesamin, previously isolated from Lindera obtusiloba. Table 4.1: 1 H and 13 C NMR spectral data of K2 N o 1 H NMR, δ (ppm), J(Hz) 13 C NMR(δ ppm) 1, 1’ - 135.1 2, 2’ 6.84, d, J=2.0 Hz 106.5 3, 3’ - 148.0 4, 4’ - 147.1 5, 5’ 6.80, m 108.2 6, 6’ 6.80, m 119.4 7, 7’ 4.72, d, J= 4.5 Hz 85.8 8, 8’ 3.05, m 54.4 9, 9’ 3.87, m, 4.23, m 71.7 10, 10’ 5.95, s 101.1 O O H H 7 9' 8' 7' 9 1 O O O O 1' 2' 8 2 6 6' 4' 4 9  K3 and K4 (Methyl cis- and trans-4-hydroxycinnamates) Their NMR spectra and data are shown in figures 4.3., 4.4 and table 4.2. HO H 3 COOC H H 1 3 1' 2' 4' Figure 4.3. 1 H NMR spectrum of K3 CDCl 3 HO H H COOCH 3 1 2 3 1' 2' 4' Figure 4.4. 1 H NMR spectrum of K4 CDCl 3 Table 4.2. 1 H and 13 C NMR spectral data of K3 and K4 No. 1 H NMR δ (ppm), J (Hz) 13 C NMR (δ ppm) HMBC K3 K4 K3 K4 1 - - 165.8 167.9 - 2 5.75, d, J=12.5 6.30, d, J= 16.0 114.1 115.3 C-1/C-3 3 6.80, d, J=12.5 7.64, d, J= 16.0 114.3 115.9 C-2 1’ - - 124.8 127.3 - 2’ 7.63 d, J=8.5 7.43, d, J= 8.5 132.5 129.9 C-3’/C-4’ 3’ 6.78 d, J=8.5 6.85, d, J= 8.5 142.8 144.6 C-2’/C-4’ 4’ - - 157.9 157.7 - 5’ 6.78 d, J=8.5 6.85, d, J= 8.5 142.8 144.6 C-4’/C-6’ 6’ 7.63 d, J=8.5 7.43, d, J= 8.5 132.5 129.9 C-4’/C-5’ 1-OMe 3.80 s 3.80, s 50.1 51.7 -  K5[5’-Methoxy-4’-hydroxy-1’-(1-methoxyprop-2-enyl)benzene] NMR spectra of K5 are shown in figure 4.5 and table 4.3. 10 Figure 4.5. 1 H NMR spectrum of K5 CDCl 3  K6 (Methyl 4’-hydroxy-3’-methoxycinnamate) NMR spectra of K6 are in figure 4.6 and table 4.3. Figure 4.6. 1 H and 13 C NMR spectra of K6 in CDCl 3 Table 4.3: NMR spectral data of K5 and K6 No. 1 H NMR δ (ppm), J (Hz) 13 C NMR δ (ppm) K5 K6 K5 K6 1 4.07, d, J=6 - 73.2 167.7 2 6.14, m 6.28, d, J= 16.0 126.6 115.2 3 6.52, d, J=16.5 7.62, d, J= 16.0 132.6 144.9 1’ - - 129.4 126.9 2’ 6.88, m 7.07, m 120.4 123.0 3’ 6.86, d, J=8.5 - 114.4 146.8 4’ - - 145.6 148.0 5’ - 6.92, d, J= 8.5 146.6 114.7 6’ 6.93, s 7.02, m 108.4 109.4 MeO-1 3.80, s 3.79, s 57.9 51.6 MeO-3’ - 3.92, s - 55.9 MeO-5’ 3.90, s - 55.9 - HO-4’ 5.50, s 5.90, s - -  K7 (Stemanthrene C) and K9 (Stemanthrene A) NMR spectra of K7 are in figure 4.7 and table 4.4. HO COOCH 3 H 3 CO 1 2 3 1' 3' 5' H 3 CO HO CH 2 OCH 3 1 2 3 1' 3' 5' [...]... 32.97 39.75 >128 59.78 23.52 16.66 CONCLUSIONS 1 2 3 4 5 This is the first time the roots of S cochinchinensis, S pierrei (collected in Savannakhet province, Laos), S tuberosa (collected in Attapu province, Laos) were studied on chemical constituents and biological activities From the roots of S cochinchinensis plants, twelve compounds have been isolated and structurall identified as β-sitosterol (K1),... Dien (2012) A cytotoxic stilbenoid and 4-hydroxycinnamates from Stemona cochinchinensis plants, growing in Savannakhet Province (Laos) J of Sciences of HNUE, Vol.57, No.7, pp.3-9 2 Vonganatha Khamko, Pham Huu Dien, Jurgen Schmidt, Dang Ngoc Quang (2012) Cytotoxic and anti-microbial constituents from the roots of Stemona cochinchinensis in Laos J of Chemistry, VAST, Vietnam, Vol 50, No.4A, pp.203-206... [M+Na]+ at m/z 135 Its molecular formula is C18H20O4 K8 is identified as 1-(3hydroxy-2-methoxyphenyl)-2-(3’-hydroxy-5’-methoxy-2’,4’-dimethylphenyl)etan Compound K8 is a new stilbenoid isolated from S cochinchinensis in Laos OH OCH3 3 6 CH3 1 OH 2" 1" 1' 4' CH3 OCH3 Figure 4.9 1H and 13C NMR spectra of K8 in CDCl3 Table 4.5: 1H and 13C NMR spectral data of K8 1 13 No H NMR δ (ppm), J C NMR δ HMBC (Hz) (ppm) . Roots of S. cochinchinensis The powder of dried roots of Stemona cochinchinensis (3.0 kg) was extracted as shown in scheme 3.1. Scheme 3.1. Isolation process of Stemona cochinchinensis . collected in Laos, including S. cochichinensis, S. pierrei and S. tuberosa plants. 3. New results from the research 1. In the first time from the roots of S.cochinchinensis plants, collected in. ext. (TH:14.76g) SKC/ n:E/ 10:1 8 4.1.1. Chemical constituents of S. cochichinensis Crude extract of S. Cochichinensis were separated as scheme 3.1 and followed by schemes 3.2, 3.3 to