Study on the isolation and biological activity of natural active compounds from plants and endophytes

NGUYEN NGOC HIEU STUDY ON THE ISOLATION AND BIOLOGICAL ACTIVITY OF NATURAL ACTIVE COMPOUNDS FROM PLANTS AND ENDOPHYTES Scientific Field: Organic Chemistry Classification Code: 62 44 01 14 DISSERTATION SUMMARY HA NOI - 2019 MINISTERY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY The dissertation was completed at: Institute of Chemistry Vietnam Academy of Science and Technology Scientific Supervisors: 1. Dr. Duong Ngoc Tu Institute of Chemistry - Vietnam Academy of Science and Technology 2. Ass. Prof. Dr. Duong Anh Tuan Institute of Chemistry - Vietnam Academy of Science and Technology 1 st Reviewer: ............................................................................................................. .............................................................................................................. .............................................................................................................. 2 nd Reviewer: ............................................................................................................ .............................................................................................................. .............................................................................................................. 3 rd Reviewer: ............................................................................................................. .............................................................................................................. .............................................................................................................. The dissertation will be defended at Institute of Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay District, Ha Noi City. At .. hour.. date.. month ..2019 . The dissertation can be found in National Library of Vietnam and the library of Institute of Chemistry, Vietnam Academy of Science and Technology. 1 I. INTRODUCTION 1. Background Vietnam is still world famous for its biodiversity potentials, with over 12,000 species of higher plants, excluding fungi, algae and mosses. Many species are endemic to Vietnam. From the treasure of folk experience, we have had a lot of experience using and ingeniously combining these diverse plant materials into very precious, special and special folk remedies. In the treatment of diseases, high health of human, protecting crops, eradicating pests, insects, harmful animals ..... With the current level of scientific and technological development, it is necessary to continue continue to research, research, select from folk experiences in combination with the support of modern technology and equipment to create new products, bringing the value of using plant resources in Vietnam to reach High new, more valuable, more efficient, highly appreciated both in terms of science and technology as well as use value. Plant endogenous fungi (endophytes) are currently being studied extensively and extensively in the world and are expected to be an unexplored resource for biotechnology and pharmaceuticals. Recent statistical results, with an estimated 51% of active compounds isolated from endophytes are new compounds, have shown great potential for research and application of the endophyte. Continuing the international cooperation program between the Institute of Chemistry (Vietnam Academy of Science and Technology) and the Institute of Biopharmaceuticals and Biotechnology (Heirich-Heine General University Duesseldorf, Germany) on the study of flora Vietnam to screen and detect natural bioactive compounds, potentially used to produce insecticides and fungal pathogens of plants, as well as expand to target new research subjects in the world as well as in Vietnam is NSTV, we propose the dissertation: "Study on the isolation and biological activity of natural active compounds from plants and endophytes". 2. Objectives and aims of the dissertation 2 The research object is 4 species of plants including Aglaia duperreana Pierre, Aglaia oligophylla Miq., Piper betle L. and Curcuma longa L. and those endophytes. The aims of the dissertation are: 1. Extracting and determining the structure of organic compounds of four plant species with potential for insecticide and fungal diseases. 2. Isolating endogenous fungi from plant samples, extracting and determining the structure of component organic compounds. 3. Testing of insecticidal and fungal activity of extracts and component organic compounds. 3. New contributions of the dissertation 3.1 For the first time in Vietnam, the relationship between plants and plant endogenous fungi on Aglaia duperreana Pierre, Aglaia oligophylla Miq., Piper betle L. and Curcuma longa L species in terms of chemical composition and biological activity has been studied in a systematic way. There were differences between the chemical composition and biological activity of plant extracts and endogenous fungi. This confirms the symbiotic and supportive relationship between host plants and endogenous fungi, as well as the potential of searching from endogenous plant fungi of alternative active ingredients to produce probiotics. 3.2 A total of 19 compounds were isolated and structurally determined including 7 compounds from A. duperreana Pierre and A. oligophylla Miq. with 6 known rocaglamide compounds (A, I, W, AB, J, rocaglaol) and 1 new compound (rocaglamide AY), 2 compounds known ar-tumeron, curcumin from C. longa L., 3 compounds known eugenol, chavicol, 4-Allylpyrocatechol from P. betle L., 2 known scopararane C compounds, diaporthein B from A. duperreana Pierre endogenous fungi (M. hawaiiensis), 4 known compounds β-sitosterol, 4R, 4aS, 9aR) -1,9a-dihydronidulalin A, 4S, 4aR, 9aR) -4a-carbomethoxy-1,4,4a, 9a- tetrahydro-4,8-dihydroxy-6-methylxanthone and (24R) -methylcholesta-7.22 - diene-3β, 5α, 6β-triol from endogenous fungi of Golden Turmeric (F. oxysporum); and ergosterol from the P. betle L. endogenous (F. solani) and identified 12 fatty 3 acids from endogenous fungi of Golden Turmeric (F. oxysporum) by the GC-MS data. 3.3 A total of 9 plant endogenous fungus have been isolated and identified. These are the first announcements about the genome of endogenous fungal strains on the Aglaia duperreana Pierre, Piper betle L. and Curcuma longa L. plants in Vietnam. 3.4 Extracts of leaves and bark of the Aglaia duperreana Pierre express 100% activity to inhibit to the growth of the Spodoptetra litura. The extracted parts of Piper betle L. and Curcuma longa L endogenous and curcumin essence inhibit 100% growth of the fungi causing the gray rot disease (Botrytis cinera). For the first time, the Golden turmeric and curcumin are systematically studied to be used as raw materials for processing biological fungicides. 4. The layout of the dissertation The dissertation consists of 141 pages and contains 159 references. The layout of the dissertation includes the following sections: Preface (4 pages), Chapter 1: Overview (32 pages), Chapter 2: Objects and methods (13 pages), Chapter 3: Experimentals (19 pages), Chapter 4: Results and discussion (44 pages), Conclusions (1 pages), Recommendations (1 page), Publications (1 page), References (16 pages), and Appendix (43 pages). 4 II. DISSERTATION CONTENTS Preface This part discusses the background, the scientific and practical significance, and the objectives of the research project. Chapter 1. Literature review 1.1. The fungi, insects harmful and the role of plant protection drugs 1.2. The trend of replacing chemical pesticides with bio-pesticides 1.3. Biological pesticides extracted from plant materials 1.4. Plant endogenous fungi and the prospect of searching for new generation biologically active substances 1.5. Introduction of the species of Aglaia duperreana Pierre, Aglaia oligophylla Miq., Piper betle L. and Curcuma longa L. Chapter 2. Methods 2.1. Isolation and purification methods Compounds were isolated and purified by using a combination of various chromatographic methods including thin-layer chromatography (TLC), column chromatography (CC) on different stationary phases such as Silicagel and Sephadex. 2.2. Methods for the determination of the chemical structures The chemical structures of isolated compounds were elucidated by a combination of physical parameters (melting point), modern spectroscopic methods (IR, UV, CD, MS, 1D-NMR, and 2D-NMR) with chemical conversion, and by comparing with literature data. 2.3. Methods for isolation and biomass of the endogenous fungi 2.4. Method for screening insecticidal and fungal activity Chapter 3. EXPERIMENTALS 3.1. Result of isolation of endogenous fungi from plant samples + Four (04) endogenous fungal strains were isolated from Curcuma longa L.: Fusarium solani, Fusarium sp., Trichoderma atroviride and Fusarium oxysporum. + Three (03) endogenous fungal strains were isolated from Aglaia duperreana Pierre: Colletotrichum gloeosporioides, Colletotrichum crassipes and Microdiplodia hawaiiensis. + Two (02) endogenous fungal strains isolated from the Piper betle L. are Colletotrichum sp. and Fusarium solani. 3.2. Result of isolation of plant compounds 3.2.1. Isolation of compounds from the Aglaia duperreana’s bark 5 3.2.1.1. Processing plant samples Bark dried samples (3kg) was extracted three times with methanol in an ultrasonic device at room temperature. Translate the total amount of distilled solvent in the pressure drop, the temperature of 45 oC obtained 115g of methanol residue. The residue of methanol is added with water and extracted with an increasing solvent of n-hexane and ethyl acetate. After removal of the solvent, obtain the residue of n-hexane (25g), ethyl acetate (20g) and methanol (65g), respectively. 3.2.1.2. Isolation of compounds from ethyl acetate residue Ethyl acetate residue (AD.E, 20g) is separated by column chromatography VLC with the solvent elution of n-hexane gradient: EtOAc: MeOH (4: 2: 1 to 0: 1: 1 solvent) 8 segments denoted from ADE1 to ADE8. Diagram 3.2.1 Diagram to isolate compounds from Aglaia duperreana’s bark Run the chromatographic column of ADE3 segment (5.4 g) on silica gel (40- 63µm) with the gradient CH2Cl2-MeOH solvent system (from 100: 0 to 0: 100) to obtain 9 segments, symbols is ADE3.1-ADE3.9. Segment from ADE3.1 (1.29 g) run column CC with solvent CH2Cl2: isopropanol obtained 9 segments (ADE3.1.1 to ADE3.1.9). Collect segments ADE3.1.4-ADE.1.1.7 (412mg) and run sephadex column with methanol solvent, collecting 36 small segments. Use TLC and HPLC to collect tubes 1-36 to obtain 6 clean substances obtained in the form of amorphous white powder. The process of separating compounds from the bark of Aglaia dupperreana Pierre is described in the diagram 3.2.1.  Compound 1: Compound 1 (3.9 mg) was isolated from the bark of the Aglaia dupperreana in white amorphous form, [α] 20D-90.5 (c, 0.25, CHCl3). UV (MeOH) λmax 219.7 and 273.0 nm. ESI-MS spectrometer (positive mode): m/z 561,1 (M+H)+, 528,4 (M+Na)+ 1H-NMR (MeOD): δ ppm 4,95 (d, J = 6.9 Hz, H-1), 4,11 (dd, J = 6.9 Hz, 13,8 Hz , H-2), 4,36 (d, J =13,8 Hz, H-3), 6,30 (d, J =1,9Hz, H-5), 6,17 (d, J =1,9 6 Hz, H-7), 7,12 (d, J=8,8 Hz, H-2’), 6,64 (d, J=8,8 Hz, H-3’), 6,64 (d, J =8,8 Hz, H-5’), 7,12 (d, J = 8,8 Hz, H-6’), 6,86 (m, H-2”), 6,98 (m, H-3”), 6,98 (m, H-4”), 6,98 (m, H-5”), 6,86 (m, H-6”), 3,81 (s, OMe-6), 3,84 (s, OMe-8), 3,66 (s, OMe-4’), 3,34 (s) & 2,86 (s) NMe.  Compound 2: Compound 2 (3,8 mg) was isolated from the bark of the Aglaia dupperreana in white amorphous form, [α]20D-80 (c, 0.45, CHCl3). UV (MeOH) λmax 209 and 279 nm. ESI-MS spectrometer (positive mode): m/z 564,1 (M+H)+, 586,4 (M+Na)+ 1H-NMR (MeOD): δ ppm 6,03 (d, J = 5,0 Hz, H1), 4,29 (dd, J = 5,0 Hz, 14,5 Hz , H2), 4,29 (d, J =14,5 Hz, H3), 6,26 (d, J =1,9 Hz, H5), 6,11(d, J =1,9 Hz, H7), 6,78 (d, J=1,9 Hz, H-2’), 6,62 (d, J =8,2 Hz, H-5’), 6,70 (d, J = 6,9 Hz, H-6’), 7,02 (m, H-2”), 6,98 (m, H-3”), 6,98 (m, H-4”), 6,98 (m, H-5”), 7,02 (m, H-6”), 3,81 (s, OMe-6), 3,73 (s, OMe-8), 3,71 (s, OMe-4’), 3,37 (s) & 2,79 (s) NMe, 1,81 (s, OCOCH3) • Compound 3: Compound 3 (2,1 mg) was isolated from the bark of the Aglaia dupperreana in white amorphous form, [α]20D-55,0 (c, 0.45, CHCl3). UV (MeOH) λmax 210 and 272,5 nm. ESI-MS spectrometer (positive mode): m/z 534,1 (M+H)+, 556,4 (M+Na)+ 1H-NMR (MeOD): δ ppm 5,99 (d, J = 6,3 Hz, H1), 3,94 (dd, J = 5,9 Hz, 14,5 Hz , H2), 4,19 (d, J =14,5 Hz, H3), 6,26 (d, J =1,9 Hz, H5), 6,12 (d, J =1,9 Hz, H7), 7,17 (d, J=8,8 Hz, H-2’), 6,61 (d, J =8,8 Hz, H-3’), 6,61 (d, J = 8,8 Hz, H-5’), 7,17 (d, J=8,8 Hz, H-6’), 6,91 (m, H-2’), 7,00 (m, H-3”), 7,00 (m, H-4”), 7,00 (m, H- 5”), 6,91 (m, H-6”), 3,74 (s, OMe-6), 3,81 (s, OMe-8), 3,65 (s, OMe-4’), 2,57 (s, NMe), 1,84 (s, OCOCH3) . • Compound 4: Compound 4 (7,2 mg) was isolated from the bark of the Aglaia dupperreana in white amorphous form, [α]20D-110,0 (c, 0.45, CHCl3). UV (MeOH) λmax 210,4 and 272,6 nm. ESI-MS spectrometer (positive mode): m/z 548,2 (M+H)+, 570,4 (M+Na)+ 1H-NMR (MeOD): δ ppm 5,95 (m, H1), 4,21 (m, H2), 4,21 (m, H3), 6,18 (d, J =1,9 Hz, H5), 6,03 (d, J =1,9 Hz, H7), 7,08 (d, J=8,8 Hz, H-2’), 6,54 (d, J =8,8 Hz, H-3’), 6,54 (d, J = 8,8 Hz, H-5’), 7,08 (d, J=8,8 Hz, H-6’), 6,80 (m, H-2”), 6,92 (m, H-3”), 6,92 (m, H-4”), 6,92 (m, H-5”), 6,80 (m, H-6”), 3,64 (s, OMe-6), 3,72 (s, OMe-8), 3,56 (s, OMe-4’), 3,27 (s) & 2,69 (s) NMe, 1,71 (s, OCOCH3) . • Compound 5: 7 Compound 5 (1,9 mg) was isolated from the bark of the Aglaia dupperreana in white amorphous form, [α]20D-41,1 (c, 0.22, CHCl3). UV (MeOH) λmax 211,3 and 278,7 nm. ESI-MS spectrometer (positive mode): m/z 509,0 (M+H)+, 531,2 (M+Na)+ 1H-NMR (MeOD): δ ppm 5,00 (d, J =5,7 Hz, H1), 3,96 (dd, J =5,7 Hz & 13,9 Hz, H2), 4,21 (d, J = 13,9, H3), 6,27 (d, J =1,9 Hz, H5), 6,15 (d, J =1,9 Hz, H7), 6,70 (d, J=1,9 Hz, H-2’), 6,64 (d, J = 8,8 Hz, H-5’), 6,64 (d, J=8,8 Hz, H-6’), 6,91 (m, H-2”), 7,00 (m, H-3”), 7,00 (m, H-4”), 7,00 (m, H-5”), 6,91 (m, H-6”), 3,81 (s, OMe- 6), 3,82 (s, OMe-8), 3,67 (s, OMe-4’), 3,61 (s, OCOCH3) . • Compound 6: Compound 6 (10 mg) was isolated from the bark of the Aglaia dupperreana in white amorphous form, [α]20D-125 (c, 0.48, CHCl3). UV (MeOH) λmax 212,8 and 272,3 nm. ESI-MS spectrometer (positive mode): m/z 457,10 (M+H)+, 890,9 (2M+Na)+ 1H-NMR (MeOD): δ ppm 4,69 (d, J =5,5 Hz, H1), 2,80 (ddd, J =6,3 Hz & 13,5 Hz, 14, 0 Hz, H-2α), 2,06 (ddd, J =1,1 Hz & 6,2 Hz, 11,8 Hz, H-2β) 3,89 (dd, J = 13,5 & 14,0 Hz, H3), 6,28 (d, J =1,9 Hz, H5), 6,17 (d, J =1,9 Hz, H7), 7,10 (d, J=8,8 Hz, H-2’), 6,61 (d, J = 8,8 Hz, H-3’), 6,61 (d, J=8,8 Hz, H-5’), 7,10 (d, J = 8,8 Hz, H-6’), 7,00 (m, H-2”), 7,00 (m, H-3”), 7,00 (m, H-4”), 7,00 (m, H-5”), 7,00 (m, H-6”), 3,87 (s, OMe-6), 3,85 (s, OMe-8), 3,81 (s, OMe-4’). 3.2.2. Isolation of compounds from leaves of Aglaia oligophylla 3.2.2.1. Processing plant samples The leaf sample of Aglaia oligophylla (3kg) was extracted 3 times with methanol in the ultrasonic device at room temperature. Translate the total amount of distillate solvent collected under reduced pressure, temperature 45 ° C, obtained 100g residue of methanol. The residue of methanol is added with water and extracted with an increasing solvent of n-hexane, dichloromethane and ethyl acetate. After removal of the solvent, obtain the residue of n-hexane (20g), dichloromethane (3.6g), ethyl acetate (18g) and methanol (55g), respectively. 3.2.2.2. Isolation of compounds from diclometane residue Diclomethan extract (AO.D, 3.6 g) conducted with VLC silicagel 60 column obtained 7 segments (AOD1 to AOD7). The OAD3 segment continues to run CC using a solvent system CH2Cl2: MeOH (10: 1) to obtain 3 segments (OAD3.1 to OAD3.3). Compound 7 is obtained by running preparative HPLC to OAD3.2 segment, detector λ = 210 nm with solvent system MeOH: H2O (3: 7). Diagram 3.2.2 Diagram to isolate compounds from leaves of Aglaia oligophylla 8  Compound 7 (New Compound) Compound 7 (3,3 mg) was isolated from the leaf of the Aglaia oligophylla Muq. in white amorphous form, [α]20D-50,5 (c, 0.45, CHCl3). UV (MeOH) λmax 210,4 and 271,1 nm. ESI-MS spectrometer (positive mode): m/z 528,1650 (M+Na)+ similar with C28H27NO8Na. Spectrometer data of Compound 7 showed at Table 4.3.1.1 3.2.3. Isolation of compounds from golden turmeric (Curcuma longa) 3.2.3.1. Processing plant samples The dried golden turmeric is finely ground, extracted with ethyl acetate solvent, then the solvent is then attracted to attract the essential oil. 3.2.3.2. Isolation of compounds Turmeric essential oil (TDN, 30.8g) is separated on silica gel column chromatography with gradient n-hexan-ethyl acetate solvent system with 12 segments. Segment 3 is re-purified by sephadex LH20 with elution solvent MeOH obtained compound 8 (2.8mg). Diagram 3.2.3 Diagram to isolate golden turmeric compounds The sludge residue after distillation entails extracting the vapors to extract the essential oil 3 times with ethyl acetate or alcohol 960. The extract is vacuumed until only a concentrated solution is left in the heat room temperature to 9 precipitate curcuminoid. After 24 hours, filter curcuminoite semi-crystalline. Coarse curcuminoid is waxed with cold alcohol and then purified in alcohol (semi-crystalline curcumin with stirring in alcohol 960 at boiling temperature), cooled to room temperature overnight to crystallize curcuminoid. The vacuum- filtered Curcuminoid mixture obtained a fine curcuminoite product. Curcumin crystals (substance 9, 12.3 mg) are purified by thin-plate preparative chromatography with the dichloromethane solvent: methanol (98: 2).  Compound 8: Compound 8 was isolated in white, oil form, UV 234-235nm. 1H-NMR data (CDCl3, 500 MHz), δH ppm 1,23 (d, 3H, J = 7 Hz, 15-CH3); 1,84 (brs, 3H, 12- CH3); 2,1 (s, 3H, 13-CH3); 2,3 (s, 3H, 4-CH3); 2,61 (m, 1H, H-); 2,69 (m, 1H, H- 8); 3,28 (m, 1H, H-7); 6,02 (s, 1H, =CH-C=0, H-10); 7,1 (m, 4H, H-2,3,5,6). 13C-NMR: (CDCl3, 125MHz) δC ppm 20,6 (C-12); 20,9 (C-15); 21,9 (C-14); 27,6 (C-13); 35,2 (C-7); 52,68 (C-8); 124,0 (C-10); 126,6 (C- 2,C-6); 129,09 (C-3, C-5); 135,5 (C-4); 143,6 (C-1); 155,0 (C- 11); 199,8 (C-9). • Compound 9: Essence of curcumin (compound 9) is purified by preparative thin-plate chromatography with dichloromethane: methanol (98: 2). NMR data show that this is a 50:50 mixture of two enol and ketone profiles of curcumin. Chemical structure of compound 9 (two forms of curcumin) 3.2.4. Isolation of compounds from Piper betle L. 3.2.4.1. Processing plant samples Samples of fresh leaves (5 kg) were extracted 3 times with methanol in ultrasonic devices at room temperature. The resulting total solution is stored in the solvent under reduced pressure, with a temperature of 45 ° C, obtained 240 g of residue of methanol. The residue of methanol is added with water and extracted with an increasing solvent of n-hexane and ethyl acetate. After removal of the solvent, obtain the residue of n-hexane (55g), ethyl acetate (50g) and methanol (130g), respectively. 3.2.4.2. Isolation of compounds from n-hexane residue The n-hexane extract (TKH, 50g) was separated by silica gel column chromatography (63-100µm) with a solvent elution system of n-hexane-ethyl acetate (100: 0 to 1: 1) obtained segments from TKH1 to TKH10. Diagram 3.2.4 Diagram to isolate compounds from P