Dissertation summary organic chemistry: Study on the isolation and biological activity of natural active compounds from plants and endophytes

The aims of the dissertation are: Extracting and determining the structure of organic compounds of four plant species with potential for insecticide and fungal diseases. Isolating endogenous fungi from plant samples, extracting and determining the structure of component organic compounds. Testing of insecticidal and fungal activity of extracts and component organic compounds.

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

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

1st Reviewer:

2nd Reviewer:

3rd 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

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

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,dihydronidulalin A, 4S, 4aR, 9aR) -4a-carbomethoxy-1,4,4a, tetrahydro-4,8-dihydroxy-6-methylxanthone and (24R) -methylcholesta-7.22 -

9a-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

acids from endogenous fungi of Golden Turmeric (F oxysporum) by the GC-MS

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)

II DISSERTATION CONTENTS

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

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 63µm) with the gradient CH2Cl2-MeOH solvent system (from 100: 0 to 0: 100) to obtain 9 segments, symbols is ADE3.1-ADE3.9

(40-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

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:

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

 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

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 3, C-5); 135,5 4); 143,6 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 Piper betle L leaves

Run the column chromatography TKH4 (1.2g) on silica gel (40-63µm) with the solvent system of n-hexane-ethyl acetate (from 100: 0 to 80:20) to obtain 4

segments, signed TKH4.1-TKH4.4 The TKH4.4 (15 mg) fraction is purified by a preparative thin plate with the n-hexan-ethyl acetate 95: 5 solvent system to obtain

a clean substance 10 (10 mg)

The TKH6 segment (3.99g) was further refined on the silica gel column with the n-hexane-ethyl acetate gradient solvents (from 95: 5 to 75:25) obtained 4

segments, denoted TKH6.1- TKH6.4 The TKH6.4 (0,797 g) segment was

repeated repeatedly on CC column to obtain clean substance 11 (150 mg)

15mg of the TKH7 fraction was purified by thin plate preparation with a solvent system of n-hexane-ethyl acetate (4: 1), which obtained a clean substance 12 (11.2

mg) The process of extracting compounds from Piper betle L leaves is showed in

3.2.5 Isolation of compound from M hawaiiensis endogenous fungi of the Aglaia

dupperreana

3.2.5.1 Sample treatmen t

Microdiplodia hawaiiensis is isolated from our bark on agar medium It was then

cultured in rice in three pots of 300g glutinous rice When the mushroom is fully developed (6 weeks), the medium containing the endogenous fungi is extracted with ethyl acetate solvent and then rotated to obtain the ethyl acetate extract (2.8 g)

3.2.5.2 Isolation of compounds from the ethyl acetate extract residue

The extract is separated on the silica gel adsorption column with a gradient of hexane solvent: ethyl acetate (1: 0 to 0: 1) with 5 segments Segment 3 continues

n-to be separated on silica gel chroman-tography column and gradient n-hexane solvent system: ethyl acetate (from 9: 1 to 0: 1) to obtain 2 clean substances denoted as substance 13 (16 , 2mg) and 14 (35.0mg) The isolation process is described in diagram 3.2.5

Diagram 3.2.5 Diagram of extracting compounds from M hawaiiensis

• Compound 13:

Compound 13 is obtained in a white needle-shaped crystal form,

Rf = 0.87 (dm: n-hexane-ethyl acetate 3: 1), melting point = 171.6

0C

NMR spectral data of compound 13 see Table 4.3.4.1

• Compound 14:

Compound 14 is obtained in a white square-shaped crystal form,

Rf = 0.33 (dm: n-hexane-ethyl acetate 3: 1), a melting point of

189.6 0C

NMR spectral data of compound 14 see Table 4.3.4.1

3.2.6 Isolate compounds from F oxysporum endogenous fungus

of Golden Turmeric

F oxysporum is isolated from Golden turmeric on agar medium It was then

cultured in rice in three pots of 300g glutinous rice When the fungus has enough growth (6 weeks), the medium containing endogenous fungus is extracted with methanol solvent and then the spinster obtained a methanol extract (10.2 g) 3.2.6.2 Dividing compounds from methanol extraction residues

The MeOH extract (NB1M, 10.2g) was separated on the silica gel adsorption column with the n-hexane solvent system: acetone (1: 0 to 0: 1) obtained 11 segments (NB1M1-NB1M11) The NB1M4 (1.1g) fraction contains a lot of oil, so

it can run GC-MS and obtain the chemical formula of 12 types of acids denoting substances 15-26 The NB1M6 (0.7g) numerical fraction continued to be

separated on the silica gel column with the n-hexane solvent system: ethyl acetate (1: 0 to 0: 1), resulting in 3 small segments (NB1M6.1-NB1M6 3) The NB1M6.1 and NB1M6.2 segments were re-refined via sephadex LH20 column with the elution solvent MeOH obtained 2 compounds 27 (18mg) and 28 (5.1mg) The NB1M6.3 fraction is separated on RP-18 reverse phase column with 3-7 MeOH-H2O eluent obtained compound 29 (6.5mg) The NB1M8 fraction is washed with acetone to obtain a soluble powder-like crystal in MeOH collecting 30 (6.6mg) The procedure for extracting 15-30 substances is described in diagram 3.2.6 Diagram 3.2.6 Diagram to isolate compounds from endogenous fungus of Golden Turmeric

Compound 27 is obtained in the white needle-shaped

crystals form, melting point is 132-133 OC Rf = 0.26

(solvent system deploys n-hexane-aceton 9: 1)

• Compound 28:

Compound 28 is obtained in white amorphous form Rf = 0.34 (solvent solvent system of dichloromethane: methanol (9: 1)

Spectral data of 28 see table 4.3.5.2

• Compound 29:

Compound 29 is obtained in white amorphous form Rf =

0.33 (solvent extraction system of dichloromethane: methanol

(9: 1)

Spectral data of 29 see table 4.3.5.2

• Compound 30:

Compound 30 is obtained in the form of white powder soluble

in hot methanol, with melting heat is 221-224oC, Rf = 0.61 in

the dichloromethane-methanol solvent system 9: 1

Spectral data of 30 see table 4.3.5.3

3.2.7 Isolation of compounds from F solani endogenous

fungus ofPiper betle L

3.2.7.1 Sample treatment

Fusarium solani (NTK) isolated from Piper betle L leaves is cultured on rice

medium in glass containers After the biomass is extracted with ethyl acetate solvent The resulting extract turns the solvent obtained with the ethyl acetate extract (NTKE, 17g)

3.2.7.2 Isolation of compounds

The extract of ethyl axetate (NTKE, 17g) is run on the silica gel column of the dichloromethane elution solvent: methanol (20: 1-1: 1), achieving 15 segments (NTK1-NTK15) Refining NTK3 (0.3g) segment through sephadex LH20 column with MeOH eluent obtained 31 (7.1mg) The extraction procedure is described in diagram 3.2.7

Diagram 3.2.7 Diagram to isolate compounds from endogenous mushrooms of

Piper betle

• Compound 31:

3.3 Screening test of insecticidal and fungal activity of extracts, fractions and clean substances

The inhibit activity to Spodoptetra litura worm and the Botrytis cinerea fungus

causing gray rot disease has been tested for the total extract of plant and plants endogenous fungus at a concentration of 1000 ppm

Chapter 4 RESULTS AND DISCUSSION

4.1 Results of plant isolation and identification of plant endogenous fungal strains 4.2 Results of insecticidal and antifungal activity tests of total extracts, fragment extracts and purifiled compounds

Samples of plant extracts and endogenous fungal extracts were examined for insecticide activity in the laboratory Experiments to test the insecticide activity of extracts were tested at the Department of Pharmacology - Institute of Chemistry The results showed that three (03) samples of extract of the Aglaia duperreana plant (leaf, bark and flower) were active at 1000 ppm concentration, in which 02 samples were 100% dead after 24 hours (including leaf and bark extracts) and 01 specimen lethal 40% deep after 48 hours (flower extract) (Table 4.2.1)

Samples of yellow turmeric extract at a concentration of 1000 ppm have activity

to inhibit 100% gray rot fungus Botrytis cinerea

The inhibitory ability of Curcumin for Botrytis cinera is very good Concentration

of 750ppm has 100% inhibitory ability, concentration of 500 ppm has the ability

to inhibit 88.75% When curcumin is reduced to 150 ppm, curcumin is also capable of inhibiting up to approximately 25% Positive control, Nystatin

antibiotic, was 100% inhibition However, ar-tumeron's (substance 8) ability to inhibit pathogenic fungi only reached 52.08% From that, it can be concluded that the ability to inhibit Botrytis cinera fungus of yellow turmeric is mainly by

curcumin

In all 3 concentrations of 1000, 750, and 500 (ppm), the n-hexane extract (TKH) segment showed 69.88% resistance to Botrytis cinera respectively; 59.38% and 43.63% The survey results of TKH4 extract (eugenol) showed that the resistance

to Botrytis cinera reached 95% Positive control, Plumbagin antibiotic, was 100% inhibition The results show that Eugenol compound has a strong inhibitory effect

on Botrytis cinera

The MeOH extract of endogenous fungal extracts of the C longa, A Dupperreana and P.betle were inhibited the growth of Botrytis cinera at 56.46%, 53.5% and 100%, respectively

From the results of this test, we can identify the target group needed to focus on