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

MINISTERY OF
EDUCATION AND
TRAINING

VIETNAM ACADEMY OF SCIENCE AND
TECHNOLOGY
GRADUATE UNIVERSITY OF SCIENCE AND
TECHNOLOGY

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


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
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1 Reviewer: .............................................................................................................

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2nd Reviewer: ............................................................................................................
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3 Reviewer: .............................................................................................................
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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

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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, 9atetrahydro-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

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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).

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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
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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 (4063µm) 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
5


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, H5”), 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:

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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, OMe6), 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

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 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
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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 vacuumfiltered 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, 12CH3); 2,1 (s, 3H, 13-CH3); 2,3 (s, 3H, 4-CH3); 2,61 (m, 1H, H-); 2,69 (m, 1H, H8); 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 (C2,C-6); 129,09 (C-3, C-5); 135,5 (C-4); 143,6 (C-1); 155,0 (C11); 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

9


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
diagram 3.2.4.
• Compound 10:
Compound 10 is obtained in liquid form, light yellow. 1H-NMR
(CDCl3) δH ppm 3.28 (d, J = 6.5 Hz; 2H, H-7), 3.83 (s, 3H, H-2OCH3), 5.03 (m, 2H , H-9), 5.93 (m, 1H, H-8), 6.65 (dd, J = 2; 8.5
Hz, 1H, H-3), 6.75 (brs, 1H, H -5), 6.77 (d, J = 2 Hz, 1H, H-6).
• Compound 11:
Compound 11 is obtained in liquid, colorless, melting temperature of
16oC. 1H-NMR spectrum: (CDCl3) (δH ppm) 3.3 (d, J = 6.5, 2H, H-7);
5.05 (m, 2H, H-9); 5.93 (m, 1H, H-8); 6.77 (m, 2H, H-2.6); 7.04 (m, 2H,
H-3.5). 13C-NMR: (CDCl3, 125MHz, δC ppm) 39.5 (C-7); 115.2 (C-9);
115.4 (C-2.6); 129.6 (C-3.5); 132.2 (C-4); 137.8 (C-8); 153.9 (C-1).
• Compound 12:
Compound 12 is obtained in liquid, colorless, boiling temperature 48 °
C. 1H-NMR spectrum: (CDCl3) (δ ppm) 2.05 (2H, d, J = 6.7 Hz,, H-7);
5.03 (m, 1H, H-9cis); 5.06 (m, 1H, H-9trans); 5.48 (1-OH); 5.53 (2OH); 5.90 (m, 1H, -CH =); 6.62 (dd, J = 2.0 and 8.1Hz, 1H, H-5); 6.70
(d, J = 2.0Hz, 1H, H-6); 6.78 (d, J = 8.1Hz, 1H, H-3).
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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 nhexane solvent: ethyl acetate (1: 0 to 0: 1) with 5 segments. Segment 3 continues
to be separated on silica gel chromatography 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
3.2.6.1. Sample treatment
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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 MeOHH2O 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

• Compounds from 15-26:
Compounds from 15 to 26 were determined by the Gas Chromatography Mass
Spectometry technique (GC / MS) at the Institute of Natural Compounds. GC /
MS results of substances 15-26 are described in Table
4.3.5.1
• Compound 27:
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:
12

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 of Piper 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:
Compound 31 (7.1mg), is a white needle-shaped
crystal, melting point mp 168-169 ° C.
Spectral data of 31 see table 4.3.6.1
13


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.

14


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
further research on chemical isolation.

15


4.3. Research results of chemical components of plants and endogenous fungal
plants
4.3.1. Chemical composition of A. dupperreana and A. oligophylla
From the residue of the ethyl acetate extract of the A. dupperreana bark , six
compounds were isolated and determined chemical structure, including flavonoidcinnamic acid amide: rocaglamide A (1), rocaglamide I (2), rocaglamide W (3),
rocaglamide AB (4), rocaglamide J (5), and rocaglaol (6). From the
dichloromethane extract residue the A. oligophylla leaves were isolated and
determined the chemical structure of rocaglamide AY (7) (new substance). The

chemical structure of these compounds is determined based on UV, MS, 1H-NMR
spectral data, and compared with previously published documents for known
compounds.
4.3.1.1. Compound 1 (Rocaglamide A)
Compound 1 (3.9 mg) was isolated from the A. dupperreana bark in white
amorphous form, [α] 20D-90.5 (c, 0.25, CHCl3). UV (MeOH) of 1 indicates λmax
219.7 and 273.0 nm. ESIMS positive mass spectra for molecular mimic ion m / z
561,1 [M + H] +, m / z 528.4 [M + Na] + corresponds to the molecular formula
C29H31NO7.
1H-NMR spectra showed the presence of 3 singlet methoxy groups in resonance
at δH 3.81 (OCH3-8), 3.84 (OCH3-6) and 3.66 (OCH3-4 '), 2 proton groups The
methyl of H-5 and H-7 in ring A was observed as the doublet-meta form at δH
6,30 and 6,17 having 1.9 spin spin constant.
Two singlet signals of N-CH3 group were observed at 3.34 and 2.86 ppm. The
very characteristic resonant signal of the spin system AA'BB 'of ring B appears at
the low field at δ = 7,12 and 6.64 ppm has shown that the B ring is substituted
para, corresponding to two proton pair H-2 '/ H-6' and H-3 '/ H-5'. The second spin
system of the phenyl mono-substituent, C-ring is observed at δH 6.86 ppm (m, H2 "/ H-6") and at 6.98 ppm (m, H-3 "/ 4"). / 5 "). In addition, the appearance of the
fourth spin system of the cyclopentan ring was observed at δH 4.95 ppm (d, 6,9),
4,36 (d, 13,8) and 4,11 (dd, 6 , 9 & 13.8Hz) are of H1α, H2α and H3β protons.

16


1H-NMR NMR data of compound 1 were obtained by using the same solvent
used for Rocaglamide A [65]. Therefore, the results are identical without adding
other spectra. Analysis of 1H-NMR, MS, UV spectra data of 1 combined with
comparison with spectral data and physical constants in reference [65] compound
1 was identified as Rocaglamide compound A. Structure chemical structure is as
follows:

4.3.1.2. Compound 2 (Rocaglamide I)
Compound 2 (3.8 mg) was isolated from the bark of Aglaia dupperreana in white
amorphous form, [α] 20D-80 (c, 0.45, CHCl3). The UV spectrum (MeOH) of 2
indicates λmax 209 and 279 nm. Electron dust mass spectra ESI-MS (positive
mode): m / z 564,1 (M + H) +, 586,4 (M + Na) + corresponds to the molecular
formula C31H33NO9.
The 1H-NMR spectrum of compound 2 shows that there are two doublet
resonance signals of the H-5 and H-7 protons in the A-position in the meta
position with a 1.9 Hz spin interaction constant at δH 6.26 ppm. and 6.11 ppm. In
addition, the signal of the methyl group appearing at the high field at 81H 1.81
ppm showed the presence of the acetoxy group at C-1. This results in a shift in the
chemical shift of proton H-1 to 6.03 ppm. In addition, 2 protons of H-2 and H-3
fatty chains were also observed on the spectrum with overlap gate signal at δH
4.29 ppm. The 1H-NMR spectrum demonstrated that a hydroxyl group at C-3
'caused a barrier effect of the aromatic proto in round B in the following order: H2 ’> H-6’> H-5 ’. The presence of hydroxyl group at C-3 changes the properties of
ring B and therefore on 1H-NMR spectrum will no longer have very characteristic
resonance signal of AA'BB 'system so para at ring B and transformed into an ABC
system. The substituent at this position also changes the position of the chemical
displacement of 3 methoxyl groups: At δH 3,73 (OCH3-8), 3.81 (OCH3-6) and at
δH 3,71 (OCH3 -4 '). Two groups of N-CH3 appeared at δH 3.34 ppm and 2.86
ppm in singlet form.
Analysis of 1H-NMR, MS, UV spectra data of 2 combined with comparison with
spectral data and physical constants in reference [65] compound 2 is defined as
compound Rocaglamide I. Structure chemistry of 2 is as follows:
4.3.1.3. Compound 3 (Rocaglamide W)
Compound 3 (2.1 mg) was isolated from the bark of Aglaia dupperreana in white
amorphous form, [α] 20D-55.0 (c, 0.45, CHCl3). UV spectra (MeOH) of 3
indicate λmax 210 and 272.5 nm. Electron dust mass spectra ESI-MS (positive
mode): m / z 534,1 (M + H) +, 556,4 (M + Na) + corresponds to the molecular
formula C30H31NO8.

17


1H-NMR spectrum of compound 2 and spectrum of compound 3 showed a loss of
N-Me signal at δH 3.34 ppm that could explain that the CONHCH3 group was
replaced at position C-2, instead of because of CON group (CH3) 2 in the case of
compound 2. Compound H-5 and H-7 of ring A are also observed as meta doublet
with J = 1.9 Hz at δH 6,26 and 6,12 ppm. Very typical spin interaction system
AA'BB 'is also observed at δH 6.61 (d, J = 8.8Hz) and 7.17 (d, J = 8.8Hz)
characterizing the para potential of ring B Similar to compound 2, the acetyl group
at C-1 resonates at δH 1.84 (s). This makes the chemical shift of H-1 shift to low
field at δH 5,99 (d, 6,3), two methyl protons resonate at δH 3.94 (dd, J = 5.9 &
14.5Hz) and 4.19 (d, J = 14.5Hz) for H-2 and H-3 respectively. The value J =
14,5Hz indicates the axial-axial interaction between H-2 and H-3. While the
interaction constant J = 5.9 Hz characterizes the axial-equatorial interaction
between H-2 and H-1.
Analysis of 1H-NMR, MS, UV spectra data of 3 combined with comparison with
spectral data and physical constants in reference [65] compound 3 is defined as
Rocaglamide W compound, with structure chemical structure is as follows:
4.3.1.4. Compound 4 (Rocaglamide AB)
Compound 4 (7.2 mg) is obtained in white amorphous form, [α] 20D-110.0 (c,
0.45, CHCl3). UV spectra (MeOH) indicate λmax 210,4 and 272,6 nm. Electron
dust mass spectrum ESI-MS (positive mode): m / z 548,2 (M + H) +, 570,4 (M +
Na) + corresponds to the molecular formula C31H33NO8. The molecular weight
of compound 4 is smaller than compound 2 of 14 dvc. The 1H-NMR spectrum of
compound 4 gives a very specific signal of the spin AA'BB 'system of the B-ring
as in compounds 1 and 3. Two pairs of protons H-2' / H-6 'and H-3' / H-5 'for
signals at 7.08 ppm 2H (2H, d, J = 8.8Hz) 6.54 ppm (2H, d, J = 8.8Hz). In
addition, the 1H-NMR spectrum gives the signals of the two N-Me singles at δH
3.27 and 2.69 ppm, just like with compound 1. On the other hand, the acetylation

at C-1 causes displacement. The chemistry of H-1 at a low field at δH 5.95 (m)
and the overlap of proton H-2 and H-3 at δH 4.21 (m) is the same as in
compounds 2 and 3. Two The proton is in the meta position of the doublet of H-5
and H-7 at ring A resonating at δH 6.18 and 6.03 ppm. Five other protons of the
mono-C ring were observed at δH 6.92 and 6.80 ppm, similar to similar
Rocaglamide compounds. Three groups of methoxyl in singlet form were
observed at δH 3,64, 3.72 and 3.56 ppm respectively for OCH3-6, OCH3-8,
OCH3-4 'groups.
Analysis of 1H-NMR, MS, UV spectra data of 4 combined with comparison with
spectral data and physical constants in reference [65] compound 4 was identified
as rocaglamide AB with chemical structure as follows:
4.3.1.5. Compound 5 (Rocaglamide J)
18


Compound 5 (1.9 mg) was isolated from the bark of Aglaia dupperreana in white
amorphous form, [α] 20D-41.1 (c, 0.22, CHCl3). UV spectra (MeOH) indicate
λmax 211,3 and 278,7 nm. Electronic atomic mass spectrometer ESI-MS (positive
mode) for m / z 509,0 (M + H) +, 531,2 (M + Na) + corresponds to the molecular
formula C28H28NO9.
On the 1H-NMR spectrum of 5, two protons of H-5 and H-7 of ring A resonate at
δH 6.27 ppm (d, J = 1.9Hz) and 6,15 ppm (d, J = 1.9 Hz) ). The hydroxyl group at
C-3 'changed the spin system of AA'BB' characteristic of the B ring in the ABC
spin rocglamide compounds as in compound 2. This group has caused a change in
the degree of transformation. Study between 3 methoxyl groups: OCH3-6 at δH
3.81 ppm, OCH3-8 at 2H 3.82 ppm and OCH3-4 'at δH 3.67 ppm. Three protons
of H-1, H-2 and H-3 resonate at δH 5.0 ppm (d, J = 5.7 Hz), 3.96 ppm (dd, J = 5.7
& 13.9 Hz) and δH 4.21 ppm (d, J = 13.9 Hz). In addition, the methoxyl group in
the singlet form of the acetate fraction at C-2 appears the resonant signal at δH
3,61 (s). 5 aromatic protons of C-ring observed at δH 6.91-7.00 ppm, are the same

as in compounds 2, 3 and 4.
Analysis of 1H-NMR, MS, UV spectra data of 5 combined with comparison with
spectral data and physical constants in reference [65] compound 5 was identified
as rocaglamide J with chemical structure as follows:
4.3.1.6. Compound 6 (Rocaglaol)
Compound 6 (10mg) was isolated from the bark of Aglaia dupperreana in white
amorphous form, [α] 20D-125 (c, 0.48, CHCl3). The UV spectrum (MeOH) of 6
indicates 212max 212,8 and 272,3 nm. Electron dust mass spectrometry ESI-MS
(positive mode): m / z 457.10 (M + H) +, 890.9 (2M + Na) + corresponds to the
molecular formula C26H26NO6.
The 1H-NMR spectrum of compound 6 shows 2 protons of H-5 and H-7 of ring A
resonating at δH 6.28 ppm (d, J = 1.9 Hz) and at δH 6.17 (d, J = 1.9Hz). Very
special AA'BB spin system 'of round B was observed at δH 7.10 (2H, d, J =
8.8Hz) and at δH 6.61 (2H, d, J = 8.8 Hz). Three groups of methoxyl in the form
of singlet have the resonant signal at δH 3.87 (OMe-6), at δH 3.85 (Ome-8) and at
δH 3.81 (Ome-4 ’). Comparing with compound 1, compound 6 has a change in the
aliphatic region. The resonance of the methylene proton appears as a pair of
multilayered geminal interactions (m) at δH 2.06 ppm (ddd, J = 1,1, 6,2 & 11,8
Hz) and 2,80 ppm (ddd , J = 6.2, 11.8 & 14.0 Hz), both protons also exhibit
vicinal interaction with methyl groups surrounded by phenyl group and hydroxyl
group at δH 3.89 and 4.69 ppm. Thus the methylene signals have a link between
the methyl protons. The signal of methylene group appeared at δH 2.06 and 2.80
ppm of H-2α and H-2β was explained with relatively small spin interaction
constant (J = 1.5 Hz) in equatorial-equatorial form and a large interaction constant
(14.0 Hz) axial-axial form for H-1β and H-3α respectively.
19


Analysis of 1H-NMR, MS, UV spectra data of 6 combined with comparison with
spectral data and physical constants in reference [65] compound 6 was identified

as rocaglaol with the chemical structure as follows :
4.3.1.7. Compound 7 (Rocaglamide AY) - new substance
Compound 7 (3.3 mg) was isolated from leaves of Aglaia oligophylla Muq. In the
amorphous white form, [α] 20D-50.5 (c, 0.45, CHCl3). UV spectra (MeOH) of 7
indicate λmax 210.4 and 271.1 nm. High resolution mass spectrometry HRESIMS for molecular dummy ion peaks at m / z = 528,1650 [M + Na] +,
corresponding to the molecular formula C28H27NO8.
1H and 13C-NMR spectra of compound 7 are similar to the spectrum of
rocaglamide J (compound 5) with the signal of 3 methoxy groups at δH 3.90 (8OMe); 3.84 (6-OMe) and 3.71 (4'-OMe), together with two fragrant proton signals
with meta-interaction at δH 6.15 and 6.26 (each signal d, J = 1.9Hz) , 5 protons of
aromatic rings were replaced once at δH 6.99 - 7.12 ppm and acetate groups (δC
57.5 and 170.0ppm). The difference of compound 7 compared to compound 5 is
shown in two points. The first is that the hydroxy group at C-1 in compound 5 has
been converted to the C-1-oxime substituent in the molecule, which is
demonstrated by the low-field shift of C-1 (δC 153.0 ppm) compared to
compound 5. Secondly, the characteristic signal appears for the spin AA'BB
'system of ring B at δH 7.13 (2H, d, J = 8.8Hz) and 6.71 (2H, d, J = 8.8Hz), this
proves that the hydroxy substituents did not exist in the compound 7. The 2DNMR spectrum analysis of substance 7 combined with the literature leads to the
conclusion that its structure is C-3'-demethoxy derivative of rocaglamide T
[48,66]. This is a new substance and is named rocaglamide AY.
Table 4.3.1.1 NMR data of compound 7 (CDCl3)
Vị trí
1
2
3

*δC
(ppm)
153,0
57,0
57,1

δC (ppm) δH
(ppm)J(Hz)
153,0
57,1
3,80 (d,13,5)
57,2
3,67 (d,13,5)

3ª
4ª
5
6
7
8
8ª

105,1
160,0
88,9
164,0
93,0
158,3
107,7

105,7
161,3
89,9
165,3
93,8

160,3
110,0

HMBC

COSY

3,8b, 9, 1”
2,3a, 9, 1’,
1”,2”/6”

3
2

6,26 (d,1,9)

4a,6,7,8a

6,15 (d,1,9)

5,6,8,8a

20


Vị trí
8b
1’
2’b
3’c

4’
5’c
6’b
1”
2”/6”
3”/5”
4”
9 (CO)
8-OCH3
6-OCH3
4’-OCH3
COOCH3

*δC
(ppm)
115,0
125,6
113,1
127,8
158,8
126,8
125,6
134,8
127,7
127,8

δC (ppm) δH
(ppm)J(Hz)
117,0
128,7

113,2
7,13 (d,8,8)
149,3
6,71 (d,8,8)
149,5
11,5
6,71 (d,8,8)
121,4
7,13 (d, 8,8)
136,7
129,4
6,99 (m)
128,7
7,12 (m)

127,8
170,0

128,0
171,7
56,1
56,3
56,3
57,5

HMBC

COSY

3a

2’,6’,4’,1”

3’/5’
2’/6’

2’/6’,4’,1”
3a

2’/6’
3’/5’

3”/5”
1”,2”/6”

3”/5”
2”/6”,
4”
3”/5”

7,12 (m)
3,90 (s)
3,84 (s)
3,71 (s)

8
6
4’
9 (C=O)

* δC: 13C-NMR spectral data of rocaglamide T measured in CDCl3 [66]

4.3.2. Chemical composition of yellow turmeric (Curcuma longa L.)
From the dichloromethane extract residue of yellow turmeric, two compounds
were isolated and determined chemical structure including ar-turmerone (8) and
curcumin (9). The chemical structure of these compounds is determined based on
1H-, 13C-NMR spectral data, and compared with previously published documents
for known compounds.
4.3.2.1. Compound 8 (Ar-turmerone)
1H and 13C-NMR spectra of substance 8 showed that the molecule contained an
aromatic ring in position 1 and 4, expressed by the signal of 4 aromatic protons at
δH = 7.1 (m, 4H, H-2, 3,5,6) and 6 aromatic carbon, including 4 CH and 2 carbon
4; 4 methyl groups include three tertiary groups [δH = 1.84; 2.1 and 2,3 (each
signal 3H, s)], a quadratic group [δH = 1.23 (3H, d, J = 7Hz), H-15CH3]. Besides,
NMR spectrum also shows the presence of a ketone group at δC = 199.8 (C-9); 1
olefinic proton in δH 6.02 (H-10) and 3 aliphatic proton resonates in the region
from 2.61 to 3.28ppm. The spectral data analyzed above are completely consistent
with the Ar-turmerone data in reference [146]. Recent research shows that this
compound exhibits the activity of repelling the two insects Sitophilus zeamais and
Spodoptera frugiperda [146].
4.3.2.2. Compound 9 (Curcumin)
21


Essence of curcumin (compound 9) is purified by preparative thin-plate
chromatography with the solvent dichloromethane: methanol (98: 2). Nuclear
magnetic resonance data show that this is a 50:50 mixture of two enol and ketone
profiles of curcumin.
4.3.3. Chemical composition of Piper betle L.
From the dichloromethane extract residue, there are 3 compounds isolated and
determined chemical structure including eugenol (10), chavicol (11), 4allylpyrocatechol (12). The chemical structure of these compounds is determined

based on 1H-, 13C-NMR spectral data, and compared with previously published
documents for known compounds.
4.3.3.1. Compound 10 (Eugenol)
On 1H-NMR spectrum of substance 10 showed that the signal of 3 aromatic
protons of one aromatic ring was replaced 3 times in δH 6.65 (dd, J = 2; 8.5 Hz,
1H, H-3), 6, 75 (brs, 1H, H-5), 6.77 (d, J = 2 Hz, 1H, H-6) and signals of the allyl
group: 3 olefinic protons in δH 5.03 (H-9); 5.93 (H-8) and a methylene group at
3.28 (H-7). There are also methyl groups attached to aromatic rings at 3.83 (s, 3H,
H-2-OCH3). Analyzing 1H-NMR spectral data in combination with comparing
spectral data reference [147] allows to confirm that compound 10 is named
eugenol.
4.3.3.2. Compound 11 (Chavicol)
Signals of 11 protons were observed on 1H-NMR spectrum of compound 11. In
which, there are 3 aromatic protons of an aromatic ring replaced twice in ortho δH
position 6.77 (m, 2H, H-2, 6); 7.04 (m, 2H, H-3,5) and signals of allyl group: 3
olefinic protons in δH 5.05 (m, 2H, H-9); 5.93 (m, 1H, H-8) and a methylene
group at δH 3.3 (d, J = 6.5, 2H, H-7). 13C-NMR spectrum of 11 indicates that
there is a total of 9 carbon molecules in the molecule, including 2 methylene
groups at δC 39.5 (C-7) and 115,2 (C-9), 5 methin groups at nhómC 115.4 (C2.6); 129,6 (C-3,5) and 137,8 (C-8), 2 carbon at level 4 at δC 132,2 (C-4) and
153,9 (C-1), there is 1 carbon at the fourth level with oxygen at δC 153.9 (C-1).
Analysis of 1H- and 13C-NMR spectral data in combination with reference
spectral data reference [148] allowed to confirm that compound 11 is named
chavicol with the chemical structure below.
4.3.3.3. Compound 12 (4-allylpyrocatechol)
Signals of 8 protons were observed on the 1H-NMR spectrum of 12. There were 3
aromatic protons of an aromatic ring at δH 6.62 (dd, J = 2.0 and 8.1Hz, 1H, H- 5);
6.70 (d, J = 2.0Hz, 1H, H-6); 6.78 (d, J = 8.1Hz, 1H, H-3) and signals of allyl
group: 3 olefinic protons in ởH 5.03 (m, 1H, H-9cis); 5.06 (m, 1H, H-9trans); and
5.90 (m, 1H, H-8) and one methylene group at δH 2.05 (2H, d, J = 6.7 Hz, H-7).
In addition, the spectrum also showed signals of two hydroxy groups at δH 5,48

(1-OH); 5.53 (2-OH) (br s, 1H signal).
22


The spectral data of 12 completely coincide with 4-allylpyrocatechol in reference
[148].
4.3.4. Chemical composition of M. hawaiiensis from Aglaia
From the residue of diclometan fragment of M. hawaiiensis, two compounds were
isolated and identified chemical structure including Scopararane C (13) and
Diaporthein B (14). The chemical structure of these compounds is determined
based on 1H-, 13C-NMR spectral data, and compared with previously published
documents for known compounds.
4.3.4.1. Compound 13 (C Scopararane)
Compound 13 is obtained in the form of a white needle-shaped crystal, Rf = 0.87
(solvent n-hexane-ethyl acetate 3: 1), melting point 171,6 0C.
1H NMR spectrum of substance 13 shows the signal of 4 olefinic protons
resonating at δH 7.04 (1H, d, J = 1.5Hz, H-14); 5.87 (1H, dd, J = 17.5; 10.5Hz, H15a) and 5.05 (1H, dd, J = 11.0; 0.5Hz, H-15b); 5.11 (1H, dd, J = 17.5; 0.5Hz, H16) of two double bonds, one double bond is replaced once and one double bond
is replaced 3 times; 4 methyl groups in the form of singlet at δH 1.20 (s, 6H, H17,20); 1.29; 1.40 (each 3H, s, H-18,19) and 10 aliphatic proton signals resonate
in the region from 1.4 to 2.0 ppm. 13C NMR spectrum of compound 13 gives the
signal of 20 carbon, including 4 groups CH3, 6 groups CH2, 2 CH groups and 8
carbon ranks 4. Besides signals suitable for 1H spectrum, 13C NMR spectrum
also gives see the presence of 1 carbonyl group (δC 181,6, C-7), 1 double bond
alternate 4 times at δC 142.7 (C-5), in which the signal is shifted toward the low
field at δC 144 , 4 (C-6) showed a double bond hydroxy substituent and a carbon
aliphatic linked to oxygen shown in signal ởC 74.1 (C-9). NMR spectral data of
13 see table 4.3.4.1. The 1H and 13C NMR spectroscopic data analyzed here are
in comparison with the data in the reference [149] to confirm that compound 13 is
called Scopararane C. The chemical structure is as follows.
4.3.4.2. Compound 14 (Diaporthein B)
Substance 14 is obtained in the form of a white square-shaped crystal, Rf = 0.33

(dm: n-hexane-ethyl acetate 3: 1), mm. 189.6 0C.
Mass spectrometry ESI-MS for molecular ion peaks at m / z 363 [M - H] -.
Combined with preliminary analysis of MS spectrum data, 1H and 13C NMR
spectra, it can be confirmed that 14 is a diterpen with molecular formula
C20H28O6.
The 1H and 13C NMR spectra of 14 are almost similar to those of substance 13
only in the following three points: The first point is the signal of the C-20 methyl
group at δH 1.20 in substance 14 replaced by the signal of the oxygen group. methylene at δH 3.71 and 4.14 (each signal d, J = 10.0Hz, H-20). The second
difference is the signal loss of 2 carbon olefinic and the emergence of 2 quadratic
aliphatic carbon attached to oxygen at 81 81.9 (C-5) and 104.1 (C-6) in substance
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