1

MINISTRY OF EDUCATION AND TRAINING
VINH UNIVERSITY

VU THI HIEN

STUDY ON CHEMICAL CONSTITUENTS
AND BIOLOGICAL ACTIVITY OF FRUITS OF
Aphanamixis polystachya (Wall.) R.Parker,
Khaya senegalensis A. Juss AND Melia azedarach L.
BELONGING TO THE FAMILY MELIACEAE IN
VIETNAM
MAJOR: ORGANIC CHEMISTRY
Code: 62.44.01.14

PhD THESIS SUMMARY

NGHE AN - 2018


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The present study has been completed at: Specialized Lab Organic
Chemistry, Vinh University

Supervisors:
1. Assoc. Prof. Dr. Vu Dinh Hoang
2. Assoc. Prof. Dr. Hoang Van Luu

Reviewer 1: Assoc. Prof. Dr. Trinh Thi Thuy

on the chemical composition and biological activity of these plants in Vietnam has
been reported. This thesis is aimed to clarify the use in folk, improve the
effectiveness and safety of medicinal plants, develop medicinal materials to use in a
more scientific way. Therefore, the study field has important theoretical and
practical significance, making an important contribution to finding natural resources,
economic value of Vietnam's natural medicinal resources. For that reason, we have
chosen the topic: "Study on chemical composition and biological activity of
fruits of Anphanamixis polystachya, Khaya senegalensis and Melia azedarach
belonging to family Meliaceae in Vietnam”
2. Objects
The research objects of the thesis are extracts from fruits of Anphanamixis
polystachya, Khaya senegalensis and Melia azedarach in Vietnam.
3. Tasks
The research tasks include:
- Selecting suitable solvents to extract the existing compounds from fruits of
Anphanamixis polystachya, Khaya senegalensis and Melia azedarach in
Vietnam.
- Separating and identifying structures of the compounds isolated.
- Assaying biological activities of the concerned compounds.
4. Methods
- Collection samples: collection, identification, cultivation for some fungi,
storage and preservation dried fungi of 2 samples at the laboratory temperature
0
(20-30 C).
Isolation of compounds by a combination of chromatographic techniques;
column chromatography (CC), thin layer chromatography (TLC) with different


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3α,12α-diacetoxy-7α-benzoyloxy-1αhydroxytrichilinin which is called trichilinin F.
- 02 flavonoid compounds: apigenin and quercetin 3-O-[-L-rhamnopyranosyl(1→6)]--D-glucopyranoside).
- 01 triterpenoid compound: taraxerol
- 02 phenolic compounds: scopoletin and acid vanillic
- 02 sterol compounds: β-sitosterol, -sitosterol-3-O--D-glucopyranoside.
4. Test of anti-inflammatory biological activity and antifungal activity that causes
plant diseases. Evaluate the antifungal activity of compounds isolated with C.
acutatum, C. fragariae, C. gloeosporioides, F. oxysporum, B. cinerea và P.
obscurans.
6. Outline of the thesis


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It is displayed in a total of 112 pages with 21 tables, 19 figures, 3 diagrams.
Its major sections include: Introduction (4 pages), overview (25 pages), methods
and experiment (20 pages), results and discussion (69 pages), conclusion (1
pages), published works (1 page). Morever, there is an appendix with 130 spectra
of the compounds.

CHAPTER 1: OVERVIEW
The thesis has conducted a literature review content:
1. Family Meliaceae
- Introduction of common plant characteristics of species belonging to the
Meliaceae family, the main chemical composition of family Meliaceae as
limonoid mono-, di-, sesqui-, and triterpenoid, coumarin, chromone, lignan,
flavonoid and phenolic compounds… Besides, the review also mentions the
biological activity and application of the family Meliaceae in agriculture,
pharmaceutical ...
2. Species Aphanamixis polystachya

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MS, HR-ESI-MS), nuclear magnetic resonance spectroscopic H-NMR, C1 1
NMR, DEPT and HSQC, HMBC, H- H COSY
2.1.4. Methods of bioactivity assay
Test anti-inflammatory activity and plant anti-fungal activity. Evaluate the
antifungal activity of compounds isolated with C. acutatum, C. fragariae, C.
gloeosporioides, F. oxysporum, B. cinerea và P. obscurans.
2.2. Chemicals and equipment
2.2.1. Chemicals: Solvents for extraction plant material are pure. The solvents
for thin layer chromatography, fast column chromatography are pure analytical
(PA).
2.2.2. Equipment: Melting temperature was determined with a Yanaco MP-S3;
The polarity was determined with a Jasco DIP -370 polarimeter. Thin layer
chromatography (TLC); column chromatography (CC); lash column
chromatography (FC); high performance liquid chromatography (HPLC);
ultraviolet (UV), infrared (IR), mass spectrometry (ESI-MS, HR-ESI-MS),
nuclear magnetic resonance spectroscopic (NMR), optical rotation and melting
points measurement;
2.3. Study on compounds from fruits of Aphanamixis polystachya
2.3.1. Collecting samples
The fruits of Anphanamixis polystachya was collected at the Vu Quang
National Park of Ha Tinh Province, Vietnam, in August 2013 and identified by
Assoc. Prof. Dr. Tran Huy Thai, Institute of Ecology and Biological Resources Vietnam Academy of Science and Technology. A voucher specimen (DHV-2013)
was deposited at the herbarium of the Department of Chemistry, Vinh University.
2.3.2. Isolating substances
The fruits of Anphanamixis polystachya (4.0 kg) were air-dried and
powdered and soaked with methanol at room temperature for 7 days, and the
combined extracts were concentrated under reduced pressure to give deep brown
syrup (290.0 g). The crude extract was suspended into water and partitioned with
cloroform and butanol, successively to afford cloroform (106 g), butanol (35.0 g),

mg).
2.4. Study on chemical constituents of fruits of Khaya senegalensis A. Juss
2.4.1. Collecting samples
The fruits of Khaya senegalensis A. Juss was collected at the Nghe An
Province, Vietnam, in January 2014 and identified by Assoc. Prof. Dr. Tran Huy
Thai, Institute of Ecology and Biological Resources - Vietnam Academy of
Science and Technology. A voucher specimen was deposited at the herbarium of
the Department of Chemistry, Vinh University.
2.4.2. Isolating substances
The fruits of Anphanamixis polystachya (4.0 kg) were air-dried and
powdered and soaked with methanol at room temperature for 7 days, and the
combined extracts were concentrated under reduced pressure to give deep brown


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syrup (285.0 g). The crude extract was suspended into water and partitioned with
n-hexane and ethyl acetate, successively to afford n-hexane (55g), butanol (95.0
g), respectively, after removal of the corresponding solvent.
The ethyl acetate soluble extracts were purified by silica gel column
chromatography eluted with n-hexane and acetone gradients (100:0; 50:1; 39:1;
30:1; 20:1; 15:1; 9:1; 4:1; 2:1; 1:1) to afford 7 fractions (F1 to F7).
Fraction F3 was subjected to silica gel column chromatography eluted with
n-hexane/acetone (20:1; 15:1; 9:1; 2:1) to afford five subfractions (F3-1 to F3-5).
Subfraction F13-3 was subjected to the silica gel column chromatography eluted
with n-hexane/acetone (9:1; 4:1) to afford KS1 (15 mg) and KS3 (29 mg).
Fraction F4 was subjected to silica gel column chromatography eluted with
chloroform:methanol (9:1) to afford KS4 (12.5 mg).
Fraction F5 was subjected to silica gel column chromatography eluted with
n-hexane/acetone (15:1; 9:1; 4:1; 2:1) to afford seven subfractions (F5-1 to F5-7).

chloroform: methanol (10:1) to afford MA4 (51 mg)
The n-butanol soluble extract was fractionated by silica gel column
chromatography eluted with chloroform and methanol gradients (30:1; 20:1;
10:1: 4:1; 2:1) to afford 5 fractions. Fraction 2 was subjected to the silica gel
column chromatography eluted with chloroform: methanol (15:1) to afford MA1
(12 mg). Fraction 3 was subjected to the silica gel column chromatography
eluted with chloroform: methanol (10:1) to afford MA2 (21.5 mg). Fraction 5
was subjected to the silica gel column chromatography eluted with chloroform:
methanol (10:1) to afford MA7 (41 mg)


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2.6. Biological activity test
Test anti-inflammatory activity and plant anti-fungal activity. Evaluate the
antifungal activity of compounds isolated with C. acutatum, C. fragariae, C.
gloeosporioides, F. oxysporum, B. cinerea và P. obscurans.
CHAPTER 3: RESULTS AND DISCUSSION
3.1. Fruits of Anphanamixis polystachya
Table 3.1: Isolated compounds from fruits of Anphanamixis polystachya
No
Symbol
Name of the compound
Mass (mg)
1
2
3
4
5
6

3
4
5
6

KS1
KS2
KS3
KS4
KS5
KS6

Seneganolide
Khayanone
Khayanolide B
6-Acetoxy-methyl angolensate
(-)-Epicatechin
Quercitrin

34
32
9
22
10
20

3.3. Fruits of Melia azedarach
3.3.1. Isolated compounds
The process of isolating the substances from the fruits of Melia azedarach
was presented in detail in the experimental section. In the course of our research

MA7
MA8

New
Apigenin
Quercetin
3-O-[-Lrhamnopyranosyl-(1→6)]--Dglucopyranoside
Scopoletin
Acid vanillic
Taraxerol
β-sitosterol
-sitosterol-3-O--Dglucopyranoside

14
12
21,5

28
31
25
128
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3.1.2. Structural elucidation of new compound MA1
Compound MA1 was isolated as optically active colorless syrup and the
HRESIMS analytical data of 1 revealed the molecular formula as C37H44O9 (m/z
655.2879, [M+Na]+). The UV absorption maxima at 205, 218, 244, and 273 nm
indicated the presence of a conjugated benzene chromophore [99]. The 1H-NMR
spectrum of MA1 (Table 3.2) displayed the characteristic resonances for four
methyl singlets [H 1.00 (6H, CH3-20, -22), 1.18 (3H, CH3-18), and 1.25 (3H,

3.56 (br s)
71.8
20
1.00 (s)
15.8
2
1.53 (m). 2.27 (m)
24.3
21
1.25 (s)
27.1
3
5.08 (br s)
72.8
22
1.00 (s)
15.4
4
42.3
23
124.6
5
2.55 (d. 12.5)
40.3
24
7.13 (br s)
140.2
6
4.27 (dd. 12.5. 3.0) 73.6
25

1’
165.0
13
40.2
2’
130.6
14
155.7
3’
8.08 (d. 7.5)
129.5
15
5.70 (br s)
122.7
4’
7.43 (t. 7.5)
128.3
16
2.32 (m)
36.6
5’
7.56 (t. 7.5)
132.9
17 2.97 (dd. 10.8. 7.5) 50.4
6’
7.43 (t. 7.5)
128.3
18
1.18 (s)
18.8

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Figure 3.21. The 13C-NMR spectra of MA1


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Figure 3.22. The 13C-NMR spectra of MA1

Figure 3.25. The DEPT spectra of MA1


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Figure 3.27. The HMBC spectra of MA1

Figure 3.28. The HSQC spectra of MA1


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Figure 3.29. The COSY spectra of MA1
3.3. Antimicrobial and antifungal activity of compounds
The food-poisoned technique-used to quantify the antibiotic activity of
compounds against Fusarium oxysporum is determined by mixing the substance
into the molten PDA environment according to the test concentration (200 ppm
and 500 ppm). Petri dishes after transplanting are incubated at 25 ° C after 3 and
7 days and measuring the diameter of the fungus. The antifungal effect is
calculated according to the formula:
CV (%) =100 × (Dc-Dt)/(Dc-4)


9
3

5

2

15

8

10

10

7

3

6

4

O

13

16


22

21
20

14

8
7

15

OAc

OAc

(AP-1) Dysobinin

(AP-2) Chisocheton compound G
O
O

23

22

21

21
12


OAc
OAc

5

25

26

8
7

6

O

O

16

14

2
8
7

20
17



29
28
21
18
19

24
23

13
9

2

17

11
8

14

10
7

HO

3

4


5'
3'

2'

O

3

4

5

23
17

13
8

16

14 15

10

6'

27
24


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The chemical compounds isolated from the fruits of Khaya senegalensis

(KS-1) Seneganolide

(KS-3) Khayanolide B

(KS-5) (-)-Epicatechin

(KS-2) Khayanone

(KS-4) 6-Acetoxy-methyl angolensate

(KS-6) Quercitrin


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The chemical compounds isolated from the fruits of Melia azedarach

(MA-1) 3α,12α-diacetoxy-7α-benzoyloxy-1α-hydroxytrichilinin

(MA-2) Apigenin

(MA-3) Quercetin 3-O-[-L-rhamnopyranosyl(1→6)]--D-glucopyranoside)

(MA-4) Scopoletin


KS4 against Fusarium oxysporum. The results showed that KS3 has strong
antifungal activity with inhibitory effect of over 80% at 500ppm concentration
and no decline after 7 days of culture.


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PUBLISHED WORKS
1. Vu Thi Hien, Tran Thi Ngoc Han, Pham Thi Hang, Tran Dinh Thang
(2013), The tertranortriterpenoid compounds from the fruits of
Anphanamixis polystachya (Wall.) Parker) (Meliaceae) in Vu Quang – Ha
Tinh, Journal of Chemistry, T.51 (6ABC), 68-71.
2. Vu Thi Hien, Nguyen Thi Thanh Phuong, Tran Dinh Thang (2014), The
limonoid compounds from the fruits of Anphanamixis polystachya (Wall.)
Parker) (Meliaceae), Journal of Science and Technology, T.52 (5A), 49-54.
3. Vu Thi Hien, Vu Dinh Hoang (2016), Chemical constituents of fruits of
Khaya senegalensis A. Juss growing in Vietnam, Vietnam Journal of
Chemistry, International Edition, 54(6) 781-785.
4. Vu Thi Hien, Nguyen Ngoc Tuan, Hoang Van Luu, Vu Dinh Hoang (2018),
Isolation and structure determination of compounds from Melia azedarach
fruits in Vietnam, Journal of Science and Technology (Technical
Universities) (Accepted)
5. Vu Dinh Hoang, Vu Thi Hien, Nguyen Ngoc Tuan, Ping-Chung Kuo,
Tian-Shung Wu, Tran Dinh Thang (2018), Chemical constituents of fruits of
Melia azedarach growing in Vietnam, Nat. Prod. Res. (submitted).