nghiên cứu thành phần hóa học và hoạt tính gây độc tế bào ung thư của cây tốc thằng cáng anodendron paniculatum wall ex roxb adc

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HUE UNIVERSITY

HUE UNIVERSITY’S COLLEGE OF SCIENCES

…………***…………

HOANG THI NHU HANH

STUDY ON CHEMICAL CONSTITUENTS AND

CYTOTOXIC ACTIVITY OF ANODENDRON

PANICULATUM (WALL. EX ROXB.) A.DC.

Major: Organic Chemistry

Code: 62.44.01.14

SUMMARY OF DISSERTATION ON

DOCTOR OF PHILOSOPHY IN CHEMISTRY

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The dissertation was completed at:

Hue University

Hue University’s College of Sciences

Scientific Supervisors: Asc. Prof. Dr. NGUYEN THI HOAI

Faculty of Pharmacy - Hue University of Medicine and Pharmacy -
Hue University

1st Reviewer:

2nd Reviewer:

3rd Reviewer:

The dissertation will be defended at Hue University at…………..

The dissertation can be found in:
-

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PREFACE

The rapid increase of many dangerous diseases which are highly
capable of infection like HIV/AIDS, cancer, SARS (Severe Acute
Respiratory Syndrome), H5N1 (avian flu), H1N1 (swine flu), Ebola

epidemic, ... is really critical with regard to community health and
also a considerable challenge for the world health system. With a
view to protecting human from theses diseases, treatments which are
not only effective but also safe for human body. Many studies from
around the world as well as in Viet Nam has shown that plants are
indeed a valuable source for drug discovery. Some typical examples
are taxol from Taxus brevifolia, vinblastine and vincristine from 
Catharanthus roseus, camptothecin from Camptotheca acuminata, 
and podophyllotoxin from Podophyllum peltatum…

In screening the cytotoxic activity in some medicinal plants of
the Pako and Van Kieu (ethnic minority groups), ten medicinal plants
which are associated with anti-cancer activity have been screened for
in vitro cytotoxic activity. Among them, Anodendron paniculatum 
(Wall. ex Roxb.) A.DC. showed inhibitory effect against five human

cancer cell lines with low IC50 values, especially on LU-1, Hep-G2,

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The objects of the dissertation are:

1. Demonstrating the main chemical constituents of Anodendron 
paniculatum (Roxb.) A.DC.

2. Evaluating the cytotoxic activity of isolated compounds 
against cancer cell lines.

Contents of the dissertation are:

1. Extraction, isolation and purification of compounds from 
Anodendron paniculatum (Roxb.) A.DC.

2. Structural determination of isolated compounds.

3. Evaluation of the cytotoxic activities of isolated compounds 
against human cancer cell lines.

New contributions of the dissertation are:

1. Four new compounds (anopaniester, anopanin A–C), together 
with sixteen known compounds were isolated from Anodendron 
paniculatum collected in Viet Nam. Among the known compounds, 
fourteen compounds, (E)-phytol, cycloartenol, desmosterol,
esculentic acid, kaempferol-3-O-rutinoside, rutin, sargentol, 4-O-β-D
-glucopyranosyl-3-prenylbenzoic acid, inugalactolipid A,
gingerglycolipid A–C, (2S)-1-O-palmitoyl-3-O-[-D
-galactopyranosyl-(1→6)-O-β-D-galactopyranosyl]glycerol and

(2R)-1-O-palmitoyl-3-O-α-D-(6-sulfoquinovopyranosyl)glycerol were
isolated from this genus for the first time.

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O-α-D-(6-sulfoquinovopyranosyl)glycerol showed inhibitory
activities against the LU-1 and MKN-7 cell lines with IC50 values

ranging from 30.89±3.60 to 72.42±8.05 µg/mL.

Chapter 1: OVERVIEW 
1.1. Introduction to the family Apocynaceae 
1.2. Introduction to Anodendron genus 
1.2.1. Botanical classification

1.2.2. Botanical characteristics and distribution 
1.2.3. Investigation of chemical constituents 
1.2.4. Investigation of biological activities

1.3. Introduction to Anodendron paniculatum (Roxb.) A.DC.

Chapter 2: OBJECTS AND METHODS 
2.1. Plant materials

The aerial parts of Anodendron paniculatum (Roxb.) A.DC.
2.2. Methods

2.2.1. Isolated and purified method

Compounds were isolated and purified by using a combination
of various chromatographic methods involving thin-layer
chromatography, column chromatography on different stationary

phases such as Silica gel, YMC RP-18, Sephadex LH-20, and Diaion
HP-20.

2.2.2. Method of determining the chemical structures

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 Method of determining the monosaccharide components 
The saponin sample was hydrolyzed by TFA 4 M at 105oC for 4

hours. The monosaccharide was then reduced to alditol with NaBH4

and acetylated with a mixture of acetic anhydride-pyridine (1: 1, v/v)
at 110oC for 2 hours. The alditol per-acetate formed was analyzed by

GC-MS method. The monosaccharide components were identified by
comparing their retention time relative to internal standard.

 Method of determining the fatty acid components of 
glycolipid

Glycolipids are dissolved in toluene and then MeOH, 8% HCl
solution (in MeOH) were added in this order. The mixture is
incubated at 45oC for two hours (partial hydrolysis) or overnight (for

complete hydrolysis). After cooling to room temperature, n-hexane 
and water were added for extraction of the methyl ester derivatives
(FAMEs). FAMEs were then analyzed by GC-MS method.

2.2.3. Biological evaluation

The in vitro cytotoxicity of isolated compounds against the

growth of eight human cancer cell lines (HL-60, LNCaP, MCF-7,
LU-1, Hep-G2, KB, MKN-7 and SW-480) was tested by
sulforhodamine B assay at Institute of Biotechnology, Viet Nam
Academy of Science and Technology.

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The dry material (2.5 kg) was extracted with MeOH (10.0 liters
x 3) at room temperature (72 hours/time). The solvent was removed
under reduced pressure to obtain MeOH extract (105 g). The MeOH
extract was dispersed in water and then extracted with chloroform
(CHCl3, 2 liters x 3 times), ethyl acetate (EtOAc, 2 liters x 3 times).

The solvents was removed under reduced pressure to obtain CHCl3

(AC, 50.7 g), EtOAc (AE, 10.2 g) and water (AW, 27.5 g).
3.2. Isolation and purification procedure

Stationary phase Mobile phase

(1) Silica gel Gradient CHCl3–MeOH (100:00:100, v/v)

(2) Silica gel Gradient n-hexan–acetone (100:04:1, v/v)
(3) Silica gel n-hexan–EtOAc (10:1, v/v)

(4) YMC RP-18 MeOH–acetone–H2O (10:5:1, v/v)

(5) Silica gel n-hexan–acetone (5:1, v/v) 
(6) Silica gel CHCl3–MeOH (20:1, v/v)

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Stationary phase Mobile phase

(1) Diaion HP-20 Gradient MeOH−H2O (0:1, 1:3, 1:1, 3:1, 1:0, v/v)

(2) YMC RP-18 MeOH–H2O (1:1, v/v)

(3) Silica gel CHCl3–MeOH–H2O (3:1:0,1, v/v)

(4) Sephadex LH-20 MeOH

(5) Silica gel CHCl3–MeOH–H2O (5:1:0,1, v/v)
(6) Silica gel CHCl3–MeOH–H2O (4:1:0,1, v/v)

(7) Sephadex LH-20 MeOH–H2O (4:1, v/v),

(8) YMC RP-18 MeOH–MeCN–H2O (3:2:3, v/v)

(9) Silica gel CHCl3–MeOH–H2O (3:1:0,1, v/v)
(10) Silica gel CHCl3–MeOH–H2O (3:1:0,1, v/v)
(11) YMC RP-18 MeOH–H2O (3:2, v/v)

(12) YMC RP-18 MeOH–H2O (6:1, v/v)

(13) Silica gel EtOAc–MeOH–H2O (3:1:0,1, v/v)

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3.3. Physical properties and spectral data of isolated compounds 
3.3.1. Compound AP1: Cycloartenol

White powder; IR (KBr) max (cm-1): 3418, 2928, 1670, 1443,

1377; Molecular formula C30H50O; M = 426; 1H- and 13C-NMR: see

Table 4.1 and Appendix 1.

3.3.2. Compound AP2: Anopaniester (New)

Colorless oil; IR (KBr) max (cm-1): 3443, 2922, 2853, 1736, 1630,

1466, 1377, 1260, 1180, 1094; UV (MeOH) max (nm): 201, 231;

HRESIMS: m/z 725.5805 [M+Na]+ (calcd forC

48H78O3Na, 725.5849);

Molecular formula: C48H78O3; M = 702; 1H-NMR (500 MHz, CDCl3)

H (ppm): 1.22 (H-1a), 1.60 (H-1b), 1.61 (H-2a), 1.74 (H-2b), 4.56 (dd,

J = 11.0, 4.5 Hz, H-3), 1.42 5), 0.79 6a), 1.57 6b), 1.08 
(H-7a), 1.31 (H-7b), 1.52 (dd, J = 12.0, 4.5 Hz, H-8), 1.11 (H-11a), 1.98 
(H-11b), 1.61 (H-12), 1.28 (H-15), 1.29 (H-16a), 1.92 (H-16b), 1.58
(H-17), 1.96 (s, H-18), 0.33 (br.d, J = 4.0 Hz, H-19a), 0.57 (br.d, J = 
4.0 Hz, H-19b), 1.38 (H-20), 0.88 (d, J = 7.0 Hz, H-21), 1.04 (H-22a), 
1.41 (H-22b), 1.86 (H-23a), 2.03 (H-23b), 5.10 (t, J = 6.0 Hz, H-24), 
1.68 (s, H-26), 1.60 (s, H-27), 0.84 (s, H-28), 0.89 (s, H-29, H-30),
2.30 (H-2), 1.61 (H-3), 1.30 (H-4), 1.30 (H-5), 1.30 (H-6), 1.36
(H-7), 2.17 (td, J = 8.0, 7.0 Hz, H-8), 5.43 (td, J = 10.5, 8.0 Hz, H-9),
5.97 (dd, J = 11.5, 10.5 Hz, H-10), 6.51 (dd, J = 15.0, 11.5 Hz, 
H-11), 5.69 (dd, J = 15.0, 6.5 Hz, H-12), 4.20 (m, H-13), 2.33 (H-14),
5.36 (td, J = 10.5, 8.0 Hz, H-15), 5.57 (td, J = 10.5, 7.5 Hz, H-16),
2.06 (m, H-17), 0.97 (t, J = 7.5 Hz, H-18); 13C-NMR (125 MHz,

CDCl3) C (ppm): 31.7 (C-1), 27.0 (C-2), 80.5 (C-3), 39.6 (C-4), 47.3

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(C-16), 52.4 (C-17), 18.1 (C-18), 29.9 (C-19), 36.0 (C-20), 18.4 (C-21),
36.5 (C-22), 25.1 (C-23), 125.3 (C-24), 131.0 (C-25), 25.9 (C-26), 17.8
(C-27), 25.6 (C-28), 15.4 (C-29), 19.4 (C-30), 173.8 (C-1), 35.0
(C-2), 25.2 (C-3), 29.1 (C-4), 29.2 (C-5), 29.6 (C-6), 29.8 (C-7), 27.8
(C-8), 133.1 (C-9), 127.9 (C-10), 126.0 (C-11), 135.2 (C-12), 72.3
(C-13), 35.4 (C-14), 123.9 (C-15), 135.3 (C-16), 20.9 (C-17), 14.4
(C-18).

3.3.3. Compound AP3: (E)-Phytol

Colorless oil; IR (KBr) max (cm-1): 3449, 2932, 1636, 1462,

1381, 1003; HRESIMS: m/z 319.2931 [M + Na]+ (calcd for

C20H40ONa, 319.2977); Molecular fomular C20H40O; M = 296; 1H-

and 13C-NMR: see Table 4.4 and Appendix 3. 
3.3.4. Compound AP4: Desmosterol

White powder; mp: 122–124oC; IR (KBr) 

max (cm-1): 3445,

2936, 1636, 1462, 1377, 1053; HRESIMS: m/z 385.3513 [M + H]+

(calcd for C27H45O, 385.3470); Molecular formula C27H44O; M = 384;
1H- and 13C-NMR: see Table 4.5 and Appendix 4.

3.3.5. Compound AP5: Ursolic acid 
White powder; 20

[ ]



D +65 (c 0.5, EtOH), mp 284–286oC, UV

(MeOH) λmax (nm): 201, 230; IR (KBr) νmax (cm-1): 3433, 2870, 1690;

ESIMS: m/z 479.2 [M + Na]+, 455.2 [M - H]-; Molecular formula

C30H48O3; M = 456; 1H- and 13C-NMR: see Table 4.6 and Appendix 5.
3.3.6. Compound AP6: Vanillin

White powder; mp 82–84oC, UV (MeOH) λ

max (nm): 204, 231, 278,

309; IR (KBr) νmax (cm-1): 3310, 1674, 1589, 1512, 1026, ESIMS: m/z

175.0 [M + Na]+; Molecular formula C

8H8O3; M = 152; 1H- and 13

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3.3.7. Compound AP7: Esculentic acid 
White powder; mp 267–268oC; IR (KBr) ν

max (cm-1): 3425, 2927,

2363, 1690, 1458, 1038; HRESIMS: m/z 511.3390 [M + Na]+ (calcd

for C30H48O5Na, 511.3399); Molecular formula C30H48O5; M = 488;

1H- and 13C-NMR: see Table 4.8 and Appendix 7.

3.3.8. Compound AP8: Kaempferol-3-O-rutinoside

Yellow powder;

[ ]



20D +11 (c 0.8, MeOH); mp 188–190oC, UV

(MeOH) λmax (nm): 203, 267, 351; IR (KBr) νmax (cm-1): 3410, 1659,

1605, 1497, 1566, 1180, 1065; ESIMS: m/z 617.1 [M + Na]+, 593.1 [M

- H]-; Molecular formula C

27H30O15; M = 594; 1H- and 13C-NMR: see

Table 4.9 and Appendix 8.
3.3.9. Compound AP9: Rutin

Yellow powder; 20

[ ]



D+4 (c 0.5, MeOH), mp 193–195oC, UV

(MeOH) λmax (nm): 204, 257, 357; IR (KBr) νmax (cm-1): 3418, 1651,

1605, 1504 , 1204, 1065; ESIMS: m/z 633.1 [M + Na]+, 609.1 [M - H]-;

Molecular formula C27H30O16; M = 610; 1H- and 13C-NMR: see Table

4.10 and Appendix 9.

3.3.10. Compound AP10: Anopanin A (New)

White powder; mp: 213–215oC; 22

[ ]



D -19.9 (c 0.1, MeOH); IR 
(KBr) max (cm-1): 3418, 2932, 1713, 1632, 145, 1383, 1265, 1070,

1030; HRESIMS: m/z 1025.5294 [M + Na]+ (calcd for C

50H82O20Na,

1025.5297); Molecular formula C50H82O20; 1H- and 13C-NMR: see

Table 4.11, 4.12.

3.3.11. Compound AP11: Anopanin B (New) 
White powder; mp: 222–224oC; 22

[ ]



D +77.7 (c 0.1, MeOH); IR 
(KBr) max (cm-1): 3443, 2930, 1730, 1647, 1460, 1377, 1252, 1074,

1028; HRESIMS: m/z 1027.5452 [M + Na]+ (calcd forC

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1027.5454); Molecular formula C50H84O20; 1H-NMR (600 MHz,

C5D5N) H (ppm): 1.11 (H-1a), 1.43 (H-1b), 1.84 (m, H-2a), 2.27 (m,

H-2b), 3.38 (dd, J = 11.4, 4.2 Hz, H-3), 1.26 (H-5), 0.68 (q, J = 12.6 
Hz, H-6a), 1.51 (H-6b), 1.03 (H-7a), 1.22 (H-7b), 1.52 (H-8), 1.06
(H-11a), 1.98 (H-11b), 1.60 (H-12a), 1.66 (H-12b), 1.48 (H-15a),
2.01 (H-15b), 5.27 (dd, J = 6.0, 6.0 Hz, H-16), 2.15 (s, CH3COO),

2.08 (dd, J = 10.2, 6.6 Hz, H-17), 1.01 (s, H-18), 0.20 (d, J = 4.2 Hz, 
H-19a), 0.46 (d, J = 4.2 Hz, H-19b), 1.78 (H-20), 1.02 (d, J = 6.0 Hz, 
H-21), 1.74 (H-22), 1.88 (H-23), 3.76 (m, H-24), 1.52 (s, H-26), 1.55
(s, H-27), 1.30 (s, H-28), 1.14 (s, H-29), 1.13 (s, H-30), 4.86 (d, J = 
7.2 Hz, H-1), 4.29 (H-2), 4.30 (H-3), 4.26 (H-4), 3.84 (ddd, J = 
9.6, 3.6, 3.0 Hz, H-5), 4.49 (H-6a), 4.53 (H-6b), 5.42 (d, J = 7.8, 
H-1), 4.10 (H-2), 4.23 (H-3), 4.34 (H-4), 3.94 (ddd, J = 9.6, 3.6, 
3.6 Hz, H-5), 4.48 (H-6a), 4.52 (H-6b), 5.16 (d, J = 7.8 Hz, 
H-1‴), 4.09 2‴), 4.26 3‴), 4.24 4‴), 4.00 (m, H-5‴), 4.32
(H-6‴a), 4.51 (H-6‴b); 13C-NMR (150 MHz, C

5D5N) C (ppm): 32.5

(C-1), 30.3 (C-2), 89.4 (C-3), 41.7 (C-4), 48.2 (C-5), 21.5 (C-6), 26.8
(C-7), 48.0 (C-8), 19.8 (C-9), 26.8 (C-10), 27.1 (C-11), 33.5 (C-12),
47.0 (C-13), 48.0 (C-14), 46.2 (C-15), 80.8 (C-16), 171.2 (CH3COO),

22.0 (CH3COO), 58.1 (C-17), 19.5 (C-18), 30.4 (C-19), 34.7 (C-20),

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3.3.12. Compound AP12: Anopanin C (New) 
White powder; mp: 218–219oC; 22

[ ]



D +66.7 (c 0.1, MeOH); IR

(KBr) max (cm-1): 3428, 2932, 1709, 1630, 1456, 1381, 1261, 1072,

1028; HRESIMS: m/z 1025.5293 [M + Na]+ (calcd for C

50H82O20Na,

1025.5297); Molecular formula C50H82O20; 1H-NMR (600 MHz,

C5D5N) H (ppm): 1.07 (H-1a), 1.42 (H-1b), 1.84 (m, H-2a), 2.27 (m,

H-2b), 3.38 (dd, J = 11.4, 4.2 Hz, H-3), 1.25 (H-5), 0.68 (q, J = 12.6 
Hz, H-6a), 1.51 (H-6b), 1.01 (H-7a), 1.23 (H-7b), 1.52 (H-8), 1.04
(H-11a), 1.92 (H-11b), 1.62 (H-12a), 1.66 (H-12b), 1.48 (H-15a),
2.01 (m, H-15b), 5.33 (dd, J = 7.8, 7.2 Hz, H-16), 2.21 (s, CH3COO),

2.51 (dd, J = 10.8, 6.0 Hz, H-17), 1.03 (s, H-18), 0.20 (d, J = 3.6 Hz, 
H-19a), 0.46 (d, J = 3.6 Hz, H-19b), 1.68 (m, H-20), 1.21 (d, J = 6.6 
Hz, H-21), 4.19 (m, H-22), 2.10 (m, H-23a), 2.29 (m, H-23b), 4.28
(H-24), 6.41 (d, J = 4.8 Hz, 24-OH), 1.31 (s, H-26), 1.53 (s, H-27), 
1.30 (s, 28), 1.14 (s, 29), 1.06 (s, 30), 4.86 (d, J = 7.8 Hz, 
H-1), 4.29 (H-2), 4.30 (H-3), 4.25 (H-4), 3.83 (ddd, J = 9.6, 3.6, 3.0 
Hz, H-5), 4.48 (H-6a), 4.53 (H-6b), 5.42 (d, J = 7.2 Hz, H-1),
4.10 (H-2), 4.23 (H-3), 4.35 (H-4), 3.94 (ddd, J = 9.6, 3.6, 3.6 Hz, 
H-5), 4.46 (H-6a), 4.51 (H-6b), 5.16 (d, J = 8.4 Hz, H-1‴), 4.09 
(H-2‴), 4.25 (H-3‴), 4.24 (H-4‴), 4.00 (m, H-5‴), 4.32 (H-6‴a),
4.50 (H-6‴b); 13C-NMR (150 MHz, C

5D5N) C (ppm): 32.5 (C-1),

30.3 (C-2), 89.4 (C-3), 41.7 (C-4), 48.2 (C-5), 21.5 (C-6), 26.7 (C-7),
48.0 (C-8), 19.8 (C-9), 26.8 (C-10), 27.1 (C-11), 33.5 (C-12), 47.1
(C-13), 48.0 (C-14), 46.2 (C-15), 80.3 (C-16), 171.0 (CH3COO), 22.1

(CH3COO), 56.2 (C-17), 19.4 (C-18), 30.4 (C-19), 38.3 (C-20), 12.8

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82.2 (C-2), 77.1 (C-3), 81.6 (C-4), 76.5 (C-5), 62.6 (C-6), 105.9

(C-1), 77.6 (C-2), 78.6 (C-3), 72.2 (C-4), 78.7 (C-5), 63.3
(C-6), 105.4 1‴), 75.3 2‴), 78.3 3‴), 71.9 4‴), 78.9
(C-5‴), 62.7 (C-6‴).

3.3.13. Compound AP13: Sargentol 
White powder; 20

[ ]



D –70 (c 0.05, MeOH), mp 271–272

oC, UV

(MeOH) λmax (nm): 207, 273; IR (KBr) νmax (cm-1): 3387, 1597, 1504,

1466, 1234, 1072; ESIMS: m/z 325 [M–H–2OCH3]-; Molecular

formula C17H24O10, M = 388; 1H- and 13C-NMR: see Table 4.17 and

Appendix 13.

3.3.14. Compound AP14: 4-O-β-D

-Glucopyranosyl-3-prenylbenzoic acid

White powder; UV (MeOH) λmax (nm): 250, 205; IR (KBr) νmax

(cm-1): 3379, 2916, 1694, 1605, 1504, 1250, 1084, 1042; HRESIMS:

m/z 391.1398 [M + Na]+ (calcd for C

18H24O8Na, 391.1369);

Molecular formula C18H24O8, M = 368; 1H- and 13C-NMR: see Table

4.18 and Appendix 14.

3.3.15. Compound AP15: Inugalactolipid A

White powder; HRESIMS: m/z 937.5842 [M + Na]+ (calcd for

C49H86O15Na, 937.5864); Molecular formula C49H86O15; M = 914; 1H-

and 13C-NMR: see Table 4.19 and Appendix 15. 
3.3.16. Compound AP16: Gingerglycolipid A

White powder; [ ]



22D +35.7 (c 0.1, MeOH); HRESIMS: m/z 
763.3423 [M+Na+4xO]+ (calcd for C

33H56O18Na, 763.3364);

Molecular formula C33H56O14; M = 676; 1H- and 13C-NMR: see Table

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3.3.17. Compound AP17: Gingerglycolipid B 
White powder; 22

[ ]



D +57.0 (c 0.1, MeOH); HRESIMS: m/z 
701.3721 [M+Na]+ (calcd for C

33H58NaO14, 701.3724); Molecular

formula C33H58O14; M = 678; 1H- and 13C-NMR: see Table 4.21 and

Appendix 17.

3.3.18. Compound AP18: Gingerglycolipid C

White powder; [ ]



22D +29.4 (c 0.1, MeOH); HRESIMS: m/z 
703.3878 [M+Na]+ (calcd for C

33H60NaO14, 703.3881); Molecular

formula C33H60O14; M = 680; 1H- and 13C-NMR: see Table 4.22 and

Appendix 18.

3.3.19. Compound AP19: (2S)-1-O-Palmitoyl-3-O-[-

D-galactopyranosyl-(1→6)-O-β-D-galactopyranosyl]glycerol

White powder; [ ]



22D +51.6 (c 0.1, MeOH); HRESIMS: m/z 
677.3746 [M+Na]+ (calcd for C

31H58O14Na, 677.3724); Molecular

formula C31H58O14; M = 654; 1H- and 13C-NMR: see Table 4.23 and

Appendix 19.

3.3.20. Compound AP20: (2R)-1-O-Palmitoyl-3-O-α-D-

(6-sulfoquinovopyranosyl)glycerol

White powder; IR (KBr) νmax (cm-1): 3418, 2932, 2367, 1713, 1383,

1265, 1070; HRESIMS: m/z 579.2797 [M+Na]+ (calcd for

C25H48O11SNa, 579.2815); Molecular formula C25H48O11; M = 556;
1H- and 13C-NMR: see Table 4.24 and Appendix 20.

3.4. Cytotoxicity of isolated compounds

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Table 3.1. Cytotoxicity ofisolated compounds against human 
cancer cell lines

Chapter 4: RESULTS AND DISCUSSTION 
4.1. The structure elucidation of isolated compounds

Twenty compounds were isolated from Anodendron 
paniculatum by using a combination of chromatographic methods 
Among these, there are 4 new compounds, 14 compounds which
were isolated from Anodendron genus for the first time. All isolated 
compounds are shown in Table 4.25. The structural elucidation of a
new compound (AP10) was described as follow:

Compound AP10 was obtained as a white amorphous powder. 
The HRESIMS of AP10 showed a quasimolecular ion peak at m/z 
1025.5294 [M+Na]+. Its molecular formula was thus determined to be

C50H82O20 by HRESIMS in conjunction with NMR data analysis. The

IR spectrum of AP10 revealed strong absorption bands corresponding

to an ester (1713 cm-1), double bond (1632 cm-1), ethers (1010, 1070

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The 1H NMR spectrum of AP10 in pyridine-d

5 showed typical

signals of seven tertiary methyl groups (each, 3H, s) at δH 1.13

(H-29), 1.25 (H-30), 1.30 (H-28), 1.57 (H-18), 1.66 (H-27), 1.67 (H-26),
and 1.69 (H-21); one acetoxy group at δH 2.06 (3H, s), and three

anomeric protons at δH 4.86 (1H, d, J = 7.2 Hz), 5.16 (1H, d, J = 7.8

Hz), and 5.41 (1H, d, J = 7.8 Hz), suggesting the presence of three 
sugar units with β-configuration. In addition, two protons of a
cyclopropyl methylene group at δH 0.22 and 0.49 (each, 1H, d, J =

4.2 Hz, H-19) were observed.

Analysis of the 13C NMR, DEPT and HMQC spectra of AP10

revealed 50 signals, of which 30 were assigned to an aglycone, 18 to
three sugar moieties, and 2 belonged to acetoxy group (δC 171.2 and

22.0). The aglycone of AP10 was deduced to be a cycloartane-type 
triterpene with two olefinic carbons of tri-substituted double bond (δC

125.1 and 132.5), three oxygenated methine carbons (δC 77.2, 79.0,

and 89.4), an oxygenated quaternary carbon (δC 76.7), and seven

quaternary methyl carbons (15.8, 18.6, 20.7, 21.2, 21.7, 26.2, and
26.4). All proton and carbon signals of AP10 were assigned by 
analyses of 2D-NMR, including HMQC, HMBC, and 1H-1H COSY.

The HMBC correlations of H3-28 (δH 1.30) and H3-29 (δH 1.13) to

C-3 (δC 89.4), C-4 (δC 41.7), and C-5 (δC 48.1) indicated the locations of

the oxygenated carbon and gem-dimethyl groups at C-3 and C-4 in 
the A ring, respectively. Similarly, the HMBC correlations between
H3-21 (δH 1.69) and C-17 (δC 56.5)/C-20 (δC 76.7)/C-22 (δC 79.0),

between H3-26 (δH 1.67)/H3-27 (δH 1.66) and C-24 (δC 125.1)/C-25

(δC 132.5) confirmed the position of two hydroxyl groups at C-20 and

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addition, the acetoxy group at C-16 (δC 77.2) was assigned from the

HMBC correlation between H-16 (δH 6.20) and carboxyl carbon at δC

171.2. Thus, the aglycone was considered to be
3,16,20,22-tetraoxygenated cycloart-24-ene.

The monosaccharide composition of sugar moiety was
determined to be D-glucose by acid hydrolysis of AP10 followed by 
reduction, per-acetylated derivatization and GCMS analysis (see
2.2.2 Section). The obtained results indicated that the single peak at
26.65 min coincide with derivative of standard D-glucose
(Sigma-Aldrich) with tR value at 26.65 min. Furthermore, the 13C NMR

signals of the sugar moiety in AP10 (Table 4.12) were closely similar 
to those of shatavarin IX. These evidences indicated the sequence of
the sugar linkages as [β-D-glucopyranosyl(1→2)][β-D
-glucopyranosyl(1→4)]-β-D-glucopyranosyl which was supported by
the HMBC correlations from H-1 (δH 5.41) to C-2 (δC 82.2), from

H-1′′′ (δH 5.16) to C-4 (δC 81.6) (Figure 4.33). The attachment of

sugar moiety at C-3 of the aglycone via glycosidic bond was deduced 
from the HMBC correlations of H-1′ (δH 4.86) to C-3 (δC 89.4), and

H-3 (δH 3.38) to C-1′ (δC 105.1).

The stereochemistry of aglycone moiety of AP10 was 
determined on the basis of the ROESY experiment. The ROESY
correlation between H-3 (δH 3.38) and H-1 (δH 1.43)/H-5 (δH

1.26)/H3-28, between H-2β (δH 1.84) and H3-29 suggested the 

-orientation for H-3, H-5 and H3-28 as well as the chair conformation

for A ring (Figure 4.34). In addition, the J values of 11.4 and 4.2 Hz 
between H-3 and H-2 (δH 1.84, 2.27) is typical for 3-axial H of

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between H-16 and H3-18/H-22 (δH 3.92), between H-17 (δH 3.19) and

H-30, and the lack of correlation between H-16 and H3-30 (δH 1.25)

revealed the -orientations of 16-OAc and H-17. The 20R

configuration was determined by the ROESY correlations of H3-21 to

H-12 (δH 1.91)/H-17, of 20-OH (δH 5.61) to H-16/H3-18 and the

lack of cross-peaks of H3-21/H3-18, 20-OH/H-17. This was

strengthened by the agreement of chemical shift of C-21 (δC 21.7)

with those of 20R,22ξ isomers [δC: 21.1 (20R,22R), 21.9 (20R,22S)],

but quite different from those of 20S,22ξ isomers of 
cholestane-3β,20,22-triol [δC: 23.8 (20S,22S), 24.2 (20S,22R)]. The absolute

configuration of C-22 chiral center was deduced by comparing δC

values of C-20 and C-22 of AP10 with those (in C5D5N) of 20R,22R

and 20R,22S forms. In 13C NMR spectrum of 20R,22R form such as

cholestane-3β,20R,22R-triol, vitexirone, frondoside A7-3, frondoside

A7-4, and stachysterone C, the carbons C-20 and C-22 resonated at

the close values of chemical shifts (C values less than 0.4 ppm). In

contrast, these values differed about 2 ppm in 20R,22S form. The 
signals of C-20, C-22 in 13C NMR spectrum of AP10 were found at

76.7 and 79.0 ppm, respectively. Therefore, the 20R,22S 
configuration was suggested for AP10. This suggestion was further

confirmed by the ROESY correlations of 22-OH (δH 5.64) to H3

-21/H-23 (δH 2.58), of H-22 to H-16, and the lack of correlations

between 22-OH and H-16/20-OH/H-23β (δH 2.93). Consequently, the

chemical structure of AP10 was elucidated to be (3β,16
,20R,22S)-16-acetoxy-20,22-dihydroxycycloart-24-ene 3-O-[β-D
-glucopyranosyl-(1→2)][β-D-glucopyranosyl-(1→4)]-β-D

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Figure 0.32. Chemical structure of AP10

Figure 0.33. Key HMBC and COSY correlations of AP10

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Table 4.11. NMR data for aglycone moiety of AP10

C δCa, b DEPT δHa, c (J, Hz) HMBC (H→C)

1 32.5 CH2 1.10 m; 1.43 m 2, 5

2 30.3 CH2 1.84 m; 2.27 m

3 89.4 CH 3.38 dd (11.4; 4.2)

4 41.7 C –

5 48.1 CH 1.26*

6 21.5 CH2 0.68 q (12.6); 1.51* 5, 10

7 26.9 CH2 1.07*; 1.23*

8 47.9 CH 1.52*

9 19.6 C

10 26.8 C –

11 27.5 CH2 1.12*; 2.06*

12 34.2 CH2 1.91*; 1.92* 11, 13, 14, 18

13 48.2 C –

14 48.2 C –

15 46.2 CH2 1.62 br.d (13.8)

2.17 dd (14.4; 8.4)

13, 14, 16, 30

16 77.2 CH 6.20 dd (7.8; 7.2) 13, 14, CH3COO-

16-OAc 171.2 C –

22.0 CH3 2.06 s CH3COO-

17 56.5 CH 3.19 d (6.6) 13, 14, 16

18 21.2 CH3 1.57 s 12, 13, 14, 17

19 30.7 CH2 0.22 d (4.2)

0.49 d (4.2)

1, 11
10, 11

20 76.7 C –

20-OH – – 5.61 s 17, 20

21 21.7 CH3 1.69 s 17, 20, 22

22 79.0# CH 3.92* 17, 23, 24

22-OH – – 5.64*

23 31.0 CH2 2.58 m; 2.93 m

24 125.1 CH 5.65* 26, 27

25 132.5 C –

26 26.4 CH3 1.67 s 24, 25, 27

27 18.6 CH3 1.66 s 24, 25, 26

28 26.2 CH3 1.30 s 3, 4, 29

29 15.8 CH3 1.13 s 3, 4, 5, 28

30 20.7 CH3 1.25 s 8, 13, 14, 15

ameasured in C

5D5N, b150 MHz, c600 MHz, *overlapping signals; #signal missing on 13C-NMR

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C δCa,b DEPT δHa,c (J, Hz) HMBC (HC)

3-Glc

1′ 105.1 CH 4.86 d (7.2) 3

2′ 82.2 CH 4.29*

3′ 77.1 CH 4.30*

4′ 81.6 CH 4.25*

5′ 76.5 CH 3.83 m

6′ 62.6 CH2 4.49*; 4.53*
2-Glc

1′′ 106.0 CH 5.41 d (7.8) 2, 2′′
2′′ 77.6 CH 4.10*

3′′ 78.6 CH 4.23*

4′′ 72.4 CH 4.32*

5′′ 78.7 CH 3.93*

6′′ 63.3 CH2 4.48*; 4.52*
4-Glc

1′′′ 105.4 CH 5.16 d (7.8) 4
2′′′ 75.3 CH 4.09*

3′′′ 78.4 CH 4.25*

4′′′ 71.9 CH 4.24*

5′′′ 78.9 CH 4.00 m
6′′′ 62.7 CH2 4.31*; 4.54*

ameasured in C

5D5N, b150 MHz, c600 MHz, glc: -D-glucopyranosyl, *overlapping signals

4.2. The in vitro cytotoxicity of isolated compounds against 
human cancer cell lines

The cytotoxicity of seventeen isolates against various cancer
cell lines (LU-1, MKN-7, Hep-G2, KB, SW-480, HL-60, LNCaP,
MCF-7) was evaluated. The results are shown in Table 3.1.
Desmosterol (AP4) exhibited moderate cytotoxicity against five cell
lines including LU-1, MKN-7, HepG2, KB, SW-480 with IC50 values

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IC50 values of 44.37±5.40, 30.89±3.60 µg/mL, respectively.

(2R)-1-O-palmitoyl-3-O-α-D-(6-sulfoquinovopyranosyl)glycerol (AP20)

exhibited low cytotoxicity against LU-1, MKN-7 cell line with IC50

values of 66.66±5.85, 72.42±8.05 µg/mL, respectively. The
remaining compounds did not show significant growth inhibitory
activities (IC50>100 mg/mL) against the tested cancer cell lines.

CONCLUSION

After the implementation process, the dissertation has achieved
two objects:

1. Chemical constituents

 From the aerial parts of Anodendron paniculatum, 20 
compounds were isolated, including 4 new compounds, 14 
compounds which were isolated from Anodendron genus for the 
first time.

 Four new compounds include anopaniester, and anopanin A-C.
 Fourteen compounds which were isolated from Anodendron 
genus for the first time include (E)-phytol, cycloartenol, desmosterol, 
esculenic acid, kaempferol-3-O-rutinoside, rutin, sargentol, 
3-prenyl-4-O-β-D-glucopyranosyloxy-4-hydroxylbenzoic acid, inugalactolipid
A, gingerglycolipid A, gingerglycolipid B, 
(2S)-1-O-palmitoyl-3-O-[-D-galactopyranosyl-(1→6)-O-β-D-galactopyranosyl]glycerol,

gingerglycolipid C, and (2R)-1-O-palmitoyl-3-O-α-D

-(6-sulfoquinovopyranosyl)glycerol.

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2. Biological evaluation

 17/20 isolated compounds were evaluated for in vitro 
cytotoxicity against the growth of human cancer cell lines (LU-1, 
MKN-7, HepG2, KB, SW-480, HL-60, LNCaP, MCF-7).

 Desmosterol exhibited moderate cytotoxicity against five cell
lines including LU-1, MKN-7, HepG2, KB, SW-480 with IC50 values

ranging from 28.11±1.95 to 41.41±2.31 µg/mL.

 Ursolic acid exhibited moderate cytotoxicity against LU-1,
MKN-7 cell line with IC50 values of 44.37±5.40.30.89±3.60 µg/mL,

respectively.

 (2R)-1-O-palmitoyl-3-O-α-D-(6-sulfoquinovopyranosyl)glycerol

exhibited low cytotoxicity against LU-1, MKN-7 cell line with IC50

values of 66.66±5.85, 72.42±8.05 µg/mL, respectively.

RECOMMENDATIONS

Based on the results of chemical composition and biological
activity of Anodendron paniculatum in Viet Nam, we have following

recommendations:

- Continue to search for cytotoxic compounds from other fractions
of this plant.

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PUBLICATIONS

1. Duc Viet Ho, Hanh Nhu Thi Hoang, Hung Quoc Vo, Hien Minh 
Nguyen, AinRaal, Hoai Thi Nguyen (2018), A new triterpene ester
and other chemical constituents from the aerial parts of Anodendron 
paniculatum and their cytotoxic activity, Journal of Asian Natural 
Products Research, 20 (2), 188-194.

2. Viet Duc Ho, Thi Nhu Hanh Hoang, Quoc Hung Vo, Van Kiem 
Phan, Tuan Anh Le, Viet Ty Pham, Minh Hien Nguyen, Takeshi
Kodama, Takuya Ito, Hiroyuki Morita, Ain Raal, Thi Hoai Nguyen
(2017), Cycloartane-type triterpene glycosides anopanins A–C with
monoacyldigalactosylglycerols from Anodendron paniculatum, 
Phytochemistry, 144, 113-118.

3. Hoang Thi Nhu Hanh, Ho Viet Duc, Tran Thi Thuy Linh, Vo 
Quoc Hung, Nguyen Thi Hoai (2016), Flavonoid and
phenylpropanoid glycoside compounds isolated from Anodendron 
paniculatum (Roxb.) A.DC., Vietnam Journal of Medicinal 
Materials, 21 (5), 304-309.

4. Hoang Thi Nhu Hanh, Vu Duc Canh, Ho Viet Duc, Nguyen Thi 
Hoai (2017), Triterpene and sterol compounds isolated from
Anodendron paniculatum (Roxb.) A.DC., Hue University Journal of 
Science: Natural Science, 126 (1B), 155-163.

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