thành phần hóa học của cây lá xuân hoa mạng pseuderanthemum reticulatum radlk họ ô rô (acanthaceae)

ĐẠI HỌC QUỐC GIA THÀNH PHỐ HỒ CHÍ MINH
ĐẠI HỌC KHOA HỌC TỰ NHIÊN





NGUYỄN PHI LINH





THÀNH PHẦN HÓA HỌC CỦA LÁ
CÂY XUÂN HOA MẠNG
Pseuderanthemum reticulatum Radlk.
HỌ Ô RÔ (Acanthaceae)



LUẬN VĂN THẠC SĨ KHOA HỌC HÓA HỌC

NGƯỜI HƯỚNG DẪN KHOA HỌC


GS. TS. NGUYỄN KIM PHI PHỤNG

Tp. Hồ Chí Minh, 2011
iv

TABLE OF CONTENTS
ABSTRACT i
ACKNOWLEDGEMENTS iii
TABLE OF CONTENTS iv
LIST OF ABBREVIATIONS vi
LIST OF TABLES viii
LIST OF FIGURES AND SCHEMES ix
Chapter 1: LITERATURE REVIEW & AIM OF STUDY 1
1.1. General information of Pseuderanthemum 1
1.1.1. Generic description 1
1.1.1.1. P. acuminatissimum (Miq.) Kuntze. 1
1.1.1.2. P. bracteatum Imlay. 4
1.1.1.3. P. carruthersii 4
1.1.1.3.1. P. carruthersii (Seem.) Guill. var. atropurpureum
(Bull.) Fosb. 4
1.1.1.3.2. P. carruthersii var. ovatifolium (Brem.) Brem. 4
1.1.1.4. P. palatiferum (Nees) Radlk. 4
1.1.1.5. P. reticulatum Radlk. 5
1.1.2. Pharmaceutical studies of Pseuderanthemum 5
1.1.2.1. In vitro assays 5
1.1.2.2. In vivo assays 6
1.1.3. Chemical studies of Pseuderanthemum 7
1.1.3.1. Eranthemum pulchellum 7
1.1.3.2. P. carruthersii atropurpureum 7
1.1.3.3. P. latifolim 8
1.1.3.4. P. palatiferum 8

1.2. Aim of study 11
v

Chapter 2: EXPERIMENTAL 12
2.1. Materials 12
2.2. Plant material 12
2.3. Extraction and isolation procedures 12
2.4. Analysis of fatty acid of two glycoglycerolipids 13
Chapter 3: RESULTS & DISCUSSION 16
3.1. Structure elucidation of compound RE-EA1 16
3.2. Structure elucidation of compound RE-EA3 16
3.3. Structure elucidation of compound RE-EA4 17
3.4. Structure elucidation of compound RE-EA6 18
3.5. Structure elucidation of two diastereomers RE-EA7 and RE-EA8,
which were isolated from RE-EA2 20
3.5.1. Structure elucidation of compound RE-EA7 21
3.5.2. Structure elucidation of compound RE-EA8 23
3.6. Structure elucidation of compound RE-Bu2 24
3.7. Structure elucidation of compound RE-Bu3 28
3.8. Structure elucidation of compound RE-Bu4 30
3.9. Structure elucidation of compound RE-PE1 34
3.10. Structure elucidation of compound RE-PE2 35
Chapter 4: CONCLUSION 40
LIST OF PUBLICATIONS 42
REFERENCES 43
APPENDICES
vi

LIST OF ABBREVIATIONS
1D/2D-NMR : One/Two Dimensional – Nuclear Magnetic Resonance

AchE : Acetylcholinesterase
ALT : Alanine aminotransferase
AST : Aspartate aminotransferase
brd : Broad doublet
brs : Broad singlet
brt : Broad triplet
Bu : n-Butanol
COSY : Correlated spectroscopy
d : Doublet
dd : Doublet of doublet
DMSO : Dimethyl sulfoxide
DEPT : Distortionless Enhancement by Polarization Transfer.
EA : Ethyl acetate
HIV : Human Immunodeficiency Virus
HSQC : Heteronuclear Single Quantum Correlation.
HMBC : Heteronuclear Multiple Bond Coherence.
HPLC : High Performance Liquid Chromatography
HR-ESI-MS : High Resolution - Electrospray Ionization - Mass Spectrometry
J : Coupling constant
GC-MS : Gas Chromatography - Mass Spectrometry
LC : Liquid Chromatography
m : Multiplet
vii

m/z : Mass to charge ratio
MDA : Malondialdehyde
Me : Methyl
NMR : Nuclear Magnetic Resonance
PE : Petroleum ether
ppm : Part per million

R
f
: Retardation factors
s : Singlet
TLC : Thin-Layer Chromatography
t : Triplet
UV : Ultraviolet
[α]
D
: Specific rotation
o
C : Degree Celcius
viii

LIST OF TABLES
Table 1: The content of some major and trace minerals in the
leaves of P. palatiferum 8
Table 2:
The NMR data of RE-EA1 and comparison of its ones
with those of tyrosol 17
Table 3:
The NMR data of RE-EA3 and comparison of its ones
with those of uracil 18
Table 4:
The NMR data of RE-EA4 and comparison of its ones with
those of stigmasterol,
β
-sitosterol and β-D-glucopyranose

20

Table 5:
The NMR data of RE-EA6 22
Table 6:
The NMR data of RE-EA8 and RE-EA7 and comparison
of their ones with those of (6S,9S)-vomifoliol 24
Table 7:
The comparison of
13
C-NMR data of RE-EA7, RE-EA8 and the
aglycon of RE-EA6 with those of the aglycons of four roseoside
diastereomers 26
Table 8:
The NMR data of RE-Bu2 and comparison of its ones with
those of salidroside 28
Table 9:
The NMR data of RE-Bu3 and comparison of its ones with
those of uridine 29
Table 10:
The NMR data of RE-Bu4 and comparison of its ones with
those of apigenin 7-O-β-D-rutinoside 33
Table 11:
The NMR data of RE-PE1 and comparison of its ones with
those of 1-linolenoyl-2-palmitoyl-3-galactosylglycerol 36
Table 12:
The NMR data of RE-PE2 39


ix

LIST OF FIGURES AND SCHEMES

Figure 1:
Xuân Hoa Nhọn, P. acuminatissimum (Miq.) Kuntze 2
Figure 2:
Xuân hoa lá-hoa, P. bracteatum Imlay. 2
Figure 3:
Xuân hoa đỏ, P. carruthersii (Seem.) Guill. var.
atropurpureum (Bull.) Fosb. 2
Figure 4:
Nấp vũm, P. carruthersii var. ovatifolium (Brem) Brem. 2
Figure 5:
P. crenulatum (Lindl.) R. Ben. 2
Figure 6:
P. eberhardtii R. Ben. 2
Figure 7:
Xuân hoa, P. palatiferum (Nees) Radlk. 2
Figure 8:
P. poilanei R. Ben.Z 2
Figure 9:
Xuân hoa mạng, P. reticulatum Radlk. 3
Figure 10:
P. tonkinense R. Ben. 3
Figure 11:
Eranthemum pulchellum 3
Figure 12:
P. alatum 3
Figure 13:
P. laxiflorum 3
Figure 14:
P. sinuatum. 3
Figure 15:

P. tuberculatum (Hook. f.) Radlk 3
Figure 16:
P. variabile 3
Figure 17:
The HMBC correlations of RE-EA6 19
Figure 18:
The HMBC correlations of Re-Bu3 27
Figure 19:
The HMBC correlations of RE-Bu4 30
Figure 20:
The HMBC correlations of RE-PE1 35
Figure 21:
The HMBC correlations of RE-PE2 37
Scheme 1:
Extraction procedure of Pseuderanthemum
reticulatum Radlk. leaves 14
Scheme 2: Isolation procedure of compounds from
the PE residue of scheme 1 14
x

Scheme 3: Isolation procedure of compounds from
the EA residue of scheme 1 15
Scheme 4:
Isolation procedure of compounds from
the Bu residue of scheme 1 15

Pseuderanthemum reticulatum Radlk. Literature review & Aim of study

1

Chapter 1
LITERATURE REVIEW & AIM OF STUDY
1.1. General information of Pseuderanthemum
Pseuderanthemum or Eranthemum
[1]
is a member of Acanthaceae. The genus
contains about 196 species, including perennial shurbs or herbs. The characteristics of
the genus are beautiful flowers and foliage, high humidity need, flowering in spring
and summer.
In Vietnam, Pseuderanthemum has nine species and two varieties
[1]
including P.
acuminatissimum (Miq.) Kuntze. (figure 1), P. bracteatum Imlay. (figure 2), P.
carruthersii (Seem.) Guill. var. atropurpureum (Bull.) Fosb. (figure 3), P. carruthersii
var. ovatifolium (Brem.) (figure 4), P. crenulatum (Lindl.) R. Ben (figure 5), P.
eberhardtii R. Ben (figure 6), P. palatiferum (Nees) Radlk (figure 7), P. poilanei R.
Ben.Z (figure 8), P. reticulatum Radlk (figure 9) and P. tonkinense R. Ben (figure 10).
In addition, we collected pictures of some species of Pseuderanthemum from
internet such as Eranthemum pulchellum (figure 11)
[32]
, P. alatum (figure 12)
[33]
, P.
laxiflorum (figure 13)
[34]
, P. sinuatum (figure 14)
[35]
, P. tuberculatum (Hook. f.) Radlk
(hình 15)
[36]

and P. variabile (figure 16).
[37]

1.1.1. Generic description
In this section, the botanic morphology of some Vietnamese Pseuderanthemum
species was described.
1.1.1.1.
P. acuminatissimum (Miq.) Kuntze.
[1],[2]

Vietnamese name: Xuân hoa nhọn (figure 1).
Perennial shrub. Stems terete in older positions, glabrous and tetragonal in
younger positions. Leaves 10-20 cm long, 5-9 cm wide; costa and lateral veins 6 pairs,
petioles 4-5 cm long; peduncle 20 cm long. Flowers in cymes, bisexual, often
zygomorphic; calyx 3.5 cm long, glabrous; corolla tube 3.5 cm long. Capsule 3.5 cm
long, glabrous.
The herb, a species of Java Island (Indonesia), has been planted in Singapore and
Vietnam (Bảo Lộc, Sài Gòn).

Pseuderanthemum reticulatum Radlk. Literature review & Aim of study

2


Figure 1:
Xuân hoa nhọn,
P. acuminatissimum (Miq.) Kuntze.

Figure 2:
Xuân hoa lá-hoa,

P. bracteatum Imlay.

Figure 3:
Xuân hoa đỏ,
P. carruthersii (Seem.) Guill. var.
atropurpureum (Bull.) Fosb.
Figure 4:
Nấp vũm,
P. carruthersii var. ovatifolium (Brem)
Brem.


Figure 5:
P. crenulatum (Lindl.) R. Ben.
Figure 6:
P. eberhardtii R. Ben.


Figure 7:
Xuân hoa, P. palatiferum (Nees) Radlk.
Figure 8:
P. poilanei R. Ben.Z
Pseuderanthemum reticulatum Radlk. Literature review & Aim of study

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Figure 9:
Xuân hoa mạng, P. reticulatum Radlk.
Figure 10:

P. tonkinense R. Ben.


Figure 11:
Eranthemum pulchellum
Figure 12: P. alatum

Figure13:
P. laxiflorum
Figure 14: P. sinuatum

Figure 15:
P. tuberculatum (Hook. f.) Radlk.
Figure 16: P. variabile


Pseuderanthemum reticulatum Radlk. Literature review & Aim of study

4

1.1.1.2. P. bracteatum Imlay.
[1]

Vietnamese name: Xuân hoa lá-hoa, Hoàn ngọc đỏ, Hồng ngọc (figure 2).
Herb, 50-60 cm tall. Stems quadrangular and ciliate in younger positions. Leaves
5-9 cm long, 3-5 cm wide; costa and lateral veins 5-6 pairs; petioles 2-3 cm long.
Peduncles
8-10 cm long; bracts similar to the leaves in shape, 2 cm long and 0.7 cm
wide, ciliate; lobes 6-7 mm long, stamens 2. Capsule 2 cm long, glabrous.
The herb has wildly growed on Dinh mountain, Bà Rịa-Vũng Tàu province.

1.1.1.3. P. carruthersii

This species has two varieties consisting of P. carruthersii (Seem.) Guill. var.
atropurpureum (Bull.) Fosb. and P. carruthersii var. ovatifolium (Brem.) Brem.
1.1.1.3.1. P. carruthersii (Seem.) Guill. var. atropurpureum (Bull.) Fosb.
[1],[2],[4]

Vietnamese name: Xuân hoa đỏ, Ô rô đỏ, Nhớt tím (figure 3).
Shrub, 1-2 m tall. Leaves, elliptic-ovate, smooth, violet-red, mottled yellow-
green, short petioles. Flowers in cymes, bisexual; corolla tube rosy-purple, 1.3 cm
long; stamens 2.
The herb has been cultivated for ornamentation in Ho Chi Minh city.
1.1.1.3.2. P. carruthersii var. ovatifolium (Brem.) Brem.
[1]

Vietnamese name: Nấp Vũm (figure 4).
Herb, different variety of P. carruthersii (Seem.) Guill. var. atropurpureum
(Bull.) Fosb. due to leaves oval, 8.5 cm long, 4.5 cm wide, costa and lateral veins 7-8
pairs.
The herb has wildly growed in Sông Bé provine and Sài Gòn.
1.1.1.4.
P. palatiferum (Nees) Radlk.
[1],[2],[5]

Vietnamese name: Xuân hoa (figure 7).
Perennial shrub, 1-2 m tall. Leaves 12-17 cm long, 3.5-5 cm wide, opposite,
decussate, lanceolate, petioles 1.5-2.5 cm long. Flowers in cymes, bisexual, often
zygomorphic; peduncles 10-16 cm long; pedicles 0-5 mm long; bracts linear-
triangular, 2.5mm long; calyx 4-5 lobed, 5-7 mm; corolla tube 25 mm long, pubescent,
5-lobed but often 2-lipped with lower lip 3-lobed and upper lip; stamens 2, attached to

corolla. Capsule woody, clavate with long, glabrous.

Pseuderanthemum reticulatum Radlk. Literature review & Aim of study

5

1.1.1.5. P. reticulatum Radlk.
[1]

Vietnamese name: Xuân hoa mạng (figure 9).
Perennial shrub, 0.5-2.5 m tall. Stems quadrangular and ciliate in younger
positions. Leaves opposite and decussate, 8-15 cm long, 5-10 cm wide, oval, glabrous;
the costa and lateral veins 9-10 pairs; petioles 1 cm long. Flowers in cymes, bisexual,

zygomorphic;
corolla tube 1.2 cm long, 5-lobed but often 2-lipped with lower lip 3-
lobed and upper lip; stamens 2; calyx 2 mm.
The shrub has wildly growed in Bà Nà mountain, Đà Nẵng province.
1.1.2. Pharmaceutical studies of Pseuderanthemum
Until now, there has only been the pharmaceutical information on three species of
Pseuderanthemum including P. carruthersii (Seem.) Guill. var. atropurpureum (Bull.)
Fosb., P. latifolim and P. palatiferum (Nees) Radlk. However, P. palatiferum (Nees)
Radlk has merely been studied on pharmaceutical by modern methods.
P. carruthersii (Seem.) Guill. var. atropurpureum (Bull.) Fosb.: leaves, roots and
flowers have been used to cure mouth disease and wounds by folk.
[2],[4]

P. latifolim: in Yunnan (China), it has been used as a medicinal herb, and has
been a potential herb in the group of species that were screened for HIV healing by
Chinese scientists.

[18]

P. palatiferum (Nees) Radlk: it was found in Vietnam since 1980s and has been
used as a medicinal herb. Now this plant has been studying on chemical constituents
and pharmaceutical by using modern methods.
In Vietnam, P. palatiferum (Nees) Radlk. has been planted in many places as a
family’s medicinal herb. According to Trần Công khánh
[3]
, the leaves of this herb can
be used as folk remedy for haemostatic, restoring of health for sick and exhausted
people, treating digestive disorder, diarrhea, dysentery, constipation, gastritis,
gastroduodenal ulcer, internal haemorrhoid, nephritis, haematuria, prostate ulcer,
regulation of hypertension.
1.1.2.1.
In vitro assays
Trần Công Khánh et al.
[5]
(1997) studied the antibacterial and antifungal activity
of crude extract of leaves of P. palatiferum. The results indicated that the crude extract
had inhibitory activity against Gram-negative bacteria (Escherichia coli, Pseudomonas
aeruginosa), Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus,
Pseuderanthemum reticulatum Radlk. Literature review & Aim of study

6

Streptococcus pyogenes), molds (Aspergillus niger, Fusarium oxysporum, Pyricularia
oryzae, Rhezoctonia solanii) and yeasts (Saccharomyces cerevisiae, Candida
albicans).
Phan Minh Giang et al.
[6]

(2005) preliminary studied the antibacterial and
antifungal activity of ethyl acetate and n-butanol extracts of P. palatiferum at the
concentration of 10 mg/ml by using disk diffusion technique. The results showed that
both extracts had inhibitory activity against Gram-positive bacteria (Bacillus subtilis,
Staphylococcus aureus), Gram-negative bacteria (Escherichia coli) and molds
(Candida albicans, Candida stellatoides); and the activity of the ethyl acetate extract
was higher than the n-butanol one. The study also exhibited that the ethyl acetate
extract had high inhibitory activity against Salmonella typhi 158 (the diameter of zone
of inhibition of 21 mm). They also studied the antioxidant activity of the extracts by
investigating the effect of these extracts on peroxydase enzyme in blood (Savron’s
method) and the results revealed that both extracts had antioxidant activity.
Trần Công Khánh et al.
[7]
(2007) published that the n-hexane extract of roots of
P. palatiferum had antibacterial activity against Staphylococcus aureus (IC
50
174.9
g/ml).
1.1.2.2.
In vivo assays
Lê Thị Lan Oanh et al.
[8]
(1999) studied the toxicity of extracts of fresh and dried
leaves of P. palatiferum on Siamese Fighting fish. The results indicated that the
extracts had no toxic effect on Siamese Fighting fish. The same results were obtained
from the studies on mice and rabits.
Trần Thị Minh Thu et al.
[9]
(1999) studied the acute toxicity and hepatoprotective
effect of crude extract of P. palatiferum on mice. The results of acute toxicity tests

indicated that the crude extract at doses of 0.83, 1.67, 3.13, 5.56, 9.19 and 11.5 g/kg
body weight did not have the acute toxicity effect on mice. The hepatoprotective effect
was tested in vivo against carbon tetrachloride (CCl
4
) induced damage in rat hepatoma
cells. The results of hepatoprotective effect test exhibited that at the toxic dose of 1 ml
CCl
4
/kg body weight, mice liver necrosed, the values of AST (aspartate
aminotransferase) and ALT (alanine aminotransferase) were very high, but the MDA
(malondialdehyde) content remarkably reduced from 14.12 nM to 10.71 nM and the
signal of liver cells recovery was not observed; at dose of 0.5 ml CCl
4
/kg body weight,
Pseuderanthemum reticulatum Radlk. Literature review & Aim of study

7

the MDA level significantly reduced from 11.57 nM to 6.25 nM, the AST, ALT levels
decreased a little, and the liver cells recovered.
Huỳnh Kim Diệu et al.
[31]
studied the effect of P. palatiferum powder on the
diarrhea of piglets. The results of treatment with P. palatiferum powder was compared
with two prevalent effective antibiotics against piglets’ diarrhea: Coli-norgen and
Cotrimxazol. The results showed that the recovered rate after three days of treatment
of P. palatiferum powder, Coli-norgen and Cotrimxazol were 92.86, 90.48 and 83.33
%, respectively; the relapsed rate were 7.14, 9.52 and 14.29 %; the duration of
diarrhea were 2.16, 2.24 and 2.03 days, respectively.
Wararut Buncharoen et al.

[19]
(2010) studied the acetylcholinesterase (AChE)
inhibitory effect of the aqueous extract of leaves of P. palatiferum on albino rats. The
results showed that only AChE activity in the hippocampus was significantly inhibited
by the extract at the dose of 0.7 and 1.0 g/kg body weight. However, AChE activity in
the serum and red blood cells of treated rats showed no significant differences from
that of controls. These results suggested that the extract could reduce the synthesis of
AchE in the rats’ brain; and the remarkably inhibitory effect of the extract indicated
the potential of this plant in the treatment of Alzheimer’s disease.
1.1.3. Chemical studies of Pseuderanthemum
Up to now, there have only been the chemical studies on four species of
Pseuderanthemum including Eranthemum pulchellum, P. carruthersii atropurpureum,
P. latifolim and P. palatiferum.
1.1.3.1. Eranthemum
pulchellum
Henrik Fischer W. Jensen et al.
[17]
(1987) isolated betaine (33) and an iridoid
glucoside eranthemoside (24) from the peduncle of Eranthemum pulchellum.
1.1.3.2.
P. carruthersii atropurpureum
Võ Thị Ngà et al.
[11]
(2007) isolated 1-triacontanol (2), a mixture of β-sitosterol
(8) and stigmasterol (9), a mixture of β-sitosterol 3-O-β-D-glucopyranoside (10) and
stigmasterol 3-O-β-D-glucopyranoside, a mixture of oleanolic acid (14) and ursolic
acid (15), indole-3-carboxaldehyde (35), uracil (34), adenine (27) and betaine (33)
from the leaves of P. carruthersii atropurpureum.
Pseuderanthemum reticulatum Radlk. Literature review & Aim of study

8

1.1.3.3. P. latifolim
Zhu Xiang-dong et al.
[18]
(2006) isolated eight compounds from the arial parts of
P. latifolim including stigmasterol (9), stigmasterol 3-O-β-D-glucopyranoside (11), an
ester of β-sitosterol 3-O-β-D-glucopyranoside sitoindoside I (13), an iridoid
anthirrinoside (25), three flavonoids 7,4'-dihydroxyflavone (28), 5,4'-dihydroxy-7-
methoxyflavone (29) and 5,6-dihydroxy-3',4',7,8-tetramethoxyflavone (30), a purine
skeleton compound allantoin (26).
1.1.3.4.
P. palatiferum
Võ Hoài Bắc et al.
[10]
(2003) analysed the major and trace mineral constituents of
leaves of P. palatiferum. The results (see table 1) showed that the content of mineral
elements including Na, K, Ca, Mg, Al and Fe in the leaves of P. palatiferum were very
high (40-900 mg/100 g fresh leaves).
Table 1:
The content of some major and trace minerals in the leaves of P. palatiferum
Major minerals
Content
(mg/100g fresh leaves)
Trace minerals
Content
(mg/100g fresh
leave)
Ca 875.5 Fe 38.75
Mg 837.6 Al 37.50

K 587.5 V 3.75
Na 162.7 Cu 0.43

Mn 0.34

Ni 0.19
Nguyễn Thị Thu Minh et al.
[12]
(2000) isolated five compounds from the leaves
of P. palatiferum including phytol (22), β-sitosterol (8), β-sitosterol 3-O-β-D-
glucopyranosid (10) and a mixture of stigmasterol (9) and poriferasterol (12).
Four compounds and two mixtures were isolated from the dried leaves of
P. palatiferum by Phan Minh Giang et al.
[13]
(2003) including 1-pentacosanol (2),
palmitic acid (5), β-sitosterol (7), stigmasterol (8), a mixture of β-sitosterol 3-O-β-D-
glucopyranoside (10) and stigmasterol 3-O-β-D-glucopyranoside (11), a mixture of 3-
methoxykaempferol 7-O-β-D-glucopyranoside (31) and apigenin 7-O-β-D-
glucopyranoside (32).
Pseuderanthemum reticulatum Radlk. Literature review & Aim of study

9

Võ Hoài Bắc et al.
[10]
(2003) anlysed the amino acids content of P. palatiferum
leaves, collected at the different times of year. The results revealed that the total
content of free and linked amino acids in the leaves, which were collected between
January and February or between September and October, was relatively high
(751-1365 mg%); especially, the isoleucine, leucine and valine contents were very

high (25-150, 46-85 and 20-1001 mg%, respectively). The best time for collecting was
Septemper and October because of the extremely high content of free amino acids
(1347 mg%).
Nguyễn Văn Hùng et al.
[14]
(2004) isolated 1-triacontanol (2), glycerol 1-
hexadecanoate (5), salicylic acid (6), palmitic acid (4) from the leaves of P.
palatiferum.
Mai Đình Trị et al.
[15]
(2005) isolated β-amyrin (17) oleanolic acid (14), β-
sitosterol (8) and stigmasterol (9), a mixture of β-sitosterol 3-O-β-D-glucopyranoside
(10) and stigmasterol 3-O-β-D-glucopyranoside (11).
Trần Kim Thu Liễu et al.
[16]
(2007) isolated squalene (23), dotriacontane (1),
phytol (22), palmitic acid (4), β-sitosterol (8), stigmasterol (9), 24-
methylenecycloartanol (20), loliolide (7), a mixture of β-sitosterol 3-O-β-D-
glucopyranoside (10) and stigmasterol 3-O-β-D-glucopyranoside (11) from the leaves
of P. palatiferum.
Six compounds including lupeol (18), lupenone (19), betulin (21), pomolic acid
(16), palmitic acid (4) and asperglaucide (36) were isolated from the roots of P.
palatiferum by Trần Công Khánh et al.
[7]
(2007).


Pseuderanthemum reticulatum Radlk. Literature review & Aim of study

10

Ursolic acid (
15
)
HO
COOH
HO
R
R COOH: Oleanolic acid (14)
RCH
3
: b-Amyrin (17)
Pomolic acid (
16
)
HO
COOH
HO





Pseuderanthemum reticulatum Radlk. Literature review & Aim of study

11

O

R
8
R
7
R
6
R
5
O
R
3
R
3'
R
4'
R
3
R
5
R
6
R
7
R
8
R
3'
R
4'
OH OH 7,4'-Dihydroxyflavone (

28
)
OH OCH
3
OH 5,4'-Dihydroxy-7-methoxyflavone (29)
OH OH OCH
3
OCH
3
OCH
3
OCH
3
5,6-Dihydroxy-3',4',7,8-tetramethoxyflavone (
30
)
OCH
3
OH OGlc OH 3-Methoxykaempferol 7-O- -D-glucopyranoside (31)
H OGlc OH Apigenin 7-O-
-D-glucopyranoside (
32
)

1.2. Aim of study
Nowadays, the advance of sciences and technologies, as well as those of
chemistry, significantly contribute to improving the quality of human life, and
especially treating diseases. A great number of medicines have been synthesized for
restoring health and curing diseases, but some of them are expensive and have side
effects. Hence, people have a tendency to return to traditonal medicine, and used

medicinal herbs for treatments. The phytochemical and pharmaceutical studies of
medicinal herbs are very important because they not only clarify the activities of
medicinal herbs, but also contribute to confirm the value of medicinal herbs and
discover new bioactive compounds for treating diseases.
In Vietnam, some Pseuderanthemum species have been used as medicinal herb
for treating digestive disorder, liver disease, restoring health, ect. However, there have
been a few studies about the phytochemistry and bioactivity of Pseuderanthemum and,
above all, no studies on P. reticulatum Radlk Therefore, the thesis on contribution to
the chemical study of leaves of P. reticulatum Radlk. has been carried out.



Pseuderanthemum reticulatum Radlk. Experimental

12

Chapter 2
EXPERIMENTAL
2.1. Materials
The solutions of different extracts were evaporated by using rotary evaporator
Buchi-111.
1
H-NMR,
13
C-NMR and 2D-NMR spectra were acquired on Bruker
Avance 500
III
(500 MHz for
1
H-NMR and 125 MHz for

13
C-NMR). HR-MS were
recorded on Bruker microOTOF Q-II. Preparative HPLC were carried out on LC-8A
Shimadzu preparative liquid chromatograph with Sulpeco C
18
column (10 µm, 25 cm x
21.2 mm) and UV-Vis Spd-20A detector at 245 nm. GC-MS was carried out on
Agilent technologies 7890A GC System, HP-5 MS capillary column (5% phenyl
methyl silox, 325 °C, 30 m x 250 µm x 0.25 µm), Agilent technologies 5975C VL
MSD with triple-Axis detector. Optical rotations were measured on a Kruss (German)
polarimeter. All instruments are in the Center Analysis of the University of Science,
National University- Ho Chi Minh City.
Solvents: Petroleum ether (60-90 °C), chloroform, ethyl acetate, n-butanol,
methanol, acetic acid.
Thin-layer chromatography (TLC) and preparative TLC was performed on silica
gel GF
254
(Merck), chemicals visualizing TLC plates: 30% aqueous solution of H
2
SO
4
.
Column chromatography was performed on silica gel (Merck) type 100
(70–230 Mesh ASTM).
2.2. Plant material
The leaves of P. reticulatum Radlk were collected in Vũng Tàu province, and the
scientific name was identified by Pharmacist Phan Đức Bình, Assistant Editor of
Medicine and Health Semimonthly. A voucher specimen (No US-A009) was deposited
in the herbarium of the Department of Organic Chemistry, Univeristy of Science.
2.3. Extraction and isolation procedures

The clean, air-dried and ground material (3.2 kg) was extracted by ethanol at
ambient temperature, and the filtrated solution was concentrated under reduced
pressure to afford ethanol residue (450.0 g). The residue was dissolved in solvent
systems of methanol: water (1: 9); then was partitioned against petroleum ether, ethyl
Pseuderanthemum reticulatum Radlk. Experimental

13

acetate and n-butanol, respectively; and the obtained solutions were evaporated to
afford corresponding residues: petroleum ether (PE) residue (120.0 g), ethyl acetate
(EA) residue (20.5 g), n-butanol (Bu) residue (30.3 g) and water residue (278.1 g).
The PE residue (120.0 g) was subjected to silica gel column chromatography,
eluted with a gradient solvent system of petroleum ether: ethyl acetate (10: 0 to 0: 10)
and then ethyl acetate: methanol (10: 0 to 0: 10) to yield five fractions: PE-A (30.5 g),
PE-B (20.8 g), PE-C (22.4 g), PE-D (8.3 g), PE-E (10.7 g).
The EA residue (20.5 g) was applied to silica gel column chromatography, eluted
with gradient solvent systems of ethyl acetate and then ethyl acetate: methanol from 1
to 100 % methanol to yield three fractions: EA-A (1.2 g), EA-B (5.5 g) and EA-C
(3.2 g).
The Bu residue (30.3 g) was subjected to silica gel column chromatography,
eluted with a gradient solvent system of ethyl acetate and methanol (10: 0 to 0:10) to
yield five fractions: Bu-A (1.2 g), Bu-B (4.1 g), Bu-C (1.0 g), Bu-D (4.0 g) and Bu-E
(4.3 g).
The EA-B residue (5.5 g) was rechromatographed many times and applied to
preparative TLC to afford a mixture of RE-EA2 (20.0 mg), a mixture of RE-EA4
(209.5 mg) and three compounds: RE-EA1 (20.0 mg), RE-EA3 (6.7 mg), RE-EA6
(5.0 mg).
The mixture RE-EA2 (20.0 mg) was subjected to preparative HPLC [Sulpeco C
18

(10 µm, 25 cm x 21.2 mm), 16% MeOH aq., 8 ml/min, UV detection (254 nm)] to
yield two compounds RE-EA7 (1.0 mg), RE-EA8 (2.0 mg).
The Bu-B residue (4.1 g) was rechromatographed many times and applied to
preparative TLC to yield three compounds RE-Bu2 (5.0 mg), RE-Bu3 (20.0 mg) and
RE-Bu4 (2.5 mg).
The PE-D residue (8.3 g) was rechromatographed many times to yield two
compounds RE-PE1 (60.0 mg) and RE-PE2 (8.9 mg).
2.4. Analysis of fatty acid of two glycoglycerolipids
[29]

Purified glycoglycerolipids ( 2.0 mg of each) was methanolysed by refluxing
with 4.0 ml solution of 5% HCl in methanol, at 95 °C for 4.5 hours. Afterward, the
reaction solution was partitioned against n-hexane. The n-hexane layer were applied to
GC-MS on HP-5 MS capillary column, programmed from 45 °C to 300 °C (10
Pseuderanthemum reticulatum Radlk. Experimental

14

°C/min), then held constant for 4 mins in order to determine the fatty acid moities of
two glycoglycerolipids.
Scheme 1:
Extraction procedure of Pseuderanthemum reticulatum Radlk.


Scheme 2:
Isolation procedure of compounds from the PE residue of scheme 1.


Ethanol residue (450.0 g)
PE residue (120.0 g)

Water extract
EA residue (20.5 g)
Water extract
Bu residue
(30.3 g)
Water extract
Water residue (278.1 g)
Ground material (3.2 kg)
- Macerate with ethanol at ambient temperature
- Concentrate in reduced pressure

- Dissolve in solvent system of methanol: water (1: 9)
- Partition against petroleum ether
- Evaporate

- Partition against ethyl acetate
- Evaporate

- Partition against n-butanol
- Evaporate

- Evaporate

PE residue (120.0 g)
PE-A (30.5 g)
PE-B (20.8 g)
PE-C (22.4 g)
PE-D (8.3 g)
PE-E (10.7 g)
RE-PE1

(60.0 mg)
RE-PE2
(8.9 mg)
- Column chromatography
- Column chromatography
- Preparative TLC

Pseuderanthemum reticulatum Radlk. Experimental

15

Scheme 3: Isolation procedure of compounds from the EA residue of scheme 1.




Scheme 4:
Isolation procedure of compounds from the Bu residue of scheme 1.





EA residue (20.5 g)
EA-A (1.2 g)
EA-B (5.5 g)
EA-C (3.2 g)
RE-EA1
(20.5 mg)
RE-EA2

(20.0 mg)
RE-EA4
(209.5 mg)
RE-EA3
(6.7 mg)
RE-EA6
(5.0 mg)

RE-EA7
(1.0 mg)
RE-EA8
(2.0 mg)
- Column chromatography

- Column chromatography
- Preparative TLC

- Preparative HPLC
Bu residue (30.3 g)

Bu-A (1.2 g)

Bu-B (4.1 g)

Bu-C (1.0 g)

Bu-D (4.0 g)

Bu-E (4.3 g)

RE-Bu2
(5.0 mg)

RE-Bu3
(20.0 mg)


RE-Bu4
(2.5 mg)

- Column chromatography
- Column chromatography
- Preparative TLC

Pseuderanthemum reticulatum Radlk. Results & Discussion

16

Chapter 3
RESULTS & DISCUSSION
3.1. Structure elucidation of compound RE-EA1
 Compound RE-EA1 was isolated as colorless oil.
 TLC experiment exhibited a yellow spot with R
f
0.5, when eluted with solvent
system of petroleum ether: ethyl acetate (1: 1) and visualized by 30% aqueous
solution of H
2
SO
4

.

1
H,
13
C and DEPT-NMR spectra (Acetone-d
6
) (Appendix 1-4): see Table 2
Discussion on the chemical structure determination
Compound RE-EA1 was a monocylic phenolic compound.
1
H-NMR spectrum
displayed four ortho-coupled protons at

7.04 (2H, d, 8.0 Hz, H-3, H-5), 6.74 (2H, d,
8.0 Hz, H-2, H-6); one oxymethylene proton at

3.67 (2H, t, 7.0 Hz, H-2’) and one
benzyl methylene proton at

2.70 (2H, t, 7.0 Hz, H-1’).
13
C-NMR and DEPT-NMR
spectra showed two substituted aromatic carbons at

156.6 (C-1), 130.8 (C-4); four
aromatic methine carbons at

130.6 (C-3, C-5), 115.9 (C-2, C-6); one oxymethylene
carbon at


64.2 (C-2’); one benzyl methylene carbon at

39.4 (C-1’). These
spectroscopic data were suitable to the published ones
[20]
so RE-EA1 was tyrosol or
4-(2-hydroxyethyl)phenol.
OH
HO
Tyrosol
1
2
3
4
5
6
1'
2'

3.2. Structure elucidation of compound RE-EA3

 Compound RE-EA3 was isolated as yellowish amorphous powder.
 TLC experiment displayed a spot with R
f
0.5, when eluted with solvent
system of chloroform: methanol (9.5: 0.5) and visualized by UV lamp.

1
H and

13
C-NMR spectra (DMSO-d
6
) (Appendix 5 and 6): see Table 3
Discussion on the chemical structure determination
Compound RE-EA3 was a monoheterocyclic compound.
1
H-NMR spectrum
showed two lactam protons at

11.01 (1H, s, H-3), 10.82 (1H, s, H-1); two olefinic
cis-coupled protons at

7.69 (1H, d, 7.5 Hz, H-6), 5.44 (1H, d, 7.5 Hz, H-5).
Pseuderanthemum reticulatum Radlk. Results & Discussion

17

13
C-NMR spectrum revealed two lactam carbons at

164.4 (C-4), 151.6 (C-2); two
olefinic carbons at

142.2 (C-6) and 100.3 (C-5). These spectroscopic data were
suitable to the published ones
[21]
so RE-EA3 was uracil.

Table 2: The NMR data of RE-EA1 and comparison of its ones

with those of tyrosol
[20]

Pos.
RE-EA1
(Acetone-d
6
)
Tyrosol
[20]

(Acetone-d
6
)

H
(J Hz)

C


C

1 - 156.6 156.8
2, 6 6.74 (d, 8.0) 130.6 130.9
3, 5 7.04 (d, 8.0) 115.9 116.1
4 - 130.8 131.0
1’ 2.70 (t, 7.0) 39.4 39.4
2’ 3.67 (t, 7.0) 64.2 64.6
3.3. Structure elucidation of compound RE-EA4

 Compound RE-EA4 was isolated as white amorphous powder.
 TLC experiment showed a pink-violet spot with R
f
0.5 when eluted with
solvent system of chloroform: methanol (85: 15), visualized by 30% aqueous
solution of H
2
SO
4
.

1
H and
13
C-NMR spectra (DMSO-d
6
) (Appendix 7-8): see Table 4
Discussion on the chemical structure determination
13
C-NMR exhibited two pairs of signals, which were typical of stigmasterol and

-sitosterol, at

140.5 (C-5a, C-5b) and 121.2 (C-6a, C-6b), 138.8 (C-22a) and 128.8
(C-23a); one anomeric carbon at

100.8 (C-1’a, C-1’b).
In
1
H-NMR spectrum, the signals at


 5.32 (2H, brd, H-6a và H-6b), 5.14 (1H,
dd, 15.0 Hz; 8.5 Hz, H-22a), 5.01 (1H, dd, 15.0; 9.0 Hz, H-23a) were characteristic of