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
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

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

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:

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]

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

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

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

[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]

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.

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

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

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 (

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

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


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

(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)


.

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

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

 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