Study on chemical constituents and biological activities of some plants of boerhaavia genus (nyctaginaceae)

VIETNAM NATIONAL UNIVERSITY - HO CHI MINH CITY
UNIVERSITY OF SCIENCE



ĐỖ THỊ MỸ LIÊN



STUDY ON CHEMICAL CONSTITUENTS AND
BIOLOGICAL ACTIVITIES OF SOME PLANTS
OF BOERHAAVIA GENUS (NYCTAGINACEAE)


Subject: Organic chemistry
Code number: 62 44 27 01


The first reviewer: Assoc. Prof. Trần Hùng
The second reviewer: Assoc. Prof. Phạm Đình Hùng
The third reviewer: Assoc. Prof. Trần Công Luận
The first independent reviewer: Assoc. Prof. Nguyễn Thị Hoàng Anh
The second independent reviewer: Dr. Nguyễn Lê Bảo Khánh




SUPERVISORS: PROF. NGUYỄN KIM PHI PHỤNG
PROF. DOMINIQUE GUILLAUME

Ho Chi Minh City, 2014
TABLE OF CONTENTS
ACKNOWLEDGEMENTS i
TABLE OF CONTENTS iii
LIST OF ABBREVIATIONS v
LIST OF TABLES vii
LIST OF FIGURES AND SCHEMES ix
Chapter 1: LITERATURE REVIEW 1
1.1. Boerhaavia genus – Generic description 2
1.1.1. Boerhaavia coccinea M. 3
1.1.2. Boerhaavia diffusa L. 4
1.1.3. Boerhaavia erecta L. 4
1.1.4. Boerhaavia repens L. 5
1.2. Boerhaavia genus- Pharmacological studies 6
1.2.1. Boerhaavia coccinea M. 6
1.2.2. Boerhaavia diffusa L. 6
1.2.3. Boerhaavia erecta L. 7
1.3. Boerhaavia genus- Chemical studies 8
1.3.1. Boerhaavia coccinea M. 8
1.3.2. Boerhaavia diffusa L. 8
1.3.3. Boerhaavia erecta L. 10
1.3.4. Boerhaavia repens L. 10
1.4 Aim of the study 15
Chapter 2: EXPERIMENTAL 16
2.1. Instrumental material 16
2.2. Plant material 19
2.3. Extraction and isolation procedures 21

iii
2.4. Biological assays 25
Chapter 3: RESULTS & DISCUSSION
3.1. Chemical structure elucidation 31
3.1.1. Chemical structure of 12a-hydroxyrotenoids 31
3.1.2. Chemical structure of dehydrorotenoids 41
3.1.3. Chemical structure of coumaronochromonoids 59
3.1.4. Chemical structure of flavonoids 68
3.1.5. Chemical structure of nitrogen compounds 78
3.1.6. Chemical structure of phenolic compounds 83
3.1.7. Chemical structure of steroids and terpenoids 101
3.1.8. Chemical structure of other compounds 110
3.2. Biological activities of extracts and isolated compounds 117
Chapter 4: CONCLUSION 121
LIST OF PUBLICATIONS 127
REFERENCES 128
APPENDICES

iv
LIST OF ABBREVIATIONS
1D/2D-NMR : One/Two Dimensional – Nuclear Magnetic Resonance
AchE : Acetylcholinesterase
B. : Boerhaavia
brd : Broad doublet
brs : Broad singlet
Bu : n-Butanol
C : Chloroform
calcd : calculated
CC : Silica gel Column Chromatography
CD : Circular Dichroism

COSY : Correlated spectroscopy
d : Doublet
dd : Doublet of doublet
ddd : Doublet of doublet of doublet
DMSO : Dimethyl sulfoxide
DEPT : Distortionless Enhancement by Polarization Transfer.
EA : Ethyl acetate
H : n-Hexane
HIV : Human Immunodeficiency Virus
HMBC : Heteronuclear Multiple Bond Coherence
HSQC : Heteronuclear Single Quantum Correlation
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
m/z : Mass to charge ratio
M : Methanol
Me : Methyl
NMR : Nuclear Magnetic Resonance
PE : Petroleum ether
ppm : Part per million
p-TLC : preparative Thin Layer Chromatography
R
f
: Retardation factors
RP – C18 : Reverse Phase silica gel C18
s : Singlet
TLC : Thin-Layer Chromatography

t : Triplet
UV : Ultraviolet
[α]
D
: Specific rotation
o
C : Degree Celcius




vi
LIST OF TABLES
Table 3.1:
1
H–NMR spectroscopic data of the known compounds 11, 21 and
the new compounds 54, 55, 56 32
Table 3.2:
13
C–NMR spectroscopic data of the known compounds 11, 21 and
the new compounds 54, 55, 56 33
Table 3.3:
1
H–NMR spectroscopic data of the known compounds 9, 10, 15,
16, 20 and the new compounds 57, 58 44
Table 3.4:
13
C–NMR spectroscopic data of the known compounds 9, 10, 15,
16, 20 and the new compounds 57, 58 45
Table 3.5:

1
H–NMR spectroscopic data of the new compounds 59, 60, 61
and the hydrolyzed aglycone of 60 57
Table 3.6:
13
C–NMR spectroscopic data of compounds 59, 60, 61
and the hydrolyzed aglycone of 60 58
Table 3.7:
1
H–NMR spectroscopic data of the known compounds 62, 18 and
the new compounds 63, 64 65
Table 3.8:
13
C–NMR spectroscopic data of the known compounds 62, 18 and
the new compounds 63, 64 66
Table 3.9:
1
H–NMR spectroscopic data of the known compounds 65, 39, 36,
67 and 31 69
Table 3.10:
1
H–NMR spectroscopic data of the known compounds 62, 36, 33
and 58 70
Table 3.11:
13
C–NMR spectroscopic data of the known compounds 65, 39,
36, 67, 31, 62, 36, 33 and 68 71
Table 3.12: NMR spectroscopic data of the known compounds 69 and 70 79
Table 3.13:
1

H and
13
C–NMR data of the known compound 71 82
Table 3.14:
1
H–NMR spectroscopic data of the new compound 72,
and the known compounds 73, 74, 75, 76, 77, 78, 79 91
Table 3.15:
13
C–NMR spectroscopic data of the new compound 72,
and the known compounds 73, 74, 75, 76, 77, 78, 79 92
vii
Table 3.16:
1
H–NMR spectroscopic data of the known compounds 80, 81, 47
and the new compound 82 98
Table 3.17:
13
C–NMR spectroscopic data of the known compounds 80, 81, 47
and the new compound 82 99
Table 3.18:

NMR spectroscopic data of the known compounds 83, 5 103
Table 3.19: NMR spectroscopic data of the known compounds 84, 85
and the new compound 86 107
Table 3.20:
1
H and
13
C–NMR data of the known compound 87 108

Table 3.21:
1
H–NMR spectroscopic data of the known compound 91 and the
new compounds 88, 89, 90 113
Table 3.22:
13
C–NMR spectroscopic data of the known compound 91 and the
new compounds 88, 89, 90 114
Table 3.23: NMR spectroscopic data of the known compounds 92 and 93 115
Table 3.24: Inhibitive activity of cytotoxic activity against three cancer cell
lines of isolated compounds 118
Table 3.25: Acetylcholinesterase inhibition of some extracts and isolated
compounds on acetylcholinesterase 119







viii
LIST OF FIGURES AND SCHEMES
Figure 1.1: Some species of Boerhaavia genus 2
Figure 2.1: Green stem-leave Boerhaavia diffusa 17
Figure 2.2: Red-purple stem-leave Boerhaavia diffusa 17
Figure 2.3: Stem-leave Boerhaavia erecta 17
Figure 2.4: Comparison of TLC the four methanolic extracts of green
stem-leave (GS), red-purple stem-leave (RS), green root (GR) and
red-purple root (RR) of Boerhaavia diffusa. 19
Figure 2.5: HPLC chromatograms of (a) red-purple and (b) green B. diffusa 19

Figure 3.1 Comparison of CD and absorption spectra of compound 59
obtained by the calculations with the experiments 64
Scheme 1: Extraction and isolation procedure for Green Boerhaavia diffusa 27
Scheme 2: Extraction and isolation procedure for Red-purple Boerhaavia
diffusa 28
Scheme 3: Extraction and isolation procedure for Boerhaavia erecta 29
ix
1

CHAPTER 1: LITERATURE REVIEW



Boerhaavia chinensis
[w1]

Boerhaavia coccinea
[w2]

Boerhaavia coulteria
[w3]




Boerhaavia diffusa
[w4]

Boerhaavia erecta
[w5]

Boerhaavia gracillima
[w6]




Boerhaavia herbstii
[w7]

Boerhaavia intermedia
[w8]

Boerhaavia linearifolia
[w9]




Boerhaavia pterocarpa
[w10]

Boerhaavia purpurascens
[w11]

Boerhaavia repens
[w12]

2



Boerhaavia scandens
[w13]

Boerhaavia tetrendra
[w14]



Boerhaavia triquetra
[w15]

Boerhaavia wrightii
[w16]

Figure 1.1. Some species of Boerhaavia genus.
1.1. BOERHAAVIA GENUS – GENERIC DESCRIPTION
Boerhaavia is a genus of 40 species, most of which are widely distributed
in tropical and subtropical areas of the world.
[19][69]
Some of the species are
treated as complex groups. They are usually coastal plants spreading in sandy
areas, gravelly plains, or on rocky slopes. Owing to the polymorphism and the
possible existence of hybrids between members of the complexes, many authors
of regional floras have indicated that the distinctiveness at the species level,
especially in the pantropical Boerhaavia diffusa complex and Boerhaavia repens
complex, were rather difficult.

[49][76][78]

The genus Boerhaavia is characterized by elongate stems with prominent
nodes, slender forking cymose or rarely racemose or spicate inflorescences with
small, scale-like free bracts, small flowers with campanulate, cylindric, funnel-
shaped perianth limb separated by a constriction from the sulcate, enlarged lower
3

portion of the perigone, and anthocarps that are clavate or oblong, often
glandular.
[19]

According to the literature, among 40 species of Boerhaavia, 6 species are
found in India. They are Boerhaavia diffusa, Boerhaavia erecta, Boerhaavia
rependa, Boerhaavia chinensis, Boerhaavia hirsute and Boerhaavia
rubicunda.
[41]
This genus Boehaavia represented in Taiwan by five species:
Boerhaavia coccinea Mill, Boerhaavia erecta L., Boerhaavia repens L.,
Boerhaavia glabrata Bl. and Boerhaavia hualienense Chen  Wu sp. nov.
[66]

In Vietnam, the generic description of this genus by the Vietnamese
botanists is not settled. The Vietnamese botanist Pham Hoang Ho
[2]
mentioned
that there were three species of Boerhaavia in Vietnam including B. diffusa L.
(Vietnamese name: nam sâm or nam sâm bò), B. chinensis (L.) Asch.  Schw.
(Vietnamese name: nam sâm trung quốc) and B. erecta L. (Vietnamese name:
nam sâm đứng). The pharmacist Do Tat Loi

[3]
wrote that there was only one
species of Boerhaavia, that was B. diffusa L., or B. procumbens Wight, or B.
repens L. The botanist Vo Van Chi
[1]
declared that there were two species and
they were B. diffusa L. (or B. repens L.) and B. chinensis (L.) Asch.  Schweinf.
(or B. repanda Willd).
Some pictures of Boerhaavia species have been found widely on the
internet (Figure 1.1).
1.1.1. BOERHAAVIA COCCINEA M.
[20][66][78]

Perennial herbs, prostrate to ascending, up to 80 cm tall. Stems terete, 2-5
mm across, green flushed with purplish red, glandular puberulent and sparsely
pilose. Leaves somewhat fleshy, opposite, in unequal pairs; blades ovate, broadly
ovate to suborbicular, upper surface yellowish green, green to bluish green,
obscurely glandular to glabrous, lower surface pale, pubescence on veins,
margins entire, cartilaginous, undulate to slightly lobed, distinctly long-ciliate ca.
1 mm long, obtuse to rounded at the apex, truncate to subcordate at the base, the
larger one 3-8 cm long and 2-7 cm wide, with a long petiole up to 4 cm long, the
4

smaller 1.5-3 cm long and 1-2.5 cm wide, with a petiole less than 1 cm long;
petioles pale flushed with purplish red, with long hairs.
1.1.2. BOERHAAVIA DIFFUSA L.
[41][66]

Perennial herbs (rarely appearing annual), sometimes slightly woody at
base; taproot fusiform, woody. Stems decumbent, ascending, or erect, usually

profusely branched throughout, 3-10 dm, glabrous or minutely pubescent basally,
glabrous or sparsely pubescent distally. Leaves mostly in basal half of plant;
larger leaves with petiole 10-30 mm, blade broadly lanceolate, ovate, or broadly
ovate, occasionally round, 20-60 mm × 15-40 mm (distal leaves smaller, often
proportinally narrower), base truncate, broadly cuneate, round, or shallowly
cordate, often oblique, margins sinuate, apex obtuse to round, abaxial surface
paler than adaxial, glabrous or minutely puberulent, sometimes glandular, usually
with few large multicellular hairs on veins, adaxial surface glabrous, rarely
sparsely puberulent, neither surface punctate. Inflorescences terminal, forked ca.
3-6 times equally, diffuse, occasionally with sticky internodal bands; branches
divergent, terminating in compact subumbellate or capitate, 2-5-flowered
clusters. Flowers pedicel shorter than 0.5 mm; bract at base of perianth quickly
deciduous, 1, lanceolate, 0.8-1 mm; perianth purplish red to reddish pink or
nearly white, campanulate beyond constriction, 1-1.5 mm; stamens 2-3, included
or barely exserted. Fruits 1-9 per cluster, gray-brown to brown, obpyramidal, 3-
4.5 mm × 1-1.2 mm, apex broadly conic, with sparse or moderately dense
stipitate-glandular hairs on ribs, less densely pubescent or glabrous in sulci
(rarely entirely glabrous); ribs 5, round, smooth; sulci 2-2.5 times as wide as base
of ribs, not rugose, not papillate.
[69]

B. diffusa was botanically reported possessing the green color of stems and
leaves.
[4][8][58]

1.1.3. BOERHAAVIA ERECTA L.
[66]

Annual herbs, slightly woody at base; taproot tappered. Stems usually
erect, sometimes decumbent, profusely branched primarily distally, 20-120 cm,

minutely puberulent with bent hair basally, usually glabrous, rarely sparsely
5

puberent distally. Leaves mostly in basal half of plant; larger leaves with petiole
6-50 mm, blade broadly rhombic-ovate, triangular-ovate, ovate, oval, or
lanceolate, 20-80 mm × 10-45 mm (distal leaves smaller, proportionately
narrower), adiaxial surface usually glabrous, sometimes minutely puberulent,
usually punctate with small patches of small brown cells, base obtuse to round,
margins entire or sinuate, usually with sticky internodal bands; branches strongly
ascending, terminating in irredular umbellate or subracemose clusters of flowers,
not all pedicels attaching at same point (flowers occasionally borne singly).
Flowers pedical 0-5 mm; bracts at base of perianth deciduous, usually 2,
narrowly to broadly lanceolate, 0.5-1 mm, apex often acuminate; perianth
whitish, usually tinged with pink or purple (bright pink) between lobes and in
tube, campanulate beyond constriction, 1-1.5 mm; stamens 2-4, slightly exserted.
Fruits 1-11 per cluster, pale greenish to straw colored or tan, narrowly obconic
2.7-4 mm × 1.2-1.5 mm, apex truncate or broadly low conic, glabrous; ribs 5,
acute, slightly rugose adjacent to sulci; sulci 0.5-1 times as wide as base of ribs,
slightly to prominently coarsely tranverse rugose, not papillate.
[69]
B. erecta occasionally forms mixed populations with B. intermedia
without apparent intergradation. Rarely, some specimens seem to combine
features of either species, particularly with regard to inflorescence structure. The
two species bloom simultaneously and are visited by small insects. Given the
presumed close relationship and weedy habitats of each, hybridization seems
possible. Usually, the two species can be distinguished by the differences in fruit
length, the appearance of a crownlike apex of the nearly mature fruits of B.
erecta (apex of ridges slightly expanded, apex of fruit slightly conic), and the
more precisely constructed terminal umbels of B. intermedia. Both species,
particularly B. intermedia, may produce entire inflorescences with branches

terminating in single flowers.
[69]
Woodson et al. (1961) suggested that B. erecta
hybridizes with the perennial B. diffusa.
[79]
6

1.1.4. BOERHAAVIA REPENS L.
[66]

Perennial herbs, prostrate, ascending to erect, up to 50 cm tall. Stems
fleshy, 2-5 mm across, green flushed with purplish red, pubescent, becoming
glabrous and woody towards the base. Leaves somewhat fleshy, opposite, in
unequal pairs; blades ovate, ovate-lanceolate to lanceolate or elliptic, upper
surface green, flushed with purplish red, puberulent or subglabrous, lower
surface pale, pubescent on veins, margins entire, cartilaginous undulate to
slightly lobed, glabrous to distinctly short-ciliate, obtuse at the apex, usually
minutely apiculate, truncate, rounded to subcordate at the base, the larger one 3-
5.5 cm long and 2-4 cm wide, with a long petiole up to 2.5 cm long, the smaller
1.5-2.5 cm long and 1-3 cm wide, with petiole less than 1.5 cm long; petioles
pale flushed with purple, pubescent, the leaves in the portion from stem to
inflorescence gradually reduced. Inflorescences of pedunculate cymes or
pseudoumbels, auxillary, or a small terminal panicle, the peduncules 1-8 cm
long, 2-4 branched, capillary at apex, green flushed with purplish red, pubescent
and glanduar to glabrous, each branch of peduncle subtended by a bract, bracts
linear lanceolate, puberulent, 1.5 mm long, caducous. Flowers 3-6, clustered at
the apex of peduncle, subsessile; bracteoles usually 2, subtending a flower, in an
unequal pair, the smaller slightly above, linear-lanceolate, 1 mm long and 0.2
mm wide, the larger below, lanceolate, 1.2 mm long and 0.3 mm wide, all those
pale yellow, flushed with purple, puberulent.

1.2. BOERHAAVIA GENUS –PHARMACOLOGICAL STUDIES
1.2.1. BOERHAAVIA COCCINEA M.
There has been no publication on the pharmacological activities of
Boerhaavia coccinea L. to the best of our knowledge.
1.2.2. BOERHAAVIA DIFFUSA L.
[5]

Rawat et al. (1997)
[59]
reported on hepatoprotective effect of B. diffusa.
Their studies also revealed that the administration of aqueous extract of B diffusa
(2 ml/kg) exhibited more effectively hepatoprotective activity than the powder
form. More importantly, in 2010, Olaleye et al.
[56]
also confirmed the
7

hepatoprotective activity of leaf extracts of B. diffusa against acetaminophen-
induced liver damage.
In 2000, Hiruma-Lima et al.
[22]
reported the remarkably analgesic property
of juice of fresh leaves of B. diffusa L.
The alkaloidal component of B. diffusa was proved to possess various
biological activities. Mungantiwar et al. (1999)
[52]
communicated that the
alkaloidal fraction exhibited immunomodulatory effect in vitro by inhibiting
SRBC-induced delayed hypersensitivity reactions in mice. Leyon et al. (2005)
[40]

communicated that aqueous methanolic 70% extract of B. diffusa inhibited the
metastasis of B16F-10 melanoma cells in C57BL/6 mice. Manu and Kuttan
(2009)
[42]
continuously reported that punanarvine isolated from B. diffusa
possessed antimetastatic property using B16F-10 melanoma cells in C57BL/6
mice due to the abilitity to suppress MMP-2, MMP-9, ERK-1, ERK-2, and
VEGF in the lung tissue of metastasis-induced animals. Manu and Kuttan
[43]
also
reported that punanarvine induced apoptosis in B16F-10 melanoma cells by
inhibiting NF-kB signaling.
In 2002, Mehrotra et al.
[47]
reported immunomodulatory effect of the
ethanolic extract of B. diffusa. The study showed that the extract exhibited
cytotoxic effect against human NK cell line in vitro, production of NO in mouse
macrophage cells, IL-2 and TNF-α human PBMCs. In addition, Mehrotra et
al.
[48]
noted that the extract also exerted the antiproliferative effect against several
lympho cell lines of mouse and human origin, i.e. mouse macrophage cells
(RAW 264.7), human macrophage cells (U937), human monocytic cells (THP-
1), mouse fibroblast cells (L929), human embryonic kidney cells (HEK293),
mouse liver cells (BNLCL.2), African green monkey kidney cells (COS-1),
mouse lymphoma cells (EL-4), human erythroleukemic cells (K562) and human
T cells (Jurkat).

Meanwhile, the methanolic extract of whole plant B. diffusa was

reported possessing antiproliferative effect against breast cancer cell lines (MCF-
7).
[70]
Bharali et al. (2003)
[10]
reported that B. diffusa possessed DMBA-induced
cancer chemopreventive activity.
8

1.2.3. BOERHAAVIA ERECTA L.
There have been few publications on the pharmacological activities of
Boerhaavia erecta L. to the best of our knowledge. Stintzing et al.
[72]
reported the
qualitative and quantitative analysis of the chemical composition of B. erecta
containing betacyanins and phenolic compounds which might be responsible for
its medicinal use in West Africa.
Hilou et al. (2006)
[21]
noted that the extract of B. erecta possessed in vivo
antimalarial activity. The plant extract showed significant antimalarial activity in
4-day suppressive antimalarial assay in mice inoculated with red blood cells
paratisized with Plasmodium berghei berghei. While the obtained value for ED
50

was 564 mg/kg, the toxicity of the plant extract was very low with the value of
LD
50
ca. 2150 mg/kg.
In 2012, Suriyavathana et al.

[74]
reported that leaf extract of B. erecta
exerted positive antimicrobial and antifungal activities against several bacterial
and fungal species.
1.3. BOERHAAVIA GENUS – CHEMICAL STUDIES
1.3.1. BOERHAAVIA COCCINEA M.
According to the best of our knowledge, there were three publications
reported for B. coccinea.
In 1986, Messana et al.
[50]
reported the presence of two 12a-
hydroxyrotenoids from roots of B. coccinea, but the authors did not offer their
chemical structures.
In 1991, Ferrari and Messana
[18]
reported the presence of coccineone A (19)
and B (20) in B. coccinea roots.
In 1998, Santos et al.
[62]
reported the isolation from the whole plant three
rotenoids coccineone C (21), coccineone D (23) and coccineone E (24).
1.3.2. BOERHAAVIA DIFFUSA L.
Up to now, although there have been many reports on Boehaavia genus,
these reports concentrated mostly on Boehaavia diffusa species.
9

There were several reports on the chemical constituents of B. diffusa
collected in India. Generally, to the best of our knowledge, the secondary
metabolites of B. diffusa mainly consisted of flavonoids including flavonols,
isoflavones, and rotenones as the major composition aside from the minor

component of steroids, triterpenoids, lignans and betanins. The very first study
on B. diffusa dates back to 1971 when Misra and Tiwari
[51]
reported the isolation
of β-sitosterol (1), ursolic acid (7), D-glucose (50), D-galactose (51), D-xylose
(52), fructose (53) from roots of B. diffusa. Eleven years later, Suri et al.
(1982)
[73]
reported the identification of β-ecdysone (3), stigmasterol 3-O--D-
glucopyranoside (6) from roots of B. diffusa. In 1986, Seth et al.
[64]
reported the
isolation of punarnavoside (46) from B. diffusa possesing antifibrinolytic activity.
In 1989, two other rotenoids boeravinone A (9) and B (10) were isolated
and identified also from roots of B. diffusa by Kadota et al
[33]
And in the same
year, Kadota et al.
[32]
reported for the first time the isolation and identification
of a rotenoid, namely boeravinone C (11), from B. diffusa roots. Since
boeravinone C belonged to a subgroup of rotenoids which was designated as 12a-
hydroxyrotenoid, this resulted in the necessity in the determination of the
stereochemistry of C-6a and C-12a. Accordingly, Lami et al. (1990)
[36]
published
a detailed report on the stereochemistry of boeravinone C suggesting that the B/C
ring junction oriented in trans-geometry. The presence of boeravinone D (12), E
(13), and F (14) from roots of B. diffusa were subsequently reported in 1991 by
Lami et al

[37]
Additionally, Lami et al. (1991)
[38]
aslo reported the isolation of
liriodendrin (48) and syringaresinol mono-β-D-glucoside (47) in which
liriodendrin posessed significant calcium (Ca
2+
) channel antagonistic effect.
A xanthone named borhavine (49) was identified in roots of B. diffusa by
Ahmed et al. in 1992.
[6]
It was not until 2005 that two other rotenoids were
reported by Borrelli et al.
[12]
when they isolated boeravinone G (15) and H (16)
along with boeravinone D and E from roots of B. diffusa. In the following year,
Borrelli et al.
[13]
continued to publish a report on the identification of 10-
demethylboeravinone C or coccineone C (21), 6-O-demethylboeravinone H (25),
10

coccineone B (20), 9-O-methyl-10-hydroxycoccineone B (22), coccineone E (24)
and 2′-O-methylabronisoflavone (38) in roots of B. diffusa. In 2007, while
Belkacem et al.
[9]
reported the presence of boeravinone I (17) and J (18) in which
the latter was a coumaronochromone instead of a rotenone as in the previous
series of boeravinones. Some other authors
[20][42]

also reported the presence of
some compounds from the whole plant of B. diffusa: stigmasterol (2),
boerhavisterol (4), -sitosterol 3-O--D-glucopyranoside (5), boerhavilanostenyl
benzoate (8), diffusarotenoid (26), eupalitin (28), kaempferol (29) and eupalitin
3-O-β-D-galactopyranoside (34).
1.3.3. BOERHAAVIA ERECTA L.
There were two publications on the chemical constituents of Boerhaavia
erecta L. to the best of our knowledge. In 2003, Stintzing et al.
[72]
reported the
qualitative and quantitative analysis of the chemical composition of B. erecta
containing flavonoids such as 5,7-dihydroxy-3’,4’-dimethoxyl-6,8-
dimethylflavone (27), quercetin 3-O-glucoside (31), isorhamnetin 3-O-rutinoside
(32), quercetin 3-O-rutinoside (33) (predominant compound), (+)-catechin (39),
(-)-epicatechin (40), procyanidin B1 (41), procyanidin B2 (42) and betalains such
as betanin (43), neobetanin (44) and amaranthine (45).
1.3.4. BOERHAAVIA REPENS L.
There was only one publication on the chemical constituents of Boerhaavia
repens. Jianxin Li et al.
[31]
reported the presence of eupalitin 3-O--D-
galactopyranoside (34), eupalitin 3-O-rutinoside (35), kaempferol 3-O-rutinoside
(36), and 6-methoxylkaempferol 3-O-rutinoside (37) in the methanolic extract of
the whole plant of this species.

11




12





13




14



AMINO ACID: Alanine, aspartic acid, glutamic acid, glycine, histidine, leucine,
methionine, proline, serine, threonine, tyrosine, valine.
FATTY ACID: Palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid.
CH
3
–(CH
2
)
14
–COOH : Hexadecanoic acid (Palmitic acid)

CH
3
–(CH
2
)
16
–COOH : Octadecanoic acid (Stearic acid)
CH
3
–(CH
2
)
18
–COOH : Eicosanoic acid (Arachidic acid)
CH
3
–(CH
2
)
20
–COOH : Docosanoic acid (Behenic acid)
CH
3
–(CH
2
)
7
–CH=CH–(CH
2
)

7
–COOH : Octadecenoic acid (Oleic acid)
15

1.4 AIM OF THE STUDY
In the view of the importance of the plants as remedy to diseases, we
chosed green stem-leaves Boerhaavia diffusa, red-purple stem-leaves Boerhaavia
diffusa and Boerhaavia erecta for chemical investigation.
The aim of this research is to isolate and structurally elucidate the
compounds and to test biological activities of the compounds from these three
species of the genus Boerhaavia, Nyctaginaceae which are grown in Vietnam.
16

CHAPTER 2: EXPERIMENTAL
2.1. INSTRUMENTATION AND MATERIALS
NMR spectra were recorded on a Bruker Avance III spectrometer 500 MHz
using residual solvent signals as internal references: chloroform-d

H
7.24,

C

77.23; methanol-d
4


H
3.31,


C
49.15; DMSO-d
6


H
2.50,

C
39.51; acetone-d
6


H

2.09,

C
206.31, 30.6; and pyridine-d
5
at

H
8.74, 7.58 and 7.22,

C
123.87,
135.91, 150.35.
The HR–ESI–MS was recorded on a HR–ESI–MS MicroOTOF–Q mass
spectrometer on a LC- Agilent 1100 LC-MSD Trap spectrometer.

Melting point of compounds was measured by a microscope hot stage,
Kofler, Polytherm A and was uncorrected. The specific rotation was measured by
a polarimeter ‒ A. Krüss Optronic, Germany. These instruments are in the
Laboratory of the Department of Organic Chemistry, University of Science,
National University - Ho Chi Minh City.
UV absorption and CD spectra were measured on a JASCO V-570
spectrophotometer and on a JASCO J-820E spectropolarimeter, respectively.
These instruments are in the Laboratory of Analytical Chemistry, Osaka
University, Japan.
TLC was carried out on precoated Silica gel 60 F
254
or Silica gel 60 RP–18
F
254
S (Merck). Spots were visualized by spraying with 20% aqueous H
2
SO
4
or
5% ferric chloride solutions followed by heating. Gravity column
chromatography was performed with Silica gel 60 (0.040 – 0.063 mm, Himedia).
The HPLC system was performed on an Agilent 1200 (USA) equipped with a
G1311A quad pump, G1322A degasser, UV-VIS detector MWD G1365D and
auto-sampler G1329A. The separation was carried out on an Eclipse XDB-C18
column (4.6 × 150 mm i.d., 5 μm) with a C-18 guard column.
Some instruments are at the Central Analysis Laboratory of the University
of Science, National University-Ho Chi Minh City and some are at the
17

Laboratory of the Department of Organic Chemistry, University of Science,

National University - Ho Chi Minh City.
Solvents used as n-hexane, chloroform, ethyl acetate, methanol, acetic acid
for TLC and CC were purchased from Chemsol. Acetic acid, water, and
acetonitrile used for HPLC analysis were purchased from Merck.
 Chemical used in the acetylcholinesterase biological activity
 Acetylthiocholine iodide (ATCI) (21.67 mg/ml) dissolved in deionized water
(Sigma)
 Acetylcholinesterase (AChE) dissolved in 0.1 % bovine serum albumin (BSA)
 Bovine serum albumin (Sigma)
 5,5’-Dithio-bis-nitrobenzoic acid (DTNB) (Sigma) 0.01 M DTNB is dissolved
in buffer containing 0.1 M NaCl and 0.02 M MgCl
2

 Galanthamine (Sigma)
 Buffer of solution Tris HCl 1M pH 8.0 (Invitrogen)
 Enzyme AChE, ATCI, galanthamine are stored at – 80
0
C.
 BSA, DTNB, Tris HCl are stored at 4
0
C
Composition of 1 well
 25 µl of the solution 15 mM acetylthiocholine iodide (ATCI).
 125 µl of the solution 5’-dithio-bis-nitrobenzoic acid (DTNB) (Sigma) 3
mM in the buffer C (50 mM tris–HCl pH 8.0, 0.1 M NaCl, 0.02 M MgCl
2
)
 50 µl buffer solution B (50 mM Tris–HCl pH8, 0.1% bovine serum albumin
(BSA).
 25 µl sample of extract or pure compound is dissolved in buffer solution

containing the maximum of 10% MeOH.
Samples were tested at 3 concentrations: 1.0 mg/mL, 0.5 mg/ mL and
0.25 mg/ mL.
 25 µl Acetylcholinesterase (AChE) 0.22 U/ml (in buffer solution B).
 Pure Galanthamine (Sigma) was used as positive control, at 3
corresponding concentrations (ten time dilution): 0.1 mg/ mL, 0.05 mg/ mL
and 0.025 mg/ mL.