Anti-tubercular agents from Ammannia baccifera (Linn.)

Article abstract of DOI:10.1007/s00044-012-9998-9

Ammannia baccifera is an important component of various traditional Chinese herbal formulations. The samples of A. baccifera were extracted with methanol and the methanolic extract was successively fractionated with n-hexane, chloroform and n-butanol. The serial chromatographic separation of chloroform and n-butanol fractions resulted in the isolation and characterization of betulinic acid (1), 4-hydroxy-α-tetralone (2), tetralone-4-O-β-d- glucopyranoside (3) and ellagic acid (4). Further, the compound 2 was chemically modified into its five semi-synthetic acyl and aryl derivatives (2A-2E) namely; 4-O-m-anisoyl-α-tetralone (2A), 4-O-benzoyl-α-tetralone (2B), 4-O-palmitoyl-α-tetralone (2C), 4-O-(3,4,5-trimethoxybenzoyl)-α- tetralone (2D) and 4-O-myricitoyl-α-tetralone (2E). The tetralone group compounds 2, 3 and semi-synthetic derivatives 2A-2E were evaluated for their in vitro anti-tubercular activity against Mycobacterium tuberculosis H37Rv by BACTEC-460 radiometric susceptibility assay. The compounds 2 and 2E have shown significant anti-tubercular activity (MIC 50 μg/ml) while 2B was moderate active (MIC 100 μg/ml) against the pathogen. The results of SAR study indicated that substitutions in alpha hydroxyl group of 4-hydroxy-α- tetralone either retards or have no increasement in in vitro anti-tubercular potential.

Full text of DOI:10.1007/s00044-012-9998-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:16–21
DOI 10.1007/s00044-012-9998-9
ORIGINAL RESEARCH
Anti-tubercular agents from Ammannia baccifera (Linn.)
•
Harish C. Upadhyay Jay Prakash Thakur
•
•
Dharmendra Saikia Santosh K. Srivastava
Received: 20 January 2012 / Accepted: 16 February 2012 / Published online: 4 March 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Ammannia baccifera is an important component
of various traditional Chinese herbal formulations. The
samples of A. baccifera were extracted with methanol and
the methanolic extract was successively fractionated with n-
hexane, chloroform and n-butanol. The serial chromato-
graphic separation of chloroform and n-butanol fractions
resulted in the isolation and characterization of betulinic acid
(1), 4-hydroxy-a-tetralone (2), tetralone-4-O-b-D-glucopy-
ranoside (3) and ellagic acid (4). Further, the compound 2
was chemically modified into its five semi-synthetic acyl and
aryl derivatives (2A–2E) namely; 4-O-m-anisoyl-a-tetra-
lone (2A), 4-O-benzoyl-a-tetralone (2B), 4-O-palmitoyl-a-
tetralone (2C), 4-O-(3,4,5-trimethoxybenzoyl)-a-tetralone
(2D) and 4-O-myricitoyl-a-tetralone (2E). The tetralone
group compounds 2, 3and semi-synthetic derivatives 2A–2E
were evaluated for their in vitro anti-tubercular activity
against Mycobacterium tuberculosis H37Rv by BACTEC-
460 radiometric susceptibility assay. The compounds 2 and
2E have shown significant anti-tubercular activity (MIC
50 lg/ml) while 2B was moderate active (MIC 100 lg/ml)
against the pathogen. The results of SAR study indicated that
substitutions in alpha hydroxyl group of 4-hydroxy-a-tetra-
lone either retards or have no increasement in in vitro anti-
tubercular potential.
Keywords A. baccifera ꢀ Lythraceae ꢀ Anti-tubercular ꢀ
SAR ꢀ MDR ꢀ Tetralone
Introduction
Tuberculosis (TB) is one of the largest killers in the world.
This contagious disease spreads through air and is caused
by M. tuberculosis, a facultative intracellular bacterium.
According to WHO, 1.7 million deaths resulted from TB
alone in 2009, of which the highest number of death was in
Africa region (WHO, 2010). The South-East Asia region
accounted for the largest number of new TB cases in 2008,
which was 35% of global incidences. The incidence per
100,000 population in sub-Saharan Africa was nearly twice
that of the South-East Asia region (WHO, 2010). Even
though improved methods of prevention, detection, diag-
nosis and treatment have greatly reduced the number of
people who contact the disease and die from it, the emer-
gence of multi drug-resistant (MDR) strains and the global
HIV pandemic have amplified the incidence of TB (Nivin
et al., 1998; Pablos-Mendez et al., 1998). The emergence
of MDR strains of tuberculosis is of special concern due to
the reported high death rates associated with it (Som-
oskkovi et al., 2001; Snider and Castro, 1998). Because of
this, there is an urgent need for new anti-TB drugs with
improved properties such as enhanced activity against
MDR strains, reduced toxicity, shortened duration of
therapy, rapid mycobactericidal mechanism of action and
the ability to penetrate host cells and exert antimycobac-
terial effects in the intracellular environment.
H. C. Upadhyay ꢀ S. K. Srivastava (&)
Medicinal Chemistry Department, Central Institute of Medicinal
and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP, Lucknow
226015, India
e-mail: santoshkumar_1955@yahoo.com
The species of genus Ammannia (Lythraceae) are annual
herbs, commonly known as ‘‘Red stems’’ and are nearly
cosmopolitan in distribution including India. A. baccifera
(Linn.) is commonly called as ‘‘blistering Ammannia,’’
J. P. Thakur ꢀ D. Saikia
Molecular Bioprospection Department, Central Institute of
Medicinal and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP,
Lucknow 226015, India
123

Med Chem Res (2013) 22:16–21
17
Extraction and isolation
‘‘monarch red stem,’’ ‘‘acrid weed,’’ ‘‘agni-buti,’’ ‘ban
mirich’ and ‘‘jaungali mehndi.’’ In India it is commonly
found in marshy places and paddy fields. A. baccifera is
widely used in traditional Chinese herbal formulations for
treatment of spinal disease (Meng, 2009), hemorrhoids
(Cao, 2009), common cold (Zhu, 2011), human female
infertility and gastroenteropathy (Upadhyay et al., 2012).
It has been reported to possess anticancer, antirheumatic,
antidiuretic, antipyretic, antisteroidogenic, antimicrobial
and rubefacient activities (Upadhyay et al., 2012). There
are a few scanty reports on the isolation of betulinic acid,
4-hydroxy-1-tetralone, ellagic acid, and lupeol from
A. baccifera (Thakkar et al., 1986; Deeseenthum et al.,
2000), which prompted us to carry out the detailed phy-
tochemical investigation of A. baccifera and bioevaluation
of the isolated phytomolecules. The present study reports
on isolation and characterization of bioactive phytomol-
ecules (1–4) from A. baccifera as well as structure
activity relationship (SAR) study of 4-hydroxy-a-tetralone
derivatives for their in vitro anti-tubercular activity. To
the best of our knowledge, the isolation and character-
ization of 3 from A. baccifera and SAR study of
4-hydroxy-a-tetralone derivatives for in vitro anti-tuber-
cular activity is being reported here for the first time.
The dried and pulverized 100 g of root and 600 g of aerial
part of A. baccifera containing leaves and stem were
extracted separately with methanol (3 9 24 h). The extract
was dried under reduced pressure which resulted in 13 and
80 g of methanolic extract from root and aerial part of A.
baccifera respectively. The TLC profile of both the extracts
was more or less same, hence they were pooled together.
The pooled and dried methanolic extract (93 g) was dis-
solved in distilled water and successively partitioned with
hexane, chloroform, and n-BuOH. The combined fractions
were separately evaporated under reduced pressure to yield
hexane (28.0 g), chloroform (1.2 g) and n-BuOH (8.3 g)
fractions.
The TLC profile of chloroform extract showed the
presence of a major compound. In order to purify, 1.0 g of
chloroform extract was subjected to column chromatogra-
phy (silica gel 60–120 mesh, 18 g, column diameter
1 9 31 cm). Gradient elution of the column was carried
out with solvents of increasing polarity using hexane,
chloroform, and methanol in various proportions. A total of
104 fractions of 50 ml each were collected and pooled
according to their similarity in behaviour on TLC plate.
Fractions 58–66 eluted with hexane–chloroform (12:88)
afforded betulinic acid (1, 22 mg) while fractions 70–92
eluted with hexane–chloroform (10:90–2:98) afforded
4-hydroxy-a-tetralone (2, 380 mg). The butanol extract
(7 g) was subjected to vacuum liquid chromatographic
(VLC, 7.5 9 7.5 cm) separation over Silica gel H (100 g),
and eluted with a stepwise gradient of chloroform and
methanol. A total of 325 fractions of 50 ml each were col-
lected and pooled on the basis of their TLC profile. Fractions
114–123 eluted with chloroform–methanol (93:7), afforded
tetralone-4-O-b-D-glucopyranoside (3, 13.0 mg) while
fractions 315–316 eluted with methanol afforded ellagic acid
(4, 8.2 mg). Structures of the isolated compounds 1–4
(Fig. 1) were deduced on the basis of their 1D and 2D NMR
spectroscopic data and the values were in agreement with
those reported in literature (Sholichin et al., 1980; Peng etal.,
1998; An et al., 2003; Fournet et al., 1994; Srivastava et al.,
2007).
Materials and methods
General experimental procedures
The 300 MHz NMR (Avance, Bruker, Switzerland) was
1
used to record H and 13C NMR with tetra methyl silane
(TMS) as internal standard. Hyphenated LC-PDA-MS
(Prominence LC and mass MS-2010EV, Shimadzu,
Japan) was used for mass spectra. Optical rotation was
measured on high sensitive polarimeter (SEPA-300,
HORIBA) with 1 dm cell. TLC plates (silica gel 60F254,
Merck) were first examined under UV illumination at 254
and 365 nm and then sprayed with vanillin-sulphuric acid
(1:5 w/v) solution in ethanol followed by heating at 95°C
for 5 min.
Derivatization of 4-hydroxy-a-tetralone (2)
Plant material
The chemical derivatization of compound 2 into acyl and
aryl derivatives was carried out according to the method of
Saxena et al. (2007) with some modifications (Scheme 1).
Briefly, for each reaction the compound 2 (50 mg) was
dissolved in dry pyridine and the respective acyl/aryl
chloride was added in 1:1.5 molar ratio. Further, catalytic
amount of 4-dimethylaminopyridine (DMAP) was added
and the reaction mixture left overnight at room temperature
The samples of A. baccifera were collected from the Fa-
izabad district of Uttar Pradesh, India in October, 2010 and
identified by Dr. D. C. Saini, Scientist, Birbal Shahni
Institute of Palaeobotany (BSIP), Lucknow and Dr. S. C.
Singh, Taxonomy and Pharmacognosy Division, Central
Institute of Medicinal & Aromatic Plants (CIMAP), Luc-
know, India. A voucher specimen (No.-9460) was depos-
ited at the CIMAP herbarium.
123

18
Med Chem Res (2013) 22:16–21
Fig. 1 Structures of the isolated
compounds (1–4)
30
20
O
29
19
21
22
1
8
5
9
2
3
7
H
12
18
11
9
O
6
17
13
14
10
25
26
4
28
1
16
(2)
OH
2
3
OH
10
8
15
27
5
OH
7
O
HO
4
3'
6
HO
O
O
7
2'
1'
4'
H
23
(1)
8
1
24
9
2
3
7
6
5'
1
5
4
6'
7'
6
10
6'
3'
4
5
HO
2
O
O
OH
5'
3
O
O
HO
HO
4'
OH
(3)
2'
1'
(4)
OH
O
O
Dry C5H5N, RCOCl,DMAP
R. T., Overnight
OR
OH
7"
OCH3
3''
2''
6''
3''
O
2''
O
2' 3'
14'
16'
4'-13'
15'
O
1''
C
CH2CH2(CH2)10CH2CH2CH3
1''
2C R=
2E R=
4''
C
1'
2B R=
1'
4''
2A R=
C
1'
5''
5''
6''
7''
OCH3
O
2''
O
3''
4''
2'
3'
13'14'
4'-12'
8''
1''
CH2CH2(CH2)9CH2CH3
C
1'
2D R=
C
1'
OCH3
9''
OCH3
6''
5''
Scheme 1 Chemical derivatization of compound 2
(30–35°C). Progress of the reaction was monitored by TLC
and after completion of the reaction, crushed ice was added
to the reaction mixture. The reaction mixture was extracted
with chloroform (3 9 25 ml) and the combined chloroform
extract was washed with 6% aqueous-HCl solution to
remove pyridine. Finally, chloroform extract was washed
with distilled water, dried over anhydrous Na2SO4 and the
solvent removed under vacuum which afforded the
respective acyl/aryl derivative of compound 2. The acyl/
aryl derivatives so obtained were separately purified by
column chromatography (Silica gel 60–120 mesh, 14 g,
column diameter 2 9 30 cm). Gradient elution of the
column was carried out with solvents of increasing polarity
using hexane and chloroform in various proportions.
123

Med Chem Res (2013) 22:16–21
19
4-O-(3,4,5-trimethoxybenzoyl)-a-tetralone (2D)
Fractions of 25 ml each were collected and pooled on the
basis of their TLC profile, which afforded the respective
derivatives, 2A–2E in 85–95% yields.
Yield 85%, amorphous solid, ESI–MS (pos. ion mode):
1
m/z = 357 [M?H]? (calculated for C20H20O6, 356). H
NMR (300 MHz, CDCl3): d 2.54 (1H, m, H-2), 2.94 (1H,
m, H-2), 2.28 (1H, m, H-3), 2.39 (1H, m, H-3), 5.22 (1H, d,
J = 6.2 Hz, H-4), 7.90 (1H, d, J = 7.5 Hz, H-5), 7.71 (1H,
dd, J = 7.5, 1.5 Hz, H-6), 7.42 (1H, m, H-7), 8.05 (1H, dd,
J = 7.5, 1.5 Hz, H-8), 7.28 (1H, brs, H-200), 7.30 (1H, brs,
Hz, H-600), 3.92 (9H, s, H-700, H-800, H-900). 13C NMR
(75 MHz, CDCl3): d 197.9 (C-1), 34.6 (C-2), 28.7 (C-3),
70.4 (C-4) 127.0 (C-5), 134.4 (C-6), 129.1 (C-7), 128.4
(C-8), 132.2 (C-9), 143.0 (C-10), 165.9 (C-10), 125.4
(C-100), 101.2 (C-200 and C-600), 153.5 (C-300 and C-500),
141.7 (C-400), 55.5 (C-700 and C-900), 60.2 (C-800).
4-O-m-anisoyl-a-tetralone (2A)
Yield 88%, amorphous solid, ESI–MS (pos. ion mode): m/
z = 297 [M?H]? (calculated for C18H16O4, 296). 1H NMR
(300 MHz, CDCl3): d 2.5 (1H, m, H-2), 2.9 (1H, m, H-2),
2.28 (1H, m, H-3), 2.39 (1H, m, H-3), 5.20 (1H, d,
J = 6.2 Hz, H-4), 7.91 (1H, d, J = 7.5 Hz, H-5), 7.70 (1H,
dd, J = 7.5, 1.5 Hz, H-6), 7.42 (1H, m, H-7), 8.05 (1H, dd,
J = 7.5, 1.5 Hz, H-8), d 7.56 (1H, s, H-200), 7.08 (1H, m,
H-400), 7.34 (1H, m, H-500), 7.62 (1H, d, J = 7.3 Hz, H-600),
3.84 (3H, s, H-700). 13C NMR (75 MHz, CDCl3): d 197.2
(C-1), 35.0 (C-2), 31.9 (C-3), 75.1 (C-4) 127.0 (C-5), 133.4
(C-6), 129.1 (C-7), 128.6 (C-8), 132.0 (C-9), 143.4 (C-10),
166.2 (C-10), 132.50 (C-100), 129.3 (C-200), 159.7 (C-300),
122.0 (C-400), 114.4 (C-500), 119.1 (C-600), 55.4 (C-700).
4-O-myricitoyl-a-tetralone (2E)
Yield 91%, amorphous solid, ESI–MS (pos. ion mode):
m/z = 373 [M?H]?1(calculated for C24H36O3, 372). 1H
NMR (CDCl3, 300 MHz): d 2.52 (1H, m, H-2), 2.86 (1H,
m, H-2), 2.29 (1H, m, H-3), 2.41 (1H, m, H-3), 5.23 (1H, d,
J = 6.2 Hz, H-4), 7.94 (1H, d, J = 7.5 Hz, H-5), 7.68 (1H,
dd, J = 7.5, 1.5 Hz, H-6), 7.40 (1H, m, H-7), 8.12 (1H, dd,
J = 7.5, 1.5 Hz, H-8) d 2.31 (2H, t, H-20), 1.62 (2H, m, H-
30), 1.26 (20H, brs, H-40 to H-130), 0.88 (3H, H-140). 13C
NMR (CDCl3,75 MHz): d 198.2 (C-1), 35.0 (C-2), 30.6 (C-
3), 70.3 (C-4) 127.9 (C-5), 133.1 (C-6), 130.1 (C-7), 129.5
(C-8), 135.3 (C-9), 142.6 (C-10), 174.6 (C-10), 33.1 (C-20),
30.7 (C-30), 30.9 (C-40), 29.7 (C-50), 30.3 (C-60 to C-120),
23.7 (C-130), 14.5 (C-140).
4-O-benzoyl-a-tetralone (2B)
Yield 90%, viscous liquid, ESI–MS (pos. ion mode):
1
m/z = 267 [M?H]? (calculated for C17H14O3, 266). H
NMR (300 MHz, CDCl3): d 2.5 (1H, m, H-2), 2.9 (1H, m,
H-2), 2.28 (1H, m, H-3), 2.39 (1H, m, H-3), 5.20 (1H, d,
J = 6.2 Hz, H-4), 7.91 (1H, d, J = 7.5 Hz, H-5), 7.70 (1H,
dd, J = 7.5, 1.5 Hz, H-6), 7.42 (1H, m, H-7), 8.05 (1H, dd,
J = 7.5, 1.5 Hz, H-8), 7.36 (3H, m, H-200, 400, 600), 7.50
(2H, m, H-300, 500). 13C NMR (75 MHz, CDCl3): d 196.6
(C-1), 35.0 (C-2), 29.0 (C-3), 69.7 (C-4) 128.9 (C-5), 131.0
(C-6), 129.5 (C-7), 128.6 (C-8), 132.7 (C-9), 142.6 (C-10),
166.5 (C-10), 134.3 (C-100), 129.4 (C-200, C-600), 128.4
(C-300, C-500), 128.6 (C-400).
In vitro anti-mycobacterial assay
M. tuberculosis H37Rv (ATCC 27294) used in this
screening was obtained from National JALMA Institute for
Leprosy and other Mycobacterial Diseases (ICMR), Agra,
4-O-palmitoyl-a-tetralone (2C)
¨
India and maintained on Lowenstein-Jansen media slant at
37°C. After 21 days of incubation, bacterial cells were
scraped from slants and transferred in 1.0 ml of BACTEC
diluting fluid and made complete homogenized suspension
by vortexing with glass beads (2 mm diameter). The sus-
pension was allowed to stand for a few minutes to permit
sedimentation of the bacterial clumps if any. The turbidity
of the homogenous suspension was adjusted to McFarland
standard 1.0 with diluting fluid. A BACTEC 12B vial
(Becton–Dickinson) was injected with 0.1 ml of this sus-
pension. This vial was used as primary inoculum after the
growth index (GI) reached a value of about 500 (approxi-
mately 1 9 106 cfu/ml). Briefly, 0.1 ml of bacterial sus-
pension from the primary inoculum culture vial (GI 500)
was injected into test compound-containing vials using
1.0 ml insulin syringe. To comply with 1% proportion
Yield 92%, viscous solid, ESI–MS (pos. ion mode):
1
m/z = 401 [M?H]? 1(calculated for C26H40O3, 400). H
NMR (CDCl3, 300 MHz): d 2.50 (1H, m, H-2), 2.82 (1H,
m, H-2), 2.29 (1H, m, H-3), 2.40 (1H, m, H-3), 5.22 (1H, d,
J = 6.2 Hz, H-4), 7.92 (1H, d, J = 7.5 Hz, H-5), 7.68 (1H,
dd, J = 7.5, 1.5 Hz, H-6), 7.40 (1H, m, H-7), 8.12 (1H, dd,
J = 7.5, 1.5 Hz, H-8), 2.34 (2H, t, J = 7.5 Hz, H-20), 1.60
(2H, m, H-30), 1.25 (24H, brs, H-40 to H-150), 0.88 (3H, brs,
H-160). 13C NMR (CDCl3,75 MHz): d 198.5 (C-1), 34.7
(C-2), 30.3 (C-3), 69.7 (C-4) 127.7 (C-5), 134.9 (C-6),
129.7 (C-7), 129.0 (C-8), 132.5 (C-9), 141.8 (C-10) 174.4
(C-10), 32.6 (C-20), 30.2 (C-30), 29.7 (C-40), 29.8 (C-50 and
C-130), 30.1 (C-60 to C-120), 25.6 (C-140), 23.3 (C-150),
17.0 (C-160).
123

20
Med Chem Res (2013) 22:16–21
Biology
method, 0.1 ml of primary inoculums was added to 9.9 ml
BACTEC diluting fluid to obtain 1:100 dilutions. From this
0.1 ml was injected into two 12B vials for two test com-
pounds containing 4.0 ml medium along with 40 ll of
DMSO. Vials were incubated at 37°C, and the GI was
recorded every 24 h in a BACTEC 460 TB instrument
(Becton–Dickinson). Once the GI of the control vial
(1:100) reached 30 then the GI values of the test (com-
pound-containing) vials were compared with that of control
vials based on difference in growth (called as DGI). The
result was interpreted as follows: If the difference (DGI) of
current GI from previous day GI in the case of drug con-
taining vials is lower than the DGI of 1:100 control vial for
the same period then the test compound is termed as active
against MTB or otherwise inactive. Twofold serial dilution
technique was used to assess the minimum inhibitory con-
centration (MIC) of the test compounds (Dhar et al., 1968).
Only broth culture was used as a positive control and media
as a negative control. The MIC (lg/ml) of each test com-
pound was determined as the lowest concentration that
inhibited growth of the organism in triplicate experiments.
Lin et al. (2005) have reported the anti-mycobacterial
activity of (-)4-hydroxy-a-tetralone (MIC 4 lg/ml)
against M. tuberculosis H37Rv by agar dilution method.
However, in our experiments, the 4-hydroxy-a-tetralone
was found to be dextrorotatory ([a]2D2 = ? 17.84
(c = 0.00127 g/ml, chloroform), and exhibited anti-tuber-
cular activity MIC at 50 lg/ml against M. Tuberculosis
H37Rv strain radiorespirometrically. The results of anti-
tubercular activities indicated that the MIC values for
compounds 2, 2B, and 2E were 50, 100, and 50, respec-
tively, while compounds 2A, 2C, 2D, and 3 were found
inactive at MIC 100 lg/ml against M. tuberculosis H37Rv.
A moderate to high lipophilicity of the compounds exhibit
better antitubercular activity due to the lipophilic nature of
the Mycobacterium cell wall. Over 60% of Mycobacterium
cell wall is lipid due to which it poses difficulty to transport
polar compounds through outer lipid layer of cell wall and
thus, the pathogen is resistant to many acidic and alkaline
compounds including antibiotics. (Saikia et al., 2010). In
present case, the compound 3 has hydrophilic glucose at
the position four of tetralone moiety and the anti-tubercular
activity has decreased in comparison to parent compound
(2) but unexpectedly, the activity also decreased in its
lipophilic acyl/aryl derivatives except in 2E where it
remains constant. Hence, the anti-tubercular potential of
compound 2 was not enhanced in both cases i.e. increase in
lipophilicity by acylation/arylation and attachment of glu-
cose moiety (hydrophilic) at position four of tetralone.
Results and discussion
Chemistry
Although isolation of ellagic acid, quercetin, betulinic acid,
lupeol, 1,4-naphthoquinone and 4-hydroxy-1-tetralone from
different parts of A. baccifera have been reported in earlier
studies (Thakkar et al., 1986; Deeseenthum et al., 2000), in
some cases the characterization of compounds was not
based on interpretations using modern spectroscopic
methods like 1D and 2D NMR, IR and ESI–MS spectro-
scopic data. Further, A. baccifera is being frequently used in
treatment of various diseases in traditional Chinese and
Indian medicine, owing to its bioactive potential, the
detailed phytochemical investigation of this plant may lead
to isolation and characterization of potent bioactive mole-
cules. The extraction of plant material, fractionation and
serial chromatographic separation of extracts resulted in
isolation and characterization of betulinic acid (1),
4-hydroxy-a-tetralone (2), tetralone-4-O-b-D-glucopyrano-
side (3) and ellagic acid (4). Since the compound 2 was
isolated as a major compound and potent anti-tubercular
activity was reported in (-)4-hydroxy-a-tetralone (Lin
et al., 2005), for SAR study, it was modified to semi-syn-
thetic acyl and aryl derivatives; 4-O-m-anisoyl-a-tetralone
(2A), 4-O-benzoyl-a-tetralone (2B), 4-O-palmitoyl-a-tet-
ralone (2C), 4-O-(3,4,5-trimethoxybenzoyl)-a-tetralone
(2D) and 4-O-myricitoyl-a-tetralone (2E).
Conclusion
The present study on A. baccifera led to isolation and
characterization of four bioactive phytomolecules (1–4)
using modern spectroscopic techniques. The preliminary in
vitro anti-tuberculosis screening of the tetralone group
compounds 2, 3 and semi-synthetic derivatives 2A–2E has
evidenced that substitutions in alpha hydroxyl group of 4-
hydroxy-a-tetralone (2) retards in vitro anti-tuberculosis
activity. Further, it may also be concluded that both dextro
and laevo rotatory forms of compound 2 are active against
M. tuberculosis H37Rv but the antitubercular potential of
later is more than former.
Acknowledgments Financial support for this research by CSIR and
UGC for providing fellowship to one of us (HCU) is gratefully
acknowledged. We also thank all of our colleagues for their excellent
assistance.
Conflict of interest The authors report no conflict of interest. The
authors alone are responsible for the content and writing of the article.
123

Med Chem Res (2013) 22:16–21
21
Saikia D, Parihar S, Chanda D, Ojha S, Kumar JK, Chanotiya CS,
Shanker K, Negi AS (2010) Antitubercular potential of some
semisynthetic analogues of phytol. Bioorg Med Chem Lett
20:508–512
References
An TY, Hu LH, Chen RM, Chen ZL, Li J, Shen Q (2003) Anti-
diabetes agents-I: tetralone derivatives from Juglans regia. Chin
Chem Lett 14:489–490
Cao Y (2009) Chinese herbal medicine for treating hemorrhoids and
preparation thereof. Chinese patent 101549115 A
Deeseenthum S, Tip-pyang S, Wattanasirmkit K, Phuwapraisirisan P
(2000) Toxic metabolites from Ammannia baccifera. Asian
Coord Group Chem 12:47–50
Dhar ML, Dhar MM, Dhawan BN, Mehrotra BN, Ray C (1968)
Screening of Indian plants for biological activity: part I. Indian J
Exp Biol 6:232–247
Fournet A, Barrios AA, Munoz V, Hocquemiller R, Roblot F, Cave A
(1994) Antileismanial activity of a tetralone isolated from
Ampelocera edentula, a Bolivian plant used as a treatment for
cutaneous leishmaniasis. Planta Med 60:8–12
Lin WY, Peng CF, Tsai IL, Chen JJ, Cheng MJ, Chen IS (2005)
Antitubercular constituents from the roots of Engelhardia
roxburghiana. Planta Med 71:171–175
Saxena M, Faridi U, Mishra R, Gupta MM, Darokar MP, Srivastava
SK, Singh D, Luqman S, Khanuja SPS (2007) Cytotoxic agents
from Terminalia arjuna. Planta Med 73:1486–1490
Sholichin M, Yamasaki K, Kasai R, Tanaka O (1980) 13C Nuclear
magnetic resonance of lupane-type triterpenes, lupeol, betulin
and betulinic acid. Chem Pharm Bull 28:1006–1008
Snider DE, Castro KG (1998) The Global threat of drug-resistant
tuberculosis. N Engl J Med 338:1689–1690
Somoskkovi A, Parsons LM, Salfinger M (2001) The molecular basis
of resistance to isoniazid, rifampin, and pyrazinamide in
Mycobacterium tuberculosis. Respir Res 2:164–168
Srivastava A, Rao LJM, Shivanandappa T (2007) Isolation of ellagic
acid from the aqueous extract of the roots of Decalepis
hamiltonii: antioxidant activity and cytoprotective effect. Food
Chem 103:224–233
Thakkar SM, Deshmukh VK, Saoji AN, Duragkar NJ (1986)
Chemical examination of Ammannia baccifera Linn. (Lythra-
ceae). J Indian Chem Soc 63:619–620
Upadhyay HC, Dwivedi GR, Darokar MP, Chaturvedi V, Srivastava
SK (2012) Bioenhancing and anti-mycobacterial agents from
Ammannia multiflora. Planta Med 78:79–81
WHO, tuberculosis factsheet No. 104 (2010) http://www.who.int/
mediacentre/factsheets/fs104/en/. Accessed Nov 2010
Zhu H (2011) Chinese medicine for treating common cold in summer
and preparation method thereof. Chinese patent 102078584 A
Meng G (2009) Chinese medicinal composition for treatment of
spinal disease. Chinese patent 101417003 A
Nivin B, Nicholas P, Gayer M, Frieden TR, Fujiwara PI (1998) A
continuing outbreak of multidrug-resistant tuberculosis, with
transmission in a hospital nursery. Clin Infect Dis 26:303–307
Pablos-Mendez A, Raviglione MC, Laszlo A, Binkin N, Rieder HL,
Bustreo F, Cohn DL, Lambregts-van Weezenbeek CSB, Kim SJ,
Chaulet P, Nunn P (1998) Global surveillance for antitubercu-
losis-drug resistance, 1994–1997. N Engl J Med 338:1641–1649
Peng C, Bodenhausen G, Qiu S, Fong HHS, Farnsworth NR, Yuan S,
Zheng C (1998) Computer-assisted structure elucidation: appli-
cation of CISOC-SES to the resonance assignment and structure
generation of betulinic acid. Magn Reson Chem 36:267–278
123