Journal of Ethnopharmacology 99 (2005) 2130

Studies of the anticancer potential of plants used in

Bangladeshi folk medicine
Letcia Veras Costa-Lotufo
a,
, Mahmud Tareq Hassan Khan
b,1
, Arjumand Ather
c
,
Diego Veras Wilke
a
, Paula Christine Jimenez
a
, Cl audia Pessoa
a
,
Maria Elisabete Amaral de Moraes
a
, Manoel Odorico de Moraes
a
a
Department of Physiology and Pharmacology, School of Medicine, Federal University of Cear a, Fortaleza Rua Cel Nunes de
Melo 1127, Caixa Postal-3157, 60430-270 Fortaleza Ce, Brazil
b
Pharmacology Research Laboratory, Faculty of Pharmaceutical Sciences, University of Science and Technology Chittagong,
Foy lake, Chittagong, Bangladesh
c
Plant Biotechnology Division, HEJ Research Institute of Chemistry, University of Karachi, Karachi, Pakistan
Received 1 November 2003; received in revised form 10 January 2005; accepted 19 January 2005
Available online 11 March 2005
Abstract
The present study evaluated the anticancer potential of 11 plants used in Bangladeshi folk medicine. The extracts were tested for cytotoxicity
using the brine shrimp lethality assay, sea urchin eggs assay, hemolysis assay and MTT assay using tumor cell lines. The extract of Oroxylum
indicum showed the highest toxicity on all tumor cell lines tested, with an IC
50
of 19.6 g/ml for CEM, 14.2 g/ml for HL-60, 17.2 g/ml for
B-16 and 32.5 g/ml for HCT-8. On the sea urchin eggs, it inhibited the progression of cell cycle since the frist cleavage (IC
50
=13.5 g/ml).
The extract of Aegle marmelos exhibited toxicity on all used assays, but in a lower potency than Oroxylum indicum. In conclusion, among
all tested extracts, only the extracts of Oroxylum indicum, Moringa oleifera and Aegles marmelos could be considered as potential sources
of anticancer compounds. Further studies are necessary for chemical characterization of the active principles and more extensive biological
evaluations.
2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Oroxylum indicum; Bangladeshi folk plants; Cytotoxicity
1. Introduction
The use of natural products as anticancer agents has a
long history that began with folk medicine and through
the years has been incorporated into traditional and allo-
pathic medicine. Several drugs currently used in chemother-
apy were isolated from plant species or derived from a
natural prototype. They include the Vinca alkaloids, vin-
blastine and vincristine, isolated from Catharanthus roseus,

Corresponding author. Tel.: +55 85 3288 8255; fax: +55 85 3288 8333.
E-mail addresses: lvcosta@secrel.com.br (L.V. Costa-Lotufo),
mthkhan2002@yahoo.com (M.T.H. Khan).
1
Present address: Department of Biochemistry and Molecular Biology,
Center for Biotechnology, University of Ferrara, Ferrara 44100, Italy.
etoposide and teniposide, the semisynthetic derivatives of
epipodophyllotoxin, isolated from species of the genus
Podophyllum, the naturally derived taxanes isolated from
species of the genus Taxus, the semisynthetic derivatives of
camptothecin, irinotecan and topotecan, isolated fromCamp-
totheca acuminata, and several others (Cragg et al., 1993,
1994; Wang, 1998). AccordingtoCraggandNewman(2000),
over 50 % of the drugs in clinical trials for anticancer ac-
tivity were isolated from natural sources or are related to
them.
Most of the research performed today focuses on the de-
velopment of new drugs to treat cancer, as well as viral and
microbial infections. The present study aims to provide data
on the cytotoxic potential of 11 extracts from Bangladeshi
plants belonging to different families (Table 1) on developing
0378-8741/$ see front matter 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2005.01.041
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Table 1
List of plants species used in this study
Plant species (family) voucher no. Time of
collection
Area of collection Dry weight
(in kg)
Part used, extraction process and
yield (%)
Some reported pharmacological
activies
References
Aegle marmelos (L.) Corr ea.
(Rutaceae) EPL-106
May 1996 Chittagong Hill
tract, Bangladesh
31.0 Stem bark; 100% ethanol (cold)
partition: ether (9.9)
Antibacterial Rani and Khullar (2004)
Anti-enteric potential Rani and Khullar (2004)
NO scavenging activity Jagetia and Baliga (2004)
Antioxidant Kamalakkannan and Prince
(2003)
Antidiabetic Sabu and Kuttan (2004), Saxena
and Vikram (2004)
Radioprotective Jagetia et al. (2004)
Antiviral activity Badam et al. (2002)
Antiproliferative Khan et al. (2002), Lambertini et
al. (2004), Lampronti et al.
(2003)
Inhibits estrogen receptor Lambertini et al. (2004)
Antidiarrheal Shoba and Thomas (2001)
Antiulcer activity Goel et al. (1997)
Antifungal activity Rana et al. (1997)
Aphanamixis polystachya (Wall.)
R. Parker (Meliaceae)
GDK-8913
July 1996 Tangail,
Bangladesh
200.0 Stem bark; 80% ethanol:water
(cold) (11.8)
Antihepatotoxic Gole and Dasgupta (2002)
Oil (2.5) and solid (8.9) Antiproliferative Khan et al. (2002)
Cuscuta reexa Roxb.
(Convolvulaceae) BC-2411
August
1996
Chittagong,
Bangladesh
3.5 Whole plant; Absolute ethanol
(cold) (10.3)
Anti-steroidogenic Gupta et al. (2003)
Analgesic Pal et al. (2003)
Antiproliferative Khan et al. (2002)
Antiviral Awasthi (1981)
Emblica ofcinalis Gaertn.
(Euphorbiaceae) BC-3823
May 1996 Chittagong Hill
tract, Bangladesh
25.0 Fruit; 100% ethanol (cold) (15.0) Antiproliferative Khan et al. (2002), Lambertini et
al. (2004), Nemmani et al. (2002)
Antipyretic and analgesic Perianayagam et al. (2004)
Antiepileptic Achliya et al. (2004a)
Hepatoprotective Achliya et al. (2004b)
Against oxidative stress Rajak et al. (2004), Bhattacharya
et al. (2002)
Esterogen receptor inhibitor Lambertini et al. (2004)
Immunomodulator Ganju et al. (2003)
Antitussive activity Nosalova et al. (2003)
Apoptocic on cancer cells Rajeshkumar et al. (2003)
Anticarcinogenic Rajeshkumar et al. (2003)
Cytoprotective Sai Ram et al. (2003), Sai Ram et
al. (2002a)
Antimutagenic Jena et al. (2003), Kaur et al.
(2002)
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2
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Table 1 (Continued )
Plant species (family) voucher no. Time of
collection
Area of collection Dry weight
(in kg)
Part used, extraction process and
yield (%)
Some reported pharmacological
activies
References
Antioxidant Bhattacharya et al. (2000),
Chaudhuri (2002), Sabu and
Kuttan (2002)
Gastroprotective Al-Rehaily et al. (2002)
Antiulcerogenic Sai Ram et al. (2002b)
Antidiabetic Sabu and Kuttan (2002)
Antihyperlipidemic Augusti et al. (2001)
Against ophthalmic disorders Biswas et al. (2001)
Hemidesmus inidicus (L.) Br. ex
Schult. (Asclepiadaceae)
BC-1083
February
1996
Dhaka, Bangladesh 2.0 Root; 80% ethanol:water (cold)
(8.9)
Antidiarrheal activity Das et al. (2003)
Inhibit hepatocarcinogenesis Iddamaldeniya et al. (2003)
Antiatherogenic Mary et al. (2003b)
Antioxidant and antithrombotic Mary et al. (2003a)
Anti-ulcerogenic Anoop and Jegadeesan (2003)
Antiinammatory Jain and Basal (2003)
Moringa oleifera Lam.
(Moringaceae) BCS-4575
November
1996
Chittagong,
Bangladesh
25.0 Root; 100% ethanol (cold) (14.5),
partition: hexane (7.4),
chloroform (5.9), methanol (9.5),
water (9.5)
Inhibits central neurotransmitters Ray et al. (2003)
Anti-HSV-1 Lipipun et al. (2003)
Antioxidant Kumar and Pari (2003),
Siddhuraju and Becker (2003)
Chemomodulator Bharali et al. (2003)
Hypocholesterolaemic Mehta et al. (2003)
Hypoglycaemic Kar et al. (2003)
Hepatoprotective Pari and Kumar (2002)
Antimicrobial activities Suarez et al. (2003)
Antiplasmodial activities Kohler et al. (2002)
Radioprotective effect Rao et al. (2001)
Nigella sativa L. (Ranunculaceae)
SDK-84
January
1996
Chittagong,
Bangladesh
12.0 Seeds; ethanol 100%
(hot-soxhlet) (25.4) partition:
ether (7.7)
Triggers apoptosis in cancer cells Gali-Muhtasib et al. (2004)
Lipid-lowering Le et al. (2004)
Insulin-sensitizing Le et al. (2004)
Antidiabetic Kanter et al. (2004), Fararh et al.
(2004)
Immunopotentiating Fararh et al. (2004)
Immunosuppressive Islam et al. (2004)
Cytotoxic Islam et al. (2004)
Protects nephrotoxicity Ali (2004)
Analgesic Hajhashemi et al. (2004)
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3
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Table 1 (Continued )
Plant species (family) voucher no. Time of
collection
Area of collection Dry weight
(in kg)
Part used, extraction process and
yield (%)
Some reported pharmacological
activies
References
Anti-inammatory Hajhashemi et al. (2004)
Tracheal relaxant Boskabady et al. (2004)
Lipid metabolic Morikawa et al. (2004a),
Morikawa et al. (2004b)
Anticonvulsant Hosseinzadeh and Parvardeh
(2004)
Liver protectant Al-Ghamdi (2003)
Anti-allergic Kalus et al. (2003)
Oroxylum indicum (L.) Kurz
(Bignoniaceae) EPL-0978
February
1996
Chittagong,
Bangladesh
10.0 Stem bark; 100% ethanol
(hot-soxhlet) (15.2)
Antiproliferative Khan et al. (2002), Tepsuwan et
al. (1992)
Against breast cancer Lambertini et al. (2004)
Estrogen receptor inhibitor Lambertini et al. (2004)
Antioxidant Jiwajinda et al. (2002)
Anti-arthritic Laupattarakasem et al. (2003)
Antimutagenic Nakahara et al. (2002), Nakahara
et al. (2001), Tepsuwan et al.
(1992)
Paederia foetida L. (Rubiaceae)
BNH-25372
May 1996 Chittagong,
Bangladesh
8.0 Whole plant; ethanol 100%
(cold) (11.9)
Anti-inammatory De et al. (1994)
Anti-shigellosis Haider et al. (1991)
Anthelmintic Roychoudhury et al. (1970)
Polyalthia longifolia (Sonn.)
Thwaites (Annonaceae)
GFS-738
February
1996
Chittagong,Bangladesh 2.1 Stem bark; 80% ethanol:water
(6.9)
Cytotoxic activity Wu et al. (1990), Zhao et al.
(1991), Chen et al. (2000)
Antimicrobial activity Faizi et al. (2003a), Faizi et al.
(2003b)
Tribulus terrestris L.
(Zygophyllaceae) EPL-012
August
1996
Dhaka, Bangladesh 4.0 Fruit; 100% ethanol (hot-soxhlet)
(17.9)
Antihypertensive Shari et al. (2003)
Sexual effects Gauthaman et al. (2003)
Inhibition of breast cancer Sun et al. (2003)
Diuretic activity Al-Ali et al. (2003)
Anthelmintic Deepak et al. (2002)
Hypoglycemic Li et al. (2002)
COX-2 inhibitor Hong et al. (2002)
Anticancer Bedir et al. (2002), Ali et al.
(2001)
Antifungal Bedir et al. (2002)
Aphrodiasic Gauthaman et al. (2002)
Antibacterial Ali et al. (2001)
All voucher specimens have been deposited in the following herbaria: BCSIR Laboratory; Jahangirnagar University at Dhaka; and Bangladesh National Herbarium.
L.V. Costa-Lotufo et al. / Journal of Ethnopharmacology 99 (2005) 2130 25
embryos of sea urchins, on tumor cell lines, erythrocytes and
brine shrimp nauplii.
2. Materials and methods
2.1. Preparation of crude extract
The roots of Hemidesmus indicus and the stem barks of
Polyalthia longifolia and Aphanamixis polystachya were ex-
tracted with 80% ethanol:water in cold extraction process.
The rst plant yielded 8.9% extract, the second, 6.87%, and
the third, which resulted in two extracts, an oily phase and a
solid mass, yielded 2.54 and 8.91%, respectively. The stem
bark of Oroxylumindicumthe, fruits of Tribulus terrestris and
the seeds of Nigella sativa were extracted in a soxhlet appa-
ratus using absolute ethanol as solvent. The rst two species
yielded 15.24 and 17.94% extracts, respectively. The ex-
tract of Nigella sativa was further partitioned in diethylether
and yielded 7.65%. The other plants, Cuscuta reexa (whole
plant), Paederia foetida (whole plant), Emblica ofcinalis
(fruits), Moringa oleifera (roots), and Aegle marmelos (stem
bark), were extracted with absolute ethanol in a cold extrac-
tion process. Cuscuta reexa, Paederia foetida and Emblica
ofcinalis yielded 10.32, 11.92 and 15.0% extracts, respec-
tively. The crude extract of Moringa oleifera, which yielded
14.54%, was further partitioned in n-hexane (7.39% yield),
chloroform (5.94%), methanol (9.46%) and water (9.48%),
and the extract derived from Aegle marmelos was partitioned
in diethylether (9.87%yield) (Khan et al., 2002). The extracts
were dried in a rotary vacuum evaporator and the residue re-
constituted in DMSO before testing. The vehicle was used as
negative control.
All of these plants were collected from various locali-
ties, including Chittagong, Chittagong Hill Tract, Tangail and
Dhaka, of Bangladesh, in different months of 1996. Table 1
shows species used in this study and the location of the de-
posit of their respective voucher specimens.
2.2. Brine shrimp assay
Brine shrimp (Artemia salina Leach) eggs were hatched
in a becker lled with seawater under constant aeration.
After 48 h the nauplii were collected by pipette and were
counted macroscopically in the stem of the pipette against
a lighted background. Ten nauplii were transferred to each
well of 24-multiwell plates containing the samples. The
extract concentration ranged from 10 to 1000 g/ml. The
plates were maintained under illumination. Survivors were
counted after 24 h of incubation and the percentage of
deaths at each dose and control (seawater plus vehicle) were
determined (Meyer et al., 1982).
2.3. Assay on sea urchins
The test was performed in 24-well plates following the
method described by Costa-Lotufo et al. (2002). Adult sea
urchins (Lytechinus variegatus) were collected at Pec em
beach, on the northeastern coast of Brazil. The gamete elimi-
nation was induced by injecting 3.0 ml of 0.5 M KCl into the
urchins coelomic cavity via the periostomial membrane. The
eggs were washed twice using ltered seawater to remove
the jelly coat surrounding the cells. Concentrated sperm was
collected with a Pasteur pipette and maintained under low
temperatures until the moment of fertilization. For fertiliza-
tion, 1 ml of a sperm suspension (0.05 ml of concentrated
sperm in 2.45 ml of ltered seawater) was added to every
50 ml of egg solution. Each well received 1 ml of fertilized
egg suspension. The extracts were added immediately after
fertilization (within 2 min) to get concentrations of 10, 30,
100, 300 and 1000 g/ml in a nal volume of 2 ml. Doxoru-
bicin (0.05858.0 g/ml) was used as positive control. The
plates were then shaken on a constant temperature water bath
at 26 2

C. At appropriate intervals, aliquots of 200 l were

xed in the same volume of 10%formaldehyde to obtain rst
and third cleavages, and blastulae. One hundred eggs or em-
bryos were counted for each concentration of test substance
to obtain the percentage of normal cells.
2.4. MTT assay
The cytotoxicity of the extracts was tested against B-
16 (murine melanoma), HCT-8 (human colon carcinoma),
CEM and HL-60 (leukemia) tumor cell lines (Childrens
Mercy Hospital, Kansas City, MO, USA). Cells were cul-
tured in RPMI-1640 medium, supplemented with 10% fetal
calf serum, 2 mM glutamine, 100 g/ml streptomycin and
100 U/ml penicillin at 37

C with 5% CO
2
. For experiments,
cells were plated in 96-well plates (10
5
cells/well for adher-
ent cells or 0.3 10
6
cells/well for suspended cells in 100 l
of medium). After 24 h, the extracts (2125 g/ml) dissolved
in DMSO (1%) was added to each well and incubated for
3 days (72 h). Control groups received the same amount of
DMSO. Doxorubicin (0.010.58 g/ml) was used as positive
control. Growth of tumoral cells was quantitated by the abil-
ity of living cells to reduce the yellowdye 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a
blue formazan product (Mosmann, 1983). At the end of 72 h
incubation, the medium in each well was replaced by fresh
medium (200 l) containing 0.5 mg/ml of MTT. Three hours
later, the formazan product of MTT reduction was dissolved
in DMSO, and absorbance was measured using a multi-plate
reader (Spectra Count, Packard, Ont., Canada). Drug effect
was quantied as the percentage of control absorbance of
reduced dye at 550 nm.
2.5. Hemolytic assay
The test was performed in 96-well plates following the
method described by Costa-Lotufo et al. (2002). Each well
received 100 l of 0.85% NaCl solution containing 10 mM
CaCl
2
. The rst well was the negative control that contained
only the vehicle (distilled water or DMSO 10%), and, in the
26 L.V. Costa-Lotufo et al. / Journal of Ethnopharmacology 99 (2005) 2130
second well, 100 l of test substance that was diluted in half
was added. The extracts were tested at concentrations rang-
ing from10 to 2500 g/ml. The serial dilution continued until
the 11th well. The last well received 20 l of 0.1% Triton X-
100 (in 0.85% saline) to obtain 100% hemolysis (positive
control). Then, each well received 100 l of a 2% suspen-
sion of mouse erythrocytes in 0.85%saline containing 10 mM
CaCl
2
. After incubation at room temperature for 30 min and
centrifugation, the supernatant was removed and the liberated
hemoglobin was measured spectroscopically as absorbance
at 540 nM.
2.6. Statistical analysis
Data are presented as mean S.E.M. The IC
50
or EC
50
values and their 95% condence intervals (CI 95%) were ob-
tained by nonlinear regression using the GRAPHPAD pro-
gram (Intuitive Software for Science, San Diego, CA).
LC
50
on brine shrimp was obtained from the 24 h counts
using the probit analysis method described by Finney (1971).
3. Results
3.1. Brine shrimp assay
The extract of Aegle marmelos was the only one that
showed a positive result in this assay, presenting a LD
50
of
17.5 2.0 g/mL.
3.2. Assay on sea urchin embryos
Table 2 shows the results for the sea urchin embryo as-
say using the plant extracts. The most active extract was the
one obtained from Oroxylum indicum, which presented an
IC
50
around 10 g/ml in all tested phases of the sea urchin
development, rst and third cleavages, and blastulae. The ex-
tract obtained from Aegle marmelos was also strongly active,
inhibiting the rst cleavage with an IC
50
of 50.5 g/ml, the
third cleavage with an IC
50
of 22.7 g/ml, and the blastu-
lae with an IC
50
of 13.2 g/ml. As it can be observed from
the IC
50
values, its efcacy increased with the time of con-
tact with the embryos. The extracts obtained from Moringa
oleifera, Emblica ofcinalis, Aphanamixis polystachya (solid
residue from the ethanolic extract), Cuscuta reexa, Tribulus
terrestris and Nigella sativa also possessed inhibitory activ-
ity on the sea urchin egg development, but with a moderate
efcacy. The other tested extracts were inactive in this assay.
3.3. MTT assay
The cytotoxicity on the tumor cell lines was evaluated us-
ing the MTT assay and the results are presented in Table 3.
The extract obtained from Oroxylum indicum was the most
active in this assay, exhibiting lower IC
50
values against
all tested cell lines, 14.2 g/ml for HL-60, 19.6 g/ml for
CEM, 17.2 g/ml for B-16 and 32.5 g/ml for HCT.8. The
extracts obtained from Aegle marmelos, Moringa oleifera,
Aphanamixis polystachya (solid residue from the ethanolic
extract), Cuscuta reexa, Emblica ofcinalis, Tribulus ter-
restris and Nigella sativa also possessed cytotoxic activity,
mainly against drug-sensitive lines like the leukemias (HL-
60 and CEM) and the melanoma. However, generally, they
were not able to block HCT-8 proliferation. The other tested
extracts were inactive in this assay.
3.4. Hemolytic assay
In order to verify whether the observed cytotoxicity is re-
lated to membrane disruption, plant extracts were tested for
their ability to induce lysis of mouse erythrocytes. The results
obtained from the hemolytic assay are presented in Table 4.
The extract obtained from Nigella sativa was the most ac-
tive in this assay (EC
50
=116.8 g/ml), followed by the ex-
Table 2
Inhibition of the cell cycle by the plant extracts on the embryos of the sea urchin Lytechinus variegatus on the rst and third cleavage and blastulae stages
Species Embryonic stage IC
50
(g/ml)
First cleavage Third cleavage Blastulae
Doxorubicin 6.3 (4.39.1) 0.3 (0.20.7) 0.5 (0.31.1)
Aegle marmelos 50.5 (41.062.3) 22.7 (20.125.6) 13.2 (11.115.6)
Aphanamixis polystachya oil >1000 >1000 >1000
Aphanamixis polystachya solid 413.0 (156.41096) 78.9 (50.591.9) 40.1 (24.765.1)
Cuscuta reexa 386.5 (168.7885.4) 400.3 (230.3695.7) 406.0 (268.5656.3)
Emblica ofcinalis 400.1 (ND) 125.9 (ND) 123.4 (32.5468.3)
Hemidesmus indicus >1000 >1000 >1000
Moringa oleifera 169.4 (155.2184.9) 119.5 (74.7190.9) 52.5 (41.067.3)
Nigella sativa 425.3 (ND) 243.2 (193.5305.8) 363.1 (303.4404.5)
Oroxylum indicum 13.5 (12.414.7) 13.0 (11.814.4) 11.5 (10.412.7)
Paederia foetida >1000 >1000 >1000
Polyalthia longifolia >1000 >1000 >1000
Tribulus terrestris 404.8 (155.61056) 195.6 (169.8225.3) 393.9 (236.4656.3)
The extract was added 2 min after fertilization of the eggs. The IC
50
and its 95 % condence interval (CI 95%) were obtained by non-linear regression. ND:
not determined. Extracts with an IC
50
value lower than 100 g/ml were considered active.
L.V. Costa-Lotufo et al. / Journal of Ethnopharmacology 99 (2005) 2130 27
Table 3
In vitro cytotoxicity of the plants extracts on four tumor cell lines measured by the MTT assay
Species Cell line IC
50
(g/ml)
HL-60 CEM HCT-8 B-16
Doxorubicin 0.02 (0.010.02) 0.02 (0.010.02) 0.04 (0.030.05) 0.03 (0.020.04)
Aegle marmelos 46.4 (42.550.8) 28.6 (23.235.3) >125 23.8 (20.328.0)
Aphanamixis polystachya oil >125 >125 >125 >125
Aphanamixis polystachya solid 87.2 (70.2108.3) 73.7 (65.982.3) >125 78.6 (55.4111.5)
Cuscuta reexa 55.0 (48.063.1) 51.5 (44.859.3) 79.4 (52.3120.7) >125
Emblica ofcinalis 70.4 (55.689.2) 33.2 (26.940.9) 66.5 (50.787.2) 63.3 (55.372.5)
Hemidesmus indicus >125 >125 >125 >125
Moringa oleifera 60.0 (59.261.4) 12.7 (8.419.4) 113.8 (ND) 28.8 (ND)
Nigella sativa 68.7 (ND) 61.0 (ND) 112.8 (ND) 81.1 (32.0205)
Oroxylum indicum 14.2 (10.918.5) 19.6 (17.821.6) 32.5 (ND) 17.2 (15.319.3)
Paederia foetida >125 >125 >125 >125
Polyalthia longifolia >125 >125 >125 >125
Tribulus terrestris 39.8 (28.755.2) 52.4 (44.461.8) >125 89.9 (52.7152.8)
The IC
50
and its 95 % condence interval (CI 95%) were obtained by non-linear regression. ND: not determined. Extracts with an IC
50
value lower than
30 g/ml were considered active.
Table 4
Hemolytic activity of the plant extracts on mouse erythrocyte (2%)
Species EC
50
(g/ml) CI 95%
Aegle marmelos 395.8 (344.8454.4)
Aphanamixis polystachya oil >2500
Aphanamixis polystachya solid >2500
Cuscuta reexa 846.9 (732.2979.7)
Emblica ofcinalis >2500
Hemidesmus indicus >2500
Moringa oleifera >2500
Nigella sativa 116.8 (95.7142.6)
Oroxylum indicum >2500
Paederia Foetida >2500
Polyalthia longifolia >2500
Tribulus terrestris 622.7 (558.8693.9)
The total hemolysis was obtained with 50 l of Triton X-100 1% and 1h
incubation. The EC
50
and 95 % condence interval (CI 95%) were obtained
by non-linear regression. Extracts with an EC
50
value lower than 200 g/ml
were considered active.
tracts from Aegle marmelos, Tribulus terrestris, and Cuscuta
reexa, which presented EC
50
values of 395.8, 622.7, and
846.9 g/ml, respectively. The other tested extracts were in-
active in this assay.
4. Discussion and conclusions
Inthis study, the antimitotic potential of 11medicinal plant
extracts from Bangladesh was investigated. The in vitro an-
timitotic potential was estimated as the ability of these ex-
tracts to inhibit tumor cell line growth and sea urchin egg
development. The toxicity to Artemia salina nauplii and the
hemolytic activity on mouse erythrocytes were also evalu-
ated.
The extract of Oroxylum indicum showed the highest ac-
tivity in the MTT assay using tumor cell lines and on sea
urchin egg development, indicating the presence of cytotoxic
compounds in this extract. IC
50
values for the four tested cell
lines and for different stages of embryo development were
around 15 g/ml, indicating that this extract did not present
any selectivity. Khan et al. (2002) also demonstrated that
the extract of the stem bark of Oroxylum indicum presented
strong in vitro antiproliferative activity on chronic myeloge-
nous leukemia K562. These authors compared the effect of
ten plant species on cell proliferation of K562 cells, and found
that the extract of Emblica ofcinalis fruits was the most
active followed by Oroxylum indicum. Interestingly, in the
present study, the extract of Emblica ofcinalis fruits was
two to three times less active than the one obtained from the
stem bark of Oroxylum indicum.
Despite this cytotoxic activity, the Oroxylum indicum ex-
tract did not possess any activity against Artemia salina nau-
plii or against mouse erythrocytes. These data suggest that
the cytotoxic activity was not related to the lytic properties
or membrane instability induced by the extract.
The inhibitory effect of Oroxylum indicum on sea urchin
egg development could be observed since the rst cleavage,
and the cells presented a homogeneous cytoplasm, indicat-
ing that the activity was related to DNA or protein synthesis
(Fusetani, 1987).
The extract obtained from Aegle marmelos showed activ-
ity in all assays, indicating the presence of cytotoxic sub-
stances. The present data suggest that the ether fraction ob-
tained from the ethanol extract of Aegle marmelos might be
toxic to tumor and normal cells.
The extracts of Moringa oleifera, Emblica ofcinalis,
Tribulus terrestris, Cuscuta reexa, Nigella sativa and the
solid fraction of Aphanamixis polystachya extract also pre-
sented moderate cytotoxic activity. Previous studies have al-
ready demonstrated the occurrence of cytotoxic activity in
some of these species, such as Emblica ofcinalis, Cuscuta
reexa and Nigella sativa (Swamy and Tan, 2000; Jose et al.,
2001; Khan et al., 2002). On the other hand, no cytotoxic
28 L.V. Costa-Lotufo et al. / Journal of Ethnopharmacology 99 (2005) 2130
activity was observed in Polyalthia longifolia, Hemidesmus
indicus and Paederia foetida extracts.
Finally, this study revealed that 8 among 11 tested species
of plants used in Bangladeshi folk medicine presented some
activity in the used assays. However according to the crite-
ria of the American National Cancer Institute, the IC
50
limit
to consider a crude extract promising for further purica-
tion is lower than 30 g/mL (Suffness and Pezzuto, 1990).
Thus, only the extracts of Oroxylumindicum(three cell lines),
Moringa oleifera (two cell lines) and Aegles marmelos (two
cell lines) could be considered as potential sources of anti-
cancer compounds. Further, studies are necessary for chem-
ical characterization of the active principles and more exten-
sive biological evaluations.
Acknowledgements
We wish to thank the Brazilian National Research Council
(CNPq), Claude Bernard Institute and the Research Support
Foundation of Cear a (FUNCAP) for nancial support in the
form of grants and fellowship awards. MTHK gratefully ac-
knowledges the travel support of the Third World Academy
of Sciences (TWAS), Italy, for his visit to Pakistan through a
SouthSouth Fellowship. The technical assistance of Sil-
vana Franca and F atima Texeira are gratefully acknowledged.
References
Achliya, G.S., Wadodkar, S.G., Avinash, K.D., 2004a. Neuropharmaco-
logical actions of panchagavya formulation containing Emblica ofci-
nalis Gaerth and Glycyrrhiza glabra Linn. in mice. Indian Journal of
Experimental Biology 42, 499503.
Achliya, G.S., Wadodkar, S.G., Dorle, A.K., 2004b. Evaluation of hep-
atoprotective effect of Amalkadi Ghrita against carbon tetrachloride-
induced hepatic damage in rats. Journal of Ethnopharmacology 90,
229232.
Al-Ali, M., Wahbi, S., Twaij, H., Al-Badr, A., 2003. Tribulus terrestris:
preliminary study of its diuretic and contractile effects and comparison
with Zea mays. Journal of Ethnopharmacology 85, 257260.
Al-Ghamdi, M.S., 2003. Protective effect of Nigella sativa seeds against
carbon tetrachloride-induced liver damage. The American Journal of
Chinese Medicine 31, 721728.
Ali, B.H., 2004. The effect of Nigella sativa oil on gentamicin nephrotox-
icity in rats. The American Journal of Chinese Medicine 32, 4955.
Ali, N.A., Julich, W.D., Kusnick, C., Lindequist, U., 2001. Screening
of Yemeni medicinal plants for antibacterial and cytotoxic activities.
Journal of Ethnopharmacology 74, 173179.
Al-Rehaily, A.J., Al-Howiriny, T.A., Al-Sohaibani, M.O., Rafatullah, S.,
2002. Gastroprotective effects of Amla Emblica ofcinalis on in vivo
test models in rats. Phytomedicine 9, 515522.
Anoop, A., Jegadeesan, M., 2003. Biochemical studies on the anti-
ulcerogenic potential of Hemidesmus indicus R. Br. var. indicus. Jour-
nal of Ethnopharmacology 84, 149156.
Augusti, K.T., Arathy, S.L., Asha, R., Ramakrishanan, J., Zaira, J., Lekha,
V., Smitha, S., Vijayasree, V.M., 2001. A comparative study on the
benecial effects of garlic (Allium sativum Linn), amla (Emblica of-
cinalis Gaertn) and onion (Allium cepa Linn) on the hyperlipidemia
induced by butter fat and beef fat in rats. Indian Journal of Experi-
mental Biology 39, 760766.
Awasthi, L.P., 1981. The purication and nature of an antiviral protein
from Cuscuta reexa plants. Archives of Virology 70, 215223.
Badam, L., Bedekar, S.S., Sonawane, K.B., Joshi, S.P., 2002. In vitro
antiviral activity of bael (Aegle marmelos Corr) upon human cox-
sackieviruses B1-B6. Journal of Communicable Diseases 34, 8899.
Bedir, E., Khan, I.A., Walker, L.A., 2002. Biologically active steroidal
glycosides from Tribulus terrestris. Die Pharmazie 57, 491493.
Bharali, R., Tabassum, J., Azad, M.R., 2003. Chemomodulatory effect of
Moringa oleifera, Lam, on hepatic carcinogen metabolising enzymes,
antioxidant parameters and skin papillomagenesis in mice. Asian Pa-
cic Journal of Cancer Prevention 4, 131139.
Bhattacharya, A., Ghosal, S., Bhattacharya, S.K., 2000. Antioxidant ac-
tivity of tannoid principles of Emblica ofcinalis (amla) in chronic
stress induced changes in rat brain. Indian Journal of Experimental
Biology 38, 877880.
Bhattacharya, S.K., Bhattacharya, A., Sairam, K., Ghosal, S., 2002. Effect
of bioactive tannoid principles of Emblica ofcinalis on ischemia-
reperfusion-induced oxidative stress in rat heart. Phytomedicine 9,
171174.
Biswas, N.R., Gupta, S.K., Das, G.K., Kumar, N., Mongre, P.K., Hal-
dar, D., Beri, S., 2001. Evaluation of ophthacare eye drops-a herbal
formulation in the management of various ophthalmic disorders. Phy-
totherapy Research 15, 618620.
Boskabady, M.H., Shirmohammadi, B., Jandaghi, P., Kiani, S., 2004. Pos-
sible mechanism(s) for relaxant effect of aqueous and macerated ex-
tracts from Nigella sativa on tracheal chains of guinea pig. BMC
Pharmacology 4, 3.
Chaudhuri, R.K., 2002. Emblica cascading antioxidant: a novel natural
skin care ingredient. Skin Pharmacology and Applied Skin Physiology
15, 374380.
Chen, C.Y., Chang, F.R., Shih, Y.C., Hsieh, T.J., Chia, Y.C., Tseng, H.Y.,
Chen, H.C., Chen, S.J., Hsu, M.C., Wu, Y.C., 2000. Cytotoxic con-
stituents of Polyalthia longifolia var. pendula. Journal of Natural Prod-
ucts 63, 14751478.
Costa-Lotufo, L.V., Cunha, G.M.A., Farias, P.A.M., Viana, G.S.B., Cunha,
K.M.A., Pessoa, C., Moraes, M.O., Silveira, E.R., Gramosa, N.V.,
Rao, V.S.N., 2002. The cytotoxic and embryotoxic effects of kaurenoic
acid, a diterpene isolated from Copaifera langsdorfi oleo-resin. Tox-
icon 40, 12311234.
Cragg, G.M., Schepartz, S.A., Suffness, M., Grever, M.R., 1993. The
taxol supply crisis. New NCI policies for handling the large-scale
production of novel natural product anticancer and anti-HIV agents.
Journal of Natural Products 56, 16571668.
Cragg, G.M., Boyd, M.R., Cardellina, J.H., Newman, D.J., Snader, K.M.,
McCloud, T.G., 1994. Ethnobotany and drug discovery experience
of the US National Cancer Institute. In: Chadwick, D.J., Marsh, J.
(Eds.), Ciba Foundation Ethnobotany and the search for new drugs
Symposium, 185. Wiley and Sons, Chichester, UK, pp. 178196.
Cragg, G.M., Newman, D.J., 2000. Antineoplastic agents from natural
sources: achievements and future directions. Expert Opinion on In-
vestigational Drugs 9, 115.
Das, S., Prakash, R., Devaraj, S.N., 2003. Antidiarrhoeal ef-
fects of methanolic root extract of Hemidesmus indicus (Indian
sarsaparilla)an in vitro and in vivo study. Indian Journal of Ex-
perimental Biology 41, 363366.
De, S., Ravishankar, B., Bhavsar, G.C., 1994. Investigation of the anti-
inammatory effects of Paederia foetida. Journal of Ethnopharmacol-
ogy 43, 3138.
Deepak, M., Dipankar, G., Prashanth, D., Asha, M.K., Amit, A.,
Venkataraman, B.V., 2002. Tribulosin and beta-sitosterol-d-glucoside,
the anthelmintic principles of Tribulus terrestris. Phytomedicine 9,
753756.
Faizi, S., Khan, R.A., Azher, S., Khan, S.A., Tauseef, S., Ahmad, A.,
2003a. New antimicrobial alkaloids from the roots of Polyalthia longi-
folia var. pendula. Planta Medica 69, 350355.
Faizi, S., Mughal, N.R., Khan, R.A., Khan, S.A., Ahmad, A., Bibi, N.,
Ahmed, S.A., 2003b. Evaluation of the antimicrobial property of
L.V. Costa-Lotufo et al. / Journal of Ethnopharmacology 99 (2005) 2130 29
Polyalthia longifolia var. pendula: isolation of a lactone as the active
antibacterial agent from the ethanol extract of the stem. Phytotherapy
Research 17, 11771181.
Fararh, K.M., Atoji, Y., Shimizu, Y., Shiina, T., Nikami, H., Takewaki,
T., 2004. Mechanisms of the hypoglycaemic and immunopotentiat-
ing effects of Nigella sativa L. oil in streptozotocin-induced diabetic
hamsters. Research in Veterinary Science 77, 123129.
Finney, D.J., 1971. Statistical aspects of monitoring for dangers in drug
therapy. Methods of Information in Medicine 10, 18.
Fusetani, N., 1987. Marine metabolities which inhibit development of
echinoderm embryos. In: Scheuer, P. (Ed.), Bioorganic Marine Chem-
istry. Springer-Verlag, Berlin, pp. 6191.
Gali-Muhtasib, H., Diab-Assaf, M., Boltze, C., Al-Hmaira, J., Hartig,
R., Roessner, A., Schneider-Stock, R., 2004. Thymoquinone extracted
from black seed triggers apoptotic cell death in human colorectal
cancer cells via a p53-dependent mechanism. International Journal of
Oncology 25, 857866.
Ganju, L., Karan, D., Chanda, S., Srivastava, K.K., Sawhney, R.C., Sel-
vamurthy, W., 2003. Immunomodulatory effects of agents of plant
origin. Biomedicine and Pharmacotherapy 57, 296300.
Gauthaman, K., Adaikan, P.G., Prasad, R.N., 2002. Aphrodisiac properties
of Tribulus terrestris extract (Protodioscin) in normal and castrated
rats. Life Sciences 71, 13851396.
Gauthaman, K., Ganesan, A.P., Prasad, R.N., 2003. Sexual effects of
puncturevine (Tribulus terrestris) extract (protodioscin): an evalua-
tion using a rat model. Journal of Alternative and Complementary
Medicine 9, 257265.
Goel, R.K., Maiti, R.N., Manickam, M., Ray, A.B., 1997. Antiulcer activ-
ity of naturally occurring pyrano-coumarin and isocoumarins and their
effect on prostanoid synthesis using human colonic mucosa. Indian
Journal of Experimental Biology 35, 10801083.
Gole, M.K., Dasgupta, S., 2002. Role of plant metabolites in toxic liver
injury. Asia Pacic Journal of Clinical Nutrition 11, 4850.
Gupta, M., Mazumder, U.K., Pal, D.K., Bhattacharya, S., 2003. Anti-
steroidogenic activity of methanolic extract of Cuscuta reexa roxb.
stem and Corchorus olitorius Linn. seed in mouse ovary. Indian Jour-
nal of Experimental Biology 41, 641644.
Haider, R., Khan, A.K., Aziz, K.M., Chowdhury, A., Kabir, I., 1991.
Evaluation of indigenous plants in the treatment of acute shigellosis.
Tropical and Geographical Medicine 43, 266270.
Hajhashemi, V., Ghannadi, A., Jafarabadi, H., 2004. Black cumin seed
essential oil, as a potent analgesic and antiinammatory drug. Phy-
totherapy Research 18, 195199.
Hong, C.H., Hur, S.K., Oh, O.J., Kim, S.S., Nam, K.A., Lee, S.K., 2002.
Evaluation of natural products on inhibition of inducible cyclooxy-
genase (COX-2) and nitric oxide synthase (iNOS) in cultured mouse
macrophage cells. Journal of Ethnopharmacology 83, 153159.
Hosseinzadeh, H., Parvardeh, S., 2004. Anticonvulsant effects of thy-
moquinone, the major constituent of Nigella sativa seeds, in mice.
Phytomedicine: International Journal of Phytoterapy and Phytophar-
macology 11, 5664.
Iddamaldeniya, S.S., Wickramasinghe, N., Thabrew, I., Ratnatunge, N.,
Thammitiyagodage, Md.G., 2003. Protection against diethylnitro-
soamine-induced hepatocarcinogenesis by an indigenous medicine
comprised of Nigella sativa, Hemidesmus indicus and Smilax glabra:
a preliminary study. Journal of Carcinogenesis 2, 6.
Islam, S.N., Begum, P., Ahsan, T., Huque, S., Ahsan, M., 2004. Immuno-
suppressive and cytotoxic properties of Nigella sativa. Phytotherapy
Research 18, 395398.
Jagetia, G.C., Baliga, M.S., 2004. The evaluation of nitric oxide scaveng-
ing activity of certain Indian medicinal plants in vitro: a preliminary
study. Journal of Medical Food 7, 343348.
Jagetia, G.C., Venkatesh, P., Baliga, M.S., 2004. Evaluation of the ra-
dioprotective effect of bael leaf (Aegle marmelos) extract in mice.
International Journal of Radiation Biology 80, 281290.
Jain, A., Basal, E., 2003. Inhibition of Propionibacterium acnes-induced
mediators of inammation by Indian herbs. Phytomedicine: Inter-
national Journal of Phytoterapy and Phytopharmacology 10, 34
38.
Jena, G.B., Nemmani, K.V., Kaul, C.L., Ramarao, P., 2003. Pro-
tective effect of a polyherbal formulation (Immu-21) against
cyclophosphamide-induced mutagenicity in mice. Phytotherapy Re-
search 17, 306310.
Jiwajinda, S., Santisopasri, V., Murakami, A., Kim, O.K., Kim, H.W.,
Ohigashi, H., 2002. Suppressive effects of edible Thai plants on su-
peroxide and nitric oxide generation. Asian Pacic Journal of Cancer
Prevention 3, 215223.
Jose, J.K., Kuttan, G., Kuttan, R., 2001. Antitumor activity of Emblica
ofcinalis. Journal of Ethnopharmacology 75, 6569.
Kalus, U., Pruss, A., Bystron, J., Jurecka, M., Smekalova, A., Lichius,
J.J., Kiesewetter, H., 2003. Effect of Nigella sativa (black seed) on
subjective feeling in patients with allergic diseases. Phytotherapy Re-
search 17, 12091214.
Kamalakkannan, N., Prince, P.S.M., 2003. Effect of Aegle marmelos Cor-
rea (Bael) fruit extract on tissue antioxidants in streptozotocin diabetic
rats. Indian Journal of Experimental Biology 41, 12851288.
Kanter, M., Coskun, O., Korkmaz, A., Oter, S., 2004. Effects of Nigella
sativa on oxidative stress and beta-cell damage in streptozotocin-
induced diabetic rats. The Anatomical Record 279, 685691.
Kar, A., Choudhary, B.K., Bandyopadhyay, N.G., 2003. Comparative eval-
uation of hypoglycaemic activity of some Indian medicinal plants in
alloxan diabetic rats. Journal of Ethnopharmacology 84, 105108.
Kaur, S., Arora, S., Kaur, K., Kumar, S., 2002. The in vitro antimuta-
genic activity of Triphalaan Indian herbal drug. Food and Chemistry
Toxicology 40, 527534.
Khan, M.T.H., Lampronti, I., Martello, D., Bianchi, N., Jabbar, S., Choud-
huri, M.S., Datta, B.K., Gambari, R., 2002. Identication of pyrogallol
as an antiproliferative compound present in extracts from the medici-
nal plant Emblica ofcinalis: effects on in vitro cell growth of human
tumor cell lines. International Journal of Oncology 21, 187192.
Kohler, I., Jenett-Siems, K., Siems, K., Hernandez, M.A., Ibarra, R.A.,
Berendsohn, W.G., Bienzle, U., Eich, E., 2002. In vitro antiplasmodial
investigation of medicinal plants from El Salvador. Zeitschrift fur
Naturforschung [C] 57, 277281.
Kumar, N.A., Pari, L., 2003. Antioxidant action of Moringa oleifera Lam.
(drumstick) against antitubercular drugs induced lipid peroxidation in
rats. Journal of Medical Food 6, 255259.
Lambertini, E., Piva, R., Khan, M.T.H., Lampronti, I., Bianchi, N., Bor-
gatti, M., Gambari, R., 2004. Effects of extracts from Bangladeshi
medicinal plants on in vitro proliferation of human breast cancer cell
lines and expression of estrogen receptor alpha gene. International
Journal of Oncology 24, 419423.
Lampronti, I., Martello, D., Bianchi, N., Borgatti, M., Lambertini, E.,
Piva, R., Jabbar, S., Choudhuri, M.S., Khan, M.T.H., Gambari, R.,
2003. In vitro antiproliferative effects on human tumor cell lines of
extracts from the Bangladeshi medicinal plant Aegle marmelos Correa.
Phytomedicine 10, 300308.
Laupattarakasem, P., Houghton, P.J., Hoult, J.R., Itharat, A., 2003. An
evaluation of the activity related to inammation of four plants used
in Thailand to treat arthritis. Journal of Ethnopharmacology 85, 207
215.
Le, P.M., Benhaddou-Andaloussi, A., Elimadi, A., Settaf, A., Cherrah,
Y., Haddad, P.S., 2004. The petroleum ether extract of Nigella sativa
exerts lipid-lowering and insulin-sensitizing actions in the rat. Journal
of Ethnopharmacology 94, 251259.
Li, M., Qu, W., Wang, Y., Wan, H., Tian, C., 2002. Hypoglycemic effect
of saponin from Tribulus terrestris. Zhong Yao Cai 25, 420422.
Lipipun, V., Kurokawa, M., Suttisri, R., Taweechotipatr, P., Pramyothin,
P., Hattori, M., Shiraki, K., 2003. Efcacy of Thai medicinal plant
extracts against herpes simplex virus type 1 infection in vitro and in
vivo. Antiviral Reseach 60, 175180.
Mary, N.K., Achuthan, C.R., Babu, B.H., Padikkala, J., 2003a. In vitro
antioxidant and antithrombotic activity of Hemidesmus indicus (L.)
R. Br. Journal of Ethnopharmacology 87, 187191.
30 L.V. Costa-Lotufo et al. / Journal of Ethnopharmacology 99 (2005) 2130
Mary, N.K., Babu, B.H., Padikkala, J., 2003b. Antiatherogenic effect of
Caps HT2, a herbal ayurvedic medicine formulation. Phytomedicine
10, 474482.
Mehta, K., Balaraman, R., Amin, A.H., Bafna, P.A., Gulati, O.D., 2003.
Effect of fruits of Moringa oleifera on the lipid prole of normal
and hypercholesterolaemic rabbits. Journal of Ethnopharmacology 86,
191195.
Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsen, L.B., Nichols, D.E.,
McLaughlin, J.L., 1982. Brine shrimp: a convenient general bioassay
for active plant constituents. Planta Medica 45, 3134.
Morikawa, T., Xu, F., Kashima, Y., Matsuda, H., Ninomiya, K.,
Yoshikawa, M., 2004a. Novel dolabellane-type diterpene alkaloids
with lipid metabolism promoting activities from the seeds of Nigella
sativa. Organic Letters 6, 869872.
Morikawa, T., Xu, F., Ninomiya, K., Matsuda, H., Yoshikawa, M., 2004b.
Nigellamines A3, A4, A5, and C, new dolabellane-type diterpene al-
kaloids, with lipid metabolism-promoting activities from the Egyptian
medicinal food black cumin. Chemical and Pharmaceutical Bulletin
52, 494497.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. Journal
of Immunological Methods 16, 5563.
Nakahara, K., Onishi-Kameyama, M., Ono, H., Yoshida, M., Trakoonti-
vakorn, G., 2001. Antimutagenic activity against trp-P-1 of the edible
Thai plant, Oroxylum indicum vent. Bioscience, Biotechnology and
Biochemistry 65, 23582360.
Nakahara, K., Trakoontivakorn, G., Alzoreky, N.S., Ono, H., Onishi-
Kameyama, M., Yoshida, M., 2002. Antimutagenicity of some edible
Thai plants, and a bioactive carbazole alkaloid, mahanine, isolated
from Micromelum minutum. Journal of Agricultural and Food Chem-
istry 50, 47964802.
Nemmani, K.V., Jena, G.B., Dey, C.S., Kaul, C.L., Ramarao, P., 2002.
Cell proliferation and natural killer cell activity by polyherbal formu-
lation Immu-21 in mice. Indian Journal of Experimental Biology 40,
282287.
Nosalova, G., Mokry, J., Khan, M.T.H., 2003. Antitussive activity of
the fruit extract of Emblica ofcinalis Gaertn (Euphorbiaceae). Phy-
tomedicine 10, 583589.
Pal, D., Panda, C., Sinhababu, S., Dutta, A., Bhattacharya, S., 2003. Eval-
uation of psychopharmacological effects of petroleum ether extract of
Cuscuta reexa Roxb. stem in mice. Acta Poloniae Pharmaceutica 60,
481486.
Pari, L., Kumar, N.A., 2002. Hepatoprotective activity of Moringa oleifera
on antitubercular drug-induced liver damage in rats. Journal of Med-
ical Food 5, 171177.
Perianayagam, J.B., Sharma, S.K., Joseph, A., Christina, A.J., 2004. Eval-
uation of anti-pyretic and analgesic activity of Emblica ofcinalis
Gaertn. Journal of Ethnopharmacology 95, 8385.
Rajak, S., Banerjee, S.K., Sood, S., Dinda, A.K., Gupta, Y.K., Gupta,
S.K., Maulik, S.K., 2004. Emblica ofcinalis causes myocardial adap-
tation and protects against oxidative stress in ischemic-reperfusion
injury in rats. Phytothrerapy Research 18, 5460.
Rajeshkumar, N.V., Pillai, M.R., Kuttan, R., 2003. Induction of apopto-
sis in mouse and human carcinoma cell lines by Emblica ofcinalis
polyphenols and its effect on chemical carcinogenesis. Journal of Ex-
perimental and Clinical Research 22, 201212.
Rana, B.K., Singh, U.P., Taneja, V., 1997. Antifungal activity and kinetics
of inhibition by essential oil isolated from leaves of Aegle marmelos.
Journal of Ethnopharmacology 57, 2934.
Rani, P., Khullar, N., 2004. Antimicrobial evaluation of some medici-
nal plants for their anti-enteric potential against multi-drug resistant
Salmonella typhi. Phytotherapy. Research 18, 670673.
Rao, A.V., Devi, P.U., Kamath, R., 2001. In vivo radioprotective effect
of Moringa oleifera leaves. Indian Journal of Experimental Biology
39, 858863.
Ray, K., Hazrai, R., Guha, D., 2003. Central inhibitory effect of Moringa
oleifera root extract: possible role of neurotransmitters. Indian Journal
of Experimental Biology 41, 12791284.
Roychoudhury, G.K., Chakrabarty, A.K., Dutta, B., 1970. A preliminary
observation on the effects of Paederia foetida on gastro-intestinal
helminths in bovines. Indian Veterinary Journal 47, 767769.
Sabu, M.C., Kuttan, R., 2002. Anti-diabetic activity of medicinal plants
and its relationship with their antioxidant property. Journal of
Ethnopharmacology 81, 155160.
Sabu, M.C., Kuttan, R., 2004. Antidiabetic activity of Aegle marmelos
and its relationship with its antioxidant properties. Indian Journal of
Physiology and Pharmacology 48, 8188.
Sai Ram, M., Neetu, D., Yogesh, B., Anju, B., Dipti, P., Pauline, T.,
Sharma, S.K., Sarada, S.K., Ilavazhagan, G., Kumar, D., Selvamurthy,
W., 2002a. Cyto-protective and immunomodulating properties of Amla
(Emblica ofcinalis) on lymphocytes: an in vitro study. Journal of
Ethnopharmacology 81, 510.
Sai Ram, K., Rao, Ch.V., Babu, M.D., Kumar, K.V., Agrawal, V.K., Goel,
R.K., 2002b. Antiulcerogenic effect of methanolic extract of Emblica
ofcinalis: an experimental study. Journal of Ethnopharmacology 82,
19.
Sai Ram, M., Neetu, D., Deepti, P., Vandana, M., Ilavazhagan, G., Kumar,
D., Selvamurthy, W., 2003. Cytoprotective activity of Amla (Emblica
ofcinalis) against chromium (VI) induced oxidative injury in murine
macrophages. Phytotherapy Research 17, 430433.
Saxena, A., Vikram, N.K., 2004. Role of selected Indian plants in man-
agement of type 2 diabetes: a review. Journal of Alternative and Com-
plementary Medicine 10, 369378.
Shari, A.M., Darabi, R., Akbarloo, N., 2003. Study of antihypertensive
mechanism of Tribulus terrestris in 2K1C hypertensive rats: role of
tissue ACE activity. Life Sciences 73, 29632971.
Shoba, F.G., Thomas, M., 2001. Study of antidiarrhoeal activity of four
medicinal plants in castor-oil induced diarrhoea. Journal of Ethnophar-
macology 76, 7376.
Siddhuraju, P., Becker, K., 2003. Antioxidant properties of various solvent
extracts of total phenolic constituents from three different agroclimatic
origins of drumstick tree (Moringa oleifera Lam.) leaves. Journal of
Agricultural and Food Chemistry 51, 21442155.
Suarez, M., Entenza, J.M., Doerries, C., Meyer, E., Bourquin, L., Suther-
land, J., Marison, I., Moreillon, P., Mermod, N., 2003. Expression
of a plant-derived peptide harboring water-cleaning and antimicrobial
activities. Biotechnology and Bioenginnering 81, 1320.
Suffness, M., Pezzuto, J.M., 1990. Assays related to cancer drug dis-
covery. In: Hostettmann, K. (Ed.), Methods in Plant Biochemistry:
Assays for Bioactivity, 6. Academic Press, London, pp. 71133.
Sun, B., Qu, W., Bai, Z., 2003. The inhibitory effect of saponins from
Tribulus terrestris on Bcap-37 breast cancer cell line in vitro. Zhong
Yao Cai 26, 104106.
Swamy, S.M.K., Tan, B.K.H., 2000. Cytotoxic and immunopotentiating
effects of ethanolic extract of Nigella sativa L. seeds. Journal of
Ethnopharmacology 70, 17.
Tepsuwan, A., Furihata, C., Rojanapo, W., Matsushima, T., 1992. Geno-
toxicity and cell proliferative activity of a nitrosated Oroxylum in-
dicum Vent fraction in the pyloric mucosa of rat stomach. Mutation
Reseach 281, 5561.
Wang, H.K., 1998. Plant-derived anticancer agents currently in clini-
cal use or clinical trials. The Investigation Drugs Journal 1, 92
102.
Wu, Y.C., Duh, C.Y., Wang, S.K., Chen, K.S., Yang, T.H., 1990. Two
new natural azauorene alkaloids and a cytotoxic aporphine alkaloid
from Polyalthia longifolia. Journal of Natural Products 53, 1327
1331.
Zhao, G.X., Jung, J.H., Smith, D.L., Wood, K.V., McLaughlin, J.L., 1991.
Cytotoxic clerodane diterpenes from Polyalthia longifolia. Planta
Medica 57, 380383.