Khan Md.

Ahad Ali et al / IJRAP 2011, 2 (4) 1186-1191

International Journal of Research in Ayurveda & Pharmacy, 2(4), 2011 1186-1191
Research Article Available online through
www.ijrap.net ISSN 2229-3566

CYTOTOXICITY AND ANTIBACTERIAL ACTIVITY OF ANDROGAPHIS PENICULATA,
EUPHORBIA HIRTA AND URGINIA INDICA
Mahmood Ayesha, Khan Md. Ahad Ali*, Chowdhury M. Mohi Uddin

Department of Pharmacy, Southern University, Bangladesh

Received on: 10/07/2011 Revised on: 02/08/2011 Accepted on: 21/08/2011

ABSTRACT
Based on the traditional uses, petroleum etheric, ethanolic and aqueous extracts of Andrographis paniculata leaves,
Euphorbia hirta leaves and Urginea indica bulbs were subjected to cytotoxicity and antibacterial activity test. In
brine shrimp lethality test, the LC
50
of aqueous extract of Andrographis paniculata leaves was found to be 600 g/ml
and that of ethanolic and aqueous fractions of Euphorbia hirta leaves were 800 and 80 g/ml respectively. Similarly,
the ethanolic and petroleum etheric extracts of Urginea indica bulbs showed significant mortality in the test shrimp
and LC
50
were found to be 600 and 100 g/ml respectively. Again, in disc diffusion test aqueous extract of A.
paniculata leaves showed significant inhibition against three bacteria, Bacillus megaterium (6 mm), Shigella
dysenteriae (9 mm) and Vibrio cholera (9 mm). Similarly, the ethanolic extract of E. hirta significantly inhibited the
growth of Bacillus subtilis (6 mm), Bacillus megaterium (7 mm) Shigella dysenteriae (13 mm) and V. cholera (9
mm). However, the aqueous extract of the plant showed no effect on the growth of the test bacteria. And in case of U.
indica bulb extracts, ethanolic fraction produced remarkable zone of inhibition against Bacillus subtilis (7 mm),
Bacillus megaterium (5 mm), Staphylococcus aureus (12 mm), Salmonella typhi (9 mm) and Vibrio cholera (11 mm).
On the other hand petroleum etheric fraction of it produced zone of inhibition only against Bacillus subtilis (7 mm)
and Bacillus cereus (9 mm).
KEYWORDS: Andrographis paniculata, Euphorbia hirta, Urgenia indica, Antibacterial, Cytotoxic.

*Author of correspondence
Md. Ahad Ali Khan, Department of Pharmcay, Southern University Bangladesh Email: [email protected]

INTRODUCTION
Medicines derived from plants have pivotal role in health
care of ancient and modern cultures. Almost 60% of
drugs approved for cancer treatment are of natural origin
like vincristine, vinblastine, etoposide, taxanes and
camptothecins are all examples of plant-derived
anticancer compounds
1
. But most of the anticancer drugs
have inevitable severe adverse reactions. Therefore, it is
an imperative need of the day to develop alternative
therapeutic measures with low risk of adverse effects
against this deadly disease. There is always a hope that
the traditional medicinal plants may provide potent and
safe remedies. Among the recent advances in cancer
chemotherapy, phytochemicals play an important role.
Similarly, plants are one of the natural sources of
antimicrobial agents. A wide range of plants those
possess various medicinal properties are used in the
preparation of herbal dugs. The different parts like root,
stem, flower, fruit, twigs, exudates and modified plant
organs are used as drugs. While some of these raw drugs
are collected in smaller quantities by the local
communities and folk healers for local use. A vast
majority of these plants have not been yet adequately
evaluated by using standard scientific methods
2
.
The people of Bangladesh especially of rural areas are
dependent on herbal treatment. Different Ayurvedic
preparations available in market throughout the country
which are used topically for skin infections and benign
tumor contain A. paniculata. Moreover, the villagers in
different areas of the country use the latex of E. hirta
against the warts and infection by injury like cuts or
burns. The traditional practitioners use different parts of
the plant in treatment of asthma, bronchitis, worm
infection, conjunctivitis and dysentery. In Yunani
medicines U. indica is used as one of the active
ingredients and these are used for the treatment of skin
diseases as well as internal pains and scabies. Some
herbal products, which are used as tumor suppressants
also contain U. indica.
Khan Md. Ahad Ali et al / IJRAP 2011, 2 (4) 1186-1191

International Journal of Research in Ayurveda & Pharmacy, 2(4), 2011 1186-1191
Considering the above magnitude of the plants as one of
the natural sources for anticancer and antimicrobial drugs
a systematic exploration was undertaken to screen
different fractions of Andrographis paniculata (Burm.f.)
Wall. (Family- Acanthaceae), Euphorbia hirta Linn.
(Family-Euphorbiaceae) and Urgenia indica Kunth.
(Family-Liliaceae) for cytotoxicity and antibacterial
activity.
MATERIALS AND METHODS
Plant materials
The fresh plant parts were collected from Chittagong,
Bangladesh. Then those were kept in the sun under a
shed till dried. Taxonomical identification of the plants
was done by he taxonomist, botanist of Bangladesh
Council for Scientific and Industrial Research
Herbarium, Chittagong, Bangladesh. And the herbarium
sheets signed by the taxonomist Mohammad Mohiuddin
and were preserved in Pharmacognosy Laboratory of
University of Science and Technology.
Preparation of extract
For the preparation of water extract the dried A.
paniculata and E. hirta leaves were ground and the
powder was packed in the thimble of a Soxhlet apparatus
and extracted with 1000 ml of distilled water at 100C
for 48 hrs. Again, the ethanol and petroleum ether extract
of E. hirta leaves and U. indica bulbs were prepared by
simple maceration in an air tight container. Fine powder
materials were merged in the respective solvents and the
homogenates were kept for 2 weeks at room temperature
(252C). The entire extracts were filtered twice. At
first, using sterile cotton and then Whatman-41 filter
paper. Petroleum ether and ethanol were completely
evaporated at room temperature by using an electric fan
facilitating the evaporation of the solvents. While
aqueous extracts were dried by using rotary vacuum
evaporator at 100
o
C, 1 atmospheric pressure in a flask
with 80 rpm.
Shrimp eggs and Microorganisms
The brine shrimp eggs for cytotoxicity assay and
bacterial strains - Bacillus subtilis, Bacillus cereus,
Bacillus megaterium, Escherichia coli, Pseudomonas
aeruginosa, Staphylococcus aureus, Shigella sonnei,
Shigella dysenteriae, Salmonella typhi and Vibrio
cholerae, for antibacterial sensitivity assay were
collected from Bangladesh Council of Scientific and
Industrial Research, Bangladesh.
Media
Nutrient agar, Nutrient broth media manufactured by
HiMedia Laboratories Pvt. Ltd. India were used as
bacterial culture media.


Chemicals
Ethanol (99.5%; Merck KGaA, Germany) was used as
solvent in maceration extraction of the plant material.
Sea salt (Sodium Chloride Crystal GR; Merck Ltd.,
Mumbai, India) was used as medium for hatching the
eggs of shrimp. In cytotoxicity test dimehyl sulfoxide
(99.9%, BioReagent, for molecular biology; Sigma-
Aldrich, India) was used as solvent to dissolve the
extracts. In antibacterial sensitivity assay amoxicillin
(Ultrafen, Tab. 50mg, Beximco Pharmaceuticals Ltd.,
Bangladesh) was used as reference standard. The water
used for hot percolation and other purposes was prepared
in laboratory by distillation.
Growth and Maintenance of Test Bacteria
Bacterial cultures of Bacillus subtilis, Bacillus cereus,
Bacillus megaterium, Escherichia coli, Pseudomonas
aeruginosa, Staphylococcus aureus, Shigella sonnei,
Shigella dysenteriae, Salmonella typhi and Vibrio
cholerae were obtained from the Microbiology
Laboratory of Bangladesh Council of Scientific and
Industrial Research, Chittagong. The bacteria were
maintained on nutrient broth (NB) at 37C
3
.
Preparation of Inoculums
Preliminary culture of the bacterial strains Bacillus
subtilis, Bacillus cereus, Bacillus megaterium,
Escherichia coli, Pseudomonas aeruginosa,
Staphylococcus aureus, Shigella sonnei, Shigella
dysenteriae, Salmonella typhi and Vibrio cholerae were
prepared in nutrient broth and kept overnight in a rotary
shaker at 37C, then centrifuged at 10,000 rpm for 5 min,
pellet was suspended in double distilled water and the
cell density was standardized spectrophotometrically
(A
610
nm)
3
.
Brine shrimp lethality test
In vitro lethality assay of the different fractions of A.
paniculata, E. hirta and U. indica were used to detect
cell toxicity
4
. Brine shrimp eggs were placed in seawater
(3.8% w/v sea salt in distilled water) and incubated at 24-
28C in front of a lamp. Eggs were hatched for 48 hrs
and a large number of larvae (nauplii) were born in the
water. Then extract solutions (50 g/ml) were prepared
by using the solvent, dimethyl sulfoxide (DMSO). Ten
sample concentrations (20 1000 g/ml) were used; 3
test tubes for each concentration. With the help of a
Pasteur pipette 10 living shrimps were taken in ten test
tubes. In control test tubes seawater and DMSO were
taken. Alive nauplii were counted after 16 hrs and the
lethal concentrations (LC
50
) were calculated from mean
percent mortality. The plot of percent mortality versus
log concentration of the extract produced an approximate
linear correlation between them on graph. From the
graph (Figure 1.1-1.5) the concentration at which 50%
Khan Md. Ahad Ali et al / IJRAP 2011, 2 (4) 1186-1191

International Journal of Research in Ayurveda & Pharmacy, 2(4), 2011 1186-1191
mortality (LC
50
) of brine shrimp nauplii occurred were
obtained. The resulting data are summarized in table 1.
Antibacterial sensitivity test
Antibacterial activity of different fractions of A.
paniculata, E. hirta and U. indica were tested by disc
diffusion method
5
. Here 100 l of suspension of entire
bacteria containing 2.010
6
cfu/ml were taken in sterile
Petri-dishes (9 mm in diameter) containing sterile
sabouraud dextrose agar medium (15 ml). The filter
paper discs (5 mm in diameter) were individually
impregnated with the crude extracts at the dose of 100
g/disc and then placed onto the agar medium in the
Petri-dishes which were previously inoculated with the
test bacteria. After that the Petri-dishes were kept at 4C
for 2 hrs in a refrigerator and subsequently incubated at
37C for 24 hrs in an incubator. The diameter of the zone
of inhibition was measured in millimeter. Amoxicillin
(30 g/disc) was used in positive control and ethanol
(solvent) was used in negative control.
The resulting data are summarized in the table 2.
Statistical analysis
The results of brine shrimp lethality and antibacterial
sensitivity test were statistically evaluated by using one
way ANOVA. Values with p<0.5 were considered
significant.
RESULTS AND DISCUSSION
In this project different fractions of Andrographis
paniculata, Euphorbia hirta and Urgenia indica were
subjected to brine shrimp lethality and antibacterial
sensitivity test. In cytotoxicity test by using brine shrimp
the extracts of the plants produced significant cell
toxicity. From table 1 it was observed that aqueous
extract of A. paniculata leaves was produced 50%
mortality (LC
50
) at the concentration of 600 g/ml. And
that of the ethanolic and aqueous fractions of E. hirta
were found to be 800 and 80 g/ml respectively.
Similarly, ethanolic and petroleum etheric extracts of U.
indica were also found to be cytotoxic with LC
50
of 600
and 100 g/ml respectively. Thus the study depicts that
the extract may contain bioactive compounds having
antitumor, anticancer or pesticide properties. However,
this cannot be confirmed without further higher and
specific tests.
On the other hand, in antibacterial disc diffusion test
different fractions of Andrographis paniculata,
Euphorbia hirta and Urginea indica were found to be
active but not against all of the test bacteria and showed
various degree of zone of inhibition. From table 2 it was
observed that aqueous extract of A. paniculata leaves
produced significant inhibition against three species such
as; Bacillus megaterium (6 mm), Shigella dysenteriae (9
mm) and Vibrio cholerae (9 mm). But there was no zone
of inhibition in entire Petri-dishes containing aqueous
extract of E. hirta while observed in naked eyes.
Nevertheless, the ethanolic extract of the plant showed
distinct zone of inhibition against Bacillus subtilis (6
mm), Bacillus megaterium (7 mm), Shigella dysenteriae
(13 mm) and Vibrio cholerae (9 mm). And among the
two fractions of U. indica bulbs the ethanolic one
produced notable zone of inhibition against Bacillus
subtilis (7 mm), Bacillus megaterium (5 mm),
Staphylococcus aureus (12 mm), Salmonella typhi (9
mm) and Vibrio cholera (11 mm). But the petroleum
etheric extract of the plant was active only against two
bacteria - Bacillus subtilis (7 mm) and Bacillus cereus (9
mm). Thus the results depicted that the extracts may
contain some biologically active compounds which have
antibacterial activity.
The results of present investigation clearly indicated that
cytotoxicity as well as antibacterial activity varied with
the species of the plant and the solvents used for
extraction. Thus the study ascertained the importance of
plants as traditional remedies of Ayurvedic, Yunani and
Herbal medicine systems which could be of considerable
interest to the development of new drugs.
ACKNOWLEDGEMENT
The authors are indebted to the director of Bangladesh
Council of Scientific and Industrial Research, Chittagong
for providing brine shrimp eggs and test bacteria.
REFERENCES
1. Grever MCB. Cancer drug discovery and Anticancer therapy
targeting the apoptotic development. In: De Vita VHS and
Rosenberg SA, editors. Cancer: Principles and Practice of
Oncology. 8th ed. Lippincott Williams & Wilkins; 2009. p.328-
339.
2. Balandrin MF, Klocke JA, Wurtele ES, Bollinger WH. Natural
plant chemicals: Sources of Industrial and Medicinal materials.
Science 1985; 228:1154-1160.
3. Mahesh B, Satish S. Antimicrobial Activity of Some Important
Medicinal Plant Against Plant and Human Pathogens. World
Journal of Agricultural Sciences 2008; 4 Suppl 1:839-843.
4. Meyer BN, Ferrigni NR, Putnam JE, Jacobson JB, Nicholas DE,
McLaughlin JL. Brine shrimp A convenient general bioassay
for active plant constituents. Planta Medica 1982; 45:31-34.
5. Latha L, Yoga S, Sasidharan Z, Zuraini S, Suryani L, Shirley S
et al. Antimicrobial activities and toxicity of crude extract of the
Psophocarpus tetragonolobus pods. Afr J Trad CAM 2007;
4(1): 59-63 .




Khan Md. Ahad Ali et al / IJRAP 2011, 2 (4) 1186-1191

International Journal of Research in Ayurveda & Pharmacy, 2(4), 2011 1186-1191
Table 1: Cytotoxic activity of different fractions of A. paniculata, E. hirta and U. indica
Sample
% Mortality
LC50 (g/ml)
Concentration (g/ml)
20 40 60 80 100 200 400 600 800 1000
Log concentration
1.3 1.6 1.8 1.9 2.0 2.3 2.6 2.8 2.9 3.0
AEAP 30 30 40 40 40 40 40 50 50 60 600
EEEH 30 40 40 40 40 40 40 40 50 50 800
AEEH 30 40 40 50 50 50 60 60 60 60 80
EEUI 20 30 30 30 40 40 40 50 50 50 600
PEEUI 20 30 30 40 50 50 50 50 60 60 100
Mean 26 34 36 40 44 44 46 50 54 56

SD 5.48 5.48 5.48 7.07 5.48 5.48 8.94 7.07 5.48 5.48
p<0.0001; calculated by using one way ANOVA where Fisher F-value = 11.08; SD = Standard deviation
UIEE = Urgenia indica ethanolic extract, UIPEE = Urgenia indica petroleum etheric extract, EHEE = Euphorbia hirta ethanolic extract, EHAE = Euphorbia
hirta aqueous extract and APAE = Andrographis paniculata aqueous extract
Table 2: Antibacterial activity of different fractions of A. paniculata, E. hirta and U. indica
Sample
(0.1 mg/disc)
Zone of Inhibition(mm)
B.su B.ce B.me E. co P.ae S.au S.so S. dy S.ty V.ch
AEAP nd nd 6 nd nd nd nd 9 nd 11
AEEH nd nd nd nd nd nd nd nd nd nd
EEEH 6 nd 7 nd nd nd nd 13 nd 9
EEUI 7 nd 5 nd nd 12 nd nd 9.5 11
PEEUI 7.0 9.0 nd nd nd nd nd nd nd nd
AM nd nd 37 nd 8 12 8.0 22 7 6
p<0.5; calculated by using one way ANOVA where Fisher F-value = 1.35; nd = Not detected
AM = Amoxicillin, B.su = Bacillus subtilis, B.ce = Bacillus cereus, B.me = Bacillus megaterium, E.co = Escherichia coli, S.au = Staphylococcus aureus,
S.so = Shigella sonnei, S.ty = Salmonella typhi, P.ae = Pseudomonus aeruginosa, S.dy = Shigella dysenteriae and V.ch = Vibrio cholera





Figure 1.1: The effect of aqueous extract of Andrographis paniculata in brine shrimp lethality assay
Khan Md. Ahad Ali et al / IJRAP 2011, 2 (4) 1186-1191

International Journal of Research in Ayurveda & Pharmacy, 2(4), 2011 1186-1191



Figure 1.2: The effect of ethanolic extract of Euphorbia hirta in brine shrimp lethality assay



Figure 1.3: The effect of aqueous extract of Euphorbia hirta in brine shrimp lethality assay




Figure 1.4: The effect of ethanolic extract of Urgenia indica in brine shrimp lethality assay

Khan Md. Ahad Ali et al / IJRAP 2011, 2 (4) 1186-1191

International Journal of Research in Ayurveda & Pharmacy, 2(4), 2011 1186-1191




Figure 1.5: The effect of petrleum etheric extract Urgenia indica in brine shrimp lethality assay

Figure 2: Antibacterial activities of different fractions of A. paniculata , E. hirta and U. indica
UIEE = Urgenia indica ethanolic extract, UIPEE = Urgenia indica petroleum etheric extract, EHEE = Euphorbia hirta ethanolic extract, EHAE = Euphorbia hirta
aqueous extract and APAE = Andrographis paniculata aqueous extract; AM = Amoxicillin, V.ch = Vibrio cholera, S.au = Staphylococcus aureus, S.so = Shigella
sonnei, S.ty = Salmonella typhi, P.ae = Pseudomonus aeruginosa, B.su = Bcillus subtilis, B.ce = Bacillus cereus, S.dy = Shigella dysenteriae, E.co = Escherichia coli,
and B.me = Bacillus megaterium

















Source of support: Nil, Conflict of interest: None Declared