Parvin et al., IJPSR, 2011; Vol.

2(11): 2786-2790 ISSN: 0975-8232

IJPSR (2011), Vol. 2, Issue 11 (Research Article)

Received on 10 June, 2011; received in revised form 26 September, 2011; accepted 28 October, 2011

IN-VITRO STUDIES OF ANTIBACTERIAL AND CYTOTOXIC PROPERTIES OF FLACOURTIA JANGOMAS

Shumaia Parvin 1, Abdul Kader 1, Gopal Chandra Sarkar 1 and Salman Bin Hosain*2

Department of Pharmacy, University of Rajshahi 1, Rajshahi, Bangladesh

University of Louisiana at Monroe, College of Pharmacy 2, Monroe, Louisiana, United States of America

ABSTRACT

Keywords: The Plant Flacourtia jangomas have shown considerable antibacterial and
Antibacterial, cytotoxicity properties. The extracts were found to have different levels of
Cytotoxicity, cytotoxicity properties in different concentration. The crude extract of the
Minimum inhibitory concentration (MIC),
plant Flacourtia jangomas was subjected to in vitro microbiological screening
Brine Shrimp lethality bioassay,
Flacourtia jangomas
for studying anti-microbial activity of the extract against a wide of Gram-
positive and Gram- negative bacteria by disc diffusion method. The result so
Correspondence to Author:
obtained was compared with that of a standard antibiotic, Amoxicillin. The
Salman Bin Hosain extract shows good activity against Shigella shiga and Bacillus megaterium
Ph. D Scholar, University of Louisiana at
and moderate activity against Bacillus cerus and poor activity against E. coli.

investigated was 31.25 g/ml and 125 g/ml for Bacillus megaterium and E.
Monroe, College of Pharmacy, USA The Minimum Inhibitory Concentration (MIC) values of the plant extract

coli respectively. For Cytotoxicity, brine shrimp lethality bioassay was used.
We used Vincristine sulfate as a standard reference. The LC50 value of
standard was 12.58 mg/ml.
INTRODUCTION: Medicinal Plants: An uninterrupted bacteria by various mechanisms than those presently
reservoir of the therapeutic principle. Therapeutic uses used antimicrobials and may have a significant clinical
of plants had in effect stored at the very beginning of value in the treatment of resistant microbial strains. As
human life on the earth then the primitive man, out of well as, the cytotoxic investigation may allow further
necessity and by nutrition, restored to use plants to research for the development of anticancer drugs. The
alleviate his sufferings from injuries and diseases. present research is therefore, an endeavor to give an
Since the beginning, plants were being used as remedy elaborated review of the literature on its
for many diverse diseases ranging from simple skin pharmacological properties.
infection to such formidable foes as heart diseases and
cancers. Plant Review: The local name of the plant is Cluster
and the taxonomy is as follows-
Since cytotoxicity and antibacterial constituent are an
important medicinal substance; recently research is Kingdom - Plantae
being continued on various plants to derive Division - Magnoliophyta
cytotoxicity and antibacterial ingredients. The rapid Class - Magnoliopsida
development of the drug resistant of microbes has led Order - Violales
to investigation of new antimicrobial lead agents Family - Flacourtiaceae
especially from plant extracts of discover new chemical Genus - Flacourtia
structures. The antimicrobial compounds from plants Species - Flacourtia jangomas (Lour.) Rausch -
extracts may inhibit bacterial growth or kill the Indian plum

Available online on www.ijpsr.com 2786

 Parvin et al., IJPSR, 2011; Vol. 2(11): 2786-2790 ISSN: 0975-8232

Chemical Constituents: The Flacourtiace has received In Vitro Antimicrobial Screening:


greater attention from chemists in the last few years
following the discovery of a series of cytotoxic Disc Diffusion Method: Disc diffusion method was
diterpenes from Casearia sylvestris 1. However, used to determine the antimicrobial activity of the
phytochemical reports are still limited to a few species crude extracts 5, against the microbial strains listed
and not much is understood about the chemistry of in Table 1. These were collected as pure cultures
the family. Studies so far have shown that the from the microbiology Laboratory of Atish Dipankar
Flacourtiaceae elaborates a diverse array if compound University of Science and Technology, Bangladesh.

such a manner so that each 20 l solution contains

classes which include terpenoids, alkaloids, flavonoids Experimental sample was dissolved in ethanol in

250 g of the plant extract. Here, Amoxicillin

and tannins, lignans and flavanolignans, glucosides,
coumarins and isocoumarins. There have also been
reports of xanthones, quinones, limonoids and (10μg/dis ) was used as the sta dard.
phenazines.
Then, for 24 hours, these plates were kept at low
Medicinal Uses: The Flacourtiace is the perhaps-best temperature (4oC) to allow maximum diffusion of
known for the seed oils produced by certain species of the test materials and Amoxicillin. To allow
the tri es Pa giae a d O o eae. Chal oogra oil is maximum growth of the organisms, these plates
obtained from the seeds of Taraktogenos kurzii and were then incubated for 24 hours, at 37.4oC. The
Hydnocarpus spices and has long been used in the test materials, which possess antimicrobial activity,
treatment of leprosy and other diseases 2. This oil has suppressed the growth of the microorganisms and
now been superseded by sulfur drugs 3. In India, dried a clear, distinct zone of inhibition was seen
leaves of Flacourtia jangomas are used to treat asthma surrounding the discs. The antimicrobial activity of
4
. the test agents was determined by measuring the
diameter of zone of inhibition, expressed in mm.


MATERIALS AND METHODS:
Collection and Identification of the Plant: The Minimum Inhibitory Concentration (MIC):
plant/plant parts of the Flacourtia jangomas were Minimum inhibitory concentration (MIC) was
collected during the month September 2010 from determined by the Serial tube dilution techniques
Booth para besides Rajshahi University, Bangladesh. or turbidimetric techniques 6 using serially diluted
Mahabubur Rahman, Taxonomist of the Department of plant extracts according to the. The stock solution
Botany, University of Rajshahi, identified the plant. was prepared by dissolving 4.00 mg of the
experimental sample i.e., chloroform extract of the
Drying and Grinding: The collected plant root was plant Flacourtia jangomas in 4 ml suitable solvent
washed with water and separated from undesirable (e.g. Dimethyl sulfoxide). Thus solutions with a
materials. It was chopped into small pieces and sun concentration of 1.00 mg/ml were obtained.
dried for two weeks. After a final drying in drier at 40°c Bacteria inocula [Escherichia coli (Gram negative),
for one hour, the plan part were pulverized into coarse Bacillus megaterium (Gram positive)] were
powder and kept in airtight containers. The container adjusted to contain approximately 106 cells/ml.
was stored in cool and dry place. The net of the The test plates were incubated at 37.5°C for 24
powder before drying and grinding was 2.5 kg. The hours.


weight of the powder after drying and grinding was 1
kg, soothe net loss of water was observed 60%. Brine Shrimp Lethality Bioassay: Brine shrimp
lethality bioassay 7, 8 technique was applied for the
Cold Extraction of Plant Material: The powdered plant determination of general toxic property of the
(750 gm) was taken in large glass bottle and extracted plant extractives. In this method, in vivo lethality in
with Ethyl acetate: Methanol (10:1) for 7 days. The a simple zoological organism (Brine shrimp nauplii)
procedure was repeated twice using same solvent is used as a favorable monitor for screening and
system for next 3 days. The extract was decanted first fractional in the discovery of new biotic natural
through a cotton plug and finally filtered through filter products.
paper to get clear filtrate.
Available online on www.ijpsr.com 2787
 Parvin et al., IJPSR, 2011; Vol. 2(11): 2786-2790 ISSN: 0975-8232

For the experiment, About 3.00 mg (table 3) of test TABLE 1: ANTIBACTERIAL ACTIVITIES OF SAMPLES OF
sample i.e. ethyl alcohol extract of plant was FLACOURTIA JANGOMAS
Diameter of Zone of inhabitation
weighed accurately and dissolved in 0.6 ml (mm) Standard
Test microorganisms
dimethyl sulfoxide (DMSO) to get the F. jangomas 10g/disc
concentration of 5 µg/µl. from which 5 types of 125g/disc 250g/disc
Gram Negative
concentration like 5 µg/ml, 10 µg/ml, 20 µg/ml, 40
Shigella shiga 8 10 10
µg/ml, 80 µg/ml respectively were made. The E. coli -- -- 25
control groups containing 10 living brine shrimp Gram Positive
nauplii in 5 ml seawater received the positive Bacillus cereus 7 8 12
Bacillus megaterium 8 11 9
control solutions.

In order to use as negative control, 20µl of DMSO In this technique a large number of sterilized test tubes
was added to each of three premarked glass test were used and each of the test tubes contained 1 ml
tube containing 5 ml of simulated seawater and 10 sterile nutrient broth medium. The tubes of nutrient
shrimp nauplii. In this case the test was considered broth medium containing graded doses of
invalid if the negative control showed a rapid experimental sample were inoculated with 10
mortality rate and therefore conducted again. microliters of the test bacteria (contain 106 cells/ ml).
Ampicillin trihydrate was used as positive control. These test tubes were incubated at 37.5°C for 24
hours.
RESULTS & DISCUSSION: The outcome found for the
antimicrobial and cytotoxic activities of Flacourtia Inhibition of growth or organisms was observed in the
jangomas are discussed in detail in this chapter. It was test tubes having minimum inhibitory concentration
shown that the specimen has shown significant (MIC) and the concentration above MIC of the test
activities for cytotoxic and antimicrobial properties. sample.

Antimicrobial Activity: The Ethanolic extract of Three control test tubes were used to perform control
Flacourtia jangomas was tested for antibacterial tests using CM (medium), CS (medium+ sample) and CI
activities against a number of both gram positive and (medium + Inoculum) where bacterial growth observed
gram negative bacteria. Standard Amoxicillin discs (30 only in Cr (medium + Inoculum) and the other two
mg/disc) were used for the comparison purpose. The were clear.
antimicrobial activities of ethyl acetate extract from
Flacourtia jangomas were tested in the present study.
The results were given in table 1.

The zone of inhibition produced by ethyl acetate crude

extract of the plant ranged from 0 to 11 mm. However,
at a concentration of 125g/disc the result of ethyl
acetate extract showed poor activity against most of
the test microorganisms (Figure 1). But at a FIG. 1: ANTIBACTERIAL ACTIVITY OF ETHYL ACETATE EXTRACT OF F.
concentration of 250g/disc the result of ethyl acetate JANGOMAS (125g/DISC) AGAINST SHIGELLA SHIGA
extract showed moderate activity of the test organisms
(Figure 2).

Out of all bacterial species, Bacillus megaterium

showed best result against the test materials and the
gram negative Bacteria are poorly sensitive to the
extract.

FIG. 2: ANTIBACTERIAL ACTIVITY OF ETHYL ACETATE EXTRACT OF F.

JANGOMAS (250g/DISC) AGAINST SHIGELLA SHIGA

Available online on www.ijpsr.com 2788

 Parvin et al., IJPSR, 2011; Vol. 2(11): 2786-2790 ISSN: 0975-8232

The minimum inhibitory concentration of chloroform g/ml respectively. The results were presented in the

coli and Bacillus megatetium were 125 g/ml & 31.25

extract of the plant investigated against Escherichia table 2 & 3.

TABLE 2: MIC OF THE ETHYL ACETATE EXTRACT OF PLANT INVESTIGATED AGAINST ESCHERICHIA COLI AND BACILLUS MEGATERIUM
No of test tubes Nutrient broth medium added (ml) Diluted solution extract (g/ml) Inoculum added (l) Observation
1 1 500 10 NG
2 1 250 10 NG
3 1 125 10 NG
4 1 62.5 10 G
5 1 31.25 10 G
6 1 15.625 10 G
7 1 7.8125 10 G
8 1 3.90625 10 G
9 1 1.9531 10 G
CS 1 500 10 NG
CI 1 00 10 G
CM 1 00 10 NG
NG= No Growth G= Growth; Escherichia coli MIC= 125 (g/ml) Bacillus megaterium MIC= 31.25 (g/ml)

Cytotoxic activity through Brine Shrimp lethality highly significant with the highest concentration
bioassay: In this method, in vivo lethality in a simple (80mg/ml). Maximum mortalities happened at a
zoological organism (Brine shrimp nauplii) is used as a concentration of 80 mg/ml, whereas least mortalities
favorable monitor for screening and fractional in the were at 5mg/ml concentration.
discovery of new biotic natural products. 2 mg of each
sample was dissolved in specific volume of DMSO So, it can be said that mortality increased gradually
(Dimethyl Sulfoxide) to obtain the desired with the increase in concentration of the test sample.
concentration of the prepared solution. The degree or The median lethal concentration, LC50 of chloroform
extent of lethality was directly proportional to the extract of plant Flacourtia jangomas and standard
concentration of the extract ranging from least ampicillin trihydrate were 12.58 mg/ml and 11.48
significant with the lowest concentration (5mg/ml) to mg/ml.

TABLE 3: THE RESULT OF BRINE SHRIMP LETHALITY BIOASSAY FOR ETHYL ACETATE EXTRACT OF THE PLANT INVESTIGATED AND FOR
STANDARD AMPICILLIN TRIHYDRATE
Conc. g/ml
g/ml
Log of No. of Nauplii No. of Nauplii Dead Average No. of Percent (%) LC50
Test samples
conc. taken V1 V2 V3 nauplii dead of Mortality
5 0.69 10 4 3 4 3.66 36.6
10 1.0 10 4 4 6 4.66 46.6
Ethylacetate extract 20 1.3 10 6 4 8 6.00 60.0 12.58
40 1.6 10 7 6 7 6.66 66.6
80 1.9 10 8 9 8 8.33 83.3
5 0.69 10 3 3 2 2.66 26.6
10 1.0 10 4 5 5 4.66 46.6
Amp Tri Std. 20 1.3 10 7 7 6 6.66 66.6 11.48
40 1.6 10 9 9 8 8.66 86.6
80 1.9 10 9 9 9 9.00 90.0
Control 20 DMSO 00 10 0 0 0 0 0 ---
V1,= Vial 1, V2= Vial 2, V3= Vial 3; Amp Tri Std. = Ampicillin Trihydrate standard. LC50= Medium Lethal Concentration

CONCLUSION: The plant has been being used for a material of the plant investigated was exhaustively
long time in herbal medicine without knowing the extracted with ethyl acetate: methanol (10:1) ratio to
exact phytopharmacological properties that works get brownish mass. The concentrated ethyl acetate
against diarrhoea, toothache, and possess stomachic & extract was fractionated with chloroform and
astringent properties. The dried coarse powdered methanol.

Available online on www.ijpsr.com 2789

 Parvin et al., IJPSR, 2011; Vol. 2(11): 2786-2790 ISSN: 0975-8232

In-vitro antibacterial screening, determination of 2. Burkill I H. A Dictionary of the Economic Plants of the Malay
Peninsula. Kuala Laumpur: Ministry of Agriculture and
minimum inhibitory concentration (MIC), and cytotoxic Cooperatives. 1966.
(Brine shrimp lethality bioassay) study were performed 3. Perry L M and Metzger J. Medicinal Plants of East and
on the ethyl acetate extract of plant. The plant can be Southeast Asia: Attributed Properties and Uses. The MIT Press
Cambridge. 1980.
further screened against various diseases in order to 4. Ahmad J, Wizarat D, Shamsuddin K M, Zaman A, Connolly J D.
find out its unexplored efficacy and can be a potential Phytochemistry 1984. 23:1269.
source of biologically important drug candidates. The 5. Bauer A W, Kirby W M M, Sherries and Tuck M. Antibiotic
susceptibility testing by a standardized disc diffusion method. J.
significant results of cytotoxic investigation allow Am. Clin. Pathol. 1966. 45:493-496
further studying for the development of anticancer 6. Roland R. Antibiotics: An Introduction. Switzerland: F.
drugs. Hoffmann La Roche and Co., Basel. 1982.
7. Meyer B N, Ferrigni N R, Putnam J E, Jacobsen J B, Nicholsand D
E, Mclaughlin JL. Brine shrimp; a convenient general bioassay
REFERENCES: for active plant constituents. Planta. Med. 1982. 45:31-34
8. Husain A, Virmani O P, Popli S P, Misra L N, Gupta M M,
1. Itokawa H, Totsuka N, Morita H, Takeya K, Iitaka Y, Schenke E P,
Srivastava G N, Abraham Z, Singh A K, Dictionary of Indian
Motidome M. Chemical and Pharmaceutical Bulletin 1990.
Medicinal Plants, CIMAP. 546
32:3384.

*********************

Available online on www.ijpsr.com 2790