A COMPREHENSIVE STUDY ON ANTIOXIDANT, ANTIBACTERIAL, CYTOTOXIC AND PHYTOCHEMICAL PROPERTIES OF AVERRHOA CARAMBOLA

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ISSN: 2278-778X
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*Corresponding Author:
Adnan Mannan
,
Assistant Professor,
Department of Genetic Engineering & Biotechnology,
University of Chittagong,
Chittagong-4331, Bangladesh.
803
A COMPREHENSIVE STUDY ON ANTIOXIDANT, ANTIBACTERIAL, CYTOTOXIC AND
PHYTOCHEMICAL PROPERTIES OF AVERRHOA CARAMBOLA
Joysree Das
1
, Zulon Datta
1
, Ayan Saha
2
, Suza Mohammad Nur
2
, Prosenjit Barua
2
, Md. Mominur Rahman
3
, Kazi Ashfak Ahmed
Chowdhury
3
, Mohammad Mostofa Chowdhury
3
, Rabiul Hossain Chowdhury
4
, Adnan Mannan
2,5*
1
Department of Pharmacy, BGC Trust University, Chittagong, Bangladesh
2
Department of Genetic Engineering & Biotechnology, University of Chittagong, Chittagong-4331, Bangladesh
3
Department of Pharmacy, International Islamic University Chittagong, Chittagong, Bangladesh
4
Department of Pharmacy, University of Science & technology Chittagong, Chittagong, Bangladesh
5
School of Pharmacy, Faculty of Health Sciences, Curtin University, Perth WA 6845, Australia
Received for publication: December 28, 2012; Revised: January 17, 2013; Accepted: March 12, 2013
INTRODUCTION
Averrhoa carambola, a tropical, drought-resistant
plant known commonly as Carambola
or Starfruit because of having a shape like a star, is
considered as a valuable species in the family
Oxalidaceae for its multifaceted medicinal properties.
According to historians, Sri Lanka and Maluku islands
were the places where cultivation of star fruit was
started for the first time. Since then, this evergreen
plant has been grown in China, Taiwan,
and Malaysia and in countries of Southern Asia
12,8
. In
fact, this plant can grow in abundance in any country
having a warm climate. The fruit of this plant - which
can taste sour in early stage of ripening and very sweet,
when fully ripe - has also been used in cooking for a
long time, especially to add extra flavor in food items
like puddings, stews, tarts and curries. On the other
hand, this species has drawn the attention of
researchers for its ability to cure diseases and
strengthen human immune system. Therefore, this
study has been carried out in order to identify and
know in more detail about the medicinal qualities that
are existent in this particular plant. We found out that
Averrhoa carambola contains chemical compounds like
saponins, steroids, alkaloids, glycosides, flavonoids and
carbohydrates, which bear clear testimony to this
plant’s high medicinal values. According to Materia
Medica, it is able to quench thirst, and has been used
for long to increase secretion of salivary glands and in
the treatment of fever
11
. Moreover, according to
Ayurvedic medicine, ripe starfruit helps in digestion and
works effectively as a tonic, but biliousness can be
caused in case of excessive intake. In India, it is
considered as one of the best cooling medicines
6,14
.
This fruit also contains antioxidants, the juice can be
used as an astringent, especially in cases of
hemorrhage, and bleeding hemorrhoids, it works
effectively to stop further loss of blood
9
. In Brazil, it
has been used as a diuretic to help patients with
bladder and urinary problems
11
. The main objectives of
this study were to determine the antioxidant,
Abstract: Experiment was conducted on petroleum ether extract of a bark part of Averrhoa carambola in order to
find out various phytochemicals in this plant and to evaluate this plant’s cytotoxicity, ability to impede bacterial
growth and ability to work against harmful molecules like free radicals with the help of antioxidants that are
present in it. To make sure that the findings are accurate, a number of samples of this plant were collected from
different places of Bangladesh. Existence of flavonoid, carbohydrate, glycoside and steroid in the petroleum
extract of Averrhoa carambola was confirmed by conducting a phytochemical screening. The disc diffusion method,
where ten pathogenic bacteria were used, confirmed that the extract is capable of showing good action against
pathogenic organisms. But among these ten bacteria, the extract’s zone of inhibitions were comparatively larger in
cases of Salmonella typhi, Pseudomonas aeruginosa, Escherichia coli and Bacillus megaterium, which clearly indicates
that the extract is able to exhibit remarkable antibacterial action against these bacteria. The cytotoxic action of the
extract was measured by using the Brine Shrimp Lethality Bio-assay method. Using this method, the value of LC50
was calculated as 19.95. The extract’s phenolic content was estimated as 62.504 mg/g GAE, and the amount of
flavonoid in it was 24.107 mg/g of quercetin equivalent. A slight increase in the DPPH radical scavenging activity of
Averrhoa carambola was observed when the concentration of the extract was enhanced. In comparison to the IC50
value of 11.20μg/mL of ascorbic acid, which was considered as a reference in the test, the IC50 value of the plant
extract was estimated as 125.429μg/mL.
Keywords: Averrhoa carambola, Phytochemical Screening, Disc Diffusion Method, Antioxidant Activity, Brine
Shrimp Lethality Bio-Assay.
Joysree et al.: Int. J. Bioassays, 2013, 02 (05), 803-807
www.ijbio.com 804
antibacterial and phytochemical properties of Averrhoa
carambola and get a clear view of how toxicity
develops in a cell in case of in vivo administration of
extract or medicinal agent of this plant. In this regard,
we used Brine Shrimp Lethality Bioassay, which was
proposed by
11
and modified by
17
, in order to assess the
cytotoxic action of the plant materials.
MATERIALS AND METHODS
Collection of sample and preparation of plant extract:
After properly cutting and slicing, the collected
sample of this species was dried in two stages – firstly,
in the sun and then by an electric dryer at 60-70C.
After that, the dry sample was turned into powder by
grinding in a mechanical grinder. The powdered sample
was preserved in a clean and closed glass container.
Later, in order to let the extraction process occur
completely, chloroform and ethanol having a total
volume of 800 ml was entered into the container and
the dry sample was left in this condition for the next 3
days. Then the plant extract was filtered in a sterilized
cotton filter and the obtained filtrate was collected in a
beaker. The solvent was completely evaporated from
the solution by placing the plant extract in a water bath
at 60°C. For the next 72 hours, the container was kept
airtight and the filtrate that accumulated during this
period was concentrated by using a rotary evaporator.
Procurement of chemicals and drugs:
We purchased DPPH (1, 1-diphenyl, 2-
picrylhydrazyl), TCA (trichloroacetic acid) and ferric
chloride from Sigma Chemical Company of USA,
Ascorbic acid from SD Fine Chem. Ltd. of India and
Ammonium Molybdate from Merck, Germany.
Presence of phytochemicals:
In order to know clearly about the various chemical
constituents of the plant extract, standard techniques
were applied at the time of conducting the
phytochemical screening. Required chemicals and
reagents were also used to testify freshly prepared
crude extracts of Averrhoa carambola. For example, Mg
and HCL were used to confirm the presence of
flavonoids. To find out whether tannin was present,
ferric chloride and potassium dichromate solutions
were used. The plant extract’s ability to produce stable
foam indicated that saponin was present. Besides,
Benedict’s reagent was used for detecting reducing
sugars and presence of steroids was confirmed by
Libermann Burchard reagent. Throughout the
experimentation, we carefully observed the change of
color at different stages
4
.
Antibacterial assay:
To assess the antibacterial activity of the dry
sample extract, ten pathogenic bacteria were used as
test organisms and disc diffusion method was followed
while conducting this antimicrobial assay. BCSIR of
Chittagong, Bangladesh gave us the required bacterial
strains. 0.5mg/disc of the sample was used to observe
the antimicrobial activity of the plant extract and to
compare with kanamycin (0.1mg/disc), which was
considered as standard. A 10 ml previously sterilized
nutrient agar media was used to inoculate the
organisms that were used in this test and after fully
mixing them with the media, they were quickly moved
to a sterile Petri dish with the help of sterile loop. A
completely aseptic condition was maintained while
performing this task. As the Petri dish was ready, we
put the prepared sample along with standard solutions
in it and then incubated the plates at 37 °C for about 12
hours. Finally, after letting them go through proper
incubation, we observed and measured the area of
inhibition surrounding the place where sample solution
was applied.
Determination of total phenolic content:
With the help of Folin-Ciocalteu method, the total
phenolic content was measured. Folin-Ciocalteu
oxidized the extract while sodium carbonate
neutralized it
16
. At this stage, blue color was noticed.
After 60 minutes, we measured the absorbance at 760
nm by using Gallic acid (GA) as standard. Total phenolic
content was expressed as mg GA equivalent/gm of
extract.
Determination of total flavonoid content:
To determine the flavonoid content, we applied
Kumaran and Karunakaran method
7
and used quercetin
as standard. 1 mg of plant extract in methanol was
mixed with 1 ml of aluminium trichloride in Ethanol (20
mg/ml) and then a drop of acetic acid was added. The
mixture was diluted up to 25 ml with ethanol and 40
minutes later, the absorbance was measured at 415 nm.
Measurement of blank samples and standard quercetin
solution (0.5 mg/ml) in methanol was done under the
same conditions.
DPPH radical scavenging activity:
With the help of DPPH, the free radical scavenging
capacity of the plant extract was determined
5,1
. A
methanol DPPH solution (0.004% w/v) was mixed with
serial dilutions (0 to 500μg) of Averrhoa carambola
extract and after 10 minutes the absorbance was taken
at 517nm using spectrophotometer. Ascorbic acid was
used as a standard. Finally, after plotting the inhibition
curve, IC50 values were found out from it.
Reducing power:
The reducing power of Averrhoa carambola extract
was found out by using the method of Oyaizu
13
.
Different concentration of Averrhoa carambola extract
in 1 ml of distilled water was mixed with phosphate
buffer (2.5 ml, 0.2 M, pH 6.6) and potassium
ferricyanide [K3Fe(CN)
6
] (2.5 ml, 1%). For the next 20
min, the mixture was incubated at 50°C. After adding
Joysree et al.: Int. J. Bioassays, 2013, 02 (05), 803-807
www.ijbio.com 805
slight amount of (2.5 ml) of Trichloroacetic acid (10%),
the mixture was centrifuged at 3000 rpm for 10
minutes. Next the upper layer of the solution (2.5 ml)
was mixed with distilled water (2.5 ml) and FeCl3 (0.5
ml, 0.1%) and then the absorbance reading was taken at
700 nm. The Ascorbic acid was reference standard and
the blank solution had Phosphate buffer in it.
Brine shrimp lethality bioassay:
At this stage of our study, we used Brine Shrimp
Lethality Bioassay to evaluate cytotoxic potential of
the extract
10,15
. A tank having a temperature of 37°C and
continuous supply of oxygen as well was used to hatch
the Brine Shrimp eggs. After 48 hours, the matured
nauplii were collected. As per requirement, extract was
dissolved in specific volume of pure dimethyl sulfoxide
(DMSO) and stock solutions were prepared. After that,
4 ml of seawater was added to each of the vials, and
later specific volume of sample was transferred from
the stock solution to the vials to obtain final sample
concentrations of 010, 20, 40, 60, 80, 100, 200, 300,
400, 500, 600, 800 and 1000μg/ml. Then same volumes
of DMSO (as in the sample vials) were taken in the
control vials. Using Pasteur pipette 10 living nauplii
were put in each of the vials and they were kept in this
condition for a day. 24 hours later, we observed their
condition, counted the number of nauplii that were
alive and calculated the percentage of lethality of Brine
Shrimp nauplii for each concentration of the extract.
RESULTS AND DISCUSSION
Phytochemical screening:
After carrying out a thorough phytochemical
analysis of the plant extract, we were able to verify
whether flavonoids, alkaloids, tannins, saponin,
steroids, carbohydrate, antranquinone, protein,
glycoside and resin were present or not. For instance,
tests confirmed that flavonoid, glycoside, alkaloid,
carbohydrates, saponin and steroid were present while
tannin, protein and resins were not. The findings have
been shown in table 1.
Table.1: Identified chemical constituents of the
Averrhoa carambola.
Compound name
Plant Parts
Bark
Alkaloid
Present
Flavono
id
Present
Tannin
Absent
Glycoside
Present
Anthraquinone
Absent
Carbohydrate
Molisch’s Test
Present
Benedict’s test
Present
Resin
Absent
Protein
Absent
Saponins
Present
Steroids
Present
Antibacterial assay:
By conducting the antibacterial assay
3,2
, we were
able to gain valuable knowledge about antibacterial
activities of Averrhoa carambola. Ten pathogenic
bacteria were used to examine the extract’s strength
of hindering bacteria from colonizing. In this case, the
area of inhibition for every bacterium was measured
and the results were expressed in millimeter (mm).
Kanamycin used as the standard antibiotic to compare
the results. The results have been shown in figure 1.
Figure.1: Antibacterial activity ethanol extract of
Averrhoa carambola stem bark.
The figure indicates that the ethanol extract of
Averrhoa carambola bark was able to kill some of the
bacteria. It is also obvious that the plant extract was
able to show antibacterial activity against S. typhi, P.
aeruginosa, E. coli and B. megateriu. On the other hand,
the extract could not show good performance in
inhibiting the growth of the rest six bacteria, which are
Vibrio cholera, Staphylococcus aureus, Shigella sonnei,
Bacillus subtilis, Bacillus cereus and Shigella dysenteriae.
In antibacterial screening the plant extract showed
average inhibitory zone 1.5-3.00 (mm) and against S.
typhi. Showed narrow inhibition property.
DPPH radical scavenging activity:
The DPPH radical scavenging activity of Averrhoa
carambola was shown in figure 2. Where increasing the
concentration of the extract, activity was found to
increase slightly and the inhibitory capacity of the plant
extract was comparatively lower than the ascorbic
acid.
Joysree et al.: Int. J. Bioassays, 2013, 02 (05), 803-807
www.ijbio.com 806
Figure.2: DPPH radical scavenging activity of
Chloroform extract of Averrhoa carambola.
In DPPH test, which is based on the ability of DPPH,
a stable free radical, to decolorize in the presence of
antioxidants, is a direct and reliable method for
determining radical scavenging action. Petroleum ether
extract of Averrhoa carambola showed good DPPH
scavenging activity. IC50 value for the plant extracts
was 125.429μg/mL. Ascorbic acid was chosen as the
reference antioxidant for this test and IC50 value for
ascorbic acid was 11.20μg/mL.
Reducing power:
By using the potassium ferricyanide reduction
method the reductive capabilities of the plant extract
was identified in comparison with ascorbic acid which
demonstrated at figure 3. The reducing power of the
extracts was moderately strong while increasing dose
it shows little increment.
Figure.3: Reducing Power of the chloroform extract of
Averrhoa carambola vs Standard.
Total phenol and flavonoid content:
The total phenol content and total flavonoid
content have been expressed in gallic acid and
quercetin equivalents respectively. We got a moderate
result (159.6014 mg/g GAE) for the phenolic contents
and amount of flavonoid was 28.3377 mg/g quercetin
equivalent. Flavonoids and phenolic acids types of poly
phenolic compounds in plants contain multiple
biological effects, including antioxidant activity which is
proven by different studies. Present studies indicate
the presence of polyphenolic compound in bark part of
Averrhoa carambola. It has been found that the
antioxidant effect of plant product is generally due to
radical scavenging activity of phenolic compounds such
as flavonoids, polyphenols, tannins, and phenolic
terpenes
9
.
Bioassay of brine shrimp lethality:
By applying Brine Shrimp Lethality bioassay, we
assessed the plant extract’s cytotoxicity. Its cytotoxic
action on Brine shrimp nauplii was closely monitored.
Here DMSO was used as a solvent. Whether DMSO had
any effect on brine shrimp lethality or not, control was
used in this study. The control group of brine shrimp
nauplii with and without DMSO exhibited no mortality.
The number of nauplii died and percent mortality was
counted for the subjected extract. The result has been
shown in table 2.
Table.2: Brine shrimp Lethality Bioassay for the ethanol
extracts of Averrhoa carambola bark.
Sample
Concentration C
(µg/ml)
Log
C
No. of viable
Shrimps
( out of 10)
%
Mortality
LC50
(ug/ml)
1000
3.00
0
100
19.95
800
2.9
0
100
600
2.77
0
100
500
2.69
0
100
400
2.6
0
100
300
2.47
0
100
200
2.3
0
100
100
2.0
2
80
80
1.90
1
90
60
1.69
1
90
40
1.30
6
40
20
1.3
8
20
10
1
10
0
LC50 (Lethal Concentration) of ethanol extract was
19.95. Therefore, there is no doubt that ethanol
extracts of Averrhoa carambola is much toxic to cell. It
has been found that here the value of Log C is 1.3.
CONCLUSION
After accomplishing this thorough study on a
sample of Averrhoa carambola, it became evident to us
that though this species does not show very strong
antibacterial activity, it possesses good antioxidant
property. In our opinion, difference was noticed in
antibacterial or antioxidant activity of the plant
extracts against different bacteria due to presence of
various active phytochemicals like flavonoid, glycoside,
alkaloid, carbohydrates, saponnines and steroid.
Another important characteristic that we also found
out about the plant extract is that it is capable of
fighting with free radicals as well as damage that is
caused by such radicals. Our study also clearly indicated
that, in terms of cytotoxicity, this species is able to
show good cytotoxic activity.
Joysree et al.: Int. J. Bioassays, 2013, 02 (05), 803-807
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Source of support: Nil
Conflict of interest: None Declared
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    In this study, the antioxidant activity of methanol extracts of five plants from the genus Phyllanthus was evaluated by various antioxidant assays, including total antioxidant, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, nitric oxide scavenging, reducing power and metal ion chelating activities. The various antioxidant activities were compared to standard antioxidants such as butylated hydroxytoluene and ascorbic acid. All the extracts showed strong antioxidant activity in all the tested methods. Among the five plants Phyllanthus debilis has been found to possess the highest activity in all tested models, the activity decreased in the order Phyllanthus debilis>Phyllanthus urinaria>Phyllanthus virgatus>Phyllanthus maderaspatensis>Phyllanthus amarus. In addition to the antioxidant activity of these plants, the total phenolic compounds, flavonoids and flavonols were measured in the extracts. A correlation between the antioxidant activity and total phenolic content was observed.