WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Nagamani et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 2.786

Volume 3, Issue 12, 692-706. Research Article ISSN 2278 4357

A STUDY ON ANTIOXIDANT AND ANTIMICROBIAL PROPERTIES

OF BOMBAX CEIBA PENTANDRA SEED EXTRACT
2
Nagamani JE*1, Vidya sri D , Syeda hajira banu2

1
Department of Biochemistry, Garden City College, Bangalore - 560 049, Karnataka, India.
2
Post graduate Department of Biochemistry, JSS College, Ooty Road, Mysore 570025,
Karnataka, India.

Article Received on
ABSTRACT
20 September 2014, Bombax ceiba pentandra belongs to the family Malavaceae, is being
Revised on 12 October 2014, largest exploited for its wide therapeutic applications in various tribal
Accepted on 03 November
2014 communities around the world. Present study investigated the
antioxidant and antimicrobial properties for solvent extracts of ceiba
*Correspondence for pentandra. Aqueous, methanol, acetone, diethyl ether, chloroform and
Author hexane extracts of seeds were used for the study. Antioxidant
Nagamani JE competence of the solvent extracts was assessed by DPPH and TBARS
Department of Biochemistry,
method. Antibacterial property was probed against five bacterial
Garden City College,
Bangalore - 560 049,
species namely Escherichia coli, Bacillus Subtilis, Staphyolococcus
Karnataka, India. aureus, Enterococcus faecalis and Alcaligenes faecalis. Antimycotic
study was performed against the fungal cultures namely Candida
albicans, Aspergillus niger, Aspergillus flaves and Aspergillu fumigatus. Among the six
solvent extracts studied Diethyl ether extract supported for the significant antioxidant
property. Acetone and methanol extract exhibited significant antibacterial and antimycotic
activity. Presence of higher concentration of alkaloids, terpenoids and Polyphenolic
compounds in the solvent extracts could be endorsed for the effective antioxidant and
antimicrobial property.

KEYWORDS: Bombax ceiba pentandra, Antioxidant, DPPH, TBARS, Antibacterial,

Antimycotic.

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1. INTRODUCTION
Free radicals have been implicated in the etiology of several degenerative disorders including
cancer, diabetes, rheumatoid arthritis, atherosclerosis, liver cirrhosis, Alzheimers disease and
[1]
other neurodegenerative disorders . Antioxidants, the compounds that can scavenge free
radicals play a significant role as they prevent damage of cell proteins, lipids, carbohydrates,
nucleic acids as well as biomembranes caused by reactive oxygen species [2].

Plants are known to be the potential sources of natural antioxidants. The herbal medicines
serve the health needs of about 80% of the worlds population, especially for millions of
people in the vast rural areas of developing countries; more than 65% of the global
population uses medicinal plants as a primary health care modality [3].

Antimicrobial agents are undeniably one of the most important therapeutic discoveries of the
20th century. Antimicrobial molecules either kill or prevent the growth of microbes.
Antibiotics are the most important weapons in fighting microbial infections benefitting the
human health. However, with the antibiotic era barely five decades old, mankind is now
[4]
faced with the global problem of emerging resistance in virtually all pathogens . In general
microbes have the genetic ability to transmit and acquire resistance to the therapeutic drugs
[5]
.

Despite the plethora of antibiotics offered, microbial diseases are still on the rise in
developing countries due to relative inefficasy of medicines and the emergence of wide
[6]
spread drug resistant microbes . Thus the search for antimicrobial compounds of plant
origin is extensively persued by many phytochemical. The secondary metabolites of higher
plants may give a new source of antimicrobial agents with possibly novel mechanism of
action.

Considering the vast potentiality of plants as better source for biological activity, the
medicinal plant Bombax ceiba pentandra, commonly known as red silk cotton tree has been
opted to screen for antioxidant and antimicrobial activity.

Ceiba pentandra is a tropical tree of the order malvales and belongs to the family
malavaceae. This tree is being largely exploited for its wide therapeutic applications in
[7]
various tribal communities around the world . Young fruits of the trees are used in the
[8]
treatment of snake bite as well as in inflammatory diseases . Stem bark decoctions are used

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in mouth washes for treating toothache and mouth problems, and taken to treat stomach
problems, diarrhea, hernia, gonorrhea, heart trouble, edema, fever, asthma, and rickets. They
are also applied on swollen fingers, wounds, sores, and leprous macules. Toxicological
studies proved that Bombax ceiba pentandra has very low toxicity profile in all the tested
[9]
animals and it is safe for oral medication . We here in report the proximate analysis for
various components, antioxidant as well as antimicrobial activities of seed extracts of B.
ceiba pentandra.

2. MATERIALS AND METHODS

Plant material
Bombax ceiba pentandra seeds were collected from herbal garden maintained by JSS
College, Mysore, Karnataka, India in the month of January and authenticated from the
Department of Botany, University of Mysore, Karnataka, India.

2.1 Preparation of plant material

Seeds were collected and dried at room temperature. The dried samples were powdered
separately. 100gm each of the sample was extracted separately with different solvents starting
with non polar to polar solvents in the order of hexane, chloroform, diethyl ether, acetone,
methanol and water. The crude residues were obtained by removing the solvents in rotary
evaporator and each of the extracts were resuspended in the respective solvents for further
study.

2.2 Proximate analysis of the solvent extracts

The proximate composition of seed extracts were carried out to determine the content of
ascorbic acid, tannins, saponins, glycosides, proteins, total phenols, flavanoids, terpenoids,
[10]
steroids as well as alkaloids. The protein content was estimated by Bradford method .
Total phenolic content was estimated using FolinCiocalteue reagent [11]. Flavonoids were
estimated following the method of Woiskey and Salatino[12]. Ascorbic acid content was
estimated using DNPH reagent [13]. Qualitative analysis of tannins, alkaloids, Saponins,
terpenoids, steroids and glycosides were performed for the different extracts[14].

2.3 Determination of Antioxidant activity

2.3.1 DPPH radical scavenging assay: 1, 1-diphenyl-2-picryl hydrazyl (DPPH) radical
[15]
scavenging activity was determined as per the method of Habila . 0.1mL of the solvent
extracts were taken in different test tubes and volume in each of the test tube was made up to

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100L using methanol. 3mL of 0.1mM DPPH in methanol was added to each of the test tube
and the mixture was shaken vigorously and allowed to stand for 20 minutes. Absorbance of
the solutions were measured at 517m using spectrophotometer (Shimadzu UV-2550).
Ascorbic acid (0.1mg/mL) was used as control for the assay.

2.3.2 Nitric oxide radical scavenging assay

Nitric oxide radical scavenging assay was conducted as per the method of Marcocci[16].
0.1mL of the solvent extracts were added to the reaction mixture containing 2.5mL of sodium
nitroprusside (10mM), 0.5mL of phosphate buffered saline (pH-7.4). The reaction mixture
was incubated at room temperature for 150 minutes and 1mL of Griess reagent was added to
all the test tubes. The reaction mixture was allowed to stand for 30 minutes at room
temperature and the absorbance of the chromophore formed was read at 546m. BHT
(0.1mg/mL) was used as positive control for the assay.

2.3.3 Hydroxyl radical scavenging assay

Hydroxyl radical scavenging activity of the extracts was estimated as per the method of
[17]
Shang . 0.1mL each of the solvent extract was added to the reaction mixture containing
0.1mL of Deoxyribose (3mM), 0.5mL of FeCl3(0.1mM), 0.5mL of EDTA (0.1mM), 0.5mL

of Ascorbic acid (0.1mM), 0.5mL of H2O2 (1mM) and 0.8mL of Phosphate buffer (20mM pH
o
7.4). The reaction mixture was incubated at 37 C for 1hour. Then 1mL of Thiobarbutiric acid
(TBA) as well as 1mL of 2.8% Trichloro acetic acid (TCA) were added and incubated at
o
100 C for 20 minutes. Thiobarbutiric acid reactive substances formed were measured after
cooling the mixture and measuring the absorbance at 532m.

2.3.4 Superoxide radical scavenging assay

The assay was done following the method of Khanna[18]. 0.1mL each of the plant extract was
added to the reaction mixture containing 50mM phosphate buffer (pH-7.6), 20g/ml
riboflavin, 12mM EDTA and 0.1mM NBT. The reaction was initiated by illuminating the
reaction mixture for 5 minutes and the absorbance was measured at 590m. Inhibition of blue
formazone formation was considered for scavenging activity. Quercitin (0.1mg/mL) was used
as positive control.

2.4 In-vitro antimicrobial assay

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2.4.1 Antibacterial Study: Pure bacterial cultures were obtained from the Department of
studies in Microbiology, University Of Mysore, Mysore, Karnataka.

Antibacterial activity was determined using 2% nutrient agar medium by well diffusion
method. 16hrs Bacterial cultures namely Escherichia coli, Bacillus Subtilis, Staphyolococcus
aureus, Enterococcus faecalis and Alcaligenes faecalis were used for the study. MIC was
determined using different solvent extracts of the seed loading 50l, 100l, 150l and 200l
to the wells incubating at 37c for 24hrs. The zone of inhibition was compared against
Amoxicillin (1ppm), a standard antibiotic.

2.4.2 Antimycotic Study

Antimycotic activity was determined using 2% PDA media by agar well diffusion method.
Fungal cultures namely Candida albicans(pathogenic strain), Aspergillus niger, Aspergillus
flaves and Aspergillu fumigates were used for the study. In-vitro antimycotic activity was
carried out for 18hrs old fungal cultures using spread plate method. MIC was determined
using different solvent extracts of the plant loading 50l, 100l, 150l and 200l to the wells
incubating at 25C for 72hrs. The zone of inhibition was compared against Flucanazole
(1ppm), a standard antibiotic.

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3. RESULTS
Table-1: Proximate analysis of different solvent extracts of Bombax ceiba pentandra seeds
Ascorbic
Extract Proteins Phenols Tannins Alkaloids Flavonoids Saponin Glycosides Steroids Terpenes
acid
Aqueous +++ + + - - - + + - -
Methanol - +++ +++ - ++ + - - - -
Acetone - +++ +++ - +++ + - - - -
Chloroform - + - + - - - - + -
Diethyl
- - - +++ - - - - + ++
ether
Hexane - - - + - - - - + +

- : absent, +: present in low concentration, ++: present in moderate concentration, +++: present in high concentration

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A
A

D
D
E B
E
B

C
C

S.aureus E. faecalis

C
A

E
B
E B
D D

A C
C

E. coli B. subtilis

E B
D

A. faecalis
A: Methanol, B: 50g/ml, C: 100g/ml, D: 200g/ml, E: Amoxicillin

Fig-1: Zone of Inhibition by Methanolic seed extract on Bacteria by well diffusion

method

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A
A

B
E C D
D B

C C

S.aureus E. faecalis

B
C

E
E
C
A D
B

D A

E. coli B. subtilis

E
B
D

A. faecalis
A: Acetone, B: 50g/ml, C: 100g/ml, D: 200g/ml, E: Amoxicillin

Fig-2: Zone of Inhibition by Acetone seed extract on Bacteria by well diffusion method

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A. faecalis
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D
E B

A: Acetone, B: 50g/ml, C: 100g/ml, D: 200g/ml, E: Flucanazole

Fig-3: Zone of Inhibition by Acetone extract on Candida albicans (pathogenic) by well

diffusion method

Table-2: Antioxidant activity of different solvent extracts of Bombax ceiba pentandra

seeds
SAMPLE DPPH radical Hydroxyl radical Superoxide radical Nitric oxide radical
(% scavenging) (% scavenging) (% scavenging) (% scavenging)
Ascorbic acid
93.480.67 95.670.51 82.760.68 89.120.07
(Positive control)
BHT
92.4+0.164 90.040.67 86.04+0.088 82.840.91
(Positive control)
a a a
Aqueous extract 54.811.071 67.360.93 54.340.58 39.211.25
a a a a
Methanol extract 66.501.16 79.580.70 77.830.99 68.260.82
a a a a
Acetone extract 62.420.48 76.280.83 70.400.92 48.550.76
Chloroform a a
extract 52.890.92 47.50.60 56.370.94 34.870.70
Diethyl ether a a a a
extract 71.840.48 69.580.83 75.830.92 58.550.76
a
Hexane extract 32.41.79 36.830.71 52.770.90 24.910.81
a
Values are means SEM; n=5, significant at p<0.0001 as compared to positive control

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Table-3: Antibacterial activity of different solvent extracts of Bombax ceiba pentandra

seeds

Zone of inhibition in mm
Diethyl
Sample Aqueous Acetone Methanol Chloroform Hexane Amoxicillin
Organism ether
g extract extract extract extract extract
extract
0 - - - - - - 0
50 - - - - - - 5
E.coli
100 - 8 - - - - 10
200 - 11 3 - - - 10
0 - - - - - - -
50 - - - - - - -
B.Subtilis
100 - 8 7 - - - -
200 - 10 10 - - - -
0 - - - - - - 0
50 - - - - - - 5
S.aureus
100 - - - - - - 11
200 - 5 7 - - - 11
0 - - - - - - -
50 - - - - - - -
E.faecalis
100 - 7 5.5 - - - -
200 - 10 7.5 - - - -
0 - - - - - - -
50 - - - - - - -
A.faecalis
100 - 5.5 6 - - - -
200 - 11 9 - - - -

Table-4: Antimycotic activity of different solvent extracts of Bombax ceiba pentandra

seeds

Zone of inhibition in mm
Diethyl
Sample Aqueous Acetone Methanol Chloroform Hexane Flucanozole
Organism ether
g extract extract extract extract extract
extract
0 - - - - - - -
50 - - - - - - -
C.albicans
100 - - - - - - 2
200 - 12 - - - - 2
0 - - - - - - -
50 - - - - - - -
A.niger
100 - - - - - - -
200 - - - - - - -
0 - - - - - - -
50 - - - - - - -
A.flavus
100 - - - - - - -
200 - - - - - - -

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0 - - - - - - -
A.fumigat 50 - - - - - - -
us 100 - - - - - - -
200 - - - - - - -

3.1 Proximate analysis of Phytochemicals

Table 1, Shows the results of proximate analysis of B. ceiba pentandra seed extracts.
Considerable amount of protein and ascorbic acid were reported in the aqueous extract.
Higher levels of flavanoids, tannins and polyphenols were present in the methanol and
acetone extracts and significant amounts of alkaloids and terpenoids were observed in diethyl
ether extract in comparison to the other solvent extracts.

3.2 In-vitro Antioxidant assay

Table 2, refers to free radical scavenging activity of the six solvent extracts of Bombax
ceiba pentandra seed extracts.

DPPH, a nitrogen centered free radical was reduced in the presence of different solvent
extracts of fruit and spike. Ascorbic acid in water, used as positive control showed 93.48%
and BHT exhibited 92.4% scavenging activity which was followed by diethyl ether, methanol
and acetone extract with 71.84% and 66.5% and 62.4% activity respectively.

Nitric oxide generated from sodium nitroprusside interacts with oxygen to form nitric ions
whose concentration was estimated using Griess reagent. Methanol extract of the seeds held
potent scavenging activity (68.26%) for Nitric oxide radical in comparison to other extracts.
Ascorbic acid and BHT showed a scavenging activity of 89.12% and 82.84% respectively.

Hydroxyl radicals were generated by the Fenton reaction and the TBA reacting substance was
estimated photometrically. Methanol and acetone extracts exhibited scavenging activities of
79.58% and 76.2% respectively. Positive control Ascorbic acid and BHT illustrated
maximum activities of 95.67% and 90.04% respectively.

The superoxide radical generated from Riboflavin and NBT in the presence of light were
scavenged by the different solvent extracts of spike and fruits. With respect to the positive
control Ascorbic acid (82.7%) and BHT (86.04%), methanol extract and diethyl ether extract
exhibited considerable scavenging activities of 77.83% and 75.40% respectively. Acetone
extract displayed a scavenging activity of 70.4%.
3.3 Antimicrobial activity

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3.3.1 Antibacterial activity

Fig 1 and 2 represents the antibacterial activity of methanol and acetone extracts against
Escherichia coli, Bacillus Subtilis, Staphyolococcus aureus, Enterococcus faecalis and
Alcaligenes faecalis inoculated on nutrient agar plates. Among the six solvent extracts of the
seeds tested against the five bacterial cultures, methanol and acetone extracts exhibited
significant growth inhibition. The zone of inhibition was noteworthy for methanol extract
against Escherichia coli, Bacillus Subtilis, Enterococcus faecalis and Alcaligenes faecalis at a
concentration of 200g/ml. Also the acetone extract revealed maximum inhibition of growth
against Escherichia coli, Bacillus Subtilis, Enterococcus faecalis and Alcaligenes faecalis at a
concentration of 200g/ml.

3.3.2 Antimycotic activity

Among all the solvent extracts used for the study, acetone extract of Bombax ceiba pentandra
seeds shown to be a potent inhibitor for the growth of the fungus, Candida albicans
(pathogenic). A maximum inhibition zone of 12mm was observed at a concentration of
200g/ml which is significant in comparison to the standard antibiotic Flucanazole.

4. DISCUSSION
Plants are the vital sources of many bioactive molecules including phenols, flavanoids,
tannins, steroids, terpenoids, alkaloids, lignins, melanins etc. Polyphenols are ubiquitously
distributed group of plant secondary metabolites which exhibit a wide range of
pluripharmacological effects including antimicrobial, anti-inflammatory, hepatoprotective
and anticarcinogenic actions[19].

Natural products are known to play an important role in both drug discovery and chemical
biology. In fact, many of the current drugs either mimic naturally occurring molecules or
have structures that are fully or in part derived from natural motifs. With the alarming
increase in the incidence of new and re-emerging infectious diseases there is continuous and
imperative need for discovery of new antimicrobial compounds with diverse chemical
structures and novel mechanisms of action[20].

This study is an attempt for the preliminary screening of antioxidant, as well as antimicrobial
activities of Bombax ceiba pentandra seeds extract. Proximate analysis of six solvent extracts
revealed the presence of higher amounts of total phenolics, Flavonoids and tannins in both

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methanol as well as acetone extracts. Considerable quantities of alkaloids and terpenoids

were found in the diethyl ether extract.

Among the six extracts tested for antioxidant activity, methanol, acetone and ethyl acetate
extracts exhibited significant scavenging activity for DPPH, hydroxyl, superoxide and nitric
oxide radicals. The potent free radical scavenging ability of methanol and acetone extracts
could be legitimated for the presence of polyphenolic compounds. From the earlier studies it
is well documented that phenols and Flavonoids serve as strong antioxidants because of high
redox properties[21]. It can be inferred that the presence of substantial quantities of alkaloids
and terpenoids in the diethyl extract attributed for the effective antioxidant property.

Presence of prominent amounts of secondary metabolities like Phenols, Flavonoids, tannins

in the methanolic as well as acetone extracts of seeds and alkaloids and terpenoids in diethyl
ether extract can contribute synergistically to the significant antioxidant potency of this plant
and thus may support the local usage for the treatment of radical related ailments.

Among the six extracts tested for anbacterial activity Methanol and acetone extract of
Bombax ceiba pentandra seeds significantly inhibited the growth of Escherichia coli, Bacillus
Subtilis, Enterococcus faecalis and Alcaligenes faecalis. Among all the solvent extracts used
against the fungal strains, acetone extract of seeds exhibited effective growth inhibition
against Candida albicans. Presence of considerable amounts of polyphenolic compounds in
the methanol and acetone extract seems to contribute for the effective antimicrobial property,
as phenols are known growth inhibitors of microbes.

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