a study on antioxidant and antimicrobial properties of bombax ceiba

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692

Nagamani et al. World Journal of Pharmacy and Pharmaceutical Sciences

A STUDY ON ANTIOXIDANT AND ANTIMICROBIAL PROPERTIES

OF BOMBAX CEIBA PENTANDRA SEED EXTRACT

Nagamani JE*1, Vidya sri D

2

, 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.

ABSTRACT

Bombax ceiba pentandra belongs to the family Malavaceae, is being

largest exploited for its wide therapeutic applications in various tribal

communities around the world. Present study investigated the

antioxidant and antimicrobial properties for solvent extracts of ceiba

pentandra. Aqueous, methanol, acetone, diethyl ether, chloroform and

hexane extracts of seeds were used for the study. Antioxidant

competence of the solvent extracts was assessed by DPPH and TBARS

method. Antibacterial property was probed against five bacterial

species namely Escherichia coli, Bacillus Subtilis, Staphyolococcus

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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VVoolluummee 33,, IIssssuuee 1122,, 669922--770066.. RReesseeaarrcchh AArrttiiccllee IISSSSNN 2278 – 4357

Article Received on

20 September 2014,

Revised on 12 October 2014, Accepted on 03 November

2014

*Correspondence for

Author

Nagamani JE

Department of Biochemistry,

Garden City College,

Bangalore - 560 049,

Karnataka, India.

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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, Alzheimer’s disease and

other neurodegenerative disorders [1]

. 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 world’s 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

faced with the global problem of emerging resistance in virtually all pathogens [4]

. 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

spread drug resistant microbes [6]

. 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

various tribal communities around the world [7]

. Young fruits of the trees are used in the

treatment of snake bite as well as in inflammatory diseases [8]

. 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

animals and it is safe for oral medication [9]

. 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,

steroids as well as alkaloids. The protein content was estimated by Bradford method [10]

.

Total phenolic content was estimated using Folin–Ciocalteue 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

scavenging activity was determined as per the method of Habila[15]

. 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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100μL 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 517ηm 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 546ηm. 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

Shang [17]

. 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 H2O

2 (1mM) and 0.8mL of Phosphate buffer (20mM pH

7.4). The reaction mixture was incubated at 37o

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

100o

C for 20 minutes. Thiobarbutiric acid reactive substances formed were measured after

cooling the mixture and measuring the absorbance at 532ηm.

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), 20μg/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 590ηm. 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 50μl, 100μl, 150μl and 200μl

to the wells incubating at 37ºc 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 50μl, 100μl, 150μl and 200μl to the wells

incubating at 25ºC 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

Extract Proteins Phenols Tannins Alkaloids Flavonoids Saponin Glycosides Ascorbic

acid Steroids Terpenes

Aqueous +++ + + - - - + + - -

Methanol - +++ +++ - ++ + - - - -

Acetone - +++ +++ - +++ + - - - -

Chloroform - + - + - - - - + -

Diethyl

ether - - - +++ - - - - + ++

Hexane - - - + - - - - + +

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



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

method

S.aureus E. faecalis

E. coli B. subtilis

A. faecalis

A: Methanol, B: 50μg/ml, C: 100μg/ml, D: 200μg/ml, E: Amoxicillin

A

B

C

D

E

E

D

C

B

A

A

C

B

C

D

E

C

A

B D

E

E D

C

B

A



699


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

S.aureus E. faecalis

E. coli B. subtilis

A. faecalis

A: Acetone, B: 50μg/ml, C: 100μg/ml, D: 200μg/ml, E: Amoxicillin

A. faecalis

C

A

A

B

C D

C

C

D

A

B

E

D B

C

E

C

B

A

E D B

A

E

D



700


A: Acetone, B: 50μg/ml, C: 100μg/ml, D: 200μg/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

(% scavenging)

Hydroxyl radical

(% scavenging)

Superoxide radical

(% scavenging)

Nitric oxide radical

(% scavenging)

Ascorbic acid

(Positive control) 93.48±0.67 95.67±0.51 82.76±0.68 89.12±0.07

BHT

(Positive control) 92.4+0.164 90.04±0.67 86.04+0.088 82.84±0.91

Aqueous extract 54.81±1.071a

67.36±0.93a

54.34±0.58a

39.21±1.25

Methanol extract 66.50±1.16a

79.58±0.70a

77.83±0.99a

68.26±0.82a

Acetone extract 62.42±0.48a

76.28±0.83a

70.40±0.92a

48.55±0.76a

Chloroform

extract 52.89±0.92a

47.5±0.60a

56.37±0.94 34.87±0.70

Diethyl ether

extract 71.84±0.48a

69.58±0.83a

75.83±0.92a

58.55±0.76a

Hexane extract 32.4±1.79 36.83±0.71 52.77±0.90a

24.91±0.81

Values are means ± SEM; n=5, significant at a

p<0.0001 as compared to positive control

E

D

C

B

A



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

seeds

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

seeds

Zone of inhibition in mm

Organism Sample

µg

Aqueous

extract

Acetone

extract

Methanol

extract

Diethyl

ether

extract

Chloroform

extract

Hexane

extract

Amoxicillin

E.coli

0 - - - - - - 0

50 - - - - - - 5

100 - 8 - - - - 10

200 - 11 3 - - - 10

B.Subtilis

0 - - - - - - -

50 - - - - - - -

100 - 8 7 - - - -

200 - 10 10 - - - -

S.aureus

0 - - - - - - 0

50 - - - - - - 5

100 - - - - - - 11

200 - 5 7 - - - 11

E.faecalis

0 - - - - - - -

50 - - - - - - -

100 - 7 5.5 - - - -

200 - 10 7.5 - - - -

A.faecalis

0 - - - - - - -

50 - - - - - - -

100 - 5.5 6 - - - -

200 - 11 9 - - - -

Zone of inhibition in mm

Organism Sample

µg

Aqueous

extract

Acetone

extract

Methanol

extract

Diethyl

ether

extract

Chloroform

extract

Hexane

extract

Flucanozole

C.albicans

0 - - - - - - -

50 - - - - - - -

100 - - - - - - 2

200 - 12 - - - - 2

A.niger

0 - - - - - - -

50 - - - - - - -

100 - - - - - - -

200 - - - - - - -

A.flavus

0 - - - - - - -

50 - - - - - - -

100 - - - - - - -

200 - - - - - - -



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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

A.fumigat

us

0 - - - - - - -

50 - - - - - - -

100 - - - - - - -

200 - - - - - - -



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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 200µg/ml. Also the acetone extract revealed maximum inhibition of growth

against Escherichia coli, Bacillus Subtilis, Enterococcus faecalis and Alcaligenes faecalis at a

concentration of 200µg/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

200μg/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



704


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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a study on antioxidant and antimicrobial properties of bombax ceiba