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WWOORRLLDD JJOOUURRNNAALL OOFF PPHHAARRMMAACCYY AANNDD PPHHAARRMMAACCEEUUTTIICCAALL SSCCIIEENNCCEESS Nagamani et al.
WWOORRLLDD JJOOUURRNNAALL OOFF PPHHAARRMMAACCYY AANNDD PPHHAARRMMAACCEEUUTTIICCAALL SSCCIIEENNCCEESS
Nagamani et al.
World Journal of Pharmacy and Pharmaceutical Sciences
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A STUDY ON ANTIOXIDANT AND ANTIMICROBIAL PROPERTIES OF BOMBAX CEIBA PENTANDRA SEED EXTRACT
A STUDY ON ANTIOXIDANT AND ANTIMICROBIAL PROPERTIES
OF BOMBAX CEIBA PENTANDRA SEED EXTRACT

Nagamani JE* 1 , Vidya sri D 2 , Syeda hajira banu 2

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 20 September 2014, Revised on 12 October 2014, Accepted on 03 November
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,
*Correspondence for
Author
Nagamani JE
Department of Biochemistry,
Garden City College,
Bangalore - 560 049,
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. www.wjpps.com Vol 3,
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, 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

20 th 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 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

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 FeCl 3 (0.1mM), 0.5mL of EDTA (0.1mM), 0.5mL

of Ascorbic acid (0.1mM), 0.5mL of H 2 O 2 (1mM) and 0.8mL of Phosphate buffer (20mM pH

7.4). The reaction mixture was incubated at 37 o 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

100 o 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

               

Ascorbic

   

Extract

Proteins

Phenols

Tannins

Alkaloids

Flavonoids

Saponin

Glycosides

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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A D E B C
A
D
E
B
C

S.aureus

C B E D A C
C
B E
D
A
C

E. coli

A D B E C
A
D
B
E
C

E. faecalis

A E B D C
A
E
B
D
C

B. subtilis

A B D E C
A
B
D E
C

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

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

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A E D B C
A
E
D
B
C

S.aureus

B E C A D
B
E
C
A
D

E. coli

A B C D C
A
B
C D
C

E. faecalis

C E D B A
C
E
D
B
A

B. subtilis

A E B D C
A
E
B
D
C

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

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

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A D E B C
A
D
E
B
C

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

Hydroxyl radical (% scavenging)

Superoxide radical (% scavenging)

Nitric oxide radical (% scavenging)

(% 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±1.071 a

67.36±0.93 a

54.34±0.58 a

39.21±1.25

Methanol extract

66.50±1.16 a

79.58±0.70 a

77.83±0.99 a

68.26±0.82 a

Acetone extract

62.42±0.48 a

76.28±0.83 a

70.40±0.92 a

48.55±0.76 a

Chloroform

52.89±0.92 a

47.5±0.60 a

   

extract

56.37±0.94

34.87±0.70

Diethyl ether

71.84±0.48 a

69.58±0.83 a

75.83±0.92 a

58.55±0.76 a

extract

Hexane extract

32.4±1.79

36.83±0.71

52.77±0.90 a

24.91±0.81

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

 

Organism

Sample

Aqueous

Acetone

Methanol

Diethyl

ether

Chloroform

Hexane

Amoxicillin

µg

extract

extract

extract

extract

extract

extract

 

0

 

- -

-

-

-

 

- 0

E.coli

50

 

- -

-

-

-

 

- 5

100

 

- 8

-

-

-

 

- 10

 

200

 

- 11

3

-

-

 

- 10

 

0

 

- -

-

-

-

 

- -

B.Subtilis

50

 

- -

-

-

-

 

- -

100

 

- 8

7

-

-

 

- -

 

200

 

- 10

10

-

-

 

- -

 

0

 

- -

-

-

-

 

- 0

S.aureus

50

 

- -

-

-

-

 

- 5

100

 

- -

-

-

-

 

- 11

 

200

 

- 5

7

-

-

 

- 11

 

0

 

- -

-

-

-

 

- -

E.faecalis

50

 

- -

-

-

-

 

- -

100

 

- 7

5.5

-

-

 

- -

 

200

 

- 10

7.5

-

-

 

- -

 

0

 

- -

-

-

-

 

- -

A.faecalis

50

 

- -

-

-

-

 

- -

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

 

Organism

Sample

Aqueous

Acetone

Methanol

Diethyl

ether

Chloroform

Hexane

Flucanozole

µg

extract

extract

extract

extract

extract

extract

 

0

- -

 

-

 

- -

-

-

C.albicans

50

- -

 

-

 

- -

-

-

100

- -

 

-

 

- -

-

2

 

200

 

- 12

-

 

- -

-

2

 

0

- -

 

-

 

- -

-

-

A.niger

50

- -

 

-

 

- -

-

-

100

- -

 

-

 

- -

-

-

 

200

- -

 

-

 

- -

-

-

 

0

- -

 

-

 

- -

-

-

A.flavus

50

- -

 

-

 

- -

-

-

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 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

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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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