Antioxidative and antibacterial effects of seeds and fruit rind of nutraceutical plants belonging to the Fabaceae family

Abstract

In the present study, the seeds and fruit rind of six plants of the Fabaceae family were selected to evaluate their potential as antioxidant and antibacterial agents. The dried powders were individually extracted with various organic solvents by the cold percolation method, were evaluated for antibacterial activity and methanol extracts used for antioxidant activities. Total phenol, protein and sugar contents were also measured. Antioxidant activities were measured by DPPH free radical scavenging activity, superoxide anion radical scavenging activity and reducing capacity assessment. Antibacterial activity was measured by the agar well diffusion method against four Gram positive and four Gram negative bacteria. The methanol extract of the fruit rind of C. indica showed the maximum DPPH free radical scavenging activity, superoxide anion radical scavenging activity, a high reducing capacity assessment and also had the highest total phenol content. There was a direct correlation between the phenol content and the antioxidant activity. The antibacterial activity of all the extracts was more pronounced on Gram positive bacteria than on Gram negative bacteria. Thus, the fruit rind of C. indica showed the best antioxidant and antibacterial activities.

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Introduction

Reactive oxygen species (ROS) are a class of highly reactive molecules derived from oxygen, and generated by metabolic processes in human beings and by external factors such as pollution, radiation or some dietary habits. ROS are detrimental to cells, as they destroy macromolecules such as DNA, protein and lipids when overproduced during conditions such as excessive exercise, hypoxia and/or in antioxidant system failure. ROS have been implicated in more than 100 diseases from malaria to hemorrhagic shock and AIDS.²

Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG) and citric acid are commonly used as antioxidants in foods to prevent or retard lipid oxidation. But, according to the toxicologists and nutritionists, synthetic antioxidants can show carcinogenic effects in living organisms,⁷ enlarge liver size and increase microsomal enzyme activity.^3,12 Thus, evaluation of the antioxidative activity of naturally-occurring substances has become a focus of interest in recent years.¹⁴ The use of plants and herbs as antioxidants in processed foods is becoming of increasing importance in the food industry as an alternative to synthetic antioxidants.²⁰

The frequency of life threatening infections caused by pathogenic microorganisms has increased worldwide, and is becoming an important cause of morbidity and mortality in immunocompromised patients in developing countries.¹ The steadily increasing bacterial resistance to existing drugs is a serious problem, and therefore there is a dire need to search for new classes of antibacterial substances, especially from natural sources. Unlike synthetic drugs, antimicrobials of plant origin are not associated with side effects and have a great therapeutic potential to heal many infectious diseases.^13,29

Any part of a plant can be therapeutically used to treat infectious diseases. There are numerous reports on the antimicrobial activity of crude plant extracts of different parts of plants, for example Punica granatum leaf,²⁵Nelumbo nucifera rhizome,³⁶Trapa natans L. fruit rind,³⁰Satureja cuneifolia,²⁸Diospyros ebenum Roxb. leaf,⁸Pistachia vera green hull³³ and Ficus carica latex,⁵etc.

The Fabaceae family is the second biggest family among the dicotyledons, and out of 12,000 species, 951 species are grown in India. The aim of this work was to analyze the antioxidant and antibacterial activities of seeds and fruit rind extracts of six nutraceutical plant species belonging to the Fabaceae family.

Materials and methods

Plant material

Fresh pods of six different plants belonging to the Fabaceae family were collected in 2008 from the local market of Rajkot, Gujarat. The ethnobotanical information⁴ of the studied plants is given in Table 1. The pods were thoroughly washed with tap water, the seeds and fruit rind separated, shade dried, crushed in a homogenizer to a fine powder and stored in air tight bottles.

Table 1 The botanical names, vernacular names and therapeutic use of the screened plants

No.	Botanical Name	Vernacular Name	Therapeutic uses
1	Cajanus indica Spr.	Tuver	Bechic, diuretic, astringent, strongly antidysenteric, laxative, vulnerant, restoring lost taste, leprosy, cough, mouth ulcers, tumors, bronchitis, heart diseases, piles, biliousness, diarrhoea, weakness and also used in increasing efficiency of liver
2	Pisum sativum L.	Vatana	Contraceptive, fungistatic and spermicidal
3	Vicia faba L.	Vaal	Hemolytic anemia, Parkinson's disease, malaria and help in controlling hypertension
4	Vigna mungo L.	Udad	Nervine disorders, debility, paralysis, facial paralysis, osteo-arthritis, liver disorders and constipation for relief from such ailments
5	Vigna radiate (L.) Wilczek	Mag	Dyspepsia, pyrexia, diarrhoea, skin diseases, leprosy, inflammations, seminal weakness, burning sensation, colic, flatulence, haemorrhages, cough, haemoptysis, agalactia, emaciation, consumption, facial paralysis, hemiplegia, vitiated condition of vata and pitta, fever and general debility
6	Vigna unguiculata (Linn.) Walp.	Chori	Astringent, appetiser, laxative, anthelmintic, anaphrodisiac, diuretic, galactogogue, liver tonic, anorexia, constipation, helmnathiasis, strangury, agalactia, jaundice and general debility

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Crude extraction

The dry powder of seeds and fruit rind (10 g) was first de-fatted with hexane (100 ml), and then individually extracted with ethyl acetate, methanol and water by the cold percolation method.³⁰ The extraction flasks were kept on a rotary shaker for 24 h at 120 rpm. Thereafter, it was filtered through 8 layers of muslin cloth and centrifuged at 5000 rpm for 15 min. The supernatant was concentrated to dryness under reduced pressure. The extraction procedure was repeated at least three times, and the dry extract was pooled, weighed and stored at 4 °C in air tight bottles.

Determination of protein and sugar content

The protein content of all the samples was measured by Lowry's method.¹⁹ The reducing, non-reducing and total sugar content of all the samples was estimated using Miller's reagent.²⁴

Determination of total phenol content

The total phenol content in ethyl acetate, methanol and water extracts of the different plants was determined by the Folin–Ciocalteu reagent method.²² 0.5 ml of sample (1 mg ml⁻¹) and 0.1 ml (0.5 N) of Folin–Ciocalteu reagent were mixed and the mixture incubated at room temperature for 15 min. Then, 2.5 ml of saturated sodium carbonate was added and the resulting mixture was again incubated at room temperature for 30 min. The absorbance of the mixture was measured at 760 nm using a spectrophotometer. The total phenol values are expressed in terms of gallic acid equivalents per gram of extracted compound. The assay was carried out in triplicate, with mean values being presented.

Antioxidant testing assays

DPPH free radical scavenging activity. The DPPH (2,2-diphenyl-1-picryl-hydrazyl) free radical scavenging activity of methanol extracts of all the plants was measured according to the method described by McCune and Johns,²³ with some modifications. Various concentrations of extracts (100–1000 μg) in methanol were added to a DPPH solution in methanol (0.3 mM). The mixture was incubated for 10 min at room temperature in the dark, after which the absorbance was measured at 517 nm. Ascorbic acid was used as a standard.

Superoxide anion radical scavenging activity. The superoxide anion radical scavenging activity of methanol extracts was measured according to the method of Robak and Gryglewski,³⁴ with some modifications. Superoxide anion radicals were generated in phenazine methosulfate (PMS)-nicotinamide adenine dinucleotide-reduced (NADH) systems by NADH oxidation and assayed by nitroblue tetrazolium (NBT) reduction. In this experiment, the superoxide radical were generated in 3 ml of Tris-HCl buffer (16 mM, pH 8.0) containing 0.5 ml of an NBT (300 μM) solution, 0.5 ml of an NADH (936 μM) solution and 0.5 ml of different concentrations of methanolic extract. The reaction was started by adding 0.5 ml of the PMS solution (120 μM) to the mixtures. The reaction mixture was incubated at 25 °C for 5 min and the absorbance measured at 560 nm against a blank sample. Gallic acid was used as a standard. The assay was carried out in triplicate and the percentage inhibition calculated. IC₅₀ values were calculated by linear regression analysis.

Reducing capacity assessment. The reducing capacity assessment of methanol extracts was determined according to the method of Athukorala et al.,⁶ with some modifications. Methanol extracts were used at different concentrations (100–500 μg). 1 ml extracts were mixed with 2.5 ml phosphate buffer (0.2 M, pH 6.6) and 2.5 ml potassium ferricyanide [K₃Fe(CN)₆] (30 mM). The mixture was incubated at 50 °C for 20 min. Then, 2.5 ml TCA (0.6 M) was added to the mixture, which was then centrifuged for 10 min at 3000 rpm. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl₃ (0.5 ml, 6 mM), and the absorbance was measured at 700 nm by a spectrophotometer. Ascorbic acid was used as a standard. The assay was carried out in triplicate. The increased absorbance of the reaction mixture is indicated.

Antibacterial assay

Microorganisms tested. The bacterial strains used to assess the antibacterial properties of the crude extracts included four Gram positive bacteria (Bacillus cereus ATCC11778, Bacillus megaterium ATCC9885, Bacillus subtilis ATCC6633 and Corynebacterium rubrum ATCC14898) and four Gram negative bacteria (Pseudomonas aeruginosa ATCC27853, Pseudomonas stutzeri ATCC17588, Pseudomonas pictoruim NCIB9152 and Klebsiella aerogenes NCTC418). The investigated bacterial strains were obtained from the National Chemical Laboratory (NCL), Pune, India. The organisms were maintained on nutrient agar (Hi Media, India) slop at 4 °C and sub-cultured before use. The bacteria studied are clinically important examples, causing several infections, and it is essential to overcome them through active therapeutic agents.

Determination of antibacterial assay. The in vitro antibacterial activity of the crude extracts was studied against eight bacterial strains by the agar well diffusion method.^32,15 Mueller Hinton agar no. 2 (Hi Media, India) was used as the bacteriological medium. The extracts were diluted in 100% dimethylsulfoxide (DMSO) at the concentration of 25 mg ml⁻¹. The antibacterial activity was evaluated at a concentration of 2.5 mg well⁻¹. The Mueller Hinton agar was melted and cooled to 48–50 °C, and a standardized inoculum (1.5 × 10⁸ CFU ml⁻¹, 0.5 McFarland) was then added aseptically to the molten agar and poured into sterile Petri dishes to give a solid plate. Wells were prepared in the seeded agar plates. The test compound (100 μl) was then introduced in the well (8.5 mm) and the plates incubated overnight at 37 °C. The antimicrobial spectrum of the extract was determined for the bacterial species in terms of zone sizes around each well. The diameter of the zone of inhibition produced by the agent was compared with those produced by the commercial control antibiotics Carbenicillin (100 μg), Imipenen (10 μg), Ceftazidine (30 μg), Ciprofloxacin (5 μg), Cefaclor (30 μg), Amikacin (30 μg), Tetracycline (30 μg), Piperacillin (100 μg), Methicillin (5 μg), Chloramphenicol (30 μg) and Azithromycin (15 μg). These are commonly used antibiotics to treat infections caused by several Gram positive and Gram negative bacteria, and were therefore selected as control antibiotics. DMSO was used as a negative control. The control zones were subtracted from the test zones, and the resulting zone diameter is shown in Table 4 and Table 5. The experiment was performed three times to minimize error, and mean values ±SEM are presented.

Results and discussion

Extractive yield

The successful determination of biologically-active compounds from plant material is largely dependent on the type of solvent used in the extraction procedure. Several parameters such as extraction temperature, solvent type and solvent concentration can influence the yield during the extraction process.¹⁷ Traditional healers primarily use water, but plant extracts from organic solvents showed better antimicrobial properties. Therefore, a number of organic solvents, such as chloroform, methanol, ethanol, acetone, dichloromethane, etc., have been used to evaluate the antimicrobial property of different plants.^31,35 In the present work, plant extraction was performed in aqueous as well as organic solvents.

The extractive yield differed in different solvents, as shown in Table 2. The extractive yield was greater in water (aqueous) than in organic solvents in both the seeds and fruit rind of all the six plants. The maximum yield was obtained in the aqueous and methanol extracts of the fruit rind of P. sativum. It appears that in the screened plants, polar compounds were greater in concentration than those of non-polar compounds. There are many reports in the literature where the extractive yield varies with different solvents.^10,15

Table 2 The extractive yield, and the total protein and sugar content of the seed and fruit rind of the screened plants

No.	Plant name	Part	% Yield (g g^−1 dry powder of the plant)				Total protein content (mg g^−1)	Sugar content (mg g^−1)
			Hexane	Ethyl acetate	Methanol	Aqueous		Reducing sugar	Non-reducing sugar	Total sugar
1	C. indica	Seed	0.77	0.24	3.23	4.44	62.40 ± 0.02	1.61 ± 0.12	22.62 ± 0.06	24.23 ± 0.09
		Fruit rind	1.4	0.49	4.98	5.56	15.51 ± 0.04	34.5 ± 0.03	64.40 ± 0.14	98.90 ± 0.13
2	P. sativum	Seed	1.48	0.40	5.39	11.03	70.20 ± 0.06	0.92 ± 0.01	17.14 ± 0.16	18.06 ± 0.09
		Fruit rind	0.47	0.46	15.71	18.67	13.18 ± 0.03	3.56 ± 0.01	1.06 ± 0.01	4.62 ± 0.02
3	V. faba	Seed	0.58	0.21	3.68	9.17	86.31 ± 0.08	0.81 ± 0.01	12.57 ± 0.03	13.38 ± 0.06
		Fruit rind	0.77	0.52	5.84	13.61	6.89 ± 0.02	7.95 ± 0.03	6.52 ± 0.02	14.47 ± 0.08
4	V. mungo	Seed	0.60	0.30	2.66	5.51	77.21 ± 0.12	0.99 ± 0.01	14.81 ± 0.07	15.80 ± 0.06
		Fruit rind	0.50	0.43	2.37	8.51	10.95 ± 0.01	1.53 ± 0.02	0.70 ± 0.02	2.23 ± 0.02
5	V. radiata	Seed	0.49	0.20	3.79	6.48	67.50 ± 0.11	2.08 ± 0.02	35.56 ± 0.16	37.64 ± 0.15
		Fruit rind	0.51	0.38	2.22	7.69	16.69 ± 0.06	3.15 ± 0.03	4.95 ± 0.03	8.10 ± 0.05
6	V. unguiculata	Seed	0.72	0.28	5.05	9.38	46.50 ± 0.09	1.41 ± 0.01	11.97 ± 0.08	13.38 ± 0.06
		Fruit rind	0.66	0.46	3.08	8.24	14.25 ± 0.02	3.04 ± 0.02	1.14 ± 0.05	4.18 ± 0.02

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Total protein and sugar content

Some chemical substances in plants like proteins, carbohydrates, vitamins and fiber also contribute to their antioxidant capacity. The protein content of seeds was more than that of fruit rind in all six plants (Table 2). The reducing sugars were less compared to non-reducing sugars in both the seeds and fruit rind of all six of these plants (Table 2). Amongst the twelve parts, the highest protein content was observed in the seed of V. faba and the highest sugar content was in the fruit rind of C. indica.

Total phenol content

Phenolics are antioxidants with redox properties that allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers. Phenolic compounds are commonly found in both edible and non-edible plants,¹⁸ like fruits and vegetables, their products, leguminous plants, herbs and spices.⁹ However, there are very few reports on fruit rind, especially plants belonging to the Fabaceae family. In all six plants, the fruit rind had a considerably greater total phenol content than the seeds (Table 3). Among the four solvents, the methanol extracts had considerably more total phenol content following aqueous extraction. Among the twelve parts, the methanol extract of the fruit rind of C. indica had the maximum total phenol content.

Table 3 Total phenol content, DPPH free radical scavenging activity (DPPH) and superoxide anion radical scavenging activity (SO) of methanolic extracts of the seeds and fruit rinds of the screened plants

Plant name	Part	Total phenol content (mg g^−1)			IC50 value (μg ml^−1)
		Ethyl acetate	Methanol	Aqueous	DPPH	SO
C. indica	Seed	4.18 ± 0.21	6.22 ± 0.09	15.62 ± 0.08	>1000	>1000
	Fruit rind	13.62 ± 0.15	158.98 ± 2.14	43.13 ± 1.43	39	420
P. sativum	Seed	6.84 ± 0.08	8.45 ± 0.12	11.12 ± 0.08	>1000	>1000
	Fruit rind	7.76 ± 0.05	11.62 ± 0.14	11.26 ± 0.07	>1000	>1000
V. faba	Seed	4.57 ± 0.03	4.35 ± 0.06	10.9 ± 0.05	>1000	>1000
	Fruit rind	11.35 ± 0.08	14.36 ± 0.07	42.96 ± 0.14	>1000	>1000
V. mungo	Seed	9.68 ± 0.03	22.7 ± 0.09	6.08 ± 0.08	>1000	>1000
	Fruit rind	16.5 ± 0.15	43.95 ± 0.23	51.99 ± 0.14	560	>1000
V. radiata	Seed	7.95 ± 0.05	16.29 ± 0.09	20.59 ± 0.12	>1000	>1000
	Fruit rind	13.78 ± 0.12	55.77 ± 0.24	74.86 ± 0.32	220	>1000
V. unguiculata	Seed	7.56 ± 0.08	8.33 ± 0.16	13.78 ± 0.11	>1000	>1000
	Fruit rind	8.67 ± 0.11	32.88 ± 0.27	47.16 ± 0.17	480	>1000

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Antioxidant testing assays

Antioxidants are known to exhibit their biochemical effects through numerous mechanisms, including the prevention of chain initiation, reductive capacity and radical scavenging mechanisms. Several methods have been used to measure the antioxidant activity of biological materials. However, the most commonly used ones are those involving chromogen compounds of radical nature, which stimulate the reductive oxygen species. These methods are popular due to their ease, speed and sensitivity.

DPPH free radical scavenging activity

The IC₅₀ values of the DPPH free radical scavenging activity of methanol extracts of seeds and fruit rinds of the six plants is shown in Table 3. The methanolic extracts of the fruit rinds showed varied levels of DPPH free radical scavenging activity, while the methanolic extracts of the seeds showed IC₅₀ values greater than 1000 μg ml⁻¹ (Table 3). The fruit rinds of P. sativum and V. faba showed the higher IC₅₀ values (>1000 μg ml⁻¹), while in the other four extracts, the value of the IC₅₀ ranged between 39–560 μg ml⁻¹. The lowest IC₅₀ value was shown by C. indica fruit rind.

Superoxide anion radical scavenging activity

The IC₅₀ values of superoxide anion radical scavenging activity are shown in Table 3. Out of all the methanolic extracts of the seeds and fruit rinds, only the fruit rind of C. indica showed a superoxide anion radical scavenging activity (IC₅₀ = 420 μg ml⁻¹), while other methanolic extracts of seeds and fruit rinds showed IC₅₀ values of more than 1000 μg ml⁻¹ (Table 3). Here, gallic acid was used as a standard (IC₅₀ = 185 μg ml⁻¹).

Reducing capacity assessment

The reducing capacity assessments of all the methanolic extracts of the seeds and fruit rinds are shown in Fig. 1. The fruit rind of C. indica showed a high reducing capacity assessment compared to all the other extracts, while seeds of V. unguiculata showed a moderate reducing capacity assessment. In the seeds, the reducing capacity assessment of the plants was in the order: C. indica > V. radiata > V. faba > V. mungo > P. sativum > V. unguiculata. In the fruit rinds, the reducing capacity assessment of the plants was in the order: C. indica > V. radiata > V. mungo > V. unguiculata > P. sativum > V. faba.

Fig. 1 Reducing capacity assessments of methanol extracts of the seed and fruit rind of the screened plants.

Phenolic compounds are considered to be the most important antioxidative plant components. They also have an ability to scavenge free radicals and active oxygen species, such as singlet oxygen, free radicals and hydroxyl radicals.¹¹ A highly positive correlation between total phenols and the antioxidant activities of many plants has been reported.^21,8,15 Natural antioxidants strengthen the endogenous defence mechanism and restore the optimal balance by neutralizing reactive oxygen species. Therefore, screening plants for total phenol content may give a clue as to their antioxidant properties. Thus, the search for crude drugs of plant origin with antioxidant properties has become a central focus of study in recent years. In this study, the methanolic extracts of the fruit rind of C. indica showed a high antioxidant activity as compared to the others that might be due to its chemical composition, which is specifically rich in phenolic compounds.

Antibacterial activity

The antibacterial activity of the various solvent extracts exhibited different levels of antibacterial activity against the selected bacterial strains (Table 4 and Table 5). All the extracts showed activity against B. cereus. The hexane and ethyl acetate extracts of the seeds and fruit rinds of all six plants, and the methanolic and aqueous extracts of the fruit rinds of all six plants, except P. sativum, showed activity against K. aerogenes. Ethyl acetate and methanol extracts of the fruit rind of C. indica showed activity against B. subtilis, B. megaterium, C. rubrum and P. pictoruim. Hexane and ethyl acetate extracts of the seeds of P. sativum showed activity against B. subtilis. The ethyl acetate extract of the fruit rind of V. faba showed activity against B. subtilis, B. megaterium, C. rubrum and P. pictoruim. The antibacterial activity of the fruit rind of C. indica against B. cereus was comparable with standard antibiotics like Amikacin, Chloroamphenical and Azithromycin (Table 6).

Table 4 The antibacterial activities of different solvent extracts of seeds of the screened plants^a

Plant name	Extracts	Zone of inhibition (mm)
		Gram positive bacteria				Gram negative bacteria
		BC	BS	BM	CR	PA	PS	PP	KA
a HE = hexane; EA = ethyl acetate; ME = methanol; AQ = aqueous; BC = Bacillus cereus; BS = Bacillus subtilis; BM = Bacillus megaterium; CR = Corynebacterium rubrum; PA = Pseudomonas aeruginosa; PS = Pseudomonas stutzeri; PP = Pseudomonas pictoruim; KA = Klebsiella aerogenes; ± = SEM value; — = no activity.
C. indica	HE	10.0 ± 0.16	—	—	—	—	—	—	9.3 ± 0.33
	EA	9.5 ± 0.16	—	—	—	—	—	—	9.7 ± 0.33
	ME	8.7 ± 0.05	—	—	—	—	—	—	—
	AQ	9.3 ± 0.05	—	—	—	—	—	—	—
P. sativum	HE	10.5 ± 0.00	—	—	—	—	—	—	9.0 ± 0.00
	EA	12.8 ± 0.33	—	—	—	—	—	—	9.0 ± 0.00
	ME	9.7 ± 0.17	—	—	—	—	—	—	—
	AQ	11.7 ± 0.17	—	—	—	—	—	—	—
V. faba	HE	10.3 ± 0.47	—	—	—	—	—	—	9.3 ± 0.33
	EA	10.3 ± 0.15	—	—	—	—	—	—	9.7 ± 0.33
	ME	9.5 ± 0.17	—	—	—	—	—	—	—
	AQ	9.5 ± 0.17	—	—	—	—	—	—	—
V. mungo	HE	8.7 ± 0.17	—	—	—	—	—	—	10.0 ± 0.00
	EA	10.3 ± 0.60	—	—	—	—	—	—	10.0 ± 0.00
	ME	8.0 ± 0.29	—	—	—	—	—	—	—
	AQ	8.1 ± 0.23	—	—	—	—	—	—	—
V. radiata	HE	8.5 ± 0.00	—	—	—	—	—	—	9.0 ± 0.00
	EA	9.3 ± 0.17	—	—	—	—	—	—	9.0 ± 0.00
	ME	8.0 ± 0.29	—	—	—	—	—	—	—
	AQ	9.8 ± 0.17	—	—	—	—	—	—	—
V. unguiculata	HE	9.5 ± 0.00	—	—	—	—	—	—	9.0 ± 0.00
	EA	11.0 ± 0.29	—	—	—	—	—	—	9.0 ± 0.00
	ME	9.5 ± 0.00	—	—	—	—	—	—	—
	AQ	9.7 ± 0.17	—	—	—	—	—	—	—

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Table 5 The antibacterial activities of different solvent extracts of fruit rinds of the screened plants^a

Plant name	Solvents	Zone of inhibition (mm)
		Gram positive bacteria				Gram negative bacteria
		BC	BS	BM	CR	PA	PS	PP	KA
a HE = hexane; EA = ethyl acetate; ME = methanol; AQ = aqueous; BC = Bacillus cereus; BS = Bacillus subtilis; BM = Bacillus megaterium; CR = Corynebacterium rubrum; PA = Pseudomonas aeruginosa; PS = Pseudomonas stutzeri; PP = Pseudomonas pictoruim; KA = Klebsiella aerogenes; ± = SEM value; — = no activity.
C. indica	HE	14.3 ± 0.17	—	—	—	—	—	—	9.0 ± 0.00
	EA	14.7 ± 0.44	9.0 ± 0.00	12.0 ± 0.0	9.3 ± 0.33	—	—	11.7 ± 0.33	9.7 ± 0.33
	ME	13.3 ± 0.17	9.0 ± 0.00	10.3 ± 0.33	10.7 ± 0.33	—	—	12.0 ± 0.00	9.3 ± 0.33
	AQ	8.5 ± 0.00	—	—	—	—	—	—	10.0 ± 0.00
P. sativum	HE	13.0 ± 0.29	11.0 ± 0.00	—	—	—	—	—	9.7 ± 0.33
	EA	13.0 ± 0.29	11.0 ± 0.00	—	—	—	—	—	9.0 ± 0.00
	ME	9.0 ± 0.29	—	—	—	—	—	—	—
	AQ	9.5 ± 0.00	—	—	—	—	—	—	—
V. faba	HE	9.5 ± 0.29	—	—	—	—	—	—	9.0 ± 0.00
	EA	12.7 ± 0.17	10.0 ± 0.00	12.0 ± 0.00	11.7 ± 0.33	—	—	10.7 ± 0.33	9.7 ± 0.33
	ME	11.7 ± 0.60	—	—	—	—	—	—	9.3 ± 0.33
	AQ	10.0 ± 0.29	—	—	—	—	—	—	10.0 ± 0.00
V. mungo	HE	9.3 ± 0.44	—	—	—	—	—	—	9.0 ± 0.00
	EA	10.3 ± 0.44	—	—	—	—	—	—	9.0 ± 0.00
	ME	11.0 ± 0.29	—	—	—	—	—	—	9.0 ± 0.00
	AQ	11.7 ± 0.17	—	—	—	—	—	—	9.0 ± 0.00
V. radiata	HE	10.0 ± 0.29	—	—	—	—	—	—	9.0 ± 0.00
	EA	11.7 ± 0.44	—	—	—	—	—	—	9.3 ± 0.33
	ME	10.3 ± 0.60	—	—	—	—	—	—	9.0 ± 0.00
	AQ	10.0 ± 0.29	—	—	—	—	—	—	9.3 ± 0.33
V. unguiculata	HE	10.7 ± 0.17	—	—	—	—	—	—	9.0 ± 0.00
	EA	10.7 ± 0.17	—	—	—	—	—	—	9.0 ± 0.00
	ME	9.7 ± 0.44	—	—	—	—	—	—	9.0 ± 0.00
	AQ	9.5 ± 0.00	—	—	—	—	—	—	9.0 ± 0.00

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Table 6 The antibacterial activities of standard antibiotics against selected Gram positive and Gram negative bacteria^a

Antibiotic	Zone of inhibition (mm)
	Gram positive				Gram negative
	BC	BS	BM	CR	PA	PS	PP	KA
a — = No activity; BC = Bacillus cereus; BS = Bacillus subtilis; BM = Bacillus megaterium; CR = Corynebacterium rubrum; PA = Pseudomonas aeruginosa; PS = Pseudomonas stutzeri; PP = Pseudomonas pictoruim; KA = Klebsiella aerogenes.
Carbennicillin	10	20	—	22	12	28	20	—
Imipenem	36	37	41	24	27	38	27	30
Ceftazidimine	18	25	—	19	17	30	12	—
Ciprofloxacin	19	30	27	16	28	30	23	10
Cefaclor	20	35	14	27	—	—	23	16
Amikacin	11	15	23	16	22	28	20	—
Tetracycline	17	21	19	20	15	25	25	10
Piperacillin	16	17	—	19	—	22	15	11
Methicillin	—	16	14	15	—	—	14	—
Chloramphenicol	10	16	14	15	34	22	25	13
Azithromycin	11	14	12	14	12	32	18	—

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The antibacterial activities of C. indica and V. faba were more pronounced on Gram positive bacteria than Gram negative bacteria. The reason for this difference in sensitivity might be ascribed to differences in the morphological constitution of these microorganisms; Gram negative bacteria have an outer phospholipidic membrane with a lipopolysaccharide component that makes the cell wall impermeable to plant extracts. Gram positive bacteria, on the other hand, are more susceptible, having only an outer peptidoglycan layer that is not as effective a permeability barrier. There are many reports in the literature that Gram positive bacteria are more susceptible to herbal extracts^26,16 and Gram negative bacteria are resistant.^27,15 The fruit rind of C. indica showed the best antibacterial activity, and therefore it can be further explored as a novel source of natural antibacterial drugs.

The present investigation suggests that fruit rind, usually a waste product that is discarded into the environment, possesses potential antibacterial and antioxidant properties. However, further isolation and preparation of bioactive compounds of the fruit rind of C. indica are required to establish in vivo antioxidant activity using different animal models. These are novel, natural and economic sources of antioxidants that can be used in the prevention of diseases caused by free radicals. However, they need to be explored as a viable, alternative source to commercially available synthetic and antibiotic drugs. This is the best use of such waste material.

Conclusion

From the above study, it can be concluded that the total phenol content, DPPH free radical scavenging activity, superoxide anion radical scavenging activity and also the reducing capacity assessment are at their maximum in the methanolic extract of the fruit rind of C. indica. The best antibacterial activity was shown by the fruit rind of C. indica. It appears that the fruit rind of C. indica could be a potential source of antibacterial and antioxidant agents. However, further studies are needed to isolate and characterize the active compounds that are responsible for the antioxidant and antibacterial activities.

Acknowledgements

The authors thank Prof. S. P. Singh, Head, Department of Biosciences, Saurashtra University, for providing excellent research facilities. M. K. is thankful to the University Grants Commission, New Delhi, India for providing financial support.

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