Comparison of Antibacterial Potential from Leaves and Fruits of Different Herbs and Shrubs of Family Solanaceae.
Bacterial infections are common source of illness in human, livestock and wildlife populations. Current study evaluated the antibacterial potential of leaves and fruits of Physalis minima, Datura inoxia, Withania somnifera and Solanum nigrum by Agar disc-diffusion method. Extracts of these plants were tested against Staphylococcus aureus, Proteus vulgaris, Escherischia coli, Pseudomonas aeruginosa, Klebsiella pneumonia and Citrobacter amalonaticus. The drug oxytetracycline was used as a positive control. The results indicate that fruits of these plants were more effective in controlling microbes in their aqueous extracts while leaves of these plants were effective in their ethanolic extracts as compared to aqueous and n-hexane extract. In comparison to leaves of these plants, the leaves of D. inoxia was rich source against antimicrobial agents, similarly in comparison of fruits, the fruit of P. minima was best in terms of antimicrobial activities.
Overall these plants were most effective against S. aureus and least effective against K. pneumonia in comparison to their antibacterial potentials.
Keywords: Bacterial strains; Agar-disc diffusion method; Oxytetracycline; Medicinal plants
The family Solanaceae comprises of approximately 84 genera and 3000 species, such as potato, petunia, nightshade and tobacco (Zygadlo et al., 1994). It is a cosmopolitan family found throughout tropical and temperate regions of the world. S. nigrum is a low branched annual herb, having triangular stems, alternate leaves and tiny white flowers. Fruit is green in color when immature and turns to purplish black when ripe (Kothekar, 1987). P. minima is also an annual herb which yields high fruit (Patel et al., 2011). It has small, round fruit and comprises of 150-300 seeds usually enclosed in bladder-like calyx (Avila et al., 2006; Peter, 2007). W. somnifera, commonly known as Indian ginseng and winter cherry, is an evergreen, erect, branching shrub having simple leaves and greenish or lurid yellow flowers and fruits are orange red when mature and are globose berries (Anonymous, 2007). D. inoxia, commonly known as devil's turnip and dhatora, also belongs to family Solanaceae (Stevens et al., 2001).
According to World Health Organization (WHO) more than 80% world's population relies on traditional medicine for their primary healthcare needs. Plants generally contain a wide range of ingredients that can be used to treat chronic as well as infectious diseases (Diallo et al., 1999). In recent years, plant derived natural products have received much attention due to diverse pharmacological properties including antioxidant and antibacterial activity (Karthikumar et al., 2007; Aidah et al., 2014). The availability, affordability, reliability and low toxicity of medicinal plants has made them important and acceptable by all religions for implementation in medical health care all over the world (Akroum et al., 2009). Presently in the developing countries, synthetic drugs are not only expensive and inadequate for the treatment of diseases but are also with damaging and side effects (Shariff et al., 2006). Therefore there is need to search for plants of medicinal value.
In the present investigation extracts of W. somnifera, P. minima, S. nigrum and D. inoxia were evaluated with the objectives to determine the antibacterial potential of four species of family Solanaceae.
Materials and Methods
Six bacterial strains were taken from bacteriology section of Bahawal Victoria Hospital and propagated in biochemistry lab at University College of Veterinary and Animal Sciences. These were S. aureus, P. vulgaris, C. amalonaticus, E. coli, K. pneumonia and P. aeruginosa. The cultures of bacteria were maintained on nutrient media and glycerol stocks were made by adding 150 mL glycerol and 850 mL cultures in the Eppendorf tubes.
Nutrient Broth: Nutrient broth was prepared by dissolving 1.3 g of it in 100 mL distilled water. Then sterilize it in the autoclave at 121 C for 20 min.
Nutrient Agar: Nutrient agar was made by dissolving 2.8 g of it in distilled water. Then sterilize it in the autoclave at 121 C for 20 min.
Extraction of Plant Materials
Ethanol Extract: 10 gm of dry plant powder was added to 100 mL of absolute ethanol in a conical flask and the mouth of flask was covered with aluminium foil to avoid evaporation of ethanol. After 42 h, the extract was filtered with common whatman filter paper 1.
n-Hexane Extract: 10 gm of dry plant powder was added to 100 mL of n-hexane in a conical flask and the mouth of flask was covered with aluminium foil to avoid evaporation of ethanol. After 42 h, the extract was filtered with common whatman filter paper 1.
Aqueous Extract by Freeze thaw Method: First of all, phosphate buffer saline (PBS) was made by dissolving sodium chloride (4 gm), potassium chloride (0.1 gm), potassium dihydrogen phosphate (0.1 gm), disodium hydrogen phosphate (1.16 gm) in 200 mL distilled water. 10 gm of plant powder was added in 100 mL PBS (phosphate buffer saline, pH=7.2) in a conical flask and mixed well and placed in freezer. After 24 h, the extract was thaw. The process of freeze and thaw was repeated three times. Finally, it was centrifuged at 4000 rpm and then filtered by using Whatman filter paper 1.
Cutting of Discs from Disc Diffusion Method
The discs were cut with the help of disc cutter from common whatman filter paper. The discs were dipped in respective plant extracts to test the antibacterial activity against cultured microbes. Negative controls were prepared using discs impregnated with 100 mL of the solvents i.e ethanol and n-hexane. Pre-soaked discs from commercially available antibiotic-oxytetracycline (positive control) were used as standards for comparison.
Bacterial strains were taken from glycerol stocks and a loop full of culture was added to the sterilized 3 mL nutrient broth. The cultures were incubated at 37 C for 24 h at constant shaking. Observe the turbidity in the test tubes. Take OD600 of cultures and bring the OD600 at 0.4. Take 1 mL of culture and spin at 12000 rpm aspirate the supernatant and left 100 mL of supernatant on pellete. Dissolve the pellete and apply on agar.
Determination of Zone of Inhibition
The antibacterial activity assay was performed by agar disc diffusion method. The molten nutrient agar was poured into sterilized petri dishes. When the media solidified, plates were inoculated with 100 mL of the respective organism by glass spreader and incubate at 37 C for 1 h. The pre-soaked discs (6 mm in diameter) in different extracts were placed on the agar medium seeded with respective micro-organisms. Pre-soaked discs with Oxytetracycline were used as a positive control and discs impregnated with solvent i.e ethanol and n-hexane were used as negative control. The plates were then incubated at 37 C for 24 h to allow maximum growth of micro-organisms. The antibacterial activity of test samples was determined by measuring the diameter of zone of inhibition expressed in millimeter. The transparently cleared zones show bactericidal activity while the cleared zones containing micro-colonies showed bacteriostatic activity (Bauer et al., 1966; Colle and Marr, 1989).
Values were meanSD (standard deviation) of three replicates. All experiments were performed at least, three times (unless indicated otherwise) and were highly reproducible. Data collected was analyzed statistically by applying one-way ANOVA using statistica software and means were separated by least significant different test at Pless than 0.05 (Steel et al., 1997).
Results revealed that the tested ethanolic, n-hexane and aqueous extracts of respective plants of family Solanaceae i.e., P. minima, S. nigrum, W. somnifera and D. inoxia possess significant antibacterial activity against various bacterial strains.
Antibacterial Assay of Leaves of P. minima
Ethanol control exhibits maximum antibacterial activity (20 mm) against P. aeruginosa while n-hexane control formed largest inhibition zone (19 mm) against S. aureus (Fig. 1). However, K. pneumonia was found to be highly resistant strain. S. aureus was highly sensitive strain when tested against ethanolic extract of P. minima leaves as maximum zone of inhibition (21 mm) was produced against it whereas P. aeruginosa, C. amalonaticus and E. coli were highly resistant strains as no zone was formed against them. In case of n-Hexane extract, highest mean value (4 mm) was observed in K. pneumonia and S. aureus and minimum in P. vulgaris and E. coli while no zone was observed against C. amalonaticus and P. aeruginosa.
Aqueous extract of P. minima leaves formed largest inhibition zone against P. vulgaris (10 mm) and E. coli (4 mm) and formed no inhibition zone against S. aureus and K. pneumonia. Moreover, standard (positive control), oxytetracycline showed significant zone of inhibition against S. aureus, measured 28 mm and P. vulgaris, measured 26 mm respectively (Fig. 9).
Antibacterial Assay of Fruit of P. minima
Ethanolic extract of fruit of P. minima showed maximum zone of inhibition against S. aureus (13 mm) while P. aeruginosa, P. vulgaris, C. amalonaticus and E. coli were revealed as highly resistant strains as no zone of inhibition was observed against them (Fig. 2). N-hexane fruit extract showed maximum inhibition zones against K. pneumonia (3 mm), E. coli (2 mm), P. vulgaris (1 mm) and C. amalonaticus (1 mm) while S. aureus and P. aeruginosa were revealed as highly resistant strains. Aqueous extract of P. minima fruit showed maximum activity against P. vulgaris (21 mm), S. aureus (14 mm) and P. aeruginosa (9 mm) while K. pneumonia was found to be highly resistant strain (Fig. 10). Statistically, it has been proved that the zones produced by the fruit of P. minima against all bacterial strains were significant.
Antibacterial Assay of Leaves of S. nigrum
Ethanol control formed maximum inhibition zones against E. coli (20 mm), S. aureus (8 mm), P. vulgaris (6 mm) and P. aeruginosa (5 mm) and formed least zones of inhibition against K. pneumonia (2 mm) and C. amalonaticus (1 mm). However, n-hexane control showed maximum activity against S. aureus (19 mm), E. coli (12 mm) and P. vulgaris (7 mm). However, it formed no inhibition zone against K. pneumonia and P. aeruginosa (Fig. 3).
Ethanolic and n-hexane extracts of S. nigrum leaves was found to be highly resistant against four bacterial strains namely S. aureus, K. pneumonia and E. coli. However, moderate antibacterial activity was observed against P. aeruginosa and P. vulgaris respectively (Fig. 11). Aqueous extract showed remarkable activity against E. coli as it formed maximum zone of inhibition (i.e. 25 mm). Least antibacterial activity (2 mm) was observed against C. amalonaticus while K. pneumonia and P. vulgaris exhibit no inhibition zone.
Results of Antibacterial Assay of Fruit of S. nigrum
Results revealed that S. aureus, P. aeruginosa and P. vulgaris were highly sensitive strains and K. pneumonia, C. amalonaticus and E. coli were highly resistant strains when tested against various extracts of fruit of S. nigrum. Ethanol control and n-hexane control showed maximum antibacterial activity against E. coli, S. aureus and P. vulgaris (6 mm) and minimum activity against K. pneumonia and C. amalonaticus, respectively (Fig. 4).
Ethanolic extract of S. nigrum fruit formed maximum zone against P. vulgaris (8 mm) and P. aeruginosa (3 mm) and n-Hexane extract showed strong antibacterial activity against P. aeruginosa (9 mm) and C. amalonaticus (7 mm). S. aureus, K. pneumonia and E. coli were highly resistant strains as no zone of inhibition when formed against them when both ethanolic and n-hexane extracts of S. nigrum fruits were tested (Fig. 12). Moreover, aqueous extract possess significant antibacterial activity against S. aureus, P. aeruginosa and P. vulgaris forming inhibition zone of 10 mm while C. amalonaticus and E. coli were proved as resistant bacterial strains. As standard (positive control), oxytetracycline possess antibacterial activity in all bacterial strains. Statistically, inhibition zones produced by all the bacterial strains were found to be highly significant.
Antibacterial Assay of Leaves of W. somnifera
Both negative controls exhibited maximum antibacterial activity (12-14 mm) against S. aureus and P. vulgaris (8.66 mm) and minimum activity against K. pneumonia. Ethanolic and n-hexane leaves extracts of W. somnifera were highly effective against E. coli and S. aureus and least effective against K. pneumonia respectively (Fig. 5). E. coli and S. aureus were revealed as highly sensitive bacterial strains (Fig. 13). Aqueous leaf extract W. somnifera formed maximum zone against P. vulgaris (18 mm) and minimum zone against P. aeruginosa (2 mm). But K. pneumonia was found to be highly resistant when tested against all extracts of leaves of W. somnifera has no inhibition zone was formed against it.
Antibacterial Assay of Fruit of W. somnifera
Ethanol control showed maximum (13 mm) antibacterial activity against P. vulgaris and least (1 mm) activity against K. pneumonia. n-hexane control exhibited maximum (14 mm) activity against S. aureus and minimum against K. pneumonia and E. coli. Moreover, ethanolic fruit extracts of W. somnifera were highly effective against S. aureus (i.e. 13 mm inhibition zone) and least effective against K. pneumonia, P. vulgaris and E. coli as no zone of inhibition was produced against them. However, n-hexane extract showed significant activity against E. coli and minimum against S. aureus, K. pneumonia, P. vulgaris and C. amalonaticus which revealed that they are highly resistant strains (Fig. 6).
Aqueous extract formed largest inhibition zone (21 mm) against P. vulgaris and minimum zone (1 mm) against K. pneumonia. But P. aeruginosa was found to be highly resistant as no inhibition zone was produced against it. As standard (positive control), oxytetracycline showed significant zone of inhibition against all selected bacterial strains. Largest zone (28 mm) of inhibition of positive control was observed against S. aureus while minimum zone (5 mm) of inhibition was observed against C. amalonaticus (Fig. 14).
Antibacterial Assay of Leaves of D. inoxia
Ethanol and n-hexane control showed maximum antibacterial activity against P. aeruginosa and minimum activity against S. aureus and K. pneumonia as no inhibition zone was produced against them (Fig. 7). Ethanolic leaves extract of D. inoxia was found to be highly effective against P. vulgaris (15 mm mean value of inhibition zone) as compared to P. aeruginosa (10 mm) and S. aureus (9 mm). C. amalonaticus was found to be highly resistant as no zone was produced against it. Similarly, n-hexane extract was also effective against P. vulgaris and P. aeruginosa while no inhibition zone was observed in case of K. pneumonia.
The largest inhibition zone formed by aqueous extract of D. inoxia leaves was against P. vulgaris (7 mm) while C. amalonaticus and K. pneumonia were found to be highly resistant species as no inhibition zone was produced against them. Moreover, oxytetracycline showed significant zone of inhibition against all selected bacterial strains (Fig. 15).
Antibacterial Assay of Fruit of D. inoxia
Ethanol control showed maximum antibacterial activity (3 mm) against C. amalonaticus and P. vulgaris and n-hexane control showed maximum activity against P. aeruginosa. However, no inhibition zones were observed against K. pneumonia and S. aureus in both negative controls. So, K. pneumonia and S. aureus were revealed as highly resistant strains (Fig. 8).
Ethanolic fruit extracts of D. inoxia was found to be highly effective against P. vulgaris (25 mm inhibition zone) and least effective against S. aureus, K. pneumonia and P. aeruginosa, as no zone was produced against them. However, n-hexane fruit extract was effective against P. aeruginosa only. Whereas S. aureus, K. pneumonia, P. vulgaris, C. amalonaticus were found to be highly resistant strains (Fig. 16). Aqueous extract also formed maximum inhibition zone against P. vulgaris (13 mm) and minimum zone against S. aureus (2 mm). However, no zones were observed against K. pneumonia and C. amalonaticus.
Antimicrobial compounds from plants represent a potentially novel source of antimicrobial substances and may thus have a clinical value in the treatment of antibiotic resistant antimicrobial strain (Eloff, 1998). Herbal medicines are considered as one of the most important fields of traditional medicine all over the world (Hamil et al., 2003). The plant extracts and their confined constituents have always been an important part of different curative systems (Vanitha and Kathiravan, 2006). Antibacterial activity was classified into highly sensitive, moderately sensitive and resistant depending on the measured values of inhibition zones. Out of different extracts, fruit extracts of P. minima and leaves extracts of D. inoxia showed most remarkable activity. Our results also correlate with the findings of Yogananth et al. (2012) who reported similar results on antibacterial activity of plants.
Aqueous fruit extracts of all plants were revealed as highly effective against all bacterial strains as compared to ethanolic and n-hexane extracts of selected plants. However, in case of leaves, ethanolic extracts were proved to be highly effective against all strains. Our findings are in line with the studies of Almazini et al. (2009); Venkatesan et al. (2009); Sridhar et al. (2011) and Shahid et al. (2013). Furthermore, our results are also in conformity with the findings of Nathiya and Dorcus (2012) who reported that S. aureus is the highly sensitive strain while P. aeruginosa and K. pneumonia is the highly resistant strain when tested by different plant extracts.
The extracts of various parts of tested plants possess a broad spectrum of activity against a panel of bacterial strains responsible for common bacterial infections. The present study forms a primary platform for further phytochemical and pharmacological investigation for the development of new potential antimicrobial compounds. In a nutshell, all these plants have a potential source of useful drugs and can be utilized in the treatment of many diseases/ailments. However, further studies are required to isolate the active principle from the crude extracts for proper drug development.
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