Antimicrobial Activity of Solanum aculeastrum.

Abstract

Solanum aculeastrum. Dunal (Solanaceae) is used in traditional medicine to treat various human and animal diseases, specifically stomach disorders and various cancers, in the Eastern Cape, South Africa. The fruit and leaf extracts of this plant were investigated for in vitro. antimicrobial activity against 10 selected bacterial and 5 fungal strains. The methanolic extracts of both the fruits and the leaves showed appreciable activity against Gram-positive and Gram-negative bacteria ranging from 4.0 to 10.0 mg/ml. Whereas the methanol extracts were the most active material, the water extracts showed the least activity against the bacteria. The methanol extracts were particularly inhibitory to the growth of the fungi with percentage inhibition ranging from 60.26% to 100% and 56.0% to 100% on Aspergillus flavus. and Pencillium notatum., respectively. The acetone extracts were active against Aspergillus flavus. (100%) and Pencillium notatum. (64.81%), and the water extract of the fruit significantly inhibited the growth of P. notatum. (69.89%). The most resistant organisms were Aspergillus niger, Candida albicans., and Fusarium oxysporum..

Keywords:

• Antibacterial
• antifungal
• antimicrobial
• medicinal plants
• Solanum aculeastrum

Introduction

There has been an increasing incidence of microbial infections in recent years, largely due to the increase in AIDS-related opportunistic fungal pathogens and the emergence of resistance microbial species (Silva et al., 2001; Afolayan et al., 2002). Although, fungal-related diseases may not be as common as other bacterial infections, when they are present, they could be difficult to eradicate, especially in immunosuppressive situations (Bryce, 1992). Mycotic infections have been observed to be the primary cause of mortality in patients with severely impaired immune mechanisms (Kelberg, 1997). Hitherto, natural products from microorganisms have been the primary source of antibiotics, but with the increasing acceptance of herbal medicine as an alternative form of health care, the screening of medicinal plants for active compounds has become very important because these may serve as promising sources of novel antibiotic prototypes (Meurer-Grimes et al., 1996; Rabe & Van Staden, 1997).

Solanum aculeastrum. Dunal (Solanaceae), known as “goat bitter-apple,” is a native of Africa and widely distributed in South Africa, mainly in Limpopo, Mpumalanga, KwaZulu-Natal, Western and Eastern Cape Provinces, and also in Swaziland. It is a thorny perennial plant that grows up to 3 m in height with white flowers and lemon-shaped berries that become yellow-green when ripe. The bitter fruits of S. aculeastrum. are used medicinally in various ways for humans as well as domestic animals (Hutchings et al., 1996). The decoction of the fruits and leaves are taken orally for the treatment of cancer, indigestion, and stomach disorders. The fresh and boiled berries are used as a cure for jigger wounds, gonorrhea, and the treatment of acne (Kokwaro, 1993; Agnew & Agnew, 1994). Previously isolated compounds such as solanculine A, β.-solanmarine, and solanmargine from the root bark and berries, respectively, have been reported to have molluscicidal activity (Alphonse et al., 2002). The antimycotic activity of S. aculeastrum. has not been reported in the literature; yet, the members of the genus Solanum. are known to be rich in steroidal glycoalkaloids and sesquiterpenoids that have antibacterial and antimycotic properties (Cipollini & Levey, 1997; Nagaoka et al., 2001; Shamim et al., 2004).

The aim of this study was to investigate the antimicrobial activity of S. aculeastrum. by preliminary bioassay screening of its extracts against 10 selected bacterial and five fungal strains. According to Mathekaga and Mayer (1998), in vitro. screening methods could provide the needed preliminary observations necessary to select crude plant extracts with potentially useful properties for further chemical and pharmacological investigations.

Materials and Methods

Plant material

The berries and leaves of S. aculeastrum. were collected from Kayalethu village in the Eastern Cape Province of South Africa. The plant was identified at the Department of Botany, University of Fort Hare, and a voucher specimen (Vedic Med 2005/16) was deposited in the Griffen Herbarium of the university.

Extract preparation

S. aculeastrum. fruits were oven-dried at 60°C overnight and the leaves were air-dried at room temperature. Dried plant material (200 g) was shaken separately in acetone, methanol, and water for 48 h on an orbital shaker. Extracts were filtered using a Buchner funnel and Whatman no. 1 filter paper, and each filtrate was concentrated to dryness under reduced pressure at 40°C using a rotary evaporator. The water extracts were freeze-dried. Each extract was resuspended in the respective solvent to yield a 50 mg/ml stock solution (Taylor et al., 1996).

Bioassays

The bacterial cultures used in this study were obtained from the Department of Biochemistry and Microbiology, Rhodes University, South Africa. They consisted of five Gram-positive and five Gram-negative strains (Table 1). Each organism was maintained on nutrient agar plates and was recovered for testing by growth in nutrient broth for 24 h. Before use, each bacterial culture was diluted 1:100 with fresh sterile nutrient broth (Afolayan & Meyer, 1997).

Table 1 Antibacterial activity of the fruits and leaves of S. aculeastrum.

		MIC (mg/ml)
		Acetone		Methanol		Water
Bacteria–species	Gram+/−	Fruit	Leaf	Fruit	Leaf	Fruit	Leaf	Chlor^a.	Strept^b.
Bacillus cereus.	+	na	4	4	4	na	na	<2	<2
Staphylococcus epidermidis.	+	na	na	4	6	na	na	<2	<2
Staphylococcus aureus.	+	na	8	6	4	na	na	<2	<2
Micrococcus kristinae.	+	na	2	6	6	na	10	<0.2	<2
Streptococcus pyogenes.	+	na	6	6	6	na	na	<2	<2
Escherichia coli.	−	na	na	6	6	na	na	<2	<2
Salmonella pooni.	−	na	6	6	4	na	na	<2	<2
Serratia marcescens.	−	na	na	10	8	na	na	<2	<2
Pseudomonas aeruginosa.	−	na	na	10	8	na	na	<20	<5
Klebsiella pneumoniae.	−	na	na	10	8	na	na	<2	<2

Minimum inhibitory concentration (mg/ml); na, not active.

^a.Chloramphenicol in µg/ml.

^b.Streptomycin sulfate in µg/ml.

Test organisms were streaked in a radial pattern on sterile nutrient agar plates containing filtered extracts at final concentrations of 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 mg/ml (Meyer & Afolayan, 1995). Plates containing only nutrient agar and another set containing nutrient agar and the respective solvents served as controls. After inoculation, the plates were incubated at 37°C for 24 to 48 h. Each treatment was performed in triplicate, and complete inhibition of bacterial growth was required for an extract to be declared bioactive. Chloramphenicol and streptomycin were used as standard control in the experiment.

Five species of fungi were used for the antimycotic investigation (Table 2). The cultures were maintained on potato dextrose agar (PDA) and were recovered for testing by subculturing on fresh PDA for 3 days. PDA plates were prepared in the usual fashion by autoclaving before the addition of the filtered extracts. Each extract was mixed with the molten agar (at 45°C) to final concentrations of 0.1, 0.5, 1.0, and 5.0 mg/ml, poured into Petri dishes, and left overnight for the solvent to evaporate. Control plates containing only PDA or PDA with the respective solvents served as controls. The prepared plates containing the extracts were inoculated with plugs obtained from the actively growing margins of the recovered fungal cultures and were incubated at 25°C for 5 days. The diameter of fungi growth was measured and expressed as percentage growth inhibition of three replicates (Afolayan & Meyer, 1997; Barreto et al., 1997; Quiroga et al., 2001). Significance differences within the means of the treatments and the controls were calculated using the LSD statistical test (Steel & Torrie, 1960). LC₅₀ (the concentration at which there was 50% inhibition of the growth of the test fungi) was calculated by extrapolation.

Table 2 Antifungal activity of the fruits and leaves of Solanum aculeastrum.

	Growth inhibition (%)
	Fruit extract				Leaf extract
Concentrations (mg/ml)	A. flavus.	A. niger.	F. oxysporum.	P. notatum.	A. flavus.	A. niger.	F. oxysporum.	P. notatum.
Acetone extracts
5.0	100.00^c.	35.19^c.	0.59^a.	9.6^a.	100.00^d.	27.75^b	0.00^a.	64.81^c.
1.0	42.29^b.	12.71^b.	0.00^a.	4.00^a.	61.06^c.	2.220^a.	0.00^a.	49.33^b.
0.5	15.40^a.	0.71^a.	0.00^a.	2.313^a.	36.86^b	2.37^a.	0.00^a.	−1.567^a.
0.1	0.11^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.
Control	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.
LC50	1.53	>5	>5	>5	0.77	>5	>5	1.17
Methanol extracts
5.0	100.00^d.	26.40^b.	24.64^c.	60.26^c.	100.00^c.	44.92^a.	1.66^a.	56.00^c.
1.0	62.13^c.	0.95^a.	−1.40^b.	15.82^b.	42.29^b.	0.900^a.	0.92^a.	18.07^b.
0.5	13.11^b.	−1.90^a.	−2.82^a.	1.52^a.	15.40^a.	−0.26^a.	1.61^a.	0.79^a.
0.1	2.53^ab.	0.00^a.	0.00^a.	0.00^a.	0.11^a.	0.00^a.	0.00^a.	0.00^a.
Control	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.
LC50	0.88	>5	>5	4.08	1.53	>5	>5	4.37
Water extracts
5.0	2.08^a.	12.44^a.	6.45^b.	69.89^d.	3.30^a.	1.38^a.	0.52^a.	−0.097^a.
1.0	0.82^a.	3.18^a.	0.00^a.	37.35^c.	2.15^a.	−0.66^a.	−0.54^a.	−0.08^a.
0.5	2.19^a.	3.81^a.	0.00^a.	26.54^b.	3.30^a.	2.05^a.	−0.54^a.	−0.17^a.
0.1	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.
Control	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.
LC50	>5	>5	>5	2.56	>5	>5	>5	>5

Values are means of percentage growth inhibition of three replicates: values within a column followed by the same superscript of the same species are not significantly different at p < 0.05 according to the LSD test. LC₅₀ values in mg/ml.

Results and Discussion

Antibacterial property

Minimal inhibitory concentration (MIC) values of ethanol, acetone, and water extracts from the fruits and leaves of S. aculeastrum. against the tested bacteria are given in Table 1. Methanol extracts inhibited the growth of both the Gram-positive and Gram-negative bacterial at MIC ranging between 4.0 and 10.0 mg/ml. There was, however, more inhibition on Gram-positive strains. Whereas the acetone and water extracts of the fruits did not show any activity against any bacterial species, the leaf extracts of the two solvents showed moderate activity against Micrococcus kristinae.. Acetone extract of the leaves showed activity against Gram-positive bacteria at concentrations of 2.0–8.0 mg/ml except Staphylococcus epidermidis., whereas there was no activity against the Gram-negative bacteria at the highest concentration tested with the exception of Salmonella pooni.. In a similar experiment, moderate activity by Solanum aculeastrum. was reported against E. coli., S. aureus., P. aeruginosa., K. pneumoniae., S. faecalis., and B. subtilis. (Alphonse et al., 2003).

Antifungal property

The results of the antifungal assay of S. aculeastrum. extracts are presented in Table 2. The majority of the extracts (33.33%) showed antimycotic activity against the tested organisms at concentrations of 5 mg/ml or lower. Extracts from the leaves were more inhibitory to the growth of the tested fungi. The acetone and methanol extracts from the leaves inhibited the growth of Aspergillus flavus. and Penicillium notatum. with inhibitory percentage ranging from 56.0% to 100.0%. Similarly, the growth of A. niger. was inhibited by methanol extract of the leaves at 5 mg/ml, which was the highest concentration used. However, it is interesting to note that both acetone and methanol extracts of the fruits completely inhibited the growth of A. flavus. (100%), whereas methanol and water extracts of the fruits suppressed the growth of P. notatam. with percentage inhibition ranging from 60.26% to 69.89%, respectively. The susceptibility of A.. flavus. to the extracts of S. aculeastrum. is noteworthy, as the fungus has recently been implicated in cases of immuno-compromised patients that frequently develop opportunistic and superficial mycosis (Ngane et al., 2000; Portillo et al., 2001; Silva et al., 2001). Acetone, methanol, and the water extracts from the fruits and leaves did not show any activity against A. niger., Fusarium oxysporum., and C. albicans..

The results of this study agreed with Portillo et al. (2001) who showed that A. niger. is resistant to dichloromethane, aqueous and methanol extracts of 14 plants used for traditional medicine in Paraguay. In this study, the acetone and methanol extracts were found to have broad-spectrum activity against the fungal species tested. Generally, these two extracts were more active than the water extracts. Traditionally, however, plant extracts are prepared with water as infusions, decoction, and poultices; therefore, it would seem unlikely that the traditional healers are able to extract those compounds which that responsible for activity in the acetone and methanol extracts. Work is in progress on the isolation, purification, and structural identification of the bioactive compounds in this plant in order to validate the claims for its use in traditional medicine by the people of the Eastern Cape, South Africa.

Acknowledgment

This research was supported by the National Research Foundation (NRF) of South Africa.