Antimicrobial activity of Alepidea amatymbica

Abstract

Alepidea amatymbica Eckl. & Zeyh (Apiaceae) is a medicinal plant traditionally used for the treatment of various diseases including asthma, influenza, and diarrhea in South Africa. The antimicrobial activities of the acetone and methanol extracts of the leaf, stem, rhizome, and root of the species were assessed in an effort to validate the traditional medicinal uses of this herb, especially for the treatment of infectious diseases. Ten bacterial and three fungal species were bioassayed using the agar dilution method. All the extracts demonstrated appreciable activities against three Gram-positive bacteria. These bacteria have been implicated in different respiratory diseases. The inhibitory activity of some of the extracts against pathogens implicated in diarrhea diseases further validated the use of the herb in traditional medicine. Except for acetone leaf extract against Aspergillus flavus and Aspergillus niger, all the extracts showed more than 50% mycotic inhibition with activity ranging from 51.39% on A. niger to 81.11% on Penicillium notatum at  5 mg mL⁻¹ which was the highest concentration tested in the study. The ability of the herb to inhibit the growth of various bacteria and fungi species is an indication of the broad-spectrum antimicrobial potential of A. amatymbica; this further validates the use of the herb for various diseases by the people of the Eastern Cape.

Keywords::

• Alepidea amatymbica
• antimicrobial
• diarrhoea
• ethnobotany
• medicinal plants

Introduction

The use of herbal medicine by an estimated 80% of the population of South Africa is well documented in literature (Van Wyk et al., 1997; Kelmanson et al., 2000). This practice may be due to easy access to traditional medication in rural communities where the majority of the people cannot afford the cost of western medication. Diarrhea is one of the diseases commonly treated using herbal medication in the Eastern Cape Province, South Africa. The disease is a potential cause of morbidity and mortality in children and young livestock in most developing countries. The WHO has encouraged the use of traditional medicine for the treatment and prevention of diarrhea diseases (Lutterodt, 1989).

Alepidea amatymbica Eckl. & Zeyh (Apiaceae) is a medicinal plant traditionally used for the treatment of various diseases including diarrhea in South Africa (Treurnicht, 1997; Somova et al., 2001). The species is found mostly in the grasslands of Eastern Cape Province, Kwazulu Natal, Mpumalanga and other southern African countries like Lesotho, Swaziland, and Zimbabwe. It is also found along the forest margins and at about 850-2500 m above sea level (De Castro & van Wyk, 1994). Traditionally, the herb is used for the treatment of cold, influenza, chest complaints, diarrhea, and wounds. The herb is also combined with ground sample of Cannabis sativa Lam. (Cannabaceae) to treat asthma. Because of its high medicinal properties, A. amatymbica has been included in the organized medicinal trade market of Kwazulu Natal (O’Connor, 2004). Previous studies on the antibacterial activity of the species only reported on the root and rhizome collected from a different ecotype (Somova et al., 2001; Stafford et al., 2005).

In a recent survey conducted on medicinal species used locally for the treatment of diarrhea among the people of the Eastern Cape Province, A. amatymbica ranked among the most frequently mentioned species (unpublished data). However, there is no scientific information on the antimicrobial activity of the leaf and stem of the species. Past studies have shown that the aerial parts of some species also possess biological activities comparative to the traditionally used underground parts (Zschocke et al., 2000; Lewu et al., 2006). The current work reports the antibacterial and antifungal activities of the acetone and methanol crude extracts of the leaf, stem, rhizome and root of A. amatymbica against some microorganisms, some of which are known to cause diarrhea in human and livestock. The findings from this study may add to the overall value of this important medicinal herb.

Materials and methods

Plant collection and preparation of extracts

Samples of A. amatymbica were collected in December 2007 from the Teaching and Research Farm of the University of Fort Hare (33°11.10´ S and 7°10.60´ E; altitude 695 m). The vegetation of the area is rich in diverse flora and has a mean annual rainfall of about  700 mm and temperature range of 13° to 25°C. The species was authenticated by D.S. Grierson of the Department of Botany, University of Fort Hare. A voucher specimen was prepared and deposited in the herbarium of the Department of Botany (Lewu Med. 2008/1).

Plant samples were separated into leaf, stem, rhizome, and root. The samples were later chopped, air-dried and pulverized before extraction. Portions (100 g each) of the pulverized materials were extracted separately for 24 h in acetone and methanol. The extracts were filtered through Whatman No.1 filter paper, evaporated to dryness under reduced pressure at 40°C using a rotary evaporator. The extracts were re-dissolved in their respective solvents to the required concentrations for the bioassay analysis.

Antibacterial assay

Bacterial species used in the study were laboratory isolates obtained from the Department of Biochemistry and Microbiology, Rhodes University, South Africa. The organisms consisted of five Gram-positive (Bacillus cereus, Staphylococcus epidermidis, Staphylococcus aureus, Micrococcus kristinae, Streptococcus pyrogenes) and five Gram-negative (Escherichia coli, Salmonella pooni, Serratia marcescens, Pseudomonas aeruginosa, and Klebsiella pneumonae). The organisms were maintained on nutrient agar plates and were revived for bioassay by culturing them in fresh nutrient broth (Biolab, Johannesburg, South Africa) for 24 h before use.

Nutrient agar (Biolab, Johannesburg, South Africa) was prepared by autoclaving and allowed to cool to about 60°C before the addition of the extracts. The agar medium containing the extracts at final concentrations of 1, 2.5, 5, 7.5 and  10 mg mL⁻¹ were poured into Petri dishes, swirled carefully until the agar began to set and left overnight for the solvents to evaporate (Afolayan & Meyer, 1997). Plates containing 1% of acetone or methanol were used as controls (Dulger & Ugurlu, 2005). Organisms were streaked in radial patterns and the plates were incubated at 37°C for 24-48 h. The minimum inhibitory concentration (MIC) values where no bacterial growth was observed were recorded. Experiments were replicated three times.

Antifungal assay

Three species of fungi: Aspergillus flavus, Aspergillus niger and Penicillium notatum, were used for the antimycotic investigation. The fungal cultures were maintained on Potato Dextrose Agar (PDA) (Biolab, Johannesburg, South Africa) and were recovered for testing by sub-culturing on fresh PDA for three days prior to bioassay.

PDA plates were prepared by autoclaving before the addition of the extracts. Each extract was mixed with the molten agar (at 45°C) to final extract concentrations of 0.1, 0.5, 1.0, and 5. 0 mg mL⁻¹ and poured into Petri dishes. Blank 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 actively growing portions of the mother fungal plates and incubated at 25°C for five days. Diameter of the fungal growth was measured and expressed as percentage growth inhibition of three replicates (Afolayan & Mayer, 1997; Barreto et al., 1997; Quiroga et al., 2001). Significant differences within the means of treatments and controls were calculated using the LSD statistical test as described by Steel and Torrie (1960). LC₅₀ (the concentration at which 50% of the growth was obtained) was calculated by extrapolation.

Results and discussion

Antibacterial assay

The results of the antibacterial activity of all the extracts tested are presented in Table 1. With the exception of Micrococcus kristinae, the extracts demonstrated moderate activity against the Gram-positive bacteria tested in this study and the MIC ranged from 2.5 to  10 mg mL⁻¹. The acetone rhizome extract showed better activity than the others especially on S. aureus and B. aereus which are known pathogens commonly associated with respiratory and diarrhea diseases (Vijoen et al., 2004). The acetone and methanol extracts also showed appreciable activity against S. epidermidis which is a predominant pathogen of skin diseases (Johannes et al., 2001). None of the Gram-negative bacteria were inhibited by the extracts tested. The result supported early studies on other species where very weak or no activity was observed on plant extracts tested on Gram-negative bacterial strains (Rabe & Van Staden, 1997; Ellof, 1998; Grierson & Afolayan, 1999; Madamombe & Afolayan, 2003; Afolayan, 2003; Lewu et al., 2006).

Table 1.  Antibacterial activity of the acetone and methanol extracts of the leaf, stem, rhizome and root of Alepidea amatymbica.

Bacterial species	Gram +/-	Minimum inhibitory concentration (mg mL^−1)
		Acetone				Methanol
		Leaf	Stem	Root	Rhizome	Leaf	Stem	Root	Rhizome
Bacillus aereus	+	na	na	5	5	na	na	10	5
Staphylococcus epidermidis	+	na	na	na	7.5	na	na	na	10
Staphylococcus aureus	+	7.5	7.5	5	2.5	10	10	10	5
Micrococcus kristinae	+	na	na	na	na	na	na	na	na
Streptococcus pyrogens	+	na	10	na	na	na	10	na	na
Escherichia coli	-	na	na	na	na	na	na	na	na
Salmonella pooni	-	na	na	na	na	na	na	na	na
Serratia marcescens	-	na	na	na	na	na	na	na	na
Pseudomonas aeruginosa	-	na	na	na	na	na	na	na	na
Klebsiella pneumonae	-	na	na	na	na	na	na	na	na

na: no activity at corresponding bacteria and extracts.

Although the inhibitory concentration was high, only the stem extracts (acetone and methanol) showed weak activity against Streptococcus pyrogens. This suggests that the stem may contain some active compounds that are absent in the other parts of the herb.

Though the extracts did not show activity against the Gram-negative bacteria tested in this study, the species demonstrated moderate activity against Staphylococcus aureus, Bacillus aereus and Staphylococcus epidermidis. The presence of antibacterial activity in the extracts of this species, especially against the above mentioned bacteria strains, validates the traditional uses of this herb for the treatments of diarrhea, respiratory and wound infections. The comparative low MIC values recorded for the rhizome extracts further supports the preferential use of the rhizome in traditional medications by the people of the Eastern Cape Province.

Antifungal assay

The result of the antifungal activity is presented in Table  2. The acetone and methanol extracts showed significant inhibition against most of the fungi species tested in this study. Except for acetone leaf extract against A. flavus and A. niger, all the extracts showed more than 50% inhibition, with activity ranging from 51.39% in A. niger to 81.11% in P. notatum at  5 mg mL⁻¹, which was the highest concentration tested in the study.

Table 2.  Antifungal activity of the acetone and methanol extracts of the leaf, stem, rhizome and roots of Alepidea amatymbi.

Concentration (mg/mL)	Growth inhibition (%)
	Leaf			Stem			Rhizome			Root
	Af	An	Pn	Af	An	Pn	Af	An	Pn	Af	An	Pn
Acetone
5	47.5^d	46.21^d	56.23^d	57.17^e	56.41^d	62.05^e	56.95^e	65.21^e	76.34^e	58.89^d	51.39^d	62.5^c
1	26.95^c	25.61^c	23.08^c	38.18^d	51.82^d	35.50^d	44.17^d	59.72^d	61.67^d	39.44^c	29.17^c	51.94^c
0.5	10.83^b	9.85^b	21.77^c	32.72^c	24.44^c	22.50^c	35.09^c	33.61^c	35.00^c	23.06^b	22.78^c	36.94^b
0.1	2.5^a	2.26^a	10.49^b	25.56^b	16.39^b	15.32^b	25.56^b	23.89^b	15.00^b	16.11^b	7.50^b	6.67^a
Acetone Cont.*	0.00^a	0.00^a	0.00^a	0.00^a	0.00^a	0.00^a	0.00^a	0.00^a	0.00^a	0.00^a	0.00^a	0.00^a
PDA 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	0.00^a	0.00^a	0.00^a	0.00^a
LC50	>5	>5	4.25	3.49	0.97	3.18	2.82	0.81	0.78	3.17	4.75	0.94
Methanol
5	54.44^e	62.80^e	57.22^d	55.28^d	53.96^d	66.39^e	56.39^e	66.52^d	81.11^d	56.11^e	51.95^d	61.78^d
1	29.45^d	41.39^d	23.33^c	36.67^c	51.95^d	33.33^d	36.67^d	57.5^d	68.05^c	48.29^d	24.91^c	39.44^c
0.5	21.67^c	23.33^c	22.50^c	31.11^b	25.95^c	26.67^c	31.11^c	30.00^c	30.84^b	33.61^c	16.39^b	23.61^b
0.1	6.67^c	8.96^b	10.56^b	27.22^b	8.61^b	21.67^b	26.67^b	12.22^b	24.17^b	6.67^b	6.96^a	18.33^b
Methanol Cont.	0.00^a	0.00^a	0.00^a	0.00^a	0.00^a	0.00^a	0.00^a	0.00^a	0.00^a	0.00^a	0.00^a	0.00^a
PDA 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	0.00^a	0.00^a	0.00^a	0.00^a
LC50	4.29	2.61	4.15	3.87	0.93	0.77	3.7	0.86	3.02	1.87	4.71	2.89

Values are means of percentage growth inhibition of three replicates. Values within a column of a treatment followed by the same superscript are not significantly different at P < 0.05 according to the LSD test. Af: Aspergillus flavus; An: Apergillus niger; Pn: Penicillium notatum. LC₅₀: values in mg mL⁻¹ were calculated by extrapolation. Cont.*: Control.

Generally, the extracts showed moderate activities against the three fungi species. However, rhizome had the highest activity (81% growth inhibition) at  5 mg mL⁻¹on P. notatum, thus supporting the predominant use of rhizome by the natives of Eastern Cape for the treatment of different diseases (Kelmanson et al., 2000). The ability of the herb to inhibit the growth of bacteria and fungi species is an indication of the broad-spectrum antimicrobial potential of A. amatymbica which further validates the use of the herb. Further laboratory and clinical assays of the active compounds present in this species are required in order to better understand the antimicrobial principles of the different parts of the herb.

Acknowledgement

The authors thank the National Research Foundation of South Africa for financial support.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.