Extracts from Pelargonium sidoides. Inhibit the Growth of Bacteria and Fungi

Abstract

Pelargonium sidoides. DC (Geraniaceae) is a medicinal plant used by the people of the Eastern Cape, South Africa, for the treatment of various diseases in man and livestock. Acetone and methanol extracts of the shoot and root of the herb were investigated for antimicrobial activity against 10 bacterial and 5 fungal species by the dilution method on solid agar medium. With the exception of Staphylococcus epidermidis., extracts obtained from both solvents demonstrated significant activity against all the Gram-positive and two of the Gram-negative bacteria tested in this study. The extracts also showed appreciable inhibitory activity against all the fungal species tested with inhibition ranging from 52.5% on Aspergillus flavus. to 82.5% on Mucor hiemalis. at 5 mg ml⁻¹, the highest concentration tested in the study. The ability of the extracts of this plant to inhibit the growth of several bacteria and fungi is an indication of the broad-spectrum antimicrobial potential of P. sidoides. that further validates the use of this plant for the treatment of various ailments by the people of the Eastern Cape.

Keywords:

• Antimicrobial activity
• ethnobotany
• medicinal plants
• Pelargonium sidoides

Introduction

Today, an estimated 80% of the black population of South Africa makes use of indigenous medicinal plants for the treatment of various ailments in man and animals (Kelmanson et al., 2000). This practice may be due to easy access to traditional medication, especially in rural areas, where the majority of people cannot afford prescribed medicines.

Pelargonium sidoides. DC (Geraniaceae) is indigenous to South Africa. It grows widely in the Eastern Cape Province where it is used for the treatment of human and livestock diseases. It is a rosette-like plant with a system of thickened underground roots that are commonly used by the traditional medical practitioners. Many of its uses relate to the treatment of microbial infections, which includes coughs, tuberculosis, and wounds in man and livestock (Van Wyk et al., 1997). Information gathered during our preliminary investigation on the local uses of the species also revealed its medicinal importance for the treatment of stomachache, dysentery, and diarrhea, especially in children. The pulverized root of the plant is mixed in fresh cow's milk and administered orally to children, and freshly harvested leaves are ground and placed on wounds in man and livestock. The pulverized root is also mixed with other herbs to treat dysentery in livestock (Batten & Bokelmann, 1966). Umckaloabo., a medicine prepared from this plant by a German company (Schwabe), is said to be effective against bronchitis in children (Van Wyk et al., 1997; Van Wyk & Gericke, 2003).

Despite the documented information on the medicinal use of the species, there is little or no information on its antibacterial property, especially in this part of the world. The work of Kayser and Kolodziej (1997) on the antibacterial activity of this plant was devoted to the European ecotype. In this paper, we report the antimicrobial activity of the acetone and methanol extracts of P. sidoides. growing in South Africa with 10 bacteria and 5 fungal species. According to Mathekaga and Meyer (1998), in vitro. antimicrobial screening has provided the preliminary observations necessary to select among crude plant extracts, those with potentially useful properties for further chemical and pharmacological investigations.

Materials and Methods

Plant collection and preparation of extracts

Shoot and root samples of P. sidoides. were collected in February 2005 from Peddie (33°11.10′S and 7°10.60′E; altitude 695 m), Eastern Cape Province, South Africa. The vegetation is rich in diverse flora, and the area has a mean annual rainfall of about 700 mm and annual temperature range of 18–25°C. The species was authenticated by Dr. D. 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. 2005/2).

Samples were 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 and evaporated to dryness under reduced pressure at a maximum of 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. Five Gram-positive (Bacillus cereus., Staphylococcus epidermidis., Staphylococcus aureus., Micrococcus kristinae., Streptococcus pyogenes.) and five Gram-negative (Escherichia coli., Salmonella pooni., Serratia marcescens., Pseudomonas aeruginosa., and Klebsiella pneumoniae.) species were used in this study. The organisms were maintained on nutrient agar plates and were revived for bioassay by culturing them in fresh nutrient broth (Biolab, South Africa) for 24 h before use.

Nutrient agar (Biolab, South Africa) was prepared by autoclaving and allowing to cool to about 60°C before the addition of the extracts. The agar medium containing the extracts at final concentrations of 1.0, 2.5, 5.0, 7.5, and 10.0 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 to 48 h. The minimum inhibitory concentration (MIC) values where no bacterial growth was observed were recorded. Experiments were replicated three-times.

Antifungal assay

Five species of fungi, Aspergillus flavus., Aspergillus niger., Fusarium oxysporium., Mucor hiemalis., and Penicillium notatum., were used for the antimycotic investigation. The fungal cultures were maintained on potato dextrose agar (PDA) (Biolab) and were recovered for testing by subculturing on fresh PDA for 3 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 of molten agar 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 5 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 activity

The results of the acetone and methanol extracts are presented in Table 1. With the exception of Staphylococcus epidermidis., all the extracts demonstrated activity against the Gram-positive bacteria tested in this study. The MIC ranged from 1 to 5 mg ml⁻¹ except for the acetone root extract against Klebsiella pneumoniae. where the value was 10 mg ml⁻¹. The extracts were more active against the Gram-positive bacteria than the Gram-negative strains. Three Gram-negative bacteria, Escherichia coli., Serratia marcescens., and Pseudomonas aeruginosa., were not inhibited by any of the extracts at the highest concentration (10 mg ml⁻¹) tested. Observations on very weak/no activity of aqueous plant extracts on Gram-negative bacteria are well documented (Eloff, 1998; Madamombe & Afolayan, 2003; Afolayan, 2003).

Table 1 Antibacterial activity of the acetone and methanol extracts of the shoots and roots of Pelargonium sidoides.

		Minimum inhibitory concentration (mg ml^−1)
		Acetone		Methanol
Bacterial species	Gram +/−	AR	AS	MR	MS
Bacillus cereus.	+	2.5	5	5	5
Staphylococcus epidermidis.	+	5	na	2.5	5
Staphylococcus aureus.	+	2.5	5	5	5
Micrococcus kristinae.	+	2.5	5	1	2.5
Streptococcus pyogenes.	+	2.5	5	5	2.5
Escherichia coli.	−	na	na	na	na
Salmonella pooni.	−	2.5	5	5	2.5
Serratia marcescens.	−	na	na	na	na
Pseudomonas aeruginosa.	−	na	na	na	na
Klebsiella pneumoniae.	−	10	na	na	na

AR, acetone root extract; AS, acetone shoot extract; MR, methanol root extract; MS, methanol shoot extract; na, no activity.

P. sidoides. has been used traditionally for the treatment of various respiratory diseases by the people of the Eastern Cape for a long time. Bacillus cereus. and Klebsiella pneumoniae. are respiratory pathogens commonly associated with colds and flu (Viljoen et al., 2004). All the extracts showed significant activity against Bacillus cereus. while acetone root extracts was active on Klebsiella pneumoniae. at 10 mg ml⁻¹. The inhibitory property of extracts from P. sidoides. against pathogens of respiratory infections might have justified the use of this plant for the treatment of cough and influenza fever by the people of the Eastern Cape, South Africa. Kayser and Kolodziej (1997) reported similar antibacterial effect of this herb against K. pneumoniae. in Europe.

The choice of acetone and methanol as solvents of extraction and the exclusion of water extracts in this analysis was based on our previous observation and other reports that water extracts of plants generally showed little or no antimicrobial activities (Meyer & Afolayan, 1995; Masika & Afolayan, 2002).

Generally, Gram-negative bacteria have been reported to be more resistant to plant extracts than the Gram-positive strains (Rabe & Van Staden, 1997; Grierson & Afolayan, 1999; Afolayan, 2003). This is also confirmed in this study.

Antifungal activity

The acetone and methanol extracts showed significant inhibition against most of the fungi tested in this study (Table 2). Except for the acetone extracts (shoots and roots) against A. niger. and the methanol extract of the roots against M. hiemalis., all the extracts showed more than 50% inhibition with activity ranging from 52.5% on A. flavus. to 82.5% on M. hiemalis. at 5 mg ml⁻¹ (the highest concentration tested in the study).

Table 2 Antifungal activity of the acetone and methanol extracts of the shoots and roots of Pelargonium sidoides.

	Growth inhibition (%)
	Shoot extract					Root extract
Concentration (mg ml^−1)	Af	An	Fo	Mh	Pn	Af	An	Fo	Mh	Pn
Acetone
5	60.83^d.	45.73^c.	65.83^d.	71.11^d.	75.56^e.	52.50^e.	49.44^d.	71.39^e.	68.06^d.	69.72^d.
1	45.00^c.	32.22^b.	42.78^c.	27.50^c.	63.89^d.	47.78^d.	43.33^c.	62.78^d.	28.06^c.	60.00^c.
0.5	43.74^c.	26.95^b.	43.89^c.	16.39^b.	47.50^c.	42.78^c.	27.78^b.	56.67^c.	13.89^b.	56.94^c.
0.1	33.34^b.	26.67^b.	40.00^b.	0.00^a.	31.11^b.	31. 39^b.	24.45^b.	31.67^b.	0.00^a.	46.95^b.
Acetone	0.00^a.	0.833^a.	0.00^a.	0.00^a.	1.94^a.	0.00^a.	0.00^a.	0.00^a.	0.00^a.	0.55^a.
PDA	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	2.26	>5	2.25	3.06	0.58	2.88	>5	0.39	3.19	0.22
Methanol
5	74.72^d.	59.17^d.	65.83^d.	82.50^c.	71.09^d.	56.95^d.	55.28^e.	66.35^e.	29.72^c.	75.56^d.
1	58.61^c.	41.11^c.	42.78^c.	6.67^b.	64.72^d.	50.28^c	46.11^d.	59.17^d.	11.11^b.	69.7^c,d.
0.5	45.28^b.	31.1^b.	43.89^c.	0.00^a.	56.66^c.	38.34^b.	38.61^c.	49.72^c.	0.00^a.	63.89^c.
0.1	41.11^b.	30.28^b.	40.00^b.	0.00^a.	42.22^b.	0.00^a.	30.28^b.	34.72^b.	0.00^a.	40.48^b.
Methanol	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	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	0.68	2.97	1.84	3.29	0.32	0.99	2.70	0.51	<5	0.26

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, Aspergillus niger.; Fo, Fusarium oxysporium.; Mh, Mucor hiemalis.; Pn, Penicillium notatum.. LC₅₀: values in mg ml⁻¹ were calculated by extrapolation.

The ability of the extracts of this plant to inhibit the growth of several bacteria and fungi is an indication of the broad-spectrum antimicrobial potential of P. sidoides. that further validates the use of this plant for the treatment of various ailments by the people of the Eastern Cape.

Acknowledgment

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