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Antimicrobial and brine shrimp activity of Acanthus pubescens root extracts

M.J. MOSHI^1*, E. INNOCENT¹, J.N. OTIENO¹, J.J. MAGADULA¹, R.S.O. NONDO¹, D.F. OTIENO², A. WENSHEIT³ and P. MBABAZI³

¹Institute of Traditional Medicine, Muhimbili University of Health and Allied Sciences, Box 65001, Dar es Salaam, Tanzania
²Department of Biological Sciences, Moi University, P. O. Box 1125, Eldoret, Kenya
³Faculty of Development Studies, Mbarara University of Science & Technology, P.O. Box 1410, Mbarara, Uganda

The root dichloromethane and ethyl acetate extracts of Acanthus puberscens (Oliv.) Engl(ACANTHACEAE)exhibited weak antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Bacillus anthracis, Salmonella typhi, Streptococcus faecalis, Streptococcus agalactiae and Pseudomonas aeruginosa, with MIC values ranging from 1.6-6.25 mg/ml. The two extracts also exhibited weak antifungal activity against Candida albicans (MIC 6.25 mg/ml). Using the brine shrimps lethality test ethanol, ethyl acetate and aqueous extracts were virtually non-toxic to brine shrimp larvae, but the dichloromethane extract (LC₅₀ 38.9 µg/ml) was mildly toxic. These results support the use of the plant in traditional medicine to treat gonorrhea, syphilis, gastroenteritis and pneumonia. Since the plant is used in combination with other plants it is difficult to make any final conclusions regarding safety and efficacy. Further work is needed to evaluate the activity of an extract made from a combination of the six plants.

Keywords: Acanthus pubescens, traditional medicine, antimicrobial activity

Acanthus puberscens (Oliv.) Engl (ACANTHACEAE) also known among the Haya people of Bukoba in Tanzania as “ Amatoju” is used in traditional medicine for treatment of syphilis and gonnorhea. The roots are cut into small pieces and boiled with water or meat broth and two bowls are administered per day, for up to a week for the treatment of gonorrhea and syphilis. The roots are boiled in a mixture with the roots of Sapium ellipticum, Trichilia emetica, Zehneria scabra, Emilia javanica and Oxygonum sinuatum. Reports from the literature indicate that a decoction of the leaves is used in Rwanda for the treatment of gastroenteritis, pneumonia and anthrax (Boily & van Puyvelde, 1986). It is also reported that, in Rwanda, a preparation of the dried leaves is used externally as a remedy for scabies (Heyndrickx et al., 1992).

An 80% ethanol extract of the leaves exhibited antifungal activity against Microsporum canis and Trichophyton mentagrophytes, a weak antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus and antiviral activity against the poliovirus and measles viruses (Vlietinck et al., 1995).

The present study is part of an ongoing initiative to evaluate ethnobotanical resources in the Kagera Region of Tanzania and ways in which these resources could contribute to poverty alleviation strategies. Plants used as anti-infectives can be developed into topical preparations, evaluated for skin hypersensitivity reactions, and put into clinical use in a short time. The need to provide data to support commercial exploitation of these plants is significant, given the amount of poverty that exists among these remotely located communities. The current study, therefore, aims to demonstrate presence of antimicrobial activity in root extracts which has not been reported before and in addition through the brine shrimp test to obtain preliminary indication of safety of the plant extracts.

Ethanol, dichloromethane, and ethyl acetate were purchased from Fisher Scientific UK Ltd (Leicestershire, LE 11 5RG, UK). Saboraud’s dextrose agar (SDA) and Tryptone soya agar and broth were purchased from Oxoid Ltd (Basingstoke, Hampshire, England), while Iodonitro tetrazolium chloride and dimethyl sulphoxide (DMSO) were purchased from Sigma (Poole, Dorset, UK). Brine shrimp eggs were bought from Aquaculture Innovations (Grahamstown 6140, South Africa). Sea salt was prepared locally by evaporating water collected from the Indian Ocean, along the Dar es Salaam coast in Tanzania.

Acanthus pubescens roots were collected from Buzi village, Bugabo Ward in Bukoba Rural District in north-west Tanzania. The plant (Voucher No. MJM 3028) was identified at the Department of Botany, University of Dar es Salaam, Tanzania. Duplicate vouchers are kept at the Herbarium of the same Department and the Herbarium of the Institute of Traditional Medicine, Muhimbili University of Health and Allied Sciences in Dar es Salaam.

The roots of the plant were ground into powder and 460 g of the powder was sequentially soaked for 48 h, with dichloromethane, ethyl acetate, ethanol and distilled water. The extracts were dried using a rotary evaporator, at 40^OC, followed by freeze drying to remove remaining water. The powders were then stored at -20^OC until the time of testing. The respective yields were 2.6 g (0.56%), 0.9 g (0.20%), 2.3 g (0.50%) and 4.4 g (0.96%) for dichloromethane, ethyl acetate, ethanol and distilled water, respectively.

Plant extracts were all dissolved in dimethyl suphoxide. Antibacterial and antifungal activities were tested by the disc-diffusion method (Singh et al., 2002). Eight standard bacteria, Staphylococcus aureus (NCTC 6571), Streptococcus faecalis (clinical isolate), Sterptococcus agalactiae (NCTC 8181), Pseudomonas aeruginosa (NCTC 10662), Salmonella typhi (NCTC 8385), Bacillus anthracis (NCTC ), Bacillus subtilis (clinical isolate), and one fungus,Candida albicans (Strain HG 392) were used. Filter paper discs (Whatman No. 1; 5 mm diameter) were impregnated with crude extracts (5 mg/disc) while standard drugs (10 μg/disc gentamicin; for bacteria) and clotrimazole (20 μg /disc for fungi) were purchased from Sigma (Poole, Dorset, UK). Discs containing DMSO alone were used as a negative control. The discs were overlayed on pre-inoculated tryptone soya agar plates (for bacteria) and Saborauld’s dextrose agar plates (for fungi) and incubated at 37 ^OC, for 24 h. The discs were tested in triplicate, including one with a solvent blank (DMSO) and one with a standard drug. The results of the disc diffusion method were only used to detect active extracts for MIC determination using the microdilution method. This was necessary to cut down on wastage of the microtitre plates.

MICs were determined using the microdilution method (Ellof, 1998). The 96 well microtitre plates were used and each plant extract was tested in duplicate at serial dilutions of 10, 5, 2.5, 1.25, 0.625, 0.31, 0.16 and 0.08mg/ml. Columns 1 and 2 were used for solvent controls and Columns 11 and 12 for positive controls. Each well was first filled with 100 µl broth followed by addition of 100 µl test drug, DMSO or standard drugs in the first well of each column and serially diluted. The bacteria suspension (100µl) was then added in each well and the plates were incubated at 37°C for 24h. After 24h, 40 µl of 2 mg/ml (0.02%) iodonitrotetrazolium chloride was added in each well and the plates were incubated for 1h at 37°C for detection of bacterial and fungal growth. MICs for fungi and bacteria were determined by visual inspection.

The brine shrimp lethality test (BST) was used to predict the presence, in the extracts, of cytotoxic activity (Meyer et al., 1982). Solutions of the extracts were made in DMSO or distilled water, at varying concentrations and incubated in triplicate vials with the brine shrimp larvae. Ten brine shrimp larvae were placed in each of the duplicate vials. Control brine shrimp larvae were placed in a mixture of artificial sea water (3.8g of sea salt per litre) and DMSO only. After 24 h the nauplii were examined against a lighted background, and the average number of survived larvae in each triplicate was determined. The mean percentage mortality was plotted against the logarithm of concentrations and the concentration killing fifty percent of the larvae (LC₅₀) was determined from the graph (Meyer et al., 1982).

The mean percentage brine shrimp mortalities were plotted against the logarithms of concentrations using the Fig P computer program (Biosoft Inc, USA), which also gives the regression equations. The regression equations were used to calculate LC₁₆, LC₅₀ and LC₈₄ values. Confidence intervals (95% CI) were then calculated using the three results (Litchfield & Wilcoxon, 1949; Meyer et al., 1982). An LC₅₀value greater than 100 mg/ml was considered to represent an inactive compound or extract

Using the disc diffusion method dichloromethane and ethyl acetate extracts of Acanthus pubescens root extracts exhibited antibacterial activity against both gram positive and gram negative bacteria. They exhibited weak antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Bacillus anthracis, Salmonella typhi, Streptococcus faecalis, Streptococcus agalacticus and Pseudomonas aeruginosa. They also showed weak antifungal activity against Candida albicans. The MICs results ranged from 1.6-6.25 mg/ml (Table 1).

Organism	MIC (mg/ml)
Organism	DCM extract	Ethyl acetate extract
Staphylococcus aureus	6.25	1.6
Salmonela typhi	-	3.12
Streptococcus faecalis	6.25	-
Bacillus subtilis	3.12	-
Bacillus anthracis	6.25	1.6
Pseudomonas aeruginosa	-	6.25
Streptococcus agalactiae	6.25	25.0
Candida albicans	6.25	6.25

The results for brine shrimp toxicity are reported in Table 2. The aqueous and ethanolic extracts were least toxic, while the dichloromethane extract was the most toxic.

Extracts	LC₅₀( μg/ml)	95% CI (μg/ml)
Dichloromethane	38.9	27.4 -55.2
Ethylacetate	136.2	92.0 – 201.6
Ethanol	>1000	-
Aqueous	>1000	-
Cyclophosphamide	16.3	10.6-25.1

Preparations of Acanthus pubescens are used in traditional medicine for the treatment of syphilis, gonorrhea, gastroenteritis, anthrax and scabies. This suggests that preparations of the plant have both antibacterial and antifungal activity. The results have shown that ethyl acetate and dichloromethane root extracts of Acanthus pubescens have weak activity against both Gram positive and Gram negative bacteria. They also have weak antifungal activity against Candida albicans. According to Aligiannis et al. (2001) MICs above 1.6 mg/ml indicate weak activity. Since all the MICs of the Acanthus pubescens extracts were 1.6 mg/ml and above then they all exhibited weak antibacterial and antifungal activity.

Traditionally the plant roots are not used alone; the roots are boiled together with the roots of Sapium ellipticum, Trichilia emetica, Zehneria scabra, Emilia javanica and Oxygonum sinuatum. The long duration of use (1 week) and the fact that the plant is not used alone shows how this plant may be effective for treatment of infections even though it does not have very good in vitro activity. However, what one can not say is on the possible metabolism of the active compounds in vivo to more active compounds, which at this point remains a speculation. It will also be interesting to test the activity of the mixture as compared to the single plants.

The brine shrimp lethality test (BST) was used to predict trends of toxicity and possible presence of potential anticancer compounds (Moshi et al., 2004). The results are interpreted as follows: LC₅₀ < 1.0 µg/ml – highly toxic; LC₅₀1.0-10.0 µg/ml – toxic; LC₅₀ 10.0-30.0 µg/ml – moderately toxic; LC₅₀ > 30 < 100 µg/ml – mildly toxic, and > 100μg/ml as non-toxic. Cyclophosphamide (LC₅₀ 16.3 μg/ml) was used as a standard so that it can allow inference to be made for potential to yield anticancer compounds.

The results show that the aqueous and aqueous ethanolic extracts have LC₅₀ values above 1000 μg/ml, indicating that they are virtuously non-toxic. The dichloromethane and ethyl acetate extracts showed LC₅₀ values of 38.9 and 136.2 μg/ml, respectively. This indicates that the ethyl acetate extract is also virtually non-toxic. However, the dichloromethane extract showed mild toxicity.

One way of preparing the plant for use is by boiling the roots with meat broth, which would effectively provide a fatty base for extraction of compounds picked up in the dichloromethane and ethyl acetate extracts. This suggests that the potential for toxicity is higher when the preparation is made with meat broth. However, in the absence of information on the mixture that is used not much can be emphasized regarding either efficacy or safety.

In conclusion extracts of Acanthus pubescens exhibited weak antibacterial activity against Gram positive and Gram negative bacteria and antifungal activity against Candida albicans. This supports the traditional medicine use and shows potential for isolation of active compounds.

We are grateful to Mr. Selemani Haji of the Botany Department, University of Dar es Salaam for his technical assistance in identifying the plant.