Microbial Load of Some Medicinal Plants Sold in Some Local Markets in Abeokuta , Nigeria

Purpose: To evaluate the microbial load on 17 randomly selected plant samples from 60 ethnobotanically collected medicinal plants from five local markets in Abeokuta, Ogun State, Nigeria. Method: The pour plate method was used to cultivate serially diluted portions of the medicinal plant samples investigated. Enumeration of bacteria was carried out on nutrient agar (NA) while that of fungi was effected on Sabouraud agar (SA). Results: The identified microbial isolates include 12 bacterial and 6 fungal genera. The mean heterotrophic bacteria counts of the different herbal samples ranged from 1.3 × 10 5 cfu/g (Cnestis ferruginea) to 6.7 × 10 6 cfu/g (Daniellia oliveri), while total fungal propagule counts ranged from 0.0 × 10 1 cfu/g (Terminalia superba, Cola gigantea, Rauwolfia vomitoria, Zingiber officinale and Argemone mexicana) to 7.1 × 10 6 cfu/g (Nesogordonia papaverifera). The synopsis and frequency (prevalence rate) of microbial species isolation showed that Bacillus spp. (82.4 %) and Mucor spp. (47.1 %) had the highest prevalence rates among bacteria and fungi, respectively. Conclusion: The findings from this study emphasized the need for constant quality assessment of herbal drugs on sale in order to ensure the production of therapeutic products suitable for human consumption.


INTRODUCTION
In the past, medicinal plants was the first line of treatment known to man and traditional medicinal practice remain an important part of the primary healthcare delivery system in most of the developing world [1].According to a World Health Organization (WHO) survey, about 70 -80 % of the world's populations, particularly in the developing countries, rely on non-conventional medicines, mainly from herbal sources, for their primary healthcare [2].
Generally, plants constitute a major source of orthodox medicines and the presence of plant secondary metabolites has been attributed for most plants' therapeutic activities [3,4].Phytomedicines have shown great promises in the treatment of intractable infectious diseases [5].The local uses of plants and their products in healthcare are much higher in those areas with little or no access to modern healthcare services [6].
The global and national markets for medicinal herbs have been growing rapidly and significant economic gains are being realized with global sales of herbal products totalling an estimated US$60 million in 2000 [7].However, the current global market for herbal medicines stands at over US$62 billion annually [8].The sale of herbal medicine is expected to reach an annual average growth rate of 6.4% [9].Several countries, despite their abundant tropical forests, earn nothing significant from the sector.For example, Nigeria is not on the list of countries that have a stake in the over $60 billion generated from herbs globally.It is at the moment generating 0.001 per cent of the revenue from herbs [10].The local markets in Abeokuta form an integral part of the life of the people.Medicinal plants traders in these markets sell large amounts of plants to indigenes and visitors who seek their help.They mostly sell the plants (barks, roots, stems and leaves) in dried forms.
Again, market surveys are an efficient means of acquiring data on local values and conservation status of indigenous species [11].An understanding of the market profile, social economic attributes influencing trade, species traded and impact of trade on plant pollution is critical for effective resources management [12].The safety and quality of raw medicinal plant materials and finished products depend on factors that may be classified as intrinsic (genetic) or extrinsic (environment, collection methods, cultivation, harvest, post harvest processing, transport and storage practice [7].
It is against this backdrop that the microbial loads of some of the ethnobotanically collected plants in Abeokuta, Nigeria were carried out.

EXPERIMENTAL Study Area
The study was carried out in the city of Abeokuta, Ogun State, Nigeria.Abeokuta is located within longitude 3 0 2 1 East and Latitude 7 0 1 1 North.The markets surveyed were Omida, Itoku, Adatan, Kuto and Lafenwa markets.Abeokuta lies within the tropical rainforest area of Nigeria with a population of about one million people and is surrounded by rocks [13].

Collection and identification of plant samples
The medicinal plant samples were bought from the five local markets surveyed in Abeokuta and aseptically collected with sterile cloves into sterile bags.The samples were immediately kept in clean, cool and dry baskets.
Most of the plant samples were identified by one of the authors (MI) while the others were identified on the basis of their local names and standard texts [14,15].Voucher specimens of the plants were kept in the Department of Plant Biology and Biotechnology Herbarium, University of Benin, Nigeria.

Preparation and sterilization of media and samples
The media used were nutrient agar (NA) and Sabouraud agar (SA) for enumeration of bacteria and fungi, respectively.They were prepared according to the manufacturer's guide and sterilized in an autoclave at 121 o C for 15 min.The dried plant samples were ground into fine particles under aseptic conditions in a surface sterilized laboratory bench.The grater was washed, dried and sterilized before use.After grinding, the samples were placed in different sterile universal containers and labelled accordingly.

Microbial analysis of plant samples
The pour plate method was used to cultivate serially diluted portions of the medicinal plant samples under investigation.Enumeration was carried out on nutrient agar (NA) for bacteria and on Sabouraud agar (SA) for fungi.Triplicate plates of appropriate dilutions were prepared.The NA plates were incubated at 37 0 C for 24 -48 h for bacterial growth while SA plates were incubated at room temperature (28 ± 2 0 C) for 48 -72 h for fungal growth.
The developed microbial colonies were counted and computed as colony forming units per gram (cfu/g) of plant material.The colonies were purified, isolated and stored for morphological and biochemical characterization.
These were further identified with to the aid of Bergey ' s Manual of Determinative Bacteriology [16] for bacteria and Illustrated Genera of Imperfect Fungi [17] for fungi.

Statistical analysis
The results were expressed as mean values ± SEM of three replicates of the total heterotrophic bacteria and fungi (cfu/g) contained in each plant sample screened.The data were analysed using Students t-test with the aid of SPSS 10 software package.
The level of significance was set at 0.05.The prevalence rate of the microbial species was also computed.

RESULTS
The results of the microbial load of the different plant materials are presented in Tables 1 -4.No significant difference was recorded in both the bacterial and fungal counts (Tables 1 and 2).The mean heterotrophic bacteria counts of the different herbal samples ranged from 1.3×10 5 cfu/g (Cnestis ferruginea) to 6.7×10 6 cfu/g (Daniellia oliveri) (Table 1), while  3 and 4, respectively.The synopsis and frequency (prevalence rate) of the microbial species isolation showed that Bacillus spp.(82.6 %) and Mucor spp.(47.1 %) had the highest prevalence rates among bacteria and fungi, respectively.The least frequently isolated bacterial species were Arizoma spp., Diphtheroids, Escherichia coli, Proteus spp., Streptococcus spp.and Pseudomonas aeruginosa with a prevalence rate of 5.9 %.The figure was the same for the fungal species (Absidia spp., Rhizopus nigrican and Saccharomyces cerevisiae) with the least prevalence rate.

DISCUSSION
Samples cultured on nutrient agar (NA) were observed to have a large growth of bacterial species while samples cultured on Sabouraud agar (SA) were observed to give few results as fungal species did not grow on some of the cultured samples, namely, Terminalia superba, Cola gigantea, Zingiber officinale, Argemone mexicana and Rauwolfia vomitoria (Tables 1 and 2).Contamination by microorganisms is   Veterinariya, 1963;43: 54-55.

Table 1 :
Heterotrophic bacterial counts (mean ± SD, n= 3) of medicinal plant samples obtained from some open markets in Abeokuta, Nigeria

Table 2 :
Total fungal propagule counts (mean ± SD, n= 3) of medicinal plant materials obtained from some open markets in Abeokuta, Nigeria

Table 4 :
Synopsis and frequency (prevalence rate) of isolation of fungi from herbal plant samples obtained from some open markets in Abeokuta, Nigeria.