Phytoconstituent screening and antimicrobial principles of leaf extracts of two variants of Morus alba ( S 30 and S 54 )

Efficacies of the two variants of Morus alba locally grown in Forestry Research Institute of Nigeria, were investigated following the recommended procedure of analysis. Phytochemical, proximate and mineral compositions of M. alba leaves were determined using a standard procedure. Antimicrobial properties of leaf extracts of M. alba were also investigated against laboratory standards of both bacteria and fungi using the disc diffusion method. Phytochemical screening showed the presence of saponins, alkaloids, tannins, oxalate and flavonoids. Trypsin inhibitors, phytate and phenolic compounds were also present in appreciable quantity. Proximate analysis revealed the presence of crude protein, crude fat and crude fibre which is an indication of the plant’s nutritional value and could find an application in feed suppliments. The moisture content of M. alba (S304.350% and S541.050%) is indicative of its nonperishability, therefore could be stored for a long period of time. Minerals are needed for some physiological functions and some of the minerals are supplied from external sources. M. alba (S30 and S54) showed the presence of potassium, calcium, phosporus, zinc and magnesium. The minerals investigated revealed that the plant has low mineral content with the exception of potassium (S3032.650 mg/100 mg and S5431.400 mg/100 mg) which was high. The antibacterial activity was evaluated against microbes by detecting zones of inhibition and minimum inhibitory concentration (MIC). The zones of inhibition were compared with standard antibiotic discs of bacitracin for bacteria and streptomycin for fungi. The MIC values of the cold water extracts of the plant were low when compared with both hot water extracts and ethanol (99.7%) extracts. This is an indication of high potency and most suitable to combat nocosomial infection. M. alba extracts could find applications in the treatment of infections as indicated by the result of the antimicrobial screening and the whole plants can also be used in feed compounding for domestic animals.


INTRODUCTION
Plants are the richest resource of drugs of traditional systems of medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediates and chemical entities for synthetic drugs (Hammer et al., 1999).
Medicinal plants are of great economical value all over *Corresponding author.E-mail: bayodele2002@gmail.com.
the world.Nature has bestowed on us a very rich botanical wealth and a large number of diverse types of plants grown in different parts of the country.Nigeria is rich in all the 3 levels of biodiversity, namely species diversity, genetic diversity and habitat diversity.Herbal medicine is still the mainstay of about 75 to 80% of the whole population, and the major part of traditional therapy involves the use of plant extract and their active constituents (Akerele, 1993).
In Nigeria, thousands of species are known to have medicinal value and the use of different parts of several medicinal plants to cure specific ailments has been in vogue since ancient times (Lawal et al., 2010).Medicinal plants also represent a rich source of antimicrobial agents.Thus, interest has revived recently in the investigation of medicinal plants to identify novel active phytochemicals that might lead to new classes of microbial drug development.The entire plant source or different parts which include root, leaf, seed, stem, flower, fruit, twigs exudates and modified plant organs can be used to identify potential active principles for various ailments either as a whole plant, crude extract, aqueous or organic extracts (Newsman and Crag, 2007;Dev, 2010;Pavithra et al., 2010).Thus, in a bid to further utilize and not only limiting the use of M. alba in sericulture practices, the antimicrobial potential was investigated.
Morus is a genus of flowering plants in the family Moraceae.The white mulberry (M.alba) is a short-lived, fast-growing, small to medium sized mulberry tree, which grows to 10 to 20 m tall.They are native to warm temperate and subtropical regions of Asia, Africa, Europe and the Americans, with the majority of the species native to Asia, most especially China.It is used for feeding silkworm (Bombyx mori L.), its dried leaves have been consumed as herb-tea beverage and food supplements.M. alba have been a rich food source, and the leaf extracts of M. alba are used for anticancer and antioxidants properties (Benalla et al., 2010;Naowaratwattana et al., 2010).A variety of medicinal properties have been attributed to the different parts of the mulberry plant (Datta, 2000), leaves are also dried and used in infusions in Asia.
Forestry Research Institute of Nigeria (FRIN) has eight varieties of M. alba in her sericulture plantation.Laboratory investigations have been carried out on the antimicrobial activities of some of the varieties of M. alba.Tirupathi et al. (2011) investigated the antimicrobial properties of M. alba; S 26 and S 36 , while Manjula and Shubha (2011) also screened the activities of total soluble protein of two varieties of M. alba; S 13 and M 5 on some pathogenic bacteria.Literature review on M. alba S 30 and S 54 revealed that no previous phytochemical and pharmacological investigations have been reported.
This study was, therefore, carried out to evaluate the antimicrobial effect of cold water, hot water and ethanol (99.7% v/v) extracts of the leaves of the plant on some selected pathogens so as to establish a scientific basis for its use in treatment of conditions related to bacterial and fungal infections.

Plant source
M. alba leaves (variants S30 and S54) were collected from Herbal Garden, Forestry Research Institute of Nigeria and taken to herbarium section for identification.The plant samples were dried and ground into powder using a laboratory milling machine and then kept away from moisture prior to extraction.Analytical grades of ethanol (99.7% v/v), sulphuric acid, methanol, sodium chloride, ferric chloride and ammonium chloride were all purchased from an authorised dealer.

Extraction and recovery
The processed plant materials (S30 and S54) were extracted with cold and hot distilled water and absolute ethanol (99.7% v/v) with constant agitation at 300 rpm for 30 min.The process was repeated three times to exhaustively extract the bioactive materials and then concentrated using rotary evaporator to obtain crude extracts free of the extracting solvents.The crude extracts were then stored under 4°C until required for analysis.

Phytochemical, mineral and proximate analysis
The crude extracts were phytochemically screened using Brain and Turner (1975) methods.Mulberry samples were analyzed for moisture, ash, crude protein, crude fiber and crude fat content using the methods described by AOAC (1990).The mineral contents of calcium, phosphorus, zinc, potassium and magnesium were determined using Ilelaboye and Pikuda ( 2009) method (Table 3).

Test organisms
Standard strains of bacteria and fungi used for the work were Escherichia coli, Pseudomonas aeruginosa, Neisseria gonorrheae and Proteus vulgaricus for Gram negative bacteria and Staphylococcus aureus and Streptococcus faecium for Gram positive bacteria.Fungal isolates; Aspergillus tamari, Aspergillus niger, Fusarium oxysporum and Penicillium oxalicum.All strains were standards obtained from the Microbiology Laboratory of the Institute of Agricultural Research and Training, Obafemi Awolowo University, Ibadan, Nigeria.All bacterial strains were cultivated in nutrient agar (NA) medium, and incubated at 37°C for 24 h, while fungi were cultivated in potato dextrose agar (PDA).These were used for the microbial activity using the disc diffusion assay method.

Antimicrobial activity test by disc diffusion method
Antimicrobial activity of the crude leaves extracts of M. alba (S30 and S54) were evaluated by the paper disc diffusion method following a procedure used by Brantner and Grein (1994) and modified by Ali et al. (1997).Paper discs impregnated with 20 µl of a solution of 10 mg/ml of bacitracin (for bacteria) and streptomycin (for fungi) as standard antibiotics were used for comparison.Antimicrobial activity was determined by the measurement of zone of inhibition around each paper disc.Three replicate trials were conducted against each organism.

Determination of minimum inhibitory concentration (MIC)
tube dilution technique (Iwaki et al., 2006;Khan et al., 2007) was used to determine MIC of the extracts against Gram positive and Gram negative bacteria and fungi.The procedure was repeated on the test organisms using the standard antibiotics (bacitracin for bacteria and streptomycin for fungal isolates).The minimum inhibitory concentration (MIC) of the extracts was determined for each of the test organisms in triplicates.Tubes containing bacterial cultures were then incubated at 37°C for 24 h, while tubes containing fungal spore cultures were incubated for 48 h at room temperature (30 to 35°C).After incubation, the tubes were then examined for microbial growth by observing turbidity.

Phytochemical, proximate and mineral compositions of the two variants of M. alba
In the present study, the two variants of M. alba (S 30 and S 54 ) used showed appreciable level of all the phytochemicals analyzed.Saponins, phenolic compounds, flavonoids and tannins were all present in the two variants of M. alba with saponins having the highest quantity (S 30 : 4.905 ± 0.04% and S 54 : 4.820 ± 0.03%) and closely followed by tannins (S 30 : 3.100 ± 0.00% and S 54 : 2.665 ± 0.01%) as shown in Table 1.Though, alkaloids, phenolic compounds and flavonoids were present in limited amount.Each of this group of compounds has been reported to possess antimicrobial activity (Ragasa et al., 2005;Cuhnie and Lamb, 2006;Soetan et al., 2006) and reportedly exert their effects by affecting the cell membrane integrity of the bacteria (Hendrich, 2006;Trombetta et al., 2005;Killeen et al., 1998).
Trypsin inhibitors, phytate and oxalate were some of the antinutrients analysed for and showed, they were present in varying levels (Table 1).Oxalate can form metal-complex with some of the trace metals essential for body functions, thereby making them unavailable for enzymatic activities and other metabolic activities.Phytate has been found to cause indigestion of food and flatulence (Maynard, 1997).Though, these antinutrients can easily be reduced to tolerable limits by proper simple processing techniques, such as soaking, cooking and frying (Ekpo et al., 2004), but care must be taken if it were to be used as food suppliments or herbal tea as practiced in China.
The plants were unusually low in crude protein, crude fat and crude fibre as indicated in Table 2 which might actually limit its use as food suppliments.Though, the protein content is relatively low but it can contribute to the proper functioning of hormones which controls a variety of body functions, such as growth, repair and maintenance of body protein (Mau et al., 1999).The low moisture contents of 4.350 ± 0.78% for S 30 and 1.050 ± 0.07% for S 54 will confer long shelf life to the plants before cultivation or consumption.
Minerals are needed by the body for some physiological functions.A meal low in calcium can result into ricket and calcification of bone.Distorted enzymatic activity and poor electrolyte balance of the blood fluid are related to inadequate Na, K, Mg and Zn; as they are the most required elements of living cells.The two variants of M. alba were low in the minerals analyzed for which could seriously limit their uses as food suppliments.Only potassium (S 30 : 32.650 ± 0.07 mg/100 g and S 54 : 31.400±0.00 mg/100 g) was present in an appreciable level which Ayoola et al.
2163   which signified that the plant could be used in potassiumdeficient food.

Antimicrobial activities and minimum inhibitory concentration (MIC) of the extracts
Plants have been used as alternative medicine and tend to be safer than the chemical-based medicine.In the present study, M. alba, which basically serve as food for silkworm (B.mori L.) in Sericulture Unit, Forestry Research Institute of Nigeria, possessed some antimicrobial properties.

Table 2 .
Proximate analysis of M. alba.

Table 3 .
Mineral compositions of M. alba.
*Bacitracin and streptomycin as standard antibiotics