Humans have long consumed all edible parts of the Moringa tree and many uses for Moringa include alley cropping (biomass production), animal forage (leaves and treated seed-cake), biogas (from leaves), domestic cleaning agent (crushed leaves), blue dye (wood), fencing (living trees), fertilizer (seed-cake), foliar nutrient (juice expressed from the leaves), green manure (from leaves), gum (from tree trunks), honey and sugar clarifier (powdered seeds), medicine (all plant parts), ornamental plantings, biopesticide (soil incorporation of leaves to prevent seedling damping off), pulp (wood), tannin for tanning hides (bark and gum) and powdered seeds for water purification (Fuglie, 1999).

Coagulation-flocculation followed by sedimentation, filtration and disinfection, often by chlorine, is used worldwide in the treatment industry before the distribution of treated water to consumer (Edzwald et al., 1989; Kawamura, 1991). Aluminium and iron salts are the most commonly used coagulants in water treatment. The costs and environmental side effects of these compounds have increased interest in the use of organic coagulants derived from plant material, such as MO seed (Jahn, 1986; Sutherland et al., 1989; Muyibi and Evison, 1995). Mo extracts have been shown to have large effects on turbidity removal (92-99% reduction) (Jahn, 1986; Muyibi and Evison, 1995). In the water treatment industry, coagulation-flocculation is followed by floc separation, sedimentation and/or filtration and then by disinfection mostly with chlorine (AWWA, 1990). During the disinfection process by chlorine, organic matter can act as a precursor of trihalomethanes which may be carcinogenic (AWWA, 1990; Babcock and Singer, 1979; UEPA, 1991). In order to use MO seeds in modern water treatment systems therefore, the above mentioned problem should be addressed by sourcing for organic source of disinfection; possibly by usage of other parts of MO plant.

The antimicrobial activity of the MO leaf on bacteria (Kurup and Rao, 1954; Das et al., 1957), viruses (Abrams et al., 1993; Prazuck et al., 1993) and helminths (Fuglie, 1999) has been documented. However, the possibility of using MO leaf for the disinfection aspect of water treatment has been scarcely investigated. The present study was aimed at exploring the possibility of using MO leaf as means of disinfecting some water samples that are commonly used as potable water in Sagamu, Southwestern Nigeria. The phytochemical screening of the leaf extract was also carried out as well as the effect of the treatment process on the chemical attributes of the treated water.

MATERIALS AND METHODS

Collection and preparation of Moringa leaves: Twigs and floral parts of MO were collected from Jericho locality in Ibadan, Nigeria. The authentication was done by a Botanist at Forest Research Institute of Nigeria (FRIN), Ibadan, Nigeria. The voucher specimen was deposited in the herbarium of the Institute. The fresh leaves of MO were washed with sterile water and room dried at 28±2°C by spreading on top of clean table which has been previously swabbed with alcohol. The dried leaves were later blended in a Waring blender and stored in a sterile, covered container.

Phytochemical screening of MO leaves: Samples of MO leaves prepared as described above were subjected to qualitative phytochemical screening using standard methods described by Harborne (1998). The phytochemicals tested for include alkaloids, tannins, saponin glycosides, anthraquinone glycosides, free anthraquinones and flavonoids.

Water samples collection and treatment: Wide-mouthed bottles were autoclaved at 121°C for 15 min. They were used for collection of water from different sources within Sagamu Township. These sources include two different river sources designated River 1 and 2 and two different wells designated Well 1 and 2. Water samples were collected aseptically and transferred to the laboratory for treatment and subsequent analysis. Modified jar method was used for the treatment of the water samples. One gram of dried, blended MO leaves was added to 100 mL of each of the water samples, stirred for 5 min and allowed to stand for 1 h and then filtered. The filtrates subsequently called treated water samples were then subjected to bacteriological and chemical analyses.

Bacteriological analysis of water samples: Both raw and treated water samples were subjected to presumptive coliform count using standard methods described by APHA (1976). Tubes that showed both acid and gas production were considered to be positive. The Most Probable Number (MPN) of the coliform count from the result of the analysis was obtained from standard probability table. Bacteria profiles of both raw and treated water were determined by isolation of bacteria present using Pour plate method. Isolates were purified by repeated streaking, characterized by standard methods (Harrigan and McCance, 1976) and identified with reference to Bergey’s Manual (Sneath et al., 1986).

In vitro antimicrobial testing of MO leaf powder on bacterial isolates: Dried MO leaves (1 g) was mixed with sterile water (9 mL) and allowed to stand for about 1 h. The in vitro antimicrobial testing was done using seeded-cup method. Each of the bacterial isolates obtained from both raw and treated water samples above was sub-cultured into different peptone water and incubated for 24 h. A one-tenth milliliter of each bacterial suspension was taken and inoculated into different molten Nutrient agar. After solidification, two wells were bored on each and two drops of MO leaves extract were added into each well. The plates were incubated at 37°C for 24 h after which the degree of sensitivity of bacteria to the extract was observed and zones of inhibition measured.

Chemical analyses of raw and treated water samples: Dissolved Inorganic Phosphate (DIP) was analyzed using phosphomolybdate-blue complex method reported by Koroleff (1983). Dissolved Organic phosphate (DOP) content was analysed using the method of Rigler (1968) whereby hydrolysable DOP compounds were converted to orthophosphates upon contact with the acidic colorimetric reagents. Total Dissolved Phosphate (TDP) was estimated by summation of DIP and DOP. The determination of chloride in both raw and treated water samples was done by employing silver nitrate as titrant and potassium chromate as the end point indicator (AOAC, 1980). Sulphate status was determined using barium chloride solution as the titrant (AOAC, 1984).

Statistical analysis: Data were expressed as mean. The statistical significance of differences was assessed using analysis of variance. A two-tailed p value of less than 0.05 was considered to be statistically significant. Values that were significantly different were separated using Duncan’s Multiple Range test using SPSS for Windows ver. 11.0 statistical package.

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RESULTS

The result for screened phytochemicals of MO leaf is shown in Table 1. Tannins, saponin glycosides and Flavonoids were present in MO leaf while anthraquinone glycosides, free anthraquinones and alkaloids were absent. The result of coliform count expressed as Most Probable Number (MPN) of both raw and treated water samples is shown in Fig. 1. For both treated and untreated water samples, the coliform counts of river water were significantly higher (p<0.05) than the well water. Subsequent to water treatment with MO leaf powder, there was no significant reduction (p>0.05) of the coliform counts in the treated water samples.

In Table 2, the bacterial profiles of both treated and untreated water samples were indicated. Bacteria isolated from the raw water samples include Escherichia coli, Klebsiella spp., Staphylococcus aureus, Pseudomonas aeruginosa and Proteus mirabilis. However, sequel to the treatment of water samples with MO leaf powder, similar bacteria except Staphylococcus aureus, was isolated from treated samples. From this result it appears that of all the bacteria present in the investigated water samples, Staphylococcus aureus was the only bacterium that was sensitive to the phytochemicals that were present in MO leaf powder. Table 3 shows the result of in vitro antimicrobial testing of MO leaf extract on bacterial isolates of investigated water samples. The result obtained depicts that at the tested concentration, only Staphylococcus aureus’ growth was inhibited by MO leaf extract while other bacteria’s growth were not inhibited.

The chemical characteristics of both raw and treated water samples are shown in Table 4. Treatment of water samples with MO leaf powder did not remove both chloride and sulphate from treated water samples. The TDP contents of raw water ranged between 8 and 11 mg L^-1 while it rose to between 19 and 30 mg L^-1 in the treated water samples. However, it is noticeable that DOP constituted between 79 and 92% of TDP in the treated water samples while the values for the raw water were between 25 and 45%.

Table 1: Screened phytochemicals from water extract of Moringa oleifera leaf

+: Present, -: Absent

Fig. 1: Most Probable Number (coliform/100 mL) of raw water and water samples treated with Moringa oleifera leaf powder

Table 2: Bacteria profiles of raw water and water samples treated with Moringa oleifera leaf powder

Table 3: In vitro antimicrobial testing of Moringa oleifera leaf extract on bacterial isolates of investigated water samples

Table 4: Chemical characteristics of raw water and water samples treated with Moringa oleifera leaf powder

+: Present, *Phosphate values are means of triplicate determinations in mg L-1, Within column values with different superscripts letters are statistically significant using Duncan’s multiple range test (p<0.05)

DISCUSSION

Various chemicals such as alkaloids, tannins, saponins, cyanoglycosides, terpenoids, phenols, coumarins, peptides, oleic and stearic acids which were naturally present in plants have been implicated in the conferment of antimicrobial activities on plant containing them (Ingham, 1973; Osbourn, 1996; Abd Elrahman et al., 2003; Putheti and Okigbo, 2008). The presence of some of these phytochemicals in MO leaf extract may have been responsible for the anti-staphylococcal activity of MO leaf extract obtained in the present study. Previous studies have reported a relationship between phytochemicals and anti-staphylococcal activity of both aqueous and ethanol extracts of Dichrostachys cinerea (Kolapo et al., 2008). The antibacterial activity of root, bark, flower, gum and leaf of MO has been exploited in the traditional treatment of dental caries/toothache, syphilis, typhoid and urinary tract infection (Fuglie, 1999). Results from the present study depicts that the antibacterial activity demonstrated by MO leaf extract is far from being broad spectrum as only the growth of Staphylococcus aureus was inhibited while the growth of other four bacteria was not inhibited. This may not be particularly strange as antimicrobial activity of some plant extracts have been shown to be microorganism specific and concentration dependent (Akande and Hayashi, 1998).

Staphylococcus aureus is a dominant skin flora and is mostly the causative organism of boil on the skin. There are reports that poultice of MO are used by folks in the treatment of skin disorders (Fuglie, 1999; Udupa et al., 1994, 1998). Result from the present study is more or less giving the scientific basis to such folklore medicinal practice. In addition, the inability of MO leaf to effect disinfection in water treatment is also being established. Hence, the search for organic source of disinfection in water treatment process should be directed to other parts of MO or other medicinal plants with strong and broad spectrum antimicrobial activity.

Previous study on the coagulation of water by the use of both shelled and unshelled MO seed depicted that both sulphate and chloride contents of the treated water were not affected while the phosphate concentration was significantly increased (Ndabigengesere and Narasiah, 1998). Results of the present study are in agreement with this. Some anions such as chloride, phosphate and sulphate are acidic and when taken in high concentrations can alter the acid-base load of the body and obviously manifest as net acid load if the diet source does not provide basic cations (such as K, Ca, Mg) that could balance it, otherwise, this tends to produce low grade chronic metabolic acidosis and hence hypercalcuria, calcium is released from the bone and osteoclastic activity sets in (Berardi, 2003). Significant increase in phosphate concentration of treated water in the present investigation is therefore suggesting that using such treated water as potable water must be coupled with a diet that is rich in supply of basic cations.

ACKNOWLEDGMENT

The authors wish to thank Mrs. Blessing Caleb, former Chief Technologist of the Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Olabisi Onabanjo University, Sagamu, Nigeria for her technical and laboratory support.