Abstract: The numerous ethno-medicinal applications of Picralima nitida plants have called for a high thorough-put investigation of all the parts of the plant including the peel that is usually discarded in order to ensure maximum utilization of the plant. In this study, nutritional evaluation, phytochemical screening and antimicrobial effects of Picralima nitida peel were carried out using standard methods, in order to determine the potentials of this discarded part of the plant. The results of proximate contents indicated the following: moisture (49.6%), ash (16.0%), crude fibre (10.5%), crude lipid (7.4%), crude protein (28.4%) and carbohydrate (37.7%) while its calorific value is 265.8 kcal/100 g. Thus, the nutritional value of Picralima nitida peel is high and as such it could be used as feed additives. The results of phytochemical screening revealed the presence of flavonoids, saponins, tannins and alkaloids and the aqueous extract had antimicrobial activities against Escherichia coli and staphylococcus aureus with varying degrees. The most potent inhibitory effect was observed with Escherichia coli. These results have revealed that the peel and its extracts have pharmacological active compounds and antibacterial effects and as such could be used in ethno-medicine for the treatment of microbial infection and other ailments.
INTRODUCTION
The ethno-medicinal and nutritional use of wild plants are as old as men as they were sources of treatment, food security and income generation (Akubugwo et al., 2007a, b; Antia et al., 2006; Ifon and Basssir, 1979). These wild plants serve not only as indispensable constituents of human diet but also as important medicinal tools for the treatment of various disease conditions (Aguwu et al., 2010; Ogunnowo et al., 2010; Suresh et al., 2008; Okorondu et al., 2006; Edmonds and Chweya, 1997; Shills and Young, 1992). Traditional societies have over the years employed medicinal plants in ethno-medicine for the treatment of various diseases without scientific knowledge of the physiologically active ingredients called phytochemicals which were responsible for the plants’ medicinal and pharmacological potentials (Aja et al., 2010; Akubugwo et al., 2007a; Adimoelja, 2000). Lai (2004) reported the use of plant extracts from Ocimum gratissimum and Azadirachta indica as a substitute for chemical pesticide for control of Sigatoka disease of banana.
Similarly, Ichor and Ekoja (2011) have shown that the methanolic extract of the plant, Anogeissus leiocarpus (Guill and Perr) inhibited the growth of Salmonella typhi, Escherichia coli and Shigella sp. isolates to varying degrees. Their findings implied that the medicinal and antimicrobial potentials of plants are as a result of their active phytochemicals contents. Their reports which they attributed to the presence of active phytochemicals were consistent with the findings of other researchers (Joseph and Sujatha, 2011; Osadebe and Ukwueze, 2004; Nweze et al., 2004; Esimone et al., 1998; Ntiejumokwu and Alemika, 1991), who demonstrated the antimicrobial potentials of numerous plants.
Picralima nitida, family Apocynaceae, (common name: Akuamma plant, Igbo: Osi-Igwe) is a species occurring in African forest region, spread through Ivory Coast to Uganda (NNMDA, 2008). Picralima nitida bears white flowers (about 3 cm long) with ovoid fruits which at maturity are yellowish in colour. The leaves are broad (3-10 cm) and oblong (6-20 cm long) with tough tiny lateral nerves of about 14 to 24 pairs. The plant has wide varied applications in Nigeria herbal medicine (NNMDA, 2008). It has been shown to possess antiplasmodial, antimicrobial, anti-inflammatory, antipyretic, as well as anti-trypanosomiasis properties. Medicinally, the bark is used to prepare remedies to treat malaria and male sexual impotence, while the fruits are used for dysmenorrhoea and gastrointestinal disorder (Fakoye et al., 2000; Ezeamuzieji et al., 1994; Iwu and Klayaman, 1992; Ansa-Asamoah and Amposo, 1986; Arens et al., 1982).
Numerous works have shown the efficacy of Picralima nitida plants extracts against skin conditions of tinea pedis (Athletes foot), tinea capitis (ringworm of the head), tinea corporis (ringworm of the body) and Trypanosoma brucei (Ezeamuzieji et al., 1994; Wosu and Ibe, 1989). Also, the hypoglycemic effects of the bark and seed extracts have been documented (NNMDA, 2008). Several researchers (Fakoye et al., 2000; Ezeamuzieji et al., 1994; Iwu and Klayaman, 1992) among have reported the medicinal potentials of this plant. However, data on the nutritional potentials and the antibacterial properties of the part of the plant usually discarded as not useful (i.e., the peel) is lacking. This study therefore was designed to assess the nutritive and antibacterial potentials of Picralima nitida peel in order to fully harvest its utility.
MATERIALS AND METHODS
Sample collection: The Picralima nitida fruits were purchased from Eke-Ukwu market, Owerri, Imo State and identified by the Curator, Department of Science Laboratory Technology, Akanu Ibiam Federal Polytechnics, Unwana, Afikpo, Ebonyi State. The Picralima nitida fruits were thoroughly washed with tap water and the peels were removed and dried under the sun. The peels were grinded to powder using a mechanical grinder.
Proximate analysis: The recommended methods of the Association of Official Analytical Chemists (AOAC, 1999) were used for the determination of moisture, ash, crude lipid, crude fibre, crude protein and carbohydrate contents.
Estimation of energy value: The sample calorific value was estimated (in Kcal) by multiplying the percentage crude protein, crude lipid and carbohydrate by the recommended factor (2.44, 8.37 and 3.57, respectively) used in vegetable analysis (Asibey-Berko and Tayie, 1999).
Phytochemical screening: A portion of the dried and grinded Picralima nitida plant sample was subjected to phytochemical screening for the presence of flavonoid, saponins, tannins, glycosides, alkaloid and phenol using the method as outlined in Harborne (1984).
Preparation of aqueous extracts: The Picralima nitida peels were extracted by maceration through the addition of exactly 200 g of the powdered sample to 300 mL of distilled water. The container was well covered and shaken vigorously to mix very well and allowed to stand for 5 days with intermittently daily shaking. After the duration, the mixture was then filtered through four (4) fold of sterile cheese cloth to remove the coarse debris. The filtrate was transferred into sterile test tubes and used for antibacterial susceptibility testing.
Test organisms: The studies were performed with staphylococcus aureus and Escherichia coli. These bacteria were laboratory strains provided by the Microbiology Unit of the Department of Science Laboratory Technology, Akanu Ibiam Federal Polytechnic, Unwana, Ebonyi State, Nigeria. The microorganism were grown in nutrient broth (Biotec, Suffolk, UK) at 37oC and maintained on nutrient agar (Biotec, Suffolk, UK) slants at 4oC. The standardized cultures of the organisms were used throughout the experiment.
Preparation and impregnation of Picralima nitida disc: Disc of diameter 6.0 mm were punched from a sheet of Whatman No. 3 filter paper (UK) by a perforator and arranged in Petri-dishes allowing a distance of 2-4 mm between each of them. The disc was sterilized in an oven at 160°C for 15 min. The extract of Picralima nitida was diluted 4 fold to 0.1, 0.2, 0.4 and undiluted. After the discs were allowed to cool (attaining laboratory temperature), the Picralima nitida disc were separately impregnated with 0.1, 0.2, 0.4 and undiluted Picralima nitida aqueous extract. The impregnated disc was arranged in separate Petri-dishes and dried by placing them in an incubator at temperature 37°C for 2-3 h.
Bacterial sensitivity test: The level of susceptibility of each of the test organism was determined using agar disc diffusion method as outlined in Suresh et al. (2008).
Statistical analysis of data: Analysis of variance (ANOVA) for the data was carried out using SPSS window version 15.1 Chicago, USA and multiple comparison performed using LSD. p-values of <0.05 were considered statistically significant.
RESULT
Proximate composition of Picralima nitida peel: The results of proximate composition of Picralima nitida Peel is shown in Table 1. The results indicated that Picralima nitida Peels contain appreciable amount of nutrients: lipid (7.4%), protein (28.4%) and carbohydrate (37.7%) as well as moisture (10.5%) and Ash (16.0%).
| Table 1: | Proximate composition of Picralima nitida peel |
| Table 2: | Phytochemical screening of Picralima nitida peel |
| Table 3: | Level of dilution of extract and mean inhibition diameter (mm) |
| Fig. 1: | Variation on the sensitivity of Picralima nitida aqueous extract to E. coli and S. aureus at different levels of dilution |
Phytochemical screening of Picralima nitida peel: The results of phytochemical screening of Picralima nitida peel is shown in the Table 2. The result revealed the presence of flavonoids, saponins, tannins, alkaloids and glycosides while phenols were absent.
Zone of inhibition: The sensitivity pattern of E. coil and S. aureus on Picralima nitida Peel aqueous extract at 0.1, 0.2, 0.4 mL and undiluted is shown in Table 3. Figure 1 shows that the zone of inhibition of E. coli by Picralima nitida Peel aqueous extract when diluted at 0.1 mL dilution was significantly lower (p<0.05) than the zone of inhibition by extract at 0.2 mL dilution. Also the zone of inhibition of E. coli by the extract at 0.4 mL dilution was significantly higher (p<0.05) when compared with the zone of inhibition at 0.1-0.2 mL and the undiluted. The zone of inhibition of the Picralima nitida Peel aqueous extract to staph aureus at 0.4 mL dilution was significantly higher (p<0.05) than the zone of inhibition at 0.1 and 0.2 mL. There was no inhibition of Staph aureus by the undiluted extract.
DISCUSSION
Proximate and phytochemical analysis is very useful in the evaluation of some bioactive and biological components of fruits and other parts of plants. Evaluation of antimicrobial property of plant is necessary in the discovery of plants with medicinal potentials. This present study assessed the proximate, phytochemical and antimicrobial potentials of Picralima nitida peel.
The results (Table 1) indicated that Picralima nitida Peels had appreciable amount of nutrients. Similar appreciable nutritional values have been reported by other researchers for similar plants (Aja et al., 2010; Akubugwo et al., 2007a, b). However, they were variations which could be as a result of plant species, genotype and other environmental factors (Gardens et al., 1996; Ezeamuzieji et al., 1994). These results have shown that Picralima nitida Peels could serve as alternative source of feed for animals and could equally be consumed by man, after quality processing to remove any toxic components.
The results of the phytochemical screening revealed presence of flavonoids, saponins, tannins, alkaloid and glycosides while phenol was found to be absent (Table 2). These findings agreed with earlier reports (Azu and Onyeagba, 2007). However, Iwu and Klayaman (1992) reported absence of glycosides while Ezeamuzieji et al. (1994) reported the presence of phenol in their findings. This could possibly be attributed to the differences in the plant species and environmental condition (Fakoye et al., 2000). Epidemiological studies have shown that flavonoids intake, are inversely related to mortality from coronary heart diseases and other incidences of heart attacks (Shills and Young, 1992). The invaluable pharmaceutical properties reported for Picralima nitida Peel may be attributed to the presence of these bioactive compounds such as flavonoid (Aguwu et al., 2010; Adimoelja, 2000; Ezeamuzieji et al., 1994).
Our result (Fig. 1) indicated variable zones of inhibition of Staphylococcus aureus and Escherichia coli by the aqueous extract of Picralima nitida disc at different dilutions. The disc diffusion method for antibacterial activity showed significant reduction in bacterial growth in terms of zone of inhibition around the disc. Among bacterial forms tested, the diameter of inhibition zone of the aqueous extract of Picralima nitida peel was more sensitive to Escherichia coli than Staphylococcus aureus (Fig. 1).
The more diluted extract has the largest zone of inhibition, hence the more the extract is diluted the more the active ingredient required to inhibit the microorganism is liberated. Nkere and Iroegbu (2005) observed similar results. That there was no zone of inhibition of S. aureus when the extract was not diluted implicated that the bioactive ingredients in the peel is ineffective in the undiluted form. Similar results have been reported by several other researchers working independently on numerous other plants extract (Ichor and Ekoja, 2011; El-Shemy et al., 2007; Zumbes et al., 2007; Nascimento et al., 2000; Singleton, 1999).
Larger zone of inhibition was observed in E. coli than Staph aureus. This agrees with the work of Nkere and Iroegbu (2005) who reported inhibition zone of 10±1, for E. coli and 8±1.75, for Staph aureus by aqueous extract of Picralima nitida peel. The differences in the zone of inhibition may be directly related to the susceptibility of test organism to the Picralima nitida peel extracts. The factors responsible for the high susceptibility of E. coli than Staph aureus may be attributed to the differences in the organisms physiology and anatomy and the presence of secondary metabolites (Omogbai and Eze, 2011; Azu and Onyeagba, 2007; Iwu et al., 1992). The inhibition of the Picralima nitida peel extracts on S. typhi and E. coli. is of great importance to the health care system since it can be used as an alternative to orthodox antibiotics in the treatment of infections due to these isolate especially as they are becoming resistant to known antibiotics (El-Shemy et al., 2007; Nascimento et al., 2000; Singleton, 1999).
The antimicrobial activity of plant extracts is, possibly, due to their ability to complex with extra cellular and soluble proteins and to complex with bacterial cell walls disrupting microbial membranes (Neuwinger, 2000; Burkill, 1985). Therefore, the level of inhibition increased as the dilution increases. This could be due to increase in the liberation of the bioactive component by the solvent (Nkere and Iroegbu, 2005). This observation supports the hypothesis that the inhibition involves phenolic compounds which the results revealed is an integral component of Picralima nitida peel extracts, because phenolic compounds sensitize the phospholipid bilayer of the microbial cytoplasmic membrane causing increased permeability, unavailability of vital intracellular constituents (Joseph and Sujatha, 2011; Fu et al., 2007; Betoni et al., 2006; Li et al., 2005; Lopez et al., 2005; Arora and Kaur, 1999). Our observation in the present study supports other findings that Picralima nitida peels have nutritional values and antimicrobial properties and as such this Picralima nitida peel extracts promise to reduce the cost of obtaining health care since the plant is readily available and the cost of preparation is relatively cheaper.
CONCLUSION
Picralima nitida is nutritionally potent and can be used in the formulation of animal feeds. Picralima nitida peel aqueous extracts have antibacterial activities against the test bacteria used in this study. Susceptibility of the bacterial varied with the concentration of extract and the organism involved. Phytochemical screening shows that the sample contains useful bioactive components (saponins, tannins, alkaloids) that contributed to antibacterial properties and hence its widely acclaimed medicinal values.