Evaluation of bioactivity of stem bark extracts of Lovoa trichiliodes (Harm) and Trichilia heudelotii Planc (Harm)

This study evaluated the bioactivity of the stem bark extracts of Lovoa trichiliodes and Trichilia heudelotii using standard methods. The highest yield of 10.20% was obtained from the stem bark extract of T. heudelotii. Qualitative phytochemical examination of the plant extracts indicated the presence of different secondary metabolites which remarkably inhibited the growth of Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Salmonella typhii, Aspergillus flavus, Candida albicans and Candida glabrata. However, the cold water extract of L. trichiliodes exhibited no activity against the test pathogens. The highest mean (22.33±0.33 mm) zone of inhibition and minimum inhibitory concentration (MIC) of 2.5 mg/ml were exhibited by the acetone stem bark extract of L trichiliodes against B. subtilis ATCC6633. The results affirmed the traditional uses of the plants in the management and treatment of numerous diseases caused by the test pathogens.


Introduction
Traditional medicine has been used for thousands of years with great contributions made by practitioners to human health particularly as primary health care providers at community level [1].In Nigeria for example, herbal medicine is the first line of treatment for 60% of children with high fever from malaria, while 85% of Nigerians use and consult traditional medicine for health care, social and psychological benefits [2].
Medicinal plants have been playing a vital role in the health and healing of man and have been reported to possess various pharmacological activities like antibacterial and antioxidant [3].Interestingly, demand for medicinal plants is progressively rising in industrialized nations as well is in developing countries.Phytochemicals are the natural bioactive compounds found in plants as secondary metabolites that work with nutrients to protect against pathogenic attack [4].Amit and Hardeep reported that phytochemicals represent the most abundant and extensively distributed substances in the plant kingdom and that several plants and herb cells produce and gather this range of medicinal phytochemicals [5].
Lovoa trichiliodes (Harm) is the only West African species of the family Meliaceae that occurs in the thickest gallery forest and is commonly found in lakeside forest of Uganda.It is a large forest tree, up to 40 m high with a dark brown crown.The bark is grayish on younger tree, but brownish, thin and scally on older trees.The slash is reddish, cedar scented and produces a little sticky sap while Trichilia heudelotii Planc (Harm), also of the family Meliaceae, is found in understory of rain forest.The tree is rarely 4.0 feet high with dense crown and wide spreading branches.The bark is

Qualitative phytochemical screening
The extracts of the different plant parts were subjected to qualitative phytochemical screening for the presence of tannins, phlobatannins, saponins, flavonoids, steroids, terpenoids, glycosides, alkaloids, anthraquinones, chalcones and phenol using standard procedures as described by Harborne [9] and Sofowora [10].

In vitro antimicrobial susceptibility test
The extracts obtained from the test plants were screened against the test organisms by agar well diffusion method [11].A 25 ml aliquot of Mueller-Hinton agar (MHA, Lab Oratorios Britania, Argentinia) was poured into each Petri plate.When the agar solidified, test organisms were inoculated on the surface of the plates (1×10 6 cfu/ml and 1 x 10 6 sfu/ml for bacteria and fungi) respectively using a sterile glass spreader, allowed to set and punched with 6 mm cork borer.A portion of 50 µl of each of the extract concentrations was introduced into the wells.Control wells containing the same volume of 30% Dimethyl sulphoside (DMSO) served as negative control, while Chloramphenicol (100 µl) and Miconazole (100 µl) were used as positive controls for bacterial and fungal plates respectively.The tests were carried out in triplicates.Bacterial plates were incubated at 37 °C while fungal plates were incubated at 25 °C for 24 h and 72 h respectively.The diameters of the zones of inhibition were then measured in millimeters.

Minimum inhibitory concentration (MIC) assay
A modified two-fold serial dilution method of Essien et al., was employed [12].The extracts were prepared in Mueller-Hilton broth and Saboraud broth for bacteria and fungi respectively to achieve a decreasing concentrations ranging from the least concentration that produced clear zone of inhibition (10to 0.156mg/ml).All tubes including the controls were labeled accordingly.Each dilution was seeded with 1ml of standardized inoculums (1.0 × 10 6 cfu/ml for bacteria and 1.0 × 10 6 sfu/ml for fungi) incubated at 37 °C for 24 h and 25 °C for 72 h for bacteria and fungi respectively.A tube containing only seeded broth (i.e.without plant extracts) was used as the positive control while the un-inoculated tube was used as negative control.The lowest concentration of each extract sample that showed a clear zone of inhibition when compared with the controls was considered as the MIC.

Data Analysis
Data were presented as mean±standard error (SE).Significance difference between different groups was tested using two-way analysis of variance (ANOVA) and treatment means were compared with Duncan's New Multiple Range Test (DNMRT) using SSPS window 7 version 17.0 software.The significance was determined at the level of p≤ 0.05.

Results and discussion
The percentage yield of the extracts ranged from 3.90 to 6.53% and 9.63 to 10.20% for the L. trichiliodes and T. heudelotii stem bark acetone, ethanol and water extracts respectively.The differences in the yield obtained could be attributed to the polarity of the solvents and types of phytochemical present [13][14].
The present study showed that presence of phytochemical in the medicinal plant extract differs depending on the nature of solvent used for extraction.The tested plant materials revealed the presence of alkaloids, saponins, phenols, tannins, anthraquinone, and glycosides in their respective extracts (Table1).However, terpenes, cardenolides, and chalcones were completely absent except in the acetone stem bark extract of T. heudelotii.The presence of these various secondary metabolites in the plant materials justified their traditional uses in the treatment of various ailments and phytomedicines [15][16][17][18].Similar phytochemicals were also reported by Essama et   The response of the tested strains to the treatment with various plant extracts varied; as it was shown to be concentration dependent as greater inhibition was observed as the concentration of the extracts increased.This may be attributed to the differences in the concentrations and the types of phytocompounds of various secondary metabolites present in the extracts as well as the extracting ability of the solvents.The results also corroborated the observations of Bharet and Vidyasagar [21], Kashariet al., [22], Guerra-Boone et al., [23] and Opawale et al., [24].The study suggests that the stem bark extracts of L. trichiliodes and T. heudeulotii have a broad spectrum of antibacterial activity, although the degree of susceptibility differed between microorganisms.Similar findings were posited by Maragathavalli et al., [25] and Raja et al., [26] on Azadiractha indica extracts.The activity of the plant materials on C. albicans, C. neoforman and T. rubrum agreed with the work of Richa and Ayushi [27] who confirmed similar activity of natural products derived from plants against dermatophytes.Aladesanmi et al., [28] had earlier affirmed the broad activity of T. heudeulotii leaf solvent extracts on E. coli and P. aeruginosa.The zones of inhibition obtained were comparable with chloramphenicol and myconazole used as antibiotic positive standards for bacteria and fungi respectively.The results of the minimum inhibitory concentration (MIC) of the extracts as presented in Table 7 revealed that the antimicrobial activity of the extracts depended on the plant materials, extracting solvent concentrations and the tested microbial strains.Interestingly, it was discovered that B. subtilis, P. aeruginosa and T. rubrum as the most sensitive strains with the lowest MIC value of 2.5 mg/ml against the acetone stem bark extracts of L. trichiliodes and T. heudeulotii.This is closely followed by S. aureus, E. coli and K. pneumoniae with MIC of 5 mg/ml.The values of MIC obtained for the tested plant materials were lower than those reported by Yusha'u [29], Maragathavalli et al., [25] and Essien et al., [12] on similar medicinal plants.
Results were in agreement with the reports of Ram et al., [30] and Oladipoet al., [31].In regard to the results of this research work, it can be deduced that L. trichiliodes and T. heudeulotii are good sources of antimicrobial agents with interesting activity on versatile multi resistant strains which might be due is the presence of different phytocompounds.

Conclusion
The stem barks of the plants have the potential to act as a source of useful drugs due to the presence of the identified phytochemicals such as alkaloids, saponins, tannins, phenols, anthraquinone and glycosides.The plant materials exhibited wide spectrum antimicrobial activities, which justified their usage in folk medicine to treat ailments such as urinary and gastrointestinal tracts infections, candidiasis and dermatophytoses.They may therefore, be exploited for discovery as development of new therapeutic agents.

Table 4
Antimicrobial activity of T. heudelotii stem bark water extract on selected human pathogens Values are Mean±S.E.M (mm).Values followed by different alphabet along the rows are significantly different at p≤0.05, NI= No inhibition, N.A= Not applicable, Chl=Chloramphenicol, S.A= Staphylococcus aureus, E.C= Escherichia coli, E.F= Enterococcus faecalis

Table 7
The MIC of theextracts on the selected human pathogens (mg/ml)