IN VITRO IMMUNE-MODULATORY POTENTIAL OF CRUDE EXTRACT OF LEAF OF ALBIZIA GUMMIFERA AGAINST STIMULATED PERIPHERAL BLOOD MONONUCLEAR AND RAW CELLS

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Introduction
Albizia gummifera is a native African herb located in Angola, Cameroon, Democratic Republic of Congo, Ethiopia, Kenya, Madagascar, Nigeria, Tanzania, Uganda, Zambia and Brazil -where it does not naturally grow.Albizia gummifera inhabits forests (low-or upland rain-forest) as well as open locations near forests.It is a 4.5 -30 m large deciduous tree, with branches apexing to a flat top.The plant is known by different names in Swahili such as mshai, mkenge and mchai mbao, and peacock flower in English.The name gummifera refers to the gum that is produced by the plant when the bark is cut (Orwa et al., 2009).
The anti-bacterial and immune-modulatory potential of various medicinal plants in Limpopo Province, South Africa has been reported for activity on leukocyte function in numerous cell lines in vitro by co-workers in the Department of Biochemistry, Microbiology and Biotechnology (BMBT), University of Limpopo (Masoko et al., 2010).New species used by traditional herbalists for treating various ailments in our province have been acquired by colleagues in the Department of BMBT (Mokgotho, et al., 2013).Out of the six plant species screened, we here report on the inflammatory potential of A. gummifera which was shown to exhibit activity in our experimental models of immunepharmacology.
There is presently an increase in research activities aimed at identifying anti-inflammatory agents.Our efforts and focus for pursuing this research activity are to identify and show proof of activity as well as expanding the ethno-botanical library of anti-inflammatory medicinal agents.

Chemicals and Reagents
Unless otherwise indicated, all chemicals and reagents were obtained from Sigma-Aldrich Co. (St.Louis, MO, USA).

Plant leaf and extraction procedure
Leaves of A. gummifera were collected from White River, Mpumalanga Province, South Africa in March 2009.The accession number UNIN 11 937 of the specimen was deposited in the University of Limpopo Herbarium.The leaf material was air-dried and extracted using acetone according to the Soxhlet extraction method (Ehrman, 1994).The plant powder (20 g) was extracted in 200 ml of acetone (w/v).

Phytochemical analysis using chemical methods
Phytochemical analysis was performed on the extract to identify chemical constituents using standard methods (Singleton et al., 1999).

Test for tannins
Water (5ml) was added to 0.5g of plant extract and heated in a water bath.The sample was cooled and to it, few drops of 0.5% iron III chloride (FeCl 3 ) was added.The appearance of green or dark blue precipitate indicated a positive reaction for the presence of tannins.

Test for alkaloids
Sample (2g) was heated in a test tube containing 25ml of 1% HCl for 15 minutes in boiling water bath.The suspension was then filtered and 5 drops of Meyer's reagent (potassium tetraiodomecurate) was added to the filtrate.The appearance of a precipitate indicated a positive reaction for the presence of alkaloids.

Test for saponins
Plants leaf extracts (0.5g) was suspended in 5ml of water and heated for 5 minutes.The sample was cooled at room temperature and agitated for 10s.The absence of persistent foam indicated a positive reaction for the absence of saponins.

Test for triterpene and steroids
Sample (0.5g) was dissolved in 3ml of chloroform and few drops of acetic anhydride and concentrated H 2 SO 4 was added.The absence of two layers upon the addition of H 2 SO 4 indicated a positive reaction for the absence of both triterpene and steroids.

RAW 264.7 macrophage cell culture
Cells were obtained from Highveld Biologicals (Johannesburg, South Africa).These cells were grown in RPMI 1640 supplemented with 10% FCS, penicillin (100U/ml) and streptomycin in a humidified atmosphere of 5% CO 2 at 37 • C .

Preparation of PBMC from human peripheral blood
PBMC were obtained by collecting venous blood (8ml) into specialised BD Vacutainer® CPT™ tubes according to the manufacturer's instructions (Beckton Dickinson, Johannesburg, South Africa).

Determination of cellular proliferative effects of leaf extract
After incubating the RAW 264.7 and PBMC cells (2 x 10 6 cells/ml) for 72 hours with or without leaf extract (5-160µg/ml) and stimulating with PMA (25ng/ml) and fMLP (1µM), proliferative activity was determined employing WST-1 assay using a commercial kit according to the manufacturer's instructions (Roche Diagnostics, Johannesburg, South Africa).

Determination of nitric oxide production by RAW cell line
Cells were incubated for 24 hours after treatment and nitric oxide measured as its end-product, nitrite, in the supernatant using the Griess assay (Bogdan, 1998).Briefly, an aliquot of 100µl of culture supernatant of RAW cells was mixed with 100µl of Griess reagent (one part of 0.1% n-(1-naphthyl) ethylenediamine dihydrochloride in water and one part sulphanilamide in 5% H 3 PO 4 ).The mixture was incubated for 10 min at room temperature in the dark and absorbance measured at 540 nm using a microplate reader (Beckman Coulter, Johannesburg, South Africa).

Determination of IL-2 production by PBMC
After treatment of cells with crude extracts for 24 hours, IL-2 production in the culture medium was measured using an enzyme-linkedimmunosorbent assay (ELISA) kit (Whitehead Scientific, Johannesburg, South Africa) according to the manufacturer's instructions.

Statistical analysis
Experimental data (repeats of 3-4 experiments performed in triplicates) was processed and analysed with GraphPad Prism Software Version 10.00 and data expressed as mean ± SEM.Statistical analysis was performed by the Student 's t test with p<0.05 considered significant.

Results
Phytochemical tests showed that the acetone extracts tested positive for flavonoids, saponins and tannins.The results of the proliferative activities of A. gummifera leaf extract on PBMCs and RAW cells are shown in Figures 1 and 2 respectively.The effects of the extract on IL-2 production by PBMCs and nitric oxide production by RAW cells are shown in Figures 3 and 4

Discussion and Conclusion
The RAW and PBMC cells were incubated with crude plant extracts of A. gummifera ranging from 5µg/ml to 160µg/ml for 72 hours.The extent of inhibition or promotion of cell growth was assessed using WST-1 assay.The results, as shown in Figure 1 and 2, indicates a dosedependent cytotoxic effect on PBMC cells and growth stimulating effect on RAW cells respectively.The observed effects were shown to be evident and significant at concentrations of 40µg/ml for PBMC cells and 20µg/ml for RAW cells and higher (p < 0.05).The observed effects may partly be related to the ability of the extract to inhibit or promote the leukocyte lipid biosynthetic lipo-and cyclooxygenase pathways, since these metabolic pathways have been implicated in the release of inflammatory mediators such as histamine, prostaglandins and leukotrienes (Serhan, 2007).
The plant extract was also shown to significantly inhibit IL-2 production by PBMCs at a concentration of 20µg/ml (p < 0.05), as represented in Figure 3. Interleukin-2 has been shown to be one of the markers of T cell activation (Boyman and Sprent, 2012), the presence of which confirms the anti-proliferative activity of the leaf extract of A. gummifera in this study.The extract was also shown to significantly (p < 0.05) promote the production of nitric oxide by RAW cells as shown in Figure 4. RAW cells are mouse derived macrophage cell line and the production of nitric oxide by phagocytes is used as a weapon against microbial pathogens (Marcinkiewics, 1997).The result is also concomitant with the growth-promoting activity of the extract shown in Figure 2.
The current findings are preliminary and further extensive research activities will be conducted with the aim of isolating and screen of chemical entities isolated from the extracts for immuno-pharmacological profiling as well as identifying the biochemical pathways involved in the pro-and anti-inflammatory activities of the extract. respectively.

Figure 1 :
Figure 1: Inhibition of cell proliferation by leaf extract of A. gummifera on PBMCs in vitro.Results are expressed as the mean percentage inhibition of cell proliferation by the extract (5 -160µg/ml) plus SEM of four different experiments conducted in tripliccates.

Figure 2 :
Figure 2: Stimulation of cell growth by leaf extract effect of A. gummifera of RAW cells in vitro.Results are expressed as the mean percentage promotion of cell proliferation by the extract (5 -160µg/ml) plus SEM of four different experiments conducted in triplicates.

Figure 3 :
Figure 3: Effects of leaf extract of A. gummifera on the production of IL-2 by PBMCs in vitro.Results are expressed as the mean percentage of IL-2 production by the extract (5 -160µg/ml) plus SEM of four different experiments conducted in triplicates.

Figure 4 :
Figure 4: Effects of leaf extract of A. gummifera on the production of nitric oxide by RAW cells in vitro.Result is expressed as the mean percentage of nitric oxide production by the extract (5 -160µg/ml) plus SEM of four different experiments conducted in triplicates.