Evaluation of Biological Activities of Extracts and Chemical Constituents of Mimusops elengi

Purpose: To isolate some compounds from the leaves and bark of Mimusops elengi, and examine them for their antibacterial and anti-inflammatory properties. Experimental: The compounds were isolated from the leaf and bark chloroform extracts using column chromatography, and characterized using physical and spectroscopic methods. The isolated compounds and their respective extracts were tested for antibacterial activity by micro-dilution antibacterial assay, and for anti-inflammatory activity by cyclooxygenase inhibitory assay. Results: of the compounds isolated include spinasterol (1), ursolic acid (2) and 3β, 6β, 19α, 23tetrahydroxyurs-12-en-28-oic acid (3) from the leaves; and taraxerol (4) and spinasterol β-Dglucopyranoside (5) from the bark. A majority of the samples showed good activity against Staphylococcus aureus (9.7 ─ 78.0 μg/mL), while moderate activity was observed against Gramnegative bacteria (78.0 ─ 156 μg/mL). Strong COX inhibition was observed for the leaf extract, and (1); selective COX-2 inhibition for (2) and (3); and selective COX-1 inhibition for bark extract, (4) and (5). Conclusion: This is the first report describing the anti-inflammatory potential of M. elengi on the basis of its isolated constituents. The results of this study support the traditional use of the plant as antibacterial and anti-inflammatory remedy.


INTRODUCTION
The family Sapotaceae comprises 35 -75 genera and approximately 800 species distributed pantropically with a few species in temperate regions.The genus Mimusops is native to the tropical parts of Africa and Asia.Mimusops elengi L. is an evergreen tree 30 feet tall, with a greyish brown fissured bark, wavy and dull green leaves, oblong berry fruit and creamy fragrant flowers.It is distributed in tropical and subtropical regions [1][2][3].
Different parts of the plant are reported to be used in traditional medicine for the treatment of microbial diseases such as diarrhoea, gum diseases, sore mouth, stomaches, ulcers, wounds and inflammation [1,3,[7][8][9].
Previous studies on M. elengi were mainly focused on the isolation and characterization of its phytochemical constituents, mostly from the stem bark, fruit, seeds and roots [10].Moreover, most of the biological activities reported for M. elengi were on the extracts rather than the active constituents which might be responsible for its observed activities [1,9].This paper reports the isolation and characterization of five constituents of the leaves and stem bark of M. elengi as well as the antibacterial and anti-inflammatory activities of the crude extracts and their isolated constituents.
Previous phytochemical investigations of M. elengi had revealed the presence of 1 in the bark, heart-wood and seeds [11], 2 in the bark and fruit [12], and 4 in the roots and bark [12] (see Figure 1).Jahan et al. [4,12] had also reported the occurrence of 1 and 2 in M. elengi but the plant part they examined was not specified.
The present study showed conclusively the occurrence of 1 and 2 in the leaves, and confirmed the presence of 4 in the bark.Interestingly, 3, previously reported from Adina rubella, Dischidia esquirolii and Guettarda grazielae [13] and found in the leaves of M. elengi in the present study, has hitherto not been reported in a member of the Sapotaceae.Compound 5 was found for the first time in the bark of M. elengi although the C-3 epimer of 5 has been reported previously for this plant [11].

EXPERIMENTAL Plant material
The plant material was collected in April 2009 from trees growing in the campus of Universiti Sains Malaysia and identified by one of the authors (F.A.).A voucher specimen (USM 9255) was deposited in the herbarium of the School of Biological Sciences, Universiti Sains Malaysia.

Identification of isolated compounds
Infrared spectra were recorded on a Perkin-Elmer 1330 spectrophotometer.Nuclear Magnetic Resonance spectra were obtained with a Bruker Avance 400 MHz spectrometer.Electron Impact and Fast Atom Bombardment mass spectra were recorded using an Agilent 5975C MSD and a Thermo Finnigan MAT95XL mass spectrometer, respectively.

Micro-dilution antibacterial assay
Serial dilution technique [14] using a 96-well micro-plate, was employed to determine minimum inhibitory concentration (MIC) as a measure of antibacterial activity.Two-millitre cultures of two Gram-positive bacteria, namely, Bacillus subtilis (ATCC6633) and Staphylococcus aureus (ATCC12600), and three Gram-negative bacteria, namely, Escherichia coli (ATCC25922), Klebsiella pneumoniae (ATCC13883) and Pseudomonas stutzeri (ATCC17588), were separately prepared and placed in an incubator overnight at 37 °C.The overnight-cultures were diluted with sterile nutrient broth (Merck) (500 μL bacteria/50 mL broth) to yield density of bacterial cells range of 10 5 -10 6 cell mL -1 .The isolated compound and crude extract samples under investigation were re-suspended to a concentration of 5 mg mL -1 with ethanol to yield a final concentration of 1.25 mg mL -1 in the assay for the first well.For each of the five bacteria used, 100 μL of the tested samples were serially diluted two-fold with 100 μL sterile distilled water in a sterile 96-well microplate.A similar two-fold serial dilution of gentamicin sulphate (1 mg mL -1 , Sigma) was used as positive control against each bacterium.One hundred μL of each bacterial culture were added to each well of the test samples, gentamicin sulphate (positive control), ethanol, water and nutrient broth (negative controls).The plates were covered and incubated overnight at 37 °C.To indicate bacterial growth, 50 μL of 0.2 mg mL -1 p-iodonitrotetrazolium violet (INT, Sigma) was added to each well and the plates incubated at 37 °C for 30 min.Bacterial growth in the wells was indicated by red colour, while clear wells indicate inhibition by the test substances.This assay was repeated three times.

Cyclooxygenase inhibitor screening assay
Evaluation of the anti-inflammatory activity of the isolated compounds was based on the inhibition of prostaglandin biosynthesis.This was assessed using COX inhibitor screening assay kit (no.560131; Cayman Chemicals, USA).The assay directly measures PGF 2α by SnCl 2 reduction of COX-derived PGH 2 produced in the COX reaction.This assay is based on the competition between PGs and a PGacetylcholinesterase conjugate (a PG tracer) for a limited amount of PG antiserum.Because the concentration of the PG tracer is held constant while the concentration of PG varies, the amount of PG tracer that is able to bind to the PG antiserum will inversely be proportional to the concentration of PG in the well.
The plate was washed to remove any unbound reagents and then Ellman's reagent (Sigma) was added to the well to yield a yellow colour.The intensity of the colour was determined spectrophotometrically at 420 nm using Micro Plate Reader.The assay for obtaining 100 % COX activity was performed with ethanol as solvent control.The test samples and indomethacin (positive control) (Sigma) were redissolved in ethanol at a concentration of 10 mg mL -1 to yield final concentrations of 5, 2.5 and 1.25 µg mL -1 .The pre-incubation time between enzyme and inhibitor was 10 min with 2-min incubation in the presence of arachidonic acid at 37 °C.Enzyme control was performed with COX-1 and 2 that had been inactivated by placing them in boiling water for 3 min.Inhibition of PGE 2 production by the test compounds and indomethacin was calculated from the standard curve using Graph Pad Prism software, version 3.00 for Windows.IC 50 values were calculated from the concentration-inhibition response curve by regression analysis using Graph Pad prism software.The values reported are the mean of triplicate experiments

Statistical analysis
The data obtained from all experiments were expressed as mean ± standard error (SEM).Statistical difference between treatments and control were evaluated by one-way analysis (ANOVA) followed by Tukey's multiple comparison test.p < 0.5 was considered to be significant.[18].

Antibacterial activity
The antibacterial activity of the leaf and stem bark extracts, and isolated compounds 1 -5, based on minimum inhibitory concentration (MIC), is shown in Table 1.Varying levels of activities were observed for the test samples against the five bacterial strains employed (Table 1).

Cyclooxygenase inhibitor screening activity:
The inhibition of prostaglandin biosynthesis are indicated in Table 2.The highest concentration used was 5 µg/mL.Therefore, all IC 50 values exceeding this value are expressed in the Table as > 5 µg/mL.All the isolated compounds were in accordance with the crude extracts in their antibacterial activity; all samples assayed showing reasonable activity against the tested strains.However, the observed activities of the leaf extract against Escherichia coli (156 μg/mL) and the bark extract against Staphylococcus aureus (9.7 μg/mL) were higher than the activity observed for the isolated compounds.This may be due to the synergistic effects of the bioactive agents in the crude extract.

CONCLUSION
The results obtained justify the reported antiinflammatory and antibacterial use of the plant in traditional medicine also and support its Ayurvedic medicinal use for ailments such as cephalalgia, diarrhoea, gum diseases, ulcers, sore teeth, stomach ache and wounds.Further study is needed to determine its mechanisms of action and structure-activity relationship.