|Year : 2012 | Volume
| Issue : 2 | Page : 105-109
Immunostimulatory, cytotoxic and antileishmanial activity of Mammea africana from Nigeria
Jude E Okokon1, Ashana Dar2, M Iqbal Choudhary2
1 Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria
2 HEJ Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan
|Date of Web Publication||20-Oct-2012|
Jude E Okokon
Department of Pharmacology and Toxicology, University of Uyo, Uyo
Introduction: Natural products from plants have been of great importance in the treatment of various disease conditions especially those involving reactive oxygen species. This study was aimed to investigate the cytotoxic, immunomodulatory and antileishmanial properties of stembark extract and fractions of Mammea africana sabine (Guttiferae) (syn. Ochrocarpus africana Oliv). Materials and Methods: The stembark extract and fractions of Mammea africana were investigated for cytotoxic activity against HeLa cells using the SRB method and DNA interaction activity using gel electrophoresis. Immunomodulatory activity of the extract in whole blood, neutrophils and macrophages was also investigated using luminol/lucigenin-based chemiluminescence assay. The extract and fractions were similarly screened for antileishmanial activity against promastigotes of Leishmania major in vitro. The GCMS analysis of the most active fraction against HeLa cells was carried out. Results: The stembark extract was found to exert significant cytotoxic activity with the dichloromethane fraction exhibiting the most pronounced effect. The crude extract and the fractions did not interact with DNA when investigated using electrophoresis. The extract prominently inhibited oxidative burst activity in whole blood, isolated polymorphonuclear cells (PMNs) and mononuclear cells (MNCs). The extract also exhibited moderate antileishmanial activity against promastigotes of Leishmania major in vitro. GCMS analysis of active fraction revealed pharmacologically active compounds. Conclusion: These results suggest that the stembark extract/fractions of M. africana possess cytotoxic, immunomodulatory and antileishmanial activities and can be exploited in primary healthcare.
Keywords: Antileishmanial, cellular antioxidant, cytotoxic, Mammea africana
|How to cite this article:|
Okokon JE, Dar A, Choudhary M I. Immunostimulatory, cytotoxic and antileishmanial activity of Mammea africana from Nigeria. J Nat Pharm 2012;3:105-9
|How to cite this URL:|
Okokon JE, Dar A, Choudhary M I. Immunostimulatory, cytotoxic and antileishmanial activity of Mammea africana from Nigeria. J Nat Pharm [serial online] 2012 [cited 2013 Dec 11];3:105-9. Available from: http://www.jnatpharm.org/text.asp?2012/3/2/105/102754
| Introduction|| |
Reactive oxygen species (ROS) have been implicated in the pathogenesis of many diseases in the body such as inflammatory diseases, cancer, AIDS, diabetes, ulcer and cardiovascular diseases.  Consequently, antioxidants that scavenge these reactive oxygen species and free radical are essential in the preventing the generation of these diseases associated with them. Owing to the preference for antioxidant from natural sources due to lack of side effect, there is a growing effort on research on plants as the sources of these desired compounds. However, pro-oxidant agents are of importance in infections  and natural products from plants have been explored for these activities.
Mammea africana sabine (Guttiferae) (syn. Ochrocarpus africana Oliv.) is a large forest tree of 50-100 feet high with bark often yellow with pale scales and resinous yellow sap.  The plant is widely distributed in tropical Africa. The stem bark of the plant is used traditionally by the Ibibios of Niger Delta region of Nigeria in the treatment of malaria related fever, diabetes, internal heat and microbial infections. The stembark is also used traditionally to treat stomach pains, rheumatism pains, scabies, cough and hypertension. , The chloroformic and ether stembark extract are reported to possess cytotoxic activity on cell culture.  Ouahouo et al.,  reported cytotoxic coumarins with antimicrobial activity against Staphylococcus aureus from the plant stembark. The stembark has been reported to posses antiplasmodial,  cardioprotective,  antidiabetic and hypolipidaemic,  vasorelaxant,  antihypertensive,  anti-inflammatory and analgesic,  antioxidant,  antidiarrheal and antiulcer activities.  The stembark has been reported to contain 5,-7-dihydroxy-8-(12-methyl-butryl) -4-N-Pentyl coumarins, ,, Mesuxanthone B.  Alkaloids have been reported to be absent in the entire plant parts. 
We, therefore, report in this study the cytotoxic, immunomodulatory and antileishmanial activities of the stembark extract of M. africana from Nigeria.
| Materials and Methods|| |
The plant material Mammea africana (stembarks) were collected in a forest in Uruan area, Akwa Ibom State, Nigeria in April, 2011. The plant was identified and authenticated by Dr. Magaret Bassey, Department of Botany and Ecological Studies, University of Uyo, Uyo, Nigeria.
The stembarks were washed and shade-dried for 2 weeks. The dried plants' materials were further chopped into small pieces and reduced to powder. The powdered material was macerated in 70% ethanol. The liquid filtrates were concentrated and evaporated to dryness in vacuo 40°C using rotary evaporator. The crude ethanolic extract (100 g) was further partitioned successively into 1 l each of n-hexane, dichloromethane, ethyl acetate and butanol to give the corresponding fractions of these solvents.
Cellular antioxidant activity
The ethanolic crude extract was screened for cellular antioxidant activities in whole blood, neutrophils and macrophages using chemiluminescence assay. Briefly, Luminol or lucigenin-enhanced chemiluminescence assay were performed as described by Helfand et al.,  and Haklar et al. Briefly, 25 μl diluted whole blood (1:50 dilution in sterile HBSS ++ ) or 25 μl of PMNCs (1×10 6 ) or MNCs (5×10 6 ) cells were incubated with 25 μl of serially diluted plant extract with concentration ranges between 6.25 and 100 μg/ml. Control wells received HBSS ++ and cells but no extract. Tests were performed in white 96 wells plates, which were incubated at 37°C for 30 minutes in the thermostated chamber of the luminometer. Opsonized zymosan-A or PMA 25 μl, followed by 25 μl luminol (7×10 5 M) or lucigenin (0.5 mM) along with HBSS ++ was added to each well to obtain a 200 μl volume/well. The luminometer results were monitored as chemiluminescence RLU with peak and total integral values set with repeated scans at 30-second intervals and 1-second points measuring time.
The growth inhibitory and cytotoxic activities of the ethanolic extracts and fractions were evaluated against HeLa cells (Cervix cancer cell) by using the sulforhodamine-B assay.  The cells (10000 cells/100 μl) in 96-well plate were incubated for 24 hours at 37°C in a humidified 5% CO 2 incubator. The stock solutions of ethanolic extract, fractions were prepared in DMSO. Various dilutions of the ethanolic extracts and fractions (0.1, 1, 10, 100, and 250 μg/ml), were added (100 μl) in each well. After 48 hours of incubation, 50 μl of cold TCA (50%) was added gently and left for 30 minutes at room temperature, followed by washing with distilled water and drying overnight. To each well, 100 μl of SRB solution (0.4% wt/vol in 1% acetic acid) was added and after 10 minutes; the unbound stain was removed by washing with acetic acid (1%), and air-dried at room temperature. The protein bound stain was solubilized with tris base (pH 10.2), and was shaken for 5 minutes. Absorbance was measured at 515 nm using a microplate reader. The absorbance of the appropriate blanks, including test substance blank, and control (without drug), was used to calculate the growth inhibition, and cytotoxicity of the test compounds, and represented as GI 50 , TGI and LC 50 (μg/ ml) values.
DNA interaction studies using gel electrophoresis
DNA protection assay was performed according to the protocol of Tian and Hua.  The reaction was carried out in an Eppendorf tube at the total volume of 15 μl containing 0.5 μg of pBR322 DNA in 3 μl of 50 mM phosphate buffer (pH 7.4), and 5 μl of tested samples (DCM fraction) at concentrations 0.1, 0.5, 1.0, 10, 50 and 100 μg/ml and standard drug, paclitaxel, 20 μg/ml. Then, the mixture was incubated at 37°C for 1 hour. The mixture was subjected to 1% agarose gel electrophoresis. DNA bands (open circular, supercoiled and linear) were stained with ethidium bromide and were analyzed qualitatively by scanning with Doc-IT computer program (VWR).
The antileishmanial activity of the extracts and fractions was evaluated against promastigotes of Leishmania major (DESTO) in culture using microplates. Leishmania major promastigotes were grown in bulk, early in a modified NNN biphasic medium, using normal physiological saline. Then the promastigotes were cultured with RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum (FBS). The parasites (Leishmania major) were harvested at log phase and centrifuged at 3000 rpm for 10 minutes. They were washed three times with saline at same speed and time. Finally the parasites were counted with the help of Neubauer chamber under the microscope and diluted with fresh culture medium to give a final density of 10 6 cells/ml. In a 96-well micro titer plate, 180 ml of the culture medium was added in different wells. The extracts and fractions were dissolved in PBS (Phospate buffered saline, pH 7.4 containing 0.5% MeOH, 0.5% DMSO) to make a stock concentration of 1000 mg/ ml. A total of 20 μl of each extract/fraction concentration was added to the wells and serially diluted to get working concentrations ranging between 1.0 and 100 μg/ml. A total of 100 ml of parasite culture (final density of 10 6 cells/ml) was added in all wells. Two rows were left, one for negative and other for positive control. Negative controls received the medium while the positive controls received Pentamidine and amphotericin B as standard antileishmanial compounds. The plate was incubated between 21 and 22°C for 72 hours. The culture was examined microscopically for cell viability by counting the number of motile cells on an improved Neubauer counting chamber and IC50 values of compounds possessing antileishmanial activity were calculated. 
GC-MS analysis of dichloromethane fraction
Quantitative and qualitative data were determined by GC and GC-MS, respectively. The fraction was injected onto a Shimadzu GC-17A system, equipped with an AOC-20i autosampler and a split/splitless injector. The column used was an DB-5 (Optima-5), 30 m, 0.25 mm i.d., 0.25 μm df, coated with 5% diphenyl-95% polydimethylsiloxane, operated with the following oven temperature program: 50°C, held for 1 minutes, rising at 3°C/min to 250°C, held for 5 minutes, rising at 2°C/min to 280°C, held for 3 minutes; injection temperature and volume, 250°C and 1.0 μl, respectively; injection mode, split; split ratio, 30:1; carrier gas, nitrogen at 30 cm/s linear velocity and inlet pressure 99.8 KPa; detector temperature, 280°C; hydrogen, flow rate, 50 ml/min; air flow rate, 400 ml/min; make-up (H 2 / air), flow rate, 50 ml/min; sampling rate, 40 ms. Data were acquired by means of GC solution software (Shimadzu).
Agilent 6890 N GC was interfaced with a VG Analytical 70-250 second double-focusing mass spectrometer. Helium was used as the carrier gas. The MS operating conditions were ionization voltage 70 eV, ion source 250°C. The GC was fitted with a 30 m×0.32 mm fused capillary silica column coated with DB-5. The GC operating parameters were identical with those of GC analysis described above.
The identification of components present in the various active fractions of the plants' extracts was based on direct comparison of the retention times and mass spectral data with those for standard compounds, and by computer matching with the Wiley 229 and Nist 21 Library, as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literature. ,
| Results|| |
Cytotoxic activity against HeLa cells
The results of cytotoxic activity of crude extract and fractions of M. africana show prominent activity with the hexane fraction exerting highest activity than other fractions and crude extract [Table 1]. The potency order was dichloromethane > butanol > ethylacetate > crude extract.
|Table 1: Cytotoxic activity of crude extract and fractions of root of Mammea africana against HeLa cells|
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DNA interaction activity
Gel electrophoresis results show that treatment of E. coli DNA with various concentrations of the dichloromethane fraction of M. africana did not produce any effect on the DNA. This effect was also observed with the standard drug used, paclitaxel [Figure 1].
|Figure 1: The effect of various concentrations of hexane fraction of Mammea africana on DNA interaction using gel electrophoresis|
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Cellular antioxidant activity
Ethanolic stembark extract of M. africana was observed to produce significant inhibitory effect on the oxidative burst activities of the whole blood, neutrophils and macrophages in a dose-dependent manner except at the lowest doses in which pro-oxidant activities were observed. The extract produced a maximum of 91.90% inhibition in whole blood, 97.50% in neutrophils when activated with zymosan-A, 77.20% in neutrophils when activated with PMA and 98.40% in macrophages [Table 2].
|Table 2: Cellular antioxidant activity of ethanolic stembark extract of Mammea africana|
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Crude extract and fractions of ethanolic stembark extract of M. africana exerted significant antileishmanial activity when tested against promastigotes of Leishmania major. Ethyl acetate fraction exerted a higher activity than other fractions and crude extract though uncomparable to the standard drugs, pentamidine and amphotericin B [Table 3].
The results of GCMS analysis of dichloromethane fraction of stembark extract of Mammea africana revealed the presence of pharmacologically active compounds as shown on [Table 4].
| Discussion|| |
Biological activities of Mammea africana have been reported to include antidiabetic, antiulcer, anti-inflammatory, anticancer and hepatoprotective among others. The pathogenesis of these diseases is linked to the generation of reactive oxygen species.  The antioxidant activity of M. africana has been reported to be associated with the presence of coumarins.  In this study, the stembark extract have been reported to possess cellular antioxidative activity in different cells of the body (whole blood, neutrophils, and macrophages) thereby inhibiting oxidative burst activities of these cells. These results correlate well with that of Nguelefack-Mbuyo et al.,  that used different models to test the activity and also isolated two coumarins; 4-phenylcoumarins and 4-n-propylcoumarins with significant antioxidant activity. The GCMS analysis revealed the presence of some phenolic compounds such as xanthones which have been implicated for many biological activities such as antioxidant, antitumoral, anti-inflammatory, antiallergy, antibacterial, antifungal, and antiviral activities. , It is probable that these compounds present in this plant may be responsible for the antioxidant activity of this plant reported in this study. The extract as well as the fractions especially dichloromethane fraction possesses a significant cytotoxic activity against HeLa cells in culture. Reactive oxygen species have been reported to be involved in the pathogenesis of cancer.  The activities of antioxidant counteract the redox state precipitated intracellularly and hence ensure cytotoxicity. The plant extract has been reported above to contain xanthones, antioxidant, and anticancer principles with reported activities against cancer cells. , These compounds may be responsible for the anticancer activity observed in this study. The study has also revealed that the extract did not cause any effect on the DNA as shown in the electrophoretic pattern similar to that of paclitaxel suggesting a mechanism different from interacting with DNA. Since the extract possesses a significant antioxidant activity, this may probably be one of the mechanisms of cytotoxic activity of this plant. The extract was also observed to possess antileishmanial activity on L. major. Antimicrobial activities are known to be promoted by proxidant state. In this study, lower doses of the extract have been observed to exhibit pro-oxidant activity. This activity has been reported to enhance antimicrobial activity.  Moreso, bioactive compounds such as xanthones which have been implicated in immune stimulation and antimicrobial activities have been reported above to be present in this extract. Xanthones have been reported to possess antileishmanial activity.  These compounds present in this plant may be responsible for the antileishmanial activity of this plant. This is the first report of antileishmanial activity of this plant.
| Conclusion|| |
From the results of these studies, it can be concluded that the stem bark extract of M. africana has cytotoxic activity against HeLa cells, antioxidative burst, and antileishmanial activities which are due to the phytochemical constituents of the extract and fractions.
| Acknowledgement|| |
Dr. Jude Okokon is grateful to TWAS for financial support for postdoctoral fellowship and ICCBS for providing research facilities
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[Table 1], [Table 2], [Table 3], [Table 4]