Abstract: This study was aimed at evaluating the effect of methanol extract of Mangifera indica on phospholipase A2 from Naja nigricollis. Phospholpase A2 was first isolated from the venom of Naja nigricollis and then partially purified using ion-exchange and gel filtration chromatography. The inhibition effect of methanol extract of Mangifera indica kernel against PLA2 was investigated. The extracted PLA2 showed purification folds of 2.85 and 13.15 for ion-exchange and gel filtration chromatography respectively while the percentage yields are 44.12 and 70% for ion-exchange and gel filtration chromatography, respectively. Extract of Mangifera indica kernel showed peak inhibition of 66.67 and 90.00% on the partially purified PLA2 using arithmetic and geometric variations in the concentrations of the extract, respectively. The concentrations of the extract (inhibitor) at which 50% of the enzyme activity was inhibited (IC50) are 6.67 and 2.5 mg mL-1 for arithmetic and geometric progression, respectively. The double reciprocal plot of the enzyme in the presence of the extract at 8 and 32 mg mL-1 revealed KM values of 0.29 and 0.28 mg mL-1 and Vmax values of 6.70 μmoles min-1 and 4.5 μmoles min-1, respectively while KM and Vmax of 0.29 mg mL-1 and 7.6 μmoles min-1, respectively were obtained in the absence of the extract which suggests non-competitive inhibition. Phytochemical analysis of Mangifera indica kernel extract revealed the presence of alkaloids, carbohydrates, tannins, flavonoids, saponins and glycosides. The results of this study suggest that Mangifera indica extract is a potential anti-venom agent against Naja nigricollis.
INTRODUCTION
Snakes are elongate legless carnivorous reptiles of the suborder serpentes that can be distinguished from legless lizards. About 15 families have been recognized comprising of 456 genera and over 2900 species (Integrated Taxonomic Information System, http://www.itis.gov). Some snakes have developed specialized glands that produce venoms (Coppola and Hogan, 1992). These venoms are composed of complex mixture of active substances mainly peptides and proteins and some of the proteins present in these venoms include enzymes like PLA2 and metallo-proteases (Sallau et al., 2005).
Phospholipase A2 (PLA2) EC 3.1.1.4 are enzymes that release fatty acids from the second carbon group of glycerol. It usually recognizes the sn-2 acyl bond of phospholipids and catalytically hydrolyzes the bond releasing arachidonic acid and lysophospholipids (Fuly et al., 2002). They are amongst the most toxic venom constituents that perform highly regulated roles in lipid metabolism. The two major modes of toxicity expressed by venom PLA2 are neurotoxicity and myotoxicity (Sallau et al., 2005).
Treatments with anti-venoms are usually effective and recommended but it is not always accessible to rural dwellers necessitating the need to search for alternatives such as ethnomedicine (Otero et al., 2000). Plants have been used in the treatment of poisonous snake bites worldwide (Pereira et al., 1994). Some of the plant extracts like Mucuna pruriens, Parkia biglobosa, Aristolochia albida and many others have already shown positive effect against snake venoms (Alam and Gomes, 2003; Asuzu and Harvey, 2003; Abba et al., 2013).
Mangifera indica, a large evergreen tree native to tropical Asia and belonging to the family Anacardiaceae, has been used widely as source of food, medicine and timber. When used for medicinal purposes, different parts are considered. Its leaves have been used locally in the treatment of diarrhea and haemorrhages. This is usually based on the bioactive principle present in it.
The methanolic extract of the bark of Mangifera indica has been used for anti-bacterial studies and has proved effective in the treatment of dysentery, gastrointestinal tract infection and respiratory and urinary tract infection (Bala, 2006).
Though medicinal plants remain largely neglected and unrecognized in most parts of the world, study has shown that they are effective in the treatment and management of diseases associated with bacterial infection, snake bite and other diseases (Toth et al., 2003). Therefore, there is need to study the efficacy of the kernel of Mangifera indica against activities of partially purified PLA2 from venom on Naja nigricollis.
MATERIALS AND METHODS
Chemicals and reagent: Important reagents and chemicals such as DEAE-cellulose, Sephadex G-50, 3, 5- Dinitrosalicylic acid (DNS) reagent and methanol were obtained from Sigma Chemical Company, St. Louis, USA. All other reagents and chemicals were obtained from reputable chemical companies worldwide.
Venom collection: Freeze dried Naja nigricollis venom was a gift from Prof. M.S Abubakar of the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Nigeria.
Plant collection and identification: Fresh kernels of Mangifera indica were obtained from the Samaru market, Sabo Gari Local Government Area, Zaria and identified at the Herbarium unit of the Department of the Biological Sciences, ABU, Zaria.
Preparation of extract/phytochemical screening: Mangifera indica extract was prepared by crushing the air dried kernels into fine powder and then macerating for 72 h using a combination of methanol and water as solvent in a 1:1 ratio. A cream coloured powder was obtained after desiccation and the extract was expressed as dry weight and phytochemical screening was carried out by standard method of Evans (2009).
Phospholipase A2 (PLA2) assay: The PLA2 activity was assayed as described by Bhat and Gowda (1989). About 25 μL of 1 mg mL-1 L-α-lecithin substrate was incubated with 10 μL of the enzyme for 10 min at 37°C. The reaction was then terminated by immersing the tube in a boiling water bath for 2 min and the amount of released free fatty acid was measured titrimetrically at pH 8.0. The activity of phospholipase A2 was defined as the amount of enzyme that hydrolyzes 1 μmol of fatty acids from L-α-lecithin per minute under standard conditions.
Determination of total protein: Here, biuret reagent (4 mL) was added into various test tubes and different volumes of albumin dissolved in distilled water was added into biuret reagent and made upto 5 mL with distilled water in 5 different test tubes. Blank was devoid of albumin and incubated at room temperature for 30 min at 37°C. The absorbance was measured at 540 nm after incubation. The same procedure was carried out for crude venom but the venom was diluted further with distilled water to reduce the sensitivity of biuret reagent. For both standard and crude venom duplicates of the reactions were done and the mean obtained. Standard curve was used to extrapolate the value of protein present in crude venom.
Enzyme purification: Two milliliter of 10 mg mL-1 of crude N. nigricollis venom was loaded onto DEAE cellulose (1.3x36 cm) pre-equilibrated with 50 mM phosphate buffer pH 6.8. The column was eluted stepwise with NaCl gradient (0.01-0.3 M) at a flow rate of 4 mL min-1. Twenty five fractions of 4 mL were collected and assayed for phospholipase A2 activity and total protein. The PLA2 active fractions were pooled together and loaded on Sephadex G-50 column equilibrated with phosphate buffer (pH 6.5). The column was eluted with the same buffer, maintaining a flow rate of 2.5 mL min-1. Fractions were collected and assayed for PLA2 and total protein concentration.
Inhibition and kinetic studies: The total assay mixture composed of 100 μL of 0.02 M phosphate buffer (pH 6.8 containing 6 mM sodium chloride), 25 μL of phospholipase A2 and extracts at concentration from 2-10 mg mL-1 arithmetically and 2-32 mg mL-1 geometrically were incubated at 37°C for 10 min. After pre-incubation, the reaction was terminated with 1.0 mL 3, 5-dinitrosalicylic acid (DNS) reagent, placed in boiling water bath for 2 min, cooled to room temperature, diluted and the amount of released free fatty acid was measured titrimetrically at pH 8.0. The control reaction representing 100% enzyme activity did not contain any plant extract. The percentage inhibition was determined as follows:
The determination of the inhibitor concentration at which 50% inhibition occurred (IC50) was extrapolated from plots of percentage inhibition versus inhibitor concentration. The IC50 values were defined as the concentration of the extract, containing the phospolipase A2 inhibitor that inhibited 50% of the PLA2 activity.
To determine the pattern of inhibition, the enzyme was assayed with varying concentrations of the substrate (0.2-1 mg mL-1), L-α-lecithin in the presence and absence of the inhibitor. Initial velocity values obtained were then used to plot Lineweaver-Burk’s plot to determine the KM and Vmax.
RESULTS
The purification scheme of phospholipase A2 from Naja nigricollis venom is summarized in Table 1. The crude enzymes had a specific activity of 2.19 μmol min-1 mg-1 which increased to 6.29 and 28.80 μmol min-1 mg-1 after passing through DEAE- cellulose and DEAE Sephadex 50 column, respectively.
Preliminary qualitative phytochemical analysis was performed to determine the active ingredients present in the extract. The results showed the presence of different types of active constituents such as tannins, saponins, flavonoids, alkaloid, glycosides, anthraquinones and carbohydrates (Table 2).
Appropriately diluted plant extract was used for enzyme inhibition assay and the activity obtained with each extract was normalized to percent relative activity from which the percentage inhibition was calculated (Fig. 1). Less than 50% PLA2 inhibition was observed in extract concentrations of 2, 4 and 6 mg mL-1 for extracts for which arithmetic progression was employed while extract concentrations of 4, 8, 16 and 32 mg mL-1 exhibited inhibitions greater than 50% on PLA2 activity for extracts for which geometric progression was employed.
| Table 1: | Purification profile of PLA2 isolated from Naja nigricollis |
| Table 2: | Phytochemical constituents of methanolic extract of Manifera indica kernel |
| Fig. 1: | Percentage inhibition of phospholipase A2 using varying concentrations (arithmetic and geometric progression) of Mangifera indica extract |
| Fig. 2: | Inhibitor concentration at which 50% inhibition occurred (IC50) of the Mangifera indica kernel extract for the two separate variations of concentrations |
| Fig. 3: | Lineweaver-Burk plot of phospholipase A2 enzyme activity in the presence and absence of extract/inhibitor exhibiting non-competitive inhibition |
The highest inhibition activities were obtained with the extract concentrations of 8 and 32 mg mL-1. The IC50 values of the arithmetic and geometric concentration were 6.67 and 2.5 mg mL-1, respectively (Fig. 2).
Figure 3 shows the Lineweaver-Burk (double reciprocal) plot of phospholipase A2 in the presence and absence of the extract/inhibitor using the concentrations with the highest inhibitions (8 and 32 mg mL-1). The double reciprocal plot of the enzyme in the presence of the extract at 8 and 32 mg mL-1 revealed KM values of 0.29 and 0.28 mg mL-1 and Vmax values of 6.70 and 4.5 μmoles min-1, respectively while KM and Vmax of 0.29 mg mL-1 and 7.6 μmoles min-1, respectively were obtained in the absence of the extract which suggests non-competitive inhibition.
DISCUSSION
Understanding the characteristics of PLA2 from venom is a serious concern for venom researchers, as it would help in designing effective therapeutic anti-venoms. This study reveals that the isolation and partial purification of PLA2 from venom of Naja nigricollis increased in specific activity of the crude PLA2 after the two-step purification which could be due to the removal of synergistically interacting components of the venom. From this study, the high percentage yield of 70% obtained was higher when compared to ostrich pancreatic PLA2 and dromedary PLA2 which could be an indication that the purification steps used here are more appropriate for this enzyme (Bacha et al., 2007).
Even though, several plants have been shown to antagonize the snake venom action, only few are reported to be effective in inhibitory hemostatic disturbance induced by the venom which is a characteristic of Viperine and Elapidae envenomation (Borges et al., 2000; Mors et al., 2000; De Almeida et al., 2004; Hung et al., 2004). This study shows that the extract of Mangifera indica kernel has inhibitory effect on the phospholipase A2 which is in agreement with the study of Abba et al. (2013). The therapeutic potential of medicinal plants is traditionally attributed to classes of active constituents including flavonoids, alkaloids, sesquiterpenes, lignins and others (Soares et al., 2005). From our study, it was evident that extract of Mangifera indica contain flavonoid, alkaloid, tannin and saponin which could be responsible for the inhibitory effect, observed on the extract. The most likely mechanism of action of antagonist activity of the extract against PLA2 could be due to the binding of the PLA2 with flavonoid and alkaloid which have been reported by Soares et al. (2005) that flavonoid and alkaloid have shown to antagonize snake venom. Since the extract contains the presences of different active constituents, the mechanism of action could also be as a result of interaction of these different active constituents with PLA2 in bringing about inhibition.
The concentrations at which 50% of the enzyme has been inhibited (IC50), suggest that the extract could inhibit phospholipase A2 at much lower concentrations. A double reciprocal plot in the presence and absence of extract of Mangifera indica revealed non-competitive pattern which suggests that sites other than the active site could be involved in the inhibition (Abba et al., 2013).
CONCLUSION
The venom of Naja nigricollis has PLA2 activities which could be inhibited by the methanolic extract of Mangifera indica kernel extract in vitro.