Evaluation of Mango Seed Kernel Methanolic Extract on Metalloproteases in Carpet Viper ( Echisocellatus ) Venom : An in Vitro Experiment Peculiar

Background: The global incidence of snakebite has become a major concern to the community. This study aimed to evaluate the effect of mango seed kernel methanol extract on metalloproteases in Carpet Viper (Echis ocellatus) venom. Methods: Mango seed kernel methanolic extract was evaluated in vitro for its anti-venom activity and inhibition of metalloproteases of Carpet Viper's (Echis ocellatus) venom. Metalloprotease portion was partially purified from the venom of E. ocellatus with a yield of 71%, a purification fold of 2.63 and a specific activity of 19.00 μmol/min/mg protein. Results: The enzyme appeared as a band on SDS-PAGE with a molecular weight of 23 kDa. The kinetic properties of the enzyme showed a Km of 0.31 mg mL and a Vmax of 9.09 μmol min. When the enzyme was incubated with the extract, kinetic studies revealed a mixed noncompetitive pattern of inhibition with Km values of 0.56 and 1.11 mg mL and Vmaxvalues of 6.67 and 4.17-μmol min for 5% and 20% inhibitor concentrations, respectively. An estimated Ki value of 0.168 mg mL was obtained from a secondary plot demonstrating that the extract had a high affinity for the partially purified enzyme; thus, could serve as an effective inhibitor. Conclusion: Methanol extract of mango seed kernel has a high affinity for the partially purified enzyme, and it might provide an inexpensive and readily available alternative to sheep serum in the management of snakebite envenomation. Therefore, further in vivo studies are necessary to assess its effectiveness and safety.


INTRODUCTION
Snakebite is a common, devastating environmental, and occupational hazard, especially in rural areas of tropical developing countries like Nigeria.One of the most common poisonous snakes in Nigeria is West African carpet viper (Echis ocellatus) from the Viperidae family.It accounts for 90% of bites and 60% of the fatalities in this country, which add up to 20% of all African cases [1][2][3][4][5][6].In humans, envenomation by E. ocellatus causes severe blistering, edema, and necrosis at the bite site, and life-threatening systemic effects including hemorrhage, coagulopathy, hemotoxicity and occasionally hypovolemic shock [2,3,5].Snake venom metalloproteases (MPs) are common components in many snake venoms, especially in Viperidae and play key roles in envenomation.Mango (Mangifera indica Linn.) is one of the most important tropical fruits in the world, including Nigeria.The kernel content of the seeds ranges from 45.7% to 72.8% and comprises about 20% of the whole fruit depending on the variety [7].Mango seed kernel extract is very rich in polyphenols and has been pharmacologically documented to have antioxidant, anti-tyrosinase, anti-inflammatory, and hepatoprotective effects as well as anti-enzymatic activities against snakes' venom [8,9].
World Health Organization has mentioned snake bite as an ignored disease occurring mostly in Asia and Africa, with an estimated 5.5 million bites each year, resulting in 2.5 million envenomations and 125000 deaths worldwide [5]."Antivenom immunotherapy which has been the only specific treatment against snakebite envenomation is burdened with various side effects such as anaphylactic shock, pyrogen reaction and serum sickness" [10].
In addition, production of antiserums in animals is time-consuming, expensive and requires ideal storage conditions.In Africa, especially in Nigeria, the availability of these antivenoms is restricted, thus, limiting the use of serum therapy [11][12][13][14].The use of natural venom inhibitors could complement or substitute sheep serum anti-venoms and could minimize the sociomedical problems of snakebites in tropical countries.Mango seeds are readily available in Nigeria as agro-waste and might provide a cheaper and more accessible alternative for snake antivenoms.
This study therefore aimed to evaluate the effect of mango seed kernel methanolic extract on metalloproteases in Carpet Viper (E.ocellatus) venom

Snake Venom
The study was conducted between March, 2014 and June, 2015 at the Department of Biochemistry, Ahmadu Bello University (ABU), Zaria-Nigeria.Freeze-dried E. ocellatus venom was obtained from the Department of Pharmacognosy and Drug Development, Ahmadu Bello University (ABU), Zaria-Nigeria.
All procedures were in accordance with ethical guidelines for care and use of laboratory animals.The study was approved by the Experimental Animals Committee of Ahmadu Bello University, Zaria Nigeria.

Seed Kernel
Mangifera indica (mango) seeds were gathered from Zaria metropolis and identified in the Ahmadu Bello University herbarium Zaria -Nigeria.

Reagents
Sephadex G-75 and DEAE-cellulose were purchased from Sigma Chemical Co.St. Louis, MO, USA.All other chemicals were obtained from reputable chemical companies.

Preparation of Extract
The kernels were manually removed from the seeds' coat and then were sun-dried and grounded to powder.Then the extraction was carried out with methanol using soxhlet apparatus.Subsequently the solvent was removed by rotary evaporator under reduced pressure to obtain the crude extract.

Partial Purification of Metalloproteases Ion Exchange Chromatography on DEAE Sephadex
DEAE-cellulose was prepared by dissolving 2 g of anion-exchanger in 20 ml of phosphate buffer, pH 7.4.The slurry was then poured into a 3.0 X 20 cm column.Crude E. ocellatus venom (100 mg) was dissolved in 10ml phosphate buffer, pH 7.4 in a beaker.This was transferred to a centrifuge tube and the insoluble components were removed by centrifugation.Then 2 ml of the recovered supernatant was loaded onto DEAE Sephadex column (3.0 x 20 cm) pre-equilibrated with 0.2 M phosphate buffer pH 7.4.The column was eluted stepwise with NaCl gradient (0.0 -0.5 M) at a flow rate of 1ml per minute.Thirty fractions were collected and assayed for metalloprotease activity and total protein.The fractions showing highest specific activities were pooled.

Gel Filtration Chromatography on Sephadex G-75
The gel was prepared by dissolving 2 g of Sephadex G-75 in 20 ml phosphate buffer, pH 7.8 for 24 h at room temperature and then mixed with a glass rod to float the swollen particles from the slurry.The slurry was then poured into a 2 by 100 cm column packed with glass wool at the bottom.The column was first equilibrated with phosphate buffer, pH 7.8 before the sample was applied.The pooled metalloprotease active fractions from the ion exchange chromatography were loaded onto Sephadex G-75 column equilibrated with phosphate buffer (pH 7.4).The column was eluted with the same buffer, maintaining a flow rate of 1ml/min.
The most active fraction was subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis according to the method of Laemmli, [15] and was prepared with silver staining.http://www.ijt.ir;Volume 11, No 5, September-October2017

Metalloprotease Assay
Metalloprotease assay was carried out according to an earlier study [16].In brief, 0.5 ml of 20 mg/ml enzyme solution with 0.05 ml of 1 mg/ml casein solution in 0.2 M phosphate buffer pH 7.5 was incubated for 30 min at 50 °C.The reaction was stopped by adding 1.0 ml of 10% TCA and the absorbance of TCA-soluble peptide was measured at 280 nm [17].A control assay, without the enzyme in the reaction mixture, was also carried out and used as the blank in all spectrophotometric measurements.

Effect of the Extract on the Partially Purified Metalloproteases
Varying concentrations of the extract preparations (0, 5, 10 and 20% w/v) were used as substrate to inhibit the partially purified metalloproteases.The initial velocity data obtained was used for double reciprocal plots and a secondary plot was obtained from the primary plot to determine the inhibition binding constant (K i ) of the extract.

Purification of Metalloprotease Enzymes from Echis Ocellatus Venom
The results of partial purification of metalloproteases from E. ocellatus venom are summarized in Table 1.The crude extract contained about 1.10 mg of protein with total and specific activities of 10.17-µmol/min and 9.25 µmol/min/mg of protein, respectively.Fractionation of crude venom on DEAE cellulose chromatography produced a specific activity of 11.71 µmol/min/mg.Subsequent gel filtration on Sephadex G-75 chromatography demonstrated an active peak (Figure1) with a specific activity of 19.00 µmol/min/mg of protein, 2.63 purification fold, and 71% recovery.
The elution profile of MPs from ion exchange chromatography on DEAE Cellulose showed four major peaks as shown in Figure 2.These fractions had the highest metalloprotease activity when the eluates were assayed for activity with casein and thus were pooled for the next purification step.
The elution profile of MPs on Sephadex G-75 pooled from DEAE Cellulose active fractions showed one prominent peak with the highest metalloprotease activity as shown in Figure 1.When the eluates were subjected to treatment with the active fraction of the mango seed kernel extract, metalloprotease activity was significantly reduced, suggesting that the active fraction of mango seed kernel extract had some antimetalloprotease activity against Echis ocellatus venom.
The purification profile (Table 1) showed that the metalloprotease specific enzyme activity for crude venom was 9.25 µmol/min/mg proteins.However, when it was subjected to purification steps, the specific enzyme activity increased to 19.00 µmol/min/mg proteins with a total yield of 71%.
The double reciprocal plot of partially purified MPs obtained from E. ocellatus venom demonstrated that the enzyme had an estimated K m of 0.31 mg/ml and V max of 9.09-µmol/min (Figure 3).

Result of SDS-PAGE for Metalloprotease
The purity and the molecular weight of the partially purified inhibitory metalloproteases were determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).Figure 4 shows the electrophoretic pattern of the sample under denaturing conditions.A faint band of the protein sample was visualized against the standard marker proteins on the gel and the molecular weight of partially purified metalloproteases (lane 3 and 4) was estimated to be 23 kDa.

Lineweaver-Burk and the Secondary Plot of the Inhibition of Partially Purified Metalloprotease Activity by the Extract
The Lineweaver-Burk (double reciprocal) plot of the inhibition of metalloprotease activity by the most active fraction of the mango seed kernel extract in the presence of the substrate suggested the presence of non-competitive inhibitory effects with K m values of 0.56 and 1.11 mg mL -1 and V max values of 6.67 and 4.17 µmol min -1 for 5% and 20% inhibitor concentrations, respectively.Whereas, the solution with 0% of inhibitor showed a K m of 0.31 mg mL -1 and a V max of 9.09 µmol min -1 .The secondary plot of the Lineweaver-Burk plot showed an estimated K i (inhibition binding constant) value of 0.168 mg mL -1 (Figure 5 and 6).

DISCUSSION
The global incidence of snakebites and their associated mortality are overwhelming and the majority occurs in rural areas of resource-poor countries; therefore, many victims do not seek hospital treatment but prefer traditional remedies [18,19].Plant extracts have been traditionally used as folk medicine in the treatment of snakebites all over the world [8,9,12,20], which is of particular importance especially in resourcepoor countries where antivenin is not readily available.As a result, efforts are being made by research groups to identify more traditionally available plant extracts that could curb the menace of snakebite envenomation [18,20].
Thus, this study has been carried out to evaluate the potency of mango (M.indica) seed kernel methanolic extract and its different solvent fractions in ameliorating snake envenomation.Crude methanolic extract of mango seed kernel has significant inhibitory activity against metalloproteases present in the venom of E.ocellatus, believed to be the major component of venom from the Viperidae family, and is responsible for the anticoagulant effect, which is the primary cause of mortality in snakebite victims.The methanolic extract of Giurera senegalensis inhibited metalloprotease and phospholipase A 2 present in E. ocellatus's venom [21].Mango's methanolic extract has inhibitory effect on enzymatic activities of Naja nigricollis venom [9].In addition, Kapok tree (Ceiba pentandra) leaves extract neutralizes E.ocellatus venom [22].
In this study, metalloproteases were partially purified from E. ocellatus venom with an increase in purification from 1.27 to 2.63 folds.The specific activity increased from 11.71 µmol/min/mg proteins to 19.00 µmol/min/mg proteins.These findings are similar to another study where an increase in purification fold and specific activity of the crude venom metalloprotease after the two purification steps could be attributed to the removal of other synergistically interacting components of the venom [23].The partially purified metalloproteases showed a distinct band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with an estimated molecular weight of 23 kDa.This result is also in line with the work of Gomes et al [24] that a metalloprotease (BthMP) from Brazilian lance head (Bothrops moojeni) venom with a molecular weight of 23.5 kDa.Furthermore, a metalloprotease with a molecular mass of approximately 23 kDa was purified from the venom of fer-de-lance (Bothropsasper) snake [25], which is similar to other previously identified metalloproteases such as B. asper hemorrhaging BH2 and BaP1 with molecular masses of 26 and 24 kDa, respectively [26].The low K m value of 0.31 mg/ml and the high Vmax value of 9.09 µmol/min obtained is an indication of high affinity of the partially purified enzyme for its substrate, which further substantiates the observed toxicities in Viperidae snakes as a result of the activities of metalloproteases [22,23].
There are several compounds with diverse chemical structures known in plants which have been accounted for with the ability to interact with peptides and proteins (enzymes) of snake venom.The mechanism of action of these compounds are still not clear and might be attributable to inactivation of molecular structures prone to chemical attacks that may block the active sites of the snake venom components.Another mechanism of action of the plant compounds is inhibition of metalloproteases present in the snake venom.This is due to the metal chelator substances in the plant extracts [27].The results in Table 2 reveal a reduction in the activity of the enzyme by 58% when treated with the crude extract while treatment with the active fraction showed a further inhibition of 63%.This suggests that the ethyl acetate fraction contained more active constituents than the methanolic extract, and thus is more effective in the inhibition of metalloproteases' activity of E. ocellatus' venom.In another study by WHO, the methanolic extract of the seed kernel of M. indica was reported to have inhibitory effect on the phospholipase A 2 activity of black-necked spitting cobra's (Najanigricolis) venom [5].The Lineweaver-Burke plot of varying concentrations of the extract on the substrate indicated that the extract inhibited the enzyme in a non-competitive manner with increasing K m and decreasing V max .In a related study [11], a non-competitive pattern of inhibition of metalloprotease, activity of saw-scaled, viper's (E.carinatus) venomwas reported by aqueous extract of Guiera senegalensis leaves that is also in consonance with Ibrahim et al. [12] findings.
In this study, the low K i (inhibition binding constant) value of the extract for the metalloproteases is an indication of a high affinity of the extract for these enzymes.Therefore, mango seed kernel could be used in designing novel E. ocellatus antivenins.http://www.ijt.ir;Volume 11, No 5, September-October2017

CONCLUSION
The results revealed a mixed noncompetitive pattern of inhibition of metalloproteases activity with an estimated K i value of 0.168 mg mL -1 , demonstrating that methanol extract of mango seed kernel has a high affinity for the partially purified enzyme.Therefore, it might provide an inexpensive and readily available alternative to sheep serum in the management of snakebite envenomation.Further in vivo studies are necessary to assess its effectiveness and safety.

Figure 2 .
Figure 2. Elution profile of ion exchange chromatography of metalloproteases on DEAE Sephadex.

Figure 6 .
Figure 6.Secondary plot of slope against inhibitor concentration showing the inhibition binding constant (K i ).

Table 2 .
The inhibitory effects of the methanol extract of Mangiferaindica seed kernel and its solvent fractions on the metalloproteases activity of Echisocellatusvenom.