Bajopas Volume 10 Number 2 Decembber, 2017 http://dx.doi.org/10.4314/bajopas.v pas.v10i2.38 Bayero Journal off Pure and Applied Sciences, 10(2): 233 - 237 Received: June, 2017 Accepted: December, r, 2017 ISSN 2006 – 6996 EFFECT OF METHANOLICSTEM CSTEM-BARK EXTRACT OF Commiphora pedunc edunculata ON PLASMA RECALCIFICATION ION TIME OF Najani gricollis VENOM TREATED ATED PLASMA 1 Abdullahi, H.L., 2Muhammed, med, H.S., 3Tajuddeen, N.,3Hamza, S.A., and 4Ibrahim, Ibrah M.A. 1 2 Department of Medica edical Laboratory Science, Bayero University, Kano, Nigeria Department of Biochemistry, Kano State University of Science and Technology, Wudil, Kano, Kano Nigeria 3 Departmentt of C Chemistry, Ahmadu Bello University, Zaria, Nigeria 4 Department of Biochemistry, Bio Ahmadu Bello University, Zaria, Nigeria Correspondence: hadeezalawal@gmail.com, had hlabdullahi.mls@buk.edu.ng ABSTRACT Several medicinal plants have e bee been used in Nigerian communities as antidotes otes for fo snakebite complications without scientific fic vali validation. In this study, the effect of the methanol thanolic extract of Commiphora pedunculata on recalc recalcification time of bovine, caprine, ovine and d came cam lid plasma treated with Najani gricollis venom was investigated. The N. nigricollis venom was found to significantly increase (p<0.05)) the p plasma recalcification time of all the plasma samples. sample However, the methanolic extract of C. pedunculata pedunc at 1, 10 and 100 µg/ml reduced the venom-associated venom increase in the plasma recalcificatio fication time in a dose dependent pattern except for camelid cam plasma where the effect of the extract ract w was non-dose dependent. This study showed d that methanolic extract of C. pedunculata could ld serv serve as a prototype for an antidote for the reversal ersal o of increase in plasma recalcification time posed sed by N. nigricollis venom. Key words: Commiphora pedunculata nculata, Najanigricollis, Plasma recalcification time. INTRODUCTION Snake bite envenomation still remains ains a neglected public health problem in most parts ts of Africa and is usually associated with impoverish verished farming populations. It is responsible for about 7,300 deaths and nearly 6,000 amputations resultin sulting from over 314,000 bites that occur annuallyy in Sub-Saharan Africa alone, with one-fifth of all cases occurring in Nigeria (Habib, 2013). Snake venomss ar are rich natural sources of biologically active molecule lecules which are mostly proteineous in nature (Calderon eron et al., 2014), and capable of affecting physiological cal processes pr such as haemostasis, the complement ent system and neurotransmission (Vyas et al., 2013). Snake venom haemotoxins affect platelet atelet function and blood coagulation (Koh et al., 2006)) whic which are usually responsible for haemorrhage characterized chara by consumption of clotting factors ors and blood incoagulability observed in envenom enomed patients (Kamiguti et al., 1998). Among the e diff different species of snakes, N. nigricollis(black necked ed sp spitting cobra) venom has been considered as one o of the highly heamotoxins-rich venom which makes akes it responsible for severe and very fast external haemo aemorrhaging and tissue necrosis around the bite area ((Naja, 2008). Antivenoms remain the only specific antidote antid to snake venom and is capable of reversing sing all systemic manifestations of envenoming when n ad administered at the right time. However, it is nott usu usually effective against the local haemotoxic effects ts of N. nigricollis venom leading to long term di disability and disfigurement. Furthermore, the scarcity arcity and cost of antivenom coupled with the triple problems of inadequacy and inaccessibility in health ealthcare had made recourse to the use of local plants lants with antivenin properties an appealing alternative ative (Sallauet al., 2015). Commiphora pedunculata (Kotschy & Peyr.) Engl. belongs to Burseraceae, a family composed com of both trees and shrubs of tropicall and sub-tropical geographical distribution. The plant lant iis used in the folkloric treatment of a number off diseases dise of public health concern but scientific validation valid on the therapeutic potentials of the plant lant has not been conducted for most of the dise diseases. Studies conducted on the plant mainly ainly focused on antimicrobial activity (Tajuddeenet al., 2014; Tajuddeen et al., 2016). Hence, despit espite the reported use of C. pedunculata for the treatmen tment of snakebiteassociated haemorrhage, there is no information on the effect of C. pedunculata extract extrac on plasma recalcification time of Najani gricollis icollis venom treated plasma. Plasma recalcification time (PRT), a measure m of time taken to clot or recalcify is a physio hysiological process involving the activities of clotting g fac factors, enzymes and calcium ions. A prolonged plasma lasma recalcification time is suggestive of coagulopathy. This study was therefore conducted cted to validate the ethnomedicinal claims for the antiophi tiophidian effects of the plant by investigating the e effects ef of the methanolic extract on the plasma recalcification reca time ofNajani gricollis venom treated plasma. asma. 233 Bajopas Volume 10 Number 2 December, 2017 Plasma recalcification time (min) MATERIALS AND METHODS Plant Collection and Identification The stem bark of C. pedunculata was collected from Basawa, Zaria, Nigeria, and authenticated at the herbarium unit of the Department of Biological Sciences, Ahmadu Bello University, Zaria, where a voucher specimen number219was deposited for reference purposes. Plant Preparation and Extraction The stem-bark sample of the plant was cleaned, air dried under shade and ground into powder. The dried powder (500 g) was extracted with methanol (2.5 L) in a Soxhlet apparatus for 48 h at 65oC. The entire process was repeated twice. The methanolic extracts were pooled together and filtered using a filter paper (Whatmann size no.1). The filtrate was evaporated to dryness in a water bath at 40ºC. The dried extract obtained was weighed and kept in an airtight bottle in a refrigerator at 4°C. Venom collection and Preparation Najani gricollis venom was obtained by milking a black-necked spitting cobra (Najanig ricollis) maintained at the Serpentarium of the Department of Veterinary Pharmacology and Toxicology, Ahmadu Bello University, Zaria. The venom was collected with the help of a skilled snake handler. The pooled venom was thereafter placed in a desiccator containing activated silica and allowed to dry at room temperature. The crystallized venom was subsequently transferred into a refrigerator and stored at -18°C. Plasma collection Fresh caprine, ovine, bovine and camelid blood samples were collected into heparinized tubes from Kano state abattoir, Fagge Local Governement, Kano, and then centrifuged at 3000rpm for five minutes to obtain the respective plasma samples. Determination of Plasma Recalcification Time The modified method described by Theakson and Reid (2003) was used to determine the effect of Najani gricollis venom on plasma recalcification time. Heparinized plasma sample (100 μl) from each of the animals was incubated in a water bath at 37°C and100 μl of crude venom dilution (1mg/ml) was added. Thereafter 100 μl of PBS(pH 7.4) and CaCl2 (25 mM) were sequentially added and the recalcification time recorded using a stopwatch. The effect of the methanol extract on the recalcification time of plasma incubated with venom was determined by replacing the PBS with 100, 10 and 1 µg/ml dilutions of the extract in PBS. Statistical Analysis All data are presented as mean ± standard deviation of three replicate determinations. Data were analyzed using students t-test and values were considered significantly different atp< 0.05. RESULTS The effect of methanolic extract of C. pedunculata on the plasma recalcification time of caprine plasma incubated with N. nigricollis venom is presented in figure 1. The recalcification time of caprine plasma treated with venom only was 25.5 ± 0.5 minutes while the recalcification time for incubated plasma treated with 100, 10 and 1 µg/ml of the methanolic extract were 7.5 ± 0.5, 8.5 ± 0.5 and 9.5 ± 1.5 minutes, respectively. The venom was found to prolong the recalcification time of ovine (figure 2), bovine (figure 3) and camelid plasma samples to 21.0 ± 1, 21.5 ± 0.5, 23.5 ± 0.5 minutes respectively. However, the methanol extract reduced the venomassociated increase in the recalcification time in a dose dependent pattern except for camelid plasma where the effect of the extract was non-dose dependent 30 25 20 15 10 5 0 P+C P+C+V P+C+E P + C + E(0.01) + V P + C + E(0.001) + P + C + E(0.0001) + V V Treatment Figure 1: Effect of methanolic extract of C. pedunculata on plasma recalcification time of N. nigricollis venom treated caprine plasma P=Plasma; C=CaCl2; PBS=Phosphate Buffer Saline; V= Venom (1g/100ml); E=Extract (1mg/ml); X=Dilution 234 Bajopas Volume 10 Number 2 December, 2017 Plasma recalcification time (min) 25 20 15 10 5 0 P+C P+C+V P+C+E P + C + E(0.01) + V P + C + E(0.001) + P + C + E(0.0001) + V V Treatment Figure 2: Effect of methanolic extract of C. pedunculata on plasma recalcification time of N. nigricollis venom treated ovine plasma P=Plasma; C=CaCl2; PBS=Phosphate Buffer Saline; V= Venom (1g/100ml); E=Extract (1mg/ml); X=Dilution Plasma recalcification time (min) 25 20 15 10 5 0 P+C P+C+V P+C+E P + C + E(0.01) + V P + C + E(0.001) + V P + C + E(0.0001) + V Treatment Figure 3: Effect of methanolic extract of C. pedunculata on plasma recalcification time of N. nigricollis venom treated bovine plasma P=Plasma; C=CaCl2; PBS=Phosphate Buffer Saline; V= Venom (1g/100ml); E=Extract (1mg/ml); X=Dilution 235 Plasma recalcification time (min) Bajopas Volume 10 Number 2 December, 2017 30 25 20 15 10 5 0 P+C P+C+V P+C+E P + C + E(0.01) + V P + C + E(0.001) + V P + C + E(0.0001) + V Treatment Figure 4: Effect of methanolic extract of C. pedunculata on plasma recalcification time of N. nigricollis venom treated camelid plasma P=Plasma; C=CaCl2; PBS=Phosphate Buffer Saline; V= Venom (1g/100ml); E=Extract (1mg/ml); X=Dilution DISCUSSION The role of medicinal plants in the treatment of snakebite has been long recognized. So far, information on the antivenom potentials of C. pedunculata has not been documented. In this study, C. pedunculata methanol extract was found to profoundly reduce the N. nigricollis venom-associated increase in recalcification time of the plasma samples from various animal models. The highest increase in plasma recalcification time was observed in caprine plasma (Figure 1); that of camelid plasma was slightly lower. This could be attributed to the susceptibility of blood clotting components of these animals to haemotoxic snake venom components. Camelid plasma is rich in clotting factor FVIII:C (Abdel Gaderet al., 2013) which serves as abundant substrate for some snake venom haemotoxins following envenomation. This effect occurs rapidly following envenomation and this possibly leads to prolonged plasma recalcification time. This finding is supported by a study conducted by Cook et al. (2010), which concluded that a camelid polyspecific IgG antivenom was not effective against the venom-induced effects of Najani gricollis venom. The methanolic extract of C. pedunculata affected ovine, bovine and caprine plasma types in a dosedependent manner (Figures 1, 2, and 3). However, PRT for camelid plasma treated with venom was decreased in a reversed dose-dependent manner (Figure 4); lowest concentration of the extract was most effective against the haemotoxic effect of the venom. This could possibly be due to the presence of a natural slow-acting competitive inhibitor of the snake haemotoxin inherent in camelid plasma which displaces the molecules of the active component of the extract from the haemotoxin’s binding site. The observed reduction of plasma recalcification time by extract indicates that the C. pedunculata is endowed with compounds such as tannins, flavonoids 236 and phytochemicals capable of binding and inhibiting the proteins and enzymes of the clotting cascade via a yet unknown mechanism. A number of phytochemicals such as tannins and flavonoids have been reported as being capable of altering the clotting cascade through binding to the relevant enzymes and proteins (Premendranetal., 2011). Interestingly, flavonoids (kaempferol, dihydrokaempferol and epicatechin) have been isolated and characterized from this methanol extract (Tajuddeen et al., 2014; 2016) and it is thus possible that, these flavonoids along with other phytochemicals mediated the observed effect on plasma recalcification time. CONCLUSION The findings of this study clearly confirm that Najani gricollis venom is capable of prolonging plasma recalcification time. This effect could however be counteracted by the methanolic extract of C. pedunculata, though the extract does not have a neutralizing effect on all venom components. This clearly lends support to the use of plants for the treatment of snakebite related complications. C. pedunculata extract could further be studied as a potential prototype for an antidote against snake venom haemotoxicity. Author’s Contributions: M.A.Ibrahim conceptualized the study; M.A.Ibrahim and H.L. Abdullahi designed the experiments; N.Tajuddeen and S.A.Hamza collected the plant material and prepared the methanol extract; H.S. Muhammed collected all the blood samples and performed the plasma recalcification time experiment together with H.L. Abdullahi,H.S.Muhammed, H.L. Abdullahi,N.Tajuddeen and S.A.Hamza conducted the statistical analysis while H.L.Abdullahi and M.A.Ibrahim drafted the manuscript to the present form. 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