Neutralisation of Local Haemorrhage Induced by the Saw-Scaled Viper Echis carinatus sochureki Venom Using Ethanolic Extract of Hibiscus aethiopicus L.

The objective of the study is to investigate the anti-snake venom activities of a local plant, Hibiscus aethiopicus L. The H. aethiopicus was dried and extracted with ethanol. Different assays were performed according to standard techniques, to evaluate the plant's acute toxicity and its antivenom activities. The results of evaluating the systemic acute toxicity of the H. aethiopicus extract using “oral and intra-peritoneal” route were normal even at the highest dose (24 g/kg) tested. All guinea pigs (n = 3) when treated with venoms E. c. sochureki (75 μg) alone induced acute skin haemorrhage. In contrast, all guinea pigs (n = 18) treated with both venom and the plant extract at a concentration between 500 and 1000 mg/kg showed no signs of haemorrhage. Moreover, all guinea pigs (n = 18) treated with venom and the plant extract below 400 mg/kg showed acute skin haemorrhage. All guinea pigs treated with venom E. c. sochureki (75 μg) alone induced acute skin haemorrhage after both 24 and 32 hours. In contrast, all guinea pigs treated with both venom and the plant extract (administered independently) at concentrations between 500 and 1000 mg/kg showed no signs of haemorrhage after 32 hours. However, after 24 hours all tested guinea pigs showed less inhibition (<60%) compared to that obtained after 32 hours. The outcome of this study reflects that the extract of H. aethiopicus plant may contain an endogenous inhibitor of venom induced local haemorrhage.


Introduction
Envenoming resulting from snake bites remains the most neglected public health issues in many countries, particularly in poor rural communities living in the tropics. E. c. sochureki causes numerous deadly bites especially in Asia [1,2]. Generally envenoming by Echis snake vipers is responsible for several clinical complications of severe systemic and local pathology.
Although an intravenous administration of antivenom, prepared from IgG of venom-immunised horses or sheep, is an effective treatment for systemic envenoming [3], the clinical consensus is that antivenom is of limited effectiveness against the effects of local envenoming that develop rapidly after a bite [4]. Such effects include severe pain, oedema, localized haemorrhage, and necrosis [5] which often results in permanent scarring and deformity. The ineffectiveness of antivenom in treating local envenoming has been attributed to the rapid activity of the toxins and the inability of antivenom IgG to cross the blood/tissue barrier [6,7]. Despite their smaller size, F(ab2)2 and Fab fragments of IgG are also ineffective against the local effects of envenoming, whether administered by intravenous or intramuscular routes [8,9]. Other limitations of the antivenom(s) also referred to the variability of snake venom. Venom constitution within the same snake species can present considerable 2 Evidence-Based Complementary and Alternative Medicine geographical variations [10][11][12], including a diverse in conformity with interpopulational, seasonal, ontogenetic, and individual factors [13]. Therefore, antivenom made against venom of an assured species may not be sufficient against envenomation of the same snake species [14]. Research to develop a treatment for local envenoming against different species as well as among the same species from different areas will be of clinical priority and should focus on the application of natural [15] or synthetic inhibitors [16] against snake venom potent molecules. Plant-derived drugs remain important resource to combat serious diseases. The present study aims to study the antisnake venom activity of a local plant, Hibiscus aethiopicus L. which is brought over 200 years ago from Africa and regrown in Bani-Hushash region, Sana'a Yemen. This plant was found to be used by traditional healers in Bani-Hushash East of Sana'a to treat some clinical complications including patients bitten by snakes and scorpions. Although Hibiscus aethiopicus L. has long been used as a medicinal plant by traditional healers, the validity of the claims made for this plant has been previously tested scientifically by our research group [17].

Evaluations of Acute Toxicity of H. aethiopicus Extract
In Vivo Using both "Oral" and "Intraperitoneal" Routes. Two in vivo assays using both "oral" and "intra-peritoneal" routes were performed to evaluate the acute toxicity and the cumulative effect "safety protection" potentialities of the H. aethiopicus composition.
2.6. Oral Route Acute Toxicity. Ten guinea pigs (300-900 g) were used in the toxicity profile. The guinea pigs were given different dosages to investigate the lethal dose. The extract solution (dissolved in water) was given orally using anaesthesia at variable dosages to reach 24 g/kg above optimum of 16 g/kg (a lethal dose according to the international standardisation for the classification of substances). The animals were observed for behaviour change, continuously for a period of two weeks after such administration. Observation was conducted hourly at day 1, and thereafter at 4-6 times per day. Subsequently, blood samples for biochemical analysis, alanine aminotransferase (ALT), aspartate aminotransferase (AST), complete blood count (CBC), and gamma glutamyl transpeptidase (GGT) were collected. At the end of the observation period, animals were sacrificed and dissected. Histopathology examination of their eyes, liver, lung, and spleen was performed.

Intraperitoneal
Route Acute Toxicity. Eighteen guinea pigs (300-900 g) of both sexes were distributed randomly into 6 groups (each group of 3 animals) and being treated i.p. with increasing dosages (250, 500, 750, 1000, 1300, and 1600 mg/kg) of H. aethiopicus extract. The fifth group served as a control and received an equivalent volume of distilled water. Guinea pigs were observed regularly over a period of 24 hours for signs of acute toxicity and death.  [19] with minor modification. Group 1 of 3 guinea pigs was injected in the right footpads with 3x minimum oedema dose (MED) (7.5 µg, previously determined) of venom sample (dissolved in 20 µL PBS, pH 7.2). The left footpads received saline, as controls. Legs were cut off at the ankle joint after 6 hours. An increase in weight due to oedema was calculated as the oedema ratio, which equals the weight of the oedematous leg × 100/weight of control leg. The MED was defined as the amount of venom sample required to cause an oedema of 100%. Group 2 (for inhibition examination) was divided into 8 equal subgroups (3 guinea pig in each) were injected subcutaneously with the venom sample that was preincubated with the extract at different concentrations (37.5-525 µg) for 30 minutes at 37 • C.

Administration of H. aethiopicus via "Oral and Intra
Peritonial" Routes Has no Acute Toxic Effect. All animals were alive after the 2 weeks of given 24 g/kg extract solution above the lethal dosage of 16 g/Kg. No abnormal behaviour was observed. Animals showed normal body weight increase during the two weeks period. Biochemical analysis showed normal range of ALT, AST, CBC, and GGT ( Table 1). Inspection of the eyes, liver, lung, and spleen showed no extraordinary signs. The results when compared to a general acute toxicity index were normal, and no acute toxicity was observed. Furthermore, guinea pigs dosed intraperitoneally with H. aethiopicus extract were initially dull with significantly reduced movement for 20-30 minutes.
However, neither death nor signs of toxicity were observed even at the highest dose (24 g/kg) tested.

The H. aethiopicus Extract Inhibits Venom Coagulation
Activity. The extract was found to inhibit, dose dependently, the procoagulant activity of the E. c. sochureki venom ( Figure 3). A progressive increased clotting time resulting in anticoagulation was recorded ( Figure 3). The anticoagulant response reached an optimum with a clotting time of 600±4, and this was attained at the extract concentration of 500 mg (P < 0.0001). No significant difference was observed between both concentration of 500 and 1000 mg as both gave the same anticoagulant response. In contrast, in the absence of the extract, the control value containing the venom sample alone revealed a clotting time of 28 ± 3 s (P ≤ 0.0001). However, when induced with Ca +2 alone, clot formation was seen at 325 ± 4 s (data not shown).

The H. aethiopicus Extract Inhibits Oedema Induction.
Toxicity studies in a mouse model revealed the following. The MED was found to be 7.5 µg (this is the amount of venom sample required to cause an oedema of 100%). The 3x MED found to cause an oedema of 190 ± 2%. The venom-induced haemorrhagic oedema was also inhibited dose dependently by the extract. At a ratio of venom to extract of 1 : 60 and 1 : 70 (w/w), the oedema of 190 ± 2.0% (caused due to 3x MED of venom) was reduced to 120 ± 2.0% which is the same as the control (i.e., leg injected with PBS), (P < 0.0001) (i.e., the foot pad that was injected with the saline alone) (Figure 4).

Discussion
Plants constitute rich sources of novel compounds with a variety of pharmacological activities. Therefore, experimental validation of the traditional use of plants is important and can facilitate the development of low-cost phytotherapeutic agents [9]. Plants used as remedy for snakebite abound in literature [20][21][22][23]. However, many of the reported studies lack detailed scientific investigation, which is needed in the development of medicinal agents from plants [20][21][22].
In this study we have used H. aethiopicus, one of the main traditional herbal plants which is used as phytotherapy practiced by a large proportion of the Yemen population for the treatment of several clinical complications including snake envenomation. To our knowledge no scientific reports on H. aethiopicus and its capacity to neutralise snake venom(s) available except a single study that was published  by our research group [17]. Therefore, this study represents the second report about H. aethiopicus and its uses as antivenom agent against E. c. sochureki.
In contrast to our results reported in the previous work [17], the oral route when compared to a general acute toxicity index showed normal with no extraordinary symptoms as well as no acute toxicity. However, both routes showed no death even at the highest tested dose (24 mg/kg) compared to the previous work. This was supported further by the biochemical analysis as shown in Table 1. However, with intra-peritoneal route doses, guinea pigs were initially dull with significantly reduced movement for about 20-30 minutes (P < 0.005).
In the subsequent experiments we used the H. aethiopicus extract to assess its efficacy to neutralise the haemorrhagic activity of E. c. sochureki venom using an in vivo minimum haemorrhagic dose (MHD). It was interesting to note that results of the evaluation assays of antisnake venom activity showed that H. aethiopicus induced noticeable and significant neutralisation capacity against venom of E. c. sochureki. This was clearly illustrated in Figure 1(A), a-k. In a guinea pig animal model, MHD was found to be 25 µg, and the 3x MED was found to cause the skin haemorrhagic area of 10 ± 2 mm. It can be seen from (Figure 1(A), e) that at concentration of 400 mg/kg which act as the end point (Figure 1(B)) showed a slight haemorrhage and/or inflammation. Therefore, above, that is, 400 mg/kg, neutralisation efficacy of 100% was achieved at a concentration of 500 mg/kg (Figure 1(A), f).
Results from the oral administration of the extract 3 hours prior to venom injection were significant and showed future promising (Figure 2(A)). In this assay we examined the neutralisation efficacy of the extract using two prospects based on time intervals of 16 and 32 hours, respectively.
In comparison with the results from above we have found that the concentration that gave 100% neutralisation was 1000 mg/kg after 32 hours rather than after 16 hours, suggesting that this difference is due to the time needed for the absorption and distribution of the active components in H. aethiopicus extract. Although such comparison between the two assays is not adequate as in the first assay, a close contact between the extract and venom was achieved to give a maximal neutralizing effect. Therefore, further investigation of the absorption rate should be performed. Moreover, this result can also suggest that having the extract before envenomation can act as a prophylactic agent.
Ecarin is an extensively characterized metalloprotease present in E. c. sochureki venom and has been found to activate prothrombin directly resulting in the pro-coagulation of citrated human plasma [24]. This property of E. c. sochureki procoagulant was found to be independent of Ca +2 requirements [25,26]. In addition, ecarin also cleaved human fibrinogen and dissolved the fibrin clot [27]. The prothrombin converting and fibrinogenolytic (thrombinlike) activity of ecarin appears to be responsible for the procoagulant activity of the venom. Interestingly in this study we found that the extract can inhibit dose dependently the procoagulant activity of the E. c. sochureki venom and progressively increases clotting time resulting in anticoagulation (Figure 2(A)). The anticoagulant response of the extract reached an optimum with a clotting time of 600 ± 4 s, and this was attained at the extract's concentration of 400 mg (P < 0.0001). However, no significant difference between both concentrations of 500 and 1000 mg was found as both gave the same anticoagulant response. In contrast, in the absence of the extract, the control value containing the venom sample alone revealed a progressively decreasing clotting time which reached an optimum at 28 ± 3 s for a venom concentration of (75 µg) of the venom sample, (P < 0.0001) (Figure 3). However, when induced with Ca +2 alone, Evidence-Based Complementary and Alternative Medicine 7 clot formation was seen at 321 ± 4 s (data not shown). Since ecarin appears to be the principal procoagulant agent of E. c. sochureki venom, the extract might induce the inactivation of ecarin. This was confirmed by early studies where several anticoagulants have been isolated and studied extensively from this venom [24,[28][29][30][31]. In addition to the coagulation assay, we examined the neutralisation of the extract against the oedema inducing activity of the E. c. sochureki venom. The result was significant, and the extract was found to inhibit dose dependently the venom-induced haemorrhagic oedema. At a ratio of venom to extract of 1 : 60 and 1 : 70 (w/w), the oedema of 190 ± 2.0% (caused due to 3x MED of venom) was reduced to 120 ± 2.0% which is the same as the control (i.e., the foot pad that was injected with the saline alone) (Figure 4), (P ≤ 0.0001). In several experimental and clinical trials, it has been demonstrated that antivenoms are of limited value to stop oedema progression within the first 12-24 hours of treatment, while they are highly efficient in restoring blood coagulation status within the same time interval [32][33][34]. Moreover, the rapid neutralization of venom in the bloodstream does not guarantee the halting of oedema progression within a short time. Therefore, in this study, we report for first time that the extract of the H. aethiopicus showed high neutralizing potency against such effect within six hours of venom injection. However, this finding was based on in vitro testing, (i.e., close contact between the extract and venom). Therefore, to confirm such efficacy, an in vivo assay has yet to be performed where, the extract has to be given orally "independent," that is, before and/or after the venom being injected.
Despite these protective effects of the plant extract of the H. aethiopicus in vitro, the results obtained from the in vivo experiment (Figure 2(A)) were highly encouraging. The extract did protect animals challenged with local haemorrhage of the E. c. sochureki venom when the extract and venom were administered independently. However, haemorrhage induction was significantly reduced (P < 0.0001) and or fully neutralised with the increase of the extract concentration and time, in contrast with the preincubation assay represented by Figure 1.
The outcome of this study reflects that the extract of H. aethiopicus plant may contain an endogenous inhibitor of venom-induced local haemorrhage. This obviously would need further investigations for both systemic and local evaluation. Further studies on the fractionation(s), isolation and characterization of the active principle/s, and its antivenom property appears promising and could contribute to the development of a potent and perhaps safe antidote against E. c. sochureki venom poisoning and might play a role in the better management of the threatening lethal bites.

Summary
Snake bite remains a public health problem in many countries even though; it is difficult to be precise about the actual number of cases. It is estimated that the true incidence of snake envenomation could exceed 5 million per year and causing about 125,000 deaths each year, predominantly within poor communities living in rural areas of countries in Southeast Asia and Africa. Although an intravenous administration of antivenom, prepared from IgG of venomimmunised horses or sheep, is an effective treatment for systemic envenoming, the clinical consensus is that antivenom is of limited effectiveness against the effects of local envenoming that develop rapidly after a bite. In addition to its high cost and other limited effectiveness, thus, there is a need to develop novel therapeutics to maximise the utility of the snakebite therapies that are available. Research to develop a treatment for local envenoming is therefore a clinical priority and has focused on the application of natural or synthetic inhibitors of snake venom potent molecules. The traditional medicine to treat snakebite victims still plays an important role in the primary health care worldwide, especially in rural areas and poor communities in the third world countries is a common practice. Because natural products of higher plants may give a new source of medication, there are many research groups that are now engaged in medicinal plants research not only for the discovery for new drugs, but possibly for discovering compounds with novel mechanisms of action that can stimulate new fields of research. Our results suggest that that Hibiscus aethiopicus L. plant may contain an endogenous inhibitor of venom-induced haemorrhage.