Naja naja karachiensis Envenomation: Biochemical Parameters for Cardiac, Liver, and Renal Damage along with Their Neutralization by Medicinal Plants

Naja naja karachiensis envenomation was found to hit more drastically heart, liver, and kidneys. 400 μg/kg of venom-raised moderate serum levels of ALT (72 ± 4.70 U/L, 0.1 > P > 0.05), AST (157 ± 24.24 U/L, 0.1 > P > 0.05), urea (42 ± 3.08 mg/dL, 0.05 > P > 0.02), creatinine (1.74 ± 0.03 mg/dL, 0.01 > P > 0.001), CK-MB (21 ± 1.5 U/L, 0.05 > P > 0.02), and LDH (2064 ± 15.98 U/L, P < 0.001) were injected in experimental rabbits. However, lethality was enhanced with 800 μg/kg of venom in terms of significant release of ALT (86 ± 5.0 U/L, 0.05 > P > 0.02), AST (251 ± 18.2 U/L, 0.01 > P > 0.001), urea (57.6 ± 3.84 mg/dL, 0.02 > P > 0.01), creatinine (2.1 ± 0.10 mg/dL, 0.02 > P > 0.01), CK-MB (77 ± 11.22 U/L, 0.05 > P > 0.02), and LDH (2562 ± 25.14 U/L, P ≪ 0.001). Among twenty-eight tested medicinal plant extracts, only Stenolobium stans (L.) Seem was found the best antivenom (P > 0.5) compared to the efficacy of standard antidote (ALT = 52.5 ± 3.51 U/L, AST = 69.5 ± 18.55 U/L, urea = 31.5 ± 0.50 mg/dL, creatinine = 1.08 ± 0.02 mg/dL, CK-MB = 09 ± 0.85 U/L, and LDH = 763 ± 6.01 U/L). Other plant extracts were proved less beneficial and partly neutralized the toxicities posed by cobra venom. However, it is essential in future to isolate and characterize bioactive compound(s) from Stenolobium stans (L.) Seem extract to overcome the complications of snake bite.


Introduction
One of the animals which represent Pakistan and India throughout the world is cobra; in particular it is rearing out of a wicker basket and dancing to the sound of turban-wearing snake charmer music. In fact, they are deadly venomous among all species of the snakes due to high rate of mortality which strongly reasoned for their notoriety [1]. At the end of the nineteenth century, all cobra populations have been merged into single species naja (formerly known as naia); however, subsequently ten subspecies of Naja naja were identified [1]. Later on, Deraniyagala classified Naja naja (India) into several subspecies and considered Naja naja karachiensis one of them widely distributed in Southern Punjab province of Pakistan [2]. They are extremely toxic due to their severe side effects in the victims. Major signs and symptoms of cobra envenomation are edema, necrosis, pain, respiratory paralysis, vomiting, headache, hypotension, cardiac arrest, coagulopathies (elevated PT, aPTT, and TT), bleeding wounds, hematuria, mucus discharge, proteinuria, 2 BioMed Research International and increased creatinine and urea levels along with altered consciousness [3,4].
Laboratory animals, particularly rabbits, have been selected previously to monitor various biochemical changes related to liver, heart, and renal toxicities [18,19]. Aspartate aminotransferase (GOT/AST) and alanine aminotransferase (GPT/ALT) are surrogate markers for liver toxicities while creatine kinase isoenzyme (CK-MB) is used exclusively to access cardiac tissue damage [20,21]. Elevated creatinine and urea levels are indicators of 75% renal damage [20], whereas high level of lactate dehydrogenase (LDH) implies general toxicity related to the liver, heart, kidneys, and skeleton muscles [22].
Diverse immunological properties of different cobras have fascinated protein chemists and other researchers to carry out efforts for the development of new antidotes. Due to this reason, for the first time, venom from Naja naja karachiensis is selected to evaluate its toxicity in vivo and to search out effective as well as cheap treatment from folk herbal remedies. Pakistan is a hub of medicinal plants where mostly rural communities rely on natural herbs to treat their problems of snake bite [6,23]. It is therefore necessary to evaluate scientifically their folklore claims as anti-snake venom in traditional system of medicine.  (Table 1).

Collection and Milking of Snake
Venom. Black Pakistani cobra snakes (Naja naja karachiensis) were collected from Cholistan desert located in southern Punjab province of Pakistan. After collection, they were properly identified by a zoologist. Venom from Naja naja karachiensis was collected by pressing their glands below their eyes in low light environment. After collection, it was lyophilized and stored in light resistant container at 2 ∘ C to 8 ∘ C. For further experiments, venom was used in terms of its dry weight [6].  Table 1. One kilogram chopped plant material was soaked in 5 L of methanol as solvent in extraction bottles. The homogenates were kept for a period of a month at optimum temperature. After filtration, filtrate was evaporated by using water bath and extracts were weighed and stored for further experimentation [24].

Experimental Animals.
Seventy-one healthy growing male rabbits (1 ± 0.5 kg) were selected for this study after getting permission from local ethical committee. Animals were acclimatized for a week in the laboratory by maintaining their standards for chow, water, and light. Subsequently, they were divided into various groups in different cages such that experimental rabbits in group I were used for baseline measurements of various biochemical parameters. Animals belonging to groups II and III were evaluated for different doses of cobra venom while group IV was served as control. Group V was redivided into twenty-nine groups such that each subgroup (V/1-V/29) was assigned to a single medicinal plant extract [25].

In Vivo Antivenom Activity of Medicinal Plant Extracts.
Venom from Naja naja karachiensis (400 g/kg and 800 g/kg) was injected subcutaneously to evaluate its toxicity to the heart, liver, and kidneys. Before injection of venom, experimental animals were anaesthetized by administration of ketamine (50 mg/kg). To determine antivenom activity of various plants' extract, they (100 mg/kg) were incubated (at 37 ∘ C for 30 minutes) with fixed amount of venom (800 g/kg) before injection while saline was used as control [26][27][28].

Biochemical Assays.
For serum analysis of various biochemical parameters, blood was collected from marginal ear artery by the use of hypodermic syringe needle after 3 hours of envenomation [25]. Separated serum was tested for alanine aminotransferase (GPT/ALT), aspartate aminotransferase (GOT/AST), urea, creatinine, creatine kinase isoenzyme MB (CK-MB), and lactate dehydrogenase (LDH) by the use of 3 Allium sativum L.

Results
Venom from Naja naja karachiensis was proved to cause severe complications in dose-dependent manner. Venom at the dose of 400 g/kg led to release of moderate serum levels of ALT (72 Complete detail about various biochemical parameters for baseline measurements different doses of cobra venom, and saline (as negative control) is summarized in Table 2.
To neutralize in vivo 800 g/kg of cobra venom, twentyeight medicinal plant extracts were evaluated. It was noticed that extract of Stenolobium stans (L.) Seem was the best antidote ( > 0.5) compared to reference standard. Allium sativum L., Althaea officinalis L., Citrullus colocynthis (L.) Schrad, Leucas capitata Desf., Pinus roxburghii Sarg, Psoralea corylifolia L., Rubia cordifolia L., and Sapindus mukorossi Gaertn were found to be valuable plants to protect liver damage ( > 0.5) as standard antidote (ALT = 52.5 ± 3.51 U/L and AST = 69.5 ± 18.55 U/L); however, required ALT and AST levels were not observed with remaining plant extracts (0.5 > > 0.05) as shown in Table 3 and comparison is shown in Figure 1. Extracts of Leucas capitata Desf. and Althaea officinalis L. were observed to be helpful ( > 0.5) as reference standard (urea = 31.5 ± 0.50 mg/dL and creatinine = 1.08 ± 0.02 mg/dL); nevertheless, remaining plants were found less valuable (0.5 > > 0.01) to minimize urea and creatinine levels to maintain kidney functions (Table 4 and Figure 2). When compared with standard antidote (CK-MB = 9.0 ± 0.85 U/L) eight medicinal plants   Table 5 and comparison is shown in Figure 3.

Discussion
Snake bite has been responsible for tens of thousands of deaths worldwide and numerous physical handicaps [29].   Generally, snake venom is an intricate mixture of various proteins (>90%) and most of them are enzymes, particularly (40%) phospholipases A 2 [30]. Among different complications, hepatic injury is one of the deadly venomous effects produced by cobra bite [31,32]. Naja naja karachiensis venom caused significant increase in ALT and AST levels (dose dependently) that are surrogate markers for liver toxicity either by direct action or immunological (hypersensitivity) reaction. The allergic reactions reactions are not dose dependent which clarify lethal effects to the cytoplasm and mitochondrial membranes of the hepatic cells [25,33]. Phospholipases enzymes, abundant in Naja naja karachiensis venom, are responsible for breakdown of membranous phospholipids and therefore resulted in cellular injury along with inflammation [24]. PLA 2 is attributed to decrease in Na + /K + ATPase activities and led to greater influx of sodium ions and water molecules into the cell. Subsequently, plasma membrane lipid bilayer disorganized and eventually resulted in hepatic cells death [33,34]. In addition, PLA 2 is also found to cause anticoagulation in victims [6]. All pharmacological effects are due to phospholipid hydrolysis or phospholipid competing biding mechanism with other coagulation factors, particularly FXa, or by both ways; however, it is very hard to pinpoint exact one [35]. Anticoagulant response posed by cobra PLA 2 (anticoagulant enzyme) may aggravate hepatic injury as many anticoagulant agents cause liver damage that are idiosyncratic in nature [36]. Cardiac injury, particularly systolic heart arrest, is one of the well-known toxicities related to Naja naja subspecies [30]. In present study, Pakistani cobra venom was found to release two cystolic enzymes (LDH and CK-MB) that are sensitive indicators of myocyte injury [37]. It is due to the presence of myotoxic PLA 2 and other cardiotoxin(s) that are salient features of cobra venom that are responsible for cellular necrosis and cytotoxicity [38,39]. Cytotoxic effect of Naja naja karachiensis venom may not be overlooked even when antisera administration is late cause of toxic components to the microvasculature and thrombus produced which poses hindrance in access of immunoglobulins to the site of snake bite [26,29,39]. Snake venom has been responsible for detrimental effects to the renal tissues [40]. Likewise, Naja naja krachiensis venom was found to cause severe renal damage by significant rise in serum urea and creatinine levels. Indeed, phospholipases enzymes are responsible for the increase in vascular permeability with hemorrhagic effects to the vital tissues in the victims. Subsequently, numerous lesions are produced related to the glomerular membrane and renal tubules either interstitial or vascular [29,41,42]. Presence of lymphocytes (white blood cells) and oedema in the cortical as well as medullary regions of renal tubules further confirmed the idea of renal damage (data has not shown) as reported previously with different snake venoms like Hemiscorpius lepturus [26,43]. Apart of it, pharmacokinetic studies of Naja naja karachiensis venom with short lived radioisotope 99m Tc confirmed that kidneys and urinary bladder are the most saturated organs (>70%) after intravenous injection in experimental rabbits (unpublished data by our group).
Medicinal plants of Pakistan are used to inhibit snake venom (PLA 2 ) enzymes [6]. Due to this effect, present study was designed to select twenty-eight medicinal plants of Pakistan to test their potentials as an antidote against toxicities produced by Naja naja karachiensis venom. It was fascinating that extract of Stenolobium stans (L.) Seem showed significant neutralization compared to that of reference standard (antisera) as reported previously by Asad et al., for inhibition of phospholipases A 2 anticoagulant activity [6,44]. Present study indicated that extract of Stenolobium stans (L.) Seem possesses an endogenous inhibitor(s) to nullify venom (PLA 2 ) induced toxicities. Extracts of these plants material are routinely used in Pakistan by simple application in the form of paste to the affected area [45]. This practice provides effective first aid treatment as tiny molecules of an antidote diffuse favorably at the site of snake bite before hospitalization and abrogated the spreading of toxins [39]. It is owing to the various secondary metabolites like phenols, flavonoids, terpenoids, xanthenes, quinonoids, and so forth, as reported earlier to mask various enzymatic actions of cobra venom [6,23]. Indeed, secondary metabolites pose hindrance in binding of different snake venom enzymes to their potential targets; therefore, antidotal effect evoked. Other plants extract were not proved significantly effective to neutralize cobra venom, therefore cannot be declared useful plants in venom therapy. It is the need of the time to isolate lethal component(s) of cobra venom attributed to its major toxicities. Furthermore, characterization of antivenom compound(s) from medicinal plant extracts would be worth full for complete and effective treatment of snake bite in the future.