In vitro Phytochemical Screening and Anti-snake Venom Activity of the Methanol Leaf and Stem Bark Extracts of Leptadenia hastata (Asclepiadaceae) against Naja nigricollis

Snake envenomation is a major cause of death and morbidity in many developing countries. Leptadenia hastata (Pers.) Decne (Asclepiadaceae) has been reportedly used in traditional medicine as an antivenom, antiulcer, antidiabetic, analgesic, cardiovascular disorders, bacterial and viral infections. This research design is to investigate the phytochemical analysis and phospholipase A2 enzyme inhibition potential of L. hastata leaf and stem bark extracts using standard procedures. Preliminary phytochemical screening revealed the presence of key constituents such as carbohydrates, tannins, flavonoids, alkaloids, triterpenes, steroids, saponins, and diterpenes. The methanol leaf and stem extracts were able to inhibit the hydrolytic action of phospholipase A2 enzyme in a concentration-dependent manner. The research findings lay credence to the folkloric claim of the leaf and stem of L. hastata as an anti-snake venom.


IntroductIon
Natural products from medicinal plants, either as pure compounds or as standardized extracts, provide unlimited opportunities for new drug discoveries due to the unmatched availability of chemical diversity. [1] It has been estimated that approximately over half of the pharmaceuticals in clinical use today are derived from natural products. [2] Some natural product-derived drugs that are a hallmark of modern pharmaceutical care include quinine, theophylline, penicillin G, morphine, paclitaxel, digoxin, vincristine, doxorubicin, cyclosporine, and vitamin A among many other examples. [2] For centuries, natural substances, particularly plants, have been used to control and treat diseases and this has culminated in the discovery of the majority of modern pharmaceutical agents. [3] Leptadenia hastata (Pers.) Decne a member of the Asclepiadaceae family are mostly herbs and shrubs with white sap comprising about 250 genera and 2,000 species, many of which are lianous and some cactus-like succulents with reduced leaves. [4] L. hastata is referred locally in Nigeria as ''yahdiya'' in Hausa and as ''hagalhadjai'' in Arabic. The plant leaves are used traditionally in the management of various ailments such as onchocerciasis, [5] scabies, [6] hypertension, catarrh, skin diseases, wound healing, snakebite, and prostate complaint. [7] Snakebite is a major cause of morbidity and mortality in Nigeria, especially in rural areas, where large numbers of envenoming and deaths are yearly reported. Venom from snake bite is largely composed of many complex compounds such as proteins, enzymes, neurotoxins, coagulants, anticoagulants, and other substances with cytotoxic effects. The venom is water-soluble, has a specific gravity of 1.03, and is acidic in nature. [8] Phospholipase A 2 enzyme (PLA 2 E) is known to stimulate the phospholipid membrane releasing arachidonic acid causing inflammation and pain at the bite site. [9] Snake venom is hemotoxic, mainly affecting the circulatory system and muscular system, causing excessive scarring, hemorrhagic, coagulant defects, hypovolemic shock, pyrogen reaction, and serum sickness. [10,11] These side effects are largely due to the action of high concentrations of non-immunoglobulin proteins present 1 in commercially available hyperimmune antivenom [12] as well as the problem of storage and short expiry dates restrict its usage. Snake venom antiserum or antivenom serum (AVS) is associated with administration problems with the exact dosage presenting a serious problem. AVS administration is also equally associated with hypersensitivity reactions, which requires further medical research. [13] Over the years, many attempts have been made for the development of snake venom antagonists, especially from plants sources; [14] hence, this study explores the antivenom efficacy of Leptadenia hastate, namely, its PLA 2 activity as a potential snakebite envenomation to scientifically or otherwise lay credence to its use in Nigeria folklore [ Figure 1].

Collection and Identification of Plant Materials
The leaves and stem of L. hastate were collected in July 2019 from Dange town, Dange Shuni local government area of Sokoto State, Nigeria. They were authenticated at the herbarium unit, Department of Pharmacognosy and Ethnomedicine, Usmanu Danfodiyo University Sokoto, Nigeria, where herbarium specimens were deposited and voucher numbers PCG/UDUS/894 and PCG/UDUS/895 were issued. The samples were shade dried at room temperature for 10 days, reduced to a fine powder using mortar and pestle, labeled and stored in an airtight glass container until ready for use.

Extraction of Plant Materials
The powdered leaves (84.23 g) and stem (64.42 g) of L. hastata were cold macerated using absolute methanol for 48 h. The extracts were concentrated using a rotary evaporator at 40°C at reduced pressure. The residues referred to as the methanol leaf and stem extracts were obtained and coded MEL and MES, respectively.

Preliminary Phytochemical Screening
Chemical tests were carried out on the MEL and MES extracts to identify the presence of various phytoconstituents such as alkaloids, flavonoids, and tannins, among others using standard procedures. [15][16][17][18]

Snake Venom Sample
The venom sample of Naja nigricollis was obtained from Dr. Amina Yusuf Jega of the Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Usmanu Danfodiyo University Sokoto. The venom with a lethal dose 99 value of 2.75 mg/kg was preserved in a desiccator at 8°C until ready for use. The venom was dissolved in 0.9% saline and centrifuged at 2000 rpm for 10 min. The venom concentration was expressed in terms of dry weight (mg/mL)

Inhibition of Venom PLA 2 Activity
Acidimetric assay for PLA 2, as described by Tan and Tan, [19] was adopted. Constant volumes of a substrate comprising calcium chloride (18 mM), sodium deoxycholate (8.1 mM), and egg yolk were mixed and stirred for 10 min to produce a homogeneous egg yolk suspension. Using 1 M sodium hydroxide solution, the pH of the suspension was adjusted to 8.0. 0.1 mL of the snake venom (0.1-8 mg) was added each to 15 mL to initiate the process of hydrolysis. About 0.9% normal saline was used as the negative control. The pH of the suspension was observed after 2 min using a pH meter. While a 1.0 unit pH decline corresponds to 133 µmol fatty acids released in the egg yolk mixture, the enzymatic activity of PLA 2 was recorded in micromoles of fatty acids released/minute.
To test, the anti-snake venom potential of L. hastate. 0.1 mg of the snake venom was pre-incubated with 0.1-0.6 mg/mL concentration of the extracts to neutralize the hydrolytic action of PLA 2 . The protection offered by the extracts that were calculated and represented in terms of percentage using the following expressions: ì mol of fatty acid released Enzyme activity Time taken in minute = Enzyme activity of the test sample % Antivenom activity 100 Enzyme activity of the control = × (2) % Enzyme inhibition activity = 100−Enzyme activity (3)

Percentage Yield
The result of the extraction yield of the leaves and stem bark of L. hastate with respect to the quantity macerated is presented in Table 1.

Phytochemical Screening
The preliminary phytochemical screening of the MEL and MES of L. hastate showed the presence of important secondary metabolites which are presented in Table 2.
The preliminary phytochemical screening revealed the presence of various bioactive phytoconstituents. The result of phytochemical screening of the MEL and MES revealed the presence of polyphenol (tannins, saponin, and flavonoids). This suggests that L. hastate leaf and stem extracts have anti-snake venom activities since polyphenols have been established to possess protein -binding and enzyme inhibiting properties, which could invariably inhibit snake venom PLA 2 activities, an enzyme presents in cobra venom. [20]

Antivenom Assay
The result of the PLA 2 of the snake venom when challenged with the MEL and MES extracts of L. hastatais presented in Table 3. PLA 2 assay is an important assay mainly used to test for the anti-snake venom of plant extracts. In this assay, the enzyme was hydrolysed and free fatty acids were released in the presence of  sodium deoxycholate. N. nigricollis venom was found to liberate fatty acids, which was measured in terms of a decrease in pH of egg yolk suspension. While the MES were inhibited the hydrolytic action of PLA 2 enzyme in a concentration-dependent manner with 48, 50, and 51% inhibition at 0.1, 0.3, and 0.6 mg/mL, MEL had an inhibition of 48, 51, and 53% at 0.1, 0.3, and 0.6 mg/mL. This shows a slightly better anti-snake venom activity of MEL compared to MES at the highest investigated dose. These results significantly blocked many of the toxic effects of N. nigricollis venom in vitro. This is possible due to the extracts acting through a mechanistic intervention rather than a direct physical interaction with the venom in vitro as this is similar to the mode of action of many polyphenolic compounds found in plant extracts. [21] This result is in agreement with the report of Ode and Asuzu [22] who used different venom concentrations different from that reported in this research with the results following the same trend, although snake venom neurotoxin content varies both qualitative and quantitative from region to region within the same species. [23,24] Further, research is currently being advanced in the fractionation assay of the L. hastate extract to identify the component(s) responsible for the inhibitory effect reported.

conclusIon
The present findings validate the tradomedicinal application of L. hastate as an anti-snake, particularly N. nigricollis venom. The plant extracts were significantly effective in neutralizing the main toxic and enzymatic effects of N. nigricollis venom. The antivenom properties of both plant extracts were potent enough to neutralize the lethality and various pharmacological activities of N. nigricollis venom. This activity is corroborated with the diverse phytoconstituents revealed to be present in the plant materials. The plant could serve as therapy for patients with snakebite envenomation.