Neutralizing potential of Rauvolfia serpentina root extract against Naja naja venom

Snake bites are a serious health hazard occurs throughout the world especially in tropical countries like India. Anti-Snake Venom Serum is the only remedy available to treat snake bite victims successfully till date. Infusion of ASV may lead to adverse reactions ranging from severe itching of the skin, hives to potentially serious allergic reactions. Considering all above difficulties research workers all over the world is constantly in search of a cheap and readily available easy formulate remedy for treating snake bite victims. In present study aqueous extract of Rauvolfia serpentina root was checked for the antidote properties against Naja naja venom by in vitro and in vivo methods. Various in vitro neutralization tests like Acetyl cholinesterase, Protease and ATPase activity of Naja naja venom were carried out and the root extract was neutralized all the toxic effects induced by the venom. The in vivo assessment of venom lethality (LD50) of Naja naja venom was found to be 0.301 μg. The aqueous root extract was effectively neutralized the venom lethality and effective dose (ED50) was found to be 12.88 mg/ 3LD50of Naja naja venom. LC-MS analysis from root extract of Rauvolfia serpentina was done for confirmation of the bioactive compounds.


INTRODUCTION
Snakebite envenoming is a neglected tropical disease which requires immediate consideration. Every year 2.5 million people are bitten by snakes with 85,000 deaths (Gutierrez et al., 2010). Agriculturists and their families living in rural areas of the country are the most affected community, thus snakebite is represented as 'disease of poverty' (Harrison et al., 2009). A total number of snake species identified is about 2,000 to date and nearly 300 species of them are venomous snakes, which prevail over all parts of the world except Antarctica (Mohapatra et al., 2011). The four major species of venomous snakes ubiquitous in India known as "Big four" are considered responsible for life-threatening envenomation around the country (Mukherjee, 2012). The most common enzymes in snake venoms are phospholipase A 2 s (PLA 2 s), serine proteinases, metalloproteinase, acetyl cholinesterase (AChEs), l-amino acid oxidases, nucleotides (5′-nucleotidase, ATPase, phosphodiesterases and DNase) and hyaluronidase. Snake venoms are the most abundant source of all these enzymes (Kang et al., 2011). The venom of Naja naja is neurotoxic, they affect the victim's central nervous system and cause heart failure. The venoms are rich in post synaptic neurotoxins called alpha-bungarotoxin and cobratoxin. Venom binds particularly to Acetylcholine receptors, prevents the interaction between Acetylcholine and receptors on post synaptic membrane result in neuromuscular blockade (Bawaskar, 2014). Antiserum is the only therapeutic agent available throughout the world. The antiserum development is a costly, time-consuming process requiring ideal storage conditions. Absolute specificity is an issue in management with antiserum. Snake venom antiserum does not provide adequate safety against venom induced local pain, local bleeding swelling and difficulty including wound necrosis, hemorrhage, necrosis and nephrotoxicity (Sarkhel et al., 2011). Over the years, many attempts have been made for the advance of snake venom antagonists particularly from plants sources since there are limitations on the expansion of anti-sera (Khan et al., 2014). In folk medicine, many botanical species are employed for the treatment of snakebites in communities that lack rapid access to serum therapy especially in developing countries. Herbal components play an important role in management and controlling of venomous snake bite (Vijaya et al., 2013).
Rauvolfia serpentina (L.) Benth. ex Kurz. (Ophioxylon serpentinum L.) is an evergreen, perennial shrub with maximum height up to 60 cm (Deshmukh, Dhanashree, Patil et al., 2012). The plant belongs to the family Apocynaceae and occurs to habitats of tropical and subtropical regions. The plant is commonly known as Sarpagandha, Chandrabagha, Snake root plant, Chotachand, Chandrika and Harkaya etc (Mallick, Jenna, Samal, 2012). The roots of Rauvolfia serpentina are used in Ayurvedic medicines as a treatment for curing hypertension, insomnia, mental anxiety, gastrointestinal disorders, anticipation epilepsy, trauma, anxiety, stimulation, schizophrenia, sedative insomnia and psychosis (Rathore et al., 2012). It is used as an antidote to snakes and bites of other toxic insects (Ghani, 1998). Rhizome and leaf decoction are orally given in snake bite in the rural areas of Kanyakumari district, India (Jeeva et al., 2006).
The present investigation explored Naja naja venom neutralizing activity of Rauvolfia serpentina root extracts by in vitro, in vivo methods and antivenom compounds were identified using LCMS analysis.

Collection and authentication of plant material:
Rauvolfia serpentina (L.) Benth. ex Kurz. (Ophioxylon serpentinum L.) belongs to the family Apocynaceae was collected from Anakkal region, Malampuzha, Palakkad district, Kerala after questionnaire with tribal people and from vaidyas in and around Palakkad district. It was authenticated by Dr. Althaf Ahamed Kabeer. Scientist 'D'.Botanical Survey of India Southern Regional Centre. Coimbatore (Herbarium voucher specimen number 1160).

Preparation of extract
20 g of powdered sample of the herb was extracted by soaking in 180 mL of distilled water in a beaker, stirred for about 6 min and left over night. Thereafter, the solution was filtered using filter paper (What man No. 1) and the extracts were evaporated to dryness under reduced pressure in 40 °C. The plant extracts were expressed in terms of dry weight.

Snake venom
The freeze-dried snake venom powders of Naja naja were obtained from Irula's Snake Catchers Industrial Co-operative Society Limited Chennai and was stored at 4 °C. Stock solution was prepared by dissolving 1 mg of lyophilized venom in 1mL of physiological saline (1 mg/mL). (Ethics committee approval number: JSSCP/ IAEC/PH.D/PH.COLOGY/02/2014-15)

Acute oral toxicity
Acute oral toxicity of all the selected plant extracts was performed as per OECD guidelines 423. A limit test of 2000 mg/kg body weight of the extracts was administered. Briefly, two thousand milligrams of the test substance per kilogram of body weight were administered to 3 healthy mice by oral gavages. The animals were observed for mortality, signs of gross toxicity, and behavioral changes at least once daily for 14 days. Body weights were recorded prior to administration and again on Days 7 and 14 (day of termination). Necropsies were performed on all animals at terminal sacrifice.

Acetyl cholinesterase activity
Acetyl cholinesterase inhibition assay was carried out according to the modified method of Ellman et al. (1961). 200 µg of venom (1 mg/mL) was pre-incubated (1 h) with different concentrations of plant extract and supernatant was added to the assay mixture which consists of 100 µL of 75 mM acetylcholine idodate in 1 mL of phosphate buffer. The activity was measured by taking the absorbance at 412 nm. Venom without plant extracts was considered as control or 100% activity Inhibition % = control-test/control x100

Proteolytic activity
Proteolytic activity was determined according to the method Satake, Murata and Suzuki (1963). Using 2% casein as substrate in 0.02 M Tris-HCl buffer (pH 8.5). Venom 200 µg (1 mg/mL) and different dilutions of plant extract were pre-incubated with 1 mL of substrate for 2 h at 37 °C. The undigested casein was precipitated by the addition of 1.5 mL of 0.44 M trichloroacetic acid (TCA) and centrifuged. The digested casein in the supernatant was determined using Folin ciocalteu's reagent. Venom without plant extracts was considered as control or 100% activity.

Inhibition % = control-test/control x100
ATPase activity ATPase activity was measured according to the modified method of Kini and Gowda (1982). Naja naja venom 200 µg (1 mg/mL) were pre-incubated with different concentrations of plant extract of Rauvolfia serpentina root for 30 minutes. To the reaction, 1 mL of assay mixture (750 µL of 0.1 M Tris pH 7.5, 100 µL of 0.1 MgCl2, 50 µL of 0.1 M ATP, and 100 µL of BSA) was added with gentle shaking at 37 °C and stopped at a certain time (1 h) by adding 1 mL of SDS solution. The inorganic phosphate formed was determined by phosphate determination method by taking 400 µL of sample along 600 µL of TCA and incubated separately for 10 min at 37 °C followed by centrifugation at 1500 rpm for 10 min. About 500 µL of supernatant was added together with 500 µL of ferrous sulphate-ammonium molybdate reagent and the absorbance was measured at 820 nm within 2 h for every 10 minutes of intervals. Reaction mixture without plant sample was referred as control or 100% activity. Inhibition reaction was calculated in terms of percentage (100%). Na,K-ATPase were mainly used.

In vivo assessment of venom toxicity and antivenom effect of plant extracts lethal toxicity
The median lethal dose (LD 50 ) of Naja naja venom was determined according to the method of Randhawa (1944).Various doses of venom in 0.2 mL of physiological saline were injected into the tail vein of mice, using groups of 3-5 mice for each venom dose. The LD 50 was calculated with confidence limit at 50% probability by the analysis of deaths occurring within 24 h of venom injection.
The anti-lethal potentials for plant extract were determined against 2LD 50 of Naja naja venom. Various amount of plant extracts (μL) were mixed with 2LD 50 of venom sample and incubated at 37 °C for 30 minutes and then injected intravenously into mice. 3-5 mice were used at each antivenom dose. Control mice received same amount of venom without antivenom (plant extracts). The median Effective Dose (ED 50 ) calculated from the number of deaths within 24 h of injection of the venom/antivenom mixture. ED 50 was expressed as μL antivenom/mouse and calculated by probit analysis (Miller, Tainter, 1944).

LC-MS analysis
Phytochemical screening for compounds present in the aqueous extract from Rauvolfia serpentina root was carried out using LC Column: Reverse Phase C-18. The chromatographic separation was performed on a Phenomenex RP C-18 (25 cm x2.5 mm ) at Column temperature: 25 °C. A gradient Electronic Spray Ionization mode was performed at a flow rate of 2 mL/mins. The mobile phase: water: methanol (40:50). The volume of injection was 10 µL. Mass spectrometry data were obtained both positive and negative ionization modes. Class V P Integrated Soft Ware was used for the MS analysis and analyzed samples were compared with METWIN 2.0 library. These antivenom compounds were detected by PASS software. The computer program PASS (Prediction of Activity Spectra for Substances) was designed to predict many kinds of biological activity simultaneously based on the structural formulae of chemical compounds (Filimonov, Poroikov, Karaicheva, 1995;Poroikov, Filimonov, 1996).

Statistical analysis
Statistical evaluation was performed using XL stat 2008 and SPSS 10 Software<0.005 was considered statistically significant.

Inhibition of Acetyl cholinesterase activity
The aqueous extract of the plant was taken in different dilutions starting from 200 µg to 300 µg with triplicate experiments. Maximum of Acetyl cholinesterase inhibition (60.29%) was occurred at 300 µg concentration of venom and aqueous extract of plant respectively. The activity was calculated in terms of percentage of inhibition compared to venom pre-incubated with different amounts of plant extract and venom with substrate. The enzyme reaction was observed for every 10 minutes intervals at 412 nm. Acetyl cholinesterase activity of the venom was considered as 100%. (Figure 1) and (Table I).

Inhibition of protease activity
To assess the in vitro antagonism of protease, the venom degrades the substrate (casein) into peptide precipitation could be observed at 600 nm. Maximum of protease inhibition (50.40%) was occurred at 300 µg concentrations of venom and aqueous extract of plant respectively. From the results it was observed that increased amount of plant extract could increase the inhibition of protease of cobra activity (Figure 2 and Table II).

Inhibition of ATPase activity
ATPase inhibition was calibrated by liberation of inorganic phosphate with of positive control of venom (200 µg) and substrate as ATP (10 µm). Different concentrations of venom and substrate were used for this reaction. The same concentration of venom 200 µg with different amounts of active aqueous extract from the plant (200 µg to 300 µg) was pre-incubated for the reaction. Maximum inhibition up to 58.76% has been noted at highest amount of plant concentration ( Figure 3 and Table III).

In vivo methods
In vivo assessment of venom toxicity (LD 50 ) of Naja naja venom was assessed by LD 50 range finding test and the median lethal dose (LD 50 ) assay using mice (18-20 g). LD 50 of Naja naja venom was calculated by Miller and Tainter method and was found to be 0.301 µg/g.    (Table IV and Figure 4).Venom neutralizing potency tested (ED 50 ) using Rauvolfia serpentina root extract was carried out by pre incubating constant amount of venom (3LD 50 ) with various dilutions of Rauvolfia serpentina root extract prior to injection. Calculation of ED 50 of Rauvolfia serpentina root of 3LD 50 of venom was found to be 12.88 µg against Naja naja venom (Table V and Figure 5). All animals survived and appeared active and healthy throughout acute oral toxicity study. There were no signs of gross toxicity, antagonistic pharmacological effects or uncommon behavior. Based on the above findings, the LD 50 of Rauvolfia serpentina root extract was > 2000 mg/kg.

LCMS analysis
Various compounds were identified by LCMS analysis which includes D-glucuronic acid, triacontanol, alppha-ionine, hydroquinone, reserpinine, reserpine, ascorbic acid, gallic acid, hydrangenol and oleic acid and their activities were determined using PASS software. Among the compounds, D-glucuronic acid, triacontanol, reserpine, gallic acid and oleic acid possess antivenom activity due to inhibition of various snake venom enzymes activities like phospholipase A 2 inhibition, ATPase inhibition, nucleotidase inhibition, 5'-nucleotidase inhibition, L-aminoacid oxidase inhibition, phosphodiesterase inhibition activity and have antidote activity (Table VI and Figure 6).

DISCUSSION
During the last few years there has been an increasing interest in the study of medicinal plants and their traditional use of different parts of India. In the recent years number of reports on the use of plants in traditional healing by either tribal people or indigenous communities of India is increasing (Namsa et al., 2009;Upadhyay, Dhaker, Kumar, 2010). The herbal medicines are mostly administered in the form of juice, decoction, paste or powder, prepared in a crude method from different plant parts such as root, bark, leaves, flowers, fruits, seeds and whole plant (Sarada et al., 2008). The aqueous extracts were selected for the study because the 'vishavaidayas' used only the aqueous extracts to treat the ill effects of snake bite. Earlier research on plants as antivenom also supports the use of aqueous extract (Houghton, Osibogun, 1993). Singh (2008) has stated the ethno remedial use of Rauvolfia serpentina plant against snake bite. Root decoction is being used as an antidote to snake venom in some tribal rich district of Orissa, India (Behera, Sahoo, Mohapatra, 2007). About 10 mL of root paste is taken orally for management of snake bite by the forest inhabitants of the Daitari range of hills of Orissa, India . Pattanaik, Reddy and Reddy (2009) have informed the use of this plant (Known as Patalgaruda locally) by the local people of Eastern Ghats, India against snakebite. In our present investigation antivenom potential for Rauvolfia serpentina aqueous root extract against Naja naja venom was studied by in vivo and in vitro methods

In vitro neutralization assays
Maximum of Acetyl cholinesterase inhibition (60.29%), protease inhibition (50.40%), and ATPase inhibition (58.76 %) was occurred for 300 µg concentration of aqueous plant extract. The inhibition of Naja naja venom enzymes by increased amounts of aqueous extract from Rauvolfia serpentina was very effective, when the extract was previously mixed with venom. There was a substantial deactivation of Acetyl cholineesterase, Protease, ATPase activities. The studies of Kadiyala (2011) have also reported the inhibition of Naja naja venom enzymes by increased amounts of methanol extracts from Andrographis paniculata.

In vivo neutralization assays
The plant extract effectively neutralized the Naja naja venom induced lethal activity. About 0.14 mg of Rauvolfia serpentina plant extract was able to completely neutralize the lethal activity of 2LD 50 of Naja naja venom. The alkaloid reserpine inhibits the action of phospholipase-A2 enzyme from Naja naja venom. In our study In vivo assessment of venom toxicity (LD 50 ) of Naja naja venom was assessed by LD 50 range finding test and the median lethal dose (LD 50 ) assayed using mice (18-20g). LD 50 of Naja naja venom was calculated by Miller and Tainter method and was found to be 0.301 µg/g .Venom neutralizing potency tested (ED 50 ) using Rauvolfia serpentina root extract was done by Miller and Tainter method and was found to be 12.88 mg against Naja naja venom. In previous report on Rajasree, Singh and Sankar (2013) the ethanol extracts from Rauvolfia serpentina plant were tested for antivenom activity against Naja naja venom. The plant extract effectively neutralized the Naja naja venom induced lethal activity. About 0.14 mg of Rauvolfia serpentina plant extract was able to completely neutralize the lethal activity of 2LD 50 of Naja naja venom. In another study of James et al. (2013) of in vivo and in vitro neutralizing potential of Rauvolfia serpentina plant extract against Daboia russelli venom,Rauvolfia serpentina plant extract was effectively neutralized the venom lethality and effective dose (ED ) was found to be 10.99 mg/ 3LD of venom The tests of determining venom lethality (LD 50 ) and antivenom neutralizing capacity (ED 50 ) are currently the only validated means of assessing venom toxicity and antivenom neutralizing potency by both manufacturers and regulatory authorities worldwide. There were no signs of gross toxicity, antagonistic pharmacological effects or uncommon behavior.

LCMS analysis of aqueous extract of Rauvolfia serpentina root
In the present study the phytochemical profile of aqueous extract from Rauvolfia serpentina root were characterized. Various antivenom compounds were detected from LCMS analysis of aqueous root extract which includes D-glucuronic acid, triacontanol, reserpine, gallic acid and oleic acid due to inhibition of various snake venom enzymes activities like phospholipase A 2 inhibition, ATPase inhibition, nucleotidase inhibition, 5'nucleotidase inhibition, L-aminoacid oxidase inhibition, phosphodiesterase inhibition activity and have antidote activity. Active phytochemical compounds like D-glucuronic acid, gallic acid, oleic acid isolated from different plant sources, have already been proven for their anti-venom potential (Butt et al., 2015). The alkaloid reserpine inhibits the action of phospholipase-A2 enzyme from Naja naja venom. Many plant extracts have been reported to possess a detoxifying effect on snake venoms (Haruna, Choudhury, 1995). The mechanism of action of the plant extracts/plant compounds are still not clear and they may be attributed to the blocking of receptorsstructure prone to chemical attack, and may block the active site of the snake venom. Hence, the presence of these anti snake venom compounds in the aqueous extract from Rauvolfia serpentina root could have contributed to its efficient antivenom activity.