Abstract
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/ 3LD50 of Naja naja venom. LC-MS analysis from root extract of Rauvolfia serpentina was done for confirmation of the bioactive compounds.
Keywords:
Snake bite; Naja naja; Rauvolfia serpentina; Antivenom 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., 2010Gutierrez JM, Williams D, Fan HW, Warrell DA. Snakebite envenoming from a global perspective: Towards an integrated approach. Toxicon. 2010;56(7):1223-1235.). 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., 2009Harrison RA, Hargreaves A, Wagstaff SC, Faragher B, Lalloo DG. Snake envenomation: a disease of poverty. PLoS Negl Trop Dis. 2009;3(12):e569.). 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., 2011Mohapatra B, Warrell DA, Suraweera W, Bhatia P, Dhingra N, Jotkar R. Snake bite mortality in India: A nationally representative mortality survey. PLOS Negl Trop Dis. 2011;5(4):e1018.). 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, 2012Mukherjee AK. Green medicine as a harmonizing tool to antivenum therapy. Indian J Med Res. 2012;136(1):10-12.). The most common enzymes in snake venoms are phospholipase A2s (PLA2s), 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., 2011Kang TS, Georgieva D, Genov N, Murakami TM, Sinha M, Kumar RP. Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis. FEBS. 2011;278(23):4544-76.). 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, 2014Bawaskar HS. Snake bite poisoning: A neglected life-threatening occupational hazard. Indian J Crit Care Med. 2014.18(1):123-4.). 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., 2011Sarkhel S, Chakravarthy AK, Das R, Gomes A. Snake venom neutralizing factor from root extract of Emblica officinalis Linn. Orient Pharm Exp Med. 2011;11(1):25-33.). 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., 2014Khan AV, Ahmed QU, Khan MW, Khan AA. Herbal cure for poisons and poisonous bites from Western Uttar Pradesh, India. APJTD. 2014;4(1):116-120.). 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., 2013Vijaya P, Ranjani R, Rao MR, Sudarsanan G. Impact of antidote medicinal plant-corallocarpus epigeus extract on lipid peroxidation induced by Naja naja-snake venom in albino rat. Int J Med Pharm Sci. 2013;3(5):23-30.).
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., 2012Deshmukh SR, Dhanashree SA, Patil BA. Extraction and evaluation of Indole alkaloids from Rauvolfia serpentina for their antimicrobial and antiproliferative activities. Int J Pharm Pharm Sci. 2012;4:329-334.). 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, 2012Mallick SR, Jenna RC, Samal KC. Rapid in vitro multiplication of an endangered medicinal plant sarpagandha( Rauvolfia serpentina). AJPS. 2012;3(2):437-442.). 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., 2012Rathore SK, Bhatt S, Dhyani S, Jain A. Preliminary phytochemical screening of medicinal plant Ziziphus Mauritiana Lam. Fruits. IJCPRF. 2012;4(3)160-162.). It is used as an antidote to snakes and bites of other toxic insects (Ghani, 1998Ghani A. Medicinal plants of Bangladesh chemical constituents and uses. Bangladesh: Asiat Soc Bangladesh; 1998; 2:36.). Rhizome and leaf decoction are orally given in snake bite in the rural areas of Kanyakumari district, India (Jeeva et al., 2006Jeeva S, Kiruba S, Mishra BP, Venugopal N, Dhas SSM, Regini GS, et al. Weeds of kanyakumari district and their value in rural life. Indian J Tradit Know. 2006;5(4):501-509.).
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.
MATERIAL AND METHODS
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.
In vitro assessment of venom toxicity and neutralization assays
Acetyl cholinesterase activity
Acetyl cholinesterase inhibition assay was carried out according to the modified method of Ellman et al. (1961)Ellman GL, Courtney KD, Andres V, Featherstone RM. A new and rapid colorimetric determination of acetyl cholinesterase activity. Biochem Pharmacol. 1961;7(2):88-95.. 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
Proteolytic activity
Proteolytic activity was determined according to the method Satake, Murata and Suzuki (1963)Satake M, Murata Y, Suzuki T. Studies on Snake venom. Biochem. 1963;53:438-447.. 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.
ATPase activity
ATPase activity was measured according to the modified method of Kini and Gowda (1982)Kini RM, Gowda TV. Studies on snake venom enzymes: Part 1 Purification of ATPase, a toxic component on Naja naja venom and its inhibition by potassium gymnemate. Indian J Biochem Biophys. 1982;19(2):152-154.. 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 anti-venom effect of plant extracts lethal toxicity
The median lethal dose (LD50) of Naja naja venom was determined according to the method of Randhawa (1944)Randhawa MA. Calculation of LD50 values from the method of Miller and Tainter. J Ayub Med Coll Abbottabad. 1944;21(3):184-5..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 LD50 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 2LD50 of Naja naja venom. Various amount of plant extracts (µL) were mixed with 2LD50 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 (ED50) calculated from the number of deaths within 24 h of injection of the venom/antivenom mixture. ED50 was expressed as µL antivenom/mouse and calculated by probit analysis (Miller, Tainter, 1944Miller LC, Tainter ML. Estimation of LD50 and its error by means of log-probit graph paper. Proc Soc Exp Biol Med. 1944;57:261.).
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, 1995Filimonov DA, Poroikov VV, Karaicheva EI. Computer-aided prediction of biological activity spectra of chemical substances on the basis of their structural formulae: computerized system pass. Exper Clin Pharmacol. (Rus). 1995;58:56-62.; Poroikov, Filimonov, 1996Poroikov VV, Filimonov DA. QSAR and molecular modelling concept, computational tools and biological applications. Barcelona: Prous Science Publishers; 1996, p.49-50.).
Statistical analysis
Statistical evaluation was performed using XL stat 2008 and SPSS 10 Software<0.005 was considered statistically significant.
RESULTS
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 (LD50) of Naja naja venom was assessed by LD50 range finding test and the median lethal dose (LD50) assay using mice (18-20 g). LD50 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 (ED50) using Rauvolfia serpentina root extract was carried out by pre incubating constant amount of venom (3LD50) with various dilutions of Rauvolfia serpentina root extract prior to injection. Calculation of ED50 of Rauvolfia serpentina root of 3LD50 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 LD50 of Rauvolfia serpentina root extract was > 2000 mg/kg.
Calculation of LD50 of Naja naja venom in mice receiving various doses of Naja naja venom by Miller and Tainter method (n=5)
Calculation of ED50 of Rauvolfia serpentina against Naja naja venom in mice by Miller and Tainter method (n=5)
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 A2 inhibition, ATPase inhibition, nucleotidase inhibition, 5’- nucleotidase inhibition, L-aminoacid oxidase inhibition, phosphodiesterase inhibition activity and have antidote activity (Table VI and Figure 6).
Activities of the compounds identified from the extract of Rauvolfia serpentina root by LC-MS analysis (PASS)
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., 2009Namsa ND, Tag H, Mandal M, Kalita P, Das AK. An ethnobotanical study of traditional anti-inflammatory plants used by the Lohit community of Arunachal Pradesh, India. J Ethnopharmacol. 2009;125(2):234-245.; Upadhyay, Dhaker, Kumar, 2010Upadhyay B, Parveen Dhaker AK, Kumar A. Ethnomedicinal and ethnopharmaco-statistical studies of Eastern Rajasthan, India. J Ethnopharmacol. 2010;129(1):64-86.). 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., 2008Sarada P Mohapatra, Gagan B Prusty, Hara P Sahoo. Ethnomedicinal observations among forest dwellers of the Daitari Range of Hills of Orissa, India. Ethnobotanical Leaflets. 2008;12:1116-23.). 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, 1993Houghton PJ, Osibogun IM. Flowering plant used against Snakiebite. J Ethnopharmacol. 1993;39(1):1-29.). Singh (2008)Singh H. Importance of local names of some useful plants in ethnobotanical study. Indian J Tradit Know. 2008;7(2):365-370. 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, 2007Behera KK, Sahoo S, Mohapatra PC. Medicinal Plant Resources for Bioprospecting and Drug Development in Tribal Rich District of Orissa, India. Ethnobot Leaflets. 2007;11:106-112.). 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 (Mohapatra, Prusty, Sahoo, 2008Mohapatra SP, Prusty GB, Sahoo HP. Ethnomedicinal observations among forest dwellers of the Daitari Range of Hills of Orissa, India. Ethnobo Leaflets. 2008;12:1116-1123.). Pattanaik, Reddy and Reddy (2009)Pattanaik C, Reddy S, Reddy KN. Ethno-medicinal survey of threatened plants in Eastern Ghats, India. Our Nature. 2009;7(1):122-128. 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)Kadiyala G. The neutralization effect of methanol extract of Andrographis paniculata on Indian cobra Naja naja snake venom. J Pharm Res. 2011;4(4):1010-1012. 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 2LD50 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 (LD50 ) of Naja naja venom was assessed by LD50 range finding test and the median lethal dose (LD50) assayed using mice (18-20g). LD50 of Naja naja venom was calculated by Miller and Tainter method and was found to be 0.301 µg/g .Venom neutralizing potency tested (ED50) 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)Rajasree PH, Singh R, Sankar C. Anti venom activity of ethanolic extract - of Rauvolfia serpentina against Naja naja(Cobra)venom. IJDDHR. 2013;3:521-524. 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 2LD50 of Naja naja venom. In another study of James et al. (2013)James T, Dinesh MD, Uma MS, Vadivelan AS, Meenathisundaram S, Shanmugam V. In vivo and In vitro neutralizing potential of Rauvolfia serpentine plant extract against Daboia russelli venom. Adv Biol Res. 2013;7(6):276-281. 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 (LD50) and antivenom neutralizing capacity (ED50) 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 A2 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., 2015Butt MA, Ahmad M, Fathima A, Sultana S, Zafar M, Yaseen G, et al. Ethno medicinal uses of plants for the treatment of snake and scorpion bite in Northern Pakistan. J Ethnopharmacol. 2015;168:164-181.). 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, 1995Haruna AK, Choudhury MK. In vivo antisnake venom activity of the furanoid diterpene from Aristolochia albida Duch. Indian J Pharm Sci. 1995;27:222-224.). The mechanism of action of the plant extracts/plant compounds are still not clear and they may be attributed to the blocking of receptors-structure 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.
ACKNOWLEDGEMENT
The authors are thankful to Principal Dr. Anirudhan, for providing the necessary facilities in the College and sincerely thanks to Dr. J. Rathinamala, Head, Department of Microbiology, Nehru Arts and Science College, Coimbatore, for her encouragement and support throughout the study.
REFERENCE
- Bawaskar HS. Snake bite poisoning: A neglected life-threatening occupational hazard. Indian J Crit Care Med. 2014.18(1):123-4.
- Behera KK, Sahoo S, Mohapatra PC. Medicinal Plant Resources for Bioprospecting and Drug Development in Tribal Rich District of Orissa, India. Ethnobot Leaflets. 2007;11:106-112.
- Butt MA, Ahmad M, Fathima A, Sultana S, Zafar M, Yaseen G, et al. Ethno medicinal uses of plants for the treatment of snake and scorpion bite in Northern Pakistan. J Ethnopharmacol. 2015;168:164-181.
- Deshmukh SR, Dhanashree SA, Patil BA. Extraction and evaluation of Indole alkaloids from Rauvolfia serpentina for their antimicrobial and antiproliferative activities. Int J Pharm Pharm Sci. 2012;4:329-334.
- Ellman GL, Courtney KD, Andres V, Featherstone RM. A new and rapid colorimetric determination of acetyl cholinesterase activity. Biochem Pharmacol. 1961;7(2):88-95.
- Filimonov DA, Poroikov VV, Karaicheva EI. Computer-aided prediction of biological activity spectra of chemical substances on the basis of their structural formulae: computerized system pass. Exper Clin Pharmacol. (Rus). 1995;58:56-62.
- Ghani A. Medicinal plants of Bangladesh chemical constituents and uses. Bangladesh: Asiat Soc Bangladesh; 1998; 2:36.
- Gutierrez JM, Williams D, Fan HW, Warrell DA. Snakebite envenoming from a global perspective: Towards an integrated approach. Toxicon. 2010;56(7):1223-1235.
- Harrison RA, Hargreaves A, Wagstaff SC, Faragher B, Lalloo DG. Snake envenomation: a disease of poverty. PLoS Negl Trop Dis. 2009;3(12):e569.
- Haruna AK, Choudhury MK. In vivo antisnake venom activity of the furanoid diterpene from Aristolochia albida Duch. Indian J Pharm Sci. 1995;27:222-224.
- Houghton PJ, Osibogun IM. Flowering plant used against Snakiebite. J Ethnopharmacol. 1993;39(1):1-29.
- James T, Dinesh MD, Uma MS, Vadivelan AS, Meenathisundaram S, Shanmugam V. In vivo and In vitro neutralizing potential of Rauvolfia serpentine plant extract against Daboia russelli venom. Adv Biol Res. 2013;7(6):276-281.
- Jeeva S, Kiruba S, Mishra BP, Venugopal N, Dhas SSM, Regini GS, et al. Weeds of kanyakumari district and their value in rural life. Indian J Tradit Know. 2006;5(4):501-509.
- Kadiyala G. The neutralization effect of methanol extract of Andrographis paniculata on Indian cobra Naja naja snake venom. J Pharm Res. 2011;4(4):1010-1012.
- Kang TS, Georgieva D, Genov N, Murakami TM, Sinha M, Kumar RP. Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis. FEBS. 2011;278(23):4544-76.
- Khan AV, Ahmed QU, Khan MW, Khan AA. Herbal cure for poisons and poisonous bites from Western Uttar Pradesh, India. APJTD. 2014;4(1):116-120.
- Kini RM, Gowda TV. Studies on snake venom enzymes: Part 1 Purification of ATPase, a toxic component on Naja naja venom and its inhibition by potassium gymnemate. Indian J Biochem Biophys. 1982;19(2):152-154.
- Mallick SR, Jenna RC, Samal KC. Rapid in vitro multiplication of an endangered medicinal plant sarpagandha( Rauvolfia serpentina). AJPS. 2012;3(2):437-442.
- Miller LC, Tainter ML. Estimation of LD50 and its error by means of log-probit graph paper. Proc Soc Exp Biol Med. 1944;57:261.
- Mohapatra B, Warrell DA, Suraweera W, Bhatia P, Dhingra N, Jotkar R. Snake bite mortality in India: A nationally representative mortality survey. PLOS Negl Trop Dis. 2011;5(4):e1018.
- Mohapatra SP, Prusty GB, Sahoo HP. Ethnomedicinal observations among forest dwellers of the Daitari Range of Hills of Orissa, India. Ethnobo Leaflets. 2008;12:1116-1123.
- Mukherjee AK. Green medicine as a harmonizing tool to antivenum therapy. Indian J Med Res. 2012;136(1):10-12.
- Namsa ND, Tag H, Mandal M, Kalita P, Das AK. An ethnobotanical study of traditional anti-inflammatory plants used by the Lohit community of Arunachal Pradesh, India. J Ethnopharmacol. 2009;125(2):234-245.
- Pattanaik C, Reddy S, Reddy KN. Ethno-medicinal survey of threatened plants in Eastern Ghats, India. Our Nature. 2009;7(1):122-128.
- Poroikov VV, Filimonov DA. QSAR and molecular modelling concept, computational tools and biological applications. Barcelona: Prous Science Publishers; 1996, p.49-50.
- Rajasree PH, Singh R, Sankar C. Anti venom activity of ethanolic extract - of Rauvolfia serpentina against Naja naja(Cobra)venom. IJDDHR. 2013;3:521-524.
- Randhawa MA. Calculation of LD50 values from the method of Miller and Tainter. J Ayub Med Coll Abbottabad. 1944;21(3):184-5.
- Rathore SK, Bhatt S, Dhyani S, Jain A. Preliminary phytochemical screening of medicinal plant Ziziphus Mauritiana Lam. Fruits. IJCPRF. 2012;4(3)160-162.
- Sarada P Mohapatra, Gagan B Prusty, Hara P Sahoo. Ethnomedicinal observations among forest dwellers of the Daitari Range of Hills of Orissa, India. Ethnobotanical Leaflets. 2008;12:1116-23.
- Sarkhel S, Chakravarthy AK, Das R, Gomes A. Snake venom neutralizing factor from root extract of Emblica officinalis Linn. Orient Pharm Exp Med. 2011;11(1):25-33.
- Satake M, Murata Y, Suzuki T. Studies on Snake venom. Biochem. 1963;53:438-447.
- Singh H. Importance of local names of some useful plants in ethnobotanical study. Indian J Tradit Know. 2008;7(2):365-370.
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Publication Dates
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Publication in this collection
06 Apr 2020 -
Date of issue
2020
History
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Received
20 Jan 2018 -
Accepted
13 Nov 2018