Objective: This study was carried out to investigate the efficacy of inhibitory effect of ethanolic root extract (ERE) of plant Coix lachrymajobi (Poaceae) on hyaluronidase and L-amino acid oxidase (LAAO) of Daboia russelii and Naja naja venom.Â Methodology: The ethanolic root extract of C. lachrymajobi is used to treat snake bite victims by traditional healers as folk medicine for centuries, was tested in-vitro to determine its ability to inhibit D. russelli and N. naja hyaluronidase and LAAO activities.Â  In-vitro studies were carried out with different doses of ERE.Results &amp; Discussion: which effectively neutralized hyaluronidase and LAAO activities of both D. russelli and N. naja venom.Â  ERE at doses of 2000Âµg successfully inhibited D. russelli (50 Âµg) hyaluronidase activity by 67.04% and LAAO activity by 77.86%. Â The hyaluronidase activity of N. naja venom (50 Âµg) was inhibited by 71.83% and LAAO activity was reduced by 74.83%.Â  From the results, It is evident that the ERE of Coix lachrymajobi effectively neutralizes important toxic enzymes of the D. russelli and Naja naja venoms.Keywords: Daboia russelii; Naja naja; Coix lacryma-jobi; Hyaluronidase; L-amino acid oxidas

BHAT K,  ISHWARA

KS, RAJESH

RAO C, VAMAN

English

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Vol 8, Issue 5, 2015 ISSN - 0974-2441INHIBITORY EFFECT OF ETHANOLIC ROOT EXTRACT OF COIX LACHRYMAJOBI ON HYALURONIDASE AND L-AMINO ACID OXIDASE OF NAJA NAJA AND DABOIA RUSSELLI VENOMRAJESH KS1, VAMAN RAO C2*, ISHWARA BHAT K31Department of Pharmacy Practice, Nitte University, Mangalore, Karnataka, India. 2Department of Biotechnology Engineering, NMAM Institute of Technology, Nitte, Mangalore, Karnataka, India. 3Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical Sciences, Nitte University, Mangalore, Karnataka, India. Email: vaman.rao@gmail.comReceived: 09 June 2015, Revised and Accepted: 16 July 2015ABSTRACTObjective: This study was carried out to investigate the efficacy of inhibitory effect of ethanolic root extract (ERE) of plant Coix lachrymajobi (Poaceae) on hyaluronidase and L-amino acid oxidase (LAAO) of Daboia russelli and Naja naja venom.Methods: The ERE of C. lachrymajobi is used to treat snakebite victims by traditional healers as folk medicine for centuries, was tested in-vitro to determine its ability to inhibit Daboia russelli and N. naja hyaluronidase and LAAO activities. In-vitro studies were carried out with different doses of ERE.Results and Discussion: This effectively neutralized hyaluronidase and LAAO activities of both D. russelli and N. naja venom. ERE at doses of 2000 µg successfully inhibited D. russelli (50 µg) hyaluronidase activity by 67.04% and LAAO activity by 77.86%. The hyaluronidase activity of N. naja venom (50 µg) was inhibited by 71.83% and LAAO activity was reduced by 74.83%. From the results, it is evident that the ERE of C. lachrymajobi effectively neutralizes important toxic enzymes of the D. russelli and N. naja venoms.Conclusion: The ERE of C. lachrymajobi has a potent inhibitory action against venom enzymes.Keywords: Daboia russelii, Naja naja, Coix lacryma-jobi, Hyaluronidase, L-amino acid oxidase.INTRODUCTIONIn the year 2009, WHO included snake bite in the list of neglected tropical disease [1]. In India, every year around 50,000 people succumb to snake bite. Number of people who are permanently disabled due to snake bite are many more [2]. Important species of snakes, which are responsible for maximum snake bite cases in India, include Naja naja, Daboia ruselli, Echis carinatus, and Bungarus caeruleus [3].Most of the snakebite deaths are avoidable with proper medical attention. Mostly, snake bite victims are farmers and the people working in estates who are generally far away from medical facility and the remoteness makes road transport connectivity very difficult. In the rural areas, non-availability of anti-venom, improper storage facility, and non-availability of trained doctors are the major reasons for deaths of snakebite victims [3,4].Traditional healers of the coastal Karnataka claim the usefulness of the aqueous root extract of Coix lachrymajobi (poaceae) in treating snakebite victims. The study was undertaken to evaluate the effectiveness of root extract of C. lachrymajobi in inhibiting the two important toxins, i.e. hyaluronidase and L-amino acid oxidase in the venom of N. naja and Daboia russelli.L-amino acid oxidase (LAAO) enzyme is highly specific for L-amino acids, and generally hydrophobic amino acids are the best substrates. Recent studies showed that LAAOs are multifunctional enzymes exhibiting edema-inducing, platelet aggregation-inducing or inhibiting, and apoptosis-inducing effects [5].Proficient distribution of toxins into systemic circulation from the bite site in the victim is very important in envenomation. Fragmentation of hyaluronan in the extracellular matrix (ECM) by snake venom hyaluronidase is a crucial element in this diffusion progression. Studies have established that hyaluronidase not only enhances the lethal potency of the venom but also enhances the injury at the bite site, causing severe morbidity [6].METHODSPreparation of extractRoots of C. lachrymajobi were collected from wildly growing plants in Udupi district of Karnataka, India, and were authenticated and voucher specimen was deposited (No: NI-5396 Blatter Herbarium, St. Xavier’s College, Mumbai).To prepare the ethanolic root extract (ERE), the fresh root of the plant C. lachrymajobi was washed and shade dried. 1 kg of dried root was ground to powder and was subjected to maceration and cold extraction in ethanol [7]. After 7 days, the ethanolic extract was filtered. The solvent from the total extract was distilled off and the concentrate was evaporated on a water bath to a syrupy consistency, and then finally evaporated to dryness and stored in a desiccator for future use.Snake venomThe lyophilized snake venom of N. naja and D. russelli was procured from Irula Snake Catchers Industrial Co-operative Society, Kanchipuram, Tamil Nadu, India and was stored at 4°C. Before use, the venom was dissolved in saline and dilutions of the required concentrations were prepared using saline.Hyaluronidase inhibition studiesHyaluronidase activity of the venom and its neutralization by the root extract was estimated by method of Mahadeswaraswamy et al., [8]. In brief, the venom sample (50 µg) was incubated with 100 µg of hyaluronic acid in 300 µl 0.2 M sodium acetate buffer, pH 5.5 containing 0.15 M NaCl at 37°C for 2 hrs. The reaction was terminated by adding 50 µl of 0.1 M potassium tetraborate and boiled for 3 minutes. 1.5 ml of coloring reagent (p-dimethyl aminobenzaldehyde [10%] in acetic acid:hydrochloric acid; 9:1; v/v) was added and incubated Research ArticleAsian J Pharm Clin Res, Vol 8, Issue 5, 2015, 206-208 Rajesh et al. 207for 30 minutes at 37°C. The absorbance was measured at 585 nm to measure the amount of N-acetyl glucosamine released. For inhibition study, one proportion of venom (50 µg) (N. naja and D. russelli) was incubated with ERE at 5, 10, and 20 proportions and above procedure was carried out. Drug free solutions (0.9% saline with 0.06% sodium carboxy methyl cellulose as suspending agent) served as control.LAAO inhibition studiesLAAO activity was determined as described by Dhananjaya et al. [9]. In brief, peroxidase (0.05 ml of 0.007% 250; NIH units/mg) was incorporated in 1 ml of 0.2 M triethanolamine buffer, pH 7.6 having 0.1% L-leucine and 0.006% o-dianisidine, later the reaction blend was incubated for 30 minutes at room temperature.Venom samples (50 µg) (N. naja and D. russelli) were incubated with reaction mixture for 30 minutes at 37°C and the increase in absorbance at 420 nm was measured. For inhibition studies, venom sample (50 mg) was pre-incubated with different amounts of ERE of C. lachrymajobi (5, 10 and 20 times the amount of venom) at 37°C for 30 minutes. Drug free solutions (vehicle only) served as control.Statistical analysisThe data are expressed as mean±standard error, analyzed by one-way ANOVA followed by Dunnett’s test. p≤0.05 was considered as statistically significant.RESULTSHyaluronidase inhibition studiesIn the present study, the ERE of C. lachrymajobi effectively inhibited hyaluronidase enzyme activity of both N. naja and D. russelli venom. The ERE at 10 and 20 times the dose of venom, significantly inhibited release of N-acetyle glucosamine by both the venoms. ERE at 5 times the dose of venom inhibited N. naja venom hyaluronidase activity significantly (Figs. 1 and 2).L-amino oxidase inhibition studiesIn the present study, ERE could effectively inhibit the LAAO activity of both N. naja and D. russelli venom (Figs. 3 and 4). At maximum dose of ERE used, i.e., 2000 µg (20 times the dose of venom), the LAAO action of N. naja was decreased to 25.16% and D. russelli venom LAAO activity was decreased to 22.12%.DISCUSSIONVenomous snakes professionally capture their prey by immobilizing them through injecting the venom into the prey by striking the prey with the fangs, and quick systemic distribution of venom in the prey [10]. The distribution of toxins inside the body is aided primarily by hyaluronidases enzyme, which degrades the elements of ECM and connective tissue surrounding the blood vessels [11-13]. Hyaluronidases are endoglycosidases, which primarily act on hyaluronan of ECM [12,14].A study carried out by Mahadeswaraswamy et al. [8] reported that methanolic extract of grape seeds (Vitis vinifera) significantly inhibited hyaluronidase activity at venom-extract ratio of 1:2. Wahby et al. [15] studied different plants for hyaluronidase inhibition property and reported that leaf extract of Rosmarinus officinalis showed 100% inhibition of hyaluronidase enzyme of Egyptian horned viper, Cerastes cerastes at venom to extract ratio of 1:10. The study also reported that Menthapeprita, Ocimum basilicum and Origanum majorana leaf extract significantly inhibited the hyaluronidase action of the same venom. In the present study, ERE of C. lachrymajobi showed significant inhibition of hylauronidase activity of N. naja and D. russelli venom in dose dependent manner. Even though, there was no 100% inhibition in activity of hylauronidase of both the venoms, percentage inhibition of hyaluronidase activity of D. russelli venom was observed to be 67.04% at 2000 µg dose and against N. naja venom, percentage inhibition was observed to be slightly better at 71.83% with the same dose of ERE.Fig. 1: Effect of ethanolic root extract of Coix lachrymajobi on release of N-acetyl glucosamine by the action of Naja naja venom hyaluronidase. The values are expressed as mean±standard error of mean, n=6 rats in one group. *p≤0.05 significant, **p≤0.01 highly significant, when compared with control groupFig. 2: Effect of ethanolic root extract of Coix lachrymajobi on release of N-acetyl glucosamine by the action of Daboia russelli venom hyaluronidase. The values are expressed as mean± standard error of mean, n=6 rats in one group. *p≤0.05 significant, **p≤0.01 highly significant, when compared with control groupFig. 3: Effect of ethanolic root extract of Coix lachrymajobi on L-amino acid oxidase activity of Naja naja venom. The values are expressed as % L-amino acid oxidase activity n=6Asian J Pharm Clin Res, Vol 8, Issue 5, 2015, 206-208 Rajesh et al. 208LAAO distributed extensively in nature, and snake venoms being the largest sources [5]. Snake venom LAAOs are very dynamic enzymes which are widely used in formulation of α-keto acids for their chemo- and stereo specificity [16,17]. α-Keto acids of essential amino acids are useful nutraceuticals as well as therapeutic agents for certain diseases. Snake venom LAAO attracts new researches in the field of biomedical studies because of its antimicrobial, anti-HIV, anticoagulant, platelet aggregation-inducing and -inhibiting, apoptosis-inducing, anticancer activities etc. [5].In a previously published work [18], authors claimed that glycyrrhizin isolated from Glycyrrhiza glabra showed protection against LAAO isolated from Habu snake. 100 µg of glycyrrhizin was pre-incubated with 4 µg LAAO, which reduced the LAAO activity to 30% of the untreated group. The study also reported glycyrrhetinic acid did not have any effect on Habu snake venom LAAO activity. In the present study, ERE of C. lachrymajobi showed significant inhibition of LAAO activity of N. naja and Daboia russelli venom in dose dependent manner. LAAO activity of D. russelli was inhibited by 77.86% which was slightly better than N. naja LAAO inhibition, which was 74.83%.CONCLUSIONIn the background of the above discussion, it can be concluded that the ERE of C. lachrymajobi has the ability to inhibit the toxic enzymes present in the D. russelli and N. naja venoms. Advanced research need to be carried out to identify the chemical components responsible for these inhibitory effect by molecular docking studies.ACKNOWLEDGMENTSWe are thankful to CSIR, Delhi, India and Nitte University, Mangalore, India, for providing financial assistance for the above research. Also, thankful to Dr. Rajendra D. Shinde, Associate Professor, Department of Botany, Blatter Herbarium, St. Xavier’s College, Mumbai-1, for the identification and authentication of the plant.REFERENCES1. World Health Organisation. WHO Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins. Fig. 4: Effect of ethanolic root extract of Coix lachrymajobi on L-amino acid oxidase activity of Daboia russelli venom. The values are expressed as % L-amino acid oxidase activity n=6Geneva: World Health Organisation; 2010.2. Warrell DA. Guidelines for the Clinical Management of Snake-Bites in the South-East Asia Region. New Delhi: World Health Organization, Regional Office for South East Asia; 2005. p. 1-77.3. Gomes A, Das R, Sarkhel S, Mishra R, Mukherjee S, Bhattacharya S, et al. Herbs and herbal constituents active against snake bite. Indian J Exp Biol 2010;48(9):865-78.4. Chippaux JP. Guidelines for the production, control and regulation of snake antivenom immunoglobulins. Biol Aujourdhui 2010;204(1):87-91.5. Nget-Hong T, Shin-Yee F. Snake venom L-amino acid oxidases. In: Mackessy SP, editor. Handbook of Venoms and Toxins of Reptiles. Boca Raton, FL: CRC Press; 2010. p. 221-33.6. Kemparaju K, Girish KS, Nagaraju S. Hyaluronidases, a neglected class of glycosidases from snake venom beyond a spreading factor. In: Mackessy SP, editor. Handbook of Venoms and Toxins of Reptiles. Boca Raton, FL: CRC Press; 2010. p. 237-54.7. Shah B, Seth AK. Text Book of Pharmacognosy and Phytochemistry. Gurgaon: Elsevier; 2010. p. 407-9.8. Mahadeswaraswamy YH, Devaraja S, Kumar MS, Goutham YN, Kemparaju K. Inhibition of local effects of Indian Daboia/Vipera russelli venom by the methanolic extract of grape (Vitis vinifera L.) seeds. Indian J Biochem Biophys 2009;46(2):154-60.9. Dhananjaya BL, Nataraju A, Rajesh R, Raghavendra Gowda CD, Sharath BK, Vishwanath BS, et al. Anticoagulant effect of Naja naja venom 5’nucleotidase: Demonstration through the use of novel specific inhibitor, vanillic acid. Toxicon 2006;48(4):411-21.10. Kini RM. Venom Phospholipase A2 Enzymes: Structure, Function and Mechanism. New York: Wiley and Sons; 1997. p. 273-8.11. Gutierrez JM, Rucavado A, Escalante T, Diaz C. 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Available from: http://www.europepmc.org/abstract/MED/9781840.

INHIBITORY EFFECT OF ETHANOLIC ROOT EXTRACT OF COIX LACHRYMAJOBI ON HYALURONIDASE AND L-AMINO ACID OXIDASE OF NAJA NAJA AND DABOIA RUSSELLI VENOM

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INHIBITORY EFFECT OF ETHANOLIC ROOT EXTRACT OF COIX LACHRYMAJOBI ON HYALURONIDASE AND L-AMINO ACID OXIDASE OF NAJA NAJA AND DABOIA RUSSELLI VENOM

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