C. parviflorum was well-studied for various pharmacological properties. Ethanolic leaf extract was neutralized in vitro and in vivo anti-cancer activity on DLA and HeLa cell lines (Prabhu et al., 2011). C. parviflorum possesses anti-oxidant activity in alloxan-induced diabetic rats (Sathishkumar et al., 2008). Canthium coromadelicum leaves posses antimicrobial and anti-HIV activity (Chinnaiyan et al., 2013).
In the present study, partial purification was carried out using TLC to identify the bioactive compound against the E. carinatus or N. naja venom. TLC separated band 5 showed maximum %inhibition compared to other bands and inhibited all the enzyme activity.
Snake venom is a complex mixture of hydrolytic enzymes and some site specific enzymes. Phospholipase A2, metalloprotenase, 5’ nucleotidase are major hydrolytic and hyaluronidase is a site specific enzymes responsible for earlier reaction of envenomation (Ushanandini et al., 2006). Hyaluronidase known as spreading factor results in membrane degradation of hyaluron lead to easy spreading of other toxic enzymes. The degradation of extracellular matrix is a continuous process and anti-venom fails to neutralize the local tissue damages (Homma and Tu, 1970; Girish et al., 2004). Lethal toxicity and pharmacological activity is combined action of all these toxic enzymes. Various plants have been studied for inhibition of these toxic enzymes effects by in vitro using various solvent extracts. In vitro inhibitory activity of Carissa spinarum leaf extracts against Krait and Viper russellitoxic snake venom enzymes was studied (Janardhan et al., 2014). Tabernaemontana alternifolia inhibits in vitroN. naja and E. carinatus venom enzyme activities (Vineetha et al., 2014).
Numerous plants species have been scientifically investigated for quest new phytoconstitute responsible for snake bite. Aristolochia species has been studied against Trimeresurus flavoviridis and Viper russellii venom effects (Vishwanath et al., 1987). Aristolochic acid, a bioactive phytochemical from Aristolochia, inhibited phospholipase which is responsible for edema formation and other pharmacological activities (Vishwanath et al., 1987). 2-OH-4-methoxy benzoic acid and lupeol acetate was isolated from Hemidesmus indicus which possesses potent inhibition activity against N. naja venom (Alam and Gomes, 1998; Chatterjee et al., 2006). A multiform of glycoprotein isolated from Mucuna pruriens seeds neutralized E. carinatus venom effects (Guerranti et al., 2004).
The sequential ethyl acetate extract of C. parviflorum exhibited enzyme activities (unpublished data). In the present study TLC separated bands of methanol root extract of C. parviflorum neutralized all the enzyme activities from both the venoms. The chromatogram of TLC separated C. parviflorum methanol root extract shows 9 prominent peak in retention time range between 6.7-35.4 min. In the previous studies, C. parviflorum ethanol leaf extract was reported 22 constituents present in the leaves and D-mannitol and squalene responsible for anti-cancer properties (Prabhu et al., 2013). GC-MS reveals presence of some active phytoconstituents which has various biological activities. Octadecanoic acid methyl ester, 13-docosenoic acid, methyl ester, (Z)-(CAS) have various biological activities present in the TLC separated methanol root extract. Presence of hexadecadecanoic acid ethyl ester compound in leaves were reported in ethanolic extract of C. parviflorum leaves (Prabhu et al., 2013). An oleic ester derivative 9-octadecanoic acid (z)-phenylmethyl ester was reported in Sauropus bacciformis stem (Jenecius et al., 2012). 11(Methylthio)canthin-6-one is an alkaloid, the probable compound in 6.7 RT is not reported in Rubiaceae family to best of our knowledge. Two canthin-6-one alkaloids isolated from Eurycoma longifolia from Simaroubaceae family have anti-cancer effects in HT-1080 human sarcoma cell-lines (Miyake et al., 2010). Quinolinealkaloid is reported in Rubiaceae family which also the probable compound at RT 4.3 min but area % is 0.1. In the presence of siloxane compound might be due to interference of silica which was used during TLC separation.
Partially purified C. parviflorum methanol root extract inhibited phosphomonoesterase, phosphodiesterase, acetylcholinesterase, hyaluronidase, protease, phospholipase A2, 5’ nucleotidase activities of E. carinatus and N. naja venom. TLC separated band 5 was promising when compare to other bands. The GC-MS analysis revealed presence of bioactive phytocompounds which might be responsible for neutralization of toxic enzymes. As per our knowledge and scientific literature survey, C. parviflorumn was not studied for antivenom properties. Therefore, the present study adds on for an additional scientific validation to the society.
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