Purification,Characterization and in vitro Anthelmintic Activity of a Neutral Metalloprotease from Laccocephalum mylittae

A neutral metalloprotease was purified from the cultured mycelia of Laccocephalum mylittae,an effective medicinal fungus widely used in anthelmintic therapy.The protease was purified to homogeneity with 31.85-fold purification and a final yield of 21.76%.The subunit molecular weight of the protease is about 40000 estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The optimum reaction pH and temperature are 7.5 and 50oC,respectively.The protease activity is largely enhanced by Ca2 +,but highly inhibited by tetrasodium ethylenediaminetetraacetate(EDTA),a metal-chelator,suggesting that the enzyme is a metalloprotease.The Michaelis-Menten constan Km and Vmax value for casein substrate are 6.09 mg·ml -1and 21.32μg·min -1·ml -1, respectively.In vitro anthelmintic tests of the protease exhibit distinct lethal effects on the third stage larvae(L3)of Ascaris suum.Scanning electron microscopy and SDS-PAGE analysis indicates that the proteolysis of larvae proteins caused by this protease may relate to the anthelmintic activity of L.mylittae.

Synthetic and biological studies on a cyclopolypeptide of plant origin

Objective:A natural cyclic peptide previously isolated from Citrus medica was synthesized by coupling of tetrapep-tide units Boc-Leu-Pro-Trp-Leu-OMe and Boc-Ile-Ala-Ala-Gly-OMe after proper deprotection at carboxyl and amino terminals followed by cyclization of linear octapeptide segment. Methods:Solution phase technique was adopted for the synthesis of cyclooctapeptide-sarcodactylamide. Required tetrapeptide units were prepared by coupling of Boc-protected dipeptides viz. Boc-Leu-Pro-OH and Boc-Ile-Ala-OH with respective dipeptide methyl esters Trp-Leu-OMe and Ala-Gly-OMe. Cyclization of linear octapeptide unit was done by p-nitrophenyl ester method. The structure of synthesized cyclopolypeptide was elucidated by FTIR,1H NMR,13C NMR,FABMS spectral data and elemental analysis. The newly synthesized peptide was evaluated for dif-ferent pharmacological activities including antimicrobial,anthelmintic and cytotoxic activities. Results:Synthesis of sarcodac-tylamide was accomplished with >78% yield utilizing dicyclohexylcarbodiimide(DCC) as coupling agent. Newly synthesized peptide possessed potent cytotoxic activity against Dalton's lymphoma ascites(DLA) and Ehrlich's ascites carcinoma(EAC) cell lines,in addition to moderate anthelmintic activity against earthworms Megascoplex konkanensis,Pontoscotex corethruses and Eudrilus sp. Moreover,cyclopolypeptide displayed good antimicrobial activity against pathogenic fungi Candida albicans and Gram-negative bacteria Pseudomonas aeruginosa,in comparison to standard drugs griseofulvin and ciprofloxacin. Conclusion:Solution phase technique employing DCC and triethylamine(TEA) as base proved to be effective for the synthesis of natural cyclooctapeptide. N-methyl morpholine(NMM) was found to be a better base for the cyclization of linear octapeptide unit in comparison to TEA and pyridine.

Anthelmintic, antimicrobial, antioxidant and cytotoxic activity of Caltha palustris var. alba Kashmir, India

The methanolic extract obtained from the root portion of Caltha palustris var. alba was evaluated for its anthelmintic efficacy against gastrointestinal nematodes of sheep under both in vitro and in vivo conditions using worm motility inhibition(WMI) assay and fecal egg count reduction(FECR) assay, respectively. The extract was subjected to antimicrobial activity using agar-well diffusion method against different bacterial strains. In addition the extract was evaluated for cytotoxic and antioxidant activity against cultured THP-1(Leukemia), A-549(Lung), HCT-15(Colon), Cervix(HeLa) and PC-3(Prostrate) cell lines by SRB and DPPH radical scavenging assays. The extract used resulted in mean %WMI of 94.44%, as observed when the worms were put in lukewarm buffer for 30 min after exposure to different treatments. The mean mortality index of the sample was 0.95. The lethal concentration(LC50) was 0.11 mg·mL-1. Cell lines were exposed to concentration of 100 μg·mL-1 of extract for 48 h, which reduced the viability of these cell lines. The same plant extract also showed 55.58% DPPH radical scavenging activity.