Cajanus indicus L, a herb, is popularly known for its hepatoprotective activity. Aqueous extract of the leaves of this plant contains hepatoprotective and hepatostimulatory molecule(s). Present study was aimed to isolate, purify and characterize the active principle(s) responsible for that activity. A hepatoprotective protein molecule has been purified to homogeneity (approximately 300 fold). Homogeneous preparation of the protein was achieved by homogenization, (NH4)2SO4 precipitation, ion-exchange chromatography, gel filtration and high performance liquid chromatography. The protein purified is composed of a single polypeptide chain having an apparent molecular mass of 43 kD as determined by SDS-PAGE and gel filtration through sephadex G-75 column. The isoelectric point of the protein determined was 4.8. Loss of biological activity after heat and protease treatment confirmed that the active molecule is a protein. Peptide fragments of the protein generated by trypsin cleavage were subjected to MALDI-TOF as well as LC-MS analyses and among the various fragments, four were very prominent and used for the determination of the amino acid sequence of the hepatoprotective protein. While one of the peptide fragment revealed strong sequence homology with plastocyanin, another fragment showed some similarity with a tomato protein present in the NCBI non-redundant database. The third peptide, on the other hand, is unique as it did not show any sequence homology with any known protein in the database. The protein showed maximum hepatoprotective activity when administered at a dose of 2 mg/kg body weight for five days after CCl4 administration. Histopathological studies also supported the hepatoprotective nature of the protein. Along with its curative property, the protein also possesses preventive role against a number of toxin induced hepatic damages.
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Abbreviations
- BSA:
-
bovine serum albumin
- PH:
-
phytohaemagglutinin
- Fip:
-
fungal immunomodulatory proteins
- (NH4)2SO4 :
-
ammonium sulphate
- SDS-PAGE:
-
sodium dodecyl sulphate polyacrylamide gel electrophoresis
- KD:
-
kilodalton
- CCl4 :
-
carbon tetrachloride
- HPLC:
-
high performance liquid chromatography
- mRNA:
-
messenger ribonucleic acid
- DEAE:
-
diethyl amino ethyl
- SGPT:
-
serum glutamate pyruvate transaminase
- DNPH:
-
dinitro phenyl hydrazine
- LC-MS:
-
Liquid Chromatography Mass Spectrometry
- MALDI-TOF:
-
Matrix Assisted Laser Desorption/Ionization-Time of Flight.
References
Akbay A., Cinar K., Uzunalimoglu O., Eranil S., Yurdaydin C., Bozkaya H., Bozdayi M. (1999). Hum. Exp. Toxicol. 18: 669–676
Basaga H., Poli G., Tekkaya C., Aras I. (1997) Cell Biochem. Funct. 15: 27–33
Bhattacharyya S. (1976). Chiranjib Banaushadi, Ananda Publishers Pvt. Ltd., Calcutta, pp. 237–240, 1
Bradford M. M. (1976). Anal. Biochem. 72: 248–254
Bruck R., Aeed H., Shirin H., Matas Z., Zaidel L., Avni Y., Halpern J. (1999). J. Hepatol. 31: 27–38
Bruck R., Aeed H., Schey R., Matas Z., Raifen R., Zaiger G., Hochman A., Avni Y. (2002). J. Hepatol. 36: 370–377
Bruck R., Aeed H., Avni Y., Shirin H., Matas Z., Shahmurov M., Avinoach I., Zozulya G., Weizman N., Hochman A. (2004). J. Hepatol. 40: 86–93
Calixto J. B., Santos A. R., Filho V. (1998). Med. Res. Rev. 18: 225–258
Chopra, R. N., Nayar, S. L., Chopra, I. C. (1986). In: Glossary of Indian Medicinal Plants. Publication and information Directorate, CSIR. New Delhi, 44
Chrungoo V. J., Singh K., Singh I. (1997). Ind. J. Exp. Biol. 35: 611–617
Edmund C. So, Wong kar-lok, Huang tian-chyuan, Tasi sheng-chou, Liu chi-feng. (2002). J. Biomed. Sci. 9: 410–414
Farghali H., Kamenikova L., Hynie S., Kmonickova E. (2000) Pharmacol. Res. 41: 231–237
Ghosh, A., Biswas, K. (1973). In: Chatterjee, A. (ed.), Bhartiya Banausadhi, Calcutta University Press, Calcutta, 2, pp 332–334
Hsu H. C., Hsu C. I., Lin R. H., Kao C. L., Lin J. Y. (1997). Biochem. J. 23: 557–565
Kirtikar, K. R., Basu, B. D., Blatter, E., Caius, J. F., Mhaskarr, R. S. (eds.) (1935). Indian Medicinal Plants, vol I, Probasi Press, Calcutta, pp 809–811
Laemmli U. K. (1970). Nature 227: 680–685
Pietrangelo A., Borella F., Casalgrandi G. (1995). Gastroenterol 109: 1941–1949
Regi, Rapheal K., Sabu M. C., Kuttan R. (2002). Ind. J. Exp. Biol. 40: 905–909
Reitman S., Frankel S. (1957). Am. J. Clin. Path. 28: 56–63
Sane R. T., Kuber V. V., Menon S. (1995) Curr. Sci. 20: 1243–1246
Sarkar K., Ghosh A., Sil P. C. (2005) Hepatol. Res. 33: 39–49
Sheweita S. A., Abd El-Gabar M. (2001). Toxicol. 165: 217–224
Simeonova P. P. et al. (1995). Toxicol. Appl. Pharmacol. 133: 43–52
Thyagarajan S. P., Jayaram S., Valliammai T. (1990). Lancet 336: 949–950
Tsoi, A. Y., Ng, T. B., Fong, W. P. (2005). J. Pept. Sci. 11(10): 665–668
Yoshikawa M., Ninomiya K., Shimoda H., Nishida N., Matsuda H. (2002) Biol. Pharm. Bull. 25(1): 72–76
Acknowledgment
The work has partly been supported by the Department of Atomic Energy (DAE), Government of India (a Grant-In-Aid to PCS, Sanction No. 2002/37/35/BRNS/2065). We are grateful to Professor Kalipada Das, Department of Chemistry, Bose Institute, for his valuable suggestions throughout the study. We would also like to thank Mr. Prasanta Pal for his technical assistance.
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Kasturi Sarkar and Ayantika Ghosh contributed equally in the study
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Sarkar, K., Ghosh, A., Kinter, M. et al. Purification and Characterization of a 43 kD Hepatoprotective Protein from the Herb Cajanus indicus L.. Protein J 25, 411–421 (2006). https://doi.org/10.1007/s10930-006-9030-7
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DOI: https://doi.org/10.1007/s10930-006-9030-7