Sains Malaysiana 52(5)(2023): 1371-1381

http://doi.org/10.17576/jsm-2023-5205-04

 

Optimisation of Protease Purification from Christia vespertilionis Leaves and Its Anti-Inflammatory Activity

(Pengoptimuman Penulenan Protease daripada Daun Christia vespertilionis dan Aktiviti Anti-Keradangan)

 

IZZAH FARHAH ZAMBARI1, SITTI RAHMA ABDUL HAFID2 & NUR AIRINA MUHAMAD1,*

 

1Institute of Biological Sciences, Faculty of Science, Universiti Malaya, 50603 Kuala Lumpur, Federal Territory, Malaysia

2Nutrition Unit, Product Development and Advisory Services Division, Malaysian Palm Oil Board, 43000 Kajang, Selangor Darul Ehsan, Malaysia

 

Diserahkan: 30 Julai 2022/Diterima: 10 Mei 2023

 

Abstract

Christia vespertilionis (L. f.) Bakh. f. is widely known for its anti-inflammatory and anti-cancer properties. However, there are no previous studies about extracting and purifying protease enzymes from C. vespertilionisleaves. Therefore, this study was conducted to extract and optimise the purification of protease from C. vespertilionis leaves and characterise its anti-inflammatory properties. The optimisation was performed using different levels of ammonium sulphate saturation (20, 40, 60, 80 and 100%). Next, dialysis was carried out for the sample with the highest specific activity (16.88 U/mg), achieved with 100% ammonium sulphate saturation. At 100% saturation, C. vespertilionis leaves showed an increase in the specific activity to 20.06 U/mg after dialysis. The findings demonstrate the successful extraction and purification of C. vespertilionis protease (CVP) with a molecular weight of 48 kDa, as proven by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. CVP also exhibited anti-inflammatory activity, with an inhibition of 45.6% in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and IC50 of 19.24 µg/mL. The HPLC test further confirmed the presence of gallic acid and quercetin compounds in C. vespertilionis, which cure inflammation. The results indicate that optimised CVP purification was achieved and its anti-inflammatory ability was proven.

 

Keywords: Ammonium sulphate; C. vespertilionis; purification; protease; SDS-PAGE

 

Abstrak

Christia vespertilionis (L. f.) Bakh. f. terkenal dengan sifat anti-radang dan anti-kansernya. Walau bagaimanapun, tiada kajian tentang pengekstrakan dan penulenan enzim protease daripada daun C. vespertilionis. Oleh itu, kajian ini dijalankan untuk mengekstrak dan mengoptimumkan penulenan enzim protease daripada daun C. vespertilionis dan untuk mencirikan keupayaannya dalam menyembuhkan keradangan. Pengoptimuman dilakukan menggunakan tahap ketepuan amonium sulfat yang berbeza (20, 40, 60, 80 dan 100 %). Kemudian, dialisis dijalankan ke atas sampel yang mempunyai aktiviti khusus tertinggi (16.88 U/mg) yang diperoleh pada ketepuan ammonium sulfat 100%. Pada ketepuan 100%, daun C. vespertilionis menunjukkan peningkatan aktiviti khusus kepada 20.06 U/mg selepas dialisis. Hasil menunjukkan pengekstrakan dan penulenan protease C. vespertilionis (CVP) dengan berat molekul 48 kDa telah berjaya diperoleh, seperti yang dibuktikan melalui ujian elektroforesis gel natrium dodesil sulfat-poliakrilamida (SDS-PAGE). CVP juga menunjukkan aktiviti anti-radang dengan perencatan sebanyak 45.6% dalam sel RAW264.7 yang dirangsang oleh lipopolisakarida (LPS) dengan IC50 pada 19.24 µg/mL. Ujian HPLC juga mengesahkan kehadiran asid galik dan kuersetin dalam C. vespertilionis yang mempunyai keupayaan untuk menyembuhkan keradangan. Ini menunjukkan CVP telah melalui kaedah penulenan yang optimum dan membuktikan keupayaannya untuk menyembuhkan keradangan.

 

Kata kunci: Amonium sulfat; C. vespertillonis; penulenan; protease; SDS-PAGE

 

RUJUKAN

Antào, C.M. & Malcata, F.X. 2005. Plant serine proteases: Biochemical, physiological and molecular features. Plant Physiology and Biochemistry 43(7): 637-650.

Belton, P.S., Sorensen, H., Sorensen, S., Bjergegaard, C. & Michelson, S. 1999. Chromatography and Capillary Electrophoresis in Food Analysis. Cambridge: The Royal Society of Chemistry.

Brien, S., Lewith, G., Walker, A., Hicks, S.M. & Middleton, D. 2004. Bromelain as a treatment for osteoarthritis: A review of clinical studies. Evidence-Based Complementary and Alternative Medicine 1(3): 251-257.

Chen, M., Qin, Y., Ma, H., Zheng, X., Zhou, R., Sun, S., Huang, Y., Duan, Q., Liu, W., Wu, P., Xu, X., Sheng, Z., Zhang, K. & Li, D. 2019. Downregulating NF-kappaB signaling pathway with triterpenoids for attenuating inflammation: in vitro and in vivo studies. Food Function 10: 5080-5090.

Chew, K.K., Ng, S.Y., Thoo, Y.Y., Khoo, M.Z., Wan Aida, W.M. & Ho, C.W. 2011. Effect of ethanol concentration, extraction time and extraction temperature on the recovery of phenolic compounds and antioxidant capacity of Centella asiatica extract. International Food Research Journal 18: 571-578.

Cheon, H., Rho, Y.H., Choi, S.J., Lee, Y.H., Song, G.G., Sohn, J., Won, N.H. & Ji, J.D. 2006. Prostaglandin E2 augments IL-10 signaling and function. Journal of Immunology 177: 1092-2100.

Choy, E.H.S. & Panayi, G.S. 2001. Cytokine pathways and joint inflammation in rheumatoid arthritis. New England Journal of Medicine 344(12): 907-916.

Drivdahl, R.H. & Thimann, K.V. 1977. Proteases of senescing oat leaves I. Purification and general properties. Journal of Plant Physiology 59(6): 1059-1063.

Esposito, M., Di-Pierro, P., Dejonghe, W., Mariniello, L. & Porta, R. 2016. Enzymatic milk clotting activity in artichoke (Cynara scolymus) leaves and alpine thistle (Carduus defloratus) flowers. Immobilization of alpine thistle aspartic protease. Food Chemistry 204(2): 115-121.

Fulzele, D.P. & Satdive, R.K. 2005. Comparison of techniques for the extraction of the anti-cancer drug camptothecin from Nothapodytes foetid. Journal of Chromatography A 1063(1-2): 9-13.

Funaro, A., Wu, X., Song, M., Zheng, J., Guo, S., Rakariyatham, K., Rodriguez-Estrada, M.T. & Xiao, H. 2016. Enhanced anti-inflammatory activities by the combination of luteolin and tangeretin. Journal of Food Science 81(5): 1320-1327.

Garnock-Jones, P.J. 1983. Note on some folklore drugs and remedies of the Lau Group, Fiji. South Pacific Journal of Natural Science 4: 4-8.

Gonzàlez, R.N., Badillo, J.A., Aranda, A.J.S. & Carmen, O.S.M. 2011. Production of plant proteases in vivo and in vitro-a review. Biotechnology Advances 29(6): 983-996.

Gupta, R., Beg, Q. & Lorenz, P. 2002. Bacterial alkaline proteases: Molecular approaches and industrial applications. Applied Microbiology Journal 59(1): 15-32.

Hofer, D., Schwach, G., Tabrizi-Wizsy, N.G., Sadjak, A., Sturm, S., Stuppner, H. & Pfragner, R. 2013. Christia vespertilionis plant extracts as novel antiproliferative agent against human neuroendocrine tumor cells. Oncology Reports 29(6): 2219-2226.

Karamaae, M.A., Kosinska, A. & Pegg, R.B. 2005. Comparison of radical-scavenging activities of selected phenolic acids. Polish Journal of Food and Nutrition Sciences 14: 165-170.

Kaur, S., Michael, H., Arora, S., Harkonen, P.L. & Kumar, S. 2005. The in vitro cytotoxic and apoptotic activity of Triphala-an Indian herbal drug. Journal of Ethnopharmacology 97: 15-20.

Kim, M., Si, J.B., Reddy, L.V. & Wee, Y.J. 2016. Enhanced production of extracellular proteolytic enzyme excreted by a newly isolated Bacillus subtilis FBL-1 through combined utilization of statistical designs and response surface methodology. RSC Advances 6(56): 51270-51278.

Kiran, K.K., Venkata, N.R. & Nagoji, K.E.V. 2012. A validated RP-HPLC method for the determination of Bedamustine hydrochloride in tablet dosage form using gemcitabine hydrochloride as internal standard. FABAD Journal of Pharmaceutical Sciences 37(3): 133-139.

Kuddus, M. 2015. Cold-active microbial enzymes. Biochemistry and Physiology 4(1): 1-2.

Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685.

Lee, J.J., Yazan, L.S., Kassim, N.K., Che Abdullah, C.A., Esa, N., Lim, P.C. & Tan, D.C. 2020. Cytotoxic activity of Christia vespertilionis root and leaf extracts and fractions against breast cancer cell lines. Molecules 25(11): 2610.

Murugesu, S., Perumal, V., Balan, T., Fatinanthan, S., Khatib, A., Arifin, N.J., Mohd Shukri, N.S.S., Saleh, M.S.M. & Hin, L.W. 2020. The investigation of antioxidant and antidiabetic activities of Christia vespertilionis leaves extracts. South African Journal of Botany 133: 227-235.

Mutalib, N.A. & Latip, N.A. 2019. Synergistic interactions between Christia vespertilionis leaves extract and chemotherapy drug cyclophosphamide on WRL-68 cell line. Asian Journal of Pharmaceutical Research and Development 7(3): 109-113.

Nam, S.H., Walsh, M.K. & Yang, K.Y. 2016. Comparison of four purification methods to purify cysteine protease from Asian pear fruit (Pyrus pyrifolia). Biocatalysis and Agricultural Biotechnology 5: 86-93.

Naidu, K.S.B. & Devi, K.L. 2005. Optimization of thermostable alkaline protease production from species of Bacillus using rice bran. African Journal of Biotechnology 4(7): 724-726.

Nikolic, K.M. 2006. Theoretical study of phenolic antioxidants properties in reaction with oxygen-centered radicals. Journal of Molecular Structure: Theochem 774: 95-105.

Nguyen-Pouplin, J., Tran, H., Tran, H., Phan, T.A., Dolecek, C., Farrar, J., Tran, T.H., Caron, P., Bodo, B. & Grellier, P. 2007. Antimalarial and cytotoxic activities of ethnopharmacologically selected medicinal plants from South Vietnam. Journal of Ethnopharmacology 109(3): 417-427.

Osman, M.S., Ghani, Z.A., Ismail, N.F., Razak, N.A., Jaapar, J. & Ariff, M.A. 2017. Qualitative comparison of active compounds between red and green Mariposa Christia vespertillionis leaves extracts. Proceedings of the American Institute of Physics Conference 1885: 020282.

Park, S.R., Lim, C.Y., Kim, D.S. & Ko, K. 2015. Optimization of ammonium sulfate concentration for purification of colorectal cancer vaccine candidate recombinant protein GA733-FcK isolated from plants. Frontiers in Plant Science Frontiers 6: 1040.

Rao, U.S.M., Ahmad, B.A. & Mohd, K.S. 2016. In vitro nitric oxide scavenging and anti-inflammatory activities of different solvent extracts of various parts of Musa paradisiaca. Malaysian Journal of Analytical Sciences 20(5): 1191-1202.

Rayburn, E.R., Ezell, S.J. & Zhang, R. 2009. Anti-inflammatory agents for cancer therapy. Molecular and Cellular Pharmacology 1: 29-43.

Ren, J., Li, L., Wang, Y., Zhai, J., Chen, G. & Hu, K. 2019. Gambogic acid induces hemeoxygenase-1 through Nrf2 signaling pathway and inhibits NF kappaB and MAPK activation to reduce inflammation in LPS-activated RAW264.7 cells. Biomedicine & Pharmacotherapy 109: 555-562.

Roy, S. & Kumar, V. 2014. A practical approach on SDS PAGE for separation of protein. International Journal of Science and Research 3(8): 955-960.

Sakdarat, S., Shuyprom, A., Pientong, C., Ekalaksananan, T. & Thongchai, S. 2009. Bioactive constituents from the leaves of Clinacanthus nutans Lindau. Bioorganic & Medicinal Chemistry 17(5): 1857-1860.

Sathya Prabhu, D., Apoorva, S., Nandita, J., Palani, C. & Devi Rajeswari, V. 2017. Purification of protease enzyme from the leaf, seed and pod samples of Vicia faba L. International Food Research Journal 25(5): 1904-1911.

Singh, M.P., Gupta, A. & Singh Sisodia, S. 2019. Qualitative analysis of gallic acid by HPLC method in different extracts of Terminalia bellerica Roxb. fruit. FABAD Journal of Pharmaceutical Sciences 44(2): 101-106.

Sun, Q., Zhang, B., Yan, Q.J. & Jiang, Z.Q. 2016. Comparative analysis on the distribution of protease activities among fruits and vegetable resources. Food Chemistry 2(13): 708-713.

Upadhyay, H.C., Sisodia, B.S., Cheema, H.S., Agrawalb, J., Pal, A., Darokar, M.P. & Srivastava, S.K. 2013. Novel antiplasmodial agents from Christia vespertilionis. Natural Product Communications 8(11): 1-4.

van der Hoorn, R.A. 2008. Plant proteases: From phenotypes to molecular mechanisms. Annual Review of Plant Biology 59: 191-223.

Viswanatha Swamy, A.H.M. & Patil, P.A. 2008. Effect of some clinically used proteolytic enzymes on inflammation in rats. Indian Journal of Pharmaceutical Sciences 70(1): 114-117.

Wardani, A.K. & Nindita, I.O. 2012. Purification and characterization of protease from protease-producing bacteria isolated from tofu whey. Jurnal Teknologi Pertanian 13(3): 149-156.

Winarti, A., Fitriyanto, N.A., Jamhari, Pertiwiningrum, A., Bachruddin, Z., Pranoto, Y. & Erwanto, Y. 2018. Optimizing of protease purification from Bacillus cereus TD5B by ammonium sulfate precipitation. Chemical Engineering Transactions 63: 709-714.

Whiting, P.A. 2007. Commercial production of Christia subcordata Moench by establishing cultural practices and by applying plant growth regulators. Dissertation. United States: The University of Georgia (Unpublished).

Yam, M.L., Abdul Hafid, S.R., Cheng, H.M. & Nesaretnam, K. 2009. Tocotrienols supress proinflammatory markers and cyclooxygenase-2 expression in RAW264.7 macrophages. Lipids 44(9): 787-797.

 

*Pengarang untuk surat-menyurat; email: nurairina@um.edu.my

 

   

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