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 MUHAMAD^1,*

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

Received: 30 July 2022/Accepted: 10 May 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

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^*Corresponding author; email: nurairina@um.edu.my