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