ZINGIPAIN, A CYSTEINE PROTEASE FROMZingiber ottensiiVALETON RHIZOMES WITH ANTIPROLIFERATIVE ACTIVITIES AGAINST FUNGI AND HUMAN MALIGNANT CELL LINES

Abstract

The objective of this study was to investigate the activity of a protein identified as cysteine protease, purified from Zingiber ottensii Valeton rhizomes, in terms of antiproliferation against fungi, bacteria, and human malignant cell lines. By means of buffer extraction followed by (NH(4))(2)SO(4) precipitation and ion-exchange chromatography, the obtained dominant protein (designated F50) was submitted to non-denaturing and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), where a single band and three bands were revealed from eletrophoretic patterns, respectively. It could be concluded at this point that the F50 was potentially a heterotrimer or heterodimer composed of either two small (∼13.8 and ∼15.2 kD) subunits or these two together with a larger (∼32.5 kD) one. In-gel digestion was carried out for the most intense band from reducing SDS-PAGE, and to the resulting material was applied liquid chromatography (LC)-mass spectroscopy (MS)/MS. The main F50 subunit was found to contain fragments with 100% similarity to zingipain-1, a cysteine protease first discovered in Zingiber officinale. The activity corresponding to the identified data, cysteine protease, was then confirmed in the F50 by azocasein assay and a positive result was obtained. The F50 then was further investigated for antiproliferation against three plant pathogenic fungi species by disk diffusion test, four bacterial species by direct exposure in liquid culture and dish diffusion tests, and five human malignant cell lines by tissue culture assay. It was found that a dose of 23.6 µg F50/0.3 cm(2) of paper disk exhibited the best inhibitory effect against Collectotrichum cassiicola, while lesser effects were found in Exserohilum turicicum and Fusarium oxysporum, respectively. No inhibitory effect against bacterial proliferation was detected in all studied bacterial strains. However, relatively strong antiproliferative effects were found against five human cell lines, with IC50 values ranging from 1.13 µg/mL (hepatoma cancer; HEP-G2) to 5.37 µg/mL (colon cancer; SW620). By periodic acid-Schiff's staining and phenol-sulfuric acid assay, the F50 was determined as a glycoprotein containing 26.30 ± 1.01% (by weight) of carbohydrate. Thus, a new glycoprotein with protease activity was successfully identified in Zingiber ottensii rhizome. The glycoprotein also contained antiproliferative activity against some plant pathogenic fungi and human cancer cell lines.

Discovery of digestive enzymes in carnivorous plants with focus on proteases

“…Microbes and animals are currently the major source of proteases with only a few commercialised plant proteases. Interest has been growing in plant proteases, which have significant commercial values due to high stability in extreme conditions ( Canay, Erguven & Yulug, 1991 ; Houde, Kademi & Leblanc, 2004 ; Karnchanatat et al, 2011 ). Examples of proteases from plant sources are listed in Table 4 .…”

Essential Oil from Zingiber ottensii Induces Human Cervical Cancer Cell Apoptosis and Inhibits MAPK and PI3K/AKT Signaling Cascades

“…Furthermore, it was shown that the pupae of Aedes aegypti and Culex quinquefasciatus mosquitoes were susceptible to ZO essential oil [28]. For an aspect of anti-cancer properties, it has reported that Zingipain, which is a cysteine protease isolated and purified from Zingiber ottensii rhizomes, possessed relatively strong antiproliferative effects against cancer cell lines including hepatocellular carcinoma, HEP-G2 (IC50 = 1.13 µg/mL), and colon cancer, SW620 (IC50 = 5.37 µg/mL) [15]. However, there had previously been no investigation for the anti-cancer activities of the essential oil from Zingiber ottensii at the signaling pathway level to explain its molecular mechanisms of action.…”

Therapeutic potential of Zingiberaceae in Alzheimer's disease

“…The species is traditionally used as a sedative remedy for convulsion and as a lumbago treatment in Malaysia. In Thailand, Z. ottensii has been traditionally used to treat external bruises and gastrointestinal ulcers (Karnchanatat et al, 2011). Rungsaeng et al (2013) investigated the inhibitory potential of AChE of the rhizome aqueous extract (obtained from ammonium sulfate), and proteases isolated from Z. ottensii.…”