ZCPG, a cysteine protease from Zingiber montanum rhizome exhibits enhanced anti-inflammatory and acetylcholinesterase inhibition potential.

A 48 kDa Zingiber montanum cysteine protease glycoprotein (ZCPG) purified previously was studied for anti-inflammatory and acetylcholinesterase inhibitory activity. The lipoxygenase inhibition by ZCPG was linear, with an IC50 value of 2.25 µM. MTT, LDH, and cell cycle analysis in THP-1 derived macrophages corroborate no significant cytotoxicity at a lower concentration. ZCPG inhibited the production of nitric oxide, reactive oxygen species, and pro-inflammatory cytokines such as interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α) in lipopolysaccharide-stimulated THP-1 macrophages. In contrast, an increase in the production of interleukin-10, an anti-inflammatory cytokine, was observed. A reverse-transcription polymerase chain reaction study further confirmed that ZCPG inhibited the expression of IL-1ß, inducible nitric oxide synthase, and TNF-α by suppressing their mRNA transcription and expression in LPS stimulated THP-1 macrophages. Furthermore, the nature of acetylcholinesterase (AChE) inhibition by ZCPG is dose-dependent, competitive, and reversible. The AChE inhibitory activity was stable in a broad range of temperatures and pH. In vitro data were further validated by molecular interaction studies with a detailed inspection of the ZCPG probable binding modes in the active sites of AChE that provides the lead to deliver the structural determinants necessary for the activity towards AChE.

Characterization and comparative studies of galactomannans from Bauhinia vahlii, Delonix elata, and Peltophorum pterocarpum.

The present investigation determines the extraction and characterization of seed galactomannans from the Leguminosae taxa of Bauhinia vahlii, Delonix elata, and Peltophorum pterocarpum. The seed galactomannans presented a total yield of 30.82, 24.01 and 25.25% with a Man/Gal ratios of 4.21:1, 2.55:1 and 3.03:1 for B. vahlii, D. elata, and P. pterocarpum, respectively exhibiting 1-4 mannose linkages with galactose side chains at C6 position. The galactomannans presented an efficient water holding capacity, solubility and emulsion properties. Intrinsic viscosity from combined Huggins and Kraemer extrapolations are 3.807, 3.424 and 3.331 dl g-1 and the viscosity average molecular weight by Mark-Houwink relationship are 5.29 × 105, 5.29 × 105 and 4.95 × 105 for B. vahlii, D. elata, and P. pterocarpum, respectively. TEM analysis of the polysaccharide showed an aggregated amorphous smooth particles, oval or tubular in shape, branched with compact surfaces. SEM micrographs of the powdered galactomannan indicate well defined amorphous material with pores and cervices on the rough surface. The thermal property studied by DSC and TG-DTA suggests good thermal stability. These findings demonstrate that the seed galactomannan of B. vahlii, D. elata, and P. pterocarpum could be explored as an effective alternative to commercial galactomannans for industrial applications.

Purification, biochemical characterization and antioxidant property of ZCPG, a cysteine protease from Zingiber montanum rhizome.

Zingiber montanum cysteine protease glycoprotein (ZCPG) was purified to homogeneity by DEAE- cellulose and Sephadex G50 resulting in sixteen fold purification and total activity of 39.4U/mg. ZCPG presented a prominent single peak in HPLC chromatogram with an estimated molecular weight of 48kDa on native PAGE. SDS-PAGE gave two subunits of ∼24.3 and ∼24.6kDa showing its heterodimeric form. Protein sequencing was studied by MALDI-TOF MS/MS. Isoelectrofocusing exhibited two isoforms with pI values of 4.8 and 5.1. Analysis of the total carbohydrate by GC-MS/MS showed the presence of glucose, mannose, fucose and xylose. The pH and temperature optimum were 9 and 60°C respectively while Km and Vmax values were 0.5±0.03µg and 13.73±2.07U/ml respectively. ZCPG was strongly inhibited by NEM indicating the cysteine-type. Substrates such as casein, azocasein, gelatin, BSA and haemoglobin showed high relative activity. Metal ions of CuCl2, CoCl2, HgCl2 and ZnCl2 showed partial inhibition at 1mM concentration. Furthermore, ZCPG exhibited promising antioxidant activity in biochemical systems as well as THP-1 cells. These findings suggested, ZCPG with significant antioxidant activity might have potential applications in therapeutic and food industry.

Fluorescence quenching, structural and unfolding studies of a purified cysteine protease, ZCPG from Zingiber montanum rhizome.

A first attempt was made to study the fluorescence quenching, structure and unfolding nature of the purified Zingiber montanum (J.Koenig) Link ex A.Dietr. cysteine protease glycoprotein (ZCPG). ATR-IR spectra showed the presences of amide groups along with carbohydrate stretch indicating the glycoprotein nature. UV-vis spectra determined the presences of peptide groups and aromatic sidechains of tyrosine, tryptophan and phenylalanine. Far UV-Circular Dichroism spectrum revealed that the secondary structure consists of 47.6% α-helix, 14.1% ß-sheet, 16.1% ß-turn, and 22.2% random coil. CD signals revealed pronounced structural stability until 70°C followed by a significant variation in the secondary structure content in the transition temperature between 80-90°C. ZCPG retained most of its secondary structure in the pH range of 3.0-10.0. The extrinsic study shows that at pH 2.0, ZCPG revealed characteristics of a molten globule-like state exhibiting strong ANS binding. The effect of GdnHCl on ZCPG evaluated by far-CD emission maximum and fluorescence emission revealed that the unfolding was incomplete determining the stability of the protein. The microenvironment of the tryptophan residues indicated the presence of relatively exposed single tryptophan residue (per monomer) with positively charged side chains.

A simple, rapid and yet less expensive method to detect chitinase in agarose plates.

Methods have been developed, in the past, to detect the chitinase activity in plate assays using fluorescent brightener Calcofluor White which is simple and rapid, but expensive. We, now, report the use of a less expensive fluorescent brightener Ranipal, which reduces 2500-times the cost of the assay. The use of Ranipal, in place of Calcofluor White, does not compromise on the sensitivity and limits of detection, but required more washes. This forms the first report on the use of Ranipal for detection of chitinases and also the least expensive method for this purpose.

Cross-linked leucaena seed gum matrix: an affinity chromatography tool for galactose-specific lectins.

A cross-linked leucaena (Leucaena leucocephala) seed gum (CLLSG) matrix was prepared for the isolation of galactose-specific lectins by affinity chromatography. The matrix was evaluated for affinity with a known galactose-specific lectin from the seeds of snake gourd (Trichosanthes anguina). The matrix preparation was simple and inexpensive when compared to commercial galactose-specific matrices (i.e. about 1.5 US dollars/100 ml of matrix). The current method is also useful for the demonstration of the affinity chromatography technique in laboratories. Since leucaena seeds are abundant and inexpensive, and the matrix preparation is easy, CLLSG appears to be a promising tool for the separation of galactose-specific lectins.