Entamoeba histolytica is the etiologic agent for amoebiasis. The excretory-secretory (ES) products of the trophozoites contain virulence factors and antigens useful for diagnostic applications. Contaminants from serum supplements and dead trophozoites impede analysis of ES. Therefore, a protein-free medium that can sustain maximum viability of E. histolytica trophozoites for the longest time duration will enable collection of contaminant-free and higher yield of ES products. In the present study, we compared the efficacy of four types of media in maintaining ≥ 95% trophozoite viability namely Roswell Memorial Park Institute (RPMI-1640), Dulbecco's Modified Eagle Medium (DMEM), phosphate-buffered saline for amoeba (PBS-A), and Hank's balanced salt solution (HBSS). Concurrently, the effect of adding L: -cysteine and ascorbic acid (C&A) to each medium on the parasite viability was also compared. DMEM and RPMI 1640 showed higher viabilities as compared to PBS-A and HBSS. Only RPMI 1640 showed no statistical difference with the control medium for the first 4 h, however the ≥ 95% viability was only maintained for the first 2 h. The other protein-free media showed differences from the serum- and vitamin-free TYI-S-33 control media even after 1 h of incubation. When supplemented with C&A, all media were found to sustain higher trophozoite viabilities than those without the supplements. HBSS-C&A, DMEM-C&A, and RPMI 1640-C&A demonstrated no difference (P>0.05) in parasite viabilities when compared with the control medium throughout the 8-h incubation period. DMEM-C&A showed an eightfold increment in time duration of sustaining ≥ 95% parasite viability, i.e. 8 h, as compared to DMEM alone. Both RPMI 1640-C&A and HBSS-C&A revealed fourfold and threefold increments (i.e., 8 and 6 h, respectively), whereas PBS-A-C&A showed only one fold improvement (i.e., 2 h) as compared to the respective media without C&A. Thus, C&A-supplemented DMEM or RPMI are recommended for collection of ES products.
Efforts in optimizing reducing agents, cysteine-HCl.H2O and sodium sulfide in order to attain satisfactory responses during acetic acid fermentation have been carried out in this study. Cysteine-HCl.H2O each with five concentrations (0.00-0.50 g/L) was optimized one at a time and followed by sodium sulfide component (0.00-0.50 g/L). Response surface methodology (RSM) was used to determine the optimum concentrations of cysteine-HCl.H2O and sodium sulfide. The statistical analysis showed that the amount of cells produced and efficiency in CO conversion were not affected by sodium sulfide concentration. However, sodium sulfide is required as it does influence the acetic acid production. The optimum reducing agents for acetic acid fermentation was at 0.30 g/L cysteine-HCl.H2O and sodium sulfide respectively and when operated for 60 h cultivation time resulted in 1.28 g/L acetic acid production and 100% CO conversion.
Garlic and garlic extracts, through their antioxidant activities, have been reported to provide protection against free radical damage in the body. This study investigated antioxidant properties of garlic compounds representing the four main chemical classes, alliin, allyl cysteine, allyl disulfide, and allicin, prepared by chemical synthesis or purification. Alliin scavenged superoxide, while allyl cysteine and allyl disulfide did not react with superoxide. Allicin suppressed the formation of superoxide by the xanthine/xanthine oxidase system, probably via a thiol exchange mechanism. Alliin, allyl cysteine, and allyl disulfide all scavenged hydroxyl radicals; the rate constants calculated based on deoxyribose competitive assay were 1.4-1.7 x 10(10), 2.1-2.2 x 10(9), and 0.7-1.5 x 10(10) M (1) second(1), respectively. Contrary to previous reports, allicin did not exhibit hydroxyl radical scavenging activity in this study. Alliin, allicin, and allyl cysteine did not prevent induced microsomal lipid peroxidation, but both alliin and allyl cysteine were hydroxyl scavengers, and allyl disulfide was a lipid peroxidation terminator. In summary, our findings indicated that allyl disulfide, alliin, allicin, and allyl cysteine exhibit different patterns of antioxidant activities as protective compounds against free radical damage.
Present study aimed to investigate the effect of adding antioxidants, cysteine and ascorbic acid on the levels of glutamic oxaloacetic transaminase (GOT), glutamic-pyruvate (GPT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and γ-glutamyl transpeptidase (GGT) enzymes of post-thawed stallion sperm. Ten ejaculates were collected each from four healthy stallions and cryopreserved using HF-20 freezing extender containing either 0 mg/ml cysteine or ascorbic acid, 0.5 mg/ml cysteine and 0.5 mg/ml ascorbic acid. All samples in freezing extender containing cysteine or ascorbic acid or none of them were assessed for sperm motility, viability, plasma membrane integrity, morphology and enzymes concentration. The ALP, LDH and GGT were significantly higher in 0-group compared with cysteine and ascorbic acid groups. The sperm motility of frozen-thawed semen with 0-group was significantly better compared with cysteine and ascorbic acid groups. The variation on viability, sperm membrane integrity and morphology were insignificant between all treated groups. Therefore, these enzymes were reduced when using antioxidants in the freezing extender. Results of the present study suggest that concentration of ALP, LDH and GGT enzymes could be used as parameters for prediction of frozen-thawed stallion semen.
S-Allylcysteine (SAC) is an extensively studied natural product which has been proven to confer cardioprotection. This potentiates SAC into many clinical relevance possibilities, hence, the use of it ought to be optimally elucidated. To further confirm this, an ischemia/reperfusion model has been used to determine SAC at 10 mM and 50 mM on cardiac function, cardiac marker, and mitochondrial permeability. Using Langendorff setup, 24 adult male Wistar rats' hearts were isolated to be perfused with Kreb-Henseleit buffer throughout the ischemia/reperfusion method. After 20 min of stabilization, global ischemia was induced by turning off the perfusion for 35 min followed by 60 min of reperfusion with either Kreb-Henseleit buffer or SAC with the dose of 10 mM or 50 mM. The cardiac function was assessed and coronary effluent was collected at different timepoints throughout the experiment for lactate dehydrogenase (LDH) measurement. The harvested hearts were then used to measure glutathione while isolated mitochondria for mPTP analysis. SAC-reperfused hearts were shown to prevent the aggravation of cardiac function after I/R induction. It also dose-dependently upregulated glutathione reductase and glutathione level and these were also accompanied by significant reduction of LDH leakage and preserved mitochondrial permeability. Altogether, SAC dose-dependently was able to recover the post-ischemic cardiac function deterioration alongside with improvement of glutathione metabolism and mitochondrial preservation. These findings highly suggest that SAC when sufficiently supplied to the heart would be able to prevent the deleterious complications after the ischemic insult.