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Myrosinases have significant scientific and medical implications. Unfortunately, detection and purification of myrosinase from microbes requires the use of highly cost substrates (glucosinolates) such as sinigrin and expensive instruments such as Fast Protein Liquid Chromatography and or ion exchange chromatography. In this work, we used only 20 mL of bacterial culture supplemented with sinigrin (10 mM) to obtain partially purified myrosinase. The crude protein extract was loaded onto native polyacrylamide gel and putative myrosinase band was identified and eluted. This step successfully minimised the numbers of protein bands of bacterial crude extracts to be further analysed. The current method describes a simple, rapid and cost effective protocol for isolation and detection of active bacterial myrosinases. Furthermore, our method can be used as a purification step.

This study was purposed to explore the synergistic amelioration effect and optimal feeding time of sesame oil and squalene on hyperlipemia-induced sexual dysfunction rats. We established the hyperlipidemia-induced reproductive damage model, the three groups of test substances (sesame oil, a mixture of sesame oil and squalene, and sildenafil) were orally administrated to those hyperlipidemic rats on day 30 and day 60. The results showed that compared with the pure sesame oil, the mixture of sesame oil and squalene can synergistically decrease concentration levels of TG, TC, and LDL-C, significantly increasing the serum testosterone level and sperm count of the epididymal tail, which the 30 days’ effect was better than the day 60. Compared with the model control (MC) group, the Organ Coefficient of penile increased significantly in the sesame oil (SO), sesame oil+ Squalene (SOS), and Sildenafil (SN) group, and no pathological changes were found in the penile and testis in above three groups at the day 30 and the day 60. In conclusion, the present results demonstrated that sesame oil and squalene have a synergistic amelioration effect on lowering blood lipid and promoting the recovery of erectile and sexual function on hyperlipemia-induced reproductive damage rats at day 30. However, further studies should be carried out to deeply elucidate the molecular mechanisms of Sesame oil and squalene in lowering blood lipids and improving sexual function in vivo.

Background: Despite its detrimental effects on health, chewing khat appears to be becoming more commonplace globally as a daily habit, particularly in the nations where it originated. As a result, it is very possible that khat users will take drugs while they chew the drug. That is high among them, increasing the likelihood of harmful khat-drug interactions. In these situations, it is critical to assess the impact of simultaneous khat and clinical medication delivery.Objective: As a preliminary study to evaluate the effect of khat (Catha edulis Forsk) chewing on serum Random Blood Sugar (RBG) level in Type 2 Diabetic Mellitus (T2DM) patients treated with metformin.Material and Method: Seventy-six male participants between the ages of 25 and 70 were included in the trial, 38 of them were already metformin-treated T2DM patients with a prior diagnosis. The remaining 38 people had no family history of diabetes, were healthy, and were not diabetic. Whether the individuals in the aforementioned two groups regularly chewed khat or not was used as a supplementary criterion for division. Using a drop of blood taken from each patient, a calibrated glucometer was used to take three readings of RBG levels.Results: The findings showed that healthy persons (non-khat chewers) had RBG levels at 4 hours after lunch that were significantly different from their corresponding values at 2 hours earlier. Using an independent sample t-test to compare the RBG levels of healthy khat chewers to healthy non-khat chewers, it was discovered that khat chewers had significantly higher RBG levels 3 and 4 hours after lunch (P = 0.042 and 0.000, respectively). RBG levels at 4 h after lunch (2 h after khat chewing) were significantly lower by -54.36 when compared to the level at 2 h after lunch of the same group of diabetic khat chewers taking metformin, whereas levels at 3 h after launch (1 h after khat chewing) were not statistically different.Conclusion: When khat was consumed, healthy volunteers' serum RBG levels went up, whereas diabetic patients receiving metformin saw no change in their RBG levels.

A recombinant β-glucosidase human gut bacterium capable of nitrile production from desulfo-glucosinolates was studied. The bgl4 gene (2,151 bp) from Enterococcus casseliflavus CP1 was cloned and overexpressed in Escherichia coli BL21(DE3) at 25 °C for 16 h in LB medium using 0.5 mM isopropyl β-D-1-thiogalactopyranoside inducer. The recombinant bgl4 enzyme (79 kDa) was purified using Ni2+ affinity column chromatography. This recombinant bgl4 enzyme of the glycosyl hydrolase 3 family did not degrade glucosinolates; however, it transformed desulfo-glucosinolates, except for desulfoglucoraphanin, to produce the corresponding pure nitriles in citrate phosphate buffer pH 7.0 and LB medium. The bgl4 enzyme activity toward pNPG in buffer was optimal at pH 7.0 and 37 °C at 23.4 U/mg, and promoted by Mn2+; however, activity was slightly deactivated by Fe2+. This provided a possible alternative metabolic route involving nitrile formation from desulfoglucosinolates by β-glucosidase in certain bacteria.

Background: Despite numerous reports regarding the associations of Khat chewing with serious health adverse effects, a significant number of people worldwide uses Khat daily, especially in its origin countries. The risk of co-administration of Khat and drugs that is high among these individuals, leading to increase probability of adverse Khat-drug interactions. Under these circumstances, it becomes important to evaluate the effect of concomitant administration of Khat and clinical drugs. Metformin is a drug of biguanide class used to the lower blood glucose concentrations in patients with type 2 diabetes mellitus (T2DM). Aim and Objectives: The aim of the study was to evaluate the beneficial or harmful effects of concomitant administration of Khat and metformin in a high fructose diet induced T2DM model. Materials and Methods: Thirty-six adult male rats weighing 120–150 g were made diabetic by feeding a high fructose diet for 4 weeks. These rats while continuing to be on high fructose diet were divided into six groups and administered orally, normal control (normal saline), normal control with Khat (1500 mg/kg), non-treated diabetic group, Khat treated group (1500 mg/kg), metformin treated diabetic (300 mg/kg), and metformin (300 mg/kg) with Khat (1500 mg/kg) treated group. All the above treatments were given for 4 weeks and their effects were studied. Results: The hyperglycemia, body weight, and lipid profile parameters were brought down significantly by Khat and metformin (P < 0.05) monotherapy; metformin being more effective than Khat. The metformin-Khat combination had a synergistic effect and resulted in much greater efficacy in ameliorating the parameters. Moreover the adverse effect of Khat like lowering of high-density lipoprotein cholesterol (HDL-C) levels was significantly mitigated by metformin-Khat combination. Conclusion: The results suggested that metformin-Khat combination had a much greater efficacy with the added advantage of reducing the adverse effects.

An oxygen insensitive azoreductase was purified from a novel bacterial strain (Staphylococcus sp. KU898286) that was isolated from an abandoned site of the textile waste discharge unit. The isolated enzyme had efficiently cleaved the azo-bonds through reductive transformation under aerobic conditions. Initial phenotypic characterization and final construction of phylogenetic tree on the basis of 16s rDNA demonstrated 99% resemblance of the isolate to Staphylococcus aureus. The purified azoreductase was found to have a broad spectrum activity that reduced RR241 at a concentration of 50mg/L with pH between 6-8 and 30°C temperature). Besides, the reactive red 241 (RR241) was reduced at extracellular level as well as NADH dependent intracellular level. Complete reduction/ decolourization of RR241 were achieved after 18 hrs of exposure. The final degradation product observed to be 2-nephthol was purified by High Pressure Liquid Chromatography (HPLC) and the molecular mass was computed by Gas Chromatography-Mass spectroscopy (GC-MS). The study revealed a cost effective and eco-friendly approach to degrade the toxic dyes into less toxic products by Staphylococcus sp. KU898286.

A Citrobacter strain (WYE1) was isolated from a UK soil by enrichment using the glucosinolate sinigrin as sole carbon source. The enzyme myrosinase was purified using a combination of ion exchange and gel filtration to give a pure protein of approximately 66 kDa. The N-terminal amino acid and internal peptide sequence of the purified protein were determined and used to identify the gene, which, based on InterPro sequence analysis, belongs to the family GH3, contains a signal peptide, and is a periplasmic protein with a predicted molecular mass of 71.8 kDa. A preliminary characterization was carried out using protein extracts from cell-free preparations. The apparent KM and Vmax were 0.46 mM and 4.91 mmol dm(-3) min(-1) mg(-1), respectively, with sinigrin as substrate. The optimum temperature and pH for enzyme activity were 25 °C and 6.0, respectively. The enzyme was marginally activated with ascorbate by a factor of 1.67.

Glucosinolate (GSL) hydrolysis is mediated by the enzyme myrosinase which together with specifier proteins can give rise to isothiocyanates (ITCs), thiocyanates, and nitriles (NITs) in cruciferous plants. However, little is known about the metabolism of GSLs by the human gut flora. The aim of the work was to investigate the metabolic fates of sinigrin (SNG), glucotropaeolin (GTP), gluconasturtiin (GNT), and their corresponding desulfo-GSLs (DS-GSLs). Three human gut bacterial strains, Enterococcus casseliflavus CP1, Lactobacillus agilis R16, and Escherichia coli VL8, were chosen for this study. GNT was metabolized to completion within 24 h to phenethyl ITC and phenethyl NIT (PNIT) by all bacteria, except for L. agilis R16 which produced only PNIT. At least 80 % of GTP and SNG were metabolized by all bacteria within 24 h to the corresponding ITCs and NITs. The pH of media over time gradually became acidic for both L. agilis R16 and E. coli VL8, while for E. casseliflavus CP1 the media became slightly alkaline with NIT and ITC production occurring between pH 3.0 and 7.5. ITC production peaked between 4 and 10 h in most cases and gradually declined while NIT production increased and remained relatively constant over time. The total percentage products accounted for 3-53 % of the initial GSL. NITs were produced from DS-GSLs suggesting an alternative metabolism via desulfation for the food based GSLs. The metal ion dependency for NIT production for GNT and its DS form was investigated where it was shown that Fe(2+) increased NIT production, while Mg(2+) stimulated the formation of ITC.

Caralluma is a plant that possessing a great therapeutic potential in folk medicine in Yemen, namely, Caralluma penicillata (C. penicillata) as antiulcer. The study aims to evaluate the anti-inflammatory properties and gastritis protection activity of C. penicillata against indomethacin in adult guinea pigs. The study was divided into four parts: firstly, the optimum dose of extract as anti-inflammatory effect was determined. Secondly, the acute anti-inflammatory effect of extract were estimated. Thirdly, the repeated doses of extract against chronic inflammation was estimated. The anti-inflammatory activity of extract was compared with indomethacin as a prototype of drug against inflammation. Fourthly, the gastritis protection properties of extract with/without indomethacin were performed. The results showed that a 400 mg/kg of 10% ethanol extract produced the maximum of anti-inflammatory effect. Also, the single dose of extract was equipotent for indomethacin (10 mg/kg), but shorter in duration with regard to acute anti-inflammatory effect. In addition, the repeated doses of extract against chronic inflammation were less potent than indomethacin with regard to ulcerogenic effect. On the other hand, extract-indomethacin combination reduced the gastritis effect of indomethacin based on ulcer index and histological study.

The degradation of glucosinolates (GLSs) are mediated by the enzyme commonly known as myrosinase (β-thioglucosidase glucohydrolase; EC 3.2.3.1). There has been limited work on bacterial myrosinases which is surprising since they must play a role in both soil and animal gut ecology. The only bacterial myrosinase that has been purified to homogeneity is that of Enterobacter cloacae (no 506) but no amino acid sequence is available and there is very limited information on the characteristics of these enzymes. In this study a bacterial myrosinase from a soil isolate Citrobacter spp was purified to homogeneity and characterised. A combination of ion exchange chromatography two steps, (Mono Q column followed by gel filtration Superdex 75) were used. The molecular mass of both denatured and native protein is approximately 66 kDa. The optimum temperature and pH of the crude extract is 25 ºC and pH 6.0 respectively. The enzyme is slightly activated by ascorbate but inhibited by glucose. Metal ions Zn2+, Ca2+, Fe3+ and Mg2+ also inhibited β-O-glucosidase activity with Fe3+ causing the largest loss of (76%) of activity followed by Mg2+, Zn2+ and Ca2+ while the metal ion chelator EDTA reduced enzyme activity by 70%. In vivo cultures of Citrobacter with sinigrin as a carbon source produced an as yet an unknown metabolite, while cell free extracts incubated with sinigrin produce 2-propenyl isothiocyanate. Sinigrin is completely degraded within 12 h of incubation of the Citrobacter in M9 medium either alone or in the presence of glucotropaeolin or gluconasturtiin. The latter two glucosinolates were metabolised differently and approximately 39% of glucotropaeolin and 20% gluconasturtiin remained in culture medium after 25 h of incubation. None of the tested β configuration substrates induced myrosinase. The enzyme hydrolysed sinigrin and glucoerucin at a faster rate than glucotropaeolin and glucoraphanin. The enzyme has both β-O-glucosidase and β-S-glucosidase activity with a greater affinity to aryl-β-glucoside with a Km of 0.0183 mM and a Vmax of 0.6 nmole.L-1.min-1 while for sinigrin a Km of 0.54 mM and a Vmax of 3.1 nmole.L-1.min-1. The enzyme activity could not be recovered from SDS-PAGE gels but was detected and eluted from native PAGE. SDS-PAGE of the eluted protein showed fewer protein bands and was comparable to the ion exchange second run in terms of number of bands on SDS-PAGE gels. The N-terminal sequence of the pure Citrobacter myrosinase shows little similarity with β-glycosidases while internal peptide analysis revealed some similarity with other O-glucosidases. However, no homology with any known myrosinases was found. Peptide sequencing of bands of 71 and 72 kDa obtained by different purification techniques (likely the same proteins) show some similarity with β-glucosidase of Citrobacter 30_2, while the peptide sequence of the secreted myrosinase band of ≈57 kDa shows similarity with glycoside hydrolase, family 3 of Ktedonobacter racemifer DSM 44963. The genome sequence of Citrobacter is required in order to allow further study such as cloning.