The effect of co-administration of artemisinin and N-acetyl cysteine on antioxidant status, spermatological parameters and histopathology of testis in adult male mice.

OBJECTIVES: This in vivo study aimed to evaluate the effect of various concentrations of artemisinin (Art) alone or together with N-acetyl cysteine (NAC) on spermatological indices, antioxidant status, and histopathological parameters of testicular tissue in adult male mice. METHODS: Six groups of five healthy male mice (25-30 g) were randomly assigned to different experimental groups. These groups received DMSO and corn oil (0.1%) as an Art solvent (Control), 50 mg kg-1 Art (Art-50), 250 mg kg-1 Art (Art-250), 50 mg kg-1 Art + 150 mg kg-1 NAC (Art-50+NAC-150), 250 mg kg-1 Art + 150 mg kg-1 NAC (Art-250+NAC-150) and 150 mg kg-1 NAC (NAC-150) for a period of 7 days. Testes and epididymis were prepared to evaluate the malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), spermatological indices, and histological parameters. RESULTS: We showed that the high dose of Art (Art-250) significantly reduced the sperm count, motility, viability, and the activity of CAT and increased the levels of MDA compared to the control group. Also, the overdose of Art caused adverse changes in testicular tissue. Co-administration of NAC with Art (Art-250+NAC-150) corrected the adverse effects of Art. CONCLUSIONS: The current study reports that a high dose of Art affects, spermatological parameters, antioxidant/stress oxidative status of the male reproductive system, and NAC is capable neutralize all adverse effects caused by Art.

Brown Seaweed (Padina australis) Extract can Promote Performance, Innate Immune Responses, Digestive Enzyme Activities, Intestinal Gene Expression and Resistance against Aeromonas hydrophila in Common Carp (Cyprinus carpio).

Eight-week oral administration of Padina australis ethyl acetate extract at 100, 200, and 400 mg/kg diets was assessed on the growth performance, tight junction proteins, intestinal immunity, and disease resistance to Aeromonas hydrophila in common carp (Cyprinus carpio). A total of 300 healthy common carp weighing around 14.8 ± 0.03 g were randomly assigned into four equal groups within 12 glass aquariums, each in three replicates (25 fish/tank), for the feeding trial experiment. The first group served as the control group and was fed an un-supplemented diet, whilst the other three groups were offered diets containing graded amounts of Padina australis ethyl acetate extract at 100, 200, and 400 mg/kg, respectively. The growth indices, including final weight, length, weight gain rate, specific growth rate, and feed conversion ratio, were meaningfully improved in fish fed with the algae at 200 and 400 mg/kg compared to the control fish (p < 0.05). Similarly, digestive enzyme activities and serum immune parameters were significantly higher in all treatments, especially 200 and 400 mg/kg fed groups, compared to the control (p < 0.05). In parallel, significant upregulation of genes related to integrity and the immune system was shown in the intestine of these treatment groups compared to control fish (p < 0.05). When fish were challenged with A. hydrophila, the cumulative survival percentages were 53.3% (p = 0.215), 70.0 % (p = 0.009), and 76.7% (p = 0.002) in fish fed 100, 200, and 400 mg/kg diets, respectively, compared to 36.7% survival in control fish (p = 0.134). These data show that the eight-week dietary administration of P. australis extract to common carp can enhance growth performance, digestive enzyme activity, immune response, and disease resistance to A. hydrophila infection.

Artemisinin and l-carnitine combination therapy alters the erythrocytes redox status.

Hematopoiesis is a sensitive target of artemisinin (ART) and its derivatives, and hemolysis is one of their commonly reported side effects. l-carnitine (LC), an amino acid derivative involved in lipid metabolism, is beneficial for hematological parameters. Sixty adult laboratory mice were randomly divided into six groups. Group I (control) received saline and corn oil; groups II and III received therapeutic (50 mg/kg) and toxic (250 mg/kg) doses of ART, respectively; groups IV and V received 370 mg/kg LC along with the 50 and 250 mg/kg ART, respectively; and group VI received 370 mg/kg LC. Drugs were administered orally for 7 consecutive days. The erythrocyte glucose 6-phosphate dehydrogenase (G6PD), catalase (CAT), and peroxidase (POX) activity, and the reduced glutathione (GSH) level were assessed by colorimetric methods. ART reduced the G6PD activity both at therapeutic and toxic doses. The therapeutic dose of ART reduced the CAT activity and the GSH level, nonsignificantly. The toxic dose of ART reduced the CAT activity and increased the POX activity. LC reduced the G6PD, CAT, and POX activities and increased GSH level. The therapeutic dose of ART and LC showed synergy in reducing the G6PD activity. LC and ART combination reduced POX activity and increased GSH level without any significant effect on the CAT activity. Inhibition of G6PD may be a potentially new mechanism of ART action. Coadministration of LC with ART or following treatment with ART may have protective effects on erythrocytes.

Evaluation of Microscopic, Flow Cytometric, and Oxidative Parameters of the Frozen-Thawed Bull Sperm in a Freezing Extender Containing Myo-Inositol.

Introduction: This research was conducted to assess the effect of myo-inositol (MYO) in the freezing extender on the semen quality and oxidative stress parameters of frozen-thawed bull sperm. Materials and Methods: Semen samples were obtained from four bulls (n = 24, six ejaculates per bull), twice a week, and diluted into four equal aliquots in freezing extenders containing different concentrations of MYO (0, 2, 3, and 4 mg/mL). After a freezing/thawing process, velocity parameters, plasma membrane integrity, apoptosis status, malondialdehyde level, and oxidative stress parameters were assessed. Results: Supplementation of freezing extender with 3 mg/mL MYO resulted in higher rapid motility (62.22% ± 2.63%), progressive motility (77.45% ± 2.65%), viability (78% ± 0.91%), plasma membrane integrity (86 ± 0.85), catalase (20.03 ± 0.39 U/mL) activity, and lower significance of lipid peroxidation (3.60 ± 0.15 nmol/dL) than those of the control group (p < 0.05). A significantly lower percentage of normal morphology and intact acrosomes were observed for frozen-thawed semen in the extender supplemented with 4 mg/mL MYO than those of the control group (p < 0.05). Freezing of the sperm in the extender containing 3 mg/mL of MYO leads to a higher percentage of live cells (38.3 ± 2.76). Beat-cross-frequency, amplitude of lateral head displacement, linearity, total antioxidant capacity, total peroxidase activity, early apoptotic status, and superoxide dismutase activities were not affected by MYO levels in the extenders (p > 0.05). Conclusion: The findings of this study suggest that the supplementation of the freezing extender with 3 mg/mL MYO resulted in a higher quality of frozen-thawed bull sperm.

The Beneficial Effects of Actinomycetales Immune Modulators in the Pancreas of Diabetic Rats.

Purpose: Type 1 diabetes mellitus (T1DM) has dramatically increased in recent years, especially in young people, and limits the life quality of the patients involved. Thus, many researchers are performing extensive studies to find alternative treatments for DM. Methods: Here, we evaluated the improvement effects of the heat-killed Actinomycetales species, including Gordonia bronchialis, and Tsukamurella inchonensis in streptozotocin (STZ)- diabetic rats by biochemical, immunological, and histopathological examinations. Results: The present findings exhibited a dramatic and progressive alteration in the serum levels of interleukin-6 (IL-6), IL-10 and tumor necrosis factor-α (TNF-α) in the diabetic group, which were related to the blood glucose and insulin levels, oxidative stress defense (evaluated by TAC and MDA activities), and the pancreas biochemical indicators (such as amylase and lipase). More importantly, the present results were consistent with the histopathological findings, which included cellular degeneration, vascular congestion, hemorrhage, focal necrosis associated with mononuclear cell infiltration. Interestingly, all of the diabetic changes in the blood serum and tissues improved remarkably in the treated groups by Actinomycetales species. Conclusion: Surprisingly, most of the current diabetic complications effectively attenuated after oral administration of both Actinomycetales species, particularly with a high dose of T. inchonensis. Thus, it is concluded that the heat-killed Actinomycetales species can prevent and improve the progression of T1DM and its various complications profoundly.

Antioxidant property of Plantago major leaf extracts reduces testicular torsion/detorsion-induced ischemia/reperfusion injury in rats.

The present study was aimed to determine the protective effects of Plantago major L (PM) leaf extracts on the testicular torsion/detorsion (T/D)-induced ischemia/reperfusion (I/R) injury in rats. Twenty-four mature male Sprague-Dawley rats, weighing 200-220 g, were selected. They were randomly divided into four groups of six animals each: Sham (sham-operated rats; all the surgical steps were performed but T/D was not induced), TDC (Control group; T/D was induced and the right testicular torsion of 720° lasting two hours was followed by detorsion), TDP50 (T/D-operated rats received 50.00 mg kg-1 of PM extract daily for seven days intraperitoneally after detorsion) and TDP100 (T/D-operated rats received 100 mg kg-1 of PM extract daily for seven days intraperitoneally after detorsion). After seven days of treatment, the right testicles were collected. Histopathological and biochemical analyses including levels of malondialdehyde (MDA) and catalase (CAT) and peroxidase activities were determined in testicular tissues of the rats. Tissue sections were taken from testis, Hematoxylin-Eosin staining was done, and the slides were examined by a light microscope. The level of MDA was significantly increased in the testes of the TDC group. The CAT activity levels were decreased significantly after I/R. The post-torsion treatment with PM, particularly at 100 mg kg-1, prevented the increase in lipid peroxidation and reduced the CAT activity levels. The PM also prevented I/R-induced cellular damage and histological changes in the testicular tissues. According to the results of the current study, PM leaf extracts had significant positive effects on the testicular T/D-induced I/R injury. The possible mechanism of reduction in biochemical and histological injuries by PM extracts could be due to antioxidant property.

Administration of grape (Vitis vinifera) seed extract to rainbow trout (Oncorhynchus mykiss) modulates growth performance, some biochemical parameters, and antioxidant-relevant gene expression.

Grape seed, as a main source of polyphenols, has many nutritional and medicinal properties in humans. In the current study, the effects of dietary ethanolic grape seed extract (GSE) on the growth performance, antioxidant activity, and some biochemical parameters in rainbow trout were investigated. Ninety fish (initial weight 78.47 g) were randomly distributed among nine cement tanks (1.8 m × 0.22 m × 0.35 m) with 10 fish per tank. Three experimental diets containing either 0, 10, or 50 g kg-1 GSE were prepared and each diet was randomly assigned to three tanks of fish for 60 days. Results showed that feeding GSE enhanced some growth parameters including the specific growth rate and condition factor in comparison with the control group. Among different serum metabolites, the glucose levels in treatment groups significantly decreased compared to the control group. The total product of lipid peroxidation indicated as malondialdehyde significantly decreased in both the GSE-added treatment groups. The gene expression related to the antioxidant enzymes, catalase, glutathione peroxidase 1, and glutathione S-transferase A, were upregulated in the intestine of fish that received a low dose of GSE. The results of the current study suggest that GSE, especially at 10 g kg-1, diet had the potential to improve (1) specific growth rate and condition factor, (2) biochemical parameters including glucose and lipid peroxidation product, and (3) upregulated the expression of antioxidant genes including catalase, glutathione peroxidase 1, and glutathione S-transferase A in rainbow trout.

The effects of coadministration of tilorone dihydrochloride and culture supernatants from Lactobacillus reuteri on the mouse hepatoma cell line.

CONTEXT: Tilorone dihydrochloride is a therapeutic agent with a different mechanism in cancer. The species of Lactobacillus have an important role in cytotoxic effect. AIMS: Because of unknown effects of tilorone and culture supernatants from Lactobacillus reuteri on hepatoma, the aim of this study is to evaluate apoptotic, cytotoxic, and therapeutic effects of tilorone on mouse hepatoma cell line with and without culture supernatants from L. reuteri. MATERIALS AND METHODS: To do so, after cell line culture, cells were divided into different groups such as negative control, treatment with four doses of tilorone, positive control of supernatant (single dose), and combination therapy groups of different doses of tilorone with supernatant (constant doses), for 48 h. All groups were studied with pathologic tests, biochemical study, tetrazolium dye (3-(4, 5- dimethylthiazol -2-yl)-2, 5-diphenyltetrazolium bromide [MTT]) assay, and absolute real-time-polymerase chain reaction (RT-PCR) were done to assess Bax and Bcl-2 genes expression, as molecular studies. RESULTS: MTT assay results revealed that the tilorone tissue culture IC50 (TCIC50) on the Hepa1-6 cell line was 50 µg/ml. RT-PCR analysis showed that tilorone dihydrochloride induced upregulation and downregulation in expression of Bax and Bcl-2, respectively. Simultaneous, antioxidant effect has also seen in a way that prevented necrosis, in biochemical analysis. These results were dose dependent and statistically significant compared to the control group. CONCLUSIONS: Based on these results, it appeared that this agent could be a good candidate for further evaluation as effective chemotherapy acting through the induction of apoptosis in hepatoma. The cell death caused through bacterial supernatant was rather necrosis than apoptosis.

Tiotropium effects on airway inflammatory events in the cat as an animal model for acute cigarette smoke-induced lung inflammation.

BACKGROUND: Chronic obstructive pulmonary disease is an inflammatory lung disease mainly caused by tobacco smoke inhalation. METHODS: Fifteen healthy adult male cats were categorized into 3 groups: (1) control group, (2) exposed to cigarette smoke (CS), and (3) exposed to CS treated with tiotropium. RESULTS: Increases in clinical signs and airway responsiveness in CS cats were found compared to control animals. The airway hyperresponsiveness and clinical signs were significantly attenuated by treatment with tiotropium. The CS-induced pulmonary release of interleukin-6, interleukin-8, monocyte chemotactic protein-1, and tumor necrosis factor alpha was reduced in the tiotropium group. Exposure to CS significantly increased total inflammatory cells number in bronchoalveolar lavage fluid, which was significantly attenuated by treatment with tiotropium. The number of macrophages, eosinophils and neutrophils and lymphocytes was increased after exposure to CS. Tiotropium significantly reduced the number of all these cells. Perivascular, peribronchiolar infiltration of inflammatory cells and Reid index increased in the CS group. Treatment with tiotropium significantly reduced these parameters to control level. Enhanced lipid peroxidation with concomitant reduction of antioxidants status was observed in the CS group. Tiotropium significantly reduced the serum, lung lavage, lung, and tracheal tissue lipid peroxides to near control levels. Tiotropium also decreased lung and tracheal protein leakage, and prevented the reduction of total antioxidant status in serum, lung lavage, lung and tracheal tissue of the CS group. CONCLUSION: Cigarette smoke increases airway responsiveness and inflammation in a cat model of CS induced lung inflammation. It can effectively be reduced by treatment with tiotropium.

A survey on anticancer effects of artemisinin, iron, miconazole, and butyric acid on 5637 (bladder cancer) and 4T1 (Breast cancer) cell lines.

CONTEXT: Anticancer properties of artemisinin and its derivatives have been shown in many experiments. AIMS: Addition of butyric acid, miconazole, and iron to this traditional drug has been done in order to enhance its anticancer potency. MATERIALS AND METHODS: Cell lines 5637 and 4T1, were cultivated and classified into 13 groups of three each. Different doses of artemisinin with constant doses of iron, miconazole and butyric acid, were added to the cultures. At the end of exposure pathological and enzymatic studies were performed. RESULTS: In four groups treated with different doses of artemisinin and iron, dose-dependent changes were observed. These changes included apoptosis and necrosis with dominance of apoptosis. The supernatant lactate dehydrogenase (LDH) level was increased in a dose-dependent manner, but there was no significant increase in the cell fraction of malonyldialdehyde (MDA) or LDH. In four other groups, which received miconazole, butyric acid and iron in addition to different doses of artemisinin, necrosis was more prominent than apoptosis, and the MDA level did not show any significant change, but LDH was increased. The groups treated with miconazole showed identical changes, with less severity compared to combination therapy groups. In butyric acid-treated groups, the only detectable changes were, mild cell swelling, few apoptosis, and rare necrosis. CONCLUSIONS: A combination therapy with artemisinin can be more effective against cancer cells than monotherapy with that. Butyric acid was not effective on cancer cells. Miconazole deviated the nature of cell death from apoptosis to necrosis and it must be used under caution.

Effects of different concentrations of artemisinin and artemisinin-iron combination treatment on Madin Darby Canine Kidney (MDCK) cells.

Artemisinin is a sesquitrepenelactone with an endoperoxide bridge. It is a naturally occurring substance from Artemisia species plants. Artemisia species have been used in oriental medicine for centuries to treat malaria, gastrointestinal helminthosia, diarrhea, and as an antipyretic and sedative agent. Antileishmanial activity of the plants has been announced a few years ago. Dogs are the most important reservoir of leishmaniasis in some parts of the world. To use it as an antileishmanial drug in dogs, its side effects on different organs, among them the kidney as the organ of elimination have to be elucidated. Artemisinin with different concentrations (0.15, 0.3, 0.6 and 1.2 µg/ml) was added to the culture of MDCK (Madin darby canine kidney) cells with and without iron (86 µg/dl). All the changes were controlled and photographed every 12 hours using an invert microscope. After 60 hours, supernatants and cell extracts were examined for LDH (lactate dehydrogenase) concentration and total protein. Also TBARS (thiobarbituric acid reactive substances) test was performed on cell extracts. Some microscopic slides were prepared from the cells and stained with hematoxylin-eosin for microscopic exams. Biochemical parameters showed cellular reaction and injury in a concentration dependent manner. Cell injury was more severe in the iron-added groups. Microscopic exams showed cell and nuclear swelling, granular degeneration, vacuole and vesicle formation, cellular detachment, piknosis, karyorrhexis, cellular necrosis and inhibition of new mitosis. On using the drug for leishmaniasis treatment in the dog, it should be done with caution and supervision.

Partial purification and characterization of rhodanese from rainbow trout (Oncorhynchus mykiss) liver.

Cyanide is one of the most toxic substances present in a wide variety of food materials that are consumed by animals. Rhodanese, a ubiquitous enzyme, can catalyse the detoxification of cyanide by sulphuration reaction. In this study, rhodanese was partially purified and characterized from the liver tissue homogenate of the rainbow trout. The enzyme was active in a broad range of pH, from 5 to 12. The optimal activity was found at a high pH (pH 10.5), and the temperature optimum was 25 °C. The enzyme was heat labile, losing > 50% of relative activity after only 5 min of incubation at 40 °C. The K(m) values for KCN and Na(2)S(2)O(3) as substrates were 36.81 mM and 19.84 mM, respectively. Studies on the enzyme with a number of cations showed that the activity of the enzyme was not affected by Sn(2+), but Hg(2+), Ba(2+), Pb(2+), and Ca(2+) inhibited and Cu(2+) activated the enzyme with a concentration-dependent manner.

Effects of Haematococcus pluvialis in maternal diet on reproductive performance and egg quality in rainbow trout (Oncorhynchus mykiss).

The aim of this study was to clarify the effects of dietary Haematococcus pluvialis (H.p) on reproductive performance in female rainbow trout and egg quality in terms of antioxidant system and biochemical parameters. 60 rainbow trout (2475.5 ± 64.4 g) were randomly assigned to 2 groups in triplicates and fed diet containing 3 g H.p kg(-1) feed equivalent to 30 mg astaxanthin kg(-1) die or control diet for 30 days. On days 20 and 30 during feeding trial, mature fish were weighed and sampled for stripping. Results indicated that supplementation of H.p did not improve total egg weight, egg number per gram and fecundity. There were few changes in triglyceride and total protein content in fish eggs. Level of glucose decreased markedly on day 30 while on day 20 of feeding trial, a non-significant decrease was shown in treatment group. On day 20, the level of malondialdehyde (MDA) indicating lipid peroxidation product significantly decreased in eggs of the treatment group. The activities of enzymes of the antioxidant system did not change during this study, even though slight increase in glutathione peroxidase in treatment group was revealed during this study. In conclusion, this study showed that female rainbow trout appear to benefit from inclusion of H.p in diet during their reproductive stages in terms of improved egg quality.

Effects of dietary Ergosan on cutaneous mucosal immune response in rainbow trout (Oncorhynchus mykiss).

The effects of dietary Ergosan on the growth performance and mucosal immunity in rainbow trout skin were investigated. 60 rainbow trout (100-110 g) were randomly assigned to 2 groups in triplicates and fed one of the experimental diet formulated with 5 g kg⁻¹ Ergosan or control diet for 50 days. Results showed that on the 45th day of feeding trial, Ergosan supplementation significantly enhanced the growth performance compared to control group. Various enzyme activities, namely lysozyme, protease, alkaline phosphatase and esterase in treatment group were also enhanced on the 45th and 50th day. Skin mucus in Ergosan-fed fish showed the agglutination of erythrocytes while in control group, no visible agglutination was shown. In addition, skin mucus in treatment group showed strong antibacterial activity against Yersinia ruckeri. In conclusion, the major immune components of rainbow trout mucus that are involved in the non-specific immunity were enhanced by administration of Ergosan in 5 g kg⁻¹.

Effects of Haematococcus pluvialis supplementation on antioxidant system and metabolism in rainbow trout (Oncorhynchus mykiss).

Effects of commercial source for astaxanthin (Haematococcus pluvialis) (H.p) on antioxidant power, specific marker enzymes, and some metabolites were examined in rainbow trout (Oncorhynchus mykiss). Fish were fed on diets containing 1, 3, and 10 g microalga kg(-1) feed for 30 days. Serum total antioxidant activity and lipid peroxidation product, indicated by malondialdehyde (MDA), significantly enhanced with different doses of administration, indicating the elevated antioxidant status in all treatment groups. In group fed with high dose of alga, significantly elevated aspartate aminotransferase activity (AST) was noted, indicating damage of normal liver function in this group. Alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were not affected in all groups. Although serum total protein remained unaffected, serum glucose level was decreased significantly in lower doses of administration. Furthermore, triglyceride and cholesterol levels showed significant decrease in 3 g kg(-1) microalga group by modulation of lipid metabolism in this group. On the other hand, in highest dose, significant increase in lipids was observed, indicating the slight dysfunction in lipid metabolism in this treatment group. The present study suggests that Haematococcus pluvialis especially in dose of 3 g kg(-1) feed administration may effectively enhance the antioxidant system and some biochemical parameters in rainbow trout.

The effects of the hydroalcohol extract of Rosa canina L. fruit on experimentally nephrolithiasic Wistar rats.

This research evaluated the possible therapeutic potential of Rosa canina (RC) as a preventive agent in experimentally induced calcium oxalate (CaOx) nephrolithiasis with ethylene glycol (1% EG) in rats. In this experiment, 50 Wistar rats were divided randomly into five groups (n = 10). These groups received tap drinking water (group I), 1% EG (group II), 250 mg/kg RC + 1% EG (group III), 500 mg/kg RC + 1% EG (group IV), or 2.5 g/kg potassium citrate + 1% EG (group V) for a period of 30 days. Blood and urine were collected for biochemical analysis, and the liver and kidneys were prepared for total lipid peroxides, calcium content and histological evaluation. The extract was analysed for total phenolics, flavonoids, ascorbic acid, citric acid and radical scavenger activity. The supplementation of the hydromethanol RC extract contributed to reducing the kidney and liver lipid peroxides to optimum levels in rats that had been treated with EG-induced CaOx lithiasis. The extract also decreased renal and urinary calcium contents, decreased the size and number of CaOx calculi in the kidneys, and significantly increased citrate excretion without changing the volume, pH, or urinary concentrations of oxalate in comparison with the control group. According to these results, RC can be useful as a preventive agent against the formation of CaOx kidney stones.

Rosa canina L. Fruit Hydro-Alcoholic Extract Effects on Some Immunological and Biochemical Parameters in Rats.

INTRODUCTION: This research investigates the possible potential of Rosa canina (RC) as an immunomodulator in rats and its effects on some biochemical parameters. METHODS: In this experiment, 45 male Wistar rats were obtained and divided into three groups (n = 15). These groups received normal saline (10 mg/kg), RC fruit extract (250 mg/kg) and RC fruit extract (500 mg/kg) as oral gavages every day for a period of four weeks, respective-ly. After obtaining blood samples (at the end of each week), differential white blood cell (WBC) counts, phagocyte activity (number), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphates (ALP) albumin and globulins levels of sam-ples were obtained. The malondialdehyde (MDA) and glutathione (GSH) levels in the se-rum were determined only in day 28 of study. The radical scavenger activity (RSA) of the RC extract was measured spectrophotometrically. RESULTS: the gamma globulin level, neu-trophil and monocyte counts and phagocyte activity increased significantly in comparison with the normal saline group. ALT, AST and ALP had not significantly differences in compared to control group. RC extract significantly increased thiobarbituric acid reactive substances (TBARS) and also decreased GSH levels in comparing to control group in day 28. CONCLUSION: the data suggest that the RC extract has been used in traditional medicine might have immunomodulatory effects.

Stepwise binding of nickel to horseradish peroxidase and inhibition of the enzymatic activity.

The incubation of horseradish peroxidase C (HRPC) with millimolar concentrations of nickel, at room temperature and at pH 4.0, induced the progressive formation of a metal-enzyme complex characterized by alterations of the enzyme Soret absorption band that were time- as well as nickel concentration- dependent. For any given incubation period between 1 and 60 min, 2 values for the apparent dissociation constant (K(d)) were found, suggesting the presence of binding sites with different affinities for nickel. The value of each K(d) dropped as the incubation time increased, indicating a progressive stabilization of the metal-enzyme complex. Hill plots suggested a cooperative binding of up to four Ni2+ ions per molecule of HRPC. The inhibition of the enzymatic activity by nickel was studied by following the H2O2-mediated oxidation of o-dianisidine by HRPC under steady-state kinetic conditions. Ni2+ was found to be either a noncompetitive or a mixed inhibitor of HRPC depending both on the duration of preincubation with the enzyme and on Ni2+ concentration. The enzyme remained active only over a limited metal concentration range and data indicated that binding of one Ni2+ affected the substrate binding site, binding of a second Ni2+ affected both substrate and peroxide binding sites, and binding of more than 2 Ni2+ per HRPC molecule led to complete loss of enzymatic activity. Results pointed to the damaging effects of prolonged exposure to heavy metals and also to the existence of a critical metal concentration beyond which immediate abolishing of enzymatic activity was observed.