A phytochemical approach to experimental metabolic syndrome-associated renal damage and oxidative stress.

The aim of this study was to evaluate the effect of DTS-phytocompound on oxidant-antioxidant balance and protein damage in the kidneys of rats administered high doses of fructose. Adult male Wistar rats were divided into four groups. Group A received a control diet, whereas groups B and C were fed a high-fructose diet (60 g/100 g), the latter with additional DTS (50 mg/kg per day) for 60 days. Lipo- and nitro-peroxidation together with α-smooth muscle actin (α-SMA) expression in the glomerular and interstitial tissue of the kidneys were measured after 60 days. Fructose-fed rats showed significantly higher lipoperoxidation, 2,4-dinitrophenol and 3-nitrotyrosine protein adducts, and upregulation of α-SMA in the kidney. DTS significantly decreased such redox unbalance in renal tissue, while partially downregulating α-SMA (p<0.01). These data suggest the potential clinical benefit of DTS in protecting the kidneys from metabolic syndrome-associated changes; gender-related analysis is under way.

Beneficial modulation from a high-purity caviar-derived homogenate on chronological skin aging.

This study tested the activity of LD-1227, which contains a caviar-derived homogenate added with coenzyme Q(10) (CoQ(10))-selenium component (CaviarLieri(®), Lab-Dom, Switzerland), in aged human skin and its potential role on skin mitochondria function. Human dermal fibroblasts were obtained from healthy donors over 70 years old and treated with LD-1227 for 72 hr. As compared to baseline, LD-1227 caused a robust (>67%) collagen type I synthesis (p<0.001) and decreased fibronectin synthesis (p<0.05) with significant fibronectin messenger RNA (mRNA) downregulation (p<0.05, r=0.78). A significant collagen mRNA overexpression occurred with LD-1227 treatment (p<0.05). Mitochondria cytosolic adenosine triphosphate (ATP) level decreased in aged skin samples (p<0.05 vs. young control), but this phenomenon was reversed by LD-1227 (p<0.01). These data show that LD-1227 may modify the extracellular matrix milieu in aged skin and also beneficially affect mitochondrial function.

Inhibiting insulin resistance mechanisms by DTS phytocompound: an experimental study on metabolic syndrome-prone adipocytes.

The present study was designed to determine whether DTS a phytocompound endowed with antioxidant properties, could beneficially modulate nitric oxide (NO) production stimulated by lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha) in adipocytes. Combined stimulation (CS-treatment) exerted by using 5 microg/ml of LPS together with 100 ng/ml of TNF-alpha significantly enhanced NO production in 3T3-L1 adipocytes. Preincubation of the adipocytes with DTS (10-30 mM) inhibited such phenomenon in a dose-dependent fashion. The production of NO was decreased by 52% at the concentration of 30mM of DTS. The decrease in NO production by DTS was associated also with a decrease in inducible nitric oxide synthase (iNOS) protein and iNOS mRNA expression. Nuclear factor-kappa B (NF-kappaB) was significantly enhanced by CS-treatment, while the pretreatment with 30 mM of DTS prevented the activity by 27%. IL-6 production in 3T3-L1 adipocytes was markedly increased by CS stimulus, and the enhanced secretion of IL-6 was suppressed in a dose-dependent manner by DTS. These results suggest that DTS regulates iNOS expression and NO production in adipocytes through the modulating activation of NF-kappaB and may have a potential clinical application within protocols designed for treating metabolic syndrome. (www.actabiomedica.it).

Improving sperm quality and spermatogenesis through a bioactive marine compound: an experimental study.

Dietary lipids may affect sperm membrane structure, fluidity and its susceptibility to oxidative phenomena which may lead to altered sperm viability and proper binding to eggs. Given the recently demonstrated beneficial effects of fish oil diets on turkey fertility and embryo viability, the aim of this study was to test a caviar-derived marine product on spermatogenesis and sperm quality. Sixty mice were divided into four different groups and fed for 3 weeks with normal chow (group A), added with LD-1227 at the dosage of either 5 mg/day (B1) or 10 mg/day (B2) while Group C received standard chow added with 10 mg of a DHA-rich mixture. At sacrifice tests/body weight ration and spermatogenesis was checked. No toxicity, histological sign or body or testes growth abnormality was noted, irrespective of the treatment. As compared to control, all supplements showed to increase sperm counting and motility although the effect of LD-1227 10 mg was significantly higher than DHA alone (p<0.05). Viability was improved by DHA (p<0.05) but not by low LD-1227 dosage while higher dosage performed better than DHA (p<0.05). Morphology was unaffected by any of the employed supplements. Taken altogether, these data suggest that LD-1227 has a remarkable effect on quali-quantitative parameters of spermiogenesis, some of them being more effective than high dosage DHA. These findings may prove to be of interest in clinical practice. (www.actabiomedica.it).

Beneficial effect of a symbiotic preparation with S. boulardii lysate in mild stress-induced gut hyper-permeability.

Increased intestinal permeability has been advocated as one of the likely causes of various pathologies, such as allergies and metabolic or even cardiovascular disturbances. Thus, the aim of the present study was to test a symbiotic preparation containing microbial lysates (KC-1317, Named, Italy) against stress-induced derangement of gut mucosa permeability. Sprague Dawley rats were allocated into control (n=20) and stress (n=20) group. Stress was implemented by 1h of water avoidance stress daily for 10 days. Body weight, food and water intake and passage of stool pellet during stress session were recorded throughout the experiment. On the 11th day, fluorescent iso-thiocyanate dextran solution was injected into small intestinal loops. One hour after the injection, rats were sacrificed. Jejunum and ileum were taken for histopathology. Blood was collected from the abdominal aorta to measure intestinal permeability. In stress group, stool pellets during stress session was significantly higher than control group (p < 0.01). Villus height (p < 0.01), crypt depth (p < 0.01), number of goblet cells in villus (p < 0.01) and crypt (p < 0.05) decreased significantly in jejunum as compared to control. These phenomena were significantly prevented by KC-1317 (p < 0.05). Ileum also showed atrophy but villus height and the number of goblet cells in the villi did not significantly differ. Plasma-concentration of brain-gut peptides (substance P, thyrotropin-releasing hormone, cholecystokinin and motilin) were affected by stress (p < 0.001) and this effect did not change during supplementation with KC-1317. Polymorphonuclear neutrophil counting was significantly higher in stress group as compared to control (p < 0.01) but this phenomenon was abolished in the ileum (p < 0.01) or partly but significantly reduced by KC-1317 supplementation (p < 0.05). Accordingly, intestinal permeability was significantly enhanced in stress group as compared to control (p < 0.01) and prevented by KC-1317 (p < 0.01) in both intestinal segments examined. While confirming that chronic mild stress in rats compromises small intestinal morphology and permeability, we showed that a symbiotic microbial lysate can partly counteract this phenomenon.

Beneficial modification of functional renal parameters in 5/6 nephrectomized rats by nutraceutical. In view of a kidney-protective intervention.

The objective of this study is to ascertain the potential beneficial effects of a novel phytoterapeutic formula (DTS, Kyotsu Jigyo, Japan) on renal function and morphological structure in 5/6 nephrectomized rats. Male Spraque-Dawley rats, 240-280 g, were divided into sham control (Group A) and nephrectomized (Group B and Group C) groups. The 5/6 nephrectomy was performed by removal of the right kidney and 2/3 ligation of left renal artery. After surgery, the animals were kept in individual cage for 6 weeks. Rats in Group A and Group B were fed with a normal protein diet only while those in Group C were fed normal protein diet added with DTS (10 mg/rat/day). The DTS supplementation was started a day after surgery. After 5 weeks, all rats were subjected to renal function study and then their left kidneys were isolated for morphological study. There were no significant differences in body weight, blood pressure, and heart rate among groups. DTS supplementation significantly increased (p<0.05) plasma creatinine concentration, glomerular filtration rate, effective renal plasma flow, and urine flow rate in nephrectomized rats when compared to sham control (Group A) and untreated nephrectomized (Group B) controls. In contrast, plasma urea concentration and morphological structure were not significantly modified by DTS supplementation in nephrectomized animals. These data suggest that feeding with a normal protein diet and DTS supplementation improves renal function without any morphological effect in 5/6 nephrectomized rats if not a slight preservation.(www.actabiomedica.it).

Progression of atherosclerotic lesions in the arteries and related gene expression: protective effect of phytonutrients.

We investigated the effect of the phytocompound Denshici-to-Chiusei (DTS) on the atherogenesis in apolipoprotein E(-/-)/low-density lipoprotein receptor(-/-) (apoE(-/-)/LDL receptor(-/-)) mice (E0). E0 mice were fed for 16 weeks with: (1) placebo or (2) 25 mg or (3) 50 mg of DTS/day. Aortic lesions were reduced by 38% (p < 0.01) in mice fed 50 mg/day, whereas peritoneal macrophages after both dosages had a 45%-60% lower (p < 0.01) capacity to oxidize LDL and to degrade it. This was associated with reduced LDL-associated lipoperoxides and a 22% inhibition (p < 0.05) in LDL aggregation. Tumor necrosis factor-alpha (TNF-alpha) expression and immunoreactivity in the aortic media increased five-fold, but this was significantly mitigated by DTS (50 mg > 25 mg) (p < 0.05). DTS significantly attenuated inflammatory mechanisms preceding atherogenesis with reduced LDL susceptibility to oxidation-aggregation.

Protective effect of a phytocompound on oxidative stress and DNA fragmentation against paracetamol-induced liver damage.

The hepatoprotective potential DTS (1.5 g/kg bw, Denshici-to-Chiusei, Kyotsu Jigyo, Tokyo, Japan) was evaluated against either toxic (1.5 g/kg bw) and sub-toxic (150 mg/kg bw) dosage of paracetamol-induced liver injury in Sprague-Dawley rat. Paracetamol intoxication caused a reduction of serum total protein and increase levels of serum alkaline phosphatase (ALP), aspartate aminotranferase (AST) and serum alanine aminotranferase (ALT) at higher extent in the toxic group. This phenomenon was paralleled by an impaired liver redox status (reduced glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) and increased MDA in both paracetamol-administered groups. Moreover, a marked reduction of ATPase and thiols together with DNA fragmentation occurred in liver tissue. Animals pretreated with DTS showed a marked mitigation of the severity of liver enzyme and of the impaired redox status of the liver. Moreover, DTS partly prevented the DNA fragmentation and the decline of liver tissue ATPase and protein thiol assay as compared with both groups treated with paracetamol alone. Although more detailed studies are awaited to ascertain the detailed mode of action of DTS, it wouls seem to be related to the prevention of formation of the reactive oxygen groups thereby preventing the damage on the hepatocytes and possibly modulating the genes responsible for synthesis of liver antioxidant enzymes thus providing marked DNA protection.

Effect of a phyto-compound on delayed gastric emptying in functional dyspepsia: a randomized-controlled study.

OBJECTIVE: The aim of the present study was to assess the efficacy of a hydro-alcoholic solution of a phytocompound based on Gentianae, Cinchonae, Absinthii and Cinnamomi on gastric emptying in Helicobacter pylori-negative dyspeptic patients. METHODS: The study group consisted of 24 patients with a diagnosis of long-standing dismotility-like functional dyspepsia. All patients were devoid of any major past or ongoing disease and dyspepsia-associated diseases were excluded. After a 2-week wash out period, the patients were randomized to a 2-week treatment in which they were given 20 gtt of the compound 30 min before meals. At the beginning and end of the study, a gastric emptying test was performed by a paracetamol absorption test using a standard meal. The global symptom index (GSI) was assessed daily by a validated questionnaire. RESULTS: Treatment with the phytocompound significantly improved the gastric emptying test (P < 0.01) which was delayed in dyspeptic patients as well as GSI (P < 0.05). CONCLUSION: This phytocompound might be an effective therapeutic option in the treatment of dismotility-like dyspepsia.

"Accelerating aging" chemotherapy on aged animals: protective effect from nutraceutical modulation.

The aim of this study was to test a novel phytocompound in an experimental model of antitumor-induced immunosuppression. Five groups of mice were considered: young (Y) and aged (A) that were given intraperitoneally 10 doses of cyclophosphamide (CPX, 25mg/kg/bw) or CPX plus (150 mg/kg/bw) of the nutraceutical DTS (Denshichi-Tochiu-Sen), and control. After sacrifice, macrophage chemotaxis and serum levels of IFN-gamma, IL-2, and GM-CSF were determined. Liver and urinary bladder were examined histologically, as were the liver and kidney for redox enzymes. CPX significantly decreased macrophage chemotaxis and all cytokines (p < 0.05, A >> Y). DTS restored macrophage function and cytokine concentration (p < 0.001) and partly improved the necro-inflammatory score and substance P receptor expression in the bladder and the redox status in liver and kidney (p < 0.05). Such data suggest that DTS effectively prevents CPX-induced immune suppression and oxidative-inflammatory damage, which are particularly enhanced in aged organisms.

In vitro study on the mechanisms of action of a novel phytotherapeutic compound against human hepatoma cells.

HepG2 human hepatoma cells were incubated for 24 or 48 h with various concentrations of YHK solution. After 24 h incubation, cell proliferation and cytotoxicity were determined by 3-(4,5-dimethylthiazol-2- yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (MTT) assay. Cytotoxicity or necrosis was expressed as lactate dehydrogenase (LDH) release. After exponential growth phase HepG2 cells were treated with different doses of YHK and apoptosis was assessed by using an Annexin V-FITC kit. Further, oxidative stress was measured by dichlorofluorescein-diacetate (DCFH-DA) assay. As compared to control, YHK-treated cultures showed a significant time-course decrease of the proliferation rate of HepG2 cell growth (p < 0.01). This is likely to be due to an enhanced cytotoxicity (MTT and LDH tests) (p < 0.001). On the other hand, YHK showed in vitro to significantly enhance the oxidative stress of HepG2 cell (p < 0.01) while also markedly increasing apoptosis at 72 h with cells G2/M phase arrest (p < 0.01). These data suggest that YHK seem to modulate the extrinsic and intrinsic regulators of apoptosis and sensitize tumour cells to apoptosis. These preliminary data are worth interest when considering that this nutraceutical has been shown in vitro and in vivo to exert protective anti-tumour effect by redox statusmodulating and immuno-regulatory actions. Given its lack of toxicity so far reported, such natural product might represent an effective nutritional supplement in a number of pathological conditions where a chemopreventive strategy is planned.

Effect of a novel phyto-compound on mucosal candidiasis: further evidence from an ex vivo study.

OBJECTIVE: To isolate, identify and determine the prevalence of yeasts in the oral cavity of individuals and to test the minimum inhibitory dilution (MID) of Kolorex against the yeasts isolated. METHODS: Twenty-nine individuals of both sexes aged on average 61.3 years were evaluated at the dental clinic in order to isolate and identify yeasts from their oral cavity, with and without lesions, and to determine the MID of the commercial phyto-product Kolorex against the strains isolated. The antifungal activity of the product tested was determined by the technique of dilution on a solid medium. Monocyte chemoattractant protein 1 (MCP-1) was measured by biotinylated antibody assay by an enzyme-linked immunosorbent assay (ELISA). RESULTS: Yeasts of the genus Candida were detected in the saliva of 45.4% of the 11 individuals with a clinically healthy mouth and in 88.2% of 17 individuals with oral lesions. In the group with oral candidiasis we isolated in tongue and lesion, respectively, for each species: C. tropicalis (5.8% and 11.7%), C. glabrata (5.8% and 5.8%) and C. parapsilosis (0% and 5.8%), in addition to C. albicans as the only species or in association with others, respectively (64.7% and 70.5%). The total clonal formation unit (CFU) (counts/mL) in the saliva showed a higher mean value in the group with oral candidiasis (158.3x10(3)) than in the control group (64.6x10(3)). Most of the 70 test strains (95.7%) were sensitive to Kolorex by presenting a MID of 1:20. Sixty percent of strains from the 70 healthy sites showed results similar to those obtained with strains from oral lesions. Different results were mainly observed among different species. Patients with oral lesions showed a significant time-course increase of the level of monocyte chemoattractant protein 1 (MCP 1) as compared to those without lesions or to healthy people in whom Candida has not been detected (P<0.05). Co-culture with Kolorex using aliquots from the same patients with oral lesions inhibited such event (P<0.05). CONCLUSION: Although this study was focused on oral cavity candidiasis, the results indicate the possibility of a broader use of the antifungal Kolorex in the prevention and treatment of mucosal candidiasis located elsewhere.

Effect of a prebiotic-enriched phytocompound in improving ovalbumin allergenicity.

OBJECTIVE: The aim of the present study was to test a prebiotic-phytotherapic compound in an experimental model of oral allergenicity. METHODS: Antigen-specific immunoglobulin E (IgE) elevated mice were prepared by injecting them intraperitoneally with 10 microg of ovalbumin. Subsequently, the mice were exposed to ovalbumin solution intranasally and blood samples were obtained on weekly intervals for 4 weeks to measure serum-ovalbumin-specific IgE and total immunoglobulin G. Mice with high titers of ovalbumin-IgE were intragastrically administered with 0.3 mL of phosphate buffered solution containing either 20 mg of ovalbumin, the same solution with 5 mL of milk, or 20 mg milk added to prebiotic-phytocompound. RESULTS: Ovalbumin administration caused a significant increase of plasma ovalbumin concentration in sensitized mice while prebiotic-phytocompound-supplemented mice showed a significantly reduced peak value (P < 0.05). Prebiotic-phytocompound added to milk exerted a significant effect in lowering the ovalbumin-IgE level and the total immunoglobulin G level as compared to control plain milk (P < 0.01). CONCLUSION: This study provides a rationale basis for a feasible non-pharmacological therapeutic strategy in food allergen hypersensitivity syndromes.

In view of an optimal gut antifungal therapeutic strategy: an in vitro susceptibility and toxicity study testing a novel phyto-compound.

OBJECTIVE: In view of the raising concern for gut fungal infection, the aim of the present research was to carry out a systematic in vitro study testing the antifungal activity and possible toxicity of a polygodyal-anethole compound (Kolorex) in several strains of Candida albicans and in other fungal pathogens. METHODS: The in vitro susceptibility tests were carried out on 4 strains of C. albicans (C. krusei, C. lipolytica, C. tropicalis, C. utilis), Aspergillus flavus and A. fumigatus. Cultures were also analyzed by varying medium, pH and inoculum size, and a time-course killing test was carried out. RESULTS: In the present study the polygodyal-anethole compound showed remarkable in vitro activity against the most common fungi, which was significantly better than polygodyal alone. Moreover, such mixture compound was shown to exert its activity against a wide spectrum of fungi, including C. lipolytica and C. tropicalis, which required significantly higher MIC of polygodyal to be unfeasible in clinical application. The activity of the polygodyal-anethole compound was significantly better than polygodyal alone with high inoculum size and low pH. Moreover, it proved to exert a significantly faster biological activity against low inoculum. CONCLUSIONS: This study suggests that the mixture compound Kolorex has a very good profile of antifungal activity in terms of effectiveness and spectrum of action while being devoid of any significant toxicity.