A 2-year Double-Blind RCT Follow-up Study with Fermented Papaya Preparation (FPP) Modulating Key Markers in Middle-Age Subjects with Clustered Neurodegenerative Disease-Risk Factors.

In recent years a number of studies have reported the significant relationship between metabolic syndrome and neurodegenerative disease. There is accumulating evidence that the interplay of combined genetic and environmental risk factors (from diet to life style to pollutants) to intrinsic age-related oxi-inflammatory changes may be advocated for to explain the pandemic of neurodegenerative diseases. In recent years a specific Fermented Papaya Preparation (FPP) has been shown to significantly affect a number of redox signalling abnormalities in a variety of chronic diseases and as well in aging mechanisms either on experimental and on clinical ground. The aim of the present study was to evaluate FPP use in impending metabolic disease patients with potentially neurodegenerative disease clustered risk factors. The study population consisted of 90 patients aged 45-65 years old, with impending metabolic syndrome and previously selected as to be ApoE4 genotype negative. By applying a RCT, double-blind method, one group received FPP 4.5 g twice a day (the most common dosage utilized in prior clinical studies) while the other received an oral antioxidant cocktail (trans-resveratrol, selenium, vitamin E, vitamin C). Then, after 21 month treatment period, a selected heavy metal chelator was added at the dosage of 3 g/nocte for the final 3 months study treatment. The parameters tested were: routine tests oxidized LDL-cholesterol, anti-oxidised LDL, Cyclophilin-A (CyPA), plasminogen activator inhibitor-1 and CyPA gene expression. From this study it would appear that FPP, unlike the control antioxidant, significantly decreased oxidized-LDL and near normalizing the anti-Ox-LDL/Ox-LDL ratio (p<0.001) although unaffecting the lipid profile per sè. Moreover, only FPP decreased cyclophilin-A plasma level and plasminogen activator-inhibitor (p<0.01) together with downregulating cyclophilin-A gene expression (p<0.01). Insulin resistance was only mildly improved. Heavy metals gut clearance proved to be effectively enhanced by the chelator (p<0.01) and this was not affected by any of the nutraceuticals, nor it added any further benefit to the biological action of FPP.

Effect of a quality-controlled fermented nutraceutical on skin aging markers: An antioxidant-control, double-blind study.

The aim of the present study was to determine whether oral supplementation with a fermented papaya preparation (FPP-treated group) or an antioxidant cocktail (antioxidant-control group, composed of 10 mg trans-resveratrol, 60 µg selenium, 10 mg vitamin E and 50 mg vitamin C) was able to improve the skin antioxidant capacity and the expression of key skin genes, while promoting skin antiaging effects. The study enrolled 60 healthy non-smoker males and females aged 40-65 years, all of whom showed clinical signs of skin aging. The subjects were randomly divided into two matched groups, and were administered FPP or antioxidant treatment of a 4.5 g/day sachet sublingually twice a day for 90 days in a double-blind fashion. The parameters investigated were: Skin surface, brown spots, skin evenness, skin moisturization, elasticity (face), redox balance, nitric oxide (NO) concentration, and the expression levels of key genes (outer forearm sample). As compared with the baseline (day 0) and antioxidant-control values, FPP-treated subjects showed a significant improvement in skin evenness, moisturization and elasticity. The two treatments improved the MDA and SOD skin concentrations, but only the FPP-treated group showed a higher SOD level and a significant NO increase, along with significant upregulation of acquaporin-3 and downregulation of the potentially pro-aging/carcinogenetic cyclophilin-A and CD147 genes (P<0.05). Progerin was unaffected in both treatment groups. In conclusion, these findings suggest that orally-administered FPP showed a consistent biological and gene-regulatory improvement in the skin, as was also demonstrated in previous experimental and clinical trials testing other tissues, while common oral antioxidants had only a minor effect.

In vitro protective effect of Celergen, a bioactive marine compound, on interleukin-6-related invasiveness of pancreatic cancer.

The purpose of this study was to assess the effect of Celergen, a marine nutraceutical, against tumor cell invasion in human pancreatic cancer cell line (PSN-I). High invasive clone (HI) and low invasive clone (LI) were established from wild type PSN-l cell line after a repeated invasion assay test. The invasive ability of HI cells and the level of IL-6 in the conditioned medium of HI cells was significantly higher than that one of LI cells but both these parameters were significantly reduced by the addition of Celergen (p<0.01). Exogenous IL-6 administration induced a dose dependent enhancement of invasive ability in both cell populations. Moreover, IL-6 receptor expression was detected in 72% of HI cells whereas this occurred only in 37% of LI cells. When co-cultured with Celergen this parameter was significantly downregulated in both cellular subsets (p<0.05). The addition of conditioned medium derived from HI cells (HCM) and LI cells(LCM) enhanced the invasive ability in both cell populations without affecting cell proliferation. The effect of HCM on the invasive ability of HI cells was partially inhibited by the addition of Celergen (p<0.01). In summary, overexpression of IL-6 and its receptor may be one relevant factor contributing to the highly invasive characteristic of the pancreatic cancer cell line we used while a significantly beneficial modulation was obtained by applying this novel marine nutraceutical. This advices to further explore the possibility of marine compounds regulation of IL-6 ligand/receptor and other possible invasive factor interaction in the therapy of this malignancy while further studies are awaited in this setting.

The hidden phenomenon of oxidative stress during treatment of subclinical-mild hypothyroidism: a protective nutraceutical intervention.

Recent studies suggest that subjects with hypothyroidism under therapy with levothyroxine (L-T4) might develop oxidative stress. The aim of this study was to test a redox-balance modulator, fermented papaya-based nutraceutical (FPP), together with subclinical (SH) or mild hypothyroidism (MH) treatment in view of biochemical changes. A total of 60 females treated for SH-MH were divided into two matched groups and received either FPP 3 grams 1 sachet three times a day (t.i.d.) or placebo for 3 months. A significant baseline increase of all oxidative markers was observed in SH-MH (p<0.05 vs. control) and even more under T4 treatment (p<0.05). FPP caused a normalization of redox markers (p<0.01 vs. placebo). Thyroid supplementation accelerates mitochondrial oxygen consumption and oxidative stress, whereas a redox-modulator therapy is advisable, given the long-lasting treatment in such cases.

Testing a novel bioactive marine nutraceutical on osteoarthritis patients.

Osteoarthritis (OA) is a slow, chronic joint disease characterized by focal degeneration of articular cartilage and alterations of the chemical and mechanical articular function and also major cause of pain and physical disability. There is clinical evidence that increasing dietary n-3 relative to n-6 may be beneficial in terms of symptom management in humans but not all studies conclude that dietary n-3 PUFA supplementation is of benefit, in the treatment of OA. Our recent studies highlight the effect of a biomarine compound (LD-1227) on MMPs, collagen metabolism and on chondrocyte inflammatory markers. Thus, the aim of the present work was to test such bioactive compound versus a common nutraceutical intervention (glucosamine/chrondroitin sulfate) in knee osteoarthritis patients. The patients population consisted of 60 subjects with a recent diagnosis of knee osteoarthririts of mild-moderate severity. Patients were randomized in a double-blind study comparing LD-1227 (group A) versus a mixture of glucosamine (500 mg), chondroitin sulfate (400 mg) (group B). Patients were allowed their established painkillers on demand. At 4, 9 and 18 weeks patients were evaluated as for: VAS score assessing pain at rest, and during physical exercise, Lequesne index, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scale and KOOS scale. Moreover, serum concentrations of IL-6, IL-ß, CRP, TNF-sR1 and TNF-sR2 were assessed. As compared to GC treatment, LD-1227 yielded a quicker and higher degree of improvement of the whole clinical indexes and a lower NSAIDs use at the end of the study. LD-1227 brought about also a more significant downregulation of the tested cytokines cascade. Taken overall, these data suggest that LD-1227 has the potential to be included in the nutraceutical armamentarium in the management of OA.

A sturgeon-derived bioactive compound beneficially modulates nuclear receptors controlling metabolic functions in patients with metabolic syndrome.

The aim of the present study was to test the possible effects of a novel sturgeon-derived compound  (LD-1227) on inflammatory markers related to metabolic nuclear receptors in patients with metabolic syndrome. The study population consisted of 76 patients with metabolic syndrome and 30 healthy subjects who were maintained to their current treatments and randomly supplemented: A) LD-1227 (n=38) or B) placebo (n=38) as compared to C) healthy controls (n=30). LD-1227 or placebo (water-soluble starch) were given daily at breakfast and dinner for three months. Levels of hs-CRP, IL-6, TNF-α, leptin and adiponectin/ resistin index were assayed at the entry, 1 month and 3 months afterwards. At the end of the study period, as compared to B group, LD-1227-treated patients showed a significant improvement of all parameters tested, irrespective of the presence of diabetes. In particular, levels of adiponectin and adiponectin/ resistin index significantly increased following LD-1227 administration. Although the metabolic syndrome remains a multifaceted condition requiring a complex approach, LD-1227 could be a potential safe therapeutic tool to be integrated into a wider treatment and preventive medicine schedule strategy.

Biomarine extracts significantly protect from ultraviolet A-induced skin photoaging: an ex vivo study.

We tested the activity of the marine nutraceutical CL-1222 added with a coenzyme Q10 (CoQ10)-lutein-selenium component (Celergen(®), Laboratoires-Dom, Switzerland) to protect human fibroblasts against ultraviolet A (UVA)-induced photoaging. Cells obtained from 22- to 39-year-old healthy donors were pretreated with CL-1222 before UV irradiation, as compared with same quantity of the CoQ10-lutein-selenium component. As compared to untreated control, UVA-irradiated samples exhibited a significant increase of secreted matrix metalloproteinase-1 (MMP-1) (p<0.001) with over four-fold MMP-1 upregulation (p<0.001). Samples treated with CL-1222, but not with the CoQ10-lutein-selenium component, showed a significant decrease of MMP-1 secretion (p<0.01) and expression decrease (>60%, p<0.01) with >54% elastase activity inhibition (p<0.01). This preliminary study shows that such marine nutraceuticals can significantly protect against UV-irradiation irrespective of the CoQ10-lutein-selenium component with a specific protective gene expression modulation amenable to novel clinical applications.

POLI-mix functional food enhances steady-state bioenergetic status independently of age: an experimental study.

BALB/c mice were divided into young, middle-aged, and aged groups, and each group was given 3 weeks of oral treatments: (1) 1 mL of VBC1-99 (a mixture of 42 fruits and vegetables extracts) or (2) 1 mL of same amount of antioxidant vitamins as control. Steady-state hepatic adenosine triphosphate (ATP) was assessed by phosphorus-31 nuclear magnetic resonance ((31)P-NMR) spectroscopy as: ß-ATP/reference peak, inorganic phosphorus (Pi)/peak and ß-ATP/Pi. As compared to untreated control, VBC1-99 significantly enhanced ß-ATP/peak and ß-ATP/Pi ratios (p<0.01) in all age groups and throughout the observation period (p<0.05) together with a significant decrease of Pi/ref peak ratio (p<0.05). However, this value in middle-aged and aged mice was comparable to antioxidant control mice. These NMR data demonstrate that VBC1-99 has a beneficial effect on hepatic energy metabolism, irrespective of age.

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.

Cardioprotective effect of a biofermented nutraceutical on endothelial function in healthy middle-aged subjects.

We tested a biofermented nutraceutical (FPP) that has been previously shown to positively modulate nitric oxide (NO). Forty-two healthy middle-aged subjects were given 3 grams of FPP three times a day for 6 weeks, and tests were repeated at 3 and 6 weeks; the control group was given a placebo. Flow-mediated dilation (FMD) was measured together with NO compounds (nitrogen oxides [NOx]: NO(2)(-)+NO(3)(-)) plasma levels and asymmetrical dimethylarginine (ADMA). In the interventional group, overall FMD significantly increased from 4.2% to 7.3% (p<0.05 vs. placebo). A significant increase in plasma NO and a decrease in ADMA were detected after consumption of FPP (p<0.01). Although larger studies are awaited, it appears that, at least in healthy individuals, such nutraceutical intervention by positively acting on significant cardiovascular parameters can be considered in the armamentarium of a proactive age-management strategy.

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).

Regulating redox balance gene expression in healthy individuals by nutraceuticals: a pilot study.

We tested the effect of a fermented papaya preparation (FPP; ORI, Gifu, Japan) on redox balance gene expression in 11 healthy nonsmoker, teetotaller individuals subjected to a detailed dietary and lifestyle questionnaire who refrained from any multivitamin supplement or fortified food. Redox status was assessed by erythrocyte and plasma parameters together with related leukocyte mRNA (glutathione peroxidase [GPx], superoxide dismutase [SOD], catalase, 8-oxoguanine glycosylase [hOGG1]) before/after 6 grams of FPP supplementation. At 2 and 4 weeks after FPP administration, plasma parameters remained unchanged, whereas FPP significantly upregulated all tested gene expression (p < 0.05). Although posttranscriptional/translation protein modifications do occur and larger and longer studies are awaited, these preliminary data suggest that a transcriptomic modification of key redox and DNA repair genes may offer further insights when attempting to interrelate "nutragenomics" to clinical phenomena.

Muscular metabolism in aged rats under exhaustive exercise: effect of a modified alkaline supplementation.

A modified alkalizing supplementation (MAS) was tested on skeletal muscle metabolism in aged rats undergoing exhaustive exercise. Aged Wistar rats were allocated into two groups: saline (A) and saline added with 16 mg of MAS (B) before treadmill exercise. Blood and gastrocnemius and soleus muscle were analyzed after exercise for succinate dehydrogenase (SDH), acetylcarnitine (ALCAR), and glycogen. Lactic acid (LA), creatin-phosphokinase (CPK), and gas analysis were tested in the blood. Exercise caused a significant increase of LA and CPK and muscle glycogen fall. Arterial desaturation at exhaustion was prevented in the B group (p < 0.05). Exercise-induced increase of SDH and ALCAR was further enhanced in B rats (p < 0.05). This study suggests that MAS can improve fast and endurance muscle metabolism in aged rats by increasing cellular acetyl group availability and tricarboxylic acid turnover.

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.