Ribodiet®

Nucleotides are low-molecular-weight intracellular compounds. They are the building blocks for nucleic acids and play a key role in many biochemical pathways. They are phosphoric nucleoside esters, made up of three components: a weakly basic nitrogenous compound, a pentose sugar, and one or more phosphate groups. They are the basic units of the nucleic acids DNA and RNA. The most important nucleotides are adenosine, guanosine, inosine, cytidine and uridine monophosphates.
Nucleotides are considered ‘semi-essential’ nutrients. This means that endogenous production satisfies requirements in the maintenance condition, but exogenous administration is needed in childhood, in stress conditions or when tissues are damaged [1].
Every new cell requires around 1 billion nucleotides in order to duplicate. Some tissues have a limited capacity for de novo nucleotide synthesis, and thus require exogenously supplied bases that can be utilized by a salvage pathway. For example, the intestinal mucosa, haematopoietic cells of the bone marrow, leucocytes, erythrocytes and lymphocytes are incapable of de novo synthesis, and thus utilize the salvage pathway, suggesting that an exogenous supply of nucleotides via the diet might be important for these cells [2].
For over 15 years, Prosol has provided a benchmark in nucleotide blends, sold under the Ribocare® brand, for infant formula application. Following this success story, the company decided to deploy its extensive knowledge in the field of yeast cell extracts and, after years of trials, has launched Ribodiet®, a combination of natural nutritional ingredients that can have positive effects on different body tissues.

Composition and technical specifications

Ribodiet® is a natural product extracted from yeast cells with a gentle, standardized and highly controlled process which is free of chemical solvents. Ribodiet® is a source of nucleotides, nucleosides, oligo nucleotides, ribonucleic acid fragments, amino acids, minerals and group B vitamins (Table 1). It is derived from Kluyveromyces fragilis or Saccharomyces cerevisiae yeast cells.

Schermata 2017-11-17 alle 11.57.23
Yeast RNA, extracted from the cell only by physical means, is concentrated, and free nucleotides are obtained via enzymatic hydrolysis. The product is then spray-dried in order to maintain its chemical and physical characteristics and make it stable and storable at room temperature. The concentration process, complete standardization, and the hydrolysis of nucleic acids allows Prosol to obtain an ingredient with a high content of free nucleotides (>40%), qualitatively and quantitatively standardized.
Ribodiet® is a gluten-free product and suitable for vegans. It is certified halal and kosher. The Ribodiet® trade mark is registered in the European Union. Table 2 summarizes the technical features of Ribodiet®.

 

Schermata 2017-11-17 alle 11.57.33

Mechanism of action

Nucleotides are mainly used in infant foods because research in human nutrition has demonstrated that the inclusion of nucleotides in parenteral and infant milk formulas improves intestinal health and the development of the immune system [3].
Nucleotides do not all have the same effect, so a mixture of nucleotides is used to provide the best result. Many studies in healthy term infants have demonstrated that nucleotide supplementation may reduce the risk of diarrhoea by approximately one quarter because immune maturation is enhanced [2].
Nucleotides have a direct effect on the maintenance of intestinal mucosal integrity. It has been demonstrated that nucleotide supplementation in young rats increases the weight of the intestinal mucosa, villus height (by 25%), and the activity of brush border enzymes (maltase, sucrase and lactase), suggesting acceleration of gut growth and differentiation [3].
Supplementation with a nucleoside–nucleotide mixture increases recovery after food deprivation, infection or protein deficiency. Small intestine atrophy and decreased activity of brush border enzymes in rats quickly resolved with nucleotide supplementation [4].
As regards microbiota health status, in vivo studies show that dietary supplementation with nucleotides improves the intestinal flora and stimulates the growth of bifidobacteria [5]. Dietary nucleotides favour the development of faecal flora with a predominance of bifidobacteria and lactobacilli and lower percentage of Gram-negative enterobacteria.
Finally, in relation to immune system modulation, nucleotides influence both humoral and cell-mediated immunity: they accelerate T-cell-dependent antibody production and seem to exert actions on T-helper cells at antigen presentation, perhaps during cognitive cell–cell interactions.
A nucleoside-nucleotide mixture stimulates the proliferation, differentiation and maturation of neutrophils [3]. Nucleotides cause a transient increase in natural killer cell cytotoxicity, interleukin-2 production and interferon-gamma secretion, and lower macrophage activation [6].
Supplementation with dietary nucleotides increases resistance to bacterial infection in animals and humans.

Efficacy

Two pre-clinical trials have been performed on Ribodiet® at the Food Chemistry and Nutraceuticals laboratories (Department of Drug Sciences – Pavia – Italy) to evaluate efficacy in modulating some parameters involved in the immune response. The cell line used in this assay was THP-1, a human monocytic cell line, which was incubated for 24 hours in a complete medium plus a non-cytotoxic concentration (1.25 mg/ml) of Ribodiet®. Before the end of the treatment, a lipo-polysaccharide (LPS), one of the best characterized macrophage-activating factors, was added to the samples to promote the inflammatory response.

The following markers of the inflammatory process were detected:

– TNF-α, a pro-inflammatory cytokine recognized as a central mediator of inflammation;
– IL-10, a cytokine that down-regulates the production of the pro-inflammatory cytokines;
– NO (nitric oxide), a compound generated endogenously as part of the inflammatory response against pathogens, such as bacteria and viruses;
– ROS (reactive oxygen species) secreted upon macrophage activation as part of the host cell defence mechanism.

The results are summarized in Fig. 1.

Schermata 2017-11-17 alle 12.06.06

Ribodiet® treatment induced a significant reduction of over 90% in TNF-α expression compared to the control group. The yeast product also modulated oxidative process markers. After inflammation was induced, the nucleotide complex reduced NO levels by 22.5% and ROS levels by 55%. However, the anti-inflammatory IL-10 cytokine was significantly increased by 23.5% compared to control.
The reported results indicate that Ribodiet®, in the investigated in vitro system, exerts an anti-inflammatory, anti-oxidant and immuno-modulatory action on the cell line stimulated with LPS.
A second pre-clinical study was carried out to evaluate Ribodiet® efficacy in combination with a source of zinc, an ingredient with European Food Safety Authority (EFSA) – approved claims regarding the immune system (Commission Regulation (EU) no.432/2012).
A THP-1 human monocyte line was treated with LPS to induce an inflammatory response at the end of 24 hours of incubation in the culture media (Fig. 2). The zinc source (0.039 mg/ml, Zn at 20%) alone reduced TNF-α levels by 16.8%. However, when combined with Ribodiet® (1.25 mg/ml), the zinc reduced TNF-α expression by 91.6% compared to control.
The results demonstrate that Ribodiet® significantly boosts the anti-inflammatory activity of zinc, thus reducing TNF-α expression induced by LPS.

Schermata 2017-11-17 alle 11.57.51

Safety

Nucleotides are ingredients generally recognized as safe. The use of nucleotides in infant formulas has not been reported to cause an increase in gastro-intestinal intolerance. Furthermore, elderly residents in a long-term care facility were fed enterally for 12 weeks with either a standard formula without any added nucleotides or an immune-modulating formula that contained 1.3 g/l of nucleotides as yeast RNA. No differences related to feeding parameters or safety measures were observed [7].

Applications and usage

In light of the above evidence, Ribodiet® is an interesting ingredient for addition to products targeted at immune system functionality.

Other potential applications are to improve:

– Gut barrier health
– Sports nutrition (recovery after performance)
– Cognition/concentration (lack of synthesis of nucleotides in brain tissue)
– Iron absorption.

The suggested daily dosage is 50-350 mg. The ingredient can be used in both solid and liquid formulations.

References

1. Sánchez-Pozo A, Gil A (2002) Nucleotides as semiessential nutritional components. Br J Nutr 87(Suppl 1):S135–137
2. Koletzko B, Baker S, Cleghorn G et al (2005) Global standard for the composition of infant formula: recommendations of an ESPGHAN coordinated international expert group. J Pediatr Gastroenterol Nutr 41:584–599
3. Jyonouchi H (1994) Nucleotide actions on the humoral immune response. J Nutr 124(1 Suppl):138S–143S
4. Belo A, Marchbank T, Fitzgerald AJ et al (2006) Gastroprotective effects of oral nucleotide administration. Gut 55:165–171
5. Uauy R (1990) Dietary nucleotides and requirements in early life. In: E. Lebenthal (ed) Textbook of gastroenterology and nutrition in infancy. Raven Press, New York, pp 265–280
6. Matsumoto Y, Adjei AA, Yamauchi K et al (1995) Nucleoside-nucleotide mixture increases peripheral neutrophils in cyclophosphamide-induced neutropenic mice. Nutrition 11:296–299
7. Hess JR, Greenberg NA (2012) The role of nucleotides in the immune and gastrointestinal systems: potential clinical applications. Nutr Clin Pract 27:281–294

Schermata 2017-11-17 alle 12.06.51

Proflora™

Apart from its primary function of nutrient absorption, the intestinal mucosa also has an important immune function. This is secondary to antigenic stress induced by food intake (with its microbial load) and by the presence of a varied flora of resident microorganisms (microbiota) which are transient or adhere to the intestinal walls. It is estimated that about 98% of the population has dysbiosis (i.e., a depletion of beneficial microbial species), which leads to various types of bowel dysfunction that may have a negative impact on health.

Proflora™ is an innovative synbiotic consisting of six micro-encapsulated probiotic strains associated with prebiotic fibre, for the natural rebalancing of the ecosystem of different parts of the intestine.


Composition and technical specifications

Proflora™ contains six exclusive, selected probiotic strains (over 2 billion /sachet) with prebiotic fibre FOS (short-chain fructo-oligosaccharides) derived from sugar beet. The probiotic strains (registered in an international culture collection) are in a gastro-protected micro-encapsulated form for maximum probiotic biological activity. It has been reported in vivo that the colonization kinetics of 1×109/CFU of probiotics in a gastro-protected micro-encapsulated form is comparable to that of 5×109/CFU of non-micro-encapsulated probiotics [1, 2].

Proflora™ is allergen free (according to Commission Directive 2007/68/EC), odourless and tasteless. Tables 1 and 2 show the ingredients and the nutritional composition of Proflora.

Schermata 2017-07-20 alle 16.48.18

Schermata 2017-07-20 alle 16.48.30

Mechanism of action

Lactobacillus rhamnosus LR06 and Lactobacillus plantarum LP02 are both able to produce active substances (bacterio cins) that limit the spread of coliform bacteria in various intestinal segments [3], which is the reason why Proflora™ is particularly recommended for patients with frequent episodes of infections of the genitourinary tract due to Escherichia coli. Each of the six microbial species, belonging to the genera Bifidobacterium and Lactobacillus, are gastro-protected and act in synergy for higher efficacy and colonization of the different intestinal segments. The effect is maintained over time because of the strong ability of Lactobacillus salivarius LS03 to adhere to the intestinal mucosa.

FOS in Proflora™ are not hydrolyzed by digestive enzymes or absorbed by the mucosa of the small intestine, and so reach the colon intact where they selectively stimulate the development of probiotic strains present in Proflora™ and beneficial lactobacilli and bifidobacteria in the resident microflora. The FOS positively affect carbohydrate and fat metabolism, improve the function of the intestinal mucosa by increasing the ‘barrier’ effect, and facilitate the absorption of certain minerals, especially calcium and magnesium.

Safety

Proflora™ is patented by Probiotical S.p.A. and is free of all allergens according to current legislation (Annex II Reg. EU 1169/2011): wheat, rye, barley, oats, spelt, kamut and hybridised strains, crustaceans and products based on shellfish, eggs and egg products, fish and seafood, peanuts, soy and soy products, milk and dairy including lactose, nuts, celery, mustard, sesame seeds, lupins, molluscs and products based on molluscs, and sulfur dioxide and sulphites at concentrations above 10 mg/kg or 10 mg/litre.

Proflora™ is recommended for children, the elderly, pregnant and breast-feeding women, and those with food intolerances, food allergies or coeliac disease.

Applications and dosage

Proflora™ can provide healthy support in case of antibiotic and/or laxative administration, diarrhoea, gastrointestinal disorders, digestive difficulties, irritable colon, uro-genital infections, respiratory allergies, adverse reactions to foods, abdominal bloating, meteorism, mental and physical stress and dry eye syndrome [4].

The suggested dosage is one sachet daily (preferably half an hour before meals) for 30 consecutive days. In cases of acute symptoms, one or two sachets should be taken each day for 8–10 days depending on symptoms.

References

1.DelPiano M, CarmagnolaS, Andorno S, Pagliarulo M, Tari R, Mogna L, Strozzi GP, Sforza F, Capurso L (2010) Evaluation of the intestinal colonization by microencapsulated probiotic bacteria in comparison with the same uncoated strains. J Clin Gastroenterol 44:S42–46

2. Del Piano M, Carmagnola S, Ballarè M, Balzarini M, Montino F, Pagliarulo M, Anderloni A, Orsello M, Tari R, Sforza F, Mogna L, Mogna G (2012) Comparison of the kinetics of intestinal colonization by associating 5 probiotic bacteria assumed either in a microencapsulated or in a traditional, uncoated form. J Clin Gastroenterol 46:S85–92

3. Bottazzi V (2009) Il microbiota intestinale. Novara, Mofin Alce Group

4. Chisari G, Chisari EM, Greco C, Madeddu R, Motta M, Chisari CG (2016) Coadministration of Lactobacillus and Bifidobacterium strains in combination with short-chain fructo-oligosaccharides reduces the ocular surface damage caused by dry eye syndrome. Minerva Oftalmol 58(2) 31-38

Schermata 2017-07-20 alle 16.48.43

Oximacro®, a natural cranberry extract with a very high content of proanthocyanidin A

Urinary tract infections (UTIs) are widespread and affect a large portion of the human population. About 150 million people worldwide develop UTIs each year, with high societal costs, and an estimated 40% of women develop at least one UTI during their lifetimes. UTIs refer to the presence of a certain threshold number of bacteria in the urine (usually >105/ml) and consist of cystitis (or lower UTIs, with bacteria in the bladder), urethral syndrome and pyelonephritis (or upper UTIs, with infection of the kidneys). Bacterial cystitis (also called acute cystitis) can occur in women and men, and some people also develop recurrent UTIs with an average of two to three episodes per year. Herpes simplex virus (HSV) infection is lifelong and its spectrum of clinical manifestations is wide, ranging from asymptomatic infection or mild mucocutaneous lesions on the lips, cornea, genitals or skin, up to more severe, and even life-threatening, infections, including encephalitis, neonatal infections, and progressive or visceral disease in immunocompromised hosts. Products extracted from the fruit of American cranberry (Vaccinium macrocarpon Ait., Ericaceae), in different formulations, are rich in compounds that are thought to exert numerous health benefits, such as the prevention of microbial infections and beneficial activity against inflammation [1]. Indeed, the berries of cranberry (Vaccinium macrocarpon Aiton) have been used for hundreds of years as a remedy for diseases of the urinary tract and have attracted attention due to their potential health benefits [2, 3]. The beneficial mechanism of cranberry was historically thought to be due to the fruit’s acids causing a bacteriostatic effect in the urine. However, recently, a group of proanthocyanidins (PACs) with A-type linkages (PAC-A) was isolated from cranberries and shown to exhibit bacterial anti-adhesion activity against both antibiotic-susceptible and antibiotic-resistant strains of uropathogenic P-fimbriated Escherichia coli bacteria, including multidrug-resistant E. coli [4–8]. Central to the efficacy of cranberry extract/juice is the determination of the optimum dose of PAC-A, which is an essential requirement in establishing botanical supplements as viable supports to conventional therapies [9]. Oximacro® is a cranberry extract produced by Biosfered (Turin, Italy) that possesses the highest content of PACs and the highest percentages of PAC-A dimers and trimers available on the market. Oximacro® has been shown to exert both antiviral and UTI prevention capability in in vitro and in preclinical studies.

Composition and technical specifications

Schermata 2017-01-13 alle 09.16.08Oximacro® is produced by Biosfered with a proprietary method of extraction. It is characterized by a high content of PACs (>36% according to the BL-DMAC method; >80% according to the Bates-Smith method; >99% according to the European Pharmacopoeia) and the highest percentages of PAC-A dimers and trimers available on the market (>85%, analysed by HPLC coupled to ESI-tandem mass spectrometry). Oximacro® fractionation (Fig. 1) shows that the extract consists of five main fractions: fraction 1 is mainly composed of delphinidin and cyanidin glycosides, and rutin; fraction 2 contains quercetin and isorhamnetin; fractions 3 and 4 are dominated by several isomers of PAC-A dimers and trimers; and fraction 5 shows no detectable compounds (Fig. 1). In vitro tests show that only the fractions containing PACs possess biological activity [10]. Oximacro® is available as both a powder extract and a fluid extract (Oximacro®-FL). The latter is standardized and titrated to provide 36 mg PAC-A per gram of product. Stress tests with increasing temperature from 22°C to 55°C show that Oximacro® is stable and that the content of PACs is not affected by temperature. The technical specifications of Oximacro® and Oximacro®-FL are reported in Table 1.

Schermata 2017-01-13 alle 09.17.20Efficacy In vitro studies and mechanism of action

We recently reported on the ability of Oximacro® to inhibit herpes simplex type 1 (HSV-1) and type 2 (HSV-2) replication in vitro [9]. Analysis of the mode of action revealed that Oximacro® prevents adsorption of HSV-1 and HSV-2 to target cells. Further mechanistic studies confirmed that Oximacro® and its PACs-A target the viral envelope glycoproteins gD and gB (Fig. 2), thus resulting in the loss of infectivity of HSV particles. Moreover, Oximacro® completely retains its anti-HSV activity even at acidic pHs (3.0 and 4.0) and in the presence of 10% human serum proteins, conditions that mimic the physiological properties of the vagina (a potential therapeutic location for Oximacro®). Figure 2 shows the interaction between the A-type PACs present in Oximacro® and HSV envelope glycoproteins.

Clinical studies

Oximacro® was shown to be effective in the prevention of UTIs and support for patients with UTIs. A balanced group of female (ranging from 19 to over 51 years of age) and male volunteers (over 51 years of age) was divided into two groups. The experimental group received one capsule containing Oximacro® (36 mg PACs-A) twice per day (morning and evening) for 7 days, while the placebo group was given the same number of capsules with no PACs. After 7 days of administration, a significant difference was found between the placebo and Oximacro® groups for both females (p<0.001; N=60) and males (p=0.016; N=10) concerning reduction of UTI symptoms (e.g., dysuria, frequency, cloudy urine and occasionally haematuria). Furthermore, CFU/ml counts from the urocultures were almost cleared and showed a highly significant difference between the experimental and the placebo groups (p<0.001) [10].

Safety

Oximacro® is produced according to strict procedures and the safety of the product is guaranteed through the use of top technology detection systems (microbiological, chemical and molecular). The product has been recently approved by the US FDA as a New Dietary Ingredient and is Generally Recognized as Safe (GRAS).

Application and use

Careful determination of the total PAC content using the BL-DMAC method and the authentication of PACs-A with mass spectrometry of Oximacro® are necessary to prepare effective doses for antiviral efficacy and UTI prevention. Our published findings indicate Oximacro® is an attractive candidate for the development of novel microbicides of natural origin for the prevention of HSV infections and UTIs and for active support for patients with UTIs. The recommended dosage of Oximacro® is 120 mg/dose, which corresponds to 36 mg PAC-A. This dosage has been demonstrated to be effective when administered twice a day for at least 7 days.

References

1. Blumberg JB, Camesano TA, Cassidy A, Kris-Etherton P, Howell A, Manach C, Ostertag LM, Sies H, Skulas-Ray A, Vita JA (2013) Cranberries and their bioactive constituents in human health. Adv Nutr 4:618–632

2. Jass J, Reid G (2009) Effect of cranberry drink on bacterial adhesion in vitro and vaginal microbiota in healthy females. Can J Urol 16:4901–4907

3. Jepson RG, Williams G, Craig JC (2013) Cranberries for preventing urinary tract infections. Sao Paulo Med J 131:363

4. Gupta K, Chou M, Howell A, Wobbe C, Grady R, Stapleton A (2007) Cranberry products inhibit adherence of P-fimbriated Escherichia coli to primary cultured bladder and vaginal epithelial cells. J Urol 177:2357–2360

5. Stapleton AE, Dziura J, Hooton TM, Cox ME, Yarova-Yarovaya Y, Chen S, Gupta K (2012) Recurrent urinary tract infection and urinary Escherichia coli in women ingesting cranberry juice daily: a randomized controlled trial. Mayo Clinic Proc 87:143–150

6. Howell AB, Reed JD, Krueger CG, Winterbottom R, Cunningham DG, Leahy M (2005) A-type cranberry proanthocyanidins and uropathogenic bacterial anti-adhesion activity. Phytochemistry 66:2281–2291

7. Howell AB, Botto H, Combescure C, Blanc-Potard AB, Gausa L, Matsumoto T, Tenke P, Sotto A, Lavigne JP (2010) Dosage effect on uropathogenic Escherichia coli anti-adhesion activity in urine following consumption of cranberry powder standardized for proanthocyanidin content: a multicentric randomized double blind study. BMC Infect Dis 10:94

8. Gurley BJ (2011) Cranberries as antibiotics? Arch Int Med 171:1279–1280

9. Terlizzi ME, Occhipinti A, Luganini A, Maffei ME, Gribaudo G (2016) Inhibition of herpes simplex type 1 and type 2 infections by Oximacro®, a cranberry extract with a high content of A-type proanthocyanidins (PACs-A). Antiviral Res 132:154–164

10. Occhipinti A, Germano A, Maffei ME (2016) Prevention of urinary tract infections with Oximacro®, a cranberry extract with a high content of type-A proanthocyanidins (PACs-A): a pre-clinical double-blind controlled study. Urol J 13:2640–2649

Schermata 2017-01-13 alle 09.17.36

Use of Microorganisms for Carotenoids Delivery

The CaroDel project lasted 2 years, ended in January 2016 and received €2 million in financial support from the EU. The aims of the project were to develop an efficient oral delivery strategy for highly active carotenoids and to evaluate the potential beneficial (probiotic) effect on health of a Bacillus delivery vehicle, with the ultimate aim of improving biomarkers associated with cardiovascular disease. The CaroDel consortium of eight partners (including five SMEs) from six European countries brought together complementary expertise and was coordinated by ProDigest BVBA, Belgium, a spin-off of Ghent University.

Characterized by their orange, yellow and red pigments, carotenoids are mainly synthesized in plants but also have been isolated from other organisms, including some bacteria and fungi. Humans, like other animals, are unable to synthesize carotenoids but absorb them from their diet. Over 700 different carotenoids have been described, but only a few have been studied in relation to their impact on human physiology. Carotenoids act as antioxidants within the body, protecting against cellular damage, ageing and even some chronic diseases (including cardiovascular disease). Plant-derived carotenoids are widely associated with cardiovascular benefits, yet their low stability and poor bioavailability (absorption in the human body) hamper successful product development.

Several bacteria from the Bacillus species were shown to produce carotenoids which are highly stable throughout the gut and show higher antioxidant activity and bioavailability than common dietary carotenoids.

These discoveries provided strong and compelling reasons for supporting further development and commercialization of these bacteria-derived carotenoids.

By combining in vitro gut models and in vivo animal studies, the CaroDel project developed an efficient oral delivery system for these highly active carotenoids, in the form of Bacillus spores which can also exert probiotic effects.

CaroDel aimed to valorize the results of the earlier FP7 COLORSPORE project, in which initial isolation and characterization of Bacillus strains producing gastric-stable carotenoids was carried out. As particular Bacillus carotenoids were shown to have better stability in the gastrointestinal tract (GIT), antioxidant activity and bioavailability than common dietary carotenoids, the COLORSPORE project provided strong and compelling evidence to support further development and commercialization of these bacteria-derived carotenoids.

The CaroDel project therefore focused on developing an efficient oral delivery strategy for these highly active carotenoids, and on evaluating the potential direct (probiotic) beneficial effects on health of the Bacillus delivery vehicle, with the ultimate goal of improving biomarkers associated with cardiovascular disease (CVD).

The study compared the effective delivery of the carotenoids in the human body following administration as (i) vegetative Bacillus cells, (ii) Bacillus spores or (iii) extracted bacterial carotenoids. In parallel, the ability of the Bacillus strain to exert bona fide effects (i.e. effects on the host microbiota, metabolism and immunity) was investigated using in vitro gut models and in vivo rat studies. Based on the results, the best delivery strategy was selected and validated in a human study, in which carotenoid bioavailability was assessed as well as endpoints related to CVD biomarkers and potential probiotic activity. In combination with a full safety assessment, a proof-of-concept production strategy and an exploitation plan, the scientific evidence compiled in this project provides a framework for efficient further commercialization of a well-characterized Bacillus carotenoid product.

The main objectives of the project were:

1. To optimize the pilot scale production process of the strain and its carotenoids;

2. To determine the best delivery approach for the carotenoids in the human GIT by means of validated in vitro gut models and in vivo rat studies;

3. To determine the effect of the carotenoids and the Bacillus strain on host endpoints and the composition/activity of the gut microbial community;

4. To demonstrate that the selected Bacillus strain is safe for human consumption, which will allow future registration under quality and patient safety (QPS) regulations;

5. To model the scale-up of the production process in order to bring the product to market;

6. To evaluate the effect of the Bacillus strain in humans in relation to CVD biomarkers, modulation of the intestinal environment and host health endpoints; and

7. To generate the necessary knowledge to develop an intellectual property (IP) protection strategy and a business model to commercialize and sustain the product.

To address these objectives, the CaroDel project was divided in two phases: Phase 1 (selection of the best formulation) and Phase 2 (translational phase for taking the product to market), by combining five research work packages (WPs).

During Phase 1 of the CaroDel project, WP2, WP3 and WP4 were run in parallel (WP1 was a management work package).

WP2 (‘Production’) was designed to optimize the production of the different carotenoid formulations that were tested in the other research WPs.

WP3 (‘Evaluation of the carotenoids’ delivery and effect in the GIT’) was designed to elucidate – through in vitro research and animal studies – the intestinal fate and bioavailability of the carotenoids when administered as purified carotenoids extracted from the Bacillus strain, when contained in the vegetative cells, or when contained in the spores of the strain. The effects of the Bacillus strain on the intestinal environment were also assessed upon administration as spores or vegetative cells. The aim of this part of the project was to determine the optimal formulation to be used in the human trial, in terms of carotenoid bioavailability profile and, if possible, potential to modulate the intestinal environment.

In parallel, in WP4 (‘Evaluation of the safety of the carotenoid-producing Bacillus strain’), all the steps necessary to demonstrate the safety of the carotenoids and the carotenoid-producing strain in relation to novel food and QPS regulations, respectively (e.g. toxicology, antibiotic resistance, genome sequencing, and characterization of the carotenoids) were performed.

In Phase 2 of the project, the best formulation identified in WP3 and supported by the data on safety determined in WP4 was tested in a human study within WP5 (‘Human intervention trial’). Analyses were conducted by the different partners to determine the bioavailability of the ingested carotenoids, the effects on the human host in terms of CVD biomarkers, and the impact on gastrointestinal microbial composition and metabolic activity.

Finally, in WP6 (‘Regulatory and life cycle assessment’), the consortium defined the regulatory strategy, evaluated consumer perception of the novel concept, identified the necessary additional R&D steps and, finally, prepared a life cycle assessment plan. In WP7, an IP strategy was developed to protect the outcomes of the project and exploit the results.

In summary, successful achievement of the above objectives has brought the Bacillus carotenoid product close to commercialization and exploitation as a unique health ingredient.

Main results achieved 

In year 1, the CaroDel consortium devoted its efforts to the production of the different formulations of carotenoids and the carotenoid-producing Bacillus strain, and to the determination of the optimal formulation to be used in the human trial. As the carotenoid-producing Bacillus strain did not encounter viability problems, the extracted bacterial carotenoids were excluded from the trial. In order to decide whether the spores or the vegetative cells of the Bacillus strain were the best option, the bioavailability and probiotic activity of both formulations were compared using in vitro gut models and in vivo animal studies. As both spores and vegetative cells exhibited probiotic activity, selection of the optimal formulation was primarily based on the best carotenoid bioavailability profile. It could be concluded that an optimized spore formulation was the better formulation in terms of bioavailability. Therefore, it was decided to use this optimized formulation of the Bacillus spores in the human trial.

In silico genomic screening and in vitro toxicity assessments suggest that the specific Bacillus spores used in this product are safe for human consumption and have an even better safety profile than other Bacilli.

Additionally, in vivo safety assays conducted in mice showed no signs of toxicity. Furthermore, the safety of oral intake was confirmed in two phase I safety studies conducted in healthy individuals, who were and were not overweight. No treatment-related adverse effects occurred with repeated intake over 2 weeks.

In year 2, a 6-week phase II efficacy study was performed in healthy, but overweight individuals. The study was designed as a randomized, placebo-controlled, double-blind, parallel study. Carotenoid analysis showed accumulation of bacterial carotenoids in the plasma of individuals treated with the Bacillus spores throughout the study period. This indicated that the bacterial carotenoids were absorbed in the human GIT and can therefore exert a systemic effect. Additionally, analysis of faecal samples showed that the bacterial strain was able to survive transit through the human GIT, potentially exerting probiotic effects. Indeed, beneficial effects were observed on some biological endpoints after intake of the spore formulation. As this was the first time that such effects were seen in humans, the results provide compelling evidence for the further development and commercialization of the CaroDel product.

A life cycle assessment showed that the CaroDel product would be environmentally competitive when compared to other products with a claimed positive effect on CVD biomarkers, making it a sustainable business. The most environmentally friendly delivery option would be to sell the product as a supplement (in capsules). Moreover, assessment of market trends, current heart health products, consumer attitudes and competitive products, showed that the CaroDel product could be successfully introduced on to the market.

CaroDel was designed to fill existing gaps between the discovery phase and the translation of those findings into a marketable product. The probiotic activity of the Bacillus strain and its effect on cardiovascular endpoints were investigated in parallel with an in vivo study of the absorption, safety and mechanism of action of the biological compound. The result was CaroDel, a new health ingredient that is different from all probiotics and carotenoids currently on the market.

ProDigest in a nutshell

ProDigest is a product leader in the development of unique laboratory models of the human and animal gastrointestinal tract. Without the need for animal or human studies, these models allow a unique insight into gut processes associated with the intestinal fate, metabolism and bioavailability of active ingredients and facilitate study of the complete gut microbiota under controlled conditions and its link with human and animal health. ProDigest is globally active as a service provider for food and pharmaceutical companies and since 2014 also installs its technology in selected R&D facilities around the world. Furthermore, ProDigest has set up a number of in-house product development projects related to microbial biotechnology and gut microbiota management and the development of novel biotherapeutics.

For information
tel +32 9 241 1190
info@prodigest.eu
www.prodigest.eu; www.carodel.eu

Nutrafoods 3 – 2016

ProGo™ salmon protein hydrolysate

The use of salmon offcuts from the salmon fillet industry to produce human grade nutritional products is an aspirational goal of sustainability for the Norwegian salmon aquaculture industry. Hofseth BioCare has developed and commercialized a novel, patented process to produce a very palatable salmon protein hydrolysate powder, ProGo™, that received the Ingredient of the Year for Weight Management NutraIngredents Award at Vitafoods 2016.

Obesity and situational anaemia are both major global health problems with over 3 million adults dying each year from obesity-related complications and over 1 billion humans suffering from situational anaemia, where haemoglobin levels are low. Overweight and obese individuals with abnormal or excessive fat accumulation are at increased risk of type 2 diabetes, cardiovascular disease and metabolic disorders, particularly cancer. Situational anaemia in infants leads to developmental problems and in adults to low energy and poor mental focus, reducing well-being and quality of life.

Most efforts to overcome obesity and anaemia have focused on developing anti-obesity and iron-containing medicines such as hunger suppressants and iron fumarate supplements. However, these preparations are often associated with serious side effects. Functional foods are considered safe for general use in the control and amelioration of chronic health conditions such as obesity and anaemia and are seeing continuous validation through better clinical studies.


Composition 
and technical specifications

ProGo™ is a salmon protein hydrolysate powder obtained from the head and backbone offcuts left after salmon filleting, which are then subjected to gentle processing using only enzymatic hydrolysis, followed by separation of the water-soluble protein phase and spray-drying the concentrate. ProGo™ contains 621 distinct oligopeptides and peptides, 100% of which have a molecular weight less than 3,000 Da, as determined by MALDI-TOF (matrix assisted laser desorption/ ionization-time of flight) analysis. Gel permeation chromatography analysis further showed that 50% of these peptides have a molecular weight of less than 1,000 Da. ProGo™ is routinely tested for the absence of persistant organic chemicals, pesticides, heavy metals and microbial contamination. The technical specifications and amino acid composition of ProGo™ are reported in tables 1 and 2, respectively.

Schermata 2016-12-21 alle 11.46.05

Schermata 2016-12-21 alle 11.46.31

Preclinical studies

Our earliest preclinical studies were carried out on elderly pointer dogs to assess the digestibility and palatability of ProGo™ as a pet food supplement.

ProGo™ has excellent digestibility (over 98%) and showed a remarkable array of bioactivity as demonstrated by serum level changes in important biomarkers such as C-reactive protein, ferritin/transferrin and myeloperoxidase. These studies were followed up with our TIM-1 study to measure the in vitro absorption rates of ProGo™ in a model human digestive system. The results showed that ProGo™ has the fastest uptake among protein powders currently commercially available [1]. These properties are being further exploited to develop a series of medical food supplements for use in a range of patients, from infants to geriatric subjects, using data from human clinical studies conducted at the Norwegian School of Sport Sciences in Oslo.

A pre-clinical proof-of-concept study has recently been published [2] that shows a statistically significant increase in serum haemoglobin levels (from 10.04 g/dl to 11.5 g/dl) following 6 weeks of supplementation with 16 g/day of ProGo™ in 24 iron deficient recipients. A larger clinical study with a lower, optimized dose of ProGo™ that will also measure increases in energy, vitality and well-being is ongoing.

Clinical studies and mechanism of action

A clinical study was recently published on the efficacy of ProGo™ to reduce body mass index (BMI) and the proposed mechanisms of action of this unique bioactive [3]. The results show that the BMI of 24 overweight subjects was significantly (p=0.005) reduced by approximately 6% after 6 weeks of daily supplementation with 16 g/day of ProGo™ salmon protein hydrolysate powder. Further results showed that metabolism-related, circulatory biomarkers – bile acid (p=0.027), adiponectin (n.s.), lipoprotein lipase (LPL) mass in preheparin serum (Pr-LPL Mass, n.s.) and interleukin-6 (p=0.038) – showed positive improvements, indicating that ProGo™ might be reducing BMI via interaction with the metabolic pathway.

Figure 1 compares the effects obtained with ProGo™ and whey protein isolate (WPI) on BMI and biomarkers. Results showed that 83% of subjects in the ProGo™ treated group showed a significant decrease in BMI, while only 25% in the WPI-treated group showed only a modest decrease. Moreover, WPI at the dose tested did not modify the examined biomarkers. This implies that the decrease in BMI obtained with ProGo™ may be related to modulation of inflammation and metabolic pathways, possibly via the presence of bioactive peptides in the salmon protein hydrolysate. Our current results clearly show that dietary supplementation with 16 g of ProGo™ per day decreases BMI in overweight individuals after only 6 weeks of treatment and also positively affects circulatory biomarkers associated with inflammation and metabolism.

Schermata 2016-12-21 alle 11.46.18

Safety

The literature has reported that protein hydrolysate powders often contain some ACE-inhibiting peptides that can cause precipitous and dangerous drops in blood pressure. Consequently, the presence of ACE-inhibiting peptides in ProGo™ was investigated using the ACE kit-WST (Dojindo Laboratories, Munich, Germany). The results showed that ProGo™ contained very little blood-pressure lowering ACE-inhibiting peptides and was thus GRAS-approved for use at up to 90 g/day. No adverse events related to the administration of ProGo™ have been reported in any clinical study.

Formulations 

ProGo™
salmon protein hydrolysate powder – over 93% pure protein

Lean Protein
salmon protein hydrolysate powder – 16 g/sachet for BMI reduction

Endurance Protein Tablets
salmon protein hydrolysate tablets – 1,000 mg per tablet 4 g/day for hemoglobin increase

Endurance Protein Drink
apricot and lemongrass flavoured sachets – 16 g/sachet

Endurance Protein Drink
blackcurrant and passionfruit flavoured sachets – 16 g/sachet

Applications and dosage 

Safety and clinical studies support the use of ProGo™ either as an unflavoured powder or in ready-to-drink flavoured formulations for a sustained reduction in weight and BMI. It can be recommended as a complementary tool in the overall management of weight loss (as ProGo™ flavoured and unflavoured powder administered at 16 g/day) as well as to improve well-being by nutritionally increasing iron (taken as four Endurance tablets/day) over 4–8 weeks of treatment.

References

1. Framroze B, Savard P, Gagnon D, Richard V, Gauthier SF (2014) Comparison of nitrogen bioaccessibility from salmon and whey protein hydrolysates using a human gastrointestinal model (TIM-1). Funct Foods Health Dis 4:222–231

2. Framroze B, Vekariya S, Swaroop D (2015) A placebo-controlled study of the impact of dietary salmon protein hydrolysate supplementation in increasing ferritin and hemoglobin levels in iron-deficient anemic subjects. J Nutr Food Sci 5:379

3. Framroze B, Vekariya S, Swaroop D (2016) A placebo-controlled, randomized study on the impact of dietary salmon protein hydrolysate supplementation on body mass index in overweight human subjects. J Obes Weight Loss Ther 6:296

Schermata 2016-12-21 alle 11.46.42

Nutrafoods 3 – 2016

Memophenol™

Cognitive function and brain health are very important: students studying for exams have to learn and retain a huge amount of information in a short period of time, active adults are constantly stretching their brains to be ever more productive and efficient, and older people, although affected by normal brain aging, want to stay mentally sharp so as to live longer and healthier lives.

Nutrition is a key factor for improving memory and preventing age-related cognitive decline. In the past decade, epidemiological studies have revealed that individuals consuming high amounts of flavonoids, and especially monomers of catechins and anthocyanins found in berries, showed better cognitive function than those with a low flavonoid intake [1, 2]. Catechins, which belong to the flavan-3-ols family, are present in high amounts in grapes, while anthocyanins are particularly found in blueberries. Both grape and blueberry intake has been shown to be positively linked to brain function. However, as the consumption of fruit and vegetables is steadily decreasing, providing food supplementation is of special interest.

Activ’Inside has developed Memophenol™ which combines French grapes (Vitis vinifera L.) rich in catechins and Canadian wild blueberry (Vaccinium angustifolium Aiton) extracts. This synergistic patent-pending formulation has been developed after 4 years of research and has clinically demonstrated ability to promote learning and memory, essential for students and active adults, and to prevent cognitive decline in older people. The main research results are summarized below.

Schermata 2016-10-25 alle 09.29.54Composition and technical specification

Memophenol™ is a blend of French grape (Vitis vinifera L.) extract and Canadian wild blueberry (Vaccinium angustifolium Aiton) extract. Memophenol™ is standardized in specific polyphenols such as flavonoids and particularly monomers of flavanols (since monomers display better bioavailability than oligomers and polymers), anthocyanins, flavonols and phenolic acids. The chemical and technical specifications are given in Table 1.

Bioavailability

Nineteen phenolic metabolites were identified in the plasma of mice administered Memophenol™, in particular catechins and their derivatives [3]. This suggests that Memophenol™ compounds are absorbed from the digestive tract before being metabolized. In a clinical study, different polyphenols and their metabolites were found in urine, with flavanols and metabolites again the main molecules identified (publication in prep.). Another important finding was the presence of polyphenols in mouse brain tissue. Other studies have demonstrated the presence of some polyphenols, including flavanol monomers and metabolites, and some phenolic acids in the brain confirming that they can cross the blood–brain barrier to act more directly on brain function [4, 5]. When the long-term effect of Memophenol™ was assessed in mice, the plasma concentration of polyphenols showed a twofold increase following chronic compared to acute administration, suggesting Memophenol™ shows better activity with long-term intake [3].

A patented synergy

An in vivo study conducted in mice highlighted the synergistic effect on bioavailability of the association of the grape and blueberry extracts contained in   Memophenol™. Compared to the administration of blueberry extract alone, administration of the blend caused a 3.0–5.5-fold increase in the plasma concentration of polyphenols  and a 2.9–6.3-fold decrease in the faecal excretion of blueberry compounds. Similarly, the association of grape and blueberry extracts increased polyphenol plasma levels after chronic consumption (15 days) compared to grape or blueberry alone (this increase was not observed following an acute dose) [3]. Other synergistic effects have been observed at physiological concentrations, especially on neuronal protection against H2O2 neurotoxicity in vitro. Although specific polyphenols from grape and blueberry extracts tested alone were unable to prevent cell death, the polyphenol mixture in Memophenol™ completely protected brain cells. Memophenol™ and its active compounds have been patented following demonstration of these synergistic effects on bioavailability and neuronal cell protection.

Mechanism of action

The mechanism of action of Memophenol™ has been carefully studied in order to better understand potential target consumers of the supplement, and which associations with other ingredients improve the effect on cognitive function.

First, to characterize the neurobiological processes underlying the effect of Memophenol™, the mRNA levels of proteins involved in synaptic plasticity were measured in an in vivo study. An increase was found in the mRNA levels of neurotrophin nerve growth factor in the hippocampus – a key region of the brain involved in memory and particularly affected in aging – in both adult and middle-aged mice supplemented with Memophenol™. Moreover, it was shown that the decrease in hippocampal calmodulin-dependant protein kinase II (CaMKII) mRNA expression in middle-aged mice can be reversed by supplementation with Memophenol™ [6] (Fig. 1). These results suggest that Memophenol™ can enhance synaptic plasticity in adult and older mice, suggesting that the same mechanism is present in humans of all ages.

Schermata 2016-10-25 alle 09.30.17

Second, Memophenol™ has been shown to improve antioxidant status in both an in vitro study on hippocampal cells and in in vivo studies. These results confirm the reduction in markers of oxidative stress in the brain previously observed in adult and aged mice after grape seed extract supplementation [7]. Oxidative stress caused by overproduction of reactive oxygen species (ROS) is involved in cell aging and in neuronal cell aging in the brain. The neuroprotective effect of Memophenol™ in the brain may be explained by its antioxidant activity.

Third, an increase in specific gene expression involved in vascularization has also been observed in an in vivo study. This has been correlated with the demonstrated vasodilating effect of grapes.

Last, Memophenol™ has been found to impact hippocampal neurogenesis in mice brain (publication in prep.) [4].

Efficacy

Preclinical studies

Schermata 2016-10-25 alle 09.30.29Improvements in learning and memory were first established through in vivo studies in mice. Spatial learning and memory have been assessed in a water-maze apparatus. Adult mice were compared to middle-aged mice in order to evaluate a memory deficit. Mice were supplemented with Memophenol™ for 8 weeks. Body weight and food intake were not affected by supplementation. During the learning phase of the water maze task, middle-aged mice performed less well than adult mice, indicating that aging induces a decrease in performance. However, after supplementation with Memophenol™, mice displayed better ability to learn than control mice irrespective of age. In particular, supplemented mice used more spatial strategies to solve the task than control mice, indicating better functioning of the hippocampus (Fig. 2). Memory evaluation 72 h after the learning session also revealed a beneficial effect of Memophenol™ on spatial long-term memory [6].

Clinical studies

The beneficial effect of berries on cognitive function has been described in epidemiological and clinical studies during the last few decades.

A clinical trial with over 200 subjects was conducted to test the effect of Memophenol™ on learning and memory. Study collaborators included the University of Bordeaux in France and the Laval University in Canada. As product efficacy on cognitive function is best assessed by measuring cognitive decline, healthy adults aged 60–70 years old were recruited to a bi-centric, double-blind, placebo-controlled intervention. The treated group received 300 mg of Memophenol™ twice a day for 6 months. Validated tests (including CANTAB tests from Cambridge Cognition) were performed to assess memory and cognition before and after 6 months of supplementation. Various biological tests were conducted and the results analysed in order to determine if there was a cause–effect relationship and to determine the safety of the product. The study was registered on ClinicalTrials.gov (Identifier: NCT02063646).

Recent results obtained from this clinical study indicate a positive effect of Memophenol™ on learning and memory. In particular, an improvement in short-term memory was observed, with the Memophenol™ group forgetting fewer words than the placebo group (in the immediate verbal recall test). Memophenol™ was also shown to enhance long-term memory, the supplemented group making two times less errors in a spatial task than the placebo group in subjects with the worst performance at baseline. This result can be quantified as a improvement of 10 years of cognitive age (comparison with normative data). The product also improved some biological parameters. The results of the study are being prepared for publication.

Safety and tolerance

The clinical trial showed the product was safe and had excellent tolerability as there were no serious adverse events at the recommended dosage. Blood analyses were also normal after 6 months of supplementation. Moreover, no adverse events were observed in any in vivo study. An in vivo tolerance study also demonstrated that long-term and chronic consumption of grape and blueberry extracts was not associated with renal or hepatic failure and can therefore be considered to be safe [8]. Finally, the longevity of aged mice was improved when they were supplemented with Memophenol™ (publication in prep.) [4].

Applications and dosage

Memophenol™ can be used in a variety of supplement formulations, alone or in combination with other ingredients to promote learning and memory in students, to support brain function in active adults, and to prevent cognitive decline in older individuals. The recommended dose is 300 mg once or twice a day, for at least 3 months.

References

1. Letenneur L, Proust-Lima C, Le Gouge A, Dartigues JF, Barberger-Gateau P (2007) Flavonoid intake and cognitive decline over a 10-year period. Am J Epidemiol 165:1364–1371
2. Kesse-Guyot E, Fezeu L, Andreeva VA, Touvier M, Scalbert A, Hercberg S, et al. (2012) Total and specific polyphenol intakes in midlife are associated with cognitive function measured 13 years later. J Nutr 142:76–83
3. Dudonne S, Dal-Pan A, Dube P, Varin TV, Calon F, Desjardins Y (2016) Potentiation of the bioavailability of blueberry phenolic compounds by co-ingested grape phenolic compounds in mice, revealed by targeted metabolomic profiling in plasma and feces. Food Funct 7:3421–3430
4. Bensalem J (2014) Effets des polyphénols de baies sur le déclin cognitif lié au vieillissement chez la souris. Thesis, University of Bordeaux
5. Bensalem J, Dal-Pan A, Gillard E, Calon F, Pallet V (2016) Protective effects of berry polyphenols against age-related cognitive impairment. Nutr Aging 3:89–106
6. Bensalem J, Servant L, Alfos S, Gaudout D, Layé S, Lafenetre P, et al. (2016) Dietary polyphenol supplementation prevents alterations of spatial navigation in middle-aged mice. Front Behav Neurosci 10:9
7. Asha Devi S, Sagar Chandrasekar BK, Manjula KR, Ishii N (2011) Grape seed proanthocyanidin lowers brain oxidative stress in adult and middle-aged rats. Exp Gerontol 46:958–964
8. Martineau A, Leray V, Lepoudere A, Blanchard G, Bensalem J, Gaudout D, et al. (2016) A mixed grape and blueberry extract is safe for dogs to consume. BMC Vet Res 12:162

Schermata 2016-10-25 alle 09.30.53

da Nutrafoods 3 – 2016

MERIVA®, building up

Over the past two decades more than 7,000 articles have discussed the molecular basis of the properties of curcumin, demonstrating that it is the most flexible and scientifically explored polyphenol. Meriva®, a formulation of curcumin with a soy lecithin delivery system, allows the health benefits of curcumin to be utilized to improve low grade chronic systemic inflammation. Supported by the findings of 24 clinical studies covering 10 health conditions, Meriva® can be widely used so that the biological benefits of turmeric can be enjoyed more extensively.

This presentation will mainly focus on the recently documented effects of Meriva® on skeletal and muscular function in sport, ageing and disease.

Composition and technical specifications

Given the poor bioavailability of curcuminoids, Indena has formulated Meriva® using Phytosome® technology to optimize absorption. Meriva® is a natural lecithin delivery system standardized to contain 18–22% of total curcuminoids. The technical specifications are reported in Table 1.

Schermata 2016-07-21 alle 10.43.20

Bioavailability

Meriva® pharmacokinetic studies in rats [1] and healthy volunteers [2] demonstrate a significant improvement in the absorption of curcuminoids. In particular, human plasma Cmax and the AUC of curcuminoids are nearly 30 times higher than those following consumption of a non-formulated turmeric extract. Remarkably, the phospholipid formulation allows better absorption of demethoxylated curcuminoids compared to curcumin, making the plasma profile of Meriva® unique.

The curcumin plasma levels obtained using Meriva® have been confirmed recently in an independent study [3].

Clinical studies

Curcumin is a pleiotropic polyphenol which can interact with many enzymes, receptors and transcription factors [4]. Meriva® harnesses this property for use in health applications for several conditions, namely eye health, prostate aging, neurological discomfort, and skeletal and muscular dysfunction. Meriva® was demonstrated to be useful in the management of osteoarthritis (OA), showing a decreased WOMAC score (the international scale for measuring exercise-related pain, stiffness and physical function) compared to best standard management. After 2 months of Meriva® daily supplementation, the median WOMAC score had decreased from 83.4 at baseline (vs 80.6 in controls) to 41.1 (vs 75.2 in controls) and had declined at 3 months to 34.8 (vs 78.8 in controls) (p<0.05). In addition, the Social and Emotional Index score decreased by two thirds, indicating a general improvement in quality of life [5, 6]. Another trial has been conducted to compare a widely used combination, glucosamine and chondroitin sulphate, with Meriva® and glucosamine [7]. Although the lack of randomization, the relatively short follow-up time and the overall limited number of subjects should be taken into account, this third study also supports the use of Meriva® in OA by suggesting that this ingredient can be considered an effective add-on treatment to glucosamine.

It is also important for patients to have good flexibility and muscle resistance to combat OA. Maintaining good muscle strength and tone is important for those who want to prevent joint aging and for those participating in sport. In this context, the benefits of Meriva® have also been evaluated for alleviating muscle soreness in runners [8] and recreational cyclists [3]. These two clinical sports nutrition studies both highlight the benefits of Meriva® on clinical symptoms and interleukin levels. In addition to the reductions in oxidative stress and C-reactive protein demonstrated in the previous OA trials, the overall changes in biomarkers observed supports a positive effect of plasma and tissue levels of the curcuminoids in Meriva® on the inflammatory response process.

Last but not least, for the first time the efficacy of the curcuminoids in Meriva® to counteract muscle wastage in healthy aging subjects has been confirmed [9]. Under normal conditions, muscle mass is maintained by a balance between protein synthesis and degradation, so muscle wasting can occur when this balance is perturbed. This is particularly true during the aging process when a physiological loss of skeletal muscle mass is observed (0.5–1% loss per year after 50 years of age). The registry study shows that the addition of Meriva®, combined or not with other nutritional supplements and standard management (balanced diet and exercise), helps to improve strength and physical performance measured using hand grip (33.9 ± 1.8 kg after supplementation vs 31.8 ± 2.0 kg in controls), weight lifting (16 ± 2 after supplementation vs 11 ± 1 in controls), time/distance before feeling tired after cycling, walking and climbing stairs, and general fitness (p<0.05 with respect to baseline and p<0.05 for supplementation compared with standard management for all parameters).

What else?

New exciting data on Meriva® will be available very soon to highlight the potential of curcuminoids in neuroprotection, liver health and cardiovascular management.

Safety and tolerance

The safety and tolerability of Meriva® are supported by long term toxicological animal studies and the lack of significant side effects has been confirmed in an extended 18 months clinical study. Furthermore, in controlled clinical studies, more than 800 subjects have taken Meriva® without reporting any significant adverse events.

Applications and dosage

Meriva® is a light yellow powder which can be used in several formulations such as tablets, capsules, powders and gel. The recommended dose is 500 mg twice/day.

References

1. Marcylo TH et al (2007) Comparison of systemic availability of curcumin with that of curcumin formulated with phosphatidylcholine. Cancer Chemother Pharmacol 60:171–177

2. Cuomo J et al (2011) Comparative absorption of a standardized curcuminoid mixture and its lecithin formulation. J Nat Prod 74:664–669

3. Sciberras JN et al (2015) The effect of turmeric (curcumin) supplementation on cytokine and inflammatory marker responses following 2 hours of endurance cycling. J Int Soc Sports Nutr 12:5–14

4. He Y et al (2015) Curcumin, inflammation and chronic diseases: how are they linked? Molecules 20:9183–9213

5. Belcaro G et al (2010) Efficacy and safety of Meriva®, a curcumin-phosphatidylcholine complex, during extended administration in osteoarthritis patients. Altern Med Rev 15:337–344

6. Belcaro G et al (2010) Product-evaluation registry of Meriva®, a curcumin-phosphatidylcholine complex, for the complementary management of osteoarthritis. Panminerva Med 52:55–62

7. Belcaro G et al (2014) Meriva®+glucosamine versus condroitin+ glucosamine in patients with knee osteoarthritis: an observational study. Eur Rev Med Pharmacol Sci 18:3959–3963

8. Drobnic F et al (2014) Reduction of delayed onset muscle soreness by a novel curcumin delivery system (Meriva®): a randomised, placebo-controlled trial. J Int Soc Sports Nutr 11:31-40

9. Franceschi F et al (2016) A novel phospholipid delivery system of curcumin (Meriva®) preserves muscular mass in healthy aging subjects. Eur Rev Med Pharmacol 20:762–766

Schermata 2016-07-21 alle 10.43.35

da Nutrafoods 15(2) 2016

A natural and effective solution to boost immune health

Modern life can cause us excessive stress and fatigue which can weaken our natural defences and leave us more sensitive to pathogens. The immune system changes as we age and so immune support also needs to evolve throughout life. While age is the first to impact on our body’s natural defences, a number of other factors, such as chronic stress, intense training or lack of sleep, can put someone at risk of increased susceptibility to infections such as the flu, common colds, gastroenteritis, etc. The common cold, for example, is one of the leading causes of days missed from school and of work absenteeism, and its economic impact on society is estimated at tens of billions of Euros. Although most adults catch a cold only two to four times a year, children and the elderly, who are more vulnerable, are likely to catch a cold up to 10 times a year. Consumers, who are aware that maintaining a strong immune system at all times is key to staying healthy and performing well, are therefore looking for solutions to enhance their natural defences. Nowadays, more and more people are familiar with the immune health benefits of ingredients like minerals, vitamins and specific strains of probiotics, defined by the World Health Organization as ‘live microorganisms which, when administered in adequate amounts, confer a health benefit on the host’.

Committed to providing the best natural and effective solutions to boost the immune system, Lesaffre Human Care partnered with academic researchers to develop LifeinU™ Bacillus subtilis CU1, a unique probiotic ingredient adapted for the whole family* and catering for those looking for natural long-term immune support.

Composition and technical specification

LifeinU™ Bacillus subtilis CU1 is a proprietary product made of an exclusive and patented strain of Bacillus subtilis registered with the French National Collection of Microorganism Cultures as CNCM I-2745. LifeinU™ Bacillus subtilis CU1 is a ubiquitous bacterium able to form spores. It is gluten-free, lactose-free, non-GMO, kosher and halal, and therefore suited for virtually any healthy individual who wants to boost their natural defences. LifeinU™ BSCU1 is a highly stable ingredient offered as a white powder that can be kept for up to 3 years as illustrated in Fig. 1.

Schermata 2016-07-21 alle 10.13.25

Clinical study

The beneficial effects of LifeinU™ Bacillus subtilis CU1 consumption were demonstrated in a 4-month clinical trial conducted in healthy seniors in collaboration with an independent expert (Pr. Philippe Marteau – Gastroenterologist, Department of hepato-gastroenterology at Saint Antoine Hospital (Paris), Head of the medico-surgical department of hepato-gastroenterology at Lariboisière Hospital (Paris) and Professor of gastroenterology at Paris 7 University) [1]. Researchers recruited 100 healthy volunteers between 60 and 74 years of age and with a history of winter infections, to participate in a randomized, double-blind, placebo-controlled study. The purpose of this trial was to investigate the effect of LifeinU™ BSCU1 on winter respiratory and gastrointestinal infections in elderly people. Seniors were specifically selected for this study as natural defences weaken as we age and sIgA concentrations decrease. Gastrointestinal and respiratory sIgA are the first line of immune defence against pathogens. They are able to detect pathogens and prevent their interaction with the epithelium, so a deficiency can lead to increased risk of infections. This trial was performed in winter over 4 months, and consisted of 10 days of consumption of LifeinU™ Bacillus subtilis CU1 (2×109 spores of the bacteria/day) or a placebo, followed by 18 days without consumption. In a subgroup of 44 participants, consumption of LifeinU™ BSCU1 was found to significantly decrease the frequency of upper respiratory tract infections versus consumption of the placebo (-45%) as shown in Fig. 2.

Schermata 2016-07-21 alle 10.13.38

In addition, a significant and sustained increase in sIgA concentrations was found with consumption of LifeinU™ Bacillus subtilis CU1 versus placebo (Fig. 3).

Schermata 2016-07-21 alle 10.13.52

According to the researchers, ‘increased SIgA levels of 87% and 45% in faeces and saliva respectively are most probably of physiological significance in ameliorating the health status of seniors receiving B. subtilis CU1’. It is worth noting that no side effects were reported during the study.

Safety

LifeinU™ Bacillus subtilis CU1 is considered a Qualified Presumption of Safety (QPS) microorganism by the European Food Safety Authority and is also self-affirmed Generally Recognized as Safe (GRAS) in the United States. Clinical research confirmed the absence of adverse effects induced by consumption of LifeinU™ Bacillus subtilis CU1 in the 100 people who participated in the clinical study.

Applications and use 

Thanks to its ability to form spores, LifeinU™ Bacillus subtilis CU1 is different from many other bacteria which are often vulnerable to manufacturing processes or gastric acid and intestinal bile. LifeinU™ BSCU1 is very stable and able to survive exposure to the human gastrointestinal tract as well as extreme conditions such as compression, high temperature and high relative humidity. It can thus be easily incorporated into both dietary supplements and a diverse range of food and beverage matrices (e.g., hot tea, cereal bars, etc.) and can deliver its health benefits even in harsh environmental conditions. LifeinU™ BSCU1 can be used alone or in combination with other ingredients to create a variety of immune support products with the potential for health claims. For example, it can be combined with nutritional yeasts fortified with vitamin B and zinc, which are known to promote the normal function of the immune system and to reduce tiredness and fatigue. The recommended dosage is 20 mg/day of LifeinU™ Bacillus subtilis CU1 (=2×109 spores daily) for at least 10 days, to be repeated as needed.

Reference

1. Lefevre M, Racedo S, Ripert G, Housez B, Cazaubiel M, Maudet C, Jüsten P, Marteau P, Urdaci M (2015) Probiotic strain Bacillus subtilis CU1 stimulates immune system of elderly during common infectious disease period: a randomized, double-blind placebo-controlled study. Immun Ageing 12:24-38

Schermata 2016-07-21 alle 10.14.04

da Nutrafoods 15(2) 2016

Casperome®

Schermata 2016-04-21 alle 12.50.55Boswellia serrata (Fig. 1) is a treasure of traditional Indian medicine Ayurveda. This is the oldest medicine system in the world and is still very popular today, with Boswellia serrata playing a significant role as a healing agent. However, we often forget that the medicinal use of natural compounds is not limited to traditional practices. Modern health science is based on natural molecules; indeed, some Western active pharmaceutical ingredients in our pharmacies are either of natural origin, or mimic natural compounds. And Casperome® is one of them.

Schermata 2016-04-21 alle 12.51.08Composition and technical specifications

Casperome® is a purified mixture of triterpenoid acids from the gum resin of Boswellia serrata. Given the poor bioavailability of these compounds, Indena has formulated the extract using the Phytosome® technology to optimize their absorption. Casperome® is one of the most recent successful products regarding solubility, permeability and absorption manufactured using the Phytosome® platform, which highlights how serum levels and the subsequent tissue distribution of boswellic acids can be markedly enhanced by this lecithin delivery form. Casperome® is standardized to contain ≥25% of triterpenoid acids as demonstrated by HPLC.

The technical specifications are reported in Table 1.

Mechanism of action and efficacy

Boswellic acids such as 11-keto-β-boswellic acid (KBA) and its acetylated counterpart (AKBA), have been proposed as selective 5-lypoxygenase inhibitors as they manage the inflammatory response function through leukotriene inhibition [1]. However, more recent findings have shifted the focus to β-boswellic acid, the major triterpenoid from the gum resin, and its ability to modulate the inflammatory response enzymes cathepsin (catG) [2] and microsomal prostaglandin E synthase (mPGES-1) [3], suggesting that β-boswellic acid plays a major role, and therefore the whole bouquet of triterpenoid acids is important for the beneficial effects of boswellia preparations. Boswellia extracts have been demonstrated to be useful for bowel-related inflammatory responses, and to improve physical function in subjects with joint disorders through significant improvements in articular cartilage status and in the airways [4].

Clinical studies

Casperome® has a specific clinical literature (four clinical studies published and two in preparation), as well as comparative animal and human pharmacokinetic studies showing its superiority compared to unformulated boswellic acids at both plasma and tissue levels.

The four completed controlled clinical studies on Casperome® (alone or in combination with other ingredients) used a repeated dosage regimen.

The first study (500 mg/day, 32 subjects) showed that Casperome® can help reduce the need for inhalation therapy with common corticosteroids and beta-agonists by up to 43% in subjects with asthma (Fig. 2) [5]. The other three studies were conducted in patients with musculoskeletal disorders. In particular, Casperome® associated with physiotherapy allows early and improved pain reduction, and better functional recovery in comparison with physiotherapy alone when administered in subjects with Achilles tendinopathy and lateral epicondylitis (250 mg b.i.d., 60 subjects) [6]. In addition, Casperome® (250 mg b.i.d., 60 subjects) in combination with R(+) thioctic acid in mild to moderate cervical and lumbar radiculopathy allows a faster and better response on pain and functional parameters, as well as on quality of life when compared to R(+) thioctic acid alone [7,8].

Schermata 2016-04-21 alle 12.51.29Safety 

Casperome® is supported by a full preclinical dossier of toxicology, including mutagenesis studies and the three months oral toxicology tests in rats.

Applications and dosage

Casperome® may be utilized in a variety of supplement formulations. The recommended dose is 250 mg once or twice/day.

References 

1. Safayhi H, Mack T, Sabierai I et al. (1992) Boswellic acids: novel, specific, nonredox inhibitors of 5-lipoxygenase. J Pharmacol Exp Ther 261:1143–1146

2. Tausch L, Henkel A, Siemoneit U et al. (2009) Identification of human cathepsin G as a functional target of boswellic acids from the anti-inflammatory remedy frankincense. J Immunol 183:3433–3442

3. Siemoneit U, Koeberle A, Rossi A et al. (2011) Inhibition of microsomal prostaglandin E2 synthase-1 as a molecular basis for the anti-inflammatory actions of boswellic acids from frankincense. Br J Pharmacol 162:147–162

4. Ernst E (2008) Frankincense: systematic review. BMJ 337:a2813

5. Ferrara T, De Vincentiis G, Di Pierro F et al. (2015) Functional study on Boswellia phytosome as complementary intervention in asthmatic patients. Eur Rev Med Pharmacol Sci 19:3757–3762

6. Lazzaro F (2014) Comparative study on Tendhyal® efficacy in Achilles tendinopathy and epicondylitis. GIOT 40:1–10

7. Lazzaro F, Loiero M (2014) Effects of R(+) enantiomer of thioctic acid and Boswellia serrata (Casperome®), in combination, in the treatment of compressive cervicobrachial and lumbar radiculopathies. GIOT 40:249–257

8. Lazzaro F, Loiero M (2015) Comparison between two treatment schedules with Destior® Bridge, a fixed combination of R(+) thioctic acid and phospholipid formulation of Boswellia serrata (Casperome®), in the treatment of cervical and lumbar spine radiculopathy. GIOT 41:80–89.

Schermata 2016-04-21 alle 12.51.52

Benolea®

Schermata 2016-04-21 alle 12.33.43Cardiovascular disease (CVD) is the main cause of death globally. According to the World Health Organization (WHO), an estimated 17.5 million people died from CVD in 2012. Two of the main risk factors for CVD are hypertension and elevated cholesterol. Hypertension affects 1 billion people worldwide, and more than one third of the adult population has raised total cholesterol levels. Although some risk factors (e.g. family history, age) cannot be modified, others such as high cholesterol, hypertension and diabetes can be prevented or decreased, resulting in an enormous reduction in CVD risk. A 10 % drop in serum cholesterol in men aged 40 has been reported to lead to a 50 % reduction in heart disease within 5 years.

As regards CVD prevention, healthy lifestyles including a balanced diet and physical activity in younger and middle ages are key factors for a life free of chronic diseases at older ages. The Mediterranean diet with its abundant use of olive oil is the gold standard for healthy nutrition and is associated with a reduced risk for CVD. Initially, the benefits were attributed to the high intake of monounsaturated fatty acids (MUFAs), but in recent years, particular attention has been focused on the beneficial properties of the ´minor´ olive components oleuropein and other polyphenols. These key components are also contained in olive leaf, in even greater concentrations. Oleuropein not only has potent antioxidant and anti-inflammatory properties [1], but also has hypoglycemic, antihypertensive and antiatherosclerotic effects [2].

With Benolea®, Frutarom has developed an olive leaf extract in which oleuropein acts in synergy with other phytochemicals in a multicomponent system (Fig. 1).

Thus it helps to reduce multiple CVD risk factors such as high blood pressure and raised blood lipids as demonstrated in scientific studies.


Composition 
and technical specification

Benolea® (EFLA®934) is an olive leaf extract manufactured from Mediterranean-grown olive leaves (Olea europeae L.) which have been carefully selected using state-of-the-art-analytics. Benolea® is standardized on its two key constituents, oleuropein and polyphenols. Frutarom has chosen oleuropein over hydroxytyrosol for standardization, since hydroxytyrosol, a breakdown product of oleuropein, has no proven benefit on blood pressure. To ensure high antioxidant activity that supports heart health, Benolea® is also standardized on the polyphenol content. Use of the innovative and patented EFLA®HyperPure technology guaranties a high degree of purity and safety. The technical specifications are reported in Table 1.

Schermata 2016-04-21 alle 12.34.01Preclinical studies: mechanism of action  

First indications for a blood pressure-lowering effect of Benolea® came from an in vivo study in which supplementation of the olive leaf extract to L-NAME (NG-nitro-L-arginine methyl ester)-induced hypertensive rats resulted in a normalization of blood pressure after 6 weeks [3]. Additionally, oral administration of the extract at the same time as L-NAME for 8 weeks showed a dose-dependent prophylactic effect against the increase in blood pressure induced by L-NAME. Researchers suggested that the antihypertensive effect of the olive leaf extract may be related to various factors leading to an improvement in vascular function. For instance, oleuropein was shown to exert vasodilatory effects in rat aorta preparations [4] and to enhance nitric oxide (NO) production [5]. NO is an important regulator of various processes in the cardiovascular system including vascular smooth muscle cell relaxation that leads to arterial vasodilation and increased blood flow. As oxidative stress is also involved in the pathogenesis of hypertension, a beneficial role may be also ascribed to the high antioxidative capacity of the polyphenolic compounds contained in the olive leaf extract. Unpublished data from Frutarom indicate that Benolea® even exceed the value of vitamin C in the ORAC assay. Additionally, the antihypertensive effects of the olive leaf extract are suggested to be associated with an inhibition of angiotensin converting enzyme (ACE) [6].

Clinical studies

The beneficial effects for cardiovascular health demonstrated in preclinical studies have also been confirmed in clinical studies during the last few years.

Schermata 2016-04-21 alle 12.34.38The first study investigating the effect of the olive leaf extract EFLA®943 on blood pressure was performed in adults with mild hypertension in an open, controlled, parallel-group design with 8 weeks of intervention [7]. Twenty monozygotic twin pairs with blood pressure values above the optimal levels (diastolic ≥80 mmHg, systolic ≥120 mmHg) were randomly assigned to different treatment groups. In the first experiment, a treatment group taking one daily tablet containing 500 mg Benolea® was compared with a control group receiving non-intervention measures (dietary recommendations, lifestyle changes). The second experiment investigated dose effects between twins supplementing with one tablet once or twice daily (500 mg vs. 1000 mg Benolea®). The duration of both experiments was 8 weeks.

Analysis of blood pressure values over the course of the study revealed a reduction during supplementation, while in controls it did not change or even increased over time. For both groups receiving Benolea®, the maximal difference in systolic pressure compared to baseline was observed after 6 weeks and reached 6 and 11 mmHg after 500 or 1000 mg/day, respectively (Fig. 2). After 8 weeks, mean systolic blood pressure remained unchanged from baseline in controls (133±5 vs 135±11 mmHg) and the low-dose group (136±7 vs 133±10 mmHg), but had significantly decreased for the high-dose group (137±10 vs 126±9 mmHg; Fig. 2).

In addition to the antihypertensive effect, a trend to reduction in LDL cholesterol (LDL-C) by 0.05 mmol/l (19.3 mg/dl) was observed in group 1 after 8 weeks compared to baseline. A significant decrease of 0.4 mmol/l (14.7 mg/dl) was seen following supplementation with 1000 mg/day Benolea® (Table 2).

Moreover, the olive leaf extract was well tolerated by the participants.

In a second study with a double-blind, parallel design, the antihypertensive effect of the olive leaf extract was compared to captopril, a standard therapy for individuals with stage 1 hypertension [8]. Additionally, the lipid-lowering effects, safety and tolerability of Benolea® were evaluated. A total of 232 participants with systolic blood pressure between 140 and 159 mmHg and diastolic blood pressure between 90 and 99 mmHg or <90 mmHg participated in the study which consisted of a 4-week run-in period (diet alone+placebo capsule) followed by 8 weeks of intervention with either the reference drug captopril or the olive leaf extract. The olive leaf extract was given orally at a continuous dose of 500 mg twice daily over the entire intervention period, while captopril was administered at a starting dose of 12.5 mg twice daily for the first 2 weeks and, if necessary, titrated to 25 mg twice daily based on the participant´s response. After 8 weeks of treatment, both groups experienced a significant decrease in systolic and diastolic blood pressure compared to baseline (Fig. 3).

Schermata 2016-04-21 alle 12.34.52

In addition to the improvement in blood pressure values, the olive leaf exact treatment resulted in a significant reduction in blood triglyceride and blood total cholesterol concentrations accompanied by a slight reduction in LDL-C. Such effects were not observed with the captopril treatment. Finally, Benolea® was safe and well tolerated by the participants.

Safety and tolerance

In the various completed studies, Benolea® showed strong compliance, high safety and excellent tolerability. Finally, Frutarom´s EFLA®HyperPure patented technology further ensures good solubility, stability, purity and removal of contaminants.

Applications and dosage

Olives and related products are good candidates for product development, since they have always been regarded as healthy. For instance, a recently published health report indicated that 80 % of respondents see olive oil as healthful, with half of them considering it to be extremely healthful [9]. Furthermore, 22 % already know that flavonoids promote heart health.

Due to the sound scientific data and its well-documented source, Benolea® enables manufacturers to offer products with various proven heart health benefits including the promotion of beneficial blood pressure values, healthy cholesterol and blood sugar levels, and vascular protection.

Thanks to its good solubility, the consistently high quality and the results of proprietary studies, Benolea® can be incorporated into various supplement and food matrices allowing the industry to create innovative products. Benolea® is stable in film-coated tablets, and is a valuable dosage form for food supplements. It is water soluble and can be used in still or carbonated beverages as well as in various foods (dairy products, breakfast cereals, bars, baked products, spread, chocolate, oils, dressings and more) at appropriate levels while taking into account national food regulations.

The recommended dosage is 500-1000mg/day.

References

1. Barbaro B, et al. (2014) Effects of the olive-derived polyphenol oleuropein on human health. Int J Mol Sci 15:18508–18524

2. de Bock M, et al. (2013) Olive (Olea europaea L.) leaf polyphenols improve insulin sensitivity in middle-aged overweight men: a randomized, placebo-controlled, crossover trial. PLoS One 8:e57622

3. Khayyal MT, et al. (2002) Blood pressure lowering effect of an olive leaf extract (Olea europaea) in L-NAME-induced hypertension in rats. Arzneimittelforschung 52:797–802

4. Zarzuelo A, et al. (1991) Vasodilator effects of olive leaf. Planta Medica 57:417–419

5. Visoli F, et al. (1998) Oleuropein, the bitter principle of olives, enhances nitric oxide production by mouse macrophages. Life Sci 62:541–546

6. Hansen K, et al. (1996) Isolation of an angiotensin converting enzyme (ACE) inhibitor from Olea europaea and Olea lancea. Phytomedicine 2:319–325

7. Perrinjaquet-Moccetti T, et al. (2008) Food supplementation with an olive (Olea euopaea L.) leaf extract reduces blood pressure in borderline hypertensive monocygotic twins. Phytother Res 22:1239–1242

8. Susalit E, et al. (2011) Olive (Olea europaea) leaf extract effective in patients with stage-1 hypertension: comparison with Captopril. Phytomedicine 18:251–258

9. IFIC 2015, What is your health worth? Food & Health Survey 2015. http://www.foodinsight.org/sites/default/files/2015%20Food%20and%20Health%20Survey%20-%20FINAL.pdf

Disclaimer: any information and documents have been provided in good faith and are believed to be accurate. The supporting data summarized in publications have not been evaluated for compliance with any regulatory requirements. Study results reported may not be generally true under other conditions or in other matrices. No warranties are made.

Schermata 2016-04-21 alle 12.35.04