Healthy Gut, Healthy Strut

[Digestive Health] Vol. 20 No. 5 July/August 2015

By Rachel Adams, Managing Editor

“The way to a man’s heart is through his stomach”—who’s heard that saying before? Interestingly, that statement is starting to ring true, and not only for men. Evolving research continues to show a healthy body (the heart included) starts with a healthy digestive tract.

Unfortunately, many consumers struggle with digestive problems such as irritable bowel syndrome, food intolerance, gas, bloating and other issues that can lead to diarrhea, constipation or other digestion-related discomforts. These issues not only affect the body’s ability to obtain nutrients, but can interfere with some of life’s simplest pleasures. Pizza, anyone?

In order to steer clear of digestive doom, consumers struggling with digestion issues can turn to a variety of natural products, including probiotics and prebiotics, enzymes and other natural ingredients backed by research to support positive effects on digestion. However, in order to successfully develop digestive health products, manufacturers must consider challenges when formulating with digestive health ingredients, and be privy to trends in the digestive health market.

A Healthy Gut for a Healthy Strut

Increasingly, consumers are turning to natural products not only to absolve digestion issues, but because research shows a healthy gut is critical to overall health and wellness.

According to David Daguet, Ph.D., scientific manager, Nexira, there are two primary reasons that explain the crucial role of the gut in overall health and wellness: 1) the essential function of the gut is to nourish the entire organism, and all the nutrients coming from the digestive tract enter into the organism following very specific and precise mechanisms; and 2) the gut is the second main barrier against the outside after the skin.

Austin Soejoto, sales representative at American Laboratories, elaborated: “With a poor functioning gut, the nutrients from consumed foods are not utilized efficiently and a number of problems can arise. By keeping the digestive tract healthy and fine-tuned, there are numerous systemic health benefits from adequate vitamins, proteins, and other nutrients to keep the body’s organs and systems well supported.”

Concerning the gut barrier, Daguet noted the issue of increased gut permeability, also called leaky gut syndrome, which can play a role in the development or progression of many health troubles or diseases. “Because the gut permeability is disrupted, ‘non-authorized’ compounds that cross the barrier and enter the organism can promote or create troubles or diseases like liver disease, chronic

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[Digestive Health] Vol. 20 No. 5 July/August 2015 fatigue syndrome, depression, diabetes, obesity, skin problems, joint pain [and] kidney failures,” he said.

What’s more, Tim Gamble, president and CEO of Nutraceutix, added that for consumers who are supplementing their diets as part of a particular regimen beyond just daily maintenance, such as athletes, “a healthy, optimized gut is key to facilitating and optimizing the absorption and utilization of both routine and specialized active ingredients, as well as deriving the most benefit from a regular diet.”

Understanding the Microbiome

To keep the gut healthy, attention is being paid to the gut microbiome—a complex microbial ecosystem that provides key functions to the body.

“The gut microbiome is one of the most exciting areas of science today, and the relevance of the microbiome to our health beyond digestion is a hot area for research,” said John Deaton, vice president of technology, Deerland Enzymes.

One critical function of the gut microbiota is its benefit to immune health. According to Nena Dockery, technical services manager at Stratum Nutrition, “These ‘friendly’ microorganisms help the immune system by targeting potentially dangerous bacteria and viruses and assisting the body in ridding itself of these pathogens before they can cause harm.”

And that’s not all—as noted by Jamie Spell, Nutraceuticals International, gut microflora is responsible for much more, including digestion of food and biosynthesis of fundamental and essential nutrients and vitamins.

Daguet attributed the increase in the scientific understanding of the intestinal microbiota to recent developments in analytical techniques to analyze the composition of microbial ecosystems. However, research is still evolving when it comes to the gut and its helpful inhabitants. Greg Leyer, Ph.D., chief scientific officer at UAS Labs, said, “The real breakthroughs will come when science can establish the causal association between modifying the ‘microbiome’ and health/disease, and the ability to harness that information into developing useful products.”

What researchers and experts do know is that gut microbiota can be promoted via probiotics and prebiotics: probiotics are bacteria ingested via foods or food supplements to colonize the gut; and prebiotics, defined as non-digestible food compounds that are selectively fermented, nourish probiotic bacteria.

Promoting Gut Health via Probiotics

According to Michael Shahani, chief operations officer at Nebraska Cultures, “good” or “probiotic” bacteria that reside in the gut are responsible for breaking down food and extracting the nutrients, producing vitamins and enzymes, and keeping unhealthy bacteria at bay to prevent sickness. “In www.naturalproductsinsider.com Page 2

[Digestive Health] Vol. 20 No. 5 July/August 2015 addition, probiotic bacteria may positively stimulate our immune system, making us even more resistant to illness,” he said.

There are several different strains of probiotics, and each strain provides different benefits, many of which are backed by research. For example, Daguet noted some strains directly impact gut health and address issues related to constipation, diarrhea, etc., while some strains indirectly affect issues related to inflammation, cognitive and mental health, etc.

The most common probiotics are Lactobacilli and Bifidobacteria, Spell said. “Lactobacilli are present at the gastric level and also in the respiratory tract and on the skin, and they are the first line of defense against the external and internal pathogens.”

A 2011 review of clinical studies on a blend of Lactobacillus helveticus Rosell-52 and Lactobacillus rhamnosus Rosell-11 (Lacidofil® by Lallemand) showed its positive effects on gut health, including several positive studies in antibiotic-associated diarrhea prevention in children and adults, H. pylori eradication, acute gastroenteritis in children, management of IBS symptoms and lactose intolerance.1

The effects of probiotic-fortified yogurt on antibiotic-associated diarrhea in children aged 1 to 12 years was evaluated in a randomized, double blind, placebo-controlled study published in 2015.2 Seventy- two children received either 200 g/d of yogurt containing Lactobacillus rhamnosus GG (LGG), Bifidobacterium lactis (Bb-12) and Lactobacillus acidophilus (La-5) or a pasteurized yogurt (placebo) for the same duration as antibiotic treatment. There were no incidents of severe diarrhea in the probiotic group, and six in the placebo group. Further, there was one episode of minor diarrhea in the probiotic group, compared to 21 in the placebo group.

Dan Lifton, president, proprietary branded ingredients group, Maypro, cited research to support Bifidobacterium longum (Morinaga® BB536 by Maypro), which improves immune function and digestive health, and has shown positive effects on B longum populations.3

In addition, Bifidobacterium infantis (Morinaga M-63 by Maypro), has shown promise with promoting the formation of a healthy microbiome in low-birth-weight infants, according to Lifton. When given to infants—as part of a three-species probiotic treatment—the probiotic strain helped establish healthier microbiota more quickly.4 Also supporting the health of low-birth-weight infants is Bifidobacterium breve (Morinaga M-16V by Maypro), which has demonstrated the ability to improve immune, digestive and respiratory health in low-birth-weight infants.5,6,7

According to Deaton, companies are increasingly seeking a multi-strain formulation of both non-spore and spore forming probiotics. “Based on this demand, more science is emerging to support the benefits of spore forming probiotic strains, such as Bacillus subtilis,” he said, adding that Deerland Enzymes’ Bacillus subtilis strain (DE111™) demonstrated in an unpublished human clinical trial the strain’s ability to control microbial populations, aid in digestion and maintain general health.

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[Digestive Health] Vol. 20 No. 5 July/August 2015

Recent research conducted using the spore forming probiotic strain Bacillus coagulans GBI-30, 6086, (GanedenBC30 by Gandeden Biotech), showed that GanedenBC30 supports protein utilization and digestive health in seniors.8,9

Another spore forming strain is Bacillus coagulans (ProDURA™ from Maypro), which demonstrated effects to help breakdown lactose and balance cholesterol levels in a study published in 2011.10

Sabinsa’s Bacillus coagulans probiotic, LactoSpore®, becomes a temporary resident of the GI tract after ingestion, and is excreted within seven days.11 Clinical research supports B. coagulans spores efficacy in conditions such as diarrhea due to acute or chronic gastroenteritis and infantile diarrhea.12,13

Prebiotics: Nourishing Probiotics

Prebiotics are substances naturally present in food that are not absorbed in the gastrointestinal (GI) tract. They arrive intact in the colon and stimulate the growth of the probiotics, Spell said, adding that common prebiotics include inulin galactooligosaccharides (GOS), fructooligosaccharides (FOS), pyrodextrin, lactulose and polydextrose.

A 2015 review on inulin-type fructans (IFTs)—indigestible, soluble, fermentable food ingredients with known prebiotic properties, and also known as inulin, oligofructose (OF) and FOS—explored the benefits of fructans and their potential to act as scavengers of reactive oxygen species (ROS).14 ROS include free radicals and nonradical molecules that attack DNA, lipids and proteins and cause cellular damage. Review authors concluded, “Fructans may act as ROS scavengers providing an increase in antioxidant defenses partially through the activation of endogenous ROS detoxification systems.”

Carol Lowry, senor food scientist at Cargill, noted a review supporting the prebiotic effects of inulin, a soluble corn fiber from chicory root, which include “modulating selectively “beneficial” gut bacteria such as bifidobacteria and improving the conditions in the gut/colon through the reduction of pH, increase of short chain fatty acids, and the reduction of metabolites from protein fermentation.”15

Nexira’s Fibregum™, a soluble dietary fiber derived from acacia gum, produced from the sap exuded from the stems and branches of acacia trees, demonstrated bifidogenic properties in a single blind controlled study performed on healthy volunteers that consumed either Fibregum at the dose of 10 g/d and 15g/d during 10 days, or sucrose at the same dose.16 Results showed concentrations of bifidobacteria, lactobacilli and total lactic acid bacteria groups were significantly increased with Fibregum at doses of 10 g/d and 15 g/d compared to control.

“Many of these health effects emanate from increased production of short-chain fatty acids (SCFAs) by the stimulated beneficial bacteria,” Daguet said, citing an in vitro study conducted using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) in 2012.17 According to the study, the fermentation pattern of Fibregum “is specific and unique in preferably producing the beneficial SCFAs propionate and butyrate,” Daguet said. www.naturalproductsinsider.com Page 4

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In a study published in 2015, partially hydrolyzed guar gum (PHGG) was associated with a significant improvement in symptoms, use of laxatives/enemas, stool form/consistency and colonic transit time (CTT) among 68 patients with irritable bowel syndrome (IBS).18 For the study, patients entered a two- week run-in period, followed by a four-week study period with PHGG. Patients completed a daily questionnaire to assess the presence of abdominal pain/discomfort, swelling and the sensation of incomplete evacuation. The number of evacuations/day, the daily need for laxatives/enemas and stool consistency-form were also evaluated.

Further, perilla extract (Perilla frutescens) improved gastrointestinal (GI) complaints in a randomized, double blind, placebo-controlled study published in 2014.19 For the study, 50 healthy subjects with GI discomfort and reduced bowel movements, aged 30 to 70 years, documented their GI symptoms, stool frequency and consistency daily during a two-week run-in phase and a four-week intervention phase with Perilla frutescens extract (as Benegut® from Maypro) or tapioca starch (placebo). All GI symptoms were significantly improved over time by Perilla frutescens extract during the intervention phase, whereas in the placebo group only abdominal discomfort was significantly improved in the placebo group.

Digesting Enzymes

“The role of digestive enzymes is to break down food-derived fats, carbohydrates and proteins into smaller substances that our bodies can use,” Deaton said. “Although the body produces its own digestive enzymes, it may not be enough to completely break down cooked or processed foods.”

Deaton added that cooking and processing can denature natural enzymes present in raw foods, while the body doesn’t produce the enzyme cellulose—an enzyme that breaks down the cell wall of plants—at all. “Those who follow a vegetarian or vegan diet are likely missing out on key nutrients from the plant-based foods they’re eating, and would most certainly benefit from an enzyme supplement,” he said.

What’s more, Soejoto noted the body slows its natural enzyme production with age, further supporting the need for supplementation with enzymes.

Soejoto added enzymes are becoming more common in various products focused in sports nutrition. “Protease, papain and bromelain help to liberate more amino acids from the main protein source in shakes, while lactase and alpha galactosidase help to ease digestive discomfort,” he said. “By adding enzymes into shakes or supplements, the athlete can benefit from more micronutrients freed for absorption and use while also enjoying a product with minimal digestive complications.”

A study published in 2015 demonstrated the positive effects of kiwifruit (as ACTAZIN® from Stratum Nutrition, which naturally contains several vitamins and minerals, soluble and insoluble fiber, polyphenolic compounds and the protein digesting enzyme, actinidin) in increasing stool frequency in healthy individuals without the side effects of laxative use.20 www.naturalproductsinsider.com Page 5

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In addition, unpublished studies revealed the product’s benefits as a prebiotic in promoting growth of Lactobacillus and Bifidobacterium species without encouraging the growth of pathogenic species of bacteria. “Both the fiber content, and possibly the polyphenols as well, work to exert a prebiotic effect helping to support the colonization of several different Lactobacillus and Bifidobacterium species,” Dockery said. “They may also contribute to benefits to gut transit time.”

Beyond fibers, other ingredients are appearing in the digestive health space. According to Daguet, “Yeasts [and] plant extracts, like mint extract, have been developed to help to deal with IBS,” he said, adding that certain amino acids are being studied in the gut health spectrum, as well.

A review published in 2014 found three weeks of prune consumption (100 g/d) improved stool consistency and frequency in constipated subjects compared to psyllium.21 In non-constipated subjects, prunes softened stool consistency in one trial and increased stool weight. Researchers concluded, “In constipation, prunes appear superior to psyllium for improving stool frequency and consistency, however, the evidence for other outcomes and the effects in non-constipated subjects is weak.”

Vitamin E, in the form of tocotrienols, protected the gastric mucosa from stress-induced injury in a rat study published in the Polish Journal of Pathology.22 For the study, animals received 60 mg/kg of annatto tocotrienol daily (as DeltaGold® by American River Nutrition), for 28 days prior to introduction of stress. Researchers measured gastric lesion score, malondialdehyde (produced when lipid membranes are damaged by oxidation), and prostaglandin E2, a mediator of inflammation that is known to render the GI tract more resistant to acute injury. Results showed that gastric lesion score was significantly lower in the tocotrienol-treated group, and that annatto tocotrienol reduced the number of lesions by 2.2-fold.

Formulating with Digestive Health Ingredients

Some digestive health ingredients, such as enzymes and probiotics, may present challenges during formulation and need to be carefully considered to ensure efficacy in finished products.

Probiotics are living organisms; in order to be effective, probiotic bacteria must survive processing methods and shelf life, and they must remain active in the gut in adequate amounts after consumption.

According to Shahani, probiotic bacteria are susceptible to heat, moisture, light and air. “All probiotic bacteria are harmed by these factors in varying degrees, depending on the strain and the manufacturing process,” he said. “However, some strains are more resistant than others to these detrimental factors, and can be successfully put in foods.” He added that most lactobacilli and bifidobacteria must be kept cool and dry.

“[Because] probiotics are highly susceptible to degradation during traditional dietary supplement manufacturing processes, during storage and distribution, and even after consumption by the consumer,” Gamble said, “many quality-conscious brands are investing in formulations and www.naturalproductsinsider.com Page 6

[Digestive Health] Vol. 20 No. 5 July/August 2015 supplement formats that heighten probiotic stability and effective delivery past the stomach to the intestinal tract, where they can provide the most benefit.”

To ensure formulation success, supplement manufacturers can utilize technologies designed to prevent die-off of probiotics during tableting. Fujicalin by Fuji Health Science is a form of dibasic calcium phosphate anhydrous that can significantly reduce probiotic strains die-off during tableting, said Xai Han, Ph.D., technical sales and support for specialty excipients, Fuji Health Science. The ingredient is made via a proprietary powdering technology that creates a spherical and porous structure. “This unique structure helps protect probiotic strains during tableting,” Han said.

When formulating functional foods with probiotics, one option is to utilize spore-forming probiotic strains, which can survive most manufacturing processes. Shahani noted his company’s ProDURA can be used successfully in some food applications.

According to Ganeden Biotech’s Michael Bush, GanedenBC30, also a spore-forming strain, can survive manufacturing processes, shelf life, stomach acids and intestinal bile, does not impact manufacturing processes and has no effect on taste or texture of a product. “GanedenBC30 can be added to virtually any product with the exception of UHT or shelf stable beverages,” he said.

Unique delivery methods can also ensure efficacy of probiotics. For example, to overcome the challenge of fortifying shelf-stable beverages with probiotics, Ganeden introduced a “telescoping straw” that contains 1 billion CFU of GanedenBC30, and it can be applied to Tetra Pak and other shelf-stable beverage containers such milk or juice boxes, pouches and bottles.

When formulating with enzymes, Soejoto advised, “Creating diverse supplements helps to ensure different enzymes will be aiding nutrient breakdown throughout various areas of the system.”

“Different enzymes from various sources will have different optimal environments for activity,” he said. “By formulating the enzymes with a systemic mind-set, products can target various pH or temperature environments with specific enzymes in a blend.” Examples include pepsin and acid-stable protease for the acidic environment of the stomach, and proteases and lipase for the neutral environment of the intestines.

Manufacturers should also keep in mind that the potency of enzymes, referred to as “activity,” is not measured in the same way as other nutritional supplements, Deaton advised. “Enzymes are not measured by weight, so the number of milligrams of a product would not describe the true potency. Plus, different enzymes use different units of measurement to determine potency.”

Further, when formulating a multi-component supplement blend that contains enzymes, challenges may be presented along the way in terms of fitting an effective dose of all the ingredients into a predefined amount of space (the capsule), Deaton continued.

“It takes extensive experience working with enzymes in order to formulate an effective enzyme-based supplement,” he said. “Specific expertise is needed in order to formulate a blend that utilizes www.naturalproductsinsider.com Page 7

[Digestive Health] Vol. 20 No. 5 July/August 2015 ingredients and enzymes that are complementary, while taking care to avoid ingredients that may detract from the efficacy of the enzymes.”

Market Trends for Digestive Health Products, Ingredients

The gastrointestinal (GI) health supplement category saw US$1.4 billion in consumer sales in 2011, which marks a 14-percent increase compared to 2010, according to a 2014 market report from Canadean Ltd. Sales are anticipated to remain strong as consumers continue to seek products that not only alleviate digestion issues, but also contribute to overall health.

“According to [Euromonitor’s] measure of dietary supplements by positioning, products positioned around digestive health have outpaced the overall dietary supplement market in the U.S. and even more strongly around the world,” said Chris Schmidt, consumer health analyst, Euromonitor. “This is due in part to a gradual shift of consumer interest away from general toward more targeted health claims, but is also being driven by health trends, such as the increasing incidence of GERD, and to a lesser extent, an increase in the awareness of the gut-microbiome’s impact on total health.”

Tom Vierhile, innovations insights director for Datamonitor Consumer, noted an array of ingredients that are being added to new products are promoted as offering digestive health benefits. Examples of ingredients being seen globally in new product launches include sprouted grains, fermented ingredients (like sauerkraut), kombucha, ginger, manuka honey and even cinnamon.

In supplements categories, Schmidt said categories haven’t seen the same type of dramatic new product launches compared to standard ingredients/over-the-counter drugs. “That said, a number of traditionally used products like probiotics have grown strongly in the last five years,” he said.

According to 2014 research from Grand View Research Inc., the probiotics market was valued at US$32.05 billion in 2013 and is anticipated to grow to $52.34 billion by 2020. Dietary supplements will represent the fastest growing application segment, the research firm noted, and is expected to grow at a compound annual growth rate (CAGR) of 7.7 percent from 2012 to 2020.

“Fiber, a category that struggled in the U.S. in the mid-to-late-2000’s, has been showing some potential for a turnaround,” Schmidt said, “with a number of producers, especially smaller producers targeting the natural and specialty retailing channels, rebranding the ingredient around the term ‘prebiotics.’”

Data from Grand View Research indicate the global prebiotics market is expected to reach US$5.75 billion by 2020, fueled by a positive outlook on dairy consumption, coupled with surging demand for sugar- and fat-free products.

Within the prebiotics category, the report noted food and beverage was the largest application segment; however, dietary supplements and animal feed are expected to be key application markets for prebiotics and are expected to grow at more than 9 percent from 2014 to 2020. www.naturalproductsinsider.com Page 8

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“One ingredient that is not new at all that seems to have a lot of appeal right now is fiber,” said Vierhile, adding that Datamonitor Consumer’s 2015 Q2 consumer survey showed 37 percent of consumers globally say they “try to eat as much as possible” of fiber.

In addition, Datamonitor Consumer’s Product Launch Analytics database of new products showed the percentage of food and non-alcoholic beverage products globally that made a “high fiber” claim grew from 2.8 percent in 2011 to 4.8 percent in 2014.

Schmidt noted that combination products, more so than new ingredients, have been generating interest from both consumers and producers. “A lot of new launches have focused on combination probiotics-fiber products, particularly in Europe, where the health claims regulations are more stringent.” He cited examples such as Yobalex by Bayer AG in Spain and Lactocare Fiber by Actavis Plc in Denmark.

References:

1. Foster et al., “A comprehensive post-market review of studies on a probiotic product containing Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011.” Beneficial Microbes. December 2011;2(4):319-334.

2. Fox MJ et al. “Can probiotic yogurt prevent diarrhoea in children on antibiotics? A double-blind, randomised, placebo-controlled study.” BMJ Open. 2015 Jan 14;5(1):e006474. DOI: 10.1136/bmjopen-2014-006474.

3. Odamaki T et al. “Effect of the oral intake of yogurt containing Bifidobacterium longum BB536 on the cell numbers of enterotoxigenic Bacteroides fragilis in microbiota.” Anaerobe. 2012;18(1):14-8.

4. Yaeshima T et al. “Effect of administration of bifidobacteria on intestinal microbiota in low-birth- weight infants and transition of administered bifidobacteria: a comparison between one- species and three-species administration.”Anaerobe. 2013 Oct;23:38-44.

5. Li Y et al. “Effects of bifidobacterium breve supplementation on intestinal flora of low birth weight infants.” Pediatr Int. 2004 Oct;46(5):509-15.

6. Ezaki S et al. “Prophylactic probiotics reduce cow's milk protein intolerance in neonates after small intestine surgery and antibiotic treatment presenting symptoms that mimics postoperative infection.” Allergol Int. 2012 Mar;61(1):107-13.

7. Van der Aa LB et al. “Synbiotics prevent asthma-like symptoms in infants with atopic dermatitis.” Allergy. 2011 Feb;66(2):170-7.

8. Edna N et al. “Effect of prebiotics on the fecal microbiota of elderly volunteers after dietary supplementation of Bacillus coagulans GBI-30, 6086.” Anaerobe. December 2014;30:75–81. www.naturalproductsinsider.com Page 9

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9. Edna N et al. “Bacillus coagulans GBI-30, 6086 Modulates Faecalibacterium prausnitzii in Older Men and Women.” The Journal of Nutrition. May 2015.

10. Sudha RM, Radkar N, Maurya A. Int J Probiotic Prebiotics. 2011 May;6(2):1-5.

11. Majeed M. and Prakash L. “LactoSpore: The Effective Probiotic.” NutriScience Publishers Inc. 1998.

12. Cui Y-L et al. “Efficacy of Bacillus coagulans tablets in the treatment of acute and chronic diarrhea.” Int. J. Immunotherapy. 2004;20(1)17-22.

13. Chandra, R.K. “Effect of Lactobacillus on the incidence and severity of acute rotavirus diarrhea in infants. A prospective placebo-controlled double-blind study.” Nutrition Research 22. 2002;65–69.

14. Franco-Robles E and Lopez M. “Implication of fructans in health: immunomodulatory and antioxidant mechanisms.” The Scientific World Journal. 2015. DOI:10.1155/2015/289267.

15. Cherbut C et al. “Acacia gum is a bifidogenic dietary fibre with high digestive tolerance in healthy humans.” Microbial Ecol Health Dis. 2003;15(1):43-50.

16. Roberfroid MB. Introducing inulin-type fructans. Br J Nutr. 2005 Apr;93 Suppl 1:S13-25.

17. Terpend K et al. “Arabinogalactan and fructooligosaccharides have a different fermentation profile in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®).” Environ Microbiol Rep. 2013 Aug; 5(4):595-603.

18. Russo L et al. “Partially hydrolyzed guar gum in the treatment of irritable bowel syndrome with constipation: effects of gender, age, and body mass index.” Saudi J Gastroenterol. 2015 Mar- Apr;21(2):104-10. DOI: 10.4103/1319-3767.153835.

19. Buchwald-Werner S et al. “Perilla Extract improves gastrointestinal discomfort in a randomized placebo controlled double blind human pilot study.” BMC Complementary and Alternative Medicine. 2014;14:173.

20. Ansell J. et al. “Kiwifruit-derived supplements increase stool frequency in healthy adults: a randomized, double-blind, placebo-controlled study.” Nutrition Research (2015)35:401-408.

21. Lever E et al. “Systematic review: the effect of prunes on gastrointestinal function.” Aliment Pharmacol Ther. 2014 Oct;40(7):750-8. DOI: 10.1111/apt.12913

22. Rodizan M et al. Pure tocotrienol concentrate proteted rat gastric mucosa from acute stress- induced injusry by a non-antioxidant mechanism.” Pol. J Pathol. 2013;1:52-58. DOI: 10.5114/PJP.2013.34604.