Bio Rex MANGOSTEEN ENG Book

Contents Introduction ………………………………………………………………………………………1 Why Nutraceutical? ………………………………………………………………………….2 Which Parts Of Mangosteen Fruit Are Used? …………………………………….4 Why Skin, Pods And Seeds? ……………………………………………………………5 Why Fermented Mangosteen? ………………………………………………………..7 How BioRex® Mangosteen is Produced? ……………………………………….10 What Active Compounds Are Important For Health Promoting Properties? ………………………………………………..12 What Kind Of Health Effects Are Boosted? …………………………………12 Anti-Cancer Effects Of Mangosteen Xanthons …………………………….13 Anti-Inflammatory Effects Of Mangosteen Xanthones …………………16 Other Health Benefits …………………………………………………………….16 How To Use And Safety Recommendations ……………………………19

INTRODUCTION The fruit of Garcinia mangostana Linn. or mangosteen, of the family Guttiferae, is usually referred to as “The queen of fruits” due to its delicious aroma, refreshing and pleasant sweet and sour taste. The purple ripe fruits consist of 6-8 seeds encased with a white and juicy pulp. The plant is originated from Southeast Asia and is mainly harvested in Thailand, India, Indonesia, Malaysia, Philippines, South China and other tropical countries. The fruit pod has been traditionally used in Asian folk medicine for the treatment of skin infections, wounds, gastro-intestinal, urinal and sexually transmitted infections, and chronic gastric ulcer. Recent reports have shown the phytochemical composition of mangosteen pod (mostly xanthones, tannins, and polyphenolics) identifying the most biologically active group as xanthones. Biological studies on cell lines and animals revealed pronounced Page 1

anti-metabolic syndrome, anti-cancer and immunomodulating effects of mangosteen. Of recent years, Mangosteen containing food supplements, beverages and functional food have been aggressively advertised as miraculous health promoting products and sold better than any other nutraceutical; hence transforming it into “The queen of Nutraceuticals”. In the United States, mangosteen products are highly popular and are ranked as the top three-selling “single botanicals”. The sales of mangosteen-containing beverages in USA alone exceeded $200 millions in 2008. But sadly, products marketed as mangosteen juice are often a blend of numerous fruit juices with mangosteen being one of the less abundant components. WHY NUTRACEUTICAL? Nutraceutical is defined as a food or a part of food that provides evidence-based beneficial health effects in the prevention and/or treatment of diseases. The health promoting plant-derived active compounds are called phytochemicals. They are relatively small organic molecules belonging to a class of secondary plant metabolites, which do not directly participate in plant growth and development. Their major function is to defend plants from various biotic and abiotic stresses such as infections, bacteria, viruses, herbivors, wounds, draught, weather changes, and solar irradiation. Intense search for natural or nature-inspired drugs and disease- preventive products led to a clear-cut conclusion that secondary Page 2

metabolites good for plant defense could be extremely helpful for human health. There are more than 8,000 secondary metabolites from fruits and vegetables that exert biological activities and can potentially be feasible as active ingredients for nutraceuticals and drugs. Some of them can be lethal against tumor cells, viruses, and microbes. Therefore, they have been under extensive investigation for possible anti-cancer and antibiotic/antiparasitic/antiviral potential. On the other hand, the content of toxic compounds in nutraceuticals of plant origin should be strictly controlled because, unlike drugs, their development and registration are not well regulated. Page 3

WHICH PARTS OF MANGOSTEEN FRUIT ARE USED? The whole fruit of mangosteen including non-edible pod (syn. pericarp or rinds) and seeds are used during our fermentation process. The pericarp of mangosteen consists of three parts having different roles in the fruit development and protection: (1) The outer layer exocarp which is called the skin or peel, purple in color, hard in texture, and too bitter to be eaten raw. It serves to protect the seeds from dangerous solar irradiation, from microbial and herbivor invasions, and from excessive heat. (2) The middle layer mesocarp is fibrous, soft, and reddish in color. It serves for additional mechanical protection of seeds and as a storage of essential nutrients necessary for seed growth and maturation like whites of the eggs. (3) The inner layer endocarp encloses the seeds. It is also called the aril or placenta of the seeds. In mangosteen it is the only edible juicy sweet-sour part of the fruit. This very small part of the fruit is crashed during the mangosteen juice preparation and the yield of mangosteen juice is very low. Mangosteen seeds are dark-brown, hard in texture, and not edible in a raw form. Page 4

Exocarp Mesocarp Endocarp WHY SKIN, PODS AND SEEDS? Representing the outer part of mangosteen fruit, the skin comes in various colors and changes during ripening turning from green to dark blue-purple. The color results from a ratio of pigments and phytochemicals such as chlorophyll, carotenoids and phenolics. Blue and purple colors of ripe mangosteen are associated with anthocyanines, resveratrol, quercetin, and ellargic acid, all of them known for numerous remarkable biological activities and pronounced health promoting effects (in detail see below). The white color of seed placenta characteristic for mangosteen pod indicates the presence of beta-glucans and lignans with specific immunomodulating and anti-cancer action. According to hundreds of scientific publications, there are two major groups of secondary metabolites attributed to the health properties in the mangosteen pericarp, the xanthones and phenolics. The most abundant xanthones in the pericarp of mangosteen fruit are α- and γ-mangostin. Of extreme importance, the Page 5

quantity of these compounds in the mangosteen pod is 10 times greater than in the edible parts of the fruit. Seeds have the most important key role in a plant life being safeguards of genetic material to maintain the existence and spreading out of the species. Even seed shells contain large amounts of highly protective secondary metabolites such as lignins, which possess anti-cancer, anti-viral, and anti-microbial properties. Within the seed, nucleic acids bearing a genetic code and a key for its decipher (DNA and RNA), and definite factors regulating stem cell activation, cell growth and development are concentrated. Page 6

WHY FERMENTED MANGOSTEEN? Although pods and seeds are often thrown away after consuming the edible part of mangosteen fruits, they contain a wealth of health promoting compounds that can be used as a source of pharmaceutical and nutraceutical products. Usually, these pod- and seeds-derived biologically active compounds are extracted by alcohols, esthers or hot water. Then, the solutions are dried under high temperature and a proper gas flow in order to prevent excessive oxidation of the active components. However, in a dried powder, admixtures of organic solvents, alcohols, esthers, and acids as well as traces of gases (nitrogen, argon, xenon or carbon dioxide) could be found. Seemingly safer hot water extracts lack, however, biologically important glycosylated and apolar molecules, which could be dissolved exclusively in organic solvents. Moreover, extraction by hot water and drying process under high temperature inevitably leads to termal and oxidative destruction of sensitive-to-temperature active compounds of fruit pod and seeds. Freshly made juices contain mechanically destroyed edible parts of fruits with good palatable properties and vitamins C, E, and A as active ingredients. Since the vitamins belong to short-living molecules subjected to fast decay in the atmosphere containing oxygen, the juices are often preserved with synthetic antioxidants and other non- natural conserving substances. To prevent bacterial contamination, Page 7

juices are sterilized under high temperature and sometimes additions of antibiotics are done. At the same time, mechanical destruction of cellular membranes of edible fruits/berries during juice preparation does not eliminate high molecular weight molecules, such as polysaccharides, glycolipids, glycoproteins, and proteins with potentially allergenic properties. Therefore people sensitive to certain plant-derived allergen(s) could develop undesired allergic reaction after fruit juice consumption. Fermentation process aimed to extract active phytonutrients from fruit pods, leaves, seeds, and edible parts has many advantages as compared to mechanical destruction (fresh juice), thermal destruction (dried products or conserved juices), or organic extraction (extracts, elixirs and decoctions). Fermentation is the most ancient and most NATURAL way of plant food processing and preservation. During fermentation (or external digestion) of plant parts, high molecular weight molecules, for example, polysaccharides and glycolipids/glycoproteins of plant cell walls, seeds, peel or pod are digested to produce low molecular weight units; just like what happens in our human gut (gastro-intestinal tract). These small molecules are readily absorbed and immediately available for a variety of metabolic processes essential to maintain the organism structure and functions. Page 8

Big polymeric molecules of DNA and RNA are also processed in the fermentation that results in the formation of nucleotides and nucleosides possessing genes regulatory effects. Of note, all biologically active agents become smaller molecules and break “free” from their fibrous polysaccharidic cages/traps; hence easily accessible for biochemical reactions. In addition, this natural way of fruit processing releases mineral micro-elements from their plant - phytate complexes. Their absorption in the gut increases substantially and are highly available for proper functioning of cells, organs, and entire organism. In addition, this highly bio-active fruit preparation is additionally enriched with natural immuno-normalizers, the membrane fragments and lipo-/glyco-peptide complexes derived from the fermenting yeasts and lactobacilli, which contribute additional probiotic nutritional and physiological value. The increasing knowledge of the benefits of lactic acid bacteria and their impact on human daily life is significant. Lactobacilli are natural members of the human gut microbiota highly beneficial to the host. Hence they have been selected for probiotic applications to combat pathogens and boost the immune system. Since ages, lactobacilli have been used all over the world for a large variety of food fermentations, such as those of milk, bread, and vegetables. The fermenting agents (yeasts and lactobacilli) in Page 9

combination with the low-molecular-weight organic mono- and dicarboxylic acid (acetic, citric, malic, glycolic, succinic acids) contribute to remarkable pro-digestive properties of the preparation. HOW BIOREX® MANGOSTEEN IS PRODUCED? BioRex® Mangosteen is a fully natural bioactive food supplement with pronounced antibacterial, immunomodulating, metabolism balancing, and cancer chemopreventive properties. The claims are confirmed by numerous in vitro and in vivo experiments. BioRex® Mangosteen is produced from wild (non-cultivars and non- genetically modified) species of tropical Garcinia mangostana Linn. fruits well-known in ethnopharmacology of South-East Asia countries. Under innovative biotechnological process developed in Japan, the mashed whole mangosteen fruits undergo the process of controlled fermentation by patented culture of Saccharomyces cerevisia yeasts and Lactobaccilus sp. within a 6-months period. Controlled enzymatic splitting is reached due to strictly regulated temperature, oxygen and nutrients supply, and frequent in-process quality tests. The fermentation process is stopped by adding honey of wild bees followed by pasteurization under 55°C. The final BioRex® Mangosteen product is a sour-sweet thick gel reddish-brown of color. To protect the final product from oxidation and bacterial Page 10

contamination, BioRex® Mangosteen gel is packed under vaccum in sterile conditions. The final fermented syrup contain a highly concentrated mixture of low-molecular weight phytonutrients entirely of plant origin with health promoting properties. BioRex® Mangosteen never contains high molecular weight allergens (fully non-allergenic) or admixtures of toxic organic solvents. Its precious thermo-labile components such as xanthones and polyphenolics remain intact, not being exposed to high temperatures. The high quality fermented nutraceuticals like the BioRex© line of fermented tropical fruits fell under the category of fully “BIOLOGICAL”or “ORGANIC” products and could be considered in full compliance with safety and high quality requirements for HALAL food since they are of plant origin, completely free from alcohols or Page 11

other synthetic toxic solvents. Moreover, they do not contain any component of animal origin, pathogenic microorganisms or preservatives. WHAT ACTIVE COMPOUNDS ARE IMPORTANT FOR HEALTH PROMOTING PROPERTIES? There is a commonly accepted view that the most biologically active compounds in mangosteen pod are xanthones. Up to now 68 xanthones have been identified in mangosteen fruits with the most abundunt representatives being alpha-, beta-, and gamma- mangostin. Other constituents are flavonoids, triterpenoids, benzophenones, etc. WHAT KIND OF HEALTH EFFECTS ARE BOOSTED? Summarizing medicinal effects of mangosteen pericarp extracts enriched in xanthones, it has been concluded that that they exhibited the following pronounced properties: ✓ Anti-tumor, ✓ anti-inflammatory, ✓ anti-allergy, ✓ anti-autoimmune, ✓ anti-bacterial, ✓ anti-viral. Page 12

Among xanthones, alpha- and gamma-mangostins are mostly effective as anti-bacterial and anti-fungal agents acting against Propionibacterium acnes, Staphylococcus epidermidis, and Candida albicans. ANTI-CANCER EFFECTS OF MANGOSTEEN XANTHONS Anti-tumor effects are mainly ascribed by xanthon, prenylated polyphenol, and anthocyanin - exerted cytotoxicity towards cultivated tumor cells (pro-apoptotic and cell cycle-inhibiting action). Their regulation of microRNA controlling tumor cell growth has been also suggested. A recent study disclosed that mangosteen xanthons inhibited sphingomyelinase, an enzyme involved in cell death, cell proliferation, and differentiation. Mangosteen xanthons have been shown to inhibit topoisomerases, which are classical targets for anti-cancer drugs such as etoposide and irinotecan. They also inhibit transformation of normal into breast cancer cells as well as the proliferation of breast and colon cancer cells in cultures. Cancer chemopreventive properties of mangosteen constituents are rather promising because they can block tumor initiation, tumor progression and promotion through several mechanisms starting from the inhibition of aromatase, an enzyme catalyzing the biosynthesis of estrogens from androgens, and thus inhibiting promotion of hormone-responsive breast cancers. Page 13

Collectively, mangosteen xanthones and polyphenolics modulate pro-apoptotic, anti-proliferative, and anti-metastatic signaling pathways in tumor cells. The effects of mangosteen xanthones on breast cancer have been examined in two studies using mammary BJMC3879 cancer cells xenografted into Balb/c mice. Subcutaneous α-MG [Shibata et al, 2011] and dietary Panaxanthone (75%–85% α-MG and 5%–15% γ- MG) [Doi et al, 2009] significantly suppressed tumor volumes and metastastic expansion in this cancer model. In vitro, α-MG induced apoptosis, cell cycle arrest, activation of caspases-3 and -9, cytochrome c release and the loss of mitochondrial potential in BJMC3879 cells. The majority of in vivo studies examining the anti-cancer activity of mangosteen xanthones have focused on colon cancer. Dietary administration of α-MG significantly inhibited the induction and development of aberrant crypt foci (ACF) in a chemically-induced rat model of colon carcinogenesis. Less dysplasia, fewer lesions and decreased cell proliferation were also detected in α-MG-treated rats [Nabandith et al, 2004]. Dietary α-MG reduced tumor mass of colon cancer HT-29 xenografts. In this study, xanthones and their metabolites were detected in serum, tumor, liver, and feces of these mice. In vitro analysis confirmed that α-MG inhibited HT-29 Page 14

proliferation and decreased BcL-2 and β-catenin expression [Chitchumroonchokchai et al, 2013]. Page 15

ANTI-INFLAMMATORY EFFECTS OF MANGOSTEEN XANTHONES Researchers examined the inhibitory effects of α-MG on the secretion of pro-inflammatory mediators by transformed and primary human cells. α-MG inhibited the secretion of IL-8 or TNF-α by human cell lines from various tissue origins challenged with a pro-inflammatory insult. Surprisingly, α-MG further stimulated the basal and LPS- stimulated secretion of TNF-α in primary cultures of human monocyte- derived macrophages cells [Gutierrez et al, 2013]. Studies showed that both intraperitoneal and oral administration of α-MG, 1- isomangostin, or mangostin triacetate had anti-inflammatory activities in several rat models of inflammation [Shankaranarayan, 1979]. Orally administered α- and γ-MG also exhibited anti-inflammatory activity in a mouse model of ovalbumin (OVA)-induced allergic asthma [Jang, 2012]. OTHER HEALTH BENEFITS The clinical research results showed that alpha-mangostin potentially possesses neuroprotective effects in a cellular model of PD. Alpha- mangostin is a small lipid-soluble molecule with the potential to pass the blood-brain barrier [Janhom & Dharmasaroja, 2015]. This study has, for the first time, demonstrated that alpha-mangostin rescues apoptosis in dopaminergic SH-SY5Y cells treated with MPP+, a cellular model of Parkinson's disease. The results suggest that cyto- protection of alpha-mangostin against MPP+-induced apoptosis may Page 16

be associated with the reduction of ROS production, modulating the balance of pro- and antiapoptotic genes, and suppression of caspase-3 activation. MPP+ is a neurotoxin used to generate animal and cellular models of Parkinson's disease. Effect of alpha-mangostin on cell viability of MPP+-treated SH-SY5Y cells. Cells were exposed to 0, 2.5, 5, and 10 μM of alpha-mangostin (α-M) in the presence of 1000 μM MPP+ for 48 hours. Data are expressed as mean ± SEM (n = 3) of percentage to untreated cells.  ∗ P < 0.01;  ∗∗ P < 0.001. BIO REX® Mangosteen can also help to enhance immune system of healthy individuals. A randomized, double blinded, placebo- controlled study was conducted in 59 healthy human subjects (40– 60 years old). The xanthone-rich mangosteen intake increased mean values for peripheral T-helper cell frequency (P = .020) and reduced the serum C-reactive protein concentration (P = .014). Increases in peripheral CD4/CD8 double-positive (DP) T-cell frequency and serum complement C3, C4, and interleukin (IL)-1α concentrations were significantly higher in the experimental group than in the Page 17

placebo group (DP, P = .038; C3, P = .017; C4, P = .031; IL-1α, P = .006). The results indicated that the intake of an antioxidant-rich product significantly enhanced immune responses and improved the subject's self-appraisal on his or her overall health status (Tang, 2009). The mangosteen pericarp water-soluble fraction containing primarily polyphenolics (proanthocyanidins) has been found a potent inhibitor of alpha-amylase, an enzyme involved in polysaccharide metabolism activity of which is highly increased in diabetes. Some papers are dedicated to anti-acne and UV-protective skin effects of alpha- mangostin. Xanthon isogarcinol extracted from mangosteen was shown to inhibit calcineurin enzyme, which plays an important role in immune regulation. On the grounds of animal studies, it has been suggested that isogarcinol could serve as non-toxic oral immuno-modulatory drug for long-term medication in autoimmune diseases (Mengqi, 2016). The research suggests that isogarcinol has the potential to be an effective therapeutic agent of low toxicity for treating MS and other autoimmune diseases. Page 18

HOW TO USE AND SAFETY RECOMMENDATIONS BioRex® Mangosteen fermented preparations are recommended in dosages 1-2 sachets twice a day. One sachet is diluted in ½ cup of warm water or can be consumed non-diluted. In the case of gastric hyperacidity the product should be dissolved. Always start new treatment in a progressive mode, with 1 sachet per day for the first three days, for optimal body adaptation. BioRex® Mangosteen should be kept in a dry, cool, and light- protected place. An open sachet should be used immediately, in order to avoid microbial contamination and oxidative degradation of active components. Analogously, diluted product should better be discarded, if not consumed immediately. Numerous animals studies and a few human trials showed complete safety of mangosteen products without any adverse or toxic effects. Although no adverse events were reported, the potential long term toxicity of such products could be exerted in definite groups of patients particularly sensitive to mangosteen fruit components. BioRex® Mangosteen contains yeasts and lactobacilii with pro-biotic properties that could be helpful in mantaining normal microbial environment in the gut. Contra-indications are documented severe food allergy, individual intolerance to the components of BioRex® Mangosteen; acute gastric Page 19

ulcer and bloating. Please stop consuming and visit your local physician if symptom persists. BioRex® Mangosteen is not recommended for children under 3 years of age. REFERENCES Chitchumroonchokchai, C.; Thomas-Ahner, J.M.; Li, J.; Riedl, K.M.; Nontakham, J.; Suksumrarn, S.; Clinton, S.K.; Kinghorn, A.D.; Failla, M.L. “Anti-tumorigenicity of dietary α- mangostin in an HT-29 colon cell xenograft model and the tissue distribution of xanthones and their phase II metabolites.” Mol. Nutr. Food Res. 2013, 57, 203–211. Doi, H.; Shibata, M.; Shibata, E.; Morimoto, J.; Akao, Y.; Iinuma, M.; Tanigawa, N.; Otsuki, Y. Panaxanthone isolated from pericarp of Garcinia mangostana L. suppresses tumor growth and metastasis of a mouse model of mammary cancer. Anticancer Res. 2009, 29, 2485– 2495. Fabiola, G.O. & Mark, L. F. “Biological Activities and Bioavailability of Mangosteen Xanthones: A Critical Review of the Current Evidence.” Nutrients 2013, 5(8), 3163-3183. Gutierrez-Orozco, F.; Chitchumroonchokchai, C.; Lesinski, G.; Suksamrarn, S.; Failla, M. “α- Mangostin: Anti-inflammatory activity and metabolism by human cells.” J. Agric. Food Chem. 2013, 61, 3891–3900. Jang, H.Y.; Kwon, O.K.; Oh, S.R.; Lee, H.K.; Ahn, K.S.; Chin, Y.W. “Mangosteen xanthones mitigate ovalbumin-induced airway inflammation in a mouse model of asthma.” Food Chem. Toxicol. 2012, 50, 4042–4050. Janhom, P., & Dharmasaroja, P. “Neuroprotective Effects of Alpha-Mangostin on MPP(+)- Induced Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells.” Journal of toxicology, 2015, 919058. Page 20

Mengqi Wang, Yufei Xie, Youxiu Zhong, Juren Cen, Lei Wang, Yuanyuan Liu, Ying Zhu, Li Tong, and Qun Wei “Amelioration of Experimental Autoimmune Encephalomyelitis by Isogarcinol Extracted from Garcinia mangostana L. Mangosteen”, Journal of Agricultural and Food Chemistry 2016 64 (47), 9012-9021, DOI: 10.1021/acs.jafc.6b04145 Nabandith, V.; Suzui, M.; Morioka, T.; Kaneshiro, T.; Kinjo, T.; Matsumoto, K.; Akao, Y.; Iinuma, M.; Yoshimi, N. “Inhibitory effects of crude α-mangostin, a xanthone derivative, on two different categories of colon preneoplastic lesions induced by 1,2-dimethylhydrazine in the rat.” Asian Pac. J. Cancer Prev. 2004, 5, 433–438. Yu-Ping Tang, Peng-Gao Li, Miwako Kondo, Hong-Ping Ji, Yan Kou, and Boxin Ou. Effect of a Mangosteen Dietary Supplement on Human Immune Function: A Randomized, Double-Blind, Placebo-Controlled Trial”, Journal of Medicinal Food.Aug 2009.755-763. Shankaranarayan, D.; Gopalakrishnan, C.; Kameswaran, L. “Pharmacological profile of mangostin and its derivatives.” Arch. Int. Pharmacodyn. Ther. 1979, 239, 257–269. Shibata, M.; Iinuma, M.; Morimoto, J.; Kurose, H.; Akamatsu, K.; Okuno, Y.; Akao, Y.; Otsuki, Y. “α-Mangostin extracted from the pericarp of the mangosteen (Garcinia mangostana Linn) reduces tumor growth and lymph node metastasis in an immunocompetent xenograft model of metastatic mammary cancer carrying a p53 mutation.” BMC Med. 2011, 9, 69 Page 21