European Review for Medical and Pharmacological Sciences 2016; 20: 2418-2424 Bilberry extracts are not created equal: the role of non anthocyanin fraction. Discovering the “dark side of the force” in a preliminary study C. GIZZI1, G. BELCARO2, G. GIZZI2, B. FERAGALLI2, M. DUGALL2, R. LUZZI2, U. CORNELLI2 1Department SMO Biotech, 2Irvine Labs, Circulation Sciences, Chieti-Pescara University, Italy Abstract. – OBJECTIVE: Several experimental Introduction studies and clinical trials support the potential of bilberry (Vaccinium myrtillus L) extracts in pro- moting eye health and circulation. Many active Bilberry or European blueberry (Vaccinium ingredients have been isolated from the berries myrtillus L.) belongs to the genus Vaccinium, which and leaves of the bilberry plant. However, antho- includes several small fruit species, like blueberries cyanins represent the most widely studied bio- (e.g., V. corymbosum, V. angustifolium), cranberry active compounds in this plant. (V. macrocarpon), and lingonberry (V. vitis-idaea). PATIENTS AND METHODS: The aim of this Bilberry grows wild in Europe and Asia, most abun- registry, supplement study was to evaluate the ® dantly in Northern and Eastern Europe, and at higher effects of Mirtoselect (standardized in 36% an- 1 thocyanins and obtained by an industrial ex- elevations in Southern Europe . The bilberry is rec- traction process that preserves the full range of ognized by traditional medicine as an herbal remedy the non-anthocyanin components, mainly natu- with interesting pharmacological activities. Besides ral sugars and polyphenols) in different types of its antioxidant and anti-inflammatory properties, retinal vasculopathies. In total, 140 patients with current research efforts are mainly focused on bil- different types of retinopathy spontaneously de- berry-based strategies for the treatment of eyes and cided to join one of the following groups: stan- 2 dard management (SM) only (n=38); SM associ- vascular disorders, and diabetes mellitus . Several ated with Mirtoselect® supplementation (n=47); active ingredients have been isolated from the ber- SM associated with a generic bilberry extract ries and leaves of the bilberry plant. These include supplementation (n=55). Retinal circulatory pa- flavonoids anthocyanosides, vitamins, sugars and rameters and flow measurements of the retinal pectins found in the berries, and quercetin, catechins, vessels were evaluated at the inclusion and af- tannins, iridoids and phenolic acids found mainly in ter 6-months supplementation. the leaves2. Among these ingredients, anthocyanins RESULTS: Overall, significant improvements in several retinal circulatory parameters such represent the most widely studied bioactive com- as retinal blood flow velocity, with respect to the pounds in this plant. Anthocyanins (from the Greek values at inclusion, were observed in both sup- anthos for flower and kyanose for blue) are flavo- plementation groups, especially in Mirtoselect® noids, water-soluble polyphenolic compounds, con- supplementation group. However, at 6 months, sisting of an anthocyanidin “core” with a sugar moi- inter-group comparison revealed a statistical ety attached (glucose, galactose, xylose, arabinose, advantage in all tested parameters for Mirtose- 3 lect® supplementation groups. No side effects or or rhamnose) at various positions . Anthocyanins tolerability concerns were reported. vary also in the number and position of hydroxyl and CONCLUSIONS: Our registry study suggests methoxyl groups attached to the core of the anthocy- that Mirtoselect® supplementation could repre- anidin structure3. Therefore, although there are less sent an effective and safe integrated approach than 20 naturally occurring anthocyanidins, there are for the treatment of different retinopathies. many hundreds of different anthocyanins. Five main Key Words: anthocyanidins are common in bilberry fruit ex- Mirtoselect®, Anthocyanins, Retinopathy, Metabolo- tracts: cyanidin, delphinidin (these two compounds mics, Bilberry, Vaccinium myrtillus, Phytoequivalent. account for more than 60% of the total anthocyanin 2418 Corresponding Author: Gianni Belcaro, MD; e-mail: [email protected] Mirtoselect® in retinopathy content), malvidin, peonidin, and petunidin. The NHS-UK grading; diabetics with initial prolifera- glycoside forms of these anthocyanidins are mainly tive retinopathy – level R3 in NHS-UK grading; 3-glucosides, 3-galactosides and 3-arabinosides4. retinal post thrombosis; hypertensive retinopathy; The amount of anthocyanins content in Vaccinium glaucoma and ischemic retinopathy) not taking myrtillus L. berries generally ranges from 300 to 698 any vascular drugs for the retinal circulation in the mg/100 g of fresh fruit. However, it varies with cul- 6 months before inclusion. tivar, growing conditions, and degree of ripeness of The retinal problems were further divided into the berry5. a predominantly “hyperflow” retinopathy group According to the European Pharmacopoeia 8.0, that presented increased systolic and diastolic the refined and standardized commercially dry ex- flow (patients with diabetic proliferative and tract of fresh bilberry fruit must contain 32.4 per non-proliferative retinopathy resulting in in- cent to 39.6 percent of anthocyanins, assessed by creased systolic and diastolic flows) and in a pre- High Performance Liquid Chromatography dominantly ischemic retinopathy group that pre- (HPLC) using specific cyanidin-3-glucoside chlo- sented decreased systolic and diastolic flow (pa- ride as absorbance reference value6. However, a tients with post-thrombotic, hypertensive, glauco- decrease in anthocyanin content can occur when ma and ischemic retinopathy, presenting a signifi- there has been an incorrect extract production cant reduction in systolic and diastolic compo- and/or storage7. Recently, there have also been re- nents at the central retinal artery). ported several cases of adulteration8,9. However, Subjects who were pregnant, breastfeeding, or even among bilberry extracts standardized to planning conception were excluded. Also, sub- guarantee a content of anthocyanins of 36%, sig- jects with any degenerative eye disorder were ex- nificant differences exist in the manufacturing cluded. process, and hence in the composition of the Cardiovascular examination indicated that pa- non-anthocyanin fraction (mainly represented by tients had no significant systemic diseases (exclud- natural sugars and organic acids). ing the risk condition of diabetes mellitus and hy- In this study we aim at evaluating the biologi- pertension). Diabetics (not using insulin) and hy- cal relevance of this non-anthocyanin natural pertensives (treated with single angiotensin-con- fraction of bilberry extracts, composed mainly of verting-enzyme inhibitors) were well compensat- sugars and polyphenolic compounds, and ac- ed, with a body/mass index <25. All subjects were counting for the 64% of the total extract. To this informed about the aim of the investigation and purpose, we compared the well known protective treatment procedure, according to the Declaration effects on retinal circulation10-13 of two typologies of Helsinki, and gave their consent. of standardized bilberry extracts obtained through Informed participants (n=140) freely decided different manufacturing processes: Mirtoselect® to be enrolled in the following groups: standard (standardized in 36% anthocyanins and obtained management (SM) only (n=38); SM associated by extracting the full range of the non-anthocyan- with Mirtoselect® supplementation (1 capsule/ in components) and a generic bilberry extract day, corresponding to 160 mg Mirtoselect®) standardized in 36% anthocyanins (produced by (n=47); SM associated with generic bilberry ex- diluting a highly purified anthocyanins-rich ex- tracts supplementation (1 capsule/day, corre- tract with maltodextrins). In particular, we as- sponding to 160 mg of bilberry extract) (n=55). sessed retinal circulatory parameters and flow SM of retinopathies includes sodium chloride and characteristics of the retinal vessels in subjects caffeine restriction, physical exercise, regular affected by retinal vasculopathy and supplement- habits recommendations (diet, sleep) and control ed with different standardized bilberry extracts. of risk factors. The following parameters were evaluated before and at the end of the observa- tional period: 1) edema; 2) capillary microaneu- Patients and Methods risms; 3) dot&blot retinal hemorrhage; 4) hard ex- udates; 5) cotton-wool spots (soft exudates); 6) ar- Patients teriolar vasoconstriction; 7) arteriovenous cross- This was an open-label, registry, supplement ing; 8) atherosclerosis, wall changes, copper wir- study (see14-17, for a complete description of these ing, silver wiring. Patient’s visual acuity was as- studies), conducted in 140 individuals affected by sessed using the standard Snellen chart. The sub- different types of retinopathy (diabetics with jects were included if their Snellen score was at non-proliferative retinopathy – level R1 and R2 in least 6/10. Eye flow, edema and blurring were al- 2419 C. Gizzi, G. Belcaro, G. Gizzi, B. Feragalli, M. Dugall, R. Luzzi, U. Cornelli so evaluated and classified with an arbitrary scale Statistical Analysis (ranging from 0 to 3) based on clinical observa- A percentage of at least 25% female subjects tion: 0 = normal; 1 = minimal alterations; 2 = im- were included in each group. Intra- and inter-group portant alterations; 3 = severe alterations. comparisons of numerical data were performed