The most characteristic compounds present in bilberry fruits are colorful polyphenols belonging to the classes of anthocyanins- and of proanthocyanidins. The pharmacological properties of these substances seem nowadays well ascertained; they are mainly linked to a strong antioxidant capacity, although associated with many other biological activities, such as anti-inflammatory, vasoactive, hypolipemic, hypoglycemic, cell-regenerating, antimicrobial, chemopreventive, etc. Almost all of these properties have been intensively studied, both in vitro and in vivo.
Bilberry anthocyanins and some related aglycones are reported to be potent scavengers of free radicals, as when tested in vitro in the superoxide anion generating system hypoxanthine/xanthine oxidase.(1,2) Anthocyanin extracts of Vaccinium myrtillus fruits have been shown to act both as scavengers against superoxide anion and as inhibitors of lipid peroxidation in rat liver microsomes.(3-5)
Cyanidin and delphinidin chlorides proved to be potent scavengers, interacting with 1, 1-diphenyl-1-picrylhydrazyl (DPPH) free radical: the IC50 values were 2.5 and 4.0 µM, respectively, comparable to that of quercetin.
Cyanidin chloride is the most active compound on CCl4 induced lipoperoxidation.(6)
Laplaud et al.(7) reported that an aqueous extract of V. myrtillus berries protected low-density lipoproteins (LDL) from copper-mediated oxidation.
As reported by Rasetti et al.(8) a proprietary Bilberry extract was capable of protecting apolipoprotein B from UV-induced oxidative fragmentation.
Ichiyanagi et al.(9) studied the activity of 11 major bilberry anthocyanins against hydroxyl radicals (OH°), superoxide anion, and singlet oxigen by using capillary zone electrophoresis. The reactivity of anthocyanins towards OH° was comparable to that of (+)-catechin used as reference substance, and was neither significantly affected by the aglycon structure nor by the conjugated sugar type. On the contrary, the reactivity towards superoxide anion and singlet oxygen were determined by the aglycon structure.
K. Nakanishi et al.(10) have demonstrated that anthocyanins-rich bilberry extracts can prevent the light-induced photo-oxidation of A2E, the major eye lipofuscin. Lipofuscins, a class of orange and fluorescent pigments, accumulate with age in retinal epithelium, and are therefore also known as “age pigments”. They have been suggested to play a critical role in the pathogenesis of age-related macular degeneration (AMD), the major cause of blindness in industrialized countries. When exposed to light, lipofuscins generate singlet oxygen, a reactive oxygen species that trigger degeneration of the visual epithelium. This process was suppressed by bilberry anthocyanins, and protection from retinal photo-oxydation might be an important mechanism for the eye beneficial effects of bilberry anthocyanins.
In a more recent study on gene expression determined by microarray analysis, it was shown that Mirtoselect® anthocyanins can attenuate the expression level of pro-inflammatory genes and restore that of anti-inflammatory genes in an inflammatory cell model, providing a rationale for the anti-inflammatory activity of bilberry anthocyanosides(11) . A catechol moiety on ring B was found critical for the anti-inflammatory activity of anthocyanins(12) . Remarkably, 80% of the anthocyanins of Mirtoselect® belong to this structural type.
Prior et al.(13) comparing the antioxidant capacity (oxygen radical absorbance capacity, ORAC) of different variety of four Vaccinium species found that V. myrtillus and V. angustifolium (low bush) exhibited potent ORAC activity (44.6±2.3 and 45.9±2.2, respectively).
A linear relationship existed between ORAC and anthocyanins (rxy = 0.77) or total phenolic content (rxy = 0.92).
Anthocyanins can also prevent the oxidation of ascorbic acid caused by metal ions by chelating the metals ions and forming an ascorbic acid (copigment)-metal-anthocyanin complex.(14) In addition the anthocyanins extract is reported to inhibit the K+ loss induced by free radicals in human erythrocytes as well as the cellular reactions induced by the oxidative compounds daunomycin and paraquat.(15,16).
Most recent studies(17) indicate that Mirtoselect® is active in protecting the kidneys form damage induced by potassium bromate in mice. Potassium bromate is an environmental pollutant, which can be formed as a by-product in the process of ozone purification of drinking water. It may form free radicals triggering harmful modifications in the kidney tissue. The protective properties of bilberry extract are due to the improved antioxidant capacity of the kidney tissue promoted by bilberry anthicyanins: the reduction of NO production and the improved ability to absorb oxygen radical (ORAC).
Li Bao et al.(18) showed that Mirtoselect® is able to alleviate stress-induced liver damage in mice by both scavenging free radicals activity and lipid peroxidation inhibitor effect. In a following study(19) the same group showed that Mirtoselect® restored alanine aminotransferase (ALT) and reactive oxygen species (ROS) to normal levels, and enhanced mitochondrial complex II activity that was lowered in the experimental conditions.
In a recent study in mice Endotoxin-induced uveitis (EIU)(20), Mirtoselect®, has been shown to reduce levels of nitric oxide and malondialdehyde in eyes and to elevate ORAC, glutathione, vitamin C, superoxide dismutase, glutathione peroxidase activity in eyes.
Moreover, Mirtoselect® increased expression of copper/zinc superoxide dismutase, manganese superoxide dismutase, and glutathione peroxidase mRNA, indicating that Mirtoselect® could attenuate inflammation-induced oxidative stress in EIU by increasing levels of antioxidants.
- Salvayre R., Braquet P., Perruchot Th., Douste-Blazy L., in « Flavonoids and Bioflavonoids, 1981 », L. Farkas, M. Gábor, F. Kállay, H. Wagner (Eds), Elsevier, Amsterdam, 1982, pp 437-442.
- Acquaviva R., Russo A., Galvano F., Galvano G., Barcellona M.L., Li Volti G., Vanella A., “Cyanidin and cyanidin 3-O-beta-D -glucoside as DNA cleavage protectors and antioxidants”, Cell. Biol. Toxicol. 19, 243 (2003).
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- Martín-Aragón S., Basabe B., Benedí J.M., Villar A.M., Phytother. Res. 12 Suppl.1, Second International Symposium on Natural Drugs, 1997, S104-S106 (1998).
- Martín-Aragón S., Basabe B., Benedí J.M., Villar A.M., Pharm. Biol. 37, 109 (1999).
- Morazzoni P., Malandrino S., Pharmacol. Res. Comms. 20, Suppl. 2, 254 (1988).
- Laplaud P.M., Lelubre A., Chapman M.J., “Antioxidant action of Vaccinium myrtillus extract on human low density lipoproteins in vitro: initial observations.”, Fundam. Clin. Pharmacol. 11, 35 (1997).
- Rasetti M.F., Caruso D., Galli G., Bosisio E., Phytomedicine 3, 335 (1996/97).
- Ichiyanagi T., Hatano Y., Matsugo S., Konishi T., “Kinetic comparisons of anthocyanin reactivities towards 2,2′-azobis(2-amidinopropane) (AAPH) radicals, hydrogen peroxide and tert-buthylhydroperoxide by capillary zone electrophoresis”, Chem Pharm Bull (Tokyo). 2004 Apr;52(4):434-8.
- Sparrow JR, Vollmer-Snarr HR, Zhou J, Jang YP, Jockusch S, Itagaki Y, Nakanishi K. A2E-epoxides damage DNA in retinal pigment epithelial cells. Vitamin E and other antioxidants inhibit A2E-epoxide formation. J. Biol. Chem., Vol. 278, Issue 20, 18207-18213 (2003).
- Chen, Jihua, Uto, Takuhiro, Tanigawa, Shunsuke, Kumamoto, Takuma, Fujii, Makoto and Hou, De-Xing. “Expression Profiling of Genes Targeted by Bilberry (Vaccinium myrtillus) in Macrophages Through DNA Microarray”, Nutrition and Cancer, 60:1,43 – 50 (2008).
- Hou DX, Yanagita Y, Uto T, Masuzaki S, and Fujii, M. “Anthocyanidins inhibit cyclooxygenase-2 expression in LPS-evoked macrophages: structure-activity relationship and molecular mechanisms involved. Biochem Pharmacol 70, 417–425, 2005.
- Prior R.L., Cao G., Martin A., Sofic E., McEwen J., O’Brien C., Lischner N., Elhenfeldt M, Kalt W., Krewer G., Mainland C.M., J. Agric. Food Chem. 46, 2686 (1998).
- Sarma A.D., Sreelakshmi Y., Sharma R., Phytochemistry 45, 671 (1997).
- Maridonneau I., Braquet P., Garay R.P. in “Flavonoids and Bioflavonoids, 1981″, L. Farkas, M. Gábor, F. Kállay, H. Wagner (Eds), Elsevier, Amsterdam, 1982, pp 427-436.
- Mavelli I., Rossi L., Autuori F., Braquet P., Rotilio G., in “Oxy Radicals Their Scavenger Syst.”, Proc. Int. Conf. Superoxide Dismutase, 3dr 1982 , G. Cohen , R.A. Greenwald , Elsevier, New York, 1983, pp 326-329.
- Bao L., Yao XS., Tsi D., Yau CC., Chia CS., Nagai H., Kurihara H., “Protective effects of bilberry (Vaccinium myrtillus L.) extract on KBrO3-induced kidney damage in mice”, J. Agric. Food Chem., 56, pag. 420-425 (2008)
- Li Bao, Xin-Sheng Yao, Chin-Chin Yau, Daniel Tsi, Chew-Sern Chia, Hajme Nagai, Hiroshi Kurihara, “Protective effects of bilberry (Vaccinium myrtillus L.) extract on restraint stress-induced liver damage in mice” J. Agric. Food Chem., Published on Wed August 9, 2008.
- Bao L, Abe K, Tsang P, Xu JK, Yao XS, Liu HW, Kurihara H. Bilberry extract protect restraint stress-induced liver damage through attenuating mitochondrial dysfunction, Fitoterapia. Volume 81, Issue 8, December 2010, Pages 1094-1101
- Yao N. Protective effects of bilberry (Vaccinium myrtillus L.) extract against Endotoxin-induced uveitis in mice. J. Agric. Food Chem., 2010, 58 (8), pp 4731–4736
Anthocyanins inhibition of cyclic nucleotide phosphodiesterases
The anthocyanins cyanidin, delphinidin and malvidin 3-O-glucosides and their aglycones are reported to inhibit phosphodiesterase (PDE) isoforms from different sources as retina, choroid, large vessels and platelets. The compounds were more active on retinal than on platelet PDEs and in particular on the retinal calmodulin stimulated enzymes. IC50 of malvidin and delphinidin 3-O-glucosides on calmodulin stimulated enzymes ranged from 5.4 to 35.6 µM. Anthocyanins appeared more active than isobutylmethylxanthine used as reference.(1-3)Expand Bibliography
- Ferretti C., Magistretti M.J., Robotti A., Ghi P., Genazzani E., Pharmacol. Res. Commun. 20, Suppl. 2, 150 (1988).
- Ferretti C., Blengio M., Malandrino S., Pifferi G., XIth International Symposium on Medicinal Chemistry, Jerusalem, Israel, September 2-7 (1990).
- Pifferi G, Malandrino S., Morazzoni P., Ferretti C., XVIth International Conference of the Groupe Polyphenols, Lisbon, July 13-16, 1992 – Polyphenols Actualities 8, 60 (1992).
Anthocyanins antiplatelet activity
Morazzoni and Magistretti (1) studied the antiplatelet activity of Bilberry 36% extract against aggregation induced by ADP, collagen and sodium arachidonate on rabbit platelet-rich plasma (PRP). Bilberry extract was a strong inhibitor of platelet aggregation with IC50 values ranging from 0.36 to 0.81 mg/mL PRP, comparable to those obtained with dipyridamole. Moreover, the Bilberry extract exerted an inhibitory effect on ADP-induced platelet aggregation in rats maintained on extracorporeal circulation. The Bilberry extract when orally administered to rats at doses up to 400 mg/kg, prolonged bleeding time for 24 h, without affecting blood coagulation pathways; administration of 400 mg/kg by oral route to mice, reduced the adhesiveness of platelets to glass micropellets. An anthocyanins extract of V. myrtillus fruits was reported to inhibit platelet aggregation in vitro when induced by ADP or adrenalin on human plasma.(2) The inhibitory effect on platelet aggregation, demonstrated in vitro, was confirmed ex vivo on ADP- and collagen-induced aggregation of platelets obtained from the blood of 30 healthy subjects treated by oral route (480 mg/day for 30-60 days).(3)
- Morazzoni P., Magistretti M.J., Fitoterapia 61, 13 (1990).
- Serranillos M.G., Zaragoza F., Alvarez P., An. Real. Acad. Farm. 49, 79 (1983).
- Pulliero G., Montin S., Bettini V., Martino R., Mogno C., Lo Castro G., Fitoterapia 60, 69 (1989).
Anthocyanins interaction with collagen, phospholipids and proteoglycans
In vitro anthocyanin extracts of V. myrtillus fruits are able to inhibit proteolytic enzymes like elastase, which are involved in the degradation of collagen and other components of the extravascular matrix in certain pathological conditions such as atherosclerosis, pulmonary emphysema, rheumatoid arthritis.(1) Anthocyanin extracts may interact with collagen metabolism, by cross-linking collagen fibres and making them more resistant to collagenase action.(2) A reduction in biosynthesis of polymeric collagen and structural glycoproteins, responsible for thickening of capillary in diabetics, has also been described.(3) Hystochemical and biochemical studies showed that anthocyanins from V. myrtillus interact with phospholipidic constituents of plasma modifying their physical chemical properties and enhancing their resistance to lesive stimuli.(4) Salmona et al.(5) studied the influence of Bilberry 36% extract on membrane viscosity of platelets and confirmed that anthocyanins were able to modify the membrane fluidity due to their high affinity for membrane phospholipids
A local stimulating effect of the anthocyanins from V. myrtillus on the biosynthesis of mucopolysaccharides in granuloma induced by foreign bodies was reported by Mian et al.(6) Mucopolysaccharides are recognized to play an important role in maintaining the integrity of both perivascular tissue and the basal membrane. In an in vitro study, using endothelial cells from human umbilical cord, Piovella et al.(7, 8) reported that anthocyanins induced active phagocytosis of pigment material and intense cell regeneration. A growth promoting activity on fibroblasts and on smooth muscle cells was also reported in the same study. Anthocyanins may facilitate the regeneration both of the cellular component of the vessel wall and of the perivascular tissues, due to their stimulating effect on mucopolysaccharides.Expand Bibliography
- Jonadet M, Meunier MT, Bastide J, Bastide P., “Anthocyanosides extracted from Vitis vinifera, Vaccinium myrtillus and Pinus maritimus. I. Elastase-inhibiting activities in vitro. II. Compared angioprotective activities in vivo”, J Pharm Belg. 1983 Jan-Feb;38(1):41-6.
- Robert A.M., Miskulin M., Godeau G., Tixier J.M. in “Frontiers of Matrix Biology”, L. Robert (Ed), Vol. 7, Karger, Basel, 1979, pp 336-349.
- Boniface R., Miskulin M., Robert L., Robert A.M., “Flavonoids and Bioflavonoids, 1985″, L. Farkas, M. Gábor, F. Kallay (Eds), Elsevier, Amsterdam-Oxford-New York-Tokio 1986, pp 293-301.
- Curri B.S., Bombardelli E., Therapia Angiol. 32, 117 (1976).
- Salmona M., Masturzo P., Cini M., Morazzoni P., Magistretti M.J., in “Flavonoids in Biology and Medicine III: Current Issues in Flavonoids Research.”, N.P. Das (Ed.), Singapore 1990, pp 475-480.
- Mian E, Curri SB, Lietti A, Bombardelli E., “Anthocyanosides and the walls of the microvessels: further aspects of the mechanism of action of their protective effect in syndromes due to abnormal capillary fragility”, Minerva Med. 1977 Oct 31;68(52):3565-81.
- Piovella C., Curri B.S., Piovella M., Piovella F., Therapia Angiol. 35, 119 (1979).
- Piovella F., Ricetti M.M., Almasio P., Feoli F.R., Pesenti Campagnoni M., Castagnola C., Min. Angiol. 6, 135 (1981).
Effect on arteriolar vasomotion of anthocyanins
The arteriolar vasomotion, a rhythmic variation of diameter of arterioles in microvascular network, influences the microvascular mechanism which regulates the formation of interstitial fluid. Colantuoni et al.(1) studied the effects of Bilberry 36% extract on arteriolar vasomotion in two experimental models: the cheek pouch of anaesthetized hamster and the skin fold window preparation (muscular type) of un-anaesthetized hamster. Bilberry 36% extract (5-10 mg/kg i.v.) induced vasomotion suppressed by anaesthetic in cheek pouch arterioles and terminal arterioles, and increased vasomotion frequency in the skeletal muscle arteriolar network. These findings indicate that the Bilberry extract may prevent or control interstitial fluid formation and contribute to control the blood flow redistribution in the microvascular network.Expand Bibliography
Recent investigations(1) on the chemopreventive properties of bilberry anthocyanins have shown that Mirtoselect® can reduce, in a dose-dependent way, the development of intestinal adenoma in rodents. The activity was related to a high concentration of anthocyanins in the target organ, sharply contrasting with very low systemic concentrations detected in plasma. A possible mechanism of action has been recently proposed(2) based on the interaction of anthocyanins with several tyrosine kinases (RTKs) believed to play a crucial role in carcinogenesis and tumor progression. Mirtoselect® showed a broad sperctum tyrosin-kinase inhibitory activity, suggesting potential chemopreventive activity. According to these data, a clinical pilot study on Mirtoselect® for colorectal cancer chemoprevention was carried out on 25 colorectal cancer patients scheduled to undergo resection of primary tumor or liver metastasis(3). The patients received 1.4, 2.4 or 5.6 grams of Mirtoselect® daily for 7 days before surgery. In tumor tissues, proliferation decreased by 7% compared to pre-intervention values. Urine, blood and target tissue levels of anthocyanins were also measured, and results are consistent to the levels detected in rodent model.Expand Bibliography
- Thomasset S, Berry DP, Cai H, West K, Marczylo TH, Marsden D, Brown K, Dennison A, Garcea G, Miller A, Hemingway D, Steward WP, Gescher AJ, “Pilot study of oral anthocyanins for colorectal cancer chemoprevention”, Cancer Prev Res (Phila Pa). 2009 Jul;2(7):625-33.
- Teller N, Thiele W, Marczylo TH, Gescher AJ, Boettler U, Sleeman J, Marko D., “Suppression of the kinase activity of receptor tyrosine kinases by anthocyanin-rich mixtures extracted from bilberries and grapes”, J Agric Food Chem. 2009 Apr 22;57(8):3094-101.
- Cooke D, Schwarz M, Boocock D, Winterhalter P, Steward WP, Gescher AJ, Marczylo TH., “Effect of cyanidin-3-glucoside and an anthocyanin mixture from bilberry on adenoma development in the ApcMin mouse model of intestinal carcinogenesis–relationship with tissue anthocyanin levels”, Int J Cancer. 2006 Nov 1;119(9):2213-20.
Anthocyanins cohort studies
In published prospective cohort study on 34 489 postmenopausal women in the US followed up for 16 years (1986 to 2003) asignificant inverse association was observed between dietary intake of anthocyanins (main anthocyanins source have been strawberries, blueberries and red wine) and Coronary heart disease, Cardiovascular disease, and total mortality(1).
Furthermore in a large prospective study(2) including 87,242 women from the Nurses’ Health Study (NHS) II, 46,672 women from the NHS I, and 23,043 men from the Health Professionals Follow-Up Study (HPFS), a higher total anthocyanin intake was significantly associated with a reduced risk of incident hypertension even after controlling for a large number of covariates including family history, physical activity, BMI, and dietary factors previously associated with blood pressure; the magnitude of the reduction was greatest in the <60-y-old participants. The authors report that among individual anthocyanins, cyanidin, malvidin, and pelargonidin were associated with reduced rates of hypertension, and results were generally stronger in participants <60 y of age (P for age interaction interaction, 0.05 for cyanidin, 0.01 for malvidin, and 0.001 for peonidin and petunidin). The 2 main sources of anthocyanins across cohorts were blueberries and strawberries. In pooled analyses of the <60-y-old group, the consumption of more than one serving of blueberries per week compared with no blueberry intake was associated with a 10% reduction in hypertension (RR: 0.90; 95% CI: 0.81, 0.98; P = 0.02). The consumption of more than one serving of strawberries per week was not significantly associated with a reduction in hypertension (RR: 0.97; 95% CI: 0.94, 1.00; P = 0.34). The results were not materially different when we restricted analyses to nonsmokers or nondrinkers.
In a last study(3) analyzing after exclusions, data from 70,359 NHS participants, 89,201 NHS II participants, and 40,420 HPFS participants, a higher consumption of anthocyanins and anthocyanin-rich fruit was associated with a lower risk of type 2 diabetes and the researchers concluded that while the data suggest an inverse association between intake of anthocyanins and anthocyanin-rich foods (eg, blueberries and apples/pears) and type 2 diabetes mellitus (T2DM) in US men and women, it is possible that these findings reflect other dietary components that co-exist in anthocyanin-rich foods, and randomized trials will be needed to establish the effects that can be specifically attributed to anthocyanins. Further research on anthocyanin-rich foods may lead to more specific recommendations on consumption of fruit, which may contribute to the prevention of T2DM.Expand Bibliography
- Pamela J Mink, Carolyn G Scrafford, Leila M Barraj, Lisa Harnack, Ching-Ping Hong, Jennifer A Nettleton, and David R Jacobs Jr “Flavonoid intake and cardiovascular disease mortality: a prospective study in postmenopausal women”, Am J Clin Nutr 2007;85:895–909.
- Aedı´n Cassidy, E´ilis J O’Reilly, Colin Kay, Laura Sampson, Mary Franz, JP Forman, Gary Curhan, and Eric B Rimm, Habitual intake of flavonoid subclasses and incident hypertension in adults, Am J Clin Nutr. 2011 Feb;93(2):338-47. Epub 2010 Nov 24.
- Wedick NM, Pan A, Cassidy A, Rimm EB, Sampson L, Rosner B, Willett W, Hu FB, Sun Q, van Dam RM., Dietary flavonoid intakes and risk of type 2 diabetes in US men and women. Am J Clin Nutr. 2012 Feb 22. [Epub ahead of print]