Berry Fruits As a Source of Biologically Active Compounds: the Case of Lonicera Caerulea

Home , Lonicera caerulea, Lonicera involucrata

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2007, 151(2):163–174. 163 © I. Svarcova, J. Heinrich, K. Valentova BERRY FRUITS AS A SOURCE OF BIOLOGICALLY ACTIVE COMPOUNDS: THE CASE OF LONICERA CAERULEA

Irena Svarcovaa*, Jan Heinrichb, Katerina Valentovaa a Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic b WALMARK a.s., Oldrichovice 44, 739 61 Trinec e-mail: [email protected]

Received: September 7, 2007; Accepted: October 12, 2007

Key words: Lonicera caerulea/Berries/Polyphenols/Biological activity

Background: Lonicera caerulea L. (blueberry honeysuckle, Caprifoliaceae) is a traditional crop in northern Russia, China, and Japan. Its fruits are little known as edible berries in North America and Europe. This review deals with the botany and chemical composition of L. caerulea and the biological activity of its main constituents, focusing on the potential health benefi ts of the berries. Methods and Results: PubMed, Science Direct and ISI Web of KnowledgeSM databases were used for this paper. Literature sources include the period 1935–2007. L. caerulea berries a are rich source of phenolic compounds such as phenolic acids as well as anthocyanins, proanthocyanidins and other fl avonoids, which display potential health promoting eff ects. Chemopreventive, antimicrobial, anti-adherence and antioxidant benefi ts, among others are described for these compounds. Conclusions: The potential of L. caerulea berries to prevent chronic diseases such as diabetes mellitus, cardiovascular diseases and cancer seems to be related above all to their phenolic content.

INTRODUCTION Caprifoliaceae family and is native to the Northern Hemisphere (Fig. 1) (ref.1). The nomenclature of honey- Lonicera caerulea L. (blue berry honeysuckle, Capri- suckles is complicated; a lot of species are sometimes clas- foliaceae) is a traditional crop used in folk medicine in sifi ed as varieties and have many synonyms. Large genetic northern Russia, China, and Japan but its fruits are little variation is common among the Lonicera genus. For exam- known as edible berries in North America and Europe1. ple 10 common species of L. involucrata are only hybrids In recent years a large number of studies have investigated of one known origin11. Many species of Caprifoliaceae the therapeutic eff ects of various fruits and vegetables in family have non-edible fruits and are cultivated only as the prevention of a range of diseases and there is increas- decorative shrubs or rambling plants12, especially for their ing interest in herbal medicine products. Berries consti- glorious blooms and ornamental fruits1. The fruit colour, tute one of the most important sources of potential health which ranges from white or yellow to scarlet or navy blue, supporting phytochemicals in the human diet2. They are is used as an indicator of plant maturity1. Honeysuckles a rich source of ascorbic acid and phenolic compounds, are also favoured because of their extreme hardiness to particularly phenolic acids, anthocyanins, proanthocya- the cold13. A few species are used in indigenous medicine nidins and other fl avonoids. These compounds provide as antipyretic, stomachic, diuretic and antidysenteric in the pigmentation of fruits and prove benefi cial to human India14. health3, 4. Their biological activities include: protection L. caerulea L., also known as blue honeysuckle, honey- against the incidence and mortality rates of cancer5, pro- berry, edible honeysuckle or sweet berry honeysuckle15 is tection against ischemic heart disease mortality6 and as native to the northern temperate zone, especially Russia well as they have antitumorigenic7, antimicrobial8, anti- (Kamchatka Peninsula, Siberia), North Eastern Asia, inlammatory-allergic9 and antimutagenic properties10. and Japan15, 16. In Europe, it occurs rarely in the Alps The aim of this paper was to review the current literature and Scandinavia3. L. caerulea is currently commercially on berry-derived biologically active compounds, with the produced in Russia and Japan, but this species was un- focus on L. caerulea. known as an edible berry in North America until very recently17. Native L. caerulea plants grow from 0.8 to 3.0 m tall, 1. BOTANICAL DESCRIPTION but under cultivation the shrubs reach 1.0 m wide to 1.8 m tall17. They do not require special soil type; even wet sandy The genus Lonicera, which includes almost 180 spe- or loamy soil is suitable, pH can range from 5 to 7 and cies of deciduous or evergreen shrubs, belongs to the the organic proportion can be higher12. Plants have strong 164 I. Svarcova, J. Heinrich, K. Valentova

and season. The highest level of sugars has been measured Lonicera caerulea: in January, and then the saccharide content decreased Kingdom: Plantae to May and after that increased again. According to the Subkingdom: Tracheobionta – Vascular plants literature18 the dominant sugar is saccharose with 50–90 % Superdivisio: Spermatophyta – Seed plants of total saccharides. The content of stachyose and raffi - Divisio: Magnoliophyta – Flowering plants nose changes dramatically during the year. While it is neg- Class: Magnoliopsida – Dicotyledons ligible from April to November, it increases rapidly from Subclass: Asteridae November to March. Fructose and glucose constitute Order: Dipsacales only small proportion of total sugars and their content Family: Caprifoliaceae does not change over the year. Accumulation of raffi nose Genus: Lonicera L. – Honeysuckle and/or stachyose has strong relationship to the freezing Species: Lonicera caerulea L. 18 – Sweetberry honeysuckle tolerance and desiccation . L. caerulea leaves comprise three glucosides, named caerulosides A, B (Fig. 2) and C. These compounds are formed from secologanin attached Fig. 1. Botanical subsumption of Lonicera caerulea L. through acetal bonds to C-4'and C-6' of the saccharide part of loganin and sweroside, respectively. Caerulosides tolerance to severe low-temperature conditions. They can are the fi rst bis-iridoids, that are composed of two units survive a temperature of –46 °C without damage17, 18. The of iridoids bound by acetal linkages22, 23. freezing tolerance of perennial plants increases in fall and winter to prevent injury under cold conditions. It is OH known as cold acclimation and seems to be connected O H H with the content and accumulation of specifi c type of H H3COOC H O carbohydrates and proteins (see below). The raffi nose H family of oligosaccharides have been shown to be poten- CH2 tial cryoprotectants because of their capacity to modify O O 18, 19 O O the freezing behaviour of aqueous solutions . Also the O O O presence of galactose-containing oligosaccharides strongly O O OH O OH H correlates with increases in freezing, as well as desiccation H OH tolerance18. Blue honeysuckle shrubs are long-lived and OH 12 COOCH3 can survive 25 to 30 years . COOCH3 H Blue honeysuckles are not self-pollinating so at least H two single plants are required. Fruits become ripe very H O early in May or June in European conditions. The plants H O can begin fruiting within one year after planting and after H2C H2C O O three years almost 500 g of fruits can be acquired from O O HO one plant12. Fruit shapes are oval to long and dark navy HO HO OH blue to purple in color15 with blue waxy coating according HO OH OH to the genotype12. They can grow up to more than 2 cm OH long and weigh more than 1.5 g17. Their fl avour is similar Fig. 2. Caerulosides A and B. to that of bilberries or black currants and it can vary from bitter to sweet15. 2.2. Lipids Blueberry honeysuckle fruit contain only 1.52 % lipids. 2. CONSTITUENTS AND THEIR The lipid fraction is mainly hydrocarbons, sterols, tria- BIOLOGICAL ACTIVITY cylglycerols and phosphatidylcholine (Table 1a) (ref.21). The weight of fatty acids is 0.89 % total weight of ber- L. caerulea berries and their juice contain sac- ries. Main acids include palmitic (38.2 %), oleic (27.6 %), charides, lipids, proteins, organic acids and polyphe- stearic (14.7 %), myristic (9.2 %), linolic (5.9 %), palmi- nols as major components and also ascorbic acid toleic (2.8 %) and lauric acid (1.6 %). The entire weight (45–93 mg · 100 g–1)(ref.15), vitamin B, magnesium, phos- of the unsaponifi able proportion (sterols, alcohols and phorus, calcium and potassium as minor compounds20. hydrocarbons) is 0.46 % of the berries, and includes α- amyrin (29.6 %), β-amyrin (24.7 %), stigmasterol (14.8 %) 2.1. Saccharides and ursolic acid (11.5 %) (Table 1b) (ref.24). Ursolic acid L. caerulea fruit contain 7.20 % saccharides. Free sac- and its 19-hydroxy derivative pomolic acid were reported charides include 3.2 % glucose and 2.9 % fructose, bound to inhibit proliferation and DNA synthesis in HL-60 saccharides are 0.8 % glucose, 0.2 % galactose and 0.1 % leukemia cell line at micromolar concentrations25. Ursolic arabinose21. Five kinds of saccharides have been identifi ed acid, β-amyrin, and glucoside of β-sitosterol inhibits the in L. caerulea shoot apices according to Imanishi et al18. growth of HCT 116 human colon cancer cells and PC-12 The content of fructose, glucose, saccharose, raffi nose adrenal pheochromocytoma cells at micromolar concen- and stachyose varies depending on climatic conditions trations26. Berry fruits as a source of biologically active compounds: the case of Lonicera caerulea 165

Table 1. Lonicera caerulea berries a) lipid fraction b) unsaponiﬁ able matter. a) b)

Compounds Relative content (%) Compounds Relative content (%) Hydrocarbons+sterols 32.0 α-Amyrin 29.6 Triglycerols 27.0 β-Amyrin 24.7 Phosphatidyl choline 21.0 Stigmasterol 14.8 Free fatty acids 7.0 Ursolic acid 11.5 Phosphatidic acid, 6.0 Triterpens 5.3 phosphatidyl serine + min. phospholipids Sitosterol 4.9 Digalactosyl diglycerol 4.0 Oleanolic acid 3.3 Phosphatidyl ethanolamine 3.0 Δ7-Stigmastenol 3.3 Hydrocarbons+sterols 3.3 Campesterol 2.0 Brassicasterol 0.6

2.3. Other components antioxidants in in vitro models, but there is lack of infor- Fruit content includes 14.62 % dry matter21, of which mation about whether they can remain a suffi cient time 14.8 % is soluble fi ber27. Organic acids (12.2 %) are repre- in effi cient chemical forms in the human body29. A large sented by citric (3.7 %), malic (18.0 %) and others (2.4 %) proportion of the ingested polyphenols from berries are (ref.20). not taken up into the circulation and pass through the up- per GIT into the large intestine where they may be trans- 2.4. Phenolic compounds formed or broken down by the indigenous microfl ora34. Small berries are one of the most important sources Phenolic compounds are metabolized by deconjugation of phenolic compounds with potential health promoting and reconjugation reactions. They are hydrolyzed to their eff ects. L. caerulea contain a huge amount of these com- free aglycones, and then they are conjugated by methyla- pounds. The phenolic content is dependent on the degree tion, sulphation, glucuronidation or their combination. of maturity, genetic diversity, preharvest climatic, posthar- The subsequent metabolic pathway is similar to that of vest storage conditions and processing20. We prepared a drug metabolism. Since drugs are usually administrated phenolic fraction from L. caerulea var. kamtschatica (4 % in hundreds of milligrams in one dose while dietary phe- of fresh fruits) containing 33.5 % of phenolics, including nolics are presented in much lower concentration, drugs anthocyanins (18.5 %), fl avonoids and phenolic acids21. usually saturate these pathways. When food phenolics are The polyphenols comprise a range of chemical class- administrated at pharmacological doses, they are found es of secondary plant metabolites that they all share the in free forms in the blood35. Large doses are metabolized ability to act as chain breaking antioxidants28. Phenolic primarily in the liver. Small doses may be metabolized in compounds are essential for the growth and reproduc- the intestinal mucosa, the liver has a secondary role in tion of plants and are produced as a response to plant their metabolism35. Hollman et al.36 proposed, based on injury by pathogens. At low concentrations they also indirect evidence, that fl avonoid glycosides actually may protect food from oxidative deterioration. At high con- be absorbed intact in the small intestine, using sodium-de- centrations they or their oxidation products may interact pendent glucose transporter 1 (SGLT1). On the one hand with proteins, carbohydrates and minerals29. The health this postulate has been confi rmed37. On the other hand it benefi ts of polyphenols are usually linked to two proper- has been demonstrated that the effi ciency of such absorp- ties: (i) inhibition of certain enzymes such as xanthine tion is dramatically suppressed by effl ux of at least some oxidase, aldose reductase, and (ii) antioxidant activity30. fl avonoid glycosides by the apical transporter multidrug Polyphenols can protect other food components such as resistance-associated protein 2 (MRP2) (ref.38). Some carotenoids and vitamin C and also digestive enzymes fl avonoid glycosides could be hydrolyzed in the small and gut epithelial cells from oxidation due to free radicals intestine39. If the fl avonoid glycosides are able to enter generated in stomach31, 32. the intestinal epithelial cells (enterocytes), which may Little is known about the bioavailability, absorp- include shredded cells, they may be hydrolyzed by broad- tion and metabolism of polyphenols in humans and it specifi c β-glucosidase (BSβG) (ref.40). For some fl avonoid is likely that single groups of fl avonoids have diff erent glycosides, lactase phloridzin hydrolase (LPH), located pharmacokinetic properties33. Phenolics are powerful in the brush border of the mammalian small intestine, 166 I. Svarcova, J. Heinrich, K. Valentova could perform this hydrolysis41. It has also been shown, no known adverse eff ect of caff eic acid in humans50,51. that phenolics, which have rhamnose in their molecule, Ferulic acid could be absorbed by passive diff usion or by cannot be absorbed through the small intestine. They are facilitated transport that appears not be saturated even at degraded by the action of rhamnosidases produced by the a luminal concentration of 50 μmol/l (ref.52). The absorp- colonic microfl ora29. tion of ferulic acid and also free cinnamic acid are controlled by the Na+/dependent carrier-mediated transport 2.4.1. Phenolic acids process in rat jejunal segments53. Some food products also Phenolic acids form approximately one third of the to- contain oxidatively coupled product of the ferulic acid, tal intake of plant polyphenols in the human diet42. Daily the diferulic acid54. Coumaric acid is a hydroxy derivative consumption of phenolic acids has been estimated as 25 of cinnamic acid. There are three isomers, o-coumaric, to 1000 mg (ref.43). They are present in free and bound m-coumaric and p-coumaric acid, that diff er by the po- forms in plant material. Bound phenolic acids may be sition of the hydroxy substitution of the phenyl group. linked through ester, ether, acetyl or other bonds42. Simple p-Coumaric acid is the most abundant isomer in nature, phenolic acids may also be formed by colonic microfl ora has antioxidant properties55 and is believed to reduce the from ingested fl avonoids44. risk of stomach cancer56. Total content of phenolic acids in L. caerulea berries ranges from 2845.8 ± 141.0 to 5418.2 ± 228.0 mg · kg–1, 2.4.2. Flavonoids dry weight (DW) (ref.45). Chlorogenic (0.42 %), caff eic Flavonoids are polyphenolic compounds, whose (0.14 %) and ferulic acid (0.10 %) were the most abun- structure is formed by the diphenyl propane skeleton dant in our phenolic fraction of L. caerulea var. kamts- (C6-C3-C6). The diff erences within each group fl ow from chatica berries; the content of protocatechuic, gentisic, variation in numbering and order of the hydroxyl groups, rosmarinic, and vanillic acids was 0.08 % in total21. Other as well as the nature and extent of alkylation and glyco- hydroxycinnamic acid and hydroxybenzoic acid deriva- sylation of these groups. The degree of hydroxylation is tives are mentioned in the literature, especially m-cou- a determinant for their tendency to degradation in the maric and p-coumaric. The amount of hydroxycinnamic colon and products formed by colonic microfl ora. The ab- acids derivatives is described as comparable to blueber- sence of hydroxyl group in the molecule prevents the deg- ries or blackcurrant46. Also dimethoxycinnamic, hydroxy- radation of the ring structure; the degree of hydroxylation caff eic, gallic, o-pyrocatechuic, protocatechuic, salicylic, magnifi es the tendency to degradation. The absence of the p-hydroxyphenylacetic and p-hydroxyphenyllactic acid are methyl group causes a decrease in the tendency to degra- reported45. dation57. Food fl avonoids are usually glycosylated mostly Free phenolic acids stand for only a minor portion with glucose or rhamnose, but galactose, arabinose, xy- of phenolic acids. Their amounts ranged from 1.7 to 4.2 lose; glucuronic acid and other sugars can also be found. % for all berries. Caff eic, ferulic and p-coumaric acids The number of sugar molecules can be one, two or three predominate. Free phenolic acids levels do not exceed in diff erent possible positions of the ring substitution. The taste threshold and they do not infl uence the taste of the glycosylation infl uences chemical, physical and biological berries45. properties of these compounds35. Bound phenolic acids are present in ester (69.7 %) The extent of absorption and bioavailability of drugs form. L. caerulea again contains m-coumaric acids as the has long been known to be aff ected by membrane transport- dominant acid. Also caff eic, ferulic, gallic, p-hydroxyphe- ers, mainly effl ux transporters, in addition to metabolism. nylacetic, p-hydroxyphenyllactic and vanillic acid occur The traditional effl ux transporter for drugs, e.g. P-glyco- in minor amounts. Phenolic acids bound by glycosidic protein, does not seem to be involved in the transport of linkages constitute 28.6 %. The majority are represented fl avonoids. Other transporters have been found to play a by hydroxycinnamic acid derivatives (61.1 %), especially role, e.g. the absorptive transporter SGLT1 (ref.38), the m-coumaric. The content of ferulic, gentisic, protocate- absorptive monocarboxylate transporter (MCT), MRP2, chuic and vanillic acids does not reach 50 mg · kg–1, DW but probably also other MRP isoforms, for glucuronide (ref. 45). and sulphate conjugates38, 58. The metabolism of fl avonoids was initially described Chlorogenic acid is formed by the esterifi cation of caf- to be mediated by cytochrome P450 (CYP) enzymes59 in feic acid with quinic acid. It can be also degraded to caf- liver microsomes from induced rats and from humans, but feic and quinic acid by esterases produced by the colonic it has never been shown to be important in vivo or in in- microfl ora47. Caff eic acid is known as an antioxidant both tact cells, where conjugative metabolism may be expected in vitro and in vivo48. While both caff eic and chlorogenic to compete with oxidation60. Glucuronic acid conjugates acid have been reported to be absorbed in humans49, caf- of fl avonoids have been well-documented with respect to feic acid absorption is nevertheless hampered when it is both the molecular site of glucuronidation and the UD esterifi ed with quinic acid. Caff eic acid is still listed un- P-glucuronyltransferase (UGT) isoforms involved61. One der older Hazard Data sheets as a potential carcinogen of the tea fl avonoids, epicatechin gallate (ECG), showed because of two early experiments on rats and mice. More only sulphate conjugation62. Other metabolic pathways recent data show that bacteria in the rat’s guts may alter include O-methylation by soluble catechol-O-methyltrans- the formation of caff eic acid metabolites. There have been ferase (COMT) (ref.63) or by bacterial enzymes. Bacteria Berry fruits as a source of biologically active compounds: the case of Lonicera caerulea 167 from faecal fl ora may be responsible for the hydrolysis of absorbed from the colon following the deglycosylation71. fl avonoids glycosides as well as fl avonoid glucuronides After intravenous dose, quercetin can be detected in urine, and sulphates. The reaction proceeds via degradation of in particular after hydrolysis with β-glucuronidase/aryl the fl avonoid backbone into numerous phenolic and car- sulphatase72. The main route of quercetin excretion is as boxylic acid products64, as well as carbon dioxide65. Many carbon dioxide, 23 – 81 % of the dose, measured by trap- of these products are absorbed and can be detected in ping exhaled air73. Apigenin is a nontoxic dietary fl avonoid human urine64. Other types of metabolites are those result- that has been shown to have anti-tumour and anti-infl am- ing from oxidation by reactive oxygen species (ROS)66. matory activities (Fig. 3). Apigenin can block the forma- Covalent binding of oxidized quercetin to DNA and cel- tion of uric acid leading to benefi cial eff ects in gout74. lular protein has been demonstrated in human cells67. Catechin and epicatechin are epimers, with (-)-epicatechin and (+)-catechin being the most common optical isomers 2.4.2.1. Flavonols, fl avons and fl avan-3-ols found in nature (Fig. 3) (ref.75). Epicatechin can reduce Flavonols (e.g. quercetin) have a similar C-ring struc- the risk of four of the major health problems: stroke, heart ture with a double bond in the 2–3 position as fl avones failure, cancer and diabetes76. Acylated fl avonoids, such as (e.g. apigenin). Flavones lack a hydroxyl group at the epicatechin are reported to be absorbed without deconju- 3-position. Flavan-3-ols (e.g. epicatechin) lack a double gation and hydrolysis77. bond in the 2–3 position. Quercetin (0.1 % of the phenolic fraction), its 3-glycoside (0.06 %) and 3-rutinoside (0.75 %) and minor quantities of epicatechin and apigenin 2.4.2.2. Anthocyanins were found in our L. caerulea var. kamtschatica phenolic Anthocyanins are secondary plant metabolites, deriva- fraction21. tives of 2-phenylbenzopyrylium, generally found in glyco- Quercetin is one of the most extensively studied fl a- sidic forms78–80. The aglycones (anthocyanidins) are rarely vonoids apropos its anticancer activity because of its found in fresh plants. They occur as 3-glycosides and 3,5- prevalence among fruits and vegetables (Fig. 3) (ref.68). diglycosides linked with glucose, galactose, rhamnose or Quercetin glucosides are resistant to hydrolysis by HCl in arabinose78. Anthocyanins represent the most important stomach69 and the absorption occurs in the small intestine group of water-soluble pigments, responsible for the blue, into enterocytes probably via active transport70,71. They purple and red colour of many plant tissues due to their can be methylated into isorhamnetin immediately after ability to associate into complexes characterized by higher absorption in the human body. Quercetin rutinosides are absorbance of light lengths, co-pigmentation and formation of complexes with metals. In aqueous solution, anthocyanins exist in a number of diff erent molecular forms a) that are in dynamic equilibrium depending mostly on pH OH (Fig. 4). The red fl avylium cation is the most abundant molecular form at pH < 2. As the pH increases, there is HO O OH rapid loss of a proton to generate the blue quinonoidal structure. At the same time, much slower hydration of the OH fl avylium cation occurs to yield the colourless hemiketal OH O form that further tautomerises to generate the chalcone form81. The positively-charged oxonium anthocyanin b) OH form may not be aff ected by MRP2 effl ux, which seri- ously limits the absorption of other fl avonoid glycosides38. HO O Anthocyanins seem to be able to be absorbed intact as glycosides82. However, the proportion of anthocyanins absorbed and excreted in the urine appears to be quite small, perhaps much less than 0.1 % of the intake83. The O OH clearance of anthocyanins from the circulation is rapid; very little is generally detected in the plasma 6 h after c) OH administration82. Some anthocyanins can be metabolized OH to colourless forms, oxidized, or degraded into other compounds. Researchers have also found that no glycoside HO O hydrolysis takes place during digestion84. The absorption of anthocyanins without removal of glycoside has also 85 OH been demonstrated . Anthocyanins and proanthocyanidins have antibacte- OH rial properties as well as the ability to inhibit adhesion of bacteria to mucosal membrane of urinary tract86. Anthocyanins also act as anti-infl ammatory and anti-muta- Fig. 3. Chemical structures of a) quercetin; b) apigenin; genic agents and provide cardioprotection by maintaining c) epicatechin. vascular permeability4. The ability to regulate the perme- 168 I. Svarcova, J. Heinrich, K. Valentova

1 1 Flavylium R Hemiketal R OH OH cation (red) (colourless) OH HO + HO O 2 + O 2 R H2O,-H R

Ogly Ogly O gly O gly + -H + -H R R1 1 -H2O O OH

O HO O O R1 R 2 R2 OH

OH Ogly OglyHO O O gly O gly R2

+ + -H 1 -H R Ogly R1 - O gly Chalcone O O (colourless) - O O O O 2 R R2 Quinonoids Ogly Ogly O gly O gly (blue)

Fig. 4. Various anthocyanin forms existing in aqueous solutions depending on pH (gly = glycoside). ability of capillary vessels has become the basis for their Cyanidin is the most common anthocyanidin, with a defi nition as vitamin P. They show protection against hep- 3', 4'-dihydroxylation of the B ring, present in 90 % of atitis A and B and also against paracetamol hepatotoxic- fruits92. Wu et al.83 detected the methylated form of an- ity87. Berry extracts rich in anthocyanins have been linked thocyanins in human urine after consumption of elder- to protective eff ects including the modulation of age-re- berries and blueberries. The cyanidin glycosides tend to lated neurological dysfunctions and improved resistance have higher antioxidant capacity than peonidin or malvi- of red blood cells against oxidative stress in vitro88. din glycosides likely due to the free hydroxyl groups on Anthocyanins are very good antioxidants due to the the 3' and 4' positions of cyanidin93. The key diff erence presence of hydroxyl groups in position 3 of the C ring, compared to other fl avonoid glucosides, is that cyanidin- which can chelate metal ions (Fe, Cu), and 3' and 4' of the 3-glucoside appears to be absorbed after oral ingestion, B ring. Antioxidant activity is also increased by acylation although to a limited extent38. Cyanidin-3-rutinoside from of sugar residues with aromatic hydroxy acids89. These sweet cherry exhibited cyclooxygenase I and II inhibitory compounds have higher antioxidative activities than vita- activities94. It showed potential in the treatment of diabe- min E, ascorbic acid or β-carotene79. tes, obesity, hyperlipidemia95 and B and C type viral hepa- The major anthocyanins in L. caerulea fruit are the titis96. Delphinidin may preserve endothelium integrity and glucosides and rutinosides of cyanidin, peonidin, dephi- protect against endothelial cell apoptosis97. Delphinidin, nidin and pelargonidin21 (Fig. 5). Quantities of these malvidin and petunidin are metabolized in the liver compounds vary depending on a number of the factors46. through the catechol-O-methyltransferase reaction in the Table 2 shows a comparison of L. caerulea phenolic frac- 3' position83. Pelargonidin itself displays an orange red tion anthocyanin content with those of Vaccinium macro- color. Pelargonidin-3-monoglucoside, isolated from frozen carpon90 and Vitis vinifera91. strawberries, protected the amino acid tyrosine from the highly reactive oxidant peroxynitrite, inhibited the growth

1 R Name R1 R2 R3 2 R Cyanidin OH OH H

+ Delphinidin OH OH OH HO O 3 R Pelargonidin H OH H Peonidin OCH OH H OH 3 Petunidin OCH OH OH OH 3

Malvidin OCH3 OH OCH3

Fig. 5. Structures of main anthocyanidins. Berry fruits as a source of biologically active compounds: the case of Lonicera caerulea 169

Table 2. Content of anthocyanins in Lonicera caerulea, Vaccinium macrocarpon and Vitis vinifera extracts.

Compounds Relative content (% w/w) L. caerulea21 V. macrocarpon90 V. vinifera91 Cyanidin-3-arabinoside – 5.5 – Cyanidin-3-galactoside – 7.1 – Cyanidin-3-glucoside 60.0 12.3 7.0 Cyanidin-3(6'-acetyl)-glucoside – – 1.8 Cyanidin-3,5-diglucoside 9.9 – – Cyanidin-3-pentoside – 2.9 – Cyanidin-3-rutinoside 7.3 – – Delphinidin – 1.8 – Delphinidin-3-glucoside 1.2 – 3.7 Delphinidin-3-rutinoside 2.0 – Pelargonidin-3-glucoside 3.3 – – Pelargonidin-3,5-diglucoside 0.6 – – Pelargonidin-3-rutinoside 0.1 – – Peonidin-3-arabinoside – 5.7 – Peonidin-3-galactoside – 10.2 – Peonidin-3,5-digalactoside – 24.1 – Peonidin-3-glucoside 5.8 60.7 18.7 Peonidin-3,5-diglucoside 8.1 – Peonidin-3(6'-coumaroyl)-diglucoside – – 1.2 Peonidin-3-rutinoside 0.5 – – Petunidin-3-glucoside – – 10.7 Malvidin-3-glucoside – – 64.6 Malvidin-3(6'-acetyl)-glucoside – – 1.0 Malvidin-3(6'-caff eoyl)-glucoside – – 1.8 Malvidin-3(6'-coumaroyl)-glucoside – – 2.5 Total anthocyanins 100 100 100 of Escherichia coli and Staphylococcus aureus, and exerted pathway and are metabolized to anthocyanidins79. They both a stimulatory and inhibitory eff ect on Lactobacillus can be considered as the fi fth class of plant biopolymers casei culture. The stimulation may be due to a decrease in besides polynucleotides, proteins, lignins and polysaccha- the oxidation-reduction potential of the media aff ected by rides. As a class, proanthocyanidins can be complex in the pigment, and/or the ability of the organism to split the structure and composition, featuring various fl avan-3-ols β-glycosyl bond and use the glucose moiety98. Peonidin has (most commonly catechin, epicatechin, and galloylated been patented for use as food colouring agent99. catechins) linked together in diff erent ways. Berry proanthocyanidins are primarily dimers, trimers and other 2.4.3. Proanthocyanidins oligomers100. These molecules may contain two types of Proanthocyanidins (condensed tannins) are oligomeric linkages between epicatechin units. The B-type (4β → 8) is and polymeric end products of the fl avonoid biosynthetic widely found in apples and grapes and A-type (4β → 8 and 170 I. Svarcova, J. Heinrich, K. Valentova

OH L. caerulea phenolics, as secondary plant metabolites, have been shown to provide defence against oxidative stress HO O OH from endogenous ROS and free radicals111. Tumor inhibi- O OH tion by the berries is likely to involve synergic activities between its phytochemicals, including fl avonols (quercetin), OH O OH proanthocyanidins and others and to be primarily based on reducing oxidation of lipoproteins, improving antioxidant status and lipid levels and mitigating the eff ect of oxi- OH 112 HO dative stress and infl ammation on the vascular system . OH Phenolics protect cardiomyocytes after ischemic episodes by inhibition of free radical formation in the process of OH reperfusion113. These compounds are able to reduce nitric oxide synthase activity and nitric oxide (NO) level114. They HO O OH inhibit the cyclooxygenase activity, adhesion and reaction of leukocytes with endothelial cells, degranulation of

R1 R2 OH mast cells and decrease in the level of interleukin (IL)-2, OH interferon (INF)-γ and tumor necrosis factor (TNF)-α HO O 115 OH (ref. ). Polyphenolics have been regarded as a potential novel, safe and nontoxic strategy for the modulation of R infl ammation dependent on the nuclear factor (NF)-κB R2 1 27 OH pathway . An extract of L. caerulea showed signifi cant anti-infl ammatory eff ects on endotoxin-induced uveitis in Fig. 6. Chemical structures of proanthocyanidin rat. The possible mechanisms for this eff ect may depend A and B type. especially on its ability to inhibit activation of NF-κB and the subsequent production of proinfl ammatory mediators 27 such as TNF-α, prostaglandin (PG)-E2 and NO (ref. ). 2β → O →7) found in cranberries can inhibit adherence Berry anthocyanins act as novel cardioprotectants by of uropathogenic P-fi mbriated Escherichia coli (Fig. 6) maintaining vascular permeability, reducing infl amma- (ref.101, 102). The stereochemistry of the linkage at C4 may tory responses and platelet aggregation, and off er superior be α or β103. The major function of proanthocyanidins in vascular protection compared to other cardioprotective plants is to provide protection against microbial patho- drugs116, 117. Bioactive compounds from L. caerulea have gens, insect pests and larger herbivores. Their deposition been also demonstrated in in vitro models to inhibit the in the endothelial layer of the seed coat appear to be an oxidation of lipoproteins. A recent study of ours showed example of a pre-formed protective barrier104. Salmonella, inhibition of copper-induced lipoprotein oxidation by Staphylococcus, Helicobacter and Bacillus are the most a phenolic fraction of L. caerulea var. kamtschatica118. sensitive bacteria to the berry phenolics. In addition, the Reduction of atherosclerotic plaques by polyphenolics has growth of Escherichia, Clostridium and Campylobacter spe- been found in animal models; reduction of carcinogenesis cies but not Lactobacillus and Listeria species are inhib- was observed in vitro. Epicatechin76 and anthocyanins, es- ited105. The main mechanisms of action are destabilization pecially delphinidin, may preserve endothelium integrity of cytoplasmic membrane, permeabilization of plasma whose damage can lead to the development of athero- membrane, inhibition of extracellular microbial enzymes, sclerosis and also cancer97. Some L. caerulea compounds direct actions on microbial metabolism and deprivation of can block mutagenesis by chemical carcinogens and en- the substrates required for microbial growth106. dogenous mutagens and have been shown to modify the process of uncontrolled cell proliferation and apoptosis in vitro119. These phytochemicals may exert their anticar- 3. TRADITIONAL USE AND PUTATIVE cinogenic eff ect by modulating the enzyme systems that HEALTH BENEFITS metabolize carcinogens or procarcinogens to genotoxins. For example CYP activity can be induced or inhibited by L. caerulea berries have long been harvested from fl avonoids. Ellagic acid inhibits mutagenesis and carcino- wild plants in regions of Russia, China and Japan where genesis by acting on both CYP xenobiotic metabolism and superior edible forms are native. Recently, research in several phase 2 detoxifying enzymes120. Berry extracts can Russia and Japan has resulted in cultivars being selected protect against carcinogenesis also in animal models121. for commercial production because of its very early matu- Numerous reports shows quercetin ability to inhibit pro- rity, unique fl avour and health benefi ts that have long been liferation of cancer cell lines in vitro, including breast, acknowledged in Russia16. Recent research has supported colon, pancreas cancer, and leukemia122. Its mechanism some of the folkloric claims for the therapeutic uses of of action includes induction of apoptosis123, inhibition blue honeysuckle berries in atherosclerosis, hypertension, of epidermal growth factor receptor expression and as- gastrointestinal disorders and bacterial infection. The sociated tyrosine kinase activity122, reduced expression of main benefi cial eff ect is due to the presence of vitamin C Ras protein in colon cancer cells and primary colorectal and high levels of polyphenolics 16, 107–110. tumors124, increased expression of endogenous inhibitors Berry fruits as a source of biologically active compounds: the case of Lonicera caerulea 171 of matrix metalloproteinases125 and phytoestrogenic ac- GIT. Soluble proanthocyanidins can inhibit pancreatic tivity involving interaction with the estrogen α- and β-re- and gastric lipase activity and therefore would be a tar- ceptors of human breast cancer MCF-7 cells68. Cyanidin get in the treatment of obesity133. Berries have also been and its 3-glycoside reduce oxidant-induced DNA strand shown to reverse age-related and oxidative stress-induced breakage in normal human lymphocytes ex vivo and are decline in brain function in rats138. as potent chemoprotectants as the fl avonols, quercetin and myricetin. Cyanidin-3-rutinoside and cyanidin-3-glucoside suppress cancer cell metastasis by inhibiting the CONCLUSION motility adhesiveness and invasiveness of metastatic human lung cancer cell lines A579 (ref.126). Cyanidin and Lonicera caerulea berries contain 7.20 % saccharides, a mixture of several of its glycosides dose-dependently in- 1.52 % lipids, 14.62 % dry matter, 12.2 % organic acids hibited HCT 116 and HT 29 colon cancer cell growth127. and 4 % phenolics, containing 33.5 % of phenolics, includ- Delphinidin, malvidin and petunidin also inhibited pro- ing anthocyanins (18.5 %), fl avonoids and phenolic acids. liferation of cancer cells derived from various tissues in- The major anthocyanins in L. caerulea fruit are gluco- cluding colon, breast, blood and lung at high micromolar sides and rutinosides of cyanidin, peonidin, dephinidin concentrations128. Peonidin has shown potent inhibitory and pelargonidin. These berries seem to be prospective and pro-apoptotic eff ects on cancer cells in vitro, notably sources of health supporting phytochemicals that exhibit human metastatic breast cancer cells99. p-Coumaric acid benefi cial activities such as anti-adherence, antioxidant is believed to reduce the risk of stomach cancer by reduc- and chemoprotective, thus they may provide protection ing the formation of carcinogenic nitrosamines56. Also against a number of chronic conditions, e.g. cancer, dia- caff eic acid and epicatechin have been shown to act as an betes mellitus, tumor growth or cardiovascular diseases. inhibitor of carcinogenesis48,76. Wild and cultivated berry These plants can be cultivated in European climatic condi- proanthocyanidin fractions demonstrated antiproliferative tions and therefore are a suitable source of economically eff ects on two models of prostate cancer: an androgen-sen- accessible nutraceutical preparations. sitive (LNCaP) and more aggressive androgen-insensitive cell line (DU145) (ref.129). Several berry derivatives have also potent anti-angio- ACKNOWLEDGEMENTS genesis properties in vitro by altering vascular endothelial growth factor (VEGF) expression and invasiveness. This study was supported by grants MSM 6198959216 Berry extracts can inhibit hydrogen peroxide and TNF-α and FT-TA3/024 that are gratefully acknowledged. induced VEGF expression in these cells and also inhibit angiogenesis in animals130. For example apigenin inhibits expression of VEGF in human ovarian cancer cells74. 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