{m ,. . 4w ‘ «um-.1853. gum. . . txfi (assaum‘rra . 3.15:5: ‘ I15 4‘ .. . flu; "0 V y I) . .. . if. r: .5 .1... a :tshizi . 3. 33...“!!! umpi, .3... {Put ‘1 -00.! NM. 1'HES!S 2., U"“f'."1.) ImmmIllil’l‘fillllilliWillHum 3 1293 01770 9142 LIBRARY Mlchlgan State University This is to certify that the dissertation entitled ANTIOXIDANT AND ANTIINFLAMMATORY COMPOUNDS IN TART CHERRIES presented by HAIBO WANG has been accepted towards fulfillment of the requirements for Ph . D . degree in Horticulture V Major professor Date 1/15/ 2 25 MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINE return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE “)3,“ APR 0 3 2 .9u571’6 004 £0350 8 gm 1 33'1" 8“ f 3009 ANTIOXIDANT AND ANTIINFLAMMATORY COMPOUNDS IN TART CHERRIES BY 'Haibo Wang A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements For the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1998 ABSTRACT ANTIOXIDANT AND AN TIINFLAMMATORY COMPOUNDS IN TART CHERRIES By Haibo Wang Tart cherry production and processing is an important industry in Michigan. Michigan's tart cherry industry produces 75% of the nation's tart cherry supply, which is about 250-300 million pounds annually. Anecdotal reports indicate that consumption of tart cherries could alleviate arthritic- and gout-related pain and reduce the incidence of cardiovascular diseases. These beneficial effects may be associated with the anthocyanins, phenolics and flavonoids present in tart cherries. In order to evaluate these claims, the bioactive components in tart cherries were isolated using antioxidant- bioassay-directed fractionation and purification. The water extracts of Montrnorency and BalatonTM tart cherries yielded three anthocyanins. Also, the results indicated that both cultivars contained identical anthocyanins. The hydrolysis of the total anthocyanins and subsequent gas chromatographic (GC) and nuclear magnetic resonance (N MR) spectral analyses suggested that the anthocyanins in Montrnorency and BalatonTM cherries were anthocyanin l [cyanidin-3-(2"-0—,B-D-glucopyranosyl-6"-0-oc-L-rhamnopyranosyl-B-D- glucopyranoside], anthocyanin 2 [cyanidin-3-(6"-0-0t-L-rhamnopyranosyl-[3-D- glucopyranoside] and anthocyanin 3 [cyanidin-3-0—[3-D-glucopyranoside]. However, BalatonTM contains approximately six times more anthocyanin than Montrnorency. In another experiment, BalatonTM cherries were extracted sequentially with hexane, ethyl acetate and methanol. Antioxidant assays indicated that ethyl acetate and methanol extracts exhibited higher antioxidant activity than hexane extract. Further purification of the ethyl acetate extract yielded chlorogenic acid methyl ester and three novel compounds, 2-hydroxy-3-(o-hydroxyphenyl) propanoic acid , 1-(3', 4'- dihydroxycinnamoyl)-cyclopenta-2,5-diol and 1-(3',4'-dihydroxycinnamoyl)- cyclopenta-2,3-diol. Similarly, the methanol extract yielded eight polyphenolic compounds, 5,7,4'-trihydroxyflavanone, 5,7,4'-tn°hydroxyisoflavone, chlorogenic acid, 5,7,3',4'-tetrahydroxyflavonol-3-rhamnoside, 5,7,4'-trihydroxyflavonol-3-rutinoside, 5,7,4'-trihydroxy-3'-methoxy-flavonol-3-rutinoside, 5,7,4'-trihydroxyisoflavone-7- glucoside and 6,7-dimethoxy-5,8,4'-trihydroxyflavone. The antioxidant activities of the purified compounds were evaluated using the Fey-induced lipid peroxidation assay. Commercial antioxidants propyl gallate, butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ) and a-tocopherol were used as positive controls. The anthocyanins and their aglycones, cyanidin, 5,8,4'-trihydroxy-6, 7- dimethoxyflavone and caffeic acid analogues from BalatonTM were the most active antioxidant compounds. The antioxidant activities of these compounds were greater than a-tocopherol and comparable to the activities of BHT and TBHQ. In the anti-inflammatory assay, which measured inhibition of the cycloxygenase (COX) activities of the prostaglandin endoperoxide H synthase-l and —2 isozymes (hPGHS-l and —2), cyanidin showed significant inhibitory activities against COX-1 and COX-2 enzymes. The ICso values of cyanidin against hPGH-l and hPGH-Z were 90 and 60 uM, respectively. Similarly, the inhibitory activities on hPGHS-l enzyme of the polyphenolics from BalatonTM tart cherries also were investigated. Genistein exhibited the highest COX-1 inhibitory activity at an ICso value of 80 uM. Other flavonoids and isoflavonoids isolated from tart cherries have ICso values greater than 400 uM concentration. ACKNOWLEDGEMENTS During the course of my dissertation work, there are a number of people who have helped me. Without their guidance, help and patience, I would have never been able to accomplish this dissertation. I would first like to sincerely thank my major professor Dr. Muraleedharan G. Nair for his advice, support and encouragement throughout my study at Michigan State University. Dr. Nair helped me consistently and constructively the entire way. He continually and rapidly read and responded my writing. Without his guidance, I am sure that I would not have been able to accomplish my goal at Michigan State University. Many thanks also go to my committee members for their support and guidance. I would like to thank Dr. Gale M. Strasburg for his assistance on my project, especially for the use of equipment in his laboratory, and for his commitments. I sincerely thank Dr. Ian J. Gray for taking time from his very busy schedule to speak to me, give me suggestion and advice, and provide a spur to my confidence. Similarly, I thank Dr. John F. Jelly and Dr. Amy F. Iezzoni for helping me improve my knowledge in horticulture and genetics, and providing valuable guidance though out these years. I would like to thank Dr. David Dewitt for providing the PGHS enzyme and the use of equipment in his laboratory. Also, I am grateful to Dr. Booren for his suggestions and advice. Evenly important are my fellow members and friends in the Department of Horticulture and Bioactive Natural Products Laboratory. My special thanks to Dr. Yu- Chen Chang, Dr. Ramsewak, Dr. Balasubramanian, Mark Kelm, Jennifer Miles, Andrew Erickson, Rafikali Momin, Clifford Laura, Di Wu, Priscilla Hockin and many of my Chinese friends at Michigan State University. Finally, I must give immense thanks to my wife Min and our son Jia—Qi (Joshua). Their love and support during long nights of work away at the lab was of immeasurable value to me. vi TABLE OF CONTENTS LIST OF FIGURES ...................................................................... IX LIST OF TABLES ...................................................................... X LIST OF ABBREVIATIONS ........................................................... XI INTRODUCTION ........................................................................ 1 CHAPTER ONE ......................................................................... 5 LITERATURE REVIEW Introdution ................................................................................. 5 Chemical Constituents of Prunus Species ............................................. 5 Anthocyanins .............................................................................. 5 Flavonoids and Polyphenolics ........................................................... 11 Other Components in Tart Cherries ..................................................... 33 Biological Activity of Prunus Species ................................................. 35 Antioxidant Activity ..................................................................... 37 Antiinflammatory Activity ............................................................... 42 Anticarcinogenic Properties of Phenolic Compounds ............................... 48 Cardiovascular Properties of Polyphenolics ........................................... 53 Antiviral Effects of Phenolic Compounds ............................................. 54 CHAPTER TWO ......................................................................... 58 Quantification and Characterization of Anthocyanins in BalatonTM Tart Cherries Abstract .................................................................................... 58 Introduction ............................................................................... 59 Materials and Methods .................................................................. 60 Cherry Fruits .............................................................................. 60 General Experimental .................................................................... 6O HPLC Condition for Anthocyanin Analysis .......................................... 61 Isolation of Crude Anthocyanins from Tart Cherry ................................. 62 HPLC Analysis of Anthocyanins in Cherry .......................................... 62 Purification of Anthocyanins 1-3 ...................................................... 62 Cyanidin, the Aglycone .................................................................. 66 Derivatization of Sugars ................................................................. 67 Characterization of Sugars by GC Analysis ........................................... 67 Results and Discussion .................................................................. CHAPTER THREE ...................................................................... 74 Antioxidant Compounds from Ethyl Acetate Extract of Tart Cherries (Prunus cerasus) Abstract .................................................................................... 74 Introduction ................................................................................ 75 Experimental Section ..................................................................... 76 General Experimental procedures ...................................................... 76 Plant Material ............................................................................. 77 Extraction and Isolation 77 vii Methylation of Compound 2 ............................................................ 79 Antioxidant Assay ........................................................................ 80 Results and Discussion .................................................................. 81 CHAPTER FOUR ........................................................................ 88 Antioxidant Polyphenolics from the Methanol Extract of Tart Cherry (Prunus cerasus) Abstract ..................................................................................... 88 Introduction ............................................................................... 89 Materials and Methods .................................................................. 90 Cherry Fruits ............................................................................... 90 General Experimental .................................................................... 90 Antioxidant Assay ....................................................................... 91 Extraction of Cherries ................................................................... 92 Purification of Antioxidant Compounds ............................................... 93 Results and Discussion .................................................................. 96 CHAPTER FIVE ......................................................................... 107 Antioxidant and Antiinflammatory Activities of Anthocyanins from Tart Cherries Abstract ................................................................................... 107 Introduction ............................................................................... 108 Materials and Methods .................................................................. 109 General Experimental ..................................................................... 109 Liposome Assay .......................................................................... 110 Cyclooxygenase Assay .................................................................. 110 Results and Discussion .................................................................. 111 CHAPTER SIX ........................................................................... 120 Antiinflammatory Activity of Flavonoids and Isoflavonoids and Their Structure-Activity Relationships Abstract .................................................................................... 120 Introduction .............................................................................. 121 Materials and Methods .................................................................. 123 Materials ................................................................................... 123 Cyclooxygenase Assay .................................................................. 123 Results and Discussion ................................................................... 124 SUMMARY AND CONCLUSIONS .................................................. 131 LIST OF REFERENCES ............................................................... 136 viii Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.5 Figure 1.6 Figure 1.7 Figure 1.8 Figure 1.9 Figure 1.10 Figure 1.11 Figure 1.12 Figure 1.13 Figure 1.14 Figure 1.15 Figure 1.16 Figure 1.17 Figure 1.18 Figure 1.19 Figure 1.20 Figure 2.1 Figure 2.2 Figure 3.1 Figure 3.2 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 5.1 Figure 5.2 Figure 5.3 Figure 5.4 Figure 6.1 Figure 6.2 LIST OF FIGURES Flavylium cation ................................................................. Anthocyanins from Prunus species .......................................... Biosynthetic pathway for flavonoids ......................................... Flavonoids from cherries ...................................................... Flavonone and flavanone glycoside in Prunus species ..................... Isoflavonoids from tart cherries .............................................. Flavones from tart cherries .................................................... Other phenolic compounds from tart cherries .............................. Other glycosides from tart cherries .......................................... Proanthocyanidins from Prunus species ..................................... Bitter principles from Prunusjamasakura and P. maximowiczii ......... Bitter principles from Prunusjamasakura and P. maximowiczii ........ Sesquilignan and neolignan from Prunusjamasakura .................. Phenolic glucoside from Prunus grayana ................................... Phenolic glucoside from Prunus grayana ................................... Phenolic glucoside from Prunus grayana ................................. Phenylpropanoid glucosides from Prunus buergeriana .................... Proanthocyanidins from Prunus species .................................... Principle carotenoids in tart cherry .......................................... Mechanism of inflammation .................................................. HPLC profile of Montrnorency and BalatonTM cherry extract ............ Anthocyanins 1-3 from BalatonTM and Montrnorency tart cherries ...... Structure of compounds isolated from ethyl acetate fraction .............. Antioxidant activities of compounds 1-4 ................................... Antioxidant activities of crude tart cherry extracts ........................ Structures of compounds 1-8 .................................................. Antioxidant activites of compounds from methanol extract ............... Percent inhibition of compounds 1-8 and their mixture .................... Anthocyanins in Montrnorency and BalatonTM cherries .................. Antioxidant efficacy of anthocyanins and commercial antioxidants. . . .. Dose response curve for the inhibition of PGHS-1 ......................... Dose response curve for inhibition of COX-1 and COX-2 ................ Structure of flavonoids tested for COX-1 inhibitory activity ............. Structure of isoflavonoids tested for COX-1 inhibitory activity .......... 6 9 12 14 15 17 18 19 20 21 23 24 25 28 ‘29 30 31 32 34 43 64 64 85 87 98 101 104 106 113 115 118 119 128 129 LIST or TABLES Table 2.1 1H NMR chemical shifts for anthocyanins and cyanidin ............ 70 Table 2.2 '3 C NMR chemical shifts for anthocyanins and their aglycone 71 Table 6.1 ICso Values of flavonoids and isoflavonoids on COX-1 ............ 130 ADP BHA BHT CD cox l3CNMR DMSO DQFCOSY dd EIMS FABMS DPA-PA HMBC HMQC 'H NMR HPLC LDL LO MOPS MPLC m/z NMR 0RD PDA PG PGHS-l PGSH-Z TBA TBHQ uv 5 J LIST OF ABBREVIATIONS Adenosine diphosphate Butylated hydroxyanisole Butylated hydroxytoluene Circular dichroism Cyclooxygenase Carbon nuclear magnetic resonance Dimethyl sulfoxide Double quantum filtered correlated spectroscopy Doublet of doublet Electron impact ionization mass spectroscopy Fast atom bombardment mass spectroscopy 3-(p-(6-phenyl)-1 ,3 ,5-hexatrienyl)phenylpropionic acid Heteronuclear multiple bond correlation Heteronuclear multiple quantum coherence Proton nuclear magnetic resonance High pressure lipid chromatography low-density lipoprotein Lipoxygenase 3-[N-morpholino] propanesulfonic acid Medium pressure lipid chromatography Mass-to-charge ratio Nuclear magnetic resonance Optical rotatory disperson Photodiode array Prostaglandin Prostaglandin endoperoxide H synthase-l Prostaglandin endoperoxide H synthase-2 2-thiobarbituric acid tert-butylhydroquinone Ultraviolet Chemical shifis Coupling constant xi INTRODUCTION Tart cherry production and processing is an important industry in Michigan. Michigan's tart cherry industry produces about 75% of the nation's tart cherry supply. Michigan produces 250-300 million pounds of tart cherries annually, which are grown on a total of 36,300 acres and having an estimated value of 50-70 million dollars. The original habitat of the red tart cherry (Prunus cerasus L.) was between Switzerland and the Adriatic Sea on the West and the Caspian Sea and somewhat northward on the East. Pomologists assumed that both sweet and red tart cherries originated in the same region and that the latter may have been derived from the former. The earliest records of the cultivation of the cherry indicated that it was first domesticated in Greece. The cherry was brought to America by the early colonists. Montrnorency, a cultivar of tart cherry, which originated from France about 300 years ago, was first studied at the Michigan Agricultural Experimental Station in 1922. Of the 270 named varieties of red tart cherries, there are only three varieties of red tart cherries grown in appreciable quantities, all of which are of European origin. They are Early Richmond (Kentish), Montrnorency and English Morello. Montrnorency represents 97% of the tart cherry acreage in the United States. Tart cherries are grown only in areas with favorable climate, soil and topography. In Michigan, tart cherries are grown in the western counties of the lower peninsula along Lake Michigan. In order to TM, was diversify the Montrnorency monoculture, a new Hungarian cultivar, Balaton introduced into the United States in 1984, and it has been tested in Michigan, Utah, and Wisconsin. BalatonTM produces fruits darker than Montrnorency, and it may be used as a source of cherry anthocyanins. Tart cherries can be used in many different ways. Tart cherry pits can be used as a source for natural benzaldehyde (Chandra and Nair, 1993). The oil from Montrnorency cherry pits has potential for cooking or frying food (Chandra and Nair, 1993). The anthocyanins from Michigan tart cherries can provide a more efficient cherry colorant (Chandra et al., 1992). Anecdotal reports indicate that consumption of cherries could alleviate arthritic- and gout-related pain (Hamel, 1975) and reduce the incidence of cardiovascular diseases. Tart cherries also can be added to ground beef to make it “lean”. Thiobarbituric acid (TBA) values for beef patties containing cherries were significantly lower when stored under refrigeration and kept for six months at -20°C than for patties which were cooked and then refrigerated for 24h (Liu et al., 1995). These beneficial effects may be associated with the anthocyanins, phenolics and flavonoids present in tart cherries. Plant polyphenolics are multifunctional and can act as reducing agents, hydrogen-donating antioxidants, and singlet oxygen quenchers. In some cases, metal chelation properties have been proposed as well. The biological, pharmacological, and medicinal properties of the flavonoids have been reviewed extensively (Cody, et al, 1986; 1988). F lavonoids and other plant are reported to have multiple biological activities, in addition to their free radical scavenging activities. (Ho et a1, 1992; Kinsella et al., 1993). These types of compounds were reported to have anticarcinogenic, antiinflammatory, antibacterial, immune-stimulating, anti-allergenic, antiviral, and estrogenic effects. They also can act as inhibitors of phospholipase A2, cyclooxygenase, and lipoxygenase (Brown, 1980, Middleton and Kandaswami, 1992; Mabry et al., 1982; Jovanvoic et al., 1992; Robak, et al., 1988; Sogawa, et al., 1993; Lindahl and Tagesson, 1993), glutathione reductase (Elliott, et al, 1992), and xanthine oxidase (Chang et al., 1993). Their effects on a variety of inflammatory processes also have been reviewed (Gabon, 1979, 1986; Farkas et al., 1986; Welton et al., 1988). The flavonoids can significantly affect the function of the immune system. A number of flavonoid compounds can affect the activity of enzyme systems which are critically involved in the immune response, and the triggering of inflammatory processes. For example, quercetin and kaempferol exhibit extensive immune response activity (Lee et al., 1982; Middleton et al., 1981; 1984). Also, quercetin was shown to inhibit the mutagenic activity of benzo[a]pyrene(BP), a representative polynuclear aromatic hydrocarbon (PAH) carcinogen, in bacterial mutagenicity studies (Ogawa et al., 1985). Quercetin also was reported to inhibit many biochemical events associated with tumor promotion (Levy et al., 1984). Anthocyanins also were regarded as naturally occurring pigments with antiinflammatory (Vlaskovska et al., 1990) and antioxidant activities (Costantino et al., 1992; Gabor, 1988). It is postulated that polyphenols, such as flavonoids, isoflavoniods, anthocyanins and anthocyanidins exist in tart cherries may act as antioxidants and possess other biological activities that are responsible for anecdotal health claims associated with tart cherry. In order to evaluate this hypothesis, we have investigated the active components in Montrnorency and BalatonTM tart cherries and evaluated their biological activities using Fey-induced lipid peroxidation and cyclooxygenase enzyme assays. Therefore, the objectives of this research are (1) Characterization and quantification of anthocyanins in BalatonTM and Montrnorency tart cherries; (2) Isolation, purification and identification of bioactive polyphenolics and flavonoids in BalatonTM and Montrnorency tart cherries using chromatographic and spectral methods, and (3) Determination of antioxidant and antiinflammatory activities of polyphenolics and anthocyanins from Montrnorency and BalatonTM tart cherries using Fe2+-induced lipid peroxidation and cyclooxygenase enzymes assay. This dissertation is organized into a series of chapters. Each chapter covers a specific aim of the study and is prepared as a manuscript with abstract, introduction, general experimental, results and discussion. l. 1.1 CHAPTER ONE Literature review INTRODUCTION Cherry is a popular temperate fruit, which belongs to the genus Prunus and the family Rosaceae. Cherries are mainly of two types, sweet (Prunus avium) and tart (P. cerasus). Many important fruits are included in the Prunus genus, such as apricot, peach, and plum. They are widely used in traditional medicine as antipyretics and useful against thirst, leprosy and leucoderma (Chopra et al, 1956). The major chemical constituents in Prunus spp. are anthocyanins, polyphenols and organic acids. In this review, major chemical components with important biological activities from Prunus genus are summarized. CHEMICAL CONSTITUENTS OF PRUNUS SPECIES Anthocyanins The anthocyanins belong to the flavonoid class of compounds consisting of a flavylium cation (Fig. 1.1). There are about 300 naturally occuring anthocyanins. The anthocyanin molecule consists of two or three portions; the aglycone moiety consisting of the flavylitun cation, a group of sugars and, often a group of acylated functionalities. The 17 anthocyanidins reported so far are listed in Table 1.1 (Harbome and Grayer, 1988). The anthocyanins occur in nature as mono-, di-, and triglycosides. The typical sugar substitutions on anthocyandins are glucose, galactose, rhamnose, arabinose and xylosc, respectively. The positions for the sugar substitutions are C3, C3', C5, C4', C5’and C7, respectively. Most of the sugar substitution present in anthocyanins are on the C3 position Fig. 1.1. Flavylium cation Table 1.1. Aglycones reported in anthocyanins Substitution Name Abbr. 3 5 6 7 3 ’ 4’ 5’ Color Apigeninidin Ap H OH H OH H OH H orange Luteolinidin Lt H OH H OH OH OH H orange Triacetinidin Tr H OH H OH OH OH OH red Pelargonidin Pd OH OH H OH H OH H orange Aurantidin Au OH OH OH OH H OH H orange Cyanidin Cy OH OH H OH OH OH H red 5-mecyandin SMCy OH OMe H OH OH OH H red Peonidin Pn OH OH H OH OMe OH H red Rosindin Rs OH OH H OH OMe OH H red 6-OHcyanidin 6OHCy OH OH OH OH OH OH H red Delphinidin Dp OH OH H OH OH OH OH blue Petunidin Pt OH OH H OH OMe OH OH blue Malvidin Mv OH OH H OH OMe OMe OH blue Pulchellidin Pl OH OMe OH OH OH OH blue Euopinidin Eu OH OMe OH OMe OH OH blue Capensinidin Cp OH OMe OH OMe OH OMe blue 2:12:33 Hirsutidin Hs OH OH OMe OMe OH OMe blue and are rarely found at C3', C5, C4', C5’ and C7 (Harbome and Grayer, 1988). Recent studies have indicated that anthocyanins can be acylated with coumaric, cafieic, ferulic, p-hydroxy benzolic, synapic acid and some aliphatic acids such as malonic, acetic, succinic, oxalic, and malic acid, respectively. However, aliphatic acylations in anthocyanins were not identified easily because of the instability of acyl linkages in methanolic HCl solution, which is a standard solvent used for pigment extraction. However, afler acetic acid was first reported to be conjugated with grape pigment, many unacylated pigments reported previously were subsequently found to have acyl groups, such as in the petals of Centaurea cyanus (Tamura, 1983). Tart cherries contain a variety of anthocyanins. Willstatter and Zollinger (1916) isolated a pigment from cherry skins, and named it as keracyanin. From Montrnorency, Li and Wagenknecht (1956) isolated and characterized two anthocyanins, cyanidin 3- rhamnoglucoside and cyanidin 3-gentioside (mecocyanin) (Fig. 1.2). These two anthocyanins were confirmed by Markakis (1960). Harbome and Hall (1964) reported this triglycoside in seven other cultivars of tart cherries. Cyanidin 3-glucoside was reported as a minor pigment of Montrnorency (Schaller and Von Elbe, 1968). In addition to cyanidin 3-glucoside and 3-glucosylrhamnoglucoside, cyanidin 3-rhamnoglucoside, cyanidin 3-sophoroside and peonidin 3-rhamnoglucoside were also identified in Montrnorency cherries (Dekazos, 1970). Also, he quantified the content of seven anthocyanins in partially and fully matured Montrnorency. Cyanidin 3-rhamnoglucoside and cyanidin 3-glucosylrhamnoglucoside were further confirmed by other authors (Von Elbe et al., 1968; Fischer and Von Elbe, 1970; Schaller et al., 1972). Chandra et a1. (1992) reported peonidin-3-galactoside in Montrnorency cherries using HPLC (Fig. 1.2). Cyanidin 3-rutinoside: R1=H, R2: rhamnosylglucose Cyanidin 3-gentioside: R1=H, R2: glucose (6 '*>1)-glucose Cyanidin 3-sophoroside: R1= H, R2: glucose (2 ~> l)-glucose Cyanidin 3-sambubioside: R1: H, R2: glucose (4 --:> 1)-glucose Cyanidin 3-(2-g1ucosyl) rutinoside: R1: H, R2: 2-g1ucosy1rutinoside Cyanidin: R]: H, R2: H Cyanidin 3-glucoside: R1: H, R2: glucose Cyanidin 3-arabinoside: R1: H, R2= arabinose Cyanidin 3-xylosylglucoside: R1: H, R2: xylosylglucose Cyanidin 3-p-coumarylglucoside: R1: H, R2: p-coumarylglucose Peonidin: R1: CH3, R2: H Peonidin 3-arabinoside: R1=H, R2=arabinose Peonidin 3-rutinoside: R1=CH3, R2=rutinoside Peonidin 3-galactoside: R1=CH3, R2=galactose Fig. 1.2. Anthocyanins from Prunus species From the varieties English Morello, Early Richmond and Meteor, Shrikhande and Francis (1973) identified cyanidin 3-glucosylrutinoside, cyanidin 3-sophoroside, cyanidin 3- rutinoside, peonidin 3-rutinoside, in addition to cyanidin 3-sarnbubioside or 3- xylosylglucoside (Fig. 1.2). Free cyanidin and peonidin were not detected. Cyanidin 3- gentiobioside and cyanidin 3-rutinoside were identified as the major anthocyanin pigments, and cyanidin 3-glucoside as a minor pigment in eight tart cherry varieties analyzed by electrophoresis and paper chromatography (Von Elbe et al., 1969). Cyanidin 3-gentioside in tart cherries was believed to be cyandin 3-sophoroside instead (Hong, V. and Wrolstad, R. E., 1990). The sweet cherry, Prunus avium L, is used commercially as a table fruit. Color is the most important indicator of maturity and quality for cherries. Cyanidin 3- rharnnoglucoside and cyanidin 3-g1ucoside were identified from ripened sweet cherries (Li and Wagenknecht, 1958). Peonidin and two of its glycoside derivatives peonidin 3- glucoside and peonidin 3-rutinoside were found in ‘Bing’ cherries (Lynn and Luh, 1964). However, Only cyanidin derivatives and no peonidin glycosides were found in varieties of P. avium (Harbome and Hall, 1964). Peonidin 3-rutinoside was identified as the main pigment in ‘Bigarreau Napoleon’ cherries (Okombi, 1980). Du et a1. (1975) studied anthocyanins present in ornamental cherry, P. sargentii, Rehd, and identified them as cyanidin 3-glucoside and cyanidin 3-diglucoside. The tomentosa cherry, P. tomentosa Thunb., is a small, hardy tree or a very large shrub grown for ornamental purposes and for its globular, light red and slight hairy fruit. The fruits contain anthocyanins such as pelargonidin and cyanidin 3-rutinosides (Ishikura, 1975). The laurel cherry, P. laurocerasus L., is an evergreen bush, seldom a small tree, nativeto 10 southeastern Europe and Iran, and grown for its ornamental value and black-purple fruit. The anthocyanins present in laurel cherry are peonidin 3-arabinoside and cyanidin 3- arabinoside (Fig. 2) (Tsiklauri, 1975). The European dwarf or ground cherry, P. fruticosa Pall, is a low-spreading bush. Only one anthocyanin was found in this fruit by Olden (1960), but its structure has not yet been elucidated. Anthocyanins in other Prunus species also were characterized mainly as cyanidin glucosides. For example, the red color in apricot, Prunus armeniaca L., is due primarily to cyanidin 3-glucoside (Joshi et al., 1986). The pigment in peach, Prunus persica, is due to cyanidin 3-glucoside (Hayashi et al., 1963). Ishikura (1975) reported the presence of cyanidin-rutinoside and cyanidin 3-glucoside in peach as 10 and 90%, respectively. The plum tree grows in temperate regions. The red pigments in ripe European plums are cyanidin 3-glucoside, cyanidin 3-rutinoside, peonidin 3-glucoside, and peonidin 3-rutinoside (Harbome and Hall, 1964; Hong and Wrolstad, 1990). From the Japanese Salicina plum (Prunus salicina Lindl.), Ishikura (1975) found only two cyanidin derivatives, cyanidin 3-glucoside and cyanidin 3-rutinoside. 1.2 Flavonoids and polyphenolics The family of flavonoid compounds includes flavanol, flavanones, anthocyanidins, flavones, and flavonols. Along with the phenylpropanoids or hydroxycinnamic acid derivatives, flavonoids are found in almost every plant (Markham, 1988; Niemann., 1988; Giannasi., 1988). The biosynthetic pathway for individual flavonoids are shown in Fig. 1.3. The 5, 7-hydroxylation pattern of the A-ring is the most common one. Similarly, dihydroxylation at the 3' and 4' positions of the B ring is also common in flavones and flavonols, followed by those with a single B ring-hydroxyl ll 3 Malonyl CoA 4-Coumaroyl CoA ’/ \ /// I/ \ \\ /// \ / tetrahydroxychalcone \\ / \_ \5‘x /,// \\ Chalcone ~14 A ~ a 3-flavene 3-h y droxy-fl a van on e i » ! 1 1 ' v flavanone —-~w ..___ ____, dihydroflavonol *L_..____ b flavonol z 4 l i 1 iv 1 ; ‘ anthocyanidin l . flavone l l y . Flavanol .L__-___- _ flavan-3,4-diol Fig. 1.3. Biosynthetic pathway for flavonoids 12 group at the 4' position. Methylation of flavonoids hydroxyl groups can be occurred at any one of these positions. However, the preferred glycosylation site on the flavonoids is at C3 and less frequently at C5 and C7, respectively. Recently, Cz', C3’, C4 'and C5' glycosides of flavonoids, were identified from several plants (Ibrahim et al., 1987). Glucose was the most common sugar moiety, but galactose, rhamnose, and xylose also were found. Another class of phenolic compounds, hydroxycinnamic acids, occur most frequently as simple esters with quinic acid or glucose. However, glycosylation often occurred at the acid group (Herrmann, 1989). Schaller and Von Elbe (1972) reported the presence of polyphenolic components in Montrnorency cherries and isolated six isomers of caffeoquuinic acid, four isomers of p-coumaroquuinic acid and two other free phenolic acids. In addition, two flavonols were identified as kaempferol 3-rhamnoglucoside and kaempferol 3-glucoside (Fig. 1.4). Olden and Nybon (1968), working with leaves of three varieties of cherries, separated the polyphenols and predicted the presence of the rutinosides and the 3-glucoside of quercetin and kaempferol (Fig. 1.4). Geissman (1956) indicated the presence of quercetin 3-glucoside in the leaves of Prunus cerasus. Shrikhande and Francis (1973) reported kaempferol 3-rhamnosylglucoside, quercetin-3-rhamnosylglucoside, quercetin 3- glucoside, quercetin 4'-glucoside and predicted the presence of three other phenolic glycosides kaempferol 3-rhamnoside-4'-galactoside, kaempferol 3-glucoside and kaempferol 4'-glucoside (Fig. 1.4). From the leaves of Prunus cerasus, Henning and Herrrnann (1980) reported quercetin 3-O-rutinosyl-7, 3’-O-diglucoside (Fig. 1.4). Also, from the bark of P. cerasus, tectochrysin 5-glucoside, naringenin, prunin, sakuranetin, sakuranin, dihydrowogonin 7-glucoside, tectochrysin (Fig. 1.5), genistein, prunetin-4'-O- 13 Quercetin: R]: H, R2: OH, R3=H, R4=H Quercetin 3-glucoside: RI=H, R2=OH, R3: glucose, R4=H Quercetin 3-rhamnosylglucoside: R1: H, R2: OH, R3: rhamnosylglucose, R4: H Quercetin 4'-glucoside: R = glucose, R = OH, R = H, R =H 1 2 3 4 Quercetin 3-rutinosyl-7,3'-diglucoside: R1= OH, R2: glucosyl, R3 = rhamnosylglucose, R4: glucose Kaempferol: R1=H, R2=H, R3=H, R4=H Kaempferol 3-glucoside: R1: OH, R2: H, R3: glucose, R4=H Kaempferol 3-rutinoside: R1=OH, R2=H, R3: rutinoside, R4=H Kaempferol 3-rhamnoside-4'-galactoside: R1: galactose, R2=H, R3: rhamnose, R4=H Kampferol 4'-glucoside: R1: glucose, R2=H, R3=H, R4=H Fig. 1.4. Flavonoids from cherries l4 Cerasinone: R1: OCH3, R2: OCH3, R3: CH3, R4: OH, R5: H, R6: H Naringenin: R1: H, R2= OH, R3= H, R4= OH, R5= H, R6: H Tectochrysin: R]: H, R2= H, R3: H, R4= OCH3, R5: H, R6= H Tectochrysin 5-glucoside: R1: H, R2: H, R3: glucose, R4: OCH3, R5 = R6: H Prunin: R]: H, R2= OH, R3: H, R4: H, R5: glucosyl, R6: H Sakuranin: R]: H, R2= OCH3, R3: glucose, R4: OCH3, R5: H, R6: H Sakuranetin: R1: H, R2: OH, R3: H, R4: OCH3, R5= H, R6: H Pinostrobin: R]: H, R2: H, R3: H, R4: OCH3, R5= H, R6: H Pinostrobin 5-glucoside: R1: H, R2: H, R3: glucose, R4= OCH3, R5: H, R6: H Dihydrowogonin 7-glucoside: R1: R2 = R3 = H, R4: glucosyl, R5: OCH3, R6: H Persicogenin 3'-glucoside: R1: R2 = CH3, R3: H, R4= CH3, R5 = H, R6: glucosyl Sakuranetin 5-xyloside: R1: H, R2= OH, R3= glucose, R4: OCH3, R5 = R6: H Isosakuranetin: R1=H, R2=OH, R3=H, R4: rhamnose, R5=H, R6=H Naringenin 4'-methyl ester 7-xylose: R1= R3= H, R2= OCH3, R4= xylose, R5 = R6 = H Fig 1.5. Flavanone and flavanone glycosides in Prunus species 15 glucoside, prunetin, pinostrobin S-B—D-glucoside, prunetin 5-glucoside, genistein 5- glucoside, pinostrobin (Fig. 1.6), apigenin 5-glucoside, chrysin, genkwanin 5-glucoside (Fig. 1.7) and neosakurarrin (Fig. 1.8) were isolated and identified (Geibel et al., 1990; 1991; 1995; Ingham, 1983). Schwab et a1 (1990) identified benzyl-B-D-glucoside, 2- phenylethyl B-D-glucoside, 6-hydroxy-2, 6-dimethyl-octa-2 (E), 7-dienyl B-D-glucoside and 2-methoxy-4-(2-propenyl)phenyl B-D-glucoside (Fig. 1.9) from fruit pulp of tart cherry by HRGC, HRGC-MS and HRGC-FTIR experiments. Nagarajan (1977) identified 7-hydroxy-5,2’,4’-trimethoxyflavanone (cerasinone) (Fig. 1.5), 2’-hydroxy-2,4,4’,6’- tetrarnethoxychalcone (cerasidin) and 2’,4’-dihydroxy-2,4,6’-trimethoxychalcone (cerasin) (Fig. 1.8) from Montrnorency cherries. Major phenolic compounds isolated from the sweet cherry, Prunus avium L, include: dihydrowogonin 7-glucoside, chrysin 7-glucoside, (-)-epicatechin, (+)-catechin (Fig. 1.10), kaempferol 3-rutinoside, 3-galactosyl-7-diglucoside, quercetin 3-rutinoside and 3-rutinosy1-4’-diglucoside, prunetin-4’-O-glucoside and neochlorogenic acid (Stohr, 1975; Ingham, 1983). Henning and Herrmann (1980) also reported quercetin 3-O- rutinosyl-7,3’-O-diglucoside, quercetin 3-O-rutinosyl-4-O-glucoside, and kaempferol 3- O-rutinosyl-4’-O-glucoside from sweet cherry. Prunin, kaempferol 3-O-rutinoside, kaempferol 3-O-glucoside, rutin, quercetin 3-O-glucoside, catechin and chlorogenic acid were characterized from P. avium leaves, (Bauer et al., 1989). Also, genistein and prunetin 5-glucosides were isolated from P. avium (Geibel et al., 1990; Khalin et al., 1939). The fruits of native cherry species in the northern hemisphere, P. jamasakura l6 Prunetin: R1= CH3, R2= H, R3: H Prunetin 4'-glucoside: R1: CH3, R2: glucose, R3: H Prunetin S-glucoside: R1= CH3, R2: H, R3: glucose Genistein: R1= H, R2: H, R3: H Genistein 5-glucoside: R1: H, R2: H, R3= glucose Genistein 7-glucoside: R1: glucose, R2: H, R3: H Fig. 1.6. Isoflavonoids from tart cherries 17 ChTYSIl’lZ R1: H, R2: H, R3: H Genkwanin: R1: OH, R2: OCH3, R3: H Genkwanin 5-glucoside: R1= OH, R2= OCH3, R3= glucose Apigenin 5-glucoside: R1: OH, R2: H, R3: glucose Fig. 1.7. Flavones from tart cherries l8 OH Rofcocu = err—"0cm, ocn3 oca3 Cerasidin: R = CH3 Cerasin: R = H COCH —— CHCHz -—.—0H Neosakuranin Fig. 1.8. Other phenolic compounds from tart cherries l9 OH H O O /0 OH H 0" CH CH 0 CH2 0H 2 2 0H 0 H Benzyl beta-D-glucoside 2—phenylethy1beta-D-glucoside 0R2 l 0“ CH3O | 0 OH OH OH / 6-hydroxy-2,6-dimethyl-octa-2(E),7-diethy1 2-methoxy-4-(2-propenyl)phenyl beta-D-glucoside beta-D-glucoside Fig. 1.9. Other glycosides from tart cherries 20 OH H. I . ,O ‘OH OH Catechin Epicatechin Fig. 1.10. Proanthocyanidins from Prunus species 21 Sieb. and P. maximowiczii Rupr are not edible because of their bitter taste. From these two species, Shimazaki et a1 (1991) isolated prunasin, (-)-epicatechin, mandelic acid (Fig. 1 .12), 1,6,2 ’ ,4 ’ ,6 ’ -O-pentaacety1-3-O-trans-p-coumaroylsucrose, 1,6,2 ’ ,3 ’,6’-O- pentaacetyl-3-O-trans-p-coumaroylsucrose, 1 ,6,2 ’,6’-O-tetraacetyl-3-O-trans-p- coumaroylsucrose, 6,2’,4’,6’-O-tetraacety1-3-O-trans-p-coumaroylsucrose, 1,2’,6’-O- tn'acetyl-3-O-trans-p-coumaroy1sucrose, 1 ,6,2 ’-O-triacetyl-3-O-tran-p-coumaroylsucrose and 6,2’,6’-O-triacetyl-3-O-trans-p- coumaroylsucrose (Fig. 1.11). The methanolic extract of the bark of P. jamasakura yielded sakuranin, neosakuranin, (i)eriodictyol, (i)- catechin, (—)-epicatechin, (i)-lyoniresinol, (2S)-5-B-D-xylopyranosyloxy-7-methoxy-4’- hydroxyflavanone (Fig. 1.5), (2,3-trans, 7",8"~erythro)-5a,5b-dihydrobuddlenol B and (i)-(2,3-trans)-2,3-dihydro-2-[3 ’ ,5 ’ -dimethoxy-4’-(1 ’ ’ ,3 ’ ’-dihydroxy-5- benzofuranpropanol (sakuraresinol) (Fig. 1.13) (Yoshinari et al, 1990). Also, 1,6,2’,4',6’- O-pentaacetyl-3-O-cis-p-coumaroylsucrose and 1 ,6,2 ’ ,6’-O-tetraacetyl-3-O-cis-p— coumaroylsucrose (Fig. 1.12) were identified from its bark (Yoshinari et al., 1990). The concentration of the flavonols, flavan 3-ols, and phenolic acids contribute to the color and flavor characteristics of apricots. The phenolic compounds of apricots, Prunus armem'aca L., were quercetin 3-rutinoside and 3-glucoside; kaempferol 3- rutinoside and 3-glucoside (Henning and Hermann, 1980); (+)-catechin; (-)-epicatechin; chlorogenic, neochlorogenic, and crytochlorogenic acid; cis- and tron-3, 4-, and 5-p- coumaroquuinic acids; cis- and tran-3-,4- and 5-feruloquuinic acids; p-coumaric acid glucoside; ferulic acid glucoside; coumarin; and scopoletin (Mosel and Hermann, 1974; Méller and Herrmann, 1983). 22 CH20R7 CH20R1 0R5 R20 0R8 Re H20R3 R4 l,6,2',4',6'-O-pentaacetyl-3-O-trans -P -coumaroylsucrose R1: AC, R2: H, R3: AC, R4: AC, R5: H, R6: AC, R7 = AC, R8: H 1,6,2',3',6'-O-pentaacetyl-3-O-trans -P -coumaroylsucrose R1= Ac, R2= H, R3= Ac, R4: Ac, R5: Ac, R6: H, R7= Ac, R8: H 1,6,2',6'-O-tetraacetyl-3-O-trans -P -coumaroylsucrose R1: AC, R2: H, R3: AC, R4: AC, R5: H, R6: H, R7 = AC, R8: H 6,2’,4',6'-O-tetraacetyl-3-O-trans -P -coumaroylsucrose R1: H, R2: H, R3: AC, R4: AC, R5: H, R6: AC, R7 = AC, R8: H 1,2',6'-O-triacetyl-3-O-trans -P -coumaroylsucrose R1: AC, R2: H, R3: H, R4: AC, R5: H, R6: H, R7 = AC, R8: H l,6,2'-O-triacetyl-3-O-trans -P -coumaroylsucrose R1: AC, R2: H, R3: AC, R4: AC, R5: H, R6: H, R7 = H, R8: H 6,2',6'-O-triacetyl-3-O-trans -P -coumaroylsucrose R1: H, R2: H, R3: AC, R4: AC, R5: H, R6: H, R7 = AC, R8 = H Fig. 1.11. Bitter principles from Prunusjamasakura and P. maximowiczii 23 HIIHO 2 H 6‘0 O ; OH ; H HO OH Mandelic acid CHZORB CH20R1 o O R OAc 1,6,2',4',6'-O-pentaacetyl-3-O-cis -p -coumaroylsucrose R1: AC, R2: H, R3: AC, R4: H, R5: AC, R6: AC, R7 = H l,6,2',6'-O-tetraacetyl-3-O-cis -p -coumaroylsucrose R1: AC, R2: H, R3: AC, R4: H, R5: H, R6 = AC, R7 =H Fig. 1.12. Bitter principles from Prunusjamasakura and P. maximowiczii 24 HO OH HO OMe (2,3-trans,7",8"-erythro )-Sa,5b-dihydrobuddlenol B HO/T OMe (2,3-trans)-2,3-dihydro-2-[3',S'-dimethoxy-4'-(1 ",3 "-dihydroxy-2"-propyloxy] -3-hydroxymethyl-7-methoxy-S-benzofuranpropanol (Sakuraresinol) Fig. 1.13. Sesquilignan and neolignan from Prunusjamasakura 25 Kaempferol 3-sophoroside from the pollen and flavones from the seed coat have been reported from P. amygdalus. in addition to persicogenin 3’-glucosdie (5,3’- dihydroxy-7, 4’-dimethoxyflavanone 3’-glucoside) (Fig. 1.4) (Rawat et al., 1995). Peach fruit, Prunus persica, is rich in hydroxycinnamic acid derivatives, especially chlorogenic and neochlorogenic acid, flavan-3-ols, and flavonol (Henning and Herrman, 1980; Mosel and Herrmann, 1974). In ripe European plums, phenolic compounds such as rutin, neochlorogenic and chlorogenic acids were found (Raynal et al., 1989). The plum tree (Prunus domestica L. ,), which grows widely in the western temperate Himalayas and is cultivated for its fruit in India, yielded seven compounds from an alcohol extract of its heartwood, and these were identified as isosakuranetin (Fig. 1.5), prudomestin, dihydrokaempferide, naringenin, 5,7,4’-trihydroxy-3-methoxyflavanone, 3,5,7-trihydroxy-6,4’- dimethoxyflavanone, and 3,5,7-trihydroxy-8,4’-dimethoxyflavanone. Also, 4-O-methyl- phloracetophenone-6-O-glucoside, 4-O-methyl, phloracetophenone and 5,7-dimethoxy-6- hydroxycoumarin (fraxinol) were identified (Nagarajan et al., 1977). The bark of plum (Prunus grayana Maxim.) has been used as a crude drug for the treatment of coughs in Europe and America. Several phenylpropanoid glucoside isolated from the bark of this plant are 2-(4-hydroxyphenyl) ethyl-(6-O-feruloyl)-B-d-glucopyranoside (grayanoside A), (2-(3,4-dihydroxyphenyl)ethyl-(6-O-feruloyl)-B-D-glucopyranoside) (grayanoside B) and (2R)-[(6-O-caffeoyl)-B--D-glucopyranosyloxy]benzeneacetonitn'le (grayanin) (Fig. 1.14). From the methanol extract of the bark of P. grayana, 2-(4-hydroxyphenyl)-ethyl- (6-O-caffeoyl)-B-D-glucopyranoside, 2-(3,4-dihydroxypheny1)-ethyl-(6-O-caffeoyl)-B-D- glucopyranoside (Fig. 1.14), a bitter tannin-related compound, 3,4,5-t1imethoxybenzoyl- 26 B-D-glucopyranoside, 2-(3',4'-dihydroxyphenyl)-ethyl-B-D-glucopyranoside and 6-0- caffeoyl-B-D-glucopyranoside (Fig. 1.15) were reported (Shimomura et al., 1987; 1989). Similarly, the heartwood of this plant gave (+)-taxifolin, dehydrodicatechin, virgaureoside, henryoside, populine, 2'-B-D-glucopyranosyloxybenzyl 2-(6-O-benzoyl-B- D-glucopyranosyloxy) benzoate (pruyanaside A) and 2'-(6-O-benzoyl-B-D- glucopyranosyloxy) benzyl 2-B-D-glucopyranosyloxy-6-hydroxybenzoate (pruyanaside B) (Fig. 1.16) (Shimomura etal., 1987). Two new phenylpropanoid glucosyl esters, 6-0- caffeoyl-l-O-p-coumaroyl-B-D-glucopyranose and three known compounds: 6-O-p- coumaroyl-D-glucopyranose, l,6-dicaffeoyl-B-D-glucopyranose (Fig. 1.17), 6-0- caffeoyl-D-glucopyranose and (2R)-[(6-O-caffeoyl)-B-D-glucopyranosyloxy] benzene acetonitrile were identified from Prunus buergeriana (Shimomura et al., 1988). Prunus spinosa is distributed throughout Europe and the Middle East. From the extracts of flowers of P. spinosa, Kolodziej et a1 (1991) reported the range of natural dimeric A-type proanthocyanidins such as ent—epicatechin-(4a-—>8; 2a—>O—>7)-catechin and ent-epiafzelechin-(4a—)8; 2a—>O—>7)-epicatechin, ent-epicatechin-(4a—>8; 2a—>O—>7)-epicatechin, ent-epiafzelechin-(4a—>8; 2a—>O—)7)-catechin and ent- epiafzelechin-(4a—>8; 2a—)O—)7) cpiafzelechin (Fig. 1.18). From the water extract of young branches of this plant, two proanthocyanidins were identified using circular dichroism (CD) and insensitive nuclei enhanced by polarization transfer (INEPT) techiques as mahuannin A and ent-epiafzelechin-(Zaa 7, 4a—)8)-epicatechin (Fig. 1.18) (Gonzalez etal., 1992) 27 HO OH / 0 R2 0 R1 0 0 OH HO OH 2-(4-hydroxyphenyl)-ethyl-(6-O-caffeoyl)-beta-D-glucoside R1=H, R2=H 2-(3,4-dihydroxyphenyl)-ethyl-(6-O-caffeoyl)-beta-D-glucoside R1: OH, R2=H 2-(4-hydroxyphenyl)-ethyl-(6-O-feruloyl)-beta-D-glucoside R1=H, R2: CH3 (Grayanoside A) 2-(3,4-dihydroxyphenyl)-ethyl-(6-O-feruloyl)-beta-D-glucoside R1: OH, R2= CH3 (Grayanoside B) O o / 0 OH HO HO OH (2R)-[(6-O-caffeoyl)-beta-D-glucosyl]benzeneacetonitrile (Grayanin) Fig. 1.14. Phenolic glucoside from Prunus grayana 28 /\/.:OH HO O O/\/.: OH HO OH HO OH 2-(3,4-dihydroxyphenyl)-ethy1-beta-D-glucoside 6-O-caffeoy1-D-glucoside HO 3,4,S-tn'methoxybenzoyl-beta-D-glucoside Fig. 1.15. Phenolic glucoside from Prunus grayana 29 OH O ”"0 OH Ho . , Taxifolin Dehydrodlcatechm A OH OH OH O 0R2 o "0 OH 0R1 OH O O ...... O o «mo—O . O O OH H HO Populine Vigaureoside A: R1: H, R2: H Henryoside: R1= H, R2: OH Pruyanaside A: R1: Benzoyl, R2: H Fig. 1.16. Phenolic compounds from Prunus grayana 30 HO o 0 HO / \ / R 0 go 0 on OH H HO 6-O-caffeoyl-1 -O-p-coumaroyl-beta-D-glucose R = H 1 ,6-dicaffeoyl-beta-D- glucose R = OH Fig. 1.17. Phenylpropanoid glucosides from Prunus buergeriana 31 ent-epicatechin-(4a >8;2a >O- 2* 7)-catechin \ \ HO 0 I I \ \ \ R \ 0H° 1 OH OH ‘~ 0 ent-epicatechin-(4a—> 8:23 —- > O - -> 7)-epicatechin R1=OH, R2=OH ent-epiafzelechin-(4a—>8;2a- ->O - -->7)-epicatechin R1=H, R2=OH ent cpiafzelechin-(4&9 8;2a—> O—~> 7)-epiafzelechin R1=H, R2=H (mahuannin A) Fig. 1.18. Proanthocyanidins from Prunus species 32 1.3 Other compounds present in Prunus species Schaller and Von Elbe (1973) reported eight carotenoids in tart cherries (P. cerasus). They are phytofluene, a-carotene, B-carotene, cryptoxanthin, cryptoflavin, lutein, zeaxanthin and mutatoxanthin (Fig. 1.19). Tart cherries also contain several organic acids such as aspartic, citramalic, citric, fumaric, galacturonic, glyceric, glycolic, glucuronic, glutamic, glutaric, isochlorogenic, lactic, malic, malonic, phosphoric, quinic, shikimic, succinic and tartaric acids. In tart cherry, malic acid represents 75-95% of the total titrable acidity (Krishna et al., 1965). Also, Tanchev (1980) reported salicylic, cinnamic, gentisic, gallic, isoferulic and p-comnaric acids in tart cherries. The main sugars reported from the fleshy part of the tart cherry are fructose, a— and B-glucose. However, sorbitol, sucrose and inositol were detected in small quantities (N eubeller et al., 1977). The volatile compounds characterized from ‘Bing’ sweet cherry fruit were propanal, butanal, ethyl acetate, 2-propanol, ethanol, pentanal, butyl acetate, hexanal, 2- methyl butyl acetate, butanol, heptanal, butyl butyrate, butyl 2-methylbutyrate, ethyl hexanoate, l-pentanol, hexyl acetate, octanal, 6-methyl 5-hepten-2-one, l-hexanol, nonanal, hexyl 2-methyl butyrate, ethyl octanoate, acetic acid, 2-ethyl l-hexanol, decanal, benzaldehyde, hexyl hexanoate and 2-methyl 2,4-pentanediol (Mattheis et al., 1992). The orange color in apricot, Prunus armeniaca L., is attributed to a- and [3- carotene, phytofluene, phytoene, cis-B-and cis-a-carotene, cryptoxanthin, lutein, and other carotenoids (Curl, 1960). Molnar et a1. (1987) isolated (3S, 5R, 6S)-5,6-epoxy-3- hydro-l2'-apo-B-carotene-3,12'-diol (persicaxanthin) and periscachrome from peach. 33 beta-carotene Zeaxanthin Fig. 1.19. Principle carotenoids in tart cherry 34 2. BIOLOGICAL ACTIVITY OF COMPOUNDS ISOLATED FROM PRUNUS SPECIES Most Prunus species fruits contain anthocyanins, pro-anthocyanidins and other phenolic compounds which are important to human health. The flavonoids have long been considered to possess antiallergenic, antiinflammatory, antiviral and anticarcinogenic activities. Gabon (1986) verified the antioxidant effects of anthocyanins based on a number of external factors such as pH, the ratio of L-ascorbic acid and oxygen, reaction time and the nature of organic acids present. It is well known that diets rich in fruit and vegetables are correlated with decreasing rask of cardiovascular diseases and cancer (Block, 1992; 1994). These protective effects of fruits have been attributed, in large part, to the antioxidants vitamin C and B-carotene, but also to the minor constituents including carotenoids and phenolics such as the flavonoids and phenylpropanoids. The fi'uits of Prunus spp have been reported to possess antipyretic activity and to be useful against thirst, leprosy and leucoderma (Chopra et al., 1956). Anecdotal reports suggested that consumption of cherries could alleviate arthritic- and gout-related pain (Hamel and Chiltoskey, 1975). P. spinosa is distributed throughout Europe and the Middle East. Its ethnobotanic use is best known in Navarra, where infusions of its branches are used in the treatment of hypertension (Fernandez, 1981). The flowers of P. spinosa were used in folk medicine in Navarra (Spain) as a mild purgative, diuretic, diaphoretic and depurative drug (Gessner, 1974). The biological activities of P. spinosa are due to the presence of numerous flavonoids including rutin and quercetrin (Rodriguez, 1986). The leaves are cited as having antidiabetic and antiasthmic properties. 35 Pharmacological studies of P. spinosa L. showed that intravenous injection of the aqueous extract produced a reduction in blood pressure. Further studies showed that the aqueous extract of the branches of P. spinosa L. diminished the response to histamine and epinephrine in guninea pig ileum (Rodriguez, 1986).. The effect of cherry-stalk (Prunus avium) extract on smooth muscle preparations was investigated. The extract, consisted of flavonoid components, was found to reverse the contractions of the rat and guinea pig uterus and counteracted oxytoxin-induced contractions (Hetényi and Vélyi-Nacy, 1969). Also, they investigated the cardiovascular effects of cherry stalk extract. The extract exhibited the reduction of heart damage in frogs caused by quinine. Blazso and Gabor (1994) reported that the stalk extract of cherry showed good antiinflammatory activity induced with capsaicin or croton oil in the mouse ear. The European dwarf or ground cherry, P. fiuticosa Pall, a low, spreading bush, with intensely flavored, somewhat bitter fruits. It is used in Europe in specialty soft drinks. The fruits and leaves of P. fruticosa also are used as a source of an aromatic oil to flavor liquors (Olden, 1960). Keishi-bukuryo-gan is a traditional Chinese herbal remedy used for the treatment of gynecological disorders such as hypermenorrhea, dysmenorrhea and infertility (Sakamoto et al., 1988). It contains five components: bark of Cinnamomum cassia, root of Paeonia lactiflora, seed of Prunus persica, carpophores of Poria cows and root bark of Paeonia sufl'ruticosa. The extract of P. mume fruits showed worrnicidal and bactericidal activities (Rhee et al., 1981). The almond (P. amygdalus) shell extract showed antimicrobial activity (Sachdev, 1968). Similarly, the seed exhibited a hypoglycemic effect in albino rabbits (Teotia and Singh, 1997). 36 The Xiaoyu Pian (XYP), containing P. persica, Carthamus tinctorius, Glycyrrhiza uralensis as the major constituent, was used to treat patients with platelet aggregation defect. The results suggested that XYP could regulate the hemostatic action and the platelet aggregation function (Shen et al., 1994). Using the Ames/Salmonella/microsome assay, Yamamoto et al (1992) examined the antimutagenic effect of the hexane extract of apricot (P. armem'aca L.), peach (P. persica Bat. ), cherry (P. avium L.), plum (P. salicina Lindle) and almond (P. dulcis Mill) seeds. Hexane extracts of apricot and peach seeds inhibited the mutagenicity of benzo[a]pyrene (b[a]P), but those from cherry, plum and almond did not. The mutagenicities of 3-amino-l,4- dimethyl-SH-pyridol[4,3-B]indole (Trp-p-l) and 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF -2) also were inhibited by the extracts of apricot and peach. It is believed that the polyphenolic compounds of Prunus species act as antioxidants contributing to anticarcinogenic, cardioprotective and other related activities. A brief survey of the various biological activities of flavonoids, which may exist in the Prunus species including tart cherries, constitutes the rest of this chapter. 2.1 Antioxidant activity Free radical formation is related to the normal metabolism of aerobic cells. The oxygen consumption inherent in cell growth leads to a series of oxygen free radicals, such as superoxide, hydroxyl, and lipid peroxides. This group of radicals may react with nucleic acids and proteins to produce oxidative reactions (Byers and Perry, 1992). Antioxidants are needed to prevent the formation and oppose the actions of reactive oxygen and nitrogen species such as nitric oxide (.NO), which are generated in vivo and 37 cause damage to DNA, lipids, proteins, and other biomolecules (Halliwell, 1996). Endogenous antioxidant defenses by superoxide dismutases, catalase, metal-binding proteins, and glutathione peroxidase are inadequate to prevent such damage completely. Therefore, the diet-derived antioxidants are important in maintaining health (Halliwell, 1996). Many dietary compounds have been proposed to have important antioxidant activities including, vitamins E, C and related plant pigments, such as flavonoids. Flavonoids have a variety of biological effects in numerous mammalian cell systems, in vitro as well as in vivo. Recently, much attention has been paid to their antioxidant properties. Hanasaki et al (1994) compared the abilities of 15 flavonoids as scavengers of active oxygen (hydroxyl radical and superoxide anion. The phenolic compounds (+)-Catechin, (-)-epicatechin, 7,8-dihydroxy flavone and rutin exhibited an 'OH scavenging activity 100-300 times superior to that of mannitol, a typical 'OH scavenger. A major part of this inhibitory effect may be due to the suppression of xanthine oxidase activity by the flavonoids. (Hanasaki et al., 1994). The antioxidant activity of a number of flavonoids in refined and bleached (RB) canola oil was compared with that of commonly used synthetic antioxidants, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) (Wannasundara and Shahidi, 1994). Among the flavonoids tested, myricetin, (-)epicatechin, naringenin, rutin, morin, and quercetin were superior to BHA and BHT in inhibiting the oxidation of canola oil. Therefore, natural flavonoids may have potential application for the stabilization of canola oil (Wanasundara and Shahidi, 1994). Cao et a1. (1997) investigated the antioxidant and prooxidant behavior of flavonoids and the related structure-activity relationships using the oxygen radical 38 absorbance capacity (ORAC) assay, which was carried out on the spectrophotometric centrifugal analyzer. The results indicated that flavones, isoflavones, and flavanones are antioxidants against peroxyl and hydroxyl radicals in the presence of Cu2+ activities (Cao et al., 1997). The higher numbers of hydroxyl substitutions produced stronger antioxidant (Cao et al., 1997). The other report also indicated that the flavonoids that contain multiple hydroxyl substitutions showed antiperoxyl radical activities several times stronger than Trolox, a water soluble alpha-tocopherol analogue (Chen et al., 1996). A single hydroxyl substitution at C5 position provided no activity, whereas the di-OH substitutions at C3'and C4' were particularly important to the peroxyl radical absorbing activity of a flavonoid (Bors et a1. 1990). The conjugation between rings A and B did not affect the antioxidant activity, but is very important for the copper-initiated prooxidant action of a flavonoid (Cao et al. 1997; Heilmann et a1. 1995). The O-methylation of the hydroxyl substitutions inactivated both the antioxidant activities of the flavonoids (Cao et al. 1997). Toumaire et al (1993) also determined the structure-activity relationship of flavonoids by the rate constants of the chemical reaction of these flavonoids with 02' determined by near-IR singlet oxygen luminescence kinetic measurements. They found that the basic element of antioxidant activity was due to the conjugation of the B-ring to the 4-oxo via a C2-C3 double bond and the presence of a C3 hydroxyl group (Tournaire et al., 1993). According to the above studies, four groups were considered to be important for determining their radical scavenging and antioxidant capacity. They are (a) the o- dihydroxy (catechol) structure in the B-ring, which confers greater stability to aroxyl radicals, possibly through hydrogen bonding which participates in electron delocalization (Bors et al., 1990 a, b); (b) the C2-C3 double bond in conjugation with C4-oxo function, 39 which are asponsible for electron delocalization from the B-ring (Tournaire et al., 1993; Bors et al., 1990a); (c) the presence of both C; and C5 hydroxyl groups for maximal radical-scavenging capacity and strongest radical absorption (Afanas’s ev et al., 1989, De Whalley et al., 1990) and (d) the number of hydroxyl groups, a higher number providing maximal radical-scavenging potential and the stronger radical absorption (Bors et al., 1990; Cao al., 1997). Saija et a1 (1995) studied the flavonoids quercetin, hesperetin, naringenin and rutin in in vitro experimental models. Quercetin, hesperetin, and naringenin interacted with dipalmitoylphosphatidylcholine (DPPC) liposomes causing different shifts of the main transition peak temperature (Tm) typical for DPPC liposomes (Saija et al., 1995). The results suggested that flavonoid capacity to modify membrane-dependent processes, such as free-radical-induced membrane lipoperoxidation, is related not only to their structural characteristics, but also to their ability to interact with and penetrate the lipid bilayers (Ioku et al., 1995). Free radicals and antioxidants also are discussed widely in the clinical literature. Bindoli et a1 (1977) demonstrated that silymarin protected liver mitochondria and microsomes from lipid peroxidation. Also, quercetin and taxifolin showed similar protective action. Jha et al (1985) described the antiperoxidation action of isoflavonoids on rat microoxidation. Eight flavonoids were investigated for their antiperoxidative activities against lipid peroxidation induced in liver cell membranes either by nonenzymic (ascorbic acid-Fe2+ system, FeAs) or by enzymic methods (arachidonic acid, AA) (Galvez et al. 1995). When lipid peroxidation was induced by F eAs and AA, the 40 order of inhibitory potency for the different flavonoids assayed was different (Galvez et a1. 1995). These flavonoids also were tested for their influence on glutathione-related enzymes, which constitutes one of the principal physiological antioxidant systems. It was concluded that the antiperoxidative effect shown by most of the flavonoids was exerted without modifying these enzymes (Ahmad et al., 1990). Low-density lipoproteins (LDL), mildly oxidized by copper ions or UV radiation exhibited a cytotoxic effect on cultured endothelial cells (Schmitt et al. 1995). A mixture of the three compounds rutin/ascorbic acid/alpha-tocopherol (4/4/1) exhibited a synergic antioxidant effect (Negre Salvayre et al., 1995). The protective effect of antioxidants was limited due to their own toxicity in the in vivo systems. The antioxidant mixture permitted a maximum cytoprotective effect when used in lower concentrations and helped to prevent the cytotoxicity (Negre Salvayre et al., 1995). In searching for new drug candidates which could help bridge the gaps between free radical oxidations, pathophysiological responses, and pharmacological treatment, a series of flavonoids was screened (Ursini et al., 1994). The most interesting compound tested was 3'-hydroxyfarrerol. This compound was an effective inhibitor of microsomal lipid peroxidation induced by either adenosine 5'-diphosphate (ADP) or carbon tetrachloride (Ursini et al. 1994). Kandaswami and Middleton (1994) proposed that flavonoids react with peroxy radicals, thus bringing about the termination of radical reactions. A number of isomeric or chemically closely related C-methylated dihydrochalcones, was isolated from the fi'uit exudate of Myrica gale L. One of the compounds myrigalone B (2',6'-dihydroxy-4'-methoxy-3',5'-dimethyldihydrochalcone) 41 showed good activity in inhibition of lipid peroxidation induced by tert-butyl hydroperoxide or bromotrichloromethane in isolated rat hepatocytes. Also, this compound inhibited enzymatic lipid peroxidation in linoleic acid by soybean 15- lipoxygenase and peroxidation induced by F 62+ ions in a cell free system with linolenic acid (Malterud et al., 1996). The biphenyl compound, 3,4,3',4'-tetrahydroxy-5,5'- diisopropyl-2,2'-dimethylbiphenyl, and a flavonoid, eriodicytol, were isolated as antioxidant components from the leaves of Thymus. These compounds inhibited superoxide anion production in the xanthine/xanthine oxidase system. Mitochondrial and microsomal lipid peroxidation induced by Fe3+-ADP/NADH or Fe3+-ADP/NADPH also were inhibited by these compounds (Haraguchi et al., 1996). Natural antioxidants, such as flavonoids, may act as reducing agents, as free-radical scavengers, as complexers of prooxidant metals, and as quenchers of the formation of singlet oxygen. However, the most important activity of these compounds is credited to their primary antioxidant activity as free radical acceptors and/or as chain-breakers (Kandaswami and Middleton, 1994). 2.2 Anti-inflammatory activity An inflammatory reaction is induced by one or several chemical or biological mediators such as arachidonic acid derivatives, prostaglandins [PG], leukotrienes [LT], thromboxanes [TX], vasoactive amines (histamine or serotonin), and oxygen free radicals (superoxide ion, 02', or hydroxyl radicals, 'OH) (Williams, 1983) (Fig. 1.20). The activity of flavonoids in antiinflammatory and antiallergenic responses was reviewed (Gabor, 1986). Also, recent studies revealed the antiinflammatory dose-dependent activity of 42 Phospholipids Triglycerides ‘1“ Phospholipases Arachidonic acid l Cyclooxygenases l Lipoxygenases v 1 PGGZ, PGH2 HETES , l TXs, PGIZ, PGE2, free radicals LTB4, LTC4 and LTD4 Fig. 1.20. Mechanism of inflammation 43 hesperidin, diosmin, and other flavonoids on the metabolism of arachidonic acid and histamine release (Gabor and Razga, 1991). These flavonoids significantly inhibited lysosomal enzyme secretion and arachidonic acid release from membranes by inhibiting lipoxygenase, cyclooxygenase, and phospholipase A2 (Gabor, 1986). The arachidonic acid released from membrane phospholipids or other sources is metabolized by the 5- lipoxygenase (S-LO) pathway to leukotrienes (LTC4, LTD4, LTE4 and LTB4) (Lewis and Austen, 1984). Butenko et al. (1993) investigated antiinflammatory properties and inhibition of leukotriene C4 biosynthesis in vitro by flavonoid baicalein, 5,6,7-trioxyflavone-7-O-B-D- glucoside. The antiinflammatory activity of baicalein was greater in the chronic inflammation model rat adjuvant arthritis than observed in the rat carrageenan-induced paw edema (Butenko et al., 1993). A study of 5-lipoxygenase (5-LO) inhibitory activity of baicalein on leukotriene C4 (LTC4) biosynthesis by rat resident peritoneal macrophages stimulated with calcium ionophore (A 23186) showed that baicalein significantly inhibited the LTC4 production. These finding suggested that inhibition of the 5-LO pathway of arachidonic acid metabolism may be one of the mode of actions of baicalein’s antiinflammatory activity (Butenko et al., 1993). Gil et al. (1994) tested four flavonoids for their influence on human recombinant synovial phospholipase A2. They showed selectivity for quercetagetin, kaempferol-3-O-galactoside, scutellarein and scutellarein-7—O-glucuronide against phospholipase A2, respectively. These flavonoids also inhibited 12-O-tetradecanoylphorbol-13-acetate-induced ear edema in mice with a potency comparable to that of indomethacin and carrageenan-induced mouse paw edema (Gil et al., 1994). The anti-inflammatory effects of Daflon, a micronized purified 44 flavonoid fraction containing 90 and 10% of diomin and hesperidin, respectively, were studied in in vivo and in vitro models (Jean and Bodinier, 1994). In a model study of inflammatory granuloma in the rat, Daflon (100 mg/kg, orally) considerably reduced edema formation and inhibited the synthesis of PGE2, a-PGF2 and TXB2 (Jean and Bodinier, 1994). Intravenous injection of Daflon reduced the hyperglycemia induced by the injection of alloxen in rats. Similarly, the mechanism of antiinflammatory activities of Daflon’s ability to scavenge active oxygen radicals was demonstrated in vitro using human neutrophils (Labrid, 1994). Another flavonoid, apigenin, demonstrated potent antiinflammatory activity in carrageenan-induced rat paw edema and inhibited IL-ch-induced prostaglandin synthesis and TNF-a-induced lL-6 and IL-8 production (Gerritsen et al., 1995). Yamarnoto et al (1984) studied the effect of several benzoquinones and flavonoids. Cirsiliol, 5,3',4'- trihydroxy-6,7-dimethoxyflavone, proved to be a potent inhibitor of 5-LO derived from rat basophilic leukemia cells and guinea pig peritoneal polymorphonuclear leucocytes (PMN) (Yamamoto et al., 1984). Selected flavonoid inhibitors significantly suppressed the 5-LO activity and LT synthesis by sensitized, challenged guinea pig lung tissue. Cirsiliol had approximately 10-fold less activity against the l2-LO enzyme and negligible effect on cyclooxygenase (COX) from bovine vesicular gland (Yamamoto et al., 1984). Partially purified mouse epidermal cell lipoxygenase (LO) was inhibited potently by hydroxyflavones but not by flavone itself (Wheeler and Berry, 1986). The partially purified 5-LO of rat basophilic leukemia cell was also inhibited by Cirsiliol (Furukawa et al., 1984). Artonin E (5'-hydroxymorusin), a naturally occurring prenylflavone, was a potent and selective inhibitor of porcine leucocyte 5-LO (Reddy et al., 1991). Hepolaetin 45 (5,7,8,3',4'-pentahydroxyflavone) proved to be a good inhibitor of rat peritoneal leucocyte S-LO whereas it was inactive as a cyclooxygenase inhibitor (Moroney et al., 1988). Swies et al. (1984) found that rarn seminal vesicle COX was stimulated by quercetin and several other flavonoids at high arachidonic acid substrate concentrations. However, quercetin showed inhibitory activities at low substrate concentrations. The activity of cyclooxygenase was affected by specific flavonoids. Baumann et al. (1980) examined the effect of several flavonoids on arachidonic acid peroxidation. Luteolin, 3',4'—dihydroxyflavone, morin, galangin and (+)-catechin were moderately active inhibitors of rat renal medulla COX enzyme. Landolfi et al. (1984) reported that flavone, chrysin, apigenin and phloretin depressed COX activity and inhibited platelet aggregation. They concluded that flavonoids offer important therapeutic potential for the treatment of a variety of inflammatory diseases such as involving an increase in leukocyte adhesion and trafficking (Landolfi et al., 1984). The effects of 24 flavonoid derivatives, reported as antiinflammatory, on lysosomal enzyme secretion and arachidonic acid release in rat neutrophils were also investigated (Tordera et al., 1994). Amentoflavone, quercetagetin-7-O-glucoside, apigenin, fisetin, kaempferol, luteolin and quercetin were the most potent inhibitors of B-glucuronidase and lysozyme release. Amentoflavone showed the highest potency for inhibition of Biglucuronidase release. Another flavonoid, 3-hydroxyflavone, was the only compound which exhibited a biphasic effect (Tordera et al., 1994). The flavonols fisetin, kaempferol and quercetagetin-7-O- glucoside, the flavones chrysin, apigenin and luteolin, as well as amentoflavone and naringenin, significantly inhibited arachidonic acid release from lipid membranes (Tordera et al., 1994). Hypolaetin-8-O-glucoside did not show significant effect on 46 lysosomal enzyme secretion, but it inhibited B-glucuronidase release in an experimental model of inflammation in rats (Barberan et al., 1987). Some structural activity relationships are also studied for the inhibition of lysosomal enzyme release. Polyhydroxylated aglycones of the flavone or flavonol types and the presence of a free hydroxyl group at C4', the keto group at C4 position and the C2- C3 double bond increased the inhibition of lysosomal enzyme release (Tordera et al., 1994). However, the glycosylation or the introduction of a free hydroxyl at C2' decreases the inhibitory activity (Tordera et al., 1994). The most active compounds were C2-C3 unsaturated, hydroxylated at C7 and C4' and with some additional hydroxy groups at C5, C3 or C3' (Tordera et al., 1994). Limasset et al. (1993) measured the inhibitory activity of 34 flavonoids or related substances on the release of reactive oxygen species by stimulated human neutrophils. They reaffirmed that ring A (C5 and C7) and ring B (C3' and C4') dihydroxylation, ring C hydroxylation at C3 and the presence of a methoxy group on ring B are important to produce high potency (Limasset et al., 1993). Flavonoids not only showed antiinflammatory activities, but also demonstrated protection against nonsteroidal antiinflammatory drug (indomethacin)-induced acute gastric damage. Blank et al (1997) reported that flavonoids could protect against acute gastric damage. The effects of 5-methoxyflavone and 5-methoxyflavanone on the gastric vasculature were compared both in vivo and in vitro on rat superior mesenteric arteries. Oral application of 5-methoxyflavone reduced indomethacin-induced macroscopic damage (Blank et al., 1997). However, the demage was not significantly reduced by 5- methoxyflavanone. Also, indomethacin-induced leukocyte adherence was inhibited to a greater extent by S-methoxyflavone than by S-methoxyflavanone (Blank et al., 1997). 47 This result suggested that the flavonoids such as 5-methoxyflavone could provide gastroprotection against nonsteroidal antiinflammatory drug-induced gastric damage (Blank et al., 1997). Some authors further suggested that scavenging of reactive oxygen species and inhibition of arachidonic acid metabolism may be related (Dehmlow et al., 1996; Hoult and Paya, 1996; Jean and Bodinier, 1994)). The effects of the flavonoid silibinin on the formation of reactive oxygen species and eicosanoids by human platelets, white blood and endothelial cells were studied. The formation of leukotrienes through the 5- lipoxygenase pathway was strongly inhibited (Dehmlow et al., 1996). This indicated that the deleterious effects of HOCl that can lead to cell death, and those of leukotrienes that are important in inflammatory reactions, can be inhibited by silibinin. There is no doubt that the flavonoids have profound effects on the function of immune and inflammatory cells as determined by a large number and variety of in vitro and in vivo studies. Ample evidence indicated that selected flavonoids, depending on structure, can affect (usually inhibit) secretory processes, mitogenesis, and cell-cell interactions including possible effects on adhesion molecule expression and function. Moreover, evidence indicated that certain flavonoids may affect gene expression and the effects of cytokines and cytokine receptors. One possible mechanism could be that flavonoids stimulate or inhibit protein phosphorylation and thereby regulate cell function. The effects of some flavonoids can certainly be attributed to their antioxidant and radical scavenging properties (Middleton and Kandaswami, 1992). 2.3 Anticarcinogenic properties of phenolic compounds 48 Recently flavonoids have attracted attention as potentially important dietary cancer chemoprotective agents (Hertog et al., 1993) and possible antitumor agents (Kandaswami et al., 1991). Flavonoids, due to their antioxidant properties and their ability to absorb UV light, may act in all stages of the carcinogenic process involving damage to the DNA (or initiation step), tumor growth (or promotion step), and invasion (or proliferative step). Due to their absorption of ultraviolet light, flavonoids can protect DNA from light damage. Recent experiments with plasmid DNA irradiated with UV—B light, showed the protective effect of naringenin and rutin against UV-induced DNA damage (Kootstra, 1994). In parallel, flavonoids are able to quench free radicals, which may promote mutations when they are generated in the vicinity of DNA. This radical scavenging ability is responsible for the protective effect of flavonoids observed in whole-body y-ray irradiated mice (Shimoi et al., 1994). Flavonoids may also protect DNA by interacting directly with carcinogens that have escaped detoxification processes, as occurs in the chromosome aberration induced by bleomycin (Heo et al., 1994). Quercetin has been shown to inhibit the mutagenic activity of benzo[or]pyrene (Bp), a representative polynuclear aromatic hydrocarbon (PAH) carcinogen, in bacterial mutagenicity studies (Ogawa et al., 1985). Castillo et al (1989) investigated the effect of quercetin on the in vitro and in vivo growth of two squamous cell carcinoma cell lines and a normal human lung fibroblast-like cell line. Quercetin caused inhibition of growth in both squamous cell carcinoma lines (Castillo et al., 1989). Effect on the fibroblast-like human lung cells was noted only at the high concentrations. Significant growth inhibition of squamous cell carcinoma was observed in implantable cell growth chambers retrieved 3 days after quercetin treatment. Quercetin appears to possess cytotoxic effect on 49 squamous cell carcinoma of head and neck origin both in vivo and in vitro (Castillo et al., 1989). The inhibitory effect of quercetin on malignant cells appears to be selective and dose-dependent. In addition, quercetin inhibited colon cancer in rats induced by azoxymethanol (Deschner et al., 1991). Apigenin, another flavonoid, was reported to suppress 12-O-tetradecanoyl- phorbol-lB-acetate (TPA)-mediated tumour promotion in mouse skin (Huang et al., 1996). F lavonoids, bilirubin and myricetin, were capable of inhibiting the mutagenicities caused by 4-nitroquinoline l-oxide and cigarette smoke (Carnoirano et al., 1994). The intake of quercetin in experimental diet lowered the incidence of colon tumors in azoxymethanol treated rats (Deschner et al., 1991) as well as fibrosarcoma in mice induced by 20-methyl colanthrene (20-MC) (Elangovan et al., 1994). The possible mode of action of quercetin may be through its influence on the initiation and promotion phases of the carcinogenic processes coupled with the enhancement of detoxification process (Elangovan et al., 1994). Non-melanoma skin cancer induced by solar UV is one of the most common cancers among humans (Bergfelt, 1993). Therefore, it is important to identify agents that can offer protection against this cancer. Katiyar et al (1997) evaluated the protective effects of silymarin, a flavonoid compound isolated from the milk thistle plant, against UVB radiation-induced nonmelanoma skin cancer in mice. The results indicated that silymarin can provide substantial protection against different stages of UVB-induced carcinogenesis, possibly via its strong antioxidant properties (Katiyar et al., 1997). Flavonoids also display an antiproliferative effect on various human neoplasic cell lines such as myeloid and lymphoid leukemia cells (Piantelli, 1993), gastric cancer cells 50 (Yoshida et al., 1990), ovarian cancer cells (Scambia et al., 1990), prostate cancer cells (Peterson and Barnes, 1993), and squamous cell carcinoma (Kandaswami et al., 1991). Flavonoids affect cell metabolism in various ways, either at the cell membrane level or on the intracellular enzymes. Flavonoid effects frequently include an inhibition of glycolysis, which is generally a very active metabolic pathway in tumor cells (Suolinna, 1975). The flavonoids may affect the activity of various enzymes involved in the transduction of mitogenic signals (kinases, phospholipases, and phosphodiesterases) and regulate other enzymes, which are critical for cell growth and proliferation. Most of the chemical carcinogens seem to require metabolic activation by DNA-reactive intermediates by cytochrome—P450-mediated mixed-function oxidase (MFOs) in order to exert their carcinogenic action (Dipple et al., 1984). The covalent binding of these reactive intermediates to cellular DNA, leading to adduct formation, is considered to be a critical event in the initiation of carcinogenesis (Miller, 1978). Flavonoids may inhibit carcinogenesis by acting as ‘blocking agents’ (Wattenberg, 1983). Blocking agents can inhibit carcinogenesis by one of several possible mechanisms such as inhibiting the metabolic activation of the carcinogen, binding to cellular targets such as DNA, RNA and protein (Wattenberg, 1983). On the other hand, flavonoids may also inhibit tumor promotional events. Almost all of the polyphenolic compounds from plants, chlorogenic acid, caffeic acid, ferulic acid, alpha- tocopherol, catechins, camosol, curcurnin, curry, mustard and synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) possess several common biological and chemical properties (Wattenberg, 1985). These included antioxidant activity, the ability to scavenge active oxygen species and electrophiles, the 51 ability to inhibit nitrosation and to chelate metals, and the capability to modulate cellular enzyme activities. These compounds are likely to be able to inhibit various steps of tumor development in experimental animals and probably in humans (Huang and Ferraro, 1992) 2.4 Polyphenolics and cardiovascular diseases Certain polyphenolic compounds have been shown to have an effects on blood platelet function, leukocyte function, blood coagulation, blood rheology, and ultimately thrombosis (Maalej, et al., 1997). Several studies indicated that certain flavonoids may have protective and therapeutic effects in coronary heart diseases. Knekt et a1 (1996) studied the association between dietary intake of flavonoids and subsequent coronary mortality. Finnish men and women between the age of 30-69 years from different parts of Finland and free from heart disease were investigated. The results indicated that people with very low intakes of flavonoids had higher risks of coronary disease. Hertog et al (1993) assessed the flavonoid intake of 805 men aged 65-84 years in 1985. Flavonoid intake was inversely associated with mortality from coronary heart disease and showed an inverse relation with incidence of myocardial infarction (Hertog et al., 1993). In order ‘ to detemrine whether flavonoid intake explains differences in mortality rates from chronic diseases between populations. Results indicated that average flavonoid intake may partly contribute to differences in coronary heart disease mortality across populations (Hertog et al., 1995). In France and other Mediterranean areas, red wine is regularly consumed with meals. Red wine and grape juice inhibited platelet activity. The reason is that red wine 52 and grape juice contain a wide variety of naturally occurring compounds including tannins, anthocyanins, and phenolics including flavonols and flavones. The antithrombotic effect of red wine and grape juice may be due to the flavonoids common in some vegetable, fruits, and herbs such as tea (Demrow et al., 1995). Besides having anti-thrombotic properties, flavonoids are also antioxidants that prevent lipid oxidization known to contribute to atherosclerosis. Studies have demonstrated that the consumption of flavonoid rich foods and beverages may have protective effects against the development of coronary artery disease and may decrease the risk of myocardial infarction due to their platelet inhibitory and antioxidant effects (Folts et al., 1997). In vitro experiments showed that flavonoids inhibit the oxidation of low-density lipoprotein (LDL) and reduce thrombotic tendencies (Maalej et al., 1997). There is ample evidence that free-radical oxidation of LDL plays an important part in atherogenesis. Flavonoids are scavengers of free radicals such as superoxide anions and peroxy radicals and thus can interrupt radical chain reactions. The oxidation of low-density lipoprotein (LDL) is thought to be a key step in the development of atherosclerosis (Mosca et al., 1997). The catechin oligomers, the procyanidin dimers and trimers were extracted, isolated and purified from grapes seeds (Kovac et al., 1992). These compounds were tested for their inhibition of LDL oxidation, along with other monomeric wine phenolics. Thus, the numerous phenolic compounds found in wine are potent antioxidants in inhibiting LDL oxidation in vitro (Kerry and Abbey, 1997). In vitro, some flavonoids inhibit the oxidative modification of LDL by macrophages, mainly by inhibiting the generation of hydroperoxides and protecting the a-tocopherol present in lipoprotein 53 oxidation. It is possible that flavonoids reduce the rate of oxidized compound, thus inhibiting the growth of atherosclerotic complications (Hertog et al., 1993). 2.5 Antiviral effects of flavonoids Naturally occurring flavonoids with antiviral activity have been recognized in the past, but only recently they are investigated for their antiviral activity. Quercetin, morin, rutin, dihydroquercetin (taxifolin), dihydrofisetin, leucocyanidin, pelargonidin chloride, apigenin, catechin, hesperitin and naringenin have been reported to possess antiviral activity against 11 types of viruses (Selway, 1986). Mucsi and Pragai (1985) demonstrated the inhibitory effect of four flavonoid compounds on virus multiplication and their influence on the intracellular cyclic AMP (cAMP) levels in cell cultures. Quercetin and quercitrin reduced the yields of human (alpha) herpesvirus 1 (HSV-l) and Suid (alpha) herpesvirus 1 (pseudorabies virus), but hesperidin and rutin had no effect. Further, quercetin and quercitrin elevated the intracellular level of cAMP, whereas hesperidin and rutin did not alter the cAMP level. Both antiviral activity and CAMP-enhancing effect were dependent on the concentrations of the flavonoids. This study suggested that a relationship existed between the antiviral effect and the CAMP-enhancing activity of flavonoids. The flavonoid quercetagetin 3'-methylether isolated from flower and leaf extracts of Centaurea rupestris L. revealed a strong antiviral activity when inoculated simultaneously with tomato bushy stunt virus in two Nicotiana species (Rusak et al., 1997). Also, the flavonoids have the ability to interfere with the initiation of virus infection (Rusak et al., 1997). Critchfield et al (1997) reported that transcription from the 54 integrated provirus is inhibited by members of two distinct classes of compounds, the flavonoids and the benzothiophenes, via an unknown mechanism, possibly involving a cellular factor (Sandoval and Carrasco, 1997). Ro-090179 (Ro), a flavonoid isolated from the herb Agastache rugosa, induced the specific swelling and disruption of the Golgi complex and strongly inhibited poliovirus infection. Also, Ro provoked the swelling and the disruption of the stacked cistemae and trans-Golgi elements without affecting the cis- most Golgi cistemae (Sandoval and Carrasco, 1997). Isoscutellarein—8-methylether (5,7,4'-trihydroxy-8-methoxyflavone) from the roots of Scutellaria baicalensis was studied on the single-cycle replication of mouse-adapted influenza viruses A/Guizhou/54/89 (H3N2 subtype) and B/Ibaraki/2/85 in Madin-Darby canine kidney (MDCK) cells. The agent suppressed replication of these viruses from 6 to 12 h after incubation in a dose-dependent manner (Nagai et al., 1995). Results suggested that this compound inhibited the replication of A/Guizhou and B/Ibaraki viruses at least partly by inhibiting the fusion of viral envelopes with the endosome/lysosome membrane which occurs at the early stage of the virus infection cycle (Nagai et al., 1995). Baicalin, 7-D- glucuronic acid-5,6-dihydroxy-flavone, was purified from the plant Scutellaria Baicalensis. The inhibitory effect of BA against human immunodeficiency virus (HIV-1) infection and replication has been studied in vitro (Li et al., 1993). The enzymatic activity of purified recombinant HIV-l/RT was inhibited by baicalin. The anti-HIV-l activity of baicalin was also observed in cultures of primary human peripheral blood mononuclear cells infected with HIV-1 in vitro (Li et al., 1993). Baicalin was also found to inhibit human T cell leukemia virus type I (HTLV-I) and reverse transcriptase activity 55 in HTLV-I-infected cells as well as the activity of purified reverse transcriptase fiom Moloney murine leukemia virus and Rous-associated virus type 2 (Baylor et al., 1992). SP-303, a natural plant flavonoid polymer, was found to have antiviral activity against two strains of type 1 herpes-type simplex virus (Barnard et al, 1993). The mode of antiviral action of this biopolymer was through inhibition of virus penetration into cells (Barnard et al., 1993). SP-303 was also found to have antiviral activity against respiratory syncytial virus (RSV) in plaque reduction assays and cytopathic-effect- inhibition assays (Barnard et al., 1993). In vivo experiments, the SP-303 was evaluated against experimentally induced influenza A (H1N1) virus infections in mice. Mice receiving SP-303 by SPA exhibited consistent but reversible hypothermia immediately after termination of treatment (Sidwell, 1994). Lophirone A, a biflavonoid, inhibited Epstein-Barr virus (EBV) activation (Murakami et al., 1991). Three structurally related flavonoids, chrysin, acacetin, and apigenin were found to inhibit HIV expression (Critchfield et al., 1996). These findings indicated that flavonoids can inhibit HIV-l activation via a novel mechanism, and that these agents are potential candidates for therapeutic strategies aimed at maintaining a cellular state of HIV-1 latency (Critchfield et al., 1996). Naturally occurring flavones, baicalein, quercetin, quercetagetin and myricetin and two catechins, (-)-epicatechin gallate and (-)-epigallocatechin gallate, were isolated from the tea (Camellia sinensis) are known inhibitors of reverse transcriptase (Nakane and Ono, 1990). They were shown to induce mammalian topoisomerase II-dependent DNA-cleavage in vitro. The flavones differed from the catechins in causing the unwinding of duplex DNA, but both classes of compound induced enzymic DNA breakage at the same sites on DNA (Austin et al., 56 1992). The structural basis for the antiviral activity of natural flavonoids was examined by Wleklik et al (1988). Hydroxylations at positions C3', C4', C3, C5 and C7 was associated with highest antiviral actvity. Isoquercitrin, 3,3',4',5,7-pentahydroxyflavone-3- B-O-glucoside, an antiviral agent from Waldsteinia fragarioides (Rosaceae) was active against herpes simplex type 1 virus (Abou Karam and Shier, 1992). Substituted y- chromones were found to weakly inhibit HIV-l proteinase, an important enzyme in the replication and processing of the AIDS virus. Chromones bearing hydroxyl substituents and a phenolic group at the 2-position (flavones) were the most active compounds (Brinkworth et al., 1992). Plant flavonoids are a large group of naturally occurring phenylchromones found in fruits, vegetables, grains, bark, roots, stems, flowers, tea and wine. Several hundred milligrams are consumed in the average western diet everyday (Hertog et al., 1993). A variety of in vitro and in vivo experiments have shown that selected flavonoids possess antiallergic, anti-inflammatory, antiviral and antioxidant activities. Moreover, particular flavonoids have been shown to exert significant anticancer activity including anticarcinogenic activities. Certain flavonoids possess potent inhibitory activity against a wide array of enzymes such as protein kinase C, protein tyrosine kinases, and phospholipase A-2. These results suggested that plant flavonoids may possess health promoting and disease-preventing attributes when ingested as dietary supplements. 57 CHAPTER TWO* Quantification and Characterization of Anthocyanins in BalatonTM Tart Cherries ABSTRACT The anthocyanin contents of BalatonTM and Montrnorency cherries were compared. The results indicate that both cherries contain identical anthocyanins. However, BalatonTM contains approximately six times more anthocyanins than does Montrnorency. Also, hydrolysis of the total anthocyanins and subsequent gas chromatography (GC) and nuclear magnetic resonance (NMR) experiments with the resulting products indicated that both varieties contain only one aglycone, cyanidin. This observation contrasts with existing reports of the presence of peonidin glycosides in Montrnorency cherry. Results of the present study suggest that the anthocyanins in BalatonTM and Montrnorency cherries are anthocyanin 1 [cyanidin-3-(2”-0—,B—D- glucopyranosyl-6”-0- a—L-rhamnopyranosyl-fl-D—glucopyranoside], anthocyanin 2 [cyanidin-3-(6"-0-a-L-rhamnopyranosyl-fl-D-glucopyranoside] and anthocyanin 3 [cyanidin-3-0—fl~D-glucopyranoside]. *Wang, H.; Nair, M. G.; Iezzoni, A. F.; Strasburg, G. M.; Booren, A. M.; Gray, J. I. J. Agric. Food Chem. 1997, 45, 2556-2560. 58 INTRODUCTION Prunus cerasus L. (Rosaceae), cv. Montrnorency is the major tart cherry (commercially) grown in the United States. In order to diversify the Montrnorency monoculture, a new Hungarian cultivar, BalatonTM tart cherry, was introduced into the United States in 1984, and has been tested in Michigan, Utah, and Wisconsin. BalatonTM produces fruits darker than Montrnorency, and may be used as a source for cherry anthocyanins. Natural pigments like anthocyanins were regarded as the index of quality in tart cherries (Mazza and Miniati, 1993). In addition, recent studies have demonstrated the strong antioxidant activity of anthocyanins such as cyanidin-3-glucoside (Tsuda et al., 1994). Antioxidants are commonly used to increase the shelf life of food products by preventing, or at least delaying, the onset of lipid peroxidation (Tsuda et al., 1994). Natural antioxidants may play an important role in the prevention of carcinogenesis. Dietary antioxidants may be effective against the preoxidative damage in living systems (Halliwell and Gutteridge, 1989; Osawa et al., 1990). Early studies revealed that Montrnorency cherry contains cyanidin -3- gentiobioside and cyanidin -3-rutinoside (Li and Wagenknecht, 1956). Cyanidin-3- glucosylrutinoside was also found in six out of seven tart cherry varieties analyzed by Harbome and Hall (1964). Cyanidin-3-glucoside is reported as a minor pigment in Montrnorency cherries (Schaller and Von Elbe, 1968; Chandra et al., 1992). Dekazos 59 (1970) reported anthocyanin pigments in Montrnorency cherry as peonidin-3-rutinoside, peonidin and cyanidin along with cyanidin-3-sophoroside, cyanidin-3-rutinoside and cyanidin-3-glucoside (Schaller and Von Elbe, 1968). However, cyanidin-3- glucosylrutinoside as well as cyanidin-3-glucoside, cyanidin-3-sophoroside and cyanidin- 3-rutinoside were identified as the main pigments in tart cherries. Using high performance liquid chromatography (HPLC) retention values, Chandra et al. (1992) reported that cyanidin-3-sophoroside and cyanidin-3-glucoside were the major and minor anthocyanins, respectively, in the Michigan-grown Montrnorency cherries. Similarly, cyanidin-3-xylosylrutinoside was detected as a minor pigment in Montrnorency cherry (Shrikhande and Francis, 1973). In addition to the comparison of anthocyanins in BalatonTM and Montrnorency, this chapter describes the isolation of anthocyanins in BalatonTM and Montrnorency cherries and their characterization by NMR, GC and mass spectroscopic (MS) methods. MATERIALS AND METHODS Cherry fruits. Pitted and frozen Montrnorency and BalatonTM tart cherries were obtained from commercial growers (Traverse City, Michigan) through the Cherry Marketing Institute, Inc. (Dewitt, Michigan). The cherries were flushed with nitrogen in freezer bags prior to their storage at -20 °C. General experimental. 1H NMR, 13C NMR and DQF COSY spectra were recorded on Varian 500 and 300 MHz spectrometers using CD3OD/DC1 (aL) solution at 60 25 °C. All chemical shifts are given in ppm relative to CD3OD (3.3ppm). GC analyses were performed on an HP 5890 II (Hewlett Packard, Palo Alto, California) using a DB-17 (30 m x 0.313 mm X 0.25 pm, J & W Scientific, Palo Alto, California) column. The temperature program used was: 150 °C, initial temperature held for 5 min, and then increased to 210 °C at 5 °C min", maintained for 5 min, and finally to 270 °C at 5 °C min". The injection port temperature was maintained at 250 °C. The flame ionization detector temperature was 300 °C and the carrier gas was helium at a linear flow velocity of 4 cm s'1 with a 1:70 split ratio. Fast atom bombardment-mass spectroscopy (FAB-MS) was carried out on a double focusing mass spectrometer in a glycerol matrix using Xe as reactant gas. HPLC conditions for anthocyanin analysis. All sample extracts (20 [LL each) were analyzed on Chemcopak and Capcellpak C-l8 columns (10XZ50 mm, 5 pm) (Dychrom; Sunnyvale, California). The mobile phase (4% aqueous H3PO4 /CH3CN (80:20 v/v) was used under isocratic conditions at a flow rate of 1.5 mL min'l. The anthocyanins were detected at 520 nm using a Waters PDA detector. Anthocyanins 1-3, 0.5mg each, were weighed and dissolved in 1 mL of H2O/CH3CN (1:1). The solutions were prepared by the serial dilution of the respective stock solutions to afford 0.25, 0.20, 0.10, 0.05, 0.025 and 0.0125 mg/mL concentrations, respectively. Quantification of anthocyanins were carried out using a Millennium 2010 chromatography manager (Waters Associates, Milford, Massachusetts). 61 Isolation of crude anthocyanins from tart cherries. The pitted cherries (400 g each of BalatonTM and Montrnorency) were homogenized separately for 10 min using a Kinematica CH-6010 (Roxdale, Ontario, Canada) homogenizer and centrifuged (Model RCSC, Sorvall Instruments, Hoffman Estates, Illinois) at 10000 g for 10 min at 4°C to separate insoluble materials from the supernatant. The supernatant (400 mL each) was applied to a XAD-2 (100 g, amberlite resin, mesh size 20-50; Sigma Chemical Co., St. Louis, Missouri) column, which was prepared as described by Chandra et a1 (1993). The column was washed with H20 (9 L) until the colorless washings gave a pH of about 7. The adsorbed pigments were then eluted with methanol (500 mL). The red methanolic solution was concentrated at 50 °C in vacuo, and the aqueous solution was lyophilized to yield an amorphous red anthocyanin powder, 0.86 and 0.54 g, respectively, for BalatonTM and Montrnorency samples. HPLC Analysis of anthocyanins in cherries. Pitted cherries (100 g) were homogenized and centrifuged as described above. The supernatant was decanted and adjusted with H2O to a final volume of 250 mL in a volumetric flask. An aliquot of 1 mL of this solution was passed through a preconditioned C-18 Sep-Pak cartridge (Waters Associates, Milford, Massachusetts). The adsorbed pigments were then washed with 2 mL of water followed by 1 mL of H20/ CH3CN ( 1:1). The eluate was stored at -20 °C prior to HPLC analysis. Purification of anthocyanins 1-3. The crude anthocyanins from BalatonTM were fractionated by C-18 medium pressure liquid chromatography (MPLC) to produce pure 62 anthocyanins. Both BalatonTM and Montrnorency showed identical HPLC profiles (Fig. 2.1). The anthocyanin mixture (350 mg) was dissolved in water (2 mL), injected into the C-18 column (40 X 500 mm) and eluted with 4% H3PO4 :CH3CN (80:20). Four fractions, 1: 125 mL, 11: 100 mL, 111: 100 mL and IV: 275 mL, were collected and evaporated under reduced pressure. The H3PO4 from these fractions was removed by passing each fraction through preconditioned C-l8 Sep-Pak (Waters Associates) with methanol, followed by 10% methanol. The adsorbed pigment was washed with 5 mL water to remove the acids and then eluted with 5 mL of H20/ methanol (1:1) to afford pure anthocyanins. The yield of anthocyanins from fractions I-IV were 53, 24, 133 and 64 mg, respectively. HPLC analysis of these fractions revealed that fraction 1 was pure and contained only anthocyanin 1. Fraction 11 contained anthocyanins l and 2, fraction 111 had anthocyanins 2 and 3 and fraction IV contained anthocyanin 3 with other phenolics as indicated by their HPLC profiles (Fig. 2.2). Since fractions 11 and III from MPLC contained all three of the anthocyanins, 40 mg of II and 30 mg of III were purified further by HPLC on Capcellpak C-l8 column (10>< 250 mm, 5 um ) to yield pure anthocyanins 2 and 3. Peaks were detected using a PDA detector at 520 and 283 nm, respectively. The mobile phase (4% aqueous H3PO4 : CH3CN , 83:17 v/v) was used under isocratic conditions at a flow rate of 2.0 mL/min. Respective anthocyanin fractions from HPLC purification of fractions II and III were combined, dried under reduced pressure and purified further using C-18 Sep-Pak to remove H3PO4_ The weights of pure anthocyanins 1-3 were 5.7, 8.9 and 2.9 mg, respectively. 63 AU a AU 043—: 0.13.: 1 043-: A 0.12-3 B 0.11: 0.11.: 0.10; 0.10.: 0.09—- 0.09-3- 0.0&-: 0.03.“. 0.07—- 0.07:- 1 I A - 0006-“ 0.06.:- 0.05-: - 0004': 0.04; 0.03-3 0,03.“ 0.02; 0.02; 0.01-3 0.01; °-° _ 0.00; —O. i I l I I I I I I . —O,G£ I I ' I I l | I 0.00 10.00 Mm 0.00 10.00 Mm Fig. 2.1. HPLC profile of Montrnorency (A) and BalatonTM (B) cherry extracts: l: Cyanidin-3-glucosylrutinoside; 2: Cyanidin-3-rutinoside; 3: cyanidin-B-glucoside. OH H HO Anthocyanin l w OH O ' O - ‘ CHZOH CH3 Anthocyanin 2 Anthocyanin 3 Fig. 2.2. Anthocyanins 1-3 from BalatonTM and Montrnorency tart cherries 65 Crude anthocyanins from Montrnorency (500 mg) were also fractionated by C-18 MPLC as in the case of BalatonTM. Three bands with red color were collected as fractions 1, II and III and removal of solvents at reduced pressure afforded 10, 30 and 20 mg of anthocyanins, respectively. Fraction I was pure and contained only anthocyanin 1. Fractions II and III were not pure by HPLC analysis and contained anthocyanins 1-3. The purified anthocyanins are red amorphous powders. Complete assignments of 1H- and l3C-NMR spectra of pure anthocyanins 1 [Cyanidin-3-(2"-0-,B-D- gluc0pyranosyl-6"-0-a—L-rhamnosyl-fl-D-glucopyranoside], 2 [Cyanidin-3-(6"-0-a'-L- rharnnopyranosyl-fl-D-glucopyranoside)] and the 1H-NMR of 3 [Cyanidin-336- Dglucopyranoside] are given in Tables 2.1 and 2.2, respectively. l3C-NMR on pure anthocyanin 3 was not performed due to its low yield. Cyanidin, the aglycone. The crude anthocyanin powder from BalatonTM (55 mg) was hydrolyzed with 3 M HCl (15 mL) for 1 h at 100 °C. The red solution was cooled to room temperature and stirred with l-butanol (20 mL). The mixture was extracted with water (3 x 20 mL). The combined water extracts were evaporated to dryness at reduced pressure to yield the sugars (30 mg). The red butanol layer was evaporated to dryness (24.3 mg). The residue was purified by silica gel preparative TLC using the solvent system, ethyl acetate : formic acid: 2 M HCl , 85: 6: 9. The single red band at Rf 0.28 was eluted with MeOH, evaporated under reduced pressure and afforded a red amorphous powder, cyanidin (11.2 mg). Similarly, pure anthocyanins (0.5 mg each) were hydrolyzed also to obtain their respective sugars for GC analysis. The aglycones from anthocyanins 66 1-3 and the crude anthocaynin gave identical Rf values and HPLC retention times. Also, all aglycones showed identical 1H- and l3C-NMR spectra (Tables 2.1 and 2.2). Characterization of sugars by GC analysis. The sugar standards rhamnose, fructose, galactose, glucose and the internal standard phenyl-fl-D-glucoside (E. Merck, Darmstadt, Germany) (1 mg each) and the sugars obtained from the hydrolysis of crude and pure anthocyanins (1-3) (1 mg each) were reacted separately with 30 mg/mL hydroxylarnine HCl in dry pyridine (2 mL). The resulting oximes were then reacted with 1.0 mL hexamethyldisilazane (HMDS) and 0.1 mL trifluoroacetic acid (TFA) to yield their silyl derivatives. The samples were then analyzed by GC using an autosarnpler. Sugars from anthocyanins 1, 2 and 3 were identified by comparing with the retention times of sugar standards. The retention times were 6.97, 9.83, 10.58, 10.90, 19.82 min, respectively, for rhamnose, fi'uctose, galactose, glucose and phenyl-fl-D-glucoside. The GC analysis of sugars yielded from the hydrolysis of anthocyanins showed that anthocyanin 1 contained a 2:1 ratio of glucose and rhamnose. Anthocyanin 2 showed a 1:1 ratio of glucose and rhamnose and anthocyanin 3 afforded only glucose. Similarly, the GC analysis of sugars from the crude anthocyanin powder indicated that it contained only rhamnose and glucose at a ratio of 2:4, respectively. RESULTS AND DISCUSSION Lyophilization of 100 g each of BalatonTM and Montrnorency cherries afforded 17.1 and 14.7 % of dry weights, respectively. The concentrations of the sugars and acids 67 in BalatonTM were about 50% more than those in Montrnorency cherries (data not presented). Similarly, total anthocyanin concentration in BalatonTM cherry is about six times greater than those in Montrnorency cherry based on anthocyanin concentrations in fractions obtained from MPLC and HPLC purifications. Prior to the isolation of anthocyanins for spectral characterization, both BalatonTM and Montrnorency cherries were analyzed by HPLC under identical conditions. HPLC profiles of the cherry extract showed that there are two major and one minor anthocyanins in both varieties as indicated by retention times 8.76, 10.58, 13.38 min, respectively, for anthocyanins 1-3 (Fig. 2.1). Also, it was evident from the marked difference in the red color between these two cherries and from HPLC profiles (Fig. 2.1) that Montrnorency contained relatively smaller amounts of anthocyanins compared to BalatonTM. The crude anthocyanins were fractionated and purified by C-18 MPLC and HPLC, respectively, to afford pure anthocyanins for spectral studies. Purification of 500 mg crude Montrnorency anthocyanins from XAD-2 yielded 60 mg of anthocyanins 1-3 (Fig. 2.2) compared to 391.43 mg from BalatonTM. This indicates that crude anthocyanins from Montrnorency obtained from the XAD-2 contained a higher percentage of other organic compounds. The presence of cyanidin and respective sugar moieties in anthocyanins 1-3 were confirmed by the comparison of their 1H- and l3C-NMR chemical shifts with published 68 data (Gl'aBgen et al, 1992; Stack and Wray, 1989). We have determined the relative configuration and nature of the sugars in anthocyanins 1 and 2 by DQFCOSY and from the vicinal and geminal IH-‘H coupling constants. The 1H NMR spectrum of 1 (Table 2.1) gave signals for three anomeric protons that appeared at 6 5.43, 4.76 and 4.64, respectively, for glucose (attached to the aglycone), glucose and rhamnose . Also, the presence of ,B-D-glucosidic linkage in 1 was confirmed by the large coupling constants for the anomeric protons (Table 2.1). The signal at 6 4.64 ppm corresponded to the anomeric proton of an L-rhamnopyranose and the 1.8 Hz coupling constant indicated an a—glycosidic linkage. The ‘3 C NMR chemical shifts observed for anthocyanins in BalatonTM and Montrnorency were similar to the published data (Agrawal et al., 1989). The O7 resonated at very low field 170.4 ppm compared to the rest of the oxygenated aromatic carbons in anthocyanins. The oxygen cation in ring C is responsible for the downfield shift of C-7 carbon. The 13C NMR signal for C-5 carbon in l at 6 69.8 confirmed the rharnnosyl moiety with an (ac-linkage to the glucose (Agrawal, 1992). The downfield shift of the C-2” proton in 1 relative to the C-2” signal of 2 (Table 2.2) was due to the glucosylation and indicated a 1,2 linkage between the two glucose units. Similarly, the downfield shifl of C-6” proton in the 1H-NMR spectrum of 1 (Table 1.1) relative to the C-6 proton signal of glucose was due to the rhamnose moiety and indicated a 1,6 linkage between the glucose and rhamnose. Anthocyanin 1 gave a molecular ion at m/z 758 [M+H]+ and the base peak at m/z 596 [M+H-C6H1005] in the 69 Table 2.1. 1H NMR chemical shifts in ppm for anthocyanins 1-3 and Cyanidin in CD3OD/DC1 (J in Hz) Proton Anthocyanin 1 Anthocyanin 2 Anthocyanin 3 Cyanidin H-4 8.89 s 8.92 8 8.985 8.625 H-6 6.67 d(l.96 ) 6.69 d(l.95) 6.71 d(l .95) 6.65 d(l.95) H-8 6.90 d(l .96) 6.91 d(l.95) 6.98 d(l .95) 6.90 d(l.95) H-2' 8.00 d(2.24) 8.02 d(2.23) 8.05 d(2.23) 8.11 d(2.23) H-5' 7.06 d(8.66) 7.01 d(8.65) 7.07 d(8.65) 7.02 d(8.66) H-6' 8.18 dd(8.66, 2.24) 8.27 dd(8.65, 2.23) 8.29 d(8.65, 2.23) 8.17dd(8.66,2.23) H-1" 5.43 d(7.29) 5.29 d(7.53) 5.40 d(7.50) H-2" 4.05 dd(9.08, 7.29) 3.67 dd(9.06, 7.53) 3.67 dd(9.00,7.50) H-3" 3.77 dd(9.28, 9.08) 3.55 dd(9.22,9.06) 3.55dd(9.22,9.00) H-4" 3.50 dd(9.53, 9.28) 3.34 dd(9.49,9.22) 3.34 dd(9.50, 9.22) H-5" 3.72 ddd(9.53, 6.41, 1.76) 3.71 m 3.71 m H-6a" 4.04 dd(l2.23, 6.41) 4.05 dd (1 1.90, 6.31) 3.91 dd(11.90, 6.30) H-6b" 3.61 dd(12.23, 1.76) 3.62 dd (1 1.90, 1.62) 3.68 dd(ll.90, 1.62) H-1"' 4.76 d(7.74) 4.65 d(l .67) H-2'" 3.19 dd(9.08, 7.74) 3.80 dd(3.35, 1.67) H—3'" 3.33 dd(9.08, 9.28) 3.63 dd(9.49, 3.35) H-4'" 3.23 t(9.28) 3.41 dd (9.49,9.21) H-5'" 2.92 dt(9.28, 3.97) 3.54 dd(9.21, 6.14) H-6'" 3.44 d(3.97) 1.15 d(6.14) 11-1"" 4.65 d(1.54) 11-2"" 3.78 dd(3.31, 1.54) 11.3”" 3.60 dd(9.50, 3.31) 11-4"" 3.27 dd(9.28, 9.50) 115"" 3.56 dd(9.28, 6.18) H-6"" 1.14 d(6.18) 70 Table 2.2. 13C NMR chemical shifts in ppm for anthocyanins 1,2 and their aglycone (Cyanidin) in CD3OD Carbon Anthocyanin 1 Anthocyanin 2 Cyanidin C-2 164.3 164.3 162.6 C-3 145.2 145.6 146.5 C-4 136.1 136.6 134.1 C-5 159.1 159.1 157.9 C-6 103.5 103.5 103 C-7 170.4 170.4 168.8 C-8 95.2 95.3 94.7 C-9 157.6 157.6 156.9 C-10 113.2 113.2 113.5 C-1' 121.2 121.2 121.9 C-2' 118.6 118.4 117.9 C-3' 147.4 147.4 147.3 C-4' 155.7 155.8 155.1 C-5' 117.6 117.5 117.2 C-6' 128.3 128.4 127.1 C-1" 104.9 103.5 C-2" 82.3 74.7 C-3" 77.2 77.4 C-4" 71.2 71.2 C-5" 77.9 78 C-6" 67.6 67.8 C-1"' 101.9 102.1 C-2'" 75.9 71.9 03'" 77.7 78.1 C-4'" 70.8 73.9 C-5'" 77.7 69.7 C-6'" 62.3 17.9 01"" 102.2 C-2"" 71.8 C-3"" 72.4 C-4"" 73.9 C-5"" 69.8 C-6"" 17.9 71 FAB MS also indicated the presence of cyanidin, two glucose and one rhamnose moieties in 1. Therefore, anthocyanin 1 is confirmed to be cyanidin-3-(2"-0-fl-D-glucopyranosyl- 6’ ’-0- a—L-rhamnopyranosyl-,6-D-glucopyranoside). 1H NMR spectrum of 2 (Table 2.1) showed signals for two anomeric protons at 6 5.29 (J= 7.53 Hz) and 4.64 (J = 1.67). This indicated the presence of a fl-D- glucose because all vicinal coupling constants were 7.53~11.9ppm. The doublet (J = 6.14 Hz) at 1.15 ppm of a methyl group confirmed one of the sugars as rhamnose in 2. The small coupling constant of 1.67 Hz for the anomeric proton suggested an a-rhamnosyl linkage. The C-6” proton of glucose at 5.5 ppm indicated a 1,6 linkage between the glucose and rhamnose. The FAB-MS of 2 gave the molecular ion at m/z 596 [M+H]+ and confirmed its structure as cyanidin-3-(6’ ’-0— a—L-rhamnopyranosyl-fl-D-glucopyranoside). The 1H NMR of anthocyanin 3 (Table 2.1) revealed only a single glucose moiety attached to the aglycone cyanidin. The structure of 3 was confirmed to be cyanidin-336- D-glucoside. Hydrolysis of crude anthocyanins and TLC of resulting products as well as 13C NMR data showed that the only aglycone present in both BalatonTM and Montrnorency cherries was cyanidin. Our results suggest that there are only three identical anthocyanins present in both BalatonTM and Montrnorency cherries. The yields of spectroscopically pure anthocyanins 1-3 in 100 g of fresh BalatonTM and Montrnorency cherries were 14.99, 6.20; 6.18, 0.97; 2.42, 0.35 mg, respectively. The amount of anthocyanins isolated from Montrnorency in 72 our studies show that it is lower than the reported yields (Dekazos, 1970). However, this may be due to varying environmental and nutritional factors. An important point to note is that when anthocyanins are monitored by HPLC at 520 nm, other phenolic compounds which absorb at 283 nm are ignored. We have isolated at least four phenolic compounds co-eluted with the anthocyanins and detected at 283 nm. Chandra et a1. (1992) reported that Montrnorency cherries grown in Michigan contain only cyanindin-3-sophoroside and cyanidin-3-glucoside. These results were confirmed by matching their retention times to those of the anthocyanins present in an authentic sample of blackberry juice described by Hong and Wrolstad (1990a, b). Also, earlier reports indicated that there are peonidin-3-glycoside and peonidin-3-galactoside present in Montrnorency cherry (Chandra et al., 1992). However, our present study indicates that Montrnorency contains the same number of anthocyanins found in BalatonTM cherries and were identical. This is the first report of the characterization of anthocyanins from BalatonTM cherries and the spectral characterization of anthocyanins from Montrnorency cherries. 73 CHAPTER THREE* Antioxidant compounds from the ethyl acetate extract of tart cherries (Prunus cerasus) ABSTRACT As indicated by a Fe (ID-induced liposome peroxidation bioassay, the ethyl acetate extract of tart cherries (Prunus cerasus L. Rosaceae) was found to have strong antioxidant activity. Purification of this extract afforded chlorogenic acid methyl ester (1) and three novel compounds, 2-hydroxy-3-(o-hydroxyphenyl) propanoic acid (2), 1-(3', 4'- dihydroxycinnamoyl)-cyclopenta-2, 5-diol (3) and 1-(3',4'-dihydroxycinnamoyl)- cyclopenta—Z, 3-diol (4), as determined by their spectral data. At a 20 11M concentration, the antioxidant activities of compounds 3 and 4 were comparable to the antioxidant activities of caffeic acid, whereas compound 1 showed activity similar to chlorogenic acid. Also, these compounds showed antioxidant activities similar to the commercial antioxidants tert—butylhydroquinone and butylated hydroxytoluene. However, compound 2 was not active when tested at a 100 11M concentration. *Wang, H.; Nair, M. G.; Strasburg, G. M. Booren, A. M.; Gray, J. I. Submitted to J. Nat. Prod. 74 INTRODUCTION Consumers are now including phytonutrients in their diet with the notion that antioxidant compounds may reduce the incidence of cancer and aging in humans. Free radicals are implicated in a number of pathological processes including aging, inflammation, reoxygenation of ischemic tissues, atherosclerosis, and cancer (Halliwell and Gutteridge, 1990). Free radicals such as hydroxyl radicals (OH') initiate a chain reaction which causes lipid peroxidation, damage to enzymes and DNA, and cell death. Naturally occurring antioxidant components such as flavonoids, can stabilize highly reactive and potentially harmful free radicals (Graf, 1992). Many common foods contain non-nutritional components such as flavonoids and are considered to reduce the incidence of chronic diseases (Cody et al., 1988; Tanaka et al., 1993). The antioxidant phenolic compounds are reported to remove free radicals and protect the structural integrity of cells and tissues (Hertog et al., 1993). Preliminary antioxidant assays revealed good antioxidant activities in the MeOH and EtOAc extracts of BalatonTM tart cherries (Prunus cerasus L. Rosaceae) while hexane extracts showed little or no activity. Anecdotal reports indicating that consumption of tart cherries can alleviate the pain of gout and arthritis (Hamel, 1975) prompted us to investigate tart cherries for biologically active compounds. We have evaluated the antioxidant efficacy of P. cerasus constituents using a Fe (ID-induced peroxidation of liposome antioxidant bioassay (Arora and strasburg, 1997). In this paper, we report the isolation and identification of three novel antioxidant compounds (2-4) from the EtOAc extracts of tart cherries. 75 EXPERIMENTAL SECTION General Experimental procedures. Commercial antioxidant TBHQ and BHT were used as positive controls. TBHQ was purchased from Eastman Chemical Products Inc., Kingsport, Tennessee. BHT was purchased from National Biochemicals Corporation, Cleveland, Ohio. Silica gel (60 mesh, 35-70 pm) used for medium pressure liquid chromatography (MPLC) was purchased from E. Merck, Darmstadt, Germany. TLC plates (GF Uniplate, Analtech, Inc., Newark, DE), after developing, were viewed under 254 and 366 nm. For preparative high pressure liquid chromatography HPLC) (LC-20, Japan Analytical Industry Co., Tokyo, Japan) purification, two Jaigel-ODS, A- 343-10 (20 mm )(250 mm, 10 um, Dychrom, Santa Clara, CA) columns were used in tandem. Peaks were detected using a model D-2500 Chromato-integrator connected with a UV detector. 1H-, ”O, DQFCOSY and HMQC NMR spectra were recorded on a Varian Unity 500 and an Inova 300 MHz spectrometers at 25°C and referenced to the residual proton solvent resonance, CD3OD at 3.30 and 49.0 ppm and DMSO-dg at 2.49 and 39.5 ppm, for 1H- and 13 C NMR, respectively. Fast atom bombardment mass spectroscopy (FABMS) were obtained on a JEOL JMS-HXI 10 using a glycerol matrix and EIMS spectra were obtained on JEOL JMS-AX505 mass spectrometers. Circular dichroism (CD) and Optical rotatory dispersion (ORD) measurements were carried out using a JASCO J-710 CD-ORD spectropolarimeter (Japan Spectrosc0pic Co., Hachioji city, Tokyo, Japan). For CD/ORD measurements, test compounds were dissolved in methanol (0.2 mg mL'l), and CD/ORD were determined under the following conditions: scan mode (wavelength), bandwidth (0.5 nm), sensitivity (50 m deg), response (1 s), wavelength range (200-400 nm for CD and 200-800 nm for ORD), step resolution (1 76 nm), scan speed (200 nm min'l), and accumulation (1). Nitrogen (99.99%) was generated by a nitrogen generator model NG-150 (Birtley Co., Durham, England) at the rate of 15 L min'l. UV spectra of compounds, in MeOH, were measured on a Shimadzu UV-visible spectrophotometer (Kyoto, Japan). Plant Material. Pitted and individually quick frozen (IQF) BalatonTM cherries (Prunus. cerasus L. Rosaceae), which were collected July, 1995, were obtained from commercial growers (Traverse City, MI) and supplied by the Cherry Marketing Institute, Inc. (Dewitt, MI). Extraction and Isolation. IQF BalatonTM tart chenies (2 kg) were lyophilized at 10°C and yielded 342 g of dried cherries. The BalatonTM dried cherries (340 g) were milled and extracted with hexane (500 mL X 3), ethyl acetate (500 mL X 3) and methanol (500 mL X 3) to yield 0.71, 2.53 and 198.9 g of extracts, respectively. The ethyl acetate extract of BalatonTM cherries (1.75 g) was fractionated by silica gel (100 g) MPLC using CHC13 and MeOH under gradient conditions starting with 100% CHC13 and ending with 100% MeOH. Fractions 1-4 (125 mL each, CHC13), 5-8 (100 mL each, CHC13-MeOH, 8:1), 9-12 (100 mL each, CHC13-MeOH, 4:1), and 12-16 (150 mL each, MeOH) were collected and combined after TLC analysis (silica gel plates developed with MeOH-CHC13, 16:1, for fractions 1-8 and MeOH-CHCl3-HCOOH, 1:4:0.2, for fractions 9-16), to yield 85, 134, 330, 910 and 225 mg each of fractions A-E, respectively. Fractions A and B did not show antioxidant activity. Fractions C-E were further purified for antioxidant compounds. Fraction C (250 mg) was purified by preparative silica TLC using MeOH-CHC13- HCOOH (4:1:0.2) as the mobile phase to yield compounds 1 (10.1 mg, Rf = 0.50) and 2 77 (8.9 mg, Rf , 0.67). lH- and 13C- NMR spectra of compound 1 was identical to the published data of chlorogenic acid methyl ester (Rumbero-Sanchez and Vazquez, 1991) Compound 2: White solid; IR (film) vmax 3316, 1728, 1590, 1406 cm'l; UV Am, (MeOH) 218 (3.04), 253 (3.42), 289 (3.66) nm; CD/ORD measurements gave straight lines indicating that compound 2 was obtained as a racemic mixture; |H NMR (CD3OD) 6 7.40 (1 H, d, J= 7.32 Hz, H-6’), 7.22 (1H, t, J= 7.57 Hz, 7,32 Hz, H-4'), 7.00 (1H, t, J = 7.57 Hz, 7.32 Hz, H-S'), 6.86 (1H, d, J = 7.57 Hz, H-3'), 4.19 (1H, m, H-2), 2.80 (2H, m, H-3); 13C NMR (DMSO-dg)! 5 178.6 (C-l), 141.6 (C-2'), 133.4 (C-6'), 128.4 (C-4'), 123.8 (C-5'), 121.3 (C-l'), 109.2 (C—3'), 73.4 (C-2), 48.6 (C-3); FABMS, m/z 183 (4) [M+H]+. Compound 3: Fraction D (900 mg) was purified using a preparative HPLC with the mobile phase being MeOH-H2O (30:70) at a flow rate of 3 mL/min, to yield compound 3 (R, = 58 min, 9.4 mg): pale yellow oily compound; IR (film) vm 3351, 2926, 1669, 1599, 1379, 1267, 1076 cm'l; UV Am“ (MeOH) 206 (3.99), 215 (4.00), 243 (3.83), 299 (3.86), 325 (3.89) nm; 0RD (m deg) 336 (75), 316 (-44), 298 (-40), 260 (35), 240 (-22) and 216 (52) nm; 1H- NMR (DMSO-d6) 5 7.45 (1H, d, J = 15.9 Hz, H-7'), 7.01 (1H, d, J= 1.8 Hz, H-2'), 6.96 (1H, dd, J: 8.1 Hz, 1.8 Hz, H-6'), 6.75 (1H, d, J= 8.1 Hz, H-S'), 6.19 (1 H, d, J: 15.9 Hz, H-8'), 5.17 (1H, m, H-l), 3.82 (1H, m, H-2), 3.54 (1H, m, H-S), 1.83 (4 H, m, H-3, H-4); 130 NMR (DMSO-dg) 5 166.1 (09'), 148.2 (C-3'), 145.5 (C-4'), 144.4 (C-7'), 125.7 (C-l'), 121.1 (C-6'), 115.8 (C-5'), 115.0 (C-8'), 114.6 (C- 2'), 70.9 (C-2, C-5), 67.5 (C-l), 35.2 (03, C-4); FABMS m/z 281 (2) [M+H]+; EIMS m/z 180 (93), 163 (100), 145 (20). 78 Compound 4: Fraction E (225 mg) was purified by preparative HPLC. The mobile phase was MeOH-H2O (40:60) at a flow rate of 4m1/min. Sub-fractions 1 (180 mg), 2 (10.8 mg), 3 (8.4 mg), 4 (8 mg), and 5 (10 mg) were collected. sub-fraction l was not active and contained malic acid, as confirmed by its lH NMR spectrum. Fraction 2 (10.8 mg) was the most active and hence purified again by HPLC under the same conditions to yield compound 4 (R, = 34 min, 9.4 mg); Oily compound; IR (film) v max 3372, 1692, 1603, 1277, 1184, 1074 cm"; UV Amax (MeOH) 203 (3.95), 215 (3.94), 243 (3.76), 299 (3.81) and 327 (3.90) nm; ORD (m deg) 314 (-58), 288 (-61) and 234 (-61) nm; 1H NMR (CD3OD) 6 7.58 (1H, d, J = 15.9 Hz, H-7'), 7.04 (1H, d, J = 1.8 Hz, H-2'), 6.93 (1 H, dd, J= 8.2 Hz, 1.8 Hz, H-6'), 6.76 (1H, d, J= 8.2 Hz, H-5'), 6.30 (1H, d, J = 15.9 Hz, H-8'), 5.35 (1H, m, H-l), 4.14 (1H, m, H-3), 3.64 (1H, dd, J=8.3 Hz, 3.1 Hz, H- 2), 2.15 (2H, m, H-4), 2.15 (1H, m, H-5a), 1.95 (1 H, m, H-5b); 13C NMR (CD3OD) 6 169.0 (C-9'), 149.4 (C-3'), 146.8 (C-4'), 146.8 (C-7'), 128.0 (C-l'), 122.9 (C-6'), 116.5 (C- 5'), 115.8 (C-8'), 115.1 (C-2'), 74.8 (C-2), 73.0 (CI), 68.3 (C-2), 41.5 (C-5), 36.7 (C-4); FABMS m/z 281 (2) [M+H]+; EIMS m/z 180 (34), 163 (100). Methylation of Compound 2: N-Nitroso-N-methylurea (1.5 g) was slowly added to 100 mL of 25% KOH and 100 mL diethyl ether mixture at 0°C and reacted for about 1 h. The yellow ether layer containing CH2N2 was separated using a separatory funnel (500 mL) and washed with cold water (100 mL) to remove excess KOH. Compound 2 (4 mg) was dissolved in methanol and mixed with excess CH2N2 reagent (5 mL) in ether. The reaction mixture was kept at room temperature for 1 h. The solvent was then evaporated to afford compound 5 (4 mg). Compound 5: White solid; 1H NMR (CD3OD) 6 7.35 ( 1H, d, J= 7.32 Hz, H-6'), 7.25 (1H, dd, J= 7.57 Hz, 7.32 Hz, H-4'), 7.01 (1H, dd, J= 7.57 79 Hz, 7.32 Hz, H-S'), 6.87 (1H, d, J = 7.57 Hz, H-3'), 4.49 (1H, dd, J = 7.32 Hz, 4.88 Hz, H-2), 2.80 (1H, d, J = 12.45 Hz, 4.88 Hz, H-3a), 2.69 (1H, dd, J = 12.45 Hz, 7.32 Hz, H- 3b), 3.69 (3H, s, OCH3), 3.47 (3H, s, COOCH3). Antioxidant Assay. All the buffers were stored in Chelex 100 to remove metal ions. A mixture containing 5 nmol of 1-stearoyl-2-linoleoyl-sn-glycerol-3- phosphocholine (Avanti Polar Lipids, Inc., Alabaster, AL) and 15 nmol of the fluorescence probe 3-[p-(6-phenyl)-1,3,5-hexatrienyl] phenylpropionic acid (Molecular Probes, Inc., Eugene, OR) was dried under vacuum. The resulting film was suspended in 500 uL of buffer (NaCl, 0.15 M; EDTA 0.1 mM; MOPS 10 mM) and was then subjected to 10 freeze-thaw cycles in an ethanol/dry ice bath. The suspension was passed 29 times through a polycarbonate membrane with a pore size of 100 nm using a LiposoFast extruder (Avestin, Inc., Ottawa, Canada). Liposomes (200 nmol) were suspended in 2 mL of buffer (100 mM NaCl, 50 mM HEPES pH 7.0) and peroxidation was initiated by addition of 4 nmol of Fe”. Control samples contained no added Fe2+ or test compound. Anthocyanins, BHT, propyl gallate or a-tocopherol were added to final concentrations of 2 11M. Fluorescent intensity of this lipid suspension was monitored for a period of 21 min with or without test compounds, immediately following addition of Fe”, using a SLM 4800 spectrofluorometer (SLM Instruments, Urbana, IL). The values of relative fluorescence were determined by dividing the fluorescence value at a given time point by that at t = 0 min. The decrease in relative fluorescence intensity with time indicates the rate of preoxidation. The percent inhibition of the lipid oxidation was calculated using the equation: Percent Inhibition ={[(Fr¢|)p|- (Frel)Fe /[(F,¢f)c - (le)pc]}>< 100, where: (Freon = relative fluorescence for the Fe (II) and test samples at the end of 21 min, (Fre1)c = 80 relative fluorescence for the control sample at 21 min, and (F,.,|)pc = relative fluorescence for the Fe (ID-containing sample at the end of 21 min (Arora and Strasburg, 1997). RESULTS AND DISCUSSION Ethyl acetate extracts of dried BalatonTM tart cherries were separated by medium pressure liquid chromatograpgy (MPLC), preparative TLC and HPLC to yield compounds 1-4. Both the 1H and '3 C NMR spectral analysis of compound 1 revealed that the chemical shifts observed were identical to the published spectral data of the known compound, chlorogenic acid methyl ester (Rumbero-Sanchez and Vazquez, 1991). Compound 2 was obtained as a white solid. The molecular formula of this compound was determined as C9H1004 by FABMS. The 1H NMR spectrum revealed two aromatic protons that appeared as doublets at 6 7.40 and 6.86, respectively. Another two aromatic protons in the molecule appeared as triplets at 6 7.22 and 7.00, respectively. This indicated that there is an ortho-substituted aromatic moiety in the molecule. The multiplets at 6 4.19 and 2.80 were assigned to oxygenated methine and methylene moieties, respectively. The '3 C NMR spectrum of 2 supported these assignments in addition to a carbonyl carbon at 6 178.6. The structure of this compound was firrther confirmed by the formation of product 5 from 2 by methylation. Methylation of 2 by CH2N2 yielded one unit each of -OCH3 and -COOCH3. These data confirmed the presence of a phenolic OH and a COOH in 2. Therefore, the NMR confirmation of the identity of compound 2 as 2-hydroxy-3-(o-hydroxyphenyl)-propanoic acid is in agreement with the methylation data. Circular dichroism (CD) studies of 2 showed that it 81 is a racemic mixture as evident from a straight line in the CD spectrum. To our knowledge, this is the first report of this compound as a natural product. Compound 3 was obtained as a pale yellow gum. The 1H NMR spectrum of 3 indicated two olefinic proton signals appeared as doublets at 6 7.45 and 6.19, respectively. A coupling constant of 15.9 Hz for these two protons suggested that they are trans oriented. The signals appeared at 6 7.02, 6.97 and 6.75 were assigned to aromatic protons of a 3, 4-dihydroxylcinnamoyl group, respectively, and were similar to the chemical shifts of chlorogenic acid. The peaks at m/z 180 and 163 in the EI-MS of 3 confirmed that it contained a caffeic acid moiety. The signals at 6 5.17, 3.82, 3.54 were assigned to three oxygenated protons, one (6 5.17) esterified, as well as multiplets at 6 1.83 integrating for four protons of two methylene groups. These oxymethines appeared at 6 70.9 (x 2) and 67.5, respectively, in the 13C NMR spectrum. Compound 3 showed only one carbonyl carbon at 166.1 ppm. The fact that compound 3 has only one carbonyl carbon and no quaternary carbon around 6 70.9 suggested that the caffeic acid moiety was not connected to a quinic acid moiety but a cyclopentane-2,5-diol moiety. From these spectral data, the structure of compound 3 was assigned as 1-(3', 4'- dihydroxylcinnamoyl)-cyclopenta-2, 5-diol. Compound 4 was obtained as a colorless oily product. The 1H NMR spectrum of 4 revealed a 3, 4-dihydroxylcinnamoyl moiety as in compound 3. However, the two multiplets in 4, appearing at 6 2.15 and 1.95 were assigned to two methylene groups, respectively. The DQFCOSY experiment showed that two CH2 protons in 4 were correlated and adjacent to each other and also coupled to other hydrogens. The 13 C NMR spectrum of this compound revealed that there were only one carbonyl carbon, eight 82 methine carbons, and two methylene carbons. Three of the methine carbons at 6 74.8, 73.0, and 68.3 were oxygenated and showed correlations to three methine protons at 6 3.64, 5.35, and 4.14, respectively, as evident from the HMQC spectrum. Also, five other methine carbons at 6 115.1, 116.5, 122.9, 146.8, and 115.8 showed correlations to three aromatic protons appearing at 6 7.04, 6.76, 6.93 and two olefinic protons at 7.58 and 6.30 ppm, respectively. Therefore, compound 4 was assigned as 1-(3’, 4’- dihydroxylcinnamoyl)-cyclopenta-2, 3-diol. CD measurements of compounds 3 and 4 did not show absorption maxima or minima. This seems to be because the cyclopentane moieties in 3 and 4 do not absorb in the UV region. However, both of these compounds gave observable peaks in their ORD spectra. Compounds 3 and 4 are novel. The antioxidant activity of compounds 1-4 was determined by fluorescence spectroscopy (Arora and Strasburg, 1997) and the activity was compared with caffeic acid, ferulic acid, chlorogenic acid, p-hydroxycinnamic acid, and two commercial antioxidants, tert-butylhydroquinone (TBHQ) and butylated hydroxytoluene (BHT), each at a 20 11M concentration. In this assay, the lipid peroxidation was initiated by Fe 2+ and the rate of decrease of fluorescence intensity reflected the rate of lipid peroxidation. The inhibitory activities of Fey-induced lipid peroxidation in the large unilarnellar vesicles (LUVs) for compounds 3 and 4 were about 80% at 20 11M. Compound 1 showed about 50% inhibitory activity. However, 2 did not show antioxidant activity even tested at a 100 11M concentration. The assay results showed that p-hydroxycinnamic acid is a weak antioxidant when compared to ferulic acid. However, the caffeic acid analogues, compounds 3 and 4, showed the highest antioxidant activity in this assay. The percent 83 inhibition of lipid peroxidation for TBHQ and BHT were > 90% at 20 uM concentration (Fig. 2.1). The variation in antioxidant activity among caffeoyl esters is dependent on the hydroxyl substitution of the aryl ring. More than one hydroxyl substitution in the aryl ring enhanced the antioxidant activity. Introduction of a second hydroxyl group in the ortho position, as in caffeic acid, also enhanced the antioxidant activity. Methylation of the hydroxyl group in the ortho position of caffeic acid, as in ferulic acid, resulted in a decrease of antioxidant activity. This result is in agreement with published studies on the effects of hydroxycinnamates on the autoxidation of fats and lipids (Shihidi and Wanasundara, 1992). Our data suggest that caffeic acid is the best antioxidant, followed by compounds 4 and 3, chlorogenic acid, and chlorogenic acid methyl ester (1). This trend in activity may be due to the difference in hydrophilicity or chelation properties of these compounds. It is interesting to note that the antioxidant activities of the novel caffeic acid analogues, 3 and 4, are comparable to the commercial antioxidants BHT and TBHQ at the concentration tested. 84 cooc:H3 ‘\\\ OH 0 "”’ OH O 0 HO OH on 2 R=H 3 R1=OI-I,R2=H s R=CH3 4 R1=H,R2=OH Fig. 3.1. Structure of compounds isolated from ethyl acetate fraction 85 Fig. 3.2. Antioxidant activities of compounds 1, 3 and 4 and some commercial antioxidants at 20uM concentration. The antioxidant activity of compound 2 was measured at 100 11M. The rate of peroxidation was monitored by a decrease in fluorescence intensity as a function of time. Relative intensity represents the fluorescence intensity at a given time divided by the initial intensity at the start of the assay. Values represent the means of duplicate measurements. 86 28 6:26.]? Bow o_._mE:oo.alnl Bow ecomobiolol 20m oEtmolll GImHIII. ._.Im _ v ucaanoo IOI m 250qu0 + N 953250 le _. ucaanooLqI ++mn_ III .9606 l 3.5 as: m 4 so aouaosaronu aagrelea 87 CHAPTER FOUR* Antioxidant polyphenolics from the methanol extract of tart cherries (Prunus cerasus) ABSTRACT Montrnorency and BalatonTM tart cherries were lyophilized and sequentially extracted with hexane, ethyl acetate and methanol. Methanolic extracts of dried BalatonTM and Montrnorency tart cherries (Prunus cerasus) inhibited lipid peroxidation induced by Fe2+ at 25 ppm concentration. Further partitioning of this methanol extract with EtOAc yielded a fraction, which inhibited lipid peroxidation by 76 % at 25 ppm. Purification of this EtOAc fraction afforded eight polyphenolic compounds 5,7,4'- trihydroxyflavanone (l), 5,7,4'-trihydroxyisoflavone (2), chlorogenic acid (3), 5,7,3', 4’- tetrahydroxyflavonol-3-rhamnoside (4), 5,7,4'-trihydroxyflavonol 3-rutinoside (5), 5,7,4'- trihydroxy-3'methoxyflavonol-3-rutinoside (6), 5,7,4'-trihydroxyisoflavone-7-glucoside (7) and 6,7-dimethoxy-S,8,4'-trihydroxyflavone (8), as characterized by 1H- and 13C NMR experiments. The antioxidant assays revealed that compound 8 is the most active, followed by quercetin 3-rhamnoside, genistein, chloregenic acid, naringenin and genistin, at 10 uM concentrations. *Wang, H.; Nair, M. G. Strasburg, G. M.; Booren, A. M.; Gray, .1. I. Submitted to J. Agric. Food Chem. 88 INTRODUCTION The Montrnorency (Prunus cerasus) variety constitutes more than 95% of tart cherry cultivations in Michigan and USA. However, BalatonTM tart cherry (P. cerasus), a new tart cherry cultivar, is being planted to replace Montrnorency in several Michigan orchards. Anthocyanin contents of Montrnorency and BalatonTM tart cherries have been reported (Wang et al., 1997; Chandra et al., 1993). However, a detailed investigation of other phenolic compounds in BalatonTM tart cherry was not carried out before. F lavonoids, a group of polyphenolic compounds, are widely distributed and have been reported to act as antioxidants in biological systems (Morel et al., 1993). Flavonoids are considered to have antioxidant activity similar to a-tocopherol, vitamin E. It is one of the most common and active naturally occurring antioxidant compounds in food because of its activity in both hydrophilic and lipophilic systems (Kiihnau, 1976). Kaempferol-3-rutinoside and kaempferol-3-glycoside were reported in the fruits of Montrnorency cherries (Von Elbe, 1970). Geissman (1956) indicated the presence of quercetin 3-glucoside in the leaves of P. cerasus. Also, various kaempferol and quercetin glucosides were identified from Montrnorency cherry (Shrikhande and Francis, 1973). From the bark of P. cerasus, tectochrysin 5-glucoside and genistein 5-glucoside, pinostrobin, naringenin, prunin, sakuranetin, sakuranin, dihydrowogonin 7-g1ucoside, chrysin, tectochrysin, genistein, prunetin and prunetin 5-glucoside were reported (Geibel et al., 1990; 1991; 1995). Isomers of caffeoquuinic acid, p-coumaroquuinic acids, caffeic and ferulic acids were characterized from Montrnorency tart cherry (Schaller and Von Elbe, 1970). Similarly, Schwab et a1 (1990) reported benzyl-B-D-glucoside, 6- 89 hydroxy-2, 6-dimethyl-octa-2 (E), 7-dienyl beta-D-glucoside and 2-methoxy-4-(2- propenyl)phenyl beta-D-glucoside from Montrnorency cherry pulp. Recently, meat products containing tart cherries are available to the consumers. Researchers have found that cooked low-fat ground beef with approximately 12 % of tart cherries had less rancidity development (Liu et al., 1995). Also, the addition of cherry fruits to ground beef before frying significantly inhibited the formation of heterocyclic aromatic amines (HAAs) (Britt et al., in press). The mechanism of this protective action may be involved in the potential antioxidant flavonoids and other polyphenolics present in cherries. Until now, researchers have not investigated the antioxidant compounds in BalatonTM and Montrnorency tart cherries. In this chapter, the isolation, identification and efficacy of antioxidant polyphenolic compounds from BalatonTM and Montrnorency tart cherries are described. MATERIALS AND METHODS Cherry Fruits. Pitted and frozen Montrnorency and BalatonTM tart cherries were obtained from commercial growers (Traverse City, MI) through the Cherry Marketing Institute, Inc. (Dewitt, MI). The cherries were flushed with nitrogen in freezer bags prior to their storage at -20°C. General Experimental. Silica gel (60 mesh size, 35-70 pm) was purchased from E. Merck, New Jersey. TLC plates (GF uniplate, Analtech, Inc., Newark, DE) after developing were viewed at 254 and 366 nm, respectively. For preparative high pressure liquid chromatography (HPLC) (LC-20, Japan Analytical Industry Co., Tokyo) purification, two JAIGEL-ODS, A-343-10 (20mm x250mm, 10 um, Dychrom, Santa 90 Clara, CA) columns were used in tandem. Peaks were detected using a UV detector equipped with model D-2500 Chromato-integrator (Hitachi, Tokyo). 1H-, 13C-, DQFCOSY and HMQC NMR spectra were recorded on a Varian UNITY 500 and an INOVA 300 MHz spectrometers at 25°C. All chemical shifts are given in parts per million relative to CD3OD and DMSO-d6 at 3.30, 49.0 ppm and 2.49 and 39.5 ppm, respectively. Fast atom bombardment mass spectroscopy (FABMS) were obtained on a JEOL JMS-HX110 using a glycerol matrix and Electrom impact ionization mass spectroscopy (EIMS) spectra were obtained on JEOL JMS-AXSOS Mass Spectrometers. Antioxidant Assay: All the buffers were stored in Chelex 100 to remove metal ions. A mixture containing 5 umol of 1-stearoyl-2-linoleoyl-sn-glycerol-3- phosphocholine (Avanti Polar Lipids, Inc., Alabaster, AL) and 15 nmol of the fluorescence probe 3-[p-(6-phenyl)-1,3,5-hexatrienyl] phenylpropionic acid (Molecular Probes, Inc., Eugene, OR) was dried under vacuum. The resulting film was suspended in 500 uL of buffer (NaCl, 0.15 M; EDTA 0.1 mM; MOPS 10 mM) and was then subjected to 10 freeze-thaw cycles in an ethanol/dry ice bath. The suspension was passed 29 times through a polycarbonate membrane with a pore size of 100 nm using a LiposoFast extruder (Avestin, Inc., Ottawa, Canada). Liposomes (200 nmol) were suspended in 2 mL of buffer (100 mM NaCl, 50 mM HEPES pH 7.0) and peroxidation was initiated by addition of 4 nmol of Fe”. Control samples contained no added Fe2+ or test compound. Anthocyanins, BHT, propyl gallate or a-tocopherol were added to final concentrations of 2 uM. Fluorescent intensity of this lipid suspension was monitored for a period of 21 min with or without test compounds, immediately following addition of Fe”, using a SLM 4800 spectrofluorometer (SLM Instruments, Urbana, IL). The values of relative 91 fluorescence were determined by dividing the fluorescence value at a given time point by that at t = 0 min. The decrease in relative fluorescence intensity with time indicated the rate of preoxidation. The percent inhibition of the lipid oxidation was calculated using the equation, Percent Inhibition ={[(F,¢.)p.- (Frel)Fe ]/[(Fr¢f)c - (Frel)p¢]} X 100, where: (F r.301» = relative fluorescence for the Fe 2+ and test samples at the end of 21 min, (Fre1)c = relative fluorescence for the control sample at 21 min, and (Fre1)1=e = relative fluorescence for the Fe 2+-containing sample at the end of 21 min (Richman et al., 1997; Arora and Strasburg, 1997) Extraction of cherries. Dried BalatonTM tart cherries (200 g) were ground and extracted sequentially with hexane, ethyl acetate and methanol (500 mL x 3) and the solvents were evaporated under reduced pressure at 40°C to yield crude extracts 0.42, 1.48 and 116.3g, respectively. Similarly, dried Montrnorency tart cherry yielded crude extracts 0.29, 0.74 and 125.4 g, respectively. The methanol extract of BalatonTM tart cherries (116.3 g) was dissolved in water (300 mL) and extracted with ethyl acetate (300 ml x 3). The ethyl acetate extract was evaporated to dryness under reduced pressure to yield fraction I (5. 3g). The aqueous layer was evaporated under reduced pressure to remove ethyl acetate, and applied to an XAD-2 column (100 g, Amberlite resin, mesh size 20-50, Sigma Chemical CO., St. Louis, MO), which was prepared as described by Chandra et a1 (1993). The column was then washed with distilled water (3 L) until the colorless washing gave a neutral pH. The adsorbed pigments were then eluted with methanol (500 mL). The red methanolic solution was concentrated at 40°C and the aqueous solution was then lyophilized to yield fraction 11 (3.5g). Since the major components of this fraction were anthocyanins, similar 92 to the components in water extract of Montrnorency and BalatonTM cherries (Wang et al., 1997), this fraction was not further purified. Purification of fraction 1. The crude solvent extracts from Montrnorency and BalatonTM cherries, fractions I and II from the methanol extract of BalatonTM tart cherry were bioassayed for antioxidant activity (Fig. 4.1). It was evident that fraction I from BalatonTM cherries contained the most active antioxidant compounds. Therefore, fraction I was further purified for antioxidant compounds. Fraction I (5.3 g) was chromatographed by MPLC (200 g) using solvent system CHC13 and methanol gradient starting with CHC13-MeOH (16:1, v/v, 1 L), CHC13-MeOH (8:1, v/v, 800 mL), CHC13-MeOH (4:1, v/v, 1 L) and finally with MeOH (1L). Sixteen fractions were collected and monitored by silica TLC plates using CHC13-MeOH (10:1) and CHC13-MeOH-HCOOH (4:1:0.1) as developing solvents. The fractions were combined to yield fiactions A-F; 740, 2500, 466, 386, 418 and 370 mg, respectively. Fractions A and B showed only weak antioxidant activity and hence, was not further purified for antioxidant compounds. Compounds 1 and 2: The fraction C (427 mg) was further purified on preparative silica gel TLC plates (20 x20 cm, 500 microns) and developed with CHC13-MeOH (15:1). The antioxidant band (26.2 mg), which showed very strong UV fluorescence at A366 and 3.254, was repeatedly purified by preparative TLC using acetone-CHC13 (1 :6) as the mobile phase. This yielded compounds 1 (Rf: 0.48, 2.4 mg) and 2 (Rf: 0.46, 2.4 mg). Compound 1: lH NMR (DMSO-dg): 612.15 (1H, s, S-OH), 10.80 (1H, s, 7-OH), 9.60 (1H, s, 4’-OH), 7.32 (2H, d, J=8.5 Hz, H-2’, H-6’), 6.81 (2H, d, J=8.5 Hz, H-3’, H- 5’), 5.90 (2H, s, H-6, H-8), 5.43 (1H, dd, J=12.7 Hz, 2.8 Hz, H-2), 3.26 (dd, J=17.1Hz, 93 12.7 Hz, H-3ax), 2.69 (dd, J=17.1Hz, 2.8 Hz, H-3eq); l3C NMR (DMSO-dg): 6196.6 (C-4), 166.8 (C-7), 163.4 (C-5), 162.1 (C-9), 157.8 (C-4’), 129.4 (C-l’), 128.8 (C-2’, C-6’), 115.6 (C-3’, 5’), 102.2 (C-10), 96.1 (C-6), 95.4 (C-8), 78.9 (C-2), 42.4 (C-3). Compound 2: 1H NMR (DMSO-dg): 12.98 (1H, s, 5-OH), 10.92 (1H, s, 7-OH), 9.60 (1H, s, 4’-OH), 8.26 (1H, s, H-2), 7.38 (2H, d, J=8.2 Hz, H-2’, H-6’), 6.82 (2H, d, J=8.2 Hz, H-3’, H-S’), 6.39 (1H, d, J=1.95 Hz, H-8), 6.21 (1H, d, J=1.95 Hz, H-6); 13C NMR (DMSO-dg): 6 180.6 (C-4), 164.4 (C-7), 163.6 (C-5), 158.1 (C-9), 157.5 (C-4’), 154.4 (C-2), 130.6 (C-2’, 6’), 122.8 (C-3), 121.7 (C-1 ’), 115.5 (C-3’, C-S’), 104.9 (C-10), 99.3 (C-6), 94.2 (C-8). Compound 3: Fraction D (155 mg) was purified by HPLC using CH3CN-H2O (25:75) as the mobile phase at a flow rate of 4 ml/min to yield active compound 3 (27.2 mg, R, = 52 min). 1H NMR (DMSO-d6): 67.45 (1H, d, J=15.9 Hz, H-7’), 7.00 (1H, d, J=2.0 Hz, H-2’), 6.95 (1H, dd, J=8.4 Hz, 2.0 Hz, H-6’), 6.75 (1H, d, J=8.4 Hz, H-5’), 6.18 (1H, d, J=15.9 Hz, H-8’), 5.16 (1H, m, H-5), 3.85 (1H, m, H-3), 3.53 (1H, m, H-4), 2.02-1.83 (4H, m, H-2, H-6); 13C NMR (DMSO-d6): 176.1 (COO'), 166.1 (C-9’), 148.2 (C-3’), 145.6 (C-4’), 144.4 (C-7’), 125.7 (C-1’), 121.1 (C-6’), 115.8 (C-5’), 115.1 (C-8’), 114.6 (C-2’), 72.9 (C-4), 71.2 (C-l), 71.0 (C-3), 67.3 (C-5), 38.8 (C-2), 35.1 (C-6). Compounds 4, 5, 6 and 7: Fraction E (418 mg) was purified by HPLC using CH3CN-H2O (30:70) as mobile phase at flow rate of 4 ml/min to yield compounds 4 (R, = 64 min, 11 mg), 5 (R.= 64 min, 8.6 mg), 6 (R, = 71 min, 13 mg) and 7 (R.= 84 min, 3.8 mg), respectively. Compound 4: lH NMR (DMSO-dg)! 67.62 (1H, d, 2.2, H-2’), 7.58 (1H, dd, 8.6, 2.2, H-6’), 6.70 (1H, d, 8.6, H-S’), 6.10 (1H, d, 2.0, H-8), 5.96 (1H, d, 2.0, H-6), 4.96 (1H, s, H-l ”), 3.82-3.22 (H-2”-H-5”), 1.15 (3H, d, 6.1, H-6”). 94 Compound s: 1H NMR (DMSO-d6): 87.94 (2H, d, J=8.8 Hz, H-2’, H-6’), 6.85 (2H, d, J=8.8 Hz, H-3’, H-S’), 6.34 (1H, s, H-8), 6.12 (1H, s, H-6), 5.24 (1H, d, 7.3, H- 1"), 4.35 (1H, s, H-l'”), 3.51(d, 10.5 Hz, H-6”), 3.37 (1H, m, H-4”), 3.28 (1H, m, H- 5“), 3.27 (1H, m, H-3’”), 3.22 (1H, dt, J=9.3 Hz, 6.0 Hz, H-S’”), 3.22 (1H, m, H-3”), 3.13 (1H, m, H-4’”), 3.13 (1H, d, J=5.1 Hz, H-2’“), 3.01 (1H, dd, J=9.6 Hz, 7.3 Hz, H- 2"), 0.94 (3H, d, J=6.0 Hz, H-6’”); 13C NMR (DMSO-d6): 8177.2 (C-4), 161.0 (C-7), 159.9 (C-S), 156.9 (C-9), 156.8 (C-2), 156.8 (C-4’), 133.4 (C-3), 131.1 (C-2’), 131.1 (C- 6’), 121.3(C-1’), 115.2 (C-3’), 115.2 (C-5’), 103.6 (010), 101.7 (C-l”), 100.9 (C-l'”), 99.4 (C-6), 94.2 (C-8), 76.4 (C-3”), 75.8 (C-S”), 74.2 (C-2”), 71.8 (C-4'”), 70.6 (C- 3'"), 70.4 (C-2’”), 70.0 (04"), 68.4 (C-S’”), 67.1 (06”), 17.9 (C-6’”); FABMS m/z 594 [M+H]+, m/z 617 [M+Na]+; EIMS m/z (% rel. Int.) 286 (100); Compound 6: 1H NMR (DMSO-dh); 87.81 (1H, d, J=2.0 Hz, H-2’), 7.48 (1H, d, J=8.4 Hz, 2.0, H-S’), 6.88 (1H, d, J=8.4 Hz, H-6’), 6.34 (1H, s, H-8), 6.12 (1H, s, H-6), 5.39 (1H, d, J=7.3 Hz, H-1 H), 4.38 (1H, s, H-l W), 3.82 (OCH3), 3.67 (1H, d, J=10.5, H- 6”), 3.37 (1H, m, H-4”), 3.31 (1H, dd, J=6.4 Hz, 5.1 Hz, H-3’”), 3.28 (1H, m, H-S”), 3.22 (1H, m, H-3”), 3.22 (1H, dt, J=9.3, 6.0 Hz, H-5’”), 3.13 (d, 5.1, H-2’”), 3.13 (1H, m, H-4’”), 3.05 (1H, dd, J=9.6 Hz, 7.3 Hz, H-2”), 0.95 (3H, d, J=6.0 Hz, H-6’”); 13C NMR (DMSO-d6): 8177.2 (C-4), 161.0 (C-7), 159.9 (C-2), 159.9 (C-S), 156.7 (C-9), 133.2 (C-3), 121.2 (C-l’), 113.4 (C-2’), 99.3 (06), 94.2( C-8), 103.6 (010), 149.4 (C- 3'), 147.0 (C-4’), 115.3 (C-S’), 122.4 (C-6’), 101.5 (C-l”), 101.1 (C-l’”), 76.4 (C-3”), 76.0 (05”), 74.3 (C-Z”), 71.8 (C-4’”), 70.6 (C-3’”), 70.4 (C-2’”), 70.2 (04”), 68.5 (C-S’”), 67.1 (C-6”), 55.9 (OCH3), 17.9 (C-6’”); FABMS, m/z 624 [M+H]+, 647 [M+Na]+; EIMS (% rel. Int.) m/z 316 (100). 95 Compound 7: lH NMR (DMSO-d6): 6 8.41 (1H, s, H-2), 7.40 (2H, d, J=8.7 Hz, H-2’, H-6’), 6.82 (2H, d, J=8.7 Hz, H-3’, H-5’), 6.72 (1H, d, J=2.1 Hz, H-8), 6.46 (1H, d, J=2.1 Hz, H-6), 5.05 (1H, d, J=7.5 Hz, H-l”), 3.91-3.30 (5H, H-2”-6”); 13C NMR (DMSO-d6): 181.7 (C-4), 164.2 (C-7), 163.8 (C-5), 158.4 (C-9), 155.8 (C-2), 155.8 (C- 4’), 131.3 (C-2’, C-6’), 123.7 (C-3), 123.7(C-1’), 116.3 (C-3’, C-5’), 101.0 (C-10), 100.7 (C-l”), 100.1 (C-6), 95.9 (C-8), 78.4 (C-S”), 77.6 (C-3”), 74.2 (C-2”), 70.7 (C-4”), 61.8 (C-6”). Compound 8: Fraction F (370 mg) was purified by preparative TLC (20 x 20 cm, 500 um) using MeOH-CHCl3-H2O (1:2:0.1, v/v) as the mobile phase. Six bands were collected and eluted with MeOH to yield bands I-VI: 9.6, 5.8, 14.5, 55.6, 131.6 and 56.2 mg, respectively. The active band, VI (56.2 mg), was further purified by preparative TLC using MeOH-CHC13 (1:8, v/v) as the mobile phase and yielded compound 8 (Rf = 0.62, 17 mg). UV kmax (MeOH): 209, 222 (sh), 290 (sh) and 303 nm; UV 714m (MeOH + AlCl3): 209, 235, 229, 321 and 360 nm; UV Mm (MeOH +AlCl3 + HCl): 209, 233, 289, 320 and 355nm; UV km“ (MeOH + NaOAc): 210 and 305nm; 1H NMR (CD3OD): 67.95 (2H, d, J=8.5 Hz, H-2’, H-6’), 6.92 (2H, d, J=8.5 Hz, H-3’, H-5’), 6.64 (1H, s, H-3), 4.02 (3H, s, OCH3), 3.91 (3H, s, OCH3); 13C NMR (CD3OD): 6 184.8 (C-4), 166.7 (C-2), 162.9 (C-4’), 149.2 (C-7), 146.4 (C-9), 142.9 (C-5), 137.8 (C-6), 132.2 (C-8), 129.8 (C- 2’, C-6’), 123.3 (C-1 ’), 117.0 (C-3’, C-5’), 107.9 (C-lO), 103.5 (C-3), 62.0 (OCH3), 61.3 (OCH3). RESULTS AND DISCUSSION 96 The methanol extract was partitioned with ethyl acetate to yield fraction I. The aqueous portion was purified on an XAD-2 column to yield fraction 11 (Wang et al., 1997). Preliminary antioxidant assay on these fractions and the crude methanol extract revealed that fraction I had the best antioxidant activity, followed by fraction II and methanol extract (Fig. 4.1). Fraction 11 contained anthocyanins 1-3 and hence was not purified further. Our preliminary antioxidant assay revealed that the percent inhibitions of F e2+ induced lipid peroxidation of hexane, EtOAc and MeOH extracts of BalatonTM tart cherry were 9.9, 28.3 and 13.6%, respectively. Similarly, the percent inhibitions of hexane, EtOAc and MeOH extracts of Montrnorency tart cherry were 11, 26.3 and 14.3%, respectively. The percent inhibitions of fraction 1 and II from methanol extract of BalatonTM tart cherry were 75.7 and 67.3%, respectively. Since Montrnorency and BalatonTM cherry extracts gave an identical chromatographic profiles, only BalatonTM extracts were further studied for antioxidant compounds. This was mainly due to larger quantities of the extracts available from BalatonTM compared to Montrnorency. Also, BalatonTM is the new variety of tart cherry grown commercially in several of Michigan cherry orchards. Purification of fraction 1 by MPLC, TLC and HPLC afforded compounds 1-8. Compounds 1 and 2 gave identical 1H and 13C NMR spectral data to that of naringenin and genistein, respectively (Harbome, 1994; Agrawal, 1989). The spectral data of 3 were identical to the 1H and 13C NMR spectral data of an authentic sample of chlorogenic acid. Similarly, compounds 4 and 7 were confirmed to be quercetin 3-rhamnoside and genistein 7-glucoside, respectively, by comparison of their 1H- and '3C NMR spectral data (Kosuge et al., 1985; Ohta et al., 1980). 97 100% 4 50% 9 % inhibition 0%. _ MeOH extract Fraction 1 Fraction II I Balaton I Montrnorency Fig. 4.1. Inhibitory effects of methanol extracts from BalatonTM and Montrnorency tart cherries and fractions I and II from BalatonTM cherry on Fe2+-induced large unilarninar vesicles (LUVs) peroxidation at 25 ppm concentrations. Fraction I contains compounds 1-8 and fraction II contains anthocyanins 1-3. The rate of peroxidation was monitored by a decrease in fluorescence intensity as a function of time. Relative intensity represents the fluorescence intensity at a given time divided by the initial intensity at the start of the assay. Data presented are mean of duplicate experiments (X i SD) 98 The FABMS and EIMS revealed a molecular formula of C27H30015 for compound 5. The 1H NMR spectrum of compound 5 gave signals for two anomeric protons that appeared at 65.24 and 4.35, respectively. These were assigned to anomeric protons of glucose and rhamnose, respectively. The 7.3 Hz coupling constant for the anomeric proton at 65.24 indicated a B-linkage of a glucose moiety to the aglycone. The doublet appeared at 0.94 ppm was assigned to a methyl group of a rhamnose sugar moiety. Therefore, The doublet at 6 4.35 corresponded to the anomeric proton of an L- rhamnopyranose. Also, the small coupling constant of <1Hz for this proton indicated an a-glycosidic linkage. The DQF-COSY spectrum of compound 5 helped to confirm the assignment of all protons in 5. The HMQC spectrum was used to assign the carbon signals in compound 5 and further confirmed glucose and rhamnose moieties in 5. The appearance of C-6 at 67.1 ppm for the glucose moiety, which was about 5 ppm firrther downfield than the normal chemical shift value of C-6 in glucose, indicated that rhamnose moiety was attached to the C-6 of the glucose moiety. The IH and 13C NMR spectra of 5 indicated a kaempferol aglycone functionality (Markham et al., 1978). The FAB-MS of 5 gave a molecular ion at m/z 594 and an ion at rn/z 617 indicating an [M+Na]+. The EI-MS gave a base peak at m/z 286, which corresponded to a kaempferol moiety. Therefore, compound 5 was assigned as kaempferol 6"-O-01-L-rhamnopyranosy1- B-D-glucopyranoside (Fig 4.2). The molecular formula of compound 6 was determined as C31H32OI6 by FABMS and EIMS, respectively. 1H and 13C NMR spectra indicated that compound 6 contained identical sugar moieties and linkages as in compound 5. The only difference was that 99 compound 6 showed the presence of a methoxy group. As in 5, H-8 and H-6 in compound 6 appeared at 6 6.34, 6.12, respectively. The chemical shifts at 67.81, 7.48 and 6.88 were assigned to protons in B ring and indicated that one methoxy group was at 3' or 4' position. The HMBC spectrum of 6 suggested that this methoxy group was attached to 3' position. The FAB—MS of 6 gave a molecular ion at m/z 624 and an ion at m/z 647 indicating an [M+Na]+. Also, the EI-MS showed a base peak at m/z 316, which corresponded to 3’-methoxy kaempferol moiety. Therefore, compound 6 was assigned as rhamnazin 6"-O-0L-L-rharnnopyranosyl-B-D-glucopyranoside. This is the first report of the isolation of this compound from tart cherries (Fig. 4.2). A flavonoid structure was revealed for compound 8 from its 1H and 13 C NMR spectral data. Compound 8 showed a carbon signal at 6 184.8. This indicated that it is a flavone with a hydroxyl group at C-5 (Agrawal, 1989). The 1H NMR spectrum of 8 showed the presence of two aromatic protons each at 67.95 and 6.92, respectively, which were assigned to H-2', H-6' and H-3', H-S', respectively. In addition, there were two OCH3 groups appeared at 62 and 61.3 ppm, respectively. The UV spectra of 8 in methanol before and after addition of aluminum chloride followed by HCl were comparable to the published value of 5,8-dihydroxy-6,7-dimethoxyflavone (Barberan et al, 1985). Further comparison with published 1H and 13C NMR spectral data of related compounds (Horie et al, 1995), compound 8 was assigned to be 6,7dimethoxy-5,8, 4’- trihydroxyflavone. Like compound 6, this is the first report of compound 8 from tart cherries (Fig. 4.2). Compounds 1, 2, 3 4, 7 and 8 were assayed at 10 um concentrations for antioxidant activity. The inhibitory effect of flavonoids on Fe2+ lipid peroxidation was 100 2: R=H 7: R=Gluc0se 0H OH O HO O \ H . C—O OH HO O 4: R1=OH, R2=rhamnose 5: R1=H, R2=rutinose 6: Rl=OCH3, R2=rutinose Fig. 4.2. Structures of Compounds 1-8 101 attributed due to their ability to chelate F e2+ with the formation of inert complexes that are unable to initiate peroxidation (Afanas’ev et al., 1989). Additionally, the Fe2+ complexes of flavonoids are considered to retain their free radical scavenging activities, therefore, can scavenge the free radical intermediate in lipid peroxidation. Also, flavonoids can act as free radical scavengers. The antioxidant activity of compound 8 was superior to the antioxidant activities of l, 2, 4 and 7 at 10 um concentrations studied (Fig. 4.3). Earlier reports suggested that the presence of ortho-dihydroxyl groups on the B ring (Bors et al. 1990), a hydroxyl group at position 3 on the C ring (Afanas’s et al., 1989; Mora et al., 1990) and a double bond at C2-C3 in conjugation with a 4-oxo functional group (Bors et al., 1990) are considered to be essential for effective radical scavenging by the flavonoids. Even though compound 8 does not posses a 3-hydroxyl group and has only one hydroxyl group on the B ring, the antioxidant activity of 8 is higher than quercetin 3-rhamnoside. Quercetin 3-rhamnoside contains an ortho- dihydroxyl group in the B ring in addition to a 3-hydroxyl group and a double bond at C2- C3 in conjugation with a 4-oxo functional group. The enhanced antioxidant activity of compound 8 was probably due to the hydroxyl and two methoxy groups in ring A. Arora et a1 (1997) reported that 7,8 -dihydroxyflavone showed similar antioxidant activity to quercetin, though it lacked any substitution on the B-ring and at 3-position. Watanabe (1998) compared the antioxidant activity of (i) catechin and (i) epicatechin with rutin and quercetin on the basis of the inhibition. The peroxyl radical-scavenging activities of these compounds were investigated by measuring the inhibition of hydroperoxidation of methyl linoleate initiated by a radical initiator, 2,2'-azobis(2,4-dimethylvaleronitrile) 102 (AMVN). The results indicated that (i) catechin and (i) epicatechin showed similar antioxidant activities to that of quercetin, even though they have C2-C3 saturated bond and no 4-oxo functional group (Rice-Evans et al., 1996). In order to evaluate which component contributed to the highest antioxidant activity in fraction 1, a mixture of compounds 1-8 was prepared, according to the ratio of their weight extracted from fraction 1. The antioxidant activities of this mixture was determined at 25 ppm concentrations, which contained compounds 1-8 0.6, 0.6, 14, 2.3, 1.1, 2.6, 0.8 and 3.5 ppm, respectively (Fig. 4.4). The antioxidant activities of individual components at 0.6, 0.6, 14, 2.3, 1.1, 2.6, 0.8 and 3.5 ppm concentrations, respectively, were also measured (Fig. 4.4) and compared with the mixture at 25 ppm concentrations. Results indicated that the mixture of compounds 1-8 gave 89.6 % of inhibition on Fe2+ induced lipid peroxidation, while compounds 1, 2, 3, 4, 5-6, 7 and 8 showed 5.1, 5.4, 76.7, 20.5, 16, 11 and 110 % of inhibition, respectively. Compounds 8 and 3 were the most active components in the mixture and probably in fraction 1. Interestingly, the sum of the antioxidant activity of individual compounds is higher than that of the mixture of these eight compounds. This suggested that in our assay system, some of the purified compounds are more effective inhibitors of lipid peroxidation when tested alone. 103 Fig. 4.3. Antioxidant activities of compounds 1, 2, 3, 4, 7, 8 and commercial antioxidants TBHQ and BHT at 10 11M concentrations. Data represent the means of duplicate experiments. 104 AEEV 0.5... OImHIOI HImi _o=coolll i ii i i A w ucaanoo+ __ n ncaanoolOl . . / h / ,, $8 4 ucaanoolXI m ucaanoolxi .\ w, / N oczanool l / F uczanoo+ III I..." ”al.,, ii . o\ooo_. ++mn_ + 1 105 uomqiuur % 100% ~ % inhibition mixture fraction I (O 'O C {U to Fig. 4.4: Percent inhibition of compounds 1-8 and their mixture on Fe2+ induced LUVs peroxidation. The mixture of compounds 1-8 was prepared, according to the ratio of their weight extracted from fraction 1. The mixture and fraction I were tested at 25 ppm concentrations. The mixture at 25 ppm concentrations contained compounds 1-8 0.6, 0.6, 14, 2.3, 1.1, 2.6, 0.8 and 3.5 ppm, respectively. Similarly, compounds 1-8 were assayed independently at 0.6, 0.6, 14, 2.3, 1.1, 2.6, 0.8 and 3.5 ppm concentrations, respectively. Data presented are mean of duplicate experiments (X i SD). 106 CHAPTER FIVE" Antioxidant and anti-inflammatory activities of anthocyanins and their aglycone from tart cherries ABSTRACT Cherries are a rich source of anthocyanins which may possess nutraceutical and phytoceutical properties. We have isolated anthocyanins and cyanidin from tart cherries and evaluated their antioxidant and antiinflammatory efficacies in vitro. The anthocyanins and cyanidin isolated from tart cherries exhibited comparable antioxidant and antiinflammatory activities to commercial products. The inhibition of lipid peroxidation of anthocyanins 1-3 and their aglycone, cyanidin, were 39, 70, 75 and 57%, respectively, at 2 11M concentrations. The antioxidant activities of anthocyanis and cyanidin were comparable to the antioxidant activities of BHA and superior to vitamin E at 2 uM concentrations. In the anti-inflammatory assay, which measured prostaglandin H endoperoxide synthase-l (PGHS-l) and and its isoform (PGHS-2) inhibitory activities, cyanidin gave IC50 values of 90 and 60 [AM for PGHS-1 and PGHS-2 enzymes, respectively. The positive controls aspirin, naproxen and ibuprofen, gave IC50 value of 1050, 11 and 25 [AM against PGHS-l enzyme. *Will be Submitted to J. Nat. Prod. 107 INTRODUCTION Public interest in phytoceuticals to inhibit chronic diseases and aging is gathering momentum. Reactive oxygen species such as hydroxyl (OH') and peroxyl radicals (R00), and the superoxide anion (02') are constantly produced as a result of metabolic reactions in living systems (Halliwell and Gutteridge, 1990). Living systems are protected from oxidative damage by these reactive species by enzymes such as superoxide dismutase and glutathione peroxidase, and by antioxidant compounds such as ascorbic acid, tocopherols, and carotenoids (Sies, 1997). However, when free radical production exceeds the antioxidant capacity of the organism, these radical species attack lipids, proteins, and DNA thus damaging structural integrity and function of cell membranes, enzymes and genetic material (Byers and Perry, 1992). A growing body of evidence indicates that various pathological conditions including cardiovascular disease, arthritis, various cancers, and Alzheimer’s disease are associated, at least in part, with the damaging effects of uncontrolled free radical production (Byers and Perry, 1992). Many foods contain non-nutritive components such as flavonoids and other phenolic compounds which may provide protection against chronic diseases through multiple effects which are as yet poorly understood (Tanaka et al., 1993). These compounds may act as antioxidants by reacting with free radicals and thus interrupting the propagation of new free radical species, or by chelating metal ions such as Fe2+ which catalyze lipid oxidation to alter their redox potentials. In addition, it has been shown that antioxidant supplements can significantly improve certain immune responses (Hertog et al., 1993). 108 Consumption of cherries was reported to alleviate arthritic pain and gout (Hamel, 1975), although there is no evidence for the active components and mode of action. These beneficial effects may be partially associated with the abundance of anthocyanins, the glycosides of cyanidin. Anthocyanins have been investigated as antioxidant substances (Costantino et al., 1992). Anthocyanins isolated from the seed coat of red beans and other sources inhibited lipid peroxidation (Tamura and Yamagarni, 1994; Tsuda et al., 1994; Kanner et al., 1994). Several anthocyanins were investigated for their antioxidant activity on human low density lipoprotein and lecithin-liposome systems (Same-Gracia et al., 1997). Commercial meat products containing cherry tissue are now available to the consumers. Cooked low-fat ground beef with approximately 12 % of tart cherries showed less rancidity development (Liu et al., 1995). The addition of cherry tissue to ground beef before frying significantly inhibited the formation of heterocyclic aromatic amines (HAAs) ( Britt et al., in press). HAAs are dietary compounds that are formed naturally during the cooking of muscle foods and are thought to arise from reactions involving creatine or creatinine, sugars and amino acids (Skog, 1993). In order to determine the nature of compounds that are responsible for these activities, we have studied antioxidant (Arora and Strasburg, 1997) and anti-inflammatory (Meade et al., 1993) activities of anthocyanins and its aglycone, cyanindin. MATERIALS AND METHODS General experimental Materials. Arachidonic acid and Ovine Prostaglandin H Synthase-l (PGHS-1) were purchased from Oxford Biomedical Research (Oxford, MI, USA). hPGHS-2 109 enzyme was obtained from Dr. David Dewitt (Department of biochemistry, Michigan State university). Anthocyanins 1-3 were purified from BalatonTM tart cherry by HPLC and were identified by lH- and 13 C NMR spectral data. Naproxen, ibuprofen and hemoglobin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Liposome Assay: All the buffers were stored in Chelex 100 to remove metal ions. A mixture containing 5 nmol of 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphocholine (Avanti Polar Lipids, Inc., Alabaster, AL) and 15 nmol of the fluorescence probe 3-[p-(6- phenyl)-1,3,5-hexatrienyl] phenylpropionic acid (Molecular Probes, Inc., Eugene, OR) was dried under vacuum. The resulting film was suspended in 500 11L of buffer (N aCl, 0.15 M; EDTA 0.1 mM; MOPS 10 mM) and was then subjected to 10 freeze-thaw cycles in an ethanol/dry ice bath. The suspension was passed 29 times through a polycarbonate membrane with a pore size of 100 nm using a LiposoFast extruder (Avestin, Inc., Ottawa, Canada). Liposomes (200 nmol) were suspended in 2 mL of buffer (100 mM NaCl, 50 mM HEPES pH 7.0) and peroxidation was initiated by addition of 4 nmol of Fe2+. Control samples contained no added Fe2+ or test compound. Anthocyanins, BHT, propyl gallate or a-tocopherol were added to final concentrations of 2 uM. Fluorescent intensity of this lipid suspension was monitored for a period of 21 min with or without test compounds, immediately following addition of Fe”, using a SLM 4800 spectrofluorometer (SLM Instruments, Urbana, IL). The values of relative fluorescence were determined by dividing the fluorescence value at a given time point by that at t = 0 min (Arora and Strasburg, 1997). Cyclooxygenase assay: Cyclooxygenase activities were measured PGHS-1 enzyme vesicles (ca. 5 mg protein/mL in 0.1 M TrisHCL, pH 7.4), a homogeneous 110 protein purified from ram seminal. Cyclooxygenase assays were performed at 37°C by monitoring the initial rate of 02 uptake using an O2 electrode (Yellow Springs Instrument Inc., Yellow Springs, Ohio). Each assay mixture contained 3.0 ml of 0.1M TrisHCL, pH adjusted to 7 by the addition of 6M HCl, 1 mM phenol, 85 ug hemoglobin and 10 nmol arachidonic acid. Reactions were initiated by adding 5 to 25 ug of microsomal protein in a volume of 15-50 11L. Instantaneous inhibition was determined by measuring the cyclooxygenase activity initiated by adding aliquots of microsomal suspensions of ' PGHS-l or PGHS-2 (10 11M O2/min cyclooxygenase activity/aliquot) to assay mixtures containing 10 11M arachidonate and various concentrations of the test substances (100- 1100 11M). The IC50 values represent the concentrations of the test compound that gave half-maximal activity under the standard assay conditions. RESULTS AND DISCUSSION About 20 fresh cherries (@ 100 g fresh tissue) contain 125 to 250 mg of anthocyanin, depending on the variety (Wang et al., 1997). The anthocyanins were assayed for antioxidant activity using the method developed by Arora and Strasburg (1997). This assay is based on the reaction of a fluorescent probe inserted into a phospholipid with free radicals generated by a pro-oxidant such as F e2+. Oxidative and reductive decomposition of peroxides, which are mediated by transition metal ions, can amplify the peroxidation process. As the reaction proceeds, the fluorescent probe is degraded and the signal declines. In the presence of an antioxidant, the rate of fluorescence decrease is reduced. Antioxidant activities of anthocyanins l, 2 and 3 and 111 of the aglycone cyanidin (Fig. 5.1) compared favorably with the commercial antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) (Fig. 2). All were better than a-tocopherol. At 2 11M concentrations, the extent of peroxidation of the sample containing a-tocopherol was indistinguishable from that of the Fe2+-containing sample with no added antioxidant (Fig. 5.2). The aglycone of anthocyanins, cyanidin, has higher efficacy than its glycosides suggesting that the antioxidant activity of anthocyanins is due to their aglycone moiety. Anthocyanins 1-3 contain 3, 2, and 1 sugar residues, respectively, and explained the lowest antioxidant activity observed for antocyanin 1 (Fig 5.2). The number of sugar residues at C3 position seem to be very important for antioxidant activity. The smaller the number of sugar units at 3-position, the higher the antioxidant activity. Bors et a1 (1990) reported that the stability of aryloxyl radical affected the antioxidant activities of compounds and may give rise to pro-oxidant effects. The antioxidant activity of cyanidin may depend on the stability of its aryloxyl radical. The mode of inhibition of oxidation by polyphenolics in general is not clear. However, some of the suggested mechanisms are: a) chelating ions via the ortho-dihydroxy phenolic structure (Afanas’ev et al., 1989); b) scavenging lipid alkoxyl and peroxyl radicals by acting as chain breaking antioxidants (Bors et al., 1990), and c) as hydrogen donors (Morel et al., 1994). The ortho-dihydroxy substitution in the B ring of anthocyanins and cyanidin is important to stabilize the resulting free radical through the 3’and 4’-OH moieties. Ortho-dihydroxy groups in anthocyanins can chelate the metal ion to prevent iron- induced lipid peroxidation. Also, the unsaturated C ring, which participates in electron delocalization, is an important factor as well (Bors et al., 1990). 112 Fig. 5.1. Anthocyanins in Montrnorency and BalatonTM cherries. Anthocyanin 1, R1=glucose, R2= rhamnose; Anthocyanin 2, R1=H, R2= rhamnose; Anthocyanin 3, R1: R2=H 113 Fig. 5.2. The antioxidant efficacy of anthocyanins and commercial antioxidants in a liposomal model system. Oxidation was initiated by the addition of ferrous ions. In the presence of test compounds, the rate of decay of fluorescence was decreased. Control samples contained no added Fe2+ and F e2+ contains no added test compounds. Other samples contained Fe2+ plus 2 11M test compound. “4 £32.: a m: 9 we a m cod aoueosaronu emelaa The anti-inflammatory assays (Meade et al., 1993) are based on the measurement the prostaglandin endoperoxide H synthase -1 and -2 isozymes (hPGHS- 1, and -2) ability to convert arachidonic acid to prostaglandins (PG) products. Cyanidin showed good PGHS-l and —2 inhibitory activities with IC50 values of 90 and 60 uM, respectively (Fig. 5.3, Fig. 5.4). In a preliminary experiment, the crude anthocyanins, 1- 3, gave hPGHS-l and hPGHS-2 activity at 33 ppm concentrations or above. However, pure anthocyanins 1-3 showed little or no activity against hPGHS-l and hPGHS-2 (data not shown). Higher concentrations of anthocyanins 1 and 2, on the contrary, increased the activity of enzyme. This is probably due to the ability of anthocyanins 1 and 2 to act as oxygen carriers at high concentration and enhance the oxygen uptake in this assay. The positive controls used in this experiment were aspirin, naproxen and ibuprofen, respectively. Aspirin had an IC50 value of 1050 uM each against hPGHS-l and hPGHS- 2 enzymes (Fig. 5.3). Naproxen and ibuprofen gave IC50 values of 11 and 25 11M against hPGHS-l enzyme, respectively (Fig. 5.3). The aglycone cyanidin inhibited hPGHS-l and hPGHS-2 enzymes at 90 and 60 HM, respectively. The ratio of IC50 values for hPGHS-l/hPGSH-2 was about 0.56 (Fig. 5.4). For measurements of time- dependent inhibition of their enzyme activities by cyanidin, hPGHS-2 isozyme suspension was pre-incubated at 37°C with 15 11M of cyanidin (one fourth of the concentration of IC50), and added to oxygen electrode chamber with arachionic acid substrate to initiate the PGHS emzyme reaction. Our results suggested that the rate of inhibition of PGSH-2 did not change with time. Further experiments should be carried out to determine the arachidonic acid metabolites in order to understand the mechanism of the action of anthocyanins and cyanidin on PGHS-1 and -2 enzymes. 116 The specific inhibition of PGHS-2 enzyme will be a major advance in anti- inflammatory therapy since it significantly reduce the adverse effects of non-steriodal antiinflammatory drugs (NSAIDs) treatment (Copeland et al., 1994). It is generally believed that ulcerogenic and other adverse properties of NSAIDs result from the inhibition of PGHS-1 whereas the therapeutically desirable effects come from inhibition of PGHS-2 enzyme (Masferrer et al., 1994). These results suggest that dietary tart cherry anthocyanins may possess numerous health benefits. Our experiments using anthocyanins and cyanidin isolated from tart cherries in model systems indicate that they possess antioxidant activity which is comparable to commercial antioxidants. Similarly, cyanidin, which may be metabolized from anthocyanins in in vivo system, showed better response than aspirin in the inflammatory assays. The antioxidant and anti-inflammatory properties of anthocyanins and cyanidin suggested that consumption of cherries containing these compounds may be beneficial in protection against chronic diseases. 117 :8 1 a? g 100% < +~Na3roxen g 50% - I O/anidin 2 j A Ibuprofen E X Aspirin E .A 10 100 1000 10000 concentration (11M) Fig. 5.3. Dose response curve for the inhibition of the human PGHS-1 enzyme by cyanidin. The anti-inflammatory properties of cyanidin were estimated by its ability to inhibit the cyclooxygenase activity of the prostaglandin H endoperoxide synthase (PGHS-1) enzyme. Cyanidin has an IC50 value of 90 11M for PGHS-l enzyme, while the non-steroidal antiinflammatory drugs, aspirin, naproxen and ibuprofen had IC50 of 1050, 11 and 25 11M, respectively. 118 100% O COX-1 . I COX-2 50% . % Initial COX Activity 0%- Concentration (um) Fig. 5.4. Dose response curve for the inhibition of COX-1 and COX-2 enzymes by cyanidin. Cyanidin has IC50 values of 90 and 60 11M for COX-1 and COX-2 enzymes, respectively. 119 CHAPTER SIX* Cyclooxygenase active bioflavonoids from BalatonTM tart cherry and their structure activity relationships ABSTRACT Five flavonoids, naringenin, quercetrin, 5,8,4’-trihydroxy-6,7-dimethoxyflavone, kaempferol 3-rutinoside and 3’-methoxy kaempferol 3-rutinoside, and two isoflavonoids, genistein, genistin, isolated from BalatonTM tart cherry were assayed for Cyclooxygenase-1 (COX-1) activity. Genistein showed the highest COX-1 inhibitory activity among the isoflavonoids tested with an IC50 value of 80 HM concentrations. Kaempferol showed the highest COX-1 activities among the flavonoids tested with an IC50 value of 180 11M concentrations. The structure-activity relationships of flavonoids and isoflavonoids revealed that hydroxyl groups at C4’, C5 and C7 in isoflavonoids were essential for a better COX-1 inhibitory activity. Also, the double bond between C2 and C3 in flavonoids are important for COX-1 enzyme inhibition. However, hydroxyl group at C3’ position in flavonoids decreased the COX-1 inhibition. *Will be submitted to Planta Medica 120 INTRODUCTION The cyclooxygenase enzyme, prostaglandin endoperoxide H synthase (PGHS), has been widely used as a tool for investigating the anti-inflammatory effects of plant products (Bayer et al., 1989; Goda et al., 1992; Wagner, 1990; Abad et al., 1994). Cyclooxygenase (COX) enzyme is the pharmacological target site of the nonsteroidal anti-inflammatory drugs (NSAIDs) (Humes et al., 1981; Rome and Lands, 1975). There are two isozymes of cyclooxygenase that catalyze the first step in prostaglandin synthesis: cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) (Hemler, et al., 1976; Dewitt et al., 1993). It is hypothesized that selective COX-2 inhibitors are mainly responsible for anti-inflammatory activities (Masferrer et al., 1994). Flavonoids have been widely investigated as anti-inflammatory substances. Arturson and Johsson (1975) first reported the activity of several semisynthetic derivatives of rutin on the prostaglandin (PG) synthase enzyme. After that, a series of flavonoids and other phenolic compounds were tested for cyclooxygenase and lipoxygenase inhibitions (Baumann et al., 1980). The structural features for cyclooxygenase (COX) and lipoxygenase (LOX) inhibitions were investigated. The 5,7-dihydroxyflavone galangin with an IC50 of 5.5 uM, was found to be the most active cyclooxygenase inhibitory flavonoid (Wurm et al., 1982). Flavonoids with an ortho-dihydroxy moiety in ring A or B were stronger inhibitors than those with a free 3-OH group (Wurm et al, 1982; Baumann et al., 1980). Certain prenylated flavonoids, such as morusin, were also active, because of their higher lipophilicity (Kimura et al., 1986). In flavonols, hydroxylation at C3 is a sufficient condition, especially if the compound possesses another hydroxyl at 5 position. In any case, the C2-C3 double bond, 121 which determines the coplanarity of the heterorings, appears to be a major determinant of COX activity (Wurm et al., 1982). These strucutural requirements have been confirmed in other systems. Also, unsubstituted flavone is a good COX inhibitor (Mower et al., 1984; Landolfi et al., 1984; Welton et al., 1986). Kalkbrenner et a1 (1992) further studied the effects of 37 flavonoids on prostaglandin endoperoxide synthase enzyme. Nonplanar flavans were more potent inhibitors than planar flavones and flavonols. Flavones with an ortho-dihydroxy structure in the B ring and flavonols with hydroxyl groups at C5 and C7 in A ring were potent prostaglandin endoperoxide synthase inhibitors. Most of the flavanones studied did not cause significant COX inhibition, except for the flavanone-3- ol, silibinin (Kalkbrenner et al., 1992). Flavones and flavanones with 3,7,4' hydroxyl groups are potent inhibitors of 5- LOX (Welton et al., 1986). The catechol structure has been proposed as an important feature for the inhibition of this enzymes (Welton et al., 1986). Also, the presence of hydroxyl at C5 increased the COX activity (Kimura et al., 1985). A study of 5- lipoxygenase inhibitory activity of baicalein (5,6,7-trioxyflavone) on leukotriene C4 (LTC4) biosynthesis showed that this compound significantly inhibited LTC4 production with an IC50 of 9.5 uM (Butenko et a1 1993). However, the anti-inflammatory activity of isoflavonids has been rarely investigated. Montrnorency and BalatonTM tart cherries were reported to alleviate arthritic- and gout-related pain in addition to antioxidant activities. The obvious antioxidant activity of flavonoid components in cherry may be responsible for the beneficial effect of chronic diseases, including inflammatory disorders. In order to evaluate anecdotal claims associated with tart cherries, we purified anthocyanins and polyphenolics from BalatonTM 122 chenies and evaluated their anti-inflammatory activity. BalatonTM tart cherry is a new cultivar being planted in Michigan orchards to replace Montrnorency cultivar. In this chapter, we have evaluated seven flavonoids and isoflavonoids from methanol extract of BalatonTM tart cherry for COX-1 enzyme inhibitory activity and compared their structure activity relationships with structurally related compounds. MATERIALS AND METHODS Materials. Arachidonic acid and Ovine Prostaglandin H Synthase-l (PGHS-1) were purchased from Oxford Biomedical Research (Oxford, MI, USA). Genistein, genistin, naringenin, quercetrin, 5,8,4’-trihydroxy-6,7-dimethoxyflavone, kaempferol 3- rutinoside and 3’-methoxy kaempferol 3-rutinoside were purified from BalatonTM tart cherry by HPLC and were identified by 1H- and 13 C NMR spectral data. Daidzein and formononetin were purchased from Research Plus, Inc. (Bayonne, New Jersey, USA). Biochanin A, kaempferol, quercetin, naproxen, ibuprofen and hemoglobin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Luteolin was purchased from Adams Chemical. Co (Round Lake, IL, USA). For measuring the effects of flavonoids and isoflavonoids, each sample was dissolved in DMSO to yield 40mM stock solution. The stock solution was further diluted to the desired concentrations according to the COX-1 inhibitory activity of each tested compound. Anti-inflammatory assay: Cyclooxygenase activities were measured PGHS-1 enzyme, a homogeneous protein purified from ram seminal vesicles. PGHS-1 enzyme (ca. 5mg protein/ml in 0.1 M Tris HCl, pH 7.4) were prepared and assayed on the same 123 day. Cyclooxygenase assay was performed at 37°C controlled by a circulation bath (Model-1166, VWR Scientific Products, Chicago, IL) by monitoring the initial rate of 02 uptake using an 5357 Oxygen electrode (INSTECH Laboratory, Plymouth Meeting, PA) (Meade et al., 1993). Each assay mixture contained 600 11L of 0.1 M Tris-HCl, pH 8.0, 1 mM phenol, 17ag hemoglobin and 1012M arachidonate and were mixed in a microchamber (INSTECH Laboratory, Plymouth Meeting, PA). Reactions were initiated by adding 1 to 5 Mg of microsomal protein (5 uL). Instantaneous inhibition of PGH Synthase isozymes were determined by measuring the cycloxygenase activity initiated by adding aliquots of microsomal suspensions of PGHS-1 in the assay mixtures containing 10 HM arachidonate and various concentrations of test compounds. The IC50 values represent the concentrations of inhibitor that gave half-maximal activity under the standard assay conditions. The kinetics of the enzyme activity was monitored by Biological Oxygen Monitor (YSI model 5300, Yellow Springs Instrument CO., Inc., Yellow Springs, Ohio) and collected in Quicklog Data Acquistion and Control computer software (Strawbeey tree Inc., Sunnyvale, CA). RESULTS AND DISCUSSION The COX-1 activity was determined by monitoring 02 uptake using an O2 electrode. The conversion of arachidonic acid to prostaglandins were initiated by adding enzyme preparations. One unit of cyclooxygenase represents the oxygenation of 1 nmol of arachidonate/min under the standard assay condition by the COX enzyme. This is a modification of the assay reported by Dewitt et a1 (1990). Km values for arachidonate 124 conversion to PGH were determined using arachidonate concentrations ranging from 2 to 50 pm. We have used 10 11M arachidonate for cyclooxygenase-1 assay, because this substrate concentration was high enough to give near-maximal cyclooxygenase activity and low enough to permit the detection of enzyme inhibition by lipophilic inhibitors (Meade et al., 1993). This methodology can also be used for cyclooxgenase-Z assay. Three known COX inhibitors, aspirin, ibuprofen and naproxen, were selected as positive controls. We compared COX-1 inhibition of flavonoids, kaemperol, quercetin, luteolin, quercetin 3-rhamnoside, 5,8,4’-trihydroxy-6,7-dimethoxyflavone, kaempferol 3- rutinoside, 3’-methoxy kaempferol 3-rutinoside and naringenin, and five isoflavonoids, genistein, genistin, daidzein, formononetin and biochanin A to the positive control used. COX-1 inhibitory activity of each compound at different concentrations was calculated by comparing the tangent of 02 uptake curves of test compounds with that of blank control. For each isoflavonoids and flavonoids, IC50 values (50% inhibitory concentrations) were calculated by linear regression analysis. The half-maximal inhibitory concentrations of aspirin, ibuprofen and naproxen, flavonoids and isoflavonoids are listed in Table 2.1. Among the flavonoids tested, kaempferol showed the highest COX-1 inhibition, followed by luteolin, quercetin, naringenin and quercetin 3-rhamnoside. In quercetin, compared to kaempferol, the presence of a hydroxyl group at C3’ position decreased the COX-1 inhibitory activity (Fig. 6.1). Kaempferol and quercetin showed varying COX inhibitory activitities under different assay conditions. (Hoult et al.,1994; Moroney et al., 1988; Kalkbrenner et al., 1992). A substitution at C3 positions also important for the COX-1 inhibitory activity. Glucosylation of hydroxyl group at C3 position removed the 125 COX-1 inhibitory activity. Comparing the COX-1 inhibitory activity of flavones (luteolin) with their corresponding flavonols (quercetin), it can be concluded that the absence of a hydroxyl group at C3 slightly enhanced the inhibitory effect on COX-1 enzyme. The quercetin 3-rhamnoside, which is not active in our studies, possessed antiinflammatory activity in vivo (Sanchez de Medina et al., 1996). It is speculated that glucoside might exhibit a COX inhibitory effect when it was metabolized to quercetin in in vivo system. The double bond between C2 and C3, which determines the coplanarity of the heteroring C, was essential for a higher inhibitory activity. Also, the saturation of C2- C3 double bond dramatically decreased the COX-1 inhibitory effect as in the case of naringenin (Fig. 6.1). This result is consistent with published reports (Wurm et al, 1982; Kalkbrenner et al., 1992). If the multiple numbers of hydroxyl and methoxyl substitution were present in A ring, the COX-1 activity was also decreased as in the case of 6,7- dimethoxy-S,8,4’-trihydroxyflavone. Among the isoflavonoids (Fig. 6.2), genistein showed the highest COX-1 inhibitory activity. When the 7-hydroxyl group in the A ring of genistein was glycosylated (genistin), the COX-1 activity was dramatically decreased. The hydroxyl group in C-4’ position was also essential for the COX-1 inhibitory activity. When 4’-OH group in genistein was methylated, the inhibitory effect of the methyl ester of genistein, biochanin A, decreased constantly (Fig. 6.2). Similarly, when 4'-OH group in daidzein was methylated, the COX-1 activity decreased dramatically. The 5-OH group in isoflavonoids is also an important factor for COX-1 inhibitory effect. The COX-1 inhibitory effect decreased when the C5 hydroxyl group in gensitein was removed to yield daidzein. These results indicated that C4’, C5 and C7 hydroxyl groups in isoflavonoids are 126 essential for COX-1 inhibitory activity. Comparison of genistein with that of kaempferol indicates that the ring B substitution at C3 position enhanced COX-1 inhibitory effect. In addition to COX-l inhibition, these isoflavonoids and flavonoids also showed good antioxidant activity as demonstrated in chapter four. The biological activities of these compounds may offer health benefits such as prevention of chronic diseases in human. Also, it suggested that the bioflavonoids present in tart cherries may be partially responsible for the anecdotal health claims such as the reduction of arthritic and gout related pain when cherries or cherry products are consumed. 127 Compound R1 R2 R3 R4 R5 R6 R7 quercetin OH OH OH OH H OH H kaempferol OH H OH OH H OH H luteolin OH OH H OH H OH H quercetrin OH OH rhamnose OH H OH H kaempferol 3-rutinoside OH H rutinose OH H OH H 3’-methoxy kaempferol OH OMe rutinose OH H OH H 3-rutinoside 5,8,4’-trihydroxyl-6,7- OH H H OH OMe OH OMe dimethoxyflavone Fig. 6.1. Structure of flavonoids tested for COX-1 inhibitory activity 128 compound R1 R2 R3 genistein OH OH OH genistin OH OH glucose biochanin A OMe OH OH daidzein OH H OH formononetin OMe H OH Fig. 6.2. Structure of isoflavonoids tested for COX-l inhibitory activity 129 naproxen on cyclooxygenase-1 enzyme Table 6.1. IC50 Values (11M) of flavonoids, isoflavonoids, aspirin, ibuprofen and Compound IC50 Aspirin 1050 ibuprofen 1 l naproxen 23 naringenin > 800 luteolin 300 quercetin 350 quercetrin not active Kaempferol 3-rutinoside not active 3’-methoxy kaempferol 3-rutinoside not active 5,8,4’-trihydroxy-6,7dimethoxyflavone not active kaempferol 180 genistein 80 genistin >400 daidzein 400 biochanin A 350 formononetin > 800 130 SUMMARY AND CONCLUSIONS It is generally believed that many common foods contain non-nutritional components such as flavonoids and anthocyanins that are considered to reduce the incidence of chronic diseases. Tart cherry is claimed to have various health benefits. In order to evaluate the anecdotal claims, we have investigated the active components from aqueous, ethyl acetate and methanol extracts of Montrnorency and BalatonTM tart cherries and determined their anticancer, antioxidant and anti-inflammatory activities. Three anthocyanins, anthocyanin l [cyanidin-3-(2"-0—fl-D-glucopyranosyl-6"- 0-a-L-rhamnopyranosyl-fl-D-glucopyranoside], anthocyanin 2 [cyanidin-3-(6”-0-a-L- rhamnopyranosyl-,B-D-glucopyranoside] and anthocyanin 3 [cyanidin-3-O-fl-D- glucopyranoside, were identified in the aqueous extracts of both Montrnorency and BalatonTM tart cherries by antioxidant assay-directed fractionation and purification. However, BalatonTM tart cherry contained approximately six times more anthocyanins than does Montrnorency tart cherries. The anthocyanins and their aglycones, cyanidin, were further tested for antioxidant activity using Fe2+-induced lipid peroxidation. The antioxidant activities of anthocyanins and cyanidin are comparable to the commercial antioxidants butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) and superior to vitamin E at 2 11M concentrations. The number of sugar moieties at C3 position is an important factor for the variation of antioxidant activity observed for anthocyanins. The anti-inflammatory activities of anthocyanins and cyanidin were assayed on prostaglandin endoperoxide H synthase-1 (PGHS-1) and -2 (PGHS-2) enzymes. Results suggested that anthocyanins did not show PGHS-1 and PGHS-2 inhibitory activities. 131 However, the aglycone cyanidin demonstrated PGHS-1 and PGHS-2 inhibitory activities at IC50 values of 90 and 60 11M, respectively. The positive controls, aspirin, ibuprofen and naproxen had IC50 values of 1050, 11 and 25 11M, respectively, against COX-1 enzyme. The anthocyanins, which are not active in in vitro assays, could be metabolized to cyanidin in biological systems and hence can act as an anti-inflammatory agent. The anthocyanins are the most abundant class of compounds in cherries and hence have the potential to substantiate the anecdotal claims such as the reduction of arthritic and gout related pain and incidence of cardivascular diseases. From the ethyl acetate extract of BalatonTM tart cherry, three novel compounds, 2- hydroxy-3-(o-hydroxyphenyl) propanoic acid, 1-(3’, 4’-dihydroxycinnamoyl)- cyclopenta-2,5-diol and 1-(3’, 4’-dihydroxycinnamoyl)-cyclopenta-2,3-diol were identified by ‘HNMR, 13C NMR, DQCOCY, HMQC, FABMS and EIMS experiments. The inhibitory activities of lipid peroxidation of 1-(3’, 4’-dihydroxycinnamoyl)- cyclopenta-2,3-diol and l-(3’, 4’-dihydroxycinnamoyl)-cyclopenta-2,5-diol were 79 and 75 %, respectively, at 20 uM concentrations. However, 2-hydroxy-3-(o-hydroxyphenyl) propanoic acid did not show activity at 100 11M. The antioxidant activities of these compounds were compared with caffeic acid analogues. It is concluded that the 3, 4- dihydroxy functionality on the aromatic ring is essential for the antioxidant activities of caffeic acid analogues. Also, the smaller the ester group, the better was the antioxidant activity. These phenolic compounds did not show anti-inflammatory activity when tested at 1000 11M concentrations. From the methanol extract of BalatonTM tart cherry, five flavonoids, 5,7,4’- trihydroxyflavanone, quercetin 3-rhamnoside, kaempferol 3- 6"-O-0L-L- 132 rhamnopyranosyl-B-D-glucopyranoside, rhamnazin 6"-O-0L-L-rhamnopyranosyl-B—D- glucopyranoside and 5,8,4’-trihydroxy-6,7-dimethoxyflavone and two isoflavonoids, 5,7,4’-trihydroxyisoflavone and 5,7,4’-trihydroxyisoflavone 7-glucoside, were identified by spectral methods. Both rhamnazin 6"-O-a-L-rhamnopyranosyl-B—D-glucopyranoside and 5,8,4’-trihydroxy-6,7-dimethoxyflavone are reported from tart cherries for the first time. 5,8.4’-trihydroxy-6,7-dimethoxyfalvone was found to be the most active antioxidant phenolic compound isolated from BalatonTM cherry. The antioxidant activity of 5,8.4’-trihydroxy-6,7-dimethoxyflavone is better than that of tert-butylhydroquinone (TBHQ) and butylated hydroxytoluene (BHT) at 10 uM concentrations. These compounds were further tested for hPGSH-l (COX-1) inhibitory activity and compared with structurally related compounds. Genistein is the most active COX-1 inhibitor among the compounds isolated from BalatonTM tart cherry. The OH groups at C4’, C5 and C7 in isoflavonoids are the essential functionality for COX-1 inhibitory activity. In the case of flavonoids, C2-C3 unsaturation determined the coplanarity of the heteroring and is essential for the increased COX-1 inhibitory activity. Similarly, the OH groups at C3’ and C7 hydroxyl groups are also important factors for COX-1 inhibitory activity. Flavonoids with high number of substitutions in A ring, such as 5,8,4’- trihydroxy-6,7-dimethoxyflavone, did not exhibit COX-1 inhibitory activity. It is possible that the phenyl group, ring B, substituted at C3 may have enhanced the COX-1 inhibitory effect. Our study indicated that a number of compounds present in tart cherries have antioxidant and prostaglandin endoperoxide H synthase (COX) enzymes inhibitory activities in vitro system. The antioxidant or free-radical scavenging action and the 133 enzyme inhibitory actions of anthocyanins and flavonoids present in tart cherries could account for many other pharmacological activities, such as antiallergic, antiviral, anticancer including anticarcinogenic activities and prevention of cardiovascular disease and aging. 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