Food Sci. Technol. Int. Tokyo, 4 (1), 48-53, 1998

Characteristics of Antioxidative Flavonoid Glycosides in Lemon Fruit

Yoshiaki MIYAKE,1 Kanefumi YAMAMOT0,1 Yasujiro MORIMITSU2 and Toshihiko OSAWA2

* Central Research Laboratory of Pokka Corporation, Ltd., 45-2 Kumanosyo, Shikatsu-cho, Nishikasugai-gun, Aichi 481, Japan 2Department of Applied Biological Sciences, Nagoya University, Nagoya 46401, Japan

Received June 12, 1997; Accepted September 27, 1997

We investigated the antioxidative flavonoid glycosides in the peel extract of lemon fruit (Citrus limon). Six flavanon glycosides: eriocitrin, neoeriocitrin, narirutin, naringin, hesperidin, and neohesperidin, and three flavone glycosides: diosmin, 6~-di- C-p-glucosyldiosmin (DGD), and 6- C-p-glucosyldiosmin (GD) were identified by high- performance liquid chromatography (HPLC) analysis. Their antioxidative activity was examined using a linoleic acid autoxidation system. The antioxidative activity of eriocitrin, neoeriocitrin and DGD was stronger than that of the others. Flavonoid glycosides were present primarily in the peel of lemon fruit. There was only a small difference in the content of the flavonoid glycosides of the lemon fruit juice from various sources and varieties. Lemon fruit contained abundant amounts of eriocitrin and hesperidin and also contained narirutin, diosmin, and DGD, but GD, neoeriocitrin, naringin, and neohesperidin were present only in trace amounts. The content of DGD, GD, and eriocitrin was especially abundant in lemons and limes; however, they were scarcely found in other citrus fruits. The content of flavonoid compounds in lemon juice obtained by an in-line extractor at a juice factory was more abundant than that obtained by hand-squeezing. These compounds were found to be stable even under heat treatment conditions (121'C, 15 min) in acidic solution.

Keywords: Iemon fruit, Citrus limon, antioxidant, fiavonoid glycoside

Lemons ( Citrus limon) are used in various food prepara- an important role in the prevention ofcarcinogenesis and in tions such as soft drinks, alcoholic drinks, and jams, and they extending the life span of animals and may offer effective are one of the most popular citrus fruits in the world. Lemons, protection against peroxidative damage in living systems which contain a number of nutrients such as citric acid, (Jacob, 1995; Cutler, 1992; Osawa et al, 1990). Therefore, ascorbic acid, minerals, and flavonoids, have been known for much attention has been focused on the importance of natural a long time as typical healthy foods. Flavonoid compounds antioxidants. are widespread in the plant kingdom and comprise a large In this study, we paid great attention to the antioxidative group of naturally occurring compounds found in all flavonoid glycosides in lemon fruit. We studied the anti- vascular plants. They are present in citrus as well as in other oxidative activity and the stability after heat treatment of the fruits, vegetables, nuts, seeds, grains, tea, and wine. Flavonoid flavonoid glycosides to examine their properties for use in compounds that exhibit beneficial effects on capillary perme- foods. We also examined for the distribution of fiavonoid ability and fragility were once known as vitamin P; they have glycosides in various sources or varieties of lemon fruit in been investigated regarding their physiological functions, other citrus fruits. such as hypotensive, antiallergic, anti-inflammatory, antiviral, anticancer, and anticarcinogenic properties (Bracke et al, Materials and Methods 1994; Middleton & Kandaswami, 1994; Mabry & Ulubelen, Materials Lemons (Citrus limon BURM. f.) and the

l 980) . other citrus fruits were purchased from a supermarket. The It has been reported that lemon juice has desmutagenic and varieties of lemon, eureka, Iisbon, and verna were obtained antimutagenic effects (Achiwa et al, 1991; Jain et al, 1987) from the Citrus Research & Education Center (Lake Alfred, in concern with the prevention of carcinogenesis. Recently, Florida, USA). The lemon juice from a juice factory was we have been involved in the isolation of antioxidative squeezed using an in-line extractor (FMC Co., Lakeland, flavonoid glycosides from lemon fruit and have identified Florida, USA). eriocitrin (eriodictyol-7-rutinoside), a flavanone glycoside Chemicals Linoleic acid and o!-tocopherol were ob- (Miyake et al, 1997a), and 6,8-di-C-~-glucosyldiosmin tained from Wako Pure Chemical Industries, Ltd. (Osaka). (DGD) and 6-C-p-glucosyldiosmin (GD), C-glucosyl flavo- Flavonoid glycosides were obtained from Funakoshi, Ltd. nes (Miyake et al, 1997b). Lipid peroxidation is known as (Tokyo). Eriocitrin, DGD, and GD were purified according one of the major factors which causes deterioration of foods to the method of Miyake et al (1997a, b.) during storage and processing. It is also suspected to be Isolation of antioxidative fuvonoid glycosides from pee/ strongly associated with aging and carcinogenesis (Yagi, of lemon fruit The peel of lemon fruit (1.50 kg) was 1987; Cutler, 1984; Harman, 1982). Recently, it was reported chopped in a homogenizer and extracted with methanol (3 l) that natural antioxidants contained in dietary plants may play at room temperature for 3 days. The extract was concentrated Characteristics of Antioxidants in Lemon Fruit 49

in vacuo. The concentrated peel extract ( I 10 g) was chromato- c,:] F~ P:i ~~ graphed on a Cosmosil 75C 18-0PN ODS column (Nacalai ~~ ~:: Tesque, Inc., Kyoto, ip37x500 mm). The column was washed with 2 1 of 20% methanol-water and was eluted with 40% methanol-water. The eluate was concentrated in vacuo, and the antioxidative fiavonoid fraction (6.00 g) was obtained. According to the method of Miyake et al (1997a), the antioxidative flavonoids glycosides were isolated from the ~ antioxidative flavonoid fraction by preparative high-per- ~1 formance liquid chromatography (HPLC). Eriocitrin (1.04 ooO C\~ g), neoeriocitrin (2.30 mg), narirutin (20.5 mg), DGD (52.0 (L) c- ~ p mg), and GD (15.8 mg) were isolated. Eriocitrin, DGD, and c:i o ~: DG were identified by MS, IH-NMR, and 13C-NMR analyses l~ c,DO (Miyake et al, 1997a, b). Neoeriocitrin and narirutin were ~:? identified from the retention time of standard reagents for < HPLC analysis. The aglycones of neoeriocitrin and narirutin t f ~~ c,~ ~il o were prepared by hydrolysis using pectinase according to the ,j IJi tJ~ [~~1 e:::~:< '0naringenin, during 20 HPLC analysis. Other flavonoid glycosides of naringin, Retention time (min) hesperidin, neohesperidin, and diosmin were identified from Fig. 1. HPLC profile for the antioxidative flavonoid fraction. ERI: eriocltrin, the retention time of standard reagents for HPLC analysis of NeoERI: neoeriocitrin, NAR: narirutin, NAN: naringin, HES: hesperidin, the antioxidative flavonoid fraction (Fig. l). HPLC (LC- NeoHES: neohesperidin, DIO: diosmin, DGD: 6,8-di-C-p-glucosyldiosmin, GD: 6- C-p-glucosyldiosmin. 10A, Shimadzu Co., Ltd., Kyoto) was carried out using a Shim-pack CLC-ODS(M) (4.6x250 mm) with a UV detector (~ =280 nm). The solvent system contained 70% of a 5% acetic acid solution and 30% methanol. The fiow rate was 1.0 ml/ min. concentration of O. 1% flavonoid glycosides was added to the A ntioxidative assay Antioxidative activity was evalu- different buffer solutions, and the solutions were heated at ated using the linoleic acid system (Osawa & Namiki, 1981). 80'C for 30 min or at 121'C for 15 min. The stability of the Each sample was added to a mixed solution of linoleic acid flavonoid glycosides after heat treatment was determined by (0.13 ml) and 99.0% distilled ethanol (lO ml) with a 50 mM the residual ratio of the flavonoid glycosides after heat phosphate buffer (pH 7.0, lOml), and the volume was treatment. The residual ratio of eriocitrin was determined adjusted to 25 ml with distilled water. The reaction mixture in from the residual peak of eriocitrin after heat treatment by a conical fiask was incubated at 40'C. At intervals during the HPLC analysis. incubation, the degree of oxidation was measured using the thiocyanate method (Mitsuda et al, 1966) by recording the Results and Discussion absorbance at 500 nm after coloring with FeC12 and thio- A ntioxidative fuvonoid compounds in lemon fruit We cyanate. c!-Tocopherol was used as the positive standard. previously reported that fiavonoid fractions prepared from Determination of fuvonoid glycosides The lemon peel extract or juice using ODS column chromatography fruit, from a Californian source, was separated into the peel, exhibited strong antioxidative activity in the linoleic acid juice, and seed in order to determine the location oferiocitrin. autooxidation system (Miyake et al, 1997a). The antiox- The peel was further separated into the flavedo (epicarp), idants were identified as eriocitrin (eriodictyol p-7-rutino- albedo (mesocarp), and pulp vesicles using a knife. Samples side), a flavanone glycoside, and as DGD and GD, C-glucosyl (5.0 g) of the subdivided peel and the seed were homogenized flavones (Miyake et al, 1997b). We further isolated another and extracted with 100 ml of methanol at 37'C for 4 days. The two antioxidative flavonoid glycosides from peel extract. pulp of the squeezed juice was removed by filtration. They were identified as neoeriocitrin (eriodictyol p-7-rham- Each citrus fruit was separated into the peel and juice by noglucoside) and narirutin (naringenin ~-7-rutinoside). hand-squeezing the fruit. The peel (5.0 g) was homogenized Figure I shows the HPLC profile of the antioxidative and extracted with 100 ml of methanol at 37'C for 4 days. The flavonoid fraction. The other flavonoid compounds were pulp of the squeezed juice was removed by filtration. identified as naringin (naringenin p-7-rhamnoglucoside), Flavonoid glycosides in the extract of the peel and in the hesperidin (hesperetin ~-7-rutinoside), and diosmin (dios- filtered juice were determined by HPLC analysis according to metin p-7-rutinoside) from the antioxidative flavonoid frac- the previous method. tion. We identified nine fiavonoid glycosides in the antiox- Stability of fuvonoid glycosides by heat treatment at idative flavonoid fraction in the peel extract of the lemon dlfferent pH The buffer solutions were at pH 2 and pH 3.5 fruit, and their structures are shown in Fig. 2. using a 100 mM Sodium citrate-HCI buffer, and at pH 5.0 and The antioxidative activity of the nine fiavonoid glycosides pH 7.0 using a 100 mM sodium phosphate buffer. The final was examined using the linoleic acid autoxidation system as 50 Y. MIYAKE et a/. shown in Fig. 3. We previously reported the antioxidative Properties of fuvonoid compounds The properties of activity of eriocitrin, DGD and GD in the linoleic acid system the flavonoid compounds in lemon fruit were examined for (Miyake et al, 1996a, b). Eriocitrin had the strongest solubility in water or ethanol. These compounds were soluble antioxidative activity among the fiavonoid glycosides in in ethanol, but GD, hesperidin, neohesperidin, and diosmin lemon fruit. Neoeriocitrin and DGD also showed antiox- were insoluble in water. The taste of these flavonoid glyco- idative activity, but the others had only a weak activity. We sides (O. I % water solution) was examined. Naringin, neoer- expect that the antioxidants in lemon fruit will provide iocitrin, and neohesperidin having the sugar chain of neohe- protection against peroxidative damage in living systems in speridose (2-0-a-L-rhamnosyl-~-D-glucose) were more bitter relation to aging and carcinogenesis. than narirutin, eriocitrin, and hesperidin having the sugar

o H OH OH OCll3 OH OH CH:oH o o o cH2 o o o Ho~Io Ho~l Hoel Ho~lo Ho~lCH2 o HO o H o o HO O OH oll OH OH OH OH Hov neoeriocitrin OH OH (NeoERI) diosmin (DIO) eriocitrin (ERI)

OH CIl~OH OCH3 OH o OCH3 OII O OH OC}ls Ho~~Io CH2 o CH:oH o o HO OH Ho~l HO:~lo H o C}izOH HO o OH OH OH HO O o OH Ho~l HO OH O neohesperidin OH OH OH hesperidin (HES) (NeoHES) 6,8-di- O-glucosyldiosmin (DGD) OH OII o O CH2 o OH OCll3 Ho~l CH20H o HO~ Ho~lo llo o cll'oll I]o o OH OH OH HO o o lro OH on o Ho~1 naringin (NAN) oll narirutin (NAR) 6 - O- glucosyldiosmin (GD) oll OH Fig. 2. Structure of flavonoid compounds in lemon fruit.

Lipid peroxidation (%) OX ZOX 40x 60~ 80x 1 OOX Contro I

100/l~ Eriocitrin

10O/l~ Neoeriocitrin

IO0/lN Narirutin

100/l~ Naringin

100/lN Hespepidin

100/l~ Neohesperidin

100/In Dioslin

100/lN DGD

10O/l~ CD

100/l~ a-Tocophrol

Fig. 3. Antioxidative activity of flavonoid glycosides. DGD6,8-di-C-fi-glucosyldi!osmin, GD 6- C- ~-glucosyldiosmin. Characteristics of Antioxidants in Lemon Fruit 51 chain of rutinoside (6-0-c!-L-rhamnosyl-p-D-glucose). These idant and unstable under high temperature in neutral solu- findings suggest that the bitterness of citrus juice is related to tion. The chemical and physical properties of flavonoid the structure of the neohesperidose portion of the flavonoid compounds in citrus fruit have been reported (Rouseffy, glycosides. l 980). It has been reported that the A and B rings in the We examined the stability of flavonoid glycosides under flavonoid molecule are stable and relatively unreactive, and conditions of sterilization because of the importance of this that the central C ring of the central C3 group is the most process in the manufacture of beverages and foods. The reactive portion of the molecule and undergoes a ring stability of fiavonoid compounds after heat treatment in opening reaction with a base or acetic anhydride (Hatori, different buffer solutions of pH 2.0, pH 3.5, pH 5.0, and pH 1962). The decrease in eriocitrin and neoeriocitrin by heat 7.0 was also examined. The heat treatment at 80'C for 30 min treatment in a neutral solution may be due to such ring assuming the sterilizatlon of yeast or mold was used, and the opening reaction. The C ring of these compounds having the heat treatment at 121'C for 15 min assuming the sterilization catecol structure of the adjacent dihydroxy groups on the of heat-stable bacteria was used. For all of the flavonoid B-ring seemed to be opened easier than that of other flavonoid glycosides, except eriocitrin and neoeriocitrin, more than 90% compounds. These results suggest that the antioxidative remained after heat treatment at 80'C for 30 min, and more flavonoid compounds such as eriocitrin and neoeriocitrin, than 80% remained after heat treatment at 1 21'C for 1 5 min. which are stable after heat treatment in the acidic condition These compounds were fairly stable under the heat treatment of pH 3.5-5.0, may be suitable for use in acidic beverages and conditions. Eriocitrin and neoeriocitrin were unstable in a foods. neutral solution (pH 7.0) at 80'C for 30 min and 121'C for 15 Flavonoid compounds in citrus fruit have generally been min, these compounds were decreased to about half of the reported to be stable under storage conditions (Ranganna et initial content after heating at 80'C for 30 min and almost al, 1983). In commercial lemon juice, the nine flavonoid completely disappeared after heat treatment at 121'C for 1 5 compounds were stable during sterilization at 80'C for 30 min min. It is suggested that these compounds having strong and under storage conditions (room temperature for 9 antioxidative activity changed by oxidation under the previ- months), and the content of ascorbic acid decreased to 60% ous conditions, as well as ascorbic acid, which is an antiox- under the same conditions (data not shown.) The stability of

Table 1. Distribution of flavonoid compounds in each variety and source of lemon juice. Concentration (mg/ 100 ml) ERl NeoERI NAR NAN HES NeoHES DIO DGD GD V ariety eureka l 5.9 N D") 05 04 5.3 TR") l .4 58 TR l .2 llsbon 13.6 ND 10 08 5.8 TR 44 TR 16.1 5.5 34 verna ND 04 05 ND TR TR Country of Produce l .3 California 12. I TR O.2 ND 8.9 ND 4.9 TR South Afrlca I 1.3 TR 0.3 ND l .7 ND 0.5 2.4 TR l.l 4.9 O, l Australla l 6.4 O. l 0.2 ND 5.8 ND O, l New Zealand l 1.0 TR 0.5 3.4 ND O.6 4.0 ND O. l Chile l I .O O. l 0.2 ND 4.2 ND 0.5 46 l .O O. l Japan 16.0 ND TR O. l 6.0 ND 42 a)not detected. b)trace: Iess than O. I mg/ 100 ml. ERI: eriocitrin, NeoERI: neoeriocitrin, NAR: narirutin, NAN: naringm HES: hesperidin, NeoHES: neohesperidin, DIO: diosmin, DGD: 6,8-di-C-p-glucosyl- diosmin, GD: 6-C-~-glucosyldiosmin.

Table 2. Distribution of flavonoid compounds in various locations in lemon fruit. Concentration (mg/ 100 ml) ERI NeoERI NAR N AN HES NeoHES DIO DG D GD I .3 4.9 J uice") l 2. l TR 0.2 ND 8.9 ND TR 33.9 5, l Peel 280 l .5 9.4 TR 173 TR 26.2 30.3 l .3 flavedo I 13 l.3 3.43 ND 2.5 0.8 40.6 l I ,8 l .O 23.6 8.9 albedo 252 0.9 0.2 227 9.6 2.7 pulp vesicles 285 NDb) 5.8 0.2 70.5 0.2 lO.8 12.3 Seed TR ND T R ') ND ND ND TR ND ND ~)The content of juice represented with flavonoid compounds (mg) of 100ml juice (mg/ 100 ml).

b)not detected. ')trace: Iess than 0.1 mg/ 100 g. ERI: eriocitrin, NeoERl: neoeriocitrin, NAR: narirutin, NAN: naringin, HES: hesperidin, NeoHES: neohesperidin, DIO: diosmin, DGD: 6,8-di-C-fi-glucosyl- diosmin, GD: 6-C-fi-glucosyldiosmln. 52 Y. MIYAKEet al . flavonoid compounds under storage conditions of the lemon content of fiavonoid compounds was fairly similar among the juice was greater than that of ascorbic acid. varieties and the sources of the lemon fruit. Determination of fuvonoid glycosides in lemon fruit The content of fiavonoid compounds in various parts of As shown in Table l, the content of fiavonoid glycosides in the lemon fruit was examined (Table 2). All of the nine the juice of different varieties of lemon fruit (eureka, Iisbon, flavonoid compounds were found to be more abundant in the and verna) and in the juice of fruit produced in different peel than in the juice, but only a small amount of these countries (California, South Africa, Australia, New Zealand, compounds was found in the seed. Eriocitrin and hesperidin Chile, and Japan) was examined. It was found that the existed abYndantly in the peel and juice. The content of

Table 3. Content of flavonoid compounds in lemon juice obtained by hand squeezing and factory squeezing. Concentration (mg/ 100 ml) ERI NeoERI NARNAN HESNeoHES DIO DGD GD Bri x pH Acidity Hand squeezing 25 03 03 ND") 8.9 ND 21 72 ND 85 25 48 In-llne extractor 98 03 08 0.3 12.2 0.2 34 97 0.2 89 24 55 ~)not detected. ERI: eriocitrin, NeoERI: neoeriocitrin, NAR: narirutin, NAN: narmgln, HES: hesperidin, NeoHES: neohesperidin, DIO: diosmin, DGD: 6,8-di- C- ~ -glucosyl- diosmin, GD: 6-C-~-glucosyldiosmin.

Table 4. Distributron of flavonoid compounds m crtrus Jruces Concentration (mg/ 100 ml) ERl NeoERI NAR NAN HES NeoHES DIO DGD GD Citrus. Iimon (lemon) 12.l TR" 0.2 NDb) 8.9 ND I.3 4.9 TR C. auranttfolia (lime) 9.7 TR 0.6 ND l0,2 ND 0.3 2.2 O. l C. sinensis (valencia orange) 0.2 TR 22.9 ND 32, l ND ND ND ND C. sinensls (navel orange) 0.3 0.2 1 4.8 O.3 37,4 ND ND ND ND C. unshiu (unshiu) 0,3 TR 26.4 TR 30. l ND TR ND ND C. iyo (iyo) ND ND l0.0 ND 12.3 ND ND ND ND C. reticulata (ponkan) O. l ND 3.6 ND 15,3 ND ND ND ND C. paradisi (grapefruit, white) O. l ND 18.6 390 0,9 2.9 ND ND ND C. paradisi (grapefrult, pink) O. l 0.2 8.5 27.0 0.5 l.5 ND ND ND C. aurantium (daidai) ND 8.4 0.6 22.3 ND I 2.9 ND ND ND C. hassaku (hassaku) O. l O.3 8.6 28.5 l .8 9,7 ND ND ND C, sphaerocarpa (kabosu) ND ND 16.4 l .8 2,8 1,l ND TR ND C. junos (yuzu) O. l O. l l 5.0 5.4 6.5 3.3 ND ND ND a)trace: Iess than O. I mg/ 100 ml. b)not detected. ERI: eriocitrin, NeoERl: neoeriocitrin, NAR: narirutln NAN narmgm HES: hesperidin, NeoHES: neohesperidin, DIO: diosmin, DGD: 6,8-di- C-fi-glucosyl- diosmin, GD: 6-C-~-glucosyldiosmln.

Table 5. Dlstribution of flavonoid compounds in each variety and source of lemon juice. Concentration (mg/ 100 ml) ERI NeoERI NAR NAN HES NeoHES DIO DGD GD Citrus. Iimon (lemon) 280 I .5 9.4 TR") 173 TR 26.2 33.9 5. 1 C. aurantifolia (lime) 94.0 3.7 2.7 O. l l 32 NDb) 10.4 25.3 8.4 C. sinensis (valencia orange) 3.4 ND ll5 O. l 277 ND l .9 ND ND C, sinensis (navel orange) 7.4 ND 121 0.7 35 l ND 3.6 ND ND C. unshiu (unshiu) 9. l ND 450 1 .2 513 ND ND ND ND C. iyo (iyo) 4.2 ND 284 O.6 394 O.9 2.5 TR ND C. reticulata (ponkan) 4.3 2.4 42 . 3 ND 415 ND 2.3 ND ND C, paradisi (grapefruit, white) 2. l 1 .O l 53 67.3 2.4 3.0 0.8 ND ND C, paradisi (grapefruit, pink) ND ND I 90 1310 2.7 13,3 ND ND ND C. aurantium (daidal) 8,6 215 14.8 835 0.5 489 28.2 ND C, hassaku (hassaku) 2, l 9.6 l 08 545 30.4 259 0.5 ND ND C. sphaerocarpa (kabosu) ND l0.3 393 l 02 1 90 7 1 .8 ND ND ND C. junos (yuzu) 4.3 3.8 193 87.8 l 18 75.8 ND ND ND ')trace: Iess than O. I mg/ 100 g. b)not detected. ERI: eriocitrin, NeoERl: neoeriocitrin, NAR: narirutin, NAN: naringm HES: hesperidin, NeoHES: neohesperidin, DIO: diosmin, DGD: 6,8-di- C-fi-glucosyl- diosmin, GD: 6- C-p-glucosyldiosmln. Characteristics of Antioxidants in Lemon Fruit 53 eriocitrin in the albedo (mesocarp, white interlor of the peel) available a greater amount of antioxidative fiavonoid com- and pulp vesicles (in part of the enveloping fiesh) was more pounds. abundant than in the flavedo (epicarp, yellow exterior of the peel), and the hesperidin content was found to be abundant in the albedo. DGD, narirutin, and diosmin existed in various parts of the peel and juice, but only small amounts of neo- References Achiwa, Y., Koda, T, and Namiki, K. (1991). Inhibition of N-nitro- eriocitrin, naringin, GD, and neohesperidin were detected. somorpholine formation by lemon juice. Nippon Shokuhin Kogyo The concentration in the juice of the lemon fruit of ascorbic Gakkaishi, 38, 826-830 (in Japanese). acid, which is used as an antioxidant in food products and has Bracke, M.E., Bruyneel, E.A., Vermeulen, S.J., Vennekens, K., Marck, biofunctional activity in vivo (Jacob, 1995), was about 45 mg V.V. and Mareel, M.M. (1994). Citrus fiavonoid effect on tumor invasion and metastasis. Food Technol., 48, 121- 124. in 100 ml of juice. Ascorbic acid is an important antioxidant Cutler, R.G. (1984). Antioxidants, aging, and longevity. In "Free in the juice of the lemon fruit but was unstable under the food Radrcals In Brology " ed by W A Pryor. Academic Press, Orlando processing conditions of heat treatment and food sterilization, FL, Vol. 6, pp. 371-423. and under food storage conditions. The total concentration of Cutler, R.G. (1992). Genetic stability and oxidative stress: common the above nine flavonoid compounds, which are stable under mechanism in aging and cancer. In "Free Radicals and Aging," ed. by I. Emerit and B. Chance. Birkhauser Verlag, Basel, Switzerland, conditions of sterilization of foods and beverages, was 37.4 pp. 31-46. mg in 100ml of juice and that of eriocitrin, the strong Harman, D. ( 1982). The free-radical theory of aglng. In "Free Radicals antioxldative flavonoid compound, it was 12.1 mg in 100 ml in Biology," ed. by W.A. Pryor. Academic Press, Orlando, FL, Vol. of juice. The flavonoid compounds seemed to be important 5, Chapter 8. Hatori. S. ( 1962). In "The Chemistry of Flavonoid Compounds," ed. antioxidants, as well as ascorbic acid, because they exist in a by T. Geissman. MacMillan, New York, pp. 286. considerable amount in the juice of the lemon fruit and are Jacob, R.A. ( 1995). The integrated antioxidant system. Nutr. Res., 15, stable under norrnal processing or storage conditions. 755-766. The content of flavonoid compounds in lemon juice was Jain, A.K., Shimoi, K., Nakamura, Y., Tomita, l, and Koda, T. (1987). examined relative to the method for squeezing the fruit by an Preliminary study on the desmutagenic and antimutagenic effect of some natural products. Curr. Sci., 56, 1266-1269. in-line extractor or by hand squeezing (as shown in Table 3). Mabry, T.J. and Ulubelen, A. (1980). Chemistry and utilization of The content of eriocitrin and hesperidin in lemon juice phenypropanoids Includlng flavonoids, coumarins and lignans. J. obtained by an in-line extractor was greater than that of the Agric. Food Chem., 28, 188-196. juice obtained by hand squeezing. We estimated that the juice Middleton, E, and Kandaswami, C. (1994). Potential health-promot- ing properties of citrus flavonoids. Food Technol., 48, I 15- 1 19. obtained by an in-line extractor contained abundant amounts Mitsuda, H., Yamamoto, K. and lwami, K. (1966). Antioxidative of eriocitrin and hesperidin because pulps, which are parts of action of indole compounds during the autooxidation of linoleic the albedo and pulp vesicle in peel, were contaminated in the acid. Eiyo to Shokuryo, 19, 210214 (in Japanese). juice. The in-line extractor method of squeezing fruit is Miyake, Y., Yamamoto, K. and Osawa, T. (1997a). Isolation of important commercially for the better yield of the juice. Using eriocitrin (eriodictyol 7-rutinoside) from lemon fruit (Citrus limon BURM. f.) and its antioxldative activity. Food Sci. Technol. Int. this method, the lemon fruit was squeezed with higher Tokyo, 3, 84-89. pressure than by hand squeezing. Because of the pressure on Miyake, Y., Yamamoto, K., Morimitsu, Y. and Osawa, T. (1997b). the fruit, parts of the peel, the albedo and pulp vesicle, in Isolation of C-glucosylflavone from lemon peel and antioxidative which eriocitrin and hesperidin are abundant, were present in activity of flavonoid compounds in lemon fruit. J. Agric. Food the juice as pulp. Chem., 45, 4619-4623. Osawa, T. and Namiki, M. (198 1 ). A novel type of antioxidant isolated Distribution of fuvonoid compounds in citrus fruits from leafwax of Eucalyptus leaves. Agric. Biol. Chem., 45, 735-739. The distribution of flavonoid compounds from the peel Osawa, T., Namiki, M, and Kawakishi, S. (1990). Role of dietary (Table 4) and juice (Table 5) of citrus fruits was investigated. antioxidants in protection against oxidative damage. In "Antimuta- Eriocitrin, DGD and GD were also shown to exist in limes as genesis and Anticarcinogenesis Mechanisms," ed. by Y. Kuroda, D.M. Shankel and M.D. Waters. Plenum Publishing, New York, pp. well as lemons, but very little was present in the other types l 39- 1 53. of citrus fruits. Little neoeriocitrin was found in lemons, but Ranganna, S., Govindarajan, V.S. and Ramana, K.V.R. ( 1983). Citrus it was found to be especially rich in daidai. Eriocitrin and GD fruits-varieties, chemistry, technology, and quality evaluation. Part were also shown to exist in limes as well as in lemons, but very II. Chemistry, technology, and quality evaluation. A. Chemistry. little was present in the other types of citrus fruits. These Crit. Rev. Food Sci. Nutr. P, 18, 313-386. . Rouseff, R.L. (1980). Flavonoid and citrus quality In "Crtrus compounds were found to be especially plentiful in lemon Nutrition and Quality," ed. by S. Nagy and J.A. Attaway. American and lime fruits and abundant in the peel obtained as a Chemical Society Publishing, Washington, D.C., pp. 83- 108. byproduct from juice factories. These results suggested that Yagi, K. (1987). Lipid peroxides and human disease. Chem. Phys. effective utilization of the peel in juice products will make Lipids, 45, 337-341 .