Dr. Kazuhiko Kubota Professor Department of Pharmacy Science University of Tokyo Tokyo, JapanCurriculum VitaeKazuhiko Kubota, Ph.D. Professor of Pharmacy Department Science University of Tokyo 1953 BS., Pharmacy, Kumamoto University 1961 Lecturer, Science University of Tokyo 1964 Ph.D., Pharmacy, Tokyo University 1964 Assistant Professor, Department of Pharmacy, Science University of Tokyo 1970—Present Professor, Department of Pharmacy, Science University of Tokyo 1988-1992 Chairman, Department of Pharmacy, Science University of Tokyo 1990 — 1993 Director, Science University of Tokyo 1989 — 1995 Professor, Research Institute of Life Science, Science University of Tokyo 1979 ~ 1980 Visiting Professor, Department of Pharmacology, Chicago Medical College 1981 - 1981 Chairman, Kanto Section of Japan Pharmaceutical Society 1989 — 1997 Member, Medicinal Advanced Technology Evaluation Committee Published Books: Pharmacy Dictionary (Japan Industrial Technology League) Pharmacology (Kodan Corporation: Scientific) Textbook of Pharmacology (Hirokawa Books) Fundamental Pharmacology Laboratories (Nanko-do) Suuummation of Clinical Medicine Inroduction (YaKuj! Nippo Corporation) Ete... Research Findings on Barley Leaf Extract: Dr. Kubota has researched barley leaf extract for many years. During the course of his study, Dr. Kubota found that barley leaf extract has many pharmacological functions, such as anti-inflammation, anti-ulcer, anti-hypercholesterol, anti-thrombosis, anti-anxiety, increasing endurance and lowering blood sugar. All of Dr. Kubota’s research findings ‘have been included in more than ten research articles published in professional journals or presented in related meetings. Research articles with respect to anti-inflammation, anti- ulcer, anti-hypercholesterol and endurance increasing activities are attached in the appendices.Barley and | By Kazuhiko Kubota, Ph.D.BARLEY AND I Kazuhiko Kubota, Ph.D. Professor of Pharmacology Department Science University of Tokyo I have a long-lasting relationship with the plants belonging to true grasses (the family of rice grass). It all started in 1959, just after graduating from the Universi I synthesized and identified the chemical formula of "Coixol," which is contained in coix, a plant belonging to the family of rice grasses. Since then, I enjoyed collecting plants of the rice-grass family and determined the content of "Coixol." I, therefore, obtained a wide knowledge about the plants belonging to the rice-qrasses family. It is one of my interests to investigate the distribution of the plants of this family when I travel abroad. ‘Taking a bus tour along the so-called "Romantishe Strasse," from Heidelberg through the medieval old town of Rotenburg in Germany, I showed tourists in the bus that the European culture has been developed by fertile, flal fields, which were scraped by Une ylaviers and Usickly covered plants belonging to the rice-grasses family, although species of the plants were limited. Why have so many mammals, along with human-kind, been relying upon the plants belonging to the rice-grasses family? The important reason is that the plants belonging to the rice-grasses family contain no alkaloids or chemical substances, showing strong medicinal action, in comparison with plants belonging to Solanacase. On the other hand, the rice-grasses family has been widely distributed worldwide ‘and are rich with a variety of species, such as one fit to high temperatures, high-humid tropical climates, and through wheat or barley, which fit to low temperatures ana ary conditions. It is evident that horses, cows, sheep, etc., can maintain their life with only rice grasses, because they are rich in vitamins, minerals, protein and in good balance. You know the Giant Panda living in China eats only bamboo leaves, which also belongs to the vice-grasces family. Additionally, thoce animals mainly eat the leaves of the plant. Taking the above facts into account, there is a person who develops a natural food spray-drying the young barley leaf juice, which is a member of the rice-grasses family. It is Dr. Yoshihide Hagiwara, ny University Senior. Since then, my relation with the rice grasses became closer through Dr. Hagiwara. We started to investigate the content of the barley lear extract dry juice, pnarmacologicailly. 10 be nonest, in tne 1beginning, I did not expect much with this investigation. By chance, when I injected a water-soluble fraction of barley leaf extract dry Juice to mice, I came to know that it nad very strong anti-inflammation activity. Then, we purified this fraction, and had succeeded in isolating a saccharoprotein, which showed a wonderful anti-inflammation activity more than 5,000 times stronger than aspiline. The mechanism of the anti-inflammation activity of this substance was different from the mechanisms of any other anti-inflammation drugs. It might be due to inhibition of activities of certain enzymes. In addition to this, we found in the barley. leaf extract dry juice a specific peroxidase which destroy mutant (chemical substance stimulate gene mutation), even in the acidic solution and detoxify its ability for mutation, also tne superoxide dismutase, wnich detoxify superoxide, is known to be a cause of inflammation or arteriosclerosis when developed too much in the body, and also many other enzymes. Moreover, both enzymes have been purified. The superoxide desmutase, it is said, might be a good index for aging, because it decreased, according to aging in animals. When we determined the barley leaf extract dry juice for prevention, from the arteriosclerosis view point, we found a kind of high molecular alcohol which lowered the cholesterol level in the blood and was confirmed through animal experiments. Even beta- cytosterol, which is now in the marketplace as an anti- arteriosclerosis drug, I determined present in the barley leaf extract dry juice. We also studied the effect for the motor ability of a mouse with the barley leaf extract dry juice. We added 2- to-4% barley leaf extract dry juice to normal feed and fed mice for 2 weeks and then gave a comparison running test to the mice. ‘The mice fed with barley leaf extract dry juice could run 150% longer than the mice fed with normal feed. We know central stimulants, such as caffeine, increase motor ability. Barley leaf extract dry juice, however, has no such function related to the central stimulant. We are now investigating the mechanism of motor stimulation of barley leaf extract dry juice. Barley leaf extract dry juice contains a wide variety of active components and, therefore, it is quite an interesting object for continued pharmacological investigation. November 29, 1994 NF/dpAppendix Isolation of Potent Anti-inflammatory Protein From Barley Leaves. Kubota, K.. Matsuoka, Y., and Seki, H., Japanese Journal of Inflammation 1983. 3(4) Studies On The Effects Of Green Barley Juice On The Endurance And Motor Activity In Mice. Kubota, K. and Sunagane, N., The 104” Annual Congress of Pharmaceutical Society of Japan 1984. . Studies on the Constituents of Green Juice From Young Barley Leaves, Anti- ulcer Activity of Fractions from Barley Juice. Ohtake, H., Yuasa, H., Komura, C., Miyauchi, T., Hagiwara, Y., and Kubota, K., Journal of the Pharmaceutical Society of Japan 1985. 105(11) Studies on the Constituents of Green Juive Frum Yury Barley Leaves, Effect on Dietarily Induced Hypercholesterolemia in Rats. Ohtake, H. Nonaka, S., Sawada, Y., Hagiwara, Y., Hagiwara, H., and Kubota, K., Journal of the Pharmaceutical Society of Japan 1985. 105(11)APPENDIX 1 Isolation Of Potent Anti-inflammatory Protein From Barley Leaves* Kazuhiko Kubota, Yutaka Matsuoka, Hirohumi Seki, Faculty of Pharmaceutical Sciences, Science University of Tokyo, 12, Ichigaya-funagawara-machi, Shinjuku-ku, Tokyo, 162, Japan. *Part of this paper has been published in the Japanese Journal of Inflammation, Vol. 3, No. 4, 1983 (1), alIntroduction It has been well established that superoxide dismutase (SOD) has potent anti-inflammatory action. Since barley leaves are abundant in superoxide dismutase, we examined anti-inflammatory activity of green juice from barley leaves and confirmed that it exerted anti-inflammatory activity. However, further examinations revealed that the anti-inflamma- tory activity of the barley juice was produced not only by SOD, but also by other protein than SOD. We isolated protein fractions named P4-D1 and D1-G1, which showed extremely strong anti-inflammatory activity. In the present paper, the authors are showing the evidence of the potent anti- inflammatory activity of the protein and barley SOD.Materials and Methods 1. Materials Barley (Horidium vulgare L. var. num Hook) was grown in Oita prefecture in Japan. The green juice obtained from the young barley leaves was brought to powder by using a spray drier in low temperature. The procedure of fractionation of the green juice was shown in Chart 1, P4-D1 and D1-G1 area kind of glycoprotein, the molecular weight of which ranged from about 20,000 to 40,000. They are considerably heat stable and highly soluble in water. The authentic SOD isolated from the green barley juice was kindly provided hy Dr. Horio, a professor of Osaka University. P4-D1 and D1-Gi were dissolved in physiological saline and aspirin was suspended in physiological saline with an addition of Tween 80. 2. Acute toxicity in mice Male ddY strain mice weighing 23 to 28 g were used. The mortality was obtained on day 4 of drug administration. LA50 value was calculated according to the method devised by van der Warden (2). 3. Carrageenin-induced edema in rats Male, Wistar strain rats, weighing 130 to 150 g were used. Carrageenin suspension was prepared by euspending carrageenin powder in a sterlized physiological saline solution at a concentration of 1% and kept for 3 to 4 days in a refrigerator before use. The carrageenin suspension (0.1 ml) was injected ouboutancouoly to the aole of the right hind paw of therate, and 0.1 ml of saline solution to the left paw. Drugs, P4-D1 and D1-Gl, were administered 30 minutes before the carrageenin injection. The paw volume of the rate was measured by the method described by Yamazaki, ct al, 3), in which a Foot Volume Meter was used before, and 1, 2, 3, 4,5,6.and 8 hours after the carrageenin injection. The edema volume was obtained as follows: (Paw volume of carragcenin injected onc) (Paw volume of saline injected one). Results The acute toxicity of D1-G1 and P4-D1 was listed in Table L Both fractions produced no toxic signe in mice when they were orally or subcutaneously administered to mice even at very high doses. They showed moderate toxicity in intraoperitoneal injection. ‘The anti-inflammatory effects of P4-D1 and D1-G1 were shown in Fig. 1 and those of barley SOD in Fig. 2. Anti-inflammatory effects of intravenous 0.1 or 0.2 mg/kg of P4-D1, D1-G1 and SOD were almost comparable to that of subcutaneous 100 mg/kg of aspirin. Anti-inflammatory activity was highest in D1-G1 among them.Although data were not shown here, the anti-inflammatory of D1-G1 was hardly reduced after heat treatment (100 C, 20 minutes) (1). While that of SOD was significantly reduced as shown in Fig. 2. D1-G1 did not inhibit prostaglandin synthesis and did not share antiacetylcholine, antiserotonin and antibradykinin activity, but inhibited enkephalinase activity (1). An oral administration of D1-G1 and P4-D1 showed no significant anti- inflammatory activity. Discussion AllofD1-G1, P4-D1 and SOD isolated from green barley juice revealed extremely potent antiinflammatory activity, especially when they were injected intravenously. They significantly suppressed the carrageenin- induced edema in rats at very low doses, such as 0.01 and 0.1 me/ke. However, the solution of SOD showed a bright yellowish color, while those of P4-D1 and D1-G1 did not, and SOD was not heat stable, while D1-G1 was heat stable. Therefore, P4-D1 and D1-G1 arechemically different from SOD. Unlike aspirin, P4-D1 and D1-G1 did not inhibit prostaglandin synthe- sis, indicating that they have quite a different mechanism of anti inflam. matory action from that of aspirin and phenylbutazone, which are potent inhibitors of prostaglandin systhesis. ‘The ratio, intravenous ED50/LD50, for D1-G1 on anti-inflammatory action is much larger than that for aspirin. This means that P4-D1 and Di G1 ocem to be much better anti-infauuualory agents than aspirin as far as they were concerned with intravenous administration. The mechanism of the anti-inflammatory action of D1-G1 and P4-D1 is unclear at present. ‘They may excrt their action through inhibiting some enzymes like pepti- dases. References 1) Y. Matsuoka, H. Seki, K. Kubota, H. Ohtake and Y. Hagiwara (1983), Japan. J. Inflamm. 3, 602. 2) BLL. van der Warden (1940), Arch, Exp. Path. Pharmak., 185, 389. 3. H. Yamasaki, ct al (1967), Polia Pharmacol. Jpn. 63, 302.Chart 1 ae aa leaves (25 kg) (1 kg) suspended in 50mM phosphate buffer (pH 7.5) ifuged at 10,000 x g for 20 min. Supernatant Hractionated by 40-100% sat. (NH,),SO, Precipitate lissolved in buffer and dialyzed DEAE Sephadex A-50 column cluvinalography [Passed solution 24D Sephadex G-100 column chromatography Di-G1 Table! Acute toxicity of P4-D1 and D1-G1 in mice. LD50 (mg/kg) P.o ip. sc. iv. Padi > 2,000 1,120 > 4,000 285 DLG1 2,830 > 4,000 224 P.o. : oral, i.p. : intraperitoneal, s.c. : subcutaneous, i.v. : intravenous08. ie Increase in Paw Volume on os: a5 Inerease in Paw Volume on Um zie eamoloeeny) Time (hr) Fig.1 Effects of intravenously administered P4-D1 and D1-G1 on carrageenin-induced edema of the rat’s paw. Each point and the vertical bar represent the mean and S.E. from 6 experiments. The ordinate denotes the increase in the paw volume. The abscissa denotes the time after the carrageenin injection. Symbols represent (O—O) Control, ( 4:--:-: ) P4-D1 0.01mg/kg iv., (a neeee@) PADI O Ima /leg i, (eee ) DG O.0img/keg os ( 7) D1-G10.1mg/kgi.v, . *: P< 0.05 and **: P< 0.01, significantly different from the control group.Increase in Paw Volume Fig. 2 Effect of aspirin and barley SOD on carrageenin-induced paw edema of rats. Each point and vertical Lar represent the mean value and standard error of 6 experiemtns. Symbols represent, 0: control, :@ aspirin 100mg/kg, Abarley SOD 0.2 mg/kg (i.v.), «heat-treated SOD 0.2 mg/kg (i.v.).APPENDIX 2 Studies On The Effects Of Green Barley Juice On The Endurance And Motor Activity In Mice* Kazuhiko Kubota and Nobuyoshi Sunagane, Faculty of Pharmaceutical Sciences, Science University of Tokyo, 12, Ichigaya-funagawara-machi, Shinjukwku, Tokyo, 162. Japan. “This paper has been reported in The 104th Annual Congress of Pharmaceutical Society of Japan, held in Sendai in 1964 MAIntroduction ‘ince the daily intake of green barley juice purportedly has an effect uf increasing stamina in humans, in the present work authors aimed at demonstrating whether such stamina increasing effect of the green barley juice could be also found in experimental animals. ‘The green barley juice has been well established to contain abundant nutrients such as protein, minerals and vitamins which may increase motor capaaity or activity in animals, However, it is also possible that the juice may contain unidentified substances which can enhance stamina in animals and humans.Materials and Methods 1. Matenals Green barley juice obtained from young barley leaves was brought to powder at a low temperature by using a spray drier. The dried green barley juice (DGBJ) used in this work was offered by Japan Pharmaceutical Development Co., Ltd., Osaka, Japan, which manufactured it in the method described above. ‘Two kinds of solid foods for mice, which contained 2% or 4% of the DGBJ in the standard food for mice, were prepared by Nippon Kurea Co., Ltd. During the processing of the solid foods, the temperature was maintained lower than 50° C. The foods were stored in a room kept at 5 + 1° C. 2. Motor activity test Male ddY mice weighing 11 to 13 g were housed in a room keptat24+1° C and 55 + 5% in the relative humidity. A control group of mice (16 mice) were fed on the standard food, and a test group of mice(16 mice) were fedon the food containing 2% of DGBJ (2% DGBJ food) for 15 days at the same period of time. A mouce fod on the standard food was placed ina wheel cage, and a mouse fed on the 2% DGBJ food in another wheel cage, and both the cages were left in the same room. The recording of the number of the revolutions of the wheel cage induced by the mice was started 20 minutes after putting the mice in the wheel cage and continued for the following 2 hours. 3. Endurance test The method of feeding the mice was similar to that described above in the motor activity test, but the test groups of mice were fed on 2%. DGBJ and 4% DGB3 foods. On day 15 of feeding, 8 mice of the control group, and the same number of mice of the test group, were placed at the same time on the treadmill belt, running at a rate of 1 km/hr, having an uphill slope of 17°, and they were forced to keep running with stimulation by electrical shock of 80 volts. The treadmill apparatus was designed by us especially for this experiment. The duration of forced running of mice in the control and test groups was compared. 4, Statistical analysis The statistical significance of difference was assessed by Student’s ttest.Results 1. Effects of DGBJ on motor activity in mice ‘The numbers of the revolution of the wheel cage induced by the control mice which were fed on the standard food and by the test group of mice fed on 2% DGBJ food were shown in Table 1 and Fig. 1. The DGBJ-fed mice revolved the wheel cage more than the control mice. The difference between the numbers of revolution of the wheel cage im the lwo groups was statistically significant. 2. Effects of DGBJ on endurance in mice ‘The duration of forced running in the test group of mice fed on the DGB food was dose-dependently prolonged. as compared with that in the control group of mice fed on the standard food. The momentum (body weight multiplied by duration) in the mice fed on 4% DGBJ food was significantly larger than that in the control mice (Fig. 2). Discussion The DGBJ-added food significantly increased the motor activity in mice. Motor activity of mice can be increased by central stimulants like caffeine and amphetamine. However, DGBJ-fed mice showed no appreciable stimulation in the central nervous system when their behaviors were compared with those of control mice, indicating that the increase in the motor activity in DGBJ-fed mice was not induced by central stimulants. This view is also supported by the fact that more increase in the motor activity was found during the period of 1 to 2 hours (Fig. 1). In general, it is not easy to enhance the endurance of animals by drugs. According to Saito (1973) (1) and Bombardelli (1983) (2), certain constituents from Panax ginseng (a Chinese herb) showed antifatigue activity in experimental animals. Although the data were not shown here, preliminary experiments revealed that 7 days feeding of mice by DGBJ-added food failed to significantly prolong forced running timein mice, but 15 days feeding could prolong it. ‘The average body welght of mice fed on DGBJ food was larger than that of mice fed on the standard food (Fig. 2), indicating that DGBJ might be better than the standard food from the nutritional point of view. Whether the effect of DGBJ to increase endurance and motor activity in mice is due to some special substances in the DGBJ remains unclear at present. The identification of the substances responsible for the effect awaits further accumulation of experiments and information. References 1) H, Saito, Metabolism, Vol. 10, p. 556 (1973), Nakayama Shoten. 2) E. Bombardelli, The Ginseng Review, Vol. 1, No. 1, p. 59 (1983). 3Fig. Effect of Green Barley Juice on Motor Activity of Mice in Wheel Cage Test, 109 200 ow su00%) contro. FT whe GB SSG [0010 CONTROL 12hr BEB SS pH 05” ‘Significantly different from CONTROL, p<0.05. Fig.2 Effect of Dried Green Barley Juice(DGB4 on Endurance of Micein Forced Treadmill Running Test. duration of running 0 300 ae 150 (min) 0 ———— ——————————— body weight ow 2% te) on a om om * ° B o Biekm ‘momentum * Significantly different from 0%, p 0.05. 0%: Standard food, 2 and 4% Standard food containing 2 and 4% of DGBJ. Momentum: Duration x body weight.Table 1 . Effect of Dried Green Barley Juice(DGB¥) on Motor Activity ofMicein Wheel Number of revolutions 1-2hr 199.0 + 64.9 22.1 + 57.1 49.8 21478" Cage Method O-1br ‘Standard food fed mice (A) 368.8 + 61.3, 2% DGBJ food fed mice (B) 463.7 #5 D/A ™ 100 (71) im paired mice 906.0 + 120.3" Each value represents the mean + S.E. of 16 experiments. * Significantly different from 100%, p<0.06.APPENDIX 3 Studies on the Constituents of Green Juice From Young Barley Leaves Anti-ulcer Actwity of Fractions from Barley Juice* Hidetoshi Ohtake, Hideo Yuasa, Chiseko Komura, Tetsuji Miyauchi, Yoshihide Hagiwara® and Kazuhiko Kubota” a) Research Laboratory, Japan Pharmaceutical Development Co., Ltd. 1-1-26, Mitsuya Minami, Yodogawa-ku, Osaka, 532, Japan. b) Faculty of Pharmaceuticl Sciences, Science University of Tokyo, 12 Ichigaya- funagawara-machi, Shinjuku-ku, Tokyo, 162, Japan. ‘This paper has been accepted for publication in Vakugaku2asshi, issued from Pharmaceutical Society of Japan. "AUIntroduction ‘The fresh green juice from vegetables has been widely accepted Wu be useful for keeping humans in good health. There have been a few reportson the effects of such green juice which were studied from the nutritional and clinical points of view (1 and 2). However, no such report can be found as pharmacologically demonstrating the evidence that the green juice could be available; for example, for preventing peptic ulcer formation. In the present paper, the authors are showing the fact that the green juice from barley (Hordium vulgare L. var. nudum Hook) can prevent stomach ulcer formation in rats. Materials and Methods The green juice was vblained frum barley which was grown in Oita prefecture in Japan and mowed when the grass height reached 15-25 cm. The green juice was then brought to dry powder by using a spray drier. The dry powder (GM-SD) was dissolved in distilled water and fractionated as shown in Chart 1. Each fraction from GM-SD and atropine sulfate(Nakarai Chem Tnd_, Japan) were suspended or dissolved in physiological saline solution. Aspirin (Sanko Pharmaceutical Co., Ltd., Japan) was suspended in CMC- Na solution. Male Wistar strain rats (Nihon Kurea) were used. 1. Stomach ulcer of Shay rats (3) Rats weighing 220 to 230 g were fasted for 48 hours and laparotomized thereafter under ether anesthesia and the stomach exposed. After ligation of the pyloric region, each fraction from GM-SD was injected into the duodenal lumen. The rats were left for 12 hours and then their stomach was excised. The stomach was cut open along the greater curvature. The longitudinal and transverse diameters of the ulcers formed in the stomach fundus were measured microscopically. The size of ulcer was expressed by ulcer index which was obtained by multiplying the two diameters. Product(mm?) 1-6 7-12 13-18 18:24 24 < or perforation UleerIndex 1 2 3 4 5 2. Stress-induced ulcer According to the method devised by Takagi and Okabe (4), rats fixed in a stress cage were immersed in water kept at 23 +1°C to the pectoral level of therats. After 8 hours immersion, therats were anesthetized with ether and the stomach was excised. The stomach was filled with 10 ml of 1% formalin solution and immersed in 5% formalin solution for 10 minutes. Then, the length of every ulcer formed in the glandular stomach mucosa was measured and stimmed np ‘The summation of the uleer length was used as the ulcer index, Green juice fractions were administered to rats in the oral route 1 hour before the exposure to stress.3. Acetic acid induces uleer (5) Rats fasted for 24 hours were laparotomized under ether anesthesia aud (he stomach was exposed. Twenty percent acetic acid (0.02 ml) was injected subserosally to the boundary region between the corpus and antrum of the stomach. Afler this procedure, the abdominal incision was sutured. On and after day 2 of the operation, the fractions from GM-SD were orally administered to the rats once a day and the stomach of the rats were excised on day 11. Thestomach fixed with one percent formalin was cut open along the greater curvature. The ulcer index was obtained by the same way as that for Shay rat ulcer. 4, Aspirin-induced ulcer The stomach of the rats fasted for 24 hours was exposed under ether anesthesia and ligated at its pylorus. After suturing the abdominal incision, 100 mg/kg of aspirin which has been suspended in 1% CMC-Na solution was orally administered to the rats. The stomach of the rats was excised 7 hours after the aspirin administration. The length of every ulcer formed in the glandular stomach was measured after the fixation of the stomach with 1% formalin and summed up for obtaining the ulcer index. The barley fractions were administered orally to the rats 1 hour before the pyloric ligation. 5. Gastro-intestinal absorption of aspirin (7) Rate received an oral administration of 500 mg/kg of barley fractions and another oral administration of 100 mg/kg of aspirin at 30 minutes thereafter. One hour after the aspirin was administered, blood samples were collected from the carotic artery of the rats with the addition of heparin. One ml of water, 0.5 ml of 6N HCl, and 30 ml of 1,2-dichloroethane were added to 0.2 ml of the serum separated centrifugally from the blood camploc. Tho mixture wac contrifugod and the cupornatant wae heated for 5 minutes in a water-bath kept at 95 C. After cooling of the supernatant, the intensity of the fluorescence, excited by a light of 303 nm wave length, was measured at the wave length of 412 nm. 6. Measurement of gastric acid ‘The rats, fasted for 24 hours, were anesthetized with ether and the pyloric region of the stomach was ligated. The stomach was excised 5 hours after the ligation under ether anesthesia. The gastric juice collected from the stomach was centrifuged and the supernatant was measured for the volume, pH, free and total acid, and pepsin activity. The pepsin activity was assayed according to hemoglobin method (8). Barley fractions were orally administered to rats 1 hour before the pyloric ligation. 7, Statistical analysis Statistical significance was assessed by Student's t-test.Results 4, Effect of GM-SD and the fractions from GM-SD on experimental ulcers 1) Effect on ulcer of Shay rat The anti-ulcer effect of GM-SD and the fractions from GM-SD which were given to Shay rats at an oral dose of 500 mg/kg was shown in Table I. GM.SN suppressed ulcer formation in Shay rate by 24%, as compared with the control, but the anti-ulcer effect was not statistically significant. The anti-ulcer effect of other fractions was not significant either. 2) Effect on stress-induced ulcer As shown in Table II, all fractions, except for a fraction GM-S. exerted significant anti-ulcer effect in the stress-induced ulcer. GM-L which is water-soluble, low molecular fraction, exhibited the highest activity. 8) Effect on acetic acid induced ulcer As shown in Table III, all fractions except for GM-Sup significantly accelerated the healing of the acetic acid induced ulcer and GM-Sup again showed the highest healing activity. 4) Effect on aspirin-induced ulcer GM-Pptand GM-SD revealed no significant anti-ulcer effect, but three other fractions, GM-P, GM-Sup and GML did reveal highly significant. anti-uloor activity (Table IV). 2. Effect on aspirin absorption and gastric secretion " Allof the fractions exerted no significant effect on the aspirin absorption, the gastric secretion of acid and pepsin activity. Discussion GM-Sup is composed of water-soluble substances, GM-Ppt of chloro- phyll, denatured protein, polysaccharides, water-insoluble substances, and GMP of water-soluble substances like amino acids, while GM-S is mainly composed of water-soluble high molecular snhstances. Chlorophyll has been claimed to have anti-ulcer activity (9), having been used for clinical purpose. However, GM-Ppt, that contains a large amount of chlorophyll, exerted no anti-ulcer action in Shay ulcer. No other fractions were found to be effective in Shay ulcer. In contrast, in the stress- induced ulcer all of the fractions except for GM-S showed significant anti- ulcer activity at an oral dose of 500 mg/kg (Table 11). All fraction except for GM-Sup significantly accelerated the healing of acetic acid induced uleer (Table IID). The aspirin-induced uleor was oignifi- cantly suppressed by three fractions, GM-Sup, GM-P and GML (Table IV). As noted above, GM-L contains amino acids, but the anti-ulcer activity of the fraction seems to be equipotent to, or a little higher than, L Glutamine (10).As shown in Tables V and VI, all fractions from GM-SD did not affect the gastric serretion and they were all ineffective in Shay uleer, suggesting that they do not reveal anti-ulcer action through inhibiting the gastric secretion or the pepsin activity. They did not inhibit aspirin absorption, indicating that their anti-ulcer action observed in the aspirin-induced ulcer is due to any other mechanism than the inhibition of aspirin absorption. On the basis of the results obtained in the present work, it is unlikely that green barley juice exerts its anti-ulcer action via affecting the attacking factors of ulcer, but suggested that the effects on the movement and blood flow in the stomach and the defense ability of the stomach mucosa are associated with its anti-ulcer activity. In the present work, it was shown that the green barley juice contained 8 variety of substances which rovealed oignificant anti-ulcer activity in various experimental ulcers and a fraction composed of low molecular weight, water-soluble substances had higher anti-ulcer activity. We are now attempting to identify the substances in the green barley juice which are responsible for the anti-ulcer action and to elucidate the mechanisms of their anti-ulcer action. References 1) Y. Hagiwara (1975), The Food Industry, 18 (8), 73. 2) T. Muto (1977), Shinyaku to Rinsho, 26, 983. 3) H. Shay, S.A. Komarov, S.S. Fels, D. Meranze, M. Grvenstein and H. Spilet (1945), Gastroenterology, 5, 43. 4) K. Takagi and S. Okabe (1968), Japan. J. Pharmacol., 18, 99. 5) K. Takagi, S. Okabe and R. Saziki (1968), Japan. J. Pharmacol., 19, 418. 6) S. Okabe, K. Takeuchi, K. Nakamura and K. Takagi (1974), Japan. J. Pharmacol., 24, 263. 7) T. Hata, N. Masuda, 8. Iwasaki, K. Koike and H. Kasahara (1979), Shindan to Chiryo, 67, 359. 8) MLL. Anson (1938), J. Gen. Physiol., 22, 79. 9) Y, Sato, T. Kuraishi, F. Ishibashi, H. Aratsuka and F. Ishida (1954), Rinsho Shokaki Byogaku, 2, 663. 10) K. Takagi, K. Takeuchi and K. Nakamura (1974), Japan. J. Pharmacol., 24, 357.Chart 1 GMSD Uke) iagolved in distilled water and centrifuged at 10,000 x for 20 min Precipitate Supernatant (GMSup) Iyephiliced setusaied wil (E1901 and centrifuged at 10,000 x g GM-Ppt (3008) for 20 min Precipitate Supernatant dialyzed to water alyzed to water tnd tyophilined GMP (200g) Dialysate Nomdialyaate 90% methanol lyophilized was added fand filtered Filtrate GMS.(2005) GML (2008) Tabie 1 Effect of Fractions from Green Juice of Young Barley Leaves on Stomach Ulcer in Shay Rats ‘Treatment Number of Dose Ulcerindex Inhibition P ‘animals (mg/kg) (mean? SE) 6 Control 8 37406 GMmsp* 6 500 28207 43 NS GM-Sup" 7 500 41-06 108 NS. GM-Ppt 6 500 30212 9° ONS. GMP 6 500 35208 54 NS. GML 6 500 37208 00 NS. Atropine sulfate 8 10 1403 2 0.02 ienificant‘Table I Effect of Fractions fram Green Juice of Young Barley Leaves on Streae induced Stomach Ulcers in Rate ‘Treatment Number of Dose Uleerindex Inhibition animals (mg/kg) (mean #5) * Control 9 216240 am-sp* 8 500 erev 486 © «0.025 GMSupt 8 500 120222 533 «0.01 GMPptt 9 500 16.7219 35 «005 Control 7 06235 GMSup" 7 500 249223 387 -<0.005 MP 7 +500 241238 46 = 001 cMs* 7 500 265240 1 NS. GML 7 500 176209 567 001 Atropine sulfate 6 1 area 33 001 Control 0 905 22.1 GMP 10 100 237230 Nea 10 500 147233, 51801 om 10 100 16234 390 «0.05 10 500 131228 5200.01 *.Qee Choate 1 NS.: Statistically no significantTable III Effect of Fractions from Green Juice of Young Barley Leaves on Acetic Acid-induced Stomach Uleersin Rats ‘Trentment Number of Dose Uloeringex _—Inibition =P ‘animals (mg/kg) (mean #3E) 3) Control 8 500 946278 GMD" 8 500 109225 685 «0.025 GMSup* 8 500 v4e27 #7 NS. GM-Pptt 8 500 151435 564 0.025 ops ® 00 every zag 00 ML 7 5 140813 395 0.0 Atropine sulfate 5 5 140813 593 01 Control 6 452435 GMP 5 100 250331 47 005 5 300 20323 513 «0.025 cML 5 100 205125 o6 aur 6 300 162215 642 © 001 +See Chart NS.: Statistically no significant Table IV. Effect of Fractions from Green Juice of Young Barley Leaves on Aspirin-induced Stomach Ulcers in Rats ‘Treatment Number of Dose Uleerindex Inhibition P animale (mg/kg) (means SE) *) Control 8 336241 GMSD" 7 +500 mast 33 (NS. GMsup" 8 ‘500 19.1226 551 -0.005 GMPpt 8 500 65241 2 NS. Control ° aeazaa GMP 8 500 102227 637 <0.005 GML 8 500 16223, 587 «0.005 *:See Chart I NS: Statistically no signi santTable V fect of Fractions frou: Green Juice of Young Barley Leaves on Gastro: intestinal Absorption af Aspirin in Rate ‘Treatment Dose Plasma salicylate Absorption (mg/kg) concentraton (amg)"? « Control 148408 100 GMsb 500 129206 872 GMSup 500 145208 980 cmp 500 47207 aa GML. 500 187207 926 Fractions from green juice of young barley leaves were given orally 30 :nutes before the administration of aspirin (100 mg/kg) 4) Each value represents the mean S.E. of 10 rats. ‘Table VI Effect of Fractions from Green Juice of Young Barley Leaves on Gastric Secretions in Pyrolus-igated Rate, Treatment Dose Volume pH ity Pepsin activity (mg/ke) (mld (uEgml) (unit/m) Control 10013 2452010 723+ 80 1013s 74 4B FISH GMsb 50092209 © 2442004 «72+ 5S 1088+ BO—_1527 2 169 omsup aw LUEUs = Zs6F0US TSF ABTS BT AT 248 GMPpt 500 1080.7 2262003 785 5G 1045+ 31979 + 156 GMP 500 120204 © 2.252005 © BATz114 —1OL3E117 1375435 Atropine 5 65220 2792019 5252 91 1050 87 —oT7#3I sulfate Bach value represents mean + of SE. of 6 rats + Significantly different from control (P<0.025)APPENDIX 4 Studies on the Constituents of Green Juice From Young Barley Leaves Effect on Dietarily Induced Hypercholesterolemia in Rats* Hidetoshi Ohtake, Suzuyo Nonaka, Yoshiko Sawada, Yoshihide Hagiwara, Hideaki Hagiwara and Kazuhiko Kubota? 4@) Research Laboratory, Japan Pharmaceutical Development Co., Lid. 1-1-26, Mitsuya Minami, Yodogawa-ku, Osaka, 532, Japan. ’) Hagiwara Institute of Health, 1173, Maruyama, Asazuma-cho, Kasai, Hyogo, 679-01, Japan. ¢) Faculty of Pharmaceutical Sciences, Science University of Tokyo, 12, Ichigaya- funagawara-machi, Shinjuku-ku, Tokyo, 162, Japan. ‘This paper has been accepted for publication in Yakugaku-zasshi, issued from Pharmaceutical Society of Japan, HYIntrodnetion In Japan today, the increased intake of fatty diets and pollution in environment have enhanced more incidence of adult diseases such as arteriosclerosis, cancer, obesity and hypertension. Cardiovascular diseases like arteriosclerosis are well known to closely associate with the impaired metabolism of cholesteral Inthe present paper, authors attempted to find some substances having hypocholesterolemic activity from the green juice from young barley leaves and identify them, since the authors have found various interesting sub- stances showing activities such as potent anti-inflammatory action (1) and antiulcer action (2) from the green barley juice.Materials and Methods 1. Isolation of substances having hypocholesterolemic activity from green barley juice The barley (Hordium vulgare L. var. nudum Hook) used in this study was grown in Oita prefecture in Japan. Fifteen to twenty-five centimeter high grass was mowed and green juice was obtained by compressing it with stainless steel rollers. The fresh green juice obtained wae brought to powder at low temperature by using a spray drier. The dried barley juice (GM-SD) was subjected to the fractionation to various samples and isolation of crystals as shown in Chart 1. Hexacosyl alcohol was isolated from Sample IV. Sample V is mainly composed of 8-sitosterol and containe a smal] amount of hexacosyl alcohol. Sample VI contains a small amount of f-sitosterol and unidentified organic compounds. The materials isolated from the barley juice and other reference drugs were suspended in olive oil or added to a high cholesterol diet at a concentra- tion of 1% and administered orally to rats or mice. The reference drugs used were nicotinic acid (Nakarai Chem. Ind., Japan), soysterol (Morishita Pharmaceutical Co., Ltd.) and 8-sitosterol (Merck & Co., Ine.). ‘The total cholesterol, free cholesterol, HDL-cholesterol, 8-lipoprotein and triglycerides were quantified by using kits (Wako Chemicals, Japan) for clinical examinations. 2. Diets and animals ‘The high cholesterol diet (HCD) was prepared by modifying the diet devised by Tensho and his co-workers (3) (Table I). Four week old male Wistar strain rats weighing 100 g were used. GM-SD, GM-Ppt, Sample! and nicotinic acid were suspended in olive oil and orally-administered to rats once a day for 6 days. The control group of rats received oral olive oil. 3. Determination of cholesterol in the serum and liver The amount of serum lipids was measured before the initiation of feeding with HCD and on days 3, 6 and 9 of feeding. About 0.6 ml of blood was taken out from the carotid vein of rats which had fasted for 16 hours (water was taken ad libitum) and the serum cholesterol in the blood was determined. The liver of the rats was excised on day 6 of feeding and 1 ¢ portion of the liver was homogenized with addition of 20 ml ethanol. The total cholesterol in the liver was determined by method of Abell (4). 4. Acute toxicity test Male ICR strain mice weighing 25-30 g received an oral administration of the test samples suspended in olive oil and fed for 7 days on the normal mouse food thereafter. 5. Statistical analysis Statistical significance was assessed by Student's t-test. 2Results 1. Hypercholesterolemia induced by HCD The feeding of rats by a HCD, devised by Tensho and his co-workers, caused good hypercholesterolemia but also produced adverse effects like diarrhea and piloerection in the rats. On the other hand, the HCD prepared by us (Table 1) induced favorable hypercholesterolemia, as shown in Fig. 1, without accompanying any observable adverse effects. 2 Anti-hypercholeste:vlemic effect uf the fractions from green barley juice Water-insoluble fractions from GM-SD were found to exert an anti- hypercholesterolemic effect. Further examinations revealed that Sample I. ahexane soluble fraction, had an anti-hypercholesterolemic effect as shown in Table I, Sample I exerted dose-dependent anti-hypercholesterolemic sffect Table ID), and showed higher activity than soysteral on day 9 (Table The entity responsible for the anit-hypercholesterolemic activity in the fractionation Sample I was found in Sample III (Table IV). The fractiona- tion of Sample III gave two fractions that showed anti-hypercholesterolemic effect (Table V). Sample VII or Crystal I, isolated from Sample IV also showed unti-hypercholesterolemic effect (Table V). The total cholesterol in the liver was lowered by Samples V and VII (Table VD. 3. Structural analysis of anti-hypercholesterolemic substances isolated from barley juice Silica gel column chromatography (Chloroform : Benzene : Ethylacetate = 7:2:1) gave Crystal I, colorless plates that showed a melting point of 78°C after recrystalization from ethylacetate. Nujol Analytical data of Crystal I were as follows: IR max cro; 3320 (OH). H-NMR (CDC1s)6; 1.15-1.46 (5H, m), 3.72 (2H, t.J=7 Hz). MS, 8.82 (M , weak), 364 (M*-H20), 336 (M*-H20-C2H). Anal. Caled. CosHs.O ; C, 81.60; H, 14.22. Found: C, 81.62; H, 14.53. On the basis of these data, it was concluded that Crystal I was n- hexacosyl alcohol having the molecular formula of CHs (CH2)a-~CHz0H. Sample V gave two kinds of crystals on silica gel column chromatog- raphy. One of them was identified to be hexacosy] alcohol and the other gave colorless needles having a melting point of 137-139°C after recrystali- zation from ethylacetate. The latter crystal was found to show exactly the same analytical characteristic as that of standard B-sitosterol produced by Merck & Co,, Inc. 4. Acute toxicity The oral administration of GM-SD, GM-Ppt and GM_R in a dose of 10 g/kg revealed no detectable toxic effect in mice (Table II).Discussion The HCD used in the present work was evidenced to be effective to produce hypercholestrolemia in rats without causing any adverse effects. ‘The serum cholesterol levels in rats fed on HCD were variable (Table II-V), However, this is because the HCD intake by rats was done spontaneously. ‘The water-insoluble fraction from barley juice was found to have anti- hypercholesterolemic effect in rats fed on HCD and significantly lowered the corum cholesterol level in the rate. Sample I extracted from the water. insoluble fraction by n-hexane had twice the anti-hypercholesterolemic activity of soysterol, which has been marketed as an anti-hypercholes- terolemic agent in Japan, and its acute toxicity was shown to be very low (Tables I] and III). In the present study, n-hexacosy] alcohol or ceryl alcohol and -sitosterol, both of which have anti-hypercholesterolemic ‘activity, were isolated from the barley juice and identified. Ton g of Sample I was found to contain 1.2 g of hexacosyl alcohol and 1 g of A-sitosterol in it. Hexacosyl alcohol was demonstrated by us to have anti-hypercholes- terolemic activity for the first time. It is of special interest that hexacosyl alcohol has equipotent hypocholesterolemic activity to that of B-sitosterol ‘and slower onset of the activity than 8-sitosterol. The hypocholesterolemic action of B-sitosterol has been reported to be due to the inhibition of the intestinal absorption of cholesterol and the acceleration of catabolism of cholesterol to bile acids (5.8). Although the mechanism of hypocholesterole- mic action of hexacosyl aleohol remains unclear at present, it may exert its effect through similar ones to those of B-sitosterol. However, its slower onset of effect suggests possible involvement of other mechanisms. Attempts of elucidating the mechanism of the hypocholesterolemic action of hexacosyl alcohol is under way. ‘The present work suggests that green barley juice may be very useful for preventing humans from vascular diseases associated with hypercholes- terolemia. References 1) ¥. Matsuoka, H. Seki, K. Kubota, H. Ohtake and Y, Hagiwara (1983), Japan. J. Inflamm. 3, 602. 2) H. Ohtake, H. Yuasa, C. Komura, T. Miyauchi, Y. Hagiwara and K. Kubota (1985), ‘Yakugaku-zassht in press. 3) A. Tensho, A. Shimizu, T. Takenawa, H. Kikuchi and M. Rokujyo (1972), Yakugaku- zasshi 92, 879. 4) L. L, Abell, B. B. Levy, B. B. Brodie and F.E. Kendall (1952), J. Biol. Chem. 195, 357. 5) S. Nakayama, K. Negishi and K. Sakamoto (1981), Folia Pharmacol. Jpn. 78, 191 6) G, Salen, E, H, Ahrens Jr,, and S. M. Grundy (1970), J. Clin. Invest. 49, 452. 7) H. Kaneda, S. Tokuda and N. Shibukawa (1971), Foods and Nutrients, 19, 439. 8) K Kornda,V Sugiva, T Oeamma, R Mignehi,T Nozawa, S Satoh, T. Takeda and T. Matsui (1971), Kiso to Rinsho, 5, 1019.Chart I GMD (1kg) suspended in 10 of water, and centrifuged at 10,000 x g for 20 min. Supernatant Precipitate [— lyophilized GM-Ppt (4508) extracted with n-hexane for 30 min. Residue Hexane extract GMR concentrated Sample I (10g) silica gel column chromatography column size: 9 x 100em silica gel - 720 g solvent : Benzene: Ethyl Acetate ay CTTTiTr ty fraction Ly jesiess am SampleII Sample III (6.3g) Gee io silica gel column chromatography ‘as noted above for Sample! SampleIV Sample V Sample VI (2.38) 4g) ley Sample VI1(1.2g) GaFtAdT or hexacosy! alcoho)TableI Composition of High Cholesterol Diet Components Tensho's Modified (%) (%) - Cholesterol 2 1 Cholie acid 1 L Casein 20 3 Sucrose 49 50 Hydrogenated fat 12 10 Cellulose 4 5 Vitamins & Minerals 4 4 Dried fish powder 8 4 Figure 1 Change in Serum Total Cholesterol Levels of Rats fed on a High Cholesterol Diet 1000 g 8 400 Serum total cholesterol (mg/dl) 8 0 a 6 9 Duration of Treatment (days) —o-: ahigh cholesterol diet shown by “modified” in Table I ©: anormal diet (CE-2, see text)Table Il Effect of Young Barley Leaves Fractions on Serum Total Cholesterol Levels of Rats Fed on a Cholesterol Diet Treatment Dose Serum total cholesterol mg/dl) Inhibition)" LD (erke Dayé (eri) Control 52622625 Msp 3 499.1 +5066) >10 GM Ppt 5 450240048) -10 Sample 1 4078 #382028" >8 GM 1 454724391139) >10 Nicotinic acid 03 s30 2671 G75" 4) Each value represents the mean = S.E. of 6 rats. *: P0065, significantly different from the control group. Table III Effect of Young Barley Leaves Fractions on Serum Total Cholesterol Levels of Rats Fed on a Cholesteral Diet mg/dl) (inhibition ®) Day ‘Treatment Dose —_Seru Control 61202265. Sample I m% 3102 2373.168)" 05% 564.8 93.8179) 0.25% 6672+ 1018(-88) 6089» 82.7(10.7) Soysterol 1% 6 228.1 (9.7) 524.3 251.1 (23.3 4) Each value represents the mean + S.E. of 6 rats by Control rats were fed on a high cholesterol diet and other groups of rats on the high cholesterol diet added sample I or soysterol in the concentrations indicated in the table + P< 0105 and *™P <0.005, significantly different from the control group,Table IV Effect of Young Barley Leaves Fractions on Serum Total Cholesterol ‘Levels ot Kats Ned on a Cholesterol Let ‘Treatment Serum total cholesterol (mg/dl) (Inhibition #8)" Days Day9 Controt 2500132 69032610 ‘Sample! 17642131296)" 43802 45.1 (96° Sample II 198.0248 (208) 502.061.8278) ‘Sample I L420 22940428)" 478.02 56.7 31.0" Besivosterl 17902351250) 348,02 70.0160.) Controls rats were fd on a high cholesterol diet and other groups ofrats ‘on the high cholesterol diet added Sample I-III or B-sitosterol at a ‘concentration of 1%, 4) Bach value represents the mean S.E. of rats. * P<0.05 and *: P< 0.005, significantly different from the control group. Table V sBttec ot Young Sarley Leaves Fractions on Serum otal Unolestero! Levels of Rats Fed on a Cholestero! Diet Trestsnent Serum total cholesterol mg/d (inbivion 5)" Dayo Day3 Day6 Days) Sample 106227.1 446.023.8241) 778.4 48.9(94.5)" SampleIV 109861 5579+ 29.4(1.6) 863.9. 33.7(25.4)" SampleV 9972115 49472349260" 895.795.9003)" Sample VI 12692111 56782 403(33) 1051.0 #81.7(11.3) Bsivosterol 11992128 3290198 440 65782 41.9144.) Control 629282 656.6192 79122164 110682796 Sample 662252 951.4 #40.3(159) 618823170154)" 739.7 268.4268" Sample VII 6352132 6646#103(-1.4) 696.32 248(4.9) 1796.1 + 35.5 (20.9)" Paitoeterol —703+190S834e161 (141) 6020428 (I7 TINY 748.6 + 50.0(25.6)" Control rats were fed on a high cholesterol diet and other groups of rats on the high cholesterol diet sdded Sample I'T-VTI or fitoateral at a concentration of 1%. 1) Each value represents the mean *S.E. of rats. ‘© P<0.05 and **: P< 0.005, significantly different from the control group.Table VI Effect of Young Barley Leaves Fractions on Liver Total Cholesterol Levels of Rats Fed on a Cholesterol Diet for 6 days Preatnent rot met might Lives wet / Duly Dn len onan & Control 152040) 53203 180285 22204 Sample! 78x04 54202 179268 22205 Sample II 69503 51203 168 +57 2ars08 Sample IIL 72202 54201 16767 23205 Sample 8103 59:02 189277 227203" Besitosterol 75202 33:01 165 +58 29:04" Control 74202 35201 tet 233 aa sor Sample 1V 17203 57403 169 +52 219203 Sample ¥ r4203 34203 133 #78 le7s05" Sample VI 10:04 51203 189 +104 243 +04 Pesitostrol 72:08 50203 108 +31 193204" Control rats were fed on a high cholesterol dit and other groups ofratson the high cholesterol diet added Sample I ~ VII or B-sitosterol at a concentration of 1%, 2) Rach value represents the mean © S.E. of € rate fad a high halastere! *: P<005 and **: p< 0.005, significantly different from the control group.APPENDIX 10 Inhibition of Malonaldehyde and Acetaldehyde Formation from Bllod Plasma Oxidation by Naturally Occurring Antioxidants Miyake, T., Shibamoto, T., J. Agric. Food Chem. 1998. 46: 3694-3697Sgr: Food Cham, 198, 40, 9094-2097 Inhibition of Malonaldehyde and Acetaldehyde Formation from Blood Plasma Oxidation by Naturally Occurring Antioxidants ‘Takashi Miyake and Takayuki Shibamoto* + Department of Environmental Toxicology, University of California, One Shields Avenue, ‘Davis, California 95616 ‘The inhibitory effect of 2”-O-glycosylisovitexin (2’-0-GIV), 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF), L-ascorbie acid (vitamin C), and 4,4isopropylidenedithio)bis(2,6-di-tert-butylphenol) (probucol) on malonaldehyde (MA) and acetaldehyde formation from horse blood plasma oxidized ‘with Fenton's reagent was determined by gas chromatography. 2’-O-GIV, DMHF, and L-ascorbic acid inhibited MA formation at a level of 100 nmol by 60, 22, and 22%, respectively; probucol did not show any inhibitory activity on MA formation. Improved inhibitory activity toward MA formation was observed when 8”-0-GIV or DMHP was mined with bie acid in equal amounts Approximately 80-90% of acetaldehyde formation was inhibited by 300 nmol of 2”-0-GIV and probucol, whereas DMHF required 1000 nmol to exhibit the same level of inhibition. Keywords: Molonaldehyde; acetaldehyde; blood plasma; natural antioxidants Food components that possess euch biological char- acteristics as anticarcinogenicity, antimutagenicity, an- oxidative activity, and antiaging activity have recently received much attention as a third functional component of foods, after nutrients and flavor compounds. Among these functions, antioxidants found in natural foodstuffs have been investigated most intensively as constituents preventing diseases associated with oxidative damage. Active oxygen species such as hydroxy radicals may lead to many biological complications, including car- cinogenesis, mutagenesis, aging, and atherosclerosis (Halliwell and Gutteridge, 1989). For example, the accumulation of cholesterol esters is reportedly caused. by the oxidation of blood plasma lipids (Retsky et al., 1993). Therefore, the oxidation of blood plasma lipids wngly astociated with atherosclerosis and endo- dysfunction (Schmidt et al., 1994; Tesfamariam, 1994), ‘The mechanisms causing these diseases by lipid peroxidation are not yet well understood. One hypoth esis is that reactive carbonyl compounds, such as malonaldehyde (MA) and acetaldehyde, formed from finid peroxidation produc abaormal adduct with boy ‘substances, including DNA and RNA (Feinman, 11988; Esterbauer et al, 1991). In fact, a strong rela: tionship between atherosclerosis and MA and acetal- dehydes formed from lipid peroxidation has been re- ported (Glavind et al., 1962). Humans are constantly exposed to reactive oxygen species produced either by natural phenomena such as ultraviolet Light or by anthropogonic activitice (for example, automobile exhaust). Therefore, supplement- ing antioxidants to scavenge undesirable reactive oxy- gen species is very important to prevent in vivo oxida- five damage. Natural plant foodstuffs are one of the ‘most important suppliers of antioxidants (Namiki, 1990). ™ Author to whom correspondence should be addressed Ulephone (680) 752-4522; fax (690) 7529904; email {shibamoto@ucdavis.edu] ‘Son2t-8561(98}00318-5 CCC: $15.00 Published In the prosant study. the inhibitory effect of natural plant antioxidants 2”-O-glycosylisovitexin (2”-0-GIV), 2 Sidimethyl-schydrory-J2e-faranone (DMEF), and L-scorbic acid (vitamin C) on acetaldehyde and MA. formation from horse blood plasma lipids oxidized with Fenton’s reagent was determined by gas chromatogra- phy (GO). A commercial antiatherosclerosis drug, probu- col {4,4’\isopropylidenedithio)bis(2,6-di-tert-butylphe- holy] was also tested according to dhe same nerd. EXPERIMENTAL PROCEDURES Materials, Butylated hydroxytoluene (BHT), sodium dode- cyl sulfate (SDS), L-aseorbie aad (vitamin C), probueol, and ‘ovine serum albumin were purchased from Sigma Chemical Co, (St. Louis, MO), Benzalkonium chloride (BC) was bought from ICN Biomesicals, Ine. Aurora, OH). DMEF, 2-me- ylpyrazine, malonaldehyde bis(diethyl acetal), N-methythy- Grazine, and ferrous chloride were obtained from Aldrich ‘Chemical Co. (Milwaukee, WI). Protein dye (Coomassie Blue) ‘was parchased from Bir Rad Laboratories (Richmond, CA). 2 (O-GIV was isolated from young green barley leaves (Hordium vulgare L. var. nudum Hook) barvested 2 weeks after germi- ‘nation according to a method previously reported (Osawa et 1, 1908). The structures of ” 0 CIV, DMP, vitamin C, and ‘probucol are shown in Figure 1 ‘Standard stock solutions (1 mL each) for GC analysis were prepared by dissolving 2-methyipyrazine and 2,4,5-trimeth- Yithiazole in dichloromethane (10 mg/mL) and stored at 5 °C ‘until used. “Authentic N-methylpyrazole (NMP) was synthe- sized according to the method reported by Umano etal. (1988), ‘and authentic 2-methylthiazolidine was synthesized according te the method reported by Yanuhara and Shibamoto (1991) ‘Sodium malonaldehyde was synthesized according to a method previously described by Lacombe etal. (1990). Blood plasma was prepared from horse blood (20-year-old ‘male quarter heres) ky contrifigation at 5000 rpm fer A0 sain fat4°C. ‘The blood plasma was frozen on dry ice immediately afer preparation and stored at ~80 °C until use. Protein Analysis in Blood Plasma. The Coomassie Blue ye-binding assay (Bio-Rad Laboratories) was used to deter- ‘mine plasma protein concentrations (Bradford, 1976). A 10 _xL aliquot of appropriately diluted plasma protein was added to the dye reagent, and then absorbance at 594 nm was (© 1998 American Chemical Society fon Web 08/1171998Inhibition of Blood Plasma Oxidation by Natural Antosidants . tem OH x 8 om h0-CV o-d HOt Ho. wor HK Hoga cH.0# DMHF Vitamin C Probucol Figure 1. Structures of chemicals used in the present study measured versus a reagent blank, using un HP 64524 divde Gray UV epectophotometer. A standard curve using havine serum albumin was used to calculate plasma protein concen- tration Oxidation of Blood Plasma with Fenton's Reagent in the Presence or Abscnes of Tooting Chomieale. Blood plasma was oxidized according. to the method reported previ- frusly (Ichinose et al, 1989). An aqueous solution (5 mL) tontaining 30 of blood plasma (B60 xg of protein), 0.25 mmol Phosphate buffer (pH 74) 3 umol of ferrous chloride. 0.5 mmol of hydrogen peroxide. 0.75 mmol of potassium chlonde. and (0.2% of surfactant BC or SDS was incubated with various amounts of 2”.0.GIV. DMBF, vitamin C, probucol, 2”.0-GIV Svitamin ©. and DMEF «+ vitamin C for 16 h at 37 °C ina 2o-mL test tube, ‘The oxidation of samples was stopped by ‘adding 50 uL of 4% BET ethanol solution. The sample tubes wwere covered with aluminum foil during incubation to avoid any influence of light. ‘Analysis of MA as 1-Methylpyrazole. MA formed in the samples was analyzed according to a GC method reported previously (Ichinose et al, 1989; Wong et al, 1994). Tue MA. Sacreacted sith Nomethvihvdrazine (NMED, and the resulting Gerivative, I-methyipyrazole, was analyzed with 2-meth- ylpyrazine as an internal standard by a GC equipped with 0 fused silica capillary column and a nitrogen~phosphorus detector (NPD), ‘The experiment was replicated three times. “Analysis of Acetaldehyde as 2-Methylthiazolidine. ‘Acetaldehyde formed in the samples was analyzed according. to.a GC method reported previously (Miyake and Shibamoto, 1999, 1995) with slight. modification. Acctaldehyde wa reacted with eysteamine at room temperature for 24h. The ‘eaction mixture was extracted with 10 ml of dichloromethane ‘using a liquid-liquid continuous extractor. After the extract twas dried over anhydrous sodium sulfate, the resulting deriva- Uve, Emethylthiazolidine, was analyzed with 2,3,0-unmetn- yithiazole as an internal standard by a GC equipped with a {fused silica capillary column and an NPD. The experiment ‘was replicated three times. Recovery Test on MA and Acetaldehyde from Blood Plasma. MA (200 nmol as sodium salt) was spiked into 50 ‘uL of horse blood plasma, and acetaldehyde (300 nmol) was ‘soiked into 30 ul, of horse blood plasma. Samples were ‘prepared and aalyzed for MA and acetaldenyde by GU as escribed above. The experiment was replicated three times. Sc 2 Age. Food Chem, Vol. 48, No, 9, 1988, 2605 Amoust of WA ot) -eteed § Foam ‘0 Be G0 ao 0 abn 1a8 1000 3600 3000 208 ‘Amount of ariondans (nmol) Figure 2 lhibitory activity of -0-GIV, DMT. robucl seb ai oward MA formation fom Bob plasm ‘upon oxidation. Instrumental Anaya A eye Packard GD)! satan Snenaan caged init ns Ooh seen em cromnorns creed i220 Oe “ass al Sorte Povo CAs tr sare seed epnibiariace. cedar Si nnd Sona pe cee SO O35 ama ee Se are a tae emp t SEP ete leasing nese he Ba cya hes carte gas ra 30 cv th SST Toa ie empeton at peed ae Sed tan ant falar eo fr Te gee i a ainda eh ene 2 go srs 1 OC mera ap BP ne ooh ns ued ct the MA Ser chaise Janeane, an Te naan tte some ae oe ee ee errs sie Ses aan 5b iad ant mare Ue sens RESULTS AND DISCUSSION In the present study, recovery efficiencios of MA and acetaldehyde from horse blood plasma were 82.1 + 1.3 ‘and 87.5 + 1.2%, respectively. The values are mean + standard deviation (n = 3). Protein concentration in the blood plasma was 11.2 ugyb. When 90 uL uf blu plasma (860 ug of protein) was oxidized, 957 + 199 nmol of MA was formed. The value is mean + standard deviation (n = 3), The control sample (860 yg of blood plasma) contained 60-80 nmol of MA, indicating that a certain amount of MA was present in the blood plasma prior to oxidation. When 30 uL of blood plasma (516 fg of protein) was oxidized. 135.0 + 7.21 nmol of ‘acetaldehyde was formed. The value is mean + stan- dard deviation (n = 3). Figure 2 shows the inhibitory activity of 2”-0-GIV, DMIF, probucol, and L ascorbic acid toward MA forma. tion in horse blood plasma upon oxidation. Results of the mixtures of 2’-O-GIV or DMHF and L-ascorbic acid in equal amounts are also shown in Figure 2. The ‘addition of 100 nmol of 2°-O-GIV inhibited MA forma- tion by 60%. On the other hand, DMHF and vitamin C inhibited MA formation by only 20% at the same level (100 nmol). When doses were increased, the activity of DMHF steadily increased, but vitamin U activity aia not change significantly. DMHF at 4000 nmol exhibited activity comparable to that of 100 nmol of 2°-O-GIV ‘Activity of 2”-0-GIV in doses higher than 100 nmol wa: not examined because of its solubility in the testing system. ‘When 2’-0-GIV and DMHF were mixed with vitamin (©, an increase in their antionidative activitios war observed. A mixture of 2°-O-GIV (50 nmol) and vitamin‘3696 J Agric. Food Chem, Vol. 46, No.9, 1996, : = I = we s 3 i. i - ; ee i, bp ‘0s 100 150 200 250 300 500 1000 2000 3000 “Amount of antioxidant (mot) ee DMF toward acetaldehyde formation from ‘upon oxidation. C (50 nmol) inhibited MA formation by 75%. The activity was ~5% higher than that of 2”-O-GIV alone. A mixture of DMHF (25 nmol) and vitamin C (25 nmol) inhibited MA formation by 63%: however, the mixture did not increase activity significantly when the total dose was increased up to 4000 nmol. These results indicate that the inhibitory activities of 2”-0-GIV and DMEF arc enhanced by the addition of vitamin C. Detailed mechanisms of these phenomena are not yet well understood. It has been known that L-ascorbie acid and a-tocopherol act synergistically to inhibit lipid peroxidation (Sharma and Buettner, 1993). Ithas been proposed that the mechanism by which this occurs is that L-ascorbie acid reacts with an a-tocopherol radical to regenerate a-tocopherol, and then the result Leaseorbic acid radical is reduced back to L-ascorbie aci by NADH (Packer et al., 1979). This set of reactions ‘may explain the results observed in the present study. ‘The results obtained from probucol were unexpected. Addition of probucol increased MA formation. The values shown in Figure 2 are Vp of actual values. When £860 xg of blood plasma was oxidized in the presence of 100 and 4000 napol of probes), 2206.8 ue 226 and 2976.5 + 194 nmol of MA were formed, respectively, suggesting that probucol may have pro-oxidative activity. How- ever, this hypothesis can be ruled out because probucol significantly inhibited acetaldehyde formation as re- ported in the following paragraph. Therefore, MA may be produced from probucol during the incubation of blood plasma. In fact, an aqueous solution of probucol (3 mol) produced 2.6 .mol of MA upon oxidation with Fenton's reagent (Miyake and Shibamoto, 1998). Figure 3 shows the respective inhibitory activity of 2°.0-GIV, probucol, and DMHF toward acetaldehyde formation from blood plasma upon oxidation. Because the structure of probucol is similar to that of BHT (Figure 1), it would be expected that probucol would have antioxidative activity and consequently inhibit the formation of lipid peroxidation products, including ac- etaldehyde and MA. As shown in Figure 3, 2”-0-GIV ‘and probucol exhibited quite similar activities. The formation of avctaldeliyde was inhibited nearly 90% in the presence of 300 nmol of either 2”-O-GIV or probucol. ‘The inhibitory activity of DMHF toward acetaldehyde formation was much less than that of either 2°-O-GIV ‘or probucol, at lower levels. However, when the level increased up to 2000 nmol, DMHF inhibited acetalde- hhyde formation by 88%, which is comparable to the activity obtained by either 2°-O-GIV or probucol at the 300 nmol level. Miyake and Shibamoto ‘The highly effective antioxidative activity of 2"-O-GIV has been shown in various lipid peroxidation systems: ethyl linoleate or saualene/Fenton’s reagent (Osawa et al., 1992; Kitta et al., 1992); ethyl te/Fenton's reagent or UV light (Nishiyama et al., 1993); phospho- lipids or fish liver oil/Fenton’s reagent (Nishiyama et al, 1994); and «3 fatty acide/Fenton’s reagent (Ogata et al., 1996), In the present study, 2°-O-GIV was also effective toward the inhibition of blood plasma oxidation. 2-O-GIV has not been reported in foods. It is present ia @ commercial Iiealth fad product prepared fv young green barley leaves. Therefore, itis possible to expect in vivo concentrations obtained through food intake, but discussion of a commercial product is not, scientifically relevant. DMHF was first found in nonenzymatic brown: reaction products in the early 1960s (Hodge et al., 1963). Later it was revorted as a characteristic flavor chémical formed in carbohydrate caramelization and dehydration reactions (Hodge, 1967). DMEF has been found as a flavor component in various fruits, such as pineapples Rodin et al., 1965), strawberries (Ohloff, 1969), and grapes (Rapp et al., 1980). More recently, it has been isolated from Swiss cheese (Preininger and Groseh, 1994); cultures of bacteria, Lactobacillus helveticus @Preininger sind Groschi, 1996), aud Maillard reaction products (Blank and Fay, 1996). Therefore, it is pre- sumed that DMHFF is ingested through the foods de- scribed above. The antioxidative activity of DMHF has never been reported prior to this study. Discovery of antioxidative activity in a flavor chemical, DMHF, is interesting because recently the antioxidative activity of flavor chemicals such as coffee flavor components has begun to receive much attention as a source of biologi- cally active substances (Singhara et al., 1998). L-Ascorbic acid (vitamin C)is a well-known naturally occurring antioxidant, and its numerous chemical and biological activities have been reported elsewhere. For example, it reportedly protected against free radical damage and cured scurvy (Krinsky, 1979). ‘This com- pound was used to compase ito activity with that of other tested compounds in the present study. Probucol was also tested in the blood plasma system because itis a commercial drug that is used widely for clinical treatment of hypercholesterolemia; it is an effective antioxidant transported in lipoproteins (U.S. Department of Health and Human Services, 1988), including LDL, and blocks the oxidative modification of LDL in vitro (Parthasarathy et al., 1986). Even though amounts of MA formed in samples with probucol are considerably different from those formed in samples with 2’-O-GIV, the results obtained from the acatalde- hyde analysis (Figure 3) are almost identical. There- fore, it is hypothesized that the naturally occurring antioxidant, 2"-O-GIV, may inhibit diseases such as atherosclerosis associated with oxidative damage as effectively as probucol. Moreover, this antioxidant might be more effective ifit is used with vitamin C. LITERATURE CITED Blank, 1.; Fay, L. B. Formation of 4-hydroxy-2,5-dimethyl- ‘12H -furanone and ¢-hydraxy-2(er 8)-ethyi-5(or 2)-methyl- ‘32H furanone through Maillard reaction based on pentoee sugars. J. Agric. Food Chem. 1996, 44, 531-536. Bradford, M. M. A rapid and sensitive method for the quan- titation of microgram quantities of protein utilizing the Principle af prtainaiye binding Anal Rischem. 1976, 72, 248-254,Innzion of Blood Plasma Oxidation by Natural Antioxidants aru, GH: Scasr, RJ Zaller, H. Chemie and ‘biochemistry of 4-hydroxynonenal, \dehyde and re- laved aldehyes. Free Radical Biol Med. 4991, 14,1125. Feinman, S. E. Structure-activity relationships of formalde: hyde. in Formaldehyde Sensitivity and Toziity, einman, SIE. Ed: ORC Press: Boca Ratan, FL, 1968; pp 197-204 Glavind, J; Hartmann, &; Clemmesen, J Jessen, KE Dam, HE Studies on the role of lipoperoxides in human pathology. I. The presence of peroxidized lipids inthe atherosclerotic sorta. Aco Pathol. Microbial. Scand. 1952, $0, 1-6. Halliwell, Bs Curteridge, J. M. C. Pree Redianls in Biology and Medicine; Clarendon Press: Oxford, UH, 199; pp, Hedge, J. Origin of flavor in foods nonenzymatic browning Teactions. In Chemistry and Physiology of Flavors: Sele, H.W. Day, E. A, Libbey, L. M., Bds; AVI Publishing: Westport, CT, 1967: pp 465-491, Hodge, J-E.; Fisher, B. E.; Nelson, E. C. Dicarbonyls, reduc ‘nes. and heterocvelies produced by the reactions of reduc ing sugars with secondary amine salts. Am. Soe. Bret Chem. Proc. 1963, 1963, 84-92. Ichinose, .; Miller, MG; Shibamoto, T. Gas chromatographic ‘analysis of free and bound malonaldehyde in rat liver homogenates. Lipids 1989. 24, 895-896. Kitta, Ki: Hagiwara, Y.: Shibamoto. T. Antioxidative activity of an isoflavonoid, 2°-O-giveasvlisovitexin isolated from zen barlerleaves. Agric Food Chem, 192. 40,1545 Kein. Cartened preteen azains oxidation, Pore Lacombe, A: Kermasha, 5; van ge Voor K, Fi: Mill, LB Preparativn and purification of malonaldebyde sodium salt J Agric. Food Chem. 1990. 38. 418-423 ‘Miyake. T.: Shibamowo, T. Quantitative analysis of acetalde- hyde in foods and beverages. J. Agric. Food Chem. 1998. Mivake, T.: Shibamow. 7. Formation of acetaldehyde from ‘Lascorbie aeid and related compounds in various oxidation systems. J Arie. Food Cem. 1995. 45. 1668~ 1672 Miyake, P: Shitsmoto. T Formation of maionaldehyde in the Bresence of pruhucol. an anti-atherorclerosis drug. Food Cem. Tasici. 1998, in press. Namiki. M. Antoxidantsantimutagens i food. Crit Re. Food ‘Set Nutr 1990.29. 273-300 Nishiyama, T Hagiwara, Ys Hagwara, H.: Shibamote, 7 Inhibition of malonaldehvde formation from lipids by an isoflavonoid isolated from voung green baries, Oil Chem. Sve, 1998. 70, 811813. Nishiyama, T: Hagiwara, Y.: Hagiwara, H: Sh . Formation and inhibition of genotoxic glvoxal and malonal- dehyde from phospholipids and fish liver oil upon lipié peroxidation. J. Agric. Food Chem. 1994, 42, 1725-1731 gta. J; Hagiwara, 1 Hagwara, H. Shibamoto,T. inhi tion of malonaldehyde formation by antioxidants from oS polyunsaturated fatty acids. J. Am. Oil Chem. Soc. 1996, 73, 653-656. Ohio, G. Chemistry of odoriferous and favoring substances. Fortschr, Chem. Forsch, 1968, 12, 185251, Osawa. T.; Katsuzaki, H.; Hagiwara, Y.; Hagiwara, H.; Shibs- ‘moto, T. A novel antioxidant isolated from young green Darley leaves. J- Agric. Food Chem. 1992, 40, 1195-1196. sabi gas AEE ee “Aa. Food Chem, Vo. 6, No 9, 18983657 Packer, J.B. Slater, TF; Wilton, RL, Direct observation of a free radical interaction between vitamin E and vitamin ©. Nanure 1979, 273, 13/~ 138. Parthasarathy, S Young, §. G.; Wirtum, J, L.; Pittman, R. C; Steinberg, D. Probucol inhibits oxidative modification of low density lipoprotein. J. Clin. Invest. 1986, 77, 641 Ga. Preininger, M.; Grosch, W. Evaluation of key odorants ofthe neutral volatiles of emmentaler cheese by the calculation of odour aetvity value. Food Si, Tecknol-Lebens-Wisten. Toohnol. 1004, 27, 297-244 Preininger, M.: Grosch, W. Determination of 4-hydroxy.2.5- dimethyi-9(2H-furanone (HDMF) in cultures af bacteria. Z Lebensm -Unters, Forsch, 1995, 201, 97-98, Rapp A: Knipeer, W ; Engel), [-; llemeyer, H.: Heimann, W. Oftflavor compounds in the berry and wine aroma. Vitis 1980, 19, 13-28, Retsky, K-L.; Freeman, M, W-: Frei, B Ascorbic acid oxidation rodacts protget human low density Kngprtein against atherogenic modification—Antoxidant rather than proox- dant activity of vitamin Cin the presence of transition metal ions. J. Biol Chem. 1998, 268, 1304-1908. Rodin. J. 0, Himel, RM; Silverstein, R. M; Leeper, R. Ws 1, W. A. Volatile Havor and aroma components of pineapple (isolation and tentative identification of 2.5- Gimethy!-4-hydroxy-8(2H-furanone. J. Food Se. 1965, 0, 250-265, Schmidt. 8M: Hon, O. srete, J: Yan, B.D: Wautier, J. Le Stern” D. Cellular receptor for advanced glveation end pragucts—implierions for induction of exam! srece and cellular dysiunction in the pathogenesis of vascular lesions, ‘Arcerionier. Phrombosis 1994, 14. 1823-1326, Sharma, MK: Buettner. G. R. interaction of vitamin-C and ‘itamin-E during free radical stress in plasma~an ESR Suds. Free Radical Biol. Med. 1993, 14. 549-653. Sins As Mach On Olibesnto, Ty Antonov aeivity tf brewed cafec extracts. in Functional Foods: Disease Prevention: Proceedings ofthe 213th National Meeting of ‘he American Chemical Society. San Francisco, CA. 1998; tn press TTesfamariam, B. Free radicals in diabetic endothelial cell dysfunction. Free Radical Biol Med. 1994. 16. 383~291 Umano. K: Dennis, Kd. Shibamoto, 7. Anaivss of free ‘nalanaliehyce sm ahooirradiatad cor ol nd beet fat Tpyramole derivative. Lipids 1966, 99, 811813 WS. Department of Health and Human Services. Report 331: National Tosiology Program: Research Triangle Fark, NC. 1988, Wong, d. W: Bbeler, 8. E: Rivkab-Isseroff R; Shibamota.T “Anslysis of malondialdehyde i biological samples by capil lary gas chromatography. Anal. Biochem. 1994, 220, 73- 81 ‘YSuhara, 4: Shibamoto, T. Determination af volo aliphatic aldebydes in the headopace of heated fod oils by dervati- zation with 2-aminoethanethiol. J. Chromatogr 1991, 547, 291-286. ‘Received for review April 2, 1998. Revised manuscript received ‘June 23, 1998. Accepted July 1, 1998. JP 9809182.ACS SYMPOSIUM SERIES 702 Functional Foods for Disease Prevention II Medicinal Plants and Other Foods Takayuki Shibamcoto, EDITOR University of California at Davis Junji Terao, EDITOR University of Tokushima Toshihiko Osawa, EDITOR Nagoya University Developed from a symposium sponsored by the Division of Agricultural and Food Chemistry at the 213th National Meeting of the American Chemical Society, San Francisco, California, April 13-17, 1997 sS American Chemical Society, Washington, DCChapter 17 Possible Inhibition of Atherosclerosis by a Flavonoid Isolated from Young Green Barley Leaves Takashi Miyake’, Yashihide Hagiwara’, Hideaki Hagiwara’, and Takayuki Shibamoto”” ‘Department of Environmental Toxicology, University of California, Davis, CA 95616 + “Hagiwara Institute of Health, 1173 Maruyama, Asazuma-cho, Kasai 679-01, Japan Young green barley leaves are known (0 possess potent pharmacological properties, including antioxidative, anti- inflammatory, antimutagenic, and antiallergic activities. In particular, an flavonoid, 2°-O-glycosylisovitexin (2"-U-G1V), isolated from an ethanol extract of young green barley leave possesses a strong inhibitory effect toward lipid peroxidation. 2". O-GIV inhibited acctaldchyde formation from LDL by 76% at a level of 1 umol/50 ug, whereas ferulic acid inhibited by 66% at the same level. In a case of a blood plasma system, 2°-O-GIV and probucol inhibited acetaldehyde formation by 89% and 94%, respectively, at a level of 3 umol. 2"-O-GIV and vitamin C inhibited MDA formation by 54% and 32%, respectively, at a level of 0.1 umol. A synergetic effect between 2"-O-GIV and vitamin C was observed. Barley has been cultivated and fed to livestock since ancient times.- Essence extracted from young green barley leaves has been reported to exhibit many biological characteristics including anti-aging, anti-carcinogenesis, anti-diabetic, and anti- arteriosclerosis (1). However, no scientific proof of these characteristics existed until a potent anti-oxidant was isolated and identified in a green barley leaf essence (2). This novel natural antioxidant, which is a flavonoid, was identified as 2"-O-glycosyi isovitexin (2"-O-GIV, Figure 1). Since the discovery of this flavonoid, the antioxidative activities of 2"-O-GIV caamined in varivus lipid peroxidation systems including squalene/UV (3), ethyl ester of fatty acids/Fenton's reagent (4), phospholipids or cod liver oil/Fenton’s reagent (5), and -3 fatty acids/Fenton's Teagent have been reported(6). Lipid peroxidation model systems have been used most commonly to investigate biological activities of chemicals because lipid peroxidation is associated with many biological complications such as carcinogenesis, mutagenesis, aging, and atherosclerosis (7-9) as well as with human immunodeficiency virus (FIV) progression (10). However, its mode of toxic action is not yet clearly understood. Lipids produce many low molecular weight carbonyl compounds upon oxidation (77) Therefore, these carbonyl compounds such as malondialdehyde (MDA), glyoxal, acrolein, acetaldehyde, and formaldehyde which directly crosslink to proteins and bind Corresponding author. 178 ©1998 American Chemical Society 8HCO, Ho CH— CHCOOH Ferulic acid HO. ss oH Probucol ou oH 2"-U-Glycosylisovitexin (2"-0-GIV) Figure 1. Structure of ferulic acid, probucol, and 2"-O-glycosyl isovitexin.180 covalently to nucleic acids (12) may possibly play an important role in the toxic effects caused by lipid peroxidation (13, 14). In addition to initiating these adverse effects, low molecular weight aldehydes formed from lipid peroxidation can be used as indicators to detect oxidation in lipids. Therefore, many studies have been conducted using these aldehydes as indicator, in particular, malondialdehyde (MDA). The formation of avculuchyde was also used 10 monitor oxidative reaction mechanisms of L-ascorbic acid (5). In the present study, the antioxidative activity of 2"-O-GIV was examined using low density lipoprotein and blond plasma oxidized with Fenton's reagent. Experimental Chemicals. L Ascorbic acid (reagent grade), butylated hydroxytoluene (BHT), Trizma® hydrochloride, Trizma® base, fatty-acid-free bovine serum albumin, probucol, and fat red 7B were purchased from Sigma Chemical Co. (St. Louis, MO). Cysteamine hydrochloride, 2,4,5-trimethylthiazole, ferulic acid, sodium dodecyl sulfate (SDS), hydrogen peroxide, and ferrous chloride were obtained from Aldrich ‘Chemical Co. (MuWaukee, WI). Hydrogen peroxide was obtained from Fisher Scientific Co., Ltd. (Fair Lawn, NJ). The standard stock solution of 2,4,5- trimethylthiazole was prepared by adding 10 mg of 2,4,5-trimethylthiazole to 1 mL of dichloromethane and was stored at 5 °C. Authentic 2-methylthiazolidine was synthesized according to the method reported previously (/6). The structures of probucol and ferulic acid are shown in Figure 1. 2".O.Glycosylisovitexin (2"-O GIV) was isolated from young green barley leaves (Hordium vulgare L. var. nudum Hook) harvested two weeks after germination by a method previously reported (2) using column chromatography with Amberlite XAD-2 nonionic polymeric absorbent. The structure of 2"-O-GIV is shown in Figure 1. Preparation of Low-Density Lipoprotein (LDL). LDL was prepared from blood sample obtained from a male quarter horse (5 yeare old) according to the method reported previously (/5). After filiration sterilization (0.45 um: Nalge Stbron) of the LDL, the protein concentration was determined by the Coomassie Blue dye- binding assay (/7). A 10-pL aliquot of appropriately diluted LDL was added to the dye reagent, the solution mixed, and absorbance at $94 nm measured versus a reagent blank, using a Hewlett-Packard 8452A diode array UV spectrophotometer. A standard curve using bovine sciuin albumin was used 1 calculate the LDL. concentration. Preparation of Blood Plasma. The blood from a male quarter horse (5 years old) was collected in a sterile, 3.5-mL tube containing 60 uL of a 7.5% EDTA solution (4.5 mg EDTA). Plasma and red blood cells were separated following centrifugation (5000 rpm for 30 min at 4 °C) and immediately frozen on dry ice. The plasma was stored at - 80°C until use. Oxidation of LDL and Blood Plasma With Fenton's Reagent With or Without Antioxidants. A 5-mL aqueous solution containing 23 uL of LDL (final concentration, 2.17 ng/mL), 0.25 mmol of Trizma® buffer (pH 7.4), 0.75 mmol of potassium chloride, | mmol of ferrous chloride, and 0.5 mmol of hydrogen peroxide was incubated with 2”-O-GIV (amounts ranging from 0 to 1 pmol, with 0.1181 pmol increments) or ferulic acid (amounts ranging from 0 to 1 pmol, with 0.1 pmol increments) at 37°C for 15h, In a separate experiment, samples containing blood plasma (516 ug of protein), 0.25 mmol of Trizma buffer (pH 7.4), 0.75 mmol of potassium chloride, 1 mmol of ferrous chloride, 0.5 mmol of hydrogen peroxide, and 10% of surfactant SDS were incubated with 2"-O-GIV (0, 0.05, 0.07, 0.1, 0.2, and 0.3 mol) or probucol (0, 0.05, 0.07, 0.1, 0.2, and 0.3 mol) at’37 °C for 15 h. In another experiment, samples containing blood plasma (860 jig of protein), 0.25 mmol of Trizma® buffer (pH 7.4), 0.75 mmol of potassium chloride, 1 mmol of ferrous chloride, and 0.5 mmol of hydrogen peroxide were incubated with 2"-O-GIV (amount ranging from 0 to 0.1 umol with 0.01 mol increment), vitamin C (amounts ranging from 0 to 0.1 yrmol, with 0.02 pmol increments), or a mixture of 2" O GIV and vitamin C (total amounts ranging from 0 to 0.1 mol, with 0.01 mol increments), in which molar ratio of 2"-O-GIV and vitamin C was 1/1. ‘While the incubation continued, the mixtures were covered with parafilm. A ‘S-mL solution containing exactly the same materials except for the ferrous chloride and the hydrogen peroxide was incubated in the same manner as_a control sample. Oxidation of the samples was stopped by adding 50 uL of 4% BHT ethanol solution, ‘The incubation system was covered with aluminum foil to avoid any influence of light on the LDL and blood plasma peroxidation systems. The all experiments were replicated three times. Analysis of Acetaldehyde and MDA. The amount of acetaldehyde was determined as a thiazolidine derivative according to the method reported previously (8). ‘The amount of MDA was measured as @ py:acule Uciivative by the method developed previously (/9). A Hewlett-Packard Model 5890 GC equipped with a nitrogen phosphorus detector (NPD) and a 30 m x 0.25 mm i.d. (dr = 1 pm) DB-1 bonded-phase fused silica capillary column (J & W Scientific, Folsom, CA) was used for quantitative analysis of acetaldehyde and MDA. The detector and injector temperatures were 250 °C. The linear velocity of the helium carrier gas was 30 cm/sec with a split ratio of 21:1. The oven temperature was programmed from 60) to 180 °C at 4°C/min and held for 10 min. GC peak areas were integrated with a Tsp SP 4400 series integrator. An HP Model 5890 series 11 GC interfaced to a HP 5971 mass spectrometer was used to confirm the identity of the thiazolidine derivative of acetaldehyde, 2-methylthiazolidine, and pyrazole derivative of MDA, 1- methylpyrazole, in the samples. The GC conditions were the same as for the GC with NPD. The mass spectra were obtained by electron impact ionization at 70 eV with an ion source temperature of 250°C. Results and Discussion Oxidative damages caused by reactive oxygen species have been known to initiate and to promote many diseases, such as cancer (20), cardiovascular disease (21), and atherosclerosis (22). A strong relationship between atherosclerosis and amounts of lipid peroxidation products in the inside wall of arteries has been reported (23, 24). Recently, oxidative damage of LDI. has received much attention as a process which implicates the development of human atherosclerosis (22). Therefore, LDL has been widely used to investigate the relationship between lipid peroxidation and atherosclerosis (25). . LDL is generally prepared from blood lipoprotein using a centrifuge. Figure 2 shows fractions of lipoprotein prepared according to their density (26). Figure 3 shows compositions of various lipoprotein fractions (26). LDL contains greatest182 Density (g/mL) Lipoprotein Chylomicrane 0.980--|———+ ‘Very-low density lipoprotein (VLDL) 1.006--| — 1.063-- Figure 2. Fractions of lipoprotein. Mi Proteins @ Phospholipids I Free Cholesterol Cholesterol Esters OF Triacylglycerots Coe VLD 2 core HOU) Figure 3. Compositions of various lipoprotein fractions.183 amount of cholesterol esters which may be associated with the development of atherosclerosis. Figure 4 shows the inhibitory effect of 2"-O-GIV and ferulic acid against LDL oxidation. Acetaldehyde was used to monitor the formation and inhibition of lipid peroxidation because MDA tends to be trapped with proteins. Over 2 nmol of acetaldehyde was formed from 50 wg of LDL. Formation of acetaldehyde decreased when the amount of either 2"-O-GIV or ferulic acid was increased. Ferulic acid inhibited acetaldehyde formation by 50% at the level of 0.3 umol/50 pg of LDL) whereas 2”-O-GIV required 0.7 umol/>0g of LOL to obtain the same level of inhibition. On the other hand, 2"-O-GIV inhibited acetaldehyde formration by 76% at the level of 1 pmol/50 wg whereas ferulic acid inhibited by 66% at the same level. Figure 5 shows the antioxidative activities of 2”-O-GIV and probucol measured in a blood plasma system. Probucol was used to examine the relative antioxidative activity of 2”-O-G1V because it is a commercial product with a million dollar salee in Japan and haz been used to treat atherosclerasis. However, probucol produced a significant amount of MDA by oxidation (T. Miyake and T. Shibamoto, unpublished data). ‘Therefore, acetaldehyde was used as an indicator of oxidation of blood plasma, The antioxidative activities of 2"-O-GIV and probucol were almost identical. When blood plasma (516 1g) was oxidized without an antioxidant, 135 nmol of acetaldehyde was recovered. Inhibitory activity of both 2"-O-GIV and probucol toward acetaldehyde formation increased greatly when their levels increased over 0.7 umol. 2°-O-GIV and probucol inhibited acetaldehyde formation by 89% and 94%, respectively, at the level of 0.3 umol). The results indicate that 2"-O-GIV may inhibit atherosclerosis. Figure 6 shows the antioxidative activities of 2"-O-GIV and vitamin C in a blood plasma system. When blood plasma was oxidized with Fenton's reagent, 1.11 nmol/jig (blood plasma) of MDA was recovered. However, no MDA was recovered from unoxidized blood plasma. In this experiment, MDA was used as an indicator of oxidation because vitamin C itself produced a significant amount of acetaldehyde by oxidation with Fenton's reagent (/5). 2"-O-GIV and vitamin C inhibited MDA formation by 54% and 32%, respectively, at the level of 0.1 pmol. On the other hand, when equal mols of 2"-O-GIV and vitamin C were mixed, a 75% inhibitory effect was obtained at the level of 0.1 umol (total). A synergetic effect between 2”-O- GIV and vitamin C was observed. ‘The antioxidative activities of flavonoid such as 2"-O-GIV are due to their ability to chelate metal ions (such as Fe2+) by means of either the 3-hydroxy, 4-keto grouping or the 5-hydroxy, 4-keto grouping, in addition to scavenging free radicals deriving from the phenolic moiety of the structure (27). Conclusion Atherosclerosis is one of the most common diseases in developed countries, such as Japan and the U.S., where people tend to eat more fatty foods. Detail mechanisms of atheroscrelosis are not yet clearly understood but there is a strong evidence that lipid peroxidation plays an important role in the initiation of atheroscrelosis, Therefore, antioxidants such as probucol have been used to treat atheroscrelosis. However, use of synthetic antioxidants (e.g., butylated hydroxy toluene) in human foods has been restricted because of their possible chronic toxicity. 2°-O-GIV is a natural plant product and large amounts of barley leaves (which contain 0.5-0.7% of 2".0-GIV) have heen consumed hy livestock for many years without any evidence of adverse effects. 2'

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