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Professor Department of Pharmacy Science University of Tokyo Tokyo,Japan

Dr. Kazuhiko Kubota

Curriculum Vitae

Kazuhiko Kubota, Ph.D.

Professor of Pharmacy Department Science 'University of Tokyo

1953 1961 1964 1964

1970 - Present 1988 - 1992 1990 -1993 1989 - 1995

1979 - 1980

1981 - 1981 1989 -1997

B.s., Pharmacy, Kumamoto University Lecturer, Science University of Tokyo Ph.D., Pharmacy, Tokyo University

Assistant Professor, Department of Pharmacy, Science University of Tokyo

Professor, Department of Pharmacy, Science University of Tokyo Chairman, Department of Pharmacy, Science University of Tokyo Director, Science University of Tokyo

Professor, Research Institute of Life Science,

Science University of Tokyo

Visiting Professor, Department of Pharmacology, Chicago Medical College

Chairman, Kanto Section of Japan Pharmaceutical Society 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 (N anko-do) Swnmation of Clinical Medicine Introduction (YaKuji Nippo Corporation)

Etc ...

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, antiulcer, anti-hypercholesterol and endurance increasing activities are attached in the appendices.

Barley and I

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 1953, just after graduating from the University, 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-grasses 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,n 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, flat fields, which were scraped by the glaciers and thickly 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 and dry 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 rice-grasses family. Additionally, these 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, my University Senior.

Since then, my relation with the rice grasses became closer through Dr. Hagiwara. We started to investigate the content of the barley leaf extract dry juice, pharmacologically. To be honest, in the

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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 had 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 the superoxide dismutase, which 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 betacytosterol, which is now in the marketplace as an antiarteriosclerosis 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 NFjdp

Appendix

1. Isolation of Potent Anti-inflammatory Protein From Barley Leaves. Kubota, K., Matsuoka, Y., and Seki, H., Japanese Journal of Inflammation 1983. 3(4)

2. Studies On The Effects Of Green Barley Juice On The Endurance And Motor Activity In Mice. Kubota, K. and Sunagane, N., The 104th Annual Congress of Pharmaceutical SOCiety of Japan 1984.

3. Studies on the Constituents of Green Juice From Young Barley Leaves, Antiulcer 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. 1 05( 11)

4. Studies on the Constituents of Green Juice From Young 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

I solation 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 ofInflammation, Vol. 3, No.4, 1983 (I), AI

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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-inflammatory activity of the barley juice was produced not only by SOD, but also by other protein than SOD. We isolated protein fractions named P4-Dl and DI-Gl, which showed extremely strong anti-inflammatory activity. In the present paper, the authors are showing the evidence of the potent antiinflammatory activity of the protein and barley SOD.

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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 are a 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 by Dr. Horio, a professor of Osaka University.

P4-D1 and D1-G1 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 suspending 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 subcutaneously to the sole of the right hind paw of the rats, and 0.1 ml of saline solution to the left paw. Drugs, P4-D1 and D1-G1, were administered 30 minutes before the carrageenin injection. The paw volume of the rats was measured by the method described by Yamazaki, et 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 carrageenin-injected one) - (Paw volume of saline-injected one).

Results

The acute toxicity of DI-G1 and P4-D1 was listed in Table 1. Both fractions produced no toxic signs 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 ofP4-Dl andD1-Gl 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 ofP4-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.

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Although data were not shown here, the anti-inflammatory of Dl-Gt 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 antiinflammatory activity.

Discussion

All ofD1-G1, P4-Dl and SOD isolated from green barley juice revealed extremely potent anti-inflammatory activity, especially when they were injected intravenously. They significantly suppressed the carrageenininduced edema in rats at very low doses, such as 0.01 and 0.1 mg/kg.

However, the solution of SOD showed a bright yellowish color, while those of P4-D1 and DI-G1 did not, and SOD was not heat stable, while DI-G 1 was heat stable. Therefore, P4-Dl and DI-G 1 are chemically different

from SOD. -

Unlike aspirin, P4-Dl and DI-Gl did not inhibit prostaglandin synthesis, indicating that they have quite a different mechanism of anti-inflammatory action from that of aspirin and phenylbutazone, which are potent inhibitors of prostaglandin systhesis.

The ratio, intravenous ED50/LD50, for DI-Gl on anti-inflammatory action is much larger than that for aspirin. This means that P4-Dl and D1-G 1 seem to be much better anti-inflammatory agents than aspirin as far as they were concerned with intravenous administration. The mechanism of the anti-inflammatory action of D'l-Gl and P4-Dl is unclear at present. They may exert their action through inhibiting some enzymes like peptidases.

References

1) Y. Matsuoka, H. Seki, K. Kubota, H. Ohtake and Y. Hagiwara (1983), Japan. J. Inflamm. 3, 602.

2) B.L. van der Warden (1940), Arch. Exp. Path. Pharmak., 185, 389.

3. H. Yamasaki, et al (1967), Folia Pharmacol. Jpn. 63,302.

P4-D1 DI-Gl

p.o

> 2,000

i.p. 1,120 2,830

s.c.

» 4,000 > 4,000

i.v.

Chart 1

Young barley leaves (25 kg)

~

GM-SD (1 kg)

--':;;pended in 50mM phosphate buffer (pH 7.5) ~centrifuged at 10,000 x g for 20 min.

Supernatant

tfractionated by 40-100% sat. (NH4hS04 Precipitate

l-russolved in buffer and dialyzed ~DEAE-Sephadex A-50 column chromatography r Passed solution

P4-D1

Sephadex G-100 column chromatography

D1-Gl

Table I

Acute toxicity ofP4-Dl and DI-Gl in mice.

LD50 (mg/kg)

285 224

p.o. : oral, i.p. : intraperitoneal, s.c. : subcutaneous, i.v. : intravenous

4

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0 ci .0 1 2 3 4 5 6 8
Time (hr)
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0.1
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0 1 2 3 4 5 6 8 Time (hr)

Fig.I Effects of intravenously administered P4-Dl and DI-Gl 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 (0-0) Control, ( A········!:i ) P4-Dl O.Olmg/kg i.o.,

( • -----. ) P4-Dl O.lmg/kg i.u., ( x········ x ) DI-Gl O.01mg/kg i.u., ('ij------\l ) DI-Gl O.lmg/kg i.u.,. *: P< 0.05 and **: P< 0.01, significantly different from the control group.

5

8 0.5 .z

o

:>

~

~

....

<I.l

~

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~

'""' 0.1

.06.

o

01234568 Time (hr)

Fig. 2 Effect of aspirin and barley SOD on carrageenin-induced paw edema of rats.

Each point and vertical bar represent the mean value and standard error of 6 experiemtns. Symbols represent, 0: control,

:. aspirin 10Omg/kg, : ~barley SOD 0.2 mg/kg (i.v.),

: A heat-treated SOD 0.2 mg/kg (i.v.).

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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, Shinjuhu-hu, Tokyo, 162. Japan.

"This paper has been reported in The 104th Annual Congress of Pharmaceutical Society of Japan. held in Sendai in 1984.

MA

Introduction

Since the daily intake of green barley juice purportedly has an effect of 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 capacity or activity in animals. However, it is also possible that the juice may contain unidentified substances which can enhance stamina in animals and humans.

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Materials and Methods

1. Materials

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% or4% 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 kept at 24 ± 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 fed on the food containing 2% of DGBJ (2% DGBJ food) for 15 days at the same period of time. A mouse fed on thestandard food was placed in a 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 30 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% DGBJ 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 afforced running of mice in the control and test groups was compared.

4. Statistical analysis

The statistical significance of difference was assessed by Student's t-test.

Results

1. Effects ofDGBJ 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. l.The DGBJ-fedmice revolved the wheel cage more than the control mice. The difference between the numbers of revolution of the wheel cage in the two 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 DGBJ 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 time in mice, but 15 days feeding could prolong it.

The average body weight 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 ofDGBJ 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).

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100

200

Fig .. 1 Effect of Green Barley Juice on Motor Activity of Mice in Wheel Cage Test.

0-1 hr

1·2 hr

300

400 (%)

CONTROL

2%GB

CONTROL

2%GB

"Significantiy different from CONTROL, p<0.05.

Fig.2 Effect of Dried Green Barley Juice (DGBJ) on Endurance of Mice in Forced Treadmill RunningTest.

0 100
0%
2%
4%
0 20
0%
2%
4%
00/0
2%
4%
0 duration of running 125

150 (min)

body weight 25

30 (g)

*

25

75 (g-Km)

50

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.

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Standard food fed mice (A) 2% DGBJ food fed mice (B)

BI A x 100 (%) in paired mice

0-1 hr 368.8 ± 61.3 463.7 ± 56.7

1 - 2 hr 199.0 ± 64.9 22.1 ± 57.1

Table 1 . Effect of Dried Green Barley Juice (DGBJ) on Motor Activity of Mice in Wheel Cage Method

Number of revolutions

335.0 ± 126.3*

349.8 ± 147.8*

Each value represents the mean ± S.E. of 16 experiments. * Significantly different from 100%, p<0.05.

: ..

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APPENDIX 3

Studies on the Constituents of Green Juice From Young Barley Leaves

Anti-ulcer Activity of Fractions from Barley Juice*

Hidetoshi Ohtake, Hideo Yuasa, Chiseko Komura, Tetsuji Miyauchi, Yoshihide Hagiuiaras! 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]chigayafunagawara-machi, Shinjuku-hu, Tokyo, 162, Japan.

*This paper has been accepted for publication in Yakugaku-zasshi, issued from Pharmaceutical Society of Japan.

AU

Producttmm-) 1-6

Ulcer Index 1

7-12 13-18 18-24 234

24 < or perforation 5

Introduction

The fresh green juice from vegetables has been widely accepted to be useful for keeping humans in good health. There have been a few reports on 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 obtained from 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 Chern. Ind., Japan) were suspended or dissolved in physiological saline solution. Aspirin (Sanko Pharmaceutical Co., Ltd., Japan) was suspended in CMCN a 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.

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 + 10 C to the pectoral level of the rats. After 8 hours immersion, the rats 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 summed up. The summation of the ulcer 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.

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3. Acetic acid induces ulcer (5)

Rats fasted for 24 hours were laparotomized under ether anesthesia and the 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. After 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. The stomach 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)

Rats 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 of6N HCI, and 30 ml of1,2-dichloroethane were added to 0.2 ml of the serum separated centrifugally from the blood samples. The mixture was centrifuged and the supernatant was heated for 5 minutes in a water-bath kept at 95 C. After cooling ofthe 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.

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Results

1. 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-SD suppressed ulcer formation in Shay rats 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.

3) 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-Ppt and GM-SD revealed no significant anti-ulcer effect, but three other fractions, GM-P, GM-Sup and GM-L did reveal highly significant anti-ulcer activity (Table IV).

2. Effect on aspirin absorption and gastric secretion

All of 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 chlorophyll, denatured protein, polysaccharides, water-insoluble substances, and GM-P of water-soluble substances like amino acids, while GM-S is mainly composed of water-soluble high molecular substances.

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 stressinduced ulcer all of the fractions except for GM-S showed significant antiulcer activity at an oral dose of 500 mg/kg (Table II).

All fraction except for GM-Sup significantly accelerated the healing of acetic acid induced ulcer (Table III). The aspirin-induced ulcer was significantly suppressed by three fractions, GM-Sup, GM-P and GM-L (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).

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As shown in Tables V and VI, all fractions from GM-SD did not affect the gastric secretion and they were all ineffective in Shay ulcer, 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 a variety of substances which revealed significant 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 juke 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. Phannacol., 18,99.

5) K. Takagi, S. Okabe and R. 8aziki (1968), Japan. J. Phannacol., 19,418.

6) S. Okabe, K. Takeuchi, K. Nakamura and K. Takagi (1974), Japan. J.

Pharmacol., 24, 363.

7) T. Hata, N. Masuda, S. Iwasaki, K. Koike and H. Kasahara (1979), Shindan to Chiryo, 67, 359.

8) M.L. Anson (1938), J. Gen. Physiol., 22, 79.

9) Y. 8ato, 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. Phannacol., 24,357.

lyophilized

Chart 1

Young barley leaves

GM-SD(Ikg)

dissolved in distilled water

and centrifuged at lO,OOO x g for 20 min.

Precipitate

I lyophilized

GM-Ppt (300g)

Supernatant (GM-Sup)

saturated with (NH.hSO. and centrifuged at 10,000 x g for 20 min.

prelciPi:::YZed to water

and lyophilized

Supernatant

dialyzed to water for 2 days

~(200g)

Dialysate

Non-dialysate

90% methanol was added and filtered

Filtrate

I

GM-L(200g)

GM-S (lOOg)

Table I

Effect of Fractions from Green Juice of Young Barley Leaves on Stomach Ulcer in Shay-Rats

Treatment Number of Dose Ulcer index
animals (mg/kg) (mean ± S.E.)
Control 8 3.7 ± 0.6
GM-SD* 6 500 2.8 ± 0.7
GM-Sup* 7 500 4.1 < 0.6
GM-Ppt* 6 500 3.0 ± 1.2
GM·P* 6 500 3_5 ± 0.8
GM·L* 6 500 3.7 ± 0.6
Atropine sulfate 8 10 1.4 ± 0.3
• : See Chart 1
N.S. : Statistically no significant Inhibition P

(%)

24.3 N.S.
-10.8 N.S.
18.9 N.S.
5.4 N.S.
0.0 N.S.
62.2 <0.025 5

Table II
Effect of Fractions from Green Juice of Young Barley Leaves on Stress-induced Stomach Ulcers in Rats
Treatment Number of Dose Ulcer index Inhibition P
animals (mg/kg) (mean :t S.E.) (%)
Control 9 27.6:t 4.0
GM-SD· 8 500 14.2:t 1.4 48.6 <0.025
GM-Sup· 8 500 12.0:t 2.2 53.3 <0.01
GM-Ppt· 9 500 16.7:t 1.9 39.5 <0.05
Control 7 40.6:t 3.5
GM-Sup· 7 500 24.9:1; 2.3 38.7 <0.005
GM-P· 7 500 24.1 :t 3.8 40.6 <0.01
GM-S· 7 500 36.5 ± 4.0 10.1 N.S.
GM-U 7 500 17.6:t 0.9 56.7 <0.001
Atropine sulfate 6 2.7:t 1.4 93.3 <0.001
Control 10 30.5:t 2.1
GM-P 10 100 23.7:t 3.0 14.8 N.S.
10 500 14.7:t 3.3 51.8 <0.01
GM-L 10 100 18.6 :t 3.4 39.0 <0.05
10 500 13.1 :t 2.8 57.0 <0.01
• ; See Chart 1
N.S. ; Statistically no significant 6

Treatment Number of Dose Ulcer index
animals (mg/kg) (mean t S.E.)
Control 9 33.6 ± 4.1
GM·SD* 7 500 22.4 ± 7.1
GM·Sup· 8 500 15.1 % 2.6
GM·Ppt* 9 500 26.5 ± 4.1
Control 9 28.1 :t 4.1
GM·P· 8 SOD 10.2 ± 2.7
GM·L* 8 500 11.6 ± 2.3
*: See Chart I
N.S. : Statistically no significant Inhibition (%)

P

Table III

Effect of Fractions from Green Juice of Young Barley Leaves on Acetic Acid-induced Stomach Ulcers in Rats

Treatment Number of Dose Ulcer index Inhibition P
animals (mg/kg) (mean :t S.E.) (%)
Control 8 500 34.6:t 7.8
GM·SD* 8 500 10.9 ± 2.5 68.5 <0.025
GM·Sup* 8 500 17.4:t2.7 49.7 N.S.
GM·Ppt* 8 500 15.1 ± 3.5 56.4 <0.025
GM·P* 8 500 15.3 ± 3.8 72.3 <0.01
GM·L* 7 5 14.0 ± 1.3 59.5 <0.01
Atropine sulfate 5 S 14.0 ± 1.3 59.3 <0.01
Control 6 45.2 ± 3.5
GM·P 5 100 2S.0 t 3.1 44.7 <0.05
5 300 22.0 t 2.3 S1.3 <0.025
GM·L 5 100 20.5 ± 2.5 54.6 <0.01
6 300 16.2 ± 1.5 64.2 <0.01
• : See Chart I
N.S. : Statistically no significant
Table IV
Effect of Fractions from Green Juice of Young Barley Leaves on Aspirin-induced Stomach Ulcers in Rats 33.3 55.1 21.1

N.S. .;;0.005 N.S.

63.7 58.7

<O.OOS <0.005

7

Control 10.01 1.3 2.4510.10
GM·SD 500 9.210.9 2.44 ~ 0.04
GM·Sup 500 11.0 ~ 0.4 2.3610.03
GM·Ppt 500 10.8 ± 0.7 2.26:1 0.03
GM·P 500 12.0:1 0.4 2.25:1 0.06
Atropine 5 6.5:1 2.0 2.79:1 0.19
sulfate 72.31 8.0 101.3 :1 7.4
77.2 :1 5.5 103.8 :1 8.0
79.51 3.9 97.1:1 1 3.7
78.5 :1 5.6 104.5 :1 6.3
84.7111.4 IOI.3:1 11.7
52.5 :1 9.1 106.01 8.7 1431 1 134 1527 i 169 1347149 13731156 1375135 1017 i 31*

Table V

Effect of Fractions from Green Juice of Young Barley Leaves on Gastrointestinal Absorption of Aspirin in Rats

Treatment Dose Plasma salicylate Absorption
(mg/kg) concentraton (mg%)s) (%)
Control 14.8 f 0.8 100
GM·SD 500 12.9:t 0.6 87.2
GM.Sup 500 14.5 i 0.8 98.0
GM·P 500 14.7:1 0.7 99.3
GM·L 500 13.7 :10.7 92.6 Fractions from green juice of young barley leaves were given orally 30 minutes before the administration of aspirin (l00 mg/kg).

a) 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-ligated Rats.

Treatment

Dose (mg /kg)

Volume (ml)

pH

Free acidity Total acidity Pepsin activity

(/.LEq/ml) (unit/ml)

Each value represents mean 1 of S.E. of 6 rats. • ; Significantly different from control (P<O.OZ5)

8

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 Hagiurarac, Hideaki Hagiuiaral» and Kazuhiko Kubota»

a) Research Laboratory, Japan Pharmaceutical Development Co., Ltd.j-j-26, Mitsuya Minami, Yodogawa-ku, Osaka, 532, Japan.

b) Hagiwara Institute of Health, 1173, Maruyama, Asazuma-cho, Kasai, Hyogo, 679-01, Japan.

c) Faculty of Pharmaceutical Sciences, Science University of Tokyo, 12, Ichigayafunagawara-machi, Shinjuhu-hu, Tokyo, 162, Japan.

"This paper has been accepted for publication in Yakugaku-zasshi, issued from Pharmaceutical Society of Japan.

HY

1

Introduction

In Japan today, the increased intake offatty 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 cholesterol.

In the 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 substances showing activities such as potent anti-inflammatory action (1) and

. antiulcer action (2) from the green barley juice.

2

Materials and Methods

1. Isolation of su bstances 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 was 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,B-sitosterol and contains a small amount of'hexacosyl alcohol. Sample VI contains a small amount of,B-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 concentration of 1 % and administered orally to rats or mice.

The reference drugs used were nicotinic acid (N akarai Chern. Ind., Japan), soysterol (Morishita Pharmaceutical Co., Ltd.) and ,B-sitosterol (Merck & Co., Inc.).

The total cholesterol, free cholesterol, HDL-cholesterol, ,B-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 I and nicotinic acid were suspended in olive oil and or . nistered to rats

once a day for 6 days. The control group 0 s received ora ~ve 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 g 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.

3

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 I) induced favorable hypercholesterolemia, as shown in Fig. 1, without accompanying any observable adverse effects.

2. Anti-hypercholesterolemic effect of the fractions from green barley juice

Water-insoluble fractions from GM-SD were found to exert an antihypercholesterolemic effect. Further examinations revealed that Sample I, a hexane soluble fraction, had an anti-hypercholesterolemic effect as shown in Table 1. Sample I exerted dose-dependent anti-hypercholesterolemic effect (Table II), and showed higher activity than soysterol on day 9 (Table III).

The entity responsible for the anit-hypercholesterolemic activity in the fractionation Sample I was found in Sample III (Table IV). The fractionation of Sample III gave two fractions that showed anti-hypercholesterolemic effect (Table V). Sample VII or Crystal I, isolated from Sample IV also showed anti-hypercholesterolemic effect (Table V).

The total cholesterol in the liver was lowered by Samples V and VII (Table VI).

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 ethyl acetate.

Analytical data of Crystal I were as follows: IR ~~I crrr";

3320 (OH). IH-NMR (CDCb)b; 1.15-1.46 (5H, m), 3.72 (2H, t,J=7 Hz). MSm/z; 3.82 (M , weak), 364 (M+ -H20), 336 (M+ -H20-C2H4). Anal. Calcd. C2sHs40 ; 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 nhexacosyl alcohol having the molecular formula of CH3 (CH2)24-CH20H.

Sample V gave two kinds of crystals on silica gel column chromatography. One of them was identified to be hexacosyl alcohol and the other gave colorless needles having a melting point of 137-139°C after recrystalization 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 ofGM-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 antihypercholesterolemic effect in rats fed on HCD and significantly lowered the serum cholesterol level in the rats. Sample I extracted from the waterinsoluble fraction by n-hexane had twice the anti-hypercholesterolemic activity of soysterol, which has been marketed as an anti-hypercholesterolemic agent in Japan, and its acute toxicity was shown to be very low (Tables II and III). In the present study, n-hexacosyl alcohol or ceryl alcohol and ,B-sitosterol, both of which have anti-hypercholesterolemic 'activity, were isolated from the barley juice and identified. Ten g of Sample I was found to contain 1.2 g of hexacosyl alcohol and 1 g of ,B-sitosterol in it.

Hexacosyl alcohol was demonstrated by us to have anti-hypercholesterolemic 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.B-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 hypocholesterolemic action ofhexacosyl alcohol 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 ofhexacosyl alcohol is under way.

The present work suggests that green barley juice may be very useful for preventing humans from vascular diseases associated with hypercholesterolemia.

References

1) Y. 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-zasshi 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. BioI. Chern. 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. Kuroda, Y. Sugiya, T. Osazuma, R. Miguchi, T. N ozawa, S. Satoh, T. Takeda and T.

Matsui (1971), Kiso to Rinsho, 5, 1019.

4

Chart I

GM·SD (lkg)

_ suspended in we of water, and centrifuged at 10,000 x g for 20 min.

f

Supernatant

PI ..

recipitate

~YOPhilized

GM·Ppt (450g)

bextracted with n-hexane for 30 min. r--------'------"

Residue Hexane extract

~ concentrated Sample I (lOg)

GM·R

1 I I

column size: 5 x lOOcm silica gel: 720 g

solvent: Benzene: Ethyl Acetate (4 : 1)

silica gel column chromatography

frLtiO]

1 2 345

Lo....-I ---<I~I I I

I I

6 7

I I

Sample II (3.6g)

Sample III (6.3g)

silica gel column chromatography as noted above for Sample I

I Sample V Sample VI

(2.4g) (l.lg)

I Sample IV (2.8g)

I

Sample VII (1.2g)

(crystal 1 or hexacosyl alcohol)

5

Table I

Components

Composition of High Cholesterol Diet

Tensho's Modified
(%) (%)
2 1
1 1
20 25
49 50
12 10
4 5
4 4
8 4 Cholesterol Cholic acid Casein

Sucrose Hydrogenated fat Cellulose

Vitamins & Minerals Dried fish powder

Figure 1

Change in Serum Total Cholesterol Levels of Rats fed on a High Cholesterol Diet

..-. 1000
"0
<,
bo 800
E
"0
'"' 600
Ql
...
(I)
Ql
"0
..c: 400
u
co
....
0 200
....
E
;:I
....
~ 0
00
0 3 6 9 Duration of Treatment (days)

~: a high cholesterol diet shown by "modified" in Table I ~: a normal diet (CE-2, see text)

6

Control Sample I

1% 0.5% 0.25% 1%

613.0 ± 26.5 51O.2:!: 37.3 (16.8)· 564.8 ± 33.8 (7.9) 667.2 ± 101.8 (-8.8) 553.6:!: 28.1 (9.7)

683.9 ± 37.9

472.3 ± 26.2 (30.9)·· 506.5 :!: 34.6 (25.9)·· 609.9 :!: 82.7 (10.7) 524.3 ± 51.1 (23.3)·

Table II

Effect of Young Barley Leaves Fractions on Serum Total Cholesterol Levels of Rats Fed on a Cholesterol Diet

Treatment Dose Serum total cholesterol (mgxdl) (Inhibition %)a) LD.~(l
(g/kg) Day 6 (g/kg)
Control 328.2 ± 62.6
GM·SD 5 493.1 ± 66.0 (6.6) > 10
GM·Ppt 5 450.2 :J: 44.6 (14.8) > 10
Sample I 4078 ± 33.2 (22.8)· > 8
GM·R 454.7:J: 43.9 (13.9) > 10
Nicotinic acid 0.5 330.1 :!: 67.1 (37.5)· a) Each value represents the mean e S.E. of 6 rats .

•. P 0.05, significantly different from the control group.

Table III

Effect of Young Barley Leaves Fractions on Serum Total Cholesterol Levels of Rats Fed on a Cholesterol Diet

Treatment

Serum total cholesterol [mg/d!) (inhibition %)a)

Day 6 Day 9

Soysterol

a) Each value represents the mean :I: S.E. ofG rats.

b) 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 < 0.05 and **:P < 0.005, significantly different from the control group.

7

Table IV

Effect of Young Barley Leaves Fractions on Serum Total Cholesterol Levels of Rats Fed on a Cholesterol Diet

Treatment

Serum total cholesterol (mg/d!) (Inhibition %)8)

Day6 Day 9

Control 250.0 t 13.2 693.3 t 61.0
Sample I 176.4 t 13.1 (29.6)'" 438.0 t 45.1 (36.8)"
Sample II 198.0 t 24.8 (20.8) 502.0 t 61.8 (27.6)
Sample III 142.0 t 29.4 (42.8)· 478.0 t 56.7 (31.0)·
.a-sitosterol 179.0 t 23.5 (28.0) 346.0 t 70.0 (50.1)" . Controls rats were fed on a high cholesterol diet and other groups of rats on the high cholesterol diet added Sample I-III or ,B-sitosterol at a concentration of 1%.

a) Each value represents the mean S.E. of rats .

• ; P < 0.05 and "": P < 0.005, significantly different from the control group.

Table V

Effect of Young Barley Leaves Fractions on Serum Total Cholesterol Levels of Rats Fed on a Cholesterol Diet

Treatment Serum total cholesterol (mgv'dl) (Inhibition %)a)
DayO Day3 Day 6 Day9
Control ll6.2 t 4.5 587.3 t 21.3 1184.6 t 95.8
Sample III 106.2 t 7.1 446.0 t 53.8 (24.1) 776.4 t 48.9 (34.5)**
Sample IV 109.8 t 6.1 557.9 t 29.4 (1.6) 883.9 t 33.7 (25.4)·
Sample V 99.7 t 11.5 434.7 t 34.9 (26.0)** 825.7 t 95.9 (30.3)·
Sample VI 126.9 t ILl 567.8 t 40.3 (3.3) 1051.0 t 81.7 (11.3)
,B-sitosterol 119.9 t 12.8 329.0 t 19.8 (44.0)** 657.8 t 41.9 (44.5)·*
Control 62.9 ± 8.2 656.6 t 19.2 731.2 ± 16.4 1106.8 ± 79.6
Sample III 66.2 t 5.2 551.4 t 40.3 (15.9) 618.8 t 31.7 (15.4)· 739.7 t 68.4 (26.5)·
Sample VII 63.5 t 13.2 664.6 t 10.3 (-1.4) 695.3 t 24.8 (4.9) 796.1 ± 35.5 (20.9)·
.a.sitosterol 70.3" 19.0 563.4 ,. 16.1 (14.1) 602.0 t 32.6 (17.7)** 748.6 t SO.O (25.6)· Control rats were fed on a high cholesterol diet and other groups of rats on the high cholesterol diet added Sample III-VII or .a-sitosterol at a concentration of 1%_

a) Each value represents the mean t S.E. oC6 rats.

*- P < 0.05 and •• ; P < 0.005, significantly different from the control group.

8

Treatment Liver wet weight Liver w~ Body Total liver cholesterol
(g) weight weight mg mg/g
(%)
Control 7.5 t 0.48) 5.3 t 0.3 180 t 8.5 24.2 :t 0.4
Sample! 7.8:t 0.4 5.4 t 0.2 179:t 6.8 23.2 t 0.5
Sample II 6.9 t 0.3 5.1 t 0.3 166 ± 5.7 24.1 ± 0.5
Sample III 7.2 ± 0.2 5.4 ± 0.1 167 ± 6.7 23.3:t 0.5
Sample 8.1 ± 0.3 5.9 ± 0.2 183 ± 7.7 22.7 ± 0.3·
.a-sitosterol 7.5 ± 0.2 5.3 ~ 0.1 165 ± 5.8 21.9 ± 0.4··
Control 7.7 ~ 0.2 5.5 dl.1 161 ± 3.3 21.1 ~ 0.1
Sample IV 7.7:t 0.3 5.7:t 0.3 169:t 5.2 21.9:t 0.3
Sample V 7.1 ± 0.3 5.4 :t 0.3 133:t 7.8 18.7:t 0.5··
Sample VI 7.0 ± 0.4 5.1 ± 0.3 169 t 10.4 24.3 :t 0.4
.a-sitosterol 7.2 ± 0.4 5.0 ± 0.3 138 ± 3.1 19.3 :t 0.4·· Table VI

Effect of Young Barley Leaves Fractions on Liver Total Cholesterol Levels of Rats Fed on a Cholesterol Diet for 6 days

Control rats were fed on a high cholesterol diet and other groups of rats on the high cholesterol diet added Sample I - VII or .a-sitosterol at a concentration of 1 %.

a) Each value represents the mean e S.E. of 6 rats fed a high cholesterol diet. *: P < 0.05 and **: p < 0.005. significantly different from the control group.

9

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

I~,~"~ '»_,

'3694'

Inhibition of Malonaldehyde and Acetaldehyde Formation from Blood Plasma Oxidation by Naturally Occurring Antioxidants

Takasbi Miyake and Takayuki Sbibamoto* •

Department of Environmental Toxicology, University of California, One Shields Avenue, Da~,California95616

The inhibitory effect of 2" -O-glycosylisovitexin (2" -O-GIV), 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF), t-ascorbic acid (vitamin C), and 4,4'-(isopropylidenedithio)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 t-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 2" -O-GIV or DMHF was mixed with L-ascorbic acid in equal amounts. Approximately 80-90% of acetaldehyde formation was inhibited by 300 nmol of 2"-O-GIV and probucol, whereas DMHF required 1000 nmol to exhibit the same level of inhibition.

~eywords: Malonaldehyde; acetaldehyde; blood plasma; natural antioxidants

Food components that possess such biological characteristics as anticarcinogenicity, antimutagenicity, antioxidative 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 carcinogenesis, 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 a1., 1993). Therefore, the oxidation of blood plasma lipids is strongly associated with atherosclerosis and endothelial dysfunction (Schmidt et a1., 1994; Tesfamariam, 1994).

The mechanisms causing these diseases by lipid peroxidation are not yet well understood. One hypothesis is that reactive carbonyl compounds, such as malonaldehyde (MA) and acetaldehyde, formed from lipid peroxidation produce abnormal adducts with biological substances, including DNA and RNA (Feinman, 1988; Esterbauer et al., 1991). In fact, a strong relationship between atherosclerosis and MA and acetaldehydes formed from lipid peroxidation has been reported (Glavind et al., 1952).

Humans are constantly exposed to reactive oxygen species produced either by natural phenomena such as ultraviolet light or by anthropogenic activities (for example, automobile exhaust). Therefore, supplementing antioxidants to scavenge undesirable reactive oxygen species is very important to prevent in vivo oxidative damage. Natural plant foodstuffs are one of the most important suppliers of antioxidants ' (Namiki, 1990).

* Author to whom correspondence should be addressed [telephone (530) 752-4523; fax (530) 752-3394; e-mail tshibamoto@ucdavis.edu].

In the present study, the inhibitory effect of natural plant antioxidants 2" -O-glycosylisovitexin (2" -O-GIV), 2.sl.dimethyl-4-hydroxy-3(2.H)-furanone (DMHF), and t-ascorbic acid (vitamin C) on acetaldehyde and MA fOrlnation from horse blood plasma lipids ~xidized with Fenton's reagent was determined by gas chromatography (GC). A commercial antiatherosclerosis drug, probucol [4,4' -(isopropylidenedithio )bis(2,6-di-tert-butylpheno1)] was also tested according to the same method.

EXPERIMENTAL PROCEDURES

Materials. Butylated hydroxytoluene (BHT). sodium dodecyl sulfate (SDS), L-ascorbic acid (vitamin C), probucol, and bovine serum albumin were purchased from Sigma Chemical Co. (St, Louis, MO), Benzalkonium chloride (BC) was bought from lCN Biomedicals, Inc. (Aurora, OH). DMHF, 2-methylpyrazine, malonaldehyde bis(diethyl acetal), N-methylhydrazine, and ferrous chloride were obtained from Aldrich Chemical Co. (Milwaukee. WI). Protein dye (Coomassie Blue) was purchased from Bio- Rad Laboratories <Richmond, CAl. 2"O-GIV was isolated from young green barley leaves (Hordium vulgare L. var, nudum Hook) harvested 2 weeks after germination according to a method previously reported (Osawa et al.,1992). The structures of2"-O-GIV, DMHF, vitamin C, and probucol are shown in Figure 1.

Standard stock solutions (1 mL each) for GC analysis were prepared by dissolving 2-methylpyrazine and 2,4,5-trimethylthiazole in dichloromethane (10 mglmL) and stored at 5 °C until used. Authentic N-methylpyrazole (NMP) was synthesized according to the method reported by Umano et al, (1988), and authentic 2-methylthiazolidine was synthesized according to the method reported by Yasuhara and Shibamoto (1991). Sodium malonaIdehyde was synthesized according to a method previously described by Lacombe et aI. (1990).

Blood plasma was prepared from horse blood (20-year-old male quarter horse) by centrifugation at 5000 rpm for 30 min at 4°C. The blood plasma was frozen on dry ice immediately after preparation and stored at -80°C until use.

Protein Analysis in Blood Plasma. The Coomassie Blue dye-binding assay (Bio-Rad Laboratories) was used to determine plasma protein concentrations (Bradford, 1976). A 10 ilL aliquot of appropriately diluted plasma protein was added to the dye reagent, and then absorbance at 594 nm was

S0021-8561(98)00318-5 CCC: $15.00 @ 1998 American Chemical Society

Published on Web 08111/1998 .

Inhibtlion of Blood Ptasma Oxidation by Natural Antioxidants

OH

2"-O-GIV

OH

o=c;n

HO-C

HO-C 0

Hf__J

HO-C;:H CH!OH

Vitamin C

DMHF

Probucol

Figure 1. Structures of chemicals used in the present study.

measured versus a reagent blank. using UTI HP b452A diode array UV spectophotometer. A standard curve using bovine serum albumin was used to calculate plasma protein concentration.

Oxidation of Blood Plasma with Fenton's Reagent in the Presence or Absence of Testing Chemicals. Blood plasma was oxidized according to the method reported previously (Ichinose et al., 1989J. AIl aqueous solution (5 ml.) containing 50 ilL of blood plasma (860 ug of protein), 0.25 mmol phosphate buffer t p'H 7.4).3 umol of ferrous chloride. 0.5 mmol of hydrogen peroxide. 0.75 rnrnol of potassium chloride. and 0.2'ii of surfactant Be or SDS was incubated 'with various amounts of2"-O-GIV. DMHF. vitamin C. probucol, 2"-O-GIV + vitamin C. and DMHF + vitamin C for 16 h at 37 °C in a 20-mL test tube. The oxidation of samples was stopped by adding 50 ,uL of 4'ii BHT ethanol solution. The sample tubes were covered with aluminum foil during incubation to avoid any influence of light.

Analysis ofMA 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). 'Ele MA was reacted with N-methylhydrazine (NMH), and the resulting derivative, I-methylpyrazole, was analyzed with 2-methylpyrazine as an internal standard by a GC equipped with a fused silica capillary column and a nitrogen-phosphorus detector (NPm. 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, 1993, 1995) with slight modification. Acetaldehyde was reacted with cysteamine at room temperature for 24 h. The reaction mixture was extracted with 10 mL of dichloromethane using a liquid-liquid continuous extractor. After the extract was dried over anhydrous sodium sulfate, the resulting derivative, 2-methylthiazolidine, was analyzed with 2,3,S-trimethylthiazole 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 spiked into 30 ,uL of horse blood plasma. Samples were prepared and analyzed for MA and acetaldehyde by GC as described above. The experiment was replicated three times.

J. Agric. Food Chem., Vol. 46, No.9, 1998_3695

~
E _c
S 100
4(
:f 600
"0
~ .tOO
::>
0
E 100
4( It 40 60 10 100 120

1000 1000 3000 4000

Amount of antioxidants (nmol)

Figure 2. Inhibitory activity of2"-O-GIV, DMHF, probucol, and t-ascorbic acid toward MA formation from blood plasma upon oxidation.

Instrumental Analysis. A Hewlett-Packard (HP) model 5890A gas chromatograph equipped with a 30 m x 0.25 mm i.d. (dr = t.,um) DB-1 bonded-phased fused silica capillary column (J& W Scientific, Folsom, CA) was used for quantitative analysis of2-methylthiazolidine, and an HP model 5890A gas chromatograph equipped with a 30 m x 0.25 mm i.d. (dr = 1 ,um) DB-Wax column was used for quantitative analysis of NMP. The NPD and injector temperatures were 250 'C. The linear velocity of the helium carrier gas was 30 ernIs with a split ratio of 20:1. The oven temperature was programmed from 60 to 180 °C at 4 'C/min with a final holding time of 10 mjn. ,Peak. a~eas were integrated with a Spectra Physics SP

4_90 integrator. _

AI, 3:P model 5890 series I' GC interfaced to an HP model 5971 mass spectrometer was Used to confirm the MA derivative. NMP, and acetaldehyde derivative, 2-methylthiazolidine, in the sample. The GC conditions were the same as for the GC described above. The mass spectra were obtained by electron impact ionization at 70 eY and an ion source tem-

perature of 250°C, ')

RESULTS AND DISCUSSION

In the present study, recovery efficiencies of MA and acetaldehyde from horse blood plasma were 82.1 ± 1.3 and Bi.5 ± 1.2'7{;, respectively. The values are mean ± standard deviation (n = 3). Protein concentration in the blood plasma was 17.2 p.g/p.L. When 50 .uL of blood plasma (860 p.g of protein) was oxidized, 957 ± 199 nmol of MA was formed. The value is mean ± standard deviation (n = 3). The control sample (860.ug of blood plasma) contained 60-80 runol of MA, indicating that a certain amount ofMA was present in the blood plasma prior to oxidation. When 30 #L of blood plasma (516 .ug of protein) was oxidized, 135.0 ± 7.21 nmol of acetaldehyde was formed. The value is mean ± standard deviation (n = 3).

Figure 2 shows the inhibitory activity of 2" -O-GIV, DMHF, probucol, and L-ascorbic acid toward MA formation 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 formation 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 C activity did not change significantly. DMHF at 4000 nmol exhibited activity comparable to that of 100 nmol of 2"-0·GIV , Activity of2"-O-GIV in doses higher than 100 nmol wa..:~ not examined because of its solubility in the testing system.

When 2" -O-GIV and DMHF were mixed with vitamin C, an increase in their antioxidative activities was observed. A mixture of2"-0-GIV (50 nmol) and vitamin

3696 J. Agric. Food Chem., Vol. 46, No.9, 1998

ISO

50 100 ISO 200 250 300 500 1000 2000 3000

Amount of antioxidants (nmol)

Figure 3. Inhibitory. activity of 2" -O-GIV, probucol, and DMHF toward acetaldehyde formation from blood plasma upon oxidation.

C (50 nmol) inhibited MA formation by 75%. The activity was -5% higher than that of 2"·O-GIV alone. A mixture ofDMHF (25 runol) and vitamin C (25 nmol) inhibited MA formation by 63%; however, the mixture did not increase activity significantly when the total dose wag. increased up to 4000 nmol. These results indicate that the inhibitory activities of 2"-O-GIV and DMHF are enhanced by the addition of vitamin C. Detailed mechanisms of these phenomena are not yet well understood. It has been known that i.-ascorbic acid and a-tocopherol act synergistically to inhibit lipid peroxidation (Sharma and Buettner, 1993). It has been proposed that the mechanism by which this occurs is that L-ascorbic acid reacts with an a-tocopherol radical to regenerate a-tocopherol, and then the resulting L-ascorbic acid radical is reduced back to t-ascorbic acid 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 1/10 of actual values. When 860 !lg of blood plasma was oxidized in the presence of 100 and 4000 nmol ofprobucol, 2296.8 ± 326 and 3875.5 ± 194 nmol ofMA were formed, respectively, suggesting that probucol may have pro-oxidative activity. However, this hypothesis can be ruled out because probucol significantly inhibited acetaldehyde formation as reported in the following paragraph. Therefore, MA may be produced from probucol during the incubation 9f blood plasma. In fact, an aqueous solution of probucol (3 !lmo}) produced 2.6!lmol of MA upon oxidation with Fenton's reagent (Miyake and Shibamoto, 1998).

Figure 3 shows the respective inhibitory activity of 2" -O-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 acetaldehyde and MA. As shown in Figure 3, 2" ·O-GIV and probucol exhibited quite similar activities. The formation of acetaldehyde was inhibited nearly 90% in the presence of 300 nmol of either 2" -O-GIV or probuco1. 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.hyde formation by 88%, which is comparable to the activity obtained by either 2" -O-GIV or probucol at the 300 nmollevel.

Miyake and Shibamoto

The highly effective antioxidative activity of2"-O·GIV has been shown in various lipid peroxidation systems: ethyllinoleate or squaleneIFenton's reagent (Osawa et al., 1992; Kitta et al., 1992); ethyl arachidonate/Fenton's reagent or UV light (Nishiyama et al., 1993); phospholipids or fish liver oillFenton's reagent (Nishiyama et al., 1994); and w·3 fatty acidslFenton'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 in a commercial health food product prepared from young green barley leaves. Therefore, it is 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 browning reaction products in the early 1960s (Hodge et al., 1963). Later it was reported as a characteristic flavor chemical formed in carbohydrate caramelization and dehydration reactions (Hodge, 1967). DMHF 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 Grosch, 1994); cultures of bacteria, Lactobacillus helueticus (Preininger and Grosch, 1995); and Maillard reaction products (Blank and Fay, 1996). Therefore, it is presumed that DMHF is ingested through the foods described above. The anti oxidative activity ofDMHF has never been reported prior to this study. Discovery of anti oxidative 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 biologically 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 compound was used to compare its activity with that of other tested compounds in the present study.

Probucol was also tested in the blood plasma system because it is 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 ofMA formed in samples with probucol are considerably different from those formed in samples with 2" -O-GIV, the results obtained from the acetaldehyde analysis (Figure 3) are almost identical. Therefore, it is hypothesized that the naturally occurring antioxidant, 2" ·O-GIV, may inhibit diseases such as atherosclerosis associated with oxidative damage as effectively as probuco1. Moreover, this antioxidant might be more effective if it is used with vitamin C.

LITERATURE CITED

Blank, 1.; Fay, L. B. Formation of 4-hyciroxy-2,5-dimethyl- 3(2H)-furanone and 4·hydroxy-2(or 5)-ethyl-5(or 2)-methyl- 3(2H}-furanone through Maillard reaction based on pentose sugars. J. Agric. Food Chem. 1996,44,531-536.

Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Bioehem. 1976, 72, 248-254.

Inhibition of Blood Plasma Oxidation by Natural Antioxidants

'of·"

Esterbauer, G. H.; Schaur, R. J.; Zollner, H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radical Bioi. Med. 1991,11,81-128.

Feinman, S. E. Structure-activity relationships of formaldehyde. In Formaldehyde Sensitivity and Toxicity; Feinman, S. E., Ed.; CRC Press: Boca Raton, FL, 1988; pp 197-204.

Glavind., J.; Hartmann, S.; Clemmesen, J.; Jessen, K. E.; Dam, a. Studies on the role oflipoperoxides in human pathology. II. The presence ofperoxidized lipids in the atherosclerotic aorta. Acta Pathol. Microbial. Scand. 1952, 30, 1-6.

Halliwell. R; Gutteridge. J. M. C. Free Radicals in Biology and Medicine; Clarendon Press: Oxford, U.K, 1989; pp 1, 51-57.

Hodge, J. Origin of flavor in foods nonenzymatic browning reactions. In Chemistry and Physiology of Flavors: Schultz, H. W., Day, E. A., Libbey, L. M., Eds.; AVl Publishing:

Westport, CT, 1967; pp 465-491.

Hodge, J .. E.; Fisher, R E.; Nelson. E. C. Diearbonyls, reductones, and heterocyclics produced by the reactions of reducing sugars with secondary amine salts. Am. Soc. Breu:

Chem. Proc. 1963, 1963, 84-92.

Ichinose, T.; Miller, M. G.; Shibamoto, T. Gas chromatographic analysis of free and bound malonaldehvde in rat liver homogenates, Lipids 1989.24, 895-898 ..

Kitta, K; Hagiwara, Y.; Shibamoto. T. Antioxidative activity of an isoflavonoid. 2"-O-glycos~'lisovitexin isolated from green barley leaves. J. Agric. Food Chern. 1992.40. 1843- 1845.

Krinsky. N. I. Carotenoid protection against oxidation. Pure .~·ljJP~·. Ciu-n.. !ti9. ;~:. ~;-;~-t)~;:_' .

Lacombe. A.: Kerrnasha. S.; van ~e Vuurt. R. F.: l\iilh;. L B.

Preparation and purification ofmalonaldehvde sodium salt. J. Agric. Food Chern, 1990.38. 41f;-423.

Miyake, T.: Shibamoto. T. Quantitativ(, analysis of acetaldehvd« in food!' and beverages. J .. 'writ'. Food Chern, 1993. 41. 1968- W70.

Mivake. T.: Shiburnoto. T. Formation of acetaldchvde from i.-ascorbic acid and related compounds in various ~xidation systems. J. lV.!ric. Food Chern, 1995. 43. 166~-1672.

Miyake, T.: Shibamotu. T. Formation of malonaldehydc in the presence of prohucol. an anti-atherosclerosis drug. Food Cliem. Toxicol. 1998. in press.

Namiki. M. Antioxidants/antimutagens in food. Crit. Rec. Food Sci. Nutr. 1990.29. 273-300.

Nishiyama. T.: Hagiwara. Y.: Hagiwara, H.: Shibamoto. T.

Inhibition of malonaldehyde formation from lipids by an isoflavonoid isolated from young green barley leaves. J. Am. Oil Chern. Soc. 1993.70,811-813.

Nishiyama. T.: Hagiwara. Y.: Hagiwara, H.: Shibamoto. T.

Formation and inhibition of genotoxic glyoxal and malonaldehyde from phospholipids and fish liver oil upon lipir' peroxidation. J. Agrie. Food Chem. 1994.42. 1728-1731.

Ogata, J.; Hagiwara. Y.; Hagiwara. H.: Shibamoto. T.lnhibition of malonaldehyde formation by antioxidants from (11-3 polyunsaturated fatty acids. J. Am. Oil Chern. Soc. 1996, 73. 653-656.

Ohlotr, G. Chemistry of odoriferous and flavoring substances.

Fortschr. Chem. Forsch, 1969. 12, 185-251.

Osawa. T.; Katsuzaki. H.; Hagiwara, Y.; Hagiwara, H.; Shibamoto, T. A novel antioxidant isolated from young green barley leaves. J. Agric. Food Chern. 1992, 40, 1135-1138.

Packer, J. E_; Slater, T. F.; Wilson, R. L_ Direct obserVation of a free radical interaction between vitamin E anl vitamin C. Nature 1979,278,737-738.

Parthasarathy, S.; Young, S. G.; Wiztum, J. L.; Pittman, R.

C.; Steinberg, D. Probucol inhibits oxidative modification oflow density lipoprotein. J. Clin. Invest. 1986, 77, 641- 644.

Preininger, M.; Grosch, W. Evaluation of key odorants of the neutral volatiles of emmentaler cheese by the calculation of odour activity values. Food Sci. Technol.-Lebens.-Wissen. Teehnol. 1994,27, 237-244.

Preininger, M.; Grosch, W. Determination of 4-hydroxy-2,5- dimethyl-3(2HHuranone (HDMF) in cultures of bacteria. Z. Lebensm-Unters. Forsch. 1995.201,97-98.

Rapp, A.; Knipser, W.; Engel, L.; Ullemeyer, H.; Heimann, W.

Off-flavor compounds in the berry and wine aroma. Vitis 1980, 19, 13-23.

Retsky, K. 1.; Freeman, M. W.; Frei, B. Ascorbic acid oxidation products protect human low density lipoprotein against atherogenic mbdification-Antioxidant rather than prooxidant activity of vitamin C in the presence of transition metal ions. J. BioI. Chern. 1993. 268, 1304-1309.

Rodin, J. 0.: Himel, R. M_; Silverstein. R. M.; Leeper, R. W.; Gortner, W. A. Volatile flavor and aroma components of pineapple (I) isolation and tentative identification of 2.5- dimethyl-4-hydroxy-3(2HI-furanone. J. Food Sci. 1965,30. 280-285.

Schmidt. A. M.: Hori. 0.; Brett. J.: Yan. D. D.: Wauitier, J. L.; Stern. D. Cellular receptors for advanced glycation end products+unplicai ions for induction of oxirinnt stress and cellular dysfunction in the pathogenesis ofvascular lesions. Arterioscler. Thrombosis 1994,14.1521-1528.

Sharma. M. K: Buettner, G. R. Interaction ofvitamin-C and vitamin-E during free radical stress in plasma-an ESR Study. Free Radical Bioi. Med. 1993. 14. 649-653.

Sinchara. A.: Macku. C.: Shibamoto. T. Antioxidative activity of brewed coffee extracts. In Functional Foods: Disease Prevention: Proceedings of the 213th National Meeting of the American Chemical Society. San Francisco. CA. 1998; in press.

Tesfamariam. R Free radicals in diabetic endothelial cell dvsfunction. Free Radical Biol. Med. 1994. 16. 383-391.

Umano, K: Dennis. K. J.: Shibamoto, T. Analysis of free malonaldehvde in photoirradiated corn oil and beef fat via a pyrazole derivative. Lipids 1988,23,811-814.

U.S. Department of Health and Human Services. Report 331; National Toxicology Program: Research Triangle Park, NC. 1988.

Wong, J. W.: Ebeler, S. E.; Rivkah-Isseroff. R.; Shibamoto. T.

Analysis of malondialdehyde in biological samples by capillary gas chromatography. Anal. Biochem. 1994. 220, 73- 81.

Yasuhara, A.; Shibamoto, T. Determination ofvoJatile aliphatic aldehydes in the headspace of heated food oils by derivatization with 2-aminoethanethiol.J. Chromatogr. 1991,547. 291-298.

Received for review April 2, 1998. Revised manuscript received June 23, 1998. Accepted July 1, 1998.

JF980318Z

ACS SYMPOSIUM SERIES

702

Functional Foods for Disease Prevention II

Medicinal Plants and Other Foods

Takayuki Shibamoto, 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

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 •

lHagiwara Institute of Health, 1173 Maruyama, Asazuma-cho, Kasai 679-01, Japan

Young green barley leaves are known (0 possess potent pharmacological properties, including antioxidative, antiinflammatory, antimutagenic, and antiallergic activities. In particular, an flavonoid, 2"-O-glycosylisovitexin (2"-O-GIV), isolated from an ethanol extract of young green barley leaves, possesses a strong inhibitory effect toward lipid peroxidation. 2"O-GIV inhibited acetaldehyde formation from LDL by 76% at a level of 1 ~mol/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 antiarteriosclerosis (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-glycosyl isovitexin (2"-O-GIV, Figure 1). Since the discovery of this flavonoid, the antioxidative activities of 2"-O-GJV examined in various 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 (.1)-3 fatty acids/Fenton'S reagent have been reponed(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 (HIV) progression (10). However, its mode of toxic action is not yet clearly understood. Lipids produce many low molecular weight carbonyl compounds upon oxidation (/ I). Therefore, these carbonyl compounds such as malondialdehyde (MDA), glyoxal, acrolein, acetaldehyde, and formaldehyde which directly crosslink to proteins and bind

JCorresponding author.

178

© 1998 American Chemical Society

,

"

179

Hleo

HO-O-- CH- CHCOOH •

Ferullo acid

HO

OH

Probucol

HO

OH

2" ·O·Glycosylisovitexin (2" -O-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 ad~ti?n to ini?ati~g these adverse effects, low molecular weight aldehydes formed from lIPId per?xIdauon can be used as indicators to detect oxidation in lipids. Therefore, many studies have been conducted using these aldehydes as indicator, in panicular, malondialdehyde (MDA). The formation of acetaldehyde was also used to monitor oxidative reaction mechanisms of L-ascorbic acid (15).

In the present study, the antioxidative activity of 2"-O-GIV was examined using low density lipoprotein and blood plasma oxidized with Fenton's reagent.

~ ~

Experimental

Chemicals. L Ascorbic acid (reagent grade), butylated hydroxy toluene (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. (Milwaukee, 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 dc. Authentic 2-methylthiazolidine was synthesized according to the method reported previously (16). 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-GJV 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 reponed previously (15). After fllrrationsterilization (0.45 11m: Nalge Stbron) of the LDL, the protein concentration was determined by the Coornassie Blue dyebinding assay (17). A lO-IlL aliquot of appropriately diluted LDL was added to the dye reagent, the solution mixed, and absorbance at 594 nm measured versus a reagent blank, using a Hewlett-Packard 8452A diode array UV spectrophotometer. A standard curve using bovine serum albumin was used to 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 J,tL of a 7.5% EDTA solution (4.5 mg EDT A). 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 ug/rnl.), 0.25 mmol of Trizma® buffer (pH 7.4), 0.75 mmol of potassium chloride, 1 mrnol of ferrous chloride, and 0.5 mrnol of hydrogen peroxide was incubated with 2"-O-GIV (amounts ranging from 0 to I umol, with 0.1

181

umol increments) or ferulic acid (amounts ranging from 0 to 1 urnol, with 0.1 urnol increments) at 37 °C for 15 h.

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 urnol) or probucol (0, 0.05, 0.07, 0.1, 0.2, and 0.3 umol) at 37 °C for IS h. In another experiment, samples containing blood plasma (860 ug of protein), 0.25 mmol of Trizma® buffer (pH 7.4), 0.75 mmol of potassium chloride, I 010101 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 umol increment), vitamin C (amounts ranging from 0 to 0.1 umol, with 0.02 urnol increments), or a mixture of 2"-O-GIV and vitamin C (total amounts ranging from 0 to 0.1 umol, with 0.01 urnol increments), in which molar ratio of2"-O-GIV and vitamin C was 111.

While the incubation continued, the mixtures were covered with parafilm. A 5-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 JlL 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 (18). The amount of MDA was measured as it pyrazole derivative by the method developed previously (19). A Hewlett-Packard Model 5890 GC equipped with a nitrogen phosphorus detector (NPO) and a 30 m x 0.25 mm i.d. (dr = I J,Lm) OB-l 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 ern/sec with a split ratio of 21 : 1. The oven ternperatu re 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 II 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 MOA, 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 reponed (23, 24). Recently, oxidative damage of LOL 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

0.950 •••.. 1-----&. - _ .

Very-low density lipoprotein (VLOL)

1.006 ···1----.+ .

182

Density (g/mL)

A \II

Lipoprotein

Chylomicrons

1.063 ···I---~ .

Low-density lipoprotein (LOL)

1.210 .•. ---~ ..•...•.•.•..•.•• -- .•...•.•...............•......••.•.

High-density lipoprotein (HDL)

Figure 2. Fractions of lipoprotein .

100

80

60

%

40

• Proteins

EI Phospholipids

II Free Cholesterol ~ Cholesterol Esters o Triacylglycerols

eM

HOL

VLDL

LDL

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 ug 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 ~mol/50 ug of LDL) whereas 2"-O-GIV required 0.7 ~mol/5()~lg of LDL to obtain the same level of inhibition. On the other hand, 2"-O-GIV inhibited acetaldehyde formation by 76% at the level of 1 J.1mol/50 J.1g 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-GIV because it is a commercial product with a million dollar sales in Japan and has been used to treat atherosclerosis. 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 J.1g) was oxidized wi thou t an antioxidant, 135 nrnol of acetaldehyde was recovered. Inhibitory activity of both 2"-0'-GJV and probucol toward acetaldehyde formation increased greatly when their levels increased over 0.7 urnol. 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-GJV may inhibit atherosclerosis.

Figure 6 shows the antioxidative activities of 2"-0-GIV and vitamin C in a blood plasma system. When blood plasma was oxidized with Fenton's reagent, 1.11 nmol/ug (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 (15). 2"-O-GIV and vitamin C inhibited MDA formation by 54% and 32%, respectively, at the level of 0.1 urnol. 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"-0- 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 peroxidarion 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% of2"-O-GJV) have been consumed by livestock for many years without any evidence of adverse effects. 2"",,:0-GIV may be useful in the treatment of atherosclerosis. Therefore, further investigation of its physiological effects should be undertaken.

184

2.5

• 2"-O-GIV E3 Ferullc acid

1.0

~ 2.0 S

c

- ~

'0

~ 1.5 Qj

:::2

~

Qj

Col -e

.... o

-

C

::I o

S -e

0.5

0.0

. - -.. ..

o 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Amount of Antioxidant (umol)

Figure 4. Inhibitory effect of2"-O-GIV and ferulic acid on LDL oxidation.

-- 160
'0
E
~
._..
Q.I 120
"0
~ -<>-- 2"·O·GJV
'Q:j
:E
~ 80 • Probucol
Q:;
f..I
<:
...
0 40
-
C
:::l
0
E 0
<:
0.0 0.1 0.2 0.3 0.4
Amount of antioxidant (umol) Figure 5. Antioxidative activities of2"-0-GIV and probucol measured in blood plasma.

---
~
E 1.4
(I)
..::
Co
-e 1.2
0
0
J:J
0.0 1.0
::1.
:;:;;
Q
E 0.8
c
-
-<:
Q 0.6
:;
...
0 0.4
-
c
::I
0
E 0.2
-e 0.00 0.02 0.04 0.06 {211-O-GIV +- ......... --r---.--~--.--~__,...-,---.----, -- Vitamin C

0.08 0.10

Amount of Antioxidant (JlmoJ)

Figure 6. Antioxidative activities of2"-O-GIV and vitaminC in blood plasma.

185

. 186

15. 16. 17. 18. 19.

20. 2l. 22.

23.

24. 25. 26.

27.

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1. 2. 3. 4. 5. 6. 7. 8. 9.

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10. 11.

12. 13. 14.

Reprinted from ACS Symposium Series 702 Functional Foods for Disease Prevention II Medicinal Plants and Other Foods

Takayuki Shibamoto, Junji Terao, and Toshihiko Osawa, Editors Published 1998 by the American Chemical Society

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