JFS Vol 65 Is 01 JAN 2000 pp 0030-0033 LIVER INJURY-PREVENTIVE EFFECT OF TEA THEANINE IN RATS.pdf

7/30/2019 JFS Vol 65 Is 01 JAN 2000 pp 0030-0033 LIVER INJURY-PREVENTIVE EFFECT OF TEA THEANINE IN RATS.pdf

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30 JOURNAL OF FOOD SCIENCEVol. 65, No. 1, 2000 2000 Institute of Food Technologists

JFS: Food Chemistry and Toxicology

Liver Injury-preventive Effect ofTea Theanine in RatsP. HE, S. WADA, N. WATANABE,AND K. SUGIYAMA

ABSTRACT: This study was conducted to isolate the constituent, which had a preventive effect on D-galactosamine-induced rat liver injury, from the 70% ethanol-soluble fraction of Japanese green tea. Theanine (glutamic acid -ethylamide) was identified as the active compound, and the liver injury-preventive effect of theanine was dose-dependent. L-Glutamic acid -ethyl ester, but not glutamine, also brought about a significantly preventive effect onliver injury when added to the diet at equimolar levels to that of 1% theanine. The results indicate that theanine isone of the effective constituents of Japanese green tea in preventing D-galactosamine-induced liver injury.

Key Words: green tea, theanine, liver injury, D-galactosamine, rats

Introduction

THEPHYSIOLOGICAL, BIOCHEMICAL, AND PHARMACOLOGICALeffects of teas or their constituents have so far been exten-

sively studied. Tea catechins (tannins or polyphenols), themajor constituents of various types of teas, have been shownto have wide range of effects, such as antioxidation (Matuzakiand Hara 1985), antimutation (Kada and others 1985), anticar-cinogenesis (Fujiki and others 1996), antibiotic action (Todaand others 1989), antihypercholesterolemia (Muramatu andothers 1986), and antihypertension (Hara and Tonooka 1990).It has also been shown that soluble polysaccharides and sa-ponins of green tea have antihyperglycemia (Shimizu andothers 1988) and antiinflammation (Sagesaka and others

1996) effects, respectively. Furthermore, we demonstratedthat green tea had a suppressive effect on D-galactosamine

(GalN)-induced liver injury in rats (Sugiyama and others1998). It has been pointed out that liver injury induced by

GalN resembles that induced by the H-1 strain virus, one of 2serologic type of the rat virus, in its symptoms, and that the

histologic signs of the experimental GalN-induced hepatitiswere similar to those of human viral hep ati tis (Ke ppl er andothers 1968). The liver injury-preventive effect of green teacould be mainly ascribed to 3 types of constituents, which

were soluble in butanol or 70% ethanol and insoluble in 70%ethanol, when green tea was fractionated into 5 fractions bysuccessive extraction with organic solvents such as chloro-form, ethyl acetate, n-butanol, and 70% ethanol (unpublishedresults). Our recent studies showed that the liver injury-pre-ventive constituents, which were soluble in butanol and insol-

uble in 70% ethanol, were identified as flavonol glycosides(Wada and others 1999) and water-soluble polysaccharides

(unpublished results), respectively. However, the entity in-cluded in the 70% ethanol-soluble fraction, which is consid-

ered to contain water-soluble compounds of low molecularweights, has not yet been identified.

This study was conducted to isolate the liver injury-preven-tive constituent from the 70% ethanol-soluble fraction of Japa-

nese green tea. The results showed that the effect of 70% etha-nol-soluble fraction could be attributable to theanine (glutamicacid -ethylamide). So, the dose-dependent effect of theanineand the effect of theanine analogues on GalN-induced liver inju-ry were also investigated in rats.

Results

Effect of 70% ethanol-soluble fractionEffects of dietary supplementation with powder of a green tea

extract at a level of 3% or the equivalent amount of 70% ethanol-soluble fraction (1.07%) on GalN-induced liver injury were com-pared. The body weight gain and food intake were slightly de-

creased by the green tea extract but not by the 70% ethanol-solu-ble fraction, as compared with GalN-injected control group (Ta-

ble 1). The decrease in the liver weight caused by GalN was pre-vented by a small, but statistically significant amount, by both

the green tea extract and 70% ethanol-soluble fraction. TheGalN-induced enhancement of plasma aminotransferase (ALT)

and aminotransferase (AST) activities were significantly sup-pressed by both the green tea extract and 70% ethanol-soluble

fraction (Fig. 2).

Effect of each fraction separated by chromatographyFour fractions (I to IV) obtained from 70% ethanol-soluble

fraction by different types of column chromatography (Fig. 1)were added to the diet based on the yield of each fraction, and

Fig. 1Fractionation procedure by various types of column chroma-tography of 70% ethanol-soluble fraction obtained from green teaextract. The yield of each fraction was indicated in parentheses.


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Vol. 65, No. 1, 2000JOURNAL OF FOOD SCIENCE 31

the effects on GalN-induced liver injury were assayed. The GalN-induced enhancement of plasma ALT and AST activities were sig-

nificantly suppressed by the fraction III but not by other 3 frac-tions (Fig. 3). The major component in fraction III was identified

as theanine from 1H and 13C-NMR analyses. In support of this,the retention time of the peak of fraction III was in accord withthat of authentic theanine in amino acid analysis using aminoacid autoanalyzer. Furthermore, the content of theanine in thefraction III was estimated to be 99% from the comparison of peakareas between fraction III and authentic theanine.

Effects of theanine and related compoundsThe dose-dependent effect of theanine and the effects of

theanine-related compounds, such as glutamine and glutamicacid -ethyl ester, on GalN-induced liver injury were assessed.The body weight gain and food intake were not affected by thesecompounds (Table 2). The liver weight was also unaffected.GalN-induced enhancement of plasma ALT and AST activities

were suppressed by dietary supplementation with theanine in adose-dependent manner (Fig. 4). In addition to theanine,equimolar level of glutamic acid -ethyl ester had significantly

suppressive effects on plasma enzyme activities, whereasequimolar level of glutamine had no significant effect (Fig. 5).

Discussion

THE PRESENT STUDY DEMONSTRATED THAT THEANINE SUP-pressed the GalN-induced enhancement of plasma ALT andAST activities. Since the extent of increase in these plasma en-zyme activities is generally thought to be parallel to that of sever-ity of liver injury, the results are taken to indicate that theaninehad a suppressive effect on liver injury induced by GalN. Thean-ine is a derivative of glutamic acid, which was first isolated bySakato (1950) from Japanese green tea (gyokuro). It is confirmedthat theanine comprises a half or more of the total free amino ac-ids of Japanese green tea, and that Japanese green tea of uppergrades contains relatively large amount of theanine, a maximum

value being about 2.7 g/100 g. The latter is associated with thefact that theanine has sweet and umami tastes. The content oftheanine in other types of teas, such as black and puerh teas, isknown to be quite low. In an earlier study, Kimura and Murata(1971) showed that theanine inhibited the convulsive action ofexcessive caffeine in mice. Recently, several reports have shownthat theanine has some other physiological effects. For instanc-es, Yokogoshi and others (1995) have demonstrated that thean-

Fig. 2Effects of dietary supplementation with green tea extractand 70% ethanol-soluble fraction of the extract onD-galactosamine-induced enhancement of plasma alanine aminotransferase (A) andaspartate aminotransferase (B) activities in rats. The column and itsbar represent the mean value and SEM, respectively. Values withdifferent letters are significantly different at P < 0.05. See Table 1for the number of rats. Abbreviations: ALT, alanine aminotrans-

ferase; AST, aspartate aminotransferase.

Fig. 3Effects of dietary supplementation with each fraction ob-tained by various types of column chromatography on D-galac-tosamine-induced enhancement of plasma alanine aminotransferase(A) and aspartate aminotransferase (B) activities in rats. The columnand its bar represent the mean value and SEM, for 7 or 9 rats, respec-tively. See Fig. 1 for fraction numbers. See Table 2 for statisticalexpression and abbreviations.

Table 1Body weight gain, food intake, and liver weight of rats fedtea extract or the 70% ethanol-soluble fraction of green tea1

Body wt gain Food intake Liver wtDiet (g/14 d) (g/14 d) (g/100 g body wt)

Control (Saline) (7) 71 2a 189 3a 4.76 0.07a

Control (GalN) (10) 70 2a 193 2a 3.54 0.02c

+ 3.0% Tea extract (8) 60 3b 180 3a 3.90 0.09b

+ 1.07% EtOH f raction (8) 74 3a 195 3a 3.76 0.06b

1 Each value is the mean SEM for the number of rats indicated in parentheses; values in acolumn with different superscript letters are s ignificantly different at P< 0.05. On 15th day, 2experimental groups and one control group of rats were injected with D-galactosamine and the

other control group of rats injected with saline. Abbreviations: GalN, D-galactosamine; EtOHfraction, 70% ethanol- soluble fraction.

Table 2Body weight gain, food intake, and liver weight of rats feddiets supplemented with graded levels of theanine or its analogues1

Body wt gain Food intake Liver wtDiet (g/14 d) (g/14 d) (g/100 g body wt)

Control (Saline) (7) 73 2ab 201 3ab 4.76 0.07a

Control (GalN) (10) 74 1ab 201 2ab 3.56 0.01b

+ 0.25% Theanine (7) 72 2ab 197 3b 3.66 0.06b

+ 0.50% Theanine (7) 70 2b 196 2b 3.65 0.06b

+ 1.00% Theanine (7) 74 1ab 202 2ab 3.65 0.03b

+ 0.83% L-Glutamine (7) 72 1ab 206 2a 3.58 0.05b

+ 1.01% L-GluOEt (7) 76 2a 205 3a 3.67 0.06b

1 Each value is the mean SEM for the number of rats indicated in parentheses; values in acolumn with different superscript letters are significantly different at P< 0.05. The amounts of0.83% L-GluOEt and 1.01% L-GluOEt were the same as 1.00% theanine on a molar basis. See

Table 1 for the experimental procedures. Abbreviations: GalN, D-galactosamine; L-GluOEt, L-glutamic acid -ethyl ester.


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32 JOURNAL OF FOOD SCIENCEVol. 65, No. 1, 2000

Tea Theanine and Liver Injury . . .

Fig. 4Effect of graded levels of dietary theanine onD-galactosamine-induced enhancement of plasma alanine aminotransferase (A) andaspartate aminotransferase (B) activities in rats. The circle and its barrepresent the mean and SEM, respectively. See Table 2 for the num-ber of rats. See Fig. 2 for statistical expression and abbreviations.

Fig. 5Comparison of the effects of dietary theanine and its ana-logues on D-galactosamine-induced enhancement of plasma alanineaminotransferase (A) and aspartate aminotransferase (B) activities inrats. The column and its bar represent the mean value and SEM,

respectively, for each group. See Table 2 for the number of rats.Abbreviations: GluOEt,L-glutamic -ethyl ester.ine had a hypotensive effect in spontaneously hypertensive rats

(SHR) when administered orally. They have also shown thattheanine increased the brain concentration of dopamine in rats(Yokogoshi and others 1998). Furthermore, Kobayashi and others(1998) have shown that theanine increased the proportion of-

waves of the total brain waves in humans. The present studyprovided evidence for another biological effect of theanine.

Since theanine is a -derivative of glutamic acid, it is interest-ing to compare the liver injury-preventive effect of theanine with

that of other -derivatives of glutamic acid. With regard to this,Katayama and others (1996) have shown that glutamine, the

most simple -derivatives of glutamic acid, had a preventive ef-fect on GalN-induced liver injury when added to the diet at a

high level (10%). In contrast, the liver injury-preventive effect ofglutamine could not be observed in the present study where theaddition level of glutamine was relatively low (0.83%). Therefore,one of the reasons for the disparity of the results between theirand our investigations might be due to difference in dietaryglutamine level. However, it is apparent that the efficacy of thea-nine in suppressing GalN-induced liver injury is higher than thatof glutamine. Yokogoshi and others (1995) have also shown thatunlike theanine, glutamine failed to reduce blood pressure inSHR rats when equal amounts of theanine and glutamine were

administered. The present study also demonstrated that in ad-dition to theanine, glutamic acid -ethyl ester had a liver injury-

preventive effect. This indicates that the structural requirementof-derivatives of glutamic acid to elicit their effect is not re-

stricted to amide bond. Yokogoshi and Kobayashi (1998) have

shown that the hypotensive effect of glutamic acid -methyla-mide tended to be greater than that of theanine in SHR rats, sug-gesting that the replacement of ethyl group of theanine by me-thyl group does not diminish the potency of the action of thean-ine. However, it is not known whether this is also the case for liverinjury preventive effect.

The mechanism by which dietary theanine suppressed GalN-induced liver injury is unclear at present. GalN is considered to

induce hepatotoxicity by inhibiting the synthesis of RNA andprotein through a decrease in cellular UTP concentration, finally

leading to necrosis of liver cells (Decker and Keppler 1974). Inmany studies, GalN has been used in combination with other

hepatotoxic substance, such as bacterial lipopolysaccharide (en-dotoxin), to cause liver injury in experimental animals, especially

in mice. Since rats are known to be more sensitive to GalN thanmice (Galanos and others 1979), we used GalN alone to cause ratliver injury. Tumor necrosis factor (TNF-) released from acti-vated macrophages is thought to play a central role in the liverinjury caused by GalN and endotoxin (Bradham and others1998). Although it is uncertain whether TNF- also plays a criticalrole in liver injury caused by GalN alone, further studies on theeffect on TNF- should help to clarify the mechanism underlyingthe liver injury-preventive effect of theanine. The effects of teaconstituents, including theanine, on other types of liver injury

models also remains to be further studied.

Materials and Methods

MaterialsJapanese green tea (sen-cha) of upper middle grade was

purchased from a market (Shizuoka City, Japan). Authentic

theanine was obtained from Tokyo Kasei (Tokyo, Japan)and glutamine from Wako Pure Chemical (Osaka, Japan). D-Galactosamine and glutamic acid -ethyl ester were ob-tained from Sigma (St. Louis, Mo., U.S.A.). The relativelylarge amounts of theanine (about 50 g) used in this study

were prepared from green tea according to the method de-scribed later. Mineral and vitamin mixtures of AIN-76 types(AIN 1977) were purchased from Oriental Yeast (Tokyo, Ja-pan).

Extraction of green teaGreen tea was extracted by adding 10 volumes (vol/wt) of

boiling water to the tea, standing for 30 min at room tempera-ture, and filtering through 5 sheets of gauze. The extract waslyophilized and powdered with a mixer. The dry matter, thus,


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Vol. 65, No. 1, 2000JOURNAL OF FOOD SCIENCE 33

extracted was 23.5 g per 100 g of green tea. The powder of thegreen tea extract was dissolved in water and extracted succes-sively with equal volume of chloroform, ethyl acetate, n-bu-

tanol, and 70% ethanol. The yield of the 70% ethanol-solublefraction was 35.7 g per 100 g of green tea extract.

Column chromatographyThe 70% ethanol-soluble fraction was further fractionated

into 4 fractions (I to IV) by column chromatography, using ac-tive charcoal (Wako Pure Chemical), silica gel 60 (Merck, Darm-stadt, Germany), and Toyopearl HW-40 ( Toso, Tokyo, Japan)(Fig. 1). In brief, the 70% ethanol-soluble fraction was appliedon active charcoal column, and the eluants were divided into 2fractions: non-adsorbed and adsorbed. The non-adsorbedfraction (fraction A in Fig. 1) was applied on Toyopearl HW-40column, and the eluants were divided into 2 fractions: sugar-rich (fraction I) and amino acid-rich (fraction II). On the other

hand, the adsorbed fraction (fraction B in Fig. 1) was appliedon silica gel column, and the eluants were divided into two

fractions (fractions III and IV ). The yield of each fraction wasalso shown in Fig. 1. Throughout the 3 types of column chro-

matography, eluants were monitored by thin-layer chromatog-

raphy (TLC) using silica gel 60. Developing solvents used werepropanol-water (85:15, by vol) or chloroform-methanol-water(2:1:0.2, by vol). Spots on the plate were visualized by vanillin-sulfate (Zweig and Sherma 1972). Since 70% ethanol-solublefraction are known to contain amino acids and sugars, ninhy-drin and phenol-sulfate were also used to visualize amino ac-ids and sugars, respectively.

Identification of fractions III and IVSince fractions III and IV gave single spots on TLC, these

compounds were analyzed by1H and 13C-nuclear magnetic

resonance (NMR) spectrograph (Model JNM-LA500, Tokyo, Ja-pan). Fraction III was analyzed by amino acid autoanalyzer

(Model 8500; Hitachi, Tokyo, Japan), since this fraction was

ninhydrin-reactive.

Animals and dietsMale rats of the Wistar strain ( Japan SLC, Hamamatsu, Ja-

pan) of 5 weeks old (90 to 100 g) were used to assess liver inju-ry-preventive effect of tea constituents. The rats were fed a

stock diet (Type MF; Oriental Yeast, Tokyo, Japan) for 3 or 4 d,and then they were given free access to the experimental diets

for 10 or 14 d in a temperature (23 to 25 C)- and humidity (40%to 60%)-controlled room with a 12-h cycle of light (06:00 to18:00) and dark. The composition of the control diet was as fol-lows (g/100 g): casein, 25; corn starch, 40.25; sucrose, 20; cornoil, 5, AIN-76 mineral mixture, 3.5; AIN-76 vitamin mixture, 1;choline bitartrate, 0.25; and cellulose, 5. Supplements wereadded to the control diet at the expense of starch. Food and

water were renewed daily, and the body weight gain and foodconsumption were also measured daily.

D-Galactosamine-induced liver injuryAfter feeding the experimental diet for 10 or 14 d, GalN was

injected intraperitoneally at a dose of 350 mg/kg body weightbetween 14:00 and 14:30 h. Rats were not starved either before

or after the injection of saline or the drug. Normal rats were in-jected with saline instead of GalN. After 22 h, the rats were killed

by decapitation between 12:00 and 12:30 h to obtain blood andliver. Plasma was separated from heparinized whole blood bycentrifugation at 2000 gfor 20 min at 4 C. The activities ofplasma alanine aminotransferase (ALT) and aspartate ami-notransferase (AST) were measured with a kit ( Transaminase CII-Test; Wako Pure Chemical), the enzyme activity being ex-pressed as I.U. (mol/min per L of plasma at 25 C). The experi-mental design was approved by the Laboratory Animal CareCommittee of the Faculty of Agriculture, Shizuoka University.

Statistical analysisData were analyzed by one-way analysis of variance, and

the differences between means were tested using Duncansmultiple range test (Duncan 1957) when the Fvalue was sig-

nificant. APvalue of 0.05 or less was considered significant.

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MS 1999-0339 received 3/23/99; revised 9/13/90; accepted 12/2/99.

This study was supported by the Program for Promotion of Basic Research Activities forInnovative Bioscience (PROBRAIN).

Authors are with the Department of Applied Biological Chemistry, Facultyof Agriculture, Shizuoka University, Ohya 836, Shizuoka 422-8529, Japan.Contact author is Kimio Sugiyama (E-mail: [email protected]).

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JFS Vol 65 Is 01 JAN 2000 pp 0030-0033 LIVER INJURY-PREVENTIVE EFFECT OF TEA THEANINE IN RATS.pdf