Indian Journal of Experimental Biology Vol. 37, March 1999, pp. 243-247

Biochemical effects of garlic protein on lipid metabolism in alcohol fed rats

C R Rajasree, T Rajamohan & K T Augusti* Department of Biochemistry, University of Kerala, Thiruvananthapuram. 695581, India.

Received on 26 November 1997; revised 1 January 1999

Garlic protein is a very good hypolipidemic agent. In the present study the water soluble protein fraction of garlic was investigated for its effect on hyperlipidemia induced by alcohol (3.76g1kg. body wt./day). The hypolipidemic action is mainly due to an increase in cholesterol degradation to bile acids and neutral sterols and mobilization of triacyl glycerols in treated rats. Garlic protein (500mg./kg body wt./day) showed significant hypolipidemic action comparable with a standard dose of gugu-Iipid (50mg.lkg. body wt./day).

Garlic [Allium siltivum Linn] is ascribed with many therapeutic effects 1.2. Garlic oil' and the sulphur containing amino acids4 present in garlic have the effect of regulating lipid metabolism. Both in man and in rats, alcohol administration increases the lipid levels viz .. triglycerides and cholesterol in blood plasmas.6 and kidney.7.8 Among all the lipids cholesterol IS the major causative factor of atherosclerosis.

Previously in our laboratory garlic protein containing diet (16% by weight) was found to produce hypolipidemic effect in cholesterol fed rats9

•

However that dose of garlic protein was too high to be recommended as a drug. In the present study we used a lower dose (500mg'/kg. body wt./day) of garlic protein in alcohol fed rats to investigate on its biochemical effects. Gugu-lipid is used as a standard drug (50mg'/kg body wt/.day) to compare the hypolipidemic effects of the protein in the present study 10.

Materials and Methods Garlic protein was prepared from fresh garlic

according to the method of Garcha et.al. 11 with some modifications. Fresh garlic yielded 5% garlic protein by weight. A dose of 18% alcohol (3.76g'/kg. body wt./day) was used for a period of 45 days to produce hyperlipidemia. Gugu-lipid, the standard drug obtained from Cipla company was used at a dose of 50 mg/.kg. body wt./day for the same period .

*Prescnt Add ress & Correspondence: Professor (If Biochemi stry. School llf Medical Education .. M G University Centre. Mellical College Campus. Kott ayam 686 008. India.

Male albino rats of Sprague Dawley strain weighing 100-120g., bred and maintained on the rat feed in our animal house were used for the study. They were divided into 4 groups of six rats each and fed as follows for 45 days.

Group I II

III

IV

Normal diet + Normal Saline solution Normal diet+ 18% alcohol (3.76 g./kg body wt/day) Normal diet+ 18% alcohol + garlic protein (500 mg/kg body wt/day) Normal diet+ 18% alcohol + gugu-lipid. (50 mg/ kg body wt/day)

Garlic protein and gugu-lipid were fed as saline suspension through a gastric tube. Alcohol was also given through a gastric tube. All the four groups were pair-fed. According to this each rat got about 50-60 mg. garlic protein and it corresponds to 1 gm. garlic. At the end of the experimental period the rats in each group were deprived of food over night, stunned by a blow at the back of the neck and sacrificed by decapitation. The blood was collected for serum separation and the tissues were quickly removed into ice cold containers for various estimations by standard methods. Stool was also collected for bile acids and neutral sterols estimations. Total cholesterol '2, triacyl glycerol" and phospholipidl 4 and free fatty acids IS in serum and tissues were determined. HDL cholesterol '6 of serum was determined. HMG-Co.A reductase activities (EC 1.1.1.34)17 both in liver and intestine were also estimated. Serum GOT (L-Aspartate:2-oxoglutarate amino transferase) (EC 2.6.1 . 1 )I ~ GPT (L-alanine: 2-

244 INDIAN J EXP BIOL, MARCH 1999

oxoglutarate amino transferase(EC 2.6.1.2)19 and alkaline phosphatase (EC 3.1.3.1)20 were determined. Lipogenic enzymes viz malic enzyme (EC. 1. 1. 1.40)21 ,

. 22 glucose 6 phosphate dehydrogenase (EC 1.1.1.49) , and isocitrate dehydrogenase (EC 1.1.1.42)23 activity also were determined. Lipoprotein lipase activity (EC 3.1.1.3)24 in heart and adipose tissues and lecithin cholesterol acyf transferase activity (EC 2.3.1.3)25.26 in plasma were detenn:in~d~ Sto.ols ~ere als9 collected from each group and were pooled and used for the estimation of bile acids27 and neutral sterols l2. All the results were analysed by analysis of variance (ANOY A). and 't' values . were. cakulate~ by­bonferroni multiple comparison procedure.

Results and Discussion Gain in body weight for alcohol group was similar

to control gr~up for 2 weeks but later decreased signficantly (P<O.OO 1). But for the treated groups increase in body weight was as regular as that of the control. The liver weight for the 1-4 groups were 4.2 ± O.15g, 5.3 ± O.14g, 4.8 ± 0.12g. and 4.6 ± 0.12g. respectively (P<O.OO I). Alcohol caused hyper

lipidemia. But on administration of garlic protein the concentration of cholesterol, triacyl glycerol and phospholipid significantly decreased (Table 1). Concommitantly concentration of HDL cholesterol increased but LDL+ YLDL cholesterol decreased by the administration of garlic protein and gugu-lipid. The atherogenic index decreased in garlic protein and gugu-lipid treated rats (Table 2). The activity of HMG-COA reductase decreased and lipogenic enzymes increased in alcohol fed group as compared to the normal (Tables 3 & 4). Free fatty acids and serum GOT, GPT and alkaline phosphatase activities

. significantly increased on alcohol feeding and these activities decreased significantly on treatment with the drugs (Tables 5 & 6). Hepatic and fecal excretion of bile acids and neutral sterols decreased in alcohol treated rats (Table 7). But it was found to be increased significantly on treatment with garlic protein and gugu-lipid . Lipoprotein lipase activity in heart and adipose tissues and lecithin cholesterol acyl transferase activity in plasma decreased in alcohol treated rats . But these activities significantly increased on treatment with the drugs (Table 8).

Table I-Concentrations of cho lesterol , triglycerides and phospholipids in serum and tissues of the control and treated rats

I Valucs. expressed as mgt I ~Og wet tissues of 100 m!. serum, are mean ± SD of 6 rats in each group I

No Group Scrum Liver Kidney Heart Aorta Brain

Cholesterol

I Normal 67.S2±0.97 447.S±II.47 S88.08±8.0S 27S±4.98 146.29±12.31 1459.7±55.22

2 N+ I X'YI . alcohol 88.42±0.73 * 514.88± 13.02* 747. I 8±20.66* 398.39±60.42* 176.9±14.25* 1821.4±62.71*

) 18% alcoho l + 69 .8±2.38* 486.01±4.54* 671.88±7.49* 213.72±7.9* 142.56±9.67* I 520.8±59.72*

. garlic protein

4 18% alcohol + 72.71±1.82* 464.7±20.46* 668.95±11.39* 231.30±10.86* 133 .34±6.91 * 1461.49±44.69*

gugulipid

Triglyceride

I Normal 12.19±0.6 1 161.03±3.03 88.41±7.69 70.54±3.87 1088.8 1 ±6.46 66.52±4.53

2 N+ 18% alcohol 14.75±0.22* 188.18±4.84* 114.58±2.34* 104.93±2.66* 1133 .23±2.64* 158.23±1.73*

3 18% alcohol + 12.2S±0.33* I 62.42±6.26* 94.35±2.22* 87.49±1.63* 1020.71±2.64* 119.36±1.97*

garlic protein

4 18% alcohol + 12.93±0.15* 165.36±4.S8* 91.21±13.23* 84. I 3±2.9G* 1019.46±2.S0* 112.34±9.77*

gugu lipid

Phospholipid

Normal 108.95±2.97 2294.71±182 2436.6±201.6 1746.75±143 .6 931 .69±3.2 4922.2±S9.4

2 N+ 18% alcohol I 77.4±1 1.6* 2958.1 ±267.3* SI65±145.80* 2103.3±163.S* 1077.2±69.2" 59 13.6±76.5*

3 18 0/,· alcohol + IS2.4±3 .1 * 23..60.2±47.2S* 4343.7±125.9* 174S.8±134.9* 946.5 1 ±25.8* S043.8±53.8*

garlic protein

4 18% alcolllli + 141.6±6.17* 2336±138.4* 4265±53.64* 1688.8±1 2 1* 93I.S±7.97* 4789±230.2*

gugu lipid

In Anova group II is compared with group I. group III and IV compared with group II , group III with group IV. The last two grou ps are not sign ificantly different.However between other groups *P<O.OOI

RAJASREE et al. : EFFECfS OF GARLIC PROTEIN ON LIPID METABOLISM 245

Tabie 2-Concentrations of cholesterol in lipoprotein fractions in ihe serum of the control and treated rats

[Values, expressed as mgllOO ml . serum, are mean ± SD 6 rats in each group]

No Group Total Cholesterol HDL-Cholesterol (LDL+VLDL) Atherogenic Cholesterol Index

1. Normal 67.52±O.975I 35.78±O.1720 31.64±O.9052 1.88±O.037

2. N+ 18% alcohol 88.42±O.7303* 32. lO±O. 1280* 56.39±1 .34* 2.75±O.027*

3. 18% alcohol + 69.88±2.38* 34.30±O.5147* 41 .15±2.56* 2.03±O.02*

garlic protein

4. 18% alcohol + 72.71±1.82* 34.39±O.4676* 39.91±3.41* 2.1I±O.03*

gugu lipid

Other details are same as in Table I . Atherogenic! index=total cholesteroVHDL cholesterol

Table 4-Activities of lipogenic enzymes in the control and treated rats [Values are mean ± SD of six rats in each group]

No Group Glucose -6 Phosphate Malic enzyme Isocitrate dehydrogenase

(units!g Protein)

Normal 117.1O±1O.0

2 N+ 18% alcohol 143.01±17.85*

3 18'% alcohol + garlic protein 119.17±23.61*

4 18% alcohol + gugu lipid 121. 75±26.57*

Other details are same as in Table I

Table 3-Activi.ty of HMG. COA reductase in liver and small intestine of the control and treated rats

No

2

3

4

[Values, are mean ± SD of six rats In each group]

Groups Ratio of HMG CoA to mevalonate Liver Intestine

Normal 1.93 ± 0.0352 2.16 ± 0.0279

N + 18% alcohol 1.61 ± 0.0554* 1.73 ± 0.0611 *

18% alcohol + garlic 1.83 ± 0.0481 * (99 ± 0.0105* protein

18% alcohol +gugu 1.85 ± 0.0376* 2.03 ± 0.0390* lipid

Other details are same as in Table I .

These results indicate that garlic protein has significant hypolipidemic effect in alcohol fed rats at a dose of garlic protein (500 mgLkg body wt/daYJ corresponding to 109. fresh garlic, i.e. each rat consumed garlic protein corresponding to 1 g. fresh garlic.

The exact mechanism of action of garlic protein is not known. Amino acid analysis9 of garlic protein showed the presence of large amount of sulfur containing amino acids, aspartic acid and glutamic acid. The sulfur containing amino acids present in garlic protein viz. cysteine and methionine an'd possibly their derivatives S. methyl cysteine sulfoxide, S-allyl cysteine sulfoxide released as

(Units!mg Protein) dehydrogenase

(Unitlmg Protein)

1.499±O.1084 1.3083±O.1017

1.969±O.0587* 1.9287±O.0454*

1.532±O.2164* 1.4383±O.2983*

1.654± 0.157* 1.4963±O.145*

Table 5-Concentration of free fatty acids in the serum and liver of the control and treated rats

[Values are mean ± SD of six rats in each group]

No Groups Serum (mgllOO ml. Liver (mgllOO g. serum) wet tissue)

I Normal 72.53 ± 1.75 258.42 ± 23.46

2 N + 18% alcohol 87.00 ± 1.35* 281.36 ± 27.38*

3 18% alcohol + garlic 74.72 ± 3.02* 264.71 ± 25.95* protein

4 18% alcohol +gugu 78.81 ± 1.41 * 267.49 ± 28.44* lipid

Other details are same as in Table I

digestion products could have counteracted the hyper lipidemic and oxidant effects of alcohol treated rats. It is also pointed out by Sanlin28 that sulfur containing amino acids have a special role as hypolipidemic

.-Kr' h k l 29 dR' h 30 agent. IC eves y et a. an aJmo an et at. reported that proteins with low ratio of lysine/ arginine affects its atherogenicity. Casein with a lysine/arginine ratio of 2 was found to be significantly more atherogenic than soy protein with lysine/arginine ratio of 0.84. But in garlic protein the lysine/arginine ratio is 1: 1.135. Sulfur containing amino acids are reported to be hypolipidemic. This factor as well as the low ratio of lysine/arginine (0.77) may play a key role for the hypolipidernic

246 INDIAN J EXP BIOL, MARCH 1999

Table 6-Activities of serum GOT, GPT and serum alkaline phosphatase

[Values are mean ± SD of six rats in each group] No Group GOT GPT Alkaline phosphatase

(K.A Units)

I Normal

2 N+ 18% alcohol

3 18% alcohol + garlic protein

4 18% alcohol + gugu-Iipid

Other details are sane as in Table I

(I . UIL Serum)

17.70±O.2847

31.88±O.3987*

18.92±O.8018*

19.86±O.5323*

(1.UIL Serum)

11.60±O.2990

14.94±O.1513*

9.26±O.9567*

9.7± 0.7949*

9 .58±O.4240

27.69±O.5682*

17.54±1.5254*

18.57±1.500S*

Table 7-Hepatic and fecal bile acids and fecal neutral sterols in control and treated rats

[Values are mean ± SD of six rats in each group]

No Group Hepatic bile acids Fecal bile acids Fecal neutral sterols (mg/IOO g wet tissue) (mg/rat/day) (mg/rat/day)

Normal 27.50±O.7548 17.85±O.535 10.52±O.36

2

3

4

N-io 18% alcohol 15.70±O.3189* 9.42±O.432* 6.43±O.24*

18% alcohol + garlic protein 24.7I±O.7171* 21 .34±O.74* 13 .73±O.84*

18% alcohol + gugu-lipid 28.08±1 .66* 29.73± 0 .84* 16.84±O.43*

Other details are same as in Table I

Table 8-Activity of lipoprotein lipase in heart and adipose tissues and lecithin cholesterol acyl transferase in plasma [Values are mean ± SD of six rats in each group]

No Group LPL (~ moles of glycerolliberated/hr/mg. protein)

Heart Adipose tissue Plasma LCAT

Normal 39.07±O.7129 158.71 ±O.8925 151.91±1.9133

2 N+ 18% alcohol 23. I 26±O.7963* 92 .3IS±I.0369S* 142.28±O.8208*

3 18% alcohol + garlic protein 49 .7117±O.7557* 188. I 367±2.2282* IS6.SSI7±2.0747*

4 18% alcohol + gugu-Iipid 43 .75 I 7±O.9639* 184.9± 1.934S* I 54.9083±2.0230*

Other details are same as in Table I

action of garlic protein. In addition to the above another suggested mechanism is that certain non protein substances accompanying the protein may have a definite role. It is well established that a number of storage albumins are glycoproteins and heterogeneous albumin constitute 75% of the total protein in garlic cloves. In the garlic protein treated group, small glycopeptides may be released as products of digestion which may be absorbed and exert some effect on lipid metabolism in liver. These are only proposed hypothesis at present. From these findings it is revealed that garlic protein is a very good hypolipidemic agent.

As drugs like tolbutamide is effective in men for a dose range of 2-3g, it is effective in diabetic rabbits only at a dose of 250mglkg31

• Therefore an extrapolation of the dose for animals to men is not correct always. Accordingly from this experiment 20-

25g garlic/day may be sufficient for men to get the benefits.

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