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Effect of garlic oil in experimental cholesterol atherosclerosis
125
Atherosclerosis,
29 (1978)
@ Elsevier/North-Holland
125-129 Scientific
Publishers,
Ltd.
EFFECT OF GARLIC OIL IN EXPERIMENTAL ATHEROSCLEROSIS
CHOLESTEROL
R.C. JAIN and D.B. KONAR Departments of Pathology Benghazi (Libya)
and Physiology,
(Received 9 July, 1976) (Revised, received 20 September, (Accepted 21 September, 1976)
Faculty
of Medicine,
University
of Benghazi,
1976)
Summary Addition of cholesterol in the diet of male albino rabbits produced hypercholesterolaemia, increased tissue cholesterol, and atheromatous changes in the aorta. Supplementation of garlic oil along with cholesterol significantly inhibited the hypercholesterolaemia, decreased tissue cholesterol and minimised the atheromatous changes in the aorta. These results show that the active constituent(s) in garlic responsible for its anti-atherogenic action is present in the oily fraction of garlic. _ ----Key words:
Aorta - Atherosclerosis agent -Serum cholesterol .___
Cholesterol
ester
-
Garlic - Hypocholesterolaemic
Introduction Administration of garlic juice to cholesterol-fed rabbits has been found to inhibit hypercholesterolaemia and atherogenesis [ 1,2]. It has been suggested that these properties of garlic were due to the active principles contained in its oily fraction [3-6]. The present study was designed to investigate the effect of garlic oil in cholesterol-fed rabbits. Materials and Methods Sixteen male albino rabbits (l-l.5 kg) were divided groups. All the animals received a stock diet containing Address for correspondence: 1451. Benghazi Libya.
Dr. R.C. Jain. M.D.,
equally into four green vegetables,
Faculty of Medicine, University of Benghazi, P.O. Box
126
soaked Bengal gram and milk powder. In addition, the diets of the different groups were supplimented as follows: Group I: 2 g cholesterol in 5 ml of olive oil/day. Group II: 2 g cholesterol in 5 ml of olive oil + 0.25 g garlic oil/day. Group III: 2 g cholesterol in 5 ml of olive oil + 0.5 g garlic oil/day. Group IV: 2 g cholesterol in 5 ml of olive oil + 1.0 g garlic oil/day. Garlic oil was obtained as follows: Garlic juice, prepared by crushing garlic bulbs, was thoroughly mixed with diethyl ether (B.P. 40-60°C) in a separating funnel. The etherial fraction was then separated and the ether was allowed to evaporate. The remaining oily material was used as garlic oil. Cholesterol suspension in olive oil, with or without garlic oil was administered by gastric intubation. Blood samples, either from an ear vein or by direct heart puncture, were collected at the beginning of the experiment and at four weekly intervals thereafter, for 16 weeks. Estimations of total and free serum cholesterol were made by the modified method of Kim and Goldberg [7], using the glacial acetic acid-acetic anhydride reagent. All the animals were sacrificed at the end of 16 weeks to study the gross pathology, histopathology and tissue cholesterol in an aliquot of the whole aorta and a piece of liver, according to the methods already described elsewhere [2] At autopsy the severity of atherosclerosis in the longitudinally opened aorta was grossly graded on a scale of 0 to 4+ to by the method described by Horlick and Duff [8]. (More modern techniques for quantitative atherosclerosis were not available at the time of the present experimental work.) Results Serum cholesterol Cholesterol feeding led to a progressive rise in total, free, and ester cholesterol in the serum of Group I animals (Table 1). The increases at 16 weeks in free (FC), ester (EC) and total (TC) cholesterol were, respectively, about 23, 36 and 33-fold. Thus though both FC and EC fractions were greatly increased by cholesterol feeding, the rise in serum EC fraction was greater than that of the FC fraction. The ratio FC/EC decreased from 0.480 to 0.335 during the 16 weeks of the experiment. Administration of garlic oil (Groups II, III, and IV) significantly decreased (P < O.OOl), but did not prevent, the diet-induced hypercholesterolaemia. The TC level in animals with the highest dose of garlic oil (Group IV) .increased by about 22-fold at, 16 weeks as compared with about 33-fold in Group I animals. The interesting point to be observed in these animals was that the FC/EC ratio, instead of being lowered, as was seen in Group I animals, increased from 0.53 to 0.66 during the 16 weeks of the experiment. This effect was also seen, to a lesser extent in other animals given garlic oil in smaller doses. Tissue The Group cantly
cholesterol cholesterol content of aorta and liver (Table 2) showed raised level in I animals. Administration of 0.25 g garlic oil (Group II) did not signifiaffect the tissue cholesterol level, but 0.5 and 1.0 g doses of garlic oil
1
OIL ON TOTAL,
FREE AND
IN SERUM
1530.0 f 165.5 489.8 f 42.4 1040.2 f 136.8 1485.5 f 179.5 505.8 + 52.2 979.7 f 149.5 1234.1 f 184.7 445.9 * 48.3 788.2 t 158.1 927.4 * 135.5 352.2 f 46.8 575.2 f 1Oa.O
884.9 f 49.5 268.5 + la.9 616.4 ? 40.8 773.4 f 52.7 251.7 * la.6 521.7 + 29.4 745.7 f 48.6 280.4 f 21.8 465.3 + 39.4 624.8 * 51.2 215.4 * 16.9 409.4 f 45.5
72.7 f 6.8 23.6 f 2.3 40.1 f 5.6
76.6 t 6.9 26.2 + 2.8 50.3 r 6.2
68.5 + 5.8 22.4 ? 2.2 46.1 f 4.2
72.5 + 5.6 25.2 ? 2.6 47.3 f 4.8
Total cholesterol Fre6 chdesteml Ester cholesterol
II
III TotaI.choIesterol Free cholesterol Ester ch&esterol
IV Total cholesterol Free cholesterol Ester cholesterol
8th Week
CHOLESTEROL
Total cholesterol Free lcholesterol Ester chdesterol
4th Week
Initial
ESTER
I
Group
Values are mean f SD (mg/lOO ml of sennd.
EFFECT OF GARLIC TEROL FEEDING
TABLE
.-___
AT DIFFERENT
1462.7 + 205.4 539.5 f 61.3 923.2 ? 188.9
1628.3 + 197.8 534.5 + 52.3 1093.8 + 168.6
1969.4 * 208.5 618.2 + 67.6 1351.2 f 192.0
2134.8 f 215.5 529.6 f 58.9 1605.2 + 186.8
12th Week
OF RABBITS
1710.5 + 198.5 680.7 * 72.5 1029.8 f 156.0
1920.5 f 207.5 665.8 f 57.2 1254.7 f 180.2
2295.5 * 216.5 656.0 + 78.8 1639.5 * 188.0
2450.5 f 230.5 610.8 * 61.2 1839.7 f 190.7
16th Week
TIME INTERVALS
AFTER
CHOLES-
128 TABLE
2
EFFECT
OF
GARLIC
ATHEROSCLEROSIS Values
are mean
OIL
ON
TOTAL
CHOLESTEROL
IN AORTA
AND
LIVER
Cholesterol
(mgjg
Aorta
wet
OF
tissue)
Liver
Atherosclerosis
I
32.9
t 4.6
96.3
? 8.5
2.90
* 0.55
II
27.6
r 4.5
92.4
? 8.6
2.60
f 0.40
P I vs. II
III
>O.l 25.3
P Group
GRADING
f SD.
Group
Group
AND
IN RABBITS
I vs. III
IV
significantly lowered and IV animals.
t 4.2
P Group I vs. IV
>O.l 76.7
>0.2 ? 1.8
the cholesterol
73.5
grading
2.10
f 0.28
co.02 -r 7.2
content
1.40
i 0.20
10.001
of aorta and liver in Groups
III
Atherosclerotic changes When aortic strips were graded for atherosclerotic lesions the mean figures + SD obtained for Group II animals were not significantly different from that of the control group (2.60 f 0.40 vs. 2.90 + 0.55). In Groups III and IV animals, however, there was a significant reduction in atheromatous lesions, the gradings being 2.10 + 0.28 and 1.40 f 0.20, respectively. Discussion It has been reported previously that garlic juice administration inhibits hypercholesterolaemia and atherogenesis in cholesterol-fed rabbits [ 1,2]. The results of the present study show‘ that these properties of garlic are present in the oily fraction extracted from raw garlic. Recently Kritchevsky [9] and Bordia et al [lo] also observed inhibiton of atherogenesis by adding garlic oil to a high-cholesterol diet. A mixture of several sulphur-containing compounds isolated from garlic have been reported to be the active constituent responsible for its various pharmacological actions [ll]. As the amount of active principle present in garlic oil is 0.3-0.4s [12], the anti-atherogenic action abserved in the present study resulted from an approximate daily dose of 1-6 mg of the active principle (present in approximately 15-60 g of garlic bulbs). The biochemical data obtained in this study correlated well with the atherosclerotic grading of the aorta in all the animals with or without garlic oil. Genesis of atheromatous lesion in cholesterol-fed animals was associated with a hypercholesterolaemia in which FC/EC was decreased. Inhibition of atherogenesis in garlic-fed animals was always associated with an alteration of the FC/EC ratio in the reverse direction. This correlation, which is significant, may
129
throw some light on the mechanism treated animals. Since it is EC that of cholesterol-fed animals, the low the protective effect of garlic could tiou. Further work is in progress on juice on the above parameters.
of the protective action evidenced in garlicpredominantly accumulated in the intima EC value in garlic-fed rabbits suggests that be due to its inhibitory action on this fracthe effect of the non-oily fractions of garlic
Acknowledgement We thank Dr. G. Barber-Riley, W.H.O. Professor and Chairmen, Department of Physiology, Faculty of Medicine, University of Benghazi for the constructive criticism of the work and going through the manuscript. References 1 Jain, R.C.. Onion and garlic in experimental atherosclerosis, Lancet. 1 (1975) 1240. 2 Jain, R.C., Onion and garlic in experimental cholesterol atherosclerosis in rabbits, Part 1 (Effect on serum lipids and development of atherosclerosis), Artery, 1 (1975) 115. 3 Jain, R.C., and Konar, D.B.. Garlic oil in experimental atherosclerosis. Lancet, 1 (1976) 918. 4 Chopra, R.N., Chopra, I.C., Handa. K.L. and Kapur, L.D. Indigenous Drugs of India, - Allium satiuum L.. 2nd edition. Dhur and Sons. Calcutta, 1951, 271 pp. 5 Wealth of India, published by the Government of India, Council of Scientific and Industrial Research, Delhi, 1948, 56 pp. 6 Jain, R.C., M.D. Thesis, University of Rajasthan, India, 1968. 7 Kim, E., and Goldberg, M., Serum cholesterol assay using a stable Liebermann-Burchard reagent, Clin. Chem., 15 (1962) 1172. 8 Horlick. L., and Duff, G.L., Heparin in cholesterol atherosclerosis in the rabbit. Arch. Path., 57 (1954) 417. 9 Kritchevsky. D., Effect of garlic oil on experimental atherosclerosis in rabbits. Artery, 1 (1975) 319. 10 Bordia, A., Arora, S.K., Kothari, L.K., Jain, K.C., Rathore. B.S., Rathore, AS., Dube. M.K. and Bhu, N., The protective action of essential oils’ of onion and garlic in cholesterol-fed rabbits. Atherosclerosis. 22 (1975) 103. 11 Cavallito. C.J.. Bailey. J.H.. and Buck, J.S., The antibacterial principle of AIlium satiuum, Part 3 (Its precursor and essential oil of garlic), J. Amer. Chem. SIX.. 67 (1945) 1032. 12 No&r. C.R.. Chemistry of Organic Compounds. 3rd edition, W.B. Saunders. Philadelphia, Pa., 1965, 316 pp.
Atherosclerosis,
29 (1978)
@ Elsevier/North-Holland
125-129 Scientific
Publishers,
Ltd.
EFFECT OF GARLIC OIL IN EXPERIMENTAL ATHEROSCLEROSIS
CHOLESTEROL
R.C. JAIN and D.B. KONAR Departments of Pathology Benghazi (Libya)
and Physiology,
(Received 9 July, 1976) (Revised, received 20 September, (Accepted 21 September, 1976)
Faculty
of Medicine,
University
of Benghazi,
1976)
Summary Addition of cholesterol in the diet of male albino rabbits produced hypercholesterolaemia, increased tissue cholesterol, and atheromatous changes in the aorta. Supplementation of garlic oil along with cholesterol significantly inhibited the hypercholesterolaemia, decreased tissue cholesterol and minimised the atheromatous changes in the aorta. These results show that the active constituent(s) in garlic responsible for its anti-atherogenic action is present in the oily fraction of garlic. _ ----Key words:
Aorta - Atherosclerosis agent -Serum cholesterol .___
Cholesterol
ester
-
Garlic - Hypocholesterolaemic
Introduction Administration of garlic juice to cholesterol-fed rabbits has been found to inhibit hypercholesterolaemia and atherogenesis [ 1,2]. It has been suggested that these properties of garlic were due to the active principles contained in its oily fraction [3-6]. The present study was designed to investigate the effect of garlic oil in cholesterol-fed rabbits. Materials and Methods Sixteen male albino rabbits (l-l.5 kg) were divided groups. All the animals received a stock diet containing Address for correspondence: 1451. Benghazi Libya.
Dr. R.C. Jain. M.D.,
equally into four green vegetables,
Faculty of Medicine, University of Benghazi, P.O. Box
126
soaked Bengal gram and milk powder. In addition, the diets of the different groups were supplimented as follows: Group I: 2 g cholesterol in 5 ml of olive oil/day. Group II: 2 g cholesterol in 5 ml of olive oil + 0.25 g garlic oil/day. Group III: 2 g cholesterol in 5 ml of olive oil + 0.5 g garlic oil/day. Group IV: 2 g cholesterol in 5 ml of olive oil + 1.0 g garlic oil/day. Garlic oil was obtained as follows: Garlic juice, prepared by crushing garlic bulbs, was thoroughly mixed with diethyl ether (B.P. 40-60°C) in a separating funnel. The etherial fraction was then separated and the ether was allowed to evaporate. The remaining oily material was used as garlic oil. Cholesterol suspension in olive oil, with or without garlic oil was administered by gastric intubation. Blood samples, either from an ear vein or by direct heart puncture, were collected at the beginning of the experiment and at four weekly intervals thereafter, for 16 weeks. Estimations of total and free serum cholesterol were made by the modified method of Kim and Goldberg [7], using the glacial acetic acid-acetic anhydride reagent. All the animals were sacrificed at the end of 16 weeks to study the gross pathology, histopathology and tissue cholesterol in an aliquot of the whole aorta and a piece of liver, according to the methods already described elsewhere [2] At autopsy the severity of atherosclerosis in the longitudinally opened aorta was grossly graded on a scale of 0 to 4+ to by the method described by Horlick and Duff [8]. (More modern techniques for quantitative atherosclerosis were not available at the time of the present experimental work.) Results Serum cholesterol Cholesterol feeding led to a progressive rise in total, free, and ester cholesterol in the serum of Group I animals (Table 1). The increases at 16 weeks in free (FC), ester (EC) and total (TC) cholesterol were, respectively, about 23, 36 and 33-fold. Thus though both FC and EC fractions were greatly increased by cholesterol feeding, the rise in serum EC fraction was greater than that of the FC fraction. The ratio FC/EC decreased from 0.480 to 0.335 during the 16 weeks of the experiment. Administration of garlic oil (Groups II, III, and IV) significantly decreased (P < O.OOl), but did not prevent, the diet-induced hypercholesterolaemia. The TC level in animals with the highest dose of garlic oil (Group IV) .increased by about 22-fold at, 16 weeks as compared with about 33-fold in Group I animals. The interesting point to be observed in these animals was that the FC/EC ratio, instead of being lowered, as was seen in Group I animals, increased from 0.53 to 0.66 during the 16 weeks of the experiment. This effect was also seen, to a lesser extent in other animals given garlic oil in smaller doses. Tissue The Group cantly
cholesterol cholesterol content of aorta and liver (Table 2) showed raised level in I animals. Administration of 0.25 g garlic oil (Group II) did not signifiaffect the tissue cholesterol level, but 0.5 and 1.0 g doses of garlic oil
1
OIL ON TOTAL,
FREE AND
IN SERUM
1530.0 f 165.5 489.8 f 42.4 1040.2 f 136.8 1485.5 f 179.5 505.8 + 52.2 979.7 f 149.5 1234.1 f 184.7 445.9 * 48.3 788.2 t 158.1 927.4 * 135.5 352.2 f 46.8 575.2 f 1Oa.O
884.9 f 49.5 268.5 + la.9 616.4 ? 40.8 773.4 f 52.7 251.7 * la.6 521.7 + 29.4 745.7 f 48.6 280.4 f 21.8 465.3 + 39.4 624.8 * 51.2 215.4 * 16.9 409.4 f 45.5
72.7 f 6.8 23.6 f 2.3 40.1 f 5.6
76.6 t 6.9 26.2 + 2.8 50.3 r 6.2
68.5 + 5.8 22.4 ? 2.2 46.1 f 4.2
72.5 + 5.6 25.2 ? 2.6 47.3 f 4.8
Total cholesterol Fre6 chdesteml Ester cholesterol
II
III TotaI.choIesterol Free cholesterol Ester ch&esterol
IV Total cholesterol Free cholesterol Ester cholesterol
8th Week
CHOLESTEROL
Total cholesterol Free lcholesterol Ester chdesterol
4th Week
Initial
ESTER
I
Group
Values are mean f SD (mg/lOO ml of sennd.
EFFECT OF GARLIC TEROL FEEDING
TABLE
.-___
AT DIFFERENT
1462.7 + 205.4 539.5 f 61.3 923.2 ? 188.9
1628.3 + 197.8 534.5 + 52.3 1093.8 + 168.6
1969.4 * 208.5 618.2 + 67.6 1351.2 f 192.0
2134.8 f 215.5 529.6 f 58.9 1605.2 + 186.8
12th Week
OF RABBITS
1710.5 + 198.5 680.7 * 72.5 1029.8 f 156.0
1920.5 f 207.5 665.8 f 57.2 1254.7 f 180.2
2295.5 * 216.5 656.0 + 78.8 1639.5 * 188.0
2450.5 f 230.5 610.8 * 61.2 1839.7 f 190.7
16th Week
TIME INTERVALS
AFTER
CHOLES-
128 TABLE
2
EFFECT
OF
GARLIC
ATHEROSCLEROSIS Values
are mean
OIL
ON
TOTAL
CHOLESTEROL
IN AORTA
AND
LIVER
Cholesterol
(mgjg
Aorta
wet
OF
tissue)
Liver
Atherosclerosis
I
32.9
t 4.6
96.3
? 8.5
2.90
* 0.55
II
27.6
r 4.5
92.4
? 8.6
2.60
f 0.40
P I vs. II
III
>O.l 25.3
P Group
GRADING
f SD.
Group
Group
AND
IN RABBITS
I vs. III
IV
significantly lowered and IV animals.
t 4.2
P Group I vs. IV
>O.l 76.7
>0.2 ? 1.8
the cholesterol
73.5
grading
2.10
f 0.28
co.02 -r 7.2
content
1.40
i 0.20
10.001
of aorta and liver in Groups
III
Atherosclerotic changes When aortic strips were graded for atherosclerotic lesions the mean figures + SD obtained for Group II animals were not significantly different from that of the control group (2.60 f 0.40 vs. 2.90 + 0.55). In Groups III and IV animals, however, there was a significant reduction in atheromatous lesions, the gradings being 2.10 + 0.28 and 1.40 f 0.20, respectively. Discussion It has been reported previously that garlic juice administration inhibits hypercholesterolaemia and atherogenesis in cholesterol-fed rabbits [ 1,2]. The results of the present study show‘ that these properties of garlic are present in the oily fraction extracted from raw garlic. Recently Kritchevsky [9] and Bordia et al [lo] also observed inhibiton of atherogenesis by adding garlic oil to a high-cholesterol diet. A mixture of several sulphur-containing compounds isolated from garlic have been reported to be the active constituent responsible for its various pharmacological actions [ll]. As the amount of active principle present in garlic oil is 0.3-0.4s [12], the anti-atherogenic action abserved in the present study resulted from an approximate daily dose of 1-6 mg of the active principle (present in approximately 15-60 g of garlic bulbs). The biochemical data obtained in this study correlated well with the atherosclerotic grading of the aorta in all the animals with or without garlic oil. Genesis of atheromatous lesion in cholesterol-fed animals was associated with a hypercholesterolaemia in which FC/EC was decreased. Inhibition of atherogenesis in garlic-fed animals was always associated with an alteration of the FC/EC ratio in the reverse direction. This correlation, which is significant, may
129
throw some light on the mechanism treated animals. Since it is EC that of cholesterol-fed animals, the low the protective effect of garlic could tiou. Further work is in progress on juice on the above parameters.
of the protective action evidenced in garlicpredominantly accumulated in the intima EC value in garlic-fed rabbits suggests that be due to its inhibitory action on this fracthe effect of the non-oily fractions of garlic
Acknowledgement We thank Dr. G. Barber-Riley, W.H.O. Professor and Chairmen, Department of Physiology, Faculty of Medicine, University of Benghazi for the constructive criticism of the work and going through the manuscript. References 1 Jain, R.C.. Onion and garlic in experimental atherosclerosis, Lancet. 1 (1975) 1240. 2 Jain, R.C., Onion and garlic in experimental cholesterol atherosclerosis in rabbits, Part 1 (Effect on serum lipids and development of atherosclerosis), Artery, 1 (1975) 115. 3 Jain, R.C., and Konar, D.B.. Garlic oil in experimental atherosclerosis. Lancet, 1 (1976) 918. 4 Chopra, R.N., Chopra, I.C., Handa. K.L. and Kapur, L.D. Indigenous Drugs of India, - Allium satiuum L.. 2nd edition. Dhur and Sons. Calcutta, 1951, 271 pp. 5 Wealth of India, published by the Government of India, Council of Scientific and Industrial Research, Delhi, 1948, 56 pp. 6 Jain, R.C., M.D. Thesis, University of Rajasthan, India, 1968. 7 Kim, E., and Goldberg, M., Serum cholesterol assay using a stable Liebermann-Burchard reagent, Clin. Chem., 15 (1962) 1172. 8 Horlick. L., and Duff, G.L., Heparin in cholesterol atherosclerosis in the rabbit. Arch. Path., 57 (1954) 417. 9 Kritchevsky. D., Effect of garlic oil on experimental atherosclerosis in rabbits. Artery, 1 (1975) 319. 10 Bordia, A., Arora, S.K., Kothari, L.K., Jain, K.C., Rathore. B.S., Rathore, AS., Dube. M.K. and Bhu, N., The protective action of essential oils’ of onion and garlic in cholesterol-fed rabbits. Atherosclerosis. 22 (1975) 103. 11 Cavallito. C.J.. Bailey. J.H.. and Buck, J.S., The antibacterial principle of AIlium satiuum, Part 3 (Its precursor and essential oil of garlic), J. Amer. Chem. SIX.. 67 (1945) 1032. 12 No&r. C.R.. Chemistry of Organic Compounds. 3rd edition, W.B. Saunders. Philadelphia, Pa., 1965, 316 pp.