Garlic And Atherosclerosis Analysis Biology Essay

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Garlic and its preparations have been widely recognized as agents for prevention and treatment of Cardiovascular and other metabolic diseases atherosclerosis, hyperlipidemia, thrombosis, hypertension and diabetes. The beneficial effects of garlic extracts may be due to its principal sulphur compounds diallyl disulphide.

A detailed study was undertaken to establish the cause of toxicity and beneficial effects in comparative study of synthetic diallyl disulphide and dipropyl disulphide in experimentally induced atherosclerosis.

Adult male albino rats were divided in to 6 groups. Normal, control, (atherogenic diet fed rats) DADS protective and curative DPDS protective and curative fed with optimum dosage of 100mg/ kg body weight maintained for 60 days. The rats were sacrificed. Lipid profile, tramsaminases, glycoprotein, calcium. LPL level in plasma as well as TBARS and SH group in aorta were estimated. The results indicate there are significant hypolipidemic and hypocholesterolemic effects seen with both DADS and DPDS synthetic compounds. The analysis of the DADS fed rats indicates the presence of acrolein, It is proposed that the acrolein, the metabolic product of DADS is responsible for free radical generation and consequent tissue damage. Hence an attempt was made to study the anti atherosclerotic effects of DPDS do not carry any allyl group hence may not produce the toxic substance acrolein thus it may be non toxic.

The possible catabolism of DPDS similar to any other disulphide leads mainly to production of a saturated thoil, which on further catabolism may give rise to a normal, non toxic metabolite-succinate.Dipropyl isulphide is more safer compared to either garlic extracts or diallyl disulphide.

Key words: diallyl disulphide dipryl disulphide, atherosclerosis, lipid, acrolein.

Introduction:

Direct anti-atherosclerosis related effects of garlic were studied using cell culture. Blood serum taken two hours after oral administration of 300mg garlic powder tablet caused substantially less cholesterol accumulation in cultured cells.This suggests that garlic powder manifests direct anti-atherogenic related action not only in vitro but also in vivo1.

An increased serum cholesterol level has been implicated as an important risk factor for the development of coronary artery disease. It is estimated that 55% of Americans have cholesterol levels > 5.17 mmol/L (200 mg/dl) and are at increased risk for the development of coronary artery disease. The population and adult treatment panel of the national institute of health predicts that the incidence and mortality from coronary disease will be reduced in future through modification of our dietary habits2.

Reports from the medical literature during the past twenty years have suggested that oral garlic supplementation maybe effective in decreasing serum cholesterol levels by as much as 15-20%. These reports however have varied widely in terms of treatment effect, study design, types of preparations, dosage and target population3.

Synthetic aromatic disulphide like dimenthol disulphide (DMDS) is not useful as hypo- lipidemic agents. The hypo- lipidemic effects are shown probably by aliphatic disulphides only.

Hypolipidemic actions of garlic homogenate in presence of conventional antihypertensive drugs, propranolol, hydrochlorothiazide and captopril : albino rats fed with normal fat diet or high fat diet were treated with garlic homogenate at three different doses (125, 250 and 500 mg/kg) orally for 30 days or in combination with propanolol (10 mg/kg), hydrochlorothiazide (10 mg/kg) and captopril (30 mg/kg) during last 7 days of garlic homogenate treatment. After the treatment, total cholesterol (TC), LDL cholesterol, triglyceride (TG) and HDL cholesterol were measured in serum and anti-atherogenic index was calculated. The result showed that moderate and high doses of garlic homogenate possessed potential anti-atherosclerotic property.(Syed MB et al. 2009)

Lipid lowering effects of garlic: many randomized clinical trials have studied the effect of garlic on lipid levels. Results from two meta analysis conducted in 1993 and 1994 of garlic's effect on total cholesterol show a significant reduction in total cholesterol levels (9 to 12%) (Ellen Tattelman M.D 2005)

Garlic was known to be effective in decreasing cholesterol and can inhibit LDL oxidation. Over 40 clinical trials have been conducted to determine the lipid lowering effects of fresh garlic and garlic supplements. Many clinical trials showed a positive effect of garlic on hyperlipidemia, atherosclerosis, thrombosis, hypertension and diabetes. (Rizwan Ashraf et al. 2005)

The aim of the investigation was to assess an effect of garlic allicor at lipid metabolism and a free radical oxidation of blood lipids anthropometric values and arterial blood pressure at patients with atherogenic dislipoprotiolemy. 112 patients (47 men and 65 women) 40 to 60 yers of age were examined. 56 patients had ischemic heart disease and or equal disorders. Another 56 patients were free of any sign of atherosclerosis but had one or more risk factors of cardiovascular pathology. Six months therapy using garlic allicor results in moderate hypolipidemic and anti- oxidative effect. (N I Gromnatskii et al. 2007)

Garlic belongs to the plant genus Allium and is known for its pungency and spiciness. Garlic is thought in particular to be linked to various beneficial health effects from reducing blood pressure and cholesterol to treating cancer. Garlic may also possess of antifungal, antibacterial, cardioprotective, antioxidant and anticancer activities. In cancer studies a host of studies provide coupelling evidence that garlic and its organic allyl sulphur components are effective inhibitors of the cancer process. Several compounds are involved in garlic's possible anticancer effects. Garlic contains allyl sulphur and other compounds that slow or prevent the growth of tumor cells. (Vidyashankar. S 2008)

Rats fed with alcohol (18%) at 3.76 g/day for 45 days showed significant reduction in body weight, glutathione content and activities of superoxide dismutase and catalase in liver. Lecithin cholesterol acyltransferase in plasma, levels of HDL in serum, hepatic bile acid production and fecal excretion of neutral sterols also showed significant reduction. Stimultaneous feeding of garlic protein or soy protein (500 mg/kg body weight/day for 45 days) to alcohol fed groups increased each of above parameters significantly towards normal values. Increase in glutathione content and catalase activity in liver was significantly higher for soy protein treated group than for garlic protein treated group. However, increase in plasma Lecithin cholesterol acyltransferase was significantly higher for garlic protein treated group than for soy protein treated group. Alcohol fed rats showed significant increase in liver weight, serum and tissue cholesterol, serum triacylglycerol, phospholipids and free fat acid levels and acivity of HMGCoA reductase in liver and intestine. Lipid peroxidation, glucose- 6 phosphate dehydrogenase, glutathione peroxidase and glutathione reductase in liver and incorporation of labeled acetate into liver cholesterol also showed significant increase. Garlic protein and soy protein treated rats showed decrease in these values towards in normal. (C R Rajashree et al 2008)

In rats rendered hyperlipidemic by maintaining them on a high fat diet (30%) for 8 weeks, inclusion of spice principles[curcumin(0.2%) or capsaicin (0.015%)] or garlic (2%) in the diet produced significant hypo triglyceridemic effect. Plasma cholesterol remained unaffected in high fat treatment. Hepatic triglyceride content was significantly higher in high fat fed rats, and this increase was effectively countered by inclusion of the hypolipidemic spice agents - curcumin, capsaicin or garlic in the diet. (Kempaiah RK et al. 2006)

Stavudine (Zerit, d4T) is widely used as an anti HIV infection drug. It prevents HIV by altering the genetic material of healthy cells but causes mutations in mitochondrial and nuclear DNA. It also produces clastogenic effects in mice. In the present investigation, comet assay test was applied to evaluate the possible genomic damage caused by stavudine and also ameliorating effects of garlic oil and vitamin E against its genotoxicity in different organs of mice. Two different doses of garlic oil (low and high dose) and vitamin E were administered to mice separately and in combination for six consecutive days followed by a dose of stavudine. The mice were sacrificed after 24, 48 and 72 hours of stavudine administration. Both the antioxidants (vitamin E and garlic oil) separately and in combination reduced the genotoxicity of stavudine. The protective effects of high doses of garlic oil were more pronounced as compared to vitamin E administered group. (Pushpindar Kaur et al. 2007)

Materials & Methods

Most researchers have attributed the hypolipidemic, hypocholesterolemic, anti-carcinogenic, hypoglycemic, anti-microbial and antifibrinolytic activity to the presence of diallyl disulphide, diallyl tri-sulphide, diallyl tetra sulphide, allicin and cyclic di-ethines. Based on these conclusions we have conducted studies on experimental animals ie., albino rats by feeding them with atherogenic diet and DADS aswell as DPDS in both protective and curative groups.

Since these studies showed more of beneficial effects, we procured the synthetic compound diallyl disulphide and dipropyl disulphide from Aldrich company , U.S.A and conducted further studies on experimental animals fed with atherogenic diet.

Study of DADS on albino rats:

Male albino rats maintained on atherogenic diet adlibitum and given 50mg, 100mg, 150mg and 200mg of diallyl disulphide as 30ml of warm aqueous solution/kg body weight respectively using gastric tube for 60 days.

Study of DPDS on albino rats:

Male albino rats maintained on atherogenic diet adlibitum and given 50mg, 100mg, 150mg and 200mg of dipropyl disulphide as 30ml of warm aqueous solution/kg body weight respectively using gastric tube for 60 days.

From the above studies it was concluded that the effective optimum beneficial dose of DADS and DPDS was 100mg. Later a detail study was undertaken to evaluate the anti- atherogenic effects of DADS and DPDS in experimental atherosclerosis ie., protective as well as curative study.

Experimental animals: 36 male albino rats of 6 to 8 weeks old weighing 150g -200g were selected for the present study from the animal house of Dr. B. R. Ambedkar Medical College. These rats were kept on stock laboratory diet. (Amruth rat feed Nava Maharastra Chakan Oils Ltd. Pune.)

Statistical analysis: Date were analyzed by ANOVA and subsequently was done by comparing the results of groups using steudent't' test. Probability values less than 0.02 were considered as significant.

Experimental induction of atherosclerosis:

A selected group of rats were fed atherogenic diet adlibitum for 60 days. All these rats were provided with water always adlibitum. The atherogenic diet was prepared by mixing whole milk powder, dalda and pure cholesterol in the ratio 1:0.5:0.1 with an extra vitamin D2 supplement of 4 mg/100 g atherogenic diet.

The rats normal as well as atherosclerotic were divided into 6 groups, consisting six animals in each group.

Group 1 (Normal group) - Consists 6 male albino rats fed stock laboratory diet and given orally 30ml of normal saline per kg body weight daily for 60 days.

Group-2 (Control group) - Consisting 6 rats fed atherogenic diet adlibitum for 60 days and given normal saline 30ml per kg body weight daily.

Group-3 (DADS Protective group)- Consisting 6 male albino rats maintained on atherogenic diet adlibitum for 60 days and given 100mg of DADS as 30ml of warm aqueous solution/kg body weight using gastric tube for 60 days.

Group-4 (DADS Curative group)- Consisting 6 male albino rats maintained on atherogenic diet adlibitum for 60 days and then given 100mg of DADS as 30ml of warm aqueous solution/kg body weight daily using gastric tube for another 60 days. During DADS feeding, the rats were maintained on stock laboratory diet, water was provided adlibitum to all these rats always.

Group-5 (DPDS Protective group)- Consisting 6 male albino rats maintained on atherogenic diet adlibitum for 60 days and given 100mg of DPDS as 30ml of warm aqueous solution/kg body weight using gastric tube for 60 days.

Group-6 (DPDS Curative group)- Consisting 6 male albino rats maintained on atherogenic diet adlibitum for 60 days and then given 100mg of DPDS as 30ml of warm aqueous solution/kg body weight daily using gastric tube for another 60 days. During DPDS feeding, the rats were maintained on stock laboratory diet, water was provided adlibitum to all these rats always.

The rats of the group 1.2,3 &5 were sacrificed by decapitation on the 61 days and the rats of group-4&6 were sacrificed by decapitation on the 121 day. Blood sample were collected using heparin as anti coagulant. Aorta was removed and put into pre weighted dry watch glass. Plasma was separated and used for estimation of Total lipids (TL)4, Triacyl glycerol (TAG)5, Total cholesterol (TC) 6 phospholipids (PL)7, HDL cholesterol 8, Free fatty acid (FFA) 9, Esterified fatty acid (EFA)9, Calcium 10, Glycoprotein11, Fibrinogen11, Lipoperotein lipase12, Aspartate Amino Transferase (AST13), and Alanine Amino Transferase (ALT 13)

A portion of aorta was immediately fixed in buffered saline and was employed for histopathological study.

A second portion of aorta was homogenized with chloroform methanol ( 1:1v/v) mixture and the extracts were used for estimation of lipid parameters.

A third portion of aorta was homogenized with 5% cold TCA and the extracts were used for the estimation of Thiobarbituric acid reactive substances (TBARS)14

A fourth portion of aorta was homogenized with phosphate buffer (pH 7.4) and the extracts were used for the estimation of total protein15 (TP)and Total sulfahydryl groups16 (SH).

Results:

Results obtained in the present study are given in table 1-2, Figure 1-6.

Table 1: Gives the mean values of TL, TAG, TC, PL, HDL cholesterol, FFA, EFA, Calcium,

Glycoprotein, Fibrinogen, LPL, AST, and ALT in Plasma in normal, control, AGE protective, AGE curative, HGE protective & HGE curative group of rats.

Table-2: Shows the aortic mean values of TL,TAG, TC, PL, TBARS, SH groups and TP (Total protein) in normal, control, AGE protective, AGE curative, HGE protective and HGE curative group of rats.

Figures 1-6 shows the histopathological findings of aortic section (H &E stain) of normal, control, DADS protective, DADS curative DPDS protective and DPDS curative group of rats. It is evident from table 1 all the lipid parameter except HDL cholesterol are increased in control group as compared to normal group. These parameters were significantly reduced in DADS protective, DADS curative, DPDS protective and HGE curative group of rats compared to control group establishing both DADS and DPDS has hypolipidemic effects. Further a raise in Glycoprotein and Fibrinogen levels seen in control group as compared to normal group. Where as feeding DADS & DPDS significantly reduces these values in protective as well as curative group as compared to control groups. The plasma AST and ALT levels are elevated in control group as compared to normal group showing a possibility of tissue damage.

There is a significant raise in aortic levels of TL, TAG, TC, PL and TP in control group as compared to normal group as seen from table 2 suggesting feeding atherogenic diet leads to accumulation of lipids and protein in aorta. These values are significantly reduced in DADS protective, DADS curative DPDS protective and DPDS curative group establishing that feeding DADS and DPDS decreases the accumulation of lipids in aorta.

The aortic TBARS levels decreased and total SH group - increased in DADS protective, DADS curative, DPDS protective & DPDS curative group as compared to control group as seen from Table 2.

It is evident from the figures that there is an accumulation of lipids in aortic walls in control group as compared to normal group. Further there is significant decrease in this accumulation in both protective as well as curative groups as seen from the fig 1-6.

Discussion:

Anti atherosclerotic effects of DADS

By the studies with DADS it is

evident that they are hypolipidemic as well as hypocholesterolemic effect in atherogenic diet fed rats and this activity may be principally due to the action of DADS.

evident that they are hypolipidemic as

well as hypocholesterolemic effect in atherogenic diet fed rats and this activity

The possible role of DADS was further evidenced by giving DADS to atherogenic diet fed rats. An optimum hypolipidemic dose of DADS (100mg /kg body wt.) was arrived at by conducting a series of feeding experiments with different doses of DADS along with atherogenic diet. This optimum dose of DADS shows a significant hypolipidemic as well as anti atherosclerotic effects in atherogenic diet fed rats, (group-3 and 4) as compared to control (group-2) which is evident from the table 1 & 2 and figures 3 & 4.

This reduction of both plasma and aortic lipid levels may be due to NADPD decreasing effects of DADS. The plasma levels of Calcium, Glycoprotien , as well as Fibrinogen are The possible role of DADS was further evidenced by giving DADS to atherogenic diet fed rats. An optimum hypolipidemic dose of DADS (100 mg/kg body wt.) was arrived at by conducting a series of feeding experiments with different doses of DADS along with atherogenic diet. This optimum dose of DADS shows a significant hypolipidemic as well as anti atherosclerotic effects in atherogenic diet fed rats, (group 3 and 4) as compared to control (group 2) which is evident from the table 1 and 2 and figures 3and 4. This reduction of both plasma and aortic lipid levels may be due to NADPD decreasing effects of DADS. The plasma levels of Calcium, Glycoprotein, as well as Fibrinogen were significantly lowered in both DADS protective (group- 3 ) and DADS curative (group- 4 ) as compared to control (group-2) suggests that DADS possibly decreases the production of these proteins by the aortic cells or may prevent infiltration of these proteins as well as Calcium into the aortic cells probably by preventing any induced membrane changes. Thus DADS may slow down the formation of atheromatous plaques as well as may favour a regression in atherosclerotic plaques (refer fig. 3 & 4)

Further increase in plasma LPL activity in both group 3 and 4 rats as compared to group 2 rats suggest DADS favours the clearance of triacylglycerol and other lipid materials probably protecting LPL through the sulphydryl exchange reactions.

DADS is a disulphide maybe metabolized in the tissues, in accordance with any other disulphides17 (Black.S 1962) utilizing NADPH to its mercaptan as given below.

This leads to the depletion of cellular available NADPH levels and decreases the synthesis of fat and cholesterol as their synthesis requires NADPH. It is evident from table 1 and 2 that there is a significant decrease in the levels of lipid parameters both in plasma and aorta whereas the HDL levels are significantly raised in plasma in DADS fed rats, group 3 and 4 as compared to control rats (group 2). Further DADS can undergo sulphydryl exchange reaction with tissue protein and enzymes similar to other disulphide.18

R1 -S- S - R1 + R2 - SH → R1 - S - S - R2 + R1 -SH

Such a possible sulfhydryl exchange reaction of DADS with fatty acid synthesize or HMG CoA reductase could bring about a conformational change in these enzymes resulting in a possible inhibition of these enzymes causing a significant reduction in fatty acid synthesis, as well as cholesterol synthesis respectively19.(Sodimuo etal,1994)

Thus the hypolipidemic effects of DADS may be due to its NADPH lowering level due to an induced conformational change in the key enzymes of lipid synthesis.

Biochemical toxic effects of DADS:

The results of anti- atherosclerotic experiments with DADS showed a significant raise in plasma transaminases as well as aortic TBARS levels with concomittent decrease in aortic total SH groups. These results probably indicate a possible tissue damage thereby causing a leakage of transaminases is observed. This is further evidenced by a raise in aortic TBARS levels. These toxic effects of DADS maybe due to acrolein generated during the breakdown of DADS. Acrolein itself is a toxic substance20 (Dunhill M.S 1982). The aliphatic disulphide, diallyl disulphide could be metabolized as follows.

The allyl mercaptan produced by an unknown may give rise to acrolein, probably by oxidation and desulphuration. Hence consumption of DADS in increased quantity is toxic and may cause tissue damage by producing free radicals.

Anti atherogenic effects of DPDS

DADS being unsaturated aliphatic disulphide having allyl groups maybe metabolized to give acrolein which is responsible for toxic effects. Hence an attempt was made to study the anti- atherosclerotic effects of saturated aliphatic disulphides, dipropyl disulphide. As this disulphide does not carry any allyl group, it may not produce the toxic substance acrolein, thus it maybe non toxic.

The results of the experiments conducted with DPDS show significant lipid lowering as well as anti- atherosclerotic effects in experimentally induced atherosclerosis which is evident from the table 1 and 2 and fig. 5 and 6.

The possible catabolism of DPDS similar to any other disulphide21 (Sheela C.G 1993) leads mainly to production of a saturated thiol, which on further catabolism may give rise to a normal non toxic metabolite- succinate.

This catabolism of DPDS requires NADPH/ NADH. This causes a decrease cellular NADPH/ NADH levels, Hence reduces synthesis of fatty acids as well as cholesterol17 (Black.S. 1962)

Similar to any other disulphides DPDS can undergo sulphydryl exchange reaction with tissue proteins and enzymes there by altering their effects.

R1 -S- S - R1 + R2 - SH → R1 - S - S - R2 + R1 -SH

Such an exchange reaction with HMGCoA reductase may decrease the activity of this enzyme there by causing a decrease in cholesterol synthesis. This is quite similar to the action of DADS.18

Similar to DADS the saturated dipropyl disulphide may decrease the

plasma levels of glycoprotein, fibrinogen and calcium probably by decreasing their production or release. Further DPDS may prevent infiltration and accumulation of these substances in the intima of blood vessels probably by preventing high lipid induced capillary membrane alterations, there by prevents accumulation of these substances along with lipid materials resulting in the prevention of high lipid induced atherosclerosis or promotes regression of atheromatous plaques in atherogenic diet induced atherosclerosis in rats (refer table 1 and 2, fig. 5 and 6)

DPDS is safer:

The results of feeding DADS as well as DPds to normal rats suggest that DPds is safer and less toxic as compared to DADS. The raise in aortic TBARS levels as well as plasma transaminases in DPDS treated rats (both protective and curative studies) ins comparatively much lower than DADS treated rats. This suggest that DPDS is safe and less toxic.

DPDS is a synthetic saturated aliphatic low molecular weight, disulphide. It has been tried and showed more effective hypolipidemic as well as anti- atherosclerotic and it is less toxic.

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