Potential of garlic and its active constituent, S-allyl cysteine, as antihypertensive and cardioprotective in presence of captopril.
The purpose of the present study was to investigate the role of fresh garlic homogenate (FGH) and its bioactive sulphur compound S-allyl cysteine sulphoxide (SACS) in potentiating antihypertensive and cardioprotective activities of captopril in rats. SACS was extracted from the fresh garlic using ion exchange resins with yield of 890 mg/kg garlic. The dose of SACS was calculated based on the amount of SACS extracted from 125 to 250 mg of FGH. Albino rats weighing 150-200 g were fed with 10% fructose in fluid for 3 weeks for induction of hypertension and subsequently administered FGH (125 and 250 mg/kg, p.o.) or SACS (0.111 and 0.222 mg/kg/day, p.o.) for the next 3 weeks in their respective groups. In CAP alone and interactive groups (GH + CAP; SACS + CAP), captopril 30 mg/kg was given during sixth week of 10% fructose in fluid. At the end of drug treatment, animals were given isoproterenol 175 mg/kg subcuta-neously for two consecutive days. Additionally, varying concentrations of SACS (4, 8, 16, 32 and 64 ng), CAP (1, 2, 4, 8 and 16 ng) and their combination (4:1) were checked for fall in blood pressure in hypertensive rats (10% fructose in fluid without pretreatment) as well as angiotensin-converting enzyme (ACE) inhibiting activity using guinea pig ileum. An isobolographic analysis was used to characterise the interaction between SACS and CAP for fall in blood pressure and ACE inhibiting evaluations. Administration of captopril, low and high doses of FGH (125, 250 mg/kg), either alone or together showed fall in fluid intake and body weight. The combined therapy of FGH 250 mg/kg and CAP was more effective in reducing systolic blood pressure, cholesterol, triglycerides and glucose. The SOD and catalase activities in heart tissue were significantly elevated in groups treated with FGH, SACS, CAP, FGH + CAP and SACS + CAP. Further, combined therapy of FGH 250 mg/kg and CAP caused significant fall in LDH and CK-MB activities in serum and elevation in heart tissue homogenate. SACS in low dose was less effective than low dose of FGH; similarly, high dose of FGH was more efficacious than high dose of SACS. Corroborating with this, combined therapy of garlic (250 mg/kg) with CAP demonstrated higher synergistic action than combination of SACS (0.222 mg/kg) with CAP suggesting the role of additional bioactive constituents apart from SACS, responsible for therapeutic efficacy of garlic. Moreover, combination of SACS and CAP exerted super-additive (synergistic) interaction with respect to fall in blood pressure and ACE inhibition. This study may represent an advertence on concomitant use of garlic or its bioactive constituent, SACS, with captopril.
S-allyl cysteine sulphoxide
[C] 2010 Elsevier GmbH. All rights reserved.
Garlic (Allium sativum) is used traditionally as a complementary therapy in the treatment of several diseases such as diabetes, several forms of cancer and neurodegenerative conditions such as ischemic stroke (Banerjee and Moulik 2002; Rahman 2003). In addition, garlic has been reported to possess range of cardiovascular effects such as lowering of plasma cholesterol; inhibition of platelet aggregation as well as reducing of arterial blood pressure (Ali and Thomson 1995). S-allyl cysteine sulphoxide (SACS), a main bioactive constituent of garlic is an organosulphur-containing amino acid (Kim et al. 2006a,b). Similar to garlic extract, SACS is reported to be antioxidative (Herrera-Mundo et al. 2006; Numagami and Ohnishi 2001); anti-cancer (Chu et al. 2007; Welch et al. 1992); anti-hepatotoxic (Hsu et al. 2006; Nakagawa et al. 1989) and can also reduce the incidence of stroke (Kim et al. 2006a,b). In the cardiac context, Padmanabhan and Prince (2006) have reported that SACS mediates cardioprotection in myocardial infarction via its antioxidative properties by decreasing lipid peroxide products. Patients with hypertension and ischemic heart disease are candidates for treatment with ACE inhibitors because administration of ACE inhibitors in the immediate post-myocardial infarction period has been shown to improve ventricular function and reduce morbidity and mortality (Hsu et al. 2006). Capto-pril (CAP), l-[(2S)-3-mercapto-2-methylpropionytl]-l-proline, is an angiotensin-converting enzyme inhibitor that is used in the treatment of hypertension and congestive heart failure (Nakagawa et al. 1989). It is known that captopril (CAP) can ameliorate the deleterious effects of elevated renin and angiotensin II levels in patients with acute myocardial infarction.
Garlic enjoys the reputation of potentiating the efficacy of concurrently administered cardiovascular therapies. Corroborating with this, we reported improved survival and cardiac function by add-on captopril (Asdaq and Inamdar 2010; Asdaq and Inamdar 2008) and propranolol (Asdaq et al. 2008) during garlic therapy in rats with myocardial infarction. Earlier reports on the drug interaction studies of garlic with calcium channel blocker indicate that it produces concentration dependent synergistic effect by its calcium blocking property (Aqel et al. 1991). Beneficial effects of combined therapy of garlic and hydrochlorothiazide was also demonstrated and confirmed in the recent past (Asdaq and Inamdar 2009a,b). With these reported interactions, we found that garlic has the enormous ability to boost the performance of concurrently administered conventional cardioprotective drugs at times of myocardial damage. Thus the present study was carried out to explore the role of garlic and its bioactive constituent, S-allyl cysteine sulphoxide, in potentiating cardioprotective and antihypertensive activities of captopril in rats.
Materials and methods
Laboratory bred female Wistar albino rats (200-250 g) and guinea pigs of either sex (300-500 g) were housed at 25 [+ or -] 5 [degrees]C in a well-ventilated animal house under 12:12 h light-dark cycle. The animals had free access to standard rat chow (Amrut Laboratory Animal feed, Maharashtra, India) containing(%, w/w) protein 22.10, oil 4.13, fibre 3.15, ash 5.15, sand (silica) 1.12, and water ad libitum. The animals were maintained under standard conditions in an animal house approved by Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA).
Preparation of garlic homogenate
Garlic (Allium sativum, family: Lilliaceae) bulbs were purchased from the local vegetable market. The cloves were peeled, sliced and ground into a paste and suspended in distilled water. Two different concentrations of the garlic homogenate (FGH) were prepared, 0.1 and 0.2 g/ml, corresponding to 125 mg and 250 mg body weight of animal (Banerjee et al. 2002). GH was administered within 30 min of preparation.
Extraction of SACS from fresh garlic homogenate (Itokawa et al. 1973)
Garlic was purchased from the local market. SACS was extracted from fresh garlic using ion exchange resins washed with 1 N HCI and deionized water. Inactivation of enzyme alliinase is achieved by boiling fresh garlic. It was then ground and extracted with 80% methanol, filtered and passed through a column of amberlite IR-120 (strong cation exchanger) so as to absorb the amino acids. The column was washed with deionized water to remove the impurities and the amino acids were eluted with [NH.sub.4]OH (2 N). Ammonia was removed by concentration of the eluates in a rotary evaporator at 40-43 [degrees]C and the concentrate was loaded onto a column of amberlite CG-120 (strong cation exchanger). The column was washed with deionized water and the amino acids eluted with 0.1 N [NH.sub.4]OH. Fractions of the effluent were tested for the presence of SACS by thin layer chromatography (TLC) as described below. The SACS containing fractions was pooled, concentrated and passed through a column of amberlite IRA-45 (weakly basic anion) so that unwanted amino acids and ammonia were absorbed onto the resin and removed. The effluent was collected and concentrated. Pure SACS was obtained from it after three recrystallization steps from 80% ethanol. It had a melting point of 164 [degrees]C. The yield of SACS was 890 mg/kg of garlic. In TLC on silica gel G, using butanol: acetic acid: water (12:3:5) pure SACS gave an [R.sub.[Florin]] value of 0.22, similar to that of an authentic sample of Biochemical Institute, Helsinki.
Female Wistar rats were divided into 11 groups of eight animals each. Group I was considered as control group, given ordinary drinking water ad libitum throughout the treatment course and the remaining groups were given 10% fructose solution to drink ad libitum (Dimo et al. 2001). Three weeks later, the fructose-treated animals were assigned the following treatment regimens - group II: fructose-fed (HF), group III: fructose plus captopril (30 mg/kg) (Kosmala et al. 1992) in the sixth week, groups IV and V were fed with fructose plus FGH 125 and 250 mg/kg, respectively, orally for 3 weeks, groups VI and VII were given 0.111 and 0.222 mg/kg of SACS orally during high fructose in fluid, groups VIII and IX were continued with fructose plus FGH 125 and 250 mg/kg respectively orally for 3 weeks as well as captopril (30 mg/kg, p.o.) in the sixth week and groups X and XI were kept on high fructose in fluid with SACS 0.111 and 0.222 mg/kg from fourth to sixth week plus captopril 30 mg/kg during sixth week. All SCS, FGH and CAP treatments were done once daily orally. Fluid intake, food intake, body weight, heart rate and systolic blood pressure (SBP) were measured every week. Concentrations of glucose, cholesterol and triglycerides (Mc Gown et al. 1983; Fossati and Prencipe 1982) were measured in plasma samples at the end of the experiment.
Isoproterenol (ISO) induced myocardial damage
At the end of treatment and recordings as mentioned above, animals of all groups except group I were administered ISO (175 mg/kg, s.c.) for two consecutive days. Blood was withdrawn from retroor-bital vein 48 h after the first dose of ISO under anesthesia and serum was separated by centrifugation for lactate dehydrogenase (LDH) and creatine phosphokinase-MB (CK-MB) measurement. After recording ECG changes, the heart was immediately isolated from each animal under ketamine (70 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anesthesia. In each group consisting of eight animals, four excised hearts were homogenized to prepare heart tissue homogenate (HTH) using sucrose (0.25 M) (Buerke et al. 1998). The activity of LDH, CK-MB, superoxide dismutase (SOD) (Erich and Elastner 1976) and catalase (Eva 1988) was measured in HTH. Microscopic slides of myocardium were prepared for histopatho-logical studies from the hearts of remaining four animals. The myocardial damage was determined by giving scores depending on the intensity as follows (Karthikeyan et al. 2007); no changes - score 00; mild - score 01 (focal myocytes damage or small multifocal degeneration with slight degree of inflammatory process); moderate - score 02 (extensive myofibrillar degeneration and/or diffuse inflammatory process); marked - score 03 (necrosis with diffuse inflammatory process).
Blood pressure and electrocardiograms measurement
As discussed above, during 6 weeks of 10% fructose in fluid, mean arterial blood pressure were measured every week in awaked animals by the non-invasive blood pressure module (NIBP pressure meter, LE 5001, V02/0402L, Panlab, Hardvard apparatus, Barcelona, Spain) and ECG was recorded in anesthetised animals [ketamine (70 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.)] by subcutaneous lead II method (Physiograph, EKG coupler, SO-02, INCO, India), QRS duration, RR interval and QT segment were measured.
Results of pharmacodynamic parameters are expressed as mean [+ or -] SEM. The statistical significance was determined using oneway analysis of variance (ANOVA) followed by Bonferroni's test. The results were considered statistically significant when P < 0.05.
ACE inhibition in the isolated guinea pig ileum
A method described by Rubin et al. (1978) was adopted in this study with slight modifications. Pieces of 3 cm length of ileum of guinea pig were cut and fixed with a tissue clamp and brought into an organ bath with Tyrode's solution at 37 C being oxygenated with [O.sub.2]. The other end was fixed to an isometric force transducer (FT-2625, RMS India) and the responses recorded on a polygraph (RMS, India). After an equilibrium time of 30min, angiotensin I (Ang I) was added in a concentration of 10 ng/ml bath solution. After allowing angiotensin I to get converted into angiotensin II for about 5 min, contraction was recorded and the angiotensin I dosage was repeated until the responses were identical. Similarly, Ang I induced contractions were taken after incubating the tissue for five minutes with SACS (4, 8, 16, 32 and 64 ng), CAP (1, 2, 4, 8 and 16 ng) and combination of SACS and CAP in 4:1 ratio. The responses were repeated for each concentration at least six times with intermittent washings in different tissue samples.
Fall in blood pressure in hypertensive rats
At the end of 6 weeks of high fructose (10%) in fluid (Dimo et al. 2001), elevated blood pressure was established. Fall in blood pressure was recorded with SACS (4, 8, 16, 32 and 64 ng), CAP (1, 2, 4, 8 and 16 ng) and combination of SACS and CAP at fixed ratio of 4:1. The therapeutic effects were noted for each dose at least six times in different hypertensive animals.
Analysis of the interaction
Dose-response curves were plotted to evaluate the ACE inhibiting activity and fall in blood pressure due to S-allyl cysteine sulphoxide and captopril at five different doses. The doses of the S-allyl cysteine sulphoxide were 4, 8, 16, 32 and 64 ng and captopril was 1, 2, 4, 8 and 16 ng. The dose that produced 50% of sedation ([ED.sub.50]) 50% of reduction in the ACE activity and fall in blood pressure were calculated using standard linear regression analysis of the log dose-response (Tallarida 2000).
An isobolographic analysis was performed to characterize the interaction between S-allyl cysteine sulphoxide and captopril. The theoretical additive doses ([Z.sub.add]) for each combination in the same component ratio (4:1) were computed from the median effective doses ([ED.sub.50]) of the single drugs, according to the method described by Tallarida (1992) to satisfy the equation:
[Z.sub.add] = fA + (1 - f)B
where A was the [ED.sub.50] of S-allyl cysteine sulphoxide and B was the [ED.sub.50] of the captopril. For a 4:1 fixed ratio, [Florin] in this case was 0.2 and (1 - [Florin]) was 0.8. The value [Florin]A = a represents the fraction of the [ED.sub.50] of the S-allyl cysteine sulphoxide in the combination and (1 - [Florin])B = b represents the fraction of [ED.sub.50] of the captopril in the combination (Tallarida 2000). [Z.sub.add] represents a total additive dose of the drugs, theoretically providing a 50% of ACE inhibition and fall in blood pressure with reference to control group. [Z.sub.exp] is an experimentally determined total dose of a mixture of two component drugs, which was administered at a 4:1 fixed ratio combination sufficient to reduce the ACE activity and fall in blood pressure by 50% with respect to the control group. The [Z.sub.exp] values were determined from the respective drug-dose effect curves of combined drugs, according to standard linear regression analysis of the log dose-response curve (Tallarida 2000).
Experimentally determined [Z.sub.exp] was statistically compared with the theoretically calculated [Z.sub.add] doses with the use of Student's t-test, according to the procedures previously described by Tallarida et al. (1989), who has proposed the use of this statistical test for analyzing the data in isobolography. [Z.sub.exp] values that were lower than [Z.sub.add] value, with a p < 0.05 for the differences in both the X and Y directions, were interpreted as a significant super-additive interaction. Graphical representation of the observed interactions in the shape of isoboles is a simple form of visualization of interactions, facilitated the interpretation of interactions between S-allyl cysteine sulphoxide and captopril. The isobologram was constructed by connecting the [ED.sub.50] of S-allyl cysteine sulphoxide on the abscissa with [ED.sub.50] of the captopril on the ordinate to obtain the additivity line (Tallarida 2000). The amounts of each component in combination (experimental ([Z.sub.exp])and theoretical additive ([Z.sub.add]) doses) were also plotted in the same graph. The theoretical additive point lies on a line connecting the [ED.sub.50] values of the individual drugs. To get a value describing of the magnitude of the interaction, a fractional analysis was performed for each combination, using the [ED.sub.50] of the S-allyl cysteine sulphoxide, captopril and their combination according to:
a/A + b/B
where A and B are the [ED.sub.50] when each drug (S-allyl cysteine sulphoxide and captopril) acts alone; a and b are the amounts when each drug acts in the combination. These total fraction values measure the divergence between the experimental dose ([Z.sub.exp]) of the combination and the theoretical ([Z.sub.add]) additive dose (Tallarida 2000). a/A + b/B was interpreted as super-additive interaction if a/A + b/B was < 1.0 and as sub-additive interaction if a/A + b/B was > 1.0 (Tallarida 2000; Wagner and Ulrich-Merzenich 2009).
Effect on fluid intake, food intake and body weight
Fluid and food intake apart from body weight of the various groups of rats are shown in Fig. 1. Significant increase in fluid intake as well as body weight and decrease in food intake was found at the end of 3 weeks of fructose in water administration when compared to normal control. Treatment of animals with CAP and different doses of FGH, SACS, alone or together, found to bring back the normal conditions in fluid intake, body weight and food intake, to significant extent. The high dose of FGH (250 mg/kg, p.o. for 3 weeks) during high fructose intake was found to be most effective in ameliorating the abnormally increased fluid intake and body weight as well as decreased food intake compared to HF control. High dose of SACS was also found to normalise these parameters at the end of three weeks with or without CAP.
[FIGURE 1 OMITTED]
Effect on systolic blood pressure, heart rate, cholesterol, triglycerides and glucose
The details about the systolic blood pressure, heart rate, cholesterol, triglycerides and glucose of all the groups are given in Figs. 2 and 3. Six weeks of HF in water resulted in significant elevation in systolic blood pressure, heart rate, cholesterol, triglycerides and glucose compared to normal control. Administration of CAP and various doses of FGH and SACS demonstrated significant fall in all the above-mentioned parameters to varying degrees when compared to HF control. The increased levels of blood pressure, heart rate, cholesterol, triglycerides and glucose was significantly lowered in animals treated with CAP (for 7 days during the sixth week of HF intake) compared to HF control. The maximum protective effect was seen in combination therapy of FGH (250 mg/kg, p.o. from fourth to sixth week of HF intake) with CAP (captopril, 30 mg/kg, p.o. during the sixth week of HF intake) when compared to HF control.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
Effect on electrocardiographic parameters
As shown in Fig. 4, significant changes in the ECG configuration were observed in the rats with HF + ISO treatment such as prolongation of QRS duration as well as significant longer QT segment and reduction in RR intervals. The CAP treated animals showed significant prolongation of QRS complex compared to normal control. The combination of high doses of FGH/SACS with CAP was found to significantly decrease the QRS complex compared to their individual treatment. There was fall in QT segment and normal RR interval in FGH 250 mg/kg and SACS 0.222 mg/kg alone or in presence of CAP compared to HF + ISO control.
[FIGURE 4 OMITTED]
Effect on biochemical parameters, antioxidants and histological scores
As evident from Figs. 5 and 6, serum LDH and CK-MB activities were increased in animals subjected to high fructose fluid for 6 weeks with subsequent two doses of ISO when compared to normal control. On the contrary, decreased activities of LDH and CK-MB were found in heart tissue homogenate (HTH) of HF + ISO animals as compared to normal control. Oral administration of CAP or different doses of FGH/SACS separately or together resulted in fall in LDH and CK-MB activities in serum and rise in HTH when compared to HF + ISO control. The best results were found with combination of CAP with FGH 250 mg/kg as there was significant difference in CK-MB activity between FGH 250 mg/kg alone and CAP + FGH 250mg/kg. Our results also demonstrated rise in CK-MB and LDH activities in HTH in animals pretreated with low and high doses of SACS. However, there was insignificant increase in these enzyme activities in FGH groups when compared to SACS groups.
[FIGURE 5 OMITTED]
[FIGURE 6 OMITTED]
Fig. 7 shows significant fall in SOD and Catalase activities with a rise in histological scores of animals subjected to high fructose intake plus isoproterenol compared to normal control. The SOD and catalase activities of all the treatment groups were found to be increased towards normal compared to HF + ISO control. Significant recovery was seen in animals treated with CAP and FGH 250 mg/kg/SACS 0.222 mg/kg compared to individual CAP group. The histological scores were found to be reverted to normal condition in all treated groups compared to HF + ISO control.
[FIGURE 7 OMITTED]
There was loss of cellular architecture, nuclear duplication and increased infiltration of leucocytes with prominent hyperchromasia in animals subjected to high fructose fluid intake as well as isoproterenol (Fig. 8a). Normal cytoarchitecture of myocardium was seen in animals treated with FGH 250 mg/kg during the last 3 weeks of high fructose fluid intake (Fig. 8b). Microphotographs of animals provided protection of CAP as well as FGH 250 mg/kg shows normal cytoarchitecture of myocardium with reduced interstitial space (Fig. 8c). There were no significant changes in histological slides of animals pretreated with either SACS 0.222 mg/kg alone (Fig. 8d) or with CAP (Fig. 8e).
[FIGURE 8 OMITTED]
ACE inhibiting activity and fall in blood pressure
Introduction of varying concentrations of SACS, CAP and their combinations at fixed ratio of 4:1 showed dose-dependent increase in percentage inhibition of ACE activity and fall in blood pressure. The values of [ED.sub.50] of SACS and CAP apart from [Z.sub.exp] and [Z.sub.add] are given in Table 1. The magnitude of interaction between SACS and CAP for inhibition of ACE activity at fixed ratio of 4:1 was less than 1 (0.58) and the Fig. 9 depict a super-additive interaction because the experimental points lay below the additive line. Similarly, magnitude of interaction in percentage fall in blood pressure between SACS and CAP was below 1 (0.86) which is evident from Fig. 10 showing super-additive effect (synergistic curve) under the additive line.
[FIGURE 9 OMITTED]
[FIGURE 10 OMITTED]
Table 1 Isobolographic determinations for interaction. Parameter Percentage inhibition Percentage fall in of ACE activity blood pressure [ED.sub.50] SACS 40.49 [+ or -] 2.23 31.28 [+ or -] 2.11 [ED.sub.50] CAP 3.32 [+ or -] 0.14 5.88 [+ or -] 0.87 [ED.sub.50] SACS + 7.35 [+ or -] 0.32 (a) 14.55 [+ or -] 1.33 (a) CAP ([Z.sub.exp]) [Z.sub.add] 33.06 [+ or -] 1.95 26.08 [+ or -] 1.55 Magnitude of 0.58 (b) 0.86 (b) interaction (a) [Z.sub.exp] value was statistically lower than [Z.sub.add] value with a p < 0.05. (b) Super-additive interaction a/A + b/B was statistically < 1.0 (p < 0.05).
Garlic contains unique organosulphur compounds, which provide its characteristic flavour and odor and most of its potent biological activity (Block 1985). In fact, over 90% of investigations on garlic's active principles have focussed on the sulphur compounds. When garlic is crushed, cut or chewed alliin is exposed to the enzyme alliinase and the thiosulphinate allicin is formed. Fresh garlic homogenate is known to possess the highest concentration of active constituent, allicin with half life up to 2.4 days when compared to normal half life of allicin, 2-16 hrs (Lawson 1988; Hayes et al. 1987). Allicin (allyl 2-propenethiosulphinate) is a reactive intermediate species that can be transformed into a variety of compounds and was earlier thought to be the principle bioactive compound responsible for the cardioprotective effect (Agarwal 1996). However, recent studies suggest that allicin is an unstable and transient compound with oxidant activity (Freeman and Kodera 1995) that is virtually undetectable in blood circulation after garlic ingestion and decomposes to form the S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC) by reacting with an enzyme alliinase or alliin lyase, which is located only in the vascular bundle sheath cells (Hayes et al. 1987). Our aim in this study was to evaluate antihypertensive and cardioprotective properties of fresh garlic homogenate in the presence/absence of captopril and also explore whether its bioactive constituent S-allylcysteine sulphoxide possesses similar potencies.
Garlic for the current research was purchased from the local vegetable market that is the most widely used form. Fresh garlic homogenate used in the study was standardised in our laboratory. It was found to be 86% water-soluble with high quantity of carbohydrates (18%). The total moisture content was 62.7%. The membrane stabilizer saponin content was 15 and tannin 2%. It was found to contain total protein of 8% and amino acid cysteine sulphoxide 6.2%. Several reports have suggested that fresh garlic extract has protective actions against cardiovascular disorders including: stroke, coronary artery disease, arteriosclerosis and hypertension (Bordia et al. 1977, 1978; Chutani and Bordia 1981). These beneficial effects have partly been attributed to its ability to inhibit platelet aggregation and thromboxane formation (Bordia 1978; Makheia et al. 1979; Samson 1982). Blood pressure lowering effect of garlic has been reported not only in spontaneously hypertensive rats (Foushee et al. 1982; Samson 1982), 2K1C rats (Al-Qattan et al. 1999) and hypertensive patients (Zheziang Institute of Traditional Chinese Medicine 1986), but also in normotensive animals (Banerjee 1979). Most of these activities were attributed to sulphur containing components that is present in abundant in fresh garlic homogenate. Hence fresh garlic homogenate was chosen for the current study. The dose of garlic was selected based on dose-dependent study reported in earlier literature (Banerjee and Moulik 2002) and later confirmed by our studies (Asdaq and Inamdar 2009a). Dose of S-allyl cysteine sulphoxide (SACS) was chosen based on the yield of the fresh garlic extract. Oral administration of FGH and SACS was initiated once hypertensive conditions were established with at least more than 150 mm Hg. The elevation in blood pressure with 10% fructose in fluid was developed by the end of three weeks. Fresh garlic homogenate and S-allyl cysteine sulphoxide were administered orally for 3 weeks (fourth to sixth week) for obtaining the best result of the treatment. Captopril was given for only 1 week, as it was sufficient to significantly reduce the rise in blood pressure. The same 7 days treatment duration was also employed in interactive group.
The use of 10% fructose in drinking water, for 3 weeks or longer appeared to be most suitable for rapid production of fructose-induced hypertension (Dai and McNeill 1995). High intake of fructose induces hypertension by potentially deleterious metabolic changes, e.g., hyperlipidemia, hyperinsulinemia, insulin resistance, hyperuricemia, hypertension, glucose intolerance and non-enzymatic fructosylation of proteins (Thorburn et al. 1989; Hwang et al. 1987; Reddy et al. 2008). In addition, excessive fructose consumption may be responsible in part for the increasing prevalence of obesity, diabetes mellitus, non-alcoholic fatty liver disease and cardiovascular diseases (Jurgens et al. 2005; Reaven 1988; Reiser 1985). Administration of animals with FGH and SACS counteracted these metabolic abnormalities. The antihypertensive ability of CAP was significantly enhanced when administered to animals under concurrent administration of SACS and FGH. The enhanced triglycerides, cholesterol and glucose levels were found to be depleted in all treated groups.
As noted earlier, both garlic (Rahman 2003) and captopril (Nakagawa et al. 1989) were found to possess potent cardioprotective efficacies. Moreover, we reported earlier improved cardioprotective efficacies of each other when given together at times of myocardial damage in normotensive rats (Asdaq and Inamdar 2010, 2008). Most of the time, it becomes mandatory to simultaneously treat both hypertension and ischemic heart diseases. Consumption of high fructose for long period might result in myocardial damage as explained in earlier literature (Reiser 1985). However, 6 weeks of 10% fructose was not able to induce any significant damage to myocardium (data not shown) and hence we opted for isoproterenol (175 mg/kg, subcutaneously for two consecutive days) mediated myocardial injury in hypertensive rats.
The pathophysiological changes following isoproterenol (ISO) administration are comparable to those taking place in human myocardial alterations (Karthikeyan et al. 2007). ISO induced myocardial damage is associated with decreased endogenous antioxidants such as superoxide dismutase (SOD) and catalase in serum which is structurally and functionally impaired by free radicals resulting in damage to myocardium. Inclination in endogenous antioxidant activities in HTH is indication for structural integrity and protection to the myocardium that were achieved by prior administration of FGH and SACS. It is interesting to note the alteration in SOD is with concomitant fluctuation in catalase after prior treatment of animals with GH. Elevated activity of catalase in HTH is more beneficial than increase in SOD activity alone because without a simultaneous increase in catalase activity, increased SOD activity may lead to intracellular accumulation of [H.sub.2][O.sub.2] with detrimental effects (Das et al. 1995). There was potent antioxidant ability of captopril that was remarkably augmented when captopril was given apart from moderate dose of FGH/SACS. The antioxidant property of CAP could be attributed to the formation S-allylmercaptocaptopril (Oron-Herman et al. 2005) which is known for enhanced cardioprotective action. The membrane of myocardium was kept intact in animals pretreated with FGH (250 mg/kg, p.o.), SACS (0.222 mg/kg, p.o.) and CAP as evident from elevated LDH and CK-MB activities in heart tissue homogenate (HTH) with depleted activities in serum. Both high and low doses of FGH and SACS were found to cause substantial fall in biomarker activities in serum and rise in HTH in CAP treated animals which was found to be more than CAP alone. Damage to cardiac musculature was also demonstrated and confirmed by histopathological scores. An increase in score is indicative of myocardial damage (Faulx et al. 2005). Pretreatment with FGH and SACS alone or with CAP substantially decreased the pathological scores and kept the myocardial integrity during ISO damage. This effect might be due to augmentation of endogenous antioxidant enzyme synthesis. The electrocardiographic parameters and haemodynamic findings were normalised in combination therapy especially RR interval and heart rate was reverted to normal conditions as well as QRS duration was reduced indicating protection from myocardial arrhythmias induced by isoproterenol. These results suggest the synergistic behaviour of CAP during FGH/SACS mediated cardioprotection.
The super-additive effect in percentage inhibition of ACE activity and percentage fall in blood pressure shown by the simultaneous administration of S-allyl cysteine sulphoxide with captopril is in agreement with our other evaluation.
Considering all the above-mentioned facts, it is clear that fresh garlic homogenate possesses strong synergistic cardioprotective potential. The efficacy of garlic homogenate is further augmented when used in combination with captopril. S-allyl cysteine sulphoxide, chief bioactive constituent of fresh garlic has marvellous ability to potentiate the action of captopril. The confirmed synergistic potential of S-allyl cysteine sulphoxide and captopril combination also validates the super-additive interaction of fresh garlic homogenate with captopril. Therefore, combination of garlic with captopril should be avoided. The commercial products of garlic and its constituent, S-allyl cysteine should be regulated. The herb-drug interactions are less important in areas where the concomitant use of garlic and drugs are less frequent.
In conclusion, concurrent administration of captopril with fresh garlic homogenate or its bioactive constituent, S-allyl cysteine produces synergistic antihypertensive and cardioprotective effects. We hope that this type of study will open new areas of research for interaction and counteraction between herb, its constituents and conventional drugs when they are taken concurrently.
The authors would like to express gratitude to Prof. Subhabrata Ray (Department of Pharmaceutics, Krupanidhi College of Pharmacy, Bangalore, India) for his stimulating hints and help during the preparation of the isobolograms. We would also like to extend our thanks to Biochemical Institute, Helsinki for a sample of S-ally cystein sulphoxide.
Agarwal, K.C., 1996. Therapeutic action of garlic constituents. Med. Res. Rev. 16, 11-124.
Ali, M., Thomson, M., 1995. Consumption of garlic, clove a day could be beneficial in preventing thrombosis. Prostaglandin Leukot. Essent. Fatty Acids 53, 221-224.
Al-Qattan, K.K., Alnaqeeb, M.A., Ali, M., 1999. The antihypertensive effect of garlic (Allium sativum L.) in the rat two-kidney-one-clip Goldblatt model. J. Ethnopharmacol. 66, 217-222.
Aqel, M.B., Gharaibah, M.N., Salhab, A.S., 1991. Direct relaxant effects of garlic juice on smooth and cardiac muscles. J. Ethnopharmacol. 33, 13-19.
Asdaq, S.M., Inamdar, M.N., 2008. Pharmacodynamic interaction of garlic with captopril in ischemia-reperfusion induced myocardial damage in rats. Pharmacologyonline 2, 875-888.
Asdaq, S.M., Inamdar, M.N., 2009a. The potential for interaction of hydrochlorothiazide with garlic in rats. Chem. Biol. Interact. 181, 472-479.
Asdaq, S.M., Inamdar, M.N., 2009b. Pharmacodynamic interaction of garlic with hydrochlorothiazide in rats. Indian J. Physiol. Pharmacol. 53, 127-136.
Asdaq, S.M., Inamdar, M.N., 2010. Pharmacodynamic interaction of captopril with garlic in isoproterenol induced myocardial damage in rat. Phytother. Res. 24, 720-725.
Asdaq, S.M., Inamdar, M.N., Asad, M., Nanjundan, P.K., 2008. Interaction of propranolol with garlic in isoproterenol induced myocardial infarction in rat. J. Pharmacol. Toxicol. 3, 414-424.
Banerjee, A., 1979. Effect of aqueous extract of garlic on arterial blood pressure of normotensive and hypertensive rats. Artery 2, 369-373.
Banerjee, K.S., Moulik, S.K., 2002. Effect of garlic on cardiovascular disorders: a review. Nutr. J. 1, 1-14.
Banerjee, S.K., Dinda, A.K., Manchanda, S.C., Maulik, S.K., 2002. Chronic garlic administration protects rat heart against oxidative stress induced by ischemic-reperfusion injury. BMC Pharmacol. 2, 16-21.
Block, E., 1985. The chemistry of garlic and onion. Sci. Am. 252, 114-119.
Bordia, A., 1978. Effect of garlic on human platelet aggregation in vitro. Atherosclerosis 29, 355-360.
Bordia, A., Joshi, H.K., Sandhya, Y.K., Bhu, N., 1978. Effects of essential oil of garlic on serum fibrinolytic activity in patients with coronary artery diseases. Atherosclerosis 28, 155-159.
Bordia, A., Verma, S.K., Vyas, A.K., Bedi, H.K., 1977. Effect of essential oil of onion and garlic on experimental atherosclerosis in rabbits. Atherosclerosis 26, 379-386.
Buerke, I., Prufer, D., Dahm, M., Oelert, H., Meyer, J., Darius, H., 1998. Blocking of classical complement pathway inhibits endothelial adhesion molecule expression and preserves ischemic myocardium from reperfusion injury. J. Pharmacol. Exp. Ther. 286, 429-438.
Chu, Q., Lee, D.T., Tsao, S.W., Wang, X., Wong, Y.C., 2007. S-allylcysteine, a water-soluble garlic derivative, suppresses the growth of a human androgen-independent prostate cancer xenograft, CWR22R, under in vivo conditions. BJU Int. 99, 925-932.
Chutani, S.K., Bordia, A., 1981. The effect of fried versus raw garlic on fibrinolytic activity in man. Atherosclerosis 38, 417-421.
Dai, S., McNeill, J.H., 1995. Fructose-induced hypertension in rats is concentration-and duration-dependent. J. Pharmacol. Toxicol. Methods 33, 101-107.
Das, D.K., Maulik, N., Moraru, I.I., 1995. Gene expression in acute myocardial stress. Induction by hypoxia, ischemia/reperfusion, hyperthermia and oxidative stress. J. Mol. Cell. Cardiol. 27, 181-193.
Dimo, T., Azay, J., Tan, P.V., Pellecuer, J., Cros, G., Bopelet, M., Serrano, J.J., 2001. Effects of the aqueous and methylene chloride extracts of bidenspilosa leaf on fructose-hypertensive rats. J. Ethnopharmacol. 76, 215-221.
Erich, F., Elastner, M., 1976. Inhibition of nitrite formation from hydroxyl ammonium chloride. A simple assay of super oxide dismutase. Anal. Chem. 70, 616-620.
Eva, M.L., 1988. Mechanism of pH dependent hydrogen peroxide cytotoxicity in-vitro. Arch. Biochem. Biophys. 365, 362-372.
Faulx, M.D., Ernsberger, P., Vatner, D., Hoffman, R.D., Lewis, W., Strachan, R., 2005. Strain-dependent [beta]-adrenergic receptor function influences myocardial responses to isoproterenol stimulation in mice. Am. J. Physiol. Heart Circ. Physiol. 289, H30-H36.
Fossati, P., Prencipe, L., 1982. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin. Chem. 28, 2077-2082.
Foushee, D.B., Ruffin, J., Banerjee, U., 1982. Garlic as a natural agent for the treatment of hypertension: a preliminary report. Cytobios 34, 145-152.
Freeman, F., Kodera, Y., 1995. Garlic chemistry: stability of S-(2-propenyl) 2-propene-l-sulfinothioate (allicin) in blood, solvents and simulated physiological fluids. J Agric. Food Chem. 43, 2332-2338.
Hayes, M., Rushman, T., Goldberg, M., 1987. Inhibition of hepatocarcinogenic responses to 1,2-dimethylhidrazine by diallyl sulfide, a component of garlic oil. Carcinogenesis 8, 1155-1157.
Herrera-Mundo, M.N., Silva-Adaya, D., Maldonado, P.D., Galvan-Arzate, S., Andres-Martinez, L, Perez-De La Cruz, V., Pedraza-Chaverri, J., Santamaria, A., 2006. S-allylcysteine prevents the rat from 3-nitropropionic acid-induced hyperactivity, early markers of oxidative stress and mitochondrial dysfunction. Neurosci. Res. 56, 39-44.
Hsu, C.C., Lin, C.C., Liao, T.S., Yin, M.C., 2006. Protective effect of s-allylcysteine and s-propyl cysteine on acetaminophen-induced hepatotoxicity in mice. Food Chem. Toxicol. 44, 393-397.
Hwang, I.S., Ho, H., Hoffman, B.B., Reaven, G.M., 1987. Fructose-induced insulin resistance and hypertension in rats. Hypertension 10, 512-516.
Itokawa, Y., Inone, K., Sasagawa, S., Fugiwara, M., 1973. Effect of S-methylcysteine sulfoxide, s-allylcysteine sulfoxide and related sulfur-containing amino acids on lipid metabolism of experimental hypercholesterolemic rats. J. Nutr. 103, 88-93.
Jurgens, H., Haass, W., Castaneda, T.R., Schurmann, A., Koebnick, C., Dombrowski, F., Otto, B., Nawrocki, A.R., Scherer, P.E., Spranger, J., Ristow, M., Joost, H., Havel, P.J., Tschop, M.H., 2005. Consuming fructose-sweetened beverages increases body adiposity in mice. Obes. Res. 13, 1146-1156.
Karthikeyan, K., SaralaBai, B.R., Devaraj, N., 2007. Cardioprotective effect of grape seed proanthocyanidins on isoproterenol-induced myocardial injury in rats. Int. J. Cardiol. 115, 326-333.
Kim, J.M., Chang, N., Kim, W.K., Chun, H.S., 2006a. Dietary S-allyl-L-cysteine reduces mortality with decreased incidence of stroke and behavioral changes in strokeprone spontaneously hypertensive rats. Biosci. Biotechnol. Biochem. 70, 1969-1971.
Kim, J.M., Lee, J.C., Chang, N., Chun, H.S., Kim, W.K., 2006b. S-allyl-L-cysteine attenuates cerebral ischemic injury by scavenging peroxynitrite and inhibiting the activity of extracellular signal-regulated kinase. Free Radic, Res. 40, 827-835.
Kosmala, M., Oledzka, K., Lewandowski, K., Filczewski, M., Milobedzka, J., 1992. A comparison of antihypertensive properties of captopril and enalapril after single and multiple oral administration in spontaneously hypertensive rats. Pol. J. Pharmacol. Pharm. 44, 437-442.
Lawson, L.D., 1988. Garlic: a review of its medicinal effects and indicated active compounds. Phytomedicines of Europe: chemistry and biological activity. In: Lawson, L.S., Bauer, R. (Eds.), ACS Symposium Series, 691. Washington, DC. American Chemical Society, pp. 176-209.
Makheia, A.N., Vanderhock, J.Y., Bailey, J.M., 1979. Inhibition of platelet aggregation and thromboxane synthesis by onion and garlic. Lancet 1, 781-785.
Mc Gown, M.W., Artiss, J.D., Strandberg, D.R., Zak, B., 1983. A peroxidase-coupled method for the colorimetric determination of serum triglycerides. Clin. Chem. 29, 538-542.
Nakagawa, S., Kasuga, S., Matsura, H., 1989. Prevention of liver damage by aged garlic extract and its components in mice. Phytother. Res. 3, 50-53.
Numagami, Y., Ohnishi, ST., 2001. S-allylcysteine inhibits free radical production, lipid peroxidation and neuronal damage in rat brain ischemia. J. Nutr. 131, 1100-1105.
Oron-Herman, M., Rosenthal, T., Mirelman, D., Miron, T., Rabinkov, A.D., Wilchek, M., Sela, B., 2005. The effects of S-allylmercaptocaptopril, the synthetic product of allicin and captopril, on cardiovascular risk factors associated with the metabolic syndrome. Atherosclerosis 183, 238-243.
Padmanabhan, M., Prince, P.S., 2006. Preventive effect of S-allylcysteine on lipid peroxides and antioxidants in normal and isoproterenol-induced cardiotoxicity in rats: a histopathological study. Toxicology 224, 128-137.
Rahman, K., 2003. Garlic and aging: new insights into an old remedy. Ageing Res. Rev. 2, 39-56.
Reaven, G.M., 1988. Banting lecture 1988. Role of insulin resistance in human disease. Diabetes 37, 1595-1607.
Reddy, S.S., Karuna, R., Baskar, R., Saralakumari, D., 2008. Prevention of insulin resistance by ingesting aqueous extract of Ocimum sanctum to fructose-fed rats. Horm. Metab. Res. 40, 44-49.
Reiser, S., 1985. Effects of dietary sugars in metabolic risk factors associated with heart disease. Nutr. Health 3, 203-216.
Rubin, B., Laffan, R.J., Kotler, D.G., O'Keefe, E.H., Demaio, D.A., Goldberg, M.E., 1978. SQ 14,225 (D-3-mercapto-2-methylpropanoyl-L-proline), a novel orally active inhibitor of angiotensin I-converting enzyme. J. Pharmacol. Exp. Ther. 204, 271-280.
Samson, R.R., 1982. Effects of dietary garlic on temporal drift on platelet aggregation. Atherosclerosis 44, 119-120.
Tallarida, R., 1992. Statistical analysis of drug combinations for synergism. Pain 49, 93-97.
Tallarida, R., 2000. Drug Synergism and Dose-Effect Data Analysis. Chapman & Hall/CRC, Boca Raton.
Tallarida, R., Porreca, F., Cowan, A., 1989. A statistical analysis of drug-drug and site-site interaction with isobolograms. Life Sci. 45, 947-961.
Thorburn, A.W., Storlein, L.H., Jemkins, A.B., Khouri, S., Kraegen, E.W., 1989. Fructose-induced in vivo insulin resistance and elevated plasma triglyceride levels in rats. Am. J. Clin. Nutr. 49, 1155-1163.
Wagner, H., Ulrich-Merzenich, G., 2009. Synergy research: Approaching a new generation of phytopharmaceuticals. Phytomedicine 16, 97-110.
Welch, C., Wuarin, L., Sidell, N., 1992. Antiproliferative effect of the garlic compound S-allylcysteine on human neuroblastoma cell in vitro. Cancer Lett. 63, A211-219.
Zheziang Institute of Traditional Chinese Medicine, 1986. The effect of essential oil of garlic on hyperlipidemia and platelet aggregation. J. Tradit. Chin. Med. 6, 117-120.
S.M. Asdaq (a), *, M.N. Inamdar (b)
(a) Department of Phaarmacology, Krupanidhi College of Pharmacy, Varthur Hobli, Chikkabellandur Village, Carmalaram Post, Bangalore 560 035, India
(b) Department of Pharmacology, Al-Ameen College of Pharmacy, Bangalore 560 027, India
* Corresponding author. Tel.: +91 80 65973260; fax: +91 80 51309161.
E-mail address: firstname.lastname@example.org (S.M. Asdaq).
0944-7113/$ - see front matter [C] 2010 Elsevier GmbH. All rights reserved.
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|Author:||Asdaq, S.M.; Inamdar, M.N.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Nov 1, 2010|
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