The antihypertensive effect of garlic (Allium sativum) in the rat two-kidney–one-clip Goldblatt model

Journal of Ethnopharmacology 66 (1999) 217 – 222

Short communication

The antihypertensive effect of garlic (Allium sati6um) in the rat two-kidney – one-clip Goldblatt model K.K. Al-Qattan, M.A. Alnaqeeb *, M. Ali Department of Biological Sciences, Faculty of Science, Uni6ersity of Kuwait, P.O. Box 5969, Safat 13060, Kuwait Received 2 July 1998; received in revised form 21 September 1998; accepted 22 September 1998

Abstract A study was designed to elucidate the antihypertensive effect of garlic in the two-kidney – one-clip (2K-1C) Goldblatt model. Since the hypertension in this model depends largely on the increasing concentrations of vasopressor agents (prostaglandins and angiotensin II) there is a strong indication that reducing these factors could contribute in controlling the pathological rise in blood pressure. In this study, single or multiple doses of 0.5 ml of aqueous extract of garlic were given orally to 2K-1C rats. The data shows that the single dose of garlic used had a maximum antihypertensive effect 2–6 h after administration. The residual effect of this single dose continued for up to 24 h. The multiple dose of garlic appeared to be effective in restraining the expected rise in blood pressure that normally occurs in 2K-1C rats. Switching multiple-dose treatments halfway through the experiment further strengthened the findings. The study suggests that garlic does have an effective antihypertensive ability, and may be used as a supplementary and natural remedy in cases of unilateral renovascular hypertension. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Garlic; 2K-1C Goldblatt model; Hypertension; Antihypertensive; Prostaglandins

1. Introduction Several reports have suggested that garlic has protective actions against stroke, coronary thrombosis and atherosclerosis (Bordia et al., 1977, * Corresponding author. Fax: +965 4847054. E-mail address: [email protected] Alnaqeeb)

(M.A.

1978; Chutani and Bordia, 1981). These beneficial effects have been attributed to its ability to inhibit platelet aggregation and thromboxane formation (Bordia, 1978; Makheia et al., 1979; Samson, 1982). Fresh garlic extracts have been found to lower blood pressure (BP) in spontaneously hypertensive rats (Foushee et al., 1982) and in anesthetized dogs where garlic extract was introduced

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via a femoral cannula, although the mechanism by which this effect is induced is not yet clear (Rashid and Khan, 1985). The hypotensive effect of garlic has also been described in various normotensive experimental animals (Banerjee, 1979) and hypertensive patients (Zheziang Institute of Traditional Chinese Medicine, 1986). The above reports in the literature all indicate that garlic is an effective agent in the control of mild hypertension in most cases. What is of interest is the use of garlic in cases of more severe hypertension, such as in unilateral renovascular hypertension (URVH). In an attempt to contain high blood pressure (BP) non-invasively in this condition, various pharmacological agents have been used to interfere with the biochemical and biophysical aspects of the renin – angiotensin system. The main aim of this step is to attenuate the actions of the active component of this system which is angiotensin II (AII), a potent vasoconstrictor octapeptide established as being the principle agent for the elevation and sustenance of high BP in URVH (DeForrest et al., 1982). Among the therapeutic techniques employed are treatments with angiotensin I-converting enzyme inhibitors (Al-Qattan and Johns, 1992), AII antagonists (Brunner et al., 1974; Huang et al., 1982) and renin antagonists (Heinrikson and Poorman, 1990). The present study investigated whether an aqueous extract of garlic administered orally to rats with 2K-1C Goldblatt model of renovascular hypertension, an experimental analogue to the ‘naturally occurring’ URVH, can control high BP.

2. Materials and methods

2.1. Animals Twenty-six normotensive female Sprague – Dawley rats with a body weight ranging between 187 and 215 g were placed individually in separate cages and randomly divided into two groups; group A (n = 10) and group B (n = 16). In both groups, the animals were numbered sequentially and retained this numerical identifica-

tion for the duration of the experiment. The rats were kept at constant physical ambient conditions (22°C, 20–30% humidity, and regular day/ night cycle) and supplied with tap water and standard diet ad libitum.

2.2. Induction of unilateral reno6ascular stenosis After 1 week and following anesthesia with an intraperitoneal injection of thiopentone sodium (7.5–10 mg/100 gram body weight; May & Baker, England), a Goldblatt type of unilateral renovascular stenosis (2K-1C) was induced in all the animals as follows. A retroperitoneal flank incision was made, and the left renal artery was exposed and cleared. Then, a 3-mm long, 1-mm wide U-shaped silver clip with an internal gap of 0.25 mm was placed around the renal artery and secured in place. The incision was sprinkled with an adequate amount of benzylpenicillin powder (Fluka Biochemika, Switzerland) and sutured. The animals were returned to their respective cages.

2.3. Garlic extract The voucher specimen number of the garlic is ALNAQEEB MA/001/1998 KTUH and the sample is kept at the Kuwait University Herbarium (KTUH). Garlic was prepared as previously described (Ali and Mohammad, 1986) and stored at − 40°C in small aliquots. Once an aliquot was thawed, the remaining extract was discarded after use. The yield of the preparation based on protein content was 93.59 2.7%. The potency of the garlic was checked every week as a function of its anti-aggregatory effect on platelet aggregation. The inhibitory effect of garlic was preserved for several months without loss of activity.

2.4. Experimental protocol 2.4.1. ‘Single-dose–short-term’ experiment One week post clipping, the even-numbered animals in group A received orally a single dose of 0.5 ml aqueous extract of garlic (50 mg/kg),

K.K. Al-Qattan et al. / Journal of Ethnopharmacology 66 (1999) 217–222

whereas the odd-numbered animals in the same group had a single dose of 0.5 ml of tap water.

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3. Results

3.1. ‘Single-dose–short-term’ experiment 2.4.2. ‘Multiple-dose – long-term’ experiment One week after clipping, the even-numbered animals in group B started to receive a single daily dose of 0.5 ml of garlic (50 mg/kg), whereas the odd-numbered animals started to receive a single daily dose of 0.5 ml of tap water. After 2 weeks, three even-numbered animals were randomly selected and switched to tap water and three odd-numbered animals were randomly selected and switched to garlic dosage. The remaining animals continued the same treatment as initiated at the start of the protocol. Forced feeding was carried out for a total of 4 weeks. 2.5. Measurements of systolic blood pressure The systolic blood pressure (SBP) of all the animals was measured the week before and the week after clipping. In the short-term experiment, the SBP of all the animals was measured at 0.5, 2, 6, 24 and 48 h after the administration of garlic or water. In the long-term experiment, the SBP was measured daily before the oral treatment and carried out such that, on every day of recording, the SBP of four garlic-fed animals and four water-fed animals was obtained. The SBP was measured using the tail-cuff technique for which all animals were pre-trained. The transducer-cuff was connected to a pressure monitor that fed into an Oscillograph (Harvard, England) calibrated against a mercury manometer.

The average of the SBP readings for the garlictreated animals was 131 915 mmHg during the week before clipping and 1329 8 mmHg during the week following clipping (Table 1). While the SBP readings of the water-treated animals was 1239 19 mmHg before compared to 135918 mmHg after clipping. The pre- and post-clipping readings for each treatment between the two groups were not significantly different. The single dose of water given to the rats caused a non-significant rise in the SBP, after 30 min of administration, which subsequently smoothed out getting closer to the pre-administration levels at 2, 6 and 24 h of recording. However, 48 h post-administration the SBP increased to 1479 13 mmHg (Table 1). Conversely, the administration of the single dose of garlic caused a drop in the SBP of the animals at 30 min, 2 and 6 h following administration. At 24 h the SBP was almost equal to the pre-administration level, but at 48 h the SBP was higher than the pre-administration reading. Nonetheless, the SBP of the garlic fed rats was lower than the SBP of the control water-fed animals at 48 h after administration (Table 1).

Table 1 The effect of a single dose of garlic (50 mg/kg) on the systolic blood pressure of clipped rats Time

Water-feda (control) (mmHg 9 S.D.)

Garlic-feda (mmHg 9 S.D.)

Pre-clipping Post-clipping (preadministration) 30 min 2h 6h 24 h 48 h

123 919 135 918

131 9 15 132 9 8

141 921 140 920 139 916 140 917 147 913

128 9 9 117 99 117 9 5* 131 96 141 915

2.6. Statistical analysis For SBP estimation, the average of three measurements was taken at the time of recording. The data presented are the means 9 S.D. Comparison between data collected before and after clipping and pre- and post-water or garlic force feeding was carried out with paired and unpaired Student’s t-test using Statgraph for Windows V1.0 (Manguistics Inc.). Values were considered significant when P B0.05.

a

Five rats were used in each group. * P50.05 compared with water treated animals at the same time.

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Table 2 The effect of multiple doses of garlic on the systolic blood pressure of clipped rats Pre-clip (mmHg 9 S.D.)

Post-clip (mmHg 9 S.D.)

Week 2 (mmHg 9 S.D.)

Week 3 (mmHg 9S.D.)

Week 4 (mmHg 9 S.D.)

91 9 13

1299 3*

136 9 17**

135 923**

175 914*,***

then garlic

104 9 10

1209 7*

123 915**

“132 9 36**

124 937**

(n =5) then water

103913 110917

1279 7* 1649 35*

131 913** 125 932**

12396** 134 96**

156 927*,*** 188 937**

Treatment

Water-fed (n= 5) Water-fed (n= 3) Garlic-fed Garlic-fed (n =3)

(control)

Treatment switching point. * P50.05 when each reading is compared with previous one; ** not significant when each reading is compared with previous one; *** P50.05 when post clip is compared with last week of treatment.

3.2. ‘Multiple-dose-long-term’ experiment Readings of the SBP of the water-treated animals were 919 13 and 1049 10 mmHg during the first week (Table 2). In the post-clipping week, the two SBP readings increased to 12993 and 120 9 7 mmHg. Thereafter, the five animals that were continuously given water showed a steady rise in SBP until the fourth week, when their mean SBP became 175914 mmHg. In contrast, the SBP of the animals that were later switched to garlic continued to increase for the first 2 weeks when they were given water. However, after switching to garlic, the rise in SBP reached a maximum of only 132936 mmHg and then dropped slightly at the end of the last week of treatment. Furthermore, when compared with the readings of animals that continued to receive water, the animals switched to garlic showed considerably lower SBP during the last week of SBP measurements (Table 2). The rats initially treated with garlic also had initially normal SBP levels (Table 2). During the post-clipping week the measured SBP was appreciably higher. The SBP of the animals that remained on garlic for the full duration was close to the post-clipping levels and increased significantly only at the end of the fourth week of treatment. In comparison, the SBP of the rats that were started with garlic then switched to water were comparable to pressure of the ‘garlic only’ fed rats at the end of the second week but exhibited an

increase in SBP after terminating the garlic treatment (1889 37 mmHg). The SBP of this group of animals was closer to the SBP of the animals that received water for the entire length of the experiment (Table 2).

4. Discussion and conclusions High BP is a reliable indicator of premature death. It is a risk factor for stroke, coronary heart disease and renal vascular disease. The control of BP through diet has been the focal point of public health and mass media attention. The method in practice to control high BP is ‘long-term’ drug therapy. Drugs have side effects that can create more clinical problems than are solved. That is why medical professionals worldwide are seeking non-drug treatment and preventative strategies. Garlic has been used as a medicine for thousands of years with no serious side effects. Here, we raise the possibility that garlic can be of significant help in BP management. In one study, aqueous extract of garlic was reported to lower BP in spontaneously hypertensive rats (Ruffin and Hunter, 1983). In another study, the ethanolic extract of garlic and onion failed to show antihypertensive effects in spontaneous hypertensive rats (Kivirata et al., 1989). The possible variation in the chemical composition of the extracts as a result of different preparation methods may be one of the reasons for the

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different results. A number of sulfur compounds have been extracted and identified from garlic extracts and have been shown to possess antithrombotic activity. The most active component responsible for the antiplatelet activity is allicin, which is formed by the action of the enzyme allinase, on the substrate allin (Block et al., 1986). Due to the volatile nature of allicin there have been controversial reports concerning the efficacy of different commercial preparation of garlic (Grunwald, 1990). The most recommended preparation of garlic as an antiplatelet agent is the aqueous extract of fresh garlic (Mohammed and Woodward, 1986) and for this reason the aqueous extract of fresh garlic was used in the present study. The comparative analysis of the SBP readings of the four groups of animals which underwent the single- and multiple-dose treatments showed that garlic had the effect of reducing the high BP development in the clipped animals. This influence, indeed, was apparent when the treatment of a number of the animals from the multiple-dose groups (chronic treatment) was reciprocally shifted while the others remained on the same treatment. The data showed that garlic had an antihypertensive effect and also minimized the pathological increases in BP that are typical of this condition. The immediate and short-term effect of garlic can be seen from the single-dose BP readings. Garlic led to a sharp and almost instantaneous fall in SBP reaching a maximum drop between 2 and 6 h after administration (Table 1). As time elapsed the potency of garlic began to vanish. The BP measurements of the multiple-dose treatment revealed that garlic had an appreciable ability of minimizing the rise in the SBP. This influence was more evident when a number of animals in the garlic-treated group were shifted to water leading to a re-surge in their SBP. In contrast, those animals that started with water and then switched to garlic exhibited an inhibition in the rise of SBP and even showed some signs of declining SBP (Table 2). The SBP of the animals that received garlic either from the start or half-way through the length of the experiment appear to have lower

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SBP than animals that had only water or had their garlic treatment terminated after 2 weeks. When comparing the SBP of animals that received water and those that received garlic, it becomes clear that if garlic is administered chronically at low doses (once a day) its hypotensive influence is not as strong compared to short-term treatment but the effect extends to almost 24 h. There are strong indications that the increase in the systemic and peripheral concentration of AII, as occurs in the case of 2K-1C Goldblatt model of hypertension, is associated with a rise in the concentration of vasoconstrictor prostaglandins (TXA2) and (PGs); thromboxane-A2 prostaglandin F2a (PGF2a ) (Dunn, 1986; Marsden et al., 1990). The possible significance of prostaglandin-I2 (PGI2) and prostaglandin-E2 (PGE2) as antihypertensive principle has been demonstrated in the blood vessels and in the kidney. Thus, locally produced PGE2 and PGI2 dilate arterial blood vessels and reduce the vasoconstrictive action of AII and norepinephrine (Vane and McGiff, 1975). Platelet thromboxaneA2 formed upon aggregation during endothelial cell damage is vasoconstrictive and could contribute to vascular resistance (Weber et al., 1981). A basis for therapeutic or preventative approach in hypertensive vascular diseases is to inhibit the thromboxane formation and favor the production of vasodilator and platelet-inhibiting prostaglandin. It has been previously shown that an aqueous extract of garlic inhibited the production of thromboxane during blood clotting in a dose-dependent manner (Ali and Mohammad, 1986). In the same study it was shown that synthesis of prostacyclin by blood vessels was not affected by any of the concentrations of garlic extract used in those experiments. These experiments suggested that it might be possible to achieve a selective suppression of thromboxane formation by platelets with sparing of vascular synthesis of prostacyclin when treating with garlic. The reduction in the BP of the clipped animals in response to the oral administration of garlic indicates its antihypertensive effect in the URVH condition. The results obtained suggest that the intake of a suitable portion of garlic per day

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could be used to maintain BP at lower levels for as long as the treatment continues.

Acknowledgements This work was supported by Kuwait University grants including grant number SB030 for which we express gratitude.

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