Clinical and Experimental Hypertension, 33(2): 9599, (2011) Copyright Informa Healthcare USA, Inc.

. ISSN 1064-1963 print/1525-6006 online DOI: 10.3109/10641963.2010.531833

Comparison of the Hypotensive and Bradycardic Activity of Ginkgo, Garlic, and Onion Extracts
LCEH

Suzana Brankovic,1 Mirjana Radenkovic,1 Dusanka Kitic,2 Slavimir Veljkovic,1 Vesna Ivetic,3 Dragana Pavlovic,2 Bojana Miladinovic2
Hypotensive Effect of Ginkgo, Garlic and Onion

Department of Physiology, Faculty of Medicine, University of Nis, Serbia, 2Department of Pharmacy, Faculty of Medicine, University of Nis, Serbia, 3Department of Neurophysiology, Faculty of Medicine, University of Novi Sad, Serbia

Abstract
The acute effect of ethanol extracts ginkgo (Ginkgo biloba L.), garlic (Allium sativum L.), and onion (Allium cepa L.) on arterial blood pressure (BP), and heart rate (HR) in anesthetized normotensive rats was examined and compared. Arterial BP was registered in the left carotid artery. The data showed that intravenous administration of the extracts produced dose-dependent and reversible hypotensive and bradycardic effects. The most effective in reducing arterial BP and HR is extract of garlic. There were statistically significant differences in bradycardic and hypotensive effects of the garlic and ginkgo extracts. Keywords: Ginkgo biloba, Allium sativum, allium cepa, rat, arterial blood pressure, heart rate

INTRODUCTION Cardiovascular diseases associated with hypertension and atherosclerosis are the greatest killers in modern society. Both clinical and epidemiologic studies have shown that hypertension is perhaps the most significant risk factor for atherosclerosis and its complications. On the other hand, the development of atherosclerosis largely depends on nutrition. Dieting is thus the first step in the prevention and suppression of atherosclerosis (1). Numerous herbal preparations are known to exert both anti-atherosclerotic and hypotensive effects, on the account of which they can be used to prevent cardiovascular diseases. Around 80% of the global population still relies on medicinal plants and several herbal medicines have advanced to clinical use in modern times (2). Some herbal drugs are providing comprehensive relief to the people suffering from cardiovascular diseases. Ginkgo (G. biloba), a living fossil, the oldest worlds living tree species, is also one of the most scientifically studied of all the herbal drugs. Ginkgo has been a staple of Chinese traditional medicine for thousand of years. Due to a wide variety of clinical conditions, the best-selling phytomedicine on the European and U.S. market is extracted from the leaves of this ancient tree. In recent years, the extract of ginkgo has been researched

extensively (3). So far, in clinical and experimental studies, the extract has been reported to be effective against ischemic brain injury, cognitive speed deficits, and peripheral vascular disease. Oral administration of ginkgo causes small improvements in claudication symptoms. Also, ginkgo benefits people with early stage Alzheimers disease and multi-infarct dementia (46). Ginkgo may have the potential to be used to reduce cholesterol levels (7). The antioxidant components are present in the nuts and leaf of the ginkgo (8). There have also been reports in the literature of ginkgo acting to reduce blood viscosity and increase cerebral perfusion in elderly people (9). Different plant formulation and several cases of combined extracts of plants are used in our traditional medicine as drugs of choice in the prevention and treatment of hypertension and atherosclerosis. Among them, the extract of garlic and onion are the most common. Garlic (A. sativum) and onion (A. cepa) belong to the group of the oldest cultivated plants. Additionally, both plants have been used for millennia as a food and medical agent in the treatment and prevention of the diseases affecting the cardiovascular system, including hypertension (10, 11). Both botanical drugs have been shown to be potentially useful as antimicrobial, antithrombotic, antitumor, hypolipidaemic,

Address correspondence to Dusanka Kitic, University of Nis, Department of Pharmacy, Bulevar Z. Djindjica 81, 18 000 Nis, Serbia. E-mail: duska@medfak.ni.ac.rs Received 25 January 2010; revised 2 June 2010; accepted 2 June 2010.

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antiarthritic, and hypoglicemic agents. Garlic and onion have beneficial effects on cholesterol levels, fybrinolytic activity, and platelet aggregation and reduce the risk of various cardiovascular diseases (1215). The oils of garlic and onion are effective antioxidants, as well as antitumor and cancer inhibition agents (16, 17). Additionally, a meta-analysis assessing the effect of garlic on hypertension showed a reduction in systolic blood pressure (SBP) and diastolic blood pressure (DBP) in patients treated with garlic compared to placebo (11, 18). Garlic intake had a protective effect on the elastic properties of the aorta related to aging in humans (19). Over the last few decades, possible application of ginkgo, garlic, and onion as alternative treatments for many cardiovascular diseases have been intensively studied. Most published scientific research papers investigate the effects of long-term application of these plants (2022). The present study was therefore undertaken to evaluate and compare the acute effects of ethanol extracts of ginkgo with garlic and onion on the BP and HR in normotensive anaesthetized rats. MATERIALS AND METHODS
Plant Material

free access to food and water. All experimental procedures with animals were done in compliance with the European Council Directive of November 24, 1986 (86/609/EEC).
Experimental Protocol

The commercial extract of Ginkgo biloba (Gingium) used in this study was produced by Hexal AG (Holzkirchen). The solution was standardized, so that 1 ml contained 40 mg of dry extract from ginkgo leaves (Ginkgo biloba leaves). Freshly peeled and chopped garlic and onion bulbs were homogenized. Ethanol extract was obtained by the extraction (20 min) in an ultrasonic bath with 96% ethanol. After the separation of solvents, the extracts were concentrated in a rotary evaporator at a reduced pressure (40C) till a constant weight was achieved. The obtained dry residue was dissolved in the ethanol in order to get 20% solution used in the experiment.
Drugs

The rats were anesthetized with intraperitoneal (i.p.) injection of sodium pentobarbital (50 mg/kg). They were implanted with carotid arterial catheter (for BP recording) and with jugular venous catheter (for the administration of test compounds). The arterial catheter was connected to a BP transducer (P-1000-A) coupled with a Narcophysiograph (NARCO Bio system, Houston, TX, USA) for arterial pressure measurement. The recording speed was set at 10 mm/s. The parameters of BP were recorded in mmHg as SBP and DBP, and were expressed as a mean arterial pressure (MBP), which was calculated as the DBP plus onethird of pulse width. After a period of equilibrium (30 min), the rats were injected intravenously with acetylcholine (1 g/kg) and noradrenaline (1 g/kg). Control responses of acetylcholine and noradrenaline were obtained. The BP and HR were recorded before and after the administration of the drugs. Following 30 min, the same animals were intravenously administrated with the plant extracts. The extracts were administrated intravenously at intervals of 1520 min. The BP and heart rate were recorded before and after the administration of the extracts. Animals were divided into three groups of six animals each. The first group of animals received ethanol extract of ginkgo at doses of 0.26 mg/kg, the second received ethanol extract of garlic (0.26 mg/kg), and the third group the extract of onion (0.26 mg/kg). Changes in BP were recognized as the difference between the steady state values before and the lowest readings after the injection. The normal response of the animal was checked through the standard compounds, acetylcholine (hypotensive), and noradrenaline (hypertensive).
Statistical Analysis

Acetylcholine and noradrenaline were purchased from Sigma Chemical Company (St. Louis, MO, USA). All drugs were dissolved in distilled water.
Animals

In this study, we used 12-week-old male Wistar albino rats, weighing 200250 g, obtained from the Animal Research Center of the Faculty of Medicine, University of Nis, Serbia. The rats were housed in stainless steel cages under standard laboratory conditions. These animals were maintained at 2024oC, with a 12-h lightdark cycle for at least 1 week before the experiment. All animals were fed a standard pellet diet and they had

The results were expressed as mean standard deviation of six determinations. Statistical evaluation was performed using the students t-test. A probability value of p < 0.05 was considered to be significant. The mean effective doses ED50, that is, the doses which elicited 50% of maximal response, were established by regression analysis. RESULTS The mean basal BP and HR of the rats were 96.45 7.91 mmHg and 293 10.76 beats/min. In order to experiment, the plant extract was further diluted with the distilled water. Ethanol in those
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Table 1. Effects of the intravenous application of the garlic, onion, and ginkgo ethanol extracts, acetylcholine, and noradrenaline on mean BP and HR in anesthetized rat Extract of Garlic (6 mg/kg) 98.56 15.37 96.45 7.91 293 10.76 48.36 9.65** 196.31 18.36** Extract of Onion (6 mg/kg) 72.01 9.65 65.24 7.36** 234.40 19.71 Extract of Ginkgo (6 mg/kg) 45.14 11.58 75.88 5.32** 246.12 21.58 Acetylcholine (1 g/kg) 85.49 10.68 79.72 8.74 194.48 46.21 Noradrenaline (1 g/kg) 110.78 13.85 168.35 9.18 428.95 38.74

Control Duration of action (s) Mean blood pressure (mm Hg) Heart rate (beat/min)

Values are the mean SEM, n = 6. *p < 0.05; **p < 0.01 (students t-test) when compared to control.

concentrations did not exhibit any hypotensive or hypertensive effects. The value of the HR and BP-lowering effects of plant extracts and acetylcholine are given in Table 1. Intravenous injection of the ethanol extract of ginkgo at doses of 0.26 mg/kg induces significant and dose-dependent decreases in MBP, with ED50 values of 42.39 7.25 mg/kg. After the hypotensive peak, the MBP increased and reached the basal value. Hypotensive effects caused by ginkgo ethanol extract (6 mg/kg) lasted 45.14 11.58 s. Intravenous administration of garlic extracts (0.26 mg/kg) in anesthetized rats immediately caused a dosedependent reduction in MBP (Figure 1) with ED50 values of 2.76 0.68 mg/kg. Hypotensive effects caused by garlic ethanol extract lasted 98.56 15.37 s. This effect became significant at the dose of 0.2 mg/kg (p < 0.05). Ethanol extracts of onion (0.26 mg/kg) induced a transient hypotensive effect with ED50 value of 11.43 2.87 mg/kg. Hypotensive effects caused by onion ethanol extract lasted 72.01 9.65 s. Maximal reduction of the arterial BP was registered after the administration of garlic extract (p < 0.01). Intravenous administration of ginkgo extract produced the smallest hypotensive effect. Comparing the hypotensive effects of plant extracts, a significant
Garlic Onion Ginkgo

Garlic
100 80
Fall (%)

Onion

Ginkgo

60 40 20 0 * 0.2 * 0.6 2 Plant extract (mg/kg) 6 ** *

Figure 2. Inhibitory response of garlic, onion and ginkgo ethanol extracts on HR in anesthetized rats. Data are expressed as the mean SEM, n = 6. *p < 0.05; **p < 0.01 (students t-test).

100 80

difference was found between the garlic and ginkgo effects (p < 0.01). Figure 2 shows the inhibitory response of ethanol extracts of ginkgo, garlic, and onion on HR in anesthetized rats. Intravenous treatment with extracts of ginkgo (0.26 mg/kg) decreased rate of contraction, with an ED50 value of 12774 189.54 mg/kg. Ethanol extracts of garlic and onion inhibited HR with an ED50 value of 57.35 9.54 mg/kg and 962.58 27.14 mg/kg, respectively. Comparing the effects of the extracts, extract of garlic caused the strongest negative chronotropic effect. DISCUSSION In this work we studied in vivo the hypotensive effect of ethanol extracts of ginkgo, garlic, and onion, used in traditional medicine for treating cardiovascular diseases. The finding of this work is that administration of ethanol extracts of ginkgo, garlic, and onion in anesthetized normotensive rats induced short-term hypotension and bradycardia in a dose-related manner. We compared the effects of different plant extracts on arterial BP. The results obtained show that there are significant differences in hypotensive effects of the studied plant extracts. As shown, the ethanol extract of garlic at a dose of 6 mg/kg produced the most pronounced effects in terms of steady hypotension. The effects of

Fall (%)

60 40 20

** * * *

0.2

0.6 2 Plant extract (mg/kg)

Figure 1. Inhibitory response of garlic, onion and ginkgo ethanol extracts on mean arterial blood pressure in anaesthetized rats. Data are expressed as the mean SEM, n = 6. *p < 0.05; **p < 0.01 (students t-test).
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ethanol onion extract are somewhat weaker, while ginkgo extract has weakest effects. According to the literature, the oral administration of garlic in animals reduced experimentally induced hypertension (2325). Moreover, increased consumption of garlic is associated with lower population incidence of hypertension (26). In spontaneously hypertensive rats, BP increase has been suppressed significantly 3 weeks after treatment by the extract of ginkgo (27). The fact that garlic induces acute, immediate hypotensive effects is an additional recommendation of garlic for hypertension treatment. The effect of the plant extracts on the HR in anesthetized rats was also compared. Heart rate decreased after the administration of ginkgo, garlic, and onion ethanol extracts. Ethanol extract of garlic induced the greatest negative chronotropic effect, while the weakest bradycardic effects were recorded with ginkgo extract. There were statistically significant differences in bradycardic and hypotensive effects of garlic and ginkgo (p < 0.01), while the respective differences in onion and ginkgo effects were not statistically significant. Since arterial BP is proportional to the minute volume of the heart and peripheral resistance, negative chronotropic effects of these extracts could be responsible for the phenomenon of arterial BP reduction. Literature reports have shown that garlic dialysate possessed cardio-depressant and anti-arrhythmic property and decreased positive inotropic and chronotropic effects of isoproterenol (28). Also, garlic extract has an effect on the HR, modulating the rate and force of contractions (29, 30). Satoh and Nishida (31) have shown that the extract of ginkgo causes in vitro a negative chronotropic effect and vasodilatation. The differences observed with the application of the studied plant extract could be caused by the presence of different classeses of active biomolecules. Garlic and onion are a rich source of several phytonutrients: thiosulfinates, volatile sulphur compounds, and polar compounds of phenolic and steroidal origin (32). Onion and garlic contain many sulphur-containing active principles with diverse activities, making these vegetables an important source of therapeutic agents (33). In garlic, alliin (converted into allicin) and ajoene can be found. Onion contains the same and some related substances-cycloalliin. These thiosulfinates, including allicin, appear to be the active substances in garlic (11). Chronic oral administration of allicin lowered BP in hypertensive rats and caused vasodilatation in the isolated rat lung (34). Allicin from garlic exerted a protective effect on the coronary endothelial function and vasoreactivity (35). The two main groups of active substances in ginkgo are flavonoids (mainly quercetin and kaempferol) and terpene lactones (ginkgolides and bilobalide) with highly specific action (36). The principal ingredient of ginkgo extract, quercetin, can activate nitric oxide

(NO) synthesis in vascular endothelial cells and induces relaxation of rat aorta (37). Terpenoids of ginkgo (bilobalide and ginkgolides A, B, and C) produce concentration-dependent vasodilatation (31). Based on the above, it could be said that ginkgo has the least acute effect on BP and HR in anesthetized rats. In contrast to that, garlic produces very strong hypotensive and negative chronotropic effects, meaning that it can be recommended as an alternative treatment of hypertension. CONCLUSION The present study demonstrates that ethanol extracts of garlic exhibited more potent hypotensive and bradycardic effects, than extracts of ginkgo, and onion in anesthetized normotensive rats. Negative chronotropic actions can be responsible of the hypotensive effect of the plant extracts. ACKNOWLEDGMENTS The authors are grateful for the financial support of the Ministry of Sciences and Technological Development of the Republic of Serbia, grant no. 145081B and III41018, and the project Training and Research in Environmental Health in the Balkans D43 TW00641 supported by the NIH/Fogarty International Center, USA.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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