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© 2006 American Society for Nutrition J. Nutr. 136:769S-773S, March 2006

Supplement: Significance of Garlic and Its Constituents in Cancer and Cardiovascular Disease

Aged Garlic Extract Improves Blood Pressure in Spontaneously Hypertensive Rats More Safely than Raw Garlic1,,2

Akiko Harauma and Toru Moriguchi3

Healthcare Research Institute, Wakunaga Pharmaceutical, Company, Koda-cho, Akitakata, Hiroshima 739-1195, Japan

3 To whom correspondence should be addressed. E-mail: moriguchi_tr{at}wakunaga.co.jp.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
We studied the effects of two garlic sources on systolic blood pressure (SBP) using spontaneously hypertensive rats (SHRs). Beginning at 12 wk of age, male SHRs were fed diets containing either aged garlic extract (AGE) or raw garlic (RG) powder for 10 wk. Both AGE and RG reduced the increase of SBP compared with the control group from 4 wk after beginning the experimental diets. The effect of AGE was accompanied by a decrease of pulse pressure (PP), suggesting an improvement of the pliability of the artery, although RG did not affect PP. However, harmful effects were observed in the RG group, including a decrease in erythrocytes, an increase in reticulocytes, and generation of papilloma in the forestomach. These results suggest that AGE may safely improve several factors related to blood vessel physiology and circulatory disease.

KEY WORDS: • hypertension • aged garlic extract • rat

Lifestyle-related diseases, such as diabetes, hyperlipemia, and hypertension, pose a serious issue in Japan's aging society due to Westernization of the diet. The phrase lifestyle-related disease has been used instead of adult disease since 1996. This terminology represents a concept for a group of diseases in which symptomatic appearance and progress are affected by living practices that include eating, exercising, rest, smoking, and drinking. The Ministry of Health and Welfare of Japan has been conducting Healthy Japan 21, a 10-year national health effort that began in 2000 to improve lifestyle-related diseases including hypertension. Hypertension, a typical lifestyle-related disease, has been considered the most important risk factor for chronic circulatory organ disease (1). The total number of hypertensive patients in Japan are estimated at 7 million or more (2). Moreover, it is estimated that there may be ~65 million hypertensive people in the United States (3). Recently, many medical supplies and health foods have been researched and developed to prevent or improve hypertension.

Garlic has been used as a traditional medicine in both chronic and acute diseases for more than 1000 years. Aged garlic extract (AGE),4 extracted for a period of >10 mo, is less irritating and does not produce the harmful aftereffects of raw garlic (3). This observation is advantageous for long-term administration to prevent or treat chronic disease. In the circulatory system, AGE and its components have been shown to improve peripheral circulation (4), protect vascular endothelial cells from oxidant injury (5–7), and reduce plasma lipid concentration (8,9).

We investigated differences in the blood-pressure (BP)–lowering effects of AGE and raw garlic (RG) in hypertensive rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Animals and study design. The experimental protocol was approved by the Animal Care and Use Committee of the Wakunaga Pharmaceutical Co. in Japan. Male spontaneously hypertensive rats (SHR/Izm) (10), at 10 wk of age, were obtained from Japan SLC Co. and fed a semi-synthetic powder diet [AIN-93G (11), Oriental Yeast] in our animal facility under conventional conditions, with controlled temperature (22 ± 1°C), humidity (50 ± 5%), and illumination (12 h; 0700–1900). At 12 wk of age, they were divided into 3 dietary groups on a pseudo-random basis, with the constraint that the 3 groups had the same mean body wt and BP. One group was switched to an AGE-diet and the second was switched to RG diet (composition below). A third control group continued to feed on a semi-synthetic powder diet without a garlic source. Each group was fed an experimental diet for 10 wk. Diet and water were provided ad libitum. Body wt, BP, and food- and water- consumption were measured weekly for the feeding period of the diet. All animals were decapitated at age 22 wk. Truncal blood and the stomach were collected and used for hematological, biochemical analysis, and histological observation.

    Preparation of garlic source and experimental diet. AGE was prepared by Wakunaga Pharmaceutical Co. To prepare it, sliced raw garlic cloves were soaked in aqueous ethanol for >10 mo in stainless-steel tanks at room temperature, then AGE was concentrated to ~50% as solid-material content and dried with a spray dryer (Pulvis GB22, Yamato Scientific).

RG was purchased from a local market. Garlic skin was peeled and cloves were sliced into 2–3 pieces. Sliced garlic was lyophilized using a freeze dryer (FRD 50P, Iwaki Glass) and was pulverized into fine powder using a laboratory blender (Waring). These powders were stored at 4°C before making the experimental diet. AGE and RG powder were used in place of a portion of cellulose and were added to the AIN93G powder diet to make an equal amount of water-soluble extract (Table 1).


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  		TABLE 1 Composition of the experimental powder diets1

 
    Measurement of blood pressure. Systolic blood pressure (SBP) and mean blood pressure (MBP) were measured by tail plethysmography (BP-98A, Softron) after a warming period in unanesthetized rats (12). The BP measurements required only a few min per individual rat and were carried out during a period of 2.5 h beginning at 1400 daily. Pulse pressure (PP) was calculated using the SBP and the MBP as follows: PP = 1.5 x (SBP – MBP).

    Statistical analysis. All data were expressed as means ± SEM and were analyzed using the STATISTICA program (Statsoft Japan Inc.). The differences between the control and experimental diet groups were analyzed by one-way or two-way ANOVA followed by Tukey's honestly significant difference test. The curves were fit to the data for Figures 1 and 2 with regression analysis using the Sigma Plot program (Hulinks).


Figure 1
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  		FIGURE 1  Alteration of SBP after beginning the garlic diet feeding. Male SHRs were fed diets containing either AGE (closed circles) or RG (closed square) for 10 wk beginning at 12 wk of age. Blood pressure was measured for the feeding period of the diet on a weekly basis. The given values for R2 represent the correlation coefficients for the fitted curves. Differences compared with the control group (open circle) were statistically significant in a two-way ANOVA [F(2, 24) = 7.179, P < 0.01; AGE, P < 0.05; RG, P < 0.01].

 

Figure 2
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  		FIGURE 2  Alterations of MBP after beginning of the garlic diet. See legend to Figure 1. Differences compared with the control group were statistically significant in a two-way ANOVA [F(2, 24) = 4.725, P < 0.05; RG, P < 0.05].

 

   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
The experimental diets using both AGE and RG produced no significant differences in body wt compared with the control diet group [F(2, 24) = 0.598, P = 0.56]. However, body wts of the RG group were slightly decreased from 5 wk after beginning the RG diet, when compared with the control and AGE groups (data not shown). At the end of the experiment (22 wk of age), the mean body wts of the control, AGE, and RG groups were 360.1 ± 6.7, 359.7 ± 4.2, and 348.0 ± 3.9g, respectively (Table 2). Both AGE and RG groups had no significant differences in food and water consumption, compared with the control [data not shown, food consumption, F(2, 24) = 0.304, P = 0.74; water consumption, F(2, 24) = 2.819, P = 0.08].


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  		TABLE 2 Effects of garlic on hematological and biochemical analysis

 
The SBPs of the control, AGE, and RG groups were 186.3 ± 3.7, 185.1 ± 2.7, and 185.4 ± 2.8 mm Hg, respectively, at the beginning of the experiment. The control group showed a continuous increase of SBP during the experiment and had increased up to 219.4 ± 3.1 mm Hg by the end. Both garlic-powder groups showed significant suppression of the SBP increase from ~3–4 wk after the beginning of the experimental diet feeding (Fig. 1). These lowering effects were significantly different compared with the control group [F(2, 24) = 7.179, P < 0.01; AGE, P < 0.05; RG, P < 0.01]. There was no significant difference between the two garlic-fed groups, although the effect of RG appeared greater than that of AGE (P = 0.472). The MBP of the RG group was significantly lower than that of the control group, although the AGE group was not different from the control group [Fig. 2, F(2, 24) = 4.725, P < 0.05; AGE, P = 0.939; RG, P < 0.05]. In the PP, which was computed from the SBP and the MBP, only the AGE group showed a lowering effect, but there was no difference in the RG group when compared with the control group [Fig. 3, F(2, 24) = 6.754, P < 0.01; AGE, P < 0.01; RG, P = 0.06]. The heart rate, which was simultaneously measured during the measurement of the SBP and the MBP, was not different among the groups [data not shown, F(2, 24) = 0.013, P = 0.99].


Figure 3
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  		FIGURE 3  Alterations of PP after beginning of the garlic diet. See footnotes to Figure 1. The differences compared with the control group were statistically significant in a two-way ANOVA [F(2, 24) = 6.754, P < 0.01, AGE, P < 0.01].

 

Figure 4
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  		FIGURE 4  Dissection view of the forestomach in the RG group. The stomachs of all animals were used histological observation. The arrowheads indicate the cell division image. K, the keratin layer; S, the squamous epithelium layer; M, the tunica muscularis.

 
Results of the hematologic and the biochemical analysis are presented in Table 2. The RG group showed a significant decrease of the erythrocytes (P < 0.01) and increase in reticulocytes (P < 0.01) compared with the control group. Administering the RG diet also led to a decrease in hematocrit, an increase of the glutamic-pyruvic transaminase (GPT) level, and decreased liver wt, although those differences were not significantly different compared with the control group. On the other hand, the AGE group had no significant differences compared with the control group in any of these parameters. There were also no significant differences in other parameters (albumin, creatinine, lipid peroxide, uric acids, urea nitrogen, sodium, chlorine, and potassium etc.) in the biochemical analysis of serum (data not shown). Also, there were no significant differences in the spleen wt among all groups.

In the stomach, many polyps were found in the forestomach of the RG group, although there were no changes of the forestomach in either the control and the AGE groups. The rate of polyp generation in the RG group was 78% (7 of 9 animals). In the histological view, intense hypertrophy of the squamous epithelium layer was observed, although there were no abnormalities observed in the keratin layer or the tunica muscularis. These polyps were judged benign papilloma, considering the nature of the cell division in the squamous epithelium layer of the stomach (Fig. 4).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
In this study, both the AGE and RG groups showed a lowering effect on the increase of SBP in the SHR rats. It is not yet clear whether the principal effective compound is the same in the two garlic preparations, although these results indicated that the two sources of garlic have a common effect on hypertension. This result is similar to the findings for hypertension in SHRs (13) and human (14). Mohamadi et al. (13) measured the SBP on SHR after administration of not only wild garlic (Allium urusimun) but also dried garlic (Kwai®) and AGE (Kyolic®). They reported that the effect of the dried garlic appeared greater than that of AGE. They indicated that the SBP-lowering effect of garlic on SHRs was due to the nitric oxide (NO) system being more active than the renin-angiotensin system, from data regarding the administration of angiotensin II or the NO synthase (NOS) inhibitors. However, the elevated SBP observed after administering the NOS inhibitor to the SHR was not different between the dried garlic and AGE groups (13). This suggested that dried garlic and AGE might have produced the same amount of NO. This finding suggests that the difference between the SBP-lowering effect of RG and AGE could not be a difference of NO production or the activation of the NOS. Also, several reports indicated that RG and AGE enhanced NO production and activated the NOS (15–18). NO promotes the suppression of contraction or the relaxation in the blood vessel and will lower the SBP promptly, because the NO affects the blood vessel directly (19,20). However, both the AGE and RG needed 3–4 wk until their action was expressed. These results suggest that the two sources of garlic have different mechanisms for their lowering effect on SBP.

PP calculated from SBP and MBP gradually increases with aging and is used as an index of heart function and artery extensibility in the circulatory system (21,22). In the fractional analysis of the blood pressure, AGE showed a lowering of the PP, only by reducing SBP and without affecting the MBP, as compared with the control group. This result suggests that chronic feeding of AGE improved the artery extensibility and prevented the stiffening of the blood vessel, a characteristic of aging. AGE and its components also have been reported to protect the vascular endothelial cells from oxidant injury (5–7). Our findings and several other studies suggest that protection and maintenance of the flexibility in the blood vessel may be one mechanism for the SBP-lowering effect of AGE. However the PP of the RG group had no significant differences compared with the control group, with respect to the lowering of both the MBP and the SBP. This suggests that the SBP lowering by RG was less effective than that of AGE on the blood-vessel extensibility.

The erythrocyte, which is the main component of blood, also fulfills a major role in the blood circulatory and BP systems. Considering the longevity (~60 d) of the erythrocyte in rats, administering the garlic source for 3–4 wk will affect half of the erythrocytes circulating in vivo. It has been shown that AGE and its components improved the peripheral circulation (4) and the erythrocyte deformability (23). This suggests that improvement of blood circulation by AGE might be one cause for the SBP-lowering effect. However, the RG group showed a significant decrease in erythrocytes and increase in reticulocytes, which are immature erythrocytes, suggesting chronic feeding of RG powder could lead to anemia (3,24). Therefore, the SBP-lowering effect of the RG group was less effective than AGE in the improvement of blood circulation. Moreover, in addition to causing an anemic condition, the RG group also had the additional complication of a high frequency of papilloma in the forestomach and a decline of body wt. This suggests that long-term feeding of the RG produced potentially harmful side effects.

In conclusion, chronic feeding of both AGE and RG prevented the increase of SBP on the SHR rats. The effect of AGE is based on the improvement in the blood circulatory system, such as the maintenance of the blood vessel flexibility and the improvement of the erythrocyte deformability. However, the SBP lowering of RG was indicated to cause harmful effects such as anemia, the decrease in body wt and the generation of papilloma in the forestomach, and long-term feeding of RG has the danger of health damage. These results suggest that AGE more safely improves several circulatory diseases related to blood vessel than raw garlic.


   ACKNOWLEDGMENTS
 
We would like to thank Professor Watanabe and Mr. Kashimoto, Research Institute for Radiation Biology and Medicine, for the histological analysis of the rat stomachs, and Dr. Kodera of Wakunaga Pharmaceutical Co., for supplying the AGE powder.


   FOOTNOTES
 
1 Published in a supplement to The Journal of Nutrition. Presented at the symposium "Significance of Garlic and Its Constituents in Cancer and Cardiovascular Disease" held April 9–11, 2005 at Georgetown University, Washington, DC. The symposium was sponsored by Strang Cancer Prevention Center, affiliated with Weill Medical College of Cornell University, and Harbor-UCLA Medical Center, and co-sponsored by American Botanical Council, American Institute for Cancer Research, American Society for Nutrition, Life Extension Foundation, General Nutrition Centers, National Nutritional Foods Association, Society of Atherosclerosis Imaging, Susan Samueli Center for Integrative Medicine at the University of California, Irvine. The symposium was supported by Alan James Group, LLC, Agencias Motta, S.A., Antistress AG, Armal, Birger Ledin AB, Ecolandia Internacional, Essential Sterolin Products (PTY) Ltd., Grand Quality LLC, IC Vietnam, Intervec Ltd., Jenn Health, Kernpharm BV, Laboratori Mizar SAS, Magna Trade, Manavita B.V.B.A., MaxiPharm A/S, Nature's Farm, Naturkost S. Rui a.s., Nichea Company Limited, Nutra-Life Health & Fitness Ltd., Oy Valioravinto Ab, Panax, PT. Nutriprima Jayasakti, Purity Life Health Products Limited, Quest Vitamins, Ltd., Sabinco S.A., The AIM Companies, Valosun Ltd., Wakunaga of America Co. Ltd., and Wakunaga Pharmaceutical Co., Ltd. Guest editors for the supplement publication were Richard Rivlin, Matthew Budoff, and Harunobu Amagase. Guest Editor Disclosure: R. Rivlin has been awarded research grants from Wakunaga of America, Ltd. and received an honorarium for serving as co-chair of the conference; M. Budoff has been awarded research grants from Wakunaga of America, Ltd. and received an honorarium for serving as co-chair of the conference; and Harunobu Amagase is employed by Wakunaga of America, Ltd. Back

2 Author disclosure: No relationships to disclose. Back

4 Abbreviations used: AGE, aged garlic extract; BP, blood pressure; MBP, mean blood pressure; PP, pulse pressure; RG, raw garlic; SBP, systolic blood pressure; SHR, spontaneously hypertensive rat. Back


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

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