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© 2006 American Society for Nutrition J. Nutr. 136:847S-851S, March 2006

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

Aged Garlic Extract Has Chemopreventative Effects On 1,2-Dimethylhydrazine-Induced Colon Tumors in Rats1,3

Takefumi Katsuki*, Keiji Hirata*,4, Hideki Ishikawa{dagger}, Nariaki Matsuura**, Shin-ichiro Sumi{ddagger},2 and Hideaki Itoh*

* Department of Surgery 1, University of Occupational and Environmental Health, Kitakyushu, Japan; {dagger} Laboratory of Hereditary Tumor, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Osaka, Japan; ** Department of Molecular Pathology, School of Allied Health Sciences, Osaka University Graduate School of Medicine, Osaka, Japan; and {ddagger} Healthcare Development Department, Wakunaga Pharmaceutical Co., Ltd., Hiroshima, Japan

4 To whom correspondence should be addressed. E-mail: hirata{at}med.uoeh-u.ac.jp.


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Garlic has been reported to have chemopreventive effects against a variety of cancers. However, different garlic preparations contain different constituents. We investigated the chemopreventive effect of aged garlic extract (AGE), an odorless product from prolonged extraction of fresh garlic, on colon carcinogenesis and cell proliferation in 1,2-dimethylhydrazine (DMH)-induced colon neoplastic rats. Rats were given weekly subcutaneous injections of DMH (20mg/kg) for 20 wk, and fed either a basal diet or one containing 4% AGE. Serum from AGE-treated rats contained detectable S-allylcysteine. The AGE diet significantly reduced the number of colon tumors and aberrant crypt foci compared to the basal diet. Cell proliferation of normal-appearing colonic mucosa was assessed by MIB-5 immunohistochemistry. AGE treatment significantly decreased the mean MIB-5-labeling index. These findings suggest AGE has a chemopreventive effect on colon carcinogenesis through suppression of cell proliferation.

KEY WORDS: • aged garlic extract • S-allylcysteine • colon carcinogenesis • chemoprevention • cell proliferation

Garlic (Allium sativum) is widely cultivated and consumed as a food in many countries and has been widely used as a popular remedy for various disorders for thousands of years. Epidemiological studies have linked increased garlic consumption with a reduced incidence of colorectal cancer in various human populations using case-control studies (1–4), cohort studies (5,6), and meta-analysis (7). Regular garlic consumption also is associated with decreased prevalence of adenomatous polyps in the colon and rectum (8). Furthermore, garlic and its organosulfur components are reported to suppress colon carcinogenesis in animal studies (9–13) and cancer-cell growth in vitro (14–23).

Garlic contains several compounds that inhibit carcinogenesis at a variety of sites in different animal species and with different initiators (24). Possible anticarcinogenic mechanisms may include the inhibition of carcinogen activation (25), mutagenesis (26–28), enhancement of detoxification (10,29), and the protection of DNA from activated carcinogens (30). However, manufacturing processes significantly affect chemical constituents of garlic preparations and some preparations may cause adverse reactions, including gastrointestinal problems (31,32).

Aged garlic extract (AGE)5 is an odorless product created through prolonged extraction of fresh garlic. It is less irritating, does not produce the uncomfortable effects of raw garlic (33), and could be suitable for long-term use. Although mechanisms of the chemopreventive effect of garlic have been investigated, it has not been reported whether aging the product inhibits colon carcinogenesis in vivo. We investigated the inhibitory effect of AGE on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Animals, diet, and materials. Five-wk-old male Sprague-Dawley rats were obtained from Seac Yoshitomi Co. The rats were maintained according to the Institutional Animal Care Guidelines and acclimated to the diet and environment for 2 wk after arrival. They were housed 3-per-rack-mounted wire cages and given tap water ad libitum. The temperature (20–22°C), humidity (45–55%), and lighting (12 h light/dark cycle) were constantly controlled. AGE was manufactured as follows: Garlic cloves were sliced and soaked in a water-ethanol mixture solution and naturally extracted for >10 mo at room temperature (34). The extract was evaporated dry and mixed with a basal powder diet at a concentration (wt:wt) of 4%. CE-2 (CLEA Japan) was used as a basal diet throughout the study. DMH was purchased from Tokyo Kasei Industries. Monoclonal antibody MIB-5 (M7248) against Ki-67 antigen was purchased from Dako Cytomation.

    Carcinogenesis protocol. The experimental protocol is shown in Figure 1. A total of 60 rats were randomly distributed into 4 groups. The rats in group 1 (n = 12) were fed a basal diet and received subcutaneous injection of normal saline (0.2 ml) once a week for 20 wk as control. Group 2 (n = 12) rats were fed a diet containing 4% AGE. Group 3 (n = 18) rats were fed a basal diet and received subcutaneous injection of DMH at a dose of 20 mg/kg weight, once a week for 20 wk. Group 4 (n = 18) rats were fed a diet containing 4% AGE and received the same DMH injections as group 3. Food intake and body weight of the rats were recorded weekly. Rats were killed at 5 wk after the last injection of DMH by ether inhalation and the weights of the liver, spleen, and kidney were measured. Venous samples were collected from hearts using a tube containing sodium citrate, centrifuged for 10 min at 3000 x g, and the supernatant fluid was stored at –20°C until analysis. At autopsies, the entire intestinal tracts were removed, flushed free of feces with saline, and cut open along the longitudinal axis. The number, size, and location of intestinal tumors were recorded.


Figure 1
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  		FIGURE 1  Experimental protocol. N.S., normal saline (0.2 mL/rat, s.c. injection x 1/wk); DMH, 1,2-dimethylhydrazine (20 mg/kg body wt, s.c. injection x 1/wk).

 
    Concentration of serum S-allylcysteine. Serum S-allylcysteine measured as a compliance marker by HPLC studies using a post-column o-phthalaldehyde derivatization ion-exchange HPLC procedure with fluorescence detection (35).

    Determination of aberrant crypt foci. For the aberrant crypt foci (ACF) analysis, colons were placed flat between two filter papers and fixed in 10% formalin in 0.01 mol/L phosphate-buffered saline (PBS; pH 7.4) for 24 h. They were briefly dipped in a 2% solution of methylene blue in distilled water for 2 min and placed on a microscope slide, with the mucosal surface up, under a dissecting microscope. At a magnification of 40, ACF were distinguished from the surrounding normal-appearing crypts by their large and elliptical luminal openings (36). The total number of ACF and the number of ACF with 4 or more aberrant crypts were counted.

    Histopathological examination. Normal-appearing colons and all protrusions were embedded in paraffin. Each specimen was cut into a 2 µm-thick section for hematoxylin and eosin and immunohistochemical staining. When the protrusion proved to be a lymph follicle by histological examination, it was excluded from the records and analysis.

    Immunohistochemistry. Immunohistochemistry using monoclonal antibody MIB-5 against Ki-67 antigen was performed as a cell-proliferation marker. Each section was deparaffinized by xylene and rehydrated with graded alcohols. The sections were retrieved in a microwave oven (300W) in citrate buffer (pH 6.0) for 10 min at 100°C. Endogenous peroxidase activity was blocked by incubation of slides in absolute methanol containing 3% hydrogen peroxidase (H2O2) for 10 min at room temperature (RT). The sections were sequentially pre-incubated with 10% normal rabbit serum for 10 min at RT to block the possible background staining by the secondary antibody. They were then incubated with antibody MIB-5 at its working dilution of 1:20. After 60 min at RT, they were treated with EnVision reagent (Dako Cytomation) for 30 min. They were washed three times with PBS after each incubation, and 3,3'-diaminobenzidine was employed as a chromogen. The nuclei were lightly counterstained with Mayer's hematoxylin solution. The primary antibodies were replaced with PBS containing 0.1% bovine serum albumin as a negative control.

For determination of immunostaining, epithelial cells of the middle colon were counted from the lowest point to the tip of the crypt (full-length crypts) by light microscopy using 400 x magnification. The number of positively stained cells in each crypt column was recorded. The results were defined as the ratio of the number of positive-stained cells to the total number of cells counted (at least 500), and then multiplied by 100. In all cases, the counts were made by 2 independent observers who were unaware of the experimental condition or other results obtained.

    Statistical analysis. Values were expressed as means ± SD All statistics were computed using a suitable program (SAS/STAT software). Unpaired Student's t test and chi-square test were used to detect statistically significant differences between groups. A P-value of < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Food intake, body weight, organ weight, and macroscopic finding. At the end of the study, no macroscopic abnormality was observed in other intra-abdominal organs except in the intestinal tract. Body, liver, spleen, kidney, relative liver weights (g/100 g body wt), and food consumption (g/day per rat) in all groups are shown in Table 1. Average food intake did not differ significantly among the groups, which indicates that AGE administration did not suppress the calorie intake.


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  		TABLE 1 Mean Food Intake, Final Body Weight, and Organ Weight of Rats1

 
    Concentration of serum SAC. Although SAC was not detected in the serum of rats in group 1, it was detected from all 6 evaluated rats in group 2 (range from 67 to 83 µg/L).

    Number, size, and location of intestinal tumors. Intestinal tumors were only recognized in DMH-treated groups. The tumor incidence (tumor-bearing rat/rat) in the colons of group 4 (66.7%) was lower than that of group 3 (77.8%), but with no significant difference. The tumor incidence in the small intestine of group 4 (33.3%) was significantly lower than that of group 3 (93.3%) (P = 0.0004). The multiplicity of intestinal tumors is presented in Figure 2. The tumor multiplicity (number of tumors/rat) in the colons of group 3 was 3.7 ± 1.0 and that of group 4 was 1.0 ± 1.0, which proved lower than group 3 (P = 0.0081). Also, the tumor multiplicity in the small intestine of group 3 was 1.6 ± 1.3, and that of group 4 was 0.4 ± 0.7, which was lower than group 3 (P = 0.0001). The mean diameter of colon tumors in group 4 was smaller than that of group 3, but with no significant difference. As to the site of the tumors, the DMH-treated rats had tumors throughout the intestinal tract, from the duodenum to the distal colon. Colon tumors in group 3 were located in the proximal colon (21.8%), middle colon (52.7%), and distal colon (25.5%). Those of group 4 were located in the proximal colon (33.3%), middle colon (66.7%), and distal colon (33.3%), in which there was no significant difference between groups.


Figure 2
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  		FIGURE 2  Multiplicity of intestinal tumor. The tumor multiplicity (number of tumors/rat) of the AGE-treated group was significantly lower than that of the basal diet group both in the small intestine (P = 0.0001) and colon (P = 0.0081).

 
    Development of ACF. Figure 3 summarizes the data on colonic ACF formation. Groups 1 and 2 showed no ACF. All DMH-treated rats in groups 3 and 4 developed ACF. In group 3, DMH induced 273.3 ± 106.0 ACF per rat, and AGE-treatment significantly reduced the number of ACF in group 4 (P < 0.0001). Also, the number of ACF with 4 or more aberrant crypts was reduced in group 4 as compared to group 3 (P = 0.0002).


Figure 3
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  		FIGURE 3  Development of DMH-induced ACF. The number of ACF was reduced in the AGE-treated group as compared to basal-diet group (P < 0.0001). Also, the number of ACF with 4 or more aberrant crypts (ACs) was reduced in the AGE treated group as compared with basal diet group (P = 0.0002).

 
    MIB-5-labeling index. Monoclonal antibody MIB-5 showed clean and distinct nuclear staining of basal proliferating cells of crypts, thus showing identical patterns as reported for MIB-1 in corresponding human tissues. The mean MIB-5-labeling indices are presented in Figure 4. MIB-5-labeling index of group 4 (22.9 ± 8.3) was significantly lower than group 3 (37.8 ± 9.0) (P < 0.0001).


Figure 4
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  		FIGURE 4  MIB-5-labeling index in normal colonic mucosa. The mean MIB-5-labeling index of the AGE-treated group was lower than that of basal diet group (P < 0.0001).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Chemoprevention has the potential to be a major component of cancer control. Accumulating evidence indicates that various food ingredients may play an essential role in colon cancer prevention (37–43). The AGE used in this study is an extract of fresh garlic that is aged over a prolonged period and contains water-soluble allyl amino acid derivatives, which account for most of its organosulfur content, stable lipid-soluble allyl sulfides, flavonoids, saponins, and essential macro- and micronutrients (44). The lipid-soluble volatile organosulfur compound allicin, which is produced enzymatically when garlic is cut or chopped, is absent in AGE. We showed that AGE given orally significantly reduced DMH-induced colon tumor and ACF without causing any apparent adverse effects. The results are mostly consistent with previous rodent chemoprevention studies using other garlic preparations or its constituents (9–13).

ACF are useful intermediate biomarkers in detecting modifying influences of natural and synthetic compounds on chemically induced colon carcinogenesis, which represents the preneoplastic lesions. Furthermore, the percentage of large ACF consisting of 4 or more aberrant crypts is considered a better intermediate biomarker of tumor occurrence than the number of ACF alone (45). Recently, Yamada et al. reported that the presence of ß-catenin accumulated crypts (ß-CAC), which appear soon after carcinogen exposure like ACF (46), and suggest that ß-CAC is more likely to be a direct precursor of colon tumors than classical ACF in rats (47). In our study, AGE lowered the number of ACF, suggesting that AGE may inhibit the growth of ACF through suppression of cell proliferation in colonic mucosa exposed to DMH, and observations support the role of ACF as precursors to colon cancer.

Cell proliferation plays an important role in multistage carcinogenesis with multiple genetic changes (48). Modulation of cell-proliferation activity in target organs is one of the important actions of cancer chemoprevention (49). The nuclear antigen, designated as the Ki-67 protein, is exclusively expressed in the nuclei of all cells in G1, S, and G2 phases and mitosis, but not in the G0 phase of the cell cycle (50), and it has therefore become useful for assessment of cell growth. MIB-1 against the Ki-67 antigen is a reliable tool for determining proliferating cells in human tissues. Recently, a novel monoclonal antibody MIB-5 was found to have the additional advantage of being able to react with the rodent-equivalent Ki-67 protein (51,52). Therefore, we used MIB-5 as an immunohistochemical proliferation marker, in which MIB-5-labeling index in normal mucosa was decreased by dietary administration of AGE; therefore, it is suggested that AGE has chemopreventive effects through inhibition of cell proliferation in the initiation period of colon carcinogenesis.

For preventive purposes, daily, long-term ingestion of product is necessary. Thus, the safety of the product should be considered seriously. Because different types of garlic preparations have different pharmacological properties, and some garlic preparations may cause undesirable effects, including gastrointestinal problems, one should be cautious about their safety as well as their effectiveness when choosing a preparation. The major unique organosulfur compounds in AGE are water-soluble SAC and S-allylmercaptocysteine, which have high content because they are produced during the process of aging (35); they are relatively non-toxic to animals when compared with other garlic volatiles, and they are likely to be more tolerable if used in human prevention studies (10).

In conclusion, this study indicates dietary AGE has chemopreventive effects on chemically induced colon carcinogenesis through modulation of cell proliferation and suggests possible applications in human clinical trials. Based on these findings, an interventional trial is being conducted in our collaborative group, in which capsules containing AGE are given to determine whether they reduce the prevalence of colon cancer or polyp.


   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: Shin-ichiro Sumi is employed by the Wakunaga Pharmaceutical Co. Back

3 This study was supported by a Grant-in Aid for Cancer Research (13,14) from the Minister of Health, Labor and Welfare (H. Ishikawa, Chairman), Japan. Back

5 Abbreviations used: ACF, aberrant crypt foci; AGE, aged garlic extract; DMH, 1,2-dimethylhydrazine; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; ß-CAC, ß-catenin accumulated crypts; RT, room temperature; SAC, S-allylcysteine. Back


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