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Recent Advances on the Nutritional Effects Associated with the Use of Garlic as a Supplement

Immunomodulatory Effects of Aged Garlic Extract1

Eikai Kyo,2 Naoto Uda, Shigeo Kasuga and Yoichi Itakura
Healthcare Research Institute, Wakunaga Pharmaceutical Company, Ltd., Koda-cho, Takata-gun, Hiroshima 739-1195, Japan.
ABSTRACT Using various kinds of models, we examined the effects of aged garlic extract (AGE) on immune functions. In the immunoglobulin (Ig)E-mediated allergic mouse model, AGE signicantly decreased the antigenspecic ear swelling induced by picryl chloride ointment to the ear and intravenous administration of antitrinitrophenyl antibody. In the transplanted carcinoma cell model, AGE signicantly inhibited the growth of Sarcoma-180 (allogenic) and LL/2 lung carcinoma (syngenic) cells transplanted into mice. Concomitantly, increases in natural killer (NK) and killer activities of spleen cells were observed in Sarcoma-180 bearing mice administered AGE. In the psychological stress model, AGE signicantly prevented the decrease in spleen weight and restored the reduction of anti-SRBC hemolytic plaque-forming cells caused by the electrical stress. These studies strongly suggest that AGE could be a promising candidate as an immune modier, which maintains the homeostasis of immune functions; further studies are warranted to determine when it is most benecial. J. Nutr. 131: 1075S1079S, 2001. KEY WORDS:

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For 5000 years, garlic has had a worldwide reputation as a formidable prophylactic and therapeutic medicinal agent (Adetumbi and Lau 1983, Essman 1984, Lau et al. 1983). More than 3000 publications in this century have provided evidence for the efcacy of this herb in the prevention and treatment of a variety of diseases, and for validating its traditional uses. Many favorable biological and pharmacologic effects with consumption of garlic preparations have been reported experimentally and clinically. Garlic has been shown to reduce risk factors of cardiovascular diseases, i.e., lowering serum cholesterol and triglycerides, inhibiting blood coagulation, improving blood circulation and lowering blood pressure (Ernst 1987, Fenwick and Hanley 1985, Kleijnen et al. 1989, Klendler 1987, Lau et al. 1983, Reuter 1995, Sendl 1995, Silagy and Neil 1994, Warshafsky et al. 1993). Many in vitro and in vivo studies have suggested possible cancer-preventive effects of garlic preparations and their respective constituents (Dausch and Nixon 1990, Dorant et al. 1993, Ip et al. 1994, Sumiyoshi and Wargovich 1989, Yang et al. 1994). In 1990, the U.S. National Cancer Institute initiated the Designer Food Pro-

1 Presented at the conference Recent Advances on the Nutritional Benets Accompanying the Use of Garlic as a Supplement held November 1517, 1998 in Newport Beach, CA. The conference was supported by educational grants from Pennsylvania State University, Wakunaga of America, Ltd. and the National Cancer Institute. The proceedings of this conference are published as a supplement to The Journal of Nutrition. Guest editors: John Milner, The Pennsylvania State University, University Park, PA and Richard Rivlin, Weill Medical College of Cornell University and Memorial Sloan-Kettering Cancer Center, New York, NY. 2 To whom correspondence and reprint requests should be addressed. E-mail:

gram to determine which foods played an important role in cancer prevention; they concluded that garlic may be the most potent food having cancer-preventive properties (Caragay 1992). In addition to the above pharmacologic activities, garlic has been shown to be a possible biological response modier. Weisberger and Pensky (1957) rst reported the augmentation of tumor immunity by garlic; subsequently a variety of immunostimulatory effects of garlic were reported. Because certain diseases can be caused by immune dysfunction, modication of immune functions by garlic may contribute to the treatment and prevention of diseases. Thus, some pharmacologic effects of garlic might be mediated through immunomodication. A unique garlic preparation, called aged garlic extract (AGE)3 has been reported to have an array of pharmacologic effects, including immunomodulation (Abdullah et al. 1989, Jain and Konar 1978, Lau et al. 1986). AGE is manufactured by Wakunaga Pharmaceutical, by the following steps: briey, garlic cloves (Allium sativum) are sliced and soaked in a water/ ethanol mixture and naturally extracted/aged for 10 mo at room temperature. AGE used for the studies contained 15% solid materials and 0.1% (calculated on the dried basis) Sallylcysteine, a marker compound for standardization. Anti-allergic effect. A number of reports have revealed the relationship between foods and allergic responses. Tanaka

3 Abbreviations used: AGE, aged garlic extract; BSA, bovine serum albumin; Con A, concanavalin A; FBS, fetal bovine serum; IFN-, interferon-; Ig, immunoglobulin; IL-2, interleukin-2; LPS, lipopolysaccharide; NK, natural killer; PFC, plaque-forming cells; PSK, polysaccharide of kawaratake; PWM, pokeweed mitogen; TNF-, tumor necrosis factor-alpha; TNP, trinitrophenyl.

0022-3166/01 $3.00 2001 American Society for Nutritional Sciences.




et al. (1992) reported that Allium vegetables, including garlic, inhibit the release of -hexosaminidase, which is correlated with histamine release, in rat basophilic leukemia cells (RBL2H3), suggesting an anti-allergic effect. Usui and Suzuki (1996) reported that the anti-allergic activity of (Z)-ajoene might be due to its inhibition of chemical mediator release. We also found that AGE has an anti-allergic property (Kyo et al. 1997). Histamine release in the rat basophil cell line RBL-2H3 was induced by mouse anti-trinitrophenyl (TNP) monoclonal antibody and the TNP-bovine serum albumin (BSA) hapten carrier complex. AGE added to the culture medium at doses of 1.25, 2.5 and 5.0 g/100 g signicantly inhibited the antigen specic histamine release by 50, 80 and 90%, respectively (Fig. 1). Oral administration of AGE (10 mL/kg) also decreased 25 45% of the ear swelling, used as an index of immunoglobulin (Ig)Emediated skin reaction, that was caused by intravenous administration of anti-TNP IgE antibody and subsequent picryl chloride painting on the ear in mice (Fig. 2). These data suggest that AGE may directly and/or indirectly modify the functions of mast cells, basophils and activated T lymphocytes, which play a leading role in allergic cascade reactions including inammation. Anti-tumor effect. AGE has a variety of pharmacologic effects, including tumor cell growth inhibition and chemopreventative effects. In rodents, AGE and its constituents have been reported to inhibit the development of chemically induced tumors in the bladder (Lau et al. 1986), mammary gland (Amagase and Milner 1993), colon (Sumiyoshi and Wargovich 1990), esophagus (Wargovich et al. 1988), lung (Sparnins

FIGURE 2 Suppression of immunoglobulin (Ig)E-mediated antigen specic skin reaction by aged garlic extract (AGE). BALB/c male mice were administered anti-trinitrophenyl (TNP) IgE antibody ascites intravenously (PCA titer was 1:4800, 0.5 mL/mouse). After 3 h, 0.3% picryl chloride (2,4,6-trinitrochlorobenzene, 15 l/ear) in the acetone and olive oil mixture (4:1) was painted on both sides of the right pinna. Because picryl chloride binds to the amino groups of protein, the conjugates work as a hapten-carrier antigen, which reacts with anti-TNP IgE antibodies that bind to the mast cell IgE receptors and subsequently induce spongiotic changes in the ear skin. AGE and oxatomide as a positive control were administered orally at 21 and 1 h before and 3 and 20 h, respectively, after the picryl chloride treatment. Thickness of the ear was measured 4 h before and at 1, 4 and 24 h after the picryl chloride treatment. Each point represents the mean SEM, n 10. Asterisks denote signicant difference from the control, *P 0.05; **P 0.01.

FIGURE 1 Inhibition of histamine release in RBL-2H3 by aged garlic extract (AGE). The rat basophil cell line RBL-2H3 (2 105 cells/mL) was inoculated in 24-well plates (Falcon). After 24 h of incubation, the basophil cells were washed twice with PBS(), and 0.3 mL of the culture supernatant containing anti-trinitrophenyl (TNP) mouse monoclonal immunoglobulin (Ig)E antibody produced by IGEL-2a mouse hybridoma was added. Because the mouse and rat IgE protein have 95% homology, anti-TNP mouse monoclonal IgE antibody can occupy the IgE receptors of RBL-2H3 cells. After 3 h of incubation, cells were again washed twice with PBS(). AGE or oxatomide (positive control) and 0.3 mL of TNP-bovine serum albumin (BSA) (10g/mL tyrode solution), as the hapten-carrier antigen to induce the histamine release, were added and incubated for one additional hour. Histamine released into the supernatant from RBL-2H3 cells was measured by the o-phthalaldehyde method. In this system, the antigen-antibody specic interaction released 116 7 ng/mL histamine into the culture medium, which corresponded to 25% of total histamine in RBL-2H3 cells. The spontaneously released histamine was 36 4 ng/mL. Values are given as the percentage of control (mean SD, n 6). Asterisks denote signicant difference from the control, *P 0.05; ** P 0.01.

et al. 1986), skin (Nishino et al. 1989) and stomach (Wattenberg et al. 1989) in rodents. Possible anticarcinogenic mechanisms of AGE and its constituents may include the inhibition of carcinogen activation (Amagase and Milner 1993), the enhancement of detoxication (Sumiyoshi and Wargovich 1990) and excretion (Tadi et al. 1990), and the protection of DNA from activated carcinogens (Tadi et al. 1991). The suppression of tumor cell growth through stimulation of immunoresponder cells has been suggested to be one of the signicant mechanisms in the prevention of carcinogenesis (Kuroki et al. 1989). Polysaccharide of kawaratake [PSK (Krestin; Kureha Chemical, Japan)], which has been approved as an anti-cancer drug by the Ministry of Health and Welfare of Japan and widely applied to cancer patients, is a protein-bound polysaccharide derived from Coriolus versicolor of the class Basidiomyvetes (Ohno et al. 1975, Tsukagoshi et al. 1984). PSK has been reported to potentiate and restore various kinds of immune reactions and to suppress the growth of allogenic or syngenic tumors even when administered before tumor inoculation (Abe et al. 1978, Ebihara and Minamishima 1984, Matsunaga et al. 1987, Tsuru and Nomoto 1983). Therefore, in this study, we investigated immunomodulation and antitumor effects of AGE in comparison with the activities of PSK as a positive drug.

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Effects on immune functions in vitro1,2
Immune functions Proliferation ConA PWM LPS IL-2 IL-2 IL-12 TNF- IFN- AGE PSK Methods MTT Assay MTT Assay MTT Assay MTT Assay MTT Assay ELISA ELISA ELISA ELISA 51Cr release Griess Assay Latex beads

Cytokines release

whereas AGE slightly increased production. Both AGE and PSK treatment strongly induced phagocytic activity of peritoneal cells. Using transplantable tumor models (Fig. 3a), we determined the effect of AGE and PSK on tumor growth. Growth inhibition by AGE and PSK was 50% in Sarcoma-180 and 20% in LL/2, and a signicant difference from the control was detected at wk 3 (Fig. 3b). Additionally, in splenic cells prepared from Sarcoma-180 bearing mice at wk 3, NK cells and killer cell activities were measured against YAC-1 and Sarcoma-180, respectively. Signicant increases in NK cell and killer cell activities were observed in splenic cells prepared from AGE-administered mice, whereas only an increase in NK

NK activity Nitric oxide Phagocytosis

1 , strong up-regulation; , weak up-regulation; , ineffectiveness. 2 Aged garlic extract (AGE) was added to the culture medium at the nal concentration of 10 l/mL; 100 mg PSK (Krestin), a positive control, was dissolved in 1 mL saline and used as the stock solution (100 mg/mL). 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay (Mosmonn 1983): C57BL/6 spleen cells (2 106 cells/well) were cultured in the presence or absence of AGE in a 96-well microtiter plate (NUNC) under 5% CO2/95% air at 37C. In the combination assay with mitogens, Concanavalin A (ConA, 2 g/mL, Honen Corporation, Japan), pokeweed mitogen (PWM, 1 g/mL, Honen), lipopolysaccharide (LPS, 1 g/mL, Sigma Chemical, St. Louis, MO) or recombinant interleukin (IL)-2 (10 ng/mL, Biosource International, CA) was added before starting cultivation. After 3 d incubation, WST-1 reagent (Dojin Corporation, Kumamoto, Japan) was added to each well and incubated further for 3 h (Ishiyama et al. 1993). The A450 of the incubated mixture was determined using a microplate reader (Bio-Rad, Tokyo, Japan). Cytokine release: C57BL/6 spleen cells (1 107 cells/ mL) were cultured in the presence or absence of AGE in a 24-well plate (NUNC) under 5% CO2/95% air at 37C. After 3 d of incubation, cytokines [interleukin (IL)-2, tumor necrosis factor (TNF)- and interferon (INF)-] released into the medium were measured using ELISA (Genzyme Duo-set ELISA bulk kit, Tokyo, Japan). Natural killer (NK) activity: 1.5 106/100 L spleen T cells and 1.5 104/50 L 51Crlabeled YAC-1 cells were cultured in the presence or absence of AGE in NUNC round-bottomed microplates. After 4 and 24 h incubation, the mixture was centrifuged; the released 51Cr in the supernatant was counted in a gamma-counter and the cytotoxicity was determined. Phagocytosis: The peritoneal cells (106 cells/200 L) were preincubated in the presence or absence of AGE for 3 h under 5% CO2 at 37C; the uorescent-labeled latex bead solution (1:100 dilution, 0.75 YG, Funakoshi, Tokyo, Japan) was added (Stewart et al. 1986). After 1 h of incubation, the cells were centrifuged, washed with PBS and xed in 2.5% buffered formalin solution. Macrophages that phagocytosized 10 latex beads were counted using the Improved Neubauer (Erma, Tokyo, Japan), and the phagocytic activity was determined as the number of activated macrophages per 100 cells (Rosenstreich et al. 1971).

Our recent ndings on immunostimulatory activities of AGE are summarized in Table 1. Both AGE and PSK enhanced the proliferation of spleen cells. AGE slightly augmented concanavalin-A (ConA)-induced proliferation of spleen cells, but did not augment the effects of the other mitogens, pokeweed mitogen (PWM) and lipopolysaccharide (LPS). PSK augmented induced proliferation and mitigated LPS-induced proliferation. Interleukin (IL)-2induced proliferation was signicantly augmented by addition of AGE and PSK. AGE signicantly increased the release of all four cytokines from mouse splenic cells. PSK increased TNF release markedly and IL-2 release slightly; however, it failed to increase interferon- (INF-) release. Both AGE and PSK treatment enhanced natural killer (NK) cell activity. PSK signicantly increased nitric oxide production of the splenic cells,

FIGURE 3 Antitumor effects of aged garlic extract (AGE). (a) Experimental procedure for antitumor assay. In the allogenic system, 106 cells of Sarcoma-180 in 100 L PBS were inoculated subcutaneously into the backs of ICR male mice. In the syngenic system, 106 cells of Lewis lung carcinoma cell line LL/2 in 100 L of PBS were inoculated subcutaneously into the backs of C57BL/6 male mice. Twenty-four hours after carcinoma cell inoculation, either water (control, 10 mL/kg) or AGE (10 mL/kg) was administered orally every other day for a total of 11 treatments. The tumor size was measured once a week using a micrometer, and the volume was calculated. Three weeks after the inoculation, natural killer (NK) and killer activities of spleen cells in Sarcoma-180 bearing mice were determined against YAC-1 cells and Sarcoma-180 cells, respectively. (b) Antitumor activities of AGE. Values are given as the percentage of control (mean SEM, n 10). Asterisks denote signicant difference from the control, *P 0.05. (c) Enhancement of NK and killer cells activities in Sarcoma-180 bearing mice by AGE. Values are given as (mean SEM, n 10). Asterisks denote signicant difference from the control, **P 0.01.

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Antipsychological stress effects of aged garlic extract (AGE)1,2
Spleen weight (mg) 87.0 2.9 70.1 2.3**a 82.9 3.8**b Number of spleen cells (106) 69.1 2.8 46.6 2.7**a 58.6 4.5*b Number of PFC cells (/ 107) 112.9 8.1 56.6 7.8**a 98.2 14.6*b

Group Normal Stress control AGE

A variety of herbs have been used traditionally to prevent and treat diseases. At present, their pharmacologic activities, particularly stimulation of immune functions, has been the focus of alternative medicine. Currently available data strongly suggest that garlic may be a promising candidate as an immune modier that maintains the homeostasis of immune function. Further studies are warranted. LITERATURE CITED
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1 Values are mean SEM, n 10; **a P 0.01 vs. normal; *b P 0.05, **b P 0.01 vs. stress control. 2 The communication box was used to provide psychological stress. The box consisted of small compartments (10 10 cm) equipped with either an electric foot-shock oor or a nonshock oor; the electric shock and the nonshock oor were placed reciprocally. A mouse (sender) placed on an electric shock oor made emotional responses when charged with electricity to the oor for 10 s at intervals of 50 s; a mouse (responder) placed in a non-shock oor was exposed only to psychological stress. The electric current for the shock was increased stepwise from 1.6 to 2.0 mA at the rate of 0.2 mA/h for 3 h. Sender mice were changed daily to naive mice to avoid reduced emotional responses to the electric shock due to adaptation to repeated exposures. Responder mice were administered daily with either AGE (10 ml/kg) or water (control) 1 h before the 3-h emotional stress for 4 d. On d 1, responder mice were immunized with sheep red blood cells (SRBC, 1 108) after the 3-h stress. After the last day of stress exposure, the spleen weight and number of spleen cells were measured, and the number of anti-SRBC plaque-forming-cells (PFC) was assayed.

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cell activity was observed in the PSK-administered mice (Fig. 3c). Current cancer therapies, such as surgical excision, radiotherapy and anti-cancer agent administration, are limited, and alternative treatments are required for patients who fail to respond to these therapies. The modication of immune function may be the most promising alternative for controlling cancer, particularly stimulation of nonspecic immune response and cell-mediated immunity, because cancer cells are indeed not recognized as alien substances by the immune system. Although further investigations are warranted, the data available to date suggest that garlic may provide a new and effective form of cancer therapy through immune stimulation. Anti-psychological stress effect. Psychological and physical stresses have been shown to suppress immune functions. Yokoyama et al. (1986) reported that an AGE preparation containing vitamins B-1 and B-12 restored physical stress induced immune suppression in mice. Recently, we determined the effect of AGE on immune suppression caused by psychological stress using a communication box. After 4 d of psychological stress, a decrease in spleen weight, spleen cells and hemolytic plaque-forming cells (PFC) was observed in the control mice. AGE signicantly prevented the decreases in spleen weight and cells, and restored the reduction of hemolytic PFC caused by the stress (Table 2). Summary Recent technologies are beginning to clarify important roles of immune functions in disease progression. Immune dysfunction may result in infectious diseases and cancer, and hyper-immune reactions may cause autoimmune diseases, including allergy and rheumatoid arthritis. Thus, the development of an immune modier that stimulates necessary functions and suppresses unnecessary functions is truly desired.

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