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© 2006 American Society for Nutrition J. Nutr. 136:870S-872S, March 2006

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

Hydroponic Cultivation Offers A Practical Means of Producing Selenium-Enriched Garlic1–3,

Tadamitsu Tsuneyoshi4, Jiro Yoshida and Takashi Sasaoka

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

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


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
Garlic enriched by selenium (Se) could be an excellent source of dietary Se for cancer chemoprevention. The production of high-Se garlic requires Se-fertilized soil, but such soil may pollute the environment. Hydroponics is a closed system that allows good control over Se fertilization without environmental consequences. We examined the effect of hydroponic cultivation on Se uptake and assimilation in garlic seedlings. Garlic bulbs were grown in the nutrient solution without Se for first 2 wk, and with potassium selenate for an additional week. Sulfate in an ordinary hydroponic solution inhibited the absorption and assimilation of selenate, but when a sulfate-free nutrient was used for Se addition, the garlic seedlings accumulated >1 mg Se, dry weight. Through HPLC inductively coupled plasma MS (HPLC-ICP-MS) analysis, Se-methlyselenocysteine (MeSeCys), {gamma}-glutamyl-Se-methlyselenocysteine ({gamma}-GluMeSeCys), selenomethionine, and nonmetabolized selenate were identified in water extracts of the garlic seedlings. The results demonstrate that hydroponic enrichment of Se in garlic seedlings could be a practical means of producing organic Se compounds for nutritional supplements.

KEY WORDS: • garlic • selenium enrichment • chemoprevention • hydroponics

Selenium (Se) is an essential trace element, and selenium deficiency in humans is associated with several health problems. It was shown that dietary Se supplements reduce the incidence of cancers in humans as well as in animal model systems (1,2). The chemopreventive efficacy of Se depends upon its chemical form (3). Organic Se compounds accumulated in Se-enriched edible plants would be an ideal nutritional supplement (4,5). Allium plants naturally accumulate sulfur-containing amino acids at higher levels, making them favorable as enrichment foods (6). Se-enriched garlic was reported to be useful as a nutritional Se supplement for cancer prevention (7,8). The production of high-Se garlic requires Se-fertilized soil, but such soil may pollute the environment. Hydroponics is a closed system and allows good control of Se fertilization. We examined the effect of hydroponic techniques on Se uptake and assimilation in garlic seedlings.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 LITERATURE CITED
 
    Garlic. A Japanese cultivar, "Hokkaido-zairai," was used for the experiments.

    Hydroponics. Small bulblets, obtained from the top of the garlic stem, were used for small-scale hydroponics; 24 bulblets were planted in a container with 200 mL of high-sulfate or sulfate-deficient nutrients (Table 1). After 14 d of cultivation, potassium selenate or potassium selenite was added to the nutrients at a concentration of 50 µmol/L, and the seedlings were cultivated for another 10 d. In addition, 21 garlic bulbs were planted in a hydroponic apparatus with 3 L of sulfate-deficient nutrient solution. After 14 d of cultivation, potassium selenate was added to the nutrients and the seedlings were cultivated for another 29 d.


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  		TABLE 1 Composition of the high-sulfate and sulfate-deficient nutrients

 
    Speciation of Se in garlic seedlings. Se-enriched garlic seedlings were vacuum-dried at 50°C for 20 h, and pulverized in a grinder mill. Ice-cold water (10 mL) was added to 100 mg of the Se-enriched garlic powder, and the samples were ground in a glass homogenizer on ice. The water extract was obtained after centrifugation (20,000 x g; 10 min) and filtration of the homogenate. Aliquots of the water extracts were analyzed by HPLC inductively coupled plasma MS (HPLC-ICP-MS)5 as previously described (9).

    Analysis of total Se in garlic seedlings and Se in the nutrients. The dried garlic powders were wet-ashed with nitric acid and perchloric acid. The diluted digest was heated with hydrochloric acid and incubated with 2,3-diaminonaphthalene. A fluorescent substance, 4,5-benzopiaselenol, that formed in the mixture was determined by HPLC as previously described (10). The quantity of selenate or selenite in the nutrient solution was determined using 2,3-diaminonaphthalene as mentioned above, without wet-ashing.


   RESULTS AND DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
LITERATURE CITED
 
    Effects of sulfate on Se absorption and assimilation in garlic seedlings. The garlic seedlings in the sulfate-free nutrients absorbed >0.4 mg Se/g of selenate, whereas the seedlings in the high-sulfate nutrients absorbed <0.2 mg Se/g of selenate (Fig. 1A). Selenate uptake in plants is thought to depend on sulfate transporters (11). In the case of garlic seedlings, the absorption of selenate was clearly inhibited by sulfate in the nutrients. Selenite was absorbed less effectively than selenate, and the uptake was not inhibited by sulfate in the nutrient (Fig. 1B).


Figure 1
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  		FIGURE 1  Effects of sulfate on Se uptake and accumulation of MeSeCys in the garlic seedlings. Sulfate conditions on precultivation and selenium addition are indicated. Selenium absorption (A, B) and MeSeCys accumulation (C, D) in the garlic seedlings grown with selenate (A, C) or selenite (B, D) are shown. Values are means ± SD, n = 3 containers.

 
The water extracts were prepared from the Se-enriched garlic powder, and Se compounds in the extract were determined by HPLC-ICP-MS analysis. Figure 2 shows the chromatogram from HPLC-ICP-MS obtained from the garlic powder enriched by selenate or selenite. The garlic enriched by selenate produced 3 distinct peaks, which corresponded to MeSeCys, {gamma}-GluMeSeCys, and nonmetabolized selenate (Fig. 2A, B). The garlic enriched by selenite produced 2 peaks, which corresponded to Se-methlyselenocysteine (MeSeCys) and {gamma}-glutamyl-Se-methlyselenocysteine ({gamma}-GluMeSeCys); no peak related to nonmetabolized selenite could be detected (Fig. 2C, D). The sulfate-deficient condition provided twice the amount of MeSeCys for garlic enriched by selenate (Fig. 1C). Conversely, the garlic precultivated in the high-sulfate nutrients contained more nonmetabolized selenate than the garlic cultivated in the sulfate-deficient condition (Fig. 2A, B).


Figure 2
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  		FIGURE 2  Speciation of Se in water extracts of Se-enriched garlic by HPLC-ICP MS. The retention time of the peaks indicated by arrows (a)–(d) corresponds to that of selenate, {gamma}-GluMeSeCys, MeSeCys, and selenomethionine, respectively. Garlic seedlings were precultivated with sulfate (B, D) or without sulfate (A, C), and were enriched by selenate (A, B) or selenite (C, D).

 
The Se-assimilation pathway is thought to overlap with sulfur assimilation in several steps (11,12). Sulfate uptake and assimilation activity are induced under conditions of sulfur starvation or high demand for sulfur metabolites (12). Hydroponics with the sulfate-deficient nutrient induced sulfur assimilation at the same time as Se assimilation in the garlic seedlings.

    Behavior of Se in hydroponic cultivation. To determine the fate of Se added to the nutrient, changes in Se concentration during cultivation and final recovery of the Se were investigated. The changes in selenate concentration in the nutrient indicated that garlic seedlings absorbed selenate in a relatively short period (Fig. 3). For comparison of Se input and recovery in hydroponics, the following kinds of measured Se were listed (Fig. 4): 1) the total reduction in Se from the nutrient solution; 2) the total amount of Se in the water extract of garlic powder determined by HPLC-ICP-MS; and 3) the total amount of Se in garlic powder determined after the wet-ashed procedure. When selenate was used for enrichment, almost all of the Se reduced from the nutrient was recovered in garlic seedlings, and the Se compounds were water soluble. In the case of selenite, more than half of the Se was missing, perhaps through volatilization during cultivation (Fig. 4). These findings agree with previous reports that the rate of Se volatilization from selenite is higher than from selenate (13,14). Our results show that the volatilized Se from selenate could be negligible in garlic hydroponics. However, further study is required to determine the precise amount of volatilized Se during cultivation.


Figure 3
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  		FIGURE 3  Changes in Se concentration during cultivation. After 21 garlic seedlings were grown in 3 L of sulfate-free nutrient, 12 mg of Se was added to the nutrient as potassium selenate on the days indicated by arrowheads.

 

Figure 4
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  		FIGURE 4  Comparison of Se recovery between selenate addition (A) and selenite addition (B). Absorption indicates the total amount of selenium lost from the nutrient solution during cultivation.

 
Hydroponic enrichment of Se in garlic seedlings could be a safe and efficient system of producing organic Se compounds in an edible plant. Se-enriched garlic contains MeSeCys and {gamma}-GluMeSeCys as the major organoselenium compounds, and would be an ideal nutritional supplement for cancer prevention.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Yasumitsu Ogra, Graduate school of Pharmaceutical Science, Chiba University for support in HPLC-ICP-MS analysis.


   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

3 Supported in part by a grant (Development of Technologies to Promote a Processed Food Supply System "Brand Nippon") from the Ministry of Agriculture, Forestry and Fisheries of Japan. Back

5 Abbreviations used: HPLC-ICP-MS, HPLC inductively coupled plasma MS; {gamma}-GluMeSeCys, {gamma}-glutamyl-Se-methlyselenocysteine; MeSeCys, Se-methlyselenocysteine. Back


   LITERATURE CITED
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 

1. Clark LC, Combs GF Jr, Turnbull BW, Slate EH, Chalker DK, Chow J, Davis LS, Glover RA, Graham GF, et al. Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group. JAMA. 1996;276:1957–63.[Abstract]

2. Ip C, Zhu Z, Thompson HJ, Lisk D, Ganther HE. Chemoprevention of mammary cancer with Se-allylselenocysteine and other selenoamino acids in the rat. Anticancer Res. 1999;19:2875–80.[Medline]

3. Ip C. Lessons from basic research in selenium and cancer prevention. J Nutr. 1998;128:1845–54.[Abstract/Free Full Text]

4. Davis CD, Zeng H, Finley JW. Selenium-enriched broccoli decreases intestinal tumorigenesis in multiple intestinal neoplasia mice. J Nutr. 2002;132:307–9.[Abstract/Free Full Text]

5. Sugihara S, Kondo M, Chihara Y, Yuji M, Hattori H, Yoshida M. Preparation of selenium-enriched sprouts and identification of their selenium species by high-performance liquid chromatography-inductively coupled plasma mass spectrometry. Biosci Biotechnol Biochem. 2004;68:193–9.[Medline]

6. Kotrebai M, Birringer M, Tyson JF, Block E, Uden PC. Selenium speciation in enriched and natural samples by HPLC-ICP-MS and HPLC-ESI-MS with perfluorinated carboxylic acid ion-pairing agents. Analyst. 2000;125:71–8.[Medline]

7. Ip C, Lisk DJ. Enrichment of selenium in allium vegetables for cancer prevention. Carcinogenesis. 1994;15:1881–5.[Abstract/Free Full Text]

8. Dong Y, Lisk D, Block E, Ip C. Characterization of the biological activity of gamma-glutamyl-Se-methylselenocysteine: a novel, naturally occurring anticancer agent from garlic. Cancer Res. 2001;61:2923–8.[Abstract/Free Full Text]

9. Ogra Y, Ishiwata K, Ruiz Encinar J, Lobinski R, Suzuki KT. Speciation of selenium in selenium-enriched shiitake mushroom, Lentinula edodes. Anal Bioanal Chem. 2004;379:861–6.[Medline]

10. Yoshida M, Kimura Y, Abe M, Ando T, Tachi H, Fukunaga K. Quantitative evaluation of selenium contained in tea by high performance liquid chromatography. J Nutr Sci Vitaminol (Tokyo). 2001;47:248–52.[Medline]

11. White PJ, Bowen HC, Parmaguru P, Fritz M, Spracklen WP, Spiby RE, Meacham MC, Mead A, Harrimanm M, et al. Interactions between selenium and sulphur nutrition in Arabidopsis thaliana. J Exp Bot. 2004;55:1927–37.[Abstract/Free Full Text]

12. Saito K. Sulfur assimilatory metabolism. The long and smelling road. Plant Physiol. 2004;136:2443–50.[Free Full Text]

13. de Souza MP, Pilon-Smits EA, Lytle CM, Hwang S, Tai J, Honma TS, Yeh L, Terry N. Rate-limiting steps in selenium assimilation and volatilization by Indian mustard. Plant Physiol. 1998;117:1487–94.[Abstract/Free Full Text]

14. Terry N, Zayed AM, De Souza MP, Tarun AS. Selenium in higher plants. Annu Rev Plant Physiol Plant Mol Biol. 2000;51:401–32.





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