An Investigation of the Immunomodulatory Activities of Garlic (Allium Sativum L.)

Chandrashekar, P. M. (2011) An Investigation of the Immunomodulatory Activities of Garlic (Allium Sativum L.). PhD thesis, University of Mysore.

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Abstract

Garlic (Allium sativum) belongs to the genus Allium of Alliaceae family. Garlic has been used throughout history for both culinary and medicinal purposes. Garlic is an herbal spice containing numerous macronutrients, vitamins, trace elements, and organosulfur compounds. In addition to its reputation as a healthy food, garlic has been shown to have anti-viral, anti-bacterial, anti-fungal, anti-atherosclerotic and anti-cancer properties. Various studies in experimental animals have demonstrated that garlic can modulate immune responses such as delayed-type hypersensitivity, T-cell proliferation, natural killer cell activity, and activation of macrophages. A processed form of garlic that is odorless and richer in antioxidants than fresh garlic is aged garlic extract (AGE), a dietary supplement. Many studies have shown that AGE is safe and effective in providing health benefits. Though studies on the effects of AGE in relation to immunomodulation have been studied moderately, there has been a lack of investigation on the individual components of AGE. The present study is focused on the modulatory effects of proteins and polysaccharides purified from AGE on the cells of the immune system. The research work carried out is presented in five chapters. The introductory chapter (Chapter 1) begins with a general account of garlic, AGE and fructans from Allium species. Garlic‟s uses since ancient times, recent studies, and biological activities with an emphasis on immunomodulation have been reviewed. Additionally, the types of immunomodulatory agents with a focus on small molecules, protein and polysaccharide immunomodulators, their sources, and the need for their development are presented. The chapter concludes with the genesis of the present study followed by the objectives and scope of the thesis. The changes occurring in the protein fraction of garlic during ageing (natural extraction) at ambient temperature is described in Chapter 2. Proteins appeared as early as 1 week during ageing, and the protein concentration remained constant after 4 weeks. Approximately 250 mg protein was obtained per kg of fresh garlic. SDS-PAGE (reducing) analysis of AGE showed a doublet around 13 kDa. Several other proteins seen in raw garlic extract (RGE) were absent in AGE. SDS-PAGE analysis of re-extracted aged garlic showed a doublet around 13 kDa, but at one-fourth the protein concentration seen during the original extraction. The purification and identification of the immunomodulatory proteins from AGE are described next (Chapter 3). Five proteins (QA-1 to QA-5) were isolated from Q-Sepharose chromatography of AGE; QA-4 and QA-5 are present in traces. QA-1, QA-2, and QA-3 displayed immunomodulatory activity (lymphocyte proliferation or mitogenicity). The identity of the two major immunomodulatory proteins, QA-2 and QA-1, has been confirmed as garlic lectins ASA I and II (ASA25; Allium sativum agglutinin I and II), respectively, based on specific hemagglutination activity and mannose-specificity. QA-3, though devoid of hemagglutination activity, exhibits mitogenic activity possibly due to the presence of an organosulfur compound. Chapter 4 covers the isolation, structural elucidation and immunomodulatory activity of fructans from AGE. The amount of fructans in AGE represents a small fraction (0.22%) of the total fructans in raw garlic. Both high molecular weight (>3.5 kDa; HF) and low molecular weight (<3 kDa; LF) fructans were isolated from AGE. 1H NMR and 13C NMR spectroscopy revealed that both HF and LF have (2→1) β-D-fructofuranosyl bonds linked to a terminal glucose at the non-reducing end and β-D-fructofuranosyl branching on its backbone. LF has been identified as a fructooligosaccharide (DP <10). Both HF and LF displayed mitogenic activity and activation of macrophages including phagocytosis. HF showed 2.5–3.5-fold increase in mitogenic activity compared to control (untreated murine splenocytes); similarly, LF showed 2-3-fold increase in mitogenic activity. Four-fold increase in nitric oxide (NO) release was seen with HF (at 1 and 5 μg/mL) compared to control (rat peritoneal macrophages). LF showed similar results at 1 μg/mL, while at 5 μg/mL the response was much higher. Percent phagocytosis was significantly higher (p < 0.001) for LF compared to HF (p < 0.01). The immunoadjuvant effect of aged garlic fructans (AGF, synonymous with HF) in vivo is presented in Chapter 5. The adjuvant activity of AGF has been investigated in BALB/c mice using ovalbumin (OVA, 30 μg) as a model antigen. AGF are non-toxic and not growth-retardant to mice. AGF, upon oral or mucosal (intranasal) administration, produced a strong humoral (serum IgG) response to OVA compared to OVA alone. A delayed response (on 50th day) was observed at a dose of 30 μg AGF by intranasal route; higher doses of AGF (>50 μg) were found to be inhibitory. The serum IgG response was seen earlier on 35th day at a dose of 100 μg AGF by oral route. The thesis ends with a comprehensive „Summary and Conclusions’ emphasizing the salient results obtained from the present study on the immunomodulatory activities of garlic.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	Garlic, Allium sativum, immune responses
Subjects:	600 Technology > 01 Medical sciences > 10 Immunology 600 Technology > 08 Food technology > 30 Spices/Condiments
Divisions:	Dept. of Biochemistry
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	14 May 2012 09:52
Last Modified:	14 May 2012 09:52
URI:	http://ir.cftri.res.in/id/eprint/10740

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