Antimicrobial properties of Allium sativum (garlic)

Although garlic has been used for its medicinal properties for thousands of years, investigations into its mode of action are relatively recent. Garlic has a wide spectrum of actions; not only is it antibacterial, antiviral, antifungal and antiprotozoal, but it also has beneficial effects on the cardiovascular and immune systems. Resurgence in the use of natural herbal alternatives has brought the use of medicinal plants to the forefront of pharmacological investigations, and many new drugs are being discovered. This review aims to address the historical use of garlic and its sulfur chemistry, and to provide a basis for further research into its antimicrobial properties.     

Agrobacterium tumefaciens-mediated transformation of leek (Allium porrum) and garlic (Allium sativum)

Transgenic leek (Allium porrum) and garlic (Allium sativum) plants have been recovered by the selective culturing of immature leek and garlic embryos via Agrobacterium-mediated transformation using a method similar to that described by Eady et al. (Plant Cell Rep 19:376–381, 2000) for onion transformation. This method involved the use of a binary vector containing the m-gfp-ER reporter gene and nptII selectable marker, and followed the protocol developed previously for the transformation of onions with only minor modifications pertaining to the post-transformation selection procedure which was simplified to have just a single selection regime. Transgenic cultures were selected for their ability to express the m-gfp-ER reporter gene and grown in the presence of geneticin (20 mg/l). The presence of transgenes in the genome of the plants was confirmed using TAIL-PCR and Southern analysis. This is the first report of leek and true seed garlic transformation. It now makes possible the integration of useful agronomic and quality traits into these crops.     

Inhibition of Helicobacter pylori by garlic extract (Allium sativum)

Abstract The antibacterial effect of aqueous garlic extract (AGE) was investigated against Helicobacter pylori . Sixteen clinical isolates and three reference strains of H. pylori were studied. Two different varieties of garlic were used. The concentration of AGE required to inhibit the bacterial growth was between 2–5 mg ml⁻¹. The concentration, for both AGE types, to inhibit 90% (MIC₉₀) of isolates was 5 mg ml⁻¹. The minimum bactericidal concentration (MBC) was usually equal to, or two-fold higher than, minimum inhibitory concentration (MIC). Heat treatment of extracts reduced the inhibitory or bactericidal activity against H. pylori ; the boiled garlic extract showed a loss of efficacy from two-to four-fold the values of MIC and the MBC obtained with fresh AGR. The antibacterial activity of garlic was also studied after combination with a proton pump-inhibitor (omeprazole) in a ratio of 250:1. A synergistic effect was found in 47% of strains studied; an antagonistic effect was not observed.     

Cadmium accumulation and oxidative burst in garlic (Allium sativum)

To investigate the temporal sequence of physiological reactions of garlic (Allium sativum) to cadmium (Cd) treatment, seedlings developed from cloves were grown in increasing concentrations of CdCl2, ranging from 1-10 mM, for up to 8 days in sand. Analysis of Cd uptake indicated that most Cd accumulated in roots, but some was also translocated and accumulated in leaves at longer exposure time (after 12h) and higher concentrations (5 and 10mM) of CdCl2. Changes in activities of antioxidative enzymes, including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), were characterized in leaves of garlic seedlings. Cd (5 and 10 mM) initially inhibited the activities of SOD and CAT but thereafter recovered or even increased compared with control plants. POD activities at 5 and 10 mM of Cd increased more than 3-4 times over control plants within 12 h and then dropped, but were still higher than controls at the end of the experiment. Otherwise lipid peroxidation enhanced with the increasing of incubation time and concentrations of external Cd. Leaves exposed to 1 mM CdCl2 showed a less pronounced response and only a small reduction in shoot growth. These results suggested that in leaves of garlic seedlings challenged by CdCl2 at higher concentrations, induction of these various enzymes is part of a general defense strategy to cope with overproduction of reactive oxygen. The possible mechanism of antioxidative enzymes changing before Cd accumulation in leaves of garlic seedlings is discussed.     

Organosulfur compounds from garlic (Allium sativum) oxidizing canine erythrocytes

The sulfurous acid ester, trans-sulfurous acid allyl ester 3-allylsulfanyl-allyl ester 8, along with two known thiosulfinates was isolated from the aqueous ethanol extract of garlic (Allium sativum). The chemical structure of 8 was determined on the basis of spectroscopic data including high resolution mass and two-dimensional NMR techniques. All of these compounds induced methemoglobin formation in a canine erythrocyte suspension in vitro resulting in the oxidation of canine erythrocytes. This is the first report of sulfurous acid ester showing oxidant activity in canine erythrocytes.     

New mannose-specific lectins from garlic (Allium sativum) and ramsons (Allium ursinum) bulbs.

Two new mannose-binding lectins were isolated from garlic (Allium sativum, ASA) and ramsons (Allium ursinum, AUA) bulbs, of the family Alliaceae, by affinity chromatography on immobilized mannose. The carbohydrate-binding specificity of these two lectins was studied by quantitative precipitation and hapten-inhibition assay. ASA reacted strongly with a synthetic linear (1----3)-alpha-D-mannan and S. cerevisiae mannan, weakly with a synthetic (1----6)-alpha-D-mannan, and failed to precipitate with galactomannans from T. gropengiesseri and T. lactis-condensi, a linear mannopentaose, and murine IgM. On the other hand, AUA gave a strong reaction of precipitation with murine IgM, and good reactions with S. cerevisiae mannan and both synthetic linear mannans, suggesting that the two lectins have somewhat different binding specificities for alpha-D-mannosyl units. Of the saccharides tested as inhibitors of precipitation, those with alpha-(1----3)-linked mannosyl units were the best inhibitors of ASA, the alpha-(1----2)-, alpha-(1----4)-, and alpha-(1----6)-linked mannobioses and biosides having less than one eighth the affinity of the alpha-(1----3)-linked compounds. The N-terminal amino acid sequence of ASA exhibits 79% homology with that of AUA, and moderately high homology (53%) with that of snowdrop bulb lectin, also an alpha-D-mannosyl-binding lectin.     

Structure of the d-galactan isolated from garlic (Allium sativum) Bulbs

Hot-water extraction of defatted garlic-bulbs yielded a mixture of polysaccharides containing a d-galactan, a d-galacturonan, an l-arabinan a d-glucan, and a d-fructan. A trace of l-rhamnose was also detected in the polysaccharide by hydrolyzate. The pectic acid was partially removed by precipitation with aqueous calcium chloride; from the remaining polysaccharide mixture a pure d-galactan containing 97.3% of d-galactose was isolated by fractional precipitation repeated chromatography through a column of DEAE-cellulose. Methanolysis and hydrolysis of the permethylated d-galactan yielded 2,3,4,6-tetra-,2,3,6-tri-, and 2,3-di-O-methyl-d-galactose in the molar proportions of 1:2:1. On periodate oxidation the d-galactan reduced 1.18 molar equivalents of the oxidant per d-galactosyl residue, and liberated one molar equivalent or formic acid per 4.13 d-galactosyl residues. Smith degradation of the d-galactan was also conducted. From these results, a structure has been assigned to the repeating unit of the d-galactan.     

A new high molecular weight agglutinin from garlic (Allium sativum)

Erythrocyte agglutination by lectins from Allium sativum was inhibited only by mannose of the sugars tested. However, asialofetuin was more effective inhibitor of agglutination as compared to mannose. This led to the use of an asialofetuin-silica affinity column to isolate agglutinins of 110 and 25 kDa (ASA110 and ASA25). While ASA25 is a dimeric protein comprising of subunits of 12.5 and 13.0 kDa, ASA110 is a glycoprotein of two identical subunits of 47 kDa. ASA110 revealed to have a high content of aspartic acid, glycine, leucine and serine but low content of cysteine and methionine. It contains 14 residues of neutral sugars in addition to 43 residues of hexosamines per mole of lectin and requires metal ions for its functional conformation. Serological cross-reactions with other species showed some common epitopes of ASA110 and ASA25 present in A. porrum, A. ascalonicum, Narcissus alba, PHA and Con A but not in A. cepa. ASA110 with CHO cells indicated it to be weakly cytotoxic with LD50 of 160 µg/ml. (Mol Cell Biochem 166: 1-9, 1997)     

Studies on the anticandidal mode of action of Allium sativum (garlic)

The mode of action of aqueous garlic extract (AGE) was studied in Candida albicans. The minimum inhibitory concentration (MIC) of AGE against six clinical yeast isolates ranged between 0.8 and 1.6 mg ml-1. Scanning electron microscopy and cell leakage studies showed that garlic treatment affected the structure and integrity of the outer surface of the yeast cells. Growth of C. albicans in the presence of AGE affected the yeast lipid in a number of ways: the total lipid content was decreased; garlic-grown yeasts had a higher level of phosphatidylserines and a lower level of phosphatidylcholines; in addition to free sterols and sterol esters, C. albicans accumulated esterified steryl glycosides; the concentration of palmitic acid (16:0) and oleic acid (18:1) increased and that of linoleic acid (18:2) and linolenic acid (18:3) decreased. Oxygen consumption of AGE-treated C. albicans was also reduced. The anticandidal activity of AGE was antagonized by thiols such as L-cysteine, glutathione and 2-mercaptoethanol. Interaction studies between AGE and thiols included growth antagonism, enzymic inhibition and interference of two linear zones of inhibition. All three approaches suggest that AGE exerts its effect by the oxidation of thiol groups present in the essential proteins, causing inactivation of enzymes and subsequent microbial growth inhibition.     

Attempts to overcome the sterility of common garlic (Allium sativum L.)

Neither the incubation of the flower scapes of garlic (Allium sativum L.) in tetracycline solution nor the extirpation of the bulblets from the inflorescences led to the development of fertile binuclear pollen. Higher tetracycline concentrations than 125 mg per litre showed a clearly phytotoxic effect. The removal of the bulblets from the inflorescences improved the survival of flowers but it enabled only the initial stages of seed development. The seeds obtained were defective and not able to germinate. The hypothesis on infectious nature of the factors causing sterility in garlic is discussed.     

Alliin lyase localization in bundle sheaths of the garlic clove (Allium sativum)

The enzyme alliin lyase (E.C. 4.4.1.4) catalyzes formation of allicin, the parent of several sulfur-containing compounds responsible for flavor, odor, and pharmacological properties of garlic (Allium sativum). Alliin lyase is a major product of the storage bud (clove), accounting for 10% of its total protein. Accumulation of this protein was characterized by locating alliin lyase deposits within the clove. Paraffin sections stained for general protein using aniline blue-black reveal dense deposits within parenchymatous bundle sheaths. Deposits are most pronounced around phloem. Remaining storage parenchyma, not in contact with bundles, appears structurally uniform, with some protein accumulating in cells near the outer surface of the clove. In freehand sections of unfixed cloves, bundle sheath cells are the only ones to show green autofluorescence when excited by blue light. Such fluorescence is consistent with the presence of pyridoxal phosphate cofactor of alliin lyase. An alliin lyase activity stain, based on detecting aminocrotonate-generating enzymes, shows activity to be restricted to bundle sheath cells in fresh material. Finally, enrichment of alliin lyase in bundle sheaths is shown by immunocytochemical staining of these areas using a polyclonal antibody generated against purified enzyme. Aliin lyase concentrates in bundle sheaths, while little if any occurs in storage mesophyll not in contact with vascular bundles. Deposits in the cloves may reflect the enzyme's role in protecting underground storage buds from decay and predation. Positioning near the phloem suggests that alliin lyase, or compounds related to its metabolism, may be translocated to and from the clove during development.     

Isolation and characterization of lectins and lectin-alliinase complexes from bulbs of garlic (Allium sativum) and ramsons (Allium ursinum)

A procedure developed to separate the homodimeric and heterodimeric mannose-binding lectins from bulbs of garlic (Allium sativum L.) and ramsons (Allium ursinum L.) also enabled the isolation of stable lectin-alliinase complexes. Characterization of the individual lectins indicated that, in spite of their different molecular structure, the homomeric and heteromeric lectins resemble each other reasonably well with respect to their agglutination properties and carbohydrate-binding specificity. However, a detailed analysis of the lectin-alliinase complexes from garlic and ramsons bulbs demonstrated that only the heterodimeric lectins are capable of binding to the glycan chains of the alliinase molecules (EC 4.4.1.4). Moreover, it appears that only a subpopulation of the alliinase molecules is involved in the formation of lectin-alliinase complexes and that the complexed alliinase contains more glycan chains than the free enzyme. Finally, some arguments are given that the lectin-alliinase complexes do not occur in vivo but are formed in vitro after homogenization of the tissue. This revised version was published online in November 2006 with corrections to the Cover Date.     

Flavor formation in tissue cultures of garlic (Allium sativum L.)

Differentiated and undifferentiated cultures of garlic (Allium sativum L.) were analyzed for the study of flavor formation in cultures. Attempts were made to correlate alliin content with free and bound amino acid contents and with enzymes like phenylanine ammonialyase (E.C. 4.1.1.5) and alliin-lyase (E.C.4.4.1.4) which play important roles in formation of the flavor percursor alliin.It was observed that in differentiating cultures showing shoot formation, there is an increase in alliin content as well as in free and bound amino acid contents. Corresponding to this there was also an increase in the activity of phenylalanine ammonialyase in differentiating cultures. Alliin-lyase activity was found to be significantly different in differentiating and undifferentiated cultures. The significance of these results is discussed.     

Efficient Agrobacterium tumefaciens-mediated transformation and regeneration of garlic (Allium sativum) immature leaf tissue

Transgenic garlic (Allium sativum) plants have been recovered directly from immature leaf material by selective culture following Agrobacterium-mediated transformation. This method involved the use of a binary vector containing the mgfp-ER reporter gene and hpt selectable marker, and followed a similar protocol developed previously for the transformation of immature onion embryos. The choice of tissue and post-transformation selection procedure resulted in a large increase in recovery of transgenic plants compared with previously confirmed allium transformation protocols. The presence of transgenes in the genome of the plants was confirmed using Southern analysis. This improvement in frequency and the use of clonal commercial “Printanor” germplasm now makes possible the integration of useful agronomic and quality traits into this crop.     

Effective concentrations of garlic distillate (Allium sativum) for the control of Tetranychus urticae (Tetranychidae)

The two‐spotted spider mite, Tetranychus urticae Koch, is a worldwide pest that feeds on a large variety of plant families. Because its resistance to acaricides is spreading rapidly, the development of new biological control tactics for population management is crucial. Plant extracts, such as garlic extract (Allium sativum Linn.), may represent viable alternatives, because they are currently considered to be minimum‐risk pesticides. Although garlic is known for its acaricidal properties, the extract concentration that provides the most efficient control has not yet been precisely determined. In this study, we conducted a series of laboratory experiments to determine the susceptibility of adult females to different concentrations of garlic extract. Fresh garlic cloves were steam‐distilled and sprayed using a Potter spray tower. Mortality and fecundity were measured upon treatment with garlic extract concentrations ranging from 0.46 to 14.4 mg/l. Female mortality increased with concentration, with LD₅₀ and LD₉₀ values of 7.49 and 13.5 mg/l, respectively. Reduced fecundity was previously observed at concentrations of 0.36 and 0.74 mg/l. The chemical composition of the Allium sativum distillate was characterized by reversed‐phase high‐performance liquid chromatography coupled with photodiode array detection, GC/MS and Fast GC‐FID against an authentic standard (Standard, Bioextract).Vinyl dithiin, diallyl disulphide, diallyl trisulphide and methyl allyl trisulphide were identified based on their mass spectra. Sesquiterpenoids were identified by their retention index.     

Sister chromatid exchanges in garlic (Allium sativum L.) callus cells

A technique is described for differential staining of sister chromatids and the study of sister chromatid exchanges (SCEs) in garlic (Allium sativum L.) callus cells. BrdU incorporation into newly synthesized DNA was ensured by culturing calli on medium containing 100 M BrdU+0.01 M FudR+1 M Urd. SCEs were visualized by FPG staining technique and their frequency was analysed. Mean frequency of SCEs in callus cells was higher than that in meristem root-tip cells. Using the same staining method, cell cycle time of callus cells was analysed. It was found that it ranges from 48 to 132 hrs. The method described represents a new approach in the study of genetic instability of plant cells cultured in vitro.Abbreviations BrdU 5-bromo-2-deoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - FPG fluorescent-plus-Giemsa - FudR 5-fluoro-2-deoxyuridine - SCE sister chromatid exchange - SSC 0.15 M NaCl + 0.015 M Na-citrate - T thymidine-containing strand of the DNA duplex - B 5-bromo-2-deoxyuridine-containing strand of the DNA duplex - Urd uridine