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.     

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.     

SSR markers development and their application in genetic diversity evaluation of garlic (Allium sativum) germplasm

Garlic (Allium sativum), an asexually propagated vegetable and medicinal crop, has abundant genetic variation. Genetic diversity evaluation based on molecular markers has apparent advantages since their genomic abundance, environment insensitivity, and non-tissue specific features. However, the limited number of available DNA markers, especially SSR markers, are insufficient to conduct related genetic diversity assessment studies in garlic. In this study, 4372 EST-SSR markers were newly developed, and 12 polymorphic markers together with other 17 garlic SSR markers were used to assess the genetic diversity and population structure of 127 garlic accessions. The averaged polymorphism information content (PIC) of these 29 SSR markers was 0.36, ranging from 0.22 to 0.49. Seventy-nine polymorphic loci were detected among these accessions, with an average of 3.48 polymorphic loci per SSR. Both the clustering analyses based on either the genotype data of SSR markers or the phenotypic data of morphological traits obtained genetic distance divided the 127 garlic accessions into three clusters. Moreover, the Mantel test showed that genetic distance had no significant correlations with geographic distance, and weak correlations were found between genetic distance and the phenotypic traits. AMOVA analysis showed that the main genetic variation of this garlic germplasm collection existed in the within-population or cluster. Results of this study will be of great value for the genetic/breeding studies in garlic and enhance the utilization of these garlic germplasms.     

Sensitivity of food pathogens to garlic (Allium sativum)

The inhibitory activity of garlic ( Allium sativum ) against Staphylococcus aureus, Salmonella typhi, Escherichia coli and Listeria monocytogenes was measured by the 'turbidity' method. Minimum inhibitory concentration (MIC) of garlic at 80% inhibition level was calculated for these bacteria. All bacterial pathogenic strains tested were inhibited by garlic; E. coli was most sensitive and Listeria monocytogenes was least sensitive. Therefore, garlic has potential for the preservation of processed foods.     

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.     

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.     

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.     

Susceptibility to purple blotch (Alternaria porri) in garlic (Allium sativum)

Experiments were conducted to study the progress of purple blotch disease of garlic caused by Alternuria porri in the field, to determine the relationship between garlic leaf age and susceptibility to Alternaria porri, and also to assess loss in bulb characters due to purple blotch of garlic. Per cent disease severity and number of purple blotch lesions on four garlic genotypes of known susceptibility, Sel-10 (highly susceptible), G-41 (highly susceptible), IC-49382 (moderately susceptible) and IC-49373 (moderate to less susceptible) were monitored from bulb formation to bulb maturity at weekly intervals. Lesions appeared early on highly susceptible cultivars, Sel-10 and G-41. Rapid progress of disease development was noticed during the last 3 wk before bulb maturity. Peak severity at the maturity of the crop was significantly higher on highly susceptible genotypes. No definite correlation could be established between number of lesions and disease severity. A logistic curve was fitted to predict the disease progress on different weeks before bulb maturity. Levels of leaf tissue found damaged by A. porri at weekly intervals from bulb initiation to bulb maturity were significantly lower on younger leaves than on older leaves. Leaves that emerged 7 wk before bulb maturity required more than a 5 wk period to reach 50% leaf damage, whereas leaves emerging 2, 3 and 4 wk before bulb maturity exceeded 50% leaf damage within a 2–3 wk period. Individual garlic leaves became more susceptible to purple blotch as they aged and emerging leaves were more susceptible the closer they emerged to bulb maturity. Per cent loss in bulb weight and bulb volume was found to be significantly higher on highly susceptible genotypes. No significant reduction in number of cloves/bulb was observed. We propose 4 wk before bulb maturity as the action threshold for initiation of fungicidal application to prevent damaging levels of disease.     

Analysis of the genetic diversity of garlic (Allium sativum L.) germplasm by SRAP

Cluster analysis and principal component analysis were used to investigate the genetic diversity of 40 garlic germplasms analyzed with 23 sequence-related amplified polymorphism (SRAP) primer combinations. A total of 130 polymorphic loci were detected among these germplasms, with an average of 5.65 polymorphic loci per SRAP primer combination. The percentage of polymorphic loci was 69.1%, whereas the mean effective number of alleles, the mean Nei's gene diversity, and the mean Shannon's information index were 1.4446, 0.2788, and 0.4365, respectively. Cluster analysis revealed that the 40 germplasms could be divided into 3 groups. The results of principal component analysis were consistent with those of unweighted pair-group method with arithmetic averages (UPGMA) clustering analysis. The Shannon-Weaver information index ranged from 0.2419 to 0.4202, indicating that the garlic germplasms had high genetic diversity.     

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)     

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.     

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.     

A quantitative assessment of the antimicrobial activity of garlic (Allium sativum)

An aqueous extract of freeze-dried garlic (Allium sativum), when incorporated into growth media, inhibited many representative bacteria, yeasts, fungi and a virus. All microorganisms tested were susceptible to garlic. Quantitative assessment of the minimum inhibitory concentrations for bacteria and yeasts showed values ranging from 0.8 to 40.0 mg garlic ml^-1. Fungal radial colony growth was inhibited by at least 25% at concentrations as low as 2.0 mg garlic ml^-1. The 50% endpoint neutralization titre for rotavirus was 2.4 to 2.8 g ml^-1. Lactic acid bacteria were the least sensitive microorganisms to the inhibitory effects of garlic. In mixed culture studies of Lactobacillus acidophilus and Escherichia coli, garlic prevented the establishment of E. coli, although the final outcome of competition was not affected.L.P. Rees, S.F. Minney and N.T. Plummer are with Interprise Ltd, Baglan Bay Industrial Park, Port Talbot SA 12 7DJ, UK. J.H. Slater and D.A. Skyrme are with the School of Pure and Applied Biology, University of Wales, Cardiff, P.O. Box 915, Cardiff CF1 3TL, UK.     

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.     

Thiol‐disulfide organization in alliin lyase (alliinase) from garlic (Allium sativum)

Alliinase, an enzyme found in garlic, catalyzes the synthesis of the well-known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold-type I family of pyridoxal-5′-phosphate-dependent enzymes. There are 10 cysteine residues per alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a S—S bridge located near the C-terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate-cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored —SH reagent N-(4-dimethylamino-3,5-dinitrophenyl) maleimide yielded chromophore-bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two —SH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that alliinase activity does not require free —SH groups. This allowed the oriented conjugation of alliinase, via the —SH groups, with low- or high-molecular-weight molecules as we showed here. Modification of the alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin.     

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