Effect of garlic powder on the growth of commensal bacteria from the gastrointestinal tract.
Growth of commensal bacteria
Garlic (Allium sativum) is considered one of the best disease-preventive foods. We evaluated in vitro the effect of a commercial garlic powder (GP), at concentrations of 0.1% and 1% (w/v), upon the viability of representative gut bacteria. In pure culture studies, Lactobacillus casei DSMZ 20011 was essentially found to be resistant to GP whereas a rapid killing effect of between 1 and 3 log CFU/ml reduction in cell numbers was observed with Bacteroides ovatus, Bifidobacterium longum DSMZ 20090 and Clostridium nexile A2-232. After 6 h incubation, bacterial numbers increased steadily and once the strains became resistant they retained their resistant phenotype upon subculturing. A colonic model was also used to evaluate the effect of GP on a mixed bacterial population representing the microbiota of the distal colon. Lactic acid bacteria were found to be more resistant to GP compared to the clostridial members of the gut microbiota. While for most bacteria the antimicrobial effect was transient, the lactobacilli showed a degree of resistance to garlic, indicating that its consumption may favour the growth of these beneficial bacterial species in the gut. Garlic intake has the potential to temporarily modulate the gut microbiota.
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The gut microbial ecosystem serves numerous important functions for the human host, including protection against pathogens, nutrient processing, stimulation and modulation of intestinal immune response and regulation of host fat storage (Backhed et al. 2005; Palmer et al. 2007). A recent study also revealed that the bacterial populations of the gut, which change rapidly after birth, are able to modulate brain developmental pathways (Heijtz et al. 2011). Although the microbial and viral communities in human fecal samples are relatively stable and remarkably resistant (Costello et al. 2009), the composition of the gut microbiota can be modified by changes in diet (Macfarlane and Macfarlane 2003). It has been shown that a Western diet low in carbohydrates and high in fat was associated with greater levels of Firmicures (Turnbaugh et al. 2008). Furthermore, specific types of gut bacteria increased in numbers in response to short-term dietary changes, including changes in resistant starch content (Martinez et at. 2010). An increasing awareness of the potential of gut microorganisms to influence human health has led to a widespread investigation of the relationship between the gut microbiota and micronutrients and their impact on the digestive system (Gibson and Roberfroid 1995).
Garlic (Allium sativum) is one of the oldest medicinal plants most often used by cultures of different countries for treatment and prevention of some diseases (Ni et al. 2002; Rivlin 2001). Several studies have confirmed the antimicrobial, immunostimulating and antioxidant properties of garlic (Fujisawa et al., 1995; Corzo-Martinez et al. 2007; Tsao and Yin 2001).
The mechanisms of action of garlic have been ascribed to its potent antioxidant property (Wei and Lau 1998), its ability to stimulate immunological responses (Reeve et al. 1993) and its modulation of prostanoid synthesis (Dimitrov and Bennink 1997), mainly due to the presence of a number organosulphur-containing compounds, phenolics, and selenium compounds.
In a normal diet, garlic is commonly used as fresh garlic on commercial preparations (garlic oils and garlic powder), and it is also a component in many prepackaged foods, soups and bread.
However, no previous work has been conducted on the action of the garlic on human gut bacteria. The present study focused on the in vitro evaluation of the effects of a commercial garlic powder upon the viability of representative members of human gut microbiota.
Materials and methods
The garlic powder (GP) used in this study was a commercial product purchased from a local supermarket (Tesco, UK). GP was left in its original glass container, closed in a black hermetic envelope and stored in the dark at room temperature. Quantitative analysis of allicin and allicin was performed using an HP1 100 HPLC system. The powder was separated on a 250 mm x 4.6 mm 5 p.m ODS column, using methanol (50%) in water at a flow rate of 1.0 ml/min. For sample preparation, GP (100 mg) dissolved in HPLC grade water was mixed on a rotomixer and clarified using a micro-centrifuge. All solvents were purchased from Rathburn Chemicals (UK).
Bacterial strains and culture conditions
Bacterial cultures used in the studies were as follows: Lactobacillus casei subsp casei DSMZ 20011, Clostridium nexile A2-232 (from Professor Flint, Rowett Research Institute, UK), Bifidobac-terium longum DSMZ 20090 and Bacteroides ovatus (IFR culture collection).
B. longum, B. ovatus and C nexile were grown statically in BHI broth plus complements (vitamin K; 0.5 mg/ml hemin; 0.02% (w/v) resarzurin and L-cysteine) pre-reduced overnight in the anaerobic cabinet (Don Whitley Scientific, Shipley, UK). L. casei was grown statically in MRS media broth supplemented with 0.5% (w/v) glucose. Cultures were incubated anaerobically (10% [H.sub.2], 10% [CO.sub.2], and 80% [N.sub.2]) at 37[degrees]C.
Effect of garlic powder on pure bacterial cultures
For each bacterial culture the corresponding media were prepared and pre-reduced overnight under anaerobic conditions (10% [H.sub.2], 10% [CO.sub.2], and 80% [N.sub.2]). GP (0.1% and 1%, w/v) was added to the fermentation tubes and inoculated with each bacterial strain (1%, v/v) corresponding to a final bacterial density of approximately 5 x [10.sup.6] CFU/ml. A negative control without garlic addition was also prepared. Cultures were incubated anaerobically at 37[degrees]C.
Samples were withdrawn every 2 h, between 0 and 2411, then serial dilutions were performed using sterile pre-reduced PBS. Enumerations of viable cells were then performed using pre-reduced fresh agar plates. Plates were incubated in the anaerobic cabinet at 37[degrees]C for 48-72 h.
The post fermentation samples from the control vessels without addition of GP and the test vessel with GP were then used as inocula (1%, v/v) to set up two new fermentation tubes containing respectively (1) pre-reduced fresh BHI media + complement; (2) pre-reduced fresh BHI media + complement with GP 1%(w/v). Samples were withdrawn at different intervals during fermentation for the enumeration of the bacteria. An experiments were performed in duplicate.
Effect of garlic powder on mixed faecal bacteria
Faecal samples were obtained from a single human individual. The volunteer was in good health, had not been prescribed antibiotics for at least 6 months before the study and had no history of gastrointestinal disorders. Samples were collected, on site, on the day of the experiment and used immediately. A 1:10 (w/v) dilution of the sample was prepared anaerobically in 0.1 M PBS, pH 7.0, and homogenized in a Stomacher (Seward, UK) for 2 min. The resulting feacal slurry was used to inoculate the batch-culture vessels. Two water-jacketed fermenter vessels (300 ml) were filled with 135 nil presterilized basal growth medium containing: peptone water (2 g/1), yeast extract (2 WI), NaCl (0.1 g/l), [K.sub.2][HPO.sub.4] (0.04g/l), [KH.sub.2][PO.sub.4] (0.040), [MgSO.sub.4] * 7[H.sub.2]O (0.01 g/l), [CaCl.sub.2]-6[H.sub.2]O (0.01 gil), [NaHCO.sub.3] (2g/1), Tween 80 (2 ml), heroin (0.05 g/l), vitamin K1 (10[micro]11), cysteine HCl (0.5 g/1), bile salts (0.5 g/l), d(+) glucose (10g/1) and distilled water, pH 7.0. The medium was sparged overnight with [O.sub.2]-free [N.sub.2] and the temperature maintained at 37[degrees]C using a circulating water bath. The pH of the medium was kept at 6.8 using a pH controller (Electrolab, UK).
Before inoculating the batch fermenters with 10% (v/v) of faecal slurry, GP was added in one vessel to give a final concentration of 1% (w/v). Each vessel was magnetically stirred and kept under anaerobic conditions. Samples were removed at intervals for bacterial enumeration.
After 24h fermentation, the sample with 1% GP was used to inoculate additional fermenter vessels exactly as described above (one control vessel and one with I% GP) and from each new vessel samples were again withdrawn after 0, 2, 5, 8 and 24 h for viable counts on specific selective agar media (Oxoid, Basingstoke, Hants, UK). Both fermentations were run in duplicate on two separate occasions.
Pure culture fermentations
GP used in this study was found to contain allicin at a concentration of 5.13 mg/g, whereas only trace of alliin was detected.
The effect of GP on the growth of Bacteroides ovatus is shown in Fig. la. At 0.1% (w/v) GP, only a slight inhibitory action on growth was observed. However, 1% (w/v) concentration of GP produced good killing with a maximum inhibition at G h (>2 [log.sub.10] CFU/ml reduction). After this time point bacterial numbers increased such that after 2411 incubation, at the stationary phase growth, the bacterial numbers were only one log less than in the control fermentation. In contrast, the growth of Lactobacillus casei DSMZ 20011 was mostly unaffected by addition of GP at both concentrations (Fig. lb). After 211 incubation, both GP samples (1%, w/v and 0.1%, w/v) caused small reduction in its growth at less than 1 [log.sub.10] CFU/ml. Beyond 8 h incubation, the growth rate was almost identical to that of the control.
In contrast, C nexile A2-232 and B. longum DSMZ 20090 were much more susceptible than L. casei or B. ovatus. With B. longum addition of GP at 0.1% and 1% (w/v) resulted in significant reduction of >2 log and >3 log, respectively, at 4 h incubation (Fig. 1c). After this time point the numbers of viable cell counts constantly increased approaching the levels observed in the control vessels by 24 h incubation. The GP concentrations tested exerted a very rapid bactericidal effect against C nexile A2-232 (Fig. 1d). A reduction of >4 log was evident after 4 h incubation with 1% (w/v) GP, and nearly 3 log reduction in cell numbers was observed with 0.1% (w/v) GP under the same conditions. in all cases we observed re-growth of the bacterial strains by 24 h. We wanted to examine if this resistant to GP was permanent. All the experiments described above were repeated with 1% (w/v) GP with one additional fermentation vessel where the inoculum originated from the 24 h culture pre grown in the presence of 1% (w/v) GP. An example of the results with C nexile A2-232 is shown in Fig. 2. Where the inoculum originated from BHI grown cells, a reduction in growth of 4 log units after 2 h of exposition time and regrowth beyond 6 h was again observed in the presence of 1% (w/v) GP. However, when the inoculum had been pre-exposed to the GP the bacteria became resistant to the addition of GP in the new culture media and the growth profile was exactly as the control obtained in the absence of GP addition.
Similar results were obtained when testing the effect of GP on B. ovatus or B. longum (data not shown).
Mixed culture fermentations
The effect of GP on mixed bacterial populations of the GI tract was tested using batch fermentation with faecal bacteria. Samples were removed at intervals and a viable count on agar plates was carried out to quantify the levels of total aerobes, anaerobes as well as specific groups of bacteria including those of sulphate reducing bacteria (SRBs). The results (Table 1) indicated that in comparison to the control vessel there was an initial decrease in the numbers of total aerobes, anaerobes and enterobacteria in response to GP (1%, w/v) after 5 and 8 h incubation time points but by 24 h their numbers had recovered to equal or higher than in the control. There was a drop of around 2 [log.sub.10] in viable cell counts of clostridia, bifidobacteria and bacteroides at 8 h incubation. Comparatively the lactobacilli were relatively more resistant with a drop of 1.4 log reduction in cell numbers. In all cases the bacterial numbers recovered except for clostridia where the recovery was significantly lower. The sulphate reducing bacteria were the least affected and by 24 h their numbers were above that of the control.
Table 1 Changes in bacterial population ([Log.sub.10] CFU/ml) in batch cultures after 24 h incubation in absence of garlic powder (control) and in the presence of 1% (w/v) of garlic powder. The vessels were seeded with 10% (v/v) fresh faecal slurry. Incubation Control GP(1%. w/v) time (h) Total aerobes 0 5.495 [+ or -] 0.19 5.495 [+ or -] 0.19 2 6.665 [+ or -] 0.077 5.39 [+ or -] 0.183 5 8.575 [+ or -] 0.1 5.155 [+ or -] 0.332 8 8.605 [+ or -] 0.19 5.725 [+ or -] 0.601 24 7.245 [+ or -] 0.162 9.3 [+ or -] 0 Enterobacteria 0 5.34 [+ or -] 0.113 5.34 [+ or -] 0.113 2 6.66 [+ or -] 0.07 5.225 [+ or -] 0.007 5 8.42 [+ or -] 0 5.115 [+ or -] 0.077 8 8.375 [+ or -] 0.289 4.99 [+ or -] 0.098 24 6.805 [+ or -] 0.473 8.855 [+ or -] 0.445 Total 0 7.94 [+ or -] 0.41 7.94 [+ or -] 0.41 anaerobes 2 8.215 [+ or -] 0.205 7.39 [+ or -] 0.184 5 8.845 [+ or -] 0.063 6.69 [+ or -] 0.24 8 9.015 [+ or -] 0.063 6.505 [+ or -] 0.403 24 8.99 [+ or -] 0.098 9.625 [+ or -] 0.516 Bifidobacteria 0 6.065 [+ or -] 0.261 6.065 [+ or -] 0.262 2 6.275 [+ or -] 0.431 5.24 [+ or -] 0.863 5 6.805 [+ or -] 0.671 4.55 [+ or -] 0.863 8 7.25 [+ or -] 0.509 4.235 [+ or -] 0.771 24 6.185 [+ or -] 0.318 7.16 [+ or -] 0.141 Bacteroides 0 7.66 [+ or -] 0.339 7.66 [+ or -] 0.339 2 7.97 [+ or -] 0.042 6.91 [+ or -] 0.17 5 8.24 [+ or -] 0.169 6.11 [+ or -] 0.156 8 8.37 [+ or -] 0.212 5.84 [+ or -] 0.269 24 8.43 [+ or -] 0.367 7.415 [+ or -] 0.078 Clostridia 0 6.955 [+ or -] 0.106 6.955 [+ or -] 0.106 2 6.915 [+ or -] 0.134 6.245 [+ or -] 0.205 5 7.18 [+ or -] 0.056 5.41 [+ or -] 0.156 8 7.515 [+ or -] 0.431 4.815 [+ or -] 0.233 24 7.47 [+ or -] 0.155 5.765 [+ or -] 0-417 Lactobacilli 0 7.405 [+ or -] 0.162 7.405 [+ or -] 0.163 2 7.575 [+ or -] 0.346 6.505 [+ or -] 0.078 5 8.18 [+ or -] 0.297 6.42 [+ or -] 0.042 8 8.49 [+ or -] 0.226 5.965 [+ or -] 0.064 24 8.645 [+ or -] 0.091 7.455 [+ or -] 0.332 Sulphate 0 7.54 [+ or -] 0.198 7.54 [+ or -] 0.198 reducing bacteria 2 7.995 [+ or -] 0.007 7.03 [+ or -] 0.226 5 8.805 [+ or -] 0.205 6.695 [+ or -] 0.134 8 8.925 [+ or -] 0.120 6.485 [+ or -] 0.049 24 8.785 [+ or -] 0.063 9.215 [+ or -] 0.007
We again examined the effect of pre-exposing the gut bacteria to 1% GP before seeding the vessels. Pretreatment of the gut microbiota with GP resulted in profiles of bacterial cell numbers that were not different to the control (Table 2).
Table 2 Changes in bacterial population ([Log.sub.10] CFU/ml) in batch cultures after 24-h incubation in absence of garlic powder (control) and in the presence of 1% (w/v) of garlic powder. The vessels were seeded with 10% (v/v) batch fermentation contents pre-exposed to 1%(w/v)GP. Incubation Control GP(1%. w/v) time(h) Total aerobes 0 8.245 [+ or -] 0.389 8.245 [+ or -] 0.389 2 8.79 [+ or -] 0.071 8.465 [+ or -] 0.191 5 10.25 [+ or -] 0.467 9.07 [+ or -] 0.368 8 9.25 [+ or -] 0.113 9.085 [+ or -] 0.049 2-1 9.08 [+ or -] 0.028 9.25 [+ or -] 0.354 Enterobacteria 0 7.83 [+ or -] 0.17 7.83 [+ or -] 0.17 2 8.555 [+ or -] 0.233 7.55 [+ or -] 0.184 5 9.26 [+ or -] 0.764 8.695 [+ or -] 0.898 8 8.605 [+ or -] 0.007 8.235 [+ or -] 0.035 24 8.365 [+ or -] 0.191 8.1 [+ or -] 0.057 Total 0 8.245 [+ or -] 0.516 8.245 [+ or -] 0.516 anaerobes 2 8.74 [+ or -] 0.17 7.975 [+ or -] 0.46 5 9.6 [+ or -] 0.537 8.885 [+ or -] 0.163 8 9.255 [+ or -] 0.106 9.08 [+ or -] 0.028 24 9.595 [+ or -] 0.474 9.21 [+ or -] 0.127 Bifidobacteria 0 6.505 [+ or -] 0.474 6.505 [+ or -] 0.474 2 6.765 [+ or -] 0.544 6.53 [+ or -] 0.467 5 7.08 [+ or -] 0.311 6.77 [+ or -] 0.481 8 7.27 [+ or -] 0.41 7.275 [+ or -] 0.587 24 7.63 [+ or -] 0.028 7.39 [+ or -] 0.523 Bacteroides 0 6.16 [+ or -] 0.141 6.16 [+ or -] 0.141 2 6.28 [+ or -] 0.085 6.66 [+ or -] 0.339 5 6.685 [+ or -] 0.035 7.055 [+ or -] 0.092 8 7.055 [+ or -] 0.233 7.325 [+ or -] 0.247 24 6.87 [+ or -] 0.41 6.95 [+ or -] 0.17 Clostridia 0 4.82 [+ or -] 0.495 4.68 [+ or -] 0.552 2 5.235 [+ or -] 0.375 4.98 [+ or -] 0.58 5 5.35 [+ or -] 0.368 5.265 [+ or -] 0.474 8 5.975 [+ or -] 0.544 5.655 [+ or -] 0.488 24 6.495 [+ or -] 0.742 7.29 [+ or -] 0.453 Lactobacilli 0 6.635 [+ or -] 0.078 6.635 [+ or -] 0.078 2 7.1 [+ or -] 0.099 7.135 [+ or -] 0.304 5 7.28 [+ or -] 0.156 7.47 [+ or -] 0.071 8 7.225 [+ or -] 0.318 7.79 [+ or -] 0.099 24 6.935 [+ or -] 0.53 7.285 [+ or -] 0.573 Sulphate 0 7.93 [+ or -] 0.424 7.93 [+ or -] 0.424 reducing bacteria 2 8.855 [+ or -] 0.064 8.495 [+ or -] 0.035 5 9.225 [+ or -] 0.233 8.905 [+ or -] 0.049 8 9.275 [+ or -] 0.205 9.13 [+ or -] 0.057 24 9.195 [+ or -] 0.035 9.3 [+ or -] 0.127
In this study we examined the influence of garlic powder on the growth of human gut bacteria. Initially, we tested the effect of GP on the growth of pure cultures of gut bacteria at two different concentrations. The inhibitory effect observed was dependent on the bacterial species. Of the four commensal bacteria tested Clostridium nexile was the most sensitive strain, whereas Lactobacillus casei was effectively resistant even at 1% (w/v) GP concentration. The chemistry of the Allium species has been dominated by many sulphur-containing compounds giving a characteristic flavour and odour and most of its potent biological activity. Key studies by Cavallito and Bailey (1944) and Stoll and Seebeck (1951) identified the compound allicin (allyl 2-propene thiosulfinate) as the main active antimicrobial agent in garlic. Allicin is formed catalytically when garlic cloves are crushed and the enzyme alliinase of the bundle sheath cells mixes with its substrate allin, (Lawson 1996). However, commercial preparations of garlic may not always contain allicin, which is very unstable and disappears during processing being quickly transformed to other types of organosulphur compounds. GP used in this study contained allicin at 5mg/g and hence may contribute to the observed in vitro biological activity of GP. Allicin is also an is an intermediate on the pathway towards other biologically important sulphur compounds. It is likely that a synergistic effect between the sulphur compounds may be responsible for the biological effects of garlic (Wagner and Ulrich-Merzenich 2009).
Previous studies with pathogenic bacteria indicated that garlic or its component had bactericidal activity against Escherichia coli, Salmonella typhimurium (Ruiz et al. 2010) and against Neisseria gon-orrhoeae, Staphylococcus aureus and Enterococcus faecalis (Ruddock et al. 2005). In contrast to the growth of C nexile A2-232, B. ovatus and B. longum DSMZ 20090 examined in this study were all initially inhibited and there was a significant drop in viable cell counts but after 4-8 h exposure all strains became resistant to GP. It is therefore likely that during exposure to GP a subpopulation of bacteria became resistant and this then started to multiply reaching the final growth similar to that observed in the absence of GP. Further subculturing experiments indicated that these bacteria which acquired resistance during the first exposure maintained this resistance phenotype during subsequent subculturing in the presence of GP.
The pure culture studies were extended to observe the effect of GP in simulated colonic fermentation using a batch fermentation model inoculated with faecal bacteria. As with the pure culture studies there was an initial selective reduction in the numbers of bacterial species. Again the clostridial group was the most sensitive with almost 2 log reduction in cell numbers at 8 h incubation time point. As with pure culture studies where L. casei was found to be least affected, in the mixed culture fermentation, lactobacilli group numbers were initially reduced by 1 log only but then recovered. However, it is important to realize that the plates selective for lactobacilli may also allow growth of other limited number of unknown species and the reduction in cell numbers observed here may reflect inhibition of these species and not necessarily only the lactobacilli. Also, there may be differences in the resistance of different lactobacilli species to GP. The sulphate reducing bacteria (SRBs) were the least affected by GP and by 24 h time point there was significant growth in their numbers compared to their growth in the control vessels. The large number of sulphur components in garlic may favour the growth of SRBs through their biotransformation by the gut microbiota (Kang et al. 2010; Iciek et al. 2009). However, as observed with the pure culture studies, the bacterial numbers in the mixed fermentation recovered again so that by 24 h the numbers were similar to or higher than at time zero. The resistant phenotype was again shown to be inherited since the use of pre-exposed fermentation culture for inoculation of fresh fermentor media with 1% GP indicated absence of initial bacteriocidal effect.
Our results indicate that garlic powder has temporal effect on the gut commensal bacteria. It is likely that if a human gut microbiota is exposed to the garlic components for the first time there will be an initial adaptation period during which lactobacilli and SRBs may have a temporary advantage. It is important to note that the donor who provided the faecal inoculum did not consume garlic products previously. It would be interesting to discover the effect of GP on gut microbiota of an individual who regularly consumes garlic product. We suspect that the temporal inhibition would not be observed. We conclude that constant exposure of garlic powder is unlikely to dramatically alter the composition of the gut microbiota. We did not measure the effect of the GP used here on specific gut pathogens. However other studies indicated that garlic inhibited pathogens such as streptococci (Ankh and Mirelman 1999; Groppo et al. 2007) and our results suggests that it should be possible to inhibit the gut pathogens by garlic supplements without adversely affecting the commensal microbial community of the human GI tract.
This research was funded by the Biotechnology Biological Sciences Research Council (BBSRC, UK) and by the University of Messina (Italy). We would like to thank Dr Gareth Evans from Neem Biotech Ltd (UK) for GP analysis.
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Angela Filocamo (a), Carmen Nueno-Palop (b), Carlo Bisignano (a), Giuseppina Mandalari (a), (c), Arjan Narbad (b), *
(a.) Pharmaco-Biological Department, School of Pharmacy. University of Messina, Vill. SS. Annunziata, 98168 Messina, Italy
(b.) Integrated Biology of the GI Tract Programme, Institute of Food Research, Norwich Research Park. NR4 7UA, Norwich, United Kingdom
(c.) Model Gut Exploitation Platform, Institute of Food Research, Norwich Research Park. NR4 7UA, Norwich, United Kingdom
Abbreviations: GP, garlic powder; GI, gastrointestinal.
* Corresponding author. Tel.: +44 1603 255131; fax: +44 1603 507723.
E-mail address: firstname.lastname@example.org (A. Narbad).
0944-7113/$-see front matter [c] 2012 Elsevier GmbH. All rights reserved.
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|Author:||Filocamo, Angela; Nueno-Palop, Carmen; Bisignano, Carlo; Mandalari, Giuseppina; Narbad, Arjan|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Jun 15, 2012|
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