Garlic & Health

The major findings of the Garlic & Health project

Introduction

Garlic (Allium sativum L.) is a crop which is used in many kitchens worldwide as a popular condiment. Also it is used as a flavouring agent in many processed foods. Furthermore garlic is thought to have health beneficial effects especially in the area of cardio-vascular diseases and possibly also in the area of cancer. The European garlic production is threatened as the prices of European garlic are significantly higher than garlic grown in China and Argentina. To protect the European garlic growers the EU issued in 1993 a regulation that only 1000 tons of garlic can be imported monthy. However this regulation will not last forever and therefore ways need to be found to revitilize the European garlic agriculture. In this context the Garlic and Health project was developed. In this project it was tried on the one hand to increase knowledge in the area of garlic biodiversity, garlic breeding systems, cultivation and the health beneficial organo-sulphur metabolism and on the other hand to increase the knowledge about the possible health beneficial effects of garlic especially in relation to cardio-vascular diseases and cancer. In this context in vitro studies, mouse/rat studies and also a human intervention trial was carried out. Furthermore a pharmaceutical study was carried out to improve the stability of garlic tablets. The outcome of the Garlic and Health project was manifold and will certainly contribute to make the European garlic agro-industry more competitive on a worldwide scale (QLK1-CT-1999-498).

Plant theme

The absence in Europe of a large collection of garlic with a high biodiversity is hindering the improvement of garlic. Therefore two collection missions took place in the area of origin of garlic, namely Central Asia (Kazahkstan, Uzbekstan; Figure 1).

Figure 1. Collection mission Central Asia 2000 and 2001. Red and blue dots indicate garlic accessions collected from market places and natural vegetations respectively.

A large collection of 300 garlic clones was sampled and rationalization of this collection took place using AFLP fingerprinting. In this way the collection was reduced to approximately 60 clones and a highly variable garlic core collection was established. This collection was further analysed for sexual fertility and health beneficial organo-sulphur traits. It was found that a large variability existed for both traits and also that environmental effects on both traits were considerable as was observed when the same clones were grown both in Israel and the Netherlands. From a conservation point of view it is of the utmost importance to halt the rapid and irreversible decline of the Central Asian garlic genepool as this precious gene-pool is under severe threat due to the rapid replacement of the traditional landraces by modern cultivars.

Until present the genetic improvement of garlic took place by means of selection of existing variation, as garlic is considered as a vegetative propagating crop. However it is possible to reproduce garlic sexually as fertile clones are present in the gene pool (Figure 2), although there is still a lot unknown about the optimal production of high quality seed.

Figure 2: Flowering garlic plant and garlic seed.

In this project we studied the sequence of morphological processes occurring during floral initiation and development and developed a model for the optimal production of high quality garlic seed. Another way to improve garlic is by means of genetic transformation, but at the start of the project this technique was not developed for garlic. In this project we showed that transformation of garlic via Agrobacterium tumefaciens is a reliable method for the transfer of genes into this crop (Figure 3).

Figure 3: Allium chromosomes showing a succesful Agrobacterium tumefaciens integration of foreign genetic material visualized via FISH imaging.
 

As a last step a good mass propagation system needs to be developed in order to multiply the elite garlic genotypes originating from sexual hybridization and genetic transformation. In this project we developed a mass propagation system based on the production of embryogenic cell suspensions from embryogenic callus induced from young leaf explants.

Next to genetic methods also the way in which a crop is cultivated can influence the phenotypic expression of traits. In this context not much was known at the onset of the project what the influence would be of environmental factors on the organo-sulphur composition of garlic. By means of field trials, greenhouse experiments and in vitro modelling, the influence of temperature, carbohydrates, light spectrum and intensity were analysed. Field trials showed that year and location had strong effects on organo-sulphur content (Figure 4).

Figure 4. Effect of climatic conditions on the alliin content of garlic cultivars cultivated in France and Spain. MES: cv. Messidrome, Morado de Quenca: cv. Morasol and PRI: cv. Printanor

Furthermore genetic differences were observed as cultivars differed in their response to climatic differences. Greenhouse and in vitro experiments showed that light spectrum and carbohydrates had a significant effect on the organo-sulphur content of garlic, however light intensity and temperature did not show this effect. Additional sulphur fertilization in the soil did indeed improve the organo-sulphur content of garlic and we showed that spring-sown cultivars were more responsive than autumn-sown ones.

As the garlic organo-sulphur metabolism was a central focus point of this project and uncertainty existed on the route the important organo-sulphur compound alliin was synthesized (serine allyl or glutathione allyl route), a study was carried out to shed more light on this problem. Crucial for the serine allyl route is the presence of an allyl cysteine synthase which can convert serine and an allyl group into S-allylcysteine. Using garlic callus cultures we indeed showed that such an enzyme is present in garlic. The enzyme was subsequently separated from the plant level by column chromatography, the peptide identified and the gene identified (Figure 5).

Expression of the gene was achieved by E. coli lysate under in vitro conditions. However the final proof whether the gene plays a crucial role in the organo-sulphur metabolism of garlic has yet to be presented. A second target in the garlic organo-sulphur biochemistry research was a plant based study on the pattern of synthesis and accumulation of alliin during development. In this context it was observed that total sulphur accumulation in the bulb took place in the end of the growing season.

Figure 5. Purification of an allyl cysteine from garlic leaves.

Health theme

Garlic has been reported to have beneficial effects on risk factors associated with cardiovascular disease, including normalization of plasma lipids, lowering of systolic blood pressure, and reduction of atherosclerosis development. In addition, claims for anti-cancer effects were substantiated by experimental results. Various studies, on the other hand, do not support these beneficial effects of garlic. This contradiction may result from differences in the design of clinical studies (e.g. low patient numbers), use of different animal models and in vitro assays, and use of different and largely uncharacterized garlic formulations and preparations. To circumvent these problems, it was one of the major goals in this study to use sophisticated and evaluated test systems and to apply only chemically well-characterized and production-controlled garlic-derived extracts and constituents to which end a sophisticated HPLC-analytic was established.

Studies in endothelial cells revealed that garlic constituents and metabolites failed toinhibit TNF-α-mediated activation of nuclear factor-κB (NF-κB) and, thus, do not directly reduce TNF-α-induced expression of adhesion molecules ICAM-1 and E-selectin. They may do so, however, indirectly as these compounds inhibited the LPS (lipidpolysaccharide)-induced liberation of pro-inflammatory cytokines IL-1β and TNF-α in a human ex vivo whole blood model, whereas the liberation of the anti-inflammatory cytokine IL-10 remained unchanged. Accordingly, blood supernatants treated with sulphur-fertilised garlic showed again lower NF-κB activity as their unfertilized counterparts. This may be a possible mechanism, contributing to the anti-inflammatory effects of garlic (Figure 6).

Figure 6.  The effects of garlic samples differing in organo-sulphur content on the activity of the nuclear transcription factor NF-kB.
 

Another indirect mechanism was observed for the inhibition of hepatocellular cholesterol biosynthesis by DADS which is mediated by the AMP-dependent kinase. As this enzyme is activated by stress, e.g. hypoxia, such conditions are a precondition for an effective action of garlic constituents. Since this signalling pathway is involved also in other diseases such as diabetes, respective actions of garlic likewise dependent on an activation of AMPK (Table 1).

Table 1. Effect of hypoxia, KCl and DADS on AMP levels, AMP-dependent kinase and cholesterol biosynthesis.

Further actions observed in vitro were the downregulation of the secretion of certain matrix metalloproteinases in endothelial cells. The in vitro experiments also provided convincing evidence that garlic grown under different conditions may vary considerably in its efficacy and that sulphur-fertilisation of the soil may improve its potency.

The possible dependence of garlic action on specific preconditions may explain at least in part why no corresponding results were found in transgenic mice. The data indicate that under conditions relevant to the human situation, the well-characterized and production-controlled garlic-derived sulphur-rich compounds and garlic powders do not display hypolipidemic properties (Figure 7).  

Figure 7. Effects of garlic preparations on lesion area and monocytes adhesion of APOE*3 Leiden mice.

In addition, the garlic powder from cultivar Printanor has no anti-inflammatory or anti-atherosclerotic properties in APOE*3-Leiden mice. These findings certainly put the in vitro results into perspective and suggest that garlic’s benefit in relation to cardio-vacular diseases may be much lower than claimed in the past.

With respect to the issue of possible cancer prevention the experimental data accumulated in the studies of this project have demonstrated a variety of biological effects for garlic or derived sulphur compounds which may be beneficial. Firstly sulphur compounds from garlic can prevent genotoxicity of carcinogens in a human cell line. They may act by scavenging ultimate metabolites and/or by modulating the activity of carcinogen metabolising enzymes (Table 2).

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Table 2. Effects of garlic on carcinogen metabolizing enzymes,CYP: cytochrome P450; GST: glutathione transferase, UGT: UDP-glucuronosyltransferase.

Secondly feeding rats with diet containing garlic inhibits the hepatocarcinogenesis initiated by chemical carcinogens. This anti-carcinogenic effect is partly due to a reduction of the genotoxicity of carcinogens and to the modulation of enzymes involved in activation or detoxification of the genotoxic compounds (Figure 8). The efficacy is correlated with the sulphur content (alliin concentration) of garlic which is itself correlated with the level of fertilisation of garlic.

Figure 8. Effects of garlic on the appearance of preneoplastic foci induced by AFB1 in the rat liver.

Thirdly the metabolism of sulphur compounds from garlic such as diallyl disulfide, have been partly elucidated. New pathways and new metabolites were identified in rat and man. It is suggested that some metabolites could be the active compounds on specific molecular or cellular processes.Taking together these findings confirm that garlic possesses strong preventive properties towards cancer notably in the first steps of carcinogenesis. As with other effects (see above) the sulphur content of garlic seems to play an essential role in the biologic effect of garlic. Increasing the sulphur levels in garlic is therefore a challenge for improving the health value of this plant.

The improvement of quality and stability of garlic powder tablets to ensure the potency and efficacy of these products for the consumers or patients, respectively, was a major aim of the pharmaceutical research of this project. Three major steps of development were undertaken: a) implementation of an analytical method to control the technical development process to film coated tablets, b) a technical process for compressing and coating tablets, and c) a packaging process to protect the final product, sufficiently. Two galenic procedures were developed that efficiently coped with this problem under strict ICH guideline conditions necessary for pharmaceuticals.  In particular a novel aqueous coated tablet form provided an economic and flexible approach combining a long shelf life with a very reasonable and environmentally friendly production process (Figure 9).

Figure 9. The  development of organic coated tablets according to EU guidelines.

The second goal was the production of the study medication (garlic dragees and identical placebo dragees) under GMP regulations. An appropriate medication produced specifically to mask the taste and smell was delivered successfully for performing the clinical human intervention study.

The human intervention study certainly was the high-light of this project. Most of the in vitro or animal results obtained were considered to efficiently design a double blind, double dummy, parallel study including ninety-two subjects with increased risk factors for cardiovascular disease. The primary endpoint was a change in C-reactive protein between the treatment groups versus placebo, while secondary endpoints were biochemical markers of endothelial function, general inflammation (fibrinogen), lipid metabolism, blood pressure, direct markers of DNA damage and others. The study was uneventful and study treatments were well tolerated. There was no effect of garlic powder on any of the efficacy parameters, whereas the positive control, atorvastatin, reduced CRP, cholesterol and triglycerides, and TNFa-concentrations and was associated with a tendency to lower plasma levels of s-selectine (Figure 10).