2Nd Annual Meeting EU Project Garlic&Health

2nd Annual Meeting EU Project Garlic & Health

Minutes Health part G & H project; April 23 2002

WP 5.1

Vollmar “Mediators of Inflammation – An overview”

An overview of the physiological pathomechanisms of inflammation an the experimental methods was given.

The tasks of the WP during the last year has been focussed on adhesion molecules (ICAM, VCAM, E-selectine), on the regulation, production and translocation of NFB, and cytokines/chemokines. For experiments a human vein endothelia cell line (HUVEC) and a kidney cell line was used. To measure the NFB action an EMSA (electrophoretic mobility shift assay) and a luciferase reporter gene assay (LRGA) is used.

Keiss “Mediators of Inflammation - Results”

NFB (EMSA Assay)

DADS: no influence on NFB translocation to the cell nucleus

other S-compounds: no influence

NFB (LRGA Assay)

An LRGA assay was established and its validity was tested and proofed.

Aqueous extracts from PRI 0 and 400 showed no effect.

Various isolated S-compounds showed no effect.

At this time it was concluded that neither garlic nor some isolated S-compounds are of influence on the NFB translocation.

Expression of adhesion molecules (ICAM, VCAM, E-Selectine)

As a experimental model a fluorescence activated cell sorting (FACS) was established, tested, and validated.

Neither isolated compounds as AM, DADS, SAC, nor aqueous garlic powder extracts of European or Chinese origin showed any significant effect and the expression of adhesion molecules.

Cytokines/Chemokines

As a experimental model the release of the interleukines IL1 (pro-inflammatory) and IL10 (anti-inflammatory) tested in whole blood. Allicin, alliin, DADS, -glutamyl allylcysteine show decreasing effects on both parameters which were not statistically significant. DMSO extracts from PRI 0 and 200 from harvest 2000 again showed not significant decreasing effects whereas aqueous extracts were of no influence. Both extracts from the harvest 2001 showed an significantly increased cytokine release at concentration levels between 10 – 100 µg/ml.

OUTLOOK and DISCUSSION

Since the used models are only a few selected ones from many more possible which are involved in the inflammation cascade an experimental look on other pro-inflammatory markers are planned (e.g. transcription factors).

From the view of the workers from this WP the garlic material used should be better chemically defined.

The suitability of the use of human vein endothelia cells as a model for arteriosclerosis (artery disease) was discussed contra dictionary.

WP 5.2

Gebhardt Cholesterol and Vessel Wall – “An overview - outlook”

An overview over the first two years of the achievements and difficulties of the health part of the project was presented. As a result the future focus should be (a) on the integration of new results within all WPs of the health part, (b) more extensive in vivo experiments for evaluation of physiological effects, and (c) intensified future dissemination of the obtained data and achievements.

Meyer “Results on the inhibition of matrix metallo proteinase (MMP) a its inhibitors (TIMP) by garlic and garlic compounds”

MMP/TIMP

Aim of this part of the WP was to estimate the influence of garlic compounds on content and formation of MMP and TIMP which are important mediators in several relevant diseases eg. atherosclerosis. For experiments beside a HUVEC cell line, a HCAEC (human aortic endothelia cells) cell line was used. The spectrum of MMP and TIMP in both cell lines were presented. The activity of various garlic compounds and different garlic powder extracts on the formation a few chosen MMP and TIMP was determined either by (a) an activity assay (MMP) or by (b) an ELISA assay (TIMP). In both cell lines, DADS showed small effects, since pure AM, SAC are of no effect. Aqueous extracts of MOR 200 (year 2000) showed no effect on both parameters. An influence of different fertilization levels can not be shown.

Gebhardt “Mechanisms of the effect of garlic compounds on cholesterol biosynthesis”

Cholesterol biosynthesis

In the past and during the project it could be shown, that the amount of activity of various isolated garlic compounds on the cholesterol biosynthesis follows: allicin>DADS>AM>vinyldithiines. Most recently a hitherto unknown stimulating effect of aqueous garlic extracts on the cholesterol biosynthesis were found. Extracts can be divided by fractioning into an activating and non-activating fraction. Nevertheless, the nature of the activating compound remains still to be unknown (it seems to be unstable during purification procedure).

To proof the hypothesis that typical garlic flavonoids are responsible for the activating effects, kaempferol, myricetine were tested for their toxic and active profile on a HEP G2 cell line. Results: kaempferol (toxic for HEP G2 cells a certain concentration levels and no effect on cholesterol biosynthesis); myricitin (not toxic and moderately stimulating effect on the cholesterol biosynthesis).

OUTLOOK and DISCUSSION

Future tasks: (a) elucidation of the mode of action of kaempferol and myricetin within cholesterol biosynthetic pathway, (b) quantitative detection of flavonoids in garlic (will be done by WP 7). The role of flavonoids as mediators were discussed in contradiction. The plenum agreed to perform further pharmacological experiments on this subject, if it can be proofed that flavonoids are present in garlic powders.

WP 6Cancer

Siess “Cancer”

Overview

The following mechanism of garlic compound are tested:

(a) inhibition of bioactivation of carcinogens

(b) prevention of genotoxicity

(c) inhibition of initiation of liver carcinogenesis

(d) mediation of apoptosis

DADS biotransformation rat

Results of the metabolism of DADS in vivo (rats), perfused rat liver and rat liver microsomes were presented. Rats were fed with 200 mg/kg DADS and the content of metabolites of DADS in stomach, liver, urine and plasma were determined after extraction with methylene chloride by GC-MS. As metabolites of DADS, AS, AMS, and the once and double oxidised form of AMS (AMSO, AMSO2) were found (for the first time since ever). Beside the precursor DADS, the allylmethyl sulfonate AMSO2 was the main metabolite in all tissues and liquids with a peak after 2-3 days after application. The elimination of AMSO2 was by urine.

DADS biotransformation perfused rat liver

The same metabolites of DADS were found after application to a perfused rat liver. Additionally, the gluthathion adducts of AS in the perfusate and tissue, but not in the bile. DADS was rapidly transformed.

DADS liver microsomes

Using rat liver microsomes DADS is transformed to allicin and again to AMS, AMSO, but not into detectable amounts of AMSO2. DADS and allicin were transformed into the gluthathion adducts either enzymatically (GST) of chemically.

Effect of garlic powder on carcinogene metabolising enzymes in living rats

The effect of garlic powder (PRI 0, 50, 200, 200 – year 2000) on rat cytochromes (CYP) isoenzymes (CYP 1A, 2B, 2E1, 3A) and on rat phase II enzymes (GST, QR, GT) were tested.

Results:

CYP 1A:  sign. for 50, 100, 200

CYP 2B:  not sign. for 0, 50, 100, 200

CYP 2E1:  sign. for 0, 50, 100, 200

CYP 3:  not sign. for 0, 50, 100, 200

GST:  sign. for 100, 200

QR:  sign. for 200

UGT:  not sign. for 0, 50, 100, 200

Outlook: check overall influence on toxicity by AMES Test.

Protection against genotoxic effects of garlic compounds

The effect of various sulphurous garlic compounds (DADS, DADSO, SAC and AM) in concentrations between 5 – 100 µM were tested by using human HEP G2 cells and rat liver tissue. The cells were incubated with the potential mutagens like peroxides, methyl methanesulfonate, quinoline oxide aflatoxin A, benzpyrene and nitrosamine and pre- and co-treated with the garlic compounds.

Results impact of carcinogens in pre-treated cells:

DADS: 

DADSO: –

AM: 

SAC: 

Results impact of carcinogens in co-treated cells:

DADS: 

DADSO: 

AM: 

SAC: 

A dose effect correlation could not be shown in any case!

Conclusion: garlic compounds could be efficient in lowering the impact of carcinogens.

Anti-genotoxic properties of garlic powder

The effect of orally applicated garlic powder (PRI 0, 100, 200 – year 2001) on the DNA of liver cells of male Wistar rats which were treated with carcinogens tested by using the COMET Assay (visualising DNA damage).

Results:

Aflatoxin B1: sign. effect using PRI 0, 100, 200

Nitrosamines: not sign. in any case

WP 7Pharmaceutics

Haffner “Development of an improved tablet”

Results

Quality and stability tests of European garlic tablets from seven countries showed that there is a major lack in quality and stability over all products. Quality and stability is not reliable for consumers, even in the case of “best before” declarations. Therefore, quality and stability has to be improved in a sense that garlic products are of “real” value for consumers who wants to do something for their health.

Two pharmaceutical approaches where presented: (a) an “economic” one solving the problem of stability and deficiency of efficacy by limited costs; (b) a “high end” one providing highest concentration/efficacy of active ingredients (e.g. garlic powder with alliinase activity + alliin rich extract) combined with odour control, integrating all major results of the project.

First results of approach (a) were presented showing a significant improvement of the stability during a period of 9 months by using very dry starting material (freeze dried garlic powder and excipients with less than 1% residual water content). The disadvantage of higher costs for pre drying could partly be overcome by a more economic tablet form (less ingredients, faster production process).

Recently the experiment for approach (b) has been started, trying to gain an alliin rich extract by different extraction methods.

WP 5.2 and 8Animal and Human Intervention Studies

Espirito-Santo “Animal atherosclerosis study on APO Leiden mice”

Results

Two garlic powders (a) MOR 200 (year 2000) and (b) LI 111 (Lichtwer Pharma) as well as DADS and DADSO were tested in a validated animal model (APO E Leiden mice). The feeding regime was presented, showing that the mice were stepwise fed with a normal, a western diet and a artherogenic diet (rich in saturated fat). The following parameters were estimated either after feeding western diet or artherogenic diet: (a) activity of alanine amino transferase, (b) cholesterol in plasma, (c) serum amyloid A, (d) van Willebrand factor, (e) platelet activation, (f) blood pressure, (g) plaque areas in aorta.

Results:

Generally, there are some effects by mice under artherogenic condition fed additionally with garlic powders and allicin on the lipid level amyloid A content but no significant effects shown under western type diet.

Outlook

Another mice study under western type diet is necessary to gain more insight in “living systems” which are more correlated to normal human conditions.

Princen “Human intervention study”

Overview

All recent human and animal studies of the clinical effect of garlic were shown. Actually, there is proof for positive effects of garlic on:

(a) Antioxidative status (in vivo, in vitro)

(b) Inhibition of platelet aggregation (in vivo, in vitro

(c) Blood vessel elasticity (in vivo human)

(d) Prevention of artherosklerosis (in vivo animals, human)

Human intervention study – plans

Two types of study designs are possible:

(a) two arm double blind study: 1. garlic powder (24 patients), 2. placebo (24 patients);
(b) three arm double blind study: 1. garlic powder low dose (27 patients), 2. garlic powder high dose (27 patients), 3. placebo (27 patients)

The following surrogate test parameters for arteriosclerosis are possible:

(a) F2-Isoprostane

(b) Content plasma lipids

(c) v. Willebrand factor

(d) Adhesion molecules (E-Selectin, VCAM, ICAM)

(e) Oxidised LDL

The following surrogate test parameters for arteriosclerosis are possible:

(a) Phase-II-enzymes

(b) DNA damage

(c) antimutagenic activity of plasma (AMES test)

(d) sulphurous metabolites

OUTLOOK and DISCUSSION

Type (which powder, which kind of tablet) and dose of the study medication remains to be clarified (Input from all participants). Suggestion: study medication from pooled Spanish PRI powder (0, 50, 100, 200) to 30 kg homogenous material (P15 is checking amounts necessary to produce study medication – 5,5 kg per arm necessary 1 –2 g garlic powder/day! suggestion: pre-dried gastric resistant film coated tablets à250 mg), additionally sugar coated for odour protection; otherwise packed under “blinded” conditions).

Minutes Plant part G & H project; April 24 2002

Garlic and Health meeting, Leipzig, April 22 – 26 2002

Wednesday April 24th Garlic

WP1: Genetic Resources

P7/10: HR outlined the work carried out over the first two years of the project. Material collected from Uzbekistan and Central Asia was quarantined for a year, but visual assessment and propagation could take place. It would therefore be possible to carry out experiments in Year 3 with material collected during this project. In the meantime, samples (115) from the 2000 collection had been sent to P1 for molecular marker analysis, and also 10 samples to P5 for CSO analysis. In addition, seeds from collections made prior to this project had been planted. High proportions had germinated in December 2001, and were now being transferred to individual pots and maintained at short day-length to prevent bulbing.

Discussion points: HR explained that the ability to flower was genetically determined, but in response to environmental cues. Once the plant had flowered, it died, but since not all cloves of an individual flowered, each line could be maintained vegetatively. The seeds required stratification but did not show dormancy. They came from open-pollination, but this year 10 – 20 plants would be put into cages for self-pollination.

P1: CK briefly reviewed garlic taxonomy. He then explained the reason for using AFLP to assess variation and identify material. Initial tests of 84 accessions (mainly from IPK Gartersleben) using 20 AFLP fragments indicated that 26 accessions represented the whole variation. This left open the question of whether duplicate or closely related accessions nevertheless contained valuable differences. Analysis of the 115 accessions from P7/10 showed they contained 87 individuals from Group II, 18 from Group I/IV and 10 from Group IV. All individuals within each group, based on 20 AFLP fragments, looked very much the same. Inspecting a further 80 AFLP fragments revealed very little additional difference leading to the conclusion that the accessions represented only 3 genotypes. Forty accessions were therefore suggested as a core collection, based on the AFLP results, together with proven fertility and some morphologically distinctive accessions. AFLP was a quick method to assess and reduce the number of accessions, but there were some contradictions between it and morphological or field observations.

Discussion points: CK pointed out that the large size of the garlic genome meant that AFLP represented a very limited sample. Similarly, mutations in a single major gene could have a profound effect on morphology. There was extended discussion of the consequences of differences between AFLP and morphological/field observations for the size of the core collection. RiK stated that it had been decided to keep all accessions in Israel for the time being, but to reduce the number to the core collection at Wageningen. In addition, since three physiological groups had now been identified, all three would be included in physiological work by P7/10. CK stated that it was difficult to assign the commercial cultivars (Printanor, Morasol etc) to a group. On the possibility of natural or artificial hybrids, he observed that the c. 10 plants that had produced seeds at Wageningen were male-sterile, or had a much reduced amount of pollen. They came from more than one physiological group. P1 intended to carry out crosses between accessions with lots or little pollen, and examine the offspring. He was unsure of the parentage of the seeds produced by Ito and colleagues, but would check.RiK said that wild seeds were unusual, so that natural hybrids were unlikely.

P5: JA stated that he could provide compounds to the Health partners upon request. In addition to alliin, alliicin and deoxyalliin, alliin methylsulphoxide and alliin methylsulphone were available. Differences in analytical protocols between P5 and P15 had been resolved by adoption of TH’s method. The bulbs sent for analysis by P7/10 had arrived in poor condition, and consequently only alliicin release potential was measured which indicated differences between accessions. A series of analyses on material from P9 had shown that there was a decrease in flavour compounds as bulbs were stored.

Discussion points: In discussion about the alliinase reaction, JA observed that alliinase did not cleave gamma-glutamyl peptides, and he detected the same amount of these peptides whether alliinase was active or not. There was extended discussion about the need to analyse bulbs from the collections, which were now growing in both Israel and Wageningen. Differences in climate and growing conditions could affect the sulphur compounds in the same accession from the two locations. RiK stated that she had carried out a pyruvate analysis on the 115 accessions collected in 2000. JA indicated that a full profile, particularly of the wild material, was needed.

WP2: Breeding systems

P7/10: RiK explained that their goal was to understand the flowering process in garlic in sufficient detail to generate fertility in garlic from all geographical regions and groups. She recapitulated their first experiment, indicating that a cold treatment (-2 to +4°C) and long days were essential to obtain bolting. This had led to a focus on light treatments in the next, on-going, experiment. All plants had received a 60 d cold treatment (4°C), and were then maintained in a phytotron at 20 or 23 °C during daylight and 12 or 15 °C during night. All plants received an initial period of short (8h) days, and then 1,2,3 or 4 weeks of long (20h) days. Control treatments with light regimens such as constant long days, constant short days etc had also been included. Although the experiment is still running, it was apparent that in short days, flowers initiated in the bulb, but the scape did not elongate. With a short period of long days, (1 week) the scape length was reduced. Light treatments also affected the balance between top-sets, vegetative buds and flowers in the inflorescence.

Discussion points: RiK indicated that they would investigate the effect of a long day treatment of less than 20 h in future. There was some discussion of the timing of scape elongation during long-day treatments, and any consequence for scape length.

P1: S-JZ stated that their goal was to develop a transformation system in garlic using Agrobacterium. He reviewed the progress that had been made during the first year of the project to obtain callus from the roots, and to regenerate these to garlic plantlets. This work had now been accepted for publication. Transformation and regeneration of plantlets had now been achieved in 4 lines, by co-cultivating calli with Agrobacterium with the entire process taking 6-8 months. The hygromycin selectable marker had been used with the GUS and GFP reporter genes. Transformation with the Cry1Ca gene had also been obtained. Callus, resistant to hygromycin, had been obtained in which reporter genes were expressed, although expression was not uniform for GFP. The callus developed shoots after 3-4 months on regeneration medium, and plantlets had now been transferred to soil. Although pcr had been used to detect the transgenes, Southern blotting would be used to confirm transformation and the copy number. Adaptor ligation PCR had indicated that there was more than one copy of a transgene in some plants.