26. Garlic and cardiovascular disease Dr HACollin
26.1. Introduction
The official wording from the Food and Nutrition Board of the National Academy,
USA, defines functional foods as any food or ingredient that may provide a health
benefit beyond the traditional nutrients it contains (Thomas and Earl 1994). The
European view states that a food can be said to be functional if it contains a
component (whether or not a nutrient) that benefits one of a limited number of
functions in the body in way that is relevant to either the state of well being and health
or the reduction of the risk of a disease or it has a physiological effect
(Clydesdale1997; Bellisle et al 1998). Garlic clearly falls into the functional food
category since it is normally eaten in small amounts either raw, cooked or as a salt. It
is also sold as a supplement in the form of powdered tablets, capsules, steam distilled
oil, or plant extract all of which can be taken on a daily basis and guaranteed not to
produce the strong smell associated with consumption of the raw garlic.
While the garlic may be taken as a pharmaceutical now, it has been used as a
flavouring and food additive for many centuries as described in detail by Rivlin,
(2001). The earliest reference is from 2600-2100 BC on Sumerian clay tablets then
subsequently its use was reported by the Egyptians and the Greeks to improve stamina
generally, but it was the Chinese who prescribed it for specific illnesses and in this
case it was for an aid to digestion and respiration and more particularly for diarrhoea
and worm infestation. In India early texts described its use in the treatment of heart
disorders. In Europe it was of value in the treatment of a wide variety of ailments,
which ranged from digestive disorders to worm infestation, kidney problems,
toothache, constipation, dropsy and plaque (Rivlin, 2001) If the modern herbalists
books are consulted fresh garlic is still prescribed for the treatment of many diseases
including the prevention of heart and circulatory problems (Grieve, 1998).
Garlic has received renewed prominence recently because of the popularity of the
Mediterranean diet, which includes the extensive use of fresh and cooked garlic. The
Mediterranean diet is thought to reduce the risk of cardiovascular disease through a
reduction in cholesterol and its consequent reduction in blood pressure. There is
extensive Web page documentation in for example www.fair-flow.com and
www.deliciousitaly.com/mediterraneancom1.htm of the benefits of the diet and in
supporting clinical trials (de Lorgeril, 1996; Laino 2003) but the exact
cardioprotective mechanism of the diet is unknown. Since the Mediterranean diet
includes a wide array of vegetables fruit, fish and oil, the protective effect is likely to
be multifactorial. Garlic is an essential component of the diet and is thought to make
an important contribution to its medical benefits.
The current enthusiasm for herbalists remedies and healthy diets has led to detailed
studies of the chemical composition of garlic and the medical importance of the
chemicals it contains. The commercial direction is now away from the fresh plant and
into a variety of garlic extracts. The long term aim of the supplement industry is either
to identify an active single compound in the extract which can then be used as a new
pharmaceutical or to use the complete plant extract in concentrated form as a food
supplement. It is assumed that since garlic has been prescribed for many centuries,
1
there is no or less need for the supplements to undergo the rigorous (and very
expensive) testing that is required for the introduction and acceptance of a new
pharmaceutical. Garlic supplements and extracts are advertised extensively on the
Web where the accompanying text claims that regular consumption of the supplement
could delay the onset of cardiovascular disease. However the supplement industry is
now under increasing scrutiny from external regulators to justify their claims, and is
looking towards the scientists to provide medical evidence that garlic can in fact
prevent the early onset of specific diseases. There have been a large number of studies
recently in which the effect of garlic on specific stages of cardiovascular disease has
been explored. As an indication of the interest in the subject there has also been a
number of reviews (Ernst,1987; Alexander et al.,1996; Bannerjee and Maulik, 2002;
Rahman,2003) describing the relationship between garlic and cardiovascular disease.
26.2. Composition of raw and cooked garlic
Raw garlic is eaten in small amounts largely as a flavouring, and while popular
especially in the Mediterranean diet, it does lead to a strong odour on the breath. The
flavour and pungency is a result of a high concentration of sulphur related compounds
in the cloves which amount to 1.0% of their dry weight. In the undamaged clove over
70% of the sulphur compounds exists as (+)-S-allyl-L-cysteine sulphoxide or alliin,
(+)-S-trans-1-propenyl-L-cysteine sulphoxide or isoalliin and S–methylcysteine
sulphoxide or methiin and γ glutamyl peptides such as γ glutamyl-S-allylcysteine and
γ-glutamyl-S-trans-1-propenyl cysteine. The sulphur is divided approximately 50%
between the cysteine sulphoxides and the γ glutamyl peptides (Mütsch-Eckner et al.,
1992). In the plant the function of the cysteine sulphoxides is probably one of
protection since their breakdown products appear to have antibiotic and fungicidal
propertie (which also contributes to their medicinal value), while the γ glutamyl
peptides appear to have a storage function for N and S. The major alkylcysteine
sulphoxide in garlic is alliin (85%), with isoalliin (5%) and methin (10%) occupying
much more minor roles (Lawson, 1996).
Garlic cloves have a slight smell when intact but on cutting or maceration a strong
smell is released rapidly. The volatile is only released on damage because there are
different parts to the flavour mechanism, a degradative enzyme, alliinase, and its
substrates, the alkylcysteine sulphoxides, which are stored in separate compartments
in the plant. Unlike the onion where there is an intracellular separation of the alliinase
and the alkylcysteine sulphoxides (Lancaster and Collin, 1981), in the garlic there is a
spacial separation based on location within different tissues of the clove (Ellmore and
Feldberg, 1994; Wen et al.,1995). The amounts of alliinase in the garlic are unusually
large amounting to at least 10% of the total protein in the clove and would explain
why there is such a rapid release of flavour and complete breakdown of the flavour
precursors upon cutting the tissue.
The chemistry of the Alliums is interesting since from a small number of original
flavour precursors a large number of compounds are produced many of which have
medicinal properties (Block, 1985, Lawson 1996). In the alliinase catalysed reaction
the first products of the reaction are the sulphenic acids which have a very short half
life before being condensed to form thiosulphinates. From the three original
2
sulphoxides, eight or nine possible thiosulphinates are formed. The major one derived
from alliin is diallyl thiosulphinate, or allicin, which represents about 70% of the total
thiosulphinates. Isoalliin does not form the lachrymatoury compound found in onion
but is rapidly converted into allyl 1-propenyl thiosulphinate and the methiin finally
degrades to largely allyl methanethiosulphinate. The thiosulphinates are colourless
liquids with a pungent smell reminiscent of fresh cut garlic of which allicin is the
main contributor to the odour. The allicin yield is important since it is used as a
measure of garlic quality in commercial preparations. It appears to be relatively stable
especially in water but in homogenates it is less stable which is a major consideration
when assessing the effectiveness of different garlic preparations.
The thiosulphinates are described as unstable and reactive and convert readily to more
stable compounds which contain the thioallyl (S-allyl) or thiomethyl group. In crushed
garlic at room temperature the reactive thiosulphinates are transformed to diallyl
sulphide, diallyl disulphide and allyl methyl trisulphide, which is also the reaction that
occurs in the mouth. In vitro studies of the thiosulphinates, allicin and methyl and
propyl thiosulphinates show that they are capable of combining with the sulphydryl
group of the amino acid cysteine to form S-allyl mercaptocysteine, S-methyl
mercaptocysteine and S-propyl mercaptocysteine thereby inhibiting the activity of
enzymes which contain cysteine as the active site. This ability to react with the
sulphydryl group of acetyl–CoA SH the building block of cholesterol and triglyceride
synthesis may help to explain its biological effect (Wills, 1956). These reactions may
have some significance in the intestinal tract during digestion as they may determine
the form in which the breakdown products of garlic enter the blood stream. There is
no conclusive data that clearly identifies the main metabolites in the blood stream
after garlic consumption (Amagase et al., 2001). Evidence has shown that allicin,
which is an unstable product of alliin breakdown and could be present in the digestive
system after consumption of garlic or garlic supplements, is rapidly converted to
diallyl disulphide in the blood ( Freeman and Kodera, 1995). This compound was
found in micromolar concentrations in the rat plasma and liver tissues after oral
administration of diallyl disulphide implicating it as a major garlic derived metabolite
in the body (Germain, 2002) but it does not seem to remain in the body for long after
consumption of raw garlic (Lawson et al 1992). The water soluble compound Sallylcysteine itself a breakdown product of garlic does seem to be a stable residue in
the blood following oral consumption (Nagae et al., 1994).
Garlic is more normally cooked so the composition will differ from the raw garlic.
Heat inactivates the alliinase and therefore inhibits the formation of allicin and other
thiosulphinates but some breakdown of the alliin occurs leading to the accumulation
of small amounts of diallyl trisulphide, diallyltrisulphide, di and tetrasulphides. In
crushed garlic the cysteine sulphoxides are largely converted to the thiosulphinates
(Lawson, 1996). After boiling in a closed container there was complete conversion of
the thiosulphinates to sulphides, whereas in an open container over 90% of the
sulphides were lost. After cooking in hot fat, most of the alliicin was lost but a much
higher proportion of the sulphides remained. In an analysis of the volatiles released
after standard periods of cooking such as oil frying, baking and microwaving the
dominant volatile was diallyl disulphide (Yu, 1993)
3
26.3. Commercial sources of garlic supplements
The commercial forms of garlic that are sold as a supplements include powdered dried
garlic cloves, oils produced upon treating chopped garlic with steam, vegetable oil or
ether, and aged extracts of chopped garlic in ethanol or water as well as garlic cloves
pickled in vinegar. An important aspect of the supplements is that the composition of
the product will vary according to variety, the year of harvest and on the processing
method. The preparation and composition of the major commercial products are
described below.
26.3.1.Garlic powder
Garlic powder such as Kwai is prepared by peeling the cloves, oven drying at 50-60o
C then grinding them to a powder. Some conversion of the flavour precursors by
alliinase does occur on cutting but the loss is minimised by reducing the amount of
cutting before drying. Alliinase retains its activity since the powder is very stable and
will only lose about 10% of its allicin yield after 5 years of storage. However it is
important that the powder is stored at no more than 4-6% water content to prevent the
alliinase being activated. The powder can be stored as tablets when again the alliicin
potential is also very stable but with an average loss of 36% alliicin over 5 years is
more variable than the powder. Garlic salt products generally have a lower yield of
alliicin than quality garlic, which probably reflect the greater degree of chopping prior
to dehydration (Lawson and Hughes, 1992).
In order to standardise the various dried products, the quality is measured by the level
of allicin. The values of some brands are 4mg/g which compares favourably with the
levels in fresh garlic. An alternative measure is the ability to form allicin from alliin
(called the alliicin potential), which depends on the presence of active alliinase in the
tablet. Alliinase is inactivated by the acid conditions of the stomach. In order to
provide the body with the pharmacologically active forms in the garlic, the activity of
the alliinase must be maintained until the pills reach the non-acid environment of the
intestine. The pills are protected from the stomach acid by being coated with cellulose
esters which require the presence of intestinal enzymes for the coating to be removed.
This has given rise to another measure, the effective allicin yield, which is obtained
by using simulated gastro intestinal conditions ie 1 hour in simulated gastric
conditions and two hours in simulated intestinal conditions (Lawson and Hughes,
1992). Those brands with an effective enteric coating were those with the largest
release after one hour. Other factors determining the amount of alliicin released will
depend on whether the tablets are consumed with or without food. A meal with a high
protein content will see the pH of the stomach rise from 1.5 to 4.0 which will be less
inhibitory to any alliinase activity. It is recommended that the tablets are taken with or
just after a meal when the pH is higher and the alliinase inactivating ability is lowered
(Blania and Spangenberg, 1991).
The other factor determining the release of allicin is the ability of the alliinase to
convert all the alliin to alliicin. This is measured by the ratio of alliicin to alliin. With
a 100% conversion this should appear as 0.46 whereas the range normally measures
from 0.32-0.42 because allyl methyl and allyl 1-proenyl thiosulphinates are also
formed. Ratios below this figure indicate a damaged alliinase. The usual reason for a
4
reduced figure is due to excessive drying of the cloves at a high temperature which
inactivates the alliinase. Significant amounts of γ glutamylcysteines are also present in
the powders and tablets (Block, 1995).
26.3.2. Oil of steam distilled garlic
Steam distilled garlic contains exclusively about 98% allylmethyl and 1-propenyl
mono- and polysulphides. The commercial product is diluted by vegetable oil so that
the final composition of sulphides represents about the same amount of allicin and
other thiosulphinates present in a similar weight of crushed garlic. This dilution
stabilizes the polysulphides and decreases the extremely strong odour of the undiluted
oil. The major compounds are the diallyl di, tri and tetra sulphides and the allyl
methyl di ,tri and tetra sulphides. The total content of different brands does vary as a
result of the different levels of dilution whereas the percentage composition does not.
The composition is also relatively stable over time as a 5 year study showed virtually
no change in the amount of the main allyl sulphides (Lawson, 1996).
26.3.3. Oil of macerated garlic
Garlic is macerated in a vegetable oil then the oil filtered or the crushed garlic is left
suspended in the oil. The product contains compounds not found in the fresh or
powdered garlic ie the ring structured vinyldithiins and the oxygenated ajoenes. The
composition seems not to vary but the total amount of sulphur compounds is
dependent on the quality of the original garlic and the amount of dilution by the oil. If
the garlic bulb is homogenized with an equal weight of oil, the highest amount of
transformation products is about 3-5mg/g. The yields rarely reach this value but can
be increased by crushing the cloves first to produce the thiosulphinates more
efficiently before addition of the oil. The oil based compounds appear to be stable
since vinyldithiins were stable over a 5 year period but the ajoenes much less so even
when enclosed in a gelatine capsule. It is recommended that oil macerate products
should be kept refrigerated to retain their ajoene concentration for more than 18
months (Iberl 1990
26.3.4. Garlic aged in dilute ethanol
Chopped garlic is incubated in 15-20% ethanol for up to 20 months at ambient
temperatures as in AGE (Lawson and Wang, 1995). The incubation medium is then
filtered and evaporated to dryness and sold in dry form as tablets or powder or liquid
forms. Analysis showed that allicin and other thiosulphinates were nearly depleted by
90 days having been transformed into diallyl and allyl methyl tri-,di- and
tetrasulphides most of which was then lost to the atmosphere. There are considerable
amounts of alliin in the extract, which had diffused out whereas the alliinase had
remained in the cells. Under these condititions the γ-glutamyl-S-allylcysteine and the
γ-glutamyl-S-1-propenylcysteine were converted to S-allylcysteinne and S-1-propenyl
cysteine under the action of an enzyme γ-glutamyl peptidase. The quantity of these
products will depend on the content of the original γ-glutamyl cysteine sulphoxides.
The range in different sources varied from 1.6-6.8 mg/g fresh weight which in turn
5
produces an amount of S-allylcysteine content of 2.7-11.3 mg/g dry weight which
represents the approximate amount that should be found in commercial products. The
amount of S-allylcysteine that is found in commercial products is much smaller than
this figure. The alliin content of commercial aged extracts varies from 0.02-0.32 mg/g
dry or fresh weight and only trace levels of the allyl sulphides have been found in
commercial extracts. The total sulphur content of the liquid form of the commercial
aged extract such as AGE was found to be 0.091% in contrast to the 0.35% typically
found in garlic cloves.
26.4. The influence of garlic compounds on cardiovascular disease
Cardiovascular disease include artheriosclerosis , hypertension and myocardial
infarcation or heart attacks and as such form the major killers in industrial societies.
Arteriosclerosis is a disease of the arteries where the inner layer becomes thickened
by fatty deposits and fibrous tissue leading to a condition known as hardening of the
arteries. Fatty streaks, which are the earliest indication of atheriosclerosis, are areas of
yellow discolouration on the inner surface of the artery but do not protrude into the
lumen or disturb the blood flow. The streaks are characterised by the subendothelial
accumulation of large foam cells filled with intracellular lipid. The foam cells, which
are derived from macrophages and smooth muscle cells are the likely precursors of
fibrous plaques, structures that form pale grey elevated lesions which may project into
the arterial wall and reduce the blood flow through the vessel. Calcification of the
fibrous plaque leads to rigidity of the artery and hypertension while rupture of the
plaque releases material into the blood stream causing a thrombus to form. Occlusion
of the vessel locally or following transport to distant sites can lead to myocardial
infarctions or strokes (Bhattacharyya and Libby 1998).
Artherosclerosis has a genetic component which is difficult to overcome, and
an environmental and diet related component which is susceptible to modification.
Regular consumption of garlic as in the so called Mediterranean diet or the use of
garlic supplements is thought to prevent early onset of artherosclerosis and
consequently delay the possibility of hypertension, stroke and heart attack. There are a
number of stages during the development of artherosclerosis at which garlic
consumption has a delaying effect. These stage are the synthesis of cholesterol,
oxidation of cholesterol, platelet aggregation, modification of the arterial cell walls
and hypertension. Investigations of the role of garlic on these stages have been based
on an in vitro approach using the response of blood cells or arterial cell cultures to
garlic supplements and an in vivo approach involving animal and human trials where
the supplement has been consumed regularly. The humans trials are difficult to
undertake since there is always the problem of achieving a satisfactory level of
replication. Decisions have to be made about the number of volunteers, their age,
gender, level of arteriosclerosis, duration of trial and type of garlic supplement. The
next part of the review provides examples of the in vitro and in vivo approach used to
examine the effect of garlic on specific stages of the disease.
26.4.1. In vitro and animal studies of cholesterol synthesis
6
The level of cholesterol in the blood is an important factor in the development of
arteriosclerosis. When fats are ingested as part of the diet, cholesterol and
triglycerides are absorbed in the intestine and finally transferred to the venous
circulation. These large molecules are hydrolysed by the enzyme lipoprotein lipase,
which releases fatty acids into peripheral tissues while the metabolic remnants
composed largely of cholesterol remain in the circulation. The liver in an endogenous
cycle of cholesterol production and metabolism releases very low density lipoprotein
(VLDL) into the circulation. Lipoprotein lipase acts on VLDL at muscle cells and
adipose tissue to release free fatty acids into the cells as before and the residue,
intermediate density lipoprotein (IDL), which contains esterified cholesterol remains
in circulation. Further processing results in cholesterol rich low density lipoprotein
(LDL) which is largely taken up by the liver. Cholesterol released back into
circulation is transported by high density lipoprotein (HDL) which returns the
cholesterol to the liver via IDL and LDL for recycling into lipoproteins or excretion in
the bile. The HDL appears to act in a protective role while elevated levels of LDL
correlates with a high incidence of arteriosclerosis. The level of cholesterol
particularly LDL is critical (Bhattacharyya and Libby, 1998) The control of
cholesterol synthesis is determined by the enzyme hydroxylmethyl glutaryl CoA
reductase (HMG CoA reductase) in the liver and other enzymes in the blood.
Inhibition of these enzymes, possibly by the presence of garlic related compounds
provides an important route for slowing down development of arteriosclerosis. The
consumption of garlic may inhibit the HMG CoA reductase for instance by interfering
with the signal transduction pathway such as the AMP dependent kinase pathway, or
adenosine–induced signalling (Gebhardt and Beck, 1996). Much of the approach has
been based on establishing an artherosclerotic effect in the blood of whole garlic
extracts or separate components of the extracts using in vitro and whole organisms
studies.
In vitro studies have concentrated on the ability of blood cells to accumulate
cholesterol as this provides the vehicle for the movement of cholesterol about the
body. The response of rats to a high cholesterol diet with and without the garlic
supplement AGE showed that plasma concentrations of total cholesterol and
triacylglycerol of the AGE supplemented rats were 15% and 30% lower respectively
than those of the non supplemented rats (Yeh and Liu, 2001) Subsequently rat
hepatocytes were used to determine the role of garlic in cholesterol biosynthesis in
vitro by measuring the incorporation of C14 acetate into cholesterol in the presence of
different crude fractions from raw garlic (Yeh and Yeh1994). These were the water
extractable fraction (WEF), methanol-extractable fraction (MEF) and petroleum
ether-extractable fraction (PEF), Kyolic a liquid form of AGE and SAC (S-allyl
cysteine). The rates of C14 acetate incorporation into cholesterol were depressed 44,
56, and 64% by MEF, PEF and WEF respectively, suggesting that water and lipid
soluble components were effective in inhibiting cholesterol production. The results
showed that water soluble compounds such as SAC were more effective than the
sulphides but maximum activity was exerted by the complete extract, Kyolic. The
effect of the individual compounds ie SAC, SEC (S-ethyl cysteine), SPC (S-propyl
cysteine), S-methylcysteine (SMC), γ-glutamyl S-allyl cysteine (GSAC), γ-glutamyl
S-methyl cysteine (GSMC), γ-glutamyl S-propyl cysteine (GSPC), S-allyl
acetylcysteine (SAAC), S-allyl sulfonylalanine (SASA), S-allyl mercaptocysteine
(SAMC) and alliin were assessed. Lipid soluble compounds were diallyl sulphide
(DAS), diallyldisulphide (DADS), diallyl trisulphide (DATS), dipropylsulphide
7
(DPS), dipropyldisulphide (DPDS) and methyl allylsulphide (MAS). The cells were
treated with C14 acetate in the presence or absence of the test compound at 0.054.0mmol/L for measurement of cholesterol synthesis as before. Among the water
soluble compounds SAC, SEC, and SPC exhibited dose dependent inhibition on the
rate of cholesterol synthesis with maximal 40-60% inhibition achieved at 2.0-4.0
mmol/L. Glutamate derivates, GSAC,GSMC,GSPC, depressed the synthesis by 2035%. Alliin SAAC and SASA had no inhibitory effect whereas the sulphides, DADS,
DATS, and DPDS diminished the rate of acetate incorporation into cholesterol at
1.0,2.0 and 4.0 mmol/L (Yeh and Liu, 2001). On the basis of these results SAC was
regarded as a major factor responsible for the cholesterol lowering effect seen in the
human intervention and animals studies.
26.4.2 Human trials of cholesterol synthesis
A typical human trial to show the cholesterol lowering effect of garlic supplements
involved a double blind randomised placebo controlled intervention study of free
living hypercholesterolemic men (34; 48.2+- 0.8 years old). The subjects were
divided into two groups to receive garlic extract or placebo as dietary supplement for
5 months. The garlic group consumed 9 capsules a day, each containing 800mg of
aged garlic extract (AGE), whereas the placebo subjects took 9 capsules each
containing 800mg of a common food ingredient. Lipids were assessed after 2, 4,and 5
months (Yeh and Liu, 2001). Plasma concentrations remained unchanged 2 months
after the supplements. After 5 months supplementation reduced the mean plasma LDL
cholesterol concentration by 10% from its baseline value. Plasma concentrations of
HDL cholesterol and triacylglycerol remained constant. Examination of food intake
and weight of subjects indicated that the changes were not due to a change in diet or
life style and were therefore due to the garlic supplement.
In a further trial using a diffent source (Kwai), garlic appeared to have a short term
and a long term effect (Orekhov, et al., 1996). Two and four hours after a single dose
of Kwai (1 tablet containing 300mg garlic powder) the atherogenicity of sera as
measured by cholesterol levels taken from patients with coronary atherosclerosis was
decreased. After 3-4 weeks of long term Kwai therapy blood serum atherogenicity
was significantly lower compared to the initial level. In the control group on a placebo
the atherogenicity of sera obtained from patients on a placebo was unchanged. The
atherogenicity was considerably reduced after 4 weeks indicating an initial rapid
reduction and a long-term decline. From this evidence garlic does appear to have an
effect on lowering the cholesterol level in the blood.
26.4.3. In vitro and animal studies of oxidised LDL
The risk factor in arteriosclerosis, such as high LDL or low HDL concentrations can
lead to excess cholesterol available being taken up by the intimal layer which is the
inner layer lining the lumen of the arteries. High LDL predisposes the arteries to
endothelial dysfunction by making them more permeable to the transport of LDL.
Once within the intima, LDL accumulates in the subendothelial space by binding to
components of the extracellular matrix. This trapping increases the residence time of
LDL within the vessel wall where the lipoprotein may undergo chemical
8
modifications. The LDL becomes oxidized by local free radicals and as oxidized LDL
it attracts circulating monocytes to the vessel wall. The modified or oxidised LDL can
be ingested by macrophages contributing to the development of foam cells. Following
oxidation of the LDL, the next stage is the attraction of leucocytes, primary
monocytes and T lymphocytes. After the monocytes have adhered to the luminal
surface they may penetrate into the subendothelial space by slipping between the
junctions. Once localized beneath the endothelium, monocytes differentiate into
macrophages, the phagocytic cells that are able to ingest oxidised LDL. The
macrophages then become lipid laden foam cells the primary constituent of the fatty
streak. More recently oxidised LDL has been recognised as playing a more important
role in vascular dysfunction leading to atherosclerosis than native LDL (Battacharyya
and Libby, 1998). Garlic seems to play a key role in this stage of the disease.
The approach has been to use an in vitro system to show whether garlic supplement
can prevent or reduce the oxidation of LDL. In the in vitro cell free system CuSO4
was used to oxidise LDL and the product, thiobarbituric acid (TBARS), measured
after 24 hours incubation in the presence and absence of the garlic supplement, AGE
(Lau, 2001). The supplement exerted a concentration dependent inhibition of Cu++
induced oxidation of LDL. All four water soluble compounds derived from garlic, Nacetyl-S allyl cysteine, S allyl cysteine, alliin and allyl mercaptocysteine showed
significant inhibition of LDL oxidation. In a further in vitro test vascular endothelial
cells, exposed to Ox–LDL showed signs of cell membrane damage by a significant
increase of lactic acid dehydrogenase (LDH) release and a decrease of methylthiazol
tetrazolium (MTT) absorbance indicating mitochondrial injury. Pretreatment of
vascular cells with AGE and SAC minimized these Ox-LDL induced parameters of
cellular injury. These garlic compounds also inhibited Ox-LDL induced lipid
peroxidation implicating lipids as the principal target in Ox-LDL mediated cellular
injury.
26.4.4. Human trials of oxidised LDL
In a small scale study (a double blind placebo controlled crossover study involving 8
subjects, 4 men and 4 women mean age 68) four subjects took 1.2g AGE three times a
day for 2 weeks then 2 weeks of no garlic (washout period) followed by two weeks of
placebo. The remaining four subjects took a placebo for the first two weeks followed
by a 2 weeks washout and 2 weeks of 1.2 g AGE three times a day (Lau, 2001). Blood
was drawn at the beginning of the experiment and at 2, 4, 6 weeks and when the
experiment was completed. Plasma LDL was isolated and the CuSO4 test repeated.
The use of garlic supplements was found to significantly increase the resistance of
LDL to oxidation.
26.4.5. In vitro and human studies of platelet aggregation
One of the consequences of the formation of foam cells and subsequently the fibrous
plaque is rupture of the plaque releasing material into the blood stream causing a
thrombus to form and platelet aggregation to occur. Aggregation is a consequence of
exposure of fibrinogen receptors on the surface of cells. These receptors bind
fibrinogen in the presence of extra cellular Ca++ and crosslink the platelets to form
aggregates. The fibrinogen is a heterodimer of the membrane glycoproteins (GP)IIb
9
and IIIa and although unstimulated platelets express the GPII-III complex at their
surface, this complex is unable to bind fibrinogen until platelets are activated for
example by ADP. The GPIIb-IIIa receptor has a high content of–SH groups and
binding of fibrinogen is inhibited by the organosulphur compound ajoene which is a
component of some garlic extracts. A reduction in aggregation would decrease the
risk of thrombosis. The approach has been to test the blood of trial volunteers for the
ability to aggregate in response to a number of chemical compounds known to
stimulate this process.
In contrast to earlier trials referred to, the importance of aggregation was investigated
in 34 normolipodemic individuals (Steiner and Li, 2001). These volunteers were
recruited for a 44 week-long double blind crossover study. No supplement were given
for the first 6 weeks then participants were selected to receive either AGE or a
placebo in a dosage of 3 capsules/d (each 800mg) for a period of 6 weeks. The dose
was then raised to 6 capsules/d for 6 weeks and finally to 9 capsules for another 6
weeks. The first intervention period was followed by a 2 week washout period. The
subjects were then switched to the other supplement that they did not receive during
the first arm of the study. A final 2 week washout period concluded the study. Blood
was sampled every 2 weeks and processed for platelet aggregation and adhesion
studies. Aggregation was tested with the following agonists which would normally
encourage aggregation: arachidonic acid, ADP, collagen, and epinephrine. Each
platelet stimulant was used in a range of concentrations to determine the threshold
levels required to induce complete aggregation. Aggregation studies using ADP as a
stimulant showed minimal increases. Collagen and epinephrine induced aggregation
was significantly inhibited by AGE. All individual consuming AGE showed an
increase in SAC in the blood, which dropped when AGE ceased to be consumed.
In a contrasting investigation on platelet aggregation in humans, 23 healthy and
normolipodemic subjects (12 men and 11 women) consumed 5ml of AGE daily for 13
weeks then blood samples were taken before and after the last dose then platelet
aggregation was measured (Rahman and Billington, 2000). The extent and rate of
aggregation in response to ADP was reduced after dietary supplementation with 5ml
of AGE. Serum lipid concentrations were not affected. It is thought that the effect of
AGE was to inhibit the ADP induced rise in cytosolic Ca++ concentrations. Both
trials suggested that regular consumption of garlic supplements would reduce the
level of aggregation in the blood and therefore the risk of thrombosis.
26.4.6. In vitro and animal studies of the arterial cell wall
The most important consequence of plaque formation is that it leads to a restricted
blood flow and hardening of the arteries. Resistance in the arterial circulation is
increased and there is the risk that the plaques might break, releasing material into the
blood stream and causing a thrombus to form and strokes to follow. Garlic
consumption appears to have a direct effect on the artery wall by reversing or
reducing wall hardening. The approach has been to examine the response of cell
cultures of arterial cells to compounds such as LDL then the arteries of animals fed a
high cholesterol diet for lesion development.
10
Free cholesterol and cholesterol esters are present in arterial cells and contribute to the
eventual formation of plaques. Uptake and metabolism of cholesterol by arterial cells
is critical to the final stages of arteriosclerosis. The control of this process has been
examined in vitro by using the response of cultured smooth muscle cells to
cholesterol. Cultures of such cells derived from a fibrous plaque of an arterosclerotic
human aorta loaded with cholesterol were incubated for 24hours with an aqueous
extract of garlic powder. Free cholesterol decreased by 30%, cholesterol esters by 3040% and triglycerides by 20% (Orekhov et al.,1995; Orekhov and Tertov, 1997). This
change is achieved because garlic appears to suppress lipid synthesis in the cells by
inhibiting the activity of acyl –CoA:cholesterol acyltransferase(ACAT), the enzyme
involved in the formation of cholesteryl esters, the main component of the excessive
fat accumulated by cells. In the untreated atherosclerotic cells, ACAT activity was 3–
fold higher than in normal cells. The aqueous extract of garlic cells decreased this
enzyme activity to normal levels. Garlic extract also stimulated cholesterol ester
hydrolase, an enzyme that degrades and therefore reduces cholesterol esters in
atheriosclerotic cells (Orekhov and Tertov, 1997).
In addition to a drop in cholesterol concentration and retardation of atherosclerotic
lesion formation, garlic enhanced the fibrinolytic activity of the blood plasma, which
is lowered as a result of cholesterol feeding. A reduction in fibrinolytic activity may
accelerate atheriosclerosis by exposing the vascular walls surfaces to recurrent
thrombi and clot association mitogens. The effect of garlic may result from an
increased fibrinolytic activity (Bordia et al., 1975). In a further correlation it was
found that in cultured cells there was a strong correlation between atherogenicity of
LDL (measured by the lipoproteins capacity to induce lipid accumulation in cultured
arterial cells and stimulate cell proliferation) and sialic acid content of LDL.
Desialylated LDL caused cholesterol accumulation in arterial cells via increased
uptake, through interaction with both the B,E-receptor and an unregulated scavenger
receptor whereas non desialylated LDL is taken up through the B,E-receptors only.
Desialylated LDL may be responsible for the onset and further development of
atherosclerotic lesions since atherogenic LDL isolated from patients blood had a
lower content of sialic acid compared with native LDL of healthy subjects. Long term
therapy with garlic tablets increased the LDL sialic content up to normal (Orekhov, et
al., 1992).
In an animal study arteriosclerosis was induced in rabbits by feeding them a
cholesterol rich diet for 2-4 months when lesions were found to have formed in the
aorta of a sample of the animals. Cholesterol feeding was withdrawn then the rabbits
given essential oil of garlic for 8-9 months or garlic homogenate for 3 months. After
cholesterol feeding was discontinued, serum cholesterol concentration decreased. In
the garlic fed animals the fact that the area of arthererosclerotic lesions had decreased
more than 50% and the lipid content of the artery wall decreased by 69% indicated a
direct effect of garlic on the arterial wall and a regression of the disease (Bordia and
Verma, 1980). Garlic supplementation also partially suppressed biosynthesis and
accumulation of collagen in the aorta of the cholesterol fed rabbits.
When the aorta of rabbits fed a standard diet was examined directly there was no fatty
lesions on the intimal surface in the presence or absence of Kyolic. In the cholesterol
fed rabbits 70% of the luminal aortic surface was covered by lipid filled lesions and
the neointima was greatly increased with a concentrated thickening of fibro-fatty
11
plaques. The Kyolic treated group had only 25% coverage of the aortic surface by
lesions, the accumulated cholesterol in the aorta was significantly reduced and the
neointima only half the size (Campbell et al., 2001). The function of smooth muscle
cells is to contract but during arteriosclerosis there is a change to a cell that can
accumulate lipid, migrate, proliferate and synthesize appreciable extra-cellular matrix.
This change to smooth muscle cells also occurs in culture but it was inhibited by the
presence of Kyolic supplement in the culture medium. Inhibition of the conversion of
the smooth muscle cell to this altered phenotype may represent a mechanism by
which garlic achieves its protective effect against arteriosclerosis under in vivo
conditions
26.4.7.In vitro studies of hypertension
Hypertension is a very common condition and one which results from
artheriosclerosis. One result of the changes during the development of artherosclerosis
is that the arterial wall tissue hardens and loses its ability to relax and contract. In
order to test the effect of garlic on the contractility of aorta, rats were fed a high
cholesterol diet with and without garlic extract then killed after 16 weeks of this
treatment. The arteries were cut into rings then these suspended between two hooks
which were able to measure the contractile response. Aortic rings were contracted
with noradrenalline and exposed to acetylcholine.or to sodium nitroprusside when
contractions had reached a plateau to test the endothelium dependent and independent
relaxation. Contractions were higher in those rings treated with garlic (Slowing, 2001)
suggesting that garlic consumption can offset the hardening effect of atherosclerosis.
Chronic hypertension is a major contributor to the development of myocardial
ischemia infarction and strokes but hypertension can be induced by factors other than
age related changes. One of the causes of induced hypertension is stress with its
accompanying depressive effect on the immune system and reduced control of
oxidative stress. Activation of the transcription factor NF-KB, which is associated with
the regulation of numerous genes encoding proteins in immune function, is an
important part of this stress response (Grimme and Baeuerle, 1993). Oxidized LDL a
product of high blood cholesterol can act as a second messenger in the activation of
NF-KB. In in vitro studies using cultured arterial endothelial cells, activation of NF-KB
by oxidised LDL led to the expression of cell adhesion factors, VCAM-1 and ICAM-1
whose formation can accelerate the development of atherogenic lesions and cell death.
Anti oxidants such as garlic derived compounds inhibited oxidant induced NF-KB
(Geng et al., 1997). The transcription factor NF-KB can also be activated by the tumor
necrosis factor (TNF)-α. When endothelial cells from the human umbilical vein
(HUVEC) were incubated with the tumour necrosis factor (TNF)-α, the cells showed
activation of NF-KB expression, whereas preincubation of HUVEC with Sallylcysteine, a major products of alliin breakdown in fresh garlic and a component of
some supplements, inhibited the activation (Ide and Lau, 2001).
Antihypertensive medications are the standard means to lower chronically elevated
blood pressure. These are diuretics, whose side effects include elevation of cholesterol
and triglyceride levels, beta blockers through a reduction in heart rate and a mild
decrease in contractility with side effects which may include a rise in serum
triglycerides, and a reduction of good cholesterol (HDL), and Ca++ channel blockers
12
which have fewer side effects but are costly (Deshmukh et al., 1998). Although the
influence of garlic on the risk factors of atherosclerosis and ultimately hypertension is
weaker than that of synthetic drugs, the broad spectrum of garlic is an important
advantage. Of equal importance is safety and here garlic has the advantage of having
being taken as a supplement for many centuries with no obvious ill effects except bad
breath and the occasional gastric complaint.
26.5. Future trends: developing new functional foods
An obvious method of increasing the uptake of active organosulphur compounds into
the body is simply to eat more garlic or consume more supplements. Another strategy
is to optimise the levels of the precursor compounds such as alliin in the existing
garlic. This both improves the amounts of precursors compounds and breakdown
products in the raw and cooked garlic and also ensures the highest possible
concentration in the garlic supplements. Increases in levels of organic sulphur
compounds in garlic can be achieved in a number of ways. These are by the addition
of sulphur fertilizer to the soil, by classical breeding techniques of selection and
crossing and by genetic manipulation.
In an example of the first approach when sulphur fertilizer was added to the soil as
3.4x10-2 kg.m2 CaSO4, it increased the total sulphur in the fresh garlic from 96.4
mmol/kg to 190mmol/kg. In the dried garlic powder from these bulbs, there was an
increase in concentration of organosulphur compounds, alliin, allicin, γ-glutamyl
propylcysteine and γ glutamyl-phenylalanine The effect of increasing soil sulphur led
indirectly to a change in levels of NF-KB in blood cells The nuclear factor, NF-KB, is
an important regulator of the immune system and is involved in artheriosclerosis
through activation of adhesion factors. Lipopolysaccharide are able to induce the
inflammatory cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α in
human blood which can activate production of NF-KB (Li and Verma, 2002).
Pretreatment of the blood cells with garlic powder extract reduced the
lipopolysaccharide production of cytokines and in turn reduced the production of the
nuclear factor NF-KB. The effect of garlic was to reduce the induction of the nuclear
factor by 25%, whereas samples treated with sulphur fertilised garlic lowered the NFKB by 41% (Keiss et al., 2003). The effect of the garlic was likely to be through
diallyl disulphide a breakdown product of alliin since Keiss et al (2003) found this
compound reduced the lipopolysaccharide activation of interleukin (IL)-1β and
(TNF)-α levels in blood.
A classical and genetic engineering approach to plant breeding to enhance the yields
of organosulphur compounds in garlic has been undertaken by participants of the
Garlic and Health grant, an EU financed project (http://www.plant.wageningen-ur.nl).
Garlic is vegetatively propagated and none of the current varieties show any seed
formation which makes it difficult to attempt varietal improvement by standard
selection and breeding techniques. The approach undertaken was to return to the
country of origin of the garlic and obtain plant varieties that showed seed formation,
in combination with high yields of alliin in the bulb. It should now be possible to
select high yielding individuals, which can be put through a standard breeding
programme involving crossing and seed production. In a genetic engineering
approach an attempt was made to alter biosynthesis of the flavour precursors by
13
genetic manipulation. This approach required identification of the pathway
intermediates and the controlling enzymes. The mechanism of alliin biosynthesis is
largely speculative but it is thought that there are two possible routes one via serine
and an allyl source and the other via glutathione and an allyl source (Fig 1) (Lawson,
1996). These two stages represent the beginning of the secondary pathways and
probably where the control of the secondary pathways occurs. Genetic manipulation
of the control enzymes would require identification of the enzymes and the genes
responsible, followed by transformation of the garlic and overexpression of the
control enzymes to overcome any metabolic limitations on the pathway.
26.6. Sources of further information and advice
While the large array of different garlic supplements available to the customer can be
confusing, the Web is now a valuable reference for both products and medical advice.
Many of the statements however have not been evaluated by regulatory bodies such as
the American Food and Drugs administration, and European Union (Thomas and Earl
1994; Clydesdale; 1997; Bellisle et al 1998), so the garlic cannot be seen as an official
cure, or treatment or for the prevention of cardiovascular disease. The evidence only
suggests that garlic may have a beneficial effect on cardiovascular disease. An
example of a helpful website is http://illness.altmedangel.com/heart.htm which is a
non profit site offering educational information and broad based research on various
health conditions, medications, supplements and therapies. It advocates the use of
garlic supplement to regulate blood pressure and to thin the blood. It claims that
consumption of the garlic will also preserve the elasticity of the arteries and delay
their stiffening. In another http://www2.vitaminconnection.com, garlic as fresh garlic
(up to one clove daily) and 400mg tablet (standardized to 1% total alliicin potential)
was shown to cause a lowering of cholesterol. There were also beneficial effects on
blood pressure, fibrinogen and LDL. In the more commercially orientated sites the
descriptions of the effects of garlic consumption are sometimes vague using such
language as cleansing cholesterol and normalizing blood pressure, helping to reduce
harmful LDL cholesterol and triglycerides, reducing blood clotting and strengthening
blood vessels.
Another site (http://www.amercanheart.org/presenter.jtml) was more critical since it
made an objective analysis of a number of clinical trials on the effect of garlic
consumption on cardiovascular disease. The analysis showed that there were a
number of modest short term benefits of garlic supplements. It maintained that the
main problem with making a comparison between the trials was the variation in the
composition of the different commercial preparations. In a larger meta-analysis the
effect of garlic on blood pressure reduction and lipid lowering was examined by
Silagy and Neil (1994a and 1994b). The conclusions from analysis of 11 randomised
controlled trials for blood pressure and 25 randomised controlled trials for serum
cholesterol was that garlic therapy may reduce both blood pressure and serum lipids.
Silagy and Neil (1994a and 1994b) suggested that this result should be treated with
caution because of shortcomings in methodology of some of the trials examined. In a
later meta-analysis (Stevinson et al,. 2000) the effect of garlic on total cholesterol in
persons with an elevated levels of cholesterol of at least 200mg/mL was analyzed in a
number of randomized double blind placebo controlled trials. The results showed that
compared with the placebo, garlic reduces the total cholesterol level in those persons
14
whose levels were elevated. The findings confirm the results of earlier studies but the
effect may be smaller than originally thought with a reduction of 4-6% of cholesterol
due to garlic. The meta-analysis included only 13 trials since it was only these that
satisfied the strict requirements of the test and of these there were serious variation in
the methodology used. One of the most significant was the unblinding of the test
because of the difficulty of disguising the garlic odour. Empirical research suggested
that trials that are not wholly double blind tend to exaggerate the treatment effects.
The other major problem in trying to establish a significant effect was the variation in
the garlic source. The test used Kwai, garlic oil and spray dried garlic powder. Of
these there was known variation in the alliicin content of the samples in addition to
the known variation between the different sources of garlic. According to Stevinson et
al (2000) what is required is to have well designed placebo controlled trials
comparing the efficiency of the different types of garlic.
The trials took essentially a short term view of the effect of garlic consumption ie the
increase in cholesterol was measured whereas it is more important to take a long term
view in terms of clinical effects, such as heart disease. Many of the other protective
effects of garlic such as reduced blood pressure, platelet inhibition, and stimulation to
blood flow have been assessed in vitro and in vivo but the clinical effects have been
implied but not measured. The results of the various meta-analyses suggested that
garlic is superior to a placebo in reducing elevated cholesterol levels but the size of
the effect is moderate. Compared with the use of pharmaceutical drugs such as the
modern statins, which can cause a reduction of 17-32%, the reduction in cholesterol
from garlic is small. Even dietary modifications can cause a 5.3% reduction over a 6
month period. The implication for clinical practice is that garlic use is not an efficient
way to decrease serum cholesterol level. Large scale and long term trials are required
to establish the association between garlic consumption and the clinical outcomes.
The one test that attempted this involved 152 patients over a four year period when it
was shown that there was deceleration of the development of artheriosclerosis in those
who included garlic in the diet (Koscielny et al., 1999). The other more long term test
for cardiovascular health is perhaps the Mediterranean diet which contains a
significant amount of garlic and which appears to have a protective effect on the
cardiovascular system.
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19
Figures
Figure1 Proposed pathways for the synthesis of alliin, isoalliin and methiin in garlic.
(Modified from Lawson 1996)
20