Effect of apple cider vinegar on plasma lipids (Model experiment in mice)

Abstract

Model experiment was carried out to investigate the effect of apple cider vinegar (ACV) on the blood and liver cholesterol (Ch), triglycerides (TG) and one of a marker of antioxidant status of blood (FRAP) in laboratory mice. Animals consumed a basal mice diet (Control) served as the control group. The same diet was supplemented either 1% cholesterol (Ch) or 1% edible sunflower oil (SFO). All groups were duplicated and their animals were supplied drinking water containing ACV (50 mg l-1)(groups: Control+ACV, Chol+ACV, SFO+ACV).The feeding and drinking was ad libitum for 21 days. At the end of experiment the animals were exterminated. Blood and liver samples were analyzed for total cholesterol (tCh), triglycerides (TG) and ferric reducing antioxidant power (FRAP). The results show that the Ch supplemented group stored higher concentration of tCh in the liver (P

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potravinárstvo
Volume 6 1 No. 1/2012
INTRODUCTION
The modern pharmaceutical industry based on synthetic
chemistry severed the historical connection between
plants, food and medicines. Nowadays food and feed
additives of natural origin, used in natural and folk
medicine with a partial predilection are coming more and
more into the front. Multicomponent botanical therapeutics
that comprise functional foods, dietary supplements and
botanical drugs hold several advantages over conventional
drugs that may earn them a more prominent place in the
medicine of the future (Raskin and Ripoll, 2004). One of
these natural substances known for hundred years and
nowadays living its renaissance is the apple cider vinegar
(ACV) which has been helping people to healthier lives.
This is claimed by advertisements in different media
(journals, TV, InterNet). They argue that ACV can help
maintain blood sugar levels in weight management, along
with a low calorie diet, by helping to lower the amount of
body fat and also helps break down the cholesterol
formations that build up on walls of blood vessel.
In the propagating literature can be found that ACV is an
essential source for several vitamins and trace elements.
It improves renal function and stops multiplication and
colonilalization of harmful bacteria (Vijayakumar and
Wolf-hall, 2002). It has a corrective effect on circulation;
it is “blood thinner”, helps healing wounds, and speeds up
metabolism.
Beneficial effects of ACV have been proved by several
practical observations, but there are only a few scientific
evidences to prove these facts right. The search for
publications in scientific data base surprisingly has only a
few scores about the biological experiments with ACV.
Practical evidences confirmed that this substance is an
outstanding fodder additive for farm animals, based on its
vitamin, free amino acid and rich mineral element content.
Apart from these and its vinegar (acetic acid) content the
substance has other acid components too, such as: citric
acid, malic acid and soluble dietary fiber: pectin
(Hellmiss, 1997) and sorbose (McComb, 1975) a non-
fermentable hexose too. Due to its pectin content ACV has
a decreasing effect on the plasma LDL cholesterol level.
Specific components in the apple juices and extracts that
contributed to antioxidant activity have found that both
fresh apple and juices inhibited copper-catalyzed LDL
oxidation (Pearson, et al., 1999).
Based on our previous experiments with Japanese quails,
which are regularly used test animals for fowls (Wilson
et al, 1961), and on turkeys getting 1:100 dilutions of the
ACV in drinking water we could state that total cholesterol
(tCh) and triacyl-glicerols (TG) had decreased in blood
(Bárdos, Kiss, 2000a, and b; Czirle and Bárdos, 2000).
Since these are primary factors in applying ACV as an
additive for foodstuffs or as a medicinal substance of
natural origin. We decided to start a model experiment on
Potravinarstvo, vol. 6, 2012, no. 1, p. 1-4
doi:10.5219/156
Received: 14. July 2011. Accepted: 19. January 2012.
Available online 15. February 2012 at www.potravinarstvo.com
© 2011 Potravinarstvo. All rights reserved.
ISSN 1337-0960 online
EFFECT OF APPLE CIDER VINEGAR ON PLASMA LIPIDS
(MODEL EXPERIMENT IN MICE)
László Bárdos, Balázs Bender
ABSTRACT
Model experiment was carried out to investigate the effect of apple cider vinegar (ACV) on the blood and liver cholesterol
(Ch), triglycerides (TG) and one of a marker of antioxidant status of blood (FRAP) in laboratory mice. Animals consumed a
basal mice diet (Control) served as the control group. The same diet was supplemented either 1% cholesterol (Ch) or 1%
edible sunflower oil (SFO). All groups were duplicated and their animals were supplied drinking water containing ACV
(50 mg l-1)(groups: Control+ACV, Chol+ACV, SFO+ACV).The feeding and drinking was ad libitum for 21 days. At the
end of experiment the animals were exterminated. Blood and liver samples were analyzed for total cholesterol (tCh),
triglycerides (TG) and ferric reducing antioxidant power (FRAP). The results show that the Ch supplemented group stored
higher concentration of tCh in the liver (P<0.01) than SFO treated animals. The cholesterol reserves were less in ACV
treated groups. The alterations of plasma tCh showed no significant changes by cholesterol or SFO supplementation and
drinking ACV containing water. The concentration of plasma and liver TG remained in the same range in all groups
independently by different treatments. Animals of SFO supplemented groups (SFO and SFO+ACV) got more fatten than
Control and Ch groups and their liver/body mass ratio (%) decreased (P<0.05). The ACV exerted a decreasing effect on the
level of plasma tCh and TG markedly (P<0.05) but only in that group (Control+ACV) which consumed the basal diet. This
lowering effect could be demonstrated only in the case of TG in the liver. The groups receiving ACV showed decreasing
FRAP values. This means a lower antioxidative capacity of plasma. The ACV can helps in the lowering of plasma lipids
(tCh and TG) and can depress their liver storage in the case of normal level of lipid consumption. When the lipid input was
elevated this benefit not occurred.
Keywords: apple cider vinegar, cholesterol, triglyceride, FRAP, mice

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Volume 6 2 No. 1/2012
mammals. This model experiment was carried out to
investigate the effect of ACV on the blood and liver
cholesterol (Ch) and triglycerides (TG) and one of a
marker of antioxidant status of blood (FRAP) in laboratory
mice.
MATERIAL AND METHODOLOGY
Animals and experimental set-up
CFLP inbreed (Charles River Ltd, Isaszeg, Hungary)
laboratory male mice were used in the experiment. Six
groups were arranged with ten-ten animal (average weight:
25 g) in each. Animals were fed ad libitum with a basic
and/or supplemented feed. Basal diet used for the mice
was laboratory mice feed. We mixed the additives with it.
After grinding this feed we mixed it with 1% cholesterol
(w/w) and with sunflower oil (v/w), respectively. From the
mixture we formed scones using cooking gelatin so we
could apply them for feeding after dehydration.
The control diet without any supplementation but it was
reformulated with gelatin too. The drinking water of ACV
treated groups was mixing with apple cider vinegar in the
ratio of 100 (water) to 1 (ACV) resulted concentration of
500 mg.l-1. The animals were fed for 21 days.
Table 1 contents the experimental and feeding set-up.
Feed additives
The experimental feed was supplemented with
cholesterol (Fluka, Germany), sunflower oil (purchased in
pharmacy) as additives. Commercial apple cider vinegar
containing 5% (v/v) acetic acid (Almaecet 5%, Buszesz
Ltd., Budapest, Hungary) was added to the drinking water.
The used gelatin for the making feed scones was
commercial edible grade.
Sampling
Six mice from each group were picked out and lege artis
sacrificed at the end of experiment. Blood samples were
drawn into tubes containing heparin. The body and liver
weights were measured. Blood plasma and liver samples
were refrigerated (-20 oC) until the analyzes.
Analytical methods
Total cholesterol (tCh) and triglyceride (TG)
concentrations of plasma were measured by enzymatic
(GPO-PAP) colorimetric methods with reagents kits
(Reanal Ltd., Budapest). Removing the total lipid content
from the tissue (Floch et al., 1957) the Ch and TG
concentrations from the homogenized liver tissue were
measured using the same methods as above. The
antioxidant capacity of plasma was characterized by FRAP
method (ferric reducing ability of plasma) (Benzie and
Strain, 1996).
Statistic
One-way ANOVA with Dunnett’s post test was
performed using GraphPad Prism version 5.00 for
Windows, (GraphPad Software, San Diego California
USA, www.graphpad.com).
RESULTS AND DISCUSSION
Our first result is that the basal diet mixed with additives
and glued with gelatin results a solid nutrient (feed scones)
again. Gelatin is a substantially pure protein food
ingredient, obtained by the thermal denaturation of
collagen, which is most common protein in the animal
kingdom. This meet with our requirements that the
additives (cholesterol and sunflower oil) must be dissolved
uniform so they can be dosed accurately. The mice
consumed this feed readily. Gelatin is not a complete
protein source because it is deficient in tryptophan and low
in methionine content, however the digestibility is
excellent. We could not calculate with the deficiency of
these amino acids because the animals were fed for three
weeks only.
The literary facts and figures concerning animals
reflected only production effects were presented with ACV
application in the diet until now. In the present
experiments we tried to find a different approach to
evaluate the beneficial physiological effects of the ACV in
the point of view of lipid metabolism. Mice treated with
ACV (Control+ACV, Ch+ACV and SFO+AVC) and its
control groups without ACV supplementation (Control,
Ch and SFO) were compared.
The group of mice consuming the feed containing Ch
and drinking ACV containing water had a little bit smaller
bodyweight and liver weight than those of the control and
Ch groups (Table 2). During dissection we found in the
mice consuming feed enriched with cholesterol large
quantities of deposited fat under the skin and in the
abdominal cavity i.e. in the mesentery, too. This is the
explanation for the smaller weight but bigger size, since fat
is lighter weight than other tissues. This phenomenon is an
evidence for the weight-reducing effect of ACV, since the
group consuming it (Control+ACV) with normal feed had
a smaller body weight than those of the control group.
Acetic acid administration inhibited the accumulation of
body fat and hepatic lipids without changing food
consumption or skeletal muscle weight. In conclusion,
Acetic acid suppresses accumulation of body fat and liver
lipids by upregulation of genes for fatty-acid-oxidation-
related proteins by mediation in the liver (Kondo et al.
2009).
In case of the group consuming cholesterol, we found
that the mice became extensively fatty. This reflects in the
weight ratio of liver/body. Animals of SFO supplemented
groups (SFO and SFO+ACV) got more fatten than Control
and Ch groups and their liver/body mass ratio (%)
decreased compared to Control (P<0.05) (Table 2).
Table 1 Experimental set-up
Groups
Control
Ch
SFO
Control+ACV
Ch+ACV
SFO+ACV
Feed
1
2
3
1
2
3
Drinking water
Tap water
Tap water containing 1% ACV
1. Basal mice feed, 2. Diet 1 containing 1% cholesterol, 3. Diet 1 containing 1% sunflower oil; ACV apple cider vinegar

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Volume 6 3 No. 1/2012
The alterations of plasma tCh showed no significant
changes by cholesterol or SFO supplementation and
drinking ACV containing water (Table 3). The plasma Ch
decreased in Control+ACV group. The results of our
experiment show that the Ch supplemented groups stored
higher concentration of Ch in the liver (P<0.01) than
Controls and SFO treated animals. The storage of Ch was
somewhat less in ACV treated groups (Table 3). These
findings can be explained by sorbose and pectin content of
ACV. One of a non-fermentable (Tamura et al., 1991)
carbohydrate constituent of ACV is the L-sorbose
(McComb, 1975). Sorbose significantly reduced plasma
cholesterol and VLDL by approximately 50%. Absolute
and relative abdominal fat weights were and fat content in
the pectoral muscle also decreased as dietary sorbose
increased (Beyers and Jensen, 1993). It was concluded
that dietary sorbose can be used as a potential regulator of
lipid deposition in broilers (Furuse et al., 1991). The TG
levels of plasma in all supplemented groups were
markedly lower (p<0.001) than in animals receiving basal
diet (Table 3). The concentration liver TG remained in the
same range in all groups independently by different
treatments (Table 3). According to Aprikian et al. (2001)
the lipoprotein profile was markedly altered in apple-fed
rats. The reduction of cholesterol in the triglyceride rich
lipoprotein fraction, together with a rise in the HDL
fraction was described. This was parallel by effects of the
apple on cholesterol absorption, which was markedly
depressed, whereas bile acid digestive balance was
unaffected. Others have demonstrated that water soluble
components of fruits have influence on lipid metabolism.
Sugar beet pulp and apple pomace dietary fibers hindered
the rise of plasma lipids in rats fed cholesterol
(Leontowicz et al., 2001). We have only information of
acidity of commercial ACV which was used in our
experiment. But there are data in the literature that among
the main organic acidic components (acetic, propionic,
malic and lactic acid) ACV contains free amino acids, non-
fermentable sugar and roughage in the forms of potash and
apple pectin (McComb, 1975; Hellmiss, 1997). One of
the water soluble dietary fibers is the pectin (Linder
1991). The supplementation of diet with apple dietary fiber
from extraction juices or alcohol-insoluble substances had
minor effects on blood serum lipids but the fecal excretion
of bile acids increased (Sembries et al., 2004). The ACV
which containing this materials exerted a decreasing effect
on the level of plasma Ch and TG
markedly (P<0.05) but only in that group (Control+ACV)
which consumed the basal diet in presented experiment.
This lowering effect could be demonstrated only in the
case of TG in the plasma. The groups receiving ACV
showed decreasing FRAP values compared with the same
supplementation without ACV (Table 3). This means a
lower antioxidative capacity of plasma because of the
direct reduction of the color-forming reagent (ferric
tripyridyltriazine), so the antioxidant capacity is
proportional to the reducing ability of plasma. These
findings are against to others’ results. According to
Aprikian et al. (2001), there was a positive effect of the
apple diet on parameters of oxidative stress prevention:
higher FRAP plasma levels than in controls, together with
a reduced MDA excretion in urine. In conclusion, their
work indicates that the supply of apples elicits interesting
effects on lipid and peroxidation parameters. Others found
that acetic acid was considered to have an impact on the
release of PUFA. For example lipid oxidation products
increased in the acid-treated oyster digestive organs.
PUFA and lipid oxidation products after treatment with the
acid were higher than that in PBS-treated ones at 37 °C
(Sajiki et al.1995). In our case presumably these effects
occurred in all ACV treated and especially in the
SFO+ACV and cholesterol+ACV supplemented groups.
CONCLUSION
The problem of the application of dry matter additives to
laboratory mice was solved by gelatin gluing of
components. The ACV can help in the lowering of plasma
Ch and TG and can depress their liver storage of TG in the
case of normal level of lipid consumption. When the lipid
input was elevated this benefit not occurred in the blood,
but a decreasing tendency of cholesterol and triglyceride
contents were determined in the liver.
We hope that our experimental results will bring us
nearer to understand of a better and more determined
utilization of the ACV, as a natural food additive,
concerning both human and animal nutrition.
Table 2 Body weight and liver weight (mean SEM)
Groups
Control
Control+ACV
Ch
Ch+ACV
SFO
SFO+ACV
Body (g)
26,621,22
26,191,20
26,472,06
26,091,16
28,122,04
26,531,91
Liver (g)
1,710,21
1,450,23**
1,860,46
1,440,15**
1,820,37
1,080,13***
Liver %
6,450,98
5,550,88
7,061,82
5,520,60**
6,431,10
4,090,59**
**p <0.01;**p <0.001 compared to control by Dunnett's Multiple Comparison Test
Table 3 Cholesterol trigliceride and FRAP values of plasma and liver (mean SEM)
Groups
Control
Control+ACV
Ch
Ch+ACV
SFO
SFO+ACV
FRAP (mmol/L)
0.640.16
0.510.1
0.660.11
0.660.11
0.470.25
0.380.17*
Plasma
tCh (mmol/L)
1.90.61
1.650.32
1.470.19
1.860.42
2.110.23
1.620.39*
TG (mmol/L)
1.510.5
1.190.16
0.690.12***
0.690.2***
0.740.1***
0.620.07***
Liver
tCh (mmol/g)
6.762.55
6.81.26
25.85.08***
24.493.84***
8.363.49
6.834.13
TG (mmol/g)
11.84.26
9.021.57
9.322.69
10.843.7
9.374.16
9.673.72
*p<0.05;**p <0.01; ***p<0.001 compared to control by Dunnett's Multiple Comparison Test

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Contact address:
László Bárdos, DVM, PhD., Department of Animal
Physiology and Health, Szent István University, 2013
Gödöllő, Hungary, E-mail: bardos.laszlo@mkk.szie.hu,
tel.: +3630-2796776,
Balázs Bender, Ing. Agr., PhD., ImmunoGenes Ltd, 2092
Budakeszi, Hungary, E-mail: benderb001@gmail.com,
tel.: +3630-976-8544.