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ABSTRACTCardiovascular
diseases (CVDs) are known as the principal cause of demise and disability
universally and also in Pakistan. In Pakistan heart diseases are the major
reason of death. CVDs are expanding step by step attributable to poor way of
life and dietary behavior. Hypercholesterolemia is considered as essential factor
for cardiovascular diseases. There are a few sorts of medications that can be
utilized for the cure of sicknesses however these medications have diverse
reactions. From the ancient times many fruits have been considered as
beneficial for health. Apple and Apple cider vinegar have been utilized since
numerous years for the treatment of various diseases including hypercholesterolemia.
The present study aimed to check the combined effect of apple and apple cider
vinegar for reducing hypercholesterolemia. Hypercholesterolemia will be induced
in rats by giving them high cholesterol diet. After the induction of hypercholesterolemia
the rats will be fed with apple juice and apple cider vinegar in their diet at
the ratio of 30ml/day and 20 ml/day respectively. The blood samples will be
taken after 30 days interval. After the treatment cholesterol, triglycerides,
LDL and HDL will be estimated from blood serum. Data obtained will be subjected
to an appropriate statistical analysis to determine the level of significance1. INTRODUCTION:CVDs are one of the major metabolic
issues that are tormenting our general public at a disturbing rate. It is the
main reason of morbidity and mortality around the world. Poor dietary decisions
and way of life are the central point in elevating the disease. CVDs contribute
to 30 percent of all deaths and 80 percent of CVDs related deaths, in lower and
middle income countries of the world (IOM, 2010). CVDs represent roughly 17.3
million deaths per year that is probably increased to more than 23.6 million by
the year 2030. Elevated cholesterol is assessed to cause 2.6 million deaths. About
85.7million, or 34
percent, of adults have high blood pressure and about 28.5 million, or 11.9
percent, of adult have total cholesterol of 240 mg/dl or higher (AHA, 2017).Hypercholesterolemia is characterized
by high level of cholesterol in blood. Cholesterol is produced by liver and is
a waxy substance that caused blockage in blood vessels. Although cholesterol is
essential for different body functions which include synthesis of membrane,
production of hormone and fat digestion substances, however a very high
cholesterol level increases the risk of heart attack (Benjamin et al., 2010). The main source of
cholesterol is our food and food products such as meat, poultry, fish and egg
yolk. These foods consist of saturated and Trans fats that increases the
synthesis of cholesterol from liver and result in hypercholesterolemia (Costa et al., 2003).The fat deposits in the arteries (that
supply blood to heart) and causes hardening of artery walls that results in
atherosclerotic plaques. These plaques act as hurdles between the blood vessel
and heart and ultimately increase the risk of heart attack. There are also some
cases in which hypercholesterolemia is inherited disease that cause excess
cholesterol to accumulate in body tissues but this case is one in five hundred
(Akishita, 2004).            Lipoproteins
are transport protein which are attached to blood and circulate cholesterol
with blood in human body. There are mainly two types of lipoprotein depending
upon the type of cholesterol they carry. Low density lipoprotein (LDL) also
known as bad cholesterol carries cholesterol from liver and transport to
different part of body. In case of its excess cholesterol accumulate in artery
walls and lead to atherosclerosis. High cholesterol lipoprotein (HDL) carries
excess cholesterol away from liver cells where it breaks down and release as a
waste product. That’s why these lipoproteins are known as good cholesterol. (CDC,
2017) .Unhealthy food containing high fat contents, obesity, diabetes, smoking,
high blood pressure and physical inactivity increase LDL level and decreases
HDL level which results in stroke and heart failure (Sangsawang et al.,
2015). Extensive use of chemical drugs causes problem that is
sometimes even more hazardous than the disease itself. As a result, some drugs
may be excluding from consideration or prescribed with extreme caution. Fear of
drug side effects brings about their improper use and rejection. Therefore, the
disease will not be desirably controlled (Beheshti et al., 2012).Because of these issues a large number of studies have
been performed to check the influence of different products on blood lipids (Shisheehbor
et al., 2008). It is proved that
apple have many protective effects against cholesterol. Apples also ranked the
second for total concentration of phenolic compounds, and perhaps more
importantly, apples had the highest portion of free phenolics when compared to
other fruits (Boyer and Lui, 2004). This means that these compounds are not
bound to other compounds in the fruits, and the phenolics may be more available
for eventual absorption into the bloodstream.            The influence of apple
cider vinegar has been investigated for hundreds of years. It was in fact first
used about 5000 years ago (Budak et al.,
2011). More valuable properties of apple cider vinegar and its ingredients,
suggesting their therapeutic effects, have been recently discovered (Fushimi et al., 2006). Apple cider vinegar
contains polyphenolic compounds that have anti oxidant flavonoid which can reduce
the deleterious effects of high cholesterol level (Verzelloni et al., 2007).            Apple products are
widely used in world. A lot of research is available on the use of apple alone,
however very little information is available regarding the effect of apple
cider vinegar towards cholesterol lowering. The main focus of this study is to
check the effect of apple juice and apple cider vinegar alone and in
combination with one another for lowering the cholesterol level (Beheshti et al.,
2012).OBJECTIVESThis study will be conducted to achieve the
following objectives.·        
To check the nutritional profile of apple and
apple cider vinegar ·        
To evaluate the significance of apple and apple
cider vinegar in hypercholesterolemic rat 2. REVIEW OF
LITERATUREThere is very close relationship
between diet and various chronic diseases. Different fruits and vegetables have
positive effect on chronic diseases due to their phenolic and antioxidant
compounds. Apple and apple cider vinegar is considered as an important fruit in
lowering the risk of chronic diseases (Cancer, CVD, Diabetes and High
cholesterol) because it contains chemical component such as flavonoids,
polyphenol and carotenoids (Boyer and Lui, 2004). Hyperlipidemia is a result of
accumulation of triglycerides and cholesterol level in blood. In Malaysia apple
cider vinegar is considered as very common home remedy to decrease weight. A
study in which hypercholesterolemia was induced in rats by giving them high fat
diet conducted by Ajaykumar et al. (2012). 1ml/day apple cider vinegar
was prescribed to rats for 14 days. Cholesterol, high density lipoprotein, low
density lipoprotein and very low density lipoprotein was measured. Results
showed that there was remarkable decrease in LDL and VLDL and increase in HDL
cholesterol. They negotiate that apple cider vinegar has significant
cholesterol lowering ability. Apple cider vinegar
has beneficial effect on lowering the serum level of cholesterol, triglycerides
and LDL after 8 weeks. A study on rats were conducted in which the consequence
of 30 ml apple cider vinegar for 60 days were evaluated and found that there
was adequate decrease in total cholesterol, LDL cholesterol, triglycerides and
increases the HDL cholesterol (Beheshti et
al., 2012).Impact of
apples on digestion of lipids on 40 male rats was deliberated. Rats were fed
with 0.3% cholesterol rich food. In the eating routine of rats, 15% lyophilized
apple was supplemented with glucose, fructose and sucrose. Blood tests were
taken after each 21 days and examined for triglyceride, HDL and cholesterol. Results
uncovered that slight but significant impact on cholesterol lowering in plasma
and in addition in liver. There was likewise decrease in triglyceride rich
lipoprotein and increase in HDL part while there was impressive impact in
cardiovascular protection effect (Aprikian et al., 2002). Effect of whole apple intake on plasma lipids was
observed. They concluded that 550g of whole apple intake for 30 days
significantly lower the low density lipoprotein cholesterol (Ravn et al.,
2013).Another examination was led to
check the Influence of apples, pears and peaches on lipids on rats .Apples,
pears and peaches were fed to rats at the rate of 10% based on dry weight and
non-oxidized cholesterol (NOC) at the rate of 1% was likewise included the
eating routine and found that apples and to a less degree pears and peaches supplemented
diet lowers the hypocholesterolemic impact in rats. The high substance of
polyphenols, phenolic acids and hypolipidemic properties of apples makes this
natural product best for dietary counteractive action of cardiovascular and
different infections (Leontowicz et al., 2002). The effect of dried apple and dried plum (75g/day) on post menopausal women was
compared. Fasting blood samples were collected at baseline, 3, 6 and 12 months
to measure serum total cholesterol and low density lipoprotein. The results
showed that there was notable reduction in serum level of total cholesterol and
high density lipoprotein cholesterol in women who consumed dried apple. This
study also concluded that daily apple consumption greatly improved atherogenic
risk factors (Chai et al.,
2012). The outcome
of dietary apple polyphenol from unripe apple on rats was determined by giving
them purified diet containing 0.5 % cholesterol and observed that dietary apple
polyphenol effectively decreases the low density lipoprotein cholesterol and
increases the high density lipoprotein cholesterol (Osada et al., 2006).
In another study effect of lyophilized apple on plasma and
tissue lipids on rats were checked. Experimental diet was equilibrated for
sugar supply contained 0.25/100 g cholesterol. Rats were hypercholesterolemic
with cholesterol accumulation in LDL and HDL fractions. Aprikian et al. (2002) concluded that apple diet
lowered plasma and LDL cholesterol (22 and 70 %) and reduced triglyceride
accumulation in heart and liver.Apple juice
also has potential to lower the hypercholesterolemia. Another study was
conducted on rabbits. They categorized the rabbits into four groups,
normal diet, high cholesterol diet, 1% cholesterol supplemented with 5 ml apple
juice and 1% cholesterol supplemented with 10 ml apple juice for 2 month. Setorki et al. (2009) conclude that
10ml apple juice significantly reduced the LDL but 5ml apple juice did not have
any effect on LDL. The effect
of apple pomace and apple juice concentrate on fat loss and lowering the cholesterol
was determined. Rats were fed with diet induced with apple pomace and apple
juice at the rate 10% each. After 5 week interval, rats were checked for their
body weight gain, adipose tissue weight, serum total cholesterol, LDL and HDL.
Results of this study showed that there was significant decrease in LDL and
increase in HDL lipoproteins. The results also indicated that both apple pomace
and juice could be helpful for reducing weight as well as improving lipid
profile (Cho et al., 2013).  Apple cider vinegar was considered as healthy
fruit for the remediation of hyperlipidemia in comparison with drug. In this
study they designed different groups in each group 25 healthy subjects were
categorized in the age of 40-60 years with hyperlipidemia. One group was
treated with apple cider vinegar while other group was given drugs. Apple cider
vinegar was given as a dose of 5cc two times a day for one month continuously
(30 min before lunch and dinner). Lovastatin drug was given at the rate of 20mg/day
for 4 weeks. Blood samples were taken before and after treatment with regular
time interval. Results of this experiment revealed that there was decrease in
the LDL and cholesterol and increase in HDL level. There was 25 and 26%
decrease in LDL while 35 and 36% increase in HDL level in blood after 4 weeks
in case of apple cider vinegar and lovastatin respectively. It is proved that
apple cider vinegar could be used as substitute for drugs with almost same
effect and with no side effect (Jaferian et al., 2006). .           The
impact of apple cider vinegar supplementation on serum triglycerides and total
cholesterol was checked and found that there was remarkable reduction in serum
lipid levels (Naziroglu et al.,
2014). The combination of apple and
apple cider vinegar with other products could also be effective for the
remediation of hyperlipidemia. They planned a study to check the combined
effect of honey and apple cider vinegar for lowering of blood lipids. Apple
cider vinegar was given as a dose of 200cc two times a day and honey was given
one tablespoon. Blood lipid levels were measured before and after treatment.
Results revealed that there was significant decrease in LDL and increase in HDL
by giving apple cider vinegar and honey in combination (Rishehri et al.,
2014). MATERIAL AND METHODSArea of ResearchThe research will be conducted in the labs of IHS and
NIFSAT, University of Agriculture Faisalabad.Procurement of
Raw MaterialApple and apple cider vinegar will be purchased from local
market of Faisalabad.Sample PreparationFresh apples will be taken and wash properly with tap water.
After drying, peel off the apple. Slice the apple into small pieces and extract
juice from electric juicer. Apple cider vinegar will be given (Aprikian et
al., 2002).  Proximate AnalysisAnalysis for moisture, ash content, crude protein, and crude
fiber, and NFE of apple juice and apple cider vinegar will be carried out
according to their respective methods (AACC 2000). And this procedure will be
conducted in the post graduate research laboratory, National Institute of Food
Science and Technology (NIFSAT), University of Agriculture Faisalabad.Moisture ContentHot air oven will be
used to determine the moisture content (AACC, 2000).Crude ProteinTo determine the protein content in sample Kjeldahl’s method
will be used (AACC,
2000).Crude
Fat  Soxhlet apparatus
will be used to check the crude fat of sample (AACC, 2000).Crude FiberThe grounded sample will be used for crude fiber
determination after fat (AACC,
2000).Nitrogen Free ExtractNitrogen free extract will also be determined by the method
described in (AACC,
2000).EFFICACY STUDYRats will be purchased from Animal Husbandry Department
University of Agriculture Faisalabad  Sample CollectionTwenty rats will be selected randomly and divide into two
groups.Ø  Group
A:  control group                                       (n=5)Ø  Group
B: Experimental group                             (n=15)Where n=number of individuals.Control group will be expressed as T1. The
experimental group will be further divide into 3 treatment groups as T2,
T3, and T4. 

Treatment                  

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Dosage

 

   Apple cider vinegar

    Apple juice

       T1

          Control

Control

       T2

         20ml/day

_______

        T3

        ________

30ml/day

        T4

       20ml/day

30ml/day

 The apple juice and apple cider vinegar will be added in
their regular diets. The study will be conducted for 60 days. Initially blood
sample will be taken from each rat and analyze at local laboratory to evaluate
the effect of apple juice and apple cider vinegar on the different biochemical
parameters.Induction of Hypercholesterolemia in RatsHypercholesterolemia will be induced in
rats by feeding them on high fat diet (banaspati ghee, corn oil, and cow ghee)
for 15 days baseline period (Tembhurne et al., 2012) Biochemical AnalysisBlood samples will be taken from each
rat at 0, 30th and 60th day and analyzed at a local
laboratory to evaluate the effect of apple juice and apple cider vinegar on
blood lipid including cholesterol, HDL, LDL, and triglycerides (Dong et al.,
2011).  Mineral Analysis
Apple juice and apple cider vinegar
will be analyzed for mineral content by wet digestion according to the methods
described in AOAC (2006).Statistical
Analysis

The data obtained for each parameter
will be analyzed statistically to determine the level of significance
(Montgomery, 2008).

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