Jacinto P. Elpa National High School
Capitol Hills, Tandag City
Science Technology and Engineering Curriculum
Phytochemical Screening and In vitro Analysis of Antihypertensive Activity of Ginger
(Zingiber Officinale) against HMG-CoA Reductase Inhibitory Activity
A Science Investigatory Project on Life Science (Team Category)
Ma. Esabel H. Llano
Samsheda C. Paudac
Glorian Iza T. Quilaton
Researchers
Ana Geran V. Millan
Research Adviser
S.Y. 2021-2022
1
Abstract
Zingiber officinale (ginger) has been used as an herbal medicine to treat various ailments
worldwide since antiquity. Hypertension is considered as the biggest single risk factor for
cardiovascular diseases, which have become the leading cause of death worldwide. According to the
World Health Organization (WHO), hypertension causes 7 million deaths every year while 1.5 billion
people suffer due to its complications. Cotton cloth was used to remove the dirt and dust from the plant
materials that was collected after they had been cleansed. The plant material was air dried before being
ground up in a blender to make up the sample (100 g), which was then macerated in ethanol at a volumeto-volume ratio of 95% for 48 hours. The testing followed standard laboratory approach to properly
measure the phytochemical contents and inhibitory impact of Ginger (Zingiber Officinale) extract. The
percentage of HMG-CoA Reductase Inhibitory Activity that each of the test drugs possessed was
analyzed using statistical methods such as mean, standard deviation, and Analysis of Variance
(ANOVA). Based on the results of the phytochemical screening, ginger (Zingiber Officinale) contains
saponins, tannins, steroids, flavonoids, and alkaloids. The percentage inhibition of 3-hydroxy-3methylglutaryl co-enzyme A (HMG-CoA) reductase infers the potential of Ginger (Zingiber Officinale)
extract and positive control as enzymatic inhibitors with similar activity as statins and are used
pharmacologically in cholesterol reduction, which can reduce the risk of coronary artery disease and
stroke. As a result of this research, we may infer that ginger (Zingiber Officinale) extract may be useful
as an inhibitor of HMG-CoA reductase. Furthermore, the results of the test revealed that ginger
(Zingiber Officinale) extract contains flavonoids. Flavonoids have been shown to reduce low-density
lipoprotein oxidation, control lipid peroxidation, and slow the evolution of atherosclerotic plaques in
patients with cardiovascular disease.
Keywords: Zingiber officinale (ginger), Hypertension, HMG-CoA, Phytochemical Screening,
Flavonoids
2
TABLE OF CONTENTS
Page
Abstract
1
Table of contents
2
Definition of key Terms
4
CHAPTER 1: INTRODUCTION
Rationale of the Study
5
Statement of the Problem
7
Research Objectives
8
Statement of the Null Hypothesis
8
Significance of the Study
8
Scope and Limitations
9
Conceptual Framework
9
Flowchart of the Study
11
CHAPTER 2: REVIEW OF RELATED LITERATURE AND STUDIES
12
CHAPTER 3: RESEARCH METHODOLOGY
Research Design
23
Sampling Design
23
Data Gathering Procedures
23
3
Statistical Tools
27
CHAPTER 4: PRESENTATION, ANALYSIS, AND
INTERPRETATION OF DATA
28
CHAPTER 5: SUMMARY, FINDINGS, AND RECOMMENDATION
33
REFERENCES
36
CURRICULUM VITAE
37
ACKNOWLEDGEMENT
41
RESEARCHERS LOG BOOK
42
4
DEFINITION OF KEY TERMS
To understand the study, the following terms are operationally defined: The following
terms are defined to provide clarity and better understanding of the words frequently used
in the following pages:
Ginger (Zingiber officinale)- a hot, fragrant spice made from the rhizome of a plant,
which may be chopped or powdered for cooking, preserved in syrup, or candied.
Phytochemical screening- refers to the extraction, screening, and identification of the
medicinally active substances found in the plant.
Hypertension- blood pressure that is higher than normal. Your blood pressure
changes throughout the day based on your activities. Having blood pressure measures
consistently above normal may result in a diagnosis of high blood pressure (or
hypertension).
5
CHAPTER 1
INTRODUCTION
Rationale of the study
Hypertension is considered as the biggest single risk factor for cardiovascular diseases
which becomes the leading cause of death worldwide. According to the World Health
Organization (WHO), hypertension causes 7 million deaths every year while 1.5 billion people
suffer due to its complications. It is also called the silent killer because it shows no direct
symptoms and many people die of the disease without understanding it (Meresa A, et.al, 2017).
Hypertension is defined as having a systolic blood pressure of > 140 mmHg and a diastolic
blood pressure of > 90 mmHg (> 140/ > 90 mmHg) (Tabassum and Ahmad, 2011). It causes
heart attack, stroke, heart failure, aneurysm or renal failure.
Drugs that are used in the treatment of high blood pressure are called antihypertensive.
Despite the availability and effectiveness of these synthetic drugs which used for the treatment
of hypertension, yet, it is believed to cause negative side effects and increased the risks of
developing new diseases (Meresa A, et.al, 2017). Due to the side effects and high prices of
antihypertensive drugs, most people directed to alternative medicines of plant origin especially
those dwelling in rural areas for their treatment of hypertension (Sara S. AlDisi, et.al) and its
complications. Over the decades, the use of medicinal plants represents the interaction between
humans and the environment.
The use of medicinal plants for treatment of hypertension is very common because these
remedies are easily available and low cost than innovative pharmaceuticals. Herbal medicines,
therefore, are gaining great demand and more importance in the treatment of hypertension
because of their wide biological and medicinal activities (Meresa A, et.al, 2017). Herbs do not
6
cause side effects like weakness, tiredness, drowsiness, impotence, cold hands and feet,
depression, insomnia, abnormal heartbeats, skin rash, dry mouth, dry cough, stuffy nose,
headache, dizziness, swelling around eyes, constipation or diarrhea and fever.
Out of the major risk factors, which include diabetes, smoking, and dyslipidemia,
hypertension is by far the most prevalent trigger for cardiovascular diseases (Sara S. Al Disi,
et.al), and its comorbidity with other risk factors is even more puissant (Yang et al., 2011;
WHO, 2013). Hypertension is responsible for around 16.5% of annual deaths worldwide
(WHO, 2013), and is indeed the main cause of morbidity and mortality associated with
cardiovascular diseases (Kizhakekuttu and Widlansky, 2010). By 2030, the annual death toll is
estimated to reach 23.5 million people (WHO, 2013). In addition to being a major player in the
onset of diseases such as atherosclerosis, stroke, peripheral artery disease, heart failure, and
coronary artery disease, hypertension can also lead to kidney damage, dementia, or blindness
(August, 2004; Freedman and Cohen, 2016).
According to the Department of Health (DOH), about eight out of ten people who had
their first stroke are diagnosed with hypertension - responsible for worsening the quality of
lives of some 14 million Filipinos. The DOH also revealed that more than 276 Filipinos die of
heart disease on a daily basis and at least one Filipino suffers from stroke every nine minutes.
Survivors have a 75 percent chance of becoming permanently disabled.
Reducing low-density lipoprotein cholesterol (LDL-C) levels lowers the risk of
consequences of cardiovascular disease. Research has confirmed these benefits in elderly
patients. The 3-hydroxy-3-methylglutaryl coenzyme A inhibitors (i.e, statins) have longstanding proven efficacy in reducing levels of LDL-C and total cholesterol (Harley, CR et.al,
2009).
7
Most people who are suffering from stroke or having high blood pressure have been
prescribed by their doctors to use statins such as atorvastatin to regulate their blood cholesterol
and blood pressure. Also known as statins, HMG-CoA (3- hydroxyl- 3- methylglutaryl
coenzyme A) reductase inhibitors work by inhibiting the synthesis of cholesterol in the liver
by the enzyme HMG-CoA reductase. These drugs, which include pravastatin, fluvastatin,
atorvastatin, simvastatin and rosuvastatin, are the mainstay of therapy for elevated LDL
cholesterol and both primary and secondary prevention of acute coronary syndrome and stroke
(Stone NJ, et al, 2013). HMG-CoA (3- hydroxyl- 3- methylglutaryl coenzyme A) reductase
inhibitors are very effective in lowering total and low- density lipoprotein cholesterol
(Hunninghake, Donald B., 1992).
Based on the above premises, the researcher intends to find out the presence of 3hydroxy-3-methylglutaryl co-enzyme A reductase inhibitory activity of inger (Zingiber
Officinale). Further, this research aims to determine if inger (Zingiber Officinale) serves as a
good source of statins which can be used as an alternative medicine to treat hypertension.
Statement of the Problem
This study seeks to answer the following questions:
1. What are the phytochemicals present in the Ginger (Zingiber Officinale) extract?
2. What is the mean percentage inhibitory activity of Ginger (Zingiber Officinale)
extract, Atorvastatin (Positive control), and distilled water (negative control) against
HMG-CoA reductase enzyme?
3. Is there a significant difference on the mean percentage inhibitory activity of Ginger
(Zingiber Officinale) extract, Atorvastatin (Positive control), and distilled water
(negative control) against HMG-CoA reductase enzyme?
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4. Which of the three test drugs significantly inhibit the HMG-CoA reductase enzyme?
Research Objectives
To carry or investigation, the following objectives were evaluated. Specifically, the study
aimed to:
1. To determine the phytochemicals, present in the Ginger (Zingiber Officinale) extract.
2. To determine the mean percentage inhibitory activity of Ginger (Zingiber Officinale)
extract, Atorvastatin (Positive control), and distilled water (negative control) against
HMG-CoA reductase enzyme.
3. To calculate the significant difference on the mean percentage inhibitory activity of
Ginger (Zingiber Officinale) extract, Atorvastatin (Positive control), and distilled
water (negative control) against HMG-CoA reductase enzyme.
4. To assess which of the three test drugs significantly inhibit the HMG-CoA reductase
enzyme.
Statement of the Null Hypothesis
1. There is no significant difference on the mean percentage inhibitory activity of Ginger
(Zingiber Officinale) extract, Atorvastatin (Positive control), and distilled water
(negative control) against HMG-CoA reductase enzyme.
Significance of the Study
This study is undertaken to determine which of the chemical compounds of Ginger
(Zingiber Officinale) extract would benefit the people who are suffering from hypertension.
9
The importance of this study is to find what flavonoids are present to respond
hypertension. This study might help future researchers and may provide them with the
knowledge of Ginger (Zingiber Officinale) can be a medication for antihypertensive..
This study has significance to the people, society, country, and the world. The
generalization of this present would be a great contribution to the vast knowledge in relation
to people's health.
Scope and Limitations
This study focuses on the phytochemical screening of Ginger (Zingiber Officinale) on
the alkaloids present as we conduct the study. The plant is to be collected at Tandag City.
This study is limited to the phytochemical screening and flavonoids present.
Conceptual Framework
The independent variables in this study are the following: Ginger (Zingiber Officinale)
extract, atorvastatin and negative control. Study showed that Ginger (Zingiber Officinale)
exhibits gut stimulatory, inhibitory and hypotensive effects mediating possibly through
cholinergic, Ca (2+) antagonist and the combination of these mechanisms respectively.
Diuretic activity adds value to its use in hypertension (Jabeen et.al, 2009). The same report also
showed that coriander fruit extracts produced dose-dependent relaxation of pre-constricted
(phenylephrine and potassium chloride) rabbit aortas, and this response was atropine and
calcium-channel dependent (Jabeen et al., 2009). Further, the same extracts showed diuretic
affects as well. The active component of which should act synergistically with the vasoactive
constituent to complement the treatment and management of hypertension (Jabeen et al., 2009).
The dependent variables of this study are the Phytochemical content, and % HMG-CoA
reductase inhibitory activity of Ginger (Zingiber Officinale) extract, atorvastatin and negative
control.
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DEPENDENT VARIABLES
INDEPENDENT
VARIABLES
Phytochemical screening of
Ginger Extract
Ginger Extract
Percentage Inhibition of three
test drugs against HMG-CoA
reductase
Atorvastatin (Positive control)
Distilled water (Negative
control)
Figure 1.
Paradigm of the Study
11
Flowchart of the Study
Plant Extraction
Phytochemical Screening
Enzyme Assay Procedures
Preparation of Extract concentration
12
CHAPTER II
REVIEW OF RELATED LITERATURE AND STUDIES
Hypertension has become one of the most principal growing health problems in
developing countries, and is an important cause of cardiovascular death in the world (Meresa
A, et.al) Globally, the overall prevalence of raised blood pressure in adults aged 25 and over
was around 40% in 2008. The proportion of the world’s population with high blood pressure,
or uncontrolled hypertension, fell modestly between 1980 and 2008. However, because of
population growth and ageing, the number of people with uncontrolled hypertension rose from
600 million in 1980 to nearly 1 billion in 2008.
Across the WHO regions, the prevalence of raised blood pressure was highest in Africa,
where it was 46% for both sexes combined. Both men and women have high rates of raised
blood pressure in the Africa region, with prevalence rates over 40%. The lowest prevalence of
raised blood pressure was in the WHO Region of the Americas at 35% for both sexes. Men in
this region had higher prevalence than women (39% for men and 32% for women). In all WHO
regions, men have slightly higher prevalence of raised blood pressure than women. This
difference was only statistically significant in the Americas and Europe.
In the Philippines, heart attack is the most common cause of death among Filipinos.
“This may be attributed to continuous neglect on the danger of hypertension and its
complications,” according to Dr. Dante Morales, President of the Philippine Society of
Hypertension (PSH). A study conducted by PSH found out that the prevalence of hypertension
in the country is increasing. In 2003, data showed that 16 percent or approximately 7 million
Filipino adults 20 years and above has hypertension. The incidence increased to 10 million or
21 percent of Filipino adults 20 years and above in 2008. Hypertension is called the “silent
13
killer” for its lack of symptoms and can go undiagnosed for years. In the Philippines, majority
of those with hypertension don’t even realize they have high blood pressure.
According to Manila Standard Lifestyle, 2016 “In the midst of government warning
that the number of Filipinos with high cholesterol has been increasing to an alarming level, it
was noted that the prevalence was higher in women compared to men. This was revealed by
Dr. Imelda Angeles-Agdeppa, assistant scientist at Food and Nutrition Research Institute
(FNRI) during the 56th Philippine Association of Thoracic and Cardiovascular Surgeons, Inc.
(PATACSI) According to Agdeppa, “A latest survey conducted by FNRI in 2013 showed that
females registered an increase of borderline cholesterol level of 51.4 percent compared to men,
which is only about 41.5 percent.” They have no study to explain why the cholesterol level is
higher in women than in men. However, she speculated it could be attributed to sedentary
lifestyles among females (Manila Standard Lifestyle, 2016).
According to the World Health Organization, about 80% of the human population
depend on alternative medicine for the primary treatment of various diseases. Medicinal plants
have been widely reported to have medicinal properties, nutritional value, and pharmacological
activities such as antioxidant, antithrombotic, anti-inflammatory, antiartherogenic, and
cardioprotective effects. (Baskaran, G. et.al, 2015).
Phytochemicals in medicinal plants have gained much interest among researchers and
the pharmaceutical and food manufacturing industries. Plant flavonoids offer significant
protection against the development of chronic illnesses such as diabetes, tumors, cancer, and
cardiovascular diseases. Flavonoids have been reported to reduce LDL oxidation, suppress
lipid peroxidation, and decrease the progression of atherosclerotic lesions in cardiovascular
diseases. The potential of medicinal plants for the treatment of hypercholesterolemia is still
14
largely unexplored and could be an alternative strategy for the progression of effective and safe
antihypercholesterolemia drugs. (Baskaran, G. et.al, 2015)
Cholesterol is a waxy, fat-like substance that’s found in all cells of the body that is
needed to make hormones, vitamin D and substances that help people digest foods. Cholesterol
travels through bloodstream in small packages called lipoproteins. These packages are made
of fat (lipid) on the inside and proteins on the outside.
Two kinds of lipoproteins carry cholesterol throughout the body: low-density
lipoproteins (LDL) and high-density lipoproteins (HDL). Having healthy levels of both types
of lipoproteins is important. LDL cholesterol sometimes is called “bad” cholesterol. A high
LDL level leads to a build-up of cholesterol in the arteries. HDL cholesterol sometimes is
called “good” cholesterol. This is because it carries cholesterol from other parts of the body
back to the liver. The liver removes the cholesterol from the body.
‘Ginger belongs to the healthiest vegetable on the planet. It is loaded with nutrients
and bioactive compounds that benefit us and it has powerful benefits for your body and
brain.”—Joe Leech, in an article published in the newsletter, Healthline. Ginger may help lower
cholesterol levels in our bodies. High levels of LDL lipoproteins (to be the “bad” cholesterol)
are linked to an increased risk of heart disease. The foods you eat can have a strong influence
on LDL levels. (Henrylito D. Tacio, 2017)
Oxidative modification of LDL is thought to play a key role in the pathogenesis of
atherosclerosis. Consumption of nutrients rich in phenolic antioxidants is associated with
attenuation of the development of atherosclerosis. Aortic atherosclerotic lesion areas were
reduced by 44% (P < 0.01) in mice that consumed 250 μg of ginger extract/day. Furthermore,
peritoneal macrophages which means the macrophages that reside in the peritoneal cavity, a
15
fluid-filled space located between the wall of the abdomen and the organs found in the abdomen
harvested from E0 mice after consumption of 25 or 250 μg of ginger extract/day had a lower
(P < 0.01) capacity to oxidize LDL (by 45 and by 60%, respectively), and to consume and
degrade oxidized LDL (by 43 and 47%, respectively). Consumption of 250 μg of ginger
extract/day may also lessen (P < 0.01) the basal level of LDL-associated lipid peroxides by
62%. The researcher concludes that dietary consumption of ginger extract significantly
attenuates the development of atherosclerotic lesions. This provides or inhibiting atherogenesis
antiatherogenic effect is associated with a significant reduction in plasma and LDL cholesterol
levels and a significant limitation in the LDL basal oxidative state, as well as their susceptibility
to oxidation and aggregation.(Bianca Fuhrman, Mira Rosenblat, Tony Hayek, Raymond
Coleman, Michael Aviram, May 2000).
The enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase is the
key enzyme of the mevalonate pathway that produces cholesterol. Inhibition of HMG-CoA
reductase reduces cholesterol biosynthesis in the liver. Synthetic drugs, statins, are commonly
used for the treatment of hypercholesterolemia. Due to the side effects of statins, natural HMGCoA reductase inhibitors of plant origin are needed (Baskaran, G. et.al, 2015). The inhibition
of HMG-CoA reductase effectively lowers the level of cholesterol in humans and most animals
by the activation of sterol regulatory element-binding protein-2, which upregulates the HMGCoA reductase and LDL receptor that lead to the reduction of cholesterol levels. Although
statins are well-known HMG-CoA reductase inhibitors, long-term consumption of statins cause
severe adverse effects such as muscle and liver damage, rhabdomyolysis, and acute renal
failure. (Baskaran, G. et.al, 2015).
The mevalonate pathway, which starts with the synthesis of mevalonate by HMGR, has
more branch pathways in plants than in most other organisms, leading to a tremendous variety
16
of isoprenoid products (Stermer BA, et.al, 1994). The enzyme 3-hydroxy-3-methylglutaryl
CoA (HMG-CoA) reductase catalyzes the NADPH-mediated reductive deacylation of HMGCoA to mevalonic acid, which is the first committed step of the mevalonate pathway for
isoprenoid biosynthesis (Campos,N., et.al, 2014)
17
Figure 2. Shows the mevalonate pathway of 3- hydroxy- 3- methylglutaryl co-enzyme A
(HMG-CoA) reductase synthesis.
18
HMG-CoA reductase inhibitors, also known as statins, selectively inhibit an enzyme
called HMG-CoA reductase (3-hydroxy-3-methylglutaryl coenzyme A reductase) that is
involved in the synthesis of mevalonate, a precursor of sterols including cholesterol. By
inhibiting this enzyme, cholesterol and LDL-cholesterol production is decreased. Statins also
increase the number of LDL receptors on liver cells, which enhances the uptake and breakdown
of LDL-cholesterol. Most of the effects of statins occur in the liver. Research has shown that
elevated levels of total cholesterol, LDL-cholesterol, and apolipoprotein B are risk factors for
developing cardiovascular disease. Statins may be used in the treatment of hyperlipidemia (also
called dyslipidemia or high cholesterol) and are most effective at lowering LDL-cholesterol.
(Oregon Health & Science University, 2009)
The result of an aqueous extract of ginger (Zingiber officinale) on serum cholesterol
and triglyceride levels as well as platelet thromboxane-B(2) and prostaglandin-E(2) production
was examined. A raw aqueous extract of ginger was administered daily. Fasting blood serum
has been investigated for thromboxane-B(2), prostaglandin-E(2), cholesterol, and triglycerides.
However, the ginger administered orally caused significant changes to the body in the serum
PGE(2) at this dose. High doses of ginger (500 mg/kg) were significantly very valuable in
lowering serum PGE(2) when given either orally or IP. A significant reduction in serum
cholesterol was observed when a higher dose of ginger (500 mg/kg) was administered to help
everyone. At a low dose of ginger (50 mg/kg), a significant depletion in the serum cholesterol
has been observed only when the ginger was administered IP. There were no significant
changes in serum triglyceride levels that have been observed upon administration of either the
low or high dose of ginger. These results advocate that ginger can be effective as a cholesterollowering, antithrombotic and anti-inflammatory agent. (Ali M, Alnaqeeb MA, Al-Qattan KK,
Al-Sawan SM, Thomson M, Khan I 2002)
19
Both gender male and female patients were enrolled. Patients have randomly divided
into two groups 30 patients were on the drug ginger pasted powder and advised to take 5 grams
in divided doses with their normal diet for three months. Thirty patients were on placebo pasted
what powder, with the same color as ginger powder, is advisable to take 5 grams in divided
doses with their normal diet for three months. Their baseline lipid profile and body weight were
recorded at the start of treatment and were advised to come for check-ups, fortnightly. When
the duration of the study was over, their lipid profile and body weight were measured and
compared statistically with pre-treatment values. Three months of treatment with 5 grams of
Ginger reduced LDL cholesterol by 17.41%, total cholesterol by 8.83%, and body weight by
2.11%. When compared with the placebo group, all changes in mentioned parameters were
significant bio-statistically.
A plant rhizome that's commonly used as a cooking spice may help lower it. Ginger has
been used as a remedy for headaches, nausea, vomiting, motion sickness, and arthritis. Ginger
may reduce inflammation, and this may be why some researchers think it's beneficial for your
heart and lowers high cholesterol. More studies are needed to prove this, however. Researchers
of a study published in the journal "Food and Function" in 2013 state that the mechanism by
which ginger may lower cholesterol is well understood by scientists and other researchers.
They specify that ginger activates an enzyme that increases your body's use of cholesterol and
lowers it. Several studies show that ginger can lower experimentally induced high cholesterol
in people, but more studies on ginger's effect on humans with high cholesterol are needed
before the substance can be touted as a treatment for high cholesterol.
Zingiber officinale (ginger) has been used as herbal medicine to treat and remove
various ailments worldwide since antiquity. Recent evidence revealed the potential of ginger
for the treatment of diabetes mellitus. Data has demonstrated the antihyperglycaemic effect of
ginger. The mechanisms underlying these actions are associated with insulin release and action,
20
and improved carbohydrate and lipid metabolism in our system. The most active ingredients
that can be found in ginger are the following: pungent principles, gingerols, and shogaol.
Ginger has shown that it has prominent protective effects on the diabetic liver, kidney, eye, and
neural system complications. (Li Y, Tran VH, Duke CC, Roufogalis BD 2012)
Ginger is an underground rhizome of the plant Zingiber Officinale belonging to the
family Zingiberaceae and now, it is considered a common constituent of diet worldwide
because of its capability (Sertie et al., 1991). Moreover, ginger is well known all over the world,
especially for its use in any abnormal sickness(Tanabe et al., 1993). It has been reported that
the ginger plant has medicinal properties against digestive disorders, rheumatism, and diabetes
(Afzal et al., 2001). The ginger extract possesses antioxidative characteristics since it can
scavenge superoxide anion and hydroxyl radicals (Krishnakantha and Lokesh, 1993). Akhani
et al. Other investigators (Sharma et al., 1996) have shown that the hypolipidemic cause of
ginger.
Ginger is an underground rhizome of the plant Zingiber Officinale belonging to the
family Zingiberaceae and now, it is considered a common constituent of diet worldwide
because of its capability (Sertie et al., 1991). Moreover, ginger is well known all over the world,
especially for its use in any abnormal sickness(Tanabe et al., 1993). It has been reported that
the ginger plant has medicinal properties against digestive disorders, rheumatism, and diabetes
(Afzal et al., 2001). The ginger extract possesses antioxidative characteristics since it can
scavenge superoxide anion and hydroxyl radicals (Krishnakantha and Lokesh, 1993). Akhani
et al. Other investigators (Sharma et al., 1996) have shown the hypolipidemic cause of ginger.
Furthermore, Bhandari et al. (1998) have reported that an ethanolic extract of ginger
prevents hypercholesterolemia and the development of atherosclerosis in cholesterol. Bhandari
et al. (2005) identified that the ethanolic extract of ginger significantly reduced serum total
21
cholesterol and triglycerides and increased the HDL-cholesterol levels; also, the extract can
preserve tissues from lipid peroxidation and exhibit significant lipid-lowering activity in
diabetic rats. Keeping in view the significant importance of ginger this research study was
conducted to investigate the effect of ginger on the blood biochemistry parameters of broiler.
In recent years, accumulating data have suggested that traditional herbs might be able
to provide a wide range of remedies for the prevention and treatment of Mets. Ginger (Zingiber
officinale Roscoe, Zingiberaceae) has been documented to improve hyperlipidemia,
hyperglycemia, oxidative stress, and inflammation. These beneficial effects are mediated by
transcription factors, such as peroxisome proliferator-activated receptors, adenosine
monophosphate-activated protein kinase, and nuclear factor κB. (Jing Wang, Weixin Ke, Rui
Bao, Xiaosong Hu, Fang Chen, 2017)
Most of the experimental studies has been supported the weight of reducing the effect
of ginger extract or powder in obese animal models, whereas the results of the available limited
clinical studies showed no changes or slight changes in anthropometric measurements and body
composition in subjects with obesity. Ginger could regulate obesity through various potential
mechanisms including increasing thermogenesis, increasing lipolysis, suppression of
lipogenesis, inhibition of intestinal fat absorption, and controlling appetite. (Vahideh
Ebrahimzadeh Attari, et. Al 2018)
Many humans experience the trials that have been carried out with garlic, onion, and
fenugreek. The mechanism underlying the hypocholesterolemic and hypotriglyceridemic
influence of spices is equitably well understood. The health implications of the
hypocholesterolemic effect of spices experimentally documented are cardio-protection,
protection of the structural integrity of erythrocytes by restoration of membrane
22
cholesterol/phospholipid profile, and prevention of cholesterol gallstones by modulation of the
cholesterol saturation index in bile. ( Srinivasan K. Food Funct. 2013)
In addition, Researchers in a study published in the journal "Food and Function" in
2013 stated that the mechanism by which ginger may lower cholesterol is well understood by
scientists and other researchers that benefit people with high cholesterol. They explain that
ginger activates an enzyme that increases your body's use of cholesterol and lowers it. Several
studies show that ginger can lower experimentally induced high cholesterol in animals, but
more studies on ginger's effect on humans with high cholesterol are needed before the
substance can be touted as a treatment for high cholesterol.
Furthermore, in a study that has been published in "Saudi Medical Journal" in 2008,
researchers split individuals with high cholesterol into two groups and gave one group 3 grams
of ginger daily split into three 1-gram capsules. They gave the other group lactose capsules
instead. At the end of 45 days, both of the groups had lower total and "bad" cholesterol, along
with higher "good" cholesterol. The ginger group experienced a significantly larger change,
however, indicating that ginger may lower cholesterol bad cholesterol and increase good
cholesterol.
23
CHAPTER 3
METHODOLOGY
This chapter outlines the research design, sampling design, and laboratory procedures,
biosafety and ethical considerations and proper waste disposal employed in the study. The
laboratory procedures include the step-by-step protocol on how to determine the phytochemical
contents and HMG- CoA reductase inhibitory activity of selected plant extracts.
Research Design
Analyzing the phytochemical constituents of Ginger (Zingiber Officinale) extract in
this study required the use of both the quantitative research design and the experimental
research design. Positive controls included the catalytic domain 3-hydroxy-3-methyl-glutarylcoenzyme A (HMG-CoA) reductase, substrate 3-hydroxy-3-methyl-glutaryl-coenzyme A
(HMG-CoA) trisodium salt, NADPH, and atorvastatin the laboratory, the extracts of the herbs
were put through a series of tests utilizing specialized laboratory equipment and kits in order
to examine and quantify the total flavonoid content of the extracts as well as the phytochemical
components of the herbs. This investigation utilized posttest true experimental study and was
carried out in vitro.
Sampling Design
In this study, a non-probability purposive sample design was utilized in the collecting
of fresh Ginger (Zingiber Officinale) in Tandag City, Surigao del Sur.
Data Gathering Procedures
A. Plant Extraction
24
Cotton cloth was used to remove the dirt and dust from the plant materials that had been
collected after they had been cleansed. The plant material was allowed to air dry before being
ground up in a blender to make up the sample (100 g), which was then macerated in ethanol at
a volume-to-volume ratio of 95% for 48 hours. Following filtering, the extracts were separated
using a rotary evaporator at a temperature of sixty degrees Celsius.
B. Phytochemical screening
B.1. Test for Tannins Following the stirring of approximately 0.5 g of each part with
approximately 10 ml of distilled water, the mixture was filtered. A few drops of a ferric chloride
solution with a concentration of one percent were added to two milliliters of the filtrate. The
presence of a blue-black, green, or blue-green precipitate confirms the presence of tannins
(Trease and Evans, 2002).
B.2. Liebermann-Burchard test for steroids After adding 2 ml of acetic acid to 0.2 g of
each component and allowing the solution to thoroughly cool in ice, concentrated hydrochloric
acid was very carefully added to the mixture. The appearance of a blue or bluish-green color
after an initial violet hue showed the presence of a steroidal ring, also known as the aglycone
component of cardiac glycoside (Sofowora, 1993).
B.3 Test for the Presence of Saponins After boiling one gram of each part in five
milliliters of distilled water, the resulting mixture was filtered. After adding another
approximately 3 milliliters of distilled water to the filter, it was then forcefully agitated for
approximately 5 minutes. The fact that foaming continued even after the temperature had been
raised was interpreted as proof of the presence of saponins (Sofowora, 1993).
25
B.4. Test for flavonoids using ferric chloride at step B.4 An equal amount of each part
was brought to a boil with distilled water, and the resulting liquid was filtered. After that, a few
drops of a ferric chloride solution that was 10 percent concentrated were added to 2 milliliters
of the filtrate. The presence of a phenolic hydroxyl group may be identified by a coloration that
was green-blue or violet (Trease and Evans, 2002).
B.5. Test for alkaloids After stirring a little bit of each component with 5 milliliters of
an aqueous solution of 1 percent hydrochloric acid on a water bath, the mixture was filtered. A
total of 1 milliliter was removed from the filtrate and placed in each of the test tubes. A few
drops of Dragendorff's reagent were added to the first portion, and the presence of an orangered precipitate was interpreted as evidence of a positive result. Mayer's reagent was applied to
the second 1 ml, and the existence of a precipitate with a buff color will serve as an indication
that alkaloids are present (Sofowora, 1993).
C. Enzyme Assay Procedures
Analytical grade substances were utilized for the entirety of the investigation's
pharmacological and chemical components. In this work, the following components were
utilized: HMG-CoA Substrate Solution (2 milliliters), HMG-CoA Reductase (Catalytic
domain) (200 microliters), Assay Buffer (50 milliliters), and Atorvastatin Inhibitor Solution
(200 micrograms).
Reaction volumes for 96 well plate samples
Sample
1 x Assay Buffer
Atorvastatin
HMG-COA Substrate
HMG
R
Blank
184 µL
12 µL
-
26
4 µL
Inhibition 181 µL
Sample
181 µL
1 µL
12 µL
4 µL
1 µL (Sample)
12 µL
4 µL
D. Preparation of Extract Concentration
The addition of the reagents to the reaction (the wells) should take place in the following
order: To begin, transfer fifty milliliters of the assay buffer into each sample container. After
that, the inhibitor, which is atorvastatin in this instance, is added to the positive control sample
that represents the inhibition. The substrate solution, which is HMG-CoA, is going to be added
to each of the samples as the next step. After that, HMG-CoA Reductase (HMGR) needs to be
added to the samples of both the Activity and the Inhibition conditions. In addition, as the very
last step, thoroughly mix the samples. Note: Before taking the initial absorbance measurement,
ensure that the plate that you will be using the plate reader on has been forcefully shaken for at
least 10 seconds before beginning the measurement process.
Biosafety and Ethical Considerations
During the course of the experiment, careful manipulation of the chemical ethanol was
observed at all times. Due to the fact that used ethanol can easily catch fire and spread flames,
it was disposed of as a hazardous waste, in accordance with DENR Administrative Order No.
29 Series 1992 and the guidelines set forth by the Environmental and Protection Agency (EPA).
Statistical Tools
In this study, the percentage of HMG-CoA Reductase Inhibitory Activity that each of
the test drugs possessed was analyzed using statistical methods such as mean, standard
deviation, and Analysis of Variance (ANOVA). These methods were used to determine
27
whether or not there was a statistically significant difference in the percentage of HMG-CoA
Reductase Inhibitory Activity that Ginger (Zingiber Officinale) extract, positive control, and
negative control all possessed.
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CHAPTER 4
PRESENTATION, ANALYSIS, AND INTERPRETATION OF DATA
The activity of Ginger (Zingiber Officinale) extract as an enzymatic inhibitor against
3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, a mevalonate pathway to
create cholesterol, is presented in this chapter together with a positive control and a negative
control. The experiment was carried out in vitro with the catalytic domain of 3-hydroxy-3methyl-glutaryl-coenzyme A (HMG-CoA) reductase, the substrate 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) trisodium salt, NADPH, with atorvastatin serving as the
positive control. Tabular and graphical representations of the overall data are provided so that
one can reduce the potential of Ginger (Zingiber Officinale) extract, positive control, and
negative control as enzyme inhibitors, with the latter two having activity comparable to that of
statins.
Phytochemical Screening
The phytochemical screening of Ginger (Zingiber Officinale) extract was done
qualitatively and results are shown in table 1.
Table 1
Phytochemical Screening of Ginger (Zingiber Officinale) extract
Phytochemicals
Trial 1
Trial 2
Trial 3
Tannins
+
+
+
Steroids
+
+
+
Saponins
+
+
+
Flavonoids
+
+
+
Alkaloids
+
+
+
29
Results of the test confirms the presence of phytochemicals tannins, steroids, saponins,
flavonoids, and alkaloids in Ginger (Zingiber Officinale) extract. The presence of these
phytochemicals may explain the potential of Ginger (Zingiber Officinale) extract as an
inhibitor against HMG-CoA reductase. Meanwhile, the findings of this study agreed to the
results of Jan et al (2022) which showed that the preliminary screening showed the presence of
tannins, phenolics, flavonoids, saponins and carbohydrates, steroids and alkaloids in all the
extracts. The phenolic and flavonoid content of dried ginger was found higher in ethanolic
extracts compared to fresh ones as revealed by HPLC.
Meanwhile, Flavonoids are phenolic chemicals that may be obtained from a wide
variety of vascular plants. There are over 8000 different flavonoids that have been identified
so far. In plants, they provide the functions of antioxidants, antimicrobials, photoreceptors,
visual attractants, feeding repellants, and screen for light. Many studies have found evidence
that flavonoids have biological effects, such as anti-inflammatory, anti-allergenic, antiviral,
and vasodilating properties. However, the majority of attention has been focused on the
antioxidant activity of flavonoids. This is because flavonoids have the potential to both inhibit
the creation of free radicals and remove existing ones from the body. In the past few years, the
ability of flavonoids to function as antioxidants in vitro has been the focus of a number of
investigations, and significant structure-activity connections regarding the antioxidant activity
have been established as a result of these studies. It is less well documented that flavonoids are
effective antioxidants in vivo, most likely because there is so little information on how
flavonoids are absorbed by people. The vast majority of flavonoids that are consumed are
significantly broken down into a variety of phenolic acids, some of which still contain the
capacity to scavenge radicals. An in vivo antioxidant activity may be displayed by the absorbed
flavonoids as well as their metabolites. This is demonstrated experimentally by an increase in
the plasma antioxidant status, a sparing effect on the vitamin E of erythrocyte membranes and
30
low-density lipoproteins, and the preservation of erythrocyte membrane polyunsaturated fatty
acids. This review offers the most up-to-date information on the structural properties and in
vitro antioxidant capacity of the most prevalent flavonoids, as well as in vivo antioxidant
activity and impacts on the body's own endogenous antioxidants (Pieta, P.2010). On the basis
of these data, one might draw the conclusion that the plant material in question has the potential
to act as an inhibitor of HMG-COA reductase activity.
HMG-COA Reductase Inhibitory Activity
The enzyme known as 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA
reductase) is the essential component of the mevalonate pathway, which is responsible for the
production of cholesterol. The production of cholesterol in the liver is slowed down when the
enzyme HMG-CoA reductase is inhibited. Positive control for the study was provided by
synthetic medications known as statins, which are routinely used for the treatment of
hypercholesterolemia. One example of such a medication is atorvastatin. There is a demand for
natural HMG-CoA reductase inhibitors derived from plants because statins might have
unwanted side effects. Ginger (Zingiber Officinale) extract was a plant that was subjected to
an experiment to determine whether or not they had the ability to act as an inhibitor to the
metabolic pathway that leads to the creation of cholesterol. In the current investigation, the
HMG-CoA reductase activity of Ginger (Zingiber Officinale) extract, the complete results are
shown in table 2.
Table 2
Percentage Inhibitory Activity of Ginger (Zingiber Officinale) extract, Positive control,
and Negative Control towards HMG-CoA reductase
Treatments
% INHIBITION
T1
T2
T3
31
Mean
S.D.
%CV
Atorvastatin
ginger (Zingiber
officinale) extract
Negative control
88.5
56.4
87.8
58.4
88.6
58.5
88.3
57.8
0.4359
1.1846
0.4936
2.05
1.2
1.4
1.2
1.3
0.1155
9.1161
Results of the test showed that the natural HMG-CoA reductase inhibitors of Ginger
(Zingiber Officinale) extract showed an inhibition of 57.8±1.1846%. Of the three test drugs,
atorvastatin a commercial antihypertensive drug, was found to have shown the highest
inhibition of 88.3±0.4359%.
To determine if there is an existing significant difference on the Ginger (Zingiber
Officinale) extract, Positive control, and Negative Control towards HMG-CoA reductase, OneWay Analysis of Variance (ANOVA) was utilized and results are shown in table 3A to 3B.
Table 3A
One-Way Analysis of Variance on the Percentage Inhibitory Activity
Of Various Plant Extract towards HMG-CoA reductase
Source of
Sum of
df
Mean
F
Variation
Squares
Square
Between
11699.336
2
5849.668
Within
3.213
6
0.536
10922.6
Total
11702.549
8
*Calculation was performed at the 0.05 level of significance
P
value
Decision*
0.000
1
Significant
Table 3
Post Hoc Multiple Mean Analysis on the Percentage Inhibitory Activity
Of Various Plant Extract towards HMG-CoA reductase
(I) Types of
Treatment
(J) Types of
Treatment
Mean
Difference
(I-J)
32
P
value
Decision*
Ginger extract
30.5
0.000
Negative control
87.0
0.000
Ginger extract
Negative control
56.5
0.000
*Calculation was performed at the 0.05 level of significance
Positive Control
Significant
Significant
Significant
Statistical analysis proved that there is an existing significant difference (p<0.05) on
the mean inhibitory activity of Ginger (Zingiber Officinale) extract, Positive control, and
Negative Control towards HMG-CoA reductase. This clearly means that the inhibition was
dependent on the type of treatment thus positive control Atorvastatin has the highest inhibition
demonstrated as compared with the plant extract and negative control.
33
CHAPTER 5
SUMMARY, FINDINGS, CONCLUSIONS AND RECOMMENDATION
Summary
This study was undertaken to examine the phytochemical components and evaluate the
inhibitory action of the Ginger (Zingiber Officinale) extract on 3- hydroxy – 3- methylglutaryl
co-enzyme A (HMG-CoA) reductase. The testing followed standard laboratory approach to
properly measure the phytochemical contents and inhibitory impact of Ginger (Zingiber
Officinale) extract.
Results of the research demonstrated that the Ginger (Zingiber Officinale) includes
saponins, tannins, steroids, flavonoids and alkaloids based on the phytochemical screening.
Percentage inhibition against 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA)
reductase infer the potential of the Ginger (Zingiber Officinale) extract and Positive control as
enzymatic inhibitor which has similar activity as statin and are used pharmacologically in
cholesterol reduction, which can reduce the risk for coronary artery disease and stroke.
Thus, this study can conclude that the Ginger (Zingiber Officinale) extract may serve
as a possible agent against HMG- CoA reductase inhibitory activity. Furthermore, findings of
the test indicated that flavonoids content is found in Ginger (Zingiber Officinale) extract.
Flavonoids have been demonstrated to minimize low- density lipoprotein oxidation, regulate
lipid peroxidation and lessen the progression of atherosclerotic lesions in cardiovascular
disorders.
34
Conclusions
After thorough investigation conducted in this research, the researcher has come
up with the following conclusions:
1. Medicinal plants and herbs like ginger (Zingiber Officinale) contain phytochemicals
like flavonoids, steroids, tannins, saponins, and alkaloids. These phytochemicals help
reduce the oxidation of low-density lipoprotein (LDL), which in turn reduces the risk
of a number of cardiovascular conditions like heart attacks and strokes. Ginger is one
of the most widely used medicinal plants and herbs.
2. The results of the statistical analysis showed that there is a significant difference
(p<0.05) between the levels of mean inhibitory activity exhibited by the Ginger
(Zingiber Officinale) extract, the Positive Control, and the Negative Control in relation
to the HMG-CoA reductase enzyme.
3. The results of the post hoc multiple mean comparison test made it abundantly evident
that the type of treatment had a direct bearing on inhibition. When compared with the
plant extract and the negative control, the positive control, which is atorvastatin, has
the highest level of inhibition that has been demonstrated.
Recommendations
After carrying out extensive research for the purpose of this study, the researcher has
arrived at the conclusions and suggestions that are as follows:
1. Patients who are afflicted with cardiovascular diseases and stroke, particularly those
who reside in rural areas and do not have the financial means to purchase
antihypertensive drugs, should look into alternative methods such as herbal remedies
for the treatment of hypertension and the complications associated with the condition.
Ginger extract, in addition to being effective in the treatment of hypertension, contains
35
flavonoids, which have antioxidant properties and provide significant protection against
the development of chronic diseases.
2. Due to the fact that they have been utilized consistently throughout the entirety of
human history, it is imperative that the significance of plants and herbs in the field of
medicine not be minimized. This research may provide an explanation for why there is
a growing interest in determining the beneficial health impacts of numerous plants and
herbs in the treatment of various ailments, including hypertension.
3. Investigate any further possible pharmacological effects that the ginger extract may
have.
36
REFERENCES
Ali M, Alnaqeeb MA, Al-Qattan KK, Al-Sawan SM, Thomson M, Khan I (2002)-The use of
ginger (Zingiber officinale Rosc.) as a potential anti-inflammatory and antithrombotic
agent.
Campos N., arro M., Ferrer A., Boronat A., 2014; Determination of 3-hydroxy-3methylglutaryl CoA reductase activity in plants. Methods in Molecular Biology 2014,
1153: 21-40. Doi: 10.1007/ 978 -1- 4939- 0606- 2-3.
Freedman B. I., Cohen A. H. (2016). Hypertension-attributed nephropathy: what's in a
name? Nat. Rev. Nephrol. 12, 27–36. 10.1038/nrneph.2015.172
Friesen, JA., and Victor W. Rodwell 2004; The 3-hydroxy-3- methylglutaryl Co-enzyme A
(HMG-CoA) reductases, Genome Biology 20045: 248./ doi.org/ 10.1186/ gb- 2004- 511-248
Kizhakekuttu T. J., Widlansky M. E. (2010). Natural antioxidants and hypertension: promise
and challenges. Cardiovasc. Ther. 28, e20–e32. 10.1111/j.1755-5922.2010.00137.
Meresa A, Fekadu N, Degu S, Tadele A, Geleta B (2017) An Ethno Botanic
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Medicinal Plants Used for the Management of Hypertension. Clin Exp Pharmacol 7:228.
doi: 10.4172/2161-1459.1000228
Ramkissoon J. S., Mahomoodally M. F., Ahmed N., Subratty A. H. (2013). Antioxidant and
anti-glycation activities correlates with phenolic composition of tropical medicinal
herbs. Asian Pac. J. Trop. Med. 6, 561–569. 10.1016/S1995-7645(13)60097-8
Stermer BA., Bianchini GM., Korth, KL, 1994; Determination of 3- hydroxy- 3- methylglutaryl
CoA reductase activity in Plants; J. Lipids Res. 1994 Jul;35(7): 1133- 40.
Stone J. D., Narine A., Shaver P. R., Fox J. C., Vuncannon J. R., Tulis D. A. (2013). AMPactivated protein kinase inhibits vascular smooth muscle cell proliferation and migration
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H369–H381. 10.1152/ajpheart.00446.2012
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WHO (2013). Cardiovascular Diseases (CVDs). Geneva: World Health Organization. Fact
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CURRICULUM
VITAE
38
PERSONAL DATA
Name: Ma. Esabel H. Llano
Address: Bongtud ,Tandag City
Birthday: Setember 04, 2006
Parents:
Father: Basilio C. Llano
Mother: Necifora H. Llano
Education Background
Elementary: Bongtud Elementary School
Bongtud Tandag City
Secondary: Jacinto P. Elpa National High School
Science Tachnology and Engeering Curriculum
Capitol Hills, Telaje, Tandag City
39
PERSONAL DATA
Name: Samsheda C. Paudac
Address: Mabua 3rd st. Tandag City
Birthday: April 23, 2006
Parents:
Father: Samsoden C. Paudac
Mother: Noraiza C. Paudac
Education Background
Elementary: Tandag Pilot Elementary School
Bag-ong Lunsod, Tandag City
Secondary: Jacinto P. Elpa National High School
Science Tachnology and Engeering Curriculum
Capitol Hills, Telaje, Tandag City
40
PERSONAL DATA
Name: Glorian Iza T. Quilaton
Address: Gamot, Tago Sds
Birthday: October 04, 2005
Parents:
Father: Flodines R. Quilaton
Mother: Jeneveb T. Quilaton
Education Background
Elementary: Tandag Pilot Elementary School
Bag-ong Lungsod Tandag City
Secondary: Jacinto P. Elpa National High School
Science Tachnology and Engeering Curriculum
Capitol Hills, Telaje, Tandag City
41
ACKNOWLEDGEMENT
To the one who always there to answer our prayers even though we cannot see him,
we know that he is there to guide us. We are very thankful to you Lord by letting us
accomplish this study. Thank you for the strength, the gift of life, and the wisdom we
received, we are very thankful for everything.
To our parents who showed their support to us financially and emotionally, we are
blissful to their kindness and hospitality. The way they make us believe in ourselves that we
had a full confidence on finishing this study.
To our research teacher Ma`am Ana Geran Millan, to our advisers Mrs. Robeleen
Valeroso and Mr. Raymond Joy Ramos, and to our consultant Sir Venchie Badong, we are
gratified to have a teachers and a consultant that supports our work even our work may not be
as better as the ones with the pro researchers. Thank you also for encouraging us to do well,
to the patience and to the knowledge you have given to us in making our study feasible.
To our ate’s and kuya’s who helped us and giving us advice by their experience. We
are glad to be receiving all your help.
To our classmates who gave their opinions and by helping us understand the things
we don’t. Thank you for sharing your jokes while we are making this study, because it means
a lot to us.
To our hardworking principal Ma`am Evelyn C. Bandoy, your strong leadership is the
foundation of school success. Thank you for inspiring us students to achieve our dreams
every single day.
To everyone who’s making a lot of efforts to give us brilliant ideas, we are so much
thankful to all of you by letting us accomplish this study.
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RESEARCHERS
LOG BOOK
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