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
volume-to-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-3-methylglutaryl 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

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 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

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

3
 Data Gathering Procedures 23

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,

6
2017). Herbs do not 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 long-

standing proven efficacy in reducing levels of LDL-C and total cholesterol (Harley, CR et.al,

2009).

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 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 3-

hydroxy-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.

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 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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 INDEPENDENT DEPENDENT VARIABLES
VARIABLES
Phytochemical screening of
Ginger Extract Ginger Extract

Atorvastatin (Positive control) Percentage Inhibition of three

test drugs against HMG-CoA
Distilled water (Negative reductase
control)

Figure 1. Paradigm of the Study

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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 killer” for its lack of symptoms and can go undiagnosed for

13
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

HMG-CoA 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 HMG-CoA 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

16
variety 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 HMG-CoA 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

19
be effective as a cholesterol-lowering, antithrombotic and anti-inflammatory agent. (Ali M,

Alnaqeeb MA, Al-Qattan KK, Al-Sawan SM, Thomson M, Khan I 2002)

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.

20
 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, 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

21
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 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)

22
 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

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-glutaryl-

coenzyme 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).

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 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

orange-red 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

Blank 184 µL - 12 µL 4 µL

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 Inhibition 181 µL 1 µL 12 µL 4 µL

Sample 181 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

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 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

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-3-

methyl-glutaryl-coenzyme A (HMG-CoA) reductase, the substrate 3-hydroxy-3-methyl-

glutaryl-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

30
E of erythrocyte membranes and 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 Mean S.D. %CV

31
 T1 T2 T3
Atorvastatin 88.5 87.8 88.6 88.3 0.4359 0.4936
ginger (Zingiber 56.4 58.4 58.5 57.8 1.1846 2.05
officinale) extract
Negative control 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,

One-Way 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 P Decision*

Variation Squares Square value
Between 11699.336 2 5849.668 Significant
0.000
Within 3.213 6 0.536 10922.6
1
Total 11702.549 8
*Calculation was performed at the 0.05 level of significance

Table 3

Post Hoc Multiple Mean Analysis on the Percentage Inhibitory Activity

Of Various Plant Extract towards HMG-CoA reductase

32
 (I) Types of (J) Types of Mean P Decision*
Treatment Treatment Difference value
(I-J)
Ginger extract 30.5 0.000 Significant
Positive Control
Negative control 87.0 0.000 Significant
Ginger extract Negative control 56.5 0.000 Significant
*Calculation was performed at the 0.05 level of significance

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,

35
 contains 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-3-
methylglutaryl 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- 5-
11-248
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 Review on
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). AMP-
activated protein kinase inhibits vascular smooth muscle cell proliferation and
migration and vascular remodeling following injury. Am. J. Physiol. Heart Circ.
Physiol. 304, H369–H381. 10.1152/ajpheart.00446.2012 
Tabassum N., Ahmad F. (2011). Role of natural herbs in the treatment of
hypertension. Pharmacogn. Rev. 5, 30–40. 10.4103/0973-7847.79097 
WHO (2013). Cardiovascular Diseases (CVDs). Geneva: World Health Organization. Fact
sheet No 317.

37
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.

42
RESEARCHERS
LOG BOOK

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