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ANTI-DIABETIC ACTIVITY OF DIPLAZIUM ESCULENTUM (FIDDLEHEAD FERN) PLANT

EXTRACTS IN REDUCING THE BLOOD SUGAR LEVELS OF HOME BASED MOUSE (MUS
MUSCULUS)

NOREEN MITZI L. LOPEZ

UNDERGRADUATE THESIS SUBMITTED TO THE FACULTY OF THE DEPARTMENT OF

BIOLOGY,
COLLEGE OF ARTS AND SCIENCES,
CENTRAL MINDANAO UNIVERSITY, IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE

BACHELOR OF SCIENCE IN BIOLOGY

JULY 2022
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ACKNOWLDEGMENET

The author would like to express her gratitude to the Biology Department for the Study of the
Anti-Diabetic Activity of Diplazium esculentum (Fiddlehead Fern) Plant Extracts in reducing the
Blood Sugar Levels of Home Based Mouse (Mus musculus) for their support and allowing the
students to explore more our knowledge.
Foremost, to our Lord and Savior Jesus Christ for giving the researcher life, wisdom,
knowledge, strength and good health. She would like to thank God for all the smiles and laughter
she experienced in conquering the battle of completing this study successfully despite the tears,
sleepless nights, sadness and problems. She offer, commit and surrender herself for without God’s
Holy Intervention, this study will not be realized.

Dr. Alma Mahogan, the researcher’s adviser for sharing her valuable time and expertise in
providing continuous assistance and pieces of advices, constant follow-up and sincere critiquing for
this study to be possible.

Mama Remedios and Papa Gonzalo, the researcher’s parents who stood with her through
ups and downs. She would like to express her sincere love and thanks to all the moral, emotional
and financial support. To the sacrifices they have experienced and done to just to provide the
researcher’s needs and fulfil her dreams.

Aubrey Jacinth Buagas, Maria Carmela Cabahug, Shenneth Mae Pastrano, and
Jennelyn Ubanan, the researcher’s one call away friends and classmates for the continuous
support and encouragements.

NOREEN MITZI L.LOPEZ

s.lopez.noreenmitzi@cmu.edu.ph
0909409374
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Copyright @ 2022 by Noreen Mitzi L. Lopez

All Rights Reserved

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ABSTRACT

ANTI-DIABETIC ACTIVITY OF DIPLAZIUM ESCULENTUM (FIDDLEHEAD FERN) PLANT

EXTRACTS IN REDUCING THE BLOOD SUGAR LEVELS OF HOME BASED MOUSE (MUS
MUSCULUS), Bachelor of Biology, Central Mindanao University, Musuan, Bukidnon, June 2022.

Thesis Adviser: Lorelie Gloria Samaniego, PhD

Diabetes is a chronic disease increasing in both prevalence and incidence worldwide.

Different doses of ethanolic fraction of Diplazium esculentum (Fiddlehead fern) will be evaluated for
reducing the blood sugar levels of normal and diabetic rabbits. The study aims to determine if
Diplazium esculentum (Fiddlehead fern) has effects on reducing the blood sugar levels of home
based mouse (Mus musculus), and to evaluate what anti-diabetic biological properties can be found
in Diplazium esculentum (Fiddlehead fern) extracts.

Keywords: Diabetes mellitus, anti-diabetic activity, Diplazium esculentum, Mus musculus, and
hypoglycaemia.
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TABLE OF CONTENTS

PAGE

TITLE PAGE i

ACKNOWLEDGMENT ii

COPY RIGHT PAGE iii

ABSTRACT iv

TABLE OF CONTENTS v

LIST OF TABLES vi

LIST OF FIGURES vii

LIST OF APPENDICES viii

CHAPTER

I. INTRODUCTION
I.1. Statement of the Problem 10
I.2. Objective of the Study 10
I.3 Significance of the Study 11
1.4. Scope and Delimitation 11
I.5. Definition of Terms 13
II. REVIEW RELATED LITERATURE
II.1. Review of related studies 14
II.2. Related Studies 23

III. METHODOLOGY
III.1. Research Design 24
III.2. Sampling Procedure 24
III.3.Method of Analysis 28

REFERENCES 32

APPENDICES 34
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LIST OF TABLES

TABLE TITLE PAGE

1 Taxonomic characteristics 28
of
Diplazium esculentum

2 Taxonomic characteristics 28
of Mus musculus

3 List of Group and Treatment 31

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LIST OF FIGURES

FIGURE TITLE PAGE

1 Sampling Site: Barangay Cagdapao, 12

Tago, Surigao del Sur
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LIST OF APPENDICES

Appendi Title Page

1 Taxonomic characteristics of 28
Diplazium esculentum
2 Taxonomic characteristics 28
of Mus musculus
3 List of Group and Treatment 31

4 Sampling Site: Barangay Cagdapao, 38

Tago, Surigao del Sur
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CHAPTER 1
INTRODUCTION

Diabetes is a chronic disease increasing in both prevalence and incidence worldwide.

Diabetes exerts a significant impact in third-world countries, particularly in the Philippines. (Tan,
2015) said that Asia will see the most significant increase in the number of people with diabetes in
2025. This increase in the burden of chronic disease in Asia will significantly affect the nation's
healthcare systems both acutely and chronically. According to the latest survey published by the
Food and Nutrition Research Institute in the Philippines (the Eighth National Nutrition Survey of
2013), the prevalence of high fasting blood glucose (> 125 mg/dL) among adults over the age of 20
was 5.4 percent, up 0.6 percent from the 2008 research. The highest prevalence rates are recorded
among the wealthiest individuals, those who live in metropolitan areas, and those aged 60 to 69 in
both sexes (Tan, 2015). The Philippine government has enacted regulations to ensure that all
Filipinos have access to affordable diabetes treatment. Non-communicable diseases (NCDs;
noninfectious or nontransmissible diseases) such as diabetes account for six of the Philippines' top
10 causes of mortality and are a significant public health concern (Tan, 2015). Globally, the
prevalence of diabetes mellitus is increasing, potentially arise to changing lifestyles and the
associated surge in obesity (King et al., 1998). Diabetes mellitus (DM) is associated with long-term
complications such as retinopathy, neuropathy, and angiopath (Kristova et al., 2008). Diabetes
affects an estimated 246 million people globally, with roughly 80 percent living in developing nations
(Mohan et al., 2008).

Plants are well known in traditional medicine for their hypoglycaemic activities (Akah et al.,
2011). Millions of people worldwide are constantly investigating the different potential of plants in
treating various diseases. People have used herbal medicine as an alternative health care system
due to its affordability and availability on a local level. Since then, plant based medicines have been
used to treat a variety of diseases. Prehistoric people used herbs as therapeutic agents and
medicaments, which they could easily procure (Ahmad et al., 2009). According to (Fiscal, 2017), on
studying the medicinal plants, he stated that the medicinal plants are frequently used internally as
decocted leaves and treat various ailments, including urinary tract infection and wound infection,
stomach ache, cough, diabetes, headache, cold, and hypertension. Throughout history, plants have
been used as sources of medicine and continue to serve as the basis for many pharmaceuticals
used today. While the modern pharmaceutical industry evolved from botanical medicine to synthetic
drug discovery (Schmidt et al., 2008). Due to their low cost, convenience of availability, and lack of
unwanted effects, the usage of plant products with anti-diabetic activity has grown in popularity
(Akah et al., 2011).Therefore, the potential for plants to act as hypoglycemic agents is constantly
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being investigated (Bailey and Day, 1989). The critical values of some plants have long been
published, but many of them remain unexplored as yet (Hussain et al., 2011).

1.1 Statement of the Problem

The locality has long recognized the importance of using modern, up-to-date technology
medicinal plants in Barangay Cagdapao. However, various problems still exist due to the lack of
medicine availability, lack of on-duty doctors, and not all people can afford the said medicine.

To be more precise, the following research problem must be addressed:

1. What is the effect of fiddle head fern on lowering the blood sugar levels home based mouse?

2. What are the anti-diabetic biological properties that can be found in fiddle head fern extracts?

3. What conditions can be seen after assessing the toxicity of fiddle head ferns plants on home
based mouse?

4. Among methanolic, and aqueous extracts what extract was found to be more potential in
lowering the blood sugar level of home based mouse?

1.2 Objective of the Study

The research's general long-term objective was to assess the anti-diabetic activity of
fiddlehead fern plants and to determine accepted, effective, and safe plants for lowering blood sugar
levels in mouse. The researcher will be able to test the different methanolic and aqueous extracts
dosage to determine if fiddlehead fern can reduce blood sugar more effectively.

The study is primarily conducted to determine the following:

1. To determine if fiddlehead fern (Diplazium esculentum) has effects on reducing the blood
sugar levels of home based mouse (Mus musculus);

2. To evaluate what anti-diabetic biological properties can be found in fiddlehead fern (Diplazium
esculentum) extracts;

3. To test the efficacy, safety, and toxicity of fiddlehead fern (Diplazium esculentum) as an
alternative medicine for reducing the blood sugar level of home based mouse (Mus
musculus);

4. To evaluate which extracts have the potential to lower the blood sugar level of diabetic mouse
(Mus musculus);
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1.3 Significance of the Study

The findings of this study will contribute to the benefit of society, considering that diabetes is
a chronic disease that arises today. This study provides the basic information about the Anti-diabetic
Activity of fiddlehead fern (Diplazium esculentum) plant on reducing the blood sugar of home based
mouse (Mus musculus). Thus, schools that apply all the recommended approach derived from the
results of this study will be able to give ideas to researchers or all healthcare workers better. Through
this study, the medicinal plant knowledge will be documented and studied well, and this expertise
will be transferred to the world's scientific community in order to find and develop new and better
pharmaceuticals for the benefit of humans.

1.4 Delimitation

This study mainly focused on open garden in Barangay Cagdapao, Tago, Surigao del Sur,
Philippines. The study limits its coverage to the highlighted, square-lined, elevated places (Fig. 1).
The researchers chose this study because it is both feasible and convenient.
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DEFINITION OF TERMS

Alloxan - used in experimental animals to induce diabetes by destroying the insulin-secreting cells
in the Langerhans islet of the pancreas.

Beta Cells - cells that produce insulin, a hormone that regulates the blood glucose level (a type of
sugar).

Diabetes – a medical term for diabetes mellitus. Diabetes is a chronic disease that happens when
the pancreas stops producing insulin or when the body cannot utilize it properly.

Diseases - are medical disorders that have specific indications and symptoms.

Ethanol – used in the synthesis of other organic chemicals as a solvent. Ethanol is also a
psychoactive component of a variety of alcoholic beverages, including beer, wine, and distilled
spirits.

Ethyl acetate – is frequently used in column and thin-layer chromatography.

Ethics - standards of right and wrong that specify what humans should do, typically in terms of
rights, obligations, societal benefits, justice, or specific virtues.

Folk remedies – herbal remedies are not prescribed by a physician.

Glucose - glucose is the primary sugar found in our blood. It is derived from the food we eat and is
the primary source of energy for our bodies.

Healthcare system – Institution or organization that provides health care services.

Homeostasis – maintain stability while adapting to the best conditions for survival.

Hyperphagia – abnormally increased appetite for food consumption is frequently associated with
hypothalamic injury.

Hypertension – is a blood pressure reading that is higher than usual.

Hypoglycemia – is a condition that can cause dizziness, tremors, and even death.

Methanol – a highly flammable alcohol that is commonly used in industry as a solvent, pesticide,
and alternative fuel source. It is also found in humans, animals, and plants naturally.

Obesity - is a complicated condition caused by excess body fat.Pharmaceuticals – are medicine

manufacturers.

Polydipsia – a medical term for the sensation of extreme thirst

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Streptozotocin – an antibiotic that destroys pancreatic islet cells malfunction in the way the body
regulates and utilizes sugar (glucose) as a fuel source

Traditional medicine – compromises a traditional knowledge on medical aspects that are local to
a country or culture.

Type I Diabetes mellitus – the pancreas either does not produce insulin at all or produces very little
insulin.

Type II Diabetes mellitus - a problem with the way the body regulates and utilizes sugar (glucose)
as a fuel source
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CHAPTER 2

REVIEW RELATED LITERATURE

PATHOPHYSIOLOGY OF DIABETES MELLITUS

Diabetes is a group of disorders characterized by elevated plasma glucose levels and

abnormal glucose metabolism. Low glucose utilization causes hyperglycemia. In most cases, the
true aetiology is still unknown. The WHO has developed several diabetes classification schemes,
the most widely accepted being the following:

Figure 2. Accepted diabetes classification schemes

TYPE 1 DIABETES MELLITUS

This type of diabetes was previously known as insulin-dependent diabetes or juvenile

diabetes. The pancreatic ß-cells are destroyed in type 1 diabetes. Several autoimmune markers
have been found in body fluids and tissues: (1) Auto-antibodies to islets (ICAs); (2) insulin antibodies
(IAAs); (3) auto-antibodies to GAD (GAD65); (4) autoantibodies to IA-2 and IA-2ß. Type 1 diabetes
mellitus is caused by an autoimmune attack on the insulin-secreting cells of the pancreatic ß-cells.
Numerous years before to clinical diagnosis and presentation, the autoimmune process begins. The
destruction must be extensive, as just 10-20% of the volume of ß-cells is required to cover clinical
signs. The pace of ß-cell breakdown varies considerably between individuals, being quick in some
(mostly newborns and children) and often gradual in adults. Without a doubt, environmental variables
have a role in the onset of diabetes. The most prevalent causes include viruses (rubella, coxsackie
virus B, and mumps), chemicals, and, in some cases, cow's milk (Palicka, 2002).

TYPE II DIABETES MELLITUS

The term type 2 diabetes mellitus (NIDDM) was previously used to refer to adult-onset
diabetes. It is far more common than type 1 diabetes, accounting for over 90% of all diabetics.
Patients are frequently older and have few symptoms. Insulin levels are usually elevated, but might
be normal or lowered. Obesity is widespread, and losing weight helps hyperglycemia. The condition
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usually strikes after 40. Oral hypoglycemic medications and dietary changes are the mainstays of
therapy; insulin is used to treat hyperglycemia. The new understanding type 2 diabetes mellitus is
represented by two disorder categories. The first is reduced insulin action on peripheral tissues. It's
called "insulin resistance". Insulin resistance is a reduced biological response to normal circulating
insulin concentrations and is the principal pathogenic mechanism. The second is pancreatic ß-cell
dysfunction, characterized by insufficient insulin production to overcome insulin resistance in
peripheral organs. Due to ß-cell malfunction, insulin production may be insufficient to counteract
insulin resistance. Insulin insufficiency is a common outcome. The major reason - insulin resistance
or insulin production derangement - was debated. The evidence suggests that insulin resistance
precedes insulin secretion dysfunction. Insulin resistance often occurs 20 years before clinical
symptoms. Insulin resistance is caused by a mix of environmental and genetic factors. The most
critical factors are body weight and activity. Obesity and type 2 diabetes are intricately linked: while
60–80% of patients with type 2 diabetes are fat, diabetes develops in less than 15% of obese
individuals. Clinical symptoms and therapeutic requirements typically improve with weight loss and
physical activity (Palicka, 2002).

GESTATIONAL DIABETES MELLITUS

Typically, gestational diabetes mellitus is asymptomatic and does not pose a threat to the
mother's life. The syndrome is related with a higher risk of neonatal morbidity, hypoglycemia in
newborns, macrosomia, and jaundice. Even normal pregnancies, particularly in the second and third
trimesters, are associated with an increase in insulin resistance. Maintaining euglycemia requires
increased insulin secretion. Gestational diabetes develops in women who are unable to enhance
insulin output. The pathogenesis of gestational diabetes mellitus is unknown, however it may include
a family history of diabetes, obesity, problems during prior pregnancy (i.e.), and advanced mother
age. Preexisting diabetes mellitus must be detected, as it has a significantly worse prognosis for the
fetus (Palicka, 2002).

Chemical Constituents Analysis and Antidiabetic Activity Validation of Four Fern Species
from Taiwan

The Pteridophyta plant family includes the about 12,000 species known as ferns. In several
nations, certain fern species are used as food or traditional medicine to treat a variety of diseases.
Ferns predominantly contain flavonoids, alkaloids, phenols, steroids, and triterpenoids; exhibit
diverse bioactivities such as antibacterial, antiosteoporosis, and anti-Alzheimer’s disease activity;
and feature hypolipidemic and hypoglycemic activities. Therefore, ferns constitute a key medicinal
resource in ethnopharmacy.
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First isolated from the bracken fern Pteridium aquilinum var., pterosin is a sesquiterpene with
a 1-indanone skeleton. The latiusculum (Pteridaceae). There have been discovered around 31
pterosins from various fern species and have leishmanicidal, smooth-muscle relaxing, and
anticancer properties. In an animal model, pterosin A was expressed against both type 1 and type
2 diabetes. In addition, other studies have shown that pterosin A can improve insulin sensitivity,
accelerate glucose absorption, and increase AMPK (adenosine monophosphate-activated protein
kinase) activation. Sesquiterpenes known as pterosins are a big category and are extensively
distributed in the Dennstaediaceae and Pteridaceae families. In addition to photochemicals from C,
we identified the seasonal changes of nine pterosins, five pterosides, six lignans, three flavonoids,
six phenolics, and one carbohydrate from four fern species. In addition to N. multiflora. Additionally,
Compounds 21 through 23 were initially recognized in H. punctata. Controls the metabolism of fatty
acids and carbohydrates. Future research may perhaps find that pterosin molecules are effective in
treating metabolic illness. The outcomes also showed that the pterosin distributions were the
outcomes also showed that the pterosin distributions werethe three aforementioned species (H.
punctata, C. thalictroide, and P. revolutum), with the exception of N. higher than the comparable
distributions of the other pterosin analogs for multiflora (Nephrolepdiaceae). Numerous triterpenes
and steroids have been identified from Nephrolepdiaceae in previous research. These results made
it very evident that N contained nonpterosin-type components. multiflora. Pterosin A may protect
pancreatic beta-cells from oxidative damage, but this is still unknown. As a result, in insulin-secreting
cells exposed to oxidative stress or lipotoxicity, the current investigation evaluated any potential
positive effects of pterosin A on cell survival and ROS production. Through the AMPK signaling
pathway, pterosin A in this study efficiently decreased the ROS-induced cell damage in the insulin-
secreting cells. According to reports, long-term FFA therapy and pancreatic aberrant glucose
metabolism can result in mitochondrial malfunction, a progressive rise in ROS generation, and β-
cell dysfunction. One of the main pterosin compounds, pterosin A, has anti-diabetic properties as
well as safeguards against -cell harm. Pterosin A could therefore be employed as a lead chemical
in the creation of type 2 diabetic medications. The lower total glucose uptake in the body was thought
to be primarily caused by the impaired glucose transport in skeletal muscles seen in type 2 diabetes
patients. The absorption of glucose is improved by both insulin stimulation and AMPK activity.

Phytochemical Properties and Effects of Crude Extracts of Sarabat Fiddlehead Fern Shoots
In Hyperglycemia Induced Normal Mice (Mus Musculus)

The current study revealed the phytochemical components of sarabat. The presence of sterol,
triterpene, flavonoids, alkaloids, saponins, glycosides, flavonoids, and tannin in the samples was
recognized and mentioned. The Sarabat fiddlehead fern extracts are consistently high in saponins
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and sterols and can therefore be effective treatments for hypercholesterolemia. The aqueous
preparations of sarabat include glycosides as well. Aqueous preparations of the same substances
also include terpernes. The turbidity in the ethanolic and methanolic extracts of Sarabat 1 indicates
a moderate amount of alkaloids, but the production of red precipitates indicates a moderate
presence of flavonoids. The bluish color shift in the ethanolic and methanolic extracts of Sarabat 2
indicates a moderate presence of sterols. All contain residues of tannins, which are identified by
variations in the color of black. Overall, it can be demonstrated that methanolic extraction produced
a good increase in the amount of phytochemical components. The aforementioned chemical
components of Sarabat are similar to some of those of Diplazium esculentum, according to Tongco
et al. (2014), even though a wider range of phytochemical components was evaluated in D.
esculentum. This is in line with the presence of phytochemical components like alkaloids, flavonoids,
particularly in the methanolic extracts, triterpenes in the aqueous extract, and phytosterols as shown.
These phytochemical components of the fern plant may work in concert to produce the desired blood
glucose lowering effect or enhance, influence, and modulate the various key physiological processes
like enzyme activity or transport of metabolites across membranes. The crude extracts of the edible
vegetable fern Sarabat (Diplazium polypodiodes Blume and Diplazium sp) were found to include
sterols, triterpenes, flavonoids, alkaloids, saponins, glycosides, and tannins, with saponins
predominating. Likewise, the aqueous extract shows a significant amount of glycosides and
triterpenes. Alkaloids and flavonoids are also observed to be present in all solvents used, with the
methanolic solvent being more strongly noticed. Oral glucose tolerance test results reveal that the
medications had effects on mice at 30 and 60 minutes, with statistical differences at 5% and 1%
levels of significance and p-values of 0.002 and 0.02 respectively. The results of additional analysis
using the Bonferroni test showed that the mice receiving Treatment 1 had significantly lower blood
glucose levels than mice receiving Induced Treatment or No Treatment. The glycalizide immediately
began to lower the glucose level at 60 minutes after treatment, especially when compared to
Treatment 4 (low dose sarabat extract), but its effect was only statistically comparable to Treatment
5. The commercial medicine had a substantial effect compared to Treatment 4 at 60 minutes after
treatment, but this benefit was not sustained at 90 and 120 minutes after treatment, as shown by
the non-statistical differences in mean glucose levels between Treatments 4 and 5 of the
experimental setup.

Diplazium esculentum, Ethnomedicinal Phytochemical, and Pharmacological Overview of the

Himalayan Ferns

Due to the high-valued bioactive substances they contain, botanicals have historically been
employed extensively to treat a variety of diseases. Search for bioactive chemicals using literature.
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Diplazium esculentum showed that various solvent systems contained alkaloids, flavonoids,
glycosides, phenolic, tannins, terpenoids, steroids, carbohydrates, lipids, and oils (Semwal et al.,
2021). (Essien et al., n.d.) Study revealed the chemical make-up of D. esculentum isolate essential
oil. Major volatile chemicals found in D. esculentum leaves included -pinene (17.2%), -pinene
(10.52%), caryophyllene oxide (7.5%), sabinene (6.1%), and 1,8-cineole (5.8 percent ). This species'
essential oil contained monoterpene hydrocarbons, oxygenated sesquiterpenoids, oxygenated
monoterpenoids, oxygenated sesquiterpene hydrocarbons, and nonterpene derivatives. D.
esculentum has a number of practical characteristics. The antioxidant, anti-inflammatory,
antibacterial, antidiabetic, and immune-modulatory properties of D. esculentum might be thought of
as perhaps enhancing the pharmacological and preventative benefits of this plant species (Semwal
et al., 2021). In the study of (Semwal et al., 2021) they concluded that a total of 30 rats were used
and treated and at the same time evaluated. It was also observed that the plant extract reduced
around (50%) of blood glucose level in STZ-induced diabetic rats.

In Vitro Callus and In Vivo Leaf Extract of Gymnema sylvestre Stimulate β-cells Regeneration
and Anti-diabetic activity in Wistar Rats

G. Sylvestre leaf and callus extracts have been shown to have antihyperglycemic properties
in alloxan induced diabetic wistar rats. Our experimental data indicate that alloxan administered
intraperitoneally to Wistar rats demonstrates diabetic action. We conclude that this investigation
confirms the efficacy of G. sylvestre leaf and callus extracts as potent herbal treatments and that
they may be capable of completely restoring pancreatic-cell activity and thus curing type I diabetes.
G. sylvestre callus extract studies have demonstrated how-cells may be produced and regenerated
in vitro, which may provide additional direction for possible IDDM medication development (Ahmed,
A. et al., 2010).

Anti-Diabetic Activity of Paspalum scrobiculatum linn. In Alloxan Induced Diabetic Rats

The ethanolic and aqueous grain extracts of Paspalum scrobiculatum were evaluated for their
antidiabetic activity. The extracts reduced FBG in alloxan-induced diabetic rats. Alloxan causes
pancreatic cell damage by producing cytotoxic oxygen free radicals. These radicals target pancreatic
DNA, causing DNA fragmentation (Shankar et al., 2007). After 60 minutes of administration of PS
extracts to glucose-loaded normal rats (OGTT), blood glucose levels were reduced. The decline
peaked at 2 h. It found a significant rise in blood glucose levels in the diabetic control group after 15
days of testing. The extracts reduced fasting blood glucose levels and increased serum insulin levels
in diabetic rats. Thus, PS may exert hypoglycemic action by increasing either pancreatic secretion
of insulin from existing beta cells or its release from bound form. Another mechanism could be PS's
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high fiber content. Dietary fibers help diabetics, especially type II diabetics, by slowing down the rate
of carbohydrate absorption from the intestine (Khan and Safdar, 2003). Lipoprotein lipase normally
hydrolyzes triglycerides. Diabetes mellitus causes hypertriglyceridemia by inhibiting this enzyme.
Fibers lower cholesterol and triglycerides (Anderson, 2000). Because Paspalum scrobiculatum
contains a high amount of fiber, it has a significant effect on serum lipid levels. Swanston-Flat et al.
(1990) and protein loss in tissues (Chatterjea and Shinde, 2002). The PS-treated rats had smaller
body weight differences than the diabetic control rats, possibly due to its protective effect in
controlling muscle wasting, reversal of gluconeogenesis, and proper glycemic control. The decrease
in hepatic glycogen content in diabetes (Whitton and Hems, 1975) is likely due to a lack of insulin in
the diabetic state, which inactivates the glycogen synthase system. These results suggest that
reactivation of the glycogen synthase system may be involved. Thus, improving glycogenesis may
be another possible anti-diabetic action (Maiti et al., 2004). The diabetic control group had higher
glycated hemoglobin levels. Increased non-enzymatic and autooxidative glycosylation may link
hyperglycemia and diabetic vascular complications (Hall et al., 1984). The extracts significantly
reduced glycated hemoglobin levels, possibly due to improved insulin secretion. Previous research
has shown that phenolic compounds can help control diabetes and other diseases (Vasco et al.,
2008; Ahmad and Mukhtar, 1999). Both extracts contained phenolic compounds. Thus, PS's
beneficial effect may be due to phenolic compounds. The data shows that PS has significant
antidiabetic activity and may be useful in the treatment of NIDDM. It's also important to do more long
term studies on chronic models to fully understand how the drug works (Jain et.al., 2010).

Hypoglycemic Effect of Scarlet Spiral flag (Costus woodsonii) to Alloxan-Induced diabetic

mice in vivo study

It was an experimental design with the restrictions of utilizing 20 male Swiss mice as the
experimental unit; Alloxan as the diabetes-inducing agent; Scarlet Spiral Flag as the treatment; and
a week of monitoring for the onset of fasting blood sugar. The study concluded that an ethanolic
extract of Scarlet Spiral Flag (C. woodsonii) has significant anti-diabetic activity in diabetic mice
induced with Alloxan. Because it belongs to the same genus as the insulin plant, it has a similar
effect on blood glucose levels. While the mice remained diabetic throughout the treatments, this
could be due to the efficiency of the Alloxan induction. It still showed a considerable hypoglycemia
effect (Magtulis, R. et al., 2020).
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Plants Having Potential Anti-diabetic Activity

Leguminoseae, Lamiaceae, Liliaceae, Cucurbitaceae, Asteraceae, Moraceae, Rosaceae,
Euphorbiaceae, and Araliaceae are hypoglycemic plant groups. Trigonella foenum graecum L.,
Momordica charantia L., Ficus bengalensis L., and Gymnema Sylvestre R. were used to make this
extract. The experiments employed a variety of methodologies. Oral glucose tolerance tests can
cause transient hyperglycemia (OGTT). The most frequently utilized diabetic models were the
streptozotocin and alloxan-induced diabetic mouse or rat. Some writers used KK Ay mice as a model
of type II diabetes with hyperinsulinemia. The majority of studies verify the benefits of medicinal
herbs with hypoglycemic properties in diabetes control. These plant extracts' activities have been
proposed in many ways. Certain hypotheses suggest that they may enhance the protective/inhibitory
impact of plant extracts against insulinase and increase insulin sensitivity or insulin-like activity. A
reduction in the glycogenic index of carbs and a reduction in glutathione's action are all possible
strategies for improving glucose homeostasis. All of these steps may help reduce or eliminate
diabetic problems. This review covered all anti-diabetic herbs used to treat diabetes mellitus. It
displays the hypoglycemic effects of the herbs mentioned above. They were often as strong as or
more potent than known synthetic oral hypoglycemic medications. However, many more plant
derived active compounds are unknown. More research is needed to determine the mechanism of
action of anti-diabetic herbs. These plants' toxicity should be clarified (Chauhan, A. et al., 2010).

Nutritional and Antioxidant Potential of Fiddleheads from European Ferns

A warning should be heeded regarding the possible toxicity of ferns while considering them
as potential widely accessible veggies. A variety of poisonous substances have been found in some
fern species. Overall, the risk of potential intoxication is reasonably reduced if fern fiddleheads are
not consumed on a daily basis and are instead consumed dried or properly cooked. This is because
we previously tested the toxicity of mature leaf extracts of all the species reported here on sheep
hepatocytes at 100 gmL1 (Dvorakova, M., 2021). Additionally, the majority of the species cultivated
in Europe appear to be free of toxicity and toxic substances (ptaquiloside and its analogs), making
those potential vegetables. As a significant source of essential fatty acids with a favorable n-6/n-3
ratio and valuable antioxidants, fiddleheads from European ferns may benefit the human diet. Our
results might act as a guide for choosing the best fern species to be raised economically as
vegetables. Polypodium vulgare, Onoclea sensibilis, or Polystichum aculeatu is an example of a
species having a high level of total phenols, strong antioxidant capacity, and a preferred n-6/n-3
ratio. A careful ingestion of fern fiddleheads along with their adequate boiling or processing before
eating could prevent potential poisoning, even though the issue surrounding the probable toxicity
still has to be addressed and evaluated via more complete assays (Dvorakova, M., 2021).
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Chemical and Biological Aspects of Extracts from Medicinal Plants with Anti-diabetic Effects
Several plants have been shown to have anti-diabetic properties in both animal and human
studies. In some circumstances, the components of these plants have greater anti-diabetic action
than traditional medications. In this recipe, you'll find Albizzia lebbeck and Aloe vera. You'll also find
Amaryllis tricolor, Azadortha indica, Bauhinia thonigii, and Caesalpinia ferrea. You'll also find Cassia
fistula, Cecropia pachystachy, Cinnamomum japonica, Cinnamomum cassia, Ficus racemosa,
Momordica charantia, Moringa oleifera, Nigella sativa, Opuntia milpa alta, Origanum vulgare, Perse
research using medicinal plants historically used to treat diabetes has shown hypoglycemic or
antihyperglycemic characteristics, validating their traditional use as anti-diabetics. The plants'
antidiabetic properties have multiple mechanisms of action. The wide range of chemical classes
suggests that different modes of action may be implicated in lowering or maintaining blood glucose
levels, such as boosting pancreatic insulin secretion or peripheral glucose uptake. Some chemicals
originating from medicinal plants may be helpful, while others may cause hypoglycemia or even be
poisonous to hepatocytes. The use of plant derived compounds with promising qualities should be
promoted in clinical practice to improve diabetes management. One should now look into the
biologically active components and how they work so that they can make therapeutic medicines
(Gushiken, L. et al., 2016).

Activity of Ethyl Acetate Extract from Chrysophyllum cainito L. Leaves in decreasing Blood
Sugar Level in Male Wistar Rats

The percentage drop in blood sugar level was based on the antidiabetic activity test of ethyl
acetate extract of C. cainito leaves. All treatment groups' blood glucose levels reduced significantly
over 7, 10, and 14 days (p 0.05). On day 3, all treatment groups demonstrated a substantial decrease
in blood glucose (p > 0). After that, the data is subjected to an LSD Pos Hoc test. Table 3 shows the
LSD Pos Hoc test results. Except for P1, all groups differed significantly from the negative control
group based on LSD findings on H3. In H7, H10, and H14, all groups differed from the negative
control group. So all the extracts are anti-diabetic. These findings are supported by the fact that all
group extracts have antidiabetic efficacy when compared to negative controls. In all dose groups, C.
cainito leaves extract lowers blood sugar levels better than the negative control group, but not as
much as metformin. The best dose for decreasing blood glucose was 75 mg/kgBW. In alloxan
induced male wistar rats, ethyl acetate extract of C. cainito leaves at 25 mg/kgBW, 50 mg/kgBW,
and 75 mg/kgBW reduced blood glucose levels. The 75 mg/kgBW ethyl acetate extract of C. cainito
leaves has the maximum reducing ability compared to the 25 mg/kgBW and 50 mg/kgBW dosages.
To reduce blood glucose in alloxan induced male wistar rats, three doses of C. cainito ethyl acetate
extract were tested (Arrijal, I. et al., 2018).
 P a g e | 22

Perspective Review on Diabetes Mellitus and the Potential Anti-diabetic Activity of Medicinal
Plants
The World Health Organization estimates that around 30 million individuals had diabetes in
1985, and that number had risen steadily to over 171 million in 2000. The number is expected to
rise to over 366 million by 2030, mostly targeting middle-aged to elderly people (45–64 years). For
example, biguanides, thiazolidinediones, and insulin were discovered to have anti-diabetic effects.
But these medications often cause nephrological issues, exhaustion, gastrointestinal issues (upset
stomach, diarrhea, vomiting), hypoglycemia, etc. Worldwide, many medicinal plants are used to cure
diabetes. They contain bioactive components such as flavonoids, tannins, phenolics, and alkaloids
that improve the performance of pancreatic tissues by increasing insulin secretion or decreasing
intestinal glucose absorption. Various anti-diabetic herbs have been pharmacologically examined
and proved to be useful in the treatment and prevention of diabetes mellitus. Some research
suggests that these plants can help prevent diabetic complications and repair metabolic imbalances.
There are also various putative processes by which these herbs can control blood glucose levels.
This plant (or its active principle) can reduce plasma glucose by interfering with processes involved
in glucose homeostasis. The current study looks at the outcomes of experimental studies on the
hypoglycemic actions of plants and their bioactive components in several Asian and other regions
of the world. The kind of diabetes is briefly defined, as are the linked physiological diseases and
available anti-diabetic herbs. Finally, this analysis summarizes hypoglycemic plant profiles found in
the literature. This may help doctors, scientists, and researchers produce evidence-based herbal
remedies or dosage formulations to treat diabetes. Extracts from many natural resources are used
to create medicines to treat hyperglycemia in diabetes mellitus. Thus, the researcher says that
medicinal herbs can be used to treat and prevent diabetes and that more research is possible
(Gargantiel, M. et al., 2021).

Effect of RRE on Different Parameters in Diabetic Rats

The hypoglycemic and hypolipidemic effects of RRE (24.11 mg/kg equivalent to 15 mg/kg RC
content), RC (15 mg/kg) and glibenclamide (600 g/kg) were studied in NA-STZ diabetic rats for 28
days. In NA-STZ diabetic rats, hyperglycemia was confirmed by polydipsia (thirst), hyperphagia
(appetite), and weight loss. Initially, non-diabetic and diabetic rats ate the same amount of water and
food. After four weeks, diabetic rats' water or food intake increased while their body weight
decreased. Oral administration of RRE (24.11mg/kg) and RC (15mg/kg) normalized diabetic rats'
water and food intake, and body weight without affecting normal rats (Shah, 2017). The current study
used NA and STZ to partially destroy the pancreas, resulting in higher fasting blood glucose and
lower serum insulin levels in diabetic rats. The FBG and insulin levels were normal in non-diabetic
rats given 24.11 mg/kg RRE and 15 mg/kg RC for 28 days. RRE (24.11mg/kg) diabetic rats RC
 P a g e | 23

(15mg/kg) or Glb (600g/kg) gradually reduced hyperglycemia and increased serum insulin over a
28-day period. RRE also reduced pancreatic beta cell destruction in diabetic rats, as shown by
pancreatic histopathological images. The pancreatic protective effect of RRE is linked to its marker
compound RC (Adam et.al. 2016). In hyperglycemia, excess sugar reacts with protein to form
gylcated hemoglobin (HbA1c), a laboratory marker of diabetes and associated risk of diabetic
complications (Shah, 2017). RRE (24.11mg/kg) and RC (15mg/kg) significantly reduced the
increased HbA1c level in diabetic rats compared to the standard drug Glb (600 g/kg). However,
normal treated rats' HbA1c levels were unaffected. The results match previous in vitro studies on
RRE and RC antiglycation potential (Shah, 2017).
 P a g e | 24

CHAPTER 3

METHODOLOGY

This chapter discusses the researcher's methodology for conducting the study. The study's
research design, sampling procedure, corpus of the study, data gathering procedure and method
analysis are discussed in detail here.

Entry Protocol

A letter signed by the thesis adviser would be presented to the barangay captain of Barangay
Cagdapao, Tago, Surigao Del Sur to request permission to get the specific plant that will used in
performing the study.

Institutional Animal Ethics Committee

A letter signed by the thesis adviser and the Department of Environment and Natural
Resources would be presented to the Institutional Animal Ethics Committee to request permission
to perform the study and gets the approval for the study.

Research Design

The current study investigated the anti-diabetic activity of fiddlehead fern (Diplazium
esculantum) plants using the experimental design.

Sampling Procedure

The researcher utilizes convenience sampling in this research. The plant will be extracted
from this territory and compiled in the Barangay Cagdapao, Tago, Surigao Del Sur; to simplify the
classification process, the researcher used taxonomic characteristics to group the information. In
Figure 1, you can see the highlighted (orange) square line, elevated and the topography of the place
where the samples are being taken (9,000 sq.) The area’s topography is defined by the coordinates
8°59'40.9 "N, 126°09'20.4 "E.
 P a g e | 25

LEGEND:
Site

Figure 1. Sampling Site: Barangay Cagdapao, Tago, Surigao del Sur

Kingdom Plantae
Phylum Pteridophyta
Class Polypodiopsida
Order Polypodiales
Family Filicopsida
Genus Diplazium
Species Diplazium esculentum
Table 1. Taxonomic characteristics of Diplazium esculentum

Kingdom Animalia
Phylum Chordata
Class Mammalia
Order Rodentia
Family Muridae
Genus Mus
Species Mus musculus
Table 2. Taxonomic characteristics of Mus musculus

Collection of Plant Material

The basic plant material for the research, fiddlehead fern whole plant, will obtained from the
Open Garden Area in Barangay Cagdapao, Tago, Surigao del Sur, Philippines. The plant can be
identified and verified by the Central Mindanao University, College of Arts and Sciences, Department
of Biology research office (botanist and taxonomist).
 P a g e | 26

Preparation of Plant Extraction

The leaves of Diplazium esculentum plants should be gathered and shadow dried to obtain
coarse powder. After securing the pulverized leaves, the researcher will prepare the aqueous and
methanol extracts. The researcher adopted the methodology of (Jain, et.al, 2009). Both extracts
were filtered, concentrated, and vacuum dried (yielding 10 g and 15 g, respectively.

Animals
Healthy adult house mouse either sex aged must be under 6 months and weighing 10-15 g it
may obtained from within the vicinity of Lopez’s residence by trapping the mouse and used for the
study. After randomization into different groups and before the initiation of the experiment, the mouse
will kept under individually in hutch, maintained under standard conditions (12 h light and 12 h dark
cycle, 25 ± 30 ◦C, 35–60% relative humidity), the animals will fed with fresh vegetables and a
standard diet and water.

Analysis of total phenolic content

Using the method of (Bozin et al., 2008) the total phenolic content was measured
colorimetrically in both extracts using the Folin-Ciocaletu (FC) reagent. To complete the reaction, a
reaction mixture containing 500 l of 0.1 percent aqueous dilutions of both extracts, 2.5 ml freshly
made 0.2 M FC reagent, and 2 ml sodium carbonate solution will maintained in the dark at ambient
temperature for 30 minutes. The absorbance of the resultant solution will be determined using a UV–
vis spectrophotometer at 760 nm in the laboratory. The total phenolic content of the extracts will
quantified as mg of gallic acid equivalents (GAE) per gram using a gallic acid reference curve (Bozin
et al., 2008). Five replicates of each measurement should be taken.

Acute Toxicity Assessment

Excellent condition of mouse of either sex were deprived overnight and placed into two groups
of five mouse each. The extracts will orally administered at escalating doses of 100, 500, 1000,
3000, and 5000 mg/kg body weight (Barik et al., 2008). According to OECD recommendations, the
study of acute toxicity was carried out. For a period of two hours, the mouse should monitored
continually under the following profiles of (Barik et al., 2008). (I) Behavior pattern. A state of
alertness, restlessness, irritation, and terror. (II) Neurological pattern. Activities that occur
spontaneously, responsiveness, touch reaction, pain response, and gait. (III) Profile of autonomic
function. The act of defecating and urinating. They were observed for lethality or death after 24
hours, 72 hours, and 14 days.
 P a g e | 27

Oral Glucose Tolerance Test

The method of (Shirwaikar et al., 2006) was imitate to test the oral glucose tolerance of normal
mouse that had been fasted overnight (18 h). Six groups of six mouse each were given 0.9 percent
(w/v) saline, 2.5 mg/kg glibenclamide, and Diplazium esculentum methanol and aqueous extracts
250 and 500 mg/kg respectively. After that glucose (3 g/kg) will fed 30 minutes after the extracts
were administered. At 0, 30, 60, and 120 minutes after orally administered to the experimental
mouse using a force feeding syringe, blood samples will be collected for the measurement of blood
glucose by puncturing the lateral tail vein. Blood glucose will be measured by using a one-touch
electronic glucometer using a glucose test strip monitoring meter kit.

Induction of Diabetes in Experimental Animals

Using the method of (Graham et al., 2011) Diabetes will be induced in mouse by a single
intraperitoneal injection of streptozotocin (140-160 mg/kg it depends) in overnight fasted mouse
weighing 10-15 g. By the method described earlier (Graham et al., 2011) the researcher used a
pharmaceutical-grade formulation of STZ to avoid impurities that may have harmful biologic
activities. The researcher followed the dose-range recommendations of the Clinical Islet Transplant
(CIT) consortium regarding procedures for the mouse bioassay in testing human islet cell
preparations to be used in clinical trials (150 to 240 mg/kg). The presence of hyperglycemia will
confirmed if the presence of elevated glucose levels after 72 hours later. Diabetic animals will be
defined as those with a blood glucose level greater than 150 mg/dl.

Experimental Design
Using the method of (Tomar and Sisodia, 2012) the mouse will be divided into seven groups
of six each. For 15 days, the extracts will be administered. Group I: Normal control mouse will try to
give saline; Group II: Diabetic control mouse administered saline; Group III: Mouse will be given
glibenclamide (2.5 mg/kg) daily for 15 days; Group IV: Diabetic mouse will be given DE methanolic
extract (250 mg/kg); Group V: Diabetic mouse will be given DE methanolic extract (500 mg/kg);
Group VI: Diabetic mouse will be given DE Aqueous extract (250 mg/kg); Group VII: Diabetic mouse
will be given DE aqueous extract (500 mg/kg). On days 0, 5, 10, and 15 following extract
administration, fasting blood glucose will be calculated.
 P a g e | 28

Group (n= 6) Treatment

I Normal Control
II Diabetic Control
III Glibenclamide (2.5 mg/kg)
IV Diabetic + DE Methanolic Ext (250 mg/kg)
V Diabetic + DE Methanolic Ext (500 mg/kg)
VI Diabetic + DE Aqueous Ext (250 mg/kg)
VII Diabetic + DE Aqueous Ext (500 mg/kg)
Table 3. List of Control and Treatment Group

Probit Analysis
These analyses are used to compare radiation tolerance across life stages and species, as
well as to assist in determining a target dose for large-scale testing. A typical quantal response
experiment involves the administration of a drug to groups of animals at varying doses. The
percentage of patients who die at each dose level is recorded. Following that, this data can be
expressed mathematically as follows: 𝑷 = 𝜶 + 𝜷 [𝒍𝒐𝒈𝟏𝟎 (𝑫𝒐𝒔𝒆)]

Where p equals five plus the response rate's inverse normal transform (called the Probit).
The five are included to eliminate the possibility of negative probits, which cause confusion when
solving the problem manually.
 P a g e | 29

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APPENDICES

A.
LEGEND:
Site

Figure 1. Sampling Site: Barangay Cagdapao, Tago, Surigao del Sur

B.

C.

D.
 P a g e | 35

E.