i

STUDIES ON SOME POTENTIAL MEDICINAL PLANTS

AS AN ALTERNATIVE TREATMENT IN REDUCING

THE RISK OF DIABETIC COMPLICATIONS

HINA AKRAM MUDASSIR

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF KARACHI

KARACHI-75270,

PAKISTAN

ii

STUDIES ON SOME POTENTIAL MEDICINAL PLANTS

AS AN ALTERNATIVE TREATMENT IN REDUCING

THE RISK OF DIABETIC COMPLICATIONS

Thesis submitted for the fulfilment of the degree of

Doctor of philosophy

BY

HINA AKRAM MUDASSIR

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF KARACHI

KARACHI-75270,

PAKISTAN

iii

Declaration

I hereby undertake that this research entitled “Studies on Some Potential Medicinal Plants

as an Alternative Treatment in Reducing the Risk of Diabetic Complications” is an

original research work of my approved synopsis from the Board of Advance Studies and

Research, University of Karachi and no part of said thesis falls under plagiarism. None of

the context of this work was previously submitted by another person which has been

accepted for the award of any degree or diploma of the university or the institute of

higher education, except where due acknowledgment has been in text.

___________________

Hina Akram Mudassir

Enrolment No. SCI/BCH/KU-30174/2010

Department of Biochemistry,

University of Karachi.

Dated: ________________

iv

Certificate

I hereby certified that Ms. Hina Akram Mudassir, (Enrolment No. SCI/BCH/KU-

30174/2010) PhD student of Department of Biochemistry, University of Karachi, has

successfully completed her research work entitled “Studies on Some Potential Medicinal

Plants as an Alternative Treatment in Reducing the Risk of Diabetic Complications”

under my supervision. The entire content is her own work and that, to the best of my

knowledge and belief, it contains no material previously published or written by anyone

else nor material which has been accepted for the award of any other degree or diploma

of the university of higher learning, except due acknowledgment made in the text.

______________________

Dr. SHAMIM A. QURESHI

Assistant Professor &

Research Supervisor

Department of Biochemistry

University of Karachi

Dated: _________________

v

Dedication

This Ph.D. thesis is dedicated to my darling daughters

Areeba, Alishba, and my close family members who did not get

enough of my time and attention during the

completion of this work.

vi

Acknowledgements

My very first feeling of thankfulness is to Almighty Allah who granted me the

courage and ability to complete this research work. Secondly, I would like to express my

special thanks and gratitude to my Supervisor Dr. Shamim A. Qureshi not only for her

guidance but also for her understandings and appreciations towards innovative ideas that

brought out new findings in this study. She is my mentor, who at every step and

situations, other than this work, has supported and encouraged me to grow much more

than just a researcher. Without her knowledge and experience, the quality of this outcome

would not have been possible.

I would like to thank Chairperson of the Department of Biochemistry, Professor

Dr. Viqar Sultana for providing the technical facilities for this research and Professor Dr.

Muhammad Iqbal Chaudary, the Director of HEJ Research Institute of Chemistry,

University of Karachi, for providing the facility of rotary evaporator in his renowned

institute of research.

I also want to thank to all my colleagues of Department of Biochemistry, Federal

Urdu University of Arts, Science and Technology (FUUAST) for their cooperation and

support. A special thank also goes to two of my Lab colleagues Ms. Tooba Lateef and Dr.

Muhammed Bilal Azmi for their unconditional support and guidance during the research

work.

My family must deserve a special expression of gratitude because without the

support of my in-laws and the prayers of my parents, I would not have achieved this

success. Recognition of constant support and understanding from my siblings and friends

is also due.

Last but not least, the appreciation and encouragement of my beloved husband

Mr. Mudassir Ahmed is countless and has always been with me as a strong support.

Hina Akram Mudassir

vii

S. No. TABLE OF CONTENTS Pg. No.

Acknowledgements

Summary

1. Chapter 1: Screening of Hypoglycemic Activity and Selection of the

Experimental Plant

1. Introduction

1.1. Worldwide Burden of Diabetes

1.2. Prevalence of Diabetes in Pakistan

1.3. Importance of OGTT and Diabetes

1.4. Minerals and Diabetes

1.5. Role of Medicinal Plants & their Constituents in the Treatment

of Diabetes

1.6. Medicinal Plants Used in Present Work

1.7. Purpose of Study

2. Material and Methods

2.1. Experimental Animal

2.2. Plant Material

2.3. Positive Control

2.4. Dimethyl Sulphoxide (DMSO)

2.5. Preparation of Aqueous & Organic Solvent Extracts of Selected

Medicinal Plants

2.6. Phytochemical Analysis

2.6.1. Qualitative Analysis

2.6.2. Quantitative Analysis

viii

2.7. Trace Mineral Analysis

2.8. Animal Grouping and Determination of Acute Toxicity

2.9. Animal Grouping and Determination of Oral Glucose Tolerance Test

(OGTT)

2.10. Determination of Percent Glycemic Change

2.11. Statistical Analysis

3. Results

3.1. Phytochemical Analysis

3.1.1. Qualitative Analysis

3.1.2. Quantitative Analysis

3.2. Trace Mineral Content in Selected Plant Extracts

3.3. Effect of Selected Medicinal Plants on OGTT and Percent

Glycemic Change

4. Discussion

5. Conclusion

Chapter 2: Effect of Ethanolic Seed Extract of Centratherum

Anthelminticum in Fructose-Induced Insulin Resistance Type 2

Diabetic Rabbits

6. Introduction

1.1. Importance of High Intake of Fructose in Relation to Diabetes

1.2. Major Complications of Type 2 Diabetes

1.3. Antidiabetic Medicinal Plants in the Treatment of fructose-

Induced Type 2 Diabetes

1.4. Plant for Present Experimental work

1.5. Purpose of Study

ix

7. Materials and Methods

2.1. Plant Material and Preparation of Ethanolic Seeds Extract (ESEt)

of Centratherum Anthelminticum

2.2. In-Vitro Investigations of ESEt of C. Anthelminticum

2.3. In-Vivo Investigations of ESEt in Fructose-Induced Type 2

Diabetic Rabbit Model

2.3.1. Experimental Rabbits

2.3.2. Positive Control and Vehicle

2.3.3. Induction of Fructose-Induced Type 2 Diabetes

2.3.4. Experimental Rabbits and their Grouping

2.3.5. Determination of Physical Parameter

2.3.6. Determination of Hematological Parameters

2.3.7. Determination of Biochemical Parameters

2.3.8. Determination of Antioxidant Parameters

2.3.9. Determination of Liver Glycogen and Total Lipids

2.3.10. Determination of Rate-Regulatory Enzyme Activity of

Cholesterol Biosynthesis

2.3.11. Determination of Trace Minerals

2.3.12. Statistical Analysis

8. Results

3.1. In-Vitro Investigations

3.2. In-Vivo Investigations

9. Discussion

10. Conclusion

11. Future Aspect of the Present Research

x

12. References

Appendices

Publication from thesis

xi

S.No List of Tables Pg.No

Table 1 Qualitative Phytochemical Analysis of Seeds/Fruits

Extract of Selected Plants

Table 2 Quantitative Phytochemical Analysis of Seeds/Fruits

Extract of Selected Plants

Table 3 Trace mineral Content in Seeds/Fruits Extracts of

Selected Plants

Table 4 OGTT of Seeds/Fruits Extracts of Selected Plants in

Rabbits

Table 5 In vitro Antiglycation and Antioxidant Activities of

AqSEt and ESEt of C. anthelminticum

Table 6 Effect of ESEt of C. anthelminticum on Percent Body

Weight Change

Table 7 Effect of ESEt of C. anthelminticum on Percent

Glycemic Change, HbA1c Fasting Insulin and FIRI

Table 8 Effect of ESEt of C. anthelminticum on HMG-

CoA/Mevalonate Ratio and CRI

Table 9 Effect of ESEt of C. anthelminticum on Liver Glycogen

and Total Lipids

Table 10 Effect of ESEt of C. anthelminticum on Biochemical

Parameters

Table 11 Effect of ESEt of C. anthelminticum on Serum Trace

Mineral levels

Table 12 Effect of ESEt of C. anthelminticum on Antioxidant

Parameters

xii

S.No List of Figures Pg.No

Figure 1 Animal grouping according to their specific treatments

Figure 2 Standard curve of Dextrin

Figure 3 Effect of ESEt of C. anthelminticum on Percent Body

Weight Change in Fructose-induced Type 2 Diabetic

Rabbits

Figure 4 Effect of ESEt of C. anthelminticum on Percent

Glycemic Change in Fructose-induced Type 2 Diabetic

Rabbits

Figure 5 Effect of ESEt of C. anthelminticum on TC and TG in

Fructose- induced Type 2 Diabetic Rabbits

Figure 6 Effect of ESEt of C. anthelminticum on Lipoproteins in

Fructose-induced Type 2 diabetic rabbits.

xiii

Summary

The incidence of type 2 diabetes is growing on every coming day globally

because of many acquired causes especially unhealthy lifestyle and inappropriate diet.

Artificial sweetener, high fructose corn syrup plays an important role in this regard and is

threatening the world by enhancing the risk of insulin resistance, one of the important

characteristics of type 2 diabetes, which ceased the action of hypoglycemic hormone

insulin in target tissues. There are number of conventional antidiabetic medicines

available for the management of this endocrine problem but they are not free from side

effects. To overcome this problem or being reluctant to usual medicines, people

especially from developing countries are prefer to use medicinal plants as an alternative

therapy for different health problems including type 2 diabetes. Keeping this view in

mind, the present study was designed and divided in two phases. Phase I study was

designed to make aqueous (AqSEt) and ethanolic seed (ESEt) extracts of medicinal plants

including Avena fatua (wild oat), Centratherum anthelminticum (black cumin), Citrus

limon (lemon) and C. paradisi (grape fruit) while aqueous (AqFEt) and methanolic fruit

(MFEt) extracts of Withania coagulans (paneer dodi) were made. After determining their

phytochemical and trace mineral contents, these aqueous and organic solvent extracts

were subjected to hypoglycemic screening in overnight fasted normal rabbits through oral

glucose tolerance test (OGTT). The particular extract of experimental plant which

showed potent hypoglycemic potential was selected to use in second phase of study.

Phase II study was associated to investigate the antidiabetic activity of selected extract of

medicinal plant in fructose-induced type 2 diabetic rabbit model. In addition, the in vitro

antiglycation and antioxidant activities of that particular extract were also determined.

In the present study, the qualitative analysis was revealed the presences of most of

the phytochemicals tested including alkaloids, cardiac glycosides, flavonoids, glycosides,

phlobatanins, resins, saponins, steroids, tannins and terpenoids in AqFEt and MFEt of

W.coagulans, followed by AqSEt & ESEt of C. anthelminticum, C. limon, and A. fatua

as compared to AqSEt & ESEt of C. paradisi. The quantitative analysis showed that seed

xiv

extracts of C. limon and C. paradisi were rich in total phenols, followed by fruit extracts

of W. coagulans, and seed extracts of C. anthelminticum. Similarly, flavonoids were

estimated high in W. coagulans, C. limon and C. anthelminticum while alkaloids found

abundant in C. limon, followed by C. anthelminticum and W. coagulans. Another

bioactive class of compound, saponins were present in large amount in C.

anthelminticum. Trace mineral determination of present study revealed that nice amounts

of cobalt (Co), chromium (Cr), iron (Fe), potassium (K), sodium (Na) and zinc (Zn) were

present in ESEt of C. anthelminticum, followed by MFEt of W. coagulans and ESEt of C.

limon.

Acute toxicity study proved that all extracts of medicinal plants were safe for oral

consumption. Among the five experimental medicinal plants tested for hypoglycemic

potential through OGTT, the most excellent hypoglycemic activity was observed by ESEt

of C. anthelminticum, followed by ESEt of C. limon and both extracts (AqFEt & MFEt)

of W. coagulans where they were found to induce glucose tolerance either at pancreatic

or extra-pancreatic level in glucose-induced hyperglycemic rabbits. In case of C.

paradisi, only ESEt showed some hypoglycemic activity whereas extracts of A. fatua

were not found effective in this regard. In the end, ESEt of C. anthelminticum was

selected for second phase of the study because of showing nice phytochemical and trace

mineral contents and most important potent hypoglycemic activity in OGTT.

Normally, high fructose intake accelerates weight gain by inducing hepatic

lipogenesis followed by hyperlipidemia, insulin resistance, hyperglycemia,

hyperinsulinemia, oxidative stress, cardiac and hepatic dysfunctions, all these constitute

type 2 diabetes. The all same were observed in fructose-induced type 2 diabetic control

rabbits that were administrated with 35% fructose solution orally in overnight fasted state

for consecutive 14 days. In the present study, three doses (200, 400 & 600 mg/kg) of

ESEt of C. anthelminticum were orally administrated in three separate fructose-induced

type 2 diabetic test groups where they were found to induce significant reduction

(p<0.05) in percent body weight gain as compared to diabetic control group. Similarly,

xv

bad lipids including triglycerides, total cholesterol, very low and low-density lipoprotein

cholesterols were found decreased and high density lipoprotein cholesterol, the good lipid

was amplified in blood of three test groups in dose-dependent manner. Inhibition in

hepatic cholesterol production was strengthened by observing improved activity of the

rate-regulatory enzyme HMG CoA reductase of cholesterol biosynthesis in test groups.

The hypolipidemic effect of ESEt in all three test groups was also confirmed by

observing decreased values of coronary risk index in same test groups.

In the present study, all three doses (200, 400 & 600 mg/kg) of ESEt were found

hypoglycemic by showing increased in percent reduction in glycemic change in their

respective test groups. Similarly, a significant drop (p<0.05) was found in glycated

haemoglobin (HbA1c) levels in test groups that indicated the antiglycated activity of

ESEt. It was also proved by examining 80% in vitro antiglycation activity of same

extract. Other important beneficial effects of ESEt were found in test groups by

observing gradual decrease in serum insulin levels and fasting insulin resistance index

(FIRI) as compared to fructose-induced type 2 diabetic control group that showed

hyperinsulinemia and high values of FIRI that clearly indicated the presence of high

insulin resistance in these diabetic control group. On contrary, ESEt was found capable of

decreasing insulin resistance and improving insulin-receptor binding. It was also verified

by observing betterment in hepatic glycogenesis in present study.

Hepato-protective property of ESEt was also established by observing normal

levels of liver-specific enzyme alanine transferase (ALT) and decreased values of total

bilirubin in serum of all ESEt treated test groups. This activity of ESEt was also

confirmed by observing the decreased levels of uric acid in test groups as compared to its

elevated levels found in fructose-induced type 2 diabetic control rabbits that indicated the

presence of hepato-necrosis, thus ESEt improved hepatocytes stability in test groups.

Likewise, improvement in levels of cardiac-specific enzyme creatine kinase (CK) was

found in all test groups indicated the cardio-protective effect of ESEt.

xvi

ESEt of C. anthelminticum also showed protective effect by improving the serum

levels of trace minerals (elements) that required in insulin metabolism and function like

zinc (Zn) and chromium (Cr) and decreasing the levels of elements that hindered the

insulin function like cadmium (Cd) and iron (Fe) in test groups. Similarly, electrolytes

including sodium (Na) and potassium (K) were also found better in ESEt treated test

groups.

ESEt of C. anthelminticum demonstrated 67% in vitro radical scavenging activity

in the present study. The same antioxidant potential of ESEt was also observed in all test

groups where all doses of ESEt (200-600 mg/kg) were found to decrease percent inhibitions

of antioxidant parameters including catalase, superoxide dismutase and reduced

glutathione while increase in percent inhibition of lipid peroxidation (an oxidative stress

indicator), as compared to fructose-induced type 2 diabetic control rabbits which

displayed entirely opposite picture of these antioxidative and oxidative parameters.

Therefore, the results conclude that ESEt of C. anthelminticum could be used as

alternative and complementary medicine in the treatment of fructose-induced type 2

diabetes as it showed hypoglycemic, hypotriglyceridemic, hypocholesterolemic,

antiglycation, antioxidant, hepato- and cardio-protective activities by improving the

serum trace mineral levels, reducing insulin resistance and enhancing insulin-receptor

sensitivity in fructose-induced type 2 diabetic rabbits. The glucose and insulin resistance

lowering effects shown by ESEt may be due to the presence of huge amount of

phytochemicals and trace minerals in same extract which could be isolated and served as

active principle of future antidiabetic medicine.

1

Screening of Hypoglycemic Activity and Selection of the

Experimental Plant

1. Introduction

Diabetes mellitus (DM) is counted as the most widespread disorder of both

developed and developing countries in today's world. The rough estimate about the

affected population from this disease is about 25% globally and till the end of 2035 it is

accepted to increase by 55% (Kavishankar et al., 2011). The disturbance in carbohydrate

metabolism is the basic factor for the progress of diabetes which is associated with

complete deficiency or low blood insulin level or insensitivity of target organs towards

insulin action (Rang et al., 2003). Frequent urination (polyuria), increased thirst

(polydipsia) and hunger (polypagia) are the alarming signs of this disease. The main

diabetic complications are ketoacidosis, strokes, cardiovascular diseases, kidney

problems and foot ulcer. Though, these should be managed by proper treatments

prescribed by diabetologists (Dixit et al., 2014).There are three main types of diabetes:

i. Type 1 Diabetes

It is an acute state in which body either completely unable to produce or fails to

produce enough insulin. Therefore, it is mentioned as "insulin-dependent diabetes

mellitus" (IDDM) as insulin is the only cure for this disease or "juvenile diabetes" as it

occurs in newborns or people less than 25 yr of age. However, its autoimmune form has

also been observed in adults of age above 30 yr and termed as LADA (latent autoimmune

diabetes of adults), which is characterized by the production of autoimmune antibodies

against beta cells of pancreas that results no insulin (Krause et al., 2014). The diminished

or insufficient insulin production affects all energy metabolism including carbohydrate,

lipid and protein (Mishra et al., 2009). 5 to 15 % population is affected with this type of

diabetes worldwide (Joshi and Shrestha, 2010).

2

ii. Type 2 Diabetes

It is a chronic condition in which body produce relatively lower amount of insulin

or target cells (hepatocytes, myocites, & adipocytes) of insulin showed resistance towards

it. This type is known as "non-insulin dependent diabetes mellitus" (NIDDM) as its basic

characteristic symptom hyperglycemia can be reversed by oral hypoglycemic medicines

that are responsible either to enhance insulin secretion (insulin secretagogues) or improve

insulin sensitivity (insulin sensitizers such as biguanides & thiazolidinediones) or reduce

glucose absorption in gastrointestinal tract (α-glucosidase inhibitors) thus improve

glucose utilization in body (Rang et al., 2003). Lack of exercise, obesity and high

carbohydrate & fat diets are the most important factors that trigger the occurrence of type

2 diabetes (T2D) in people of age group 35 yrs and above (American Diabetes

Association, 2007). It is reported that only 20% of the total T2D cases depends on

hereditary condition whereas 80% depends on lifestyle (Norris et al., 2005). In addition,

many metabolic (hypertension, hyperlipidemia, etc) and endocrine (gigantism,

acromegaly, etc) disorders are also contributing its prevalence in the world (Rang et al., 2003;

Jordan and Jordan, 2012). Interestingly, many medicines (anti-inflammatory glucocorticoids,

interferon, etc) are reported to induce insulin resistance (Ripsin et al., 2009). Therefore,

T2D constitutes about 80-90 % of total diabetic cases globally (Vijan, 2010). The patients

of this type do not entirely depend on insulin injections but in worst condition it becomes

necessary for their survival and it covers around 30% of total T2D cases (Ebesunun et al.,

2012). Beside these, controlled dietary plan and exercise also play an important role in

the management of this health hazard. Because of inability of body to utilize glucose,

triglycerides become an alternate source of energy in diabetic patients, results in

accelerating the process of beta oxidation of fatty acids that leads to increased production

of acetyl CoA which cannot be easily handled by tricarboxylic acid (TCA) cycle for its

complete catabolism into CO2 and water. Therefore, this large amount of acetyl CoA

diverts in creating diabetic dyslipidemia by increasing the concentrations of all bad

factors including cholesterol, ketone bodies, triglycerides, low-density lipoproteins that

lead to oxidative stress and many chronic micro- and macro-vascular complications in

3

body (Zoppini et al., 2012). It has been estimated that 70-80 % of casualty rates in

diabetic patients is due to the vascular complications (Gulliford and Charlton, 2009).

iii. Gestational Diabetes

It is classified as a third type of diabetes and appears only during pregnancy

(especially in the 3rd

trimester of gestation) without any earlier record of diabetes. During

pregnancy, hormonal fluctuations interfere with insulin sensitivity that produces

hyperglycemic condition of variable severity and results in complications in child birth

(Pereira et al., 2014). After child delivery, this type of diabetes usually normalized but

the probability of having diabetes in newborn babies of gestational diabetic mothers is

still questionable and these mothers are also at high risk of diabetes in future (Hoffman et

al., 1998).

Therefore, as diabetes is affecting all age groups of life, a multipurpose drug

would be an ideal for the treatment of diabetes that possesses hypoglycemic property

along with other preventive effects (Wadkar et al., 2008). The available synthetic agents

for treating diabetes have some serious limitations and their long-term usage bring out

adverse conditions like unconsciousness, liver and kidney dysfunction and of course use

of few anti-diabetic medicines are risky during pregnancy. Another important factor

regarding treatment is the insulin injections that are the only possible life-saving way for

severe diabetic patients but as it is protein in chemical nature so care should be required

for its handling and storage plus it cannot be given orally.

1.1. Worldwide Burden of Diabetes

The frequency of diabetes among adults has growing rapidly. There are more than

285 million people present around the world with this disorder, and this value is expected

to increase up to 438 million by the year 2030 (Shaw et al., 2010). Studies showed that

South Asian people are more prone to type 2 diabetes (Mather and Keen, 1985) which is

very clear through the estimated occurrence of diabetes from the year 2000 to 2030 that

4

was found above 151% in South Asian region (Jayawardena et al., 2012). Interestingly,

in the developed countries, 42% rise in incidence of this disease has been seen whereas

developing countries are more threatened by this disease and showed 70% increase (Patel

et al., 2006). Statistically, it is calculated that diabetic casualties will take 7th

position by

the end of 2030 if urgent action will not be taken in this regard (Deshpande et al., 2008).

1.2. Prevalence of Diabetes in Pakistan

Pakistan belongs to high prevalence area of diabetes where this disorder is a

major challenge for healthcare professionals. Currently, there are 6.9 million people are

affected with this problem and our country is at the 7th

position in the list of highest

diabetic populated countries, and expected to gain 5th

position by the end of year 2025

(Qidwai and Asfaq, 2010). A survey reported that the overall diabetes frequency in

Pakistan is 11.1% and the incidence of type 2 diabetes among adults is more than 10%.

The same survey also described that in urban areas of Pakistan 5.1% males and 6.8%

females have been identified with diabetes whereas rural areas showed that 5.0% men

and 4.8% women were suffered with the same problem in 2008 (Aziz et al., 2009).

International Diabetes Federation (IDF) included Pakistan among the top 10

countries where diabetes affects people of age group ranging from 20-79 yrs (Ansari et

al., 2015). A national survey also described that people with age 40+ are the most

affected ones (Qureshi et al., 2011). The foremost cause behind the occurrence of type 2

diabetes in Pakistan among middle aged people of both genders are lack of physical

activity and unhealthy eating habits. Furthermore, low ranked health care system to

manage diabetes also contributed to enhance its frequency (Reza et al., 2015).

1.3. Importance of OGTT and Diabetes

The normal blood glucose concentration (70-110 mg/dl) is the reflection of

intestinal absorption of carbohydrates, hepatic yield and muscular acceptance of glucose.

Fasting value of blood glucose is the hepatic turnover of glucose which describes the

5

liver function of an individual. For the diagnosis of pre- or post-diabetic condition in an

individual, blood glucose level in fasting and random (after 120 minutes) states are

crucial. The oral glucose tolerance test (OGTT) describes the glucose tolerance status in

an individual and it is performed by giving 75-100 g of glucose orally in fasting state,

followed by monitoring the blood glucose level from 0 to 120 minutes with 30 minute

interval. It indicates proper utilization of glucose in body by showing its rapid clearance

from the blood. OGTT is normally used to diagnose diabetes, insulin resistance and

disorders of carbohydrate metabolism (Lindahl et al., 2009).

1.4. Minerals and Diabetes

Trace elements (micro-minerals) including iron, copper, chromium, cobalt,

fluoride, iodine, selenium, and zinc are essential for human nutrition and health as same

as macro-minerals viz., calcium, potassium, magnesium, sodium and sulphur but required

in very small amount like less than 200 mg/day (Fraga, 2005). Minerals have vital

position in biological system like these are involved in regulating humoral & cellular

immunities, membrane potential, muscle contractions, transmissions of nerve impulses,

mitochondrial performance and these are also an integral part of many enzymes like

cytochromes, etc (Matsumura et al., 2000). The minerals and vitamins work together to

augment their effects in body and are indispensable for maintaining healthy human life

(Siddiqui et al., 2014). Interestingly, new strategies used in the treatment of diabetes

described that both macro- and micro-minerals reduces the risk of insulin resistance (type

2 diabetes) either by playing their roles as cofactor of various enzymes involved in

carbohydrate metabolism or enhancing insulin release or activating its receptors thereby

increasing insulin-receptor sensitivity (Akhuemokhan et al., 2013). In addition, trace

elements also act as antioxidants thereby preventing lipid peroxidation which is the gate

way of many chronic complications of diabetes especially cardiovascular diseases

(Rajpathak et al., 2004). Several studies described the intimate beneficial and adverse

associations between minerals and diabetes like chromium, magnesium, vanadium, zinc,

manganese, molybdenum and selenium are reported to potentiate the action of insulin in

6

maintaining blood glucose level (Fraga, 2005). Whereas dietary iron overload involved in

the development of diabetes by acting as a pro-oxidant and damaging beta-cell of

pancreas, though the same mineral is a part of many enzymes and protein which are

essential for health, growth, and different metabolism especially DNA (Simcox and

McClain, 2013). Similarly, alteration in calcium flux also produce adverse effect on beta-

cell secretory function and insulin release, thereby enhancing the risk of diabetic

complications (Pittas et al., 2007).

Many beneficial impacts of trace elements in the prevention of diabetes have been

reported like cobalt (as cobalt chloride) showed glucose lowering effect either by

decreasing glucose production through inhibiting gluconeogenesis or enhancing glucose

uptake in tissues via increasing expression of (glucose transporter 1) GLUT 1 (Saker et

al., 1998). Similarly, copper has an insulin-like activity whereas its deficiency induced

glucose intolerance and hypercholesterolemia which in turn increases the risk of heart

problems like atherosclerosis (Kazi et al., 2008; Ekmekcioglu et al., 2001). Type 2

diabetic patients have higher rate of excretion of chromium than its cellular burden in

tissues. However, being a component of glucose tolerance factor (GTF), chromium

supplement is highly suggested in the treatment of diabetes and its related complications.

Chromium is not only important for insulin action but also for sustaining carbohydrate,

protein and lipid metabolism (Cefalu and Hu, 2004; Nsonwu et al., 2006).

Magnesium plays a crucial role in preventing insulin resistance, as it is necessary

for insulin secretion and activity. It (sometime replaced by manganese) also acts as a

cofactor of many enzymes involved in carbohydrate metabolism (Tosiello, 1996; Volpe,

2008). Zinc is another cation acts as a cofactor for many intracellular enzymes involved

in all metabolism especially glucose and antioxidant system. It is not only involved in

glucose uptake in tissues but also in the regulation of signal transdation initiated by

insulin and synthesis of its receptor (Tang and Shay, 2001). Normally, Zn is involved in

immune functions and taste alertness which are reported to be disturbed in diabetes due to

its deficiency (Yahya et al., 2011).

7

In diabetes, hyperglycemia-induced glycation decrease the activity of membrane

bound enzyme (pump) Na-K-ATPase which in turn altered the electrolyte balance across

the membrane which has been reported as the basic cause of cellular injury in

hyperglycemic patients (Mimura et al., 1992; Al-Rubeaan et al., 2011). Sodium, along

with calcium, influences the insulin secretion from pancreatic beta cells (Reza et al.,

2015). Few studies reported that vanadium mimics the action of insulin and produced

positive effect on carbohydrate metabolism (Cam et al., 2000) whereas selenium has

controversial role in the prevention and progression of diabetes (Durak et al., 2010; Bleys

et al., 2007).

Polyuria is the basic feature of diabetes, which not only induced dehydration but

also loss of electrolytes and other essential ions, thus create deficiency of vital minerals

in body which give warm welcome to diabetic complications (Bhanot et al., 1994).

Therefore, supplements and diet rich in minerals are the best way to slows down the

harmful impacts of diabetes. In this regard, treatment of diabetes with medicinal plants

has dual positive effects like besides being hypoglycemic agent they can provide

deficient micronutrients to diabetic patients as plants are rich in variety of minerals and

trace elements.

1.5. Role of Medicinal Plants and their Constituents in the

Treatment of Diabetes

Medicinal plants have been using as an innate remedy for the treatment of many

diseases including diabetes from a long time (Badoni and Badoni, 2001). A report

described that about 80% population especially from developing countries depends on

phytomedicines for different ailments (Patel et al., 2012; Tiwari et al., 2013). Actually,

plant-derived medicines offer easy accessibility, negligible side effects and low cost that

make them prominent among number of synthetic medicines in commercial market and

are widely recommended (Pitchai et al., 2010). More than 1200 medicinal plants have

been testified for antidiabetic constituents (Arif et al., 2014). World Health Organization

8

(WHO) also approved the use of plant derived medicines for the treatment of many

problems like asthma, eczema, arthritis, migraine, menopausal problems, fatigue,

constipation and diabetes (Singh, 2011). Interestingly, the active principle of metformin

(commonly used antidiabetic medicine) has also been derived from plant Galega

officinalis before it was synthesized (Bailey and Day, 2004).

The globally held in vivo and in vitro trials established the role of medicinal plants

in the treatment of diabetes (Jung et al., 2006), like Brassica juncea (mustard) seeds are

reported to have a potent hypoglycemic activity (Thirumalai et al., 2011). Experimental

models and clinical studies of Eugenia jambolana (Jamun) revealed its hypoglycemic

role in diabetes (Ravi et al., 2004). Similarly, leaf extract of Catharanthus roseus

(sadabahar) (Ohadoma and Michael, 2011) and Coccinia grandis (baby water melon)

(Ajay, 2009) are claimed to reduce blood glucose level in diabetic animal models. The

root extract of Rauwolfia serpentina (snake root) is also reported to involve in reducing

hypertriglyceridemia and hyperglycemia in alloxan-induced diabetic mice model (Azmi

and Qureshi, 2012a). The fruit juice of Momordica charantia (bitter melon) possesses

hypoglycemic effect which was testified both in animals and in type 1 and 2 diabetic

human volunteers (Welihinda et al., 1986).

A single plant is a store house of variety of phytochemicals including terpenoids,

alkaloids, flavonoids, phenols, etc which alone or jointly ameliorate blood glucose level

like three constituents viz., charantin, polypeptide-p (plant insulin) and alkaloids of M.

charantia are reported as hypoglycemic agents (Mohammady et al., 2012). Similarly, the

hypoglycemic effect of Allium sativum L. (garlic) is due to Allin (S-allylcysteine

sulfoxide) which is actively confined in garlic cloves (Younas and Hussain, 2014). A.

sativum lowers blood sugar level either by its capacity to excite the pancreas to boost

insulin synthesis, or positively affects insulin-receptors (Augusti and Sheela, 1996).

Alkaloids mainly decrease glucose transport through the intestinal epithelium by

inhibiting alpha-glucosidase activity (Bhushan et al., 2010). Some important alkaloids

include mycaminose I isolated from Syzygium cumini (Linn.) seed (Gupta and Saxena,

9

2011), mahanimbine II, a carbazole alkaloid isolated from Murraya koenigii (Linn.)

leaves (Nayak et al., 2010), arecoline III, isolated from Areca catechu (Prabhakar and

Doble, 2011), tecomine IV isolated from flower extract of Tecomastans (Linn.), yellow

bell-shaped flowering plant (Aguilar-Santamaría et al., 2009), berberine V isolated from

Tinospora cordifolia (Singh et al., 2003), aegeline VI, an alkaloidal-amide isolated from

Aegle marmelos leaves (Narender et al, 2007), and catharanthine VII isolated from C.

roseus (Chattopadhyay, 1999), all are reported to possess antidiabetic potential.

Other phytochemicals include flavonoids which are widespread naturally and

possess anti-diabetic activity (Brahmachari, 2009; Qi et al., 2010). They act on pancreatic

beta cells to increase the release of insulin as well as increase hepatic glucokinase activity

that helps to normalize glucose concentration (Bhushan et al., 2010). Similarly, phenols

like trigonelline (VIII) isolated from fenugreek seed (T. foenum-graecum Linn.) were

reported to inhibit α-amylase activity, thus decreased glucose absorption in gut

(Suryanarayana et al., 2004; Alauddin et al., 2009). Glycosides such as anthocyanin

(XLVII) isolated from Vaccinium arctostaphylos (Linn.) do the same job (Nickavar and

Amin, 2010). Saponins are natural detergents that are reported to stimulate insulin release

and regulate blood glucose level, the prominent saponins are triterpenoid, steroid, and

steroidal glycol-alkaloid (Bhushan et al., 2010).

1.6. Medicinal Plants Used in Present Work

1.6.1. Avena Fatua L.

A. fatua is commonly called common wild oats (family: Poaceae / Gramineae)

and it is indigenous to Asia, America, Australia, Europe, North Africa and different

islands. This plant is used as a nerve tonic and stimulant. The seeds of this plant are

diuretic, emollient and refrigerant in nature. However, common cultivated oats named

Avena sativa belongs to the same family has reported to have various pharmacological

activities like it is effective in case of nervous, skin, and gastrointestinal disorders (Khare

CP, 2007). The same oats also has beta-glucan, a polysaccharide which found beneficial

10

in the treatment of type 2 diabetes, hyperlipidemia and cardiovascular diseases where it

acts as an antioxidant and anti-inflammatory agent (Andersson et al., 2013; Al-Snafi,

2015).

1.6.2 Centratherum Anthelminticum (L.) Kuntze

C. anthelminticum belongs to the family Asteraceae. The alternative name of this

plant is Vernonia anthelminticum whereas it is popularly known as wild cumin/worm

seed and in Urdu it is termed as kali zeeri (Fatima et al., 2010). The seeds of this plant

possess unpleasant bitter taste and are blackish brown in colour. These are used in

number of disorders including vitiligo (white patches on skin), kidney problems, ulcers,

liver diseases and asthma. They also have antipyretic and diuretic properties (Galani and

Panchal, 2014). The scientific assessments of organic solvent and aqueous extracts of

these seeds revealed their medicinal role as antibacterial, larvicidal, antiviral, antifungal,

anticancer, antidiabetic, antioxidant, pain-relieving, anti-inflammatory and wound

soothing agents (Paydar et al., 2013). The ethanolic seed extract of this plant also

reported to be involved in improving the lipid profile in high-fat induced hyperlipidemic

rabbits (Lateef and Qureshi, 2013). Besides having significant amount of fixed and lesser

amount of volatile oils, the seeds of this plant contain number of phyto-constituents (Patel

et al., 2012). Different flavonoids, sterols and steroids have also been isolated and

reported from these seeds (Tian et al., 2004; Amir and Koey, 2011).

1.6.3. Citrus Limon (L.) Burm.f.

C. limon (family: Rutaceae), is normally known as lemon. Due to the presence of

polyphenolic compounds, lemons are globally used as a remedy for reducing obesity,

hyperlipidemia, hyperglycemia and insulin resistance (Naim et al., 2012). The ripe fruits

of this plant are reported to have anti-scorbutic, antiemetic, tonic, stomachic (Rozza et

al., 2011), antimicrobial, antiviral, hepato- and cardioprotective properties (Shefalee et

al., 2007; Viuda et al., 2008; Dhanavade et al., 2011; Mohanapriya et al., 2013). Studies

claimed that lemon fruit juice was found useful in treating nephrolithiasis (Kang et al.,

11

2007) and its seeds were in piles and inflammation (Shrivastava et al., 2010). The

different parts of Citrus plants are a good source of essential oils and other chemical

constituents like protopine and corydaline alkaloids, lactons, polyacetylene, acyclic

sesquiterpenes, hypericin and pseudohypericin which have broad spectrum activity

against various gram-negative and positive bacteria (Sah et al., 2011; Hindi and Chabuck,

2013). Studies on C. limon peel discovered the presences of polymethoxyflavones (PMF)

including hesperidin, hesperetin, naringin, etc, which are proved as anticancer agents

against colon cancer in experimental models, also found effective in reducing 10% risk of

gastric and breast cancer in women plus they also produced positive improvement in

patients with Parkinson‟s disease (Chun et al., 2008; Wang et al., 2014; Akhila et al.,

2015). Other studies reported that flavonoids from C.limon include quercitin, eriocitrin,

didymin, etc, are found effective in preventing oxidative stress by improving the status of

antioxidant enzymes (catalase, superoxide dismutase, glutathione transferase) and lipid

peroxidation (Nijveldt et al., 2001; Street et al., 2013). Naringin and hesperidin (PMFs)

also reported to have antidiabetic activity (Jung et al., 2004; Pari and Suman, 2010).

Citrus peels were also found efficient to control hyperthyroidism (Naim et al., 2012). In

fact, due to the presence of higher amount of vitamin C, folate, carotenoid and phenols,

C.limon are unavoidable for maintaining the healthy life.

1.6.4. Citrus Paradisi Macfad

C. paradisi (family: Rutaceae) is commonly known as grape fruit. It usually

grows on a 3-5 meter high trees, looks big, round, yellow coloured fruits with slightly

bitter taste. Different parts of this plant including seeds possess essential nutrients and

phyto-chemicals that are involved in maintaining health (Uckoo et al., 2012). Its pulp

provides ascorbic acid, pectin fiber, phenols and the beneficial antioxidants, β-carotene

and lycopene that produced vitamin A and helps to reduce cholesterol in body (Oboh and

Ademosun, 2006). Citrus fruit peel is 1000 times more sweeter than sucrose (Surana et

al., 2006) but is a good source of essential oil having antibacterial and antioxidant

activities (Gupta et al., 2011; Kamal et al., 2013) plus it can induce apoptosis in human

12

leukemic (HL-60) cells (Hata et al., 2003). C. paradisi like other fruits of this genus rich

in flavanone glycosides and polymethoxyflavones, a class of flavonoids, for example, it

possess naringen which showed anticancer (So et al., 1996), antiatherogenic (Gupta et al.,

2011), and gastroprotective activities (Zayachkivska et al., 2005). In addition, the same

flavones also reduce the risk of myocardial infarction by inhibiting platelet aggregation

(Folts, 2002). Another compound, 6, 7-dihydoxy bergamottin present in grape fruit juice

found effective against AIDS virus (Kupferschmidt et al., 1998; Chinsembu & Hedimbi,

2010). Similarly, grape fruit juice reported to have antihypertensive (Diaz-Juarez et al.,

2009), anti-inflammatory (Ojewole, 2004) and hepatoprotective properties (Alveraz-

Gonzalez, 2011). On the other hand, aqueous and organic solvents extracts of different

parts of C. paradisi have antibacterial (Sharma & Sharma, 2010), antianxiolytic,

antidepressant (Gupta et al., 2011), antidiabetic and hematopoietic effects (Adeneye,

2008; Mallick and Khan, 2015). In fact, C. paradisi is a good source of polyphenolic

compounds with wide spectrum of medicinal properties.

1.6.5. Withania Coagulans Dunal

W. coagulans belong to the family Solanaceae or nightshade and it is known as

paneer dodi in Urdu. It is widely cultivated in the Eastern Mediterranean region and

South Asia (Datta et al., 2013). Various parts including seeds, flowers, fruits and leaves

of this plant have been used traditionally for different purposes like its fruit are widely

used in wound healing, coagulating milk and diabetes (Kirtikar and Basu, 1996; Bown

1995; Jaiswal et al., 2009; Gupta and Keshari, 2013). The fruits of this plant have

numerous pharmacological properties like antimicrobial, anthelmintic, antifungal, liver

protective, hypoglycemic, hypolipidemic, cardiovascular, free radical scavenging, anti-

inflammatory, antitumor, immunosuppressive (Maurya et al., 2008; Lateef and Qureshi,

2014) which are due to its chemically active compounds called withanolides (steroidal

lactones). About 24 withanolides and a dimeric lignin bispicropodophyllin glucoside

have been isolated from W. coagulans (Khodaei et al., 2012). Similarly, nine different

13

compounds including withanolides, ergosta-5, 25-diene-3β, 24-diol and sitosterol-β-D-

glucoside have been isolated from fruits of this plant (Mathur and Agrawal, 2011).

1.7. Purpose of the Study

The main objective of this study was to assess and compare the hypoglycemic

activity of five medicinal plants including aqueous and organic solvent seed extracts of A.

fatua, C. anthelminticum, C. limon, C. paradisi and fruits extracts of W. coagulans in

experimental rabbits. In addition, phytochemical screening and trace mineral content

determination of these extracts have also been done. The plant extract which give best

hypoglycemic potential will be selected for further investigation in fructose induced type

2 diabetic animal model.

14

2. Materials and Methods

2.1. Experimental Animals

Male albino rabbits (1-1.5 kg) were bought from the breading house of Dow

University of Health Sciences (DUHS), Karachi and kept under usual management

condition in conventional animal house of Department of Biochemistry, University of

Karachi. They were given standard laboratory diet with free excess to water ad libitum.

2.2. Plant Materials

The fruits of Citrus limon, C. paradisi were purchased from local market to

collect their seeds personally whereas seeds of Avena fatua & Centratherum

anthelminticum and dried fruits of Withania coagulans were purchased from Hamdard

Dawakana, Sadar, Karachi. All these samples were identified by expert of Department of

Botany, University of Karachi. The voucher specimen of each sample has been kept in

Department of Biochemistry (KU/BCH/SAQ/05-09).

2.3. Positive Control

Glibenclamide (Daonil) purchased from Sanofi-Aventis Pakistan Ltd. and used as

positive control in a dose of 5 mg/kg.

2.4. Dimethyl Sulphoxide (DMSO)

DMSO of analytical reagent grade was purchased from Fisher (UK) and its 0.05%

concentration was used as vehicle for administering the doses of ethanolic/methanolic

seed/fruit extracts in experimental rabbits.

2.5. Preparation of Aqueous and Organic Solvent Extracts of

Selected Medicinal Plants

The collected seeds /fruits were washed thoroughly under running tap water, dried

15

and grind to powder. For preparing aqueous extract, 40 g of seeds /fruits powder of each

plant was boiled in 1L of distilled water at 100 ⁰C for 30 minutes, filtered through

Whatmann No. 42 (125 mm), and concentrated till dryness through freeze dryer

(lyophilizer). The resulting brown residue termed as aqueous seeds/fruits extracts

(AqSEt/AqFEt). On the other hand, same amount of each sample was soaked in 1L of

ethanol (methanol was used for W. coagulans) separately for overnight at room

temperature, filtered and concentrated at 40 ⁰C till dryness by using rotary vacuum

evaporator. Finally residue was obtained and termed as ethanolic/methanolic seeds/fruits

extracts (ESEt/MFEt) (Azmi and Qureshi, 2012b).

2.6. Phytochemical Analysis

2.6.1. Qualitative Analysis

The seeds /fruits extracts of selected plants were screened for the qualitative

determination of major constituents including alkaloids, flavonoids, carbohydrates,

glycosides, resins, saponins, steroids, tannins, triterpenoids, etc, by standard methods

(Harborne, 1973; Kondongala et al., 2010).

2.6.1.1. Tests for Alkaloids

i. Hager’s Test

Few drops of Hager‟s reagent were mixed with 1ml of extract in a test tube,

resulted in the appearance of yellow precipitates that confirmed the presence of alkaloids.

ii. Wagner’s Test

1 ml of extract was acidified with hydrochloric acid (HCl), followed by the

addition of few drops of Wagner‟s reagent, led to the formation of yellowish brown

precipitates that indicated the presence of alkaloids.

2.6.1.2. Borutrager’s Test for Anthraquinones

1 ml of FeCl3 (10%) and 0.5 ml of concentrated HCl were added to 1 ml of

16

extract, followed by boiling in a water bath for few minutes and filtered. The filtrate was

treated with diethyl-ether and concentrated ammonia. Appearance of deep pink colour

reflected the presence of anthraquinones.

2.6.1.3. Tests for Carbohydrates

i. Benedict’s Test

1ml of extract was mixed with 9 ml of distilled water, filtered. The filtrate was

concentrated till half of its original volume, then same volume of Benedict‟s reagent was

added and boiled for 5 minutes. Brick red coloured precipitates were appeared within 5

minutes, indicated the presence of carbohydrates.

ii. Fehling’s Test

The initial step of this method was as same as in Benedict‟s test, however the

concentrated filtrate was mixed with 1 ml of each of Fehling‟s solution A and B, boiled

for few minutes and production of brick red coloured precipitates confirmed the presence

of reducing sugar.

2.6.1.4. Ammonia Test for Flavonoids

A small piece of filter paper was dipped in 1ml of extract then exposed to

ammonia vapours. Instantly, yellow spots were appeared on filter paper to confirm the

presences of flavonoids.

2.6.1.5. Test for Glycosides and Cardiac Glycosides

i. Mohlisch’s Test

2 ml of extract was mixed with 8 ml of distilled water and filtered. The filtrate

was concentrated. To this, 2 drops of freshly prepared alcoholic solution of α- naphthol

(20%) and 2 ml of concentrated sulphuric acid (H2SO4) were added to observe a red

violet ring that indicated the presence of carbohydrates mainly glycosides.

ii. Keller-Killani Test

17

This test is specific for detecting cardiac glycosides. In this, 5 ml of extract was

allowed to react with 2 ml of glacial acetic acid and one drop of ferric chloride solution

followed by the addition of 1ml of concentrated H2SO4 led to the formation of brown ring

at the interface (indicated a deoxy sugar characteristic of cardenolides) and violet ring

below it.

2.6.1.6. Test for Resins

Equal volume of extract and acetone were mixed in a test tube then this mixture

was transferred into a beaker containing 2 ml distilled water. Turbidity showed the

presences of resins.

2.6.1.7. Liebermann-Burchard’s Test for Steroids

0.5 ml of extract heated with acetic anhydride till boiling and cooled, followed by

the addition of 1ml of concentrated H2SO4 along the sides of test tube that brought green

colouration which indicated the presences of steroids.

2.6.1.8. Test for Saponins

5ml of extract and 1-2 drops of sodium bicarbonate solution were mixed shaked

and left for 3 minutes. Honeycomb like froth was appeared at the bottom of test tube to

confirm the presences of saponins.

2.6.1.9. Salkowski’s Test for Triterpenoids

5 ml of extract and 2 ml of chloroform were mixed, followed by the addition of 3

ml of H2SO4, slowly by the sides of the test tube. Reddish brown colouration confirmed

the triterpenoids.

2.6.1.10. Test for Tannins

2 ml of extract and few drops of FeCl3 (5%) solution were mixed in a test tube

which produced green colouration to confirm the presences of gallotannins while

18

pseudotannins was confirmed by brown colouration.

2.6.1.11. Test for Phlobatannins

1ml of extract was boiled with HCl (1%) in a test tube, red precipitates appeared

due to the presences of phlobatannins.

2.6.2. Quantitative Analysis

2.6.2.1. Determination of Alkaloids

Finely ground seeds/fruits powder (5 g) of selected plant was mixed with 200 ml

of 10% acetic acid in ethanol. This mixture was kept for 4 hours in a covered conical

flask at room temperature. After filtration, the filtrate was condensed to one-fourth of its

actual volume by boiling on water bath. Ammonium hydroxide (concentrated) was added

to the extract drop wise till thorough precipitation was done. The solution was kept to

settle the precipitates which were collected, washed with dilute ammonium hydroxide

and then filtered. The alkaloid residue was then dried and weighed (Harborne, 1973).

2.6.2.2. Determination of Flavonoids

10 g of finely ground seeds/fruits powder of selected plant was extracted twice

with 100 ml of 80% aqueous methanol and filtered through Whatman filter paper No.42

(125 mm). The filtrate was kept in a crucible for evaporation on water bath till dryness.

This evaporation continues until constant weight was achieved (Boham and Kocipai,

1974).

2.6.2.3. Determination of Saponins

In a conical flask, 20 g of finely ground seeds/fruits powder of selected plant and

200 ml of aqueous ethanol (20%) were taken, followed by heating in a water bath at 55°C

for 4 hours along with continuous stirring. The mixture was filtered and the residue was

re-extracted with another 200 ml of 20% aqueous ethanol. The combined extracts were

19

reduced to 40 ml over water bath at about 90 °C. The concentrate was transferred into a

250 ml separatory funnel and 20 ml of diethyl-ether was added and shaken vigorously.

The aqueous layer was recovered while the ether layer was discarded. The purification

process was repeated. Then 60 ml of n-butanol was added. The combined n-butanol

extracts were washed twice with 10 ml of 5% aqueous sodium chloride. The remaining

solution was heated in a water bath. After evaporation, the samples were dried in the

oven till constant weight was achieved (Obadoni and Ochuko, 2002).

2.6.2.4. Determination of Total Phenols

1 ml of Folin-Ciocalteu reagent (1:10 diluted with distilled water) was mixed with

0.1 mg of extract in a test tube. After 3 minutes, 3 ml of sodium carbonate (2%) was

added, mixed and incubated for 2 hours. Absorbance of sample was read by using

spectrophotometer at 760 nm against blank. For the preparation of standard curve 0.01–

0.1mg of gallic acid was used. The phenolic content was calculated in milligrams per

gram of starting material (Slinkard and Singleton, 1977).

2.7. Trace Mineral Analysis

Trace mineral content of ESEt of C. limon, C. paradisi and MFEt of W.coagulans

was analyzed by using atomic absorption spectrophotometer (PG990).

2.8. Animal Grouping and Determination of Acute Toxicity

Overnight fasting normal rabbits were randomly divided into different groups

including control and test groups. Doses of each seed / fruit extracts were individually

administered orally to the rabbits of their respective test groups, whereas rabbits in

control group were treated only with distilled water (1 ml/kg) orally. The rabbits in both

control and test groups were then allowed free access to food and water ad libitum, and

their activity was observed over a period of 12-24 h for acute toxicity in terms of

20

behaviour (sedative or not), mortality rate and any other side effects such as itching,

ruffled hair, clumping together, etc.

The following are the doses of seed/ fruit extracts used in the present study for

determining acute toxicity,

i. AqSEt / ESEt of A. fatua from 10–600 mg/kg

ii. AqSEt / ESEt of C. anthelminticum from 10–3000 mg/kg

iii. AqSEt / ESEt of C. limon from 10–1000 mg/kg

iv. AqSEt / ESEt of C. paradisi from 10–600 mg/kg

v. AqFEt / MFEt of W. coagulons from 10–3000 mg/kg

2.9. Animal Grouping and Determination of Oral Glucose

Tolerance Test (OGTT)

Overnight fasted experimental rabbits were divided into different groups

(6/group) according to the treatments including normal control group treated with distilled

water 1 ml/kg, negative control treated with 0.05% DMSO 1ml/kg, positive control treated

with glibenclamide 5 mg/kg and test group, which was further divided into 6 sub-groups

(3 groups for aqueous and other 3 for organic solvent extract) for each plant. Each extract

was given in doses of 200, 400 and 600 mg/kg to their respective test groups.

Each group, after receiving its respective treatment orally, was immediately

administrated with glucose load (2 gm/kg) from the same route. Blood glucose level of

each rabbit belongs to each group was monitored by pricking the ear vein at 0, 30, 60 and

120 minutes with the help of glucometer (Optimum Xceed, Diabetes Monitoring System

by Abbot)

2.10. Determination of Percent Glycemic Change

Percent glycemic change between control and test groups administrated with

different doses of plant extracts was determined by using the following formula

21

Percent glycemic change (gain/loss) =

Where Go = mean blood glucose level of control group at different time intervals

and Gx = mean blood glucose levels of each of test and positive control (glibenclamide)

groups at different time intervals respective to control (Azmi and Qureshi, 2012b).

2.11. Statistical Analysis

Results are expressed as mean ± SEM (Standard Error Mean). One way Analysis

of Variance (ANOVA) followed by LSD (Least Significant Difference) test were

performed (SPSS, version 17.0) to analysis data. The differences between control and test

groups were found significant at p<0.05 and p<0.01.

22

3. Results

3.1. Phytochemical Analysis

3.1.1. Qualitative Analysis

The qualitative screening of five selected plants including A. fatua,

C. anthelminticum, C. limon, C. paradisi, and W. coagulans showed the presence of large

number of medicinally active constituents. Out of these plants, AqSEt & ESEt of

W. coagulans, followed by C. anthelminticum, C. limon, and A. fatua found rich in most

of the constituents tested including alkaloids, carbohydrates, cardiac glycosides,

flavonoids, glycosides, phlobatanins, resins, saponins, steroids, tanins and triterpenoids as

compared to AqSEt & ESEt of C. paradisi (Table 1).

3.1.2. Quantitative Analysis

The total phenol content in AqSEt and ESEt of C. anthelminticum was found as

18.5 and 21.5 mg/g respectively. Whereas, the ground seeds powder of same plant

showed 116.5 mg/g of alkaloids, 12 mg/g of flavonoids and 170 mg/g of saponins.

Similarly, the total phenols in AqSEt and ESEt of C. limon were found as 32.5 and 190

mg/g respectively, whereas alkaloids and flavonoids were found as 410 and 100 mg/g of

dried seeds powder of C. limon. The AqSEt and ESEt of C. paradisi contained the total

phenol content as 177 and 80 mg/g respectively. MFEt of W. coagulans showed the total

phenols around 125 mg/g while flavonoids 200 mg/g and 15 mg/g of each of alkaloid and

saponins were present in dried fruits powder of same plant (Table 2).

23

Table 1: Qualitative Phytochemical Analysis of Seeds / Fruits Extracts of Selected Plants

Phytoconstituents

C. anthelminticum C. limon C. paradise W. coagulans A. fatua

ESEt AqSEt ESEt AqSEt ESEt AqSEt MFEt AqFEt ESEt AqSEt

Alkaloids + + + + - - + + + +++

Flavonoids + + + + - - + + +++ +

Resins + + + - - - + + +++ -

Saponins + + - - - - + + - -

Steroids + + + ++ + +++ + + + +

Cardiac glycosides NA NA ++ + - - + + NA NA

Carbohydrates - + ++ - - + + + - +++

Tanins + + + + - - + + +++ ++

Triterpenoids + NA + + - - + + + -

Anthraquinone NA NA + + - + NA NA +++ +++

Phlobatanins + + NA NA NA NA + + + -

Glycosides NA NA - + NA NA + + NA NA

+ = present, ─ = absent, NA = not available.

24

Table 2: Quantitative Phytochemical Analysis of Seeds / Fruits Extracts of Selected Plants

Plants Extracts Phytoconstituents (mg /g of plant material)

Total phenols Alkaloids Flavonoids Saponins

C. anthelminticum AqSEt 18.5 116.5* 12* 170*

ESEt 21.5 - - NA

C. limon AqSEt 32.5 410* 100* NA

ESEt 190 - - NA

C. paradisi AqSEt 177 NA* NA NA

ESEt 80 - NA NA

W. coagulans MFEt 125 15* 200* 15*

*mg/g of seeds or fruits powder of selected plant

NA = not available

25

3.2. Trace Mineral Content in Selected Plant Extracts

ESEt of C. anthelminticum was found to rich in iron, potassium and zinc.

However, it showed to have sufficient amount of cobalt, chromium, and sodium. On the

other hand, MFEt of W. coagulans found as a good source of cobalt, chromium, and

sodium. In addition, it also showed a nice content of iron, potassium, and zinc as

compared to ESEt of C. limon (Table 3).

3.3. Effect of Selected Medicinal Plants on OGTT and Percent

Glycemic Change

3.3.1. Effect of AqSEt and ESEt of C. anthelminticum on Glucose

Tolerance in Rabbits

AqSEt of C. anthelminticum in dose of 200 mg/kg was found effective as

compared to other two doses (400 & 600 mg/kg) of same extract and produced significant

(p<0.05) percent reduction (-23 to -30%) in blood glucose level in experimental rabbits

on 30, 60 and 120 minutes after glucose load in comparison to control and positive

control groups. However, AqSEt in doses of 400 and 600 mg/kg was found effective

(p<0.05) particularly on 120 minutes whereas the medium dose (400 mg/kg) also showed

some hypoglycemic effect (-6%) on 30 minutes (Table 4). On the other hand, all doses

(200, 400 and 600 mg/kg) of ESEt of C. anthelminticum found efficient (p<0.05) in

producing percent reduction in blood glucose level on 60 minutes from -12 to -24 % and

on 120 minutes from -30 to -43% as compared to control and negative control groups. In

addition, both high doses (400 & 600 mg/kg) also produced significant hypoglycemic

effect (-10 to -24%) after 30 minutes of glucose load as compared to positive control

(Table 4).

26

Table 3: Trace Mineral Content in Seeds / Fruits Extracts of Selected Plants

Trace Minerals

(ppm)

ESEt of

C. anthelminticum

ESEt of

C. limon

MFEt of

W. coagulans

Cobalt (Co) 0.484 0.367 0.524

Chromium (Cr) 0.182 bdl 0.608

Iron (Fe) 6.0609 0.892 4.562

Potassium (K) 72.139 43.489 63.776

Sodium (Na) 380.93 245.54 486.095

Zinc (Zn) 4.487 1.456 2.372

bdl = below detection limit

27

3.3.2. Effect of AqSEt and ESEt of C. limon on Glucose Tolerance in

Rabbits

ESEt of C. limon @ 200 mg/kg produced a significant (p<0.05) hypoglycemic

activity (-13 to -34%) after 30, 60 and 120 minutes as compared to control groups. The

same dose at 30 minutes also reduced blood glucose level significantly (p<0.05) than

negative and positive control groups. ESEt @ 400 mg/kg showed significant

hypoglycemic effect from 0 to 120 minutes by showing percent reduction in blood

glucose level from -27 to -47% as compared to control, negative and positive control

groups. The same extract @ 400 mg was found more effective (p<0.05) at 60 and 120

minutes than AqSEt @ 400 mg of this plant. However, ESEt @ 600 mg significantly

(p<0.05) decreased blood glucose level at 0, 60 and 120 minutes (-26 to -47%) than

control, negative and positive control groups. ESEt @ 600 mg at 60 and 120 minutes

found more hypoglycemic, when compared with same dose of AqSEt of this same plant.

On contrary, AqSEt of same plant did not show any hypoglycemic activity from 200 to

600 mg/kg (Table 4).

3.3.3. Effect of AqSEt and ESEt of C. paradisi on Glucose Tolerance in

Rabbits

Out of all three doses tested, only ESEt of C. paradisi @ 400 mg/kg was found to

have hypoglycemic activity from 0 to 120 minutes after glucose load by showing percent

decrease in blood glucose level from -25 to -57% as compared to all three control groups.

The same dose was also observed better than AqSEt @ 400 mg of same plant. On the

other hand, AqSEt of same plant was appeared ineffective in hypoglycemic aspect (Table

4).

28

3.3.4. Effect of AqFEt and MFEt of W. coagulans on Glucose Tolerance

in Rabbits

All doses (200-600 mg) of MFEt (-43 to -55%) and AqFEt (-29 to -51.6%) of

W. coagulans produced significant (p<0.05) decrease in glycaemia after 120 minutes as

compared to control and negative control groups. Excluding, 120 min, MFEt and AqFEt

in a dose of 400 mg were also found effective at 0, 30 & 60 min whereas same extracts in

dose of 600 mg produced decrease in blood glucose level at 60 minutes (Table 4).

3.3.5. Effect of AqSEt and ESEt of A. fatua on Glucose Tolerance in

Rabbits

All doses of AqSEt and ESEt of A. fatua did not show any significant reduction in

blood glucose level during OGTT, whereas non-significantly ESEt @ 400 mg and AqSEt

@ 200 and 400 mg were found to reduce blood glucose level after 30 minutes as

compared to control, negative and positive control groups (Table 4).

29

Cont…

Table 4: OGTT of Seeds / Fruits Extracts of Selected Plants in Rabbits

Groups Treatments 0 min 30 min 60 min 120 min

Control d.H2O (1ml/kg) 89.5±3.40 181±11.88 194.25±5.45 178±4.0

Negative control 0.05%DMSO

(1ml/kg) 89.25±7.31 187.5±23.11 185.5±14.33 169.25±8.75

Positive control Glibenclamide

(5mg/kg) 93.5±5.17(4.46%) 208.5±7.23(15.19%) 198.5±4.90(2.18%) 130.75±8.04ab(26.54%)

ESEt of

C. anthelminticum

200mg 87± 3.87(-2.79%) 193.25±31.03 (6.76%) 170.25±39.69 (-12.35%) 107±8.21abc (-39.88%)

400mg 87.75±9.83 (-1.95%) 161.75±9.38c (-11.04%) 146.5±11.97ab (-24.58%) 101.25±4.32abc (-43.25%)

600mg 93.75±8.61 (4.74%) 161.25±12.16c (-10.91%) 158.25±11.14ab (-18.53%) 123±4.37abc (-30.89%)

AqSEt of

C. anthelminticum

200mg 83.75±6.06 (-6.42%) 137.75±26.01 (-23.89%) 136±34.64a (-29.98%) 124.75±19.22abc (-29.91%)

400mg 90±2.30 (0.55%) 170±14.4 (-6.07%) 214±2.0 (10.16%) 127±9.7abc (-28.65%)

600mg 80.25±5.17 (-10.33%) 193.5±2.17 (6.9%) 211.75±6.93 (8.62%) 128±1.91abc (-28.08%)

ESEt of C. limon

200mg 80±6.23(-10.61%) 120±3.36abc(-33.7%) 168.25±13.26(-13.38%) 136.25±7.50ab(-23.45%)

400mg 60.5±9.27abc(-32.4%) 130.5±16.83abc(-27.76%) 138.25±5.66abcd(-28.82%) 95.5±7.08abd(-46.34%)

600mg 65.5±5.56abc(-26.81%) 186±4.67(2.76%) 125.75±15.83ab(-35.26%) 94.5±19.6ab(-46.91%)

AqSEt of C. limon

200mg 103.25±14.3(15.36%) 228.75±30.28(25.96%) 220.75±28.25(9%) 146.75±21.63(-17.55%)

400mg 120.25±1.84(34.35%) 197±9.89(8.83%) 214±6.13(8.23%) 148.75±24.12(-28.08%)

600mg 111±7.14(24.02%) 213.5±18.24(17.95%) 206.5±14.56(9.9%) 137.25±28.39a(-22.89%)

30

Groups Treatments 0 min 30 min 60 min 120 min

ESEt of C.

paradisi

200mg 105.25±8.69 (17.59%) 197±32.98 (9.11%) 186±20.37 (-4.27%) 132±11.78ab (-25.84%)

400mg 66.5±11.40abc(-25.69%) 131±11.81abc(-27.62%) 107.75±10.12abcd(-44.53%) 76.5±5.19abcd (-57.02)

600mg 98.75±6.0(10.33%) 181.75±5.45(0.414%) 157.75±10.77ac(-18.79%) 144±9.24(-19.10%)

AqSEt of C.

paradisi

200mg 94.25±1.70(5.30%) 232.75±7.25(28.59%) 221.75±11.70(14.15%) 223.25±1.65(25.42%)

400mg 93.75±2.59(4.74%) 166±9.94c(-8.28%) 214±14.74 (10.16%) 170±8.71(-4.49%)

600mg 108.75±5.80(21.5%) 176±10.29(-2.76%) 206.5±14.77(6.30%) 135.5±12.73a(-23.87%)

MFEt of W.

coagulans

200mg 80.5±5.63 (-10.05%) 207.5±10.9 (14.64%) 138.25±13.73abcd(-28.82%) 85.75±14.27abd(-51.82%)

400mg 79±9.94 (-11.73%) 173.5±11.60 (-4.14%) 173.25±19.79 (-10.81%) 97.5±3.96ab(-45.22%)

600mg 91.25±3.7 (1.95%) 185.75±7.81 (2.62%) 189.5±13.59 (-2.44%) 100±3.08ab(-43.82%)

AqFEt of W.

coagulans

200mg 93.25±6.60 (4.18%) 180±12.58 (2.20%) 148±0.70ac(-23.8%) 101.5±8.09ab(-42.97%)

400mg 82±7.52 (-8.37%) 174.75±4.02 (-3.45%) 141±3.48abcd(-27.41%) 86±8.13abcd(-51.68%)

600mg 71.75±3.44c(-20.67%) 182.5±5.18 (0.828%) 174±5.43 (-10.16%) 126.75±10.82ab(-29.21%)

ESEt of A. fatua

200mg 92.00±4.898 (2.79%) 200±8.41 (10.49%) 236±3.08 (21.49%) 222.5±8.29 (25%)

400mg 92.25±4.60 (3.07%) 167.5±12.39 (-7.45%) 217.5±20.11 (11.7%) 228±21.97 (28.08%)

600mg 103±2.67 (15.08%) 190.5±6.75 (4.97%) 252.25±13.45 (29.72%) 285.75±4.95 (60.11%)

AqSEt of A. fatua

200mg 91.25±6.36 (1.95%) 159.5±9.67 (-11.87%) 253±21.42 (30.24%) 210.25±20.27 (18.11%)

400mg 97.75±3.47 (9.21%) 164.25±12.59 (-9.25%) 200.25±1.93 (3.08%) 193±28.56 (8.42%)

600mg 93.75±5.48 (4.74%) 183.5±27.10 (1.38%) 257.5±22 (32.3%) 212±35.55 (19.1%)

a p<0.05, when compared with control group,bp<0.05, when compared with negative control group cp<0.05, when compared with positive control group, dp<0.05 when compared between test groups

All values are expressed as mean ±SEM (n=4).Values in parenthesis show percent decrease (-) / increase (+) in blood glucose level

38

4. Discussion

Diabetes is an international epidemic that affects both genders of all age groups

(Olokoba et al., 2012). Fasting hyperglycemia (FH) and impaired glucose tolerance (IGT)

are the important and predictive states of classical diabetes (Maitra and Abbas, 2005).

FH can be measured by monitoring blood glucose levels in fasting and random states

whereas IGT by conducting oral glucose tolerance test (OGTT) in which after oral

administration of glucose (75-100 g), blood glucose (BG) level is measured at 0, 30, 60

and 120 minutes in order to check whether the BG level back to normal within 120

minutes (2 hours) or not (Kawamori et al., 2009). There are number of therapies available

for the management of diabetes including alternative and complementary medicines like

herbs, medicinal plants or plant derived products, homeopathic medicines, etc (Ríos et

al., 2015). From the roots of mankind, medicinal plants have been using as curative

agents for different ailments including diabetes. In this regard, many controlled

investigations in animal models and patients have been conducted to validate the

therapeutic potential of many medicinal plants for diabetes so far.

The presences of bioactive compounds of different chemical classes like

alkaloids, flavonoids, steroids, etc, in medicinal plants make them more appropriate for

the treatment of diseases with negligible side effects (Kumar et al., 2014). In the present

study phytochemical analysis of experimental plants including Avena fatua,

Centratherum anthelminticum, Citrus limon, C. paradisi, and Withania coagulans

showed that they are rich in phytochemicals like AqSEt and ESEt of W. coagulans,

followed by C. anthelminticum, C. limon, and A. fatua showed the presence of most of

the constituents tested including alkaloids, carbohydrates, cardiac glycosides, flavonoids,

glycosides, phlobatanins, resins, saponins, steroids, tannins and triterpenoids as compared

to AqSEt and ESEt of C .paradisi. Whereas the quantitative analysis notified that high

total phenol content was found in C. limon, followed by in C. paradisi, W. coagulans,

and C. anthelminticum. Similarly, flavonoids, an important class of phenols, were

39

estimated high in W. coagulans, C. limon and C. anthelminticum. An entirely different

picture was observed in case of alkaloids which indicated that their high amount was

present in C. limon, then in C. anthelminticum and W. coagulans. Saponins, another

bioactive class of compounds, were present in large amount in C. anthelminticum than W.

coagulans. Different studies described that all these tested chemical classes of

constituents are found biological active in the treatment of diabetes like hypoglycemic

action of alkaloids of M. charantia was reported (Mohammady et al., 2012), alkaloid rich

methanolic extract of whole plant of Vica rosea also displayed antidiabetic and

antihyperlipidemic activities in alloxan-induced diabetic rats (Ahmed et al., 2010).

Similarly, alkaloid rich antihypertensive plant Rauwolfia serpentina has demonstrated

inhibition in postprandial BG in normoglycemic mice (Azmi and Qureshi, 2012b) and

antihyperglycemic potential in alloxan-induced type 1 diabetic mice (Azmi and Qureshi,

2013; 2014).

Many published scientific papers have described the importance of flavonoids

rich extracts of medicinal plants in normalizing the BG levels like flavonoid rich extract

of Trichlia emetic and Opilia ametacea displayed antidiabetic, antihypertensive and free

radical scavenging activities in type 2 diabetic animal model (Konate et al., 2014),

optimal extracted flavonoids from Ipomoea batatas leaf has also reported to decrease BG,

TG and TC in alloxan-induced diabetic mice (Li et al., 2009). Steroids, water insoluble

class of chemical compounds, also exhibited hypoglycemic effect in experimentally

induced diabetic animal models like steroid rich chloroform extract of Carica papaya leaf

showed antidiabetic activity in streptozotocin-induced diabetic rats (Juarez-Rojopa et al.,

2014).

Now-a-days, trace minerals as supplements are gradually gaining fame in the

treatment of diabetes in medical practice as it has been proved that tissue levels of trace

minerals are reduced in diabetic patients by several epidemiologic studies (Zheng et al.,

2008). Although these minerals required in minute amount but they are very essential in

all body metabolisms either by enhancing the release of any hormone like insulin or

40

neurotransmitters or modulating their action, accelerating the activity of any enzyme as a

cofactor or being a part of prosthetic group, regulating membrane potential and

mitochondrial performance, etc (Welch and Graham, 2004). In this regard, balance diet is

required to maintain the homeostasis of trace minerals in body. Medicinal plants are also

rich in these minerals and are using worldwide as substitute of trace mineral provider

(Mahmud et al., 2012). The experimental plants used in the present study especially C.

anthelminticum, followed by W. coagulans and C. limon are found rich in cobalt (Co),

chromium (Cr), iron (Fe), potassium (K), sodium (Na) and zinc (Zn). Interestingly, Zn

and Cr play an important role in insulin metabolism (Zheng et al., 2008; Cefalu and Hu,

2004). Disturbance in glucose metabolism and electrolyte imbalance are also correlated

and well-established with K & Na deficiencies which can be occurred due to polyuria in

diabetic patients (Reza et al., 2015).

OGTT has a predictive value and is used to diagnose impaired glucose tolerance

in an individual either due to complete or relative insulin deficiency or insulin

insensitivity (resistance) or any other problem related to saccharide metabolism (Kim et

al., 2014). This test is also important for investigating the hypoglycemic potential of

medicinal plants in glucose-induced hyperglycemic animal model (OGTT). The same has

been done in the present study in order to investigate and compare the hypoglycemic

activities of five medicinal plants to select any one of them to investigate its effect on

fructose-induced type 2 diabetes in rabbits. Among the five medicinal plants tested, best

hypoglycemic activity was demonstrated by C. anthelminticum, followed by C. limon, W.

coagulans and C. paradisi where they were found to ameliorate glucose tolerance in

glucose-induced hyperglycemic rabbits. However, there wasn‟t any good hypoglycemic

result observed by A. fatua in OGTT. Interestingly, A. sativa (the common oat) was

reported with hypoglycemic activity by showing its ability to reduce post-prandial BG

level (Ahmed et al., 2010) while its wild species A. fatua did not prove to be

hypoglycemic in nature in the present study.

41

During OGTT, when hypoglycemic activity of AqSEt and ESEt of C.

anthelminticum in glucose-induced hyperglycemic rabbits was compared, It was found

that ESEt produced much better result than aqueous extract like all doses (200-600

mg/kg) of ESEt induced percent decrease in BG level ranging from -12 to -24% on 60

min and from -30 to -43% on 120 min. High doses (400 & 600 mg/kg) of same ESEt also

showed significant (p<0.05) glucose lowering effect from -10 to -24% on 30 min after

glucose load. On the other hand, AqSEt of C. anthelminticum in dose of 200 mg/kg was

found helpful (p<0.05) in decreasing BG level from -23 to -30% in experimental rabbits

on all time intervals whereas high doses (400 & 600 mg/kg) of same aqueous extract also

showed some hypoglycemic effect but only on last time interval (120 min). In case of C.

limon, again ESEt was found better than AqSEt. In fact, aqueous seed extract did not

produce any fruitful hypoglycemic effect in OGTT. On contrary, a significant (p<0.05)

hypoglycemic activity was demonstrated by all doses of ESEt of C. limon like its small

dose (200 mg/kg) produced percent reduction in BG level from -13 to -34%, median dose

(400 mg/kg) from -27 to -47% and high dose (600 mg/kg) from -26 to -47% on all time

intervals in OGTT as compared to hyperglycemic and positive control groups.

All doses of MFEt and AqFEt of W. coagulans (200-600 mg/kg) produced

significant (p<0.05) decrease in BG level from -43 to -55% and -29 to -51.6%

respectively on 120 min after glucose load as compared to control groups. Beside 120

min, MFEt and AqFEt in a dose of 400 mg/kg were found effective in reducing BG

levels at 0, 30 and 60 min from -4 to -11% and -3 to -27% respectively while the high

dose (600 mg/kg) of same both extracts also produced hypoglycemic effect on 60 min.

Interestingly, only single dose (400 mg/kg) of ESEt of C. paradisi was found effective

(p<0.05) in lowering BG level from -25 to -57% on all time intervals (0 to 120 min) after

glucose load in OGTT. On the other hand, AqSEt of same plant was not found

hypoglycemic in function. Similarly, all doses of AqSEt and ESEt of A. fatua did not

show any significant reduction in BG level of glucose-induced hyperglycemic rabbits

during OGTT.

42

It has been suggested that experimental plants including C. anthelminticum, C.

limon and W. coagulans which have found potent hypoglycemic agents in the present

study can capable of inducing glucose tolerance in glucose-induced hyperglycemic

rabbits, which might be due to by enhancing the release of insulin at pancreatic level

which in turn normalizes the BG level or at extra-pancreatic level by enhancing the

glucose uptake in insulin target tissues including liver, muscles and adipose tissues or by

inhibiting glucose absorption in intestine via inactivating carbohydrate hydrolyzing

enzymes or by acting as insulin receptor modulators (Qureshi et al., 2009; Coman et al.,

2012; Al Snafi et al., 2015). Many plants have reported with insulin releasing action like

allicin, a sulfur containing compound in Allium sativum (garlic), found to enhance insulin

release from β-cells of normal rats in vitro and thought to be involved in hypoglycemic

activity of same plant in alloxan and streptozotocin-induced diabetic rats (Mustafa et al.,

2007). Similarly, the mechanism of hypoglycemic action of Trigonella foenum-graecum

(methi) was elucidated by in vitro experiments that described the insulinotropic effect and

inhibition of carbohydrate hydrolyzing enzymes by leaf and seed extracts of same plant

(Patel et al., 2012). On the other hand, few alkaloids, isolated from roots of R. serpentina,

were found as potent activators of insulin receptors (Ganugapati et al., 2012).

Interestingly, crude methanolic extract of C. anthelminticum found to increase insulin

secretion from β-TC6 cell line (Arya et al., 2012) and also showed some effect against

salivary amylase and glucosidase activities in vitro (Amir and Chin, 2011) which could

be involved in hypoglycemic activity demonstrated by this plant in alloxan-induced

diabetic rats (Bhatia el al., 2008) and in present study. The steroids and alkaloids of W.

coagulans like withanolid F and coagulin L showed antihyperglycemic activity in

streptozotocin-induced diabetic rats and transgenic mice (Maurya et al., 2010; Kumar et

al., 2014) but evaluation of exact mechanism is still in progress. Hexane extract of lemon

peel was also demonstrated an acute and a brief hypoglycemic activity in alloxan-induced

diabetic rats (Naim et al., 2012). However, ethanolic seeds extract (ESEt) of lemon is

first time used in the present study for investigating the hypoglycemic activity through

OGTT where it showed significant glucose lowering activity. The observed

43

hypoglycemic activity of ESEt of lemon may be due to the presence of large amounts of

total phenols, flavonoids and alkoloids in that extract as these classes of compounds are

well-reported with antioxidant activities and inhibiting glucose absorption in intestine

(Yang et al., 2008; Russo et al., 2012). Recent studies have suggested that combine

therapy with antidiabetic medicines and antioxidants would produce better results in the

management of diabetes (Tabatabaei-Malazy et al., 2013).

Therefore, a single medicinal plant is the store house of phytochemicals, trace

minerals, vitamins and hormones (Hosseini and Abdollahi, 2012; Hassani-Ranjbar et al.,

2010) which are not only essential for normal growth and function of the body but also

for correcting the faults occurred in metabolic pathways in different diseases. In the end,

on the basis of potent hypoglycemic activity and nice content of phytochemicals and

trace minerals, ethanolic seeds extract (ESEt) of C. anthelminticum was selected to

investigate its effect in fructose-induced type 2 diabetic rabbits in second phase of the

present study.

44

5. Conclusion

The results conclude that presences of biologically active chemical compounds

and trace minerals make medicinal plants more appropriate for the treatment of diabetes.

In the present study, out of five experimental plants tested, ethanolic seeds extracts

(ESEt) of C. anthelminticum, C. limon, and methanolic fruits extract (MFEt) of W.

coagulans were found to produce significant percent glycemic reduction in oral glucose

tolerance test thereby promoting glucose tolerance and metabolism in glucose-induced

hyperglycemic rabbits. In the end, because of the potent hypoglycemic activity showed

by ESEt of C. anthelminticum, it was selected to check its effect in fructose-induced type

2 diabetic rabbits in the second phase of the present study.

45

Effect of Ethanolic Seeds Extract of Centratherum

anthelminticum in Fructose-Induced Insulin Resistance

Type 2 Diabetic Rabbits

1. Introduction

Type 2 diabetes (T2D) has turn out to be one of the most important health

affecting chronic endocrine problems globally and it is an increasing cause of disability

and premature death, mostly through cardiovascular diseases (Liu et al., 2010). It has

been reported that the figure of adults suffering from this disease will reach 439 million

in 2030 and mostly belong to T2D cases (Shaw et al., 2010; Olokoba et al., 2012).

Pakistani population is also threatening by this health hazard especially the middle-aged

community of both genders probably due to adopting unhealthy lifestyle such as

increased intake of fat with decreased intake of vegetable, fruits and whole grain, plus

lack of physical activity, all these factors open the gate for obesity which is the best

initiator of insulin resistance and high prevalence of type 2 diabetes in our country

(Ansari, 2009; Ansari et al., 2015). Moreover, lack of awareness towards risk factors of

diabetes in population also contributed its high rate in Pakistan (Ulvi, 2009).

1.1. Importance of High Intake of Fructose in Relation to Diabetes

Fructose is a monosaccharide found in fruits, honey, sucrose (table sugar) and

high-fructose corn syrup which is produced due to the isomerization of dextrose in the

presences of biocatalyst (Elliott et al., 2002). From last 30 years, artificial sweeteners

especially fructose consumption has been accelerated which enhanced the risk of many

endocrine or metabolic disorders like obesity, diabetes, hypertension and kidney diseases

(Johnson et al., 2007; Segal et al., 2007). It has been reported that high fructose intake is

the main factor for the growing rate of type 2 diabetes (Thomas et al., 2015). Fructose

has found to be a principal keto-sugar which is equally available with glucose in sucrose

46

and it is more rapidly glycolyzed by liver than glucose. Normally, fructose does not

generate endocrine signals that are involved in the long-term regulation of energy balance

as glucose does (Havel, 2005). Literature described that intake of large amount of

fructose can rapidly induce postprandial hypertriglyceridemia, which ultimately become

the cause of high blood pressure in individuals as compared to starch-based diet (Brown

et al., 2008; Stanhope et al., 2008). Moreover, it is an important risk of fatty liver disease

(Ouyang et al., 2008). Other reports described that fructose can cause metabolic

syndrome by promoting hepatic lipogenesis, intracellular ATP depletion, oxidative stress,

uric acid generation and endothelial dysfunction (Segal et al., 2007; Havel, 2005).

Administration of glucose activate insulin release, which triggers leptin secretion

from adipocytes and the inhibition of ghrelin from intestine in order to regulates satiety

and energy homeostasis at central nervous system (CNS) level whereas fructose does not

require insulin, which would attenuate leptin and ghrelin responses (Teff et al., 2004).

The fructose uptake into hepatocytes requires a sodium-independent monosaccharide

transporter (GLUT-5) which is insulin-independent where it encourages cholesterol and

triglyceride synthesis and in turn induced insulin resistance (T2D) accompanied with

hyperinsulinemia (Sievenpiper et al, 2009). High fructose intake actually affects many

insulin stimulated processes like phosphorylation of tyrosine in insulin receptor, weakens

phosphorylation of insulin receptor substrate-1 (IRS-1) and its relation with

phosphotidylinositol-3 kinase in muscles and liver, thus disturb insulin signalling cascade

and induce insulin resistance (Havel, 2005).

1.2. Major Complications of Type 2 Diabetes

(i) Oxidative Stress

The reactive oxygen species (ROS) are decomposed into inactive and harmless

species by endogenously synthesized antioxidant enzymes like catalase (CAT),

superoxide dismutase (SOD), glutathione reductase (GR) and others, these act as free

radical scavenger (Kumar and Anandan, 2007; Elinasri and Ahmed, 2008). SOD is an

47

important enzyme in the enzymatic antioxidant defence system that eliminates superoxide

anion by converting into hydrogen peroxide (H2O2) and prevents the body from toxic

effects of this radical. Similarly, CAT is widely present in animal tissues and hydrolyzed

H2O2 thereby protecting the cells from reactive hydroxyl radicals (Ragini et al., 2011).

Similarly, glutathione reductase and peroxidase regulate the amount of antioxidant

protein glutathione in body tissues (Hisalkar et al., 2012). It has been proposed that high

fructose catabolism similar to glucose, amplifies the free radical generation that induced

oxidative stress and weakened the body defence system (Reddy et al., 2009).

In diabetes, many factors are involved in the generation of ROS especially

glycation reaction (non-enzymatic glycation of protein) that leads to the generation of

advanced glycation end products (AGEs). These AGEs can induced harmful effects like

inactivation of enzymes by altering their structure and function, blocked vasodilating

effect of nitric oxide and activate NF-KB that may lead to beta cell destruction and

accelerate insulin depletion (Maritim et al., 2003; King and Loeken, 2004). Increased

glucose oxidation through polyol / sorbitol pathway also contribute oxidative stress, in

which two main enzymes including aldose reductase and sorbitol dehydrogenase convert

glucose into sorbitol and then fructose which later involved in the formation of ROS by

activating NADH oxidase (mitochondrial enzyme) and AGEs by forming fructose-3-

phosphate and 3-deoxy-glucosone (strong glycating agents) that may lead to the

development of eye and kidney problems in diabetic patients (Chung et al., 2003). A

study described that cataract development is directly proportional to the aldose reductase

expression in transgenic diabetic mice by showing increase level of sorbitol and

malondialdehyde and decrease level of glutathione in lenses (Lee et al., 1995). Similarly,

aldose reductase induced oxidative stress in nerves produced lesions in sciatic nerves

thereby reducing nerve conduction velocity (Ho et al., 2000). Another study showed that

increase intracellular level of sorbitol in nerve cells promote necrosis and degeneration of

neurons that may leads to diabetic peripheral neuropathy (Zhang et al., 2013).

48

Other contributors of oxidative stress in diabetes include protein kinase C (PKC)

and hexosamine pathways. Activation of PKC pathway accelerate the generation of cell

adhesion molecules, transforming growth factors 2β, endothelin-1 and vascular

endothelial growth factors (VEGF), plasminogen activator inhibitor-1 and inhibit nitric

oxide synthase activity, all these direct to vascular occlusion that impair cell permeability

and induced damage to tissues (Geraldes et al., 2010). Similarly, many studies correlate

the relationship of hyperglycemia-induced activation of hexoamine pathway (HP) with

diabetic complications by observing increased level of UDP-N-acetylglucosamine, which

activates O-linked N-acetylglucosamine transferase that later induced post-translational

glycosylation of many proteins or enzymes (Buse, 2006). Another worst marker of

diabetic oxidative stress is the lipid peroxidation. It is caused by either enzymatic via

activation of phospholipase A2 or non-enzymatic via the involvement of mitochondrion

in generation of ROS (Arora et al., 2013). Lipid peroxidation is a free radical

(superoxide)-mediated process leading to oxidative deterioration of polyunsaturated

membrane lipids that become the reason of many macro-vascular problems (Thomas et

al., 2015).

(ii) Dyslipidemia

The liver is an essential organ that adjusts glucose and lipid balance with the

assistance of insulin. However, insulin resistance induced hyperglycemia by reducing

hepatic glycogenesis and accelerating gluconeogenesis (de Castro et al., 2013). A high-

fructose diet greatly accelerates the production of pyruvate and glycerol-3-phosphate in

liver, which in turn stimulates de novo lipogenesis, by up-regulating the lipogenic

enzymes that increase the synthesis, esterification, secretion and accumulation of

cholesterol and fatty acids in hepatocytes which promotes the development of

hepatomegaly and dyslipidemia (Schwimmer et al., 2008; Feinman and Fine, 2013).

Diabetic dyslipidemia associated with the decrease in HDL-c, the size/density of

LDL-c and increase in VLDL-c and triglycerides (TG) levels (Dixit et al., 2014). The

49

lipid abnormalities are common in diabetes because insulin resistance adversely affects

lipid metabolism by altering the activities of important enzymes (Taskinen, 2002). In

particular, many processes are affected like apolipoprotein B (the major component in

LDL and VLDL) synthesis, actions of lipoprotein lipase & cholesteryl ester transferase,

etc (Mooradian, 2009). A study described the presence of small dense LDL (LDL

phenotype pattern B) in diabetic patients and found that it is more atherogenic than LDL

present in non-diabetic individuals (Chehade et al., 2013). Another study described that a

gradual reduction in LDL size is in accordance to increase in stages from impaired

glucose tolerance to diabetes. Dyslipidemia is associated with increase in TG rich

lipoproteins, result in keeping the VLDL and chylomicron both in blood for longer time,

that accelerate the transfer of cholesterol ester and formation of TG-rich LDL which in

turn activates hepatic lipase and leads to the formation of small dense LDL (sdLDL).

This small LDL gets easily oxidized and binds with proteoglycans in arterial wall and

accelerates the risk of endothelial dysfunction and atherosclerosis. On the other hand,

catabolism of HDL becomes increased, resulting in decrease in HDL-c levels in diabetic

patients (Goldberg, 2001; Taskinen, 2002). In fact, hyperglycemia, weight gain and

insulin resistance are associated with the development of atherosclerosis and other

cardiovascular diseases (Regmi et al., 2009). About 70-80% deaths are reported due to

the vascular diseases in diabetic patients. Therefore, an ideal treatment for diabetes would

be a drug that not only controls the blood sugar level but also prevents other vascular

complications (Upendra et al., 2010).

1.3. Antidiabetic Medicinal Plants in the Treatment of Fructose-

Induced Type 2 Diabetes

Current researches are inclined towards the use of improved, safe and natural

antidiabetic plant-based products which is the result of traditional treatment because of

their chemopreventive action, low cost and non-toxic effect during long-term oral

administration along with their easy availability at large scale (Udoamaka et al., 2014).

Many plant secondary metabolites including alkaloid, flavonoids, steroids, etc have been

50

reported to improve biological processes and reduce the risk of chronic complications in

diabetic patients (Gupta et al., 2008).

High fructose fed animal model provide similar clinical characteristics as

developed in insulin resistance (de Moura et al., 2009) because excess fructose intake

stimulates de novo lipogenesis results in increase TG and VLDL in blood (Jung et al.,

2010). The high content of systemic lipids not only masked the insulin receptors to

induce insulin resistance but also produced oxidative stress that worsen the condition and

subject body to different diabetic complications.

Medicinal plants represent a worthwhile substitute to treat diabetes mellitus as

dietary supplements or alternative medicines. Traditionally, leaves of Murraya koenigii

are reported as hypoglycemic agent (Harish et al., 2012). Later (Kumar et al., 2010) a

compound called mahanimbine has been isolated from M.koenigii possessed alpha

amylase inhibitory effect and supposed to be the base of hypoglycemic activity showed

by same plant in streptrozotocin-induced wistar rats. Seeds of fenugreek (Trigonelia

foenum-graecum Linn) (Alauddin et al., 2009) were also reported to produce anti-

hyperglycemic effects in alloxan-generated diabetic rats. The fresh slices of Allium cepa

(red onion) produced hypoglycemia in type 1 and type 2 diabetic patients in a clinical

study (TajEldin et al., 2010)

Many studies reported the positive influence of herbal medicine in insulin

resistance caused by high intake of fructose like garlic (Allium sativum), with amino acid

L-cysteine has been found to be antilipidemic (Hasimun et al., 2011) and antioxidative

agent in minimizing the insulin resistance in high fructose fed hyperglycemic rats

(Thomas et al., 2015). Another study revealed that the intake of green, black, and puerh

(Camellia sinensis) tea leaves improved the fructose-induced hyperlipidemia and

hyperleptinemia by lowering fatty acid synthase (FAS) activity in liver and increased

AMP-protein kinase phosphorylation in rats (Huang & Lin, 2012). Aqueous extracts of

three medicinal plants included Urtica dioica, Trigonella foenum-graecum and Fumaria

officinalis were reported to produce hypoglycemia by improving insulin resistance in

51

21% fructose fed diabetic rats for 2 months (Goodarzi et al., 2013). Similarly, a

hydroalcoholic leaf extract of Urtica dioica was alone found to improve type 2 diabetes

by decreasing serum glucose, leptin and FIRI values in 10% fructose-fed diabetic rats in a

long-term treatment for 8 weeks (Ahangarpour et al., 2012). A herbal medicine named

Gastrodia elata and its ethanolic extract (100 mg/kg/day for 8 weeks) were also found

effective in reducing dyslipidemia, hypertension, and insulin resistance in 65% high-

fructose diet rats (Kho et al., 2014). The antihyperglycemic and antihyperlipidemic

activities of aqueous extract of Moringa oleifera leaves (200 mg/ kg) were observed in

both insulin resistant and insulin deficient rat models by reverting fructose fed insulin

resistance in 60 days and restoring the blood glucose level near to normal along with

normal plasma lipid contents (Divi et al., 2011). Recently another study reported that

treatment with Mimusa pudica leaf extract significantly improved the complications

related to type 2 diabetes including body weight, insulin sensitivity, dyslipidemia and

liver damage and brought nearly normal in high fructose diet fed rats in 14 days

(Sundaresan and Radhiga, 2015).

1.4. Plant for Present Experimental Work

Centratherum anthelminticum Kuntze (Kali zeeri) belongs to family Asteraceae

(Ngeh and Rob, 2013).The seeds of this plant are acrid and enriched with a variety of

phyto-chemicals such as alkaloids, carbohydrates, flavonoids, fatty acids, phenols,

steroids, resin, etc (Amir and Koay, 2011). Nine steroid hydrocarbons (stigmastane-type)

called vernoanthelcin A to H and I along with two steroidal glycosides named

vernoantheloside A and B were reported to be involved in the production of estrogen

from human ovarian granulosa-like cells (Srivastava et al., 2014). The seeds also possess

antimicrobial activities (Lei et al., 2012). The anti-cancerous potential of vernodalidimers

A and B have also been reported from the seeds of this plant (Srivastava et al., 2014).

The prominent flavonoids were isolated from the seeds of C. anthelminticum include

tetrahydroxychalcone, tetrahydroxyflavone and butin (Guilian et al., 2004). Beside these,

their polyphenolic constituents like quercetin glycoside, naringenin-7-O-glucoside and

52

kaempferol isolated and proved with anti-diabetic, anti-oxidant, and anti-inflammatory

activities (Paydar et al., 2013). Traditionally, seeds of C. anthelminticum have been used

as astringent, anthelmintic, diuretic, stomachic, tonic and reported in the treatment of

fever, skin diseases, leucoderma, ulcers, asthma, kidney troubles, hiccough, intestinal

colics, achings and eyes infections (Lei et al., 2012; Singh et al., 2012).

The hypoglycemic property of the methanolic extract of C. anthelminticum seeds

was reported in streptozotocin (STZ)-induced diabetic rat model (Arya et al., 2012).

Petroleum ether and alcoholic extracts of same C. anthelminticum seeds showed

analgesic, antiurolithiatic and antipyretic activities (Purnima et al., 2009). Stone forming

elements like oxalate, calcium and phosphate were also reported to be lowered by using

alcoholic seed extract of same plant (Purnima et al., 2009; Galani and Panchal , 2014).

Recently, the role of ethanolic seed extract (ESEt) of C. anthelminticum was also testified

in experimentally induced hyperlipidemic rabbits where improvement in lipid profile was

reported significantly (Lateef and Qureshi, 2013).

1.5. Purpose of the Study

This chapter is the first attempt to examine the effect of ethanolic seeds extract

(ESEt) of Centratherum anthelminticum in fructose-induced insulin resistance type 2

diabetic rabbits. In addition, in vitro antiglycation and antioxidant activities of same

extract have also been investigated.

53

2. Materials and Methods

2.1. Plant Material and Preparation of Ethanolic Seeds Extract

of Centratherum anthelminticum

The seeds of C. anthelminticum were purchased from Hamdard Dawakhana,

Sadar, Karachi, identified by expert of Department of Botany, University of Karachi and

kept in Department of Biochemistry of same university with voucher specimen No.

KU/BCH/SAQ/08). The preparation of ethanolic seeds extract (ESEt) of C.

anthelminticum was done by soaking 40 g of ground seeds powder in 1L of ethanol

(95%) for overnight, filtered and concentrated at 40 ⁰C till dryness by using rotary

vacuum evaporator (Azmi and Qureshi, 2012a).

2.2. In-Vitro Investigations of ESEt of C. anthelminticum

2.2.1. Antiglycation Assay

Principle

The method is based on the fructose-mediated production of fluorescent advanced

glycated end products (AGEs) on human serum albumin (HSA) and used to determine

antiglycation activity of test extract (Singh et al., 2001).

Reagents

i. Compound / extract (1 mM).

ii. Human serum albumin (HSA) fraction V (AdventBio, USA).

iii. Sodium azide (BDH VWR international, USA).

iv. Sodium dihydrogen phosphate (NaH2P04) and disodium hydrogen phosphate

(Na2HP04) were obtained from Sigma Aldrich, USA and used to prepare

54

phosphate buffer (pH 7.4).

v. DMSO (Fisher Scientific, UK).

vi. Rutin (standard inhibitor) of Alfa Aesar, Germany.

vii. 96-well polystyrene solid black plate (Corning Life Sciences, USA).

Procedure

i. Extract was dissolved in DMSO in a concentration of 1 mM.

ii. HSA was used as a model protein to be glycated at 10 mg/ml with 0.5 M

fructose as glycating agent.

iii. Test: Extract (0.5 mg/ml) was incubated in triplicates in 96-well plate with

10 mg/ml HSA, 0.5 M fructose, 0.1 M sodium azide (bactericidal agent) and

0.1 M phosphate buffer (pH 7.4) for 7 days at 37°C.

iv. Positive control: Fructose, HSA and phosphate buffer were incubated

with same concentrations and condition with absolute DMSO.

v. Rutin was used as standard with IC50 of 54.5 ± 0.05 μM.

vi. After 7 days of incubation, the 96-well plate was observed for fluorescence at

330-440 nm on micro-titer plate spectrophotometer (Spectra Max M2,

Molecular Devices, USA).

vii. The percent inhibition of test extract and standard was calculated by using

following formula:

Percent Inhibition =

The test sample that showed 50% or above percent inhibition, was processed

for calculating median inhibitory concentration (IC50) value with the help of Ez-fit

software provided by Perrella Scientific, (USA).

55

2.2.2. DPPH Radical Scavenging Assay

Principle

DPPH (2, 2-diphenyl-l-picrylhydrazyl radical), consists of stable free-radical

molecules that looks like a dark-coloured crystalline powder. DPPH is used for

antioxidant investigations in a scientific research. The ethanol solution of DPPH (exist in

radical state) appeared as deep violet in colour and has maximum absorbance at 517 nm.

On reaction with an antioxidant (e.g., vitamin C) radical state of DPPH get reduced to

molecular form DPPH which seemed as pale-yellow in colour with decrease in

absorbance. Therefore, radical scavenging power of the test sample can be measured by

computing the change in absorbance (Thadahani et al., 2011).

Reagents

i. Standard (0.5 mM) in DMSO

ii. Crude Sample (extract 0.5 mg/ml) in DMSO

iii. DPPH (Wako Chemicals USA, Inc.) solution (0.3 mM) in ethanol

Procedure

The ethanolic solution of DPPH (95/µl, 300/µM) and test sample (5/µl,

500/µM) were mixed and incubated for 30 minutes at 37 ºC. Absorbance was read

by using multiplate (SpectraMax 340) at 517 nm. When the colour turned violet to pale-

yellow indicated the reduction of DPPH. The DMSO was used as control and used to

56

compare with test sample for the evaluation of percent radical scavenging activity (%

RSA). A reduction in the early DPPH concentration by 50% was denoted as IC50 value.

The IC50 value of test sample was calculated by using the Ez-Fit Enzyme kinetics

software program (Perrella Scientific Inc. Amherst, MA, USA). N-acetylcysteine,

ascorbic acid and butylated hydroxyanisole (BHA) were used as the reference

compounds.

2.3. In-Vivo Investigations of ESEt in Fructose-Induced Type 2

Diabetic Rabbit Model

2.3.1. Experimental Rabbits

Male albino rabbits, weighing from 1 to 1.2 kg, were purchased from the breading

house of Dow University of Health Sciences (DUHS), Karachi, kept and handled

according to the international guidelines in animal house of Department of Biochemistry,

University of Karachi. They have free access to standard laboratory diet and water.

2.3.2. Positive Control and Vehicle

Pioglitazone (Zolid; 15 mg) was purchased from Getz Pharma, Pakistan Ltd. and

used as positive control whereas dimethyl sulphoxide (DMSO; 0.05%) of Fisher

Scientific (UK) was used as vehicle for administering the doses of ESEt of

C. athelminticum in experimental rabbits.

2.3.3. Induction of Fructose-induced Type 2 Diabetes

Type 2 diabetes was induced by giving 35% fructose in drinking water to

experimental rabbits after overnight fasting for 14 days consecutively (Neeharika et al.,

2012).

57

2.3.4. Experimental Rabbits and Their Grouping

Overnight fasted experimental rabbits were divided in two major groups including

normal control (which were given distilled water 1ml/kg) and fructose-induced type 2

diabetic (giving 35% fructose solution) groups. The diabetic group was further divided

into fructose-induced type 2 diabetic control, positive control and test groups according

to the treatments (Figure 1). All treatments were done orally in early morning before diet

once in a day for 14 days uninterruptedly. On completion of 14 days trial, rabbits were

sacrificed on 15 day in order to collect blood, serum and liver tissues for biochemical

analysis.

Figure 1. Animal Grouping According to their Specific Treatments

58

2.3.5. Determination of Physical Parameter

2.3.5.1. Percent Body Weight Change

Percent change in body weight of each rabbit in each group was calculated by

using below mentioned formula after measuring their weights on initial and final day of

trial with the help of weighing balance (Azmi and Qureshi, 2012a).

Percent body weight change = (Final day weight - Initial day weight) × 100

(Initial day weight)

2.3.6. Determination of Hematological Parameters

2.3.6.1. Glycosylated / Glycated Hemoglobin (HbA1c)

The value of HbA1c is assessed through Turbidimetric Inhibition Immunoassay

(TINIA) by using automatic clinical analyzer (Roche/Hithachi 902). The principle of this

assay is based on the reaction of HbA1c with anti-HbA1c antibody to form soluble

antigen-antibody complexes. Since only one specific HbA1c antibody site is present on

HbA1c molecule so formation of insoluble complexes does not take place. Addition of

polyhaptens react with excess anti-HbA1c antibodies to form an insoluble antibody-

polyhapten complex which can be determined turbidimetrically. Whereas, hemoglobin

(Hb) concentration is determined in a second channel, in which liberated Hb is converted

to a derivative having characteristic absorption spectrum and measured bi-chromatically.

The final result is expressed as percentage of HbA1c.

2.3.7. Determination of Biochemical Parameters

2.3.7.1. Percent Glycemic Change

Percent glycemic change in each rabbit of each group was calculated by using

below mentioned formula after measuring fasting blood glucose (FBG) levels through

59

glucometer (Optimum Xceed, Diabetes Monitoring System by Abbott, Pakistan) by

pricking their ear veins on initial and final day of the trial (Perfumi and Tacconi, 1996).

Percent glycemic change =

Where, IFBG = blood glucose level at initial day and FFBG = blood glucose level at final

day of trial.

2.3.7.2. Fasting Insulin Level

Electro-chemiluminescence immunoassay (ECLIA) technique is used to evaluate

serum fasting insulin level with the help of Cobas e411 automated analyser. The principle

of this method is based on the development of sandwich complex of insulin between

biotinylated monoclonal insulin-specific antibody and a monoclonal insulin-specific

antibody labelled with a ruthenium complex. These complexes bind to the solid phase by

means of biotin-streptavidin-coated paramagnetic micro-particles. The reagent mixture is

shifted to the measuring cell, where the micro-particles are magnetically captured to the

electrode surface by applying voltage, producing chemi-luminescent emission and

measured by a photomultiplier. The automatic display of result is instrument specific and

determined through a calibration curve (Sapin et al., 2001).

2.3.7.3. Fasting Insulin Resistance Index (FIRI)

The fasting insulin resistance index (FIRI) was calculated by using following

formula (Duncan, 1995).

FIRI = Fasting insulin (µU/ml) × Fasting glucose (mg/dl)

25

2.3.7.4. Lipid Profile

The complete lipid profile including total cholesterol (TC), triglycerides (TG) and

high density lipoprotein cholesterol (HDL-c) was determined by commercially available

60

enzymatic kits (Randox, United Kingdom). Whereas low- (LDL-c) and very low-density

lipoprotein (VLDL-c) cholesterols were calculated by authentic formulae.

I. Total Cholesterol by Enzymatic Endpoint Method (Roeschlau et al, 1974)

Reaction Principle

Cholesterol ester + H2O Cholesterol + Fatty acids

Cholesterol + O2 Cholestene-3-one + H2O2

2H2O2 + phenol + 4·Aminoantipyrine Quinoneimine + 4H2O

Reagents

i. Buffer Reagent (pH 6.8): 4-Aminoantipyrine 0.30 mmol/l, Phenol 6 mmol/l,

Peroxidase ≥ 0.5 U/ml, Cholesterol esterase ≥ 0.15 U/ml, Cholesterol oxidase

≥ 0.1U/ml, Pipes Buffer 80 mmol/l.

ii. Standard: Cholesterol 200 mg/dl.

Procedure

Pipette into cuvette:

Reagent Blank (μl) Standard (μl) Sample (μl)

Distilled H20 10 - -

Standard - 10 -

Sample - - 10

Buffer Reagent 1000 1000 1000

Cholesterol esterase

Cholesterol oxidase

Peroxidase

61

Mixed and incubated for 5 min at 37 ºC. The absorbance of sample was measured at

500 nm against the reagent blank within 60 minutes

Calculation

II. Triglycerides by GPO-PAP Method (Tietz, 1990)

Reaction Principle

Triglycerides + H2O Glycerol + Fatty acids

Glycerol + ATP Glycerol-3-phosphate + ADP

Glycerol-3-phosphate + O2 Dihydroxyacetone+phosphate + H2O2

2H2O2 + 4-Aminophenazone + 4 Chlorophenol Quinoneimine + HCl + 4H2O

Reagents

i. Buffer (R1a): Pipes Buffer 40 mmol/l, 4-Chloro-phenol 5.5 mmol/l, Magnesium-

ions 17.5 mmol/l.

ii. Enzyme Reagent (R1b): 4-Aminophenazone 0.5 mmol/l, ATP 1.0 mmol/l,

Lipases ≥ 150 U/ml, Glycerol-kinase ≥ 0.4 U/ml, Glycerol-3-phosphate oxidase ≥

1.5 U/ml, Peroxidase ≥ 0.5 U/ml

iii. Reagent (R1): One vial of R1b was dissolved with 15 ml of R1a before used.

iv. Standard: Triglycerides 194 mg/dl

Glycerol-3-phosphate oxidase

Glycerol kinase

Peroxidase

Lipase

62

Procedure

Pipette into test tubes:

Reagent Blank (μl) Standard (μl) Sample (μl)

Sample - - 10

Standard - 10 -

Reagent (R 1) 1000 1000 1000

Mixed and incubated for 5 minutes at 37 °C. Absorbance of the sample (Asample)

and standard (Astandard) against the reagent blank was measured at 500 nm within 60

minutes.

Calculation

III. High-Density Lipoprotein Cholesterol (HDL-c) by Precipitant Method (Lopes-

Virella et al., 1977)

Principle

Low density lipoproteins (LDL and VLDL) and chylomicron fractions are

precipitated quantitatively by the addition of phosphotungstic acid in the presence of

magnesium ions. After centrifugation, the cholesterol concentration in the HDL (high

density lipoprotein) fraction, which remains in the supernatant, is determined.

Reagents

i. HDL-Cholesterol Precipitant (R1): Phosphotungstic acid 0.55 mmol/l.

ii. Diluted Precipitant (R2): R1 was diluted in 4:1 ratio with re-distilled water before

used.

63

iii. Magnesium chloride(MgCl2): MgCl2 25 mmol/l

Procedure for Precipitation

Pipette into centrifuge tubes:

Semi Micro

Sample/Standard 200 μl

Diluted Precipitant (R2) 500μl

Mixed, kept for 10 minutes at room temperature and centrifuged for 15 minutes at

3,000 rpm to separate clear supernatant. Determination of cholesterol content was done as

same as in the method described for TC estimation.

Calculation

HDL-c (mg/dl) = ∆ASample

× Conc. of standard (mg/dl) ∆AStandard

IV. Low-Density Lipoprotein Cholesterol (LDL-c)

LDL-c was calculated by using Friedewald formula (Friedewald et al., 1972)

which was given on Randox kit, as

LDL-c (mg/dl) = TC – (TG /5) – HDL-c

V. Very Low Density Lipoprotein-Cholesterol (VLDL-c)

VLDL-c was calculated by using Friedewald formula (Friedewald et al., 1972),

given below

VLDL-c = TG (mg/dl) /5

64

VI. Coronary Risk Index (CRI)

Coronary risk index (CRI) was calculated by using following formula (Misra and

Fridovich, 1972),

CRI = TC (mg/dl) / HDL-c (mg/dl)

2.3.7.5. Total Bilirubin by Colorimetric Method (Jendrassik and Grof, 1938)

Principle

This method is based on the reaction between bilirubin (conjugated) and

diazotized sulphanilic acid in alkaline medium gives blue coloured complex. Its

absorbance can be measured by spectrophotometer at 278 nm. Total bilirubin is

determined in the presence of caffeine, which releases albumin bound bilirubin, after

reaction with diazotized sulphanilic acid.

Reagents

i. Reagent 1 (R1): Suphanilic acid 29 mmol/l, Hydrochloric acid 0.17N

ii. Reagent 2 (R2): Sodium nitrite 38.5 mmol/l

iii. Reagent (R3): Caffeine 0.26 mol/l, Sodium benzoate 0.52 mol/l

iv. Reagent 4 (R4): Tartrate 0.93 mol/l, Sodium hydroxide 1.9 N

Procedure

Pipette into a cuvette

Blank Sample

R1 200μl 200μl

R2 - 1 drop (50μl)

R3 1000μl 1000μl

Sample 200μl 200μl

65

Mixed and incubated for 10 minutes at 20-25 °C. Then R4 was added and again

incubated for 5-30 minutes at 20-25 °C. Finally, the absorbance of the sample against the

blank (ATB) was read at 578 nm.

Calculation

The total bilirubin concentration was calculated by using factor given in Randox

kit, as

Total Bilirubin (mg/dl) = 10.8 × ATB (578 nm)

2.3.7.5.1. Direct Bilirubin

Procedure

Pipette into a cuvette

Blank Sample

R1 200μl 200μl

R2 - 1 drop (50μl)

NaCl (0.9 %) 1000μl 2000μl

Sample 200μl 200μl

R1, R2, NaCl and sample were mixed and incubated for 10 min at 20-25 °C.

Absorbance of the sample against the blank (ADB) was read at 546 nm.

Calculation

The direct bilirubin concentration was calculated by using factor given in Randox

kit, as

Direct bilirubin (mg/dl) = 14.4 × ADB (546 nm)

66

2.3.7.6. Uric Acid (UA)

Principle

Uric acid is oxidized into allantoin and hydrogen peroxide by the action of uricase.

The hydrogen peroxide reacts with 3, 5-dichloro-2-hydroxybenzene sulfonic acid and 4-

aminophenazone in a reaction catalyzed by peroxidase to produce a coloured product

(Fossati et al., 1980).

Uric acid + O2 + 2H2O

Allantoin + CO2+ H2O2

2H2O + 3, 5-dichloro-2-hydroxybenzenesulfonic acid + 4 aminophenazone

N-(4-antipyryl)-3-chloro-5-sulfonate-p-benzo-quinoneimine

Reagents

i. R1a. Buffer: Hepes buffer (50 mmol/l, Ph 7.0), 3,5-Dichloro-2-hydroxy benzene

sulfonic acid (4mmol/l).

ii. R1b. Enzyme Reagent: 4-Aminophenazone (0.25 mmol/l), Peroxidase ( ≥1000 U/l),

Uricase (≥ 200 U/l)

Procedure

Pipette into a test tubes

Reagent Blank (μl) Sample (μl) Standard (μl)

Sample - 20 -

Standard - - 20

Reagent (R1) 1000 1000 1000

Mixed and incubated for 5 minutes at 37 °C. The absorbance of sample (Asample)

and standard (Astandard) was measured against reagent blank within 30 minutes at 520 nm.

Calculation

Uric acid (mg/dl) = Standard conc. (mg/dl) ×

Uricase

Peroxidase

Asample

Astandard

67

2.3.7.7. Liver- and Cardiac-Specific Enzymes

Liver and cardiac-specific enzymes including alanine aminotransferase (ALT) and

creatine kinase (CK) were estimated in serum by using commercially available kits of

Randox (UK) and method described by IFCC, 1980 and Szasz et al., 1976.

I. Alanine Aminotransferase (ALT)

Reaction Principle

α-oxoglutarate + L-alanine ALT

L-glutamate + pyruvate

Pyruvate + NADH + H+ LD

L-Iactate + NAD+

LD = Lactate dehydrogenase

Reagents

i. R1a. Buffer/Substrate: Tris buffer (100mmol/l, pH 7.5), L-Alanine (0.6 mol/l).

ii. R1b. Enzyme/Coenzyme/α-oxoglutarate: (15 mmol/l), LD (≥1.2 U/ml),

NADH (0.18 mmol/l).

Procedure

Pipette into cuvette:

Micro

Sample 0.1 ml

R 1: Enzyme/Coenzyme/a-oxoglutarate 0.1 ml

Mixed and absorbance was read after 1, 2, & 3 minutes at 340 nm.

Calculation

ALT activity was calculated by using the factor given in Randox kit, as

U/L=1746 x ∆A 340 nm/min

68

II. Creatine Kinase (CK)

Reaction Principle

Creatine phosphate + ADP Creatine + ATP

Glucose + ATP Glucose-6-P + ADP

Glucose-6-P + NADP+

Gluconate-6-P + NADPH + H+

CK = Creatine kinase

HK = Hexokinase

G 6-PDH = Glucose 6 phosphate dehydrogenase

Reagents

i. R1a. Buffer/Glucose: Imidazole buffer 0.10 mol/l, pH 6.7, Glucose 20 mmol/l, Mg-

acetate, EDTA 2.0 mmol/l.

ii. R1b. Enzymes/Coenzymes/Substrate: ADP 2.0 mmol/l, AMP 5.0 mmol/l,

Diadenosine pentaphosphate 10 μmol/l, NADP 2.0 mmol/l, HK ≥ 2.5 U/ml, G-6-PDH

≥1.5 U/ml, N-acetylcysteine 20 mmol/l, Creatine phosphate 30 mmol/l.

Procedure

Pipette into cuvette:

Semi Micro (37 ºC)

Enzymes/Coenzymes/Substrate 1.0 ml

Sample 0.02 ml

Mixed and incubated for 1 min at 37 °C. The absorbance was measured after 1, 2

and 3 minutes simultaneously.

CK

HK

G 6-PDH

69

Calculation

CK activity was calculated by using the factor given in Randox kit, as

U/I = 8095 x ∆A 340 nm/min

2.3.8. Determination of Antioxidant Parameters

I. Reduced Glutathione (GSH)

Principle

This method is based on the development of a yellow chromogenic compound (5-

thio-2-nitrobenzoic acid, TNB), when DTNB (5,5'- dithiobis-2- nitrobenzoic acid) reacts

with reduced GSH in the presence of glutathione reductase. The colour intensity can be

measured by using spectrophotometer at 412 nm (Ellman, 1959).

Reagents

i. Phosphate Buffer (0.01 M; pH 7.0): 1.735 g disodium hydrogen phosphate and

sodium dihydrogen phosphate was dissolved in 1L of distilled water.

ii. Liver Homogenate (0.5%): 0.50 g liver tissue was homogenized in 100 ml of

distilled water.

iii. Ellman’s Reagent: 19.8 mg of 5,5‟ dithio (bis) nitrobenzoic acid was dissolved

in 100 ml of 1% sodium citrate.

Procedure

0.5 ml of liver homogenate was precipitated with 2 ml of TCA (5 %) and

centrifuged at 3000 rpm for 10 minutes. Then 1 ml of supernatant was taken and mixed

with 0.5 ml of Ellman‟s reagent and 3 ml of phosphate buffer. Standards were treated in

the same way. Finally, developed colour was read at 412 nm for 3 minutes

simultaneously.

70

Calculation

Abs of control – Abs of test

Abs of control

II. Catalase (CAT)

Principle

CAT

2H2O2 2H2O + O2

The CAT activity is directly proportional to the decomposition of H2O2 which is

reflected by decrease in absorbance measured at 620 nm for 3 minutes in the presence of

colour developer dichromate-acetic acid reagent (Pari and Latha, 2004).

Reagents

i. Phosphate Buffer (0.01 M pH 7.0): 1.735 g disodium hydrogen phosphate and

sodium dihydrogen phosphate was dissolved in 1L of distilled water.

ii. Liver Homogenate (0.5%): 0.50 g liver tissue was homogenized in 100 ml of

distilled water.

iii. Hydrogen Peroxide (H2O2 2 M): 6.8 ml H2O2 was dissolved in 93.2 ml of

distilled water.

iv. Dichromate-Acetic acid Reagent: 5% potassium dichromate and glacial acetic

acid were freshly mixed in ratio of 1:3 before used.

× 100 Percent inhibition of GSH =

71

Procedure

Reagents Control Test

Phosphate buffer 1.0 ml 1.0 ml

Distilled H2O 0.1 ml -

Homogenate - 0.1 ml

Hydrogen peroxide 0.4 ml 0.4 ml

Dichromate-acetic acid 2.0 ml 2.0 ml

Calculation

Abs of control – Abs of test

Abs of control

III. Superoxide Dismutase (SOD)

Principle

The conversion of superoxide ion (O2-) into oxygen and hydrogen peroxide is

catalyzed by SOD. This superoxide radical is involved in autoxidation of epinephrine at

high pH. This method is based on the ability of SOD to inhibit the autoxidation of

epinephrine at alkaline pH which can be reflected by rise in absorbance at 480 nm (Misra

and Fridovich, 1972).

Reagents

i. Liver Homogenate (0.5%): 0.50 g liver tissue was homogenized in 100 ml of

distilled water.

ii. Ethylenediamine tetraacetic acid (EDTA; 0.6 mM): 0.175 g EDTA was

dissolved in 1L of distilled water.

iii. Carbonate-Bicarbonate Buffer (0.1 M; pH 10.2)

× 100 Percent inhibition of CAT =

72

iv. Epinephrine (1.8 mM): 0.329 g of epinephrine was dissolved in 1L of distilled

water.

v. Ethanol (95%)

vi. Chloroform.

Procedure

Reagents Control Test

Ethanol 0.75 ml 0.75 ml

Chloroform 0.15 ml 0.15 ml

Homogenate - 0.1 ml

Centrifuged for 10 minutes at 3000 rpm

Supernatant - 0.5 ml

EDTA (0.6 mM) 0.5 ml 0.5 ml

Buffer (pH 10.2) 1 ml 1 ml

Epinephrine (1.8 mM) 0.5 ml 0.5 ml

The increase in absorbance was measured at 480 nm for 3 min.

Calculation

Final absorbance - Initial absorbance

3

Abs of control – R

Abs of control × 100

Test absorbance rate (R) =

Percent inhibition of SOD =

73

IV. Lipid Peroxidation (LPO)

Principle

In biological system polyunsaturated fatty acids are attacked by free radicals

which results in the formation of sequence of reactions to produce conjugated dienes and

lipid peroxides. The end products of lipid peroxidation are malondialdehyde (MDA),

TBARS (Thiobarbituric acid reactive substances) and lipid hydroperoxides. Lipid

hydroperoxides are broken down into aldehydes in the presence of iron or other metals

complex to form aldehydes. Hence, the TBARS assay can be conducted to assess the

level of lipid peroxidation (Niehaus and Samuelson, 1968; Alam et al., 2011).

Reagents

i. Liver Homogenate (0.5%): 0.50 g liver tissue was homogenized in 100 ml of

distilled water.

ii. TBA-TCA-HCl reagent: thiobarbituric acid (TBA 0.37%), trichloroacetic acid

(TCA 15%) and HCl (0.25 N ) were mixed in 1:1:1 ratio.

Procedure

0.1 ml of liver homogenate (0.5 % w/v) was treated with 2 ml of TBA-TCA-HCl

reagent in test tube. Then it was placed in a boiling water bath for 30 minutes, cooled and

centrifuged. The amount of malondialdehyde formed was assessed by measuring the

absorbance of clear supernatant at 535 nm against reference blank.

Calculation

Percent inhibition of LPO = Abs of control – Abs of test

Abs of control

× 100

74

75

2.3.9. Determination of Liver Glycogen and Total Lipids

I. Liver Glycogen by Phenol-Sulphuric Acid Method

Principle

The reaction between monosaccharides, oligosaccharides, polysaccharides and

their by-products (like methyl ethers with free or potentially free reducing groups) and

phenol along with concentrated sulphuric acid give an orange–yellow colour. Its

absorbance can be read at 490 nm (Dubois et al., 1956).

Reagents

i. Stock Dextrin Solution (1mg/ml): 0.1 g of dextrin was dissolved in 1L of distilled

water.

ii. Working Dextrin Solution (0.1 mg/ml): stock solution was diluted in 1:10 ratio.

iii. Sulfuric Acid (concentrated).

iv. Phenol Reagent (5 %)

Procedure

Preparation of Sample

0.5g of liver tissue was homogenized in 100 ml of distilled water. Then 0.5 ml of

homogenate was diluted up to 10 ml with distilled water. Finally 0.1ml of diluted sample

was taken in a test tube marked as test and subjected to colour development.

76

Preparation of Blank, Standards, Test and Colour Development

S1 S2 S3 S4 S5 B T C

Diluted homogenate _ _ _ _ _ _ 0.1 ml 0.1 ml

Dextrin Solution 0.2 ml 0.4 ml 0.6 ml 0.8 ml 1 ml _ _ _

Distilled water 0.8 ml 0.6 ml 0.4 ml 0.2 ml _ 1 ml 0.9 ml 0.9 ml

Phenol reagent 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml

Sulphuric acid 5 ml 5 ml 5 ml 5 ml 5 ml 5 ml 5 ml 5 ml

Mixed well and incubated all the tubes at room temperature for 30 minutes.

Absorbance was read at 490 nm.

Calculation

The concentration of liver glycogen (gm /gm of liver tissue) was calculated from

standard curve of dextrin which was prepared by using its concentration from 0.02-0.12

mg (Figure 2).

Figure 2: Standard Curve of Dextrin

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 0.05 0.1 0.15 0.2 0.25

Ab

sorb

an

ce

Concentration of dextrin

77

II. Total Lipids by Gravimetric Method

Principle

The lipid including both free and bound as lipoproteins comprise complex

mixture of different classes of compounds. Lipids occurring in the human tissue are

hydrocarbon, alcohol, fatty acids, neutral glycerides (mono-, di-, triglycerides), sterols

(cholesterol), glycerol-phospholipids and sphingolipids. Organic solvent are used to

extract lipids. Substances other than lipids are removed by washing the extract with

aqueous salt solution (Folch et al., 1956).

Procedure

Liver tissue (0.5 g) was homogenized with 100 ml of chloroform: methanol (2:1)

mixture, filtered and evaporated the filtrate at room temperature until the residues were

remained either on the walls or at the bottom of test tube. Finally, the residues were

weighed as total lipids (g /g of liver tissue).

Calculation

W = W2 – W1

W = Weight of total lipids (g / 0.5 g of liver tissue).

W1 = Weight of empty test tube.

W2 = Weight of test tube containing residues.

Finally, results are expressed as g of total lipids/g of liver tissue.

78

2.3.10. Determination of Rate-Regulatory Enzyme Activity of

Cholesterol Biosynthesis

3-Hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoAR) is the rate-

regulatory enzyme of cholesterol biosynthesis. The reductase activity was estimated in

term of HMG-CoA / mevalonate ratio in liver tissue (Rao and Ramakrishnan, 1975).

Principle

The concentrations of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) and

mevalonate in liver homogenate are estimated by taking the values of absorbance

spectrophotometrically. The ratio between the HMG-CoA and mevalonate is considered

as an index for reductase activity which catalyzes the conversion of HMG-CoA to

mevalonate. The high ratio shows decreased activity of this enzyme in liver (for example

in fasting and high cholesterol intake states) whereas low ratio shows increased activity

of same enzyme (for example after taking triton injection or phenobarbital treatment).

Reagents

i. Liver Homogenate (10%): 10 g of liver tissue was homogenized with 100 ml of

saline arsenate solution (1%).

ii. Sodium Arsenate Solution: 1 g of sodium arsenate was dissolved in 1L of

physiological saline.

iii. Dilute Perchloric Acid (PCA): 50 ml of PCA was dissolved in 1L of distilled

water.

iv. Physiological Saline: 0.9 g of NaCl was dissolved in 100 ml of distilled water.

v. Hydroxylamine Hydrochloride Reagent (2 mol/l): 138.98 g of hydroxylamine

hydrochloride was dissolved in 1 L of distilled water.

79

vi. Hydroxylamine Hydrochloride Reagent for Mevalonate (pH: 2.1-2.2): equal

volumes of hydroxylamine hydrochloride reagent and water were mixed before

used.

vii. Hydroxylamine Hydrochloride Reagent for Mevalonate (pH: 5.5-5.9): equal

volumes of hydroxylamine hydrochloride reagent and NaOH solution were mixed

before used.

viii. Sodium Hydroxide (4.5 M): 180 g of NaOH was dissolved in 1L of distilled

water.

ix. Ferric Chloride Reagent: 5.2 g of TCA and 10 g of ferric chloride were

dissolved in 50 ml of 0.65 M HCl and volume was made up to 100 ml with

distilled water.

x. Hydrochloric acid (HCl 0.65 M): 53.6 ml of concentrated HCl was dissolved in

946.4 ml of distilled water.

Procedure

Preparation of Sample

Liver homogenate (10 %) was prepared in sodium arsenate (0.1%) solution and

filtered. The filtrate was used for the estimation of HMG-CoA and mevalonate

concentrations.

80

Preparation of Tests

Reagent HMG-CoA Mevalonate

Filtrate 1.0 ml 1.0 ml

PCA (diluted) 1.0 ml 1.0 ml

Incubated for 5 min and centrifuged for 15 min at 3000 rpm

Supernatant 1.0 ml 1.0 ml

Hydroxyamine HCl reagent (2M, pH 5.5) 0.5 ml --

Hydroxyamine HCl reagent (2M, pH 2.1) -- 0.5 ml

Incubated for 5 min at room temperature

FeCl3 reagent 1.5 ml 1.5 ml

Mixed well and incubated for 10 min at room temperature

Absorbance was read at 540 nm against saline arsenic blank that contained 1 ml

distilled water in place of supernatant.

Calculation

HMG-CoA reductase activity was calculated in term of HMG-CoA/Mevalonate ratio

and as described by (Rao and Ramakrishnan, 1975).

2.3.11. Determination of Trace Minerals

Blood samples were digested by the conventional wet acid method (Memon et al.,

2007). In which, 0.5 ml of sample (whole blood / serum) was taken in a Pyrex flask then

3 ml of freshly prepared mixture of concentrated nitric acid and hydrogen peroxide

[HNO3 - H2O2] in 2:1 ratio was added and placed for 10 minutes in covered state (using

watch glass). This was subjected to digestion at 60 - 70 ºC for 1-2 hours by adding 2 ml

nitric acid and few drops of H2O2 and kept on hot plate at about 80 ºC until a clear

81

digested solution was achieved. The excess acid mixture was evaporated to semi-dry

mass, cooled and diluted with 0.1 ml of nitric acid. This was shifted into 100 ml

volumetric flask and diluted with triple distilled water up to mark mentioned on flask. A

blank (without the sample) was prepared by the same procedure using triple distilled

water. The mineral content in both test samples and blank were analyzed by atomic

absorption spectrophotometer (PG990). The concentration of mineral was expressed in

ppm.

2.3.12. Statistical Analysis

Results are expressed as mean ± SEM (Standard Error Mean). Difference between

treated and diabetic groups was considered significant at p<0.05 when data were

analysed by using one-way ANOVA followed by LSD (least significant difference) test

(SPSS version 18).

82

3. Results

3.1. In-Vitro Investigations

3.1.1. Antiglycation and DPPH Radical Scavenging Activities of

C. anthelminticum

ESEt and AqSEt of C. anthelminticum showed good (80-82 %) in vitro

antiglycation activity which was not very far from the same activity showed by standard

rutin (Table 5). On the other hand, ESEt demonstrated moderate in vitro DPPH- radical

scavenging activity than standard gallic acid whereas AqSEt was found completely

inactive in this regard (Table 5).

3.2. In-Vivo Investigations

3.2.1. Effect of ESEt of C. anthelminticum on Physical Parameter of

Fructose-induced Diabetic Rabbits

The fructose-induced diabetic control group demonstrated 25 % gain in body

weight after daily intake of 35% fructose solution for 14 days consecutively in overnight

fasting state. Whereas pioglitazone (15 mg/kg) and ESEt in doses of 400 and 600 mg/kg

made a significant decrease (p<0.05) in the percent body weight gain in positive control

and two test groups as 19.15, 17.04 and 13.32% respectively. On contrary, ESEt in dose

of 200 mg/kg showed much increase in body weight gain in its respective group as

compared to fructose-induced diabetic control group (Table 6; Figure 3).

3.2.2. Effect of ESEt of C. anthelminticum on Percent Glycemic Change,

HbA1c, Fasting Insulin and FIRI in Fructose-induced Diabetic Rabbits

All doses (200-600 mg/kg) of ESEt of C. anthelminticum induced significant

(p<0.05 & p<0.001) glycemic reduction from -1.80 to -11.18% in their respective test

groups as compared to fructose-induced diabetic control and positive control groups that

showed about 21 to 26 % increase in glucose level on final day of animal trial (Table 7,

Figure 4).

83

Table 5: In vitro Antiglycation and Antioxidant Activities of AqSEt and

ESEt of C. anthelminticum

Samples

Antiglycation Activity

(0.5 mg/ml) Antioxidant Activity(mg/ml)

Percent

Inhibition

IC 50 ± SEM

[μg/ml]

Percent Radical

Scavenging Activity

IC 50 ± SEM

[mg/ml]

AqSEt 82.5 169.28 ± 6.8 - Inactive

ESEt 80.5 217.6 ± 5.2 67.36 321.31 ± 0.66

Rutin 94.5 27.0 ± 0.15 - -

Gallic acid

(0.094ng/ml) - - 93.13 4.3 ± 0.43

84

Table 6: Effect of ESEt of C. anthelminticum on Percent Body Weight

Change

Groups

Body Weight (kg) Percent

BodyWeight

Change 1st day 14

th day

Control 1.15 ± 0.196 1.047 ± 0.134 -8.71 ± 0.06

Diabetic control 0.798 ± 0.04 0.998 ± 0.05 25.06 ± 0.01

Positive control 0.851 ± 0.001 1.014 ± 0.015 19.15 ± 0.01 d

ESEt 200mg 0.984 ± 0.08 1.367 ± 0.164 38.9 ± 0.08

ESEt 400mg 0.663 ± 0.03 0.776 ± 0.06 a 17.04 ± 0.03

d

ESEt 600mg 0.848 ± 0.05 0.961 ± 0.07 13.32 ± 0.02 d

All values are mentioned as mean ± SEM (n=4). ap<0.05, dp<0.0001,when test groups compared

with diabetic control.

85

Figure 3: Effect of ESEt of C. anthelminticum on Percent Body Weight

Change in Fructose-induced Type 2 Diabetic Rabbits

All bars are representing the mean ± SEM (n=4). dp< 0.0001,when test groups compared with

diabetic control.

-20

-10

0

10

20

30

40

50

Control Diabetic

control

Positive

control

ESEt

200mg

ESEt

400mg

ESEt

600mg

Control

Diabetic control

Positive control

ESEt 200mg

ESEt 400mg

ESEt 600mg

Bod

y W

eigh

t C

ha

ng

e (%

)

d d

d

86

Similarly, all doses ESEt (200-600 mg/kg) of extract and pioglitazone in their

respective test and positive control groups were found effective (p<0.0001) in decreasing

the values of HbA1c as compared to diabetic control group (Table 7). In addition, all test

doses of ESEt (200-600 mg/kg) along with pioglitazone showed significant reduction

(p<0.05) in values of fasting serum insulin as compared to fructose-induced diabetic

control groups (Table 7). Therefore, the values of fasting insulin resistance index (FIRI)

were also improved (p<0.0001) in the test groups especially treated with 400 and 600

mg/kg of extract as same as pioglitazone showed the same positive result in positive

control group (Table 7).

3.2.2. Effect of ESEt of C. anthelminticum on Lipid Profile and CRI in

Fructose-induced Diabetic Rabbits

The lipid profile exhibited significant decline by showing decrease in serum

levels of TC from 141 to 160 mg/dl, TG from 110 to 122 mg/dl, LDL-c from 22 to 38

mg/ dl and VLDL-c from 22 to 24 mg/dl in all the test groups treated with ESEt in doses

of 200 to 600 mg/kg (Figure 5 & 6) as compared to fructose-induced diabetic control

group whereas a prominent rise (p<0.05) was observed in the values of HDL-c from 145

to 164 mg/dl in all same test groups. Similarly, pioglitazone-treated positive control

groups also showed a significant decrease in TG, TC, LDL-c, VLDL-c and increase in

HDL-c levels (Figure 5 & 6). Therefore, a significant decrease (p<0.0001) was observed

(p<0.0001) in the values of CRI in all test groups treated with ESEt while fructose-

induced diabetic control group showed marked increase up to 3.01 in the same ratio

(Table 8).

87

Table 7: Effect of ESEt of C. anthelminticum on Percent Glycemic Change, HbA1c, Fasting Insulin and FIRI

Groups

FBG (mg/dl)

Percent

Glycemic

Change

HbA1c

(mg/dl)

Fasting Insulin

(pmol/l) FIRI

Initial Day Final Day

Control 112.5 ± 11.79 100.75 ± 6.49 -8.51 ± 7.25 4.45 ± 0.14 0.22 ± 0.00 0.78 ± 0.04

Diabetic control 102.75 ± 3.59 125.25 ± 1.18 21.59 ± 4.39 6.67 ± 0.13 0.807± 0.31 4.06 ± 1.59

Positive control 100 ± 16.41 119.25 ± 1.7 26.28 ± 14.7 5.4 ± 0.20 d 0.37 ± 0.08

a 1.79 ± 0.41

a

ESEt 200mg 111.75 ± 4.77 115.5 ± 2.59 -1.807 ± 0.80 a 5.87 ± 0.01

d 0.35 ± 0.03

a 1.66 ± 0.18

ESEt 400mg 114 ± 4.14 103.25 ± 3.32 bc

-9.26 ± 2.55 c 5.97 ± 0.09

c 0.31 ± 0.03

a 1.28 ± 0.16

d

ESEt 600mg 115.5 ± 2.02 102.5 ± 3.8 bc

-11.18 ± 3.49 c 5.62 ± 0.11

d 0.34 ± 0.03

a 1.45 ± 0.17

d

All values are mentioned as mean ± SEM (n=4). ap<0.05, bp<0.01, cp<0.001, & dp<0.0001, test groups compared with diabetic control

88

Figure 4: Effect of ESEt of C. anthelminticum on Percent Glycemic

Change in Fructose-induced Type 2 Diabetic Rabbits

All bars are representing the mean ± SEM (n=4). ap<0.05 & cp<0.001, test groups compared with

diabetic control.

-20

-10

0

10

20

30

40

50

Control Diabetic

control

Positive

control

ESEt

200mg

ESEt

400mg

ESEt

600mg

Control

Diabetic

controlPositive

controlESEt 200mg

ESEt 400mgGly

cem

ic C

han

ge

(%)

a c

89

Figure 5: Effect of ESEt of C. anthelminticum on TC and TG in

Fructose- induced Type 2 Diabetic Rabbits

All bars are representing the mean ± SEM (n=4). dp<0.0001, test groups compared with diabetic

control.

0

50

100

150

200

250

Control Diabetic

control

Positive

control

ESEt

200mg

ESEt

400mg

ESEt

600mg

TC(mg/dl)

TG(mg/dl)

d

d

d

d

d

d

d

d

90

Figure 6: Effect of ESEt of C. anthelminticum on Lipoproteins in

Fructose-induced Type 2 diabetic rabbits

All bars are representing the mean ± SEM (n=4). cp<0.001, &dp<0.0001, test groups compared

with diabetic control.

0

50

100

150

200

250

Control Diabetic

control

Positive

control

ESEt

200mg

ESEt

400mg

ESEt

600mg

HDL-c

LDL-c

VLDL-

c

Lip

op

rote

ins

(mg/d

l)

c

d

d

d

d d

d

d

d

d

d

d

91

3.2.3. Effect of ESEt of C. anthelminticum on HMG-CoA/ Mevalonate

Ratio

HMG-CoA/ mevalonate ratio was non-significantly increased in all test groups

treated with ESEt (200-600 mg/kg) as compared to fructose-induced diabetic control

group (Table 8).

3.2.4. Effect of ESEt of C. anthelminticum on Liver Glycogen and Total

Lipids

Liver glycogen (g /g of liver tissue) was found improved in all test groups treated

with ESEt (200-600 mg/kg) and positive control group treated with pioglitazone (15

mg/kg) as compared to fructose-induced diabetic control group. Similarly, total lipid

content in same tissue in test groups was also found increased as compared to same

diabetic control group (Table 9).

92

Table 8: Effect of ESEt of C. anthelminticum on HMG-CoA/

Mevalonate Ratio and CRI

Groups HMG-CoA / Mevalonate ratio CRI

Control 2.11 ± 0.80 1.14 ± 0.05

Diabetic control 1.06 ± 0.08 3.01 ± 0.5

Positive control 1.7 ± 0.36 1.49 ± 0.13

ESEt 200mg 1.21 ± 0.04 0.95 ± 0.06 d

ESEt 400mg 2.53 ±1.01 0.99 ± 0.01 d

ESEt 600mg 1.63 ± 0.14 0.922 ± 0.12 d

All values are mentioned as mean ± SEM (n=4). dp<0.0001, test groups compared with diabetic

control.

93

Table 9: Effect of ESEt of C. anthelminticum on Liver Glycogen

and Total Lipids

Groups Total lipids Glycogen

g/g of liver tissue

Control 0.15 ± 0.02 0.069 ± 0.007

Diabetic control 0.21 ± 0.005 0.097 ± 0.004

Positive control 0.175± 0.02 d 0.111 ± 0.023

ESEt 200mg 0.25 ± 0.02 0.124 ± 0.019

ESEt 400mg 0.25 ± 0.02 0.132 ± 0.026

ESEt 600mg 0.22± 0.02 0.102 ± 0.028

All values are mentioned as mean ±SEM (n=4). dp<0.0001, compared with diabetic control.

94

3.3. Effect of ESEt of C. anthelminticum on Liver- and Cardiac-

Specific Enzymes in Fructose-Induced Diabetic Rabbits

I. Alanine Aminotransferase (ALT)

All doses of ESEt of C. anthelminticum (200-600 mg/dl) in test and pioglitazone

(15 mg/kg) in positive control groups revealed a drop (p<0.01) in the values of serum

ALT (U/l) in comparison with fructose-induced diabetic control group (Table 10).

II. Creatine Kinase (CK)

The ESEt in a dose of 600 mg/kg induced a significant fall (p< 0.05) in values of

CK of test group in comparison with fructose-induced diabetic control group and two

other test doses (200 & 400) of same extract. Even pioglitazone did not show any

significant reduction in CK level of positive control group (Table 10).

3.3.1. Effect of ESEt of C. anthelminticum on Serum Bilirubin and Uric

Acid Levels

The total bilirubin was found increased in fructose-induced diabetic and positive

control groups whereas ESEt (200-600 mg/kg) slightly decreased the same parameter in

test groups by improving the levels of direct and indirect bilirubin (Table 10). Similarly,

increased concentration of uric acid was observed in fructose-induced diabetic control

group which significantly (p<0.01) lowered up to 9 mg/dl in all test groups treated with

ESEt. However, Pioglitazone also showed slightly increased level of same parameter

(Table 10).

95

Table 10: Effect of ESEt of C. anthelminticum on Biochemical Parameters

Groups

Enzymes (U/I) Bilirubin (mg/dl)

Uric Acid

(mg/dl) CK ALT Total Bilirubin Direct/conjugated

Bilirubin

Indirect

Bilirubin

Control 18.21 ± 2.02 5.67 ± 1.09 1.36 ± 0.19 0.81 ± 0.20 0.73 ± 0.34 10.39 ± 0.29

Diabetic control 56.66 ± 10.45 17.89 ± 2.79 2.13 ± 0.17 1.76 ± 0.15 0.37 ± 0.09 14.3 ± 1.89

Positive control 36.42 ± 7.75 4.36 ± 0.87 d 2.48 ± 0.59 2.31 ± 0.29 1.09 ± 0.34 a 11.61 ± 1.57

ESEt 200mg 46.54 ± 11.44 5.23 ± 0.71 d 1.45 ± 0.16 1.28 ± 0.11 0.17 ± 0.03 9.78± 0.27 b

ESEt 400mg 34.4 ± 7.66 5.67 ± 1.31 d 1.88 ± 0.15 1.46 ± 0.05 0.43 ± 0.11 9.86 ± 0.13 b

ESEt 600mg 22.26 ± 3.87 b 6.54 ± 2.06 d 1.72 ± 0.2 1.21 ± 0.12 0.516 ± 0.13 9.43 ± 0.29 b

All values are mentioned as mean ± SEM (n=4). ap<0.05, bp<0.01, & dp<0.0001, compared with diabetic control.

96

3.3.2. Effect of ESEt of C. anthelminticum on Serum Trace Mineral

Content

The serum Na concentration showed a significant decrease (p<0.05) in test groups

treated with ESEt in doses of 400 and 600 mg/kg as compared to fructose-induced

diabetic control group. The highest dose (600 mg/kg) of same extract was also found

effective in decreasing K level in its respective group. All doses of ESEt (200-600

mg/kg) and pioglitazone in test and positive control groups showed a significant decrease

in values of serum Zn (p<0.0001), Fe

and Cr

(p<0.05) levels in comparison with same

diabetic group. Whereas, the serum Cd level was gradually decreased in test groups with

increased in dose of ESEt (Table 11).

3.3.3. Effect of ESEt of C. anthelminticum on Antioxidant Enzymes

I. Percent Inhibition of CAT, SOD and GSH Activities

All doses of ESEt of C. anthelminticum 200-600 mg/ kg) and pioglitazone in test

and positive control groups, showed a significant decrease (p<0.0001 & p<0.001) in

percent inhibition of CAT as compared to fructose-induced diabetic control group (Table

12).

Similarly, significant (p<0.0001) percent inhibition of SOD was observed in dose-

dependent manner in all test groups and of course positive control group showed

significant improvement in SOD activity (Table 12). ESEt (200-600 mg/kg) of C.

anthelminticum also found effective in improving the levels of reduced glutathione

(GSH) in all test groups by lowering (p< 0.05 & p<0.01) its inhibition from 34 to 16 %

from smallest to highest dose of same extract as compared to fructose-induced diabetic

control group. However, positive control group did not show any positive result in this

regard (Table 12).

97

II. Percent Inhibition of Lipid Peroxidation (LPO)

LPO was significantly inhibited in all test groups treated with ESEt (200-600

mg/kg) of C. anthelminticum as compared to fructose-induced diabetic control group.

Pioglitazone also improved the percent inhibition of LPO in positive control group but

non-significantly (Table 12).

98

Table 11: Effect of ESEt of C. anthelminticum on Serum Trace Mineral Levels

Groups

(mg/dl) (µg/l)

Na K Zn Fe Cd Cr

Control 19.86 ± 0.22 18.02 ± 0.42 18.4 ± 0.32 16.16 ± 0.311 0.77 ± 0.10 0.59 ± 0.11

Diabetic control 19.93 ± 0.74 19.0 ± 0.4 21.5 ± 1.30 16.40 ± 1.56 0.72 ± 0.07 0.84 ± 0.04

Positive control 19.42 ± 0.60 18.57 ± 0.47 15 ± 1.14

d 12.95 ± 0.86 0.81 ± 0.06 0.59 ± 0.18

ESEt 200mg 18.43 ± 0.47 18.87 ± 0.41 13 ± 0.41

d 11.43

± 1.26

b 0.81 ± 0.03 0.422

± 0.07

a

ESEt 400mg 18.1 ± 0.34

a 18.95 ± 0.56 15.07

± 1.0

d 11.67

± 1.77

a 0.73 ± 0.07 0.68 ± 0.11

ESEt 600mg 17.1 ± 0.68

b 16.22

± 0.35

d 14.80

± 1.15

d 13.16 ± 1.69 0.64 ± 0.07 0.30

± 0.10

b

All values are mentioned as mean ± SEM (n=4). ap<0.05, bp<0.01, & dp<0.0001, compared with diabetic control.

99

Table 12: Effect of ESEt of C. anthelminticum on Antioxidant Parameters

Groups

Percent Inhibition

CAT SOD GSH LPO

Control 8.23 ± 1.10 c 23.29 ± 2.07

d 16.48 ± 0.16

b 27.1 ± 4.32

Diabetic control 62.52 ± 3.68 96.09 ± 2.22 57.5 ± 13.14 10.16 ± 2.88

Positive control 32.78 ± 11.87c 29.2 ± 1.55

d 48.25 ± 10.86 23.85 ± 3.65

a

ESEt 200mg 10.6 ± 1.55 d 19.86 ± 4.64

d 34.27 ± 3.04 34.1 ± 5.95

b

ESEt 400mg 7.07 ± 3.50 d 17.85 ± 5.89

d 30.5 ± 11.25

a 43.62 ± 9.13

c

ESEt 600mg 3.54 ± 0.52 d 11.34 ± 0.94

d 16.9 ± 2.66

,b 51.17 ± 8.33

d

All values are mentioned as mean ± SEM (n=4). ap<0.05, bp<0.01, cp<0.001, & dp<0.0001, compared with diabetic group

100

4. Discussion

The incidence of type 2 diabetes is increasing day by day globally; the biggest

contribution in this regard is given by developing countries (Ansari et al., 2015). Many

acquired causes especially adaptation of unhealthy lifestyle and consuming imbalance

diet are accelerating 80% risk of this endocrine disorder in population. High fat and high

carbohydrate diets are playing important role in the development of hyperlipidemia and

unstable hyperglycemia in both genders; even children are not safe from these metabolic

problems. These altered lipidemia and glycemia actually accelerate the generation of

oxidative stress and glycation in body which in turn open the gate for insulin resistance or

type 2 diabetes (Brouwers et al., 2010). Artificial sweeteners including high fructose corn

syrup are the major silent life threatening component present in desserts, canned food,

candies, etc. Interestingly, fructose is a lipogenic in characteristic. Its absorption in

gastrointestinal tract (GIT) does not require the assistance of insulin as the absorption of

glucose does. Fructose can easily absorbed in GIT then entered in hepatocytes with the

help of insulin independent glucose transporters (GLUT-5) where it accelerates the

synthesis of TG and TC thereby creating hypertriglyceridemia and hypercholesterolemia

in body (Armato et al., 2015). This increased amount of TC and TG contribute in the

development hypertension, hyperlipidemia and masking of insulin receptors on target

cells including hepatocytes, myocytes and adipocytes thus interfere the insulin-receptor

sensitivity and induced insulin resistance (Azmi and Qureshi, 2012).

Fructose is a fat inducer. After absorption in liver, it immediately converts in

fructose-1,6-bisphosphate that hydrolyzed into two three-carbon containing compounds

named glyceraldehyde-3-phosphate (GA3P) and dihydroxyacteone phosphate (DHAP).

On one hand, GA3P and DHAP accelerate glycolysis to produce more and more pyruvate

and acetyl CoA which cannot be easily handled by tricarboxylic acid (TCA) cycle for

energy production thus divert in the synthesis of cholesterol and create hypercholesterolemia.

On the other hand, both of these compounds accelerate the synthesis of TG in liver and

create hypertriglyceridemia (Tirosh et al., 2008). This fructose-induced hypercholesterolemia

101

and hypertriglyceridemia encouraged overweight or obesity which is the initiator of

insulin resistance (type 2 diabetes) that generate hyperglycemia and hyperinsulinemia

(Barnes and Miner, 2009; Monnier et al., 2009; Wilson and Islam, 2012). The same was

observed in the present study that oral intake of 35% fructose solution by rabbits in

overnight fasted state for 14 days consecutively induced 25% gain in their original body

weights. However, three doses (200, 400 & 600 mg/kg) of ethanolic seeds extract (ESEt)

of C. anthelminticum found effective in reducing the body weight gain in dose-dependent

manner in their respective treated test groups. This beneficial weight controlling effect of

ESEt may be due to accelerating the activity of lipase enzyme thereby promoting the

process of lipolysis. There are many medicinal plants reported with lipolytic activity like

Murraya koeingii (curry leaf), and Brassica juncea, (mustard), Cudrania tricuspidata

(leaves), and jasmine tea and green tea (Khan et al., 1995; Kim et al., 2012; Okuda et al.,

2001; Koo and Noh, 2007).

The overweight in fructose-induced type 2 diabetic control rabbits was also

accompanied with hyperlipdemia which described the existence of all bad lipids (TC, TG,

LDL-c, VLDL-c) in high levels and decreased level of good one HDL-c in blood, which

confirmed the mode of action of fructose to induce hyperlipidemia and obesity in high

fructose-intake population (Hsieh et al., 2013). All three doses of ESEt of C.

anthelminticum were also found to decrease TC, TG, LDL-c, VLDL-c and increase in

HDL-c levels in test rabbits. The relationship between hyperlipidemia and cardiovascular

diseases (CVDs) is well-established and surveys reported that CVDs are the biggest

reason of death worldwide (Haffner et al., 1998). The cholesterol and LDL-c lowering

effect of ESEt of C. anthelminticum may be due to inhibiting the activity of HMG Co

reductase, the rate-limiting enzyme of cholesterol biosynthesis as the statins (synthetic

hypolipidemic group of medicines) normally do which automatically decrease the LDL-c

levels (Kawai et al., 2005) or by inhibiting enterohepatic recirculation of bile as bile acid

binding resins can do (Adisakwattana et al., 2012). Similarly, TG and VLDL-c reducing

effect of ESEt of C. anthelminticum may be due to by inhibiting the fructose absorption

in liver cells via down regulating the activity of GLUT-5 transporters thereby inhibiting

102

TG biosynthesis in liver (Armato et al., 2015) or by inhibiting the activity of malonyl

CoA carbooxylase, the rate-regulatory enzyme of fatty acid biosynthesis (McGarry et al.,

1977) or by accelerating the activity of lipoprotein lipase which in turn degrade TG and

release free fatty acids thereby stimulating β-oxidation as the fibrates produced their

hypotriglyceridemic effect (Zhang et al., 2008) or by enhancing the excretion of TG in

bile and feces (Insull, 2006).

In order to verify one of the possible mechanism of action of cholesterol lowering

effect of ESEt, the HMG CoA reductase activity in terms of HMG CoA/ mevalonate ratio

was determined in fructose-induced type 2 control and test rabbits in the present study.

The values of this ratio were gradually increased in three test groups with respect to

increase in doses of ESEt which indicated the inhibition of HMG CoA reductase activity

in all three test groups as compared to fructose-induced type 2 diabetic control rabbits.

Similarly, one of the suggested mechanisms of hypotriglyceridemic action of ESEt was

also verified by observing accelerated activity of lipase, TG hydrolyzing enzyme, in high

fat induced hyperlipidemic rabbits (Lateef and Qureshi, 2014). The lipid lowering

property of ESEt is also strengthens by observing the decrease in coronary risk index

(CRI) values in all three test groups treated with ESEt (200-600 mg/kg). This index is

important in determining the risk of heart problems and its high values are alarming and

indicative for the risk of atherosclerosis and other CVDs (Jacobson et al., 2007).

The hyperglycemia is actually the secondary effect of hypertriglyceridemia

induced by high fructose consumption (Stanhope and Havel, 2008). The accumulated TG

could disturb insulin receptor binding by depositing on receptors thereby impairing

glucose utilization in body which results in persistent hyperglycemia and hyperinsulinemia,

that developed an accurate picture of insulin resistance type 2 diabetes. The same was

observed in fructose-induced type 2 diabetic control rabbits which showed high percent

glycemic gain up to 21% after consumption of 35% fructose solution for 14 days

consecutively in early morning before diet whereas treatment with ESEt in doses of 200

to 600 mg/kg induced significant percent reduction from -1.8 to -11.18% in BG levels of

103

all three test groups in dose-dependent manner. Even the synthetic antidiabetic medicine

pioglitazone was not found effective in lowering BG level in diabetic positive control

group. The hypoglycemic property of ESEt might be due to the inhibition of fructose

absorption in intestine. It could be true as methanolic seed extract of same plant was

reported to inhibit the activities of glucose hydrolyzing enzymes including α- amylase

and glucosidase in vitro (Ani and Naidu, 2008; Amir and Chin, 2011). This activity must

be associated with the presence of total phenols, flavonoids and alkaloids in the same

extract observed in first phase of the present study which are reported to have

normoglycemic properties in many studies (Hashim et al., 2013; Shih et al., 2012;

Wilson and Islam, 2012).

Hyperglycemia is also associated with non-enzymatic glycation of proteins

especially haemoglobin (Hb) and formed HbA1c (glycated Hb) in blood. The presence of

HbA1c represents the relative amount of glucose in blood (Farhan et al., 2012). The same

was observed in the present study where fructose-induced hyperglycemia was

accompanied with high amount of HbA1c in type 2 diabetic control rabbits. On contrary,

the HbA1c levels were gradually decreased with respect to doses in all three ESEt treated

test groups. Therefore, ESEt could be beneficial in the treatment of type 2 diabetes as it is

not only hypoglycemic in nature but also has antiglycation activity which was also

proved by in vitro method conducted in the present study where ESEt showed 80%

inhibition of protein glycation that was very closed to the antiglycation activity showed

by rutin (flavonoid), the standard used in the same method.

The most important characteristic of insulin resistance type 2 diabetes is

hyperinsulinemia (Wilson and Islam, 2012) which was clearly observed in fructose-

induced type 2 diabetic rabbits in the present study. The hyperinsulinemia is directly

proportional to the degree of insulin resistance present in type 2 diabetes which can be

corrected as soon as the insulin-receptor sensitivity increased (Monnier et al., 2009). The

recovery of fasting insulin levels in test groups treated with ESEt is another noteworthy

finding of the present study and it may be secondary to hypolipidemic effect of ESEt of

104

C. anthelminticum thereby uncovering the receptors and make them available for insulin

binding. This property of ESEt was also confirmed by observing the decreased values of

fasting insulin resistance index (FIRI) in all three test groups as compared to its high

value observed in fructose-induced type 2 diabetic control rabbits. FIRI is an important

indicator of insulin resistance; its decrease values represent the low degree of resistance

(Pratley et al., 2000). This would be the new addition in antidiabetic mechanism of action

of C. anthelminticum that it is capable to normalize insulin levels by reducing insulin

resistance in type 2 diabetes. However, the previous finding stated that the methanolic

seed extract of same plant has insulin secretagogue property on β-TC6 cell line (Arya et

al., 2012).

Development of fatty liver is one of the reported bad consequences of high

fructose intake (Vos and Lavine, 2013). This finding supports the possibility that

fructose-induced TG biosynthesis could be involved in masking of insulin receptors on

hepatocytes. This possibility is also fortified by observing the inhibition of insulin-

dependent glycogenesis in liver of fructose-induced type 2 diabetic control rabbits

whereas the same process was found reversed and improved in all test groups treated with

ESEt from 200-600 mg/kg in present study. The improved hepatic glycogenesis in test

rabbits also supports the hypoglycemic effect of ESEt of C. anthelminticum. However,

the process of hepatic lipogenesis was found almost similar in both diabetic control and

test groups.

Fructose-induced obesity or hyperlipidemia reflect the inhibition of lipoprotein

lipase and hormone sensitive lipase that results in fats accumulation in tissues especially

liver that leads to hepatocytes dysfunction like impaired cell membrane fragility which in

turn increased the level of liver-specific enzyme ALT (Farswan et al., 2009). The ALT

activity is the mirror image of liver function like mild increase in ALT levels is

frequently reported in type 2 diabetic patients that is the indication of liver dysfunction

(Harris, 2005; Wannamethee et al., 2005). The same was observed in fructose-induced

type 2 diabetic control group whereas ESEt was found to normalize the level of same

105

enzymes in all three test groups. Beside ALT, total bilirubin (direct and indirect

bilirubins) is another important parameter used to assay liver function and its

accumulation in blood and tissues leads to jaundice (Han et al., 2010). The total bilirubin

can be elevated in number of cases including high fructose consumption which actually

accelerated the synthesis of conjugated bilirubin (direct bilirubin) that could easily

diffuse in circulation and elevated the level of total bilirubin. The same was observed in

the present study that showed the elevated levels of total & direct bilirubin as compared

to indirect bilirubins in fructose-induced type 2 diabetic rabbits. This picture was

gradually improved in ESEt treated three test groups by observing decreased levels of

total and direct bilirubins in present study. Interestingly, pioglitazone was not found

effective in this regard. The beneficial effect of ESEt in restoring the levels of ALT and

total bilirubin constitute the hepatoprotective effect of same extract of C. anthelminticum.

Uric acid is the end product of purine metabolism (Murray et al., 2000). High uric

acid levels in blood increases the chances of many metabolic diseases including insulin

resistance type 2 diabetes (Mahmood, 2007). High fructose intake actually accelerates its

metabolism in liver which required adenosine triphosphate (ATP) largely and depletes

this energy currency of the body. This depleted amount of ATP affects every metabolic

pathway including protein biosynthesis and reduce the half-life of cells which results in

the formation of uric acid that cannot be fully excreted through kidneys and ultimately its

levels become increased in blood (Johnson et al., 2013). The same situation was observed

in fructose-induced type 2 diabetic rabbits which showed elevated levels of uric acid up

to 14 mg/dl whereas its reported normal levels are within the range of 3-7mg/dl (Safi et

al., 2004). On the other hand, ESEt slowly decreased the level of uric acid up to 9 mg/dl.

This is one more important significant finding of present study which indicated that ESEt

is capable to restore cell stability. ESEt of C. anthelminticum was also found cardio-

protective by observing the normal levels of CK in all three test groups in present study

as compared to elevated level of same enzyme displayed by fructose-induced type 2

diabetic control group. CK is a cardiac-specific enzyme, any kind of injury in cardiac

106

muscle or heart attack causes the release of this enzyme in blood stream (Frank and

Finsterer, 2012).

One of the basic symptoms of diabetes is polyuria that induced depletion of the

body stores of trace minerals which one way or other participates in insulin metabolism

(Akhuemokhan et al., 2013). Zn plays an important role in insulin release, structure and

stability (Saper and Rash, 2009). In the present study, high amount of Zn was observed in

fructose-induced type 2 diabetic control groups that confirmed the presence of

hyperinsulinemia and insulin resistance in these diabetic rabbits whereas the Zn levels

was gradually decreased in ESEt treated test groups reflected the improvement in insulin-

receptor sensitivity. Similarly, reduced levels of Cr levels were also found in ESEt treated

test groups. This observation was supported by many studies like Cr-supplement was

reported to improve glucose clearance in Cr-deficient rats (Striffler et al., 1995). In

addition, Cr with cinnamon can reduce insulin resistance in type 2 diabetes (Haase and

Carmicheal, 2012). On the other hand, high amounts of Fe and Cd are reported to have

negative impact on insulin production & function and positive impact on insulin

resistance (Trevino et al., 2015). Two separate clinical trials also demonstrated the high

levels of Cd and Fe in type 2 diabetic patients (Akinloye et al., 2010; Kundu et al., 2013).

The same was observed in fructose-induced type 2 diabetic rabbits which showed high

amounts of Fe and Cd that definitely correlated with the existence of insulin resistance in

these diabetic rabbits. Interestingly, levels of both minerals were decreased in ESEt

treated test rabbits which showed better insulin-receptor sensitivity in liver and muscles.

Similarly, Na and K levels were also found better in ESEt treated test groups which

confirmed the ability of seed extract in regulating the electrolyte balance in blood.

In insulin resistance type 2 diabetes, chronic hyperglycemia and dyslipidemia

induced hepatic inflammation and oxidative stress by inducing mitochondrial dysfunction

thereby accelerating the generation of ROS (hydrogen peroxide, superoxide, hydroxyl

radicals) and affecting antioxidant enzymes system (Waggiallah and Alzohairy, 2011;

Kumawat et al., 2013). The accumulated ROS also induced oxidative damage to

107

macromolecules especially polyunsaturated membrane lipids that constitute lipid

peroxidation (Malini et al., 2011). The relationship between hyperglycemia and

suppression of antioxidant protein and enzymes was also observed in the present study by

observing greater percent inhibition of antioxidant enzymes including CAT, SOD and

protein reduced glutathione (GSH) and lesser percent inhibition in lipid peroxidation in

fructose-induced type 2 diabetic control rabbits. However, a significant reduction was

observed in percent inhibitions of CAT, SOD, GSH and increased in percent inhibition of

lipid peroxidation in liver tissues of ESEt treated test groups. Amazingly, the seeds

extract of C. anthelminticum was found much better than positive control pioglitazone in

antioxidant aspect. The antioxidant potential of same ESEt was also confirmed by

observing its 67% in vitro radical scavenging activity in present study. The antioxidant

effect of ESEt is also verified the hypoglycemic and hypolipidemic effects of same

extract observed in present study. Second thought is that the presences of total phenols

and flavonoids in this extract might be involved in elevating the performance of

antioxidant enzymes and normalizing the status of GSH in hepatocytes like polyphenols

in grapes juice are reported to prevent oxidative stress and improved the performance of

antioxidants enzymes in human volunteers (Hokayem et al., 2013).

108

5. Conclusion

The present study proved that ESEt of C. anthelminticum have weight controlling,

hypoglycemic, antiglycated, hypotriglyceridemic, hypocholesterolemic, antioxidant,

cardio- and hepato-protective effects in fructose-induced type 2 diabetic rabbits by

reducing insulin resistance and enhancing insulin-receptor binding. This protective

character of ESEt might be due to the presence of phytochemicals (total phenols,

flavonoid, etc) and trace minerals in the same extract. Therefore, ESEt of C.

anthelminticum could be used as an alternative and complementary medicine in the

treatment of fructose-induced type 2 diabetes. In addition, this seed extract could be used

as a source of active principle having hypoglycemic activity that can be isolated and used

in the formulation of future antidiabetic medicine.

109

Future Aspect of the Present Research

The present study verified the importance of ethanolic seeds extract of C.

anthelminticum in the management of fructose-induced type 2 diabetes by lowering

insulin resistance and accelerating insulin-receptor binding. The following are the

possible suggestions for future aspect of this research work,

Fractionation of crude ethanolic seeds extract (ESEt) of C. anthelminticum should

be done by using different organic solvents with least to high polar one.

Evaluation of antidiabetic activity should be done in each of the prepared fraction

of crude ESEt of C. anthelminticum which may help in isolating an active

principle involved in antidiabetic activity of this seed extract and that could be

used in the preparation of antidiabetic medicine with negligible side effect.

Evaluation of possible mechanisms of antidiabetic action (suggested in the present

study) of ESEt should be done.

Evaluation of antidiabetic activity of ESEt of C. anthelminticum in type 2 diabetic

patients should be done which help in establishing its use as an alternative and

complementary medicine for the treatment of type 2 diabetes and allow its use

either alone and in combination with commercially available antidiabetic

medicines.

110

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