The National Ribat Universityrepository.ribat.edu.sd/public/uploads/upload/repository/final thesis... · to finish my M.Sc degree. III Abstract The purpose of this study was to investigate

The National Ribat University

Faculty of Graduate Studies and Scientific Research

Hypoglycaemic Effect of Solenostemma argel in

Type II Diabetic patients in

Jaber Abo Aleiz Specialized Center for Diabetes Mellitus

A Thesis Submitted in Fulfillment of Partial Requirement of

Master Degree in Human Nutrition and Dietetics

By:

Hanadi Elyas Elawad Mohammed

Supervisor:

Professor Omer Musa Izzeldin Othman

Co-Supervisor:

Association Professor Khanssa Mohammed Elamin Osman

March, 2014

I

Dedication

It is with my deepest gratitude

and warmest affection that

I would like to dedicate this thesis

to my beloved husband

Mutasim Khidir Abdalla Kanon

and to my cherished children

Mohamed,

Rugiadan,

Rinad

and

Rubeen

II

Acknowledgement

I would like to express my deepest appreciation and thanks to my supervisor

Professor Omer Musa Izzeldin, for encouraging and guiding me to accomplish my

research. Special thanks to my co-supervisor Assn. Professor Khanssa Mohammed

Elamin for her brilliant comments and suggestions. Special gratitude to our master

program coordinator Professor Bahieldin Ibrahim Magboul for giving me

additional knowledge.

In addition, thanks to Jaber Abo Aleiz Specialized Center for Diabetes

Mellitus for giving me permission to organize my experimental study. Thanks to

Prof Mahdi Mohammed for guiding me. Furthermore, I acknowledge with

appreciation Dr. Nagwa Abdulrahman Fatout for her suggestions and help. Also

thanks to the patients who agreed and cooperates to be the subjects of my study.

Thanks to Ms. Salma Bakheet who has been there to assist me in recruiting patients

and gathering data. Special thanks to all of my friends for their support.

My deepest appreciation to Ms. Elena Dariagan who helped me to

coordinate my project especially in writing this research. My acknowledgement to

Ms. Sara Ahmed who extended her help in many ways.

A special thanks to my family. No words can express how grateful I am for

all the support. To my dearest father and mother, your prayers have sustained me

this far and gratitude for taking care of my kids. To my adorable children thank

you so much for the love and inspiration you have given me. Last but not the least,

my sincere appreciation to my beloved husband who has supported me all the way

to finish my M.Sc degree.

III

Abstract

The purpose of this study was to investigate the effects of Solenostemma

argel to reduce blood glucose level in type II diabetic patients in Jabir Abo Aleiz

Specialized Center for Diabetes Mellitus. A three months before-after study was

conducted in 56 uncontrolled type II diabetic patients on oral hypoglycaemic

agent. The patients selection was carried out following inclusion and exclusion

criteria. The patients received (2g) of S.argel once/day in the form of water extract.

Glycated haemoglobin (HbA1c) and fasting blood glucose (FBS) were determined

at the beginning and the end of the study. Blood samples were collected monthly

for measurement of fasting blood glucose levels. The results showed a significant

decrease in HbA1c and FBS levels. The mean level of HbA1c was reduced from

8.602 to 7.45 and mean level of FBS was reduced from 188.13 to 157.67mg/dl

after three months. On the basis of the results of this study, it is concluded that

S.argel has significant antidiabetic activity as it lowers the HbA1c and FBS in type

II Diabetic patients.

IV

خــــــالصة األطـــــزوحة

ذد .الدم خافضة لمستوى سكزنبات الحزجل العشبي كماده فعالية هدفث هذه الدراسة لتقييم

67عه عذد إشرهد .انذراسح ثالثح اشزيذج ,انذراسح تزكز جاتز أت انعز انرخصص نزظ انسكز

.يسرخذي انحثب انخفعح نهسكز ي نذو غيز يرظ سكز ا انثاي يزيط سكز ي انع

جى ف انيو ي انحزجم ف صرج ٢ذال كم يزيط إخريار انزظ ذثعا نعاييز يحذد,ذى

يذ يسر ذرحن نكم يزيط ذى فحص يسر سكز انذو قثم تعذ اراء انذراسحيسرخهص يائ .

عياخ دو شزيا نكم يزيط نرحذيذ سكز انذو صيا تاالظاف نسحةيذ يسر انيغهتي انسكز ذحذ

. يسر سكز انذو صيا

يرسط أظزخ ريجح انذراسح اخفاض يهحظ ف يسرياخ سكز انذو عذ انزظ .حيث اخفط

يرسط اخفط 2.76± 8.56 إن 2.66 ± 9.706ي نذ انزظ تي انسكزيسر انيغه

يهجزاو نكم ديس :70.21 ±268.78 إن 69.86±299.21ي نذ انزظ يسر سكز انذو صيا

تاءا عه ريجح ذ انذراسح خهص ان أ ثاخ انحزجم ن قذر عه خفط يسر سكز انذو. نيرز.

V

List of Contents

No Title

Page

No.

Dedication …………………………………………………. I

Acknowledgement …………………………………………. II

Abstract (English) ………………………………………….. III

Abstract (Arabic) …………………………………………... IV

List of Content ……………………………………………... V-VI

Appendices VII

List of Abbreviations ………………………………………. VIII

List of Table ……………………………………………….. IX

List of Figures ……………………………………………… X

Chapter One

1 Introduction ………………………………………………... 2-3

1.1 Justification ………………………………………………... 3

1.2 Objectives ………………………………………………… 4

1.2.1 General Objective ………………………………………….. 4

1.2.2 Specific Objective …………………………………………. 4

1.3 Hypothesis …………………………………………………. 4

Chapter Two

2 Literature Review 6

2.1 Classification of Diabetes Mellitus ………………………... 6

2.1.1 Type I Diabetes ……………………………………………. 6

2.1.2 Type II Diabetes …………………………………………... 6

2.1.3 Other Specific Types ………………………………………. 7

2.2 Gestational Hyperglycaemic & Diabetes ………………….. 7

VI

2.3 Management of Type II ……………………………………. 7-8

2.4 Anti-diabetic plants ………………………………………... 8-9

2.5 Solenostemma argel ………………………………………... 10

2.5.1 Active ingredients present in S.argel ………………………. 10-12

2.5.2 Effect of water extract of S.argel in the induced-diabetic

rats ………………………………………………………….

12

2.5.3 Traditional medicinal uses ………………………………… 13-14

2.5.4 Toxicity ………………………….………………………… 14-15

Chapter Three

3 Subjects and Methods 17

3.1 Study Design ………………………………………………. 17

3.2 Study Area …………………………………………………. 17

3.3 Study Duration …………………………………………….. 17

3.4 Subjects ……………………………………………………. 17

3.4.1 Sample Size ………………………………………………... 17-18

3.4.2 Criteria 18

3.4.2.1 Inclusion Criteria ………………………………………….. 18

3.4.2.2 Exclusion Criteria ………………………………………….. 18

3.5 Plant Example ……………………………………………... 19

3.6 Procedures …………………………………………………. 19

3.7 Statistical Analysis ………………………………………… 19

3.8 Ethical Clearance for research ...…………………………… 20

Chapter Four

4 Results ……………………………………………………... 22-35

Chapter Five

5 Discussion …………………………………………………. 37-39

Chapter Six

VII

6.1 Conclusion ………………………………………………… 41

6.2 Recommendation ………………………………………….. 41

References …………………………………………………. 43-52

Appendices

Appendix A Questionnaire ………………………………………………. 54-55

Appendix B Concept and Permission forms …………………………….. 56-59

Appendix C Figures and Tables …….……..…………………………... 60-66

Appendix D Images of Solenostemma argel …………………………….. 67

VIII

LIST OF ABBREVIATIONS

ADA - American Diabetes Association

ALP - Alkaline phosphate enzyme

AST - Aspartate aminotransferase

BMI - Body Mass Index

dl - decilitre

FBS - Fasting blood sugar

g - gram

GIT - Gastro Intestinal Tract

GOT - Glutamic-oxalacetic transaminase

GPT - Glutamic pyruvate transaminase

HbA1c - Glycated Haemoglobin

IDDM - Insulin dependent diabetes mellitus

kg - kilogram

mg - milligram

Mo. - Month

Mos. - Months

NIDDM - Non-insulin dependent diabetes mellitus

No.(s) - Number(s)

P Value - Probable Value

S.argel - Solenostemma argel

SD - Standard deviation

WHO - World Health Organization

β-cell - Beta-cell

IX

LIST OF TABLE

Table 1

The main characteristics of study population …… 23-24

Table 2 Effects of duration of disease on HbA1c and FBS

levels before and after treatment of S.argel………. 29

Table 3 Effect of number of drugs on HbA1c and FBS

levels before and after treatment of S. argel……… 30

Table 4 Effect of gender on HbA1c and FBS levels before

and after treatment of S. argel……………………. 32

Table 5 Effect of age group on HbA1c and FBS levels

before and after treatment of S. argel…………….. 33

Table 6 Effect of occupation on HbA1c and FBS levels

before and after treatment of S. argel………….…. 34

Table 7 Effect of education on HbA1c and FBS levels

before and after treatment of S. argel…………..… 35

Table 8 Effect of 3 months treatment with S.argel on

HbA1c levels of study group………………….…. 60

Table 9 Effect of 3 months treatment with S.argel on FBS

levels of study group…………………………….... 60

Table 10 Effect of BMI cut off points on HbA1c and FBS

levels before and after treatment of S.argel…….… 61

Table 11 Effect of comorbid conditions on HbA1c and FBS

levels before and after treatment of S.argel……….. 62

X

LIST OF FIGURES

Figure 1 Effect of 3 months treatment with S.argel on

HbA1c levels of study group…………………. 25

Figure 2 Effect of 3 months treatment with S.argel on FBS

levels of study group………………………….. 26

Figure 3 Effect of BMI cut off points on HbA1c levels

before and after treatment of S.argel…………. 28

Figure 4 Effect of comorbid condition on HbA1c levels

before and after treatment of S.argel…………. 31

Figure 5 The main characteristics of study population.. 63

Figure 5-1 Gender ………………………………………... 63

Figure 5-2 Age/Year ……………………………………… 63

Figure 5-3 Educational Level ……………………………. 64

Figure 5-4 Occupation …………………………………… 64

Figure 5-5 Duration of Disease ………………………….. 65

Figure 5-6 Number of Drugs …………………………….. 65

Figure 5-7 Comorbid Condition …………………………. 66

Figure 5-8 BMI cut off points ……………………………. 66

1

Chapter One

Introduction

2

1. Introduction

The term "diabetes mellitus" describes a metabolic disorder of multiple

etiology characterized by chronic hyperglycaemia with disturbances of

carbohydrate, fat and protein metabolism resulting from defects in insulin

secretion, insulin action, or both. The effects of diabetes mellitus include long-term

damage, dysfunction and failure of various organs (WHO, 1999).

The world prevalence of diabetes among adults (aged 20-79 years) will be

affecting 285 million adults in 2010 (6.4%), and will increase to 439 million adults

by 2030 (7.7%). Between 2010 and 2030, there will be a 69% increase in numbers

of adults with diabetes in developing countries and a 20% increase in developed

countries (Shaw et al., 2010).

More than 400 traditional plant treatments for diabetes mellitus have been

recorded, but only a small number of these have received scientific and medical

evaluation to assess their efficacy. Traditional treatments have mostly disappeared

in occidental societies, but some are prescribed by practitioners of alternative

medicine or taken by patients as supplements to conventional therapy. However,

plant remedies are the mainstay of treatment in under developed regions. A

hypoglycaemic action from some treatments has been confirmed in animal models

and non-insulin-dependent diabetic patients and various hypoglycaemic

compounds have been identified. A botanical substitute for insulin seems unlikely,

but traditional treatments may provide valuable clues for the (Bailey and Day,

1989).

3

Renewed attention to alternative medicines and natural therapies has

stimulated a new wave of research interest in traditional practices and the World

Health Organization expert committee on diabetes has listed as one of its

recommendations that traditional methods of treatment for diabetes should be

further investigated (Watt and Wood, 1988; WHO, 1980).

Solenostemma argel (Del) Hayne is known locally in Sudan as „hargal‟, and

belongs to the family Asclepiadaceae. Other members of the family include S.

Oleifolium (Nectoux) Bullocket Bruce and S. Triste (Nees) K. Muelli. It is an erect

shrub reaching a height of 60-100cm, with many velvety, pubescent branches from

the base. It is distributed in Saudi Arabia, Egypt, Libya, Chad and Palestine. In

Sudan, it is indigenous in the northern regions between Barbar and Abu Hamad

(ElKamali, 1991). Sudan is regarded now as the richest source of this plant

(Organgi, 1982; El-Ghazali, 1997 and Ahmed, 2003). S.argel leaves were at one

time, used to adulterate Khartoum Senna (Trease and Evans, 1989).

1.1 Justification:

A larger number of studies indicate a growing burden of diabetes,

particularly in developing countries. The traditional anti diabetic plants might

provide a useful source of new oral hypoglycaemic compounds for development as

pharmaceutical entities, compare to allopathic medicine which can cure a wide

range of diseases, but its high prices and occasional side-effects are causing many

people to return to herbal medicines.

4

1.2 Objectives

1.2.1 General objective:

To assess the Solenostemma argel effect to reduce blood glucose level in

type-2 diabetic patients.

1.2.2 Specific objective:

To evaluate the appropriate dose of S.argel.

To determine the side effect of S.argel.

To determine effect of comorbid condition HbA1c and FBS levels

before and after treatment of S.argel.

To determine effect of BMI cut off points on HbA1c and FBS levels

before and after treatment of S.argel.

1.3 Hypothesis:

S.argel acts as a hypoglycaemic medicinal plant that reduces blood glucose

level in type II diabetic patients. That based on experimental work which was

carried on albino rats affect blood glucose level.

5

Chapter Two

Literature

Review

6

2. Literature Review

2.1 Classification of Diabetes Mellitus:

2.1.1 Type 1 diabetes

(β-cell destruction usually leading to absolute insulin deficiency) Immune-

mediated diabetes. This form of diabetes, which accounts for only 5-10% of those

with diabetes previously encompassed by the terms insulin dependent diabetes

(IDDM), type I diabetes or juvenile-onset diabetes results from a cellular-mediated

autoimmune destruction of the β-cells of the pancreas (ADA, 2008). In which

“insulin is required for survival” to prevent the development of ketoacidosis, coma

and death (WHO, 1999). Again (Willis et al., 1996) stated that individual with this

form of type I diabetes often become dependent on insulin for survival eventually

and are at risk for ketoacidosis.

2.1.2 Type 2 diabetes

(Ranging from predominantly insulin resistance with relative insulin

deficiency to predominantly an insulin secretory defect with insulin resistance).

This form of diabetes which accounts for 90-95% of those with diabetes (ADA,

2008). Previously encompassed by the terms non-insulin dependent diabetes

(NIDDM), type II diabetes or adult-onset diabetes referred to individuals who have

insulin resistance and usually have relative (rather than absolute) insulin

deficiency. At least initially and often throughout their lifetime, these individuals

require insulin for control, i.e. metabolic control rather than for survival (WHO,

1999).

7

2.1.3 Other specific types:

Other specific types are currently less common causes of diabetes mellitus,

but are those in which the underlying defect or disease process can be identified in

a relatively specific manner. They include for example, fibrocalculous

pancreatopathy a form of diabetes which was formerly classified as one type of

malnutrition–related diabetes mellitus (WHO, 1999).

2.2 Gestational hyperglycaemia and diabetes

Gestational diabetes is carbohydrate intolerance resulting in hyperglycaemia

of variable severity with onset or first recognition during pregnancy. It does not

exclude the possibility that the glucose intolerance may antedate pregnancy but has

been previously unrecognized. The definition applies irrespective of whether or not

insulin is used for treatment or the condition persists after pregnancy (WHO,

1999).

2.3 Management of type II

Dietary control of diabetes is fundamental to the management and treatment

of NIDDM. In the last few decades, a number of studies have indicated the value

of plant fibre or complex carbohydrates including highly viscous soluble fibre such

as guar gum and B-glucan, for control of blood glucose concentrations (Edwards et

al., 1988; Groop et al., 1993; Japan and Pitts, 1985 and Jenkins et al., 1978).

(Clark, 1998) stated that while external insulin is necessary for control of

type I diabetes mellitus, the use of drug therapy in type 2 diabetes is initiated only

after dietary and lifestyle modifications. Similarly, (Zhang and Moller, 2000)

reported that the available therapies for diabetes include insulin and oral

8

antidiabetic agents such as sulfonylureas, biguanides and α-glycosidase inhibitors.

Many of these oral antidiabetic agents have a number of serious adverse effects.

In addition (Cristina et al., 2012) stated that type II diabetes is the most

commonly encountered type of diabetes. Current antidiabetic therapy is based on

synthetic drugs that very often have side effects. For this reason there is a

continuous need to develop new and better pharmaceuticals as alternatives for the

management and treatment of the disease. Natural hypoglycaemic compounds may

be attractive alternatives to synthetic drugs or reinforcements to currently used

treatments. Their huge advantage is that they can be ingested in everyday diet.

Recently, more attention is being paid to the study of natural products as potential

antidiabetics. Also (Bnouham et al., 2006) stated that uncontrolled diabetes leads

to many chronic complications such as blindness, heart failure and renal failure. In

order to prevent this alarming health problem the development of research into

new hypoglycaemic and potentially antidiabetic agents is of great interest.

2.4 Anti-diabetic plants:

In the last years there has been an increasing demand for natural products

with antidiabetic activity mainly due to the side effects associated with the use of

insulin and oral hypoglycaemic agents (Cunha et al., 2008). Moreover bioactive

drugs isolated from plants having hypoglycaemic effects showed antidiabetic

activity equal and sometimes even more potent than known oral hypoglycaemic

agents used in clinical therapy. (Bnouham et al., 2006)

Gallegaofficinalis is now well established that its hypoglycaemic and

insulin-sensitizing potential is associated with its guanide compound, galegine.

This plant is still of great importance today despite the fact that the guanide

9

compounds were discovered to be toxic for the human body. Related compounds

such as the biguanide metformin molecule were later developed and are still

widely used in antidiabetic therapy (Goldstein and Wieland, 2008).

The mode of action of the extracts from these plants is uncertain however,

many antidiabetic plants act at least in part through their fibre, vitamin or mineral

contents and some secondary metabolites (Day, 1998).

A large number of hypoglycaemic compounds have antioxidant properties.

(Goldstein and Wieland, 2008). While polyphenolic compounds especially

flavonoids are among the classes of compounds that have received the most

attention (Soumyanath, 2006) with regard to their antidiabetic properties.

Flavonoids are natural polyphenolic molecules of plant origin known for their

antioxidant, anti-inflammatory and anti-carcinogenic properties and dietary intake

of flavonoids might prove to be important for alternative diabetes treatments or

reduction of the risk of the disease (Pinent et al., 2008). Unfortunately, many of

these compounds are alkaloids, flavonoids and glycosides which do not lend

themselves readily to pharmaceutical development (Day, 1995).

In addition (Edwards et al., 1988) claimed that inclusion of viscous

polysaccharides in the diet decreased postprandial blood glucose concentrations in

subjects with type II diabetes.

10

2.5 Solenostemma argel:

2.5.1 Active ingredients present in S.argel

Solenostemma argel, belongs to the Asclepiadaceae family. This family

includes many wild growing medicinal plants (e.g. Calotropisprocera, S.argel,

Leptadineaspp) (El Tigani and Ahmed, 2009) similarly, (Ahmed, 2003) claims

that S.argel is considered to be medicinally important in the Sudan, Libya and

Chad. Also, S.argel is a plant or plant part of valued for its medicinal, aromatic or

savory qualities. Herb plants produce and contain a variety of chemical substances

that act upon the body (Shayoub, 2003).

In addition, it was found that tissue cultures have produced compounds

previously undescribed and cultures of higher plant cells may provide an important

source of new economically important compounds (Butcher, 1977; Constabel and

Tyler, 1994). Moreover, chemical investigations, chromatographic screening and

phytochemical as well as tissue culture studies of S.argel leaves, stems and flowers

revealed the presence of numerous biochemical ingredients such as pyrgene

glycosides, flavonoids, kaempferol, quercetin, rutin, flavonols, flavanones,

chalcones and alkaloids. (Eltigani and Ahmed, 2009; Shafek and Michael, 2012

and Plaza A et al., 2005). In their report on S.argel, (Khalid et al., 1974) showed

the presence of kaempferol and steroidal glycosides in leaves of hargel also they

found that the flavanoids can be detected at (_290-368 nm).

Again (Mohamed et al., 2012) argued that the solenostemma argel contain

flavonoids, kaempferol, quercetin, rutin, flavonols, flavanones, chalcones and

alkaloids in S.argel. Also they contain pregnane ester glycosides in S.argel

extracts. S.argel was found to include some flavanoids saponins alkaloids (Khalid

et al., 1974). Moreover there are 2000 flavonoid found in S.argel found in as

11

methoxil or hydroxile group, further studies were needed to investigate this

flavonoid. S.argel can be used medically in kidney disease, liver, respiratory

system. Leaves of S.argel can be used an anti-inflammatory, antiseptic,

vasodilatory and hypotensive (Koca et al., 2011).

Phytochemical studies of the leaves, stems and flowers revealed the presence

of -amyrin and -sitosterol, 7-methoxy-3-22-dihydroxy-stigmastene, ethoxy

derivative of vangurolic acid, an unidentified sterol. Moreover, they detected the

presence of flavonoids and saponins in the different organs and alkaloids and/or

nitrogenous bases in the leaves, stems and flowers (Khalid et al., 1974). Kamel

(2003) proved that it contained acylated phenolic glycosides.

While (Mahran and Saber, 1964), (Mahran et al., 1976) isolated -amyrin, -

sitosterol-containing rutin and quercetin from S.argel. In addition, ElTohami, MS

(1996) claimed that S.argel. Solenostemma argel contains an acidic resin,

glycoside, choline, phytosterols and amyrins.

Many previous studies have reported the presence of monoterpenes,

pregnane glycosides and acylated phenolic glycosides in the leaves. In addition,

there is an occurrence of four new pregnane glycosides from the pericarps of

S.argel (Plaza et al., 2003). Also Kamel (2003) proved that it contained acylated

phenolic glycosides. Another study in its chloroform extract showed that it had an

anti-inflammatory activity and it contained a new pregnene glycoside (solenoside

A) and a known one besides kaempferol 3-O-glucoside and 3-O-rutinoside

(Innocenti et al., 2005).

12

From the previous phytochemical studies, it was found that its leaves

characterized by high carbohydrates (64.8%) and low crude fiber (6.5%), 15 %

protein, 1.6% crude oil, 7.7% ash, and 4.4% moisture content. It contained high

potassium (0.54%), calcium (0.06%), magnesium (0.03%) and sodium (0.01%),

but it characterized by low cupper (0.0001%), ferrous (0.002%), manganese

(0.002%) and lead (0.001%). The protein fractionation of leaf characterized by

high Albumins (16.7%), Non-Nitrogenous Protein (15.3%), Prolamine (11.7) and

low Globulins (8.7 %) and Glutulin (6.2%). Leaf contained phytic acid (3.2 g/100g

and tannin content (0.4%) (Murwan et al., 2010).

While (Plaza, et al., 2003) reported that the protein, sugar, fiber and vitamins

are present with minerals Na+, K+, Ca+2, Mg+2, Ni+3, P+3.

On the other hand, the presence of biologically active components such as

phytates and phenolic compounds are found to have adverse effects on intrinsic

properties of protein (Khalid et al., 1974 and Yagoub, 2003). Similarly, Phytic acid

represents a complex class of naturally occurring organic form of phosphorus

compounds that can significantly influence the functional and nutritional properties

of foods (Goldstein and Swain 1963).

2.5.2 Effects of water extract of S.argel on the induced-diabetic rats

Albino rats were fasted for 12 hours, then they were given water extract

225mg/kg the reduced glucose was measured at initial time 0hr. then after 2hrs.

and then after 4hrs., the best results were obtained after 4hrs where the reduce

glucose was 82.40mg/dl while, the standard Dawnil was 83.28mg/dl the control

was 93.40 (Izzeldin et al.,2012).

13

2.5.3 Traditional medicinal uses of S.argel

Some uses Solenostemma argel in folkloric medicine as treatment of GIT

(Gastro Intestinal Tract) disturbances, hypercholesterolemia and diabetes mellitus;

and externally in poultice form as anti-inflammatory and anti-rheumatic and

inhalation of its smoke for the treatment of measles and cold. The stem is generally

used as antispasmodic and to treat cough. Moreover, „Hargal‟ infusion is used to

treat jaundice, urinary tract infection and the disturbance of the menstrual cycle

(ElKamali and Khalid, 1996).

It is also used to cure stomach ache, anti-colic, remedy for suppurating

wounds and anti-syphilitic when used for prolonged period of 40 to 80 days

(Boulos, 1983; Hammiche and Maiza, 2006). Also anti-inflammatory and anti-

rheumatic agent (Shayoub et al., 2013). Again leaves are used as an antispasmodic,

carminative and as an anti-diabetic (Kamel et al., 2000 and Hassan et al., 2001).

In addition, it is used in indigenous medicine as an effective remedy for

cough. The infusion of its leaves is used for gastro-intestinal cramps and infections

of the urinary tract (ElTohami, 1996). It is an effective remedy for bronchitis and

is used to treat neuralgia and sciatica (Tharib et al., 1986).

While (Mudawi, 2003) reported that the chloroform extract (600 – 800)

mg/kg induced a delayed and gradual decrease in amplitude of the spontaneous

contractions of pregnant or non-pregnant uterus. In a similar (ElTahir et al., 1987)

studied the pharmacological activities of S.argel, including spasmolytic and uterine

relaxant activities.

14

(Plaza et al., 2005) found that pregnane glycosides isolated from this plant

were reported to reduce cell proliferation. Also the plant has antimicrobial activity

(Mohamed et al., 2012). Again Ross and their co-workers (1980) illustrated the

presence of antibiotic substances in the ethanol extracts of Hargel plant. Similarly

it was reported to have antimicrobial properties as well as antibacterial and

antioxidant activity (Shafek and Michael, 2012; Mahalel, 2012).

Moreover many studies confirmed that the S.argel had remedial effect

against numerous diseases and health problems such as diabetes mellitus (Trojan,

et al., 2012) and cancer (Amr, et al., 2009; Hanafi and Mansour, 2010).

2.5.4 Toxicity

From (Osman et al., 2014) argued that S.argel had incurred hepatorenal

toxicity in the experimental animals. Also in a feeding test with chicken a diet

containing 10 leaves of solemenstomma argel caused a depression in growth and

hepatotoxicity (EL-sanusi and Adam, 2007).

In addition (Osman et al., 2014) finding that human use of S.argel, it could

be of significance to propose for those seeking S.argel for treatment, to use the

plant with the dose far below 600 mg/kg and to monitor closely the levels of

creatinine, urea, alkaline phosphatase (ALP) and aspartate aminotransferase (AST)

during the course of treatment.

On the other hand, (Shayoub et al., 2013) finding that the different types

(leaves, extracts or alkaloids) of Solenostemma argel tablets showed a very good

therapeutic effectiveness (71%-100%) and a great margin of safety (98%-100%).

No side effects or adverse reactions were recorded and the patients did not

15

complain of any undesirable or intolerable toxic or adverse effects of these

preparations of Solenostemma argel.

16

Chapter Three

Subjects and

Methods

17

3. Subjects and Methods

The methodology of this study is a modification of two studies that had used

of antidiabetic plants on human (Huseini et al., 2006; Bunyapraphatsara et

al.,1996).

3.1 Study design:

Before - after case study.

3.2 Study area:

Jabir Abo Aleiz Specialized Center for Diabetes Mellitus in Khartoum,

Sudan

3.3 Study duration:

From April to August, 2014

3.4 Subjects:

3.4.1. Sample size

Prevalence of antidiabetic plants is 50%.

n = z 2. p q / d

2

n = Sample size

z = Standard normal deviate = 1.96

p = Proportion of the characteristic under study estimated in the target

population

q = 1- p

d = Error allowed = 0.05

18

= = = 384

Due to limited time and budget a total of 46 patients with type 2 diabetes

enrolled in this study.

The patients had been selected from cases of type II diabetes mellitus at the

Jabir Abo Aleiz Centre, who are treated with oral hypoglycaemic agent using

the following inclusion and exclusion criteria.

3.4.2 Criteria

3.4.2.1 Inclusion criteria

Uncontrolled type-II diabetic patients.

HbA1c above 6.5.

Aged 40-70 years.

Freely consented to participate in the study.

3.4.2.2 Exclusion criteria

Liver disease patients.

One of the functions of liver is alkaline phosphates enzyme which

affect carbohydrate metabolism, therefore all liver disease patients

were not included.

Kidney disease.

Disfunctioning of kidney affected cholesterol level which reflected on

blood glucose level.

19

3.5 Plant Sample:

S. argel powdered leaves packed in tea bags (1g each) were obtained from

the local market. The dose calculation based on dose translation from animal

to human (Reagan-Shaw et al., 2007).

3.6 Procedures:

The study group received 2g of S.argel once/day in the form of water

extract (pour 100ml of boiling water over two bags) in the morning in

combination with oral hypoglycaemic agent for three months.

Blood samples withdrawn monthly for measurement of fasting blood

glucose levels.

HbA1c was measured two times, the first one at the beginning of the

study and the second one by the end of three months.

Every patient was well educated about how to use the S.argel tea

bags, besides receiving an advice about importance of using S.argel.

The patients follow ups was done in two ways:

Telephone call weekly.

Each patient was given a form to tick on the days which he/she use

the tea bags. This will confirm the follow up procedures.

3.7 Statistical analysis:

Data was recorded and analyzed using SPSS computer program.

Appropriate statistical methods were employed.

20

3.8 Ethical Clearance for Research:

Ethic Committee of Scientific Research of Ribat University gave the

permission and had been accepted by the administration of Jabir Abo Aleiz

Specialized Center for Diabetes Mellitus.

The researcher considers ethical clearance at both the application and

implementation stages.

21

Chapter Four

Results

22

4. Results

A total of 46 patients had been enrolled in this study. Table 1 shows the

main characteristics of the study population. Females were more than males, 31

compared to 15, a percentage of 67.4% and 32.6% respectively. The age group of

40-50 years old represents 43.5% (20 patients), which is the highest values;

followed by 51-60 years 41.3% (19 patients). The group of 61-70 years old

represents the lowest value 15.25% (7 patients). In case of education levels

secondary group was 30.4% (14 patients), followed by primary and high secondary

23.9% (11 patients). The illiterate group ranked last 21.70% (10 patients).

Unemployed represents 54.3% (25 patients) while the employed was 45.7% (21

patients).

The patients who have <10 years duration of disease correspond to 71.1%

(33 patients) the highest value, followed by 23.9% (11 patients) for 10-20 years

duration of disease and 4.3% (2 patients) for >20 years duration of disease (the

lowest percent). Monotherapy (received one hypoglycaemic agent) represents

lower than multiple therapy (received more than hypoglycaemic agent) 26.1% (12

patients) compared to 73.9% (34 patients).

Patients with diabetes only represents 58.7% (27 patients) compared to

41.3% (19 patients) for comorbid conditions.

For BMI cut off points, the overweight patients has the highest value of

43.5% (20 patients) followed by Normal BMI of 23.9% (11 patients). Obesity

grade 1 was 21.7% (10 patients), followed by obesity grade II 6.5% (3 patients)

23

and 2.2% (1 patient) for both underweight & morbid obesity.

Table 1 - The Main Characteristic of Study Population.

Patients No.(s) %

Gender:

Male

Female

15

31

32.6

67.4

Age/years:

40-50

51-60

61-70

20

19

7

43.5

41.3

15.2

Education Level

Illiterate

Primary

Secondary

High Secondary

10

11

14

11

21.7

23.9

30.4

23.9

Occupation

Employed

Unemployed

21

25

45.7

54.3

Duration of Disease:

<10

10-20

>20

33

11

2

71.7

23.9

4.3

Number of Drugs

*Mono Therapy

** Multiple Therapy

12

34

26.1

73.9

24

„cont.

***Other Diseases

Yes

No

19

27

41.3

58.7

****BMI cut off points:

Underweight (<18.5)

Normal (18.5-24.9)

Overweight (25-29.9)

Obesity grade I (30-34.5)

Obesity grade II (35-39.9)

Morbid Obesity >40

1

11

20

10

3

1

2.2

23.9

43.5

21.7

6.5

2.2

*Hypoglycaemic Agent one drug.

**Multiple drug hypoglycaemic agent

***Hypertension, Asthma, Hyper cholestramia, Others

****Cut off points of the BMI (Mahan and Escott-Stump, 2008)

25

After treatment of S.argel for three months there is statistically significant

difference in the main parameters, the mean level of HbA1c = 8.602± 1.52 and

after the treatment it was reduced to 7.45±1.62; the P value was (.000) Figure 1.

Figure 1 – Effect of 3 months treatment with S.argel on HbA1c levels of study

group.

26

Observed FBS (Figure 2) after one and three months (FBS2 & FBS4) from

the beginning of treatment: there is statistically significant difference (P value

<0.05) which is positive correlation to the HbA1c.

Before treatment the mean level of FBS was 188.13mg/dl and after three

months it was reduced to 157.67 mg/dl. After one month it was reduced to 163.48

mg/dl, after two months it was reduced to 165.89mg/dl but statistically

insignificant result was obtained (P value>0.05).

Figure 2 - Effect of 3 months treatment with S.argel on FBS levels of study

group.

27

28

Figure (3) shows that after treatment of S.argel for three months there is a

significant difference within the BMI cut off points, (P value < .05) the P value =

.008. The more response happened in obesity grade II they reduced HbA1c from

8.1 to 5.96, followed by overweight from 8.52 to 7.07, next obesity grade I from

8.64 to 7.67, lastly normal BMI group from 8.93 to 8.54. (The normal BMI

decrease of HbA1c was .39, the overweight 1.45, obesity grade I .97 and obesity

grade II 2.14). In FBS no significant difference within BMI cut off points.

Figure 3 - Effect of BMI points on HbA1c levels before and after treatment of

S.argel.

29

No significant difference within the duration of disease group concerning HbA1c before and after treatment

of S.argel (table 2). The same result regarding FBS excluding before treatment there was significant difference (P

value .004).

Table 2 - Effect of the duration of disease on HbA1c and FBS levels before and after treatment of S.argel.

Duration of the

Disease

Patients No.(s)

HbA1c 1st

HbA1c 2nd

FBS 1 FBS2 FBS3 FBS4

<10 33 8.49±1.16 7.33±1.63 189.64±55.28 156.88±33.65 157.97±65.38

151.21±46.51

10-20 11 8.77±2.28 7.51±1.47 162.18±46.34 176.9±49.85 186.18±53.18

172.36±90.59

>20 2 9.4±2.55 9.15 ±2.19 306±28.28 198.5±30.41 185.±41.01

183±79.09

Over All 46 8.6 7.45 188.13 163.48 165.89 157.67

P Value .664 .307 .004 .143 .397 .505

Values are given as mean ±SD from the number of patients in each group.

30

Table (3) shows no significance difference between monotherapy and multiple therapy regarding HbA1c

before and after treatment of S.argel. Looking into the first two months (FBS1 & FBS2) after treatment of

S.argel, there was significant difference (P value .015, .014) respectively. The highest response was in patients who

received only one hypo glycaemic agent (mono therapy), the FBS2 & FBS3 were decreased by (35.7, 48.3). While

the multiple therapy FBS2 & FBS3 decreased by (20.8 and 13.4). In the last month (FBS3) there was no

significant difference (P value =.721).

Table 3 - Effect of the number of drugs on HbA1c and FBS levels before and after treatment of S.argel.

Type of

Medication

Patients

No.(s)

HbA1c

1st

HbA1c

2nd FBS 1 FBS2 FBS3 FBS4

Mono Therapy

12 7.89±1.07 6.91±1.13 176±55.93 140.33±21.01 128.7±31.43 152.25±34.198

Multiple Therapy

34 8.85±1.59 7.64±1.73 192.41±60.15 171.65±40.57 179±65.32 159.59±67.29

Over All 46

P Value .059 .181 .412 .015 .014 .721

Values are given as mean ±SD from the number of patients in each group.

31

In case of patients with diabetes only and comorbid conditions figure (4),

regard with HbA1c there was a significance difference before and after treatment

of S.argel (p value < .05). The patients with diabetes only, were more controlled

than comorbid conditions before and after treatment. The decrease of HbA1c (by

1.15) was equal in patients with diabetes only compare to patients with comorbid

conditions. View the FSB there was no significance difference after three month

duration of treatment of S.argel.

Figure 4 - Effect of comorbid conditions on HbA1c levels before and after

treatment of S.argel.

32

Table (4) shows FBS. For HbA1c no significant difference after treatment only before there was significance

difference (P value <.05), males were more controlled than females (7.95 for males before and after treatment no

significant difference between males and females regarding compare to 8.92 for females).

Table 4 - Effect of the Gender on HbA1c and FBS levels before and after treatment of S.argel.

Gender Patients

No.(s)

HbA1c 1st

HbA1c 2nd

FBS 1 FBS2 FBS3 FBS4

Male 15 7.95±1.00 6.83±1.14 182.33±53.15 152.07±46.18 155.13±55.93 137.07±40.10

Female 31 8.92±1.64 7.75±1.74 190.94±61.92 169.0±34.21 171.10±65.24 167.65±66.02

Over All

46

P Value .043 .069 .647 .168 .421 .107

Values are given as mean ±SD from the number of patients in each group.

33

Table 5, 6 and 7 tells us that there was no significance difference (P value >.05) within the group of ages,

occupation and education levels consider HbA1c and FBS.

Table 5 - Effect of age group on HbA1c and FSB levels before and after treatment of S.argel.

Age/

Years

Patients

No.(s)

HbA1c

1st

HbA1c

2nd FBS 1 FBS2 FBS3 FBS4

40-50 20 8.54 ±1.23 7.45 ±1.92 187 ±50.05 173.65±32.57 172.15±71.461 180.55±60.18

51-60 19 8.97 ±1.86 7.52 ±1.48 195.95±67.65 154.42±38.57 163.47±62.55 147.42 ±58.89

61-70 7 7.77 ±1.01 7.13 ±1.16 170.14±60.41 159±53.12 154.57±28.39 120.14 ±39.12

Over

All 46 8.6 7.5 188.13 163.48 165.89 157.67

P Value .199 .855 .617 .293 .801 .042

Values are given as mean ±SD from the number of patients in each group.

34

Table 6 - Effect of the Occupation on HbA1c and FBS Levels before and after treatment of S.argel.

Occupation Patients

No.(s)

HbA1c

1st

HbA1c

2nd FBS 1 FBS2 FBS3 FBS4

Employed 21 8.69

±1.77

7.46

±1.73 191.14 ±59.37 157.81±38.46 168.33±63.17 151.86±60.11

Unemployed 25 8.52

±1.31 7.44 ±156 185.60±59.32 168.24±39.29 163.84±62 60 162.56±60.95

Over All 46

P Value .708 .979 .754 .370 .810 .554

Values are given as mean ±SD from the number of patients in each group.

35

Table 7 - Effect of Education level on HbA1c and FBS levels before and after treatment of S.argel.

Education

Level

Patients

No.(s) HbA1c

1st

HbA1c

2nd FBS 1 FBS2 FBS3 FBS4

Illiterate 10 8.13 ±8.7 7.17±1.64 170.8 ±64.44 161.2 ±46.63 173.7 ±76.26 149.40±78.30

Primary 11 9.3 ±2.25 7.37±1.80 175.27±71.55 156 ±42.74 168.09±60.19 145.55±64.12

Secondary 14 8.59 ±1.26 7.52 ±1.87 208.5 ±53.80 170.07±38.86 174.50±66.49 166.93±52.35

High Level 11 8.35 ±1.33 7.69 ±1.21 190.82±42.9 164.64±30.20 145.64±46.69 165.55±51.61

Over All 46 8.6 7.45 188.13 163.48 165.89 157.67

P Value .315 .905 .386 .846 .672 .775

Values are given as mean ±SD from the number of patients in each group.

36

Chapter Five

Discussion

37

5. Discussion

The present study investigated the effect of S.argel on the glucose profile in

patients with type II diabetes. The results show that statistically significant

differences in the main parameters between before treatment of S.argel the HbA1c

= 8.602± 1.52 and after the treatment of S.argel (duration of three months) the

HbA1c was reduced to 7.45±1.62; the P value was (.000).

There are not too many studies that have investigated the anti-diabetic

activity of S.argel, except for one study: Effects of water extract of S. argel. on

the induced-diabetic rats (Izzeldin et al., 2012). The result found in the present

study is in agreement with the finding from this study.

The mechanism underlying the glucose lowering effect of S.argel is not

clear. S.argel contains a wide number of active constituents including flavonoids

(Eltigani and Ahmed, 2009; Shafek and Michael, 2012; Plaza A et al., 2005;

Khalid et al. 1974; Mohamed et al., 2012 and Koca et al., 2011). Flavonoids are

natural polyphenolic molecules of plant origin known for their antioxidant, anti-

inflammatory and anti-carcinogenic properties and dietary intake of flavonoids

might prove to be important for alternative diabetes treatments or reduction of the

risk of the disease (Soumyanath, 2006; Pinent et al., 2008; Day, 1995). S.argel has

antioxidant properties (Shafek and Michael, 2012; Mahalel, 2012) and a large

number of hypoglycaemic compounds have antioxidant properties (Goldstein and

Wieland, 2008). Many antidiabetic plants act at least in part through their fiber,

vitamin or mineral contents (Day, 1998) and S.argel contain protein, sugar, fiber

and vitamins with minerals Na+, K+, Ca+2, Mg+2, Ni+3, P+3 (Plaza, et al. 2003

and Murwan et al., 2010) .

38

After treatment of S.argel for three months there is significant difference

within the BMI cut off points. The normal BMI decrease of HbA1c was .39, the

overweight 1.45, obesity grade I .97, obesity grade II 2.14, that means with

exceptional of obesity grade I, we found that the more response associated with

increasing of obesity. This mean there are good effect in this group may be due to

decrease weight; so the good response to their treatment plus the effect of S.argel.

Regarding the effect of number of medications we found that before and

after treatment of S.argel no significant difference in HbA1c and FBS4 between

mono therapy and multiple therapy, but on other hand there was significant

difference after treatment of S.argel in FBS2 and FBS3. The highest response was

in patients who received only one hypoglycaemic agent (mono therapy) because

their diabetes is not so bad from the beginning.

Duration of disease (>10, 10-20, <20) years there were no significant

difference before and after treatment of S.argel. We expect that group III (<20

years) has less response. This group must be more resistant to the plants extract

because diabetic patients must have had more complication in the long duration

of disease.

The age, educational level, occupation and gender table show no significant

differences regarding HbA1c and fasting blood glucose level before and after

treatment of S.argel with exception of gender in which there was significant

difference before treatment. We expect this result.

Usually hypertension and cardiovascular are the most common diseases

associated with diabetes in addition to others like asthma, intestinal diseases and

39

stomach diseases. Significant difference occurred in HbA1c before and after

treatment of S.argel. In comorbid conditions the decrease of HbA1c was equal to

patients with diabetes only. This contradicts our exception that more response will

occur in patients with diabetes only because the comorbid conditions have more

complications hence, the response to treatment of plant extract might not give good

result. This result is a good support to the claim that S.argel can remedy widely

diseases (ElKamali and Khalid, 1996; Boulos, 1983; he and Hammic Maiza, 2006;

Hassan et al., 2001; ElTohami, 1996 and Tharib et al., 1986).

40

Chapter 6

Conclusion

and

Recommendation

41

6.1 Conclusion

In this study, evidence is presented that the Solenostemma argel, a

traditional medicinal plant, can reduce blood glucose level in type II diabetic

patients. This significant positive result is due to active ingredient components

present in S.argel.Thus, the traditional medicine use of S.argel for the control of

diabetes may be supported by this study, which is the same result approved on

induced-diabetic rats.

Currently, there is a dramatically worldwide increase in the number of

people suffering from diabetes, particularly in developing countries. Beside high

prices and occasional side-effects of hypoglycaemic agents are causing many

people to return to herbal medicines.

6.2 Recommendation

More research must be carried on S.argel to find the active ingredients

which reduce blood glucose in diabetes type II.

Liver functions like (ALP) alkaline phosphate enzyme, (GPT)

glutamic pyruvate transaminase, (GOT) glutamic-oxalacetic

transaminase, Billirubin and kidney functions like (ammonia, urea,

creatinine, total protein), these tests must carried out to show there is

no any toxic effect for human body in case of treatment of diabetes

patient with active ingredient of S.argel.

Pharmaceutical and chemical formulation of the accurate doze of

S.argel active ingredient must be formulated and manufactured to give

it as prescription for these patients.

42

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Appendices

54

Appendix (A)

Questionnaire

Hypoglycaemic effect of Solenostemma argel in Type II diabetic patients in

Jaber Abo Aleiz Specialized Center for Diabetes Mellitus

Serial No:……………………………………………………

Name: ………………………………………………………

Address of use: ……………………………………………..

File No: ……………………………………………………..

1\General Information:-

1. Gender : male female

2. Age: 40-50 51-60 61-70

3. Education level:

Illiterate Primary Secondary

High level

4.Occupation:

Employee Unemployed

2\Medical History:

1. Duration of Disease: <10 10-20 >20

55

2. Number of Drugs: Mono Therapy

Multiple Therapy

1. Comorbid Conditions: Yes No

3\Nutrition Assessment:

1. Weight: …………………..

2. Height: ……………………

3. BMI: ………………………

4\ Investigation:

1. HbA1c 1st

2nd

2. F.B.S: 1st

2nd

3rd

4th

56

Appendix (B)

57

58

59

60

Appendix (C)

Table 8 – Effect of 3 months treatment with S.argel on the HbA1c levels of the

study group.

Beginning After 3 months P Value

HbA1c 8.602± 1.52 7.450 ±1.620 .000

Values are given as mean ±SD from the number of patients in the study group.

Table 9 - Effect of 3 months treatment with S.argel on the FBS levels of the

study group.

Beginning After 1 month After 2 months

After 3

months

FBS 188.13± 58.75 163.48±38.838 165.89 ±62.197 157.67± 60.139

P Value .009 .061 .009

Values are given as mean ±SD from the number of patients in the study group.

61

Table 10 - Effect of BMI cut off points on HbA1c and FBS levels before and after treatment of S.argel.

BMI cut off

points

Patients

No.(s)

HbA1c

1st

HbA1c

2nd FBS 1 FBS2 FBS3 FBS4

Normal

(18.5-24.9) 12 8.93 ±1.97 8.54 ±1.19 211.92 ±61.57 180.42±49.43 185.58 ±65.06 171.58 ±52.92

Overweight

(25-29.9) 20 8.52 ±1.33 7.07 ±1.14 186.55 ±52.82 158.4±30.46 156.75±56.27 155.8 ±63.73

Obesity

Grade I

(30-34.5)

9 8.64±1.47 7.67±2.39 179.33±75.15 154.44±36.93 167.9±81.99 154.22±78.45

Obesity

Grade II

(35-39.5)

5 8.1 ±1.34 5.96 ±0.93 153.2 ±21.16 159.4±43.85 151.6±39.36 138 ±16.98

Over all 46 8.6 7.49 188.13 163.48 165.89 157.67

P Value .775 .008 .272 .376 .606 .759

Values are given as mean ±SD from the number of patients in each group.

62

Table 11 - Effect of comorbid conditions on HbA1c and FBS levels before and after treatment of S.argel.

Other Diseases

Patients No.(s)

HbA1c 1st

Hb1c 2nd

FBS 1 FBS2 FBS3 FBS4

Yes 19 8.03±1.12 6.88±1.33 156.95±45.35 164.89±43.24 152.84±51.19 158.63±69.89

No 27 9.00±1.65 7.85±1.71 210.07±57.79 162.48±36.24 175.07±68.33 157±53.63

Over All 46

P Value .031 .046 .002 .838 .237 .929

Values are given as mean ±SD from the number of patients in each group.

63

Figure 5 - The main characters of study population

5-1 Gender:

5-2 Age/Years:

64

5-3 Education Level:

5-4 Occupation:

65

5-5 Duration of Disease:

5-6 Number of Drugs:

66

5-7 Comorbid Conditions:

5-8 BMI Cut Off Points:

67

Appendix (D)

Images of Solenostemma argel