The National Ribat University

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1 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

2 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 I

3 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. II

4 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 to 7.45 and mean level of FBS was reduced from to mg/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. III

5 خالصة األطزوحة هدفث هذه الدراسة لتقييم فعالية نبات الحزجل العشبي كماده خافضة لمستوى سكز الدم. ذ د انذراسح ت زكز جاتز أت انعز انرخصص ن زظ انسكز, يذج انذراسح ثالثح اش ز. إشر هد عه عذد 67 يزيط سكز ي ان ع انثا ي غيز ي رظ سكز انذو ي يسرخذي انحث ب ان خفعح نهسكز. ذى إخريار ان زظ ذثعا ن عاييز يحذد, ذ ا ل كم يزيط ٢ جى ف اني و ي انحزجم ف ص رج يسرخهص يائ.ذى فحص يسر سكز انذو قثم تعذ ا ر اء انذراسح نكم يزيط نرحذيذ يسر ان ي غه تي انسكز ذحذيذ يسر سكز انذو ص يا تاالظاف نسحة عي اخ دو ش زيا نكم يزيط نرحذيذ يسر سكز انذو ص يا. أظ زخ ريجح انذراسح ا خفاض يهح ظ ف يسر ياخ سكز انذو ع ذ ان زظ.حيث ا خفط ير سط يسر ان ي غه تي انسكز نذ ان زظ ي ± 2.66 إن 8.56 ± 2.76 ا خفط ير سط يسر سكز انذو ص يا نذ ان زظ ي 69.86± إن ± : يهجزاو نكم ديس نيرز. ت اءا عه ريجح ذ انذراسح خهص ان أ ثاخ انحزجم ن قذر عه خفط يسر سكز انذو. IV

6 List of Contents No Title Dedication. Acknowledgement. Abstract (English).. Abstract (Arabic)... List of Content... Appendices List of Abbreviations. List of Table.. List of Figures Page No. I II III IV V-VI VII VIII IX X Chapter One 1 Introduction Justification Objectives General Objective Specific Objective Hypothesis. 4 Chapter Two 2 Literature Review Classification of Diabetes Mellitus Type I Diabetes Type II Diabetes Other Specific Types Gestational Hyperglycaemic & Diabetes.. 7 V

7 2.3 Management of Type II Anti-diabetic plants Solenostemma argel Active ingredients present in S.argel Effect of water extract of S.argel in the induced-diabetic 12 rats Traditional medicinal uses Toxicity Chapter Three 3 Subjects and Methods Study Design Study Area Study Duration Subjects Sample Size Criteria Inclusion Criteria Exclusion Criteria Plant Example Procedures Statistical Analysis Ethical Clearance for research Chapter Four 4 Results Chapter Five 5 Discussion Chapter Six VI

8 6.1 Conclusion Recommendation.. 41 References Appendices Appendix A Questionnaire Appendix B Concept and Permission forms Appendix C Figures and Tables Appendix D Images of Solenostemma argel.. 67 VII

9 LIST OF ABBREVIATIONS ADA ALP AST BMI dl FBS g GIT GOT GPT HbA1c IDDM kg mg Mo. Mos. NIDDM No.(s) P Value S.argel SD WHO β-cell - American Diabetes Association - Alkaline phosphate enzyme - Aspartate aminotransferase - Body Mass Index - decilitre - Fasting blood sugar - gram - Gastro Intestinal Tract - Glutamic-oxalacetic transaminase - Glutamic pyruvate transaminase - Glycated Haemoglobin - Insulin dependent diabetes mellitus - kilogram - milligram - Month - Months - Non-insulin dependent diabetes mellitus - Number(s) - Probable Value - Solenostemma argel - Standard deviation - World Health Organization - Beta-cell VIII

10 LIST OF TABLE Table 1 The main characteristics of study population Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 Effects of duration of disease on HbA1c and FBS levels before and after treatment of S.argel. 29 Effect of number of drugs on HbA1c and FBS levels before and after treatment of S. argel 30 Effect of gender on HbA1c and FBS levels before and after treatment of S. argel. 32 Effect of age group on HbA1c and FBS levels before and after treatment of S. argel.. 33 Effect of occupation on HbA1c and FBS levels before and after treatment of S. argel.. 34 Effect of education on HbA1c and FBS levels before and after treatment of S. argel.. 35 Effect of 3 months treatment with S.argel on HbA1c levels of study group.. 60 Effect of 3 months treatment with S.argel on FBS levels of study group Effect of BMI cut off points on HbA1c and FBS levels before and after treatment of S.argel. 61 Effect of comorbid conditions on HbA1c and FBS levels before and after treatment of S.argel.. 62 IX

11 LIST OF FIGURES Figure 1 Figure 2 Figure 3 Figure 4 Effect of 3 months treatment with S.argel on HbA1c levels of study group. 25 Effect of 3 months treatment with S.argel on FBS levels of study group.. 26 Effect of BMI cut off points on HbA1c levels before and after treatment of S.argel. 28 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 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 X

12 Chapter One Introduction 1

13 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 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). 2

14 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 cm, 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. 3

15 1.2 Objectives General objective: To assess the Solenostemma argel effect to reduce blood glucose level in type-2 diabetic patients 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. 4

16 Chapter Two Literature Review 5

17 2. Literature Review 2.1 Classification of Diabetes Mellitus: Type 1 diabetes (β-cell destruction usually leading to absolute insulin deficiency) Immunemediated 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 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). 6

18 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 7

19 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 8

20 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. 9

21 2.5 Solenostemma argel: 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 (_ 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 10

22 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). 11

23 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) 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 (Izzeldin et al.,2012). 12

24 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 antirheumatic 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 ( ) 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. 13

25 (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) 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 14

26 complain of any undesirable or intolerable toxic or adverse effects of these preparations of Solenostemma argel. 15

27 Chapter Three Subjects and Methods 16

28 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, Subjects: 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 =

29 = = = 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 Criteria Inclusion criteria Uncontrolled type-ii diabetic patients. HbA1c above 6.5. Aged years. Freely consented to participate in the study 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. 18

30 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. 19

31 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. 20

32 Chapter Four Results 21

33 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 years old represents 43.5% (20 patients), which is the highest values; followed by years 41.3% (19 patients). The group of 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 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) 22

34 and 2.2% (1 patient) for both underweight & morbid obesity. Table 1 - The Main Characteristic of Study Population. Gender: Male Female Age/years: Education Level Illiterate Primary Secondary High Secondary Occupation Employed Unemployed Duration of Disease: < >20 Number of Drugs *Mono Therapy ** Multiple Therapy Patients No.(s) %

35 cont. ***Other Diseases Yes No ****BMI cut off points: Underweight (<18.5) Normal ( ) Overweight ( ) Obesity grade I ( ) Obesity grade II ( ) Morbid Obesity > *Hypoglycaemic Agent one drug. **Multiple drug hypoglycaemic agent ***Hypertension, Asthma, Hyper cholestramia, Others ****Cut off points of the BMI (Mahan and Escott-Stump, 2008) 24

36 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. 25

37 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 mg/dl and after three months it was reduced to mg/dl. After one month it was reduced to mg/dl, after two months it was reduced to mg/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. 26

38 27

39 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 (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. 28

40 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 < ± ± ± ± ± ± ± ± ± ± ± ±90.59 > ± ± ± ± ± ±79.09 Over All P Value Values are given as mean ±SD from the number of patients in each group. 29

41 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 Multiple Therapy ± ± ± ± ± ± ± ± ± ± ± ±67.29 Over All 46 P Value Values are given as mean ±SD from the number of patients in each group. 30

42 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. 31

43 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 ± ± ± ± ± ±40.10 Female ± ± ± ± ± ±66.02 Over All 46 P Value Values are given as mean ±SD from the number of patients in each group. 32

44 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 FBS ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±39.12 Over All P Value Values are given as mean ±SD from the number of patients in each group. 33

45 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 ± ± ± ± ± ±60.11 Unemployed ± ± ± ± ± ±60.95 Over All 46 P Value Values are given as mean ±SD from the number of patients in each group. 34

46 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 ± ± ± ± ± ±78.30 Primary ± ± ± ± ± ±64.12 Secondary ± ± ± ± ± ±52.35 High Level ± ± ± ± ± ±51.61 Over All P Value Values are given as mean ±SD from the number of patients in each group. 35

47 Chapter Five Discussion 36

48 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, antiinflammatory 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 and Murwan et al., 2010). 37

49 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 38

50 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). 39

51 Chapter 6 Conclusion and Recommendation 40

52 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. 41

53 References 42

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64 Appendices 53

65 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: Education level: Illiterate Primary Secondary High level 4.Occupation: Employee Unemployed 2\Medical History: 1. Duration of Disease: < >20 54

66 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 1 st 2 nd 2. F.B.S: 1 st 2 nd 3 rd 4 th 55

67 Appendix (B) 56

68 57

69 58

70 59

71 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± ± 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 ± ± ± ± P Value Values are given as mean ±SD from the number of patients in the study group. 60

72 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 ( ) Overweight ( ) Obesity Grade I ( ) Obesity Grade II ( ) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±16.98 Over all P Value Values are given as mean ±SD from the number of patients in each group. 61

73 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 ± ± ± ± ± ±69.89 No ± ± ± ± ± ±53.63 Over All 46 P Value Values are given as mean ±SD from the number of patients in each group. 62

74 Figure 5 - The main characters of study population 5-1 Gender: 5-2 Age/Years: 63

75 5-3 Education Level: 5-4 Occupation: 64

76 5-5 Duration of Disease: 5-6 Number of Drugs: 65

77 5-7 Comorbid Conditions: 5-8 BMI Cut Off Points: 66

78 Appendix (D) Images of Solenostemma argel 67