University of Nigeria

Research Publications

OKONTA, Jegbefume Matthew

Author

PG/Ph.D/99/27325

Evaluation of the Antidiabetic Activity of the Seed

Title Extract of Picralima Nitida Stapf (Apocyanaceae)

Pharmaceutical Sciences Faculty Faculty

Pharmacology and Toxicology Department Department

January, 2007 Date

Signature Signature

EVALUATION OF THE ANTIDIABETIC ACTIVITY OF THE SEED EXTRACT OF Picralima nitida STAPF (APOCYANACEAE)

OKONTA, JEGBEFUME MATTHEW (PG/Ph.D/99/27325)

DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA, NSUKKA

JANUARY 2007 EVALUATION OF THE ANTIDIABETIC ACTIVITY OF THE SEED EXTRACT OF Picralima nitida STAPF (APOCYANACEAE)

OKONTA, JEGBEFUME MATTHEW

(PGlPh.Dl99127325)

A THESIS SUBMITTED TO THE DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF DOCTOR OF PHILOSOPHY (Ph.D) DEGREE IN PHARMACOLOGY

SUPERVISOR PROF*CJY-AGUWA DEPARTMENT OF CLINICAL PHARMACY AND PHARMACY MANAGEMENT, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA. CERTIFICATION

Okonta Jegbefume Matthew, a postgraduate student with registration number

PG/Ph.D/99/27325 has satisfactorily completed the requirement for the award of the

degree of Doctor of Philosophy (Ph. D) of the Department of Pharmacology and

Toxicology, Faculty of Pharmaceutical Sciences.

The research embodied in this thesis is original and has not been submitted in part or in

full for the award of any other Diploma or Degree of this or any other University

Head of Departmknt, ?

External Examiner . ,. 4. <.-1. , ., ,. ' DEDICATION

To

My precious mother in law, Late Madam Lucy Nka;

All that suffered Diabetes mellitus;

All that is suffering Diabetes mellitus,

All that will suffer Diabetes mellitus,

And

To my beloved wife, Lillian and my son, Chidera

For their understanding, support and prayers. ACKNOWLEDGEMENT

I am sincerely obligated to Prof. C. Nze Aguwa, my supervisor, whose patience, fatherly guidance and timely material assistance immensely contributed to the completion of this research work. In his numerous trips within and outside the country, he brought all he deemed needful for this research work; these were evidence of his commitment to my successful completion of this work. Thank you, Sir.

May I also express my sincere gratitude to Prof.I.U.Asuzu of Dept of Vet.

Pharmacology and Physiology for the free access to his academic materials especially scientific papers; Prof.A.A.Ubachukwu of Dept of Physics and Astronomy and Prof.

M.1.Uguru of Dept of Crop Sciences for their spiritual and academic assistance. Thank you for those your 'how far have you gone with your work? And we are praying for you' after every church service.

My beloved brother, indeed and in need, Dr Vincent Chikwendu Ejere, I am not unmindful of your vested interest in my well being. In the days of my sojourn in Central

America, you stood out for my young family and me. My siblings: Vincent, Andy,

Mabeje, Nelzo and Catho, I aq gat:@ 49 you for all your encouraging calls and text messages. God bless all of you.

1 cannot forget my colleagues, Drs Okoli, Obonga, Ofokansi, Esimone, Okorie,

Odoh and Nwodo; and Pharmacists Udeogaranya, Obitte, Ndu, Ezea, Omeje, the Okoyes,

Nworu and Madu for both their spoken and unspoken encouragements.

The encouragements that I got from my senior colleagues and my teachers in the persons of Prof. P A. Akah, Dr C.V. Ukwe, Prof. M.U. Adikwu, Prof. S.I. Ofoefule, Dr C.E. Ibezim, Dr C.O. Ezugwu and my precious friend, Dr V.C. Okorie cannot go without acknowledgement. I thank you all.

The earnest contributions of Mr Tochi Okonkwo and Igboeme Sabastin in the laboratory work stage of this research is worthy of mention. Only God will reward you enough for the sleepless nights we shared together in the Laboratory.

I am grateful to Dr Q. Zao of the Xavier-Tulane Coordinated Instrumentation

Facility, Tulane University, New Orleans, USA for the 500 mHz NMR Spectra that formed a part of the Spectra data described in this thesis.

I am very grateful to my soul mate, my precious wife and friend, Lillian for all her moral, emotional, and academic support while this research work lasted. Also my son

Chidera is not left out in this shower of appreciation. His curiosity gave me great joy as a father.

Finally, I am also grateful to my God for the health he granted my family and I, and peace that past all understanding even during some very trying moments, while this work lasted.

Okonta, J.M. University of Nigeria, Nsukka.2007 ABSTRACT

This study investigated the antidiabetic effect of the seed extracts of Picralima nitida in rats treated with alloxan monohydrate at 120 mgkg intraperitoneally. Methanol extract (ME, 5 gkg) caused 20.0 % maximal reduction of mean fasting blood glucose levels in normoglycemic rats while glyburide (5 mgkg) caused 25.9 % maximal reduction. In the alloxanized rats, ME caused 49.7% maximal reduction in the mean fasting blood glucose levels while glyburide caused 71.9% maximal reduction in the mean blood glucose levels after per oral administration. The blood glucose lowering effect of the extract commenced at the first hour and climaxed at twelve hour.

The alkaloids extract of Picralima nitida seed given i.p. caused increase in fasting blood glucose levels while the glycosides extract reduced the blood glucose levels in normoglycemic and hyperglycemic rats though not in dose-dependent pattern.

Glycosides extract caused significant (p < 0.05) reduction of 38.6 % (250 mgkg) and

22.9% (500 mgkg) of the fasting blood glucose levels in normoglycemic, and 64.4%

(250 mgkg) and 39.0 % (500 mgkg) in the hyperglycemic rats. The glycosides extract maintained reduced fasting b1~~)d,.glqcoselevels for 24 h which is indicative of long duration of action. On subchronic (10 days) treatment of hyperglycemic rats, glycosides extract (250 mgkg) caused 82.99 % while glyburide (5 mgkg) caused 60.81% reduction of mean blood glucose levels.

The n-butanol fractions (F7>F5>F4>Fs) showed consistent reduction of the fasting blood glucose levels of diabetic rats in the following order, 84.58 %, 80.88 %, 76.83 % and 66.92% while the glyburide caused 86.14 %. These fractions that exhibited significant blood glucose lowering action for 24 hours and F7 has the most persistent hypoglycemic effect. The fraction E of F7 caused 89.17 % maximal reduction of the mean fasting blood glucose levels of treated rats while the standard drug, glyburide caused 79.66% reduction.

The peak mean blood glucose concentration achieved after glucagon administration was reduced by the glycosides fractions like glybwide did. They respectively caused reduction in mean blood glucose levels in the following order

E>F7>F5.

From the fraction E of the seed extract of Picralima nitida, that had the highest hypoglycemic activity, was isolated a hydrophilic triterpenoidal saponin; 2a, 3a 16a, 23- tetrahydroxyloleana-5, 13 (1 8)-dien-28-oic acid which may be the enatiomer of Mimusic acid obtained from the Mimusops elengi.

In conclusion, it may be stated that the high hypoglycemic activity of the seed extract of Picralima nitida resides in the hydrophilic triterpenoidal saponins which may have achieved this through reduction of hepatic overproduction of glucose or increase in glucagon catabolism, and inhibition of gastric emptying in diabetic conditions. .. 11 .. . 111 iv v vii ix xiv xvi xix CHAPTER TWO: MATERIALS AND METHODS

2.3.1 Fractionation of Alkaloids and Glycosides from methanolic extract------47 2.4.3 Chemical identification of the respective spots of Fraction F7 bE------49

2.4.4 Determination of the Nuclear Magnetic Resonance (NMR) and Mass Spectrophotometry (MS) of Fraction F7 bE spots------49

2.6.2 Effects of methanol (ME) and chloroform (CHE) extracts of Picralima nitida seed on mean fasting blood glucose levels in normal rats------5 1

2.6.6 Effect of alkaloids (AKF) and glycosides fractions of methanol extract of Picralima nitida on mean fasting blood glucose levels in normal rats------53

2.6.8 Effect of aqueous (AQP) and chloroform (CHP) phases of glycosides fraction of Picralima nitida on mean fasting blood glucose levels in alloxanized rats---- 54

2.6.9 Prolonged treatment effect of q-Bum01 extract (nBF) of glycosides fraction of Picralima nitida on mean fasting blood glucose levels in alloxanized rat--- 54

2.7.1 Effects of chromatographic fractions (F4-8) of nBF of Picralima nitida on mean fasting blood glucose level in alloxanized rats------55

2.7.2 Effects of chromatographic fractions (F4,+8) of nBF of Picralima nitida on mean fasting blood glucose levels in glucagon-induced hyperglycemic rats------56 2.8 Preparation of Calibration Curve of Phenol Red in Glucose solution------58

CHAPTER THREE: RESULTS

Phytochemical analysis of extracts and fractions------60

Effect of methanol (ME) and chloroform (CHE) extracts of Picralima nitida on mean fasting blood glucose levels in normal rats------72

Effect of methanol (ME) extract of Picralima nitida on mean fasting blood glucose levels in normoglycemic and alloxanized rats------72

Effect of methanolic extract of Picralima nitida seeds on mean blood glucose levels of rats orally fed with glucose...... 73

, . .. Effect of alkaloids (Alk) and glycosides (gly) fractions of Picralima nitida seed on mean fasting blood glucose levels of normoglycemic and hyperglycemic rats-84

Effect of chromatographic fractions of n-butanol extract of Picralima nitida seed on mean fasting blood glucose levels of hyperglycemic rats ...... 94

3.1 1 Effect of extract of Picralima nitida seed on Gastric emptying rate in normal and hyperglycemic Rats------95

LIST OF FIGURES

Figure 1b: Some diterpenoids and Sesqueterpenoids with antidiabetic activity------22

Figure 1c: Some of the Triterpenoids with antidiabetic activity------23

Figure Id: Some monoterpenoids with antidiabetic activity------24

Figure 3: Extraction and screening procedure of Picralima nitida seed------70

Figure 5a: Effect of F7bE and ME of Picralima nitida seed on gastric emptying and blood glucose levels in rats fed with 40% glucose test meal.------105

Figure 5b: Effect of F7 bE and ME of Picralima nitida seed on gastric emptying and blood glucose levels in glucose pretreated rats ...... 107

Figure 6: Structure of Mimusic acid (2a, 3a 16a, 23-tetrahydroxyloleana-5,13(1 8)-dien- 28-oic acid) fiom S2 in Fraction E of Picralima nitida seed------111 LIST OF TABLES

Table 1: Plants with confirmed antidiabetic activity within the tropical vegetation ----- 28

Table 3: Extractive yields of extract and fractions of P.nitida seed------6 1

Table 4a: Phytochemical constituents of the Picralima nitida seed extracts. AKF- alkaloids extract, CHP- chloroform phase of glycosides extract and AQP- aqueous phase of glycosides extract...... 63

Table 4b: Phytochemical Constituents of the nBF fractons of Picralima nitida seed. F4,5,7 8 are fractions of n-Butanol Fraction (nBF), and F7 bE------65

Table 6: Effect of methanol (ME) and chloroform (CHE) extracts of Picralima nitida seed on mean fasting blood glucose levels of normoglycemic rats------74

Table 7: Percentage reduction of the mean fasting blood glucose levels in normal rats by different doses of methanol (ME) extract of Picralima nitida seed------76

Table 8: Effect of methanol extract of Picralima nitida seed on mean fasting blood

Table

Table 12: Effect of glycosides (gly) fraction of methanol extract of Picralima nitida seed on mean fasting blood glucose levels of alloxanized rats------88 Table 13: Effect of aqueous and chloroform phase of glycosides fraction of methanol extract on mean fasting blood glucose levels in alloxanized rats------90

Table 14: Effect of n-butanol extract of aqueous phase of Picralima nitida seed extract on mean fasting blood glucose levels of alloxan-induced diabetic albino rats after prolonged treatment ...... 92

Table 15: Effect of chromatographic fiactions of n-Butanol extract of P.nitida on mean fasting blood glucose levels in alloxanized rats------97

Table 16: Effect of chromatographic fractions of F7 of n-butanol extract of Picralima nitida seed on mean fasting blood glucose levels in diabetic rats------99

Table 17: Effect of chromatographic fractions of n-butanol extract of P.nitida seed on Glucagon-induced hyperglycemla. in. rats-- ...... 101 LIST OF APPENDICES

Appendix 1 UV Spectra scan of sample S1 in chloroform solvent------132

Appendix 2 UV Spectra scan of sample S2 in chloroform solvent------133

Appendix 3 UV Spectra scan of sample S3 in chloroform solvent------134

Appendix 4 UV Spectra scan of sample S1in ethanol solvent------135

Appendix 5 UV Spectra scan of sample S2in ethanol solvent------136

Appendix 6 UV Spectra scan of sample S3 in ethanol solvent------137 PUBLICATION ON PICRALIMA NITIDA 1,

CHAPTER ONE

INTRODUCTION

,> .I. 1. i. t DIABETES MELLITUS

1.1 Epidemiological estimation of Diabetes mellitus incidence

Diabetes mellitus is an endocrine and metabolic disease that is on the rise worldwide and being considered to be at an epide'mic level by the World Health

Organization (Petal and Rybczynski, 2003). Diabetes mellitus has been differentiated into two forms: Insulin-dependent diabetes mellitus, (IDDM) or Type I and non-insulin- dependent diabetes mellitus (NIDDM) or the Typc I1 diabetes (Ozturk, et al, 1996; Judd and Raman, 1999).

I) An estimate of 140 million people worldwide have diabetes mellitus and the projection for the year 2025 is 300 millim (Smith and Tadayyon, 2003); Type 2 diabetes is estimated to be about 90% of the estimated 140 million global diabetes mellitus population. It was aIso documented that the number of people diagnosed with Type I1 diabetes mellitus globally is estimated to be at 2 -3 .% of the world population and rising at a rate of 4 - 5% per year (Petal and Rybczynski, 2003). This trend describes the health crisis of this century because it .will~doubtlesslyburden the healthcare systems globally if not promptly addressed. This projected explosion of diabctes mellitus globally can be If attributed to ageing population, increasing prevalence of obesity, sedentary life styles and increase in the consumption of western' diets by both the urban and rural populations especially in developing countries of the world (King et al, 1998). b

1.2 Definition and Etiology of Diabetes mellitus

Diabetes mellitus is a complex metabolic disorder (Petal and Rybczynski, 2003;

Ozturk, et nl, 1996) that involves chronic alterations in the carbohydrate, fat, and protein metabolism, basically resulting from the secretion of dysfunctional and /or insufficient endogenous insulin by the B-cells of the pancreas. Diabetes mellitus is characterized by elevated blood glucose concentration, In the untreated state it is accompanied by symptoms of severe thirst, profuse urination, polyphagia, weight loss, and/or stupor

(Ozturk, et h1, 1996; Bamidele et al, 2002). The high glucose level in the blood and other biochemical abnormalities seen in diabetic patients result from the insufficient production or secretion of insulin due to the dysfunction or destruction of the p-cells of the pancreas

(Judd, and Raman, 1999) and/or insensitivity to insulin in target cells (Ozturk, et nl,

1996).

Though the exact etiology of diabetes mellitus has not been fully established but infections like mumps, congenital rubella and cytomegalovirus (Barret, 1985),

autoimmunity (Al-Sakkaf et a1, .1989.),.genetic and environmental factors, point mutations

k' in insulin gene (Ozturk, et al, 1996), and altered prostaglandin metabolism in the

pancreatic tissue has been suggested to cause impairment in insulin secretion in diabetes

mellitus. The decreased sensitivity of target cells to insulin in diabetic patients has been

attributed to changes in the cellular prostaglandin metabolism, decrease in the number of

insulin receptors in the target cells and altered insulin receptor tyrosine kinase activity

(Ozturk, et 01, 1996). Endocrine disorders that cause increased production of growth

hormones, glucocorticoids, catecholamines, glucagon and somatostatin, and genetic disorders contribute to the p-cell dysfunction and the attendant hyperglycemia (Aneela and Vincent, 2005). 1' 1.3 Complications of Diabetes mellitus

In normal individual, most of the glucose at the cellular level is converted to glucose-6- phosphate by hexokinase. The unconverted minor amount enters the polyol pathway, the glucose metabolism alternative route that leads to the production of sorbitol from the glucose. In diabetics, the hexokinase is saturated with glucose, and the increased influx of glucose through the polyol pathway causes increase in glucose turnover thereby creating glucose imbalances in the tissues that are not dependent on insulin for uptake of glucose. T$is metabolic perturbation provokes the early tissue damage in the target organs of diabetic patients thereby producing complications (Chihiro, Yabe-

Diabetics are particularly prone to various acute andlor chronic complications

(Petal and Rybczynski, 2003; Ozturk et al, 1996; Judd and Rarnan, 1999; Moller, 2001).

The acute complications include hyperglycemia, diabetic ketoacidosis (DKA) for Type I diabetics, hyperosmotic hyperglycernic nonketotic (HNNK) diabetic coma for Type 2 ., ,? . ..-, 9,' , ,'J diabetics. The chronic complications include micro- and macroangiopathies, atherosclero.sis, congestive heart failure, hyperlension, retinopathy, nephropathy, neuropathy, fecal incontinence and diabetic.diarrhea, erectile dysfunction and impotence.

Others include decrease fertility in males and increased incidence of spontaneous abortions in females, urinary bladder dysfunction and urinary tract infections (Judd and In hyperglycemic state, nonenzymatic glycation of structural proteins is enhanced and the glycation end products accumulate in diabetic tissues. The nonenzymatic glycation alters the structure and function of various macromolecules in the tissues thus

t; causing basement membrane thickening, demyelination and impaired axonal transport

(Scott, 2006).

Magnesium deficit in the body has been associated with diabetes mellitus chronic complications. The intracellular free magnesium levels have been observed to be lower in patients with diabetes mellitus than in the general population. Studies have shown that the mean plasma levels of magnesium ions are loyer in diabetes mellitus patients than the non-diabetic subjects. Magnesium deficiency is associated with insulin resistance and increased platelet reactivity, and may result in increase in cardiovascular mortality and

I' morbidity in diabetics (De Valk, 1999).

1.4 Pathogenesis of diabetes mellitus

The development of diabetes mellitus especially the non-insulin dependent or the

Type 2 diabetes mellitus has been linked to numerous metabolic defects such as P-cell . ,,. ..I .: * dysfunction, peripheral insulin resistance, and increased rate of endogenous hepatic glucose production (glycogenolysis and gluconeogenesis) (Judd and Raman, 1999;

Mahler and Alder, 1999). ,, _ . I' p-cell dysfunction in Type 2 diabetes mellitus reduces glucose transport. Insulin secretion depends upon transmembranous transport of glucose and the glucose coupling to the glucose sensor. The complexing of glucose/glucose sensor induces an increase in glucokinase by stabilizing the protein and impairing its degradation. This glucokinase induction serves as link between the insulin secretory apparatus and intermediary metabolism. As glucose transport in jp-cells in Type 2 diabetics is greatly reduced, the control of insulin secretion is removed from glucokinase to the glucose transport system

(Porte, 1991).

The'liver has a critical role in regulating endogenous glucose production from de

now synthesis or catabolism of glycogen. Increased rates of hepatic glucose production

are largely responsible for the development of overt hyperglycemia particularly in Type 2

diabetics (Moller, 2001). A relative decrease in insulin level, or reduced hepatic

responsiveness to insulin, can lead to increased output of glucose by the liver. Thus, the

liver in Type 2 diabetes is known to overproduce glucose (Mahler and Alder, 1999).

Type 1 or insulin dependent diabetes mellitus is a catabolic disorder in which

circulating insulin is very low or absent, plasma glucagon is elevated, and the pancreatic

ti p-cells fail to respond to all insulin-secretory stimuli. Type 1 diabetic patients need

exogenous insulin to reverse this catabolic condition, prevent ketosis, decrease

hyperglucagonemia, and normalize lipid and protein metabolism.

Type 1 or Insulin dependent diabetes mellitus is an autoimmune disease. The ,, . ..<. ..' insulin deficiency in Type 1 diabetes mellitus occurs due to lymphocytic infiltration into

the pancreas and the destruction of insulin-secreting p-cells of the islets of Langerhans

(Ozturk, et al, 1996). Most patients have circulating islet cell antibodies, and the majority

also has detectable anti-insulin antibodies before receiving insulin therapy. Most islet cell

k, antibodies are directed against glutamic acid decarboxylase (GAD) within pancreatic P-

cells (Ganong, 1999; Aneela and Vincent, 2005). Type 1 diabetes mellitus may also result

from damage to pancreatic p-cells by infectious or environmental agents (Ozturk, et al, induction serves as link between the insulin secretory apparatus and intermediary metabolism. As glucose transport in p-cells in Type 2 diabetics is greatly reduced, the control of insulin secretion is removed from glucokinase to the glucose transport system

(Porte, 199 1).

The'liver has a critical role in regulating endogenous glucose production from de

novo synthesis or catabolism of glycogen. Increased rates of hepatic glucose production

are largely responsible for the development of overt hyperglycemia particularly in Type 2

diabetics (Moller, 2001). A relative decrease in insulin level, or reduced hepatic

responsiveness to insulin, can lead to increased output of glucose by the liver. Thus, the

liver in Type 2 diabetes is known to overproduce glucose (Mahler and Alder, 1999).

Type 1 or insulin dependent diabetes mellitus is a catabolic disorder in which

circulating insulin is very low or absent, plasma glucagon is elevated, and the pancreatic

p-cells fail 'to respond to all insulin-secretory stimuli. Type 1 diabetic patients need

exogenous insulin to reverse this catabolic condition, prevent ketosis, decrease

hyperglucagonemia, and normalize lipid and protein metabolism.

Type 1 or Insulin dependent diabetes meliitus is an autoimmune disease. The - ,? .*I. ." . '> insulin deficiency in Type 1 diabetes mellitus occurs due to lymphocytic infiltration into

the pancreas and the destruction of insulin-secreting p-cells of the islets of Langerhans

(Ozturlc, et al, 1996). Most patients have circdlating islet cell antibodies, and the majority

also has detectable anti-insulin antibodies before receiving insulin therapy. Most islet cell t' antibodies are directed against glutamic acid decarboxylase (GAD) within pancreatic P-

cells (Ganong, 1999; Aneela and Vincent, 2005). Type 1 diabetes mellitus may also result

from damage to pancreatic p-cells by infectious or environmental agents (Ozturk, et al, C. Random blood glucose test (RBG) Blood sample is withdrawn from the patient and analyzed at any time of the day irrespective of meal that was taken and when it was taken. When the blood glucose level is above 250 mg%, the patient is further tested with a method for diagnosis.

I1

1.5 .3 Monitoring test This method is mainly employed to monitor the therapeutic outcomes in management of diabetes mellitus and to enable the health personnel to choose the right drug(s) especially in ambulatory patients.

A. Glycosylatcd haemoglobin measurement: , Glucose has been found to bind to proteins irreversibly and non-enzymatically thus cause chemical alteration in the proteins. The non-enzymatic glycosylation of the

1, proteins occurs by direct reaction between the aldehyde groups of the reducing sugars and primary amino groups in proteins to form Schiff bases that is rearranged to form stable protein ltetoarnine derivative. This contributes to diabetes complications because it

is an oxidative process (Odukoya and Ogbeche, 2002). In normal individual, the .).7.1. - glycosylated haemoglobin (HbAIC)is between 3-6 'XI while in a diabetic patient, the level

may be as high as 18-20 %. It is used to mopitor therapy compliance in diabetics

considering their blood glucose control. .

1.6 ~heraplkuticAgents in Diabetes Mellitus management Diabetes mellitus though incurable could be managed reasonably to enable the

patient live normal life. Due to the variation in the forms of diabetes, the treatments of

diabetes vary appreciably. In Type 1 or the insulin dependent diabetes mellitus there is absolute lack of insulin hence this condition can only be handled pharmacologically with exogenous insulin (George, 1992; Judd and Raman, 1999; Aguwa, 2004). In the Type 2 diabetes mellitus, there is relative lack of insulin and this condition can be managed with oral hypoglycemic agents and exogenous insulin in stressful circumstances like surgery, infections andlor pregnancy (Aguwa, 2004). The sole aims of treatment of diabetes inellitus are?

1. To alleviate symptomatic hyperglycemia and improve quality of life while avoiding hypoglycemia.

2. To avoid ketoacidosis and infections in diabetic patients

3. To keep the fasting blood glucose as close to normal as possible to reduce tendencies of development of complications (Bosseri, 2002).

1.6.1 INSULIN

Thei'islet of Langerhans in the pancreas produces insulin; it secretes about 40-60 units daily in normal human being. It is a polypeptide made up of two chains of amino acids linked by disulfide bridges, and the physiologic effects are enormous and complex . ,. 4.1" (Ganong, 1999). Exogenous Insulin types are conveniently classified according to their actions: Rapid acting insulin includes regular insulin, lispro, and aspart insulin. They increase transport of glucose, amino acids and K+ into insulin- sensitive cells.

Intermediate acting insulin include; Neutral Protamine Hagedoi-n (NPH) insulin, which contains a mixture of regular and protamine zinc insulin, and lente insulin, which

t' contains 30% semilente insulin and 70% ultralente insulin in an acetate buffer; they stimulate protein synthesis, inhibits protein degradation, activates glycolytic enzymes and glycogen synthase; delayed or ultralente acting insulin that increase the messenger RNAs for lipogenic and other enzymes (Ganong, 1999; Aguwa, 2004). Type 1 diabetes mellitus patients (Aneela and Vincent, 2005) require insulin therapy to control initial hyperglycemia and maintain serum electrolytes and hydration. Initiation of insulin therapy in adults is calculated depending on level ,of blood glucose and the weight of the patient. This dose usually is divided so that one half is administered before breakfast, one-fourth before dinner, and one-fourth at bedtime. After selecting the initial dose, 1, adjusting the amounts, types and timing depend on plasma glucose levels.

The insulin dose often is adjusted in increments of 10% at a time, and the effects

are assessed over 3 days before making any further dose changes. More frequent

adjustments of regular insulin can be made if risk of hypoglycemia is present. Initiation

of insulin therapy in children with moderate hyperglyceniia but without ketonuria or

acidosis may be started with a single daily subcutaneous injection of 0.3-0.5 Ulkg of

intermediate-acting insulin alone. While in children with hyperglycemia and ketonuria

but without acidosis or dehydration may be started on 0.5-0.7 Ulkg of intermediate-acting I, insulin and subcutaneous injections of 0.1 Ukg of regular insulin at 4- to 6-hour . ,, ."...:. intervals.

Insulin schedules include multiple subcutaneous insulin injections administered to control

hyperglycemia after meals and to maintqin normal plasma glucose levels throughout the

day. This may increase the risks of hypoglycemia. Therefore, patients should be well

educated about their disease and about self-monit'oring of plasma glucose levels. About

25% of the total daily dose is administered as intermediate-acting insulin at bedtime, with

additional gases of rapid-acting insulin before each meal (4-dose regimen). These patients may need additional intermediate- or long-acting insulin in the morning for all- day coverage. Patients should adjust their daily dosage(s) based on their self-monitoring of glucoses before each meal and at bedtime.

1.6.2 ORAL HYPOGLYCEMIC AGENTS

Oral hypoglycemic agents are particularly useful in the management of Non-

Insulin dependent diabetes mellitus or Type 2 diabetes mellitus. They are effective in the lowering of blood glucose levels especially when there is no acute or chronic source of

stress such" as surgery, infections, and/or pregnancy (Aguwa, 2004). These oral liypoglycemic agents include:

1.6.2.1 Sulphonylureas

They form the major class of drugs used in the treatment of Type 2 diabetes mellitus. Sulphonylureas are primarily insulin secretagogue that increase circulating

insulin level in the body through direct stimulation of the pancreatic P-cell mediated by

potassium ATP-sensitive channel (Ganong, 1999, Canadian Pharmaceutical Specialties,

2000). These agents reduce glucose by increasing insulin secretion from pancreatic P- , .:..+ I cells in pafients with residual P-cell function. All the sulphonylureas may cause mild

liypoglycemia but severe hypoglycemia is uncommon (Scott, 2006).

These sulphonylureas are classified into first generation (like tolbutamide,

chlorpropamide, tolazamide and acetohexamide), second generation, which are more

potent, longer acting, and less frequently administered (like glyburide, glipizide and

gliclazide) and third (like glimepiride) generation (Judd and Raman, 1999; Nolte and

Karam, 2004). The various generations and formulations of sulphonylureas differ in potency and duration of action, but no study has been conducted to show clear advantage of one formulation over another (Judd and Raman, 1999). The sulphonylureas, a class of

It oral hypoglycemic agents, have troublesome concern pertaining to cardiac safety. ATP-

dependent potassium channels are present in p-cells, myocardium and vascular smooth

muscles. The sulphonylureas bind to particularly receptors in p-cell resulting in the

closure of potassium ATP channels and the opening of calcium channels leading to

increase in cytoplastic calcium that effect the insulin secretion (Mahler and Alder, 1999).

The attachment of sulphonylureas to these channels inhibit the response of ischemic

lesions to treatment thus could delay the recovery of contractile function and increase

infarct size in myocardial infarction (Leibowitz and Cerasi, 1996). (l; Repaglinide is a new agent that stimulates the secretion of insulin from the

pancreatic p-cells like the sulphonylureas but is structurally different hence do not bind to

the same receptors at the p-cells (Mahler and Alder, 1999). It is taken preprandially and

has rapid onset of action.

1.6.2.2 BIGUANIDES ,. 4.7 .. T The most clinically used of this group of oral hypoglycemic agents is metformin.

This drug is known to reduce hepatic glucose output and decrease peripheral insulin I' resistance in Type 2 diabetes (Smith and Tadayyon 2003); it also slows the absorption of

glucose from the intestine and reduces blood glucagon levels (Nolte and Karam, 2004).

Metformin, from post market survey, has been found to reduce body weight unlike the

sulphonylureas and insulin (Judd and Raman, 1999). It has been proved to be more

effective than sulphonylureas and insulin in reducing rates of any diabetes-related end- points like mortality and stroke (Smith and Tadayyon, 2003). It also has cardioprotective actions that stemmed from its ability to mildly impact insulin resistance, such as its impact on lipoprotein profile, iniproves fibrinolysis through reduction of plasminogen activator inhibitor (PAI-1 improves vascular reactivity and reduces platelet aggregation (CPS, 2002; Snlith and Tadayyon 2003). The only worrisome side effect of metformin is the lactic acidosis, which is exacerbated by renal impairment (Gan et al,

1992). The drug should be kept off male patients with serum creatinine levels >1.5 mgdl-' and females with > 1.4mgdle' (Judd et al, 1999).

1.6.2.3 TEIIAZOLlDINEDlONES

The thiazolidinediones are insulin sensitizers exemplified by rosiglitazone and pioglitiazode, which are not currently used as monotherapy. The thiazolidinediones were discovered by serendipity (Smith and Tadayyon, 2003). They improve hyperglycemia by

enhancing insulin sensitivity without stimulating insulin secretion. They appear to reduce

cellular resistance by the activation of Peroxisome proliferator-activated receptor gamma

(PPAR-y), a member of a family of transcription factor receptors that regulate several . ,, . 4.1. 3,. > ,I+- . genes transcription involved in pre-adipocyte differentiation and insulin mediated glucose

uptake in peripheral tissues. The PPAR-y receptors, in human, are found in target tissues

for insulin action such as adipose tissue, skeletal n~usclesand liver (DPIC 2000; Nolte

1, and Karam, 2004).

By activating PPAR-y, thiazolidinediones enhance expression of GLUT4 glucose

transporters, resulting in increased insulin-stimulated glucose uptake. It is agreed, based

on the numerous clinical studies that the glucose-lowering action of thiazolidinediones is mediated primarily by decreasing insulin resistance in skeletal m~~scleand evidence of insulin sensitization in the liver (Smith and Tadayyon, 2003).

It has been clinically proved that free fatty acid elevation in Type 2 diabetes mellitus patients induce insulin resistance by inhibiting glucose disposal by muscle and stimulating glucose production by the liver. Thiazolidinediones reduce circulating free bl fatty acids, primarily by restoring insulin suppression of adipose tissue lipolysis

(Mayerson et al, 2002).

1.6.2.4: ALPHA-GLUCOSIDASE INHIBITORS

Alpha-glucosidase inhibitors act by inhibiting the enzyme alpha-glucosidase found in the brush border cells that line the small intestine, which cleaves more complex carbohydrates into sugars. Because they inhibit the breakdown and subsequent absorption

I' of carbohydrates from the gut following meals, these drugs impact on postprandial hyperglycemia more reasonably and modestly on fasting plasma glucose levels. They have been associated with a reduction in HbA,.

. . . ,P The serious side effects observed with these agents are gastrointestinal effects such as abdominal discomfort, bloating, flatulence and diarrhea. Acarbose treatment has been linked to elevation in serum transaminase 1e;vels and the use of this agent is contraindicated in patients with liver cirrhosis. The alpha-glucosidase inhibitors are contraindicated in patients with a serum creatinine level more than 2.0 mg per dL and

tl patients with inflammatory bowel disease or a history of bowel obstruction. 1' 1.6.2.5: GLUCAGON- LIKE PEPTIDES (INCRETINS)

The glucagon peptide subfamily includes the gastric inhibitory polypeptide

(GIP) and glucagon-like peptide- 1 (GLP- 1).

Gastric inhibitory polypeptide (GIP). It is a single 42-amino acid peptide synthesized in and secreted by enteroendocrine K-cells particularly located in the duodenum and proximal jejunum and rarely scattered all over ,the intestine (Meier et al, 2002). Its secretion is commonly stimulated by the ingestion of carbohydrate and lipid-rich meals, and following the meal ingestion the plasma concentration increases by 10-20 folds .It t> reaches the peak plasma concentrations 15-30 minutes following the ingestion of oral glucose or lipids, even before the absorption of nutrients into the gut (Cataland et al,

1974). It is inactivated by dipeptidyl peptidase IV (DPP-IV). The GIP receptor is expressed in the pancreatic islet, as well as, the gut, adipose tissue, heart, pituitary,

adrenal cortex and the brain (Usdin et al, 1993).

GIP infusion results in insulin secretion only in the presence of elevated glucose concentrations; and is not glucagonotropic in normal humans during euglycemic or

hyperglyceinic conditions. It.. only.I? gppgars to be effective in postprandial state than in 1' fasting state (Elahi, et a1 1979).

Glucagon-like peptide 1 (GLP-1)

It is a 32- amino acid peptide that was first identified as a gastrointestinal

hormone and a cleavage product of the preproglucagon precursor (Smith and Tadayyon,

2003). It has a role in the regulation of metabolism especially food intake and glucose

homeostasis as it synergises with GIP as incretins and glucose to stimulate insulin

secretion, inhibit gastric motility and induce satiety. Its circulation increases as soon as

tf food is ingested. Insulin resistance and progressive decline of insulin secretion is the underlying "etiology of impaired glucose tolerance and Type 2 diabetes. 50% of the postmeal insulin surge in normal subject has been attributed to incretin response (Rask et al, 2001); this action is reduced in insulin-resistant nondiabetics and maybe totally absent in Type 2 diabetics (Lugari et al, 2000). This implies that the inadequate response of incretin may be contributory to the poor P-cell insulin secretion and may lead to the development of impaired glucose tolerance and its progression to Type 2 diabetes mellitus.

The ability of GLP-1 to stimulate insulin secretion in a glucose-dependent manner

I makes it an attractive treatment strategy in Type 2 diabetes especially as it has been found to be effective when diet, sulphonylureas or metformin has failed to control the blood glucose level (Smith and Tadayyon, 2003). It has been found to improve glucose and C-peptide profiles, reduced free fatty acids, increased p-cell function and insulin sensitivity and produced a 1.3 % reduction in HbAlc levels on chronic administration. lncrctin Mimctics: These are a new class of antidiabetic agents. The first of this class is

.*1.+' > esenatide. Incretins are hoimones produced from the gastrointestinal tract that act to enhance th~~normalrelease of insulin after the oral ingestion of carbohydrates. (Nauck et a/, 1986; Drucker, 2003). They also slow the gastric absorption of nutrients and act to promote a feeling of satiety that can lead to weight loss in overweight individuals. These agents work to lower glucose levels without causing hypoglycemia, but with gradual weight loss due to decrease in caloric intake. Exenatide augment the hypoglycemic effects of sulphonylureas when co administered but on its own, will not cause hypoglycemia, and do not when used in combination with metformin. Liraglutide is a human long-acting form of glucagon-like peptide-1 (GLP-1) that is similar in action to exenatide.

t, 1.7 Phytotherapy and Diabetes mellitus management

Plants and many of their derivatives are being used as natural remedies and folk medicines for the treatment of diabetes globally (Akah et al, 2002). More than 1200 plant-derived compounds have been tested for their ability to lower blood sugar levels.

Many have been found to contain chemical constituents that have hypoglycemic activity in animal model (Li et al, 2004) as shown in Table 1.

Currently, the utilization of alternative medicine for different disease conditions is on the incr6ase globally particularly in the developing world. A number of herbs have been used traditionally to stabilise blood sugar and address excess weight gain by appetite suppression or other means. Additionally, scientific inquiry into these botanicals yields increasing knowledge about how they work.

1.7.1 Plants and plant-derived compou.nds with antihyperglycemic activity . , .. . a Plants have always been important source of drugs and many of the currently available drugs have been derived directly or indirectly from them. A survey of literature has shown ;hat a large variety of compounds obtained from several plants were found to possess antihyperglycemic activity. These compounds include terpenoids, polysaccharides, flavonoids, sterols, alkaloids and other miscellaneous compounds (Li et nl, 2004). Terpenoids, a class of glycosides, consist of many compounds with antihyperglycemic activity especially triterpenoid saponins such as ginsenosides, senticoside A (Ni et al, 1998), momordiun from Boussingaultia baselloides, timosaponin,

3, 6, 9-trihydroxylurs-12-en-28-oicacid and 2, 3-dihydroxylurs-12-en-28-oic acid from

Eriobotrya japonica (De Tommasi et al, 1Wl), Oleanolic acid from Bouvardia termflora

(Perez et al, 1998), gymnemagenin, gymnemasaponin, gymnemic acid and gyrnnemoside

from G. sylvestre (Li et al, 2004), P-Sitosterol-D-glucoside and 5,25-Stigmastadiene-3-

P-ol-D-glucoside from Momordica charantia (Sanjay, 2002). Scopoletin, a steroidal

saponinpeptide ester from Trigonella foenum-graecum has been reported to have

hypoglycemic activity (Sanjay, 2002).

The diterpenoids and sesquiterpenoids include trans-dehydrocrotonin from Croton

cajucara (liarias et nl, 1997), Steviol (Jose Barbosa Filho et al, 2005) and stevioside

(White et al, 1994) from Stevia rebaudiana.

The monoterpenoids include iridoid glycosides such as rehmannioside A, ByC

and D, and Catalpol (Li et al, 2004) from Rehmannia glutinosa. Betberine

,. Vindoline

Fig.la: Some alkaloids with antidiabetic activity (Li et a1,2004) (f-+i0

,

H3C 'COO ,,; Stevioside

Steviol

0

Vig. lb: Some diterpenoids and Sesqueterpenoids with antidiabetic activity (Li et nl, 3,6,9-Trihydroxyurs-12-en-28-oic Acid

OH Gy mnemagen~nAcid

Fig. lc: Some of the Triterpenoids with antidiabetic activity (Li et nl, 2004)

I3 Rehmannioside Catapol

Rehrnannioside C

Rehmannioside D

.,,, . 4 "1. .:' , . t '

Fig. Id: Some monoterpenoids with antidiabetic activity (Li et al, 2004)

,' Flavonoids that have hypoglycemic effect include 5,7,3-trihydroxy-3, 6-4-

I' trimethyflavone from Brickellia veronicaejolia (Perez et al, 2000a) and glycoside isoorientin from Cecropia obtusifolia (4ndrade Cetto and Wiendenfield, 2001). Others include Kalconien and 7-(6-O-malonyl-~-D-glucopyranosyloxy)-3-(4-hyroxyphenyl)-4H-

1 -benzopyran-4-one from P. lobata (Hirakura et al, 1989), Flavone C-glycoside from P. temata (Li et al, 2004), sappanchalcone and Protosappanin A from C. sappan (Moon et al, 1986), and magniferin from A. asphodeloides (Li et al, 2004). (-) Epicatechin,

Myricetin and quercetin from Pterocarpus marsupium, as well as Marsupsin, Pterosupin and pterostilbene from this plant's heartwood showed hypoglycemic activity in

I' Streptozotocin-diabetic rats.

Alkaloids that have documented hypoglycemic activity are relatively few. Some of these compounds include berberin and vindoline from Stephania tetradra (Lei et al,

2004), Trigonelline from Trigonella foenum-graecum, ergot alkaloids from Claviceps purpurea (Sanjay, 2002) and Leurosine of Catharanthus rosea (Jose, Barbosa Filho et al,

2005).

Polysaccharides of different types have been isolated from traditional medicines . ,... % t... , ,J for antidiabetes activity, most of them showed good hypoglycemic effect. The isolated h' ones include Panaxan, laminaran, coixans, trichosan, pachymaran, anemarn, moran, lithosperman, ganoderans, saciharan, (ephedran, atractan (Li, et al, 2004) and, peptidoglycan galactomannans (Sanjay, 2002).

Others Compounds that show excellent antidiabetic effect are numerous. These include polypeptides and amino acids such as the p-insulin from M.charantia, ginseng glycopeptides, and S-alkyl cysteine sulfoxide from A. sativum, hypoglycine A from Blighia sapida (Li et al, 2004; Jose Barbosa Filho et al, 2005). Sterol like charantin from

Ad charuntia; unsaturated fatty acids like linoleic acid from Bombyx mori and

t3 trihydroxyljecoric acid from Bombyx alba L, edyson from Morus alba L and sodium

oxaloacetate from Euonyrnzls alatus (Li et al, 2004). 0 H

\ R' = C-beta-D-xy lopy ranosy l R' OH^,OH 0 R" = C-beta-D-galactopyranosyl Flavone C glycoside

C-H20H - 0 H OH OH 0 0;;\ 0 H \ 0 H

Sappanchalcone

Magniferin

o/N/O- /.

0 ---. OH 0 Kakonem Fig. le: Some flavonoids with antidiabetic activity (Li et nl, 2004) Table 1 Plants with confirmed antidiabetic activity within the tropical vegetation 2 9

Table 1

Plant species and Part of Confirmed active Reference family plant used constituent Sailjay, 2002. Momordica charantia Fruits Ij-sitosterol-D-glucoside Nadkami, 1994; (Cucurbitaceae) and 5,254gmasten-3- Ij- Jayasooriya, 2000 01-D-glucoside Charantin, Welihinda, 1986 foetidin and inomordin I, Leung and Foster, 'Trigonella foenum Seeds 4-hydroxyisoleucine 1996 graecum Molham and (I .eguminosae) Trigonclline Rainan1 998; Broca, 1999 Sharma et al, 1990

Vernonia amygdalina Leaves Alltaloids and glycosides Akah et al, 1992 (Compositae) Gyang, et al, 2004

Meletis and Lagerstroemin Leaves Corosolic acid Bramwell, 2001 ; speciosa Okada, 2003 Kaltuda, 1996; Miyaji 1999 Tacoma saqs Leaves and Tecomine and Altah et al, 2002 (Bignoneaceae) bark tecostamine Garcia and Colis, 1926 Jain and Sharma, Musa sapientum Flowers and Anthocyanins 19G7; Hood and (Musaceae) peels Triterpenes Lowburry, 1954; .,,q..*l..>' Altah et al, 2002

Anarcardium Bark and Quercetol and kaempferol Esimone et al, 2001 occidentale leaves glycosides Curie and Tiinell, (Anacardiaceae) 1959 Akah et al, 2002

Azadirachta indica Fruits, Nimbine Akah et al, 2002 (Meliaceac)' leaves and bark Ociinum gratissimum Leaves Unknown Altah et al, 2002 (Lamiaceae) Manske, 1960; Dioscorea Tubers Dioscoretine Watt and Breyer- dumetorurn brandwijk, 1962. (Dioscoreaceae) Iwu, 1992 Akubue and Undie,

Allium sativum Bulb Ally1 propyl 200 1; (Liliaceae) disulfide Sheela and Augusti, and S-allyl-cysteine 1992; sulfoxide, Bailey and Day, 1989 I' Gymnema sylvestre Leaves Gurrnarin and Shanmugasundarain (Ascelipiadaceae) gymnemic acid et al, 1990 Persaud et al, 1999

Aloe vera Aloe gel and Sap Glucomannan, and Pandey et al, 1995 ; (Liliaceae) from leaves laxative Ajabnoor, 1990 anthraquinone Ginsenosides, Dey et al, 2002 Panax genus Root panaxans, fatty Konno et al, 1985 (Araliaceae) acids, polypeptides, Shibata, 2001 polyacetylenic Ng and Yeung, alcohol, and 1985 polysaccharides Bridelia ferruginea Roots, bark and Tannins, flavonoids Iwu, 1980 Benth, Ling. leaves And Kaempterol

Moinordica foetida Fruits , . ., . Foetidin Marquis et al, 1977 Hordeum vulgare Radicle of growing Alkaloid hordenine Dhar et al, 1968 (Barley) seeds and gramine Lochnerine Catharanthus roseus Leurosine, Svobada, 1964 (Apocynaceae) Leaves . Vindoline and 0 ' Isovindoline

Phaseolus vulgaris Bean seed Stigrnasterin, Sachser, 196 1 (Kidney bean) Quercetin and glucoronic acid

Argyreia cuneate Leaves Flavonal Jain and Sharma (Rivea leavks) heterosides ,1967 Coccinia grandis Tuberous roots Caffeic acid, Jain and Sharma, (Coccinia roots) Quercetin,B- 1967, sitosterol

Blighia sapida Seeds Hypoglycin A and B Goldner, 1958 (Sapindaceae) Ucciani et al, 1964

Morus alba Leaves Glycosides, Moran Sharaf and (Moraceae) A and maracetin Monsour, 1964

Eugenia jambolana Fruits Peptydoglycan and Sanjay, 2002 (Myrtaceae) Triterpenoids

Pterocarpus Leaves (-) Epicatechin, Sanjay, 2002 rnnrsupium Myricetin, (Leguminosae) Quercetin, Marsupsin, Pterosupin and Pterostilbene Securidaca longepedunculata Leaves Undetermined Onyechi and (L) (Polygalaceae) Kolawole, 1 2005

.4lliurn cepa (Onion Bulb Organic sulphur Augustin et a1 bulbs) (Liliirceae) compounds (1974) , ,, . 4 "1. .:' . -

Gyinnospori a Leaves J3- arnyrin, oleanolic Joshi et a1 (1981) rnontana acid

1' Mucuria pruriens Seeds Proteins, Dhar et a1 (1968) mucunine and

Triterpenes, Murraya koenigii L Seeds, bark and root alkaloids, steroids, Barbosa-Filho et a1 (Kutaceae) carbohydrate (2005) 1) Opuntia streptacantha Fruits and Stems Fiber and Pectin Frati et a1 (1990.) (Cataceae)

Coccinia indica Whole plant Unknown Grover et a1 (2002)

Brickellia Entire plant 5,7,3-Trihydroxy- Barbosa-Filho et a1 veronicaefolia 3,6-4- (2005) (Asteraceae) Triniethoxyflavone

Stevia rebaudiana Stem, Leaves Steviol, Isoesteviol Barbosa-Filho et a1 (Asteraceae) (2005) If

Croton cajucara Not stated Diterpene Farias et a1 (1 997) (Euphorbiaceae) Trans- dehydrocrotonin

Salacia reticulate Root and stem Kotalanol and Yoshikawa et a1 (Hippocrateaceae) Salacinol (1 997)

Bauhinia forficate Leaves Kaempferol-3,7-0- Barbosa-Filho et a1 (Fabaceae) dirhaminoside (2005) .,, . 4 "1. .' r'

Bouvardia terniflora Stem bark Oleanolic acid and Barbosa-Filho et a1 (Rubiaceae) ursolic acid (2005)

Cecropia obtusifolia Leaves Isoorientin Barbosa-Filho et a1 (Ceropiaceae) Chlorgenic acid (2005) Phyllatus embira Leaves Fagasterol Barbosa-Filho et a1 (Euphobiaceae) (2005)

Astragalus Roots Polysaccharides Li et al, 2004 membranaceus (Leguminosae)

Plantago psyllium Pods Mucilage Frati-Munari et a1 (Plantaginaceae) (1 985)

Rehinannia Roots Polysaccharides, Li et al, 2004 glutinosa Rehrnannioside A- (Scorphula14aceae) D, Iridoids

Trichosanthes Roots Glycans: Trichosans Li et a1 (2004) ltirilowii A,B, C, D and E (Cucurbitaceae)

Cuminum nigrum Seeds Flavonoids Gilani et a1 (2000)

Pueraria lobata Roots Flavonoids: Li et a1 (2004) (Leguminosae) Kakonien .,,,. .*7, .?' , ,.+

Polygonatup Rh'izomes Spirostanol Li et a1 2004 sibiricum glycoside PO-2 (Liliaceae)

Polygonatum Rhizomes Saponins Li et al, 2004 odoratum (Liliaceae)

Cornus officinalis Pulps Ursolic acid, Li et al, 2004 (Cornaceae) Oleapolic acid Atractylodes Rhizomes Atractans A, B, C japonica (Compositae)

Coptis chinesis Rhizomes Berberine (Ranunculaceae)

Anemarrhena Rhizomes Magniferin Li et a1 (2004) asphodeloides (Liliaceae)

Flavpnoids: Psidium guajava L Leaves Strictinin, Li et a1 (2004) (Myrtaceae) isostrictinin, pedunculagin Table 2

Some bioactive constituents of antihyperglycemic plants and their modes of action Table 2

Modes of action Plants bioactive Reference constituents Charantin, R-sitosterol-D- Sanjay, 2002; Inhibition of glucose absorption glucoside and 5,25- Nadkarni, 1994; in the gut, stimulation of insulin stigmasten-3- 13-01-D- Jayasooriya, 2000 secretion, and the stimulation of glucoside, Foetidin, Welihinda, 1986 hepatic glydbgen synthesis momordin and vicine of Delays gastric emptying A4 charantiu

4-hydroxyisoleucine of Marles and Trigonella foenum Farmsworth, 1995 graecum

Fiber (glucomannan), Evans and aloins, anthraquinones, Rushakoff, 2002 barbaloin, polysaccharides, salicylic acids of Aloe Vera

Increased glucose uptake and Gymnemic acids and Ye11 et al, 2003 utilization, ,, increased insulin Gurmarin of Gylnnerna secretion, and increased p-cell sylveslre number, decreases fasting glucose and HbAlc.

Extract inhibits hyperglycemia Quercetol and kaempferol Esimone et al, 4". . .' by conlpetively blocking ifie glycosides of 2001 Curie and glucose associated receptors on Anarcardium occidentule Timell, 1959 fl-cell membranes, and may also Akah et al, 2002. act by increasing the resistance of I3-cells through activating- the superoxide radical scavenger (I superoxidc dismutase (SOD) Mechanisms of action include Triterpenoid saponin decreased rate of carbohydrate glycosides (ginsenosides Shane-McWhorter absorption into the portal or panaxosides) from (2001) hepatic circulation, increased Panax ginseng, glucose transport and uptake Eleutherococcus

mediated by nitric oxide, senticosus + and increased glycogen storage, and P.quiquefolius. modulation of insulin secretion.

Increased secretion or slowed Ally1 propyl disulfide, a Shane-McWhorter degradation of insulin, increased volatile oil, and S-allyl- (2001); Bailey and glutathione peroxidase activity cysteine sulfoxide, a sulfur Day (1989) and improved liver glycogen containing amino acid storage. from Allium sativum and cepa .

Stimulation of insulin secretion Stevioside from Steviu Barbosa-Filho et a1 by direct action on p-cells of rebaundina (2005) pancreatic islet

Regenerate atrophied pancreatic Alltaloids and L-ephedrine Li et a1 (2004) islets and restore secretion of of Ephedra distachya and insulin Prunella vulgar is

Inhibition of Aldose Reducta~e.,,. ,,.Lipans from Schisandra Li et a1 (2004) and protein tyrosine kinase. sphenanthera and Sappanchalcone of Sanjay (2002) Caesalpinia sapa. Myricetin and quercetin of

-- - Pterocaqms marsupiunz. I1 Increase GLUT4 mRNA and Glycosides gf Cornus protein expression by promotion oflicinalis Li et a1 (2004) of pancreatic islets proliferation 'and increase postprandial glucose transport t ' Modulation of intracellular Forskolin from Coleus Sanjay (2002) secondary messenger such as forskoli causing ATP dependent enhancement ofglucose stiinulation of insulin secretion and activation of adenylate cyclase that increases CAMP biosynthesis. ).' 1.7.2 Pharmacognostic profile of Picrnlima nitida

'I'axonomy

Phyllum - Angiospermae

Subphyllum - Dicotyledon

Order - Gentianales

Family - Apocynaceae

Subfanlily - Plumeroideae

Genus -Picralima IT Species - nitida Stapf

Common Names: Akuamma tree (Ghana), motoko-toko (Cameroon), Osu, osu-igwe

(Igbo), otosi (Idoma), and Erin (Yoruba).

Picralima nitidn (Oliver, 1960 and Iwu, 1993) is a deciduous tree that is distributed in the tropical Africa. It is cultivated and found also in the forest.

The tree is about 5-12m in height and 45cm in girth with dense evergreen crown.

The bark is usually grayish, fibrous yielding rather scanty latex when cut. The leaves are

oblong -1apceolate to broadlyr.oblong-elliptic,, ,, .". shortly acuminate, blade rounded to slightly cuneate. The leaves are dark-green, leathery and glossy glabrous with many parallel venation. The tree flowers twice yearly. The flower is about 5 cm wide, borne on terminal inflorescence that is entirelmd glabrous. The individual flowers possess tubular calyx, slender corolla -tube with narrow elongated lobes.

The plant fruits twice in the year; they are found in pairs hanging down on the long stalk. They are broadly ovoid, smooth, glabrous and leaf-green in colour but turn orange when ripe. They could be 15 cm long and 10 cm wide, and contain numerous

k' disc-like shaped seeds embedded in gelatinous pulp. Their fruits are harvested when their

colour change from green to yellowish orange colour (Dalziel, 1958).

1.7.2.1 Ethnomedicinal uses of Picralima nitida

The aqueous extract of Picralima nitida (the "akuamma" tree) is used in West

Africa as painkiller. In Ghana, pulverized and encapsulated seeds are sold for medicinal

purposes in the market (Neuwinger, 1996). Extracts of various parts of the plant are used

for different types of fever and malaria. In central Africa, it is used for the treatment of

primary hypertension, malaria and jaundice (Iwu, 1993). The crushed seeds are eaten for

stoinachachk and pneumonia. The extracts of all the parts of the plant are used for

venereal disease and as vermifuge. In some West African countries, the dried seeds, fruit

rind and stem bark are highly valued as antimalaria agent and remedy for sleeping

sickness (Iwu, 1993). The seeds of the plant have been reported to cure respiratory tract

infections and as enema in Ghana (Dalziel, 1958). It is used for blood sugar reduction in

western Nigeria and as antimalaria in eastern Nigeria.

1.7.3 Pharmacological studies on Picralima nitirln extracts

.,,,, .*-. .:' , .? ' Extracts of the plant have been reported to possess opioid analgesic activity tt (Menzies et al, 2004). The alltaloids extracted from the plant possess local anaesthetic

effect that is three times the effect of cocaine hydrochloride (Iwu, 1993), and varying 0 degrees of agonist and antagonist activity at opioid receptors but possess neither high

affinity nor selectivity for mu-, delta- or kappa receptors or opioid receptor-like 1-

receptor (Menzies et al, 2004). Akuammidine has been showed to have a sympatholytic

action and hypotensive effect that is weaker than that of yohimbine (Iwu, 1993). The major alkaloids of the tree were shown in in-vitro studies to be active against drug-resistant and sensitive strains of Plasmodium falciparium, clinical isolates of

Leishmania promastigotes and Trypanosomia brucei (Water Reeds Army Institute of

Research Technology for Licensing notice, 1994: Hamet, 1940). The alkaloids posses also central nervous system depressant effect as well as intestinal smooth muscle spasmogenifi activity (Aguwa et al, 2001). The whole extract of the seed has been used as an aphrodisiac (Ayensu, 1978). The alkaloids of Picralima nitida have been reported to inhibit an enzyme, P-D-glucosidase extracted from the local snail, implicated to enhance infectivity of HIV-1. This is indicative of the possible relevance of Picralima nitida in the treatment of human immunodeficiency virus infection (Akpan and Umoh, 2004). The ethanol whole extract of the seed, stem bark and root of the plant showed antibacterial activity against a wide range of bacteria that cause diseases in both humans and animals

(Nkere, and Ireogbu, 2005).

Thetiaqueous extract of the seed of.this plant has been reported to lower blood glucose levels in normoglycenlic and hyperglycemic rabbits (Aguwa el al, 2001).

1.7.4 Chemical constituents.of Picf(~I&zn'nitida.

Alkaloids form the highest percentage of the secondary metabolites found in the plant parts. They include the indole and dihydroindole alkaloids: akuammiline, akuammidine, aluainmine, akuammigine, pseudo-akuammigine, akuarnmicine, echitamine, picraline, picratidine, picracine, picraphylline (Iwu, 1992); the non-alkaloid compounds include acidic saponin, P-amyrin, isolated from the stem bark and many t' others yet to be discovered. 1.8 Aim and Scope of study

'l'he short supply, side effects and cost of orthodox drugs used in the management

t ' of diabetes mellitus, high cost of hospital management of the disease, non availability of competent health personnel in the mansgement of diabetes and long distance to health facilities made many diabetic patients in the economically distressed countries of the world to turn to Phytotherapy for the management of their pathologic conditions ( Akah et al, 2002; Gyang et al, 2004)

A preliminary study on aqueous extract of Picralima nitida seeds showed reduction of mean fasting blood sugar levels in alloxanized laboratory animals. This earlier findings stimulated my research interest into this plant which the natives of Ihiala use for the I' treatment of malaria and high blood sugar among other disease conditions.

The aims of this research therefore are:

I. To evaluate the blood glucose lowering effects of the Picrdinza nitida seed extract.

2. To isolate and phytochemically identify the bioactive constituent(s) responsible for the blood glucose lowering activity.

,,,..v ,..?' 9 .a ' 3. To propose the possible mechanism(s) of action of the plant's phytochemical constituents in the blood glucose lowering effect using laboratory animals. Figure 2a :Photograph of Picralima nitida plant f' . .. Figure 2a :Photograph of Picralima nitida plant Figure 2b: Photograph of Picralima nitida seeds I'

CHAPTER TWO

MATERIALS AND METHODS 2.0 MATERIALS AND METHODS

2.1 CHEMICALS AND REAGENTS I" 'The following chemicals were used in the course of this research: phenol red, alloxan inonohydrate (Sigma, USA), glucagon injection (Eli lily, USA), glibenclamide (NGC,

Nigeria), Glucose (Fluka, Germany), n-butanol, diethyl ether, n-Hexane, trichloromthane

(Sigma- Aldrich, Germany), ethyl acetate, methanol, chloroform, hydrochloric acid,

Trichloroacetic acid, sodium bicarbonate, ammonia.hydroxide ( Merck, Germany), Silica

gel (60-200mesh, Burbiges & Co, India), acetone,' Accu Check blood glucose estimation

kit (Roche, Germany), Sodium hydroxide, sulphuric acid (BDH, England).

tr 2.2 COLLECTION OF PLANT MATERIAL

The pods of the Picralima nitida were collected from Ihiala, Anambra state,

Nigeria in February 2004 by a herbalist and authenticated by Mr. J. A. Ekekwe,

Department of Botany, University of Nigeria, Nsulcka. The voucher sample was

deposited in the departmental herbarium.

2.3 EXTRACTIONS . ,. .. .I. ... .

The matured pods were broken open; seeds were collected and air-dried for six

weeks. The,seeds were pulverized and 2 kgof the powdered seeds was macerated in 5 L

of chloroform at room temperature for 72 h with intermittent shaking. It was filtered and

the marc was air dried for 2 h and macerated with 5 L of 90 % methanol for 7 days. The

methanol extract (ME) and chloroform extract (CHE) were concentrated to solid residues

at room temperature, and stored in the refrigerator until evaluated for blood glucose

lowering activities and phytochemical tests done ( Harbone,1984). 2.3.1: Fractionation of Alkaloids and Glycosides from methanol extract

A. Alkaloids fractionation

The methanol extract (ME) of the seed was weighed (130.2 g) and dissolved in

70% methanol and made alkaline with ammonia hydroxide (pH > 7). The mixture was

continuously shaken gently for one hour and extracted with equal ratio mixture of

chloroform after acidifying with dilute HCL (0.1N). The chloroform phase was basified

with ammonia hydroxide (pH >7) and dried to constant weight by heating over a water

bath at 45°C to obtain the alkaloids (AKF) (Brain and Turner, 1975). Dragendorff

reagent, Wagner's reagent and Hager's reagent were used to confirm the presence of

alkaloids (Evans, 1989).

B. Glycosides fractionation

1. The methanol extract (ME) of the seed was weighed (1 50.6 g) and dissolved in 70%

methanol; this was vigorously shaken for 10 minutes and warmed on a water bath at 45OC

for 5 minutes. After cooling, 5 ml of 5 % lead acetate was gradually added and filtered

until no mQre precipitate was formed. The filtrate was pooled and concentrated into

syrupy paste over a water b~itli"at"45~~60~C(Gyang et al, 2004). The paste was partitioned

into aqueous (AQP) and chloroform (CHP) phases and tested for glycosides (Sofowora,

1993).

2. n-Butanol extract of aqueous soluble phase of glycosides

The aqueous phase (AQP) from the glycosides fraction was reduced to half the

volume using rotary evaporator and extracted with 200 ml of n-butanol repeatedly until

n-butanol fraction was clear. The n-butanol fraction (nBF) was concentrated to pasty

t; residue, and tested for glycosides (Sofowora, 1993). tl 3. Column chromatography of nBF of Picrnlima nitida extract

The solution of n-butanol fraction was mixed with 20 g silica gel (60-200 mesh) and allowed to dry into a free flowing powder. The powder was packed on top of dry- packed Silica gel (500g) colun~nand eluted with chloroform: methanol: water (65:20:2);

40 bands of 160 mL aliquots were collected. The bands were pooled together based on the RFvalues of their constituents in preparative thin layer chromatography plate (20 x 20 cm) coated with silica gel 60 (60-200pm) using chloroform: methanol (1:6) into eight

fractions and tested for hypoglycemic activities (Jalalpure et nl, 2006).

11 The most potent hypoglycaeinic fraction, (1 5.21g) was further subjected to silica

gel 210 g, 60-200 mesh) column chron~atographyeluting with 11-hexane, chloroforin,

ethylacetate, acetic acid acidified acetone and methanol to generate six bands of 50 mL

aliquots. They were further tested for hypoglycemic activities (Jalalpure et al, 2006) and

the phytochemical constituents of the bands determined.

2.4 ClIARACTERIZATION

2.4.1 Thin layer chromatography. ,+ .... (.TLC).of Fraction F7bE w According to Niederwieser et a1 (1966) modified method, the fraction was subjected

to thin layer chromatography using preparative thin plates 20 x 20 cm coated to 0.75 min

with silica gel (Kiesegol) 6OG slurry, and activated in the oven at 10.5' C for 1 11.

Fraction F7 bE was dissolved in methanol and applied manually, using capillary tube, 2

cm from the edge of each plate. The plates were developed with methanol: chloroforn~

(5:2) mixture for 4.5 minutes and air-dried. The' spots were visualized with UV lamp

under short and long wavelengths and Rf values were determined. 2.4.2 Determination of Wavelength of maximum absorption (A,,,) of the respective I' spots of Fraction F7bE

Each of the marked and scrapped out spots was placed in clean empty bottle and redissolved in undiluted chloroforin and ethanol respectively (Scott, 1964). The hMaxand absorbance was determined using UV spectrophotometer (Hach DlU4OOU

Spectrophotometer, USA). Undiluted chloroform and ethanol were used as blank.

2.4.3 Chemical identification of the respective spots of Fraction F7 bE

The respective spots (SI-S3)were then subjected to chemical identification (Scott 1964;

Harbornc, 1984; Evans 1989). t) 2.4.4 Determination of the Nuclear Magnetic Resonance (NMR) and Mass

Spectrophotometry (MS) of Fraction F7 bE spots

Nuclear magnetic resonance (NMR proton and carbon) and MS for the respective spots of F7 bE were done.

2.5 ANIMAL STOCKS

Albino mice (20-358) and rats (185-2008) of either sex, bred in the animal unit, . ,, a,,. .' a Departinen(, of Pharmacology, University qf Nigeria Nsuklta were used for this study.

They had access to water before and during the experimental stages and were fed with

standard feed from Pfizer Plc, Lagos. he animals were kept for one week prior to

experimentation at room temperature under standard 12-h lightldark cycle. 2.6 In vivo PHARMACOLOGICAL EXPERI~ENTS

2.6.1 Acute Toxicity studies: The acute toxicity test of the methanol extract, LDSo,was determined prally and intraperitoneally in mice using Lorlte's method (1 983).

A. LDSo using oral route in mice: The study was divided into two stages. In the first stage, the mice were divided into four groups of three mice per group. The mice were given the following doses of the methanol extract of P. nitidu seed: 10 mglkg

(group I), 100 mg/kg (group 2) and 1000 mg/kg (group 3) while the control group received 3 mllkg normal saline (group 4) after fasted for 12 hours. The number of nice

1 dead per group within 24 hours was nil hence the second stage was commenced.

Fresh sets of mice were divided into four groqs of three mice per group and

Casted for 112 hours. The mice given 2000 mglkg (group I), 4000 mgtkg (group 2), and

8000 mglkg (group 3) of methanol extract of Picrali~nanitidu seed, and the control group received 5 mllkg of normal saline. None of the animals treated with the extract died after

24 hours.

13. LDS0using intraperitoneal route in mice: The study was done like that of the

oral route above using the following doses of methanol extract of P.nitidu seed: group 1 - , 4 1. d' . ' received 100 mglkg, group 2 received 500 mg/kg, group 3 received 1000 mgikg and

control group received 3mllkg normal saline. All the animals in group 3 died while none

in group 2 died. The LDjowas calculated aceording to modified Lorke's method (1983). 2.6.2 Effects of methanol (ME) and chloroform (CHE) extracts of Picralimn nit& seed on fasting blood glucose levels in normal rats Eight normal rats of either sex were fasted for 24 h but had access to water. They were then divided into two groups of four rats per group. Blood sample was withdrawn from the tail vein of each animal and the blood glucose level determined using

Glucometer (Lifescan, USA). Fifteen minutes after the blood withdrawal, each of the 4 rats in group 1 received methanol extract 3 g/kg orally, while the group 2 received chloroform extract 3 g/kg orally also. The blood glucose levels were determined at fixed time intervals for 12 hr after the drugs were administered. Another set of nine rats were grouped into three of three rats per group, and treated with 1.67 g/kg (group I), 2.5 g/kg

(group 2) and 5 g/kg (group 3) of methanol extract.

I' 2.6.3 Effect of methanol extract (ME) of Picralima nitidfc seed on fasting blood glucose levels in normal rats The normoglycemic rats were fasted for 24 11 but had access to water ad libitum through out the experiment. Three groups of 6 rats per group were used, one group each for methanol extract (5 g/l

first group r~eceivedmethanol extract 5 g/l& the second group was given 5 mglkg of

glyburide (NGC) and the third group received 3 mllkg distilled water. Blood samples

were withdrawn from the animals at fixed time intervals (111, 2 h, 4 h, 8 h, 12 and 24 h)

after the administration of the respective drugs and the blood glucose levels determined. 2.6.4 Effect of methanol extract (ME) of Picrcxlima niiinn on fasting blood glucose levels in alloxanized rats

Normal rats of either sex, with blood glucose levels between 58-100mgIdl before fast, were fasted for 12 h before they were used for this study. The animals were given

120 mglkg of alloxan monohydrate (Sigma, USA) intraperitoneally. The alloxanized animals were kept for 7 days for hyperglycemia to develop with free access to food and water (Aguiwa et al, 2001). The animals were fasted on,the day 8 for 12 h and their blood glucose levels were determined using the glucometer kit (Lifescan, USA). The rats with blood glucose levels above 150 mgldl were divided into three groups of 4 animals per group. The animals in the groups were respectively given methanol extract (5 glkg),

glyburide (5 mglkg) and distilled water (3 mllkg). All the drugs were administered orally.

At fixed tin'ie intervals for 24 h, the blood samples were collected and blood sugar levels

determined using Glucometer.

2.6.5 Effect of methanol extract (ME) of Picrnlima nitida seed on oral glucose load in normal rats

Normal rats were fasted .for 24 h and their weight taken before the , ,, . .., .: .' i coinmencement of the experiment. They were grouped into four of 4 rats per group with

access to water ad libitum before and during the evaluation. The blood glucose levels of

the animals were determined before the exiracts and glucose were administered. The

bl anirnals in the first group were given methanol extract (5 glkg), second group received

glyburide (5 mglkg) and the control gronps, the third group received glucose (1 gkg) and

the forth group received distilled water (3 mlkg). The extract and glyburide were

administered one hour before glucose load (1 glkg). ,At fixed time intervals, after the drugs and glucose administration, the blood glucose levels were determined for three hours (1 .O, 1.5,2.0, 2.5 and 3.0 h) using Georgette et a1 (2005) procedure.

2.6.6 Effect of alkaloids (AKF) and glycosides fractions of methanol extract of Picmlima nitidn on fasting blood glucose levels in normal rats Norpal rats of both sexes were fasted for 24 11 but had access to water ad libitum throughout the experiment. Four groups of four rats per group were used, groups one and two for alkaloids fraction at doses of 250 mgkg-' and 500 mgkg-', and groups three and four for glycosides fraction at doses of 250 mgkg-'and 500 mgkg-'; all the animals received the drugs ip. At the end of the fast, blood was withdrawn from the tail vein of the animals and the blood glucose levels were determined as the 0 h level. After the 0 h blood withdrawal, the animals in all the groups received the respective doses of their drugs. Blood samples were withdrawn from the animals at fixed time intervals (1,2,4, 8,

12 and 24 h) after the administration of the respective drugs and the blood glucose levels determined using Cilucometer kit (Life scan, USA).

2.6.7 Effect of alkaloids and glycosides extracts of Picralimn nitidn on fasting blood glucose levels in alloxanized rats Albino rats of either sex with blood glucose levels between 78-1 10 mgldl were , ,,. dh,. 3. , fasted for 12 h before use. They were given 120-mglkg of alloxan monohydrate (Sigma,

USA) intraperitoneally. The alloxanized animals were kept for 7 days for hyperglycemia to develop and stabilize but had free. access to food and water (Aguwa et al, 2001). The animals were fasted on the day 8 for 12 h and their blood glucose levels were determined

,i using the glucometer kit (Lifescan, USA). The rats with blood glucose levels above 150 mgldl were sorted into four groups of 4 animals per group while the normal rats formed the iifih group (Jalalpure, 2005). Group 1 and 2 were given 250 mglkg and 500 mglkg of glycosides fraction respectively, group 3 received glyburide (5 mglkg) and group 4 received distilled water (3 mllkg), the fifth group received 250 mgllcg of alkaloids. All the drugs were administered ip. At fixed time intervals (lh, 2 h, 4 11, 8 h, 12 h and 24 h), the blood samples were collected and blood glucose levels determined.

2.6.8 Effect of aqueous (AQP) and chloroform (CHP) phases of glycosides fraction of Picrnlima niticln on fasting blood glucose levels in alloxanized rats Twenty albino rats of either sex, with blood glucose levels between 78-1 10 mgldl were fasted for 12 11 before use. Sixteen animals were given 120-ingllcg of alloxan monohydrate (Sigma, USA) intraperitoneally. The alloxanized animals were kept for 7 days for hyperglycemia to develop and stabilize but had free access to food and water.

The animals were fasted on the day 8 for 12 h and their blood glucose levels were determined using the glucometer kit (Lifescan, USA). The rats with blood glucose levels above 150 mgldl were sorted into four groups of 4 animals per group while the normal rats formed the fifth group. Group 1 and 2 were given 250 mgkg-' aqueous extract and

250 mgkg bf the chloroform phase of glycosides fraction respectively, group 3 received glyburide (5 mglcg -I), group 4 received distilled water (3 ml kg -') and the fifth group was the normal rats. All the drugs were administered ip. At fixed time intervals (1, 2, 8 and 24 h), the blood samples,.werct collected and blood glucose levels determined in accordance with Jalalpure et al(2006)

2.6.9 Prolonged treatment effect of n-Butanol extract (nBF) of glycosides fraclion of Picrnlima nitirkr on fasting blood glucose levels in alloxanized rat

Twenty albino rats of both sexes were fasted for 12 h on the first day of the experiment land sixteen of the rats were treated ip with 120 mgkg alloxan monohydrate

(Sigma, USA) and tested for hyperglycemia after 7 days. Animals with blood glucose level above 150 mgldl were considered hyperglycemic. They were grouped into five of fbur rats per group. Group 1 received n-butanol extract of aqueous glycosides fraction at

2.7.2 Effects of chromatographic fractions (F4,5,7-8)of nBF of Picrnlinza rzitida on fasting blood glucose levels in glucagon-induced hyperglycemic rats

Normal rats of both sexes fasted for 24h and their weights taken before the coininencement of the experiment. The rats were randomly divided into eight groups of 4 rats per group with access to water ad libitum before and during the evaluation. The blood glucose levels of the animals were determined before the drugs were administered.

Glucagons, 0.4 yglg, (Eli Lily, USA) was administered intraperitoneally to each of the animals onk' hour post administration of 250 mgkg of fractions F4, Fj, F7,F 8, E, ME and glyburide, and one group was given glucagon alone. The animals in the first group were given F4, (250mglkg with 0.4 pglg glucagon), second group received Fj (250 mglkg with

0.4 pglg glucagon), the third group received F7 (250 mglkg with 0.4 pglg glucagon), the fourth group received F8 (250 mglkg with 0.4 pglg glucagon), the fifth group received E

(250 mgkg with 0.4 pglg glucagon), the sixth group received ME (250 mglkg with 0.4 pglg glucagon), the seventh group received glyburide (5 mglkg with 0.4 pg/g glucagon) and the eighth group received glucagon alone . At fixed time intervals (5, 10, 20, 30, 60

I' and 120 minutes) the blood glucose levels were determined using the blood glucose

., ,, a"?. ,!. . ,? ' estimation kit.

2.7.3 Effect of Picralima nitida seed extract on Gastric emptying in normal and hyperglycemic Rats

Measurement of gastric emptying: Rats were fasted for 24 11 prior to the experiments but had access to water ad libitum. Gastric emptying was determined by modification of

Matsuda et a1 (1999a) method. Forty percent glucose solution containing 0.05% phenol red was given to the rats p.o (OSmlIrat). The rats were sacrificed by cervical dislocation I' 30 minutes later. The abdominal cavity of each rat was opened, and the stomach clamped ti at the esophageal and pyloric junctions were removed. The removed stomach was placed in 20 1111 of 0.1N NaOH and homogenized. The suspension was allowed to stand for 1 h at room temperature, and 5 ml of thz supernatant was added to 0.5 ml of 20 %

Trichloroacetic acid (wlv) and centrihged at 3000 rpm for 20 minutes. The supernatant was mixed with 4 ml of 0.5 N NaOH, and the amount of phenol red was determined from the absorbance at 560 nm. The phenol red recovertd from the rats sacrificed immediately after administration of 40% glucose meal p.o was used as standard test. The gastric emptying in 30 minutes period was calculated according to the equation below and serum ki glucose was determined by the glucose oxidase method (Accu Check,).

TS.4 GE(%)= (1 - ----)x100 ; GE=Gastric emptying (%); TSA= amount of test sample; SSA= ss* amount of standard

Gastric emptying of Test mcal (40 O/u Glucose) in normal rats: Three groups (A-C) of four rats per group were used. Groups B and C received methanol extract (ME, 250 mglkg) and fraction E (250 mglkg) respectively one hour before the administration of the test meal (40% glucose), and K'fkceived test meal only. The gastric emptying and blood

glucose levels were determined 30 minutes after administration of test meal. The drugs

were administered intraperitoneally.

,I Gastric emptying in glucose (i.v.)-Induced hyperglycemic rats: A 10% glucose saline

solution (10 mllkg, i.v.) was administered to fasted rats. The rats were randomly

distributed into three groups (A-C) of four rats pei group. The group A received the test

meal only, while B and C received test meal one hour after the drugs administration. The gastric emptying and serum glucose levels were determined 30 minutes after test meal administratibn.

Gastric emptying in Alloxan-induced hyperglycemic rats: Alloxan monohydrate, 120 mglkg, was injected i.p to rats and allowed to develop diabetes 72 h before administration of the drugs. Rats with fasting blood glucose levels of above 150 mgldl were used for the study. Groups B and C received 250 mgkg of ME and fraction E respectively while A received test meal only. Gastric emptying and blood glucose levels were determined 30 minutes after administration of test meal.

2.8 Preparation of Calibration Curve of Phenol Red in Glucose solution: Various ti concentrations of 0.05% Phenol red solution were mixed with 40 % glucose solution to form a series of standards consisting of 50.0 pglml, 25.0 pglml, 15.0 pglml, 7.5 pglml, and 3.5pglml. To each of the concentration was added 20 ml of 0.1N NaOH and homogenized. The suspension was allowed to stand for 1 h at room temperature, and 5 ml of the supernatant was added to 0.5 ml of 20 % Trichloroacetic acid (wlv) and centrifuged at 3000 rpm for 20 minutes. The supernatant was mixed with 4 ml of 0.5 N

NaOH, and the absorbafice 'df 'th'e"iiifferent concentrations of phenol red was determined at 560 nln (Matsuda eta1 , 1999a).

I1 2.9 STASTISTICAL ANALYSIS

Results are given as mean blood glucose levels * SEM (standard error of mean).

One way ANOVA with post hoc Dunnett's multiple comparison tests. P values of 0.05 and less were taken to imply statistical significance between the means. Analysis was done using Statistical Programme for Social sciences (SPSS) version 7.5 tl CHAPTER THREE

RESULTS

. ,, .hl 5.' ? +I

3.1 Extractive Yields

The yield of the different extracts and fractions was expressed in percentage of the powdered seeds material as shown in Table 3. ME had the highest yield and F7 bE had the least yield.

3.2 Phytochemical analysis of extracts and fractions The result of the qualitative phytochemical tests of the chloroform (CHE) and methanol (ME) extracts, and the alkaloids (AKF), aqueous (AQP) and chloroform (CHP) phases of dycosides are shown in Table 4a, while RBF and F7 bA-F chromatographic fractions are shown in Table 4b. From Table 4a, alkaloids, glycosides, tannins and carbohydrates are present in both the CHE and ME while saponins, sterols1 terpenes and flavonoids are either in trace amount or absent in the CHE; and the AQP contained every

secondary metabolites found in ME except tannins.

From table 4b, the fractions F7 and F7bE:were found to be rich in triterpenoid saponins..

3.3 Acute toxicity tests I]

- ,,,.*I. 3:. , .'> The methanol extract caused no death in mice after oral administration even

above 5 gkg. But when intraperitoneal route was used, the acute toxicity test (L&)) was

calculated to be 707.1 1 + 32 mg /kg. ,I . .. Table 3

Extractive yields of extract and fractions of P.nilida seed. ME-methanolic extract; CHE-chloroform extract; AKF-alkaloids extract; CHP- chloroform phase of glycosides extract; AQP- aqueous phase of glycosides extract; nBF-

kt butanol extract, Fq, F5, F7. Fs, F7bE fractions of nBF extract. Table 3

EXTRACTIFRACTION PERCENTAGE YIELD (%) ME 12.10

CHE " AKF CHP AQP nBF F4 Table 4a Phytochemical constituents of the Picralima nitida seed extracts. AKF-alkaloids extract, CHP- chloroform phase of glycosides extract and AQP- aqueous phase of glycosides extract 1'

Table 4a

Phytochemical constituents ME CHE AQP CHP AKF Alkaloids ++ ++ + + + G lycosides ++ + ++ - -

Saponins ++ ' f + + f -

Carbohydrates + + + f - Fats/ oils - + - - -

3-+ Moderately present, + Present, h trace, - absent Table 4b

Phytochemical Constituents of the n-bmanol fractions (F4, F5, F7,Fs and F7 bE) of

Picralimu nitidn seed Table 4b

Phytochemical constituents I F4 F5 h F8 h bE Alkaloids + - f f -

Glycosides +.+ + + +

Sterols f f f k -

Triterpenes + + + + + ,' Flavonoids + + + + f

Carbohydrates + + + + +

++ Moderately present, + Present, =k trace, - absent;

. ,, r*, ..' 3 3.4 CIIARACTERISTICS OF F7bE SPOTS (S1-S3) The fraction F7 bE spots characteristics under the short and long wavelengths and their

Rf-values die shown in Table 5. The spots gave fluorescent blue, green and gray

colorations under the different wavelengths which were indicative of the presence of

some phenol compounds (Touchstone, 1978).

The wavelength of maximum absorption for S1was 239 nm and absorbance of 0.376; S2

was 245 nnl and 0.883 and S3 was 272 nm and 0. 195 in chloroform as solvent system.

But in the ethanol solvent system, S1wavelengths: were 212, 224, 239, 344 and 371 nm;

S2 was 242, 347 and 356 nm; and S3 233, 344 and 353 nm. Their respective wavelength

scans are shown in Appendix 1,2,3,4,5 and 6. The 'H and I3cNMR for S2 are shown in

I1 Appendix 8 and 9. Table 5 Ultraviolet characteristics of the F7bE spots showing the existence of phenolic compounds Table 5

Rf values

I Sample (Methanol: chloroform 5: 2) UV characteristics

Long wavelength (gray) 1

Long wavelength (green) I Normal light ( yellow)

Long wavelength (blue) Normal light (faint yellow) Figure 3 Extraction and screening procedure of Picralima nitida seed POWDE 1.DSEED METHANOL CHLOROFORM EXTRACT (ME) EXTRACT

$I PRELIMINARY PH~LRMACOLOGICALSCREENING TESTS

FRACTION 1 PHARMACOLOGICAL SCREENING TESTS I

First column chromatography

+ PHARMACOLOGICAL SCREENING TESTS

Second column chromatography

1 1: v 1 1

PHARMACOLOGICAL SCREENING TESTS Figure 3' 3.5 Effect of methanol (ME) and chloroform (CHE) extracts of Picralima nitida on fasting blood glucose levels in normal rats. The methanol extract (ME) at 3 gtkg caused the maximal reduction in the mean fasting blood glucose levels from 54.3k0.95 at 0 h to 44.3i 1.26 mgldl at 12 h when administered orally while CHE did not cause reduction in mean fasting blood glucose levels (Table 6). The percentage reduction of mean fasting blood glucose levels of the

ME was 18.4% at the twelfth hour

3.6 Effect of methanol (ME) extract of Picralima nitida on fasting blood glucose levels in normoglycemic and alloxanized rats The ME extract caused reduction in the fasting blood glucose levels in a dose- dependent manner with 5 gkg showing a significant reduction (pC0.05) which translated into 24.2% maximum reduction of mean fasting blood glucose levels in normal rats at the twelfth hour (Table 7). The extract caused increase in mean blood glucose levels in the first hour before it caused maximal reduction that amounted to

20.0% maximal reduction of the mean fasting blood glucose level (Table 8) while the positive standard, glyburide caused 25.9% maximal reduction at the same time after it caused similar rise in mean fasting blood glucose levels in the first hour.

In the alloxanized rats, 5 gkg dose of the ME lowered the mean fasting blood glucose levels (Table 9). Its, ,, blood. "I. .: ,glucsse lowering effect commenced within the first hour and climaxed at 12 h. The effect of glyburide followed the same pattern with the maximal reduction of mean fasting blood glucose level at the eighth hour. The percentage maximal reduction in Tean. blood.. glucose levels produced by glyburide and ME were 71.9% and 49.7% respectively which were significant (p<0.05) when compared to that of distilled water (0.9%). 3.. 7 Effect of methanol extract of Picrnlima nitida seeds on mean blood glucose levels of rats orally fed with glucose.

In the group of rats fed with glucose alone (1 g/kg), their peak blood glucose concentrations were reached in 90 minutes post administration and thereafter decreased gradually. The mean peak blood glucose concentration (81.50 % 2.6 mgldl) achieved after administration of the glucose alone was reduced by methanol extract (38.65 %) and glyburide (47.24 %) respectively when administered one hour before glucose administration (Table 10). The methanol extract and the standard drug, glyburide, reduced the mean blood glucose concentrations of the treated rats below their basal values at zero hour; this is indicative of the blood glucose lowering activity of the

Picralima nitida extract and possible improvement of glucose tolerance though not as

effective as glyburide.

The percentage reduction of the peak blood glucose concentration was determined with the method adopted by Georgette et a1 (2005). Table 6 Effect of methanol (ME) and Chloroform (CHE) extracts of Picralinza nitida seed on fasting blood glucose levels of normoglycetnic rat. Animals per group=4; Standard error ofmean (* SEM) and number in parenthesis is representative of glucose levels % reduction. Table 6

Mean fasting blood glucose levels (mgdl-'h SEM) in normal rats Extract ...... gk ...... Time (h)...... 0 1 2 4 8 12 24

CHE 46.71k0.9 62.4Ik1.8 63.21k2.2 72.6Ik2.5 49.5Ik1.2 51.3Ik1.7 56.7h1.1 (3 .O) (0.0) (-33.6) (-35.4) (-55.7) (I) (-10.0) (-2 1.5) Table 7 Effect of different doses of methanol extract of Picralima nitida on fasting blood glucose levels of normoglycemic rats.Animals per group (n)=4; Standard error of mean (* SEM) and number in parenthesis is representative of glucose level % reduction ; * p< 0.05 level of significance vs 0 h of same dose. Table 7

Mean fasting blood glucose levels (mgdl-'2t SEM) in normal rats Extract ...... dkg ...... Time (h)...... 0 1 2 4 8 12 24 Table 8

Effect of methanol extract of Picralima nitida seed on fasting blood glucose levels in normoglycemic rats. Ratslgroup (n) = 4; standard error of mean (* SEM) and number in parenthesis represent glucose level % reduction.. Table 8

Mean fasting blood glucose levels (mgdl-'k SEM) in normal rats Drugs ...... mgk ...... Time (h)...... 0 1 2 4 8 12 24

Glybu 48.2k0.7 52.2*1.1 48.0k0.8 42.7k1.1 39.8k0.6 35.7*0.7 41.6k1.2 ride (5.0) (0.0) (-8.3) (0.4) (1 1.4) (1 7.4) (25.9) (20.2)

Dist. 51.7*0.5 55.3k0.8 56.3k0.4 50.0*0.5 58.8k0.6 54.3k0.7 55.1*1.4 water (0.0) (-6.9) (8.9 (0.7) (21 (-5.0) (-0.9) (3 mu Table 9 Effect of methanol extract (ME) of Picralima nitida seed on fasting blood glucose levels of alloxanized rats. Animals per group (n) = 6; Standard error of mean (* SEM); and number in parenthesis is representative of blood glucose % reduction. * p < 0.05 level of sig.vs 0 h of the same dose levels. " p< 0.05 level of sig. vs Dist. Water. Table 9

Drug Mean fasting Blood glucose Levels (mgdl-'4 SEM) in diabetic rats 1'

Glyburide 418.0 372.2 305.2a 281.1 117.4" 122.8 132.8 (5 mglkg) k21.9 f14.7 k12.5 +20.9* +12.7* k11.3" k12.7

Dist. 502.3 513.0 554.3 509.8 497.7 505.8 507.2 water k1.O k0.5 k1.3 +1 .O k1.8 k1.8 k12.9 (3 mllkg) (0.0) (-2.1) (-10.4) (-1.5) (0.9) (-0.7) (-2.5) . ,, .-1. .t. a ' Table 10

Effect of methanol extract on blood glucose level of glucose fed normal rats. Animals per group = 4; Standard Error of the mean (* SEM). All values of mean fasting blood glucose levels are in mg/dl. Table 10

Drug Mean Blood glucose Levels (mgdl-'* SEM) in glucose fed rats

1' ME (5 g/kg)+ 52.30*4.6 53.0W2.9 50.00*7.9 48.50*2.6 49.00*3.7 40.05*3.6 Glucose ( 1 gw

Glyburide (5 mglkg) +Glucose 52.0W3.9 46.51*4.7 43.00*3.7 41.50*3.8 36.00*4.5 28.80*4.7 (1 dkg)

Glucose (1 glkg ) 42.30*2.5 74.OO*l.9 81 SOk2.6 76.00*3.7 64.80*4.8 5332.4

Dist. water 5 1.08*4.6 52.04k3.6 5 1.00*2.8 5 1.98*2.5 52.04*1.4 5 1.99*2.6 (3 mllkg) . ,, .!' + ' - 84 b

3.8. Effect of alkaloids (AKF) and glycosides (gly) fractions of Picrnfima nitida seed on fasting blood glucose levels of normoglycemic and hyperglycemic rats.

The rats treated with 250 mglkg of alkaloids fraction showed increase in fasting blood glucose levels above the 0 h blood glucose baseline value while some rats treated

with 500 mglkg of alkaloids fraction died of hyperglycemia at the 4 11. The rats treated

with lower dose of glycosides (250 mglkg) fradtion experienced consistent reduction

(p<0.05) in the mean fasting blood glucose levels while those treated with 500 mglkg

showed lower reduction in mean fasting blood glucose levels (Table 11). The 250 mglkg

and 500 rnglkg doses of glycosides fraction of Picralima nitida seed extract caused

maximal reduction of 38.6% and 22.9 % respectively in normoglycemic rats.

In hyperglycemic rats, 250 mgkg dose of glycosides significantly reduced

(p<0.05) the mean fasting blood glucose levels while the 500 mglkg dose caused blood

glucose level reduction from 425.83k 4.2 at 0 h to 259.58k12.5 mgldl at 12 h. The blood

glucose reduction by glycoside commenced from the first hour and climaxed in the

twelveth hour like the standard drug, glyburide (Table 12). The 250 mglkg dose oi

glycoside cqused 64.4 % reduction in mean blood glucose level of diabetic rats while the

,, .,I. .' r glyburide caused 65.8 % reduction.

The aqueous and chloroform phases of the glycosides fraction were used to treat

hyperglycemic rats in accordance with Jalalpure et a1 (2006) method at same dose level

(250 mglkg). The aqueous phase caused significant reduction (p < 0.05) in fasting blood

glucose levels from 164.0 *4.56 mgldl at 0 h to 77.01- 1.01 mgldl at 8 h that translated

into 53.1 % reduction of mean fasting blood glucose levels while the chloroform phase

caused reduction of mean fasting blood glucose levels from 21 5.75*6.40 at 1 h to 133.0*

7.98 at 8 hwhich amounted to 38.35 % reduction (Table 13), and the glyburide caused 63.9% reduction of the mean fasting blood glucose levels. Thus, the aqueous phase caused significant hypoglycemia in diabetic rats that is significantly different from that of the diabetic control group.

The 11-butanol fraction of the aqueous phase caused significant (p < 0.05) reduction of mean blood glucose levels from 393.00 + 12.54 mgldl on day one to 66.85 2

6.51 mgldl on day ten that amounted to 82.99% reduction on prolong treatment. The methanol extract caused reduction of mean blood glucose levels from 307.75 2 21.84 mgldl on day one to 60.00 2 10.06 mgldl on day ten that amounted to 80.5% reduction while the standard drug, glyburide, caused the reduction of mean blood glucose levels from 203.50 2 18.52 mgldl on day one to 79.75 2 12.29 mgldl on day ten that amounted to 60.81 % (Table 14). All the groups treated with extracts and standard drug had significantl$ different (p < 0.05) mean blood glucose levels from the diabetic control group on the tenth day of treatment. Table 11 Effects of alkaloids and glycosides fractions of Picralima ~titidaseed extract on fasting blood glucose levels of normoglycemic rats. Number of animals per group (n) = 4. Numbers in parenthesis represent % reduction of mean fasting blood glucose levels. Standard error of mean (* SEM). * P < 0.05 level of sig. vs. Oh of the same dose. Table 11

Mean fasting blood glucose levels (mgdl-'+ SEM) in normal rats Drugs ......

mgk ...... Time (h) ...... 0 I 2 4 8 12 24

Alkal 70.0+2.1 95.5+l.O 1 O2.2+2.3 79.2+5.0 74.2+2.8 75.8+5.2 76.4+4.5 oids (250) (0.0) (-36.4) (-46.0) (-13.1) (-5.9) (-8.3) (-1 8.2)

Glyco 108.3+2.0 110.8+2.9 94.7+4.6 91.3+4.2 79.2+2.7 67.2+2.9 86.5+5.7 sides (250) (0.0) (-2.3) (12.6) (1 5.7) (26.9)* (38.6)* (29.5)

Glyco 94.7+5.1 88.8+3.7. *q-480:8+6.7' 82.2+2.5 75.5+6.2 73.0+7.6 98.7+3.4 sides (0.0) (6.2) (14.6) (13.2) (20.3) (22.9) (1 9.7) (500) Table 12

Effect of glycosides (gly) fraction of methanol extract of Picralima nitida seed on mean fasting blood glucose levels of alloxanized rats. Number of animals per group = 4; Numbers in,,parenthesis represent percentage reduction of fasting blood glucose levels "P< 0.05 level of significance against 0 h of the same dose of drug Table 12

Mean fasting Blood glucose Levels (mgdl-'h SEM) in diabetic rats

4 16.67 390.42 290.42 21 1.67 195.00 148.33 178.91 Glycosides 250 mglkg 517.0 527 54.9* 521.5* +23.9* h21.3* h3.7 (0.0) (6.3) (30.3) (49.2) (53.2) (64.4) (54.2)

Glycosides 425.83 328.75 295.00

Glyburide 267.75 153.75 119.00 5 mgkg Table 13

Effect of aqueous and chloroform phase of glycosides fraction of methanol extract on mean fasting blood glucose levels in alloxanized rats (mean * SEM). CHE =chloroform phase; WE = water phase; DC = diabetic control and NC = Normal control. Number of animals per group = 4. Numbers in parenthesis represent percentage reduction of fasting blood glucose levels.* p < 0.05 level of sig. vs. 0 h of the same dnxg dose "p < 0.05 level of sig. vs. DC Table 13

Mean fasting Blood glucose Levels (rngdl-'i SEM)

Drug

CHE 215.8Zt6.4 189.3Zt6.3 156.8& 7.6 153.0*7.9* 170.0i7.2*a 250 mglkg k' (0.0) (1 2.3) (27.4) (3 8.4) (10.5)

Glyburide 267.8f2.6 153.84~6.3~11 9.O~t4.9~96.fU~2.7~ 194.5423.4 5 mglkg (0.0) (42.6) (55.6) ' (63.9) (27.4) Table 14 Effect of n-butanol extract of aqueous phase of Picralima nitida seed extract on fasting blood glucose levels of alloxan- induced diabetic albino rats after prolonged treatment ; number of animals per group (n) = 4. * p< 0.05 sig. vs. the 1'' day glucose level of the same drug. ap< 0.05 sig. vs. DC; Nd = not determined. Table 14

Blood Glucose level mgldl (mean + SEM) Group Dose Day 1 Day 10 % Red

Non- diabetic Dist. Water 3 mlkg b. w.

Diabetic Dist. Water 2 17.3 + 40.0 20 1.8 +32.6 Nd control 3 mlkg b. w

n-butanol 250 mglkg b.w 393.0 k 12.5 66.9 +6.5 82.9 fraction

Methanol 250 mglkg b.w. 307.8 k 2 1.8 60.0 + 10.1 80.5 extract 3.9 Effect of chromatographic fractions of n-butanol extract of Picralima niiida seed on fasting blood glucose levels of hyperglycaemic rats.

The nBF fractions, except Fj and F6, showed consistent percentage maximal reduction in fasting blood glucose levels of treated diabetic rats in the following order: F7> F5> F 4 > F8. The F7 caused reduction of mean fasting blood glucose level from 372.9 k 6.4 mg/dl at 0 h to 57.5 k 0.9 mgldl at 8 h which amounted to 84.6 %; the F5 caused maximal reduction of the mean fasting blood glucose levels from

464.2k6.3 mgldl at 0 h to 88.8 k 1.7 mgldl at 12 h that translated to 80.9%. The F4 caused maximal reduction of fasting blood glucose level of 41 5.4k9.5 mg/dl at 0 h to

96.3 k4.4 mg/dl at 12 h that amounted to 76.8% reduction while F8 caused reduction of the mean fasting blood glucose level of 497.5 k3.9 mg/dl at 0 h to 164.6k7.7 mg/dl at 4 h, that amounted to 66.9% reduction (Table 15). These fractions that exhibited significant blood glucose lowering action commenced their hypoglycaemic activities from the first hour through twelve hours of the 24 hours evaluation like glyburide.

Glyburide caused reduction of mean fasting blood glucose levels from 350.1 k1.5 mgtdl at 0 h to 48.5 k1.4 mgldl at 8 h which translated into 86.1%. F7 has the most

,, . .-4. ..' persistent effect than even the standard'drug because at the twenty fourth hour it still had 67.3 % reduction on the blood glucose level while glyburide was 54.7%.

The chromatographic fractions of F7, F7bC and F7bE, produced the significant lowering effect on the fasting blood gl&ose ~kve~sof the treated alloxanized rats. The fraction F7bE caused the lowering of the fasting blood glucose levels of treated rats from 546.3k10.9 mg/dl at 0 h to 59.2k6.9 mg/dl at 12 h thus produced percentage reduction of 89.2% and F7bC caused fasting blood glucose levels reduction from 350.12% 1.45 mgldl at 0 h to 48.51k 1.42 mgldl at 8 h that amounted to

69.1 % while glyburide caused 79.7% reduction in the 12 h (Table 16).

3.10: Effect of chromatographic fractions of n-butanol extract of P. nitidn seed on Glucagon-induced hyperglycemia in rats. In tile group of nornlal rats injected with glucagon (Eli Lily, USA) alone (0.4 pglkg), their peak mean blood glucose concentration (159.17k 3.58 nigldl) was attained in 15minutes post administration and thereafter decreased gradually. The peak mean blood glucose concentration (159.17* 3.58 mgldl) achieved after administration of the glucagon alone was reduced by the fractions in the following order: F7 > F7 bE >

F5>Fs>ME > F4 (Table 17). The F7, F7 bE, and glyburide caused the reduction of the peak mean blood glucose concentration when administered one hour prior to glucagon administration (Figure 4).

I( 3.11: Effect of extract of Picralima nitida seed on Gastric emptying rate in normal and hyperglycemic Rats

The test meal caused the gastric emptying of 95.7% in normal rats after 30 minutes post administration of-the'tkSt mehl (Fig 5A). The F7 bE and ME at 250 mgfkg , caused 24.4% and 10.8% inhibition of gastric emptying induced by test meal respectively. The serum glucose levels were significantly decreased by ME and FE in

,I . . 40% glucose test meal fed rats (p < 0.05).

From Fig 5B, the prior treatment of rats with intravenous glucose in saline

solution (lo mllkg, i.v.) caused the gastric emptying of 95.1%. The ME and F7bE

inhibited the gastric emptying caused by glucose iv solution by 19% and 30%

respectively. The effect of the parenteral glucose solution did not significantly affect the 1 serum glucose levels and the ME and F7bE did not affect the serum blood glucose levels 1 significantly also. 1 The prior treatment with alloxan (120 mg/kg,, i.p.) increased the plasma glucose 1 levels by about 3-folds, and caused gastric emptyiqg of 76.1 % (FigSC). The control (C) ( group in alloxanized rats had attenuated gastric emptying when compared to the gastric

emptying in normal rats in Fig. 5A. The ME and F7bE, inhibited the gastric emptying in I, the alloxanized rats by 1 1 .O% and 19.5% respectively. The ME and F7bE significantly

reduced the blood glucose levels in the alloxanized rats. Table 15 Effect of chromatographic fractions, F4, 5, 7 and 8 of n-butanol extract of Picralima nitida seed on fasting blood glucose levels in alloxanized rats. Number of rats per group (n) = 4, ** p < 0.01, * p< 0.05 sig. vs. 0 h of same dose treatment; " p< 0.05 sig. vs. DC. Numbers in parenthesis represent the % reduction of fasting blood glucose levels. Table 15

Mean fasting blood glucose levels (mgldl* SEM) in diabetic rats treated with n- butanol fractions of Picralima nitida seed extract Drug Time (h) 0 1 2 4 8 12 24

F5 464.2 236.7a 232.8*a 194.2*a 120.0*a 88.8**a 1 34.5*a 250 h6.3 *8.5 *5.6 h5.9 *0.9 h1.7 h5.9 mglkg (0.0) (49.0) (48.9) (58.2) (74.2) (80.8) (43.8) F7 372.9 330.0 300.0 153.3*~ 57.5**a 79.2**a 142.1*a 250 h6.4 h9.9 *8.9 *4.8 h0.9 *1.5 h6.8 mglkg (0.0) (1 1.5) (19.6) (58.9) (84.6) (78.8) (67.3)

Glyburide 350.1 215.7 172.8 103.5 48.5 54.3 123.8 5 mgkg h 1.5 * 4.8 * 2.5 * 5.9 h 1.4 4.9 4.7 (0.0) (38.4) (50.6) (70.4) (86.1) (84.5) (54.7) Table 16 Effect of the chromatographic fractions of F7 of n-butanol extract of Picralima nitida lr seed on mean fasting blood glucose levels in diabetic rats ( mean h SEM) ; number of rats per group (n) = 4 ; number in parenthesis represent % reduction of fasting blood glucose level ; * p < 0.05 sig. vs. 0 h of the same dose and, a p < 0.05 sig. vs. DC group. Table 16

Mean fasting blood glucose levels (mgldl * SEM) in diabetic rats treated with n- butanol fractions of Picralima nitida seed extract Drug Time (h) 0 1 2 4 8 12 24 A 422.2 503.8 504.2 472.5 424.7 514.2 520.6 250 2~2.7 *7.6 +3.7 *10.5 *4.3 i6.2 * 6.2 mglkg (0.0) (-1 9.3) (-19.4) (-1 9.9) (-0.6) (-2 1.8) (-29.2)

Glyburide 244.2 215.0 157.2 94.7 80.0 49.7 95.0 5 mg/kg *5.7 * 6.3 * 7.5 * 3.2 *4.9 * 5.9 * 4.3 (0.0) (11.9) (35.6) (61.2) (67.2) (79.7) (68.2) Table 17 Effect of chromatographic fractions of n-butanol extract of of Picralima nitida seed on Glucagon-induced hyperglycaemia in rats. All values of fasting blood glucose levels are in mg/dl * SEM. Numbers in parenthesis represent percentage reduction of fasting blood glucose levels. Table 17 - Effect of n-butanol fractions of P.nitida seed extract on mean blood glucose levels of glucagon-induced hyperglycaemia in rats Drug 5min 10 min 15 min 20 min 30 min 60 min 120 min F4 (250mglkg) 98.33 126.33 13 1.67 134.67 1 10.00 120.83 94.1 7 +Glucagon * 2.01 * 4.83 h5.72 h4.21 h4.16 h5.86 h3.12 (0.4Pdg) (0.0) (-28.5) (-33.9) (-36.9) (I1.9) (-22.9) (4.2)

(250mglkg) 84.17 104.17 120.00 115.00 96.67 93.83 85.83 +Glucagon * 4.67 h 4.78 * 5.83 *6.31 *2.13 *3.21 *3.68 00.4 I (0.0 - 14.9) (-1 1.5 -1.9 E (250mg/kg)+ 100.00 103.33 103.33 88.83 95.83 88.83 81.33 Glucagon h 2.90 h 4.74 * 4.56 *5.67 h 2.89 h 3.25 *2.13

(250mglkg) 101.67 105.5 121 33 108.00 100.50 98.00 95.00 + Glucagon k 4.68 h 3.46 h3.21 h 4.01 * 6.09 * 1.03 h 3.06 -,- -- (0.4Pgk) (0.0) (-3.8) (-19.3) (-6.2) (1.2) (3.6) (6.6) . 4 GI y buride t (5 mglkg) + 92.17 84.67 91.33 93.83 87.50 78.00 73.33 - c

Glucagon *3.21 *3.24 k8.53 h3.78 *5.75 k 6.10 * 7.30 -! a'.# (0.4PgIg) (0.0) (8.1) (0.9) (-1.8) (5.1) (15.4) (20.4) .,,, . ..I, .*. . > ' Glucagon 111.33 133.00 159.17 141.33 138.00 135.83 129.67 (0.4Pdg) h 6.98 h 4.01 G.58 *5.78 * 3.85 *4.01 h2.93 (0.0) (-1 9.5) (-43 .O) (-26.9) (-24.0) (-22.0) (- 16.5) Figure 4

Percentage reduction of Glucagon-induced hyperglycemia in rats by F7, and F7bE of n-butanol extract of P. nitida seed (Mean * SEM, n = 4) Time (min)

+F7 tglucagon +E + glucagon 4 Glyburide tglucagon - 4 - Glucagon alone

Figure 4 I' Figure 5A

Effect of F7bE (250 mglkg) and ME (250 mglkg) of Picralima nitida seed on gastric emptying and blood glucose levels in rats fed with 40% glucose test meal. (Mean &S.E.M,n =4) C is untreated group. C ME Treatment grp

:B Gastric emptying(%) +- Blood gluc Figure 5A Figure 5 B

Effect of F7 bE (250 mglkg) and ME (250 ~ngfkg)of Picralima nitida seed on gastric emptying and blood glucose levels in glucose - pretreated (10 mllkg i.v.) rats,(Mean *S.E.M, n =4) C is untreated group. Treatment grp

,I . .. Gastric emptying +Blood glucose

Figure 5B Figure 5C

Effect of FE (250 mglkg) and ME (250 mg/kg) of Picralima nilidu seed on gastric I emptying and blood glucose levels in alloxan petreated (120 mlkg i.p.) rats, (Mean *S.E.M, n =4) C is untreated group. C ME Treatment groups

Gastric emptying *Blood glucose

Figure 5C Figure 6: Structure of Mimusic acid (2a, 3a 16a, 23- tetrahydroxyloleana-5,13 (18)-dien-28-oic acid) from S2 in Fraction E of Picralima nitida seed v Figure 14.

Figure 6: 2a, 3a 16a,23-tetrahydroxyloleana-5,13(18)-dien-28-oic acid CHAPTER FOUR

I+ DISCUSSlON AND CONCLUSION

The screening procedures of Picralirna nitida seed extracts showed that the phytochemical constituents that posses hypoglycemic activity are those highly soluble in polar solvents like water, and these polar solvents have higher extractive yields than the non polar solvents. This observation corroborated. the findings of Nkere and Iroegbu

(2005) that water solvent system extract more than nonpolar solvents. The yield values of the chloroform extract and methanol extract, and chloroform phase and aqueous phase of glycosides fraction of Picralima nitida seed were 5.10% and 12.10%, 1.57% and 3.27%

11 respectively. The higher extractive yield of the methanol extract and aqueous phase of glycosides depicts that efficient extraction of the secondary metabolites requires the use of solvents of relatively higher polarity such as water (Aguwa et al, 2001; Okoli et al,

2002) and alcohol which are commonly used in ethnomedicine.

The acute toxicity test (LDS0)in mice recorded no death in oral administration of the methanol extract even above 5 glkg. But when intraperitoneal route was used, the acute toxicity test (LD50) was cahcu~dtbbe 707.1 1 f 32 mg /kg. Since the toxic dose of the methanol extract was above 5 glkg per oral, it shows the relative safety of the oral administration of the plant extract. In folk medicine, the plant decoction is mainly administered orally. Thus the oral use of the plant aqueous and lor alcohol extract in folk medicine is safe (Lorke, 1983).

The phytochemical analysis revealed the presence of the following metabolites: alkaloids, glycosides, flavonoids, saponins and tannins in both methanol and chloroform extracts. The methanol extract had the following metabolites: terpenes, sterols, proteins and carbohydrates while chloroform extract contained resins and fats. The phytochemical constituents of methanol and chloroform extracts may be similar in class

I' but different in polarity for them to exhibit the observed difference in hypoglycen~ic activities. The soluble forms of those ~hytochemicalconstituents are more in the polar solvent extract, thus, the hypoglycemic activity of seed extract of Picralima nitida is resident in the polar phytochemical constituents. The other extracts like aqueous phase of glycosides, and fractions F4, 5, 7 and 8, and F7 bC and F 7 bE that showed high hypoglycemic activities contain very polar phyhochemical constituents of methanol extract. The fraction F7 bE from methanol extract that showed higher percentage maximal reduction of the mean fasting blood glucose levels in alloxanized rats than glyburide, has

I' lriterpenoid saponins that had Rr value ranges of 0.72 - 0.97 in chloroform: methanol

(25) mobile phase in silica gel 60 G preparative thin layer chromatography (TLC).

In other plants used in folk medicine for diabetes mellitus management, pliytochemical constituents like alkaloids, glycosides, flavonoids, and tannins have variously been reported to be contributory (Akah et al, 2002; Gyang et al, 2004; Akubue el LI~,1986). The blood glucose., ,,Iqygingeffect of the seed extract of Picralima nitida was found to be more with the methanol extract and fraction F 7 b E. These were found to be rich in saponins glycosides and the phytochemical constituent chemically identified in t ' fiaction F7 bE was the triterpenoid saponib among others. This type of saponins has been known to be biologically active (Suttisri el al, 1993; Li et al, 2004) probably through decreased rate of carbohydrate absorption into the portal hepatic circulation, increased glucose transport and uptake mediated by nitric oxide, increased glycogen storage, and modulation of insulin secretion (Shane-McWhorter, 2001). Matsuda et al. (1999a) did not find insulin-like or insulin-releasing activity in rats given oleanolic acid glycosides, a triterpenoid saponin, hence proposed that the hypoglycemic action of the compound may be due to inhibition of gastric emptying and the inhibition of glucose transport across the

(.i brush border of the small intestine.

In the oral glucose tolerance test, normal rats fed with glucose after treatment with methanol extract of Picralimn nitidn seed at a dose of 5 glkg (p.0.) showed reduction of blood glucose when compared to that of glucose alone (negative control) by 38.65% while the standard drug, glyburide (positive control) was 47.24%. This action could be due to the direct stirnulation of the secretion of insulin through enteroinsular axis

(Onyemelultwe and Baltari, 2002; Georgette et al, 2005), increased insulin sensitivity

(Ohnishi et al, 1996), reduced glucose absorption (Onomura et al, 1999) or a combination

I) of these modes.

The ME and fractions were screened for blood glucose lowering effect on both fasted normal and alloxanized rats. In the normoglycemic fasted rats, the methanol extract administered per oral maximally reduced the mean fasting blood glucose levels at

the twelveth hour. In the hyperglyse,~jc,,, w rats also, the orally administered methanol extract maximally reduced the mean fasting blood glucose levels from first hour and climaxed in the eight hour. The methanol extract and glyburide exhibited persistent hypoglycemic effect in a similar fashion. The variation in the blood glucose level I, reduction between glyburide and the extract may be based on differences in their extent of extra-pancreatic and pancreatic influences. The percentage maximal reduction of mean fasting blood glucose levels by methanol extract maybe because of the saponins that reduce protein digestibility probably by the formation of sparingly digestible saponin- protein complexes (Potter et al, 1993) that reduces their absorption.

The alkaloids fraction elevated the mean fasting blood glucose levels of ilorrnoglycernic rats in a dose dependent pattern. The higher dose caused severe

I1 hyperglycemia that resulted in the death of the animals; this maybe due to the inflammation and necrosis of liver hepatocytes (Fakeye et al, 2005) as hepatic damage can lead to insulin resistance (Yeh et al, 2003). The glycosides fraction reduced the mean blood glucose levels though not in a dose dependent' fashion. The glycosides fraction produced noticeable reduction in the mean fasting blood glucose levels in normoglycemic rats at the twelveth hour post administration. In the hyperglycemic rats, the blood glucose reduction by glycoside doses commenced from the first hour till the twelveth hour of treatment just like the standard glyburide treated group.

P All the effective hypoglycemic fractions of the n-butanol extract commenced their blood glucose lowering effects, in hyyerglycenlic rats, from the first hour and their twenty fourth hour mean fasting blood glucose levels were still lower than their zero hour

values; this was similar to the.,,,.+wl. observatjons ., of Inya-Agha el a1 (2006) that extracts of

Picrnlinza nitida have prolonged blood glucose lowering effect in diabetic rats for upward of twenty four hours.

Alloxan is a known diabetogenic agent that selectively and permanently destroys the pancreatic p-cells through production of free radicals and excessive calcium I' concentrations in cell cytoplasm. (Ozturk et al, 1996; Szltudelski, 2001; Aguwa et al,

2001). This pancreatic p-cells destruction invariably leads to hyperglycemia through absolute insulin deficiency, hepatic glucose over production and reduced muscles uptake of glucose (Mahler and Alder, 1999). Since the methanol extract, glycoside fractions and fraction F7 bE reduced the hyperglycemia in alloxanized rats, it implies that the extracts have extra pancreatic effects (Esimone et al, 2001). But when used in Type 2 diabetes mellitus condition where insulin is relatively deficient, this extract may reduce the blood glucose by stimulation of insulin production from the pancreatic since p-cells are

(1 relatively conlpromised.

Glucagon is a known hormone secreted by a-cells in the pancreas that plays a central role in the maintenance of normal glucose homeostasis (Cherrington, 1999). It acts on enzymes that stimulate glycogen phosphorylase. In fasting conditions, approximately half of total hepatic glucose output is dependent upon the maintenance of normal basal glucagon levels or production of glucose by liver is activated by a low insulin to glucagon ratio to meet the body needs and inhibition of basal glucagon secretion causes a profound reduction in endogenous glucose production and decline in

I' plasma glucose concentration (Baron et al, 1987). Thus glucagon acts only on liver glycogen converting it to glucose (CPS, 2002). In hyperglycemic animals, increased rates

of hepatic glucose production .are ,, . .largely,responsiblewl. . for the worsening of hyperglycemia

(Moller, 2001). A relative decrease in insulin level and overt elevation in plasma glucagon concentration, or reduced hepatic responsiveness to insulin, can lead to this

hepatic overproduction of glucose. 11 . -.

Rats with glucagon-induced hyperglycemia were treated with methanol extract, glycosides fractions, fraction F7 bE and glyburide; the fractions caused reduction in peak 1, blood glucose levels when administered one hour prior to glucagon administration. Since the hyperglycemia induced by glucagon was due to the hepatic overproduction of glucose, and was reduced by the extract and fractions, it implies that Picralima nitida seed extracts may also be acting by inhibiting hepatic glucose overproduction by increasing hepatic glycogen storage (Shane-McWhorter, 2001) thus reversing the effects of glucagon.

The speed of gastric emptying is important in the regulation of glucose homeostasis (Horowitz et al, 1993). Gastric emptying delay is common in diabetic

b' conditions and is basically caused, in part, by vagal activity impairment due to prolonged hyperglycemia that often precipitate neuronal dysfunction in the enteric nervous system

(Matthew, 1998; Ishiguchi et al, 2002). The usual condition with 30-50% of diabetics is delayed gastric emptying (Horowitz et al, 2002); this has been confirmed by the reduced gastric emptying in the alloxan-induced hyperglycemic rats. But it has been reported that gastric emptying occurs faster in some type 1 and 2 diabetics, in obese diabetics, and diabetic rodents (Matsuda et al, 1999b) than in healthy controls.

An inverse relationship between the rate of gastric emptying and the blood

I' glucose concentration has been proposed, such that gastric emptying is slower during hyperglycemia and faster during.,, ...hypoglycemia (Horowitz et al, 1996). The results of this study corroborated this proposal since the percentage gastric emptying decreased as the blood glucose levels of the respective control groups of the treatment categories of rats increased in accordance with Horowitz et a1 (1996), while in each treatment group the gastric emptying cannot be said to have inverse relationship. The Picralima nitida seed extract inhibited the gastric emptying in rats loaded with 40 % glucose test meal, and significantly inhibited gastric emptying in rats with parenteral glucose induced- 1' hyperglycemia as well as in alloxanized rats. These results suggest that Picralima nitida seed extract inhibits gastric emptying and blood glucose levels in hyperglycemic condition.

The rate of gastric emptying is a major determinant of post-prandial glycaemic excursions in healthy subjects, as well as in Type 1 and Type 2 patients. A number of therapeutic agents like metformin (Nolte and ,Karam, 2004), and plants bioactive constituents like 4-hydroxyisoleucine, charantin and polysaccharides (Marles et al, 1995) are used to improve postprandial glycaemic control by modulating the rate of delivery of I' nutrients to the small intestine. The triterpenoid saponin, fraction F7bE, has greater gastric emptying inhibitory effect than the methanol extract of the seed in the respective treatment categories. The extract reduced blood glucose levels in hyperglycemic rats by inhibiting the gastric emptying thus prolong the postprandial absorption of glucose from iood delivered into the small intestine and enabling the body to handle it appropriately.

The thin layer chromatography of the F~~Eyielded three spots, SI-S3.The S2 had higher absorbance at the wavelength of 242 nm in ethanol solvent system that indicated the presence of triterpenoid saponins and authenticated through chemical identification IS (Harbone, 1984). The NMR (g~,ot~n,and,carbon-13)of the S2 led to the identification of a

novel triterpenoid saponin compound, Mimusic acid that has not been previously

identified in Picralima nitida, except in seeds of Mimusops elengi. It is a P- aniyrane

saponin because of six quaternary cdi-bons' absorbing in the range of 34.7-54.8 ppm

(Kilonda et al, 2003) and glycosylated at C-28 hence a monodesmoside (Hua et al, 2006).

Tt is a derivative of oleanane group of saponins that has been known to posses

hypoglycemic activity but Mimusic acid has not been evaluated for hypoglycemic

activity in, the literatures. The structure was elucidated as 2P, 3P, 16a, 23- tetrahydroxj;loleana-5,13(18)-dien-28-oicacid by NMR spectra studies (Sahu et al, 1997) in Minzusops elengi but that which was isolated from Picralinza nitida was 2a, 3a 16a,

23-tetrahydroxyloleana-5,13(18)-dien-28- acid which may be the enatiomer of that obtained from the Mimzrsops elengi .

In conclusion, it may be stated that the hypoglycemic activity of the seed extract of PicraIima nitida resides in the hydrophilic triterpenoidal saponins of the oleanane group and the P- amyrane derivative (Hua et al, 2006) that are known to lower gastric emptying thus prolong the postprandial absorption of nutrient glucose. The extract may have achi;bed this blood glucose lowering effect through reduction of hepatic overproduction of glucose or increase in glucagon catabolism, and inhibition of gastric emptying in diabetic conditions. REFERENCES Aguwa C.N., Omole, K.M. Diabetes mellitus In: Therapeutic basis of Clinical Pharmacy in tfe Tropics. Aguwa C.N Ed, 3rd Ed, pp 302-327 (2004).

Aguwa, C.N., Ukwe, C.V., Inya-Agha, S.I., Okonta, J.M. (2001). Antidiabetic effect of Picralima nitida aqueous seed extract in experimental rabbit mode1.J Nat. Remed, 1,2:135-139.

Akhtar MS Ahar MA, Yaqub M (1981): Effect of Momordica charantia on blood glucose levels of normal and alloxan-diabetic rabbits. Planta Medica 42:205-221.

Ajabnoor, M.A (1990). Effect of aloes on blood glucose levels in normal and alloxan diabetic mice. J Ethnopharmacol. 28:215-220.

Akah, P.A., Okoli, C.O.and Nwafor, S.V (2002): Phytotherapy in the management of diabetes mellitus. J.Nat. Remedies 2,l:l-10

IJ Akali P.A and Okafor, C.L (1992): Blood sugar lowering effect of Vernonia amydalina Del, in experimental rabbit model.Phytotherap. Res. 6: 171 -1 73.

Akpan J.E and Umoh I.B. (2004) Inhibitory activity of seed extract from P.nitida (Staph) on P-D-glucosidase. Biokemistri 16; 2: 72-78.

b Altubue, P.1 and Undie, A.S. (1 986): Pharmacological evaluation of Dioscorea dume&tim tuber used in Traditional antidiabetic therapy. JEthanopharmacol. 15: 133-144

Al-Saltkaf, L., Pozzili P., Tarn A.C., Schwarz G.(1989). Persistent reduction of CD41CD8 lyn~phocyteratio and activation before the onset of type1 diabetes. Diabetologia 32:322-325.

. ,,.. "1. ... .a ' Andrade-Cetto, A., Wiedenfeld, 13.(2001): Hypoglycemic effect of Cecropia obtusifolia on Streptozotocin diabetic rats. J Ethnopharmacol 78:145-149. I' Antonio, C. (2005). Postprandial Hyperglycemia and Diabetes complications. Diabetes 54,l: 1-7. It . .. Augustin,K.J. and Benaim, M.E. (1974): Effect of essential oil of Onion (allylpropyldisulphide) on blood glucose,free fatty acids and insulin levels of Normal subjects. Clinical Chimica Acta, 6O:l21 7123.

Ayensu, E.S. (1978). Medical plants in West Africa. Reference Publications 1nc.Algonac Michigan.pp330 Bamidele, T.V., Josiah, S., Odetola, A.A and Okunade W.G (2002). The modulatory effect of Momordica charantia on the Plasma and intestinal mucosa ~a+ATPase in normal and alloxan induced diabetic rabbits.Nig.J PPhnrm Res. 1: 1,2629.

Bailey, c.J.' and Day, C. (1989). Traditional plant medicines as treatments for diabetes. Diahetes Care l2:553-564.

Barbosa-Filho, J.M., Vasconcelos T.H.C, Alencar A.A., Bartista L.M. et a1 (2005): Plants and active constituents from South, Central and North America with hypoglycemic activity. Braz. J Pharmacogl5,4:392-413.

Barret C.E. (1985). Is insulin dependent diabetes mellitus caused by Sackie virus B infection? A review of the epidermologic evidence. Review of Infectious Disease. 7: 207-21 5.

Baron A.D., Schaeffer, L., Shragg, P., I(olterman, O.G. (1987): Role of hyperglucagonemia in maintenance of increased rates of hepatic glucose output in type I1 diabetics. Diabetes. 36:274-83.

Bosseri, S.4. (2002): Obstacles to and solutions for prevention of diabetic foot ulcers. Diahetes International 12, 2: 51-54. l3raii1, K.R. and Turner, T.D. (1975): The Practical evaluation of Phytochemicals. Saunders and Company.NY, U.S.A. Pp 98-102

Broca, C. (1999): 4 hydroxyisoleucine: experimental evidence of its insulinotropic and antidiabetic properties. Am J Physiol. Oct; 277:4 [Abstract].

Brown D. (1 995) The Herb Society of America Encyclopedia of Herbs and their Uses. 1st ed. New Yorlt: Dorling Kindersley .pp234

. , 4:.r' Campillo J.E., Perez, C., Ramiro, J.M., Torres, M.D. (1991): Hypoglycemic activity of an aqueous extract from Ficus carica in streptozotocin diabetic rats. Diabetologia 34 (Suppl.2):A-181.

Canadian Pharmaceuticals and Specialties, 2002 , . . Cataland, S., Crocltett, S.E., Brown, J.C., Mazzaferri, E.L (1974): Gastric inhibitory polypeptide stimulation by oral glucose in man.J Clin.Endocrino1.Metab. 41: 260- 265.

Cherrington AD (1999): Control of glucose uptake and release by the liver in vivo. Diabetes 48: 11 98-1 21 4, Chihiro, Yabe-Nishimura (1998). Aldose Reductase in Glucose toxicity: a potential target for the prevention of diabetic complications. Pharmacological Reviews; 50:I; 21- 31.

Curie A.L and Time11 T.E. (1959). Constitution of methylglucuron-oxylan from Kapok Ceiba petandra. Cunad. J. Chern., 3 7:922-959.

I1

Ilalziel, J.M. (1958). Useful plants of West Tropical Africa, Crown Overseas Agents for Colonies; London; pp 65.

13ey L., Attele A.S., Yuan C.S. (2002): Alternative therapies for Type 2 diabetes..Altern.Med. Rev.7:45-58.

De Tommasi, N., De Simone, F., Cirino, G., Cicala, C., Pizza, C. (1991): Hypoglycen~ic effects of sesqueterpene glycosides and polyhydroxylated triterpenoids of Ei-iobolya jaconica. Plantamedica5 7:44 4-416

De Valk HW, (1 999). Magnesium in Diabetes mellitus. Neth. J. Med. %:I 39-146

Dhar, M.L., Dhar, M.M., Bhawan, B.N., Mehrothra, B.B., Ray, C. (1 968): Screening of 1ndib plants for biological activity Part 1. Indian J. Explal Biol. 6: 222-230

DPIC March -April 2000: 19: 2.

Drucker D.J.(2003). Enhancing incretin action for the treatment of type 2 diabetes. Diabetes Care. 26:2929-2940.

Elahi, D., Andersen, D.K., Brown, J.C. (1979): Pancreatic alpha -and beta-cell responses

to GIP infusion in norm4..,, . qnn.&z.J h . .. Physol. 23 7: El85-El91 [Abstract].

Esimone C.0, Okonta J. M, Ezugwu C.O. (2001). Blood sugar lowering effect of Anacrri-durn occidentale leaf extract in experimental rabbit model. J. Nat. Remed., 1, 1: 60-63.

Evans W.C. (1989) Trease and Evans Textbook of Pharmacognosy 13"' edition London: Bailliere Tindall, pp 3 15-679

Evans J.L., Rushaltoff K.J. (2002): Oral Pharmacological Agents for Type 2 Diabetes: Sulphonylureas, Meglitinides, Metformin, Thiazolidinediones, a-Glucosidase Inhibitors, and Emerging Approaches. In Endotext.com: Diabetes and Carbohydrate Metabolism. Degroot L, Goldfine ID, Rushakoff RJ, Eds. http://www.medtext.com, MDtext.com. Retrieved in 12/3/2006. Fakeye T. O., Awe S. O., Odelola H. A., Ola-Davies 0. E., Itiola 0. A., and Obajuluwa, T. (2005): Evaluation of Valuation of Toxicity Profile of an Alkaloidal Fraction of the Stem Bark of Picralima nit id^^ (Farn. Apocynaceae). JHerbul Med Plzarmacotherap.Innov. Clin. and Applied Evidence-Based Herbal A4ed. 4; 3:37-45.

Farias, R.F.A., Rao, V.S.N., Viana, G.S.B., Silviera,E.R. Maciel, M.A.M., Pinto, A.C. (1997): Hypoglycemic effect of trans-dehydrocrotonin , a nor-clerodane Dite~penefrom Croton cajucara. Planta Med 63:558-560

Frati A.C., Diaz N.X., Altaniirano, P., Ariza, R., Lopez-Ledesnia, R. (1991): The effect of two sequential doses of Opuwtiu streptacantha upon glycemia. Arch. Iwest. Med. (Mex)22:333-336. Frati, A.C., Gordillo, B.E., Altamirano, P., Ariza, C.R., Cortes-Franco, R., Chavez- Negrete, A, (1990): Acute hypoglycemic effects of Opuntia streptacantha L in NIDDM. Diabetes Care 13:455-45.

Frati-Munari, A.C., Castillo-Insunza, M.R., Riva-Pinal, H., Ariza-Andraca, C.R., Banales-Ham, M. (1985): Effect of Plantago psyllitlnz mucilage on the glucose tolerance test. Arch. Invest. Med. (Mex.) l6:l9l-l97.

Gan S. C., Barr J., Arieff A.I., Pearl R.G. (1992): Biguanides associated lactic acidosis. Arch Intern Med. 15.2: 2333-2336

Ganong, WcF. Review of Medical Physiology, 20th ed, McGraw Hill, NY.pp334-335 (1 999).

Garcia, F. and Colis, J (1926): Studies on antidiabetic properties of Tecoma mollis. Preliminary Report. JAmer. Pharm. Asso. l5:556-560.

., , . ."1. d' . a ' George, E.F . Diabetes mellitus In: Pharmacotherapy Pathphysiologic Approach.Dipiro JT et al, ~d.2"~ed, Elsevier Sci.Publ.Co, NY, pp 1 121-1 l27(l992) .

Georgette Nyah Njilte, Pierre Watcho, Tdlesphore Benoit Nguelefack and Albert Kamanyi. (2005): Hypoglycaemio activity of the leaves extracts of Bersama englerianain rats. Afr. J. (Trad.; Compl. Altern. Med. . 2, 3: 215-221

Gilani, A.H., Malik, T., Althtar, S.M and Ahmed, M (2000): Hypoglycemic action of Flavonoid fraction of Cunzinum nigrum seeds Phytotherap. Res. 14,2:103-106. I' Goldner, M.G. (1958): Oral hypoglycemic agent past and present. Arch. Int. Med. 102:830-840.

Grover, J.K., Yaday, S., Vats, V. (2002): Medicinal Plants of India with antidiabetic potential. JEthnoparnzacol. 81:8l-lUO. Gyang, S.S., Nyam, D.D., Sokomba, E.N. (2004). Hypoglycemic activity of Vernonia arnygdalina (chloroform extract) in normoglycemic and alloxan-induced hyperglycemic rats. J. Pharm. Bioresources I, 1: 61-66.

Hamet, L. Medicinal plants in Nigeria, Nigeria College of Arts, Science and Techno1ogy;Ibadan; pp77-78 (1940).

Harbome J.B. Phytochemical Methods. Chapman and Hall, London.pp. 166-226 (1984 )

Hiraltura, K., Nakajima, K., Sato, S., Mihashi, H. (1989): Isolation of 7-(6-O-malonyl-P- D-glucopyranosyloxy)-3-(4-hydroxypheny1)-4H-1 -benzopyran-4-one from Puefaria lobata Ohwi as Aldose Reductase inhibitors and pharmaceutical formulations. Japan Kokai Tokkyo Koho (Patent). Patent number: JP 01 146894

Hood, A; Lowburry, M. (1954). Anthocyanins in banana. Nature, London 173:402-403

Horowitz, M.,Edelboelt,M.A.L., Wishart, J.M., Straathof, J.M. (1993):Relationship betweem oral glucose tolerance and gastric emptying in normal healthy subjects.Diabetologia 36:85 7-862.

Horowitz, M., Wishart, J.M., Jones, K.L., Hebbard, G.S. (1996): Gastric emptying in diabetes: an overview. Dinbet Med.Sep;13 (9 Suppl5), 112-1IS.

13orowitz,M., O'Donovan, D. , Jones, ILL., Feinle,C., Rayner,C.K., Samsom,M. (2002): Gastric emptying in diabetes: clinical significance and treatment. Diabet. Med. 19, 177-194 . ha, s.u.G; Wei-Shuo, F.A.N.G.; Wen-Zhao, WANG; Chun HU (2006): Structure- activity relationships of Oleanane- and ursane type triterpenoids.Botanica1 studies 47: 339-368.

.,,,...I..,. ... ' Inya-Agha, S.I., Ezea, S.C., Odukoya, O.A. (2006): Evaluation of Picralima nitida: Hypoglycemic activity, Toxicity and analytical standards. Intern. J. Pharmacol. 2.5:5 74-5 78. lshiguchi, T., Tada, H., Nakagawa, K., Yamamura, T., Takahashi, T. (2002): Hyperglycemia impairs antro-pylbric coordination and delays gastric emptying in conscious rats. Auton. Neurosci. 10; 95(1-2): 112-20

Iwu, MM (1992). Traditional Igbo Medicine. Institute of African Studies, University of Nigeria, Nigeria .lO4.

Iwu, M.M. ,,Handbool

Jalalpure S.S., Bamme Shivaji, Patil M.B (2006): Anti-diabetic activity of Holarrhsna c~ntidysenterica(Linn) Wall, bark on alloxan induced diabetic rats.J.Nat.Remed. 6.1 :26-30.

Jayasooriya, A.P.(2000). Effects of Moinordica charantia powder oil serum glucose levels and various lipid parameters in rats fed ,with cholesterol-free and cholesterol- enriched diets. J Ethnopharmacol Sep; 72:l-2, 331-6.

Judd, R.L.; Raman, P. (1999). Pharmacological Management of Type 2 Diabetes Mellitus: Current and Future therapy. Pharm. Times Oct. 85 - 93.

(1 Kakuda, T, (1996). Hypoglycemic effect of extracts from Lagerstroemia speciosa L. leaves in genetically diabetic KK-Ay mice. Bioscience, Biotechnology, Biochemistry, 60, 2b

Kilonda, A., Babady-Bila, Osomba, L., Toppet, S., Compernolle, F. (2003): Acetylation products of pentacyclic triterpene glucosides from Combritum psidioides. Arkivoc 4:3-21.

King, H., Aubert, R.E.and Herman, W.H. (1998). Global burden of diabetes, 1995- 2025:prevalence, numerical estimates and pro-jections. Diabetes Care, 21:1414- 1431.

Konno C, Murakami M, Oshima Y, Hikino H. (1985) Isolation and hypoglycemic activity of panaxans Q, R, S, T and U glycans of Panax ginseng roots.J. Elhnopharmacol. 14:69-74. I'

Leibowitz, G., Cerasi, E. (1996);. ,,.. $ulghonylurea treatment of NIDDM patients with cardiovascular disease: a mixed blessing? 39503-5 14.

Leung, A.Y and Foster, S. Encyclopedia of Common Natural Ingredients Used in Food, Drugs and Cosmetics, 2nd ed, New Yorlc John Wiley & Sons Inc 1996; pp

Li W.L., Zheng H.C., Bukuru J., De (Kinrpe N. (2004):Natural Medicines used in the traditional Chines medicinal system for therapy of Diabetes mellitus. J. Ethnopharmacol. 92: 1-21.

Lugari, R., Dell'anna, C., Ugolotti, D. (2000). Effect of nutrient ingestion on GLP-1 secretion in human Type 1 and type -2 diabetes.Hormon. mctabol.Res. 32: 424- 428. Mahler R.J%nd Alder, M.L. (1999): Type' 2 Diabetes Mellitus: Update on Diagnosis, Pathophysiology, and Treatment. J. Clin. Endocrinol. and Metnbolisnz, 84; 4:ll65-ll7l

Manske, R.H.F. (1960). The Alkaloids, Vol. VI, Academic Press, NY, pp 169-17 1.

Marles, R.J., Farnsworth, N.R. (1995): Antidiabetic plants and their active constituents. Phylomed 2:13 7-189

Marquis, V.O., Adanlowo, T.A., Olaniyi, A.A. (1997) The effect of foetidin from Momordica foetida on blood glucose level of albino rats. Planta Medica 31:367- 3 74.

Matsuda, H., Li, Y., Yamahara, J., Yoshikawa, M (1999a): Inhibition of Gastric Emptying by Triterpene saponin, Momordin Ic, in Mice: Roles of blood glucose, Capsaicin-Sensitive sensory nerves ,and Central Nervous system. JPharmacol. ~X~lalTherap. 289,2: 729- 734.

Matsuda, H.; Li, Y.H.; Murakami, T; Yamahara, J. and Yoshikawa, M. (1999b) Structure-related inhibitory activity of oleanolic acid glycosides on gastric emptying in mice. Bioorg. Medicinal Chem. 7, 323-327

Matthew, J.0: Diabetes Mellitus and related disorders In: The Washington Manual of Medical Therapeutics.Carey, CF et a1 Ed; 29th ed. Lippincott Williams & Wikinson pp.398 (1998).

Mayerson, A.B; Hundal, R.S. and Dufour S (2002): The effects of rosiglitazone on insulin sensitivity, lipolysis and hepatic and skeletal muscle triglyceride content in patients with Type 2 diabetes; Diabetes; 51:797-802).

Meletis, C.D., Brainwell, B.. ,I [2091).,,w Natural approaches to the prevention and inanfigement of diabetes mellitus. Alternative and Complementary Therapies, Elsevier Science, Amsterdam.

Meier, J.J, Nauck, M.A, Schmit, W.E, Gallwitz, B (2002): Gastric inhibitory polypeptide: the neglected incretin revisited. Regulatory Peptidesl07: 1-13.

Menzies, J.R., Paterson, S.J., Duwiejua, M., Corbett,' A.D. (2004). Opioid activity of alkaloids extracted from P.nitidn; Medline Abstract.

Miyaji N, (1 999). Influence of banaba-kuwa extracted powder on plasma glucose level in rat. J Trad Med . l6,5/6: 208-1 1. ,' Molham, A. 1. H., Raman, A (1998). Antidiabetic and hypercholesterolaemic effects of fenugreek. Phytother Res; 12:4. Moller, D.E. (2001). New drug targets for type 2 diabetes and metabolic syndrome. Natuve 41 4:821-827.

I' Nadkarni, A. (1994). Dr KM Nadkarni's Indian Materia Medica. 3rd ed. Popular Pralcashan Bombay, India. pp 805-807

Nauck, M.A., Homberger, E., Siegel, E.G. (1986). Incretin effects of increasing glucose loads in man calculated from venous insulin and C-peptide responses. J Clin Endocvinol Metab.;63:492-498.

Neuwinger, H. D. (1996). African Ethnobotany. Poisons and Drugs. Chapman & Hall, Weinheim, pp 123.

Ng,T.B.,Yeung,H. W. (I 985) Hypoglycemic conitituents of Panax ginseng. Gen Pharmacol.16: 542-552.

Ni,J.S., Wu,J.X., Xiao,R.Q., Shi, B.,Wang, X.X. and Liu, S.X (1998). Effect of total saponins of radix of Acanihopanax senticosus on diabetic rat serum lipid peroxide and SOD activity .J. Novman Bethune Univ.of Medical Sciences 24, 33-34.

Niederwieser, A. and Honegger, C.C. (1966). Chromatographic Techniques In: Advances in Chromatography, Vo1.2, Giddings, J.C. and Keller, R.A. Eds, Marcel Dekker, NY, pp125-147.

Nlcere, C.K. and Iroegbu, C.U. (2005): Antibacterial. Screening of the root, seed and stembark extracts of Picvalima nitida. Afi. J. Biotech .4,6:522-526.

Odukoya, A.O.and Ogbeche, K.A.(2002).Sorghum flavonoids antioxidant effects on haemoglobin glycosy1ation:potential antidiabetics.Nig. J. Pham. Res. 1:1;39-40.

Ohnishi Y, Takagi S, Miura T,. Usami,, - . *I. + I$ Kako M, Ishihara E, Yano H, Tanigawa K, Seino Y (1 996): Effect of ginseng radix on GLUT2 protein content in mouse liver in normal and epinephrine-induced hyperglycemic mice. Biol Phavm Bull 19: 1238-1 240.

Olcada C.Y, (2003). A new triterpenoid isolated from Lagerstronemia speciosa (L.) Pers. Chem Phavm Bull Tokyo, 51: 4: 452-4: -.

Olcoli, A.S., Okeke, M.I., Iroegbu, C.U., Ebo, P.U. (2002): Antibacterial activity of Harungana madugascariensis leaf extracts.Phytothev. Res. 16:174-179.

Oliver B. (1960): Encyclopedia of Medicinal Plants. College of Arts, Science and Tech. Ibadan. Onomura My Tsukada H, Fukuda K, Hosokawa M, Nakamura H, Kodama MyOhya M, Seino Y (1999): Effects of ginseng radix on sugar absorption in the small intestine. Am J Chin Med 27: 347-354.

Onyechi, O.C., Kolawole, J.A.(2005): Preliminary screening of aqueous extract of the lea& of Secrrridnca longepedz~nculata(linn) for anti-hyperglycemic property. Nig. J. Phnrn?. Rex 4,2:18-21

Onyemelukwe,G.C.; Bakari,A.G. (2002): Insulin secretion in Type 2 diabetes - A review. Diab. Intern. (Africa/ Middle East) 12,2: 41 - 43

Ozturk, Y.; Attan, V.M. and Yildizoglu-Ari, N. (1996). Effects of Experimental diabetes and insulin on smooth muscle functions. American Soci. Pharmacol. and Exptcrl Therapeut. 48: 1,69-I 01.

Pandey, V.N., Rajagopalan, S.S., Chowdhary, D.P.(1995). An effective Ayurvedic hypoglyceic forn~ulation.J. Res. Ayurveda Siddha XVI: 1-14,

Petal, M.and Rybczynslti, P (2003). Treatment of non-insulin dependent diabetes mellitus. Expert Opin.Inveslig. Drugs 12:4,623-633.

I{ Perez C, Dominguez E, Ramiro A, Campillo J, Torres MD (1996): A study on the glycemic balance in streptozotocin diabetic rats treated with an aqueous extract of Ficus carica leaves. Phytotlzer. Res. 10: 82-86.

Perez, R.M., Perez, C., Perez, S., Zavala, M.A. (1998): Effect of triterpenoids of Bouvardia ternzji'ora on blood sugar levels of normal and alloxan diabetic mice. Phytomed.5:4 75-484.

Pcrez, R.M., Cervantes, H., Zavala, M.A., Sanchez, S.J., Perez, S., Perez, C. (2000): Isolation and hypoglycemic activity of 5,7,3-trihydroxy-3, 6,4 - trimethoxyflavone from 'linckellia4 @I. 3' venonicqefolia. Phytomed. 7:25-29.

Persaud, S.J., Al-Majed, H., Raman, A., Jones, P.M. (1999) Gymnema sylvestre stimulates insulin release in vitro by increasing membrane permeability. J Endocrinol l63:2O 7-21 2.

Id (1 Porte D. (1 991): p-cells in Type 2 diabetes mellitus. Diabetes .do: 166-1 80.

Potter S.M., Jimenez-Flores R., Pollack J., Lone T.A., Berber-Jimenez M.D. (1993) Protein saponin interaction and its influence on blood lipids. J.Agric. Food Chem. 41: 1287-1 291

Rask, E., Olsson, T., Soderberg, S. (2001). Impaired incretin response after a mixed meal is associated with insulin resistance in non-diabetic men. Diabeles Care 24:1640- 1645. Sachser, J.A. (1961): An AA oxidase inhibitor system in beans pods.Am. J. Bot. 48:820- 831.

Sal~u,N.P., Koike, K., Jia, Z., Nilcaido, T. (1997): Triterpenoid saponins from Mimusops elengi. Phytochemist. 44,6: 1145-1149.

Sanjay, M.J. (2002): Herbal Drugs as Antidiabetics: An Overview. CRIPS 3:2:9-13. I; Scott M.G (2006): Drug therapy of metabolic syndrome: minimizing the emerging crisis in polypharmacy. Drug Discovery 5:4; 295-305.

Scott, LA. (1964). Interpretation of the Ultraviolet spectra of Natural products. Vo1.7 Pergamon Press NY, pp 34 1-343.

Shane-McWhorte, L.( 2001). Biological complementary therapies: a focus on botanical products in diabetes. Diabet. Spectrum 14:199-208.

Shanmugasundaram, E.R.B., Rajeswari, G., , Baskaran, 1 Kumar, B.R.R., Shanmugasundaram, K.R.,, Ahmath, B.K. (1990:) Use of Gymnema sylvestre leaf extract in the control of blood glucose in insulin-dependent diabetes mellitus. J Ethnopharmacol. 30:28l-2 94, [Medline abstract].

Sharaf, A., Mansour, M.Y. (1964): Pharmacological studies on the leaves of Marus alba with"special reference to its hypoglycemic activity. Planta medica 12: 71- 72.

Sharina RD (1986): Effect of fenugreek seeds and leaves on blood glucose and serum insulin responses in human subjects. Nutr. Res. 6: 1353-1 364.

Sharma, R.D., Raghuran,T.C., Rao, N.S. (1990): Effect of fenugreek seeds on blood glucose and serum lipids in type of diabetes.Eur: J. Clin. Nutr.44:301-306

..,,. rll. d' 'i ' Sheela, C.G. and Augusti K.T (1992). Antidiabetic effects of S-ally1 cysteine sulphoxide isolated from garlic Allium sativum Linn. Indian J. Exp Biol. 30.523-526.

Shapiro K, Gong WC (2002): Natural products used for diabetes. J. Am. Pharm. Assoc. 42 :2 17-226

Shibata S. (2001) Chemistry and cancer preventing activities of Ginseng saponins and somg related triterpenoid compounds. J Korean Med Sci.; 16(Suppl): S28-S37

Smith, S.A. and Tadayyon (2003). Insulin sensitization in the treatment of type-2 diabetes. Expert Opin.Investig. Drzlgs .12:3,307-324.

Sofowora.(l993).Medicinal plants and traditional remedies in Africa. Spectrum Books Ltd, Ibadan, Nigeria.ppl3-27 Suttisri R, Lee IS, and Kinghorn D, Plant-derived triterpenoid sweetness inhibitors, Journal of Ethnopharmacology 1995; 47: 9-26. Srivastava, Y. (1993). Antidiabetic and adaptogenic properties of Momordica charantia extract: an experimental and clinical evaluation. Phytotherap. Res. 7:285-289).

Svoboda, G.H., Gorman, M., Root, M.A. (1964):~lkaloidsof Vinca rosea, a preliminary report on hypoglycemic activity, Planta medica 27:361-363.

Szltudelslti, T (2001): The mechanism of alloxan and streptozocin action in p-cells of the rat pancreas.Physio1.lks. 50: 6; 537-46.

Torres MD, Dominguez E, Romero A, Campillo JE, Perez C (1993): Hypoglycemic and hypolipidemic activity of an aqueous extract from Ficus carica in streptozotocin diabetic rats. Diabetologia 36 (Suppl. 1):A-181.

Touchstone, J. C.(1978): Practice of Thin Layer Chromatography; 3rd ed, Wiley- Interscience Pub. NY. pp 173.

IJccciani, E., Detretin, P.P., Boutoux, M., Busson,F. (1964): The proteins and lipids of Bighia sapida. Oleagineux 19,18-19:563-569.

Usdin, T.B., Mezey, E., Button, D.C. (1993). Gastric inhibitory polypeptide receptor, a member of the secretin -vasoactive intestinal peptide receptor family, is widely distributed in peripheral organs and the brain. Endocrinology 133:2861- 287U[a bstract] . I' Welilinda, J. (1986) of Momordica charantia on the glucose tolerance in maturity onset diabetes. J Ethnopharmacol. 17.277-82.

Walter Reeds Army institute of Research Technology for licensing notice, 1994

. ,,...,..!. ..,> ' Watt, J.M and Beyer-Brandwijk, M.G. (1962). The medicinal and Poisonous Plants of Southern and Eastern Africa, Livingstone, Edinburgh, pp 385-386.

White Jr J.R., Kramer, J., Campbell, R.K., Bernstein, R. (1994): Oral use of topical preparation containing an extract of Stevia rebundiana and the chrysanthemum flower in bbthe management of hyp'erglyceinia .Diabetes Care 17:940-941.

Yell, Y., Eisenberg, D.M., Kaptchuk, T.J., Philips, R.S. (2003): Systematic Review of Herbs and Dietary Supplements for Glycemic Control in Diabetes. Diabetes Care 26.1277-1294.

Yoshikawa,M., Nishida N., Shimoda H., Takada M., Kawahara Y. and Matsuda H. (2001): Polyphenol constituents from Salacia species: quantitative analysis of magniferin with alpha-glucosidase and aldose reductase inhibitory activities. Yakugaku zasshi l21;5:371-378. Test Report Test Date: 08-03-2007 User Name: Okonta JM Test Mode: SCANNING Graph's Name: UVNlS Scan of S1 Start Wavelength: 200.0 nm End Wavelength: 900.0 nm Scan Interval : Inm nm

peak: I WLOl=239.O Ab~0.376 , ,, 4.1 ." 1 i

%, Test Report Test Date: 08-03-2007 User Name: Okonta JM Test Mode: SCANNING Graph's Name: UVlVlS Scan of S1 Sdrt Wavelength: 200.0 nm End Wavelength: 900.0 nm Scan Interval : Inm nm

2: UV SFECl'ilii SCAN OF SAMPLE S2

I peak: WLOl=245.O Abs=0.883 Test Report Test Date: 08-03-2007 User Name: Okonta JM Test Mode: SCANNNG Graph's Name: UVNlS Scan of SJ, Smrt Wavelength: 200.0 nm E, -J Wavelength: 900.0 nm Scan Interval : Inm nm

Abr APPENDI.: 3: UV SPECTRA SC;iN OF SAEIPLE S3 IN CHI~OROFORM SOLVENT

peak: WL01=272.0 Abs=0.195 , .1 .> . ) ' Test Report Test Date: 812012007 User Name: Okonta : Test Mode: SCANNING ' Graph's Name: Sample scan of S1 F'?rt Wavelength: 200.0 nm €7Wavelength: 401.0 nm Scan Interval : 3nm nm

Abs APPENDIX 4: UV SPECTRA SCAN OF SAMPLE S1 IN ETHANOL SOLVENT f' 3.000 t

peak: WLO1=212.0 Abs=1.114 i WL02=224.0 Abs=1.519 . ,( d.1. . . I WL03=239.0 Abs=1.796 i WL04444.0 Abs=0.352 Page 1 Test Report Test Date: 8120/2007 User Name: Okonta Test Mode: SCANNING Graph's Name: Sample scan of S2 31~rfWavelength: 200.0 nm End Wavelength: 401.0 nm Scan Interval : 3nm nm

APPENDIX 5: UV [SPECTRA SCAN OF SAKPLE S2 TN E'I'HANOL SOLVENT

peak: WL01=242.0 Abs=3.000

WL02=347.0 Abs=0.544lq " ." ' WL03=356.0 Abs0.477 Page 2

Test Report r. ;t Date: 812012007 Jser Name: Okonta rest Mode: SCANNING F-aph's Name: Sample scan of S3 start Wavelength: 200.0 nm End Wavelength: 401.0 nm Scan Interval : 3nm nm

Abs APPENDIX 6: SPECTRA SCAN OF SAMPLE S3 IN ETI~IANOL SOLVENT

DO0

500

.ooo

.SJO

1 .OOO

3 500

,000

peak: WLOl=233.O Abs=1.237 . 1.. .', ' WL02=344.0 Abs=O. 109 WL03=353.0 Abs=O. I38 0 10 20 30 10 50 60 Conc. of phenol red (mcgll)

Relative Abundance APPENDIX lo: NMR data for Minusic acid

NMR data for rnlmuslc acid (1)

Cn~bonN,, 8, (ppiil IH r I1C' HETCOR 'H "I?IlMRC 'H r 'H NOESY 4 45.7 < 144.3 s 4n. z 10 33.5 I! I359 14 45.0 17 54.8 18 127.8 2" 34.7 2 S 170 1 C' H 2 m.9 4.52 (11. J = 4 147. H-IaJ 3 74.4 4 !? (d. J = 3.6 HI. H-k) 6 12fl.5 5.91 (dd. J = 24.48 Hz H-6) 9 48.4 1.96 (-'. H-91) 16 711,s 5OI (dd, J = If1.l. 4.3 HI. H-161, CH, I 45.7 1.55 (--. H-17) 2.53 (dd. J = 14. 4.2 Hz H-It) 1.96 (--'. H-7 81 2.51 (d. J = I6 5 HI. H-1:rj 1.66 (111. H-I IPI H-01. H.12~ i.sn(-: H-113) 2. I !C-: H-12,~) H-I 1P 2.82 (III, H-IIP) - 1.76 f-'. H-15zb H-16p. 3H-26 2.4fI (Jd. J = 14. 4.2 117. H-l PI - 2.U (d. J = l3.l HI. H-196) H-218. !H-iV. !H-!rl 2.70 ((1. J = 13.1 HI. H-IVz! 1.55 (--. H-2lf1 H-1%. ?H-Zq, !H-31) 2.1 3 G-.. H-21 7) 2. 11 {--. H-2 hi H-141'. H-211 2.78 (n~.H-2281 4.13 !d. J = v1.R HI. H,-2.1) H-!I. TH-24 4.20 (d, J = ll!S tlr. H,-L!) CH, 24 21 8 2 5 23.5 H-If'. 3H-26. H-I lb. 2 6 20.4 IH.25. H-IIP. H-158. H-lr?.,6 2 7 27,! H-71. H-1 11. H-15s 29 25.0 H-191. H-211. !H-?h 30 ?2 8 H-Iil. H-lap. n-2:~

ngure 2. Key NOE relatlomhlp observed for mlmusic acid (1) in a NOESY. Antidiabetic effect of Picralima nitida aqueous seed extract in experimental rabbit model

C. N. Aguwa'. C.V. Ukwe', S. I. Inya-Agha2, J. M. Okontal*

1. Department of Clinical Pharmacy and Pharmacy Management, University of Nigeria. Nuskka, Nigeria. 2. Department of Pharmacognosy, University of Nigeria, Nsukka, Enugu State, Nigeria.

Received 2000: Revised and Accepted 8 June 2001

Abstract

Objective: The blood sugar lowering effect of P. nitida aqueous seed extract was investigated on normoglycaemic and hyperglycaemic rabbits. Materials and methods: The hypoglycaemic effect of P. nitida was evaluated using alloxan (80 mglkg body weight, i.p) induced hyperglycaemic rabbits. The extracts potency was compared with standard drug, tolbutamide and distilled water. The LD,,, was determined using mice. Results: A dose of 648 mglkg body weight of extract caused maximum lowering of blood sugar levels in both normal and alloxanized rabbits. The mean fasting blood sugar in the normoglycaemic rabbit was reduced by 19.46% within 3 h, while in alloxanized rabbits blood sugar level was reduced by 75.5% within 6 h. Thc LD,,, of the extract in mice was 1601.2 + 60.5 mg/kg body weight when given i.p. Conclusion: t? nitida, though a crude drug, exhibited a faster onset of action and more persistent in hyperglycaemic situation than tolbutamide standard controls. This qualifies it to be used in ethnomedical diabetic management. . 1: .'a Keyword: Picrnlinla nitida, Antidiabetic activity, Alloxan.

1. Introduction

A large number of medicinal plants are used in " rainforest of Africa [7, 81. The fruit is broadly ethnomedicine globally as palliative therapy of abovoid, smooth and glabrous measuring about diabetes dellitus like Dioscorea dumentorunt, [l, 15cm long and l0cm in diameter. Each fruit 21 Bridelia ferruginea [3], Allium sativunz [4], contains three flattened seeds embedded in Vernonia amygdalia [5] and Anacardium pulp[8]. The seeds of R nitidn are used as quinine occidentale [6]. substitute in ethnomedical treatment of malaria Picralima nitida (Apocynaceae) Staph is a [9]. It has also been reported to have curative effect in respiratory infections and as enema in deciduous tree of about 20m in height with dense Ghana [lo]. crown and widely distributed in the tropical 136 C. N. Aguwn er al. /Journal of Natural Remedies, Vol. 1/2 (2001) 135 - 139

Almost all parts of the plant are used in the rabbits (0.6 1-0.8 1 kg) were used in the treatment of ailments; the root bark, seeds and experiments. The animals were kept under leaves are used in the treatment of all types of room temperature with access to water and fever, as antitussive, for wound healing, as food for 1 week before the commencement aphrodisiac [I 11, trypanosomiasis treatment and of experiments. as local anaesthetic comparable to cocaine [12]. 2.4 Acute toxicity test Some of its alkaloids posses antibacterial and analgesic properties [131, central nervous system The LD,,, of the extract was determined in mice depressant effect as well as intestinal smooth ip using the method of Tainter and Miller [16]. muscles spasmogenic activity [14]. 2.5 Determination of blood sugar lowering effect It is widely used plant in West Africa for different of extract purposes but very little is known or said about 2.5.1 Using nornzoglycaemic rabbits the effect on blood sugar level to the best of our knowledge. For this purpose, this study was The animals were fasted for 12 h, but were designed to evaluate the blood sugar lowering allowed access to water before and while the effect of the commonly used aqueous extract of experiment lasted. At the end of the fasting l? nitida seed using experimental animals. period, taken as zero time (0 h), blood was withdrawn from the marginal ear vein, blood 2. Materials and methods sugar level were determined by 0-toluidine 2.1 Plant materials method [17]. Animals having blood sugar The pods of P. nitida were collected through a concentrations of 84-102mg% after the 12 h herbalist at Orlu, Imo State Nigeria and identified fasting were grouped into three of five animals by Dr. (Mrs.) S.I. Inya-Agha of Department of per group. Pharmacognosy, University of Nigeria, Nsukka The group A received 648mg/kg extract in 1999. A voucher sample of the pod was (maximum effective dose observed from deposited in the Departmental Herbarium. preliminary work), B received 500mg/kg 2.2 Preparation of the aqueous extract tolbutamide, and C received 3ml/kg Distilled water. The tolbutamide group and distilled The pods were broken open, the. sleds Were' water group were the respective control groups. obtained and air-dried for 2 weeks. The seeds All administered drugs were through were ground to fine. About 150 g of the intraperitoneal route. powdered seed was maceraled in 350 ml of distilled water at room temperature for 24 h 2.5.2 Using Ityperglycaetnic rabbits with intermittent shaking. The material ,,was In alloxanized group, normal adult rabbits filtered and freeze - dried to solid residue having blood sugar levels of 84 - 102mg% after (21.6%). The extract was chemically tested for the 12 h fasting were used. The animals were the presence of different chemical constituents injected intravenously with 80mg/kg body using standard methods [15]. weight of alloxan monohydrate (Sigma, USA), 2.3 Animals freshly prepared in distilled water. The animals Wistar albino mice (20 - 41g) bred in the were fed for 7 days. Department of Pharmacology Animal Unit On the day 8, the survivors were fasted for 12 of the Universitv and local strain of adult h and their blood sugar levels determined by C. N. Aguwa et d. / Journal of Natural Remedies, Vol. 112 (2001) 135 - 139 137

0-toluidine method as above. Only animals and alloxanized rabbits. This extract dose with blood sugar level above 300 mg% were significantly (P < 0.05) lowered the blood used for the experiment. The diabetic rabbits sugar level in fasted normal rabbit from a mean were divided into 3 groups of five animals each value of 99.2 + 5.2 mg% (Oh) to 78.4 2 3.4 and treated as above but in day 9. mg% (3h) (Table 1) while beyond that hour, the blood sugar level rose continually. The 2.5.3. Collection of blond and estimation of effect of tolbutamide at 500mglkg followed blood sugar almost the same fashion, with a maximum At fixed time interval (0, 1, 3, 6, 9h) after reduction at 6h. Normal distilled water caused treatment, blood samples were withdrawn from no significant change in the blood sugar level. the marginal ear vein of the animals and their blood sugar levels were expressed as mg% 2 The effects of the extract, tolbutamide and SEM and the student's t - test was used to test distilled water on the blood sugar levels of the significance of difference between treated alloxanized rabbits are shown in Table 2. The groups and distilled water control. and between extract reduced the mean fasting blood sugar the blood sugar levels at 0 h and at various time levels from 404.3 mg% in 0 h to 99.0 mg% at intervals in each treated group with P=0.05. 6 h while tolbutamide caused blood sugar levels reduction from 337.5 mg% in 0 h to 104.7 mg% 3. Results in 3 h. The percentage reductions in blood sugar 3.1 Chemical Constituents of Pnitida extract levels produced by the extract in the fasted The aqueous extract of the seed gave positive normal and alloxanized diabetic rabbits were chemical reactions for glycosides, saponins, 19.46% and 75.5% respectively. tannins, alkaloids, proteins, and carbohydrattx.,. .I, ' 4. Discussion 3.2 Acute toxicity test The pharmacological investigations of the Administered intraperitoneally, the LD,, of the extract in mice was 1601.2 + 60.5 mg/kg. extract of P nitidn showed that the plant extract caused significant reduction in the blood 3.3 Hypoglycaemic egect of P nitida extract sugar.levels in hyperglycaemic rabbits. In the The maximum reduction in blood sugar alloxan-induced diabetic rabbits, the extracts occurred at a dose of 648mg/kg of both normal produced marked reduction in blood sugar level

Table 1 Effects of l? niticla aqueous seed extract, tolbutamide and distilled water on mean fasting blood sugar of rabbits Drug Doses Fasting blood sugar (mg%) Percentage Max. lh 3h 6h 9h Reduction 138 C. N. Aguwa et (11. I Journal of Natural Remedies, Vol. 112 (2001) 135 - 139

Table 2. Effects of I? nirida seed extract, tolbutamide and distilled water on mean fasting blood sugar of alloxanized rabbits Drug Doses Fasting blood sugar (mg%) Percentage -- (mgW Max. Oh lh 3h 6h 9h Reduction Extract 648mglkg 404.3 + 6.3 174.4 * 3.4* 121.2 k 7.0* 99.0 -+ 1.0** 191.7 * 3.7* 75.5 Tolbutamide 5001nglkg 337.5 + 4.7 212.3 * 7.0 104.7 * 6.2** 160.2 * 5.1 168.1 * 2.9 69.0 Distilled water 3 mllkg 356.3 r 6.5 365.7 * 5.8 355.9 k I .0 356.7 * 3.0 356.7 * 6.0 - p- Values are expressed as mean i SEM; * P < 0.05 vs respective control; ** P < 0.01 vs respective control; n=5.

within 1 h post administration which climaxed hypoglycaemic activity in alloxan-induced in the sixth hour post administration. The diabetic animals. This points LO a mechanism hypoglycaemic effect of the extract did not of action different from that of tolbutamide, persist beyond the first eight hours. and not related to insulin secretion from pancreatic @-cell. When compared with tolbutamide treated controls, the extract produced noticeable In diabetes, causes and sites of intervention in percentage maximal reduction in the mean the biochemical processes are many and include fasting blood sugar levels in normoglycaemic the action of hormones and chemical mediators

animals. The extract achieved its maximum. . t as well as vascular modifications of the pancreas blood sugar level lowering effect on the and insufficient insulin production [5]. Alloxan normoglycaemic animals faster than is known to permanently destroy the pancreatic tolbutamide treated controls but the latter p-cells [6] but the extract lowered the blood sugar experienced more persistent control of the mean levels in alloxanized rabbits which is an indication blood sugar level than the extract and a more thatethe extract has extra pancreatic effects. I' . .. significant percentage maximal reduction of the These may have been caused by the presence of blood sugar level which invariably showed that several biologically active secondary the extract has lesser hypoglycaemic effect in metabolites although the plants seed has been normoglycaemic rabbits than tolbutamide. known to be very rich in indole alkaloids highly Rut in the hyperglycaemic animals, the extract of implicated in the inhibition of phosphoenol P nitidcl seed has significant blood sugar level pyruvate carboxykinase, an important enzyme lowering activity even within 1 h post in glyconeogenesis, thus producing administration aid the maximal reduction of blood hypoglycaemia [ 181. sugar level highest in 6 h post administration while Inspite of these tentative mechanisms for the the maximal reduction of blood sugar level by L -.-,.,. i :- -A:..:*-. -c n c..-. L-- C. N. Aguwa el al. /Journal of Natural Remedies,Vol. 112 (2001) 135 - 139 139

References

I. Undie AS, Akubue PI. (1986) J. 10. Dalziel JM. (1358) Useful plants of West Ethnopharmucol. 15: 133 - 144. Tropical Africa, Crown Overseas Agents for Colonies: London; 65. 2. Iwu MM,Okunji CO, Akah PA, Tempesta MS, Corley DG. (1990) Plartta Med. 56: 1 19 - I I. Iwu MM. (1982) Ethrtomed. 7: 387 120. 12. Hamet C. (1940) In: Medicinal plants iit Nigeria, Nigeria College of Arts, Science 3. Iwu MM. (1980) Planfa Med. 39: 247 and Technology: Ibadan: 77 - 78. 4. Reiter HD (1995) Phyzomed. 2 (10): 78 - 91 13. Arens M, Borde HO, Ulbrich B, Stockigt J. (1982) Pfar~taMed. 46: 210 - 214. 5. Akah PA, Okafor CL. (1992) Phytother: Res. 6: 171 - 173. 14. Levy J, Lemen J, Jannot MM. (1963) Tetrahedr: 19: 1265. 6. Ezugwu CO, Okonta JM, Esimone CO. (2000) 15. Trease GE, Evans WC. ( 199 1 ) Pliarmncogrtosy, J. Nar. Remed. 1 (I): 60 - 63 13th edn, Bailliere Tindall: London; 167 - 7. Keay RWJ, OnochieCFA, Stemfield DD. (1964) 197. 386. Nigerian Trees, Ibadan Federal Dept. of 16. Tainler MC, Miller LC. (1944) Proc. Soc. Expt. Forest Rcs: Ibadan; 2, 378 - 396. Bid. Med. 57: 26 1.

8. Irvine FR. (1961) Woody plant of Gharra, Oxf. 17. Stroev EA, MakarowaVG. (1989) Lab. Manual University Press: London; 629 - 630. in Biochem, Mir Publishers: Moscow; 143.

9. Francoise G, Ake-Assi-I, Holenz J, Bringmann 18. Webb Leyden J. (1966) Enzyme and Metabolic G. (1996) J. Ethrtophamtacol. 54: 2 - 3, 1 13 Inhibitors, Academic Press: N.U; (3) 321 - - 117. 326. Intamnational Journal of Pharmacology 3 (6):505-509,2007 ISSN 181 1-7775 6 2007 Asian Network for Scientific Information

Evaluation of Hypoglycemic Activity of Glycosides and Alkaloids Extracts of Picralima fiitida Stapf (Apocynaceae) Seed

J.M. Okonta and C.N. Aguwa Department of Clinical Pharmacy and Pharmacy Management, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, Nigeria

Abstract: The blood glucose lowering effect of the seed extract of Picralima nitida has been suggested to be. due to its rich indole alkaloids; this study, therefore, is aimed at evaluating the hypoglycemic activity of the alkaloids and glycosides extracts of the Picralima nitida seed The alkaloids extract of Picralima nitida seed (Apocynaceae) given i.p. caused increase in mean fasting blood glucose levels while the glycosides extract reduced the blood glucose levels in normoglycemic and hyperglycemic rats. Glycosides extract caused significant @<0.05) percentage maximal reduction of 38.6% (250 mg kg-') and 22.9% (500 mg kg-') of the mean fasting blood glucose levels in normoglycemic and 64.4% (250 mg kg-') and 39.0% (500 mg kg-') in the hyperglycemic rats. The glycosides extract maintained low mean fasting blood glucose levels throughout the 24 h duration of study in hyperglycemic rats. On subchronic treatment of hyperglycemic rats, glycosides extract (250 mg kg-') caused 82.99% while glyburide (5 mg kg-') caused 60.81 % reduction of mean blood glucose levels. Thus the hypoglycemic activity of seed extract of Picralima nitida may be resident in the glycosides of the seed extract.

Key words: Picralima nitida, alkaloids, glycosides, hyperglycemia, hypoglycemia

INTRODUCTION (Gyang et al., 2004, Akah and Okafor, 1992), Musa sapientum (Jani and Sharma, 1967) and many others Diabetes mellitus is a complex metabolic disorder are used in the folklore management of diabetes mellitus. (Petal and Rybczynski, 2003; Ozturk, et al., 19%) that The seed extract of Picralima nitida (commonly named involves chronic alterations in the carbohydrate, fat and Akuamma seed in Ghana and Osi-Igwe seed in Igboland, protein metabolism, basically resulting from the secretion Eastern Nigeria) has been evaluated for blood glucose of dysfunctional andlor insufficient endogenous insulin lowering activity and has been proved effective for such by the p-cells of the pancreas. It is characterized by pharmacological action (Aguwa et al., 2001; Inya-

elevated blood glucose concentration that. may be , Agha et al., 2006). It has been suggested that the .. 4 "r. . ,.,, .?+ accompanied by severe thirst, imnatlon, hypoglycemic activity of this plant extract is due to polyphagia, weight loss, or stupor (Ozturk et al., 1996; the rich indole alkaloids (Inya-Agha, 1999; Bamidele et al., 2002). Aguwa et al., 2001). Since Picralima nitida extract is also The number of people that are suffering from th~s richly endowed with glycosides, which include scourge is on the increase and the orthodox antidiabetic biologically active compounds such as saponins, agents are relatively expensive andlor unavailable. These flavonoids, steroids and triterpenes (Vladimir and Ludmila, reasons have favoured the utilization of altematiye ,2001), the aim of this study, therefore, is to evaluate the medcine to stabilize the blood glucose level in most hypoglycemic activity of the alkaloids and glycosides diabetic patients of some developing countries of the extracts of the Picralima nitida seed so as to propose the world. Other factors include high cost of hospital class of the secondary metabolites that has the management of the disease, non-availability of competent hypoglycemic activity. health personnel and the long distance patient has to walk to health facilities (Gyang et al., 2004; Akah et al., 2002). MATERIALS AND METHODS In West Africa, plants like Momordica charantia (Nadkarni, 1994; Sanjay, 2002), Bersama engleriana Plant collection and extractions: The pods of the plant (Watcho et al., 2005), Vernonia amygdalina were collected from Ihiala, Anambra state, Nigeria in 2004

Corresponding Author: J.M. Okonta, Department of Clinical Pharmacy and Pharmacy Management, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, Nigeria Tel: +234 8076241044, +234 806472485 505 through a herbalist and authenticated by Dr (Mrs.)Inya- glucose levels were determined as the 0 h level. After the Agha of Department of Pharmacognosy, University of 0 h blood withdrawal, the rats in all the groups received Nigeria, Nsukka. The seeds were extracted from the pods, their drugs and blood samples were withdrawn at fixed air-dried for six weeks in the tropical sunlight and time intervals (1, 2, 4, 8, 12 and 24 h) and the blood pulverized. The powdered seed (301.89 g) was defatted glucose levels determined using Glucose meter kit with chloroform and macerated with 1 L of 90% methanol (Life scan, USA). for 7 days with intermittent shaking. The filtrates were concentrated to solid residues at room temperature. Effect of alkaloids and glycosides extracts of Picralirna Alkaloids extraction: The methanolic extract (9.6 g) nitida on mean fasting blood glucose levels in alloxanized was weighed out and dissolved in 70% methanol and rats: Twenty rats of either sex, with blood glucose levels made alkaline with ammonia hydroxide (basicity confirmed between 78-1 10 mg dL-' were fasted for 12 h before use. with litmus paper). The mixture was continuously shaken They were given 120 mg kg-' of alloxan monohydrate gently for one hour and extracted with equal ratio mixture (Sigmq USA) intra peritoneally. The alloxanized rats were of chloroform after acidifying with enough dilute HCl kept for 7 days for hyperglycemia to develop and stabilize (0.1N). The chloroform phase was basified with enough but had free access to food and water. The rats were ammonia hydroxide (pH >7) and dried to constant weight fasted on the day 8 for 12 h and their blood glucose by heating over a water bath at 45°C (Brain and Turner, levels were determined using the glucose meter kit 1975). The standard phytochemical tests were carried out (Lifescan, USA). The rats with blood glucose level above on the chloroform extract in accordance with Harborne 150 mg dI-' were randomly distributed into four groups (1 984) procedures. of 4 rats per group while the normal rats formed the fifth group. Two groups were given 250 and 500 mg kg-' of Glycosides extract: The methanolic extract (1 4.9 g) was glycosides extract respectively, one group received dissolved in water (60 mL) and mixed with 200 mL of glyburide (5 mg kg-'), one group of the rats received n-butanol and left to partition in a separating funnel 250 mg kg-' of alkaloids and one other group received overnight. The butanol phase was percolated through distilled water (3 mL kg-'). All the drugs were activated carbon bed (2 g). The n-butanol phase was administered ip after the blood samples for 0 h blood concentrated in a rotary evaporator into solid residue, glucose levels were collected. At fixed time intervals (1, 2,4, 8, 12 and 24 h), the blood samples were collected tested for glycosides (Harbome, 1984) and stored until and blood glucose levels determined. evaluated. Subchronic treatment effect of glycosides extract of Animals: Albino mice (20-35 g) and rats (85-200 g) of PicraIima nitida on mean fasting blood glucose levels in either sex, bred in the animal unif Department of alloxanized rat: Sixteen rats of either sex were fasted Pharmacology, University of Nigeria Nsukka and handled , for 12 h on the first day of the experiment after according to the stated guidelines of-,the rE+hicwl? twelve of the rats were treated ip with alloxan Committee, were used for these studies. They had access monohydrate, 120 mg kg-' (Sigma, USA) and tested for to water before and during the experimental stages, fed hyperglycemia 72 h post induction. Animals with blood with standard feed from Pfizer Plc, Lagos. The animals glucose levels above 150 mg a-' were considered were kept for one week prior to experimentation at room hyperglycemic. They were grouped into four of four rats temperature in the Department of Pharmacology, per group, one group received glycosides extract of University of Nigeria, Nsukka, Nigeria where the research ,250 mg kg-', one group received glyburide (5 mg kg-' was conducted in 2006. positive control) and a group received distilled water (3 mL kg-' negatwe control) and the last group received Effect of alkaloids and glycosides extracts of Picrdima distilled water but were normoglycemic rats; all the nitida on mean fasting blood glucose levels in normal animals receivedtheir drugs i.p for ten days. At the end of rats: Wistar albino rats of either sex were fasted for 24 h the fast in the fust day, blood was withdrawn from the tail but had access to water ad libitum. Five groups of four vein of each the animals and the blood glucose levels rats per group were used, two groups for alkaloids extract were determined as the first day level. After the blood at doses of 250 and 500 mg kg-', two groups for withdrawal, the animals recelved single dose of the glycosides extract at doses of 250 and 500 mg kg-' and respective drugs for the next ten days. Blood samples one group received glyburide at 5 mg kg-'; all the animals were withdrawn from the animals again on the tenth day received their drugs ip. At the end of the fast, blood was and the blood glucose levels determined using Glucose withdrawn fiom the tail vein of each animal and the blood meter kit (Life scan, USA). Int. J. PharmacoL, 3 (6): 505-509.2007

Acute toxicity test: Of the methanolic extract, LD,,, was baseline value while some of rats of the 500 mg kg-' determined intra peritoneally in mice using Lorke's treated group died of severe hyperglycemia at the 4th h. method (1 983). This maybe due to the inflammation and necrosis of liver hepatocytes (Fakeye et al., 2005) a5 hepatic damage can Statistical Analysis: Results are given as mean blood lead to insulin resistance (Yeh et al, 2003). The glucose levels * SEM (standard error of mean). One-way no~moglycemicrats treated with lower dose of glycosides ANOVA with post hoc Dunnett's multiple comparison (250 mg kg-') experienced consistent reduction (pc0.05) tests and student t-test. p values of 0.05 and less were in the mean fasting blood glucose levels from 108.3354.2 taken to imply statistical significance between the means at 0 h to 67.17f 2.9 mg dI-' at 12 h, while those treated with 500 mg kg-' showed reduction in mean fasting blood RESULTS AND DISCUSSION glucose levels from 94.67G.1 at 0 h to 73.0a7.6 mg &-' at 12 h (Table 1). The 250 and 500 mg kg-' doses of The acute toxicity test (LD,,) of methanolic extract, glycosides fraction of PicraIima nitida seed extract the parent extract of the alkaloids and glycosides, in caused percentage maximal reduction of 38.6 and 22.9% mice through intraperitoneal administration was calculated respectively in nonnoglycemic rats. to be 707.1 1532 mg kg-'. In hyperglycemic rats, 250 mg kg-' dose of The qualitative phytochemical analysis of extracts glycosides significantly reduced (p

Table 1: Effects of alkaloids and glycosides fractions on blood glucose levels of nmoglycmic rats (meanSEW 0 1 2 4 8 12 24 Drug Alkaloids 250 mg kg-' 70.01i2.1 95.5W1.0 102.17i2.3 79.17i3.5 74.1 7i2.8 75.83*5.2 76.42M. 5 (0.00) (-36.40) (-46.00) (-13.10) (-5.90) (-8.30) (-9.50) Gly cosldes 250 mg kg-' 108.33*4.2 110.83*2.9 94.67M.6 91.33H.2 79.1 7i2.7. 67.17*29* 86.45*5.7 (0.00) (-230) (12.60) (15.70) (26.91) (38.60) (20.20) 500 mg kg-I 94.67*5.1 88.83*3.7 80.83*6.7 8217*25 75.5B6.2 73.00*7.6 98.65*3.4 (0.00) (6.17) .,,G4.Q?),,.,,...:.,(13.20) (20.25) (22.90) (-4.20) Glyburlde 5 mg kg-I 84.75*2.4 79.23*4.1 73.45i2.3 64.51M.8 57.32i5.2 53.45*3.2 82.31M.1 (0.00) (6.511 (13.33) (23.88) (32.37) (36.90) (2.88) No. of animals per group = 4. No. in parenthesis represent percentage blood glucose reduction. Standard Emor of the Mean (*SEM). All values of mean fasting blood glucose levels were in mg dP,*: p

Table 2' Effect of glycosides My) exbact of Picralima nib& seed on mean fasting blood glucose levels of alloxanized rats (meanSEM, n = 4) 0 1 2 4 8 12 24 Drugs Glycosldes extract 250 mg kg-! 41 6.67i17.0 390.42*27 290.42i4.9* 211.67*21.5a* 195.0&23.9'** 148.33*21.3'** 178.91f3.7 (0.00) (6.30) (30.20) (49.20) (53.20) (64.40) (57.06) 500 mg kg-I 425.83*4.2 328.75i8.9 295.00112.8 316.25i14.2 327.5a2.5 259.58*12.5* 267.56*3.9 (0.00) (22.80) (30.72) (25.73) (23.09) (39.00) (37.17) Glyburlde (5 mg kg-') 267.75*26 153.75*6.3 119.00*4.9 96.75327 94.5W8.4 91.60*125 104.5e2.4 (0.00) (42.58) (55.56) (63.87) (64.71) (65.80) (60.95) DM.water (3 mL kg-') 186.25a4.7 183.75i2.1 197.50S.9 190.0B18.3 187.37*11.3 189.15*7.8 198.W6.9 (0.00) (1.34) (-6.04) (-201) (-0.06) (-1.56) (-6.79) Alkalolds 250 mg kg-' 234.26a8.3 238.01i17.4 241.97*5.7 248.54M.7 253.26a6.9 241.46*13.8 245.45*15.3 (0.00) (-1.60) (-3.29) (-6.10) (-8.1 1) (-3.07) (4.78) **: pc0.01 and *: pc0.05 level of significance against 0 h of the same dose of drug, ? FO.05 significant against Dist.water treated gnp. No. in parenthesis represent percentage reduction in mean fasting blood glucose levels Int. J. PharmacoL, 3 (6):505409,2007

Table 3: Effect of glycosides extract of Picrdma nitidz seed extract cm (Esimone et al., 2001 ;Inya-Agha, 1999) lke the glyburide. mean fasting blood glucose levels of alloxan-induced diabetic These pancreatic and extra-pancreatic effects on the -- albmo rats after subchronic treatment Blood Glucose level (mg dL-') (meaniSEM) blood glucose levels could be through prevention of hepaac glucose overproduction, increase in glucose Groups Day 1 Day 10 (YO)Max. red. uptake by the muscle, inhibition of gastric emptying Non-diabetic 89.OW.58 86.0M3.46 3.37 3 mL kg-' b.wt (Nolte and Karam, 2004) and/or increase in glucose Distilled water permeability of plasma cell membrane (Mahler and Alder, Diabetic control 21 7.25*40.03 20 1.75i32.63 7.13 1999; Shane-McWhorter, 2001 ). 3 mL kg-I b.wt Distilled water Since the subchronic treatment of hyperglycemic rats Glybwide 203.5W18.52 79.75*12.29*' 60.81 with the glycosides extract of Picralima nitida seed 5 mg kg-I b.wt. effected higher percentage maximal reduction than the Glycosides extract 393.00+12.54 66.85+6.51* 82.99 250 mg kg-' b.wt standard antidiabetic drug, glyburide, it may be of use in No. of animal per group =4, p

Bamidele, T.V., S. Josih A.A. Odetola and Nadkarni, A,, 1994. Dr KM Nadkarni's Indian Materia W.G. Okunade, 2002. The modulatory effect of Medica. 3rd Edn. Popular Prakashan Bombay, India, Momordica charantia on the Plasma and intestinal pp: 805-807. mucosa Ca+ ATPase in normal and alloxan induced Nolte, S.M. and J.H. Karam, 2004. Pancreatic Hormones diabetic rabbits. Nig. J. Pharm. Res., 1: 26-29. and Antidiabetic Drugs. In Basic and Clinical Brain, K.R. and T.D. Turner, 1975. The Practical Pharmacology, Katzung, Lange, B. (Ed.). 9th Edn., Evaluation of Phytochemical. Saunders and pp: 693-7 12. Company. NY., USA., pp: 98-1 02. Ozturk, Y., V.M. AttanandN. Yilctwglu-Ari, 1996. Effects Esimone, C.O., J.M. Okonta and C.O. Ezugwu 2001. Blood of Experimental ctabetes and insulin on smooth sugar lowering effect of Anacardium occidental muscle functions. Am. Soc. Pharmacol. Exp. Ther., leaf extract in experimental rabbit model. J. Natl. 48: 69-1 01. Remedies, 1: 60-63. Pari, L. and A. Satheesh, 2004. Antidiabetic activity of Fakeye, T.O., S.O. Awe, H.A. Odelola, O.E. Ola-Davies, Boerhaavia dzma L: Effect on hepatic key enzymes O.A. Itiola and T. Obajuluwa, 2005. Evaluation of in experimental diabetes. J. Ethnopharmacol., Valuation of Toxicity Profile of an Alkaloidal 91: 109-113. Fraction of the Stem Bark of Picralima nitida Petal, M. and P. Rybczynski, 2003. Treatment of non- (Fam. Apocynaceae). J. Herbal Med. Pharmacother. insulin dependent diabetes mellitus. Expert Opin. Innovations Clin. Applied Evidence-Based Herbal Invest. Drugs, 12: 623-633. Med., 4: 37-45. Sanjay, M.J., 2002. Herbal Drugs as Antidiabetic: An Gyang, S.S., D.D. Nyam and E.N. Sokomba, 2004. Overview. CRIPS, 3: 9-13. Hypoglycemic activity of Vernonia amygdalina. Shane-McWhorter, L., 2001. Biological complementary (chloroform extract) in normoglycemic and alloxan- therapies: A focus on botanical products in diabetes. induced hyperglycemic rats. J. Pharmacy Bioresour., Diabetes Spectrum, 14: 199-208. 1 : 61 -66. Smith, S.A. and Tadayyon, 2003. Insulin sensitization in Harbome, J.B., 1984. Phytochemical Methods. Chapman the treatment of type-2 diabetes. Expert Opin. Invest. and Hall, London, pp: 166-226. Drugs, 12: 307-324. Inya-Agha, S.I. 1999. The hypoglycemic properties of Szkudelski, T., 2001. The mechanism of alloxan and Picralima nitida. Nig. J. Natl. Prod. Med., 3: 66-67. streptozocin action in kcells of the rat pancreas. Inya-Agha, S.I., S.C. Ezea and O.A. Odukoya, 2006. Physiol. Res., 50: 537-546. Evaluation of Picralima nitida: Hypoglycemic Vladunir, K. and M. Ludmila, 2001. Glycosides in medicine: activity, Toxicity and analytical standards. Int. J. The role of glycosidic residue in biological Pharmacol., 2: 574-578. activity. Curr. Med. Chem., 8: 1303-1328. Jani, S.R. and S.N. Sharma, 1967. Hypoglycemic drugs of Watcho, P., G. Nyah Njike, T. Benoit Nguelefack and Indian Indigenous origin. Planta Med.; 16.. 439-442:' A. Kamanyi, 2005. Hypoglycemic activity of the Lorke, D., 1983. A new approach to practical acute toxicity leaves extracts of Bersama engleriana in rats. Afr. J. testing. Arch. Toxico. l., 54: 275. Trad. Compl. Altemat. Med., 2: 21 5-221. Mahler, R.J. and M.L. Alder, 1999. Type 2 diabetes Yeh, Y., D.M. Eisenberg, T. J. Kaptchuk and R.S. Philip, mellitus: Update on diagnosis, pathophysiology 2003. Systematic review of herbs and dietary and treatment. J. Clin. Endocrinol Metabolism, supplements for glycemic control in diabetes. 84: 1165-1171. Diabetes Care, 26: 1277-1294.