The mechanisms of action of some of these extracts include their ability to restore the function of pancreatic tissues or in protecting β-cells and smoothing out fluctuation in glucose levels^5,6 .

In general, there is very little biological knowledge on the specific modes of action in the treatment of diabetes, but most of the plants have been found to contain substances like glycosides, alkaloids, terpenoids, flavonoids etc., that are frequently implicated as having antidiabetic effects⁷. Many of these substances can also activate PPARs which control carbohydrate metabolism⁸.

Despite the great strides that have been made in the understanding and management of diabetes, the disease and disease related complications are increasing unabated⁹. Inspite of the presence of known antidiabetic medicine in the pharmaceutical market, remedies from medicinal plants are used with success to treat this disease¹⁰. Plant drugs⁷ and herbal formulation ^5-6 are frequently considered to be less toxic and freer from side effects than synthetic one. Based on the WHO recommendations, hypoglycemic agents of plant origin should be used in traditional medicine¹¹.

In human diabetics many have received such recognitions, examples include Fenugreek¹², Gymnema sylvestre¹³, and Gymnema yunnanense¹⁴. Lipid peroxidation is high in diabetics¹⁵. Few of the plant extracts have both antidiabetic and antilipidemice properties. These are Vinca Rosea leave^16,17, and Cinnamon which has been in the news lately because of its effects on improving insulin, blood sugar and blood lipid metabolism for the past 20 years¹⁸.

These results suggest that oxidative stress plays a key role in diabetes and treatment with herbal are useful in controlling not only glucose and lipid levels but other associated diseases of diabetes mellitus. Many plant extracts have been used to reduce the hyperglycemic blood level associated with diabetes but few have been found to be used in management of dyslipidemia in diabetes. We therefore deem it necessary to evaluate the glycemic and antilipidemic effects of the alkaloids of this plant in dithizone-induced diabetes. This is done to see whether this extract could be characterized and used in the management of diabetes and associated lipidemia.

2. MATERIALS AND METHODS:

2.1 Sample Preparation:

Fresh leaves of Emelia sonchifolia were collected from the Forest Reserve in University of Port Harcourt, Nigeria. The leaves were rinsed in water and dried in an open air. The dried leaves were blended with a high speed blender. The ground leave was stored dry and used throughout the work. Five hundred grams (550gms) of the leave was soaked in chloroform-methanol (2:1) and extracted for 18 hrs in a container on a flask shaker (Gallen Kamp). The mixture was filtered and the filtrate was extracted with equal volume of water and evaporated to dryness. A known weight of the extract was subjected to column and thin layer chromatography using Sephadex LH₂₀ (BDH) and silica gel (BDH) respectively. Elution was done with a mixture of chloform:methanol (2:1and 1:1) and finally with 95% methanol at a flow rate of 2mls/5mins and 130 fractions were collected. The elution pattern was monitored using thin layer chromatography. Fractions were tested for the presence of alkaloid with Dragendoff’s reagent.

The fractions that showed the presence of alkaloid (Plate 1) were pulled together and used as crude alkaloid extract. Chlorpropamide (Pz), a known antidiabetic drug was bought from Phamaceuticals and used as positive control. The drug was ground into powder and 48mg/kg body weight was given orally.

2.2 Treatment of Animal:

Male Wistar albino rats were bred in Department of Biochemistry animal house until they were 10 wks old. They were transferred to the experimental laboratory and housed for 1 week in a temperature-controlled room at 34 ± 1°C and given free access to standard laboratory diet and water before the experiment. The rats were treated according to the Ethical Guidelines of the Animal Center, University of Port Harcourt, Choba and the experimental protocol was approved by the Animal Studies Committee of University of Port Harcourt, Nigeria. Three groups the male rats (n=3) with average weight of 300-350g were used.

2.3 Induction of Experimental Diabetes:

The normal glucose levels of all the rats were tested before induction of diabetes. Diabetes was induced by intraperitoneal injection of dithizone (5mg/kg body weight) to the rats. They were allowed for 72 hrs for development of diabetes. After 72 hrs, hyperglycemic glucose levels were determined.

2.4 Oral Administration of the Extract:

Oral administration of 16, 32, and 48mg/kg body weight of the alkaloidal extract (AF) were given to groups A, B and C respectively. Group D was given 48mg/kg body weight of (chlorpropamide (CP) (diabinese), at the same time. Group E was non-treated diabetic rats that received 5 ml of normal saline and hence served as negative control (CO).

2.5 Collection and analysis of blood:

Blood was drawn before and after 72hrs of dithizone injection. Further collection of blood was done after 06, 12, 18 and 24hrs of oral administration of AF, CP and CO. Blood glucose concentration was determined using the glucose oxidase method of Trinder¹⁹. Cholesterol and triacylglycerol levels were determined using the methods of Braun²⁰ and Carstensen et al.²¹ respectively.

2.6 Statistical analysis:

Statistical analysis was done using ANOVA. Values were considered significant at p<0.05 and LSD was used to compare the means.

Table 1: Effects of Alkaloidal Extract of Emelia sonchifolia on Blood Glucose in Dithizone-Induced Diabetic Rat.

	Glucose Concentration (mmol/l)
	Gluc. Level before DTZ. Injectn	Glucose Level After 72hrs of DTZ injection (Hypgly. Level)	Hours After Administration of Samples (Hrs)
	Gluc. Level before DTZ. Injectn		(06)	(12)	(18)	(24)
Group A ( Ak Ext -16mg/Kg Body Weight)	3.02 +0.23^t	4.47 +0.25^t	4.26 +0.25^at	4.19 +0.36^mt	4.03 +0.47^ft	3.61 +0.22^agt
% Difference from Hyperglycemia Level	32.43	0	4.7	6.3	8.5	19.2
Group B ( Ak Ext -32mg/Kg Body Weight)	3.09 +0.15^v	4.67 +0.65^v	3.31 +0.09^vmf	4.36 +0.08^fmv	3.60 +0.57^v	4.30 +0.20f^v
% Difference from Hyperglycemia Level	33.83	0	29.1	6.7	22.9	7.9
Group C ( Ak Ext -48mg/Kg Body Weight)	3. 02 +0.43^q	4.56 +0.23^q	3.25 +0.03^qaf	4.44 +0.03^qfm	3.92 +0.48^q	4.09 +0.09^qfa
% Difference from Hyperglycemia Level	33.77	0	28.7	2.6	14.0	10.3
Group D(CP) (Chlorpropermide- 48mg/kg Body Weight)	3.09 +0.34^y	4.87 +0.29^y	4.14 +0.81^yf	3.89 +0.28^yfm	3.93 +0.17^y	3.99 +0.17^yfg
% Difference from Hyperglycemia Level	36.55	14.9	15.0	20.1	19.3	18.1
Group E(CO) (5 ml Normal Saline)	3.12 +0.09^b	4.46 +0.23^b	4.41 +0.03	4.45 +0.12	4.49 +0.06	4.43 +0.09
% Difference from Hyperglycemic Level	32.45	0	0.03	0.01	0.04	0.02

Values represent Mean+ Standard deviation of three samples in a group (n=3). Statistical analysis was done using ANOVA. Values were considered significant at p<0.05 and LSD was used to compare the means. Values with the same superscript are significant.

Table 2: Effects of Alkaloidal Extract of Emelia Sonchifolia on Cholesterol Level in Dithizone-Induced Diabetic Rat.

	Cholesterol Concentration (mg/dl)
	Chol. Level Before 72hrs of Induced Diabetes	Cholesterol Level After 72hrs of Induced Diabetes (Hypchol. Level ) (0)	Hours After Administration of Samples (Hrs)
	Chol. Level Before 72hrs of Induced Diabetes		(06)	(12)	(18)	(24)
Group A ( Ak Ext -16mg/Kg Body Weight)	2.04 +0.01^o	2.58 + 0.06^op	2.51 + 0.16^pn	2.57 + 0.03^opn	2.59 + 0.11^opn	2.52 + 0.01^opn
% Difference from Hypchol. Level	20.31	0	0.79	0.80	1.03	1.56
Group B ( Ak Ext - 32mg/Kg Body Weight)	2.09 +0.07^b	2.57 + 0.03^b	2.59+ 0.02^bdf	2.52 + 0.05^b	2.45 + 0.07^b	2.41 + 0.02^b
% Difference from Hypchol. Level	18.67	0	0.77	1.94	4.67	6.23
Group C ( Ak Ext - 48mg/Kg Body Weight)	2.02 +0.06ⁿ	2.55 + 0.07ⁿ	2.41+ 0.09^nbcd	2.58 + 0.03ⁿ	2.52 + 0.10ⁿ	2.40 + 0.01ⁿ
% Difference from Hypchol. Level	20.78	0	5.49	1.17	1.17	5.40
Group D(CP) (Chlorpropermide- 48mg/kg Body Weight)	2.21 +0.04^s	2.56 + 0.08^s	2.20+ 0.01^sfbcd	2.61 + 0.03^s	2.54 + 0.01^s	2.63 + 0.03^s
% Difference from Hypchol. Level	13.67	0	10.16	1.95	0.78	2.73
Group E(CO) (5 ml Normal Saline)	2.23 +0.05^c	2.41 + 0.01^c	2.40+ 0.01^df	2.41 + 0.02	2.42 + 0.10	2.41 + 0.09
% Difference from Hypchol. Level	15.3	0	1.34	0.89	0.45	0.89

Table 3: Effects of Alkaloidal Extract of Emelia sonchifolia on Triacylglyceride in Dithizone-Induced Diabetic Rat.

	Triacylglyceride(TAG) Concentration (mg/dl)
	TAG Level Before 72hrs of Induced Diabetes	TAG Level After 72hrs of Induced Diabetes (Hyptri. Level) (0)	Hours After Administration of Samples (Hrs)
	TAG Level Before 72hrs of Induced Diabetes		(06)	(12)	(18)	(24)
Group A ( Ak Ext -16mg/Kg Body Weight)	0.97 +0.07	1.05 + 0.12^k	0.96 + 0.16	0.85 + 0.05^ak	0.89 + 0.09	0.95 + 0.05
% Difference from Hyptri. Level	7.62	0	7.60	19.04	15.23	7.59
Group B ( Ak Ext -32mg/Kg Body Weight)	0.89 +0.12^m	1.06 + 0.19^m	0.85 + 0.05^m	1.02 + 0.12^c	0.95 + 0.15	0.93 + 0.05
% Difference from Hyptri. Level	16.03	0	19.8	3.78	10.37	12.26
Group C ( Ak Ext -48mg/Kg Body Weight)	1.00 +0.08^f	1.13 + 0.18^f	0.95 + 0.15	0.85 + 0.05^acf	0.83 + 0.10	0.83 + 0.01af
% Difference from Hyptri. Level	11.50	0	15.92	24.77	26.54	26.54
Group D(CP ) (Chlorpropermide 48mg/kg Body Weight)	0.87 +0.07	1.09 + 0.16	0.90 + 0.10	0.93 + 0.03	0.94 + 0.01	1.00 + 0.01^a
% Difference from Hyptri. Level	20.18	0	17.43	14.67	13.76	8.26
Group E(CO) (5 ml Normal Saline)	1.01 +0.01^p	1.14 + 0.03^p	1.09 + 0.01	1.14 + 0.01^a	1.08 + 0.02	1.12 + 0.03
% Difference from Hyptri. Level	12.4	0	0.99	0.00	0.97	0.05

3.2: Effects of Alkaloidal Extract of Emelia Sonchifolia on Cholesterol Level in Dithizone-Induced Diabetic Rat:

The effect of AK extract on cholesterol level was shown on Table 2. Cholesterol level was increased to a percentage range of 13.67- 20.78% after 3 days of dithizone injection. In Group A, cholesterol was significantly reduced by the extract after 12 and 18 hrs when compared to the hypercholesterol level of 2.58±0.06 mg/dl. After 6hrs of extract administration, Group C significantly reduced cholesterol level by 5.49% compared to 0.79 and 0.77% in Groups A and B respectively. Chlorpropamide in Group D significantly (p< 0.05) reduced blood cholesterol only after 6hrs of administration. In Control group E cholesterol remained almost at the same hypercholesterol level throughout the time of investigation.

3.3 Effects of Alkaloidal Extract of Emelia sonchifolia on Triacylglyceride in Dithizone-Induced Diabetic Rat.

The result in Table 3 shows that dithizone was able to increase the level of triacylglycerol from a normal range of 0.87±0.07–1.01±0.18mg/dl to a hypertriaylglycerol level of 1.05±0.12 – 1.13±0.18mg/dl. The highest percentage increase in triacylglycerol level after three days of inducing diabetes was 20.18%. There were decreases in triacylglycerol after 6, 12, 18 and 24hrs of extract administration in all the groups with exception of group E. After 6hrs of extract administration the highest decrease in triacylglycerol was found in Group B with percentage decrease of 19.8%. Increase in concentration of the extract decreased the triacylglycerol. Thus group C with 48mg/kg body weight of the extract has 26.54% decreases when compared with that of chlorpropamide, the decrease was more with the extract especially at 48mg/kg body weight. High levels of triacylglycerol were observed throughout the study in group E.

DISCUSSION:

Hypercholestrolemia and hypertriglyceridemia have been reported to occur in diabetic rats²². In normal condition, insulin increases the receptor-mediated removal of LDL-cholesterol and decreased activity of insulin during diabetes thereby causing hypercholestrolemia. This observation was found in the present study where dithizone was used to induce diabetes. There were increases in cholesterol and triacylglycerol by percentage ranges of 13.67–20.78% and 7.6–20.18% respectively. The increased concentration of cholesterol could result in a relative molecular ordering of the residual phospholipids resulting in a decrease in membrane fluidity²³. Accumulation of triglycerides is one of the risk factors in Coronary Heart Disease (CHD). The significant increase in the level of triglycerides in liver and kidney of diabetic control rats may be due to the lack of insulin. Since under normal condition, insulin activates the enzyme lipoprotein lipase and hydrolysis triglycerides²⁴.

Dithizone induced diabetes increased the level of triacylglycerol from normal level to hyperlipidemic level by 7.62–20.18%. Similar increase in triacylglycerol level in experimental diabetes with diabetogenic agents like alloxan and streptozotocin has been reported²⁵.

The extract reduced the glucose, cholesterol and triacylglycerol levels. This reduction is concentration dependent, ie the higher the concentration, the greater the percentage reduction. Few plant extracts have been used to reduce the blood glucose, cholesterol and triacylglycerol level and these compare well with the result of the present study. Aqueous extract of Piper nigrum seeds and Vinca rosea flowers were administered orally to alloxan induced diabetic rats once a day for 4 weeks. These treatments lead to significant lowering of blood sugar levels and reduction in serum lipids. They also found out that oxidative stress plays a key role in diabetes, and thus could be one of the reasons for the dyslipidemia associated with diabetes.¹⁵ Dianex, a polyherbal formulation produced significant (p<0.05) hypoglycemic and reduced triglycerides, and cholesterol levels activity at 250-500 mg/kg doses in normal and streptozotocin induced diabetic mice²⁶, Many other plant extracts have been used in treatment of diabetes. Daily amounts of 1, 3, or 6 grams of cinnamon reduced glucose by 18-29% following 40 days of treatment; a continued reduction in glucose levels even at the 60-day mark with 16% reduction was observed²⁷. In the present study, 24hrs was used as this is a preliminary study. Extracts from banana also showed a 30% reduction in blood glucose levels²⁸. Another extracts that lowered cholesterol and blood glucose at the same time like in the present study was guggul which also lessened serum turbidity²⁹. The cholesterol-lowering effects have been seen in monkeys kept on a high cholesterol diet, with results comparable to those of Atromid-S.3. Garlic extract also has been found to reduce total cholesterol more than the placebo (p< 0.01). Mean total cholesterol decrease was 15.7mg/dl³⁰.

Dithizone is a metal chelator, and has been extensively used in the study of islets for transplantation^31,32 depending on concentration. Tissues selected on the basis of dithizone staining was shown to contain insulin-positive cells and to accumulate insulin in the medium during a subsequent period in tissue culture. Dithizone is therefore used for identification of islets in transplantation and also used in induction of diabetes in experimental animals. Partial destruction of islets in dogs, cats and rats³³ has been demonstrated and this compares well with the present study where dithizone raised the blood glucose level by 32.45–36.55%. Rerup, (1970)³⁴ in his review reported that dithizone diabetes shows similar phasic fluctuation with selective damage of insulin producing cells. The phasic glycemia comprises an initial hyperglycemia, followed by hypoglycemia and finally a permanent hyperglycemia. This is achieved in the present study by allowing the development of diabetes for 72hrs. This is similar to the method of inducing diabetes with alloxan³⁴. Development of diabetes was shown to be connected with its ability to chelate metals like zinc. The dynamics of changes of the Zn content in the pancreatic islets after dithizone injection showed that the amount of the metal in the islets reduced due to destruction of the cells and under the effect of high blood plasma glucose concentration. The insulin producing cells were devoid of zinc in diabetes of long duration with high hyperglycemia³⁵.

Much studies on Emelia have not been reported, neither have there been any report of its antidiabetic or antilipidemic properties. The few reports on the herb include the antipyretic effect and its remedy for influenza, cough, and bronchitis³⁶. Other members of Aster family used in management of diabetes inludes Vernonia amygdaline (bitter leave)³⁷, Milk thirstle^38-39 and Psacallium decompositum⁴⁰. Infusions of asteracea like Carqueja has been documented to lower blood glucose levels in human and animal studies². The infusion is prepared with 5 g (about a teaspoon) of dried herb to 4-6 ounces water and infused for 10 minutes; the present study used alkaloid instead of infusion.

Sulfonylureas have significant side effects like hypoglycemia and weight gain (typically 4 to 6 kg)^41,42. They have no effects on lipids. This lack of benefit on cardiovascular complications may be related to the fact that they cause hyperinsulinemia, which is associated with the metabolic syndrome⁴³. The chlorpropamide (sulphurnylurea) significantly reduced the glucose level after 12, 18 and 24hrs. This is in consonance with the sulphurnylureas action, however, the reduction did not go below the normal level or into hypoglycemia. The chlorpropamide reduced the cholesterol level after 6hrs when compared with the highest concentration (48mg/kg body weight) of the extract. On the contrary, the extract decreased the triacylglycerol more than the chlorpropamide, especially at 48mg/kg body weight of the extract. This is also a confirmation of the fact that sulfonylureas do not affect the lipids excessively.

CONCLUSION:

The present study shows that the alkaloidal extract of Emelia reduced the glucose level of dithizone- induced diabetes which induced a partial destruction of the pancreas, showing partial presence of insulin. The extract proved to be effective in reducing both the high cholesterol and triacylglycerol levels associated with diabetes. Chlorpropamide, which is a known antidiabetic drug, reduced the blood glucose more than the extract, but the extract reduced cholesterol and triacylglycerol more than the drug. The control group did not show any significant reduction from the diabetic levels. The alkaloidal extract of Emilia sonchifolia could be further refined tested and used in management of diabetes and lipidemia.

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Received on 20.07.2010

Accepted on 11.08.2010

Research J. Science and Tech. 2(3): May –June. 2010: 51-56