Tragia tennifolia Benth. (Euphorbiaceae) is an herbaceous, mostly twining
or trailing plant found in Ghana and other West African countries. In Ghanaian
traditional medicine, the leaf-mash is used to treat sores, as an abortifacient
or promote child delivery (Burkill, 2004; Irvine,
1967) other parts have also been used as remedy for impotence (Dokosi,
1998), rheumatism and arthritis (Irvine, 1967); an
infusion of the warmed leaves are drunk as remedy for diarrhea, colitis or dysentery.
The plant has been reported to decrease the anti-diabetic drug dosage when used
together as infusions or fresh juice (Stuart, 1979).
The prevalence of diabetes for all age-groups is projected to rise to 366 million
in 2030 in developed countries (Rathmann and Giani, 2004;
Shaw et al., 2010; Wild
et al., 2004). This estimate is expected to double in urban populations
in developing countries (Wild et al., 2004). In
developing countries, high cost of effective medicines and lack of appropriate
health facilities have led to the poor management of life threatening and chronic
diseases like diabetes. Thus a greater percentage of the population relies on
traditional medicines (Calixto, 2000; Okwu
and Uchegbu, 2009; Sofowora, 1993). More than 400 plant
species and 100 polysaccharides isolated from plants have been reported to have
hypoglycaemic effects (Ernst, 1997; Wang
and Ng, 1999; Yuan et al., 1998). Despite
the global and local need of cost effective drugs for the management of diabetes,
only a few of these plant sources have been explored.
The present study reports of the hypoglycaemic effects of T. tennifolia on models of hyperglycaemia in Sprague-Dawley rats, thus supporting its local use in the management of diabetes mellitus.
MATERIALS AND METHODS
Plant sample collection: Quantities of the whole plant of Tragia tennifolia were collected from the botanical garden at the Faculty of Pharmacy, KNUST in September 2000 and identified by the Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, KNUST, Kumasi, Ghana.
Preparation of extract: Whole plants of Tragia tennifolia were collected; air-dried and powdered. The powdered material (300 g) was Soxhlet extracted with 70% v/v ethanol. The extract was then evaporated to a brown semi-solid mass under reduced pressure and kept in a desiccator until required. This is subsequently referred to as the extract or TTE. Suitable quantities of the dried product were dissolved in normal saline for use.
Animals: Sprague-Dawley rats of either sex (200-300 g) were purchased
from Noguchi Memorial Institute for Medical Research, University of Ghana, Legon
and maintained in the Animal House of the Department of Pharmacology, Kwame
Nkrumah University of Science and Technology (KNUST), Kumasi. The animals were
housed in groups of six in stainless steel cages (34x47x18 cm) with soft wood
shavings as bedding, fed with normal commercial pellet diet (GAFCO, Tema), given
water ad libitum and maintained under laboratory conditions (temperature
22-24°C, relative humidity 60-70% and 12 h light-dark cycle). All procedures
and techniques used in these studies were in accordance with the National Institute
of Health Guidelines for the Care and Use of Laboratory Animals (NIH, Department
of Health Services publication No. 83-23, revised 1985). The Departmental Ethics
Committee approved the protocols for the study.
Drugs: The drugs and chemicals used in this study include Glibenclamide and metformin (Hovid Pharmaceuticals, Belgium) suspended in 1% w/v tragacanth; insulin (Novo-Nordisk A/S, Denmark).
Oral Glucose Tolerance Test (OGTT): Twenty Sprague-Dawley rats were fasted overnight and randomly put into six groups (n = 5) as follows; TTE (26.5, 53, 106 mg kg-1), Metformin-treated (12.14 mg kg-1), Glibenclamide-treated (0.14 mg kg-1, p.o.) and vehicle-treated (0.2 mL normal saline, p.o.). Thirty minutes after extract/drug/vehicle treatment, the rats received a loading dose of glucose (1.75 gk g-1, p.o.). The plasma glucose concentration of each rat was then measured after 0.5, 1.0, 1.5, 2.0, 3.0 and 4.0 h using a Glucostix® reagent strip (Bayer Diagnostics, Ames, USA) according to the manufacturers instructions. Briefly, a drop of blood expressed from the tail vein was placed unto the Glucostix® test pads. The test strip was then inserted into the Glucometer® II after drying in air for 90 sec and the plasma glucose read. Urine glucose and ketone were semi-quantitatively determined using Uriquick TM CLINI-9SG (Stanbio lab. Inc., San Antonio Texas, USA) in accordance with the manufacturers instructions.
Fasting Plasma Glucose Test (FPGT): Twenty rats (200 -250 g) were fasted overnight but allowed free access to water and then their fasting plasma glucose concentrations were determined. The animals were randomized into four groups (n = 5) and treated with vehicle, glibenclamide (0.14 mg kg-1), metformin (12.14 mg kg-1) or TTE (53 mg kg-1), respectively. Plasma glucose concentrations were measured at hourly intervals for up to 5 hours as described in OGTT.
Alloxan-induced DM: Rats were put in individual metabolic cages and
allowed five days to acclimatize. Food and water intake, urine and faeces output,
bodyweight and plasma glucose concentration were then measured daily over a
period of 7 days to establish the normal (control) conditions. DM was induced
with alloxan monohydrate (140 mg kg-1, intraperitoneally.)
as previously described (Panneerselvam and Govindaswamy,
2002). After a week, rats with plasma glucose concentration of at least
19.4 mmol L-1 (350 mg dL-1) and having glycosuria (indicated
by Uriquick TM CLINI-9SG) were considered diabetic. Changes in food and water
intake, urine and faeces output, bodyweight and plasma glucose concentration
were recorded daily for 10 days (the diabetic stage). The effects of insulin
(8.5 units/kg/day), glibenclamide (0.14 mg kg-1), metformin (12.14
mg kg-1) and TTE (53 mg kg-1) on the parameters above
in the diabetic rats were recorded for 7 days (the treatment stage). Treatment
was then withdrawn and the animals observed for a further 4 days.
Acute toxicity studies: TTE was administered orally to 5 groups of rats (n = 6) at dose levels of 0.1, 0.2, 0.3, 0.5 and 1.0 g kg-1. Control group received vehicle (1 mlk g-1, p.o.). The animals were observed for up to 24 h (acute) and subsequently for 7 days to observe possible delayed toxicity.
Statistical analysis: Data are presented as Mean±SEM. (n = 5-10) and analysed by One-way ANOVA followed by Newman-Keuls test for column graphs. GraphPad Prism Version 5.0 for Windows (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses. The p≤0.05 was considered statistically significant in all analysis. The graphs were plotted using Sigma Plot for Windows Version 11.0 (Systat Software Inc., Germany).
Oral Glucose Tolerance Test (OGTT): After the glucose load (1.75 gk g-1), plasma glucose level peaked 4.3 fold (2.37-12.54 mmol L-1) within 1 h (Fig. 1a). Glibenclamide (0.14 mg kg-1) and metformin (12.14 mg kg-1) decreased this peak to 6.39 mmol L-1 (51% reduction) and 5.89 mmol L-1 (47% reduction), respectively (p≤0.01). TTE (26.5, 53 and 106 mg kg-1, p.o.) also reduced the peak plasma glucose value by 2.63 mmol L-1 (21% reduction), 5.89 mmol L-1 (47% reduction) and 6.45 mmol L-1 (51.5% reduction), respectively (Fig. 1). The AUC values for drug-treated rats compared with vehicle-treated rats are as shown in Table 1. TTE (53 mg kg-1) reduced the AUC under the OGTT curve by 45%.
Fasting Plasma Glucose Test (FPGT): In the vehicle-treated control group,
there was no significant change in plasma glucose throughout the experimental
||Effect of (a) TTE (26.5-106 mg kg-1) and (b) Gilbenclamide
(0.14 mg kg-1) and Metformin (12.14 mg kg-1) on plasma
glucose concentration of normal rats in an oral glucose tolerance test.
Values are Means±SEM (n = 5). *p≤0.05, ** p≤0.01, ***p≤0.001
compared to vehicle-treated group (Two-way ANOVA followed by Bonferronis
post hoc test)
|| The area under the curve (AUC) values for curves in the OGTT
|OGTT: Oral glucose tolerance test, FPGT: Fasting plasma glucose
test and AUC: area under curve
Metformin (12.14 mg kg-1) had a similar effect to the vehicle-treated
control. Fasting plasma glucose in glibenclamide (0.14 mg kg-1) and
TTE-treated (53 mg kg-1) rats however decreased significant (p≤
0.01) after 2-3 h of drug administration (Fig. 2). The AUC
values for TTE-treated rats compared to vehicle-treated reduced by 14.8% (Table
Alloxan-Induced DM: Plasma glucose concentration increased significantly (p≤0.001) from 4.7±0.32 to 21.35±0.20 mmol L-1 after induction of DM (urine glucose also increased from trace quantities to 1500 mg dL-1). Treatment of the diabetic rats with insulin (8.5 units kg-1) caused a 3 fold reduction (p≤0.001) in glycemic levels from 21.35±0.20 to 6.55±1.24 mmol L-1 (Fig. 3a). However, plasma glucose concentration increased significantly again to 19.81±0.96 after insulin withdrawal. Glibenclamide (0.142 mg kg-1), metformin (12.14 mg kg-1) and TTE (53 mg kg-1) did not reduce significantly (p≤0.05) plasma glucose concentration during treatment (Fig. 3a). Urine glucose levels were between 1500-2000 mg dL-1.
||Effect of TTE (53 mg kg-1), Gilbenclamide (0.14
mg kg-1) and Metformin (12.14 mg kg-1) on plasma glucose
concentration of normal rats in an oral glucose tolerance test. *p≤0.05,
**p≤0.01, ***p≤0.001 compared to vehicle-treated group (Two-way ANOVA
followed by Bonferronis post hoc test)
Insulin reduced food intake on treating diabetic rats but glibenclamide, metformin
and TTE did not reduce significantly food intake (Fig. 3b).
Daily water intake increased (p≤0.001) 3 fold (32.3 to 86.8 mL) and urine
output increased (p≤0.001) 6 fold (11.1 to 63.6 mL) compared to vehicle-treated
control. Aside insulin which significantly decreased (p≤0.001) water intake
and urine output, there were no significant changes (p≤0.05) on treating
with glibenclamide, metformin and TTE (Fig. 4a, b).
There was however, no significant changes in body weight before and after induction
of diabetes and treatment with all the drugs (Fig. 5).
||Changes in (a) plasma glucose concentration (mmol L-1)
and (b) food intake (g) with respect to normal rats, alloxan-induced diabetic
rats and the results of treatment of the diabetic rats with TTE (53 mg kg-1),
Gilbenclamide (0.14 mg kg-1) and Metformin (12.14 mg kg-1)
and post-treatment effects. Each bar represents the mean±SEM. (n
= 5). ***p≤0.001 (One-way ANOVA followed by Newman-Keuls post hoc test)
||Changes in (a) water intake (mL) and (b) urine output with
respect to normal rats, alloxan-induced diabetic rats and the results of
treatment of the diabetic rats with TTE (53 mg kg-1), Gilbenclamide
(0.14 mg kg-1) and Metformin (12.14 mg kg-1) and post-treatment
effects. Each bar represents the mean±S.E.M. (n = 5). ***p≤0.001
(One-way ANOVA followed by Newman-Keuls post hoc test)
||Changes in weight (g) with respect to normal rats, alloxan-induced
diabetic rats and the results of treatment of the diabetic rats with TTE
(53 mg kg-1), Gilbenclamide (0.14 mg kg-1) and Metformin
(12.14 mg kg-1) and post-treatment effects. Each bar represents
the Mean±SEM. (n = 5). *p≤0.05 (One-way ANOVA followed by Newman-Keuls
post hoc test)
Acute toxicity: There was no convulsions, labored breathing, diarrhea, constipation, emaciation, skin eruptions, abnormal posture, bleeding (from the eyes, nose, or mouth), sedation, ataxia, flaccid paralysis polyuria, polydipsia, polyphagia and anorexia rhinorrhoea/nasal congestion, loss of autonomic reflexes, decreased locomotory activity, neuromuscular incoordination and collapse hyperesthesia, hypothermia, twitching, spasticity, writhing, respiratory depression, ocular discharge, prostrate posture and mortality.
The significant reduction in AUC values caused by glibenclamide, metformin
and TTE in OGTT indicates hypoglycaemic effect. Glibenclamide inhibits ATP-dependent
potassium channels on pancreatic beta cells which augment insulin secretion
and increases sensitivity to insulin of the insulin-dependent tissues i.e.,
the liver, muscles and adipose tissues resulting in glucose utilization while
metformin enhances glucose utilization by peripheral tissues and also a delay
in the absorption of glucose (Sethu et al., 2002).
Metformin stimulates the hepatic enzyme AMP-activated protein kinase (Zhou
et al., 2001). If insulin secretion is induced before glucose loading,
plasma glucose concentration will not rise significantly above normal ranges
2 h postprandial. Plasma glucose concentration falls rapidly as the extent of
utilization would be high and could even reduce to within fasting plasma glucose
ranges in relatively short hours postprandial. TTE could possibly be exerting
its hypoglycaemic effect by mechanisms similar to that of glibenclamide and
The degree of hypoglycaemia produced by glibenclamide and TTE in fasted rats
increases significantly with time over the dose administered. This effect was
however insignificant with metformin. The maintenance of blood glucose levels
in the fasting state is largely dependent on hepatic glucose production. Any
agent that reduces hepatic glucose production therefore, will produce a hypoglycaemic
effect (El-Shabrawy and Nada, 1996). Glibenclamide enhances
insulin secretion from pancreatic β-cells which cause hepatic glucose utilization
i.e., glycogen formation. Insulin release antagonizes the activity of glucagon
with a resultant reduction in fasting plasma glucose (Sethu
et al., 2002).
Direct stimulation of glycogen formation, reduction in gluconeogenesis, slowing of glucose absorption and induction in glucagon secretion takes place only when plasma glucose levels are rising. Metformin which exerts its hypoglycaemic effect mainly by these mechanisms will insignificantly affect fasting plasma glucose levels. Since TTE lowers fasting plasma glucose significantly, it may have suppressive action on hepatic glycogenolysis as the maintenance of blood glucose in the fasting state depends largely on hepatic glucose production.
The effect of TTE, glibenclamide and metformin on plasma glucose concentration
in the alloxan-induced diabetes suggested that TTE may not be useful in insulin-dependent
DM. However, insulin produced significant reduction in blood glucose levels.
Alloxan is a diabetogenic agent known to induce diabetes mellitus by destroying
pancreatic β-cells of the islet of Langerhans where insulin is produced
and secreted (Singh and Gupta, 2007). There could therefore
be a partial or complete lack of insulin resulting in the metabolic disorder
characterized by hyperglycaemia. When insulin is lacking, uptake and utilization
of glucose by insulin-sensitive sites are shut down. Glucose therefore persists
in plasma (hyperglycaemia) and spills into the urine (glucosuria). There is
energy and water loss mainly through urine as renal threshold for glucose conservation
(about 180 mg dL-1) is exceeded. There is osmotic diuresis resulting
in polyuria, dehydration and thirst (Ganong, 1995).
The results confirm observations made that in insulin-dependent diabetes mellitus
there is hyperglycaemia, glucosuria, polyphagia, polydipsia, polyuria (English
and Williams, 2004). Insulin treatment showed significant reduction and
reversal in hyperglycaemia, glycosuria, polyphagia and polyuria when alloxan-induced
diabetic rats were treated with exogenous insulin. Injected insulin mimics the
activity of endogenous insulin and causes a reversal of the catabolic features
of insulin deficiency and enhances anabolic activity in the liver. It also enhances
protein and glycogen synthesis in the muscles and increases triglyceride formation
from glucose, to be stored in adipose tissues: Hence, the fall in plasma glucose
concentration (Herfindal and Gourley, 1996). It is clear
from the results obtained that glibenclamide and metformin do not significantly
reverse the condition of hyperglycaemia and hence glycosuria, polyphagia and
polyuria in alloxan-induced DM. This is consistent with findings on the mechanism
of action of glibenclamide and metformin in insulin-dependent DM. TTE had effects
similar to glibenclamide.
The present study demonstrates that TTE produces hypoglycaemic effects in the oral Glucose Tolerance Test (OGTT) and Fasting Plasma Glucose Test (FPGT) but had no significant effect in the alloxan-induced diabetes. Thus TTE is useful in insulin-independent diabetes rather than insulin dependent type. The results of the acute toxicity studies suggests that TTE is safe in rats up to a dose of 0.5 g kg-1.
The Authors wish to express their gratitude to Mr. Thomas Ansah of the Department of Pharmacology, KNUST, Kumasi for his technical assistance.