INTRODUCTION:

Diabetes mellitus is an endocrine disorder that is characterized by hyperglycemia¹. The pharmaceutical drugs are either too expensive or have undesirable side effects. Treatment with sulphonylureas and biguanides are also associated with side effects². However, for a number of reasons, complementary medicine has grown in popularity in recent years. Dietary measures and traditional plant therapies as prescribed by Ayurvedic and other indigenous systems of medicine were used commonly in India. Many indigenous Indian medicinal plants have been found to be useful to successfully manage diabetes and some of them have been tested and their active ingredients isolated^3,
4. The World Health Organization (WHO) has also recommended the evaluation of the plants effectiveness and conditions where we lack safe modern drugs⁵.

In recent years, much attention has been focused on the role of oxidative stress, and it has been reported that oxidative stress may constitute the key and common event in the pathogenesis of secondary diabetic complications⁶. Free radicals are continuously produced in the body as a result of normal metabolic processes and interaction with environmental stimuli. Oxidative stress results from an imbalance between radical-generating and radical-scavenging systems that has increased free radical production or reduced activity of antioxidant defenses or both. Implication of oxidative stress in the pathogenesis of diabetes mellitus is suggested not only by oxygen free-radical generation but also due to non-enzymatic protein glycosylation; auto-oxidation of glucose, impaired glutathione metabolism, alteration in antioxidant enzymes and formation of lipid peroxides^7-10. In addition to reduced glutathione (GSH), there are other defense mechanisms against free radicals, such as the enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), whose activities contribute to eliminate superoxide, hydrogen peroxide and hydroxyl radicals¹¹.

Many of the complications of diabetes mellitus, including retinopathy and atherosclerotic vascular disease, the leading cause of mortality in diabetes mellitus, have been linked to oxidative stress, and antioxidants have been considered as treatments¹². Plants often contain substantial amounts of antioxidants, flavonoids and tannins and the present study suggests that antioxidant action may be an important property of plant medicines associated with the hypoglycemic effect on diabetes mellitus¹³.

Morinda tinctoria (MTR) belongs to the family of Rubiaceae that grows wild and is distributed throughout Southeast Asia. It is commercially known as Nunaa and is indigenous to tropical countries.MTR is considered as an important folklore medicine. The tribes of Australia have used the ripe fruits of MTR for the treatment of respiratory infections¹⁴. Further, it has been reported to have abroad range of therapeutic and nutritional values¹⁵.There is a greater demand for its fruit juice in treatment for arthritis, cancer, gastric ulcer and heart diseases¹⁶. The major components identified in the Nunaa plant include octoanic acid, potassium, vitamin C, terpenoids, scopoletin, flavones glycosides, lineoleic acid, anthraquinones, morindone, rubiadin, and alizarin^17-20 Therefore, this study was designed to investigate the protective effect of Morinda tinctoria (MTR) on lowering the blood glucose level, tissues lipid peroxides and enzymic antioxidants in STZ-induced diabetic rats.

MATERIALS AND METHODS:

Preparation of aqueous extracts of MTR:

The aqueous extract was prepared by cold maceration of 250 g of the shade-dried fruits powder in 500 ml of distilled water allowed to stand overnight, and boiled for 5-10 minutes till the volume was reduced to half its original volume. The solution was then cooled, filtered, concentrated, dried in vacuum (yield 36 g) and the residue stored in a refrigerator at 2-8°C for subsequent experiments.

Induction of diabetes:

The animals fasted overnight and diabetes was induced by a single intraperitoneal injection of freshly-prepared STZ (55 mg/kg body weight of rats) in 0.1 M citrate buffer (pH 4.5)²¹. The animals were allowed to drink 5% glucose solution overnight to overcome the drug-induced hypoglycemia. Control rats were injected with citrate buffer alone. The animals were considered as diabetic, if their blood glucose values were above 250 mg/dL on the third day after the STZ injection. The treatment was started on the fourth day after the STZ injection and this was considered the first day of treatment. The treatment was continued for 30 days.

Experimental procedure:

The rats were divided into four groups comprising eight animals in each group as follows:

Group 1: Control rats given only buffer.

Group 2: Diabetic controls (STZ 55 mg/kg body weight of rats).

Group 3: Diabetic rats treated with protamine-zinc insulin i.p. injection (6 units/kg body weight of rats/day)²².

Group 4: Diabetic rats treated with Morinda tinctoria (MTR) (300 mg/kg body weight of rats/day) in aqueous solution orally for 30 days.

After completion of treatment, the animals were sacrificed. Blood was collected in tubes containing potassium oxalate and sodium fluoride. Plasma was used for the estimation of glucose using the O-Toluidine method reported by Sasaki el al²³. The levels of haemoglobin and glycosylated haemoglobin were estimated using the methods of Drabkin and Austin²⁴, and Nayak and Pattabiraman²⁵, respectively. Plasma insulin level was assayed by enzyme-linked immuno sorbent assay kit (ELISA, Boerhringer Mannheim, Germany).

The liver and kidney tissues were excised and rinsed in ice-cold saline. Tissues were cut into small pieces and homogenised with a Potter-Elvehjem tight-fitting glass-Teflon homogeniser in Tris-HCl buffer (pH 7.4). The homogenate was centrifuged and the supernatant was used for various measurements. The following analyses were carried out: serum total cholesterol (TC), high density lipoprotein (HDL-C) and triglycerides (TG) were estimated using the standard kit of Ranbaxy laboratories, New Delhi, India. Low density lipoprotein (LDL-C)²⁶, thiobarbituric acid reactive substances (TBRAS) (lipid peroxides) and hydroperoxides were estimated according to the methods of Ohkawa et al²⁷ and Jiang et al²⁸, respectively.

GSH was estimated using the method of Sedlak and Lindsay²⁹. The activity of SOD was assayed using the method of Marklund and Marklund³⁰. The activity of GPx was assayed using the method of Lawrence and Burk³¹. CAT activity was assayed using the method of Aebi³². Protein was estimated using the method of Lowry et al³³. All spectrophotometric measurements were carried out in a Camspec UV-Visible (Camspec M330B, UK) spectrophotometer.

Statistical analysis:

All the grouped data were statistically evaluated using the Statistical Package for Social Sciences (SPSS) version 7.5 (Chicago, IL, USA). Hypothesis testing methods included one way analysis of variance (ANOVA) followed by least significant differences test. p-values of less than 0.05 were considered to indicate statistical significance. All the results were expressed as mean ± standard deviation (SD) for eight animals in each group.

RESULTS:

A significant increase in the level of blood glucose and a decrease in body weight were observed in diabetic rats when compared to control rats. Administration of Morinda tinctoria (MTR) and insulin to diabetic rats significantly decreased the level of blood glucose and increased body weight gain to near control level. The diabetic rats showed a significant decrease in the levels of total haemoglobin and a significant increase in the level of glycosylated haemoglobin (HbA1c) and plasma insulin level as compared to diabetic rats. Administration of Morinda tinctoria (MTR) or insulin to diabetic rats restored the total haemoglobin and HbA1c to almost control levels (Table I).

The serum lipid profile is shown in Table II. The values of TC, HDL-C and LDL-C of those treated with Morinda tinctoria (MTR) extract returned to values nearing that of the control group. This showed that treatment with Morinda tinctoria (MTR) significantly improved the lipid profile in diabetic animals. Table III shows the concentration lipid peroxidation and hydroperoxides in the liver and kidneys of both control and experimental groups of rats. There was a significant elevation in tissue lipid peroxidation and hydroperoxides in diabetic rats. Administration of Morinda tinctoria (MTR) or insulin to diabetic rats decreased the levels of tissue lipid peroxidation and hydroperoxides to normal levels. The concentration of tissues SOD, CAT, GSH and GPx were significantly decreased in diabetic rats when compared to the control group. Administration of Morinda tinctoria (MTR) extract and insulin to diabetic rats tend to bring the activities of these enzymes to near normal level (Tables IV and V).

DISCUSSION:

Currently-available drug regimens for management of diabetes mellitus have certain drawbacks and therefore, there is a need for safer and more effective antidiabetic drugs^2-4. This study was undertaken to assess the antidiabetic effect of Morinda tinctoria (MTR) fruits. In the present study, the oral treatment of Morinda tinctoria (MTR) fruits extract decreased the blood glucose levels in diabetic rats. It has been reported that using medicinal plant extract to treat STZ-induced diabetic rats results in activation of ß-cells and insulinogenic effects³⁴

Morinda tinctoria (MTR) may also have brought about hypoglycaemic action through stimulation of surviving ß-cells of islets of Langerhans to release more insulin. This was clearly evidenced by the increased levels of plasma insulin in diabetic rats treated with Morinda tinctoria (MTR). Since the percentage fall in plasma glucose levels was different in models with varying intensity of hyperglycaemia, it implies that the anti hyperglycaemic effect of that plant is dependent on the dosage of diabetogenic agent, which in turn leads to ß-cell destruction³⁵. A number of other plants have also been observed to exert hypoglycaemic activity through insulin-release stimulatory effects^36,37.

The decreased level of total haemoglobin in diabetic rats is mainly due to the increased formation of HbA1c. HbA1c was found to increase in patients with diabetes mellitus and the amount of increase is directly propotional to the fasting blood glucose level³⁸. During diabetes mellitus, the excess glucose present in the blood reacts with haemoglobin to form HbA1c³⁹. HbA1c is used as a marker for estimating the degree of protein glycation in diabetes mellitus. Administration of Morinda tinctoria (MTR)fruits to diabetic rats reduced the glycosylation of haemoglobin by virtue of its normoglycaemic activity and thus decreases the levels of glycosylated haemoglobin in diabetic rats. This normalisation of glycosylated haemoglobin indicates decreased glycation of proteins

The concentrations of lipids, such as cholesterol, TG, LDL-C and HDL-C, were significantly higher in diabetic rats than in the control group. A variety of derangements in metabolic and regulatory mechanisms, due to insulin deficiency, are responsible for the observed accumulation of lipids⁴⁰. The impairment of insulin secretion results in enhanced metabolism of lipids from the adipose tissue to the plasma. Further, it has been reported that diabetic rats treated with insulin shows normalised lipid levels⁴¹. Thus, the results indicate that Morinda tinctoria (MTR) shows insulin-like action by virtue of its lipid lowering levels.

Oxidative stress has been shown to play a role in the causation of diabetes mellitus. Antioxidants have been shown to have a role in the alleviation of diabetes mellitus⁴². In diabetes mellitus, oxygen free radicals (OFRs) are generated by stimulating H2O2 in-vitro, as well as in-vivo, in pancreatic ß-cells⁴³. OFR-scavenging enzymes can respond to conditions of oxidative stress with a compensatory mechanism that increases the enzyme activity in diabetic rats⁴⁴. In our study, concentrations of lipid peroxides and hydroperoxides were increased in liver and kidneys of diabetic rats, indicating an increase in the generation of free radicals. Increased lipid peroxidation in diabetes mellitus can be due to increased oxidative stress in the cell as a result of depletion of antioxidant scavenger systems. The present finding indicates significantly increased lipid peroxidation of rats exposed to STZ and its attenuation by Morinda tinctoria (MTR) treatment. This suggests that the protective role of Morinda tinctoria (MTR) fruits extracts could be due to the antioxidative effect of flavonoids present in the leaf, which in turn act as strong superoxide radicals and singlet oxygen quenchers.

Numerous studies have revealed lowered antioxidant and enhanced peroxidative status in diabetes mellitus⁴⁵. In the current study, the SOD, CAT and GPx activities were significantly reduced in the liver and kidneys of diabetic rats. These observations emphasise the critical importance of maintaining the antioxidant potential of the pancreatic ß-cell in order to ensure both its survival and insulin secretion capacity during times of increased oxidative stress. The decreased activities of SOD and CAT in both liver and kidneys during diabetes mellitus may be due to the production of reactive oxygen free-radical that can themselves reduce the activity of these enzymes.

Reduced glutathione is a potent-free radical scavenger GSH within the islet of ß-cell and is an important factor against the progressive destruction of the ß-cell following partial pancreatectomy⁴⁶. Depletion of GSH results in enhanced lipid peroxidation.

Table I. Effect of treatment Morinda tinctoria (MTR) fruits extract for 30 days on blood glucose, body weight, total haemoglobin, glycosylated haemoglobin and plasma insulin of control and experimental groups of rats.

Group	Blood glucose (mg/dL)		Change in body weight (g)	Total haemoglobin (g/dL)	Glycosylated haemoglobin (Hb %)	Plasma insulin (μU/ml)
Group	Initial	Final	Change in body weight (g)	Total haemoglobin (g/dL)	Glycosylated haemoglobin (Hb %)	Plasma insulin (μU/ml)
Control	76.0±5.6	87.0±5.7	32.4 ± 2.3	15.21 ± 1.12	7.4 ± 1.37	16.21 ± 0.69
Diabetic	269.8±8.0*	296.0±8.6*	-34.1 ± 2.0*	11.56 ± 0.71*	15.3 ± 1.56*	5.42 ± 0.31*
Diabetic + insulin	260.7±7.7*	90.6 ± 6.0*	20.0 ± 1.1*	14.66 ± 0.68*	8.2 ± 0.93*	1.30±0.52*
Diabetic + Morinda tinctoria (MTR)	264.4±8.5*	98.1 ± 5.9*	21.6 ± 1.3*	15.20 ± 0.80*	8.5 ± 1.12*	12.11 ± 0.65*

Values are given as mean ± SD for groups of eight animals each. Values are statistically significant at *p<0.05.

Diabetic rats were compared with control rats; Morinda tinctoria (MTR) -treated diabetic rats were compared with diabetic rats; insulin-treated diabetic rats were compared with diabetic rats.

Table II. Effect of treatment Morinda tinctoria (MTR) fruits extract for 30 days on serum lipid profile of control and experimental groups of rats.

Parameter	Control	Diabetic	Diabetic + insulin	*Diabetic + Morinda tinctoria* (MTR)**
TC	129.2 ± 10.3	279.5 ± 13.2*	155.0 ± 10.5*	168.2 ± 7.8*
LDL-C	60.2 ± 4.5	185.4 ± 8.3*	73.1 ± 6.2*	78.7 ± 9.0*
HDL-C	42.4 ± 5.1	58.6 ± 4.2*	66.4 ± 6.8*	64.1 ± 5.6*
TG	88.0 ± 7.6	187.2 ± 10.5*	103.3 ± 9.5*	109.2 ± 10.6*

Values are given as mean ± SD for groups of eight animals each. Values are statistically

Significant at *p<0.05.

Diabetic rats were compared with control rats; Morinda tinctoria (MTR) treated diabetic rats were compared with diabetic rats; insulin-treated diabetic rats were compared with diabetic rats.

Table III. Effect of treatment Morinda tinctoria (MTR) fruits extract for 30 days on level of TBARS and hydroperoxides in liver and kidney of control and experimental groups of rats.

Group	TBARS (mM TBARS/100 g of wet tissue)		Hydroperoxides (mM hydroperoxides/100 g of wet tissue)
Group	Liver	Kidney	Liver	Kidney
Control	0.93 ± 0.07	1.30 ± 0.14	73.4 ± 3.4	56.4 ± 4.6
Diabetic control	1.71 ± 0.45*	2.28 ± 0.31*	99.2 ± 5.2*	80.3 ± 3.4*
Diabetic + insulin	1.05 ± 0.31*	1.59 ± 0.11*	76.2 ± 4.5*	69.1 ± 2.1*
Diabetic + Morinda tinctoria (MTR)	0.97 ± 0.05*	1.41 ± 0.10*	73.1 ± 3.9*	71.20 ± 3.0*

Values are given as mean ± SD for groups of eight animals each. Values are statistically significant at *p<0.05.

Diabetic rats were compared with control rats; Morinda tinctoria (MTR) treated diabetic rats were compared with diabetic rats; insulin treated diabetic rats were compared with diabetic rats.

Table IV. Effect of treatment Morinda tinctoria (MTR) fruits extract for 30 days on superoxide dismutase, catalase, glutathione peroxide and reduced glutathione in livers of control and experimental groups of rats

Group	Control	Diabetic	Diabetic + insulin	*Diabetic + Morinda tinctoria* (MTR)**
SOD (U/mg protein)	22.56 ± 1.76	15.63 ±1.38*	18.52 ± 2.00*	18.34 ± 1.45*
CAT (U/mg protein×103)	0.231 ± 0.025	0.117 ±0.014*	0.179 ±0.022*	0.201 ± 0.018*
GPx (U/mg protein)	0.195 ± 0.042	0.138 ±0.031*	0.163 ±0.047*	0.179 ± 0.060*
GSH (mg/100 g tissue)	55.6 ± 3.00	30.3 ± 2.34*	54.4 ± 3.20*	51.0 ± 1.64*

Values are given as mean ± SD for groups of eight animals each. Values are statistically significant at *p<0.05.

Diabetic rats were compared with control rats; Morinda tinctoria (MTR) treated diabetic rats were compared with diabetic rats; insulin-treated diabetic rats were compared with diabetic rats.

Table V. Effect of treatment Morinda tinctoria (MTR) fruits extract for 30 days on superoxide dismutase, catalase, and glutathione peroxide and reduced glutathione in kidneys of control and experimental groups of rats.

Group	Control	Diabetic	Diabetic + insulin	*Diabetic + Morinda tinctoria* (MTR)**
SOD (U/mg protein)	13.14 ± 1.61	9.24 ±1.28*	14.15 ± 1.16*	13.16 ± 1.40*
CAT (U/mg protein×103)	0.121 ± 0.19	0.080 ± 0.008*	0.116 ± 0.027*	0.134 ± 0.030*
GPx (U/mg protein)	0.064 ± 0.007	0.044 ± 0.006*	0.058 ± 0.009*	0.051 ± 0.005*
GSH (mg/100 g tissue)	40.3 ± 2.25	29.0 ± 1.26*	36.2 ± 1.95*	38.7 ± 2.17*

Values are given as mean ± SD for groups of eight animals each. Values are statistically significant at *p<0.05.

Diabetic rats were compared with control rats; Morinda tinctoria (MTR) treated diabetic rats were compared with diabetic rats; insulin-treated diabetic rats were compared with diabetic rats.

This can cause increased GSH consumption and can be correlated to the increase in the level of oxidised glutathione (GSSG). Treatment of Morinda tinctoria (MTR) resulted in the elevation of the GSH levels, which protect the cell membrane against oxidative damage by regulating the redox status of protein in the membrane⁴⁷. SOD, CAT and GPx are enzymes that destroy the peroxides and play a significant role in providing antioxidant defences to an organism. GPx and CAT are involved in the elimination of H2O2. SOD acts to dismutate superoxide radical to H2O2, which is then acted upon by GPx. The functions of all three enzymes are interconnected and a lowering of their activities results in the accumulation of lipid peroxides and increased oxidative stress in diabetic rats. Treatment of Morinda tinctoria (MTR) increased the activity of these enzymes and thus may help to avoid the free radicals generated during diabetes mellitus

The study suggested that diabetic animals are exposed to oxidative stress and Morinda tinctoria (MTR) can partially reduce the imbalances between the generation of reactive oxygen species (ROS) and the scavenging enzyme activity. According to these results, Morinda tinctoria (MTR) could be a supplement, as an antioxidant therapy, and may be beneficial for correcting the hyperglycaemia and preventing diabetic complications due to lipid peroxidation and free radicals. The Morinda tinctoria (MTR) fruit is not only similar to insulin in having a hypoglycaemic effect; it also controls the antioxidant level and could be used to improve the lipid metabolism. Longer duration studies of Morinda tinctoria (MTR) and its isolated compounds on chronic models are necessary to develop a potent antidiabetic drug.

CONCLUSION:

It can be concluded from the data that Morinda tinctoria (MTR) fruits extract supplementation is beneficial in controlling the blood glucose level, improves the lipid metabolism and prevents diabetic complications from lipid peroxidation and antioxidant systems in experimental diabetic rats. This could be useful for prevention or early treatment of diabetic disorders.

ACKNOWLEDGEMENT:

The authors are thankful to the V.HAZEENA BEGUM, professor and Head, Department of Siddha medicine Tamil University for providing the necessary lab facilities to carry out the work successfully.

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Received on 06.05.2011 Accepted on 13.06.2011

Asian J. Pharm. Tech. 1(2): April-June 2011; Page 34-39