Author: Murugan, P
Date published: April 1, 2010
Journal code: JCLT
Collagen is the most abundant protein present in the connective tissue, which is affected during diabetes. Collagen plays a major role in contraction, relaxation and other activities of the organs in general . Modifications of this protein may play a critical role in the complications of diabetes .Abnormalities in connective tissues that contain Type I collagen are common in human diabetes and in animal models. These defects include poor wound healing , stiffening of arteries , toughened skin , limited joint mobility , decreased bone formation , and decreased lung expansion volume . Such defects predict the development of more serious diseases such as neuropathy and retinopathy [6,9].
The cross-linking of collagen by the non-enzymatic advanced glycated endproducts (AGEs) formation or the enzymatic glucose incorporation is probably one of themainmechanisms underlying the increased arterial stiffness in diabetic patients or diabetic complications in general . The solubility of collagenous tissues decreases due to diabetes [8-l0], the tensile strength of the tissues increases [11-12], and the non-enzymatic glycation (NEG) of collagen increases [8,13].
Hyperglycemia results in increased NEG of proteins from many tissues of diabetics . NEG is the first in a series of reactions known as Maillard-browning . This process can promote cross-linking of proteins, in vitro via 6-NH, groups of lysine or hydroxylysine. Maillard-browning might occur in vivo and be enhanced due to diabetes, especially in tissues where protein turnover is slow. It has been proposed that fluorescent compounds accumulate in tendons .
The common name Tanner's cassia (Cassia auriculata L.), belonging to the family Ceasalpinaceae is a shrub that has attractive yellowflowers, commonly used for the treatment of skin disorders and body odour. It is a native plant present in different parts of India. Indigenous people use various parts of the plant for diabetes mellitus. It is widely used in Ayurvedic medicine as a "Kalpa drug" containing five parts of the shrub (roots, leaves, flowers, bark and unripe fruits). They are taken in equal quantity, dried and then powdered to give "Avarai Panchaga Choornam", for the control of sugar levels and reduction of symptoms such as polyuria and thirst in diabetes [16,17].
Aliterature survey showed that a decoction of leaves, flowers, and seeds of the Tanner's cassia mediates an antidiabetic effect . The available literature reveals that very little work has been done with respect to Tanner's cassia flowers, other than its hypoglycemic properties. In earlier publications from our laboratory, we have already reported that this extract shows antiperoxidative activity, improves plasma insulin, decrease glucose levels, scavenges free radicals in diabetic rats [18,19]. Tanner's cassia extract also showed antihyperlipidemic effect . The aim of the present study was to examine the influence of oral administration of Tanner's cassia flower extract on some biochemical parameters and properties of collagen content in the tail tendon of streptozotocin (STZ) induced diabetic rats.
MATERIALS AND METHODS
Animals: All the experiments were carried out with male Wistar rats aged seven to eight weeks (180- 200 g), obtained from the Central Animal House, Rajah Muthiah Medical College, Annamalai University. The animals were housed in polypropylene cages and provided with water and standard pellet diet (Karnataka Agro Food Corporation Limited, Bangalore, India) ad libitum. The experiment was approved by ethical committee,AnnamalaiUniversity.
Chemicals: STZ was obtained from Himedia Laboratory Limited,Mumbai, India.All other reagents used were of analytical grade.
Plant Material: Fresh flowers of Tanner's cassia were collected fromNeyveli, Cuddalore District, Tamil Nadu. The plant was identified and authenticated at the Herbarium of Botany Directorate in Annamalai University. A voucher specimen (No.231) was deposited in the Botany Department of Annamalai University.
Preparation of plant extract: 500 g of Tanner's cassia flowers were extracted with 1,500 ml of water by the method of continuous hot extraction at 60 °C for six hours and evaporated. The residual extract was dissolved in water and used in the study .
Induction of experimental diabetes: A freshly prepared solution of streptozotocin (45 mg/kg i.p/bw) in 0.1Mcitrate buffer, pH 4.5 was injected intraperitoneally in a volume of 1 ml/kg. After 48 hours of STZ administration, rats with moderate diabetes having glycosuira and hyperglycaemia (i.e. with a blood glucose of 200- 300 mg/dl) were chosen for the experiment .
Experimental procedure: In the experiment, a total of 30 rats (24 diabetic surviving rats, six normal rats) were used. The rats were divided in to four groups of six rats each as follows:
Group 1: Normal untreated rats.
Group 2: Diabetic control rats.
Group 3: Diabetic rats given Tanner's cassia flowers extract (TCFEt) at a dose of 0.45 g/kg body weight in 1 ml of aqueous solution daily using an intragastric tube for 45 days .
Group 4: Diabetic rats given glibenclamide (600 ìg/ kg body weight) in 1 ml of aqueous solution daily using an intragastric tube for 45days.
After 45 days, the animals were deprived of food overnight and sacrificed by decapitation. Blood was collected in tubes containing potassium oxalate and sodium fluoride mixture for the estimation of blood glucose. Plasma was separated by centrifugation for determination of glucose and insulin assay. Plasma glucose was estimated colorimetrically using commercial diagnostic kits (Sigma Diagnostics Pvt Ltd, Baroda, India) . Plasma insulin was assayed by ELISA using a Boehringer-Mannheim kit with an ES300 Boehringer analyzer (Mannheim, Germany). Total haemoglobin was estimated using the Cyanmethaemoglobin method described by Drabkin and Austin . Glycosylated haemoglobin was estimated according to the method of Sudhakar Nayak and Pattabiraman  with modifications according to Bannon .
Analytical methods: The tail was removed and stored frozen at -80 °C and tail tendon tissue was prepared as described below. Weighed tail tendon tissue was hydrolyzed in 6.0 N HCl for 18 hrs at 110 °C. The collagen content was determined by measuring hydroxyproline, as described byWoessner . The extent of glycation was determined by the method described by Rao and Pattabiraman . AGE-linked fluorescence was measured by the method of Monnier et al. . The levels of hydroperoxides (lipidperoxiodation) in the insoluble collagen was assessed by the method of Jiang et al. . The solubility pattern of tail tendon collagen was determined as described by Miller and Rhodes . The supernatants were pooled and an aliquot was used for the assay of hydroxyproline . The data for various biochemical parameters were analyzed using analysis of variance (ANOVA), and the group means were compared by Duncan's multiple range test (DMRT). Values were considered statistically significant if p < 0.05 .
Figure 1 shows the level of plasma glucose and plasma insulin of different experimental groups. The diabetic control rats showed a significant increase in the level of plasma glucose with significant decrease in the level of plasma insulin. Oral administration of Tanner's cassia flowers extract (TCFEt) to diabetic rats significantly reversed the above biochemical changes. In our previous study, [10,23] we have reported that TCFEt at 0.45 mg/kg body weight showed better effect than 0.15 and 0.30 mg/kg body weight, therefore, 0.45 mg/kg body weight was used in this study. In agreement with these results, the present study also showed that the administration of TCFEt and glibenclamide significantly improved the plasma glucose and plasma insulin levels. TheTCFEt administration was more effective than glibenclamide.
Figure 2 shows the level of total haemoglobin and glycosylated haemoglobin of different experimental groups. The diabetic control rats showed a significant decrease in the level of total haemoglobin and significant increase in the level of glycosylated haemoglobin. Oral administration of TCFEt and glibenclamide to diabetic rats significantly restored the levels of total haemoglobin and glycosylated haemoglobin. In the case of normal rats, the level of haemoglobin and glycosylated haemoglobin remained unaltered.
Table 1 shows the increased levels of extendent of glycation,AGE-linked fluorescence, lipid peroxidation and total collagen in tail tendon of diabetic rats, when compared with normal rats. Treatment with TCFEt and glibenclamide has reversed the levels to near normal. The effect of TCFEt was more potent than that of glibenclamide.
The levels of acid, pepsin and neutral soluble collagens of tail tendon of control and diabetic rats are represented in table 2. Diabetic rats have decreased levels of acid, pepsin and neutral soluble collagens in the tail tendon as compared with normal rats. Treatment with TCFEt and glibenclamide has reversed the levels of acid, pepsin and neutral soluble collagens to near normal. The effect of TCFEt was more prominent compared with that of glibenclamide.
We determined the influence of TCFEt on collagen content and characteristics in diabetic rats. The capacity of the TCFEt to decrease the elevated blood sugar to near normal levels is an essential trigger for the liver to revert to its normal homeostasis during experimental diabetes. The possible mechanism by which the plant extract exerts its antihyperglycemic action in diabetic rats may be by potentiating the plasma insulin effect by increasing either the pancreatic secretion of insulin from the existing ßcells or its release from the bound form, as demonstrated by the significant increase in insulin levels induced by the plant extract in diabetic rats. In this context, a number of other plants have also been reported to have antihyperglycaemic and insulin release stimulatory effects [35,36].
Glycation is a chronic, non-enzymatic process directly related to glucose levels, which causes marked accumulations of irreversible reaction products (advanced glycation end-products). It has been attractive to propose its involvement in diabetic complications .
Glycated hemoglobin significantly increased in diabetic control rats, and this increase is directly proportional to fasting blood glucose .Anemia is a much more common disease in type 2 diabetic patients, contributing to the pathogenesis of diabetic complications. In the present study, the decreased concentration of hemoglobin indicates the anemia in STZ diabetic rats, reflects the excess glucose transport in the blood reacting with hemoglobin to form glycated hemoglobin. In this context, a number of other plants showed decreased level of glycated hemoglobin [39,40].
Streptozotocin-induced diabetes mellitus characterized by hyperglycemia caused a significant increase in hydroxyproline levels and collagen content. The correlation between collagen and intracellular degradation is of interest and may have a role in the regulation of collagen content. The increased degradation of newly synthesized collagen during streptozotocin-induced diabetes might contribute to collagen deposition in the early stages . Diabetic rats treated with the TCFEt and glibenclamide showed a significant decrease in total collagen content when compared to untreated diabetic rats. This decreasemay be attributed to the significant decrease in blood glucose and consequent decrease in nonenzymatic glycation and deposition of collagen in diabetic rats treated with the TCFEt and glibenclamide. The effect of the extract was more pronounced than glibenclamide.In the present study, an increase in the extent of glycation was observed in the tail tendon of diabetic rats, probably due to exposure of the tissues to glucose in the diabetic state. Earlier studies have also reported that glucose is directly involved in the accelerated cross-linking of collagen in the diabetic state.Many reports have also established that collagen glycation is increased during exposure to high glucose levels in vitro and in vivo . Flavonoids were reported to have antiglycating property . The decrease in the extent of glycation in diabetic rats treated with the plant extract could be due to the antiglycating property of the flavonoids, phenolic compounds and tannins present in the extract .
The percentage of neutral salt, acid and pepsin soluble collagen decreased in the tail tendon of diabetic rats. As cross linking proceeds, the solubility of collagen in neutral buffer and acid solution also changes. Highly cross-linked collagen becomes less soluble in the above solutions and can be released only by limited pepsin digestion . It has been proposed that free radicals and reactive carbonyls generated during diabetes may contribute significantly to the increased cross-linking of collagen [46,47]. Fromthe solubility patterns obtained for collagen of tail tendon tissues of TCFEt treated and untreated diabetic rats, it can be seen that the plant extract treatment resulted in increased solubility in neutral, acid and pepsin digestion. This is an indication of decreased levels of cross-linking in the collagen of the treated groups. The reduction in the advanced glycation and crosslinking of collagen in diabetic rats treated with the plant extract may be due to its antiperoxidative activity , since lipid peroxidation products have been shown to directly influence collagen crosslinking and advanced glycation end product (AGE) formation [49,50]. In addition, AGE were also reported to induce the upregulation of the expression of type I collagen genes that could result in excess deposition of collagen in diabetes .
The increase in AGE levels in diabetic rats observed in the present study could be responsible for the up regulation of collagen gene expression which results in the increased deposition of collagen and consequent increased cross-linking in streptozotocin-diabetic rats. The TCFEt is reported to be rich in flavonoids  which contribute by their protective action to the reduction of collagen cross-linking in treated diabetic rats. Collagen obtained fromthe tail tendon of diabetic animals showed increased fluorescence, which is a strong indication of increased advanced glycation. Previous studies have also documented an overall increase in the fluorescence of diabetic tissue collagen [38, 52]. It has been shown that, in addition to glucose, free radicals and lipid peroxides also play an important role in the development of collagenlinked fluorescence . It appears that the reactive radicals formed during glycation and oxidation reactions can also have an influence on the development of fluorescence.
Administration of the TCFEt and glibenclamide significantly reduced the intensity of fluorescence in diabetic rats. This may be due to the significant reduction in blood glucose and consequent decreased glycation, and to the significant scavenging of free radicals generated during diabetes by flavonoids present in Tanner's cassia flower extract.
In conclusion, our study suggests TCFEt has a good glycaemic control and beneficial effect on collagen and its characteristics in the tail tendons of diabetic rats. This reflects the protective effect of TCFEt from the risk of diabetic complications.
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Department of Biochemistry, Faculty of Science, Bharthidarsan University College,
Perambalur District, Tamil Nadu 621 107. E. mail: firstname.lastname@example.org
Received: February 19, 2010; Accepted: March 15, 2010