The Mechanisms of PLANT Rhizome Curcuma Longa Action on Carbohydrate Metabolism in Alloxan - Induced...

Aizman R.I., Koroshchenko G.A., Gajdarova A.P., Lukanina S.N., Subotyalov M.A.

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The Mechanisms of PLANT Rhizome Curcuma Longa Action on Carbohydrate Metabolism in Alloxan - Induced Diabetes Mellitus Rats

Aizman R.I.1,, Koroshchenko G.A.1, Gajdarova A.P.1, Lukanina S.N.1, Subotyalov M.A.1

1Department of Anatomy, Physiology and Life Safety FSBEI HPE «Novosibirsk State Pedagogical University», Novosibirsk

Abstract

In experiments on rats with the alloxan-induced diabetes model the mechanisms of the turmeric (Curcuma longa) rhizomes influence on the various links in carbohydrate metabolism were studied. For this purpose, the glucose concentration in whole blood, titer hormones of insulin, C-peptide and cortisol in plasma, content of glycogen in the liver, structural and functional organization of the islet apparatus of the rats pancreas were studied. Under the influence of the turmeric rhizomes intake in rats with diabetes, as compared with the same animals on a standard diet, there were less expressed increasing of the glucose concentration in blood, increasing of the insulin and C-peptide concentration in plasma, lowering the concentration of cortisol and reliable increasing of the glycogen level in the liver. In the study of the microstructure of samples of pancreatic tissue of experimental animals on the background of the turmeric intake, the most preservation of the islet apparatus in comparison with a group of animals in conditions of a standard diet was marked. The results indicate the positive impact of the Curcuma longa rhizomes on the homeostatic mechanisms of the carbohydrate metabolism regulation in the alloxan-induced diabetes.Aim. The effects of the plant rhizome Curcuma longa as a food additive were studied on different processes of carbohydrate metabolism: glucose concentration in blood, concentration of hormones - insulin and C-peptide in plasma, content of glycogen in the liver, structural and functional organization of the islet apparatus of the pancreas in rats with alloxan-induced diabetes mellitus. Materials and methods. The concentration of glucose in blood and perfused solution was determined with the picricacid method by intensity of colour reaction on a spectrophotometer. The concentration of hormones (insulin, C-peptide) was determined by the immunoenzyme method with standard sets on a tablet spectrophotometer. The morphological structure of the pancreas was studied by a method of light microscopy. The content of glycogen in the liver was measured by means of a SHICK-reaction on the Mac-Manus method with measurement of colour intensity on a spectrophotometer. Results. Under the influence of the turmeric rhizomes intake in rats with diabetes, as compared with diabetic animals on a standard diet, a lower increase of the glucose concentration in blood, the tendency of decrease of glucose absorption in the gut, higher concentration of the insulin and C-peptide in plasma and the significant increase of glycogen content in the liver was found. The microstructure of pancreatic tissue samples of experimental animals using turmeric intake, was characterized by the better preservation of the islet apparatus in comparison with a group of animals on a standard diet. Conclusion. The results indicate the positive effect of the Curcuma longa rhizomes on the homeostatic mechanisms of the carbohydrate metabolism regulation in the alloxan-induced diabetes mellitus rats.

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Cite this article:

  • R.I., Aizman, et al. "The Mechanisms of PLANT Rhizome Curcuma Longa Action on Carbohydrate Metabolism in Alloxan - Induced Diabetes Mellitus Rats." American Journal of Biomedical Research 3.1 (2015): 1-5.
  • R.I., A. , G.A., K. , A.P., G. , S.N., L. , & M.A., S. (2015). The Mechanisms of PLANT Rhizome Curcuma Longa Action on Carbohydrate Metabolism in Alloxan - Induced Diabetes Mellitus Rats. American Journal of Biomedical Research, 3(1), 1-5.
  • R.I., Aizman, Koroshchenko G.A., Gajdarova A.P., Lukanina S.N., and Subotyalov M.A.. "The Mechanisms of PLANT Rhizome Curcuma Longa Action on Carbohydrate Metabolism in Alloxan - Induced Diabetes Mellitus Rats." American Journal of Biomedical Research 3, no. 1 (2015): 1-5.

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1. Introduction

Increased interest to use phytogenesis means for the correction of various pathological conditions [1, 2] makes it necessary to study the mechanisms of their actions. Data has appeared in recent years on the biological effects of turmeric Curcuma longa, such as antimicrobial [3], anti-inflammatory [4], antioxidant [5], etc., making it a promising to study the mechanism of its action at different pathological states, and, in particular, at diabetes mellitus (DM) [6].The specific action of Curcuma longa on various organs and tissues has been found: skin, the gastrointestinal tract, liver, respiratory system [7]. In acute and long lasting studies in mice, rats, guinea pigs and monkeys, it has been shown that a powder of turmeric rhizomes is not toxic to the organism [8].

To study the effect of turmeric and mechanisms of its action we have chosen diabetes, as according to WHO statistics [9], there are now 347 million diabetic patients in the world and their growth rate is 609,000 cases per year. Therefore, the use of this phytopreparation in the complex drug therapy for the correction of carbohydrate metabolism in diabetes mellitus patients is of great theoretical and practical value.

Therefore, the purpose of the present study was to elucidate the mechanisms of plant Curcuma longa rhizomes action on the carbohydrate metabolism in rats with an experimental model of diabetes mellitus.

2. Material and Methods

To achieve the goal mentioned above experiments on adult male9-10weekold Wistar rats (weight: 190-210 g) were performed. All animals were divided into four groups. The first (n = 19) and second (n = 19) groups were intact healthy animals. Rats of the third (n = 22) and the fourth (n = 21) groups were injected with 10 % alloxan solution in dose 0.1 ml/100 g body weight into the interscapular region to get the DM model, while animals of the first and the second groups received the same volume of saline solution. All animals were fed on a commercially available diet and allowed free access to drink water. Animals of the second and the fourth groups werefed additionally with turmeric rhizome powder at the weight of 2% of the food.

In all rats on the 1-st, 3-rd and 6-th days after injection, 0.2 ml of blood samples from the tail notch were collected for glucose measurement. The glucose concentration in the blood was determined with the picricacid method by the spectrophotometer “Spekol” (Germany) at a wavelength of 560±5 nm.

The amount of glucose absorption in the intestine was determined through in vivo experiments on rats by filling the immobilized segment of the small intestine of length ~ 20 cm with 2 ml of 30 % glucose solution for an hour, with preservation of its innervation and blood supply, under anaesthesiaby barbitalom sodium of 0.1 ml/100 g body weight injected intramuscularly according [10]. The difference between the infused amount of glucose and that left in the intestine after one hour was indicated as the absorbed amount.

At the end of the experiment (on the 6-th day after the alloxan or saline injection) from animals under ether anaesthesia the blood samples in a volume of 5 mL from vena cava inferior were taken in chilled tubes for determining the concentration of the hormones - insulin and C-peptide by ELISA using standard kits for the tablet spectrophotometer “Power Wave” (USA). For morphological study samples of pancreatic tissue were collected. The glycogen was determined in liver tissue using a PAS- reaction according to McManus and measuring the intensity of colouring on the spectrophotometer “Spekol” (Germany) at a wavelength of 430±5 nm.

Statistical analysis of the results was carried out by determining the arithmetic mean (M ) and their errors (±m). Differences between obtained results were evaluated by methods of variation statistics using a non-parametric Wilcoxon-Mann-Whitney test for independent samples and were considered significant at p ≤ 0,05. Calculations were made ​​by conventional formulas using the standard software package Statistica 7.0.

All experiments were performed in accordance with international guidelines for biomedical research involving animals, adopted by the International Council of Scientific Societies (CIOMS) in 1985, with Article XI of the Helsinki Declaration of the World Medical Association (1964) and laboratory practice regulations in the Russian Federation (MoH Order from 19.06.2003, № 267).

3. Results

Previously, it has been shown [11, 12] that after alloxan injection the blood glucose concentration in animals on the 1-st day of observation was already significantly higher than in controls, indicating the development of diabetes (Table 1). However, during the entire period of observation the blood glucose concentration in animals of the 4-th group was significantly lower compared with those in the rats of the 3-rd group, fed a standard diet, while a little higher than in the control animals. It should be noted that in intact rats receiving powder rhizome of Curcuma longa (group 2) blood glucose concentration at days 3 and 6 of the experiment was also significantly lower than in animals of the 1-st group.

Table 1. Glucose concentration in the blood of rats of different groups (mmol /L) ( M ± m)

Consequently, the intake of Curcuma longa rhizome powder resulted in the decrease of blood glucose concentration in control rats (group 2), while in animals with alloxan-induced diabetes there occurred a less significant increase and more rapid normalization of blood glucose.

One of the reasons for decreasing blood glucose after ingestion of turmeric may be a reduction of the absorption rate of carbohydrates in the gastrointestinal tract due to inhibition of the Na+-glucose cotransporter [13, 14]. To test this hypothesis, the experiment with perfusion of immobilized small intestine with 30 % glucose solution was performed. It can be seen that in the diabetic rats the rate of glucose absorption was more than 2-fold higher than in control ones(Table 2), which may be due to the activation of the Na+-glucose cotransporter [14]. Rhizomes of Curcuma longa powder even in control animals contributed to reducing the absorbed amount of glucose, while in diabetic rats of the 4-th group receiving Curcuma longa there was observed only the tendency toward the reduction of the absorption rate. Turmeric, probably, inhibits the Na+- glucose cotransporter (SGLT), and similarlysynthesized blockers [15], but under the conditions of diabetes this effect was less pronounced.

Table 2. Glucose absorption in the gut of rats of different groups (M ± m)

The glycogenesis process may play an important role in maintaining the glucose concentration in plasma may play.

To elucidate the mechanisms causing the significantly lower hyperglycemia in rats treated with turmeric, the glycogen content in the liver tissue was analyzed.

In alloxan-induced diabetic ratsthe glycogen content in the liver was significantly lower than in the control group (Table 3). This is probably due to an impaired glycogen synthesis during diabetes as a result of reduced activity of glycogensintetaseand the decrease of glucose oxidation processes due to a defect in the pyruvate dehydrogenase complex [16]. However, in rats of the4-th group, the glycogen content in the liver was increased almost to the control level and significantly differed from that of animals of the 3-rd group. Note, that in healthy rats, while inducing turmeric, glycogen content in the liver was also significantly lower than in the control group 1.Probably,this was due to a decrease in the blood glucose concentration in animals of this group (N 2), owing to that there was a redistribution of carbohydrates between the organ-depot (liver) and the blood for maintenance of the glucose concentration in plasma on normal values.

Since atalloxan-induced diabetes the endocrine function of the pancreas is impaired, it was important to evaluate changes in the concentration of basic glucose regulating hormones.

Table 3. Glycogen content in the liver of rats (mg/100 g wet weight) ( M ± m)

Analysis of the results showed that in diabetic rats (group 3) plasma insulin concentration was significantly lower than in control animals, that coincides with data obtained previously and confirms the development of diabetes type I [11, 17]. A similar trend was observed concerning the level of C-peptide. However, in animals receiving turmeric (group 4) the concentration of these hormones in plasma increased (Table 4).

Table 4. Concentration of hormones in the blood of rats with alloxan diabetes (M ± m)

As increasing the endocrine activity of the pancreas under the influence of Curcuma longa may be due to structural changes in the islet apparatus of the gland, the morphology of the pancreas in rats with alloxan-induced diabetes has been investigated.

The morphometric analysis of samples from the pancreas of animals has been demonstrated that the area of the islets in rats with alloxan-induced diabetes was almost 2 times larger than similar samples from the control group (Table 5). These results do not agree with the data of other researchers, showing reduction of the area of the pancreatic islets in the model of alloxan diabetes [18]. To resolve this contradiction we applied a method of morphological analysis, which allowed for visualizin the morphology of the pancreatic islets.

Table 5. Area of the Langerhans’islets, µm ²

Preparations at the light level of the pancreas had marked differences in the group of rats with an alloxan diabetes model from similar samples of the control group (Figure 1A, Figure 1B ).

Figure 1B shows that intercellular substance of gland stroma both in the periphery of islets of Langerhans, and in endocrine part of the gland have symptoms of disorder. On histologic sections pays attention plethora of blood vessels and perivascular edema of the intercellular substance of space. A typical feature of rat’s pancreas samples of this group was the sludge phenomenon of erythrocytes in the lumen of the capillaries. In the cytoplasm of endocrine cells there are numerous small and large optically clear vacuoles (Figure 1B). At the periphery of the islands are visible cells with signs of lethal damage. Thus, increase of the islets of Langerhans area was resulted from tissue edema.

Morphologic analysis of the pancreas samples of rats treated with turmeric demonstrated reduction of structural damage compared to the samples in the second group of rats (Figure 1C). Signs of edema of the intercellular substance of connective tissue stroma was visible only in insular part of the gland and was absent in the intercellular substance that separated the exocrine part from the endocrine, as was the case in similar samples of gland in rats of the second group. Therefore, the insula part of the gland had significantly less area compared with those in the rats of the 2-nd group and did not differ from the intact animals. In samples of rat’s pancreas in the 3-d group was observed multicellularity in periphery of endocrine part of the gland (Figure 1D). The cells in this compartment had various morphology. Among the identified cells there were cells with low and high level of differentiation as well as with sublethal damage. The signs of owater - salt homeostasis disorders in the structures of the gland had been revealed.

Figure 1. pancreas of control group rat (A), with alloxan diabetes model (B, C), and after intake of the rhizome turmeric powder (D).

These signs of structural and functional disorders of the gland, as known, can reflect one of the pathogenetic mechanisms of diabetes - free radical damage. Swelling of the intercellular substance of the stroma, numerous optically clear vacuoles in the cytoplasm of endocrine cells are probably the result of damage in membrane organelles and their protein carriers that maintain water and electrolyte homeostasis [14].

 In the group of animals consuming turmeric, stabilization of structural and functional disorders of the pancreas manifested itself in the absence of signs of stromal edema and hypercellularity at the periphery of the islets of Langerhans. From our point of view, it reflects the positive impact of the active ingredients of turmeric, in particular curcuminoids [4, 5, 6], on erythrocytes, capillaries, and endocrinocytes. The absence of sludge phenomenon of erythrocytes proof this point. Increased levels of low-differentiated cells at the periphery of the gland can be a sign of the migration of stem cells from stem niches to this part of the gland by hematogenous way [20], which is necessary for the formation of new endocrine cells.

These effects could be due to the weakening of the alloxan action on the insular apparatus of the pancreas under the influence of rhizome of turmeric powder, and (or) the influence of turmeric on homeostatic mechanisms of regulation of carbohydrate metabolism. We suggest that the first effect hardly occurs because the turmeric reduced glucose absorption from the gastrointestinal tract and its concentration in blood not only in rats with alloxan -induced diabetes mellitus, but also in the control animals. In addition, we have clinical observations that turmeric causes decreased glycosylated hemoglobin and plasma glucose in patients with type 2 diabetes who were on combination therapy with sulfonylurea and biguanides (unpublished data). Therefore, it is difficult to assume that the observed effects described in this paper due to the direct effect of turmeric on alloxan.

4. Conclusion

Thus, intake of Curcuma longa provides the hypoglycemic effect in alloxan -induced diabetic rats as a result of: partially regeneration of β - islet cell, reducing the structural damage of cells and the intercellular substances, improving blood supply, resulting in stimulation of insulin and C - peptide secretion, the activation process of glycogenesis in the liver, and reducing the rate of glucose absorption in the small intestine. These data gives good impact for clinical using of Curcuma longa rhizomes powder in complex therapy of patients with diabetes mellitus.

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