Brazilian Journal of Biological Sciences, 2018, v. 5, No. 11, p. 683-698. ISSN 2358-2731 https://doi.org/10.21472/bjbs.051107
Antidiabetic effect of Coccinia grandis (L.) Voigt (Cucurbitales: Cucurbitaceae) on streptozotocin induced diabetic rats and its role in regulating carbohydrate metabolizing enzymes
Meenatchi Packirisamy¹,², Purushothaman Ayyakkannu² and Maneemegalai Sivaprakasam¹,*
¹Department of Biochemistry. Bharathidasan University Constituent College for Women. Orathanadu 614 625. Thanjavur-District. Tamil Nadu. India. Email: [email protected]. ²Post-Graduate & Research Department of Biochemistry. Mohamed Sathak College of Arts and Science. Chennai 600 119. Tamil Nadu. India.
Abstract. Coccinia grandis (L.) Voigt (Cucurbitales: Cucurbitaceae) is a climbing perennial herb, growing throughout Received India and it is widely used in the traditional treatment of September 4, 2018 diabetes. The aim of present study was to evaluate the antidiabetic potential of the mature unripe of Coccinia grandis in Accepted November 23, 2018 streptozotocin (STZ)-induced diabetic rats with special reference to carbohydrate metabolizing enzymes. The optimum Released dose of Coccinia grandis extract (GCE) was determined by oral December 31, 2018 glucose tolerance test. The effects of CGE were compared with glibenclamide. Oral administration of Coccinia grandis extract at Full Text Article a concentration of 250 mg/kg body weight once daily to diabetic rats for the period of 30 days resulted in significant reduction in the levels of plasma glucose and glycosylated hemoglobin. Administration of CGE showed a significant increase in the levels of glycolytic enzymes and glycogen content and decrease in the levels of gluconeogenic enzymes in the liver of diabetic treated rats. The anti-hyperglycemic effect of the extract was comparable with glibenclamide, a known hypoglycemic drug. Present findings provide experimental evidence that the fruits of
C. grandis have potential antidiabetic activity which might be
used as a functional food and safe remedy for the treatment of diabetes and associated complications. 0000-0003-2540-9008 Keywords: Coccinia grandis; Streptozotocin; Antihyperglycemic Meenatchi effect; Glibenclamide; Carbohydrate metabolizing enzymes. Packirisamy 0000-0001-5018-5515 Purushothaman Ayyakkannu 0000-0002-8932-4540 Maneemegalai Sivaprakasam
ISSN 2358-2731/BJBS-2018-0063/5/11/7/683 Braz. J. Biol. Sci. http://revista.rebibio.net 684 Packirisamy et al.
Introduction charantia (Bitter Melon) (Welihinda et al., 1986; Ali et al., 1993). Diabetes mellitus is a metabolic Liver is an insulin dependent disease affecting millions of individuals tissue which is severely affected during worldwide, characterized by absolute or diabetes mellitus, Decreased activities of relative deficiencies in insulin secretion glycolytic and pentose phosphate and/or insulin action associated with pathway enzymes with concomitant chronic hyperglycemia and disturbances increase in the activities of of carbohydrate, lipid and protein gluconeogenic and glycogenolytic metabolism (Wild et al., 2004; Araki et enzymes have been reported (McAnuff et al., 2017). WHO estimates that, globally, al., 2005) in diabetes mellitus. The 422 million adults aged over 18 years glucose homeostasis is one of the pivotal were living with diabetes in 2014. therapeutic modality in the management Worldwide, the number of people with diabetes. Acarbose, voglibose and diabetes has substantially increased miglitol are the inhibitors of between 1980 and 2014, rising from 108 carbohydrate metabolizing enzymes million to current number (422 million). have been used clinically to control Forty per cent of this increase is postprandial hyperglycemia in diabetics estimated to result from population (Subramanian et al., 2008). Recently growth and ageing, 28% from a rise in there has been a growing interest in age-specific prevalences and 32% from hypoglycemic agents from natural the interaction of the two (WHO 2016). products, especially those derived from According to WHO report 2016, the total plants. Plant sources are usually burden of deaths from high blood considered to be non‐toxic, with fewer glucose in 2012 has been estimated to side effects than synthetic sources. Many amount to 3.7 million. This number medicinal plants have been found to be includes 1.5 million diabetes deaths, and useful for the successful management of an additional 2.2 million deaths from diabetes (Stamp, 2003). cardiovascular diseases, chronic kidney Coccinia grandis (L.) Voigt disease, and tuberculosis related to belongs to the Cucurbitaceae Family and higher-than-optimal blood glucose. grows abundantly in India. It is a Therefore, novel concepts in the climbing perennial herb, growing management of diabetes have aroused a throughout India especially in warmer curiosity among researchers throughout and humid climatic conditions. It is the world. In countries such as India and widely used in traditional treatment of China, use of herbal medicines is a very diabetes (Venkateswaran and Pari, 2002) common practice from ancient time, and The fruits are used for culinary purposes herbal medicines are considered to be as a vegetable. Scientific investigations much safer and less expensive have supported the efficacy of leaf and therapeutic strategies for the treatment root extracts in amelioration of diabetic of various diseases. A proper scientific complications (Venkateswaran and Pari, investigation of the traditional herbal 2003; Akhtar et al., 2007). Coccinia indica remedies can provide valuable leads for leaves have been reported to stimulate the development of alternative drugs and insulin secretion in diabetic rats (Kumar strategies for the management of et al., 1993). diabetes (Ambady and Chamukuttan, Earlier studies from our 2008). Role of herbs in the management laboratory provided experimental and control of diabetes has emerged fast evidence that the fruits of Coccinia over the years with the discovery of grandis have potential antidiabetic hypoglycemic effect of Mormodica activity presumably by its antioxidant and antiglycation potential and its
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insulinotrophic properties in RINm5F al. (2015) with minor modifications. The cells in vitro (Meenatchi et al., 2017). dried, ground plant material was Therefore, the present study was extracted by maceration with methanol. designed to investigate the effect of Fifty g of plant material was soaked with mature unripe Coccinia grandis on 500 mL of the solvent for 24 h at room glucose utilization pathways and on temperature in a shaker. The sample was hepatic glucose production since both of filtered through filter paper. The residue them contributes significantly to plasma from the filtration was extracted again, glucose levels. For this, the activities of twice, using the same procedure. The key enzymes of carbohydrate filtrates obtained were combined and metabolism (glucose utilization/ then evaporated to dryness using a production) are measured in rotary evaporator at 40 °C. The obtained streptozotocin-induced diabetic rats. The Coccinia grandis extract (GGE) was results were also compared with stored in sterile sample tube at -20°C. glibenclamide as a reference drug. Phytochemical analysis of CGE Materials and methods Preliminary phytochemical analyses of the CGE were done using Source of chemicals standard procedures of Sofowora (1993) Streptozotocin (STZ) and other and Harbourne (1998). fine chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA). All Determination of total other chemicals, reagents and solvents phenolic content used were of good quality and analytical The amount of total phenolics in grade and obtained from SISCO Research CGE was determined by the method of Laboratories (SRL) and SD fine Singleton et al. (1999) with minor chemicals, Mumbai, India. modifications. One hundred mL of crude extract (20 mg/mL) was mixed with 0.2 Plant material mL of Folin-Ciocalteu reagent, 2 mL of Coccinia grandis (L.) Voigt distilled water and 1 mL of 15% Na2CO3. mature unripe whole fruits were The mixture was measured at 765 nm collected from Southern part of India using UV-Visible spectrophotometer (Kancheepuram District, Tamil Nadu, (T60U, PG Instruments Limited, UK) after India) during the month of July 2017 and 2 h at room temperature. Gallic acid was the pharmacognostic authentication was used as a standard and the total done by Dr. K. N. Sunil Kumar, RO and phenolics were expressed as mg of gallic HOD, Pharmacognosy and Dr. M. Kannan, acid equivalent (mg GAE) per g of extract RO (Siddha) and In Charge, Siddha (dry weight). Central Research Institute, Arignar Anna Government Hospital Campus (Central Determination of total Council for Research in Siddha, flavonoid content Department of AYUSH, Ministry of Health Total flavonoid content was and Family Welfare, Government of determined using the method of Chang et India), Chennai-600 106 (Voucher al. (2002) with some modifications using Specimen O17112402B). The fruits were quercetin as the standard. A calibration cut in to small slices, air dried under curve of quercetin was prepared in the shade, pulverized to fine powder using a range of 0-200 mg/mL. Briefly, extract laboratory scale cutting mill. (0.5 mL) and standard (0.5 mL) were placed in different test tubes and to each Extraction procedure 10% aluminum chloride (0.1 mL), 1 M The extract was prepared using potassium acetate (0.1 mL), 80% the methods described by et methanol (1.5 mL) and distilled water
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(2.8 mL) were added and mixed. A blank Diabetes was induced in was prepared in the same manner where overnight fasted experimental rats by a 0.5 mL of distilled water was used single intraperitoneal injection of instead of the sample or standard, and streptozotocin (40 mg/kg body weight) the amount of aluminum chloride was dissolved in freshly prepared citrate also replaced by distilled water. All tubes buffer (0.1 M, pH 4.5). Streptozotocin were incubated at room temperature for injected animals were allowed to drink 30 min. The absorbance was taken at 415 20% glucose solution overnight to nm using UV-Visible spectrophotometer. overcome the initial drug-induced The flavonoid content was expressed as hypoglycemic mortality. Control rats mg quercetin equivalent (QE) per g of were injected with same volume of extract. citrate buffer alone. The animals were considered as diabetic, if their blood Determination of saponins and glucose values were above 250 mg/dL on dietary fiber content the third day after STZ injection. The Saponins in CGE extract was treatment was started on the third day determined by standard method as after STZ injection and continued for 30 described by Anhawange et al. (2004) days at 24 h intervals during the entire The saponins were calculated as mg per period of the experiment. g of extract (dry weight). The dietary fiber content was analyzed by the Oral glucose tolerance test enzymatic-gravimetric method of Asp et Oral glucose tolerance test was al. (1983) and expressed as g per 100 g performed according to the method of of plant material. (Joy and Kuttan, 1999). After overnight fasting, 0 min blood sample (0.2 mL) was Animals studies taken from control and experimental Male albino Wistar rats weighing rats. Without delay, a glucose solution 200-220 g were used in this study. The (2 g/kg body weight) was administered rats were housed in clean polypropylene orally. Blood was withdrawn from the cages, maintained in the air-conditioned retro orbital sinus at 30, 60, 90, 120 and animal house with a constant 150 min interval. All the blood samples photoperiod of 12 h light/dark cycle with were collected with potassium oxalate the light cycle from 6:00 h to 18:00 h and and sodium fluoride solution for the the dark cycle from 18:00 h to 6:00 h. estimation of glucose. They were maintained at an ambient temperature of 25±2 °C and 12/12 h of Experimental design light/dark cycle. Animals were given The animals were divided into standard commercial rat chow and water seven groups of six animals in each. ad libitum. The experiments were Different doses of CGE were conducted according to the ethical norms administered orally using an intragastric approved by the Ministry of Social tube for the period of 30 days. Justices and Empowerment, Government Antidiabetic drug glibenclamide was of India and Institutional Animal Ethics dissolved in distilled water and used as a Committee (IAEC) Guidelines. Animal standard drug. welfare and the experimental procedures were carried out strictly in accordance Group I : Normal control with the Guide for Care and Use of Group II : Diabetic control Laboratory Animals. Group III : Diabetic + CGE (125 mg/kg body weight) Experimental induction of Group IV : Diabetic + CGE diabetes (250 mg/kg body weight)
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Group V : Diabetic + CGE and Bergmeyer (1984), respectively. (500 mg/kg body weight) Glycogen content was determined as Group VI : Diabetic + CGE described by Morales et al., (1975). The (750 mg/kg body weight) estimation of protein was carried out by Group VII : Glibenclamide the method of Lowry et al., (1951). (5 mg/kg body weight) Histopathological analysis After 30 days of treatment, the Formalin-fixed liver tissues from animals were deprived of food overnight all groups were paraffin embedded, and sacrificed by cervical decapitation. sectioned (3 mm thickness) and placed Blood was collected in a dry test tube on glass slides. Paraffin-embedded and allowed to coagulate at ambient sections of tissue were deparaffinised, temperature for 30 min. Serum was rehydrated with graded alcohol and separated by centrifugation at 2000 × g stained with Harris’ haematoxylin and for 10 min and used for the biochemical eosin (Dako, Glostrup, Denmark) in a estimations. Blood was collected in tubes Leica Autostainer (Wetzlar, Germany) with a mixture of potassium oxalate and and examined under a microscope. sodium fluoride (1:3) for the estimation of plasma insulin, glucose, and Statistical analysis ethylenediamine tetra acetic acid (EDTA) Data were analyzed with SPSS for the estimation of hemoglobin, Version 16 software. Hypothesis testing glycated hemoglobin. Liver was methods included one-way analysis of immediately dissected out and washed in variance (ANOVA) followed by LSD. The ice-cold saline to remove the blood. values are expressed as mean ± S.D. and Tissues was minced and homogenized the results were considered significantly (10% w/v) with 0.1 M Tris–HCl buffer different if P-values less than 0.05. (pH 7.4) in ice cold condition. The Statistically significant variations are homogenates were centrifuged at compared as follows: Normal control rats 1000×g for 10 min then the supernatants versus drug control rats (CGE alone were separated and used for enzyme treated rats), control versus diabetic assays. control, diabetic rats versus CGE treated diabetic rats and CGE treated diabetic Biochemical analysis rats versus glibenclamide treated Plasma glucose was estimated by diabetic rats. the Method of Trinder using a reagent kit Trinder (1969). Hemoglobin (Hb) and Results glycated hemoglobin (HbA1c) were estimated by the method of Drabkin and Phytochemical studies Austin (1932) and Sudhakar and The ethanol extract of Coccinia Pattabiraman (1981), respectively. The grandis (L.) Voigt were subjected for plasma insulin was measured by the phytochemical screening, which reveals method of Bürgi et al., (1988). the presence of different compounds in Glucokinase, glucose 6-phosphatase, plant extract such as alkaloid, glycoside, fructose 1,6-bisphosphatase and glucose- flavonoid, saponin, carbohydrate, fixed 6-phosphate dehydrogenase were oil and fat, and tannins (Table 1). The assayed in the tissues by the methods of percent yields of ethanol extract was Brandstrup et al., (1957), Koide and Oda found to be 6.87% ± 0.47%. (1956), Gancedo and Gancedo (1971)
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Table 1. Preliminary phytochemical screening of ethanol extract of mature unripe Coccinia grandis (L.) Voigt.
Test Observation Inference Alkaloids Brown precipitate formed. + Flavonoids Reddish pink color was observed. + Saponins Layer of foam did not form. + White precipitate did not form. Carbohydrates A brown ring was observed. + Phenols Blue green color was observed. + Terpenoids Reddish violet color was observed. + A purple color was not observed. Phytosterols Bluish green color was observed. + Red color was observed. Glycosides Red color was observed. + Pink color was observed. + indicates presence; - indicates absence.
In this study, the total phenolic and Total phenolics, flavonoids, flavoniod contents were quantified and saponins and dietary fiber found to be 14.72 ± 1.65 mg GAE per g, It is well known that phenolic 7.35 ± 0.67 mg QE per g of extract (dry compounds belong to the bioactive weight), respectively. The saponins and components of plant products and have dietary fiber contents were found to be good health-promoting activities. 0.092 ± 0.02 mg per g of extract and Saponins are the glycosidic compounds 41.97 g per 100 g of plant material, found in most of the plants and have respectively. (shown in Table 2). been reported to possess anticarcinogenic and antifungal activity.
Table 2. Total phenolic, flavonoid, saponin and fiber contents in ethanol extract of Coccinia grandis (L.) Voigt. Parameter Concentration Total Phenolic Contentsa 14.72 ± 1.65 Total Flavonoid Contentsb 7.35 ± 0.67 Total Saponinsc 0.092 ± 0.02 Soluble Dietary Fiber* 9.65 ± 0.08 Insoluble Dietary Fiber* 32.32 ± 1.89 Each value is expressed as mean ± SD from minimum of three independent experiments. a Data expressed as milligram of gallic acid equivalent (mg GAE) per g of extract (dry weight). b Data expressed as milligram of quercetin equivalent (mg QE) per g of extract (dry weight). c Data expressed as milligram per g of extract (fresh weight). * Data expressed as gram per 100 g of plant material.
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Oral glucose tolerance test in case of diabetic rats treated at a dose of diabetic rat 250 mg/kg body weight showed Results of oral glucose tolerance significant decrease in blood glucose test conducted on normal and level (183.15 ± 11.4 at 90 min; 174.53.5 experimental rats are shown in Figure 1. ± 9.9 at 120 min and 158.58 ± 8.2 at 150 The blood glucose level in both normal min) which were similar to that of and drug alone treated control rats glibenclamide treated diabetic rats. The showed a high peak value at 30 and 60 maximum glucose lowering effect of the min after glucose load and decreased to extract was observed at a dose of 250 near normal at 120 min. In diabetic rat mg/kg body weight than the other two the blood glucose levels reached the peak doses. Therefore, further studies were value at 30 min, 60 min and remain carried out with this dose. higher even after 120 min. But in the
Figure 1. Oral glucose tolerance test (GTT).
Effect of CGE on Body weight weight significantly decreased in diabetic changes and levels of glucose, insulin, rats compared with normal control. hemoglobin and glycosylated Administration of CGE or glibenclamide hemoglobin to diabetic rats resulted in significant The changes in the body weight increase in body weight. The levels of and the levels of plasma glucose, insulin, plasma glucose and HbA1c were Hb, and HbA1c in normal and diabetic significantly increased whereas the rats were depicted in Table 3 and Table levels of insulin and Hb were 4, respectively. Food intake was significantly decreased in the diabetic significantly increased whereas the body rats compared control rats. On treatment
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with CGE or glibenclamide reversed normoglycemic rats showed no these values to near normal in diabetic significant changes in the above said rats. Administrations of CGE to parameters.
Table 3. Effect of CGE on body weight in control and experimental animals during 30 days treatment. Body weight (g) Animal Groups Change (%) Initial Final Normal control 202.6 ± 4.28 229.8 ± 3.55 a 13.43 Normal + 250 mg CGE 206.5 ± 3.76 235.2 ± 3.58 12.93 Diabetic control 205.7 ± 5.14 190.3 ± 4.62 b -7.49 b Diabetic + 250 mg CGE 211.3 ± 4.17 225.8 ± 3.56 c, d 6.86 c,d Diabetic + 5 mg GBE 208.5 ± 4.87 224.4 ± 2.78 f 7.63 f Values are given as mean ± S.D. for six animals in each group. Values are considered significantly different at P < 0.05 with post hoc LSD test. a Control vs CGE treated normal control rats. b Control rats vs Diabetic rats. c Control vs CGE treated diabetic rats. d Diabetic rats vs CGE treated diabetic rats. e CGE treated diabetic rats vs Glibenclamide. f Glibenclamide vs Diabetic rats.
Table 4. Effect of CGE on the levels of glucose, insulin, hemoglobin and glycosylated hemoglobin in control and experimental animals during 30 days treatment. Glucose Insulin Hb HbA1c Animal Groups (mg/dL) (μU/mL) (g/dL) (mg/g of Hb) Normal control 107.65 ± 2.86 a 14.28 ± 0.62 14.58 ± 1.36 0.53 ± 0.042 Normal + 250 mg CGE 101.24 ± 3.25 13.86 ± 0.46 13.69 ± 1.48 0.55 ± 0.036 Diabetic control 289.65 ± 22.63 b 5.85 ± 0.43 b 9.45 ± 0.87 b 1.26 ± 0.027 b Diabetic + 250 mg CGE 154.36 ± 4.64 c,d 8.87 ± 0.52 c,d 10.80 ± 1.06 c,d 0.62 ± 0.039 c,d Diabetic + 5 mg GBE 138.45 ± 4.59 e,f 11.57 ± 0.72 f 11.67 ± 1.21 f 0.59 ± 0.023 f Values are given as mean ± S.D. for six animals in each group. Values are considered significantly different at P < 0.05 with post hoc LSD test. a Control vs CGE treated normal control rats. b Control rats vs Diabetic rats. c Control vs CGE treated diabetic rats. d Diabetic rats vs CGE treated diabetic rats. e CGE treated diabetic rats vs Glibenclamide. f Glibenclamide vs Diabetic rats.
Effect of CGE on hepatic key phosphate dehydrogenase and glycogen enzymes content were significantly decreased in Table 5 shows the activities of the liver of diabetic rats when compared carbohydrate metabolizing enzymes and to control rats. Oral administration of the hepatic glycogen content in the liver CGE or glibenclamide reversed these of control and experimental rats. The parameters to near normalcy. activities of hexokinase, glucose-6-
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Table 5. Effect of CGE on hexokinase, glucose-6-phosphate dehydrogenase and glycogen content in the liver of control and experimental animals during 30 days treatment. Glucose-6-phosphate Animal groups Hexokinase Glycogen content dehydrogenase Normal control 108.79 ± 5.65 a 2.65 ± 0.18 53.64 ± 4.13 Normal + 250 mg 2.72 ± 0.22 56.47 ± 3.96 112.62 ± 4.92 CGE Diabetic control 87.53 ± 3.57 b 1.38 ± 0.14 b 13.60 ± 2.78 b Diabetic + 250 mg 1.72 ± 0.13 c,d 38.14 ± 2.85 c,d 98.64 ± 4.63 c,d CGE Diabetic + 5 mg GBE 107.56 ± 3.87 e,f 2.23 ± 0.26 f 43.42 ± 4.21 f Values are given as mean ± S.D. for six animals in each group. Values are considered significantly different at P < 0.05 with post hoc LSD test. a Control vs CGE treated normal control rats. b Control rats vs Diabetic rats. c Control vs CGE treated diabetic rats. d Diabetic rats vs CGE treated diabetic rats. e CGE treated diabetic rats vs Glibenclamide. f Glibenclamide vs Diabetic rats.
Table 6 depicts the activities of significantly increased in diabetic rats. gluconeogenic enzymes in the liver of Oral administrations of CGE or control and experimental rats. Glucose-6- glibenclamide to diabetic rats reversed phosphatase and fructose 1,6- the activities of these hepatic enzymes to biphosphatase activities were near normal.
Table 6. Effect of CGE on activities of Glucose-6-phosphatise and fructose-1,6- bisphosphatase in the liver of control and experimental animals during 30 days treatment. Animal Groups Glucose-6-phosphatase Fructose-1,6-bisphosphatase Normal control 0.12 ± 0.02 a 4.88 ± 0.33 Normal + 250 mg CGE 0.12 ± 0.01 5.27 ± 0.52 Diabetic control 0.31 ± 0.03 b 8.26 ± 0.41 b Diabetic + 250 mg CGE 0.08 ± 0.04 c,d 3.72 ± 0.36 c,d Diabetic + 5 mg GBE 0.11 ± 0.03 e,f 4.12 ± 0.24 f Values are given as mean ± S.D. for six animals in each group. Values are considered significantly different at P < 0.05 with post hoc LSD test. a Control vs CGE treated normal control rats. b Control rats vs Diabetic rats. c Control vs CGE treated diabetic rats. d Diabetic rats vs CGE treated diabetic rats. e CGE treated diabetic rats vs Glibenclamide. f Glibenclamide vs Diabetic rats.
Histopathological analysis cellular architecture of liver. Diabetic Figure 2 shows the induced group show hepatocytes with histopathological examination of liver loss of architecture, necrosis, fatty sections of control and experimental changes and are looking pale. Diabetic group of animals, stained with rats treated with 250 mg and 5 mg haematoxylin and eosin, viewed at 10X. glibenclamide treated rats showed Normal control group and normal + 250 normal cellular architecture with distinct mg CGE treated rats showed normal hepatic cells and sinusoidal spaces.
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Figure 2. Histopathological examination of liver sections of control and experimental group of animals. Stained with haematoxylin and eosin (10X). A. Normal control group rats shows normal cellular architecture of liver; B. Normal + 250 mg CGE treated shows normal cellular architecture of liver; C. Diabetic induced group showing hepatocytes with loss of architecture, necrosis, fatty changes and are looking pale. D. Diabetic + 250 mg CGE treated; and E. Diabetic + glibenclamide treated rats show normal cellular architecture with distinct hepatic cells and sinusoidal spaces.
Discussion the presence of phytochemicals such as phenols, tannins, flavonoids, saponins, Phytochemical screening is of steroids, and alkaloids. The phenolic paramount importance in identifying compounds are one of the largest and new source of therapeutically valuable most ubiquitous groups of plant compounds having medicinal metabolites and attracted a great significance, to make the best and attention in relation to their potential for judicious use of available natural wealth beneficial effects on health (Mourya et (Mungole et al., 2010). This study has al., 2017). Thus, the anti hyperglycemic focused on both the phytochemical activity of the CGE as recorded in this screening and antidiabetic effect of C. study might be attributed to the presence grandis. Screening of the CGE revealed of high phenolic and flavonoid contents.
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The saponins content was found to be doses. This dose was as effective as that 0.092 ± 0.02 mg per g of extract. The of synthetic drug glibenclamide dietary fiber content of CGE was especially during oral glucose tolerance analyzed by the enzymatic-gravimetric test (OGTT) and is an essential trigger for method and the extract showed good the liver to revert its normal homeostasis amounts of both soluble and insoluble during experimental diabetes. CGE might fibers (41.97 ± 1.62 g per 100 g of plant bring about glucose lowering action material). Pectin is a soluble fiber that affects blood glucose levels either by cells or decreasing the transit time or indirectly Langerhansthrough stimulation to release of moreeither insulin surviving from β through the production of short chain the pancreas.regenerated This was β clearly cells of evidenced islets of fatty acids (SCFAs). Propionate, butyrate by increased levels of plasma insulin in and acetate are the commonly produced diabetic rats treated with CGE. Earlier SCFAs obtained by anaerobic findings from our laboratory have also fermentation of dietary fiber components reported the insulinotrophic (insulin by the microflora in the large intestine. secretory) properties of Coccinia grandis Thus, the high dietary fiber content of extract in RINm5F cells in vitro CGE recorded in the present study (Meenatchi et al., 2017). In addition to correlate the beneficial effects of CGE in insulinotrophic property, other possible the diabetic condition (Smith et al., 1998; mechanism of action of CGE could be Gao et al., 2009). correlated with reminiscent effect of In our study, initial and final body hypoglycemic sulphonylurea that weight of control and experimental rats promotes insulin secretion by the closure were measured whereas the food and of K+-ATP channels, membrane fluid intake were evaluated on daily depolarization and stimulation of Ca2+ basis. Rise in insulin levels upon influx, an initial key step in insulin treatment with CGE or glibenclamide in secretion from th diabetic animals resulted in improved from regenerated glycemic control which prevented the (Fuhlendorff et al.,e 1998 remnant). Saponins β cells and or loss of body weight and excess of food alkaloids (present inβ the cells extract) of pancreas had this and fluid intake. Streptozotocin-induced protective effect by scavenging ROS diabetic animals displayed the following species, which destroys the cells in the characteristics polyuria, increased water pancreas. The restorative effect o - intake, dehydration, weight loss and cells of due to the action ofβ flavonoids muscle wasting, excessive hair loss and present could speculate to normal bloodn β scaling, diarrhea, cataracts and increased glucose level, and this was also observed food intake (Wei et al., 2003). Decrease in the study of Tiware and Roa, (2002) in body weight of diabetic rats is due to using flavonoid extract of Pterocarpus catabolism of fats and proteins. Due to marsupium. The flavonoids have also insulin deficiency, the protein content is been reported to inhibit aldose reductase decreased in muscular tissue by activity, which is beneficial in mitigating proteolysis (Babu et al., 2007). the glucose autoxidation, glycation and The streptozotocin-induced acts against the major contributor ROS diabetic rats showed significant and other free radical (Tiware and Roa, reduction in plasma glucose and significant increase in insulin levels on cells and led to the normal levels of blood treatment CGE. The antihyperglycemic glucose2002). This in wouldSTZ- inducedlead to regenerationdiabetic rats β action of the extract was observed in a treated with CGE. dose dependent manner. As far as the Advanced glycation end-products most effective dose is concerned, 250 (AGEs) are the final products derived mg/kg body weight of CGE was found to from the Maillard reaction, which is a be more effective than the other two non-enzymatic glycation of free amino
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groups by sugars and aldehydes. AGE either be due to insulin deficiency or loss formation begins under hyperglycemic of insulin receptors (Saravanan and or oxidative stress conditions as Pugalendi, 2005). One of the key observed in STZ-induced diabetic rats enzymes in the metabolism of glucose is and is characterized by conversion of hexokinase, which phosphorylates reversible Schiff-base adducts to glucose into glucose-6-phosphate (Pari covalently bound Amadori products, and Rajarajeswari, 2009). The activity of which undergo further rearrangements this enzyme was decreased in the liver of that terminate in the formation of STZ- diabetic rats. Administration of CGE irreversibly bound compounds known as to STZ- induced rats resulted in an AGEs (Thornalley, 2005; Nenna et al., increased activity of liver hexokinase. 2015) The interaction of AGEs with The increased activity of hexokinase can receptors for AGEs (RAGE) directly cause increased glycolysis and increased activates multiple intracellular signaling, utilization of glucose for energy gene expression, and the secretory pro- production (Jayanthi et al., 2010). CGE inflammatory molecules accompanied by has been observed to reduce the levels of increasing free radicals that contribute glucose in the blood. The decrease in the towards pathologic complications related concentration of blood glucose in STZ- to diabetes (Caengprasath et al., 2013). induced rats treated with CGE may be The glycosylated hemoglobin (HbA1c) due to increased glycolysis (increased levels are monitored as a reliable index liver hexokinase activity). The activity of of glycemic control in diabetes and it was glucose-6-phosphate dehydrogenase was found to increase in patients with decreased which slows down the pentose diabetes mellitus due to glycosylation of phosphate pathway in diabetic hemoglobin and the amount of increase conditions. In our study, administration was directly proportional to the fasting of CGE significantly increased the activity blood glucose levels (Babu et al., 2007). of glucose-6- phosphate dehydrogenase During diabetes, the excess glucose in diabetic state. It provides hydrogen, present in blood reacts with hemoglobin. which binds NADP+ and produces NADPH Therefore, the total hemoglobin level is and enhances the synthesis of fats from decreased in diabetic rats (Ananda et al., carbohydrates, i.e., lipogenesis (Bopanna 2012). Administration of GGE and et al., 1997) finally the plasma glucose glibenclamide prevented a significant levels were decreased. elevation in glycosylated hemoglobin Liver plays a vital role in thereby increasing the level of total buffering the postprandial hemoglobin in diabetic rats. This could hyperglycemia and is involved in be due to the result of improved glycemic synthesis of glycogen. Diabetes mellitus control produced by CGE. is known to impair the normal capacity Liver is a general metabolic organ of the liver to synthesize glycogen (Sirag, that plays a pivotal role in glycolysis and 2009). Synthase phosphatase activates gluconeogenesis. A partial or total glycogen synthase, resulting in deficiency of insulin causes derangement glycogenesis. This activation step in carbohydrate metabolism that appears to be defective in STZ- induced decreases activity of several key enzymes diabetic rats (Kirana and Srinivasan, including glucokinase, hexokinase, 2008). Diabetic rats treated with CGE phosphofructokinase and pyruvate had liver glycogen brought back to near kinase, resulting in impaired peripheral normal levels, which could be due to glucose utilization and increased hepatic increased secretion of insulin, which glucose production. In our study, the enhances glycogenesis. activities of hexokinase and glucose-6- The hepatic gluconeogenic phosphate dehydrogenase have been enzymes (glucose-6-phosphatase and decreased in diabetic rats, which may fructose-1, 6-bisphosphatase) were
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significantly increased in diabetic state and Catharanthus roseus (Linn.) in alloxan- (Shulman, 2000). The activities of the induced diabetic rats. Research Journal of two enzymes may be due to the Medicine and Medical Sciences, v. 2, no. 1, increased synthesis of enzymes p. 29-34, 2007. contributing to the increased glucose Ali, L.; Khan, A. K.; Mamun, M. I.; production during diabetes by the liver Mosihuzzaman, M.; Nahar, N.; Nur-e-Alam, (Pari and Saravanan, 2005).The M.; Rokeya, B. Studies on hypoglycemic gluconeogenic enzyme glucose-6- effects of fruit pulp, seed, and whole plant of phosphatase is a crucial enzyme of Momordica charantia on normal and diabetic glucose homeostasis because it catalyses model rats. Planta Medica, v. 59, no. 5, p. 408-412, 1993. https://doi.org/10.1055/s- the ultimate biochemical reaction of both 2006-959720 glycogenolysis and gluconeogenesis (Singh and Kakkar, 2009). Increased Ambady, R.; Chamukuttan, S. Early diagnosis hepatic glucose production in diabetes and prevention of diabetes in developing mellitus is associated with impaired countries. Reviews in Endocrine and , v. 9, no. 3, p. 193-201, suppression of the gluconeogenic Metabolic Disorders 2008. https://doi.org/10.1007/s11154-008- enzyme fructose-1,6-bisphosphatase. 9079-z Activation of gluconeogenic enzymes is due to the state of insulin deficiency, Ananda, P. K.; Kumarappan, C. T.; Sunil, C.; because under normal conditions, insulin Kalaichelvan, V. K. Effect of Biophytum on streptozotocin and functions as a suppressor of sensitivum nicotinamide-induced diabetic rats. Asian gluconeogenic enzymes. Asian Pacific Journal of Tropical Biomedicine, v. 2, no. 1, p. 31-35. 2012. Conclusion https://doi.org/10.1016/S2221-1691(11) 60185-8 Based on this study, it can be Anhawange, A. A.; Ajibola, V. O.; Oniye, S. J. concluded that the culinary plant Chemical studies of seeds of Moronga oleifera Coccinia grandis (L.) Voigt represent as a (Lam) and Detarium microcarpum (Guill and good candidate for alternative and/or Sperr). Journal of Biological Sciences, v. 4, complementary medicine in the p. 711-715, 2004. https://doi.org/10.3923/ management of diabetes mellitus. Since, jbs.2004.711.715 it exhibited anti-hyperglycemic potential Araki, E.; Haneda, M.; Kasuga, M.; Nishikawa, through improving insulin secretion and T.; Kondo, T.; Ueki, K.; Kadowaki, T. New modulating the carbohydrate glycemic targets for patients with diabetes metabolizing enzymes. However, as this from the Japan Diabetes Society. Journal of is only preliminary study, further studies Diabetes Investigation, v. 8, no. 1, p. 123- are in progress to identify the active 125, 2017. https://doi.org/10.1111/jdi. constituents and their molecular 12600 mechanisms in vivo. Asp, N. G.; Johansson, C. G.; Hallmer, H.; Siljestroem, M. Rapid enzymic assay of Conflicts of interest insoluble and soluble dietary fiber. Journal of Agricultural and Food Chemistry, v. 31, Authors declare that they have no no. 3, p. 476-482, 1983. https://doi.org/ conflict of interests. 10.1021/jf00117a003 Babu, P. S.; Prabuseenivasan, P; Ignacimuthu, References S. Cinnamaldehyde: A potential antidiabetic agent. Phytomedicine, v. 14, p. 15-22, 2007. Akhtar, M. A.; Rashid, M.; Wahed, M. I. I; https://doi.org/10.1016/j.phymed.2006.11.0 Islam, M. R.; Shaheen, S. M.; Islam, M. A.; 05 Amran, M. S.; Ahmed, M. Comparison of long Bergmeyer, U. Glucose 6-phosphate term antihyperglycemic and hypolipidemic dehydrogenase. In: Bergmeyer, U. effects between Coccinia cordifolia (Linn.) Methods of enzymatic analysis. 2. ed. New York:
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Braz. J. Biol. Sci., 2018, v. 5, No. 11, p. 683-698.