Aqueous extract of Digitaria exilis grains ameliorate diabetes in streptozotocin-induced diabetic male Wistar rats

Journal Pre-proof Aqueous extract of Digitaria exilis gra4ins ameliorate diabetes in streptozotocininduced diabetic male Wistar rats Dele Moses Adams, Musa Toyin Yakubu PII:

S0378-8741(19)33242-8

DOI:

https://doi.org/10.1016/j.jep.2019.112383

Reference:

JEP 112383

To appear in:

Journal of Ethnopharmacology

Received Date: 15 August 2019 Revised Date:

3 November 2019

Accepted Date: 10 November 2019

Please cite this article as: Adams, D.M., Yakubu, M.T., Aqueous extract of Digitaria exilis gra4ins ameliorate diabetes in streptozotocin-induced diabetic male Wistar rats, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/j.jep.2019.112383. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Digitaria exilis Streptozotocin induces: ↑Blood glucose, serum albumin, urea, creatinine, cholesterol, liver glucose-6phosphatase, liver fructose-I, 6-bisphosphatase, feed and water intake

Diabetic rat

& ↓pancreatic insulin, body weight, weight of pancreas, liver glycogen, red blood cells, white blood cells, liver hexokinase and phosphofructokinase

Digitaria exilis aqueous extract and metformin ameliorated streptozotocin-treated related changes in the biochemical parameters

Antidiabetic activity of D. exilis grains via glucose uptake and utilization and insulin secretion

Metformin (Reference drug)

Aqueous extract of Digitaria exilis gra4ins ameliorate diabetes in streptozotocin-induced diabetic male Wistar rats 1,2

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Adams, Dele Moses and *1Yakubu, Musa Toyin Phytomedicine, Biochemical Toxicology and Reproductive Biochemistry Research Laboratory, Department of Biochemistry, University of Ilorin, Ilorin, Kwara State, Nigeria 2 Phytomedicine, Biochemical Toxicology and Clinical Biochemistry Research Unit, Department of Biochemistry, Bingham University, Karu, Nasarawa State, Nigeria

Abstract Ethnopharmacological relevance: The absence of scientific data on the age long folkloric use of Digitaria exilis grains by sufferers of diabetes prompted the present investigation. This study was aimed at evaluating the antidiabetic activity of aqueous extract of Digitaria exilis grains in streptozotocin (STZ)-induced diabetic rats. Material and Methods: Forty two male rats (166.43 ± 3.32 g) were completely randomized into six groups (A-F) of 7 animals each. Animals in group A (control) were administered 0.5 ml of distilled water while those in groups B, C, D, E and F which were induced with diabetes mellitus (by intraperitoneal administration of 60 mg/kg body weight of STZ) were also administered distilled water, 50 mg/kg body weight of metformin (a reference antidiabetic drug), 50, 100 and 200 mg/kg body weight of aqueous extract of D. exilis grains respectively, twice daily for 14 days. Blood glucose levels and some relevant biomolecules were determined 14 days postadministration. Results: Alkaloids, flavonoids, saponins, tannins, anthraquinones, terpenoids, cardiac glycosides, phlobatannins, phenolics and cardenolides were detected in the extract with alkaloids (30.20 mg/ml) occurring the most and phlobatannins (0.22 mg/ml) the least. Streptozotocin significantly (p<0.05) increased the levels of blood glucose, serum albumin, urea, creatinine and cholesterol, activities of glucose-6-phosphatase and fructose-1,6-bisphosphatase in the liver and 1

intake of feed and water. Body weight, weight of pancreas, pancreatic insulin, liver glycogen content, red blood cell and white blood cell and their related indices, liver hexokinase and phosphofructokinase activities were significantly reduced by STZ. In contrast, the extract significantly reversed all those STZ-treatment induced changes with the 200 mg/kg body weight of the extract producing profound values that compared favourably with the distilled water treated non-diabetic animals and metformin treated diabetic animals. Conclusion: Overall, this study revealed that Digitaria exilis grains possess antidiabetic activity via increased insulin secretion, as plasma concentrations of insulin were not determined, enhanced activities of hexokinase and phosphofuctokinase and repletion of hepatic glycogen content. Keywords: Digitaria exilis, Poaceae, Diabetes mellitus, Insulin secretion, Metformin.

*Corresponding Author: Email address: [email protected]; Phone number: +2348037544437

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Introduction Diabetes mellitus is a chronic disorder of carbohydrate metabolism caused by insufficient or complete cessation of insulin synthesis or secretion and/or peripheral resistance to insulin action. In 2017, the prevalence of diabetes worldwide and Nigeria (20-79 years old) were 425 million and 1.70 million cases respectively and has been projected to 642 million and 3.40 million by 2040 (IDF, 2017). This worldwide public health problem, currently affecting mankind, regardless of socioeconomic status and geographic location (Barcelo and Rajpathak, 2001; AlShamsi et al., 2004), may be caused by repeated consumption of diets high in starch, physical inactivity and obesity, increasing intake of saturated fat and endocrine system disorders like hyperthyroidism and polycystic ovary syndrome (Ullah et al., 2016). Although, most of the antidiabetic drugs are effective in giving long term glycaemic control, they are not also free from some adverse effects like gastrointestinal irritation, nausea, diarrhoea, cramps and flatulence (Nayak et al. 2011). Therefore, it is imperative to explore options in medicinal plants for the management of the disease. Digitaria exilis (family: Poaceae), otherwise known as Hungry Rice (English), acha (Hausa, Northern Nigeria), ukwu (Igbo, Eastern Nigeria) and suuru (Yoruba, Southwestern Nigeria) (Morales-Payan et al. 2002), is annual, herbaceous plant that is free-tillering, erect, slender and glabrous culms and grows up to 80 cm high (Adoukonou-Sagbadja, 2010). It is widely grown in Nigeria in the cool region of Plateau State, part of Kaduna State, Bauchi, Kebbi, Taraba, and Niger States where it is used in various forms as salads, stew, porridges, bread or other recipe made from its flour. Generally, D. exilis grain is not just consumed by human being as food, but is also a suitable, healthy food for the poorest population as well as the coeliacs (Taylor et al., 2006). Jideani (1999) reported that traditional foods like thick and thin porridges, steam cooked 3

products like couscous and non-alcoholic and alcoholic beverages can be obtained from D. exilis and D. iburua (Iburu) whereas technologically, the grains can be utilized in ways similar to rice, and for quick cooking (cookies, crackers and popcorn), non-conventional food products like weaning foods of low bulk density and breakfast cereal with good fiber content. Furthermore, in the northern part of Nigeria, D. exilis grains has been touted to have a long use as a diabetic food and is believed to control blood sugar level in the body (Jideani, 2012). In addition, D. exilis grains has also been claimed to reduce the risk of heart diseases and stroke (Taylor et al., 2006). Chukwu and Abdul-kadir (2008), Temple and Bassa (1991), Jideani and Akingbala (1993) and Glew et al. (2013) have reported, in separate studies, the physiochemical properties, fatty acid, amino acid and mineral element constituents and antioxidant activities of D. exilis grains. Other reports on D. exilis grains included the microstructure and nutritional composition (Irving and Jideani 1997; Ballogou et al., 2013), adaptability and yield of some of the accessions of D. exilis and D. iburua (Dachi and Gana, 2008), glycaemic index and glycaemic load of D. exilis in healthy and diabetic subjects (Alegbejo et al., 2011), changes in chemical composition of treated and untreated D. exilis grains (Echendu et al. 2009) and immobilization of α-amylase from D. exilis (Egwim and Oloyede, 2008). Olagunju et al. (2018) have equally reported that supplementation of D. exilis flour with Cajanus cajan flour (70:30) exhibited the highest inhibitory activity against α-amylase and α-glucosidase and thus can be used as snacks in the management of hyperglycemia and prevention of associated degenerative diseases. Until now however, studies that have comprehensively substantiated the anti-diabetic activity of the aqueous extract of D. exilis grains in STZ-induced diabetic male rats appear not to exist in the open scientific literature. It is in the light of this that the present study was designed to

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provide scientific data that will substantiate or refute the opinion of Jideani (2012) on the antidiabetic activity of aqueous extract of D. exilis grains. Materials and methods Plant materials and authentication Fresh D. exilis grains was obtained from a herb seller at Kawo Market in Kaduna, Nigeria. The plant was authenticated at the Herbarium Unit of the Department of Plant Biology, University of Ilorin, Ilorin, Nigeria. A voucher specimen was prepared and deposited under the reference number UILH 001/1201. Experimental animals Male Wistar rats (Rattus norvegicus) weighing 166.43 ± 3.32 g were obtained from the Animal House of the National Veterinary Research Institute, Vom, Jos, Plateau State, Nigeria. The animals were housed in aluminium cages that were placed in well ventilated Animal House under the controlled temperature (28-31°C), photoperiod (12 hours light and dark) and humidity (50-55%). The animals were allowed unrestricted access to rat pellets (Vital Feed®, Grand Cereals Ltd, Jos, Plateau State, Nigeria) and tap water. Glucose meter and test strips The Accu-Chek® Active Compact Plus Glucometer and the Accu-Chek® Active Test Strips were products of Roche Diagnostics, Mannheim, Germany. Assay kits and drugs The assay kits for albumin and cholesterol were products of Randox Laboratories Ltd., CoAntrim, UK. Streptozotocin (STZ) and metformin were manufactured by Santa Cruz Biotechnology, Dallas County, Texas, USA and Jiangsu Ruinian Qianjin Pharmaceuticals

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Company, Ltd, Jiangsu Province, China, respectively. All other chemicals used were of analytical grade obtained from Sigma-Aldrich, Mannheim, Germany. Preparation of aqueous extract of Digitaria exilis grains The grains were oven-dried at 40ºC before being pulverized with an electric blender (Kenwood, Model BL490, England, UK). A known amount of the powder (2500 g) was extracted in 25 L of distilled water for 48 hours at 28ºC. The filtrate was then freeze-dried (Labconco Freeze Drier, Model 64132, Kansas City, Missouri, USA) to yield 45.90 g. The resulting extract was reconstituted in distilled water to give the doses of 50, 100 and 200 mg/kg body weight that were used for the present antidiabetic study. The 50 and 100 mg/kg body weight corresponded respectively, to a table spoon and a handful of the plant powder estimated to be consumed by an adult of 70 kg as a remedy for diabetes. The 200 mg/kg body weight which is quadruple-fold of the least dose was used to account for cases of ‘abuse’ by the users. Screening for secondary metabolites The procedure described by Trease and Evans (1983) was adopted for the screening of the secondary metabolites present in the plant extract. Quantification of the detected secondary metabolites were carried out by adopting the procedures described for alkaloids (Adeniyi et al, 2009), saponins (Obadoni and Ochuko, 2001), flavonoids (Boham and Kocipai, 1974), terpenoids (Van-Burden and Robinson, 1981), cardiac glycosides, cardenolides, phlobatannins and anthraquinones (El-Olemy et al., 1994), tannins (Swain, 1979) and phenolics (Makkar et al., 1997). Induction of diabetes After eight hours of fast (without food, but water) and before the administration of STZ, the levels of the fasting blood glucose of the animals were determined by placing an aliquot (0.6 µL) 6

of the blood collected from an incised tail on the test strip that had been inserted into the glucometer. The reading on the glucometer was taken as the concentration of glucose in the blood of the animals. Thereafter, diabetes mellitus was induced in the animals by intraperitoneal administration of 60 mg/kg body weight of STZ solution (prepared in sodium citrate buffer, 0.1 M; pH 4.5) (Akbarzadeh et al. 2007). One hour after the administration of STZ, the animals were given 5% dextrose solution by oral intubation, to enable the male rats overcome the early hypoglycaemic phase (Mundargi et al., 2011). The glucose concentration in the blood of the male rats was again determined at 48 hours of administration of STZ. Rats that displayed blood glucose concentration higher than the baseline of 200.00 mg/dL were declared diabetic and included in the subsequent study (Kim et al., 2014). Animal grouping A total of forty two male rats (166.43 ± 3.32 g) were completely randomized into six treatment groups (A-F) of seven rats each as follows: Group A - normoglycaemic that received 0.5 ml of distilled water (distilled water only) Group B – STZ treated animals that received 0.5 ml of distilled water Group C - STZ treated animals that were administered 0.5 ml equivalent of 50 mg/kg body weight of metformin (reference drug) Group D - STZ treated animals that were administered 0.5 ml equivalent of 50 mg/kg body weight of the extract of D. exillis grains Group E - STZ treated animals that were administered 0.5 ml equivalent of 100 mg/kg body weight of the extract of D. exillis grains Group F - STZ treated animals that were administered 0.5 ml equivalent of 200 mg/kg body weight of the extract of D. exillis grains 7

The distilled water, metformin and the D. exilis grains extract were orally administered, twice daily, for 14 days according to the posology obtained from ethnobotanical survey on the use of plants for managing diabetes conducted within the locality of the authors. This study was carried out after ethical approval from University of Ilorin Ethical Review Committee (UERC) which was communicated to the authors by a letter dated 12th March 2015 under UERC Approval Number UERC/ASN/2015/048. Monitoring of Body weight, feed and water intake of the animals The weight of the feed and volume of water were determined each time they were to be given to the animals. Twenty four hours after, the left over feed were weighed and the volume of water determined. The difference between the initial and final weight of the feed and volume of water, computed for each day were noted as the daily feed intake and water intake. In addition, the animals were weighed individually prior to the commencement of the experiment and at intervals of 5 days for the 15 days experimental period to obtain the change in body weight of the rats. Preparation of serum and tissue supernatants Under ether anaesthesia, 5 ml of blood from the male rats was collected into centrifuge tubes from cut made on the jugular vein. The blood was then left to clot for 10 minutes and thereafter centrifuged (High Speed Centrifuge, Model YXJ-2, Essex, England) at 685 x g for 10 minutes to obtain the serum. In addition, an aliquot of the blood was also collected in the sample bottles containing ethylenediamine tetraacetic acid and used for the analyses of the haematological parameters. The pancreas and liver were excised from the dissected animal, blotted in blotting paper, weighed, homogenized in 0.25M sucrose solution (1:5 w/v) and centrifuged at 1398 x g for 15 minutes to obtain their respective supernatants. Both the serum and the tissue supernatants were used for the biochemical analyses within 12 hours of preparation. 8

Determination of full blood count The blood was analysed for full blood count (red blood cell, packed cell volume, mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration, red cell distribution width, white blood cell, platelet, lymphocytes, basophils, eosinophils, neutrophils and monocytes) using Albacus Junior Haematology Analyzer (Model 111478, Diatron GmbH, Vienna, Austria). Determination of levels of some metabolites and enzyme activities The levels of some biochemical metabolites were determined by adopting the procedures described for serum creatinine (Jaffe, 1941), serum urea (Rosenthal, 1955), serum albumin (Doumas et al., 1971), serum total cholesterol (Fredrickson et al., 1967), hepatic glycogen (Kemp et al., 1954) and pancreatic insulin (Burgi et al., 1988). The activities of hexokinase, phosphofructokinase, glucose-6-phosphatase and fructose-1,6-bisphosphatase in the liver of the male rats were determined according to the procedures described by Brandstrup et al. (1969), Castano et al. (1979), Koide and Oda (1959) and Gancedo and Gancedo (1971) respectively. Data analysis Results were expressed as the mean ± SEM of seven determinations. Analysis of Variance and Duncan’s Multiple Range Test at 5% confidence level were employed to determine the statistical signficance of the data. The statistical analysis was carried out with SPSS Version 23.0 Software (Statistical Package for Social Sciences, Inc., Chicago, IL, USA). Results Screening of the secondary metabolite constituents of aqueous extract of D. exilis grains revealed that 10 secondary metabolites (alkaloids, flavonoids, saponins, anthraquinones, terpenoids, tannins, phlobatannins, phenolics, cardiac glycosides and cardenolides) were present 9

whereas steroids were not detected (Table 1). Furthermore, alkaloids 30.20 mg/ml) was the most abundant of the secondary metabolites whereas phlobatannins (0.22 mg/ml) were the least abundant (Table 1). Against the 67.31 mg/dL basal blood glucose level, administration of 60 mg/kg body weight of STZ elevated the blood glucose level after 48 hours by 3.8 fold (Table 2). The blood glucose levels were persistently and progressively increased till the end of the experimental period. Although, all the doses of the extract (50, 100 and 200 mg/kg body weight) significantly (p<0.05) and progressively lowered the blood glucose levels of the animals, the decrease by the 50 mg/kg body weight did not manifest until after day 6 of treatment. By the end of the 15 days experimental period, the 100 and 200 mg/kg body weight of the extract had lowered the blood glucose content of the animals to levels that compared favourably (p>0.05) with the basal glucose level and those of STZ-treated animals that received metformin (Table 2). Administration of STZ to the male rats increased the amount of feed consumed and water intake by 67 and 509% respectively (Table 3). In contrast, the extract significantly (p<0.05) reduced the amount of feed consumed with that of 200 mg/kg body weight of the extract treated diabetic animals comparing favourably (p>0.05) with the normoglyceamic animals that received distilled water (Table 3). Furthermore, administration of metformin to the diabetic animals significantly (p< 0.05) increased the amount of feed consumed by male rats when compared with the normoglycaemic rats that received distilled water. Although, the administration of the extract to the STZ-treated male rats reduced (p<0.05) the volume of water intake by the animals like those STZ-treated rats that received metformin, the reduction in the volume of water intake did not compare (p>0.05) favourably with the normoglyceamic rats that received distilled water (Table 3). 10

Streptozotocin generally reduced the body weight of the male rats when compared with the normoglyceamic rats that received distilled water (Table 4). In contrast, administration of the extract significantly and progressively increased (p<0.05) the body weight of the animals and by the end of the experimental period, the 50, 100 and 200 mg/kg body weight of the extract had increased the body weight of the animals by 12, 8 and 17% respectively as against the 15% produced by the metformin (Table 4). Compared with the normoglyceamic rats that received distilled water, STZ administration significantly (p<0.05) reduced the weight of the pancreas, pancreatic insulin and the glycogen contents in the liver of the male rats (Table 5). All the doses of the extract produced weight of pancreas and hepatic glycogen concentration that were not significantly (p>0.05) different from the normoglyceamic male rats that received distilled water and STZ-treated rats that were administered metformin. Although the levels of pancreatic insulin increased after the administration of all the doses of the extract and metformin when compared with the STZ-treated animals, the insulin levels were significantly (p<0.05) lowered when compared with the normoglyceamic rats that received distilled water. The increases in the levels of creatinine, albumin and total cholesterol after STZ administration were significantly reversed by all the doses of the extract in a manner similar to those of STZ-treated rats that received metformin. Furthermore, the elevated level of urea by the STZ was significantly (p<0.05) and dose dependently reduced by the extract (Table 5). It is worthy of note that the level of urea produced after the administration of 200 mg/kg body weight of the extract to the STZ-treated male rats compared favourably (p>0.05) with the STZ-treated rats that were administered metformin and the normoglyceamic rats that received distilled water (Table 5).

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Streptozotocin significantly (p>0.05) reduced the activities of hepatic hexokinase and phosphofructokinase whereas it increased the activities of glucose-6-phosphatase and fructose1,6-bisphosphatase when compared with the distilled water treated normoglyceamic control rats (Table 6). In contrast, all the doses of the extract increased the STZ-treatment related decreases in the activities of hexokinase and phosphofructokinase. Furthermore, the elevated glucose-6phosphatase and fructose-1,6-bisphophatase activities by STZ were reduced (p<0.05) by all the doses of the extract. The extract treatment-related changes in the hepatic enzymes in the present study were not dose related but similar to those of STZ treated animals that received metformin (Table 6). Administration of STZ significantly (p<0.05) reduced the levels of Hb, PCV, RBC, MCV, MCH, MCHC, RCDW, WBC, platelets, lymphocytes, basophils, eosinophils neutrophils and monocytes in the male rats (Table 7). In contrast, all the doses of the extract increased (p<0.05) the Hb levels in the STZ-treated animals. Although, the levels of Hb produced by metformin compared well (p>0.05) with the normoglyceamic distilled water treated rats, the extract produced Hb levels that were higher (p<0.05) than the normoglycemic control animals (Table 7). Furthermore, the levels of PCV produced by all the doses of the extract compared favourably (p>0.05) with those of normoglyceamic distilled water treated animals. The pattern of PCV produced by the extract was not significantly (p>0.05) different from that of metformin (Table 7). The extract also reversed the STZ-treatment related decreases in RBC, MCV, MCH, MCHC, RCDW, WBC, platelets, lymphocytes, basophils, eosinophils neutrophils and monocytes in the male rats (Table 7).

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Discussion Streptozotocin has been accepted as the first line choice for the induction of diabetes in experimental animals (Lenzen, 2008). It is selectively accumulated in the β-cells of the pancreas via the low-affinity GLUT2 glucose transporter in the plasma membrane. The transfer of the methyl group from streptozotocin to the DNA molecule results in a cascade of event, including activation of poly ADP-ribosylation which depletes NAD+ and ATP and generation of superoxide, hydrogen peroxide and hydroxyl radicals from the enhanced dephosphorylation of ATP. In addition, other toxic amount of nitric oxide generated by STZ also inhibits aconitase activity and participates in DNA damage. Protein glycosylation may be an additional damaging factor. All these inhibit the secretion of insulin that eventually gives rise to insulin-dependent diabetes mellitus. Normally, cells of the pancreas maintain blood glucose levels within a narrow limit by modulating the activity of the β-cells of the Islets of Langerhans through secretion of insulin. Therefore, the elevated blood glucose (hyperglycemia) in the male rats after the administration of STZ may be adduced to impairment in the release of insulin as a consequence of the destroyed βcells of Islets of Langerhans in the pancreas. In contrast, the lowered blood glucose levels and the ameliorative effects of the extract at 100 and 200 mg/kg body weight suggests that the extract increased the functional activity of the pancreas to secrete insulin. The enhanced secretion of insulin by the extract of D. exilis grains could be linked to the increase in pancreatic insulin in the present study. The extract-induced increase in the secretion of insulin by the pancreas as a response to the STZ-induced hyperglycermia might have promoted the up-take of glucose from the blood for energy production and thus restored the glycemic state of the animals. Therefore, the aqueous extract of D. exilis grains might possess an insulin-like effect or stimulate insulin 13

secretion from β-cells. The findings with respect to the extract normalising hyperglycemia induced by STZ is similar to that reported by El-Quady et al. (2019). Nagmoti et al (2015) have reported that STZ-induced diabetes is characterized by hyperglycemia (elevated blood glucose), polyphagia (excessive eating or appetite), polydipsia (excessive water intake) and loss of body weight. The STZ-treatment related increases in feed and water intake, occasioned by enhanced secretion of ghrelin and a reduction in the activity of leptin receptor and/or insulin insufficiency were reversed and/or ameliorated by the doses of the extract. Although, the extract ameliortated the STZ-induced high feed intake in the animals, the amount of feed consumed by STZ-treated animals that received metformin was the highest. Furthermore, the increase in the body weight of STZ-treated animals that received the extract is quite understandable since improvement in glycemic control via the enhanced production and secretion of insulin (insulin sufficiency) would have restored the otherwise STZ-treatment related reduction in the metabolism of glucose, increased fat metabolism and proteolytic breakdown of structural proteins into amino acids that provided the alternative source of energy since there was reduced and/or impaired uptake of glucose (Kammlakkannan and Prince 2006). The effects of the extract on body weight of diabetic animals in the present study implied that the extract restored efficient metabolic homeostasis and good health in the animals. The findings in the present study with respect to body weight changes are similar to those previously reported by Sharma and Gupta (2017) on anti-hyperglycemic activity of aqueous extract of some medicinal plants on Wistar rats. In addition, the reversal of the STZ-treatment related decrease in the absolute weight of the pancreas by the extract my not be unconnected with restoration of the normal architecture of the secretory granules that led to insulin sufficiency and glycemic control

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in the animals. This findings and the school of thought in the present study agrees with the earlier reports of Campbell-Thompson (2012). Complications of diabetes mellitus have been widely reported to manifest among others as elevation in the levels of albumin, urea, creatinine and cholesterol as well as reduction in hepatic glycogen level (Hu et al. 2016; Gad-Elkareem et al. 2019). In the present investigation, the STZinduced increases in serum albumin and urea suggests impaired synthesis of the albumin and/ or diminished protein intake and increased break down of muscles and other tissues proteins into amino acids due to enhanced proteases activity respectively (Kalaiselvi et al., 2015). Increased creatinine levels after STZ administration could be ascribed to STZ-induced nephropathy whereas the elevated levels of cholesterol may be due to inactivation of lipoprotein lipase which is required for the hydrolysis of lipids (Kreisberg, 1998). All these biochemical changes are consequences of deranged metabolic and regulatory mechanisms, due to insulin deficiency, in the animals. The extract through the enhancement of insulin secretion have restored the metabolic and regulatory mechanism of the diabetic animals with respect to protein and lipid metabolism. The present study demonstrated that disturbances of protein and lipid metabolism characteristic of diabetes were ameliorated by the aqueous extract of D. exilis grains. The in vivo regulation of glycogen metabolism depends on the important roles played by the multifunctional enzymes, glycogen synthase and glycogen phosphorylase (Diaz-Lobo et al. 2015). Therefore, depleted hepatic glycogen concentration after the administration of STZ in this study may be because of decreases in activities of insulin-dependent and insulin-sensitive hexokinase, as it is in the current study, and glycogen synthase. In contrast, the repletion of the hepatic glycogen after the administration of the extracts may not be unconnected with the its ability to stimulate the secretion of insulin from the pancreas which further restored the activities 15

of hexokinase and glycogen synthase. Such restoration of hexokinase activity by the extract as evident in this study will enhance phosphorylatation of glucose to glucose-6-phosphate for further utilization by the cells and the synthesis of glycogen in the hepatocytes of the animals. Insulin has been reported to stimulate the activation of both hexokinase and phosphofrucktokinase activities, which are important regulators of storage and disposal of glucose (Tanner et al. 2018). Therefore, since STZ was reported earlier in this study to reduce the secretion of insulin from the pancreas, the lowering of the activity of these enzymes is quite understandable. The lowering of activities of hexokinase and PFK will slow down the rate of glycolysis in the liver of the animals and consequently increasing the level of glucose to a hyperglycemic situation.

However, the increase in the activities of these enzymes by the

components of the extract of D. exilis grains in the present study, like those of the metformin treated animals, will promote glycolysis that will be accompanied by decreased hepatic glucose levels and enhanced production of energy via glycolysis (Tanner et al. 2018). Consequently, this will assist in the restoration of glycemic control as evident in the present study.

Furthermore,

insulin has been reported to reduce gluconeogenesis by lowering the activities of key enzymes like glucose-6-phosphatase, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase (Hatting et al. 2018). The reduction in the activities of glucose-6phosphatase and fructose-1,6-bisphosphatase by the extract of D. exilis grain which contrast that of the STZ-treated animals may be due to its ability to restore insulin in the animals.

The

changes in the activities of the liver enzymes investigated in the present study might have restored glycemic control in the animals via enhanced peripheral glucose utilization by stimulating liver glycolysis and limiting the formation of gluconeogenic compounds (Koti et al. 2011). The findings with respect to the key carbohydrate metabolizing enzymes in this study are 16

consistent with those reported by Chetna et al. (2019) after the oral administration of citral to high-fat-diet- and STZ-induced diabetic rats. Studies have shown that diabetes is associated with reduction in the levels of RBC, Hb, PCV, RCDW, MCV, MCH, MCHC, WBC and indices relating to it (Demirtas et al. 2015; Okorie et al. 2019). The reduction in the levels of these haematological parameters was also obtained in the present study after STZ was administered and indicates anemia and inflammation which are common pathophysiology of diabetes mellitus (Goji et al. 2017). It is also an indication of abnormal synthesis of heamoglobin, failure with respect to osmoregulation, plasma osmolarity. The occurrence of anemia in diabetes has been linked to increased non-enzymatic glycosylation of RBC membrane proteins (Oyedemi et al. 2011). Although, glycolsylated haemaoglobin was not investigated in the present study, the increase in the levels of RBC, Hb and PCV by the extract may not be unconnected with reduced non-enzymatic glycosylation of RBC membrane proteins, suppressing haemolysis through osmoregulation and biomembrane stabilization (Takagi et al. 2011; Bashir et al. 2015). The extract might have also reduced the inflammation that is an important in the progression to diabetes. Mamun-or-Rashid et al. (2014) reported that bioactive agents commonly used for managing diabetes were the flavonoids, tannins, phenolic, and alkaloids. Alkaloids have been reported to enhance the activities of hexokinase and phosphofuctokinase, resulting in glucose transport, carbohydrate digestion and absorption ((El Barky et al., 2017) whereas both alkaloids and saponins regenerate pancreatic β cells and insulin secretion and inhibit their degradation (Bharti et al. 2018). Furthermore, flavonoids and phenolics have been reported to be involved in insulin secretion, free radical scavenging and insulinonematic activity ((Bharti et al. 2018). Therefore, the presence of alkaloids, flavonoids, phenolics and tannins may be responsible for the 17

antidiabetic activity of Digitaria exilis grains in the present study as evident from the increased insulin content of the pancreas and enhanced activities of hexokinase and phosphofuctokinase. Conclusion This study has scientifically substantiated the folkoric use of Digitaria exilis grains in the management of diabetes via increased insulin secretion, as plasma concentrations of insulin were

not determined, enhanced activities of hexokinase and phosphofuctokinase and repletion of hepatic glycogen. The presence of alkaloids, flavonoids, phenolics and tannins might have conferred the desired antidiabetic activity on Digitaria exilis grain. Acknowledgements The authors wish to acknowledge Mr Raymond O. Jonathan and Mrs Anuoluwapo V. Aluko for their technical assistance. Competing interests The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. Authors’ contributions Conception and Design: ADM and YMT Acquisition of Data: ADM Analysis and Interpretation of Data: ADM and YMT Drafting the Manuscript: ADM Revising for Intellectual Content: YMT Final Approval of the Completed Article: ADM and YMT.

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References Adeniyi, S.A., Orjiekwe, C.L., Ehiagbonane, J.E., 2009. Determination of alkaloid and oxalates in some selected food samples in Nigeria. African Biotechnology 8, 10-112. Adoukonou-Sagbadja, A.H., 2010. Genetic Characterization of Traditional Fonio Millets (Digitaria exilis, D. iburua STAPF) Landraces from West-Africa: Implications for Conservation and Breeding. PhD thesis submitted by M.Sc Hubert Adoukonou-Sagbadja to Justus-Liebig University, Giessen, Germany from University of Abomey-Calavi, BP 526, Cotonu, Benin Republic with summaries in English, German and French, pp 107. Akbarzadeh, A., Norouzian, D., Mehrabi, M.R., Jamshidi, S.H., Farhangi, A., Allah-Verdi, A., Mofidian, S.M., Lame Rad, B., 2007. Induction of diuabetes by streptozotocin in rats. Indian Journal of Clinical Biochemistry 22(2), 60-64. Alegbejo, J.O., Ameh, D.A., Ogala, W.M., Ibrahim, S., 2011. Glycaemic index and load of Acha (Fonio) in healthy and diabetic subjects. Journal of Pure and Applied Microbiology 5(1), 117-122. AlShamsi, M., Amin, A., Adeghate, E., 2004. Beneficial effect of vitamin E on the metabolic parameters of diabetic rats. Molecular and Cellular Biochemistry, 261(1-2), 35-42. Ballogou, V.Y., Soumanou, M.M., Toukourou, F., Hounhouigan, J.D., 2013. Structure and Nutritional Composition of Fonio (Digitaria exilis) Grains: A review. International Research Journal of Biological Science 2(1), 73-79. Barcelo, A., Rajpathak, S., 2001. Incidence and prevalence of diabetes mellitus in the Americas. Revista Panamericana de Salud Publica 10(5), 300-308. Bashir, L., Shittu, O.K., Busari, M.B., Sani, S., Aisha, M.I., 2015. Safety evaluation of giant African land snails (Archachatina maginata) haemolymph on hematological and biochemical parameters of albino rats. Journal of Advanced Medical and Pharmaceutical Sciences 3, 122-130. Bharti, S.K., Krishnan, S., Kumar, A. & Kumar, A., 2018. Antidiabetic phytoconstituents and their mode of action on metabolic pathways. Therapeutic Advances in Endocrinology and Metabolism 9(3), 81-100. Boham, B.A., Kocipai, A.C., 1974. Flavonoids and condensed tannins from leaves of Hawaiian vaccinium vaticulatum and V. calycinium. Pacific Science 48, 458-463. Brandstrup, N., Kirk, J.E., Bruni, C., 1969. The hexokinase and phosphoglucoisomerase activities of aortic and pulmonary artery tissue in individuals of various ages. Journal of Gerontology 12,166-171.

19

Burgi, W., Briner, M., Franken, N., Kessler, A.C., 1988. One step sandwich enzyme immunoassay for insulin using monoclonal antibodies. Clinical Biochemistry 21(5), 311314. Campbell-Thompson, M., Cliv Wasserfall, M. S., Emily, L., Montgomery, B.S., Atkinson, M.S., Kaddis, J.S., 2012. Pancreas organ weight in individuals with disease-associated auto antibiotics at risk of type 1 diabetes. Journal of the American Medical Association, 308(22), 2337-2339. Castano, J.G., Nieto, A., Felui, J.E., 1979. Inactivation of phosphofructokinase by glycogen in rat hepatocytes. Journal of Biological Chemistry 254, 5576-5579. Chetna, M., Monowar, K., Naazmin, F., Babita, S., Dinesh, T., Mohammad, W., Abbas, A.M., 2019. Effects of citral on oxidative stress and hepatic key enzymes of glucose metabolism in streptozotocin/high-fat-diet induced diabetic dyslipidemic rats. Indian Journal of Basic Medical Sciences 22(1), 49.57. Chukwu, O., Abdul-kadir, A.J., 2008. Proximate chemical composition of Acha (Digitaria exilis and Digitaria iburua) grains. Journal of Food Technology 6(5), 214-216. Dachi, S.N., Gana, A.S., 2008. Adaptability and yield evaluation of some Acha (Digitaria exilis and Digitaria iburua Kippis Stapf) accessions at Kusogi-Bida, Niger State, Nigeria. African Journal of General Agriculture 4(2), 73-77. Demirtes, L., Degirmenci, H., Akbas, E.M., Ozcicek, A., Timuroglu, A., Gurel, A., Ozcicek, F., 2015. Association of haematological indices with diabetes, impaired glucose regulators and microvascular complications of diabetes. International Journal of Clinical and Experimental Medicine 8(7), 11420-11427. Diaz-Lobo, M., Garcia-Amoros, J., Fita, I., Velasco, D., Guinovart, J.J., Ferrer, J.C, 2015. Selective photoregulation of the activity of glycogen synthase and glycogen phsophorylase, two key enzymes in glycogen metabolism. Organic & Biomolecular Chemistry 13, 72827288. Doumas, B.T., Watson, W.A., Biggs, H.G., 1971. Albumin standards and measurement of serum albumin with bromocresol green. Clinical Chemistry Acta 31, 87-92. Echendu, C.A., Obizoba, I.C., Anyika, J.U., Ojimelukwu, P.C., 2009. Changes in chemical composition of treated and untreated hungry rice acha (Digitaria exilis). Pakistan Journal of Nutrition 8, 1779-1785. Egwim, E.C., Oloyede, O.B., 2008. Immobilization of α-amylase from Acha (Digitaria exilis) on different cellulose fibre materials. Asian Journal of Biochemistry 3(3), 169-175.

20

El Barky, A.R., Hussein, S.A., Alm-Eldeen, A.A., Hafez, Y.A., Mohamed, T.M., (2017). Saponins and their potential role in diabetes mellitus. Diabetes Management 7(1), 148-158. El-Olemy, M.M., Al-Muhtadi, F.J., Afifi, A.A., 1994. Experimental Phytochemistry. A Laboratory Manual Saudi Arabia: King Saud University Press Riyadh, pp. 3-137. El-Quady, F., Lahrach, N., Ajebli, M., El-Haidani, A., Eddouks, M., 2019. Antihyperglycemic effect of the aqueous extract of Foeniculum vulgare in normal and streptozotocin-induced diabetic rats. Cardiovascular & Haematological Disorders-Drug Target (E-pub ahead of print) DOI:10.2174187152577666190612121516. Fredrickson, D.S., Levy, R.I., Lees, R.S., 1967. Fat transport in lipoproteins- An integrated approach to mechanisms and disorders. North England Journal of Medicine 276, 148-156. Gad-Elkareem, M.A.M., Abdelgadir, E.H., Badawy, O.M., Kadir, A. 2019. Potential antidiabetic effect of ethnaolic and aqueous-ethanolic extracts of Ricinus communis leaves on streptozotocin-induced diabetes in rats. Peer Journal 7, e6441. http://doi.org/10.7717/peerj.6441. Gancedo, J.M., Gancedo, C., 1971. Fructose-1,6-diphosphatase, phosphofructokinase and glucose-6-phosphate dehydrogenase from fermenting and non-fermenting yeasts. Archives of Microbiology 76, 132–138. Glew, R.H., Laabes, E.P., Presley, J.M., Schulze, J., Andrews, R., Wang, Y., Chang, Y., Chuang, L., 2013. Fatty acid, amino acid, mineral and antioxidant contents of acha (Digitaria exilis) grown on the Jos Plateau, Nigeria. International Journal of Nutrition and Metabolism 5(1), 1-8. Goji, A.D.T., Mohammed, A., Tanko, Y., Kawu, M.U., Isah, A.D., 2017. Effects of folic acid and magnesium co-administration on some haematological parameters in streptozotocininduced type 1 diabetic Wistar rats. American Journal of Medicine and Medical Sciences 7(8), 313-317. Hatting, M., Tavares, C.D.J., Sharabi, K., Rines, A.K., Puigserver, P., 2018. Insulin regulator of gluconeogenesis. Annals of the New York Academy of Sciences 1411(1), 21-35. Hu, X., Cheng, D., Zhang, Z., 2016. Antidiabetic activity of Helicteres angustifolia root. Pharmaceutical Biology 54, 938-944. IDF (2017). International Diabetes Federation. Diabetes Atlas ed. Brussels: International Diabetes Federation. Irving, D., Jideani, I.A., 1997. Microstructure and composition of Digitaria exilis Stapf a potential crop. Cereal Chemistry 74(3), 224-228.

(acha):

Jaffe, M., 1941. The measurement of creatinine in plasma and urine. Nature 148 (3743), 110. 21

Jideani, I.A., 1999. Traditional and possible technological uses of Digitaria exilis (acha) and Digitaria iburua (iburu): a review. Plant Foods and Human Nutrition 54(4), 363-374. Jideani, I.A., 2012. Digitaria exilis (acha/fonio), Digitaria iburua (Iburu/fonio) and Eluesine ciracana (tamba/finger millet)-Non-conventional cereal grains with potentials. Scientific Research and Essay 7(45): 3834-3843. Jideani, I.A., Akingbala, J.O., 1993. Some physiochemical properties of acha (Digitaria exilis Stapf) and Iburua (Digitaria iburua Stapf) grains. Journal of Science and Food Agriculture 63, 369-371. Kalaiselvi, A., Reddy, G. A., Ramalingam, V., 2015. Ameliorating effect of ginger extract (Zingiber officinale Roscoe) on liver marker enzymes, lipid profile in aluminium chloride induced male rats. International Journal of Pharmaceutical Science and Drug Research 7, 52-58. Kammlakkannan, N., Prince P.S.M., 2006. Antihyperglycaemic and antioxidant effect of rutin, a polyphenolic flavonoid, in streptozotocin-induced diabetic Wistar rats. The Nordic Pharmacology Society 98: 97-103. Kemp, A., Adrienne, J. M., Heijningen, K.V., 1954. A colorimetric micromethod for the determination of glycogen in tissues. Biochemical Journal 56(4), 646-648. Kim, J., Choi, J., Lee, M., Kang K., Paik, M., Jo, S., Jung, U., Park, H., Yee, S., 2014. Immunomodulatory and antidiabetic effects of a new herbal preparation (HemoHIM) on streptozotocin-induced diabetic mice. Evidence-Based Complementary and Alternative Medicine 4(3), 461-665. Koide, H., Oda, T., 1959. Pathological occurrence of glucose-6-phosphatase in serum in liver diseases. Clinica Chimica Acta 4, 554-561. Koti, B.C., Gore, A., Thippeswamy, A.H.M., Viswanatha, S., Kulkarni, R., 2011. Alcoholic leaf extract of Plectranthus amboinicus regulates carbohydrate metabolism in alloxan-induced diabetic rats. Indian Journal of Pharmacology 43(3), 286-290. Kreisberg, R.A., 1998. Diabetic dyslipidemia. American Journal of Cardiology 82(12A), 67U73U. Lenzen, S. 2008. The mechanisms of alloxan- and streptozotocin-induced diabetes. Diabetologia, 51(2):216-226. Makkar, H.P.S., Baker, K., Abel, H., Pawelzik, E., 1997. Nutrient content, rumen protein degradability and nutritional factors in some colour-and-white flowering cultivars of Vicia faba beans. Journal of Science and Food Agriculture 75, 511-520.

22

Mamun-or-Rashid, A., Hossain, M.S., Naim Hassan, B., Kumar Dash, M., Sapon, A., Sen, M.K., 2014. A review on medicinal plants with antidiabetic activity. Journal of Pharmacognosy and Phytochemistry 3(4), 149-159. Morales-Payan, J.P., Richard O.J., Julio C.T., Francisco T.S., 2002. Digitaria exilis as a crop in the Dominican Republic. Supplement to: Trends in new crops and new uses. J. Janick and A. Whipkey (eds.). Alexandria, VA: ASHS Press. Mundargi, R.C., Rangaswamy, V., Aminabhavi, T.M., 2011. pH-sensitive oral insulin delivery systems using Eudragit microspheres. Drug Development and Industrial Pharmacology 37(8), 977-985. Nayak, B.S., Marshall, J.R., Isitor, G., Adogwa, A., 2011. Hypoglycemic and hepatoprotective activity of fermented fruit juice of Morinda citrifolia (Noni) in diabetic rats. EvidenceBased Complementary and Alternative Medicine Volume 2011, Article ID 875293, 5 pages Nagmoti, D.M., Kothavade, P.S., Bulani, V.D., Gawali, N.B., Juvekar, A.R. 2015. Antidiabetic and antihyperlipidemic activity of Pithecellobium dulce (Roxb.) Benth seeds extract in streptozotocin-induced diabetic rats. European Journal of Integrative Medicine 7(3), 263273. Obadoni, B.O., Ochuko, P.O., 2001. Phytochemical studies and comparative efficacy of the crude extracts of some homeostatic plants in Edo and Delta States in Nigeria. Global Journal of Pure and Applied Sciences 8b, 203-208. Okorie, H., Obeagu, E.I., Anaebo, Q.B.N., 2019. Investigation of some haematological parameters in pregnant women with gestational diabetes at Federal Medical Centre, Owerri, Imo State, Nigeria.Annals of Clinical and Laboratory Research 7(2), 305. Olagunju, A.I., Omoba, O.S., Enujiugha, V.N., Aluko, R.E., 2018. Development of value-added nutritious crackers with high antidiabetic properties from blends of Acha (Digitaria exilis) and blanched Pigeon pea (Cajanus cajan). Food Science and Nutrition 6, 1791-1802. Oyedemi, S.O., Yakubu, M.T., Afolayan, A.J. (2011). Antidiabetic activities of aqueous leaves extract of Leonotis leonurus in streptozotocin induced diabetic rats. Journal of Medicinal Plant Research, 5: 119-125. Rosenthal, H.L., 1955. Determination of urea in blood and urine with diacetyl monoxime. Annals of Chemistry 27, 35. Sharma, A.K., Gupta, R., 2017. Antihyperglycemic activity of aqueous extracts of some medicinal plants on Wistar rats. Journal of Diabetes and Metabolism 8, 752. doi:10.4172/2155-6156, 1000752.

23

Swain, T., 1979, Tannins and Lignins, In: G.A. Rosenthal and D.H. Janzen, eds. Herbivores: Their interaction with secondary plant metabolites. Pp. 657-682. Academic Press, New York. Takagi, S., Murata, H., Goto, T., Hatate, H., Endo, M., Yamashita, H., Miyatake, H., Ukawa, M. (2011). Role of taurine deficiency in inducing green liver symptom and effect of dietary taurine supplementation in improving growth in juvenvile red sea bream (pragrus major) fed non-fishmeal diets based on soy protein concentrate. Fisheries Science, 77: 235-244. Tanner, L.B., Goglia, A.G., Wei, M.H., Sehgal, T., Parsons, L.R., Park, J.O., White, E., Toettcher, J.E., Rabinowitz, J.D., 2018. Four key steps control glycolytic flux in mammalian cells. Cell Systems 7, 49-62. Taylor, J.R.N., Schober, T.J., Bean, S.R. 2006. Novel food and non-food uses for sorghum and millets. Journal of Cereal Science 44: 252-271. Temple, V.J., Bassa, J.D., 1991, Proximate chemical composition of ‘acha’ (Digitaria exilis) grains. Journal of Science, Food and Agriculture 56, 561-563. Trease, G.E., Evans W.C., 1983. A Textbook of Pharmacognosy, 12th edn. Bailliere-Tindall Ltd., London, pp. 343-383.

Ullah, A., Khan, A., Khan, I., 2016. Diabetes mellitus and oxidative stress- A concise review. Saudi Pharmaceutical Journal 24(5), 547-553. Van-Burden, T.P., Robinson, W.C., 1981. Formation of complexes between protein and tannin acid. Journal of Agriculture and Food Chemistry 1, 77.

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Table 1: Secondary metabolite content of aqueous extract of Digitaria exilis grains Secondary Metabolites Alkaloids Tannins Flavonoids Steroids Terpenoids Phlobatannins Anthraquinones Cardiac glycosides Phenolics Saponins Cardenolides

Observation Cream colour with Mayer’s reagent; Reddish-brown with Wagner’s reagent Greenish-brown precipitate Dark yellow precipitate with NH3 Absence of violet to blue or green colour with H2SO4 Reddish-brown colour with H2SO4 Reddish precipitate with HCl Bright pink colour with NH3 Brown violet ring at the interface Greenish precipitate with FeCl3 Stable, persistent froth with distilled water Turbid brown ring at the interface

Data are mean ± SEM; n = 3

Concentration (mg/ml) 30.20 ± 0.08 0.55 ± 0.05 7.61 ± 0.04 Not detected 0.93 ± 0.02 0.22 ± 0.00 15.38 ± 0.06 24.32 ± 0.05 0.30 ± 0.00 9.31 ± 0.03 5.30 ± 0.08

Table 2: Blood glucose level of streptozotocin-induced diabetic rats after oral administration of aqueous extract of Digitaria exilis grains Blood glucose levels (mg/dL) Treatment Days Treatment group

Basal blood glucose

3

6

9

12

15

65.95±2.31a

Blood glucose prior to treatment 64.78±2.21a

Distilled water only

65.48±2.32a

63.95±95a

64.16±2.14a

65.25±1.99a

64.84±1.99a

STZ + Distilled water

67.31±2.62a

258.98±3.79b

264.75±2.56b

294.73±4.07b

315.14±2.37b

320.97±2.34b

325.19±0.94b

STZ + metformin

65.81±3.55a

243.04±5.92c

240.47±5.95c

192.37±2.21c

109.32±2.37c

79.41±2.68c

64.06±2.16a

STZ + 50 mg/kg body of extract

65.12±3.45a

262.98±4.01b

260.99±4.61b

227.94±3.43d

147.44±6.44d

78.72±2.10c

70.60±2.56c

STZ + 100 mg/kg body of extract

66.34±3.92a

254.89±6.12c

251.68±6.20c

226.29±6.06d

135.50±3.27e

80.16±2.38c

67.15±2.16a

STZ + 200 mg/kg body of extract

68.70±2.72a

259.85±2.66b

248.17±2.12c

209.35±4.90e

123.82±3.21f

76.20±1.81c

64.43±1.60a

Data were mean ± SEM; n = 7. Values with superscripts b, c, d, e and f different from their respective control, a, down the column are significantly different (p<0.05); STZ = Streptozotocin

Table 3: Feed and water intake of streptozotocin-induced diabetic rats after oral administration of aqueous extract of Digitaria exilis grains Parameter

Distilled water only

STZ + Distilled water

STZ + metformin

STZ + 50 mg/kg body of extract

Feed intake (g)

118.26±0.73a

197.94±0.88b

173.82±1.81c

66.66±1.07d

STZ + 100 mg/kg body of extract 80.55±1.27e

Water intake (ml)

74.07±1.63a

450.77±1.68b

142.80±1.20c

181.70±1.35d

347.80±1.62e

STZ + 200 mg/kg body of extract 119.19±0.95a 235.59±1.34f

Data were mean ± SEM; n = 7. Values with superscripts b, c, d, e and f different from their respective control, a, across the row are significantly different (p<0.05) STZ = Streptozotocin

Table 4: Body weight of streptozotocin-induced diabetic rats after oral administration of aqueous extract of Digitaria exilis grains

Treatment group Distilled water only

Initial weight 156.21±0.98a

1 160.88±1.19a

STZ + Distilled water

146.52±8.22a,b

141.24±7.14b

STZ + metformin

142.40±4.12a,b

STZ + 50 mg/kg body of extract

Body weight (g) Treatment Days 5 179.43±0.66a

10 186.48±1.01a

15 198.15±0.93a

136.54±6.16b

131.61±2.57b

126.77±0.49b

145.83±3.83c

148.85±4.53c

154.48±4.08c

164.11±3.10c

137.96±4.26b

141.71±3.83b

145.40±3.63c

149.43±3.17c

154.80±2.30d

STZ + 100 mg/kg body of extract

155.18±1.48a

157.83±1.14a

160.65±0.79d

165.34±0.74d

167.62±0.70c

STZ + 200 mg/kg body of extract

149.03±3.30a,b

152.80±2.98d

158.76±2.17d

165.53±2.47d

173.84±1.33e

Data were mean ± SEM; n = 7. Values with superscripts b, c, d and e different from their respective control, a, down the column are significantly different (p<0.05) STZ = Streptozotocin

Table 5: Effect of aqueous extract of Digitaria exilis grains on selected biomolecules of streptozotocin-induced diabetic rats Treatment Group

Pancreatic insulin (µIU/ml)

Urea (mg/100 ml)

Creatinine (mg/100 ml)

Albumin (g/L)

Total cholesterol (mmol/L)

Distilled water 0.95±0.72a only

19.97±0.30a

137.35±0.64a

1.93±0.16a

9.82±0.21a

6.53±0.10a

Liver glycogen (mg/100 mg of glucose) 64.00±1.05a

STZ + 0.68±0.03b Distilled water

2.47±0.20b

215.19±1.63b

9.59±0.29b

20.13±0.38b

14.25±0.05b

38.43±1.21b

0.88±0.05a

12.89±0.12c

138.19±0.73a

2.00±0.11a

9.56±0.28a

6.10±0.98a

63.43±1.25a

STZ + 50 0.82±0.05a mg/kg body of extract

10.77±0.28d

147.49±0.71c

2.17±0.15a

9.86±0.18a

6.35±0.20a

67.60±0.82a

STZ + 100 0.96±0.08a mg/kg body of extract

17.44±0.14a

144.51±1.59c

2.18±0.13a

10.46±0.66a

7.51±0.12a

65.86±1.14a

STZ + 200 0.91±0.48a mg/kg body of extract

14.91±0.19c

136.99±0.52a

2.17±0.12a

9.81±0.45a

6.98±0.11a

62.71±1.02a

STZ + metformin

Weight of pancreas (g)

Data were mean ± SEM; n = 7. Values with superscripts b, c, and d, different from their respective control, a, down the column are significantly different (p<0.05) STZ = Streptozotocin

Table 6: Hepatic carbohydrate metabolizing enzyme activities of streptozotocin-induced diabetic rats after oral administration of aqueous extract of Digitaria exilis grains Treatment Group

Hexokinase (units/g protein)

Phosphofructokinase (mMol/min/mg protein)

Glucose-6-phosphatase (U/mg protein)

Distilled water only

12.23±0.17a

28.58±0.30a

7.80±0.18a

Fructose-1, 6bisphosphatase (U/mg protein) 12.74±0.27a

STZ + Distilled water

9.19±0.02b

21.41±0.21b

33.89±0.21b

73.71±0.27b

STZ + metformin

18.76±0.19c

29.75±0.29a

12.20±0.13c

18.99±0.24c

STZ + 50 mg/kg body of 19.98±0.23c extract

48.18±0.45c

14.68±0.28c

29.19±4.25d

STZ + 100 mg/kg body of extract

15.20±0.21d

35.53±0.17d

18.25±0.23d

56.47±0.25e

STZ + 200 mg/kg body of extract

23.36±0.36e

42.04±0.22e

25.71±0.24e

14.48±0.26a

Data were mean ± SEM; n = 7. Values with superscripts b, c, d and e different from their respective control a, down the column are significantly different (p<0.05) STZ = Streptozotocin

TABLE 7: Haematological parameters of streptozotocin-induced diabetic rats after oral administration of aqueous extract of Digitaria exilis grains Parameters

Haemoglobin (g/dL) Packed Cell Volume (%) Red Blood Cell (x106/µL) Mean Corpuscular Volume (fl) Mean Corpuscular Haemoglobin (pg) Mean Corpuscular Haemoglobin Concentration (g/dL) Red Cell Distribution Width (%) White Blood Cell (x103/µL) Platelets (x103/µL) Lymphocytes (x103/µL) Basophils (%) Eosinophils (%) Neutrophils (%) Monocytes (%)

Distilled water only

STZ + Distilled water

STZ + metformin

STZ + 50 mg/kg body of extract

STZ + 100 mg/kg body of extract

STZ + 200 mg/kg body of extract

24.19±0.27a

19.40±0.2b

23.29±0.46a

27.34±0.29c

27.84±0.29c

28.87±0.08c

0.93±0.02b

0.50±0.00b

0.84±0.01a

0.79±0.02a

0.85±0.01a

1.03±0.01a

0.16±0.02c

0.08±0.01b

0.16±0.00a

0.28±0.00c

0.27±0.01c

0.22±0.00d

60.04±0.43a

45.86±0.30b

59.81±0.24a

59.39±0.04a

63.37±0.28a

61.27±0.34a

12.79±0.10a

9.29±0.18b

13.39±0.04a

12.23±0.44a

14.91±0.33a

13.01±0.36a

23.50±0.13a

15.71±0.14b

22.73±0.31a

24.16±0.13a

23.81±0.09a

20.82±0.25a

14.80±0.19a

11.79±0.16b

13.86±0.18a

13.33±0.27a

15.31±0.17a

14.81±0.14a

25.71±0.24a

5.94±0.02b

10.80±0.10c

14.66±0.17d

13.82±1.52e

17.97±0.23f

638.13±1.32a

79.20±0.38b

153.19±0.95c

184.35±1.60d

242.18±0.99e

113.65±0.58f

22.35±0.09a

7.47±0.17b

15.39±0.17c

17.42±0.02d

14.98±0.10e

11.98±0.14f

0.91±0.02a 13.09±0.09a 33.82±0.24a 21.75±0.46a

0.56±0.02b 3.28±0.14b 7.35±0.17b 1.39±0.01b

0.08±0.04b 5.12±0.14c 15.17±0.13c 19.98±0.17c

0.32±0.01c 4.90±0.10c 20.29±0.19d 16.63±0.20d

0.20±0.04d 7.76±0.23d 17.91±0.17e 7.70±0.16e

0.36±0.05c 12.88±0.01a 24.99±0.04f 10.45±0.18f

Data are mean ± SEM; n = 7. Values with superscripts b, c, d, e and f different from their respective control a, across the row are significantly different (p<0.05) STZ = Streptozotocin