Evaluation of the Antidiabetic Activity of Hydromethanolic Roots Extracts of Rumex abyssinicus Jacq: (Polygonaceae) in Swiss Albino Mice

Introduction Diabetes Mellitus isa chronic metabolic disorder that required long-lasting treatment. In Ethiopian traditional medicine practices, abundant plants have been used for the treatment of diabetes mellitus for a long period. The root of Rumex abyssinicus is employed for the treatment of diabetes mellitus by Ethiopians. This study aimed to investigate the antidiabetic activity of the crude extract of Rumex abyssinicus root in Swiss albino mice. Methods Cold maceration technique and hydro methanolic (80% methanol) solvent with occasionally shaking were employed during the crude extraction processes. To evaluate the antidiabetic activity of the crude extract, normoglycemic, glucose-loaded, and streptozotocin-induced diabetic models were used. In each model, the overnight fasted mice were randomly divided into five groups for normoglycemic and glucose-loaded models as a negative control, positive control, and three tested groups, whereas, in streptozotocin-induced diabetic models, the mice were grouped into six groups like one diabetic and one normal negative control groups, three diabetic tested groups, and one diabetic positive group. Each group comprised six mice. For all models, the tested groups received the crude extract at 100, 200, and 400 mg/kg doses, both diabetic and nondiabetic negative control groups received 10 ml/kg distilled water, and positive groups received 5 mg/kg glibenclamide. Results The crude extract of the plant did not show any sign of toxicity up to 2000 mg/kg dose. In normoglycemic and oral glucose tolerance tests, the crude extract significantly (p < 0.01) reduced the blood glucose level at 200 and 400 mg/kg doses. In the streptozotocin-induced diabetes models, a significant effect was observed at all tested doses. Conclusion The finding of this study revealed that the crude extract of the plant owned antidiabetic activity and supports the traditional use of Rumex abyssinicus root for the treatment of diabetes mellitus.


Introduction
Diabetes mellitus (DM) is a metabolic disorder of carbohydrates, proteins, and fats. It characterizes by persistent hyperglycemia and develops as a consequence of a defect in insulin secretion or impairment of its action [1][2][3]. e signs and symptoms of DM include polyuria, polyphagia, polydipsia, calorie loss, generalized weakness, poor wound healing, numbness, blurry vision, and weight loss. e secondary complication of DM includes retinopathy, vascular complications, peripheral neuropathy, renal failure, vascular complications, and limb amputations [4]. Now a day, DM is a serious health issue for both developed and developing countries. Based on the estimation of the International Diabetes Federation (IDF) 2021, the global prevalence of DM was 10.5% (536.6 million people) in 20-79 years old. By 2045, this figure is expected to be 12.2% (783.2 million) by a 46% increment. In 2021, the prevalence of DM was higher in urban areas (12.1%) as compared to the rural area (8.3%), in high-income (11.1%) countries than in lowincome countries (5.5%). During this year, the global expenditure for DM was 966 billion United States Dollars (USD) and is expected to be raised by 1,054 billion USD by 2045 [5]. In Ethiopia, 1.3 million people (20-79 years old) lived with DM and the prevalence was 2.9%. is figure is projected to be 1.8 million by 2030 [6]. e impact of DM is still a challenge for humankind. It impairs the quality of life and decreases the daily income of individuals and the development of countries [7]. Currently, numerous antidiabetic drugs are available on the market, but there was no satisfactory antidiabetic drug for all diabetic patients. ey are not easily accessible at an affordable price and cause dangerous side effects like weight gain and hypoglycemia with sulfonylureas, lactic acidosis, metallic taste, and weight loss in metformin [8]. Besides this, no antidiabetic drugs produce a curative effect [9]. Consequently, looking and searching for safe and effective antidiabetic drugs become a day-to-day issue in the pharmaceutical science and medical world. Across the world, the majority of people still depend on the use of plants for the treatment of DM and its complication [10]. e reason for this dependency include easily accessible of plants with affordable price, ease for self-administration and use, people's belief that plants have fewer side effects, and they have high cultural acceptance [6]. Rumex abyssinicus (R. abyssinicus) is categorized under a family of Polygonaceae and distributed in tropical Africa, particularly in Central and Eastern Africa [11]. In Ethiopia's folklore medicine, R. abyssinicus has a great role in the treatment of different human ailments. Traditionally, R. abyssinicus is used for the treatment of DM, and hepatitis [12]. e root and the stem were used for the treatment of diabetes, hypertension, wounds, cancer [13,14], tuberculosis, teeth infections, abdominal problems, stomach pain [15,16], headache and toothache [17], asthma, liver problems, common cold, inflammation, and painful conditions [12]. e leaf part is also used for the treatment of ascariasis and headache [17], and malaria [18].
Several in vitro and in vivo studies have been conducted on different parts of R. abyssinicus. e root extract exhibited good antioxidant [19], antibacterial and chemopreventive potential on preneoplastic lesions [20], antibacterial activity on Streptococcus pyogenes and Staphylococcus aureus, and antiviral against Coxsackie virus B3 and influenza A virus, and anti-inflammatory activities [21]. e methanolic extract of the rhizomes showed diuretic and analgesic activity [22], wound healing and anti-inflammatory activity [11], and hepatoprotective and radical scavenging activities while the leaves extract demonstrated anti-malarial activity [23]. e methanolic extract of bulbs of R.abyssinicus also revealed antihelminthic activity [24]. Furthermore, some of the Rumex species also showed a glucose-lowering activity. e ethanolic root extract of R.nepalensis demonstrated in vitro α-amylase inhibition, in vivo hypoglycemic activity in normal and glucose-loaded rats [25]. e phytochemical screening test on the root extract revealed the presence of saponins, flavonoids, tannins, steroids, anthraquinones, alkaloids, quinones, phenol, and glycosides, but the absence of terpenoids [26]. e folklore antidiabetic uses, in vitro, and in vivo studies provide significant clues to conducting this experiment. Moreover, until this time, the antidiabetic activity of the root extract of R.abyssinicus was not conducted. erefore, this study aimed to evaluate the safety and antidiabetic efficacy of hydro methanolic root extract of R.abyssinicus in streptozotocin-induced diabetic mice.

Plant Material.
e fresh root of R.abyssinicus was collected from the South Gondar Zone of the Amhara region, Northwest Ethiopia, and wrapped with plastic sheets during transportation. e plant was identified and authenticated by a Botanist at the University of Gondar and the voucher number ED001/2011 was deposited in the Herbarium of Biology Department, Faculty of Natural and Computational Science, University of Gondar.

Preparation of Plant Extract.
e root of R.abyssinicus was thoroughly washed with tap water to remove dirt and derbies. en, the root was dried under a shadow in the pharmacy laboratory room. After drying, the root of the plant was subjected to size reduction using a mechanical miller. A mass of 1.6 kg coarse powdered was macerated in a sufficient amount of 80% methanol for 72 h with occasional stirring and agitation. Following 72 h maceration, the extract was separated from the mark using filter paper (Whatman No.1). e marc was re-macerated two times with fresh 80% methanol. e filtrates obtained from the three successive filtrations were combined and concentrated using Rotary Evaporator (Yamato, Japan) set at 40°C. Finally, the semidried residues were frozen in the refrigerator overnight and then, lyophilized to completely dry using a lyophilizer (Lab freeze, China) [27]. Finally, 188.8 g (11.8%) of a dark red powder of crude extract was obtained. e obtained crude extract was stored in an airtight bottle in the refrigerator until it was subjected to the experimental procedure.

Experimental Animals.
To conduct this experiment, 6-8 weeks of age and 20-30 g body mass of Swiss albino male mice were used for all experimental models (oral glucose loaded, normoglycemic, and STZ-induced diabetic models). Relative to male mice, female mice are more resistant to developing DM secondary to STZ administration [28,29]. ereby, the female mice were excluded from this study. e mice were gained from the animal house of the Department of Pharmacology, School of Pharmacy, and the University of Gondar. e mice dwelled in a clean metabolic cage that was bedded with a chip of wood under a standard condition (12 h light and 12 h dark cycle) with free access to a commercial pellet diet, which was purchased from the Ethiopian Public Health Institution (EPHI), Addis Ababa, Ethiopia, and water ad libitum. Before initiation of the experiment, the mice were acclimatized to the laboratory condition for seven days. e mice were handled according to the international standard guidelines set for the Care and Use of Laboratory Animals [30]. e ethical approval permission was requested and gained from the research and Ethics Committee of the Department of Pharmacology, the University of Gondar with ethical license number sopy/2311.

Grouping and Dosing of Animals.
For normoglycemic and oral glucose-loaded models, the overnight fasted mice were arbitrarily assembled into five categories with six mice in each category. Group I(negative control) was treated with 10 ml/kg of distilled water; Groups II-IV (the tested mice) and received 100, 200, and 400 mg/kg of the crude extract of plant respectively, and Group V (positive control) was treated with the standard drug, glibenclamide (GLC) 5 mg/ kg based on the method illustrated by Belayneh et al [31]. In STZ-induced diabetic model, the overnight fasted mice were arbitrarily allocated into six groups with six mice in each group (five diabetic groups and one nondiabetic group). Group I and II were given 10 ml/kg of distilled water and served as normal negative control and diabetic negative control, respectively. Groups III-V (tested group) were administered the plant extract at doses of 100, 200, and 400 mg/kg respectively, whereas group VI (positive) was treated with the standard drug GLC 5 mg/kg [32]. e doses of the plant extract were determined based on the result of the acute oral toxicity test as explained by Desta et al. [33]. Accordingly, the middle dose was calculated as one-tenth of the limit dose of the plant extracted (200 mg/kg), and the lower and the higher doses were calculated as half and twice the middle dose (100 and 400 mg/kg) respectively. Each volume of the treatment doses was calculated based on Organization for Economic Cooperation and Development (OECD) guidelines that specified as 1 ml/100 g body weight of the rodents [34]. Since, the plant material is taken in the acclaimed area by oral route [14], the mice also received the doses of each treatment (crude extract of the plant, distilled water, and glibenclamide) through oral route using oral gavage. Both the crude extract of the plant and the standard drug GLC were dissolved in DW just before administration.

Induction of Experimental Diabetes. Streptozotocin
(STZ) is the most commonly used diabetogenic chemical to evaluate the antidiabetic activity of plant extract in experimental diabetes animal models. Intravenous or intraperitoneal (IP) high dose of STZ 100-200 mg/kg selectively destroyed the islet β cell of the pancreas by saving extrapancreatic cells of the mice and induced a rapid and permanent hyperglycinemia in mice [35,36]. Before launching the experiment, STZ was dissolving in a 0.1 M citrate buffer (the pH was adjusted at 4.5) immediately before being administered to the experimental mice. e mice were fasted overnight (14 h) and injected freshly prepared STZ at a dose of 150 mg/kg via the IP route. Following 30 min of administration of STZ, the mice were permitted free access to both food and water. After 6 h administration of STZ, the mice were administered 1 ml/kg of 5% glucose solution for the next 24 h to prevent hypoglycemic shock and death secondary to massive and rapid β-cell necrosis that results in the release of large amounts of insulin. After three days of post-STZ administration, the mice were screened for diabetes. Mice with a fasted BGL greater than 200 mg/dl were incorporated in the study as diabetic mice [28,[37][38][39].

Collection and Measuring of Blood Glucose Level.
At the time of the experiment, the blood sample was taken aseptically from the tip of the fasted mice's tail after cutting with a surgical blade. e BGL was measured three times using a blood glucose meter and test strip. e measurement was performed three times and an average of the triplicates value was taken. e percentage of blood glucose reduction was calculated based on the following formula [40].
where G 0 is blood glucose level at 0 h and G p is posttreatment blood glucose level.

Hypoglycemic Effects of the Crude Extract on Normoglycemic Mice.
For the normoglycemic model, the mice fasted overnight (14 h) from food with free access to water. en, the mice were grouped indiscriminately into five Evidence-Based Complementary and Alternative Medicine groups and treated as elucidated in the dosing and grouping section. Following grouping, the BGL was measured just before giving the treatment of each dose (at 0 h as baseline). After administration of the respective treatment dose, the blood sample was collected and BGL was determined at 1, 2, 3, and 4 h post-treatment. e procedure was done according to the method established by Belayneh, et al [31].

Antihyperglycemic Effects of the Crude Extract on Oral Glucose Tolerance Tests in Normoglycemic Mice.
e method for the determination of the effect of the crude extract on the oral glucose tolerance test was run based on the method utilized by Kifle et al [41]. e mice were grouped randomly and treated as described in the grouping and dosing section. e mice fasted from food overnight for 14 h and then the BGL was measured, as a baseline, just immediately before the treatment of the doses (at 0 h). en, the mice received their respective treatment doses. After 0.5 h treatment; each mouse was loaded 2 g/kg glucose solution (40% w/v) orally. e blood sample was taken from the tip of each mouth tail aseptically and the BGL was measured at 0.5, 1, and 2 h post glucose administration. is procedure was done based on the methods established by Alene, Cherie Melaku et al [42].

Antidiabetic Activity of Single Dose of Crude Extract on
Streptozotocin-Induced Diabetic Mice. As depicted in the grouping and dosing section, overnight fasted (14 h) mice were grouped randomly into six groups with six mice per group. e mice received their respective doses as stated in the grouping and dosing section and the blood sample was collected.
e BGL was measured immediately before treatment (0 h as a baseline), and 1, 2, 4, and 6 h after the respective treatment doses. e procedure followed the method described by Alene et al [43].

Antidiabetic Activity of Repeated Daily Dose of Crude
Extract on Streptozotocin-Induced Diabetic Mice. Following induction of diabetes by STZ, the mice were grouped into six groups (six mice in each group) as illustrated in the grouping and dosing part and the method for running the procedure was based on Alene et al [43]. e respective doses of the treatment (GLC, distilled water, and the tested doses of the crude extract) were provided to each experimental mouse daily for a total of 14 days. e overnight fasted (14 h) BGL was measured just before administration of the respective dose for each mouse (day 0) as a baseline, and then at the 7 th and 14 th days of post-treatment [44].

Preliminary Phytochemical Screening.
e crude extract of the R. abyssinicus root was subjected to a qualitative phytochemical screening test for the presence or absence of phenols, flavonoids, tannins, terpenoids, alkaloids, saponins, steroidal, and anthraquinones according to the following respective methods and procedures.
(i) Test for Phenols Ferric Chloride Test e crude extract (50 mg) was dissolved in 5 ml of distilled water and the 3 drops of 5% ferric chloride solution were added. e formation of a dark green/bluish-black color indicated the presence of phenols [45][46][47]. Lead Acetate Test: e crude extract (50 mg) was dissolved in 5 ml of distilled water and the 3 ml of 10% lead acetate solution was added to this mixture. e formation of a white precipitate indicated the presence of phenols [46].

(ii) Test for Flavonoids
Lead Acetate Test About 1 ml of the crude extract was mixed with a few drops of 10% lead acetate solution. e formation of yellow color precipitate showed the presence of flavonoids [46][47][48]. Alkaline Reagent Test About 2 ml of the crude extract was mixed with 2 ml of 2% of sodium hydroxide solution in a test tube. e formation of an intense yellow color was turned colorless when 2 drops of dilute hydrochloric acid were added. is is an indication of the presence of flavonoids [46,47,49].
(iii) Test f'or Tannins Ferric Chloride Test To 1 mL of crude extract, an equal amount of ferric chloride solution was mixed. e formation of greenish-black color indicated the presence of tannins [50]. Gelatin Test e crude extract was dissolved in 5 ml of distilled water.
en, 1% gelatin solution containing 10% sodium chloride was added to the crude extract. e formation of white precipitate indicated the presence of tannins [46,47,51].
(iv) Test for Alkaloids Mayer's Test About 50 gm of the crude extract was mixed with a few ml of diluted hydrochloric acid and then filtered. e filtrate was treated with 1-2 drops of Mayer's reagent (along the sides of the test tube). e formation of white creamy precipitate indicated the presence of alkaloids [46,47]. Wagner's Test About 50 gm of the crude extract was mixed with a few ml of diluted hydrochloric acid and then filtered. About 2 drops of Wagner's reagent were added to the filtrate (along the sides of the test tube). e appearance of brown/reddish precipitate indicated the presence of alkaloids [45,47,52].  Evidence-Based Complementary and Alternative Medicine e crude extract (2 ml) was dissolved in an equal amount of chloroform. About 2 ml of concentrated sulfuric acid and 2 ml of acetic acid were added to the Mixture. e formation of green-colored precipitate indicated the presence of steroids [50,53].
(VI) Test for Terpenoids Salkowski Test About 50 mg of the crude extract was dissolved in 5 ml of methanol and the extract was mixed with 2 ml of chloroform. e mixture was warmed and cooled. en, 3 ml of concentrated sulfuric acid was added slowly along the sides of the test tubes. e formation of a radish browncolored precipitation at the interface indicates the presence of terpenoids [54].

(VII) Test for Saponins
Foam Test: e crude extract (50 mg) of the plant was diluted with DW up to 20 ml suspension. e suspension of the crude extract was shaken for 15 minutes. e presence of a 1 to 2 cm foam layer indicated the presence of saponins [45].

(VIII) Test for Anthraquinones
Borntrager's Test e crude extract (6 g) was soaked in 10 ml of benzene for 10 minutes in a conical flask and the filtrate was filtrated. en, 10 ml of 10% ammonia solution was added to the filtrate and shaken vigorously for 30 seconds. e observation of pink, violet, or red-colored in the ammonical solution or at the lower phase was an indication of the presence of anthraquinones [49]. Ammonium hydroxide Test e crude extract (10 mg) was dissolved in isopropyl alcohol. One drop of concentrated ammonium hydroxide solution was added to the dissolved crude extract. e observation of a red color after 2 minutes indicated the presence of anthraquinone [49,55].

Acute Toxicity
Study. An acute oral toxicity test was carried out according to OECD 2008 guideline 425 [56]. One female Swiss albino mouse fasted for 4 h on the first day of the test. en, 2 g/kg of the extract was given orally and observed strictly for physical and behavioral changes for the next 24 h and special attention was given during the first 4 h. Based on the results from the first mouse, another four female mice were recruited and fasted for 4 h and then were given 2 g/kg as a single dose and observed for 14 days.

Statistical Analysis
Statistical Package for the Social Sciences (SPSS) Version 23 Software was used for statistical analysis. e results of the study were expressed as mean ± standard error of the mean. Means of all parameters among and within groups were compared using One-way Analysis of Variance (ANOVA) followed by Tuckey's post hoc multiple comparisons. e p -value <0.05 was considered statistically significant.

Preliminary Phytochemical Screening.
e qualitative phytochemical screening tests of the crude extract of the R.abyssinicus root illustrate the presence of phenols, flavonoids, tannins, steroids, saponins, alkaloids, anthraquinones, and terpenoids. Table 1 presents the results of qualitative phytochemical screening tests of the crude extract of the root of R.abyssinicus.

Acute Oral Toxicity Study.
After loading 2 g/kg of a single dose of the crude extract of R. abyssinics root, the mice were carefully observed within the first 24 h up to 14 days in their cage. During 14 day follow-up period, neither death nor any signs of toxicity such as a change in breathing rate, paw licking and shivering, restlessness, motor activity, and diarrhea were observed in any of the mice. is indicated that the median lethal dose (LD 50 ) of the root extract, a dose requires to kill 50% of the experimental animal, is greater than 2 g/kg.

Hypoglycemic Effects of the Crude Extract on Normoglycemic Mice.
e hypoglycemic effect of the crude extract of R.abyssinics root on normally overnight fasted mice presents in Table 2. ere was no significant difference in fasting BGL across the group at baseline BGL. As compared to the negative control group, there was a significant effect on BGL in mice which received 200 (p < 0.01) and 400 mg/kg (p < 0.01) of the crude extract at 4 th h with 30.94% and 49.65% BGL reduction from the baseline, respectively, while the standard drug, glibenclamide, showed a significant reduction on the normal fasted BGL starting from at 1 st (p < 0.01), 2 nd (p < 0.001), 3 rd (p < 0.001), and 4 th h (p < 0.001). Mice received 100 mg/kg crude extract failed to show a significant reduction in BGL at all times.

Antihyperglycemic Effects of the Crude Extract on Oral Glucose Tolerance Tests in Normoglycemic Mice.
e effect of crude extract of R. abyssinicus on oral glucose tolerance test in normoglycemic mice is presented in Table 3. e peak BGL was achieved within 0.5 h post-loading of 2 g/kg glucose to mice receiving distilled water. Mice received 400 mg/kg showed a significant reduction in BGL after 1 and 2 h loading of oral glucose, while 200 mg/kg at 2 h relative to mice received distilled water. Mice that received the standard drug (GLC 5 mg/kg) showed a significant reduction of the BGL at 0.5, 1, and 2 h after challenging the oral glucose as compared to the negative group.

Antihyperglycemic Activity of the Crude Extract on Single
Dose in Streptozocin-Induced Diabetic Mice. All the tested crude extract doses of the root and glibenclamide 5 mg/kg showed a significant BGL (p < 0.001) reduction at any time Evidence-Based Complementary and Alternative Medicine as compared to the baseline BGL. Moreover, administration of crude extracts at all tested doses and glibenclamide 5 mg/ kg significantly reduced the BGL (p < 0.001) as compared to the diabetic control (Table 4).
Results are expressed in mean ± SEM; n � 6 for each treatment. a Compared to baseline fasting BGL, b compared to diabetic control (DC), c compared to RA 100 mg/kg, d compared to RA 200 mg/kg, 1 p < 0.05 2 p < 0.01, and 3 p < 0.001,   Results are expressed in mean ± SEM; n � 6, a compared to the negative control, b compared to 100 mg/kg, c compared to 200 mg/kg, d compared to 400 mg/kg. 1 p < 0.05, 2 p < 0.01, and 3 p < 0.001, NC � negative control (received 10 ml/kg distilled water), RA � Rumex abyssinicus, GLC � Glibenclamide, SEM � standard error of mean. Evidence-Based Complementary and Alternative Medicine NC � negative control (received 10 ml/kg distilled water), DC � diabetic control (received 10 ml/kg distilled water), RA � Rumex abyssinicus, GLC � Glibenclamide, SEM �standard error of mean.

Antihyperglycemic Activity of Repeated Daily Doses of the Crude Extract in Streptozotocin-Induced Diabetic
Mice. e outcome of the daily repeated doses of the crude extract of R. abyssinicus root on the BGL in diabetic and normal mice is summarized in Table 5.
ere was no significant discrepancy across the group on the BGL at the baseline. All three treatment doses of the root extract of R. abyssinicus revealed a significant (p < 0.001) reduction in the fasting BGLs on the 7 th and 14 th days as compared to the diabetic and normal control groups.
e GLC treated group also showed a significant (p < 0.01) reduction in BGL on the 7 th and 14 th days when compared to the diabetic and normal control groups, but there was no a significant difference between GLC treated groups and groups treated with all the three root extracts of R. abyssinicus. ere was no considerable discrepancy among the three doses of the crude extract treated groups.

Discussion
Evaluation of acute oral toxicity test of the crude extract of R. abyssynicus verified that 2000 mg/kg dose did not show any sign of toxicity and death during 14 days of observation and confirmed that the folklore use of the root part is safe. e animal model is an important tool to assess the pathophysiology mechanism of DM and the antidiabetic action of plant remedies. e induction of DM using STZ is a common one for the evaluation of the antidiabetic activity of medicinal plants in animal models [28,57]. e chemical structure of STZ contains a glucose and methyl nitrosourea moiety. e glucose moiety guides the transport of STZ into the pancreatic β-cells, while the nitrosurea moiety causes the production of pancreatic β-cells toxicity. e β-cells of the islet take up STZ via glucose transporter 2 (GLUT2). e STZ selectively accumulated in pancreatic β-cells of islet and cells lacking this glucose transporter is resistant to STZ. Once inside β-cells, STZ causes cytotoxicity on β-cells via méthylation of deoxyribonucleic acid (DNA) strand and results in the fragmentation of the DNA. STZ also induces the activation of poly adenosine diphosphate ribosylation and nitric oxide release; thereby exerting oxidative stress through free radical generation. As a consequence, β-cells are destroyed and unable to secrete insulin which results in the development of DM hyperglycemia [28,58,59]. Previously studied research indicated that the pancreas contains stable (quiescent) β-cells which can restore themselves after being damaged by STZ and alloxan. is enhances insulin secretion and improves hyperglycemia [60,61]. Plant extract from Spondias pinnata (Linn. f.) Kurz, Coccinia grandis (L.) Voigt and Gmelina arborea revealed the regeneration of β-cell in rats that developed diabetes secondary to alloxan [62].
In the evaluation of the hypoglycemic effect of R. abyssinicus, the crude extract of the root showed a significant dropping in the BGL in time and dose-dependent fashion in normoglycemic mice. is indicated that the root part of R. abyssinicus owned a hypoglycemic activity. is hypoglycemic effect was in line with the hypoglycemic effect of the root extract of Rumex nepalensis, in which the two plants are classified under the same family [25]. e oral glucose tolerance test is an important tool to assess the release of insulin and insulin resistance in clinical and research areas. e test is used to measure the ability of the body to use and clear glucose from the bloodstream and is used to diagnose pre-diabetes and diabetes cases [63]. In impaired glucose tolerance, the body required a long period to clear the challenged glucose. e impairment of glucose tolerance decreases the uptake of glucose by muscle, and fat, thereby the maintenance of blood glucose homeostasis is impaired [64]. Hence, the oral glucose tolerance test was done to evaluate the effect of the crude extract of R. abyssinicus on glucose homeostasis. Even though there was an increase in BGL after glucose loading from the baseline glucose level, 200 and 400 mg/kg crude extract and glibenclamide reduced significantly from peak level to baseline within 2 h compared with negative control. From previous studies, plant extracts lowered BGL by increasing insulin secretion, enhancing insulin sensitivity and glucose uptake at peripheral tissue, escalating glucose excretion, and inhibiting glycogenolysis, and gluconeogenesis [65,66]. Nevertheless, the exact mechanism of action is unknown; the glucose-lowering action of the crude extract of R. abyssinicus root might be mediated via insulin secretion, enhancing insulin sensitivity and glucose uptake at peripheral tissue, escalating glucose excretion, inhibition of glycogenolysis and gluconeogenesis.
In STZ-induced diabetic mice, the crude extract of R. abyssinicus root showed a significant reduction in BGL as compared to diabetic control. e lower dose (RA 100 mg/ kg) of the extract failed to show a reduction of BGL in normal and glucose-loaded animals, but there was a considerable reduction in BGL in STZ-induced diabetic mice. A comparable effect was also observed from the leaves of Caylusea abyssinica [37]. e finding of this study suggested that the lower dose of the crude extract was unable to overcome the physiological counter-regulatory mechanism.
is inability might be due to the presence of low amounts and types of phytochemical constitutes of the crude extract of the root. e lower concentration of the phytochemical constitutes induces hypoglycemia in normoglycemic and glucose-loaded normal mice or the hypoglycemic nature of the lower dose would be apparent when there is an alteration in normal blood glucose regulatory systems by diabetes [37]. e reduction of BGL was in a dose and time-dependent manner. e preliminary phytochemical screening test of the crude extract of R. abyssinicus root showed the presence of phenols, flavonoids, tannins, saponins, anthraquinones, Evidence-Based Complementary and Alternative Medicine alkaloids, and terpenoids. Phytochemical constitutes are the responsible bioactive compounds for the antidiabetic activity of medicinal plants as reported previously studies [67,68]. Saponin, isolated from Tribulus terrestris L extract, demonstrated a significant BGL reduction in alloxan-induced diabetic mice [69]. Alkaloids from the leaf extract of Catharanthus roseus (L.) G. Don, showed hypoglycemic activity in vitro study [70]. Rutin, is a flavonoid, that also demonstrated antidiabetic activity [71]. e antidiabetic mechanism action of these phytochemical constitutes mediates through reduction of glucose production in the liver, increase the expression of glucose transporter isoform 4 (GLUT4), enhancement of β-cell regeneration that increases the release of insulin, exerting an antioxidant effect, blocking of glucose absorption in the lumen of intestine, decreasing the activity of glucokinase enzyme [72][73][74]. e antihyperglycemic effect of R. abyssinicus root extract might be mediated through these phytochemical constituents either in an individually or synergistically action. Flavonoids, isolated from Nelumbo nucifera leaves, inhibited the activity of α-glucosidase, lipase, and α-amylase in vitro study [75]. Quercetin, which is a flavonoid, also showed a significant reduction in blood glucose levels in STZ-induced diabetic rats through regeneration of the pancreatic islets cell [76]. Alkaloids isolated from leaves of Catharanthus roseus (L.) G. Don demonstrated antioxidant and antidiabetic activities in vitro [70]. Tannin, which was presented in the crude extract of R.abyssinicus root, demonstrated antidiabetic activity by inhibiting alpha-amylase and alpha-glucosidase. Tannin also exerted its antidiabetic activity secondary to its antioxidant action on pancreatic β-cells [77]. Another study revealed that saponins from plant extract demonstrated antidiabetic activity in the alloxaninduced diabetic rats. An in-vitro study from previously studied indicated that alkaloids from Catharanthus roseus (L.) G. Don demonstrated antidiabetic activity through increasing glucose uptake [70]. e previous investigations revealed that the related species Rumex vesicarius showed significant antidiabetic activity in a dose-dependent fashion [78]. Rumex rothschildianus, another species under the family Polygonaceae, also showed antidiabetic activity in vitro study [79]. Since these two planets are in the same family and also support the observed antidiabetic activity of R. abyssinicus root extract. Although the exact antidiabetic mechanism action of R. abyssinicus is unknown, the possible antidiabetic mechanisms action of the R.abyssinicus root may be enhancements of insulin action at the target tissue, inhibition of carbohydrate absorption in small intestine, augmentation of insulin secretion in pancreatic β-cell as result of restoration of β-cell, and inhibition of gluconeogenesis.

Conclusion
e Hydromethanolic crude extract of the root of R. abyssinicus possesses significant glucose-lowering activity both in normal and STZ-induced diabetic mice in a dose and time-dependent manner. is study confirmed that the root part of R. abyssinicus is owned with antidiabetic activity and supported the folklore use of the root for the treatment of DM. e higher dose of the root part of the plant showed a significant blood glucose-reducing activity. Based on the acute oral toxicity study, the study plant showed a wide margin of safety.

Abbreviations
BGL: Blood glucose level DM: Diabetes mellitus GLC: Glibenclamide LD50: Median lethal dose OECD: Organization for economic coperation and development OGTT: Oral glucose tolerance test STZ: Streptozotocin.

Data Availability
All the data that were used during the process of the experiment are available from the corresponding author and proved upon reasonable request.
Ethical Approval e care and handling of animals were under internationally accepted Ethical Guidelines for Laboratory Animals, National Institutes of Health (NIH) Publication No. 86-23, Revised 1985, and the ARRIVE guidelines. e experimental protocol for laboratory animal use and care was requested and approved by the Ethics Committee of the Department of Pharmacology, School of Pharmacy, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia with reference:"sopy/2311."

Conflicts of Interest
e authors declare that they have no competing interests.