Urtica dioica (Gazaneh) distillate restores glucose metabolism in diabetic rats

Fahimeh Zamani-Garmsiri; Masoumeh Akmali; Ali Gohari; Fatemeh Zal; Atefeh Seghatoleslam

doi:10.4103/ijpvm.ijpvm_293_21

ORIGINAL ARTICLE

Year : 2023 | Volume : 14 | Issue : 1 | Page : 68

Urtica dioica (Gazaneh) distillate restores glucose metabolism in diabetic rats

Fahimeh Zamani-Garmsiri, Masoumeh Akmali, Ali Gohari, Fatemeh Zal, Atefeh Seghatoleslam
Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

Date of Submission	27-Jun-2021
Date of Acceptance	27-Oct-2022
Date of Web Publication	27-May-2023

Correspondence Address:
Atefeh Seghatoleslam
P. O. Box: 71348-45794, Shiraz
Iran

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/ijpvm.ijpvm_293_21

Abstract

Background: Diabetes has become an important health problem in the world. Natural agents, with antidiabetic property, are potential candidates for improving diabetes. Urtica Dioica Distillate (UDD) or Araghe Gazaneh is widely used for the treatment of diabetes as per traditional medicine. Despite the tremendous use of UDD as an antidiabetic compound in folk medicine, the antidiabetic effects of UDD has been neglected by medical scientists. In this study, we aimed to evaluate the effects of UDD on the glucose metabolism in diabetic rats. Methods: A total of 24 male rats were divided equally into four groups, two treatment and two control groups, each containing normal or Streptozotocin (STZ)–induced diabetic rats. During 4 weeks, control and treatment rats received water or UDD, respectively. Fasting blood sugar (FBS), HbA1c, serum creatinine, blood urea nitrogen, and specific activities of hepatic enzymes including glucokinase (GK), hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD), and muscle glucose transporter type 4 (GLUT4) and liver phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels were measured. Results: FBS and HbA1c increased in diabetic groups. Treatment with UDD significantly lowered FBS and prevented weight loss. Decreased FBS level was associated with higher activity levels of GK and HK in UDD-treated diabetic rats. G6PD-specific activity decreased in diabetic control rats compared to nondiabetic ones, but UDD treatment improved it to the normal levels. A significant decrease in the expression level of GLUT4 was observed in diabetic control rats compared to nondiabetic ones, but UDD increased it to the normal levels. Conclusions: These findings suggest that UDD might exert therapeutic effects against diabetes by improving glucose metabolism and can be used as an alternative or complementary medicine for the treatment of diabetic patients.

Keywords: Diabetes mellitus, diabetic rats, hepatic enzymes, Urtica Dioica distillate

How to cite this article:
Zamani-Garmsiri F, Akmali M, Gohari A, Zal F, Seghatoleslam A. Urtica dioica (Gazaneh) distillate restores glucose metabolism in diabetic rats. Int J Prev Med 2023;14:68

How to cite this URL:
Zamani-Garmsiri F, Akmali M, Gohari A, Zal F, Seghatoleslam A. Urtica dioica (Gazaneh) distillate restores glucose metabolism in diabetic rats. Int J Prev Med [serial online] 2023 [cited 2023 Oct 2];14:68. Available from: https://www.ijpvmjournal.net/text.asp?2023/14/1/68/377667

Introduction

Traditional Iranian medicine includes a diversity of knowledge and practice used for health maintenance and diagnosis, prevention, and management of disorders.^[1] World Health Organization has emphasized that the combination of traditional medicine, as a complementary and/or alternative medicine with conventional drugs, is a suitable, safe, and effective strategy to solve some health issues.^[2]

Given the severe long-term complications, diabetes has become a serious problem in modern countries.^[3] The increase in the prevalence of diabetes from 2010 to 2030 is predicted to be 69% in developing countries and 20% in developed countries.^[4] Treatment of diabetes mellitus using traditional plants as alternative medicines has been reported to have lower side effects.^[3],[5]

Urtica dioica (U. dioica) belongs to the Urticaceae family which has been introduced as a hypoglycemic medicinal plant by Avicenna in ancient medicine.^[6] The hypoglycemic properties of the plant have been already studied^[7] and its antidiabetic effects are also well known in folk medicine.^[8],[9] There are several methods for using plants in herbal therapy mostly including extraction, purification, fractionation, concentration, fermentation, and distillation.^[10] The oldest method is water distillation. It is the most appropriate method to use fresh and dry plant parts which are not damaged by direct heating.^[11] Plant distillates (also recognized by other names such as herbal/essential/floral water, hydrolate, and hydrosol) are currently being used in Turkey, Egypt, and Iran widely.^[10] The distillate obtained from different medicinal plants (locally named Aragh) is known as a common beverage in different parts of Iran, mostly used to relieve health problems.^[12],[13]

Despite the antidiabetic effects of U. dioica extract,^[6],[7],[8] a previous study has shown that there are some side effects for the extract of this plant such as nephrotoxicity and hepatotoxicity.^[14] On the other hand, folk medicine has targeted the use of herbal distillates in therapeutic interventions; their pharmaceutical and side effects have not yet been studied and tailored to the need by medical researchers. Therefore, the present study aimed to investigate the effect of UDD on weight gain, blood glucose, glycosylated hemoglobin (HbA1c), serum creatinine, and blood urea nitrogen (BUN) and the specific activity of the hepatic enzymes involved in glucose metabolism such as glucokinase (GK), hexokinase (HK), and glucose-6-phosphate dehydrogenase (G6PD) in streptozocin (STZ)–induced diabetic rats. The expression levels of the muscle glucose transporter type 4 (GLUT4) and liver phosphoenolpyruvate carboxykinase (PEPCK) genes were also evaluated in the experimental rats.

Materials and Methods

Preparation of UDD

U. dioica plant was collected from the jungles around Sari (Safar Abad region, at 2014 May), Mazandaran province, Iran, and identified by Sari Agricultural Sciences and Natural Resources Office. To obtain 1 liter of UDD, we washed 3.75 Kg of the fresh plant, put it into the upper partition of the steam boiler, and added 1.5 liters of water. The procedure was performed as described by Seghatoleslam et al.,^[13] keeping the temperature at 65°C-70°C. The steam passing through the condenser duct was cooled and UDD or Aragh was collected and conserved at 4°C until use.

Experimental animals

Eight-twelve–week-old male Sprague–Dawley rats (200-250 g) were provided. The animals were kept under standard conditions, 24 ± 2°C, 12 h light/dark cycle.^[15] The rats (n = 24) were randomly divided into four groups of six, including nondiabetic control (group I), UDD-treated nondiabetic (group II), diabetic control (group III), and UDD-treated diabetic (group IV). [Table 1] displays a summary of initial weights and initial fasting blood sugar (FBS). For the randomization process, four empty cages were provided and named as the first, second, third, and fourth groups. Then, the 12 nondiabetic rats were randomly but equally divided into the first and second cages, and the 12 diabetic rats were placed in the same way into the third and fourth cages, accordingly. Diabetes was induced in overnight-fasting rats by a single intraperitoneal (IP) injection of STZ (50 mg/kg) (Sigma Chemical Company, St. Louis, MO, USA) dissolved in a vehicle (0.1 M citrate buffer, pH 4.5),^[16] whereas the rats in nondiabetic groups only received the vehicle. Fasting blood glucose was determined 72 hours after STZ injection in tail vein blood samples by an Accu-Check Active glucometer (Roche Diagnostics GmbH, Hannheim, Germany).^[17] Diabetes was confirmed by fasting blood glucose levels >250 mg/dL. The rats in groups II and IV received 12.5 mL/kg/day (once a day) UDD via intragastric gavage and groups I and III were administered 12.5 mL/kg/day distilled water for 27 days.^[18] During 4 weeks of the study, all rats received the same diet (standard chow diet). The body weight and FBS of the animals were measured every 9 days (0^th, 9^th, 18^th, and 27^th). On the 28^th day, the nonfasting rats were anesthetized by CO₂ and blood samples were collected in EDTA-containing tubes by heart puncture to evaluate HbA1c. The liver and skeletal muscle (soleus muscle) tissues were also isolated and stored at -70°C until use.

Table 1: Baseline characteristics

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Measurement of HbA1c, creatinine, and BUN

The level of HbA1c in the whole blood was determined by a kit-based enzymatic and colorimetric assay (Diazyme Laboratories, USA).^[19] Serum creatinine and BUN were determined using Man Company Kit.

Hematoxylin and eosin (H & E) staining

After fixing in 10% (v/v) buffer formalin and dehydrating in a graded alcohol series, we embedded the liver specimens in paraffin, cut them at 5-μm sections, and stained them with H & E. The slides were, then, examined by a pathologist and the results were analyzed.

Assessment of enzyme activities

To determine the total HK and GK activities, we minced 1 gram of frozen liver tissue and homogenized it at 4°C in 9 mL of 50 mM HEPES buffer, pH 7.4 (Sigma Chemical Company, St. Louis, MO, USA), using Potter Elvehjem Homogenizer. The supernatant was prepared by centrifugation of the homogenate at 12,000 × g for 1 hour at 4°C for further enzyme assays. The activities of GK and total HK were determined based on the coupled enzyme assay of Davidson and Arion^[20] and Ferre et al.^[21] The formation of NADH was measured by the increase in the absorbance at 340 nm in 25°C to evaluate the enzyme activity.^[22]

To investigate the level of G6PD activity, we homogenized the liver samples in 4 volumes of cold 0.154 M KCl. The homogenate was centrifuged at 27,000 × g for 25 minutes at 4°C and the supernatant was collected for enzyme assay.^[22],[23] G6PD activity was measured as per the method of Bottomley et al.,^[24] using glucose 6-P and NADP+ as substrates.^[25] Enzyme-specific activities were expressed in mU per mg protein which was determined by the Bradford method.^[26]

Evaluation of gene expression

Liver and muscle tissues were homogenized in BIAZOL total RNA extraction reagent (Bioflux, Japan). Total RNA was extracted as per the manufacturer's instructions and cDNA was synthesized using reverse transcriptase (Thermo Fisher Scientific Company, USA). The levels of GLUT4 and PEPCK mRNA expression were assessed in the skeletal muscle and liver by RT-PCR, using ABI-applied Thermal Cycler (ABI 7500 real-time PCR system, USA) and Eva Green PCR Master Mix (Yekta Tajhiz Azma, Iran), respectively.

All amplifications were performed as duplicates for each sample. Primer sequences were as follows: β-actin: F-5′CCACACCCGCCACCAGTTCG-3', R-5'CTAGGGCGGCCCACGATGGA-3', PEPCK: F-5'CAGGAAGTGAGGAAGTTTGTGGAA-3', R-5'GGAGCCGTCGCAGATGTGAATA-3', GLUT4: F-5'ATGTTGCGGATGCTATGG-3', R-5'TTAGGAAGGTGAAGATGAAGAAG-3'. Expression levels were normalized to β-actin and relative levels were calculated by 2^-^ΔΔ^Ct.

Statistical analysis

The data were expressed as mean ± SEM as per the statistics consultant's suggestion. Statistical analysis was performed using SPSS 24 and the graphs were designed by GraphPad prism 8. As the values were not normally distributed, the nonparametric Kruskal-Wallis and Mann-Whitney tests were used for a pairwise comparison. P < .05 was considered statistically significant.

Results

Effect of UDD on FBS

To evaluate the possible effects of UDD on STZ-induced diabetes, we measured the FBS levels in the experimental rats during the treatment period. As shown in [Table 2], the diabetic rats showed a significant increase (P < .01) in the FBS levels in a time-dependent manner compared to nondiabetic rats. The treatment of diabetic rats with UDD significantly decreased the FBS levels (P < .01) up to the day 9 and kept it without significant changes until the day 27. However, the FBS level in nondiabetic rats was not changed by the administration of UDD.

Table 2: Effects of UDD on Blood Glucose levels (mg/dl) in the experimental rats

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Effect of UDD on the level of glycosylated hemoglobin

STZ-induced diabetes resulted in a significant increase in HbA1c levels compared with those in the nondiabetic rats (P < .01). As per our data, the treatment of diabetic animals with UDD did not exert any significant changes on HbA1c levels compared to the diabetic control rats. The level of HbA1c was not changed significantly in UDD-treated nondiabetic rats compared to the nondiabetic control ones [Table 3].

Table 3: Effects of UDD on HbA1c levels in four study groups

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Effect of UDD on the level of serum creatinine and BUN

To investigate the effect of UDD administration on renal function, we measured the levels of creatinine and BUN. STZ administration elevated creatinine and BUN levels to 0.65 ± 0.02 and 29.37 ± 1.66 mg/dL, respectively, compared to their levels in nondiabetic control (0.55 ± 0.02 and 18.83 ± 0.98 mg/dL, respectively) [Table 4]. Treatment with UDD decreased the levels of BUN in the diabetic group compared to the control diabetic group, but this decrease was not statistically significant. Notably, creatinine and BUN levels of nondiabetic rats after treatment with UDD did not significantly differ from the levels in the nondiabetic control group.

Table 4: Effects of UDD on creatinine and BUN levels in all experimental groups

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Histopathological findings

Microscopically, the livers of nondiabetic control rats displayed the normal histological structure of hepatic lobules, as shown in [Figure 1]. There was no abnormal histopathological finding in the diabetic rat liver under the experimental condition, which was similar to the previous study.^[27] Notably, nondiabetic and diabetic rats who received the UDD showed apparently normal liver architecture with no histopathological changes.

Figure 1: Effect of UDD on the liver tissue. H and E staining for liver sections (X200 magnification). The livers of nondiabetic control rats displayed the normal histological structure of hepatic lobules. Diabetic rats showed apparently normal liver architecture with no histopathological changes. There was no abnormal histopathological finding in the diabetic rat liver who received the UDD

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Effect of UDD on the weight gain of the experimental rats

In [Figure 2]a, the body weight changes are shown after injection of STZ or treatment with UDD. The average weight differences between the first day and the 27^th day in each experimental group are shown in [Figure 2]b. As expected, the body weight was significantly decreased in the diabetic rats compared to the nondiabetic control group after 27 days (P = 0.002). The results showed that treatment with UDD significantly reversed these changes (P = 0.004). However, UDD-treated nondiabetic rats revealed no significant increase in the body weight compared to the nondiabetic control ones.

Figure 2: Effect of UDD on the body weight in experimental rats. The changes in the body weight during the study (a) and the average weight differences between the first day and the 27^th day (b) are presented. Data are represented as mean ± SEM (n = 6). ^**P < .01

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Effects of UDD treatment on the specific activities of hexokinase, glucokinase, and G6PD in the liver

To investigate the effectiveness of using UDD on glucose metabolism, we measured the specific activities of hepatic HK, GK, and G6PD in the experimental groups. When compared with nondiabetic group, the hepatic specific activities of these enzymes were significantly lowered (P = 0.002 for HK and GK and P = 0.003 for G6PD) in the diabetic group [Figure 3]a, [Figure 3]b, [Figure 3]c. After 4 weeks of UDD administration, the hepatic HK-specific activity of nondiabetic (P =.01) and diabetic groups (P =.045) was significantly increased compared to the control groups. As illustrated in [Figure 3]b, UDD consumption had no significant effect on the hepatic GK activity in nondiabetic rats, but it significantly increased the GK activity (P =.045) in the diabetic rats. UDD administration also significantly elevated the G6PD activity in diabetic rats (P =.005) compared to diabetic control ones. Meanwhile, an increase in the G6PD activity in the UDD-treated normoglycemic rats was observed which was not statistically significant under the experimental conditions.

Figure 3: Effect of UDD on the specific activities of hexokinase (a), glucokinase (b), and G6PD is shown in the liver. Data are represented as mean ± SEM (n = 6). ***P <.001, **P <.01, *P <.05

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Effect of UDD on GLUT4 gene expression in the skeletal muscle

To further insight into the molecular mechanisms of the antidiabetic effects of UDD, we assayed the expression levels of muscle GLUT4. As shown in [Figure 4]a, the expression of GLUT4 was lower in the muscle tissue of the diabetic control group compared to the nondiabetic control group (P =0.04). The administration of UDD increased the GLUT4 gene expression of diabetic rats. However, as shown in [Figure 4]a, this increase was not significant (P = 0.58). No significant change was also observed in the expression of GLUT4 gene in nondiabetic rats treated with UDD compared to nondiabetic control ones (P = .88).

Figure 4: Effect of UDD on GLUT4 gene expression in muscle (a) and PEPCK gene expression (b) in the liver is presented. Values are expressed as mean ± SEM (n = 6). *P <.05

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Effect of UDD on PEPCK gene expression in the liver

To analyze the efficacy of UDD on gluconeogenic-related genes, we measured the mRNA levels of PEPCK. As per our data which are shown in [Figure 4]b, an increased expression of PEPCK was detected in the muscle of diabetic rats compared to nondiabetic control ones; however, the increase was not statistically significant. Furthermore, administration of UDD in diabetic rats did not reveal any significant decrease in the PEPCK gene expression.

Discussion

In the folk medicine of various countries, U. dioica is considered to possess different therapeutic effects. Previous studies have described the hypoglycemic properties of this plant through the improvement of glucose intolerance,^[28] and enhancement of insulin secretion and pancreatic function.^{[7],[23],[24]}

The possible mechanisms of U. dioica antidiabetic activities could be classified into pancreatic and extra-pancreatic mechanisms. Pancreatic mechanisms could function through stimulation of insulin release from β-cells^[29] and/or the regenerations of beta cells.^[30] Extra-pancreatic mechanisms might work through inhibiting glucose intestinal absorption,^[31] alpha-amylase activity,^[32] and also forming unique glucose penetrable pores to facilitate the glucose uptake.^[33]

However, although antidiabetic effects of U. dioica extract are documented by many studies,^[6],[7],[8] the hypoglycemic properties and the potential toxicity of UDD have not been examined yet. The widespread use of herbal distillates in traditional medicine makes it a necessity for further study to improve their clinical application.

There is evidence showing that STZ-induced diabetes leads to pancreatic islets destruction, reduction of insulin secretion, and hyperglycemia.^[18] Conversely, U.dioica, specially its extract, is known for having antidiabetic properties. In this study, we showed that UDD consumption decreased the FBS levels by about 50% without any effects on the levels of HbA1c. Our result is in agreement with that of Qujeq et al.'s^[7] and Golalipour et al.'s^[34] studies, reporting that the administration of U. dioica extract in diabetic rats increased the secretion of insulin by β-cells. Furthermore, our previous reports showed that the UDD could induce β-cell regeneration and increase the serum insulin level in STZ-induced diabetic rats.^[18] Therefore, the results of our experiments not only confirmed the previous results but also revealed a mechanism for the hypoglycemic effect of UDD. Measurements of HbA1c are used both as an index of mean hyperglycemia and a risk factor for the development of diabetic complications.^[35] However, the ameliorative effects need a longer time to be revealed at the level of HbA1c.

Studies have shown that diabetes is associated with a reduction in GK.^[21] GK regulates the rate of glucose metabolism in the liver.^[36] To further determine the mechanism of the antidiabetic effects of UDD, the specific activity of GK and HK was assessed in the liver tissues. Our data demonstrate that the UDD inhibits a diabetes-induced decrease in the specific activity of hepatic enzymes, suggesting that the UDD administration exerts the antidiabetic effect by activation of major hepatic enzymes involved in the glucose metabolism.

Regarding to distillate was used in recent study, which ingredients might be responsible for these findings. G6PD, the rate-limiting enzyme in the pentose phosphate pathway, has an important role in the antioxidant defense system by producing NADPH which regulates the cellular redox state against oxidative stress by maintaining reduced glutathione and H₂O₂ detoxifying enzyme of glutathione reductase.^{[25],[37],[38],[39]} Data showed that the levels of G6PD were increased about 1.9 folds after treatment with UDD in STZ-induced diabetic rats. In the present study, creatinine and BUN levels were not changed in nondiabetic rats after treatment with UDD. Meanwhile, UDD administration did not produce alterations in the hepatocytes, sinusoids, and the portal triads. Therefore, under experimental conditions, no nephro/hepato toxicity was observed in the rats who received UDD. The activities of HK and G6PD were also increased in UDD-treated normal rats, indicating its antioxidant effects. These findings are in agreement with those of Golalipour's study reporting that U. dioica extract has antioxidant activities and free radical scavenging properties.^[40] However, this outcome is not in accordance with the results of Güneş et al.'s^[14] study which revealed that U. dioica extract not only had no effect on the diabetes but also had toxic effects on the kidneys and liver. The discrepancy between the latter study and ours might be due to the differences in the dose and duration of the treatment and the selection of the different methods of herbal drug preparations (i.e., distillate vs. extract).

Insulin resistance in the muscle is formed by a glucose transport defect. The main insulin-sensitive glucose transporter in the muscle is the insulin-sensitive GLUT4, which removes glucose from the circulation followed by insulin stimulation.^[41] In a study by Kadan et al.,^[42] it was revealed that exposing L6-GLUT4 myc cells to U. dioica extract almost doubled the GLUT4 translocation and improved about 1.6 folds in the insulin-stimulated state which was approximately in line with the result of our study in the diabetic group.

PEPCK is a key enzyme of gluconeogenesis, a primary metabolic pathway leading to the production and release of glucose in the liver. At the gene transcription level, insulin downregulates the mRNAs encoding PEPCK.^[43] Previous investigations have indicated that oxidative stress impairs the hepatic PEPCK gene expression by an insulin-independent mechanism. In a parallel study, we showed that UDD had antioxidant activities (data not published yet). Our data showed that UDD administration had no significant effects on PEPCK gene expression in the liver but increased the GLUT4 gene expression in the diabetic rat muscles compared to diabetic rats not receiving UDD; however, this increase was not statistically significant. Although there was no evidence of UDD impact on the PEPCK gene expression in the literature, perhaps prolonged treatment with UDD could be more effective.

This study had potential limitations which include the use of a limited number of male Sprague–Dawley rats as an animal model of diabetes induced by STZ. In addition, there is a possibility that the observed effects of UDD in animals differ from how humans would present. In the future investigations, we will aim to identify the most effective and safe dose for human consumption and evaluate the effect of UDD on diabetes at the clinical research phase using larger population samples. However, in the category of plant distillate consumption by human, the standardization, dosage, efficacy, and the stability of the distillates require further investigation.

[TAG:2]Conclusion [/TAG:2]

This article is the first report of hypoglycemic effects of UDD or Aragh Gazaneh administration on diabetic rats. The investigation on the actual chemical components of UDD is under way; meanwhile, it is known that herbal distillates contain essential oil compounds and organic acids and other water soluble components.^[18] Further experiments are also necessary to evaluate the various aspects of the antidiabetic effects of UDD and the active constituents involved in the observed effects.

The last but not the least, it can be concluded that the administration of UDD in STZ-induced diabetic rats not only lowered the plasma glucose but also improved the liver carbohydrate metabolism through increasing the specific activities of the related key enzymes. Therefore, due to the beneficial effects and lack of toxicity under experimental conditions, it could be recommended as a complementary or alternative medicine to reduce blood sugar in diabetic patients. Moreover, simultaneous administration of UDD and antidiabetic drug might be regarded by the physicians to avoid synergic effects.

Ethics statement

The study protocol was approved by the National Institutes of Health guide for the care and use of Laboratory Animals (NIH Publications No. 8023, revised 1978) modified by the Institutional Animal Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran (IR.SUMS.REC).

Abbreviations

FBS, Fasting blood sugar; GK, glucokinase; G6PD, Glucose-6-phosphate dehydrogenase; GLUT4, Glucose transporter type 4; HK, hexokinase; PEPCK, Phosphoenolpyruvate carboxykinase; STZ, Streptozocin; UDD, Urtica Dioica Distillate.

Financial support and sponsorship

The present article was extracted from the MSc thesis written by Fahimeh Zamani-Garmsiri and supported by grant number 93-7232 from the Office of the Vice Chancellor for Research, Shiraz University of Medical Sciences, Shiraz, Iran.

Conflicts of interest

There are no conflicts of interest.

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