Anti-diabetic potential of Urtica Dioica: current knowledge and future direction

Abstract

Aim

This review summarizes studies on the anti-diabetic effect of Urtica Dioica (UD) in Type-2-diabetes.

Materials and methods

We studied worldwide traditional medicines, old texts, and published literature for anti-diabetic effect of UD. Electronic databases comprising PubMed, Web of Science, Science Direct, Scopus and Google Scholar were searched to collect articles published between 1990 and 2021 years.

Results

Our literature investigation suggests UD as a glucose lowering, blood lipid regulating, anti-inflammatory and anti-oxidation plant.

Conclusions

UD’s anti-diabetic properties make it potential traditional therapeutics for lowering the clinical manifestations of T2DM through affecting hyperglycemia and therefore suggest it as a proper medication with no or limited side effects.

Introduction

Diabetes mellitus (DM) is a chronic disease caused by lack of insulin secretion, insulin resistance, or both. According to the International Diabetes Federation (IDF), the prevalence of DM is rising with around 415 million adults in 2015 between ages of 20 to 79 years being affected. The numbers are expected to rise by 200 million in 2040 [1]. Based on the statements of the World Health Organization (WHO), about 80–90% of all diabetes cases are Type-2-diabetesmellitus (T2DM) [2]. In T2DM, the response to insulin is defective and insulin resistance occurs due to decreased insulin sensitivity in adipose tissues, skeletal, liver and muscles. T2DM is seen in adults and younger people who refer to energy-dense diets, increasing levels of obesity, and physical inactivity [3]. Despite the development in the controlling of diabetes by hypoglycemic medications, exploration for novel treatments is an active area of research mainly since the existing anti-diabetic medications have limitations such as absence of effectiveness and detrimental side effects [4]. The main chemical drugs for T2DM are thiazolidinedione (TZD) (ex. pioglitazone), biguanide (ex. metformin), meglitinide (ex. repaglinide and nateglinide), sulfonylureas (ex. glyburide and glipizide) and Sodium-glucose Cotransporter-2 (SGLT-2) inhibitors ( ex. canagliflozin, dapagliflozin and empagliflozin). These chemical medicines show side effects, for instance TZD leads to obesity and fat accumulation [5]. Lactic acidosis in patients with cardiac and liver diseases is one of the side effects of metformin treatment [6]. It has been shown that sulfonylureas cause hypoglycemia in older diabetic patients [7]. Thus, researchers focus on finding other sources of medications with lower side effects including herbal remedies.

Plants are considered as probable sources of antioxidants [8] and hypoglycemic agents to control and treat diabetes [9]. In addition, seeking for innovative anti-diabetic treatments from herbs is very attractive ascribed to their low side effects, low cost, easy accessibility and high efficiency even when their biologically active composites are unidentified [10]. Herbal drug preparation typically includes fractionation, concentration, purification, fermentation extraction and distillation. The most regular used forms of the herbal drugs are plant extracts [7]. Plants like Aloe vera, Swertia Mentha, Zingiber are shown to be effective for controlling hyperglycemia [11].

Urtica Dioica (UD) is a plant which has glucose lowering, blood lipid regulating, anti-inflammatory, and anti-oxidation properties with great promises for diabetes treatment [12–14]. UD leaves have shown to be effective for insulin secretion stimulation [15] and/or acting as insulin imitator [16]. Other studies also described that UD extract has cell proliferation ability [17] and could stop alpha-glucosidase enzymes activities[18]. However, some studies indicate that the use of UD hydro-alcoholic extract has no hypoglycemic impact and has no effect on β-cell renewal in diabetic rats (16). Therefore; in this review we discuss these studies on the anti-hyperglycemic effect of UD and its therapeutic effects on T2DM as a traditional medicine.

Urtica Dioica

Urtica Dioica (UD) is a plant with thin hairs on the stems and leaves. The name Urtica is derived from the Latin verb “Urere'' that stands for, “to burn” due to the stinging hairs. Also, the word “Dioica” means “two houses'' since the plant typically reproduces with either female or male flowers [14]. This species consists of six subspecies. Trichrome or stinging hairs on the leaves and stems, which perform like hypodermic needles, perfusing histamine, choline and serotonin that cause irritation when they meet humans and other animals. UD medicinal products are generally made from the stems and leaves, and rarely from the roots [19]. This herb constitutes iron, calcium, potassium, manganese, and vitamins A and C. UD in its harvest season, comprises 25% protein, dry weight, which is a high amount for this green vegetable [20]. Drenching UDs in water or the heat from cooking eliminates the stinging factors from the plant, which causes it to be used and eaten without injury. UD has been used in traditional medicine for a long time. Studies show that it has numerous helpful effects on diabetes and rheumatoid arthritis [21]. UD showed to prevent liver tissue damage caused by carbon tetrachloride induced hepatotoxicity in rats [22]. In traditional medicine, UD has been used for treating iron deficiency ascribed to its high amount of iron and used to lessen the extreme menstrual bleeding, nosebleeds, and hematuria. The root of UD has been consumed for remission of asthma [23]. Furthermore, UD demonstrates an antioxidant role in diabetes. Unpaired electrons are factors that can damage several tissues in the body by their oxidative power[21]. Hence, aggregate oxidative stress can build up risk factors of cardiovascular occurrence and other difficulties in patients with diabetes [24, 25]. Reactive oxygen species (ROS) includes hydrogen peroxide, hydroxyl, and superoxide radicals in diabetes are generally associated with cell impairment. Besides, it is well verified that oxidative stress is a key metabolic irregularity in both cardiovascular disorders and diabetic vascular problems [26]. In addition, UD has been shown to have a defensive performance against hepatic ischemia–reperfusion[27], hypercholesterolemia [28], and hyperglycemia [29]. Also, it is established that supplementation of UD leaves has been shown to have a protective effect against liver injury which takes root from deficiency in tricarboxylic acid cycle Furthermore, it’s obvious that ceramides stimulate cytosolic protein phosphatase 2A (PP2A) and akt gets inactivated by PP2A. It has been demonstrated that UD could increase ceramide and so on akt gets inactivated and this causes improvement in insulin sensitivity in skeletal muscle [30, 31]. Also, despite the buildup of ceramides, akt phosphorylation increases and this could reduce insulin resistance arising from FFA in myotubes [32].

Clinical trials investigating the effect of UD on diabetes

Components and mechanisms

Thirteen studies comprising the Clinical trials, which were investigating the effect of UD on diabetes, was reviewed, Table 1. It is believed that UD causes an increase in glucose uptake by adipose tissues and skeletal muscles and this mechanism might lower the blood glucose concentrations [33]. It has also been shown that UD has the anti-inflammatory effect that can protect the pancreatic β-cells, insulin release and glucose uptake by the cells and eventually lowering the blood glucose [17]. Studies indicate that it may work as a factor which could improve secretion, or as a Peroxisome Proliferator-Activated Receptor (PPAR) agonist. PPAR agonists are therapeutics that reduce blood glucose levels by enriching insulin excretion by Langerhans Islets [34]. Special flavonoids and phenolic compounds which are the building materials of UD look like to make antioxidant effects and take part in stabilizing lipid peroxidation [35]. Quercetin 2-(3, 4-dihydroxyphenyl)-3, 5, 7-trihydroxy-4H-chromen-4-one is the main flavonoids in UD. These factors created the antioxidative feature of UD in patients with T2DM [36].

Table 1.

Anti-diabetic effects of UD Clinical Trial Studies

Sample size and groups	UD dose and type of extract	Duration of study	outcome	References
Intervention group (24 = 11 F + 13 M) control group (11 M + 10 F = 21)	100 mg/kg capsule 3 portions a day -Hydroalcoholic extract	8 weeks	Interleukin-6/ CRP↓	[49]
Intervention group (n = 27) control group (n = 23)	100 mg/kg -Hydroalcoholic extract	8 weeks	TAC↑ SOD↑ No significant change in MDA and GPX	[21]
Intervention group (n = 46) control group (n = 46)	one 500 mg capsule every 8 h	3 months	FBS↓ 2 h' postprandial glucose↓ HbA1c ↓	[50]
1-aerobic training (Ae) (n = 10 M) 2- UD (n = 10 M) 3- (Ae + UD) (n = 10 M) 4-control group (n = 10 M)	10 g of UD powder was dissolved in yogurt 15 min before breakfast	8 weeks	FBS↓ FBS was different in each of the 3 groups	[50]
1-aerobic training (Ae) (n = 10 F) 2-UD (n = 10 F) 3- (Ae + UD) (n = 10 F) 4-control group (n = 10 F)	-	-	FBS↓, HDL-c ↓	[51]
Intervention (n = 25) control group (n = 24)	100 mg/kg/day	8 weeks	Insulin↑, FBS↓	[52]
Intervention group (n = 30) control group (n = 30)	200 mg powder in a capsule per day	90 days	TG ↓ no effect on cholesterol and blood pressure	[53]
Intervention group (n = 25 F) control group (n = 25 F)	5 ml UD 3 portions a day -hydro alcoholic extract	8 weeks	FPG and SGPT↓ HDL, NO and SOD ↑	[54]
Intervention group (n = 24) control group (n = 21)	100 mg per day -hydro alcoholic extract	8 weeks	TG, plasma atherogenic factors (LDL-C / HDL-C and Total Cholesterol / HDL-C) and SBP ↓, (HDL-C) ↑ No changes in DBP, TC and LDL-C	[55]
interval training [IT] (n = 10 M) [IT + UD] (n = 10 M) control group (n = 10 M)	10 g UD powder 3 portions a day	8 weeks	FBS↓, Blood pressure↓	[56]
Intervention group (n = 22) control group (n = 22)	20 mg kg-1 day-1 -alcoholic extract	8 weeks	blood glucose levels↓, AMPK and Insulin ↑	[57]
Intervention group (n = 25) control group (n = 25)	100 mg kg-1 3 portions a day	8 weeks	FBS and HbA1c↓	[58]
Intervention group (n = 42) control group (n = 44)	500 mg capsule Every 8 h	12 weeks	HbA1c↓	[50]

CRP, C-reactive protein; TAC, Total antioxidant activity; SOD,Superoxide dismutase; MDA, Malondialdehyde; GPX,Glutathione Peroxidase; FBS, Fasting Blood Sugar; HbA1c, Hemoglobin A1c.HDL-c, High-density lipoprotein-c; TG, triglyceride; NO, Nitric Oxide; SGPT, Serum glutamic pyruvic transaminase, SBP, Systolic blood pressureAMPK, AMP-activated protein kinase; DB, diastolic blood pressure; TC, Total Cholesterol.

UD effects on Liver and heart disease

Quercetin derived from UD could decrease total cholesterol, plasma glucose concentration and insulin resistance and also it could raise plasma adiponectin, HDL-cholesterol and hepatic GSH-Px activity. Furthermore, hydro-alcoholic extract of UD is a promising source of bioactive complexes and may decline the diabetes-related risk factors of cardiovascular disease in patients with diabetes mellitus [37]. The risk of atherosclerosis and related cardiovascular disorders is increased when cholesterol level in the blood plasma is high. [38]. Additionally, it has been shown that UD might not cause changes in the Serum glutamic pyruvic transaminase (SGPT) blood levels and subsequently liver dysfunction might not occur in UD consumers [39]. Also, it has been found that UD causes SGPT reduction in hyperlipidemia induced rats [40]. Studies showed that high sugar consumption is associated with general adiposity but also with increased liver fat accumulation which is expected to raise SGOT and SGPT factors [2]. Likewise, it has been proposed that nettle protects the liver from oxidative damage in T2DM, which is imputable to its antioxidant effects [17].

UD consumption is shown to considerably reduce LDL, leptin and LDL/HDL ratio in serum and it might reduce the risk of heart disease caused by diabetes. It was also shown that UD might have an improving effect on metabolic syndrome and blood lipid profile [41]. Leptin is a hormone that participates in an oxidative stress process which causes vascular smooth muscle hypertrophy and vascular inflammation, Also leptin takes part in the pathogenesis of atherosclerosis, T2DM, hypertension, and coronary heart disease [42]. Leptin production by adipocytes is induced by insulin [43]. It is confirmed that UD could reduce insulin, and this could lessen the leptin concentration in the blood and thus leads to improvement in diabetes and heart diseases [44]. Likewise, UD could regulate blood pressure. Endothelium-derived relaxing factor or NO [45], which is secreted from vascular endothelium, is an important part of the insulin-signaling pathway which implements micro vascular vasodilation [46]. It is expected that UD could inhibit liver dysfunction in patients with T2DM. It is commonly known that after nitric oxide (NO) is released, a soluble enzyme named guanylate-cyclase is activated and might cause secretion and enhancing of the c-MP factor and followed by that vascular vasodilation is accrued [47]. It has been shown that after 8 weeks consuming UD by diabetic patients, an increase in NO concentration was detected in comparison with the placebo group. Furthermore, another study determined that UD could cause hypotension via vasorelaxation power produced by the secretion of endothelial NO and activation of potassium channels [48]. It has been demonstrated that hydro-alcoholic extract of UD declined IL-6 and high-sensitivity C-reactive protein (hs-CRP) in T2DM patients after 8 weeks of consumption. Thus, hydro-alcoholic extract of UD might have a protective effect on CVD in patients with type-2-diabetes by reducing specific inflammatory elements [47] (Table 1).

Anti-Diabetic effect of UD in vivo

Anti-hyperlipidemic effect of the UD in T2DM rats showed that the aqueous extract of UD, deactivates the lipoprotein lipase enzyme and consequently lowers the cholesterol and triglyceride levels in type 2 diabetic rats [59]. It is confirmed that after 8 weeks treating diabetic rats with UD root extract, the LDL concentration in blood plasma is reduced by deactivating HMG-COA reductase enzyme [54]. On the other hand, Das et al. indicated that aqueous extract of UD reduced FBS and cholesterol concentration and in contrast to the other studies had no effect on triglyceride and LDL in T2DM rat models [60]. UD has antioxidative compounds which participate in removing active free radicals and can contribute to antidiabetic properties[61]. UD hydro-alcoholic extract like polyphenols including tannin, cafe oil, fumaric, kaempferol, myricetin, quercetin, rutin, p-coumaric, naringin, isorhamnetin, ferulic, syringic, vanillic, anthocyanin, chlorogenic and ellagic acid. Probably UD has antioxidant activity and participates in stabilizing lipid peroxidation [12, 29, 35, 36]. UD causes enhancement in pancreatic beta cells proliferation; it is estimated that this result correlates to the antioxidant properties of UD extract which can hinder or scavenge the effects of free radicals and this might be headed to the rise of insulin secretion[17]. Table 2 presents an overview of various animal studies done for the effect of UD on diabetes (Table 2).

Table 2.

Anti-diabetic effects of UD animal studies

Duration (Days)	Sex	Sample Size	Species	Induced Rout of Admn	Diabetes induced	UD Dosage	outcome	Country	References
84	male	36	C57BL/6 J mice	P.O	STZ	-	no effect on the constituents of the insulin signaling pathway (AKT, IRS proteins, PI3K, GLUT4, and insulin receptor) FIAF in adipose and skeletal muscle, Ppar-α and FOXO in muscle and liver, and Cpt1 in liver ↑	USA	[62]
72	male	40	Witar rat	P.O	STZ	50 (mg/kg/day) hydroalcoholic extract	GPx, SOD, CAT and GSH activities in the hippocampus↑	Iran	[63]
98	male	48	Sprague–Dawley rat	P.O	STZ	200 (mg/kg/day) Aqueous-alcoholic extract	Blood glucose ↓	Afghanistan	[64]
28	male	40	Wistar rat	I.P	STZ	100 (mg/kg/day)	no hypoglycemic impact no effect on β-cell renewal	Iran	[17]
30	male	30	Wistar-Albino rat	I.P	STZ	0.40–0.60 ml	TG↓	Iran	[33]
14	male	40	Wistar rat	I.P	Fructose	200 mg hydroalcoholic extract	glucose, LDL/HDL ratio, LDL, leptin, FIRI and insulin ↓ AST, VLDL and TG ↑ did not change ALP	Iran	[39]
8	Both sex		Long-Evans rats	P.O	STZ	1.25 g/kg water extract	TG and glucose ↓, no significant effect on CRP	Iran	[60]
14		45		I.P	Alloxan	50 mg/kg/day	glucose↓ insulin, ACC and NDPK activity ↑	Iran	[65]
5	male	30	Wistar rat	I.P	STZ	100 mg /kg	Preserved the morphometric characteristics in the periportal and perivenous zones of the liver lobule	Iran	[61]
-	male	-	Wistar rat	I.P	-	24 mg/kg/h Water extract	diuresis and natriuresis↑, so arterial blood pressure were reduced by 28%	Morocco	[66]
49	male	50	Wistar-Albino Rat	P.O	-	200 mg kg-1	oxidative stress↓ amends the lymphocyte DNA damage	Turkey	[67]
40	male	-	Wistar rat	P.O	-	300 mg kg-1 Water extract	TC, TG, LDL/HDL-ratio, LDL and TNH-CHOL ↓ No impact on HDL ALT, AST, LDH, and γ-GT↓ bilirubin levels↓ total protein and albumin levels↑	Nigeria	[40]
30	-	-	Sprague–Dawley Rat	P.O	-	150 mg/kg/day	apoB, total cholesterol, LDL / HDL cholesterol ratio, LDL cholesterol ↓	Lebanon	[68]
-	-	-	Wistar rat Swiss mice	P.O	Alloxan	250 mg-1 kg-1 Water extract	Insulin ↓	Morocco	[69]

FIAF, fasting induced adipocyte factor; Ppar-α, peroxisome proliferator-activated receptor-α; FOXO, forkhead box protein; Cpt1, Carnitine palmitoyltransferase I; FIRI, fasting insulin resistance index; AST, aspartate aminotransferase;VLDL,Very low-density lipoprotein; ALP serum Alkaline phosphatase; ACC, Acetyl coenzyme A carboxylase; NDPK, Nucleoside diphosphate kinase; TNH-CHOL, Total Non-HDL cholesterol; HDL, high density lipoprotein cholesterol; γ-GT, Gamma-glutamyl transferase; apoB, Apolipoprotein B; P.O., per os; P.I., per injection.

Anti-diabetic effects of UD in vitro

Study on human myoblast showed that the treatment of these cells with UD extract causes glucose uptake and this might be an anti-diabetic action. Another study, Table 3, evaluated the effect of UD on myotubes [32]. Obanda et.al. confirmed that UD affects the homeostasis of plasma glucose and influences the Akt pathway and reduces PP2A activity which causes attenuation in the capability (Table 3).

Table 3.

Anti-diabetic effects of UD in vitro

UD Concentration	Type of cell line	Outcome	Country	References
50, 100 and 200 mg/mL	Rat RIN5F pancreatic beta cells	No effect on insulin and insulin sensitivity in muscle cells	Iran	[70]
3 ml of condensed vapor	glucose‐responsive MIN6 clonal beta‐cells	did not stimulate insulin	Canada	[16]
3 ml of condensed vapor	L6‐GLUT4 myc myoblast cells	significantly enhanced glucose uptake (∼1.5‐fold)	Canada	[16]
80 μg	C2C12 myotubes	Enhanced plasma glucose homeostasis improved skeletal muscle insulin sensitivity without influencing body weight UD lessened the capacity of FFA to cause insulin resistance in myotubes UD caused PP2A hyperactivity without altering PP2A expression and ceramide accumulation UD cause decrease in Ak dephosphorylation and follow by declined PP2A activity	USA	[32]

GLUT,glucose transporter; FFA, free fatty acids; PP2A, Protein phosphatase 2.

UD Advantage over current chemical medications

UD has shown to function as an alpha-glucosidase inhibitory agent and could have glucose and triglyceride lowering effects in patients with diabetes mellitus [50]. Intestinal α-glucosidase is the enzyme that takes part in the digesting and catalyzing starch via hydrolyzing the α-1, 4-glucoside linkages, hence, the hindrance in the function of this enzyme notably reduces uptake and digestion of carbohydrates and subsequently lessening the postprandial blood glucose amount in patients with non-insulin-dependent diabetes [50]. Medications like voglibose, miglitol, and acarbose are the chemical drugs which are used as α-glucosidase and α-amylase inhibitors. The major problem of these drugs is that their hypoglycemic consequence is lower than other oral antidiabetic drugs like sulfonylureas. Diarrhea and bloating are the other disadvantages of these drugs. Extreme inhibition of the pancreatic α-amylase might cause fermentation of carbohydrates via intestinal bacteria and this might lead to diarrhea in patients who consume the drug [71]. It has been claimed that traditional drugs like UD had low inhibitory impact on α-amylase and high inhibitory influence on α-glucosidase thus it could be used as an applicable drug to diminish postprandial hyperglycemia with least adverse effects [72]. Based on research, the UD extracts displayed a similar inhibitory form like acarbose (an identified α-glycosidase inhibitor). According to surveys on UDs effective dose, it's confirmed that 0.4 mg/ml of UD leaf extract could inhibit 60% of α-amylase activity [41].

Safety and adverse effects

Using the proper dose of the UD had positive effects on the kidneys and increased urination and thus lowered blood pressure, but using a higher dose might be toxic and caused reduction of hematocrit in rats [66]. Also, UD has been recognized to have diuretic properties. Since the UDs diuretic properties, it may increase the influence of blood pressure medicines like calcium channel blockers [73]. Also, this herb could amplify the effects of other diuretics, comprising loop diuretics and thiazides so increase the risk of electrolyte disturbances and dehydration [74]. Additional irregular side effects comprise sweating, fluid retention, mild stomach upset, rash, and diarrhea. Touching UD might cause allergic rash, hence handling carefully is essential. UD should not have contact with open wounds [41, 75].

Discussion

The blood glucose lowering effect of UD is mentioned in Avicenna’s ancient scripts. Studies showed that this effect is mainly due to increased insulin levels in pancreatic islets. However, UD seems to decrease the glucose level by three main mechanisms. First by improving glucose uptake of the muscle cells; second, by stimulating the insulin production by pancreatic β cells, and finally by hindering the alpha-amylase action. Alpha-amylase is one of the most important inhibitors of carbohydrate hydrolysis, which helps to keep blood sugar levels in check. Other studies showed the antioxidant and anti-inflammatory effects of UD extract, higher glucose uptake by the cells and lower intestinal glucose absorption. In vitro studies on GLUT4 expressing myoblast cells showed that UD increases glucose uptake by these cells [16]. Bnouham et al.also demonstrated that the intestinal absorption of glucose increases following UD treatment [69]. Obanda et al. found that insulin sensitivity is improved in myotubes [32] but Mobassseri and Shabani et al. achieved the opposite results which might be due to difference in cell types between studies [16, 70]. It is confirmed that UD treatment decreased risk factors of cardiovascular disease in T2DM patients by reducing effect on FPG, TG, and SGPT [22, 67] and incremental effect on SOD, NO and HDL blood levels [54]. In another study, Namazi et al.demonstrated that UD at the dose of 100 mg/d for 8 weeks changed systolic blood pressure in T2DM, with no significant difference in diastolic blood pressure [55]. Moreover, UD could affect the lipid profile. All lipid factors except HDL-C decreased after UD treatment [55], while this finding was dismissed by another study where they could not see the same effect [40]. These contradictory results could be due to differences in the dose and methods of trials and type of UD extract. Also, animal study designed by Daher et al. indicated the reducing effects of UD on LDL/HDL-C, LDL-C and TC concentration in rat’s blood [40, 68]. Although in one study AST enzyme was shown to increase after UD consumption [39], another study showed AST was decreased and showed that UD does not have an adverse effect on liver function [40].There was no study to investigate possible complications of UD on liver tissue and function by histological evaluation. Also, the direct effect of UD on pancreatic beta cells is not fully studied yet, so it suggests that further studies are needed to evaluate the effect of UD on regeneration of beta cells. In the future research, it is needed to establish the effects of UD on regulation of AMPK and PPAR signaling pathways as the main mechanism for glycemic regulation. Moreover, anti-inflammatory and antioxidant effects should be studied with higher doses of UD and larger sample size in humans. Since the UD exact side effects are not completely known, the evaluation of probable side effects is an active area of research in the future.

Conclusion

According to these data, UD could reduce blood pressure, insulin resistance and oxidative stress in T2DM. Also, it has been shown that UD could regulate the blood glucose and lipid profile. Hence this inexpensive herb might be a proper supplement to reduce the complications of the people suffering from T2DM glycemic dysregulation.

Authors' contributions

Conceptualization: Mostafa Araj-Khodaei.

Data curation: Mostafa Araj-Khodaei, Reza Yarani.

Formal analysis: Flemming Pociot, Tannaz Novin Bahador.

Funding acquisition: Mostafa Araj-Khodaei.

Investigation: Tannaz Novin Bahador, Zahra Yousefi.

Methodology: Anita Chehri, Seyed Kazem Shakouri.

Project administration: Mostafa Araj-Khodaei, Reza Yarani.

Resources: Majid Mobasseri, Alireza Ostadrahimi.

Supervision: Mostafa Araj-Khodaei, Reza Yarani.

Validation: Anita Chehri.

Writing – original draft: Anita Chehri, Tannaz Novin Bahador.

Writing – review & editing: Mostafa Araj-Khodaei, Reza Yarani.

Funding

This work was supported by Tabriz University of Medical Sciences [grant number: 64989, Code of Ethics: IR.TBZMED.1399.093].

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On behalf of all authors, the corresponding author states that there is no conflict of interest.