- Research article
- Open Access
Protective effects of methanolic extract of Juglans regia L. leaf on streptozotocin-induced diabetic peripheral neuropathy in rats
BMC Complementary and Alternative Medicine volume 17, Article number: 476 (2017)
Oxidative stress has a pivotal role in the pathogenesis and development of diabetic peripheral neuropathy (DPN), the most common and debilitating complications of diabetes mellitus. There is accumulating evidence that Juglans regia L. (GRL) leaf extract, a rich source of phenolic components, has hypoglycemic and antioxidative properties. This study aimed to determine the protective effects of Juglans regia L. leaf extract against streptozotocin-induced diabetic neuropathy in rat.
The DPN rat model was generated by intraperitoneal injection of a single 55 mg/kg dose of streptozotocin (STZ). A subset of the STZ-induced diabetic rats intragastically administered with GRL leaf extract (200 mg/kg/day) before or after the onset of neuropathy, whereas other diabetic rats received only isotonic saline as the same volume of GRL leaf extract. To evaluate the effects of GRL leaf extract on the diabetic neuropathy various parameters, including histopathology and immunohistochemistry of apoptotic and inflammatory factors were assessed along with nociceptive and biochemical assessments.
Degeneration of the sciatic nerves which was detected in the STZ-diabetic rats attenuated after GRL leaf extract administration. Greater caspase-3, COX-2, and iNOS expression could be detected in the STZ-diabetic rats, which were significantly attenuated after GRL leaf extract administration. Also, attenuation of lipid peroxidation and nociceptive response along with improved antioxidant status in the sciatic nerve of diabetic rats were detected after GRL leaf extract administration. In other word, GRL leaf extract ameliorated the behavioral and structural indices of diabetic neuropathy even after the onset of neuropathy, in addition to blood sugar reduction.
Our results suggest that GRL leaf extract exert preventive and curative effects against STZ-induced diabetic neuropathy in rats which might be due to its antioxidant, anti-inflammatory, and antiapoptotic properties.
Protection against neuropathy
Peripheral neuropathy is one of the most common long term complications of diabetes mellitus which affects all peripheral nerves, including pain fibers . Almost 12% of all diabetic patients are affected with symptomatic painful diabetic neuropathy . In this regard, several mechanisms account for the development of diabetic neuropathy such as metabolic disorders, microvascular damages, neurotrophic support deficit, alternation in neuro-immune interactions, neural and glial cell apoptosis, and inflammation [2, 3]. It is well known that most of these factors result from increased oxidative stress which has a pivotal role not only in the development of diabetic neuropathy but also in the resistance of it even after good glycemic control, might be due to accumulated free radicals that are not easily removed . Increased oxidative stress after hyperglycemia is caused mainly due to autooxidative glycosylation, advanced glycation end-product (AGE) formation, and increased polyol pathway activity . Therefore, it has been postulated that the use of antioxidant supplements may offer some protection against this complication through scavenging of the free radicals. In recent decades, a rapidly growing number of natural phenolic compounds with free radical scavenging properties have been described. The seeds, green husks, and leaves of the Persian or common walnut (Juglans regia L.), the best-known member of the Juglans genus, are a rich source of these molecules which have been traditionally used in Iranian folk medicine for treatment of several diseases such as infections, inflammations, and diabetes and its complications [5, 6]. Interestingly, these effects were confirmed according to the recent scientific studies [7, 8]. Flavonoids, phenolic acids, and naphtoquinones are considered as major phenolic compounds in Juglans regia L. leaves [9, 10]. Pharmacologically, there is accumulating evidence that attributed the beneficial effects of Juglans regia L. leaf extract to a variety of biological activities, including anti-oxidative [8, 10], anti-inflammatory , anti-carcinogenic , anti-microbial , and anti-fungal properties . Recently, there are a few experimental studies on the hypoglycemic effect of Juglans regia L. leaf extract in diabetes mellitus (15–18). These studies documented that administration of Juglans regia L. leaf extract significantly reduced fast blood sugar (FBS) and hemoglobin A1c (HbA1c) compared to control groups [15,16,17,18]. Moreover, results of two clinical trial studies have shown that fast blood glucose (FBG) and HbA1c significantly decreased after consumption of 100 mg Juglans regia L. leaf extract for 3 months  and 200 mg Juglans regia L. leaf extract for 2 months  compared to placebo groups. An in vitro study also reported that walnut leaf extract inhibits protein tyrosine phosphatase 1B (PTP1B) and enhances glucose-uptake . In this study, we investigated the beneficial effects and the underlying molecular mechanisms of Juglans regia L. leaf extract in diabetic neuropathy as a common serious complication of diabetes, according to the traditional claims and the existing scientific evidences regarding hypoglycemic and antioxidative properties of Juglans regia L. leaf extract.
Extract preparation and gas chromatography-mass spectrometry (GC-MS).
Fresh leaves of Juglans regia L. were collected during July–August 2014 from cultivated trees in Khorramabad (Lorestan province, western Iran) and authenticated by Natural Resources Research Center of Lorestan Province. It was registered under the voucher number 8763. Briefly, the leaves were dried and pulverized, and then stored in dark at room temperature. Methanol was added to the pulverized leaves for 72 h and then filtered through filter paper. The obtained extracts were concentrated under reduced pressure at 40 °C. GC-MS was carried out using a Hewlett-Packard 6859 with a quadrupole detector, on a HP-5 column, operating at 70 eV ionization energy, using the same temperature programme and carrier gas as above. Retention indices were calculated by using retention times of n-alkanes that were injected after the extract at the same chromatographic to Van Den Dool method .
Male adult Spargue-Dawley rats were used (250–275 g) (laboratory animal research center, Sari, Iran) in this study. They were kept in the laboratory under constant conditions of temperature (23 ± 1 °C) and light/dark cycle (12 h/12 h) for at least 7 days before and over the course of the experiment. All experimental procedures and protocols were approved by ethical committee of Health Sciences, Mazandaran University of Medical Sciences (Ethics approval references number: IR.MAZUMS.REC.95.2266) to minimize the animal’s suffering. These guidelines were in accordance with the internationally accepted principles for the care and use of laboratory animals.
Induction of diabetes and experimental design
Diabetes was induced by an intraperitoneal injection of 55-mg/kg  single dose of streptozotocin (Santa Cruz Biotechnology) diluted in 0.1 M citrate buffer with PH-4.5. Blood samples were collected from tail vein ~48 h after streptozotocin administration and estimated plasma glucose levels using a commercial glucometer and test strips (Accu-Chek® Active test meter). Rats with plasma glucose level more than 250 mg/dLwere considered as diabetics and were further considered for study. Juglans regia L. leaf extract was administered by oral gavages (200-mg/kg/day). The doses and treatment schedules were based on previous studies [15, 17, 22,23,24] and pilot experiments in our laboratory.
The animals were randomly allocated in five groups, each containing 7 rats: (Ι) Streptozotocin (STZ) treated group, which received an intraperitoneal injection of 55-mg/kg single dose of streptozotocin diluted in 0.1 M citrate buffer and then received isotonic saline as the same volume of GRL leaf extract; (ΙΙ) Preventive group, which received STZ and then received orally administration of 200-mg/kg/day Juglans regia L. leaf extract from the 1st week and continued up to the end of the 8th week after induction of diabetes; (III) Curative group, which received STZ and then received orally administration of 200-mg/kg/day Juglans regia L. leaf extract from the 5th week and terminated at the end of the 8th week after induction of diabetes; (IV) Vehicle group, which received only buffer citrate as the same volume of STZ (non-diabetic rats); (V) Juglans regia L. leaf extract treated group (GRL group, non-diabetic rats) which received only 200-mg/kg/day Juglans regia L. leaf extract. All animals in the experimental groups survived until the end of the experiments, except one rat of the Streptozotocin treated group (Ι). Blood glucose level and body weight were measured on weeks 0, 1, 4, and 8 after STZ injection.
The formalin test was carried out in all groups on weeks 0, 1, 4, and 8 after STZ or vehicle injection. The rats were given a subcutaneous injection of 2.5% formalin into the plantar surface of one hind paw and scoring of nociceptive behaviours began immediately after formalin injection (each 3 min time block) and continued for 60 min. Average nociceptive score, ranging from 0 (the injected paw is not favored) to 3 (the injected paw is licked, bitten or shaken), was measured . At the end of the eighth week, the rats immediately after the last formalin test were euthanized with an injection of sodium pentobarbital and then sciatic nerves were harvested for histopathological, immunohistochemical, and biochemical assessments.
The obtained samples (the left side sciatic nerves) were thoroughly cleaned of blood, then were immediately frozen and stored in a − 80 °C freezer for assays of tissue malondialdehyde (MDA) and Glutathione (GSH) levels, catalase (CAT) and superoxide dismutase (SOD) activities. The estimation of MDA level was done by measuring thiobarbituric acid reactive substances (TBARS) which was determined spectrophotometrically by the absorbance at 535 nm . The concentration was expressed as micromoles per milligram of protein. The estimation of GSH level was done with 10% trichloroacetic acid as described by Akerboom and Sies  which was determined spectrophotometrically by the absorbance at 412 nm. The concentration was expressed as micromoles per milligram of protein. The estimation of CAT activity was determined spectrophotometrically by following the decrease in absorption at 240 nm in a reaction medium containing phosphate buffer and hydrogen peroxide  and expressed as nanomoles trolox equivalent per milligram of protein. The estimation of SOD activity was based on the inhibition of superoxide radical reaction with pyrogallol  which was determined spectrophotometrically by the absorbance at 420 nm and expressed as nanomoles trolox equivalent per milligram of protein.
The obtained samples (the right side sciatic nerves) were immediately fixed in 10% formaldehyde and embedded in paraffin. Three-micrometer serial sections were taken from the paraffin-embedded blocks by microtome. Some tissue sections were deparaffinized and stained with hematoxylin and eosin (H&E) or luxol fast blue (LFB), and studied by light microscopy to assess the histopathological changes. Morphometric analysis of the sciatic nerve was done using ImageJ software (MacBiophotonics ImageJ 1.41a) . All the histological assessments were done in a blinded fashion.
For immunohistochemistry, some sections were incubated in Goat normal serum (in order to block non-specific site), and then with anti-caspase 3 rabbit polyclonal antibody (1:100 in PBS, v/v, ABCAM ab4051), anti-COX 2 rabbit polyclonal antibody (1:100 in PBS, v/v, ABCAM ab15191), anti-S100B rabbit polyclonal antibody (1:1000 in PBS, v/v, ABCAM ab868), and anti-iNOS rabbit polyclonal antibody (1:100 in PBS, v/v, ABCAM ab15323) overnight at 4 °C. Sections were washed with PBS and then incubated with secondary antibody conjugated with horseradish peroxidase (goat anti-rabbit IgG, ABCAM ab205718) for 2 h and detected by diaminobenzidine tetrahydrochloride for 5 min. After wards, they were dehydrated and mounted. For negative controls, primary antibodies were omitted. For quantitative analysis, immunohistochemical photographs (n = five photos from each samples collected from all rats in each experimental group) were assessed by densitometry using Image J software. Data are expressed as a percentage of total tissue area.
Statistical analysis was carried out in SPSS (Version 15, Chicago, IL, USA). Results were presented as mean values (±SD). The K-S test was used in order to evaluate the normality of the data. One-way ANOVA followed by Tukey′s post-hoc tests were used to compare each two groups and data among the groups, respectively. Two-way ANOVA followed by Bonferroni’s post-hoc tests were used for multiple comparisons. A value of p < 0.05 was considered significant.
The various chemical constituents identified in Juglans regia L. leaf extract are shown in Table 1. A total of 28 constituents were identified in the leaf extract. The principal components of the extract were 2-ß-Pinene (17.09%), α-Pinene (13.29%), trans-Caryophyllene (10.58%), and Germacrene D (8.90%). Other minor identified constituents were dl-Limonene (3.85%), Terpine-4-ol (3.70%), ß-Slinene (3.25%), and Methyl salicylate (3.07%).
Blood glucose levels and body weight
The histogram of the fasting blood sugar (FBS) levels for all groups is shown in Fig. 1. Administration of STZ in the STZ group produced a significant elevation (p < 0.001) in FBS level and the hyperglycemia was maintained throughout experimental period compared to vehicle group. At the end of the experiment, the FBS levels in the Preventive and Curative group were significantly lower than that those in the STZ group (p < 0.001), while the differences between Preventive and Curative were not significant (p > 0.05).
Body weight of 8th week diabetic rats was significantly (p < 0.001) lower than vehicle rats (Table 2), while the differences between STZ and Preventive or Curative treatment group were not significant (p > 0.05).
The nociceptive rating scores in the five groups have been presented as mean value ± SD (Fig. 2). The nociceptive response between the all groups before and one week after induction of diabetes were not significant. The nociceptive response of the STZ-treated diabetic rats at the end of the fourth week was found to be significantly (p < 0.001) increased as compared to vehicle rats, while treatment with the leaf extract in Preventive group significantly (p < 0.001) decreased the nociceptive score compared to the STZ-treated group. There was a significant difference (p < 0.001) in the nociceptive score at the end of the study (8th week) between STZ and Preventive or Curative treatment group, while the differences between Preventive and Curative were not significant (P < 0.05).
Malondialdehyde (MDA) levels for all groups at the end of the experiment are shown in Table 3. Administration of STZ in the STZ group produced a significant elevation (p < 0.001) in lipid peroxidation level compared to vehicle group. The MDA levels in the Preventive and Curative group were significantly lower than that those in the STZ group (p < 0.01), while the differences between Preventive and Curative group were not significant (p > 0.05).
Glutathione (GSH) levels for all groups at the end of the experiment are shown in Table 3. Administration of STZ in the STZ group produced a significant (p < 0.001) decrease in GSH level compared to vehicle group. The GSH levels in the Preventive and Curative group were significantly (p < 0.01) higher than that in the STZ group, while the differences between Preventive and Curative group were not significant (p > 0.05).
Catalase (CAT) activity levels for all groups at the end of the experiment are shown in Table 3. Administration of STZ in the STZ group produced a significant (p < 0.001) decrease in CAT activity compared to vehicle group. The CAT activities in the Preventive and Curative group were significantly (p < 0.01) higher than that in the STZ group, while the differences between Preventive and Curative group were not significant (p > 0.05).
Superoxide dismutase (SOD) activity levels for all groups at the end of the experiment are shown in Table 3. Administration of STZ in the STZ group produced a significant (p < 0.001) decrease in SOD activity compared to vehicle group. The SOD activities in the Preventive and Curative group were not significantly (p > 0.05) higher than that in the STZ group.
Histological examination of the sciatic nerves in the STZ-induced diabetic animals (Fig. 3a) revealed that the myelin sheath of the myelinated nerve fibers was thin, loose, and disorganized. Meanwhile, numerous inflammatory cells infiltrated among the separated nerve fibers. Treatment with Juglans regia L. leaf extract reduced the changes, so that only focal loss of myelin sheath and a few scattered inflammatory cells were detected in Preventive and Curative group (Fig. 3b). No detectable injury was shown in vehicle and GRL group. Computer-aided morphometric analyses of the myelinated nerve fibers in the experimental groups are shown in Table 4.
Figures 4, 5, 6, and 7 show the immunohistochemical staining of caspase-3, COX-2, iNOS, and S100B, respectively. Administration of STZ in the STZ group increased the expression of caspase-3 (Fig. 4a), COX-2 (Fig. 5a), and iNOS (Fig. 6a), while Juglans regia L. leaf extract treatment in the Preventive and Curative group reduced the degree of positive staining for caspase-3 (Fig. 4b), COX-2 (Fig. 5b), and iNOS (Fig. 6b) compared to the STZ group. Administration of STZ in the STZ group decreased the expression of S100B (Fig. 7a), while Juglans regia L. leaf extract treatment in the Preventive and Curative group increased the degree of positive staining for S100B (Fig. 7b) compared to the STZ group. Quantitative analysis assessed by densitometry method using ImageJ software. The histograms of the quantitative analysis of caspase-3, COX-2, and iNOS, and S100B positive staining in the experimental groups are shown in Figs. 4c, 5c, 6c, and 7c respectively.
The hypoglycemic effects of Juglans regia L. leaf extract in this study, have been previously proven by the experimental [15,16,17,18] and human clinical trial studies [7, 19]. In this regard, the activity was attributed to the antioxidant capacity of the polyphenols present in walnut leaves , its effects on glucose-uptake due to inhibition of protein tyrosine phosphatase 1B , and its effects on beta cells regeneration and its anti-inflammatory properties . Diabetic neuropathy is the most common complication of diabetes mellitus. On the other hand, free radical induced oxidative stress has been implicated to play an important role in the pathogenesis of diabetic neuropathy . Meanwhile, studies have shown that diabetic neuropathy is a progressive process, despite adequate control of blood sugar, which might be due to accumulation of reactive oxygen species in neural tissue . In diabetic rats, we observed a significant increase in lipid peroxidation, a significant reduction of catalyses and superoxide dismutase activity, and a significant reduction of glutathione content in the sciatic nerve. At the end of the study, treatment with Juglans regia L. leaf extract in Preventive and Curative group ameliorated lipid peroxidation and improved antioxidant status in the sciatic nerve of diabetic rats, while the differences between Preventive and Curative were not significant. In addition to hypoglycemic effects, the results indicate that the use of this extract has antioxidative effect even after the onset of neuropathy, considering that oxidative stress after onset of diabetic neuropathy is a progressive process despite adequate control of blood sugar . Free radicals reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated as a potential contributor to the pathogenesis of neurodegenerative diseases such as diabetic neuropathy [33, 34], which seems appropriate for therapeutic interventions such as the use of free radical scavengers. Juglans regia L. leaves contain a large amount of phenolic compounds, well-known free radicals scavengers. Phenolic acids, naphthoquinones, and flavonoids are the main phenolic compounds in fresh Juglans regia L. leaves [10, 31, 35, 36]. In this regard, study of antioxidant activity of Juglans regia L. leaf extract by the reducing power assay and the scavenging effect on DPPH radicals revealed that walnut leaves cultivars have high antioxidant properties . In vitro study indicated that flavonoids from Juglans regia L. leaves could reduce the reactive oxygen species level in RAW264.7 cells . Carvalho et al. documented that Juglans regia L. leaf extract significantly protected AAPH-induced oxidative hemolysis of human erythrocytes in a time- and concentration-dependent manner . In another study, the antioxidant potential of ethanolic extract of Juglans regia L. leaves was measured and the highest ability to chelate Fe2+, high reducing power, high antiradical activity, and relatively low prevention of lipid oxidation documented . Results of an in vivo study demonstrated that administration of walnut leaf extract increased the antioxidant enzymes superoxide dismutase and catalase against CCI4-induced oxidative damage in rat liver .
Several lines of evidence obtained in experimental and clinical studies demonstrated that inflammation is one of the major reasons behind various deficits seen in diabetic neuropathy. In this regard, it was well documented that the enhanced inflammatory response was mediated by NF-kB axis activation in peripheral nerve disorders caused by diabetes . The NF-kB activation induces cyclooxygenase-2 overexpression, which is implicated in neuropathic changes . For instance, some studies reported that treatment with a COX-2-selective inhibitor and COX-2 gene inactivation prevents functional and biochemical peripheral nerve deficits in the diabetic animals [41, 42]. On the other hand, upregulation of inflammatory mediators of COX-2, iNOS, and TNF-α reported in the course of diabetic neuropathy, blocking of these mediators lead to a marked reduction in pain intensity . Meanwhile, activation of poly (ADP-ribose) polymerase (PARP), a nuclear enzyme which is activated by strand break in DNA, contributes to upregulation on inducible nitric oxide synthase (iNOS) and to nitrosative stress, leading to peroxynitrite formation . Our immunohistocehmical assessments showed that increased COX-2 and iNOS expression in diabetic rats significantly attenuated after treatment with Juglans regia L. leaf extract in Preventive and Curative group, while the differences between Preventive and Curative were not significant. In addition to hypoglycemic effects, the results indicate that the extract has anti-inflammatory effects even after the onset of neuropathy, noting that inflammatory response after onset of diabetic neuropathy is a cardinal and progressive process despite adequate blood glucose control . Hosseinzadeh et al. documented that the aqueous and ethanolic extracts of Juglans regia L. leaves have anti-inflammatory effect against xylene-induced ear swelling in mice, which is mediated by membrane-stabilizing effect that reduces capillary permeability and/or release of inflammatory mediators . In the other study, it was shown that Juglans regia L. leaf extract exhibited anti-inflammatory activity against carrageenan-induced hind paw edema model in mice, the mechanism of the anti-inflammatory activity were not determined .
Apoptosis is a key mechanism of degenerative diseases, which is triggered by some factors such as hyperglycemia toxicity. In vivo and in vitro studies revealed that hyperglycemia affected the cell survival and induced apoptotic changes in dorsal root ganglion neurons and Schwann cells [45, 46]. Our immunohistochemical results showed that administration of STZ considerably increased the expression of caspase-3, which plays a critical role in apoptosis. On the contrary, S100B expression, as a Schwann cell marker, decreased in diabetic peripheral neuropathy. Also, our results showed that these up- and down regulations significantly attenuated after Juglans regia L. leaf extract consumption in Preventive and Curative group, while the differences between Preventive and Curative group were not significant. Meanwhile, in addition to hypoglycemic effects, our results indicate that the extract has antiapoptotic effects even after the onset of neuropathy, considering that glial apoptosis after onset of diabetic neuropathy is a progressive process despite adequate control of blood sugar . Javidanpour et al. documented the proliferative effects of Juglans regia L. leaf extract on pancreatic ß-cells in STZ-induced diabetic rats . Results of another study demonstrated that walnut leaf extract has a hepatoprotective effect against carbon tetrachloride-induced cell death . On the contrary, some studies demonstrated that walnut leaf extract showed a higher antiproliferative efficiency than green husk and seed extracts against various cancer cell line such as human renal, oral, breast and colon cancer cell lines [12, 47], which is more likely related to its phenolic constituents. Also, it was found that walnut extracts suppressed proliferation and induced apoptosis in a dose- and time dependent manner by modulating expression of apoptosis-related genes namely Bax, caspase-3, Bcl2, and tp53 [48, 49]. Sensitized peripheral nociception is a common symptom associated with diabetic neuropathy. Although the exact mechanism of this reaction is unclear, but different mechanisms may account, including peripheral receptor sensitization, ischemic tissue injury, alternation in dorsal root ganglia cells, and sprouting fibers with ectopic activity [50, 51]. In the present study, we used of the formalin-induced nociceptive test because of the induced pain is continuous and, meanwhile it is more similar to the clinical pains versus other nociceptive tests . Acute chemonociception mediated by transient receptor potential vanilloid 1(TRPV1)-expressing nociceptors and prolonged pain provoked by damage of sensory afferents are two distinct mechanisms through which formalin evokes nocifencive behaviors . We observed a significant reduction in nociception after Juglans regia L. leaf extract treatment in Preventive and Curative group, while the differences between Preventive and Curative were not significant. Erdemoglu et al. documented that treatment with Juglans regia L. leaf extract showed antinociceptive activity against p-Benzoquinone-induced abdominal contraction test in mice .Also, antinociceptive activity of Juglans regia L. leaf extract is further supported by the study of Hosseinzadeh et al. , where in central and peripheral antinociceptive effects were documented. These effects may be mediated through non-opioid receptors or inhibition of cyclooxygenase enzyme .
The main findings of the current study showed that administration of Juglans regia L. leaf extract attenuates criteria of neuropathy in STZ-induced diabetic rats, in addition to hypoglycemic effects. More importantly, results of this study showed that these beneficial effects are even after the onset of peripheral diabetic neuropathy. In other words, consumption of Juglans regia L. leaf extract has preventive and curative effects against STZ-diabetic neuropathy which might be due to its antioxidant, anti-inflammatory, and antiapoptotic properties.
BCL2 Associated X Protein
B-cell lymphoma 2
diabetic peripheral neuropathy
advanced glycation end-product
fast blood glucose
fast blood sugar
Gas chromatography-mass spectrometry
- GRL :
Juglans regia L.
hematoxylin and eosin
inducible nitric oxide synthase
luxol fast blue
poly (ADP-ribose) polymerase
Phosphate buffered saline
protein tyrosine phosphatase 1B
reactive nitrogen species
reactive oxygen species
S100 calcium-binding protein B
tumor necrosis factor alpha
tumor protein p53
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This work was supported financially by Molecular and Cell Biology Research Center, Faculty of Medicine, Mazandaran University of Medical Sciences (grant number, 2266).
This work was solely financed by Mazandaran University of Medical Sciences. No external funding was provided.
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The datasets analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate
All experimental procedures and protocols were approved by ethical committee of Health Sciences, Mazandaran University of Medical Sciences (Ethics approval references number: IR.MAZUMS.REC.95.2266) to minimize the animal’s suffering. These guidelines were in accordance with the internationally accepted principles for laboratory used and care.
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Nasiry, D., khalatbary, A.R., Ahmadvand, H. et al. Protective effects of methanolic extract of Juglans regia L. leaf on streptozotocin-induced diabetic peripheral neuropathy in rats. BMC Complement Altern Med 17, 476 (2017). https://doi.org/10.1186/s12906-017-1983-x
- Diabetic neuropathy
- Juglans regia L. leaf