Effect of Nobiletin on Diabetic Neuropathy in Experimental Rats

Research Article

Austin J Pharmacol Ther. 2014; 2 (5). 1028

Effect of Nobiletin on Diabetic Neuropathy in Experimental Rats

Parkar N1,2 and Addepalli V2*

1Department of Pharmacology, Bhanuben Nanavati College of Pharmacy, India

2Department of Pharmacology, NMIMS University, India

*Corresponding author: : Addepalli V, Department of Pharmacology, SPP School of Pharmacy and Technology Management, NMIMS University, Vile Parle (W), Mumbai, 400 056, India

Received: June 11, 2014; Accepted: July 31, 2014; Published: Aug 04, 2014


Diabetic neuropathy (DN) is a microvascular complication of diabetes that leads to allodynia, nerve conduction slowing and progressive sensory loss. Despite the prevalence and severity of DN, currently there is no treatment available for DN. Objective of present study was to evaluate efficacy of nobiletin in the treatment of diabetic neuropathy in rats. Diabetes was induced in rats using a single dose of streptozotocin (50mg/kg i.p.). Four weeks after the induction of diabetes, treatment with nobiletin (10mg/kg and 25mg/kg) was given for further four weeks. At the end of eight weeks, the nociception latency was measured using hot plate and tail flick test. Further the nerve conduction velocity was measured and the histopathology of the sciatic nerve was studied. The results indicated that nobiletin caused improvement in nerve conduction velocity at a dose of 25mg/kg (42.58 ± 2.02** vs. control 30.00 ± 1.51) and sciatic nerve histology. The nociception latency also was improved. Thus, the study showed efficacy of nobiletin in the treatment of diabetic neuropathy in rats.

Keywords: Diabetic neuropathy; Nobiletin; Streptozotocin


DM: Diabetes Mellitus; DN: Diabetic Neuropathy; STZ: Streptozotocin; BSL: Blood Sugar Levels; PKC: Protein Kinase C; PARP: Poly ADP Ribose Polymerase; MMP-2: Matrix Metalloproteinase–2; MMP-9: Matrix Metalloproteinase–9; GOD/POD: Glucose Oxidase-peroxidase; NORMO: Normal; NOB: Nobiletin; MINO: Minocycline; MNCV: Motor Nerve Conduction Velocity; SD: Standard Deviation; ANOVA: Analysis of Variance; CMC: Carboxy Methyl Cellulose


Diabetes mellitus (DM) is a chronic metabolic condition affecting a large majority of population worldwide. Several factors like changing patterns of diet and physical activity, sedentary lifestyles and increase in obesity are responsible for the increasing incidence of diabetes. People with prolonged hyperglycemia develop several vascular complications that are responsible for increase in the ratio of morbidity and mortality of affected individuals. Diabetic peripheral neuropathy (DN) is a multifaceted and potentially severe complication of diabetes affecting more than 50% of diabetic individuals and is the leading cause of non-traumatic amputation and anatomic failure [1,2]. Hyperglycemia triggers various alternate pathogenetic pathways for the circulating glucose like aldol reductase [3], non-enzymatic glycation [4], protein kinase C (PKC) [5], mitogen activated protein kinases [6] and poly ADP ribose polymerase (PARP) [7] to name a few. The activation of these alternate pathways leads to the production of several toxic metabolites that cause deleterious effects on different biological systems of the affected individuals. Early disorders of nerve function include slowing in nerve conduction velocity followed by axonal degeneration, paranodal demyelination and loss of myelinated fibers [8]. Long term neuropathy leads to more severe effects like severe pain, loss of sensation, foot ulceration and amputation, burns, infection, cellulites, sleep disorder, impaired daily functioning, mood disorders, gangrene, involvement of different systems such as cardiovascular, gastrointestinal and reproductive systems [9,10].

Despite efforts to make an early diagnosis and to stop the progression of DN, currently very few drugs are available to cure this disease and the others only provide symptomatic relief [11]. Several combination strategies of drugs with natural molecules like vitamin E have been tried to reduce the neuropathic pain [12]. Report of ethnobotany suggested that about 800 medicinal plants possess antidiabetic potential and the bioactive compounds such as glycosides, alkaloids, terpenoids and flavonoids (phenols) are effective drugs both in preclinical and clinical studies [13,14]. Flavonoids are a class of secondary metabolites from natural sources that studied for their various activities [15]. Nobiletin is a flavonoid present in peels of citrus fruits and is found to be a potential molecule possessing several biological activities including inhibition of MMP-2 and MMP-9 in cancer cells [16]. Since MMPs are involved in the pathogenesis of diabetic vascular complications, we hypothesize that nobiletin can be a potential molecule in ameliorating the diabetic complications. The present study aimed at evaluating the effect of nobiletin in diabetic neuropathy using STZ diabetic rats.

Materials and Methods

Chemicals and drug solutions

All the reagents and chemicals used for the study were of analytical grade. Nobiletin was purchased from Baoji Hongyuan Biotechnology Co. Ltd. (Baoji City, China). Streptozotocin (STZ) was purchased from Sigma Aldrich (USA). Minocycline gift sample was available from US Vitamins (Mumbai, India). All the biochemical diagnostic kits were procured from Erba Diagnostics, Mumbai, India. Nobiletin and Minocycline were suspended in freshly prepared 0.5% CMC (Carboxy methyl cellulose) solution before use. STZ was dissolved in freshly prepared ice cold Citrate Buffer (pH 4.5) before use.

Animals and Experimental Protocol

Wistar rats (Male, 190–240g) were used for the study. The animals were purchased from Haffkine Institute, Lower Parel (Mumbai, India). Animals were caged in clean environment and maintained at a temperature of 25 ± 1°C, RH 45-55%. 12 hr light/ dark cycle was maintained in the animal house and the animals had free access to food and water ad libitum.

For induction of diabetes, the animals were fasted overnight for 12 hrs. Diabetes was induced with a single dose of STZ (50mg/kg, i.p.) freshly dissolved in ice cold citrate buffer pH 4.5. After STZ injection, the animals were allowed free access to feed and water. Diabetes was checked after 48 hrs by estimating the blood sugar levels (BSL) using a GPD/POD kit. The animals with a BSL of >300mg/kg were considered diabetic and used further for the study.

The doses of nobiletin were calculated on the basis of results of earlier reported in vitro studies and pharmacokinetic data. The doses calculated were such that they would produce a plasma concentration of nobiletin required to inhibit MMP-2 and MMP-9. In the eight week study period, the treatment phase of the study was the last four weeks daily p.o. Grouping of animals was done randomly and animals were divided into five groups of six animals each. Group I served as normal control (NORMO), Group II served as Vehicle Control and received 0.5% CMC solution (1 ml/kg); Group III and IV were the treatment groups and received nobiletin (NOB) at a dose of 10mg/ kg (NOB10) and 25mg/kg (NOB25) respectively; Group V served as standard and received minocycline (MINO) at a dose of 50mg/kg. The BSL and body weight of the animals was estimated weekly during the experiment.

Thermal nociceptive response

The thermal nociceptive response of the animals was measured by determining the hot plate and tail flick latency of the experimental animals on a weekly basis till the end of eight week study protocol. For hot plate latency , the animals were placed into a glass cylinder on a hot plate (IITC, Inc., Model 35-D) adjusted to 54.5 ± 1°C to induce thermal hyperalgesia. The time in seconds, from placing the rat on the hot plate to either licking of the hind paw or attempting to jump out of the cylinder cage was recorded for each rat. The cut off time set for the test was 30 s. The tail flick latency was measured using a 20 amp analgesiometer (INCO, India). A 6 amp current passed through the naked nichrome wire. The reaction cut off time was set at 10s. The heat source and the tail skin were placed 1 cm apart and the tail flick latency was measured in seconds.

Motor Nerve Conduction Velocity (MNCV)

At the end of eight week study period, the motor nerve conduction velocity of the animals was measured in the sciatic-posterior tibial conducting system using a data acquisition system (Iworx data acquisition system, USA) as reported previously. The rats were anesthetized using pentobarbital sodium (45mg/kg, i.p.). The body temperature of the animal was measured using a rectal probe and maintained at 37 ± 1°C throughout the procedure. MNCV was measured in anesthetized rats by stimulating the sciatic and the tibial nerve using 26 gauge bipolar needle electrodes with 8 V single stimuli [17,18]. The receiving electrodes were placed on the foot muscle. MNCV was calculated by subtracting the distal latency from the proximal latency, and the result was divided into the distance between the stimulating and recording electrode.

Sciatic nerve histology

The rats were sacrificed and the sciatic nerve was carefully removed from each animal. The nerve tissue was washed with saline and fixed in 10% formalin for histopathological study. Slides were prepared by embedding the nerve in paraffin and staining with hematoxylin and eosin. The histology of sciatic nerves was studied using a bright field microscope and images were taken for reference.


All data were expressed as mean ± S.D. Statistical analysis was performed using Graph Pad Prism (version 4.0, Gra.ph Pad Inc., San Diego, (CA) software. For multiple comparisons, one-way analysis of variance (ANOVA) was used. In case ANOVA shows significant differences, post-hoc analysis was performed with Dunnet test, p<0.05 was considered statistically significant.


The experimental animals showed hyperglycemia and marked reduction in bodyweights on induction of diabetes using STZ (Table 1). Diabetes produced four fold increases in blood sugar levels of the animals. The increase in BSL was consistent throughout the study period. Four week treatment with nobiletin or minocycline did not produce any effect on the body weight and blood sugar levels of the animals (Table 1).