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Oleo gum resin of Ferula assa-foetida L. ameliorates peripheral neuropathy in mice Farshad Homayouni Moghadam, Maryam Dehghan, Ehsan Zarepur, Reyhaneh Dehlavi, Fatemeh Ghaseminia, Shima Ehsani, Golnaz Mohammadzadeh, Kazem Barzegar

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S0378-8741(14)00273-6 http://dx.doi.org/10.1016/j.jep.2014.03.069 JEP8725

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Journal of Ethnopharmacology

Received date: 30 October 2013 Revised date: 11 March 2014 Accepted date: 30 March 2014 Cite this article as: Farshad Homayouni Moghadam, Maryam Dehghan, Ehsan Zarepur, Reyhaneh Dehlavi, Fatemeh Ghaseminia, Shima Ehsani, Golnaz Mohammadzadeh, Kazem Barzegar, Oleo gum resin of Ferula assa-foetida L. ameliorates peripheral neuropathy in mice, Journal of Ethnopharmacology, http: //dx.doi.org/10.1016/j.jep.2014.03.069 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Oleo gum resin of Ferula assa-foetida L. ameliorates peripheral neuropathy in mice Farshad Homayouni Moghadama, Dehlavic,

b*

, Maryam Dehghanb, Ehsan Zarepurc, Reyhaneh

Fatemeh Ghaseminiac, Shima Ehsani c, Golnaz Mohammadzadehc, Kazem

Barzegard a: Department of Physiology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. b: Neurobiomedical Research Center, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. c: Student Research Committee, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. d: English Language Department, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

*Corresponding author information: Farshad Homayouni Moghadam Department of Physiology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Address: Shahid Sadoughi University of Medical Sciences, Shohadaye Gomnam Blvd., 8915173143, Yazd, Iran, Tel.: +98 351 8202630; fax: +98 351 8203414. E-mail address: [email protected]

Abstract Ethnopharmacological Relevance: According to the Chinese, European, Iranian and Indian traditional medicines, oleo gum resin of Ferula assa-foetida (asafoetida) has therapeutic effects on different kinds of diseases. Some of these effects are related to the diseases of nervous system such as hysteresis and convulsion. In recent studies, some anti-epileptic and neuroprotective roles were also considered for it and we examined its possible role on treatment of peripheral neuropathy. Material and Methods: In vitro studies were carried out to identify the response of isolated sciatic nerves to different concentrations of oleo gum resin of asafoetida solved in Lock’s solution. Then, in vivo studies were conducted to evaluate its effect on amelioration of peripheral neuropathy in mice. Peripheral neuropathy was induced by intraperiotoneal injection of high doses of pyridoxine in adult Balb/c male mice. Tail flick tests were performed to identify the incidence of neuropathy in animals. After 10 days treatment with asafetida, the efficiency of treatment was assessed by behavioral, electrophysiological and histological studies. Results: In vitro experiments confirmed that incubating the nerves in aqueous extract of oleo gum rein of asafoetida increased the amplitude and decreased the latent period of nerve compound action potential (CAP). Nerve conduction velocity (NCV) and amplitude of CAP also improved in asafoetida treated animals. Histological and behavioral studies showed that asafoetida was able to facilitate the healing process in peripheral nerves. Conclusions: In vitro experiments showed that asafoetida is a nerve stimulant and its administration in neuropathic mice exerted neuroprotecting effects through stimulating axonal regeneration and remyelination and decrement of lymphocyte infiltration. Key words: Asafoetida; Peripheral Neuropathy; Neuroprotective.

1. Introduction Ferula assa-foetida L. belongs to the Apiaceae (Umbelliferae) family of plants and its oleo gum resin is known as asafoetida and people of some countries still consume it as a spice and medicinal herb (Iranshahy and Iranshahi, 2011; Kavoosi and Rowshan, 2013). Asafoetida has different names in different languages such as Hing or Hingra in hindi, Stinkasant or Teufellsdreck in German, Assafoetida in Italian, Anghouzeh in Persian, A Wei in Chinese, and ase-fetide in French (Iranshahy and Iranshahi, 2011). This plant is commonly cultivated in the lands of the central Asia (Kanani et al., 2011). According to the Chinese, Iranian, European, American, and Indian traditional medicines, asafoetida has multiple usages for treatment of different diseases such as epilepsy, hysteresis, infection, impotency, asthma, stomachache, and flatulence (Iranshahy and Iranshahi, 2011). Recent researches indicated that it exerts antispasmodic, hypotensive, anti-inflammatory, antioxidative, sedative, anti-ulcer, anti-diabetic, and neuroprotective effects (Mahendra and Bisht, 2012). Furthermore, there is some evidence about its usage as a nerve stimulating factor among people of United States of America (Iranshahy and Iranshahi, 2011). In the pilot experiments of the present study, we also found that it has a nerve stimulating effect on isolated mice sciatic nerves. This finding supports its nerve stimulating effect and is against the reports on its anti-convulsion and sedative effects. Moreover, it was recently stated that oleo gum resin of asafoetida has a neuroprotective effect on cultured neurons (Moghadam et al., 2013b). Reports from traditional usages and these new findings suggest that asafoetida can exert some effects on the function of the nervous system but its exact effect is not clear. Because of both neuroprotective and nerve stimulating effects, we thought it might be a useful substance for studying the treatment of peripheral neuropathy.

Peripheral neuropathy has a high prevalence because it is a common side effect of several diseases such as infections, diabetes mellitus, autoimmune disorders and malignancies (Broyles et al., 2013; Carozzi et al., 2013; Sindic, 2013; Vincent et al., 2013). Among these diseases, incidence of peripheral neuropathy in diabetic patients is highly remarkable, and almost 50% of diabetic patients are suffering from peripheral neuropathy (Xu et al., 2012). Also, several chemotherapy drugs are neurotoxic, and peripheral neuropathy is their major side-effect that limits their therapeutic dose and treatment period (Geber et al., 2013). Several drugs, neuroprotective factors, neurotrophins, and antioxidants were tested to find a cure for neuropathy, but there is conflicting evidence about their efficacy (Schröder et al., 2013). So, there is not any effective and conventional medication for the treatment of neuropathy (Schröder et al., 2013). Studies about the effect of herbal treatments on neuropathy are growing and a lot of substances are considered to be effective but their exact efficacy is not identified yet. It has been suggested that the best treatment protocol might exert a combination of neuroprotective, neuroregenerative and anti-inflammatory effects (Schröder et al., 2013; Tofthagen et al., 2013). So, regarding the reported neuroprotective, anti-oxidative, nerve stimulating and antiinflammatory effects of asafetida, we studied its effect on amelioration of pyridoxine-induced peripheral neuropathy in mice. 2. Materials and Methods 2.1. Animals Animals used in this study were male Balb/c mice (Pasteur institute, Tehran, Iran). For in vitro studies, sciatic nerves of ten adult male Balb/c mice were isolated and their electrical responses were monitored under exposure to different concentrations of asafoetida. For in-vivo set of experiments, fifty adult male Balb/c mice with an average body weight of 40-50 grams were

used. Animals were housed with a 12-hour light/dark cycle with free access to water and food. Temperature and humidity of animal house were adjusted to 25º C and 45-55% , respectively. All experimental protocols were approved by our University Ethical Committee. 2.2. Preparation of aqueous extract of oleo gum resin of ferula assa-foetida L Oleo gum resin was collected from Ferula assa-foetida L. (Iranian Biological Resource Center (IBRC, Tehran, Iran) P1006636; the herbarium code is for the seed of the plant), a type of ferula family which naturally grows in Mehriz area in Yazd province of Iran. Oleo gum resin of this plant was collected in June by making some small incisions (1-5 cm) on its stem near the roots giving a resin with high quality called Ashki asafoetida by local people (Ashki is a Persian word meaning: the purity of tears and is an indication for clearness and good quality). Ashki asafoetida is a type of asafoetida with the best quality but less quantity. It is different from regular type of asafoetida which is collected by complete cessation of stem from root giving a type of asafoetida with less quality but high quantity. After collection, oleo gum resin was cut into small pieces and placed under hood until it dried (1 week). For the preparation of asafoetida injection solution, the specified amount (10 mg, 100 mg and 1g) of dried oleo-gum-resin of asafoetida was solved in 10 mL distilled water and filtered by 0.02 μm filters. 2.3. Induction of neuropathy and asafoetida treatment Animals were divided randomly into 5 groups: 1: Normal (non-neuropathic), 2: Control (Neuropathic), 3: Group 1 (Neuropathic and asafetida-treated 0.1 mg/kg), 4: Group 2 (Neuropathic and asafetida-treated 1 mg/kg), and 5: Group 3 (Neuropathic and asafetida-treated 10 mg/kg). Groups 2-5 were subjected to induction of peripheral neuropathy by treatment with high doses (350 mg/kg, ip) of pyridoxine twice daily for 8 days. Tail flick tests were performed for the assessment of neuropathy incidence in animals before and after neuropathy induction.

After the establishment of incidence of neuropathy in animals, asafoetida treatment was initiated and continued for 10 days. The doses of injection were calculated according to the body weight of each animal belonging to each treatment group (0.1 mg/kg, 1mg/kg and 10 mg/kg). The same volumes of distilled water were injected to the animals of control group. For in-vitro study, different amounts of oleo gum resin of asafoetida were solved in Lock's solution to prepare concentrations at 0.001, 0.01, 0.1, 1, 10, 100, 1000, 10000 g/ml. Then, the prepared solutions were filtered and their pH was adjusted to 7.4. 2.4. Tail flick test Tail-flick test is a behavioral assay for determining the tolerance of animal to pain. Pain was induced by heating up the distal part of the tail by placing it under the focused light and the time lapse between the light projection and flicking the tail out of the light was recorded. Fifteen seconds cut-off time was calibrated on the device to avoid tissue damage. Tail-flick tests were performed three times: before neuropathy induction, after neuropathy induction, and after asafoetida treatment. 2.5. Electrophysiological analysis Electrophysiological studies were performed in two different sections. At first, for finding the nerve stimulating concentration of asafoetida, its different concentrations (0, 0.001, 0.01, 0.1, 1, 10, 100, 1000, 10000 g/ml) were applied on isolated sciatic nerves and their electrical function was recorded by the following method. Asafoetida solutions were poured on nerves by syringe from fewer concentrations to the higher concentrations. Between the intervals, nerves were washed by Lock's solution and a 15-minute break was taken before testing the next concentration. In the second part of electrophysiological studies, asafoetida treatment was done on neuropathic mice. After ten days of treatment, animals were killed by cervical dislocation.

The sciatic nerves were removed and placed in modified Lock's solution (pH 7.4), which consisted of 140 mM NaCl, 1.2 mM MgCl2, 5.6 mM KCl, 2.2 mM CaCl2, 10 mM Tris buffer (tris- (hydroxymethyl) amino-methane), and 10 mM glucose. During electrophysiological studies, to avoid tissue drying, the nerves were placed in humidified chamber. We used C.E.P.T.U instrument (Complete Electrophysiology Teaching Unit, Washington Bioscience) to record compound action potentials. Properties of exerted stimulus to the nerve by stimulator were 1 v, delay: 0 and width: 0.05 ms. The responses of nerves were amplified by the gain of 1000 by amplifier. To monitor the response curve digitally, output wire of the amplifier was connected to the microphone entry port of the sound card of the computer and data were converted to digital format by digital sound card oscilloscope software (Virtins Sound Card Multi-Instrument Software, version 2.1). The Images of compound action potentials were recorded and analyzed by Image-J software to compute their conduction velocity, duration, and voltage (Moghadam et al., 2013a). 2.6. Tissue preparation and staining After electrophysiological experiments, sciatic nerves were fixed with 10% paraformaldehyde. Then, they were washed with saline and dehydrated by incubation in different concentrations of ethanol and rinsed with xylene. Next, they were embedded in melt paraffin and 5ߤ݉ thickened cross-sections were prepared from them by microtome (Olympus). Three sections were selected from the beginning, middle, and the end of each paraffin block and sections were stained with Masson’s trichrome. Stained sections were photographed under light microscope (Nikon). Three images were captured from each nerve and the number of axonal degeneration, axonal demyelination and lymphocyte infiltration were counted in the images.

2.7. Determination of Free Ferulic Acid, Umbelliferone and Foetidin concentrations in oleo gum resin of ferula assa-foetida by high performance liquid chromatography (HPLC) technique Since Ferulic acid is one of the best known bioactive components of asafetida, we measured its concentration. In addition, concentrations of Umbelliferone and Myrcene, two other important bioactive compounds of asafoetida were determined. 2.7.1. Reagents and solvents Ferulic acid (Cat. No. 46278), Umbelliferone (Cat. No. 93979) and Myrcene (Cat. No.M100005) were obtained from Sigma. High performance liquid chromatography grade of methanol was obtained from Merck (Cat. No.106018). 2.7.2. High performance liquid chromatography (HPLC) Conditions A system of HPLC consisted of a Shimadzu LC-10ADVP pump, SIL-HTA/HTC autosampler, SPD-10AVP/10AVVP UV detector, and CLASS-VP Ver.6.1 system manager was used (Shimadzu Co., Kyoto, Japan). Separation was achieved by a COSMOSIL Cholester Packed column (4.6 mm I. D. x 150 m, 5 m, Biotic, Taiwan, catalog number: 05976-61). A mobile phase composed of Methanol/H2O (80/20) was used throughout the analysis. Flow rate was 1.0 mL/min and UV absorbance was set at 290-310 nm. All injections (10 μl) were performed in triplicate. For the preparation of asafoetida sample, one milligram of dried oleo gum resin of ferula assa-foetida was solved in distilled water and filtered through 0.22 μm filter. For the preparation of standard concentration curve for each compound, known quantities of Ferulic acid, Myrcene and Umbelliferone were solved in distilled water (1-100 μg/ml). Standard curves were prepared by injection of known quantities of Ferulic acid, Myrcene and Umbelliferone to HPLC system. Linearity was determined for all standard concentrations. The concentration of each component in the test sample was determined according to its standard curve.

2.8. Statistical analysis The statistical analysis was performed using SPSS software version 21. Data of electrophysiological experiments were evaluated using one-way analysis of variance (ANOVA) followed by Dunnett’s t-test. The results of histological assays were compared using KruskalWallis test. P-values less than 0.05 were assumed to be statistically significant. 3. Results 3.1. In-vitro experiments Results of in-vitro experiments (Figure 1) showed that asafoetida can change the latent period (nerve conduction velocity) and amplitude of isolated sciatic nerves. As shown in Figure 1, D, by increasing the concentration of asafetida, the response of nerve to the stimulus became faster and stronger. These changes were initiated at the concentration of 0.1g/ml and continued at the higher concentrations up to the final concentration (10 mg/ml). The level of changes was prominent at the concentrations of 0.1g/ml and 1g/ml. For both concentrations, there were significant changes at the latent period, and the amplitude of compound action potential (CAP) was compared to the vehicle-treated group. These changes were significantly remarkable at the concentration of 1g/ml compared to the 0.1g/ml (P

Oleo gum resin of Ferula assa-foetida L. ameliorates peripheral neuropathy in mice.

According to the Chinese, European, Iranian and Indian traditional medicines, oleo gum resin of Ferula assa-foetida (asafoetida) has therapeutic effec...
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