Article Neuroprotective Effects of Dried Tubers of napellus

Ambreen Shoaib 1,2 , Hefazat Hussain Siddiqui 2, Rakesh Kumar Dixit 3, Sahabjada Siddiqui 4, Badrud Deen 2,*, Andleeb Khan 5 , Salman H. Alrokayan 6, Haseeb A. Khan 6 and Parvaiz Ahmad 7,* 1 Department of Clinical Pharmacy, Faculty of Pharmacy, Jazan University, Jazan 45142, Saudi Arabia; [email protected] 2 Department of Pharmacology, Faculty of Pharmacy, , 226026, India; [email protected] 3 Department of Pharmacology, King George Medical University, Lucknow, 226003, India; [email protected] 4 Department of Biotechnology, Era’s Lucknow Medical College & Hospital, , Lucknow 226003, India; [email protected] 5 Department of Pharmacology & Toxicology, Faculty of Pharmacy, Jazan University, Jazan 45142, Saudi Arabia; [email protected] 6 Department of Biochemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia; [email protected] (S.H.A.); [email protected] (H.A.K.) 7 Botany and Microbiology Department, College of Science, King Saudi University, Riyadh 11451, Saudi Arabia * Correspondence: [email protected] (B.D.); [email protected] (P.A.); Tel.: +91-0522-2890730 (P.A.)

 Received: 30 November 2019; Accepted: 18 February 2020; Published: 11 March 2020 

Abstract: The present study was designed to explore the neuroprotective properties of Aconitum napellus (). The detoxification was done using either water, or cow or goat milk as per the Ayurvedic shodhana method. The evaluation of the neuroprotective role of A. napellus was performed on diabetic neuropathy induced by streptozotocin in Sprague Dawley (SD) rats. Body mass, blood sugar level, oral glucose tolerance test, hyperalgesia, cold allodynia, motor co-ordination test, and locomotor activity, oxidative biomarkers (TBARS, reduced glutathione, catalase and superoxide dismutase) and sciatic nerve histomorphology were assessed. The in vitro studies were done on human neuroblastoma cell line SHSY-5Y and used an MTT assay to assess the antiproliferative activity of different extracts. Results suggest that the goat milk treated chloroform extract has less percentage of . After administration of the detoxified chloroform extract to the diabetic animals, there was a significant improvement in the myelination and degenerative changes of the nerve fibers along with behavioral changes (p < 0.05 as compared with diabetic control group). The findings of the in vitro research show an effective neuroprotective role of A. napellus. This suggests that A. napellus should be further investigated for its effect in diabetic pathology.

Keywords: Aconitum napellus; neuropathy; sciatic nerve; histopathology; neuroblastoma cell line; cell viability

1. Introduction Neurodegeneration is the serious side effect of acute injuries or chronic irregular incidents of ischemia and hypoxia, which can generate oxidative stress and neuro-inflammation, eventually leading to neuropathy [1]. Neuronal degradation after a long-term of non-treated or uncontrolled diabetes may lead to diabetic neuropathy (DN). DN is a disorder mainly coupled with diabetes mellitus, which may result in numerous clinical manifestations. There is a symmetrical degeneration of nerve fibers that

Plants 2020, 9, 356; doi:10.3390/plants9030356 www.mdpi.com/journal/plants Plants 2020, 9, 356 2 of 16 affects motor and autonomic system. About 50% of the diabetic population develops neuropathy if left untreated or blood sugar levels are not controlled [2]. Oxidative nervous tension and construction of cytokines along with dysfunction in vascular tissues are significant aspects responsible for DN [3]. Currently, the management of this complication relies on supplementation of vitamin B-complex and anti-epileptics or anti-depressant drugs. These allopathic compounds have their own limitations due to the high propensity of drug side effects [4]. Also, the treatment is not satisfactory every time. Therefore, there is a need to discover alternative treatments, which can be safe enough and retain optimum efficacy. Many studies have revealed the medicinal potential of herbal products [5,6]. Traditionally, A. napellus has been used as an arrow for hunting purposes due to its toxic nature. In Chinese medicine, A. napellus is used to prevent from cold, general weakness and ‘Yang’ deficiency and as an antidote for several [7]. It is used in folklore medicine for the management of facial paralysis, joint pain, inflammation, gout, pyrexia and pericardiatis [8] for sciatica and rheumatism [9]. Aconite tubers contain aconitine, benzoylaconitine, mesaconitine; isoaconitine; benzaconitine; aneopelline; eoline; napelline; ipaconitineconine [10]. A. napellus is also used as a constituent of Ayurvedic, Unani medicinal preparations and polyherbal formulations to treat diabetes and as nerve tonic with anti-oxidant properties [11,12]. Aconite is considered to be a useful approach for the improvement of subjective symptoms such as numbness, sensation of cold and pain in the extremities [13], which are associated with diabetic neuropathy. Neuroprotection intends to prevent the neuronal degeneration, and to minimize the damage and maximize the recovery of a neural system after acute toxicity or during chronic insult [14]. There are various animal models used for the estimation of neuroprotective activity, but widely used is diabetic neuropathy induced by streptozotocin (STZ). As STZ is highly toxic, it causes alkylation of β cells and produces nerve degeneration and hypoalgesia [4]. Many in vitro models have been developed to analyze the cellular mechanism of diabetes and its complications by using PC 12 cell line, dorsal root ganglion, Schwann cell and neuroblastoma cell line [15]. The present in vitro study was done on neuroblastoma cell line (SHSY-5Y) because it has capability to reproduce in culture medium devoid of any infectivity [16,17]. We have observed the neuroprotective properties of A. napellus extract through in-vivo study against STZ-induced diabetic neuropathy along with in-vitro studies on human neuroblastoma cell line (SHSY-5Y) treated with normal and high glucose levels.

2. Materials and Methods

2.1. Chemicals and Reagents Eagle’s minimal essential medium fetal bovine serum, penicillin, streptomycin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and other biochemical materials were purchased from Himedia (Mumbai, India). Chloroform, acetonitrile (HPLC grade) and aconitine ( 95% purity) were procured from Sigma Aldrich (ST. Louis, MO, USA). All other reagents used in this ≥ experiment were of analytical grade.

2.2. Collection of Plant Material The dried Aconitum napellus tubers were procured from the local market of Kashmir, J&K, India. Their authentification was further confirmed from the National Botanical Research Institute, Lucknow, India (NBRI/CIF/383/2013).

2.3. Detoxification and Extraction Detoxification of the tubers was done using either water, or cow or goat milk according to the traditional Ayurvedic method i.e., shodhana samaskara [18,19] and extraction was performed using chloroform as per our previous study [12]. Different solvents e.g., n-hexane, chloroform, methanol Plants 2020, 9, 356 3 of 16 and water were used for extraction and chloroform extract was found to contains maximum percentage yield hence was used for further investigation [20].

2.4. HPLC Analysis The extracts were filtered over a 0.45 µm filter paper, and the 50 µL filtrate was injected for quantitative HPLC analysis. HPLC analysis (Prominence LC-20 model with a photodiode array detector, Shimadzu, (California, USA) was achieved at 1 mL/min flow rate and using acetonitrile: 10 mM ammonium bicarbonate buffer as the mobile phase [21].

2.5. Preparation of Standard Solutions The aconitine standard was precisely weighed (1 mg) and dissolved in methanol (1 mL) to prepare a stock solution. The solution was employed for the preparation of calibration standards. A calibration curve was plotted in the range of 10–80 µg/mL by further diluting the stock solution with methanol.

2.6. Animals Sprague Dawley (SD) rats (Male; 250 g–280 gm) were purchased from the Central Drug Research Institute (CDRI, Lucknow, India). The accommodation of animals was as per standard laboratory condition in animal house of Integral University (Lucknow, India) and they were fed with a standard pellet diet. For acclimatization, they were housed (five animals/cage) in the restricted conditions of 25 2 C. Ethical consent (IU/IAEC/16/27) was obtained from the Institutional Animal Ethical ± ◦ Committee (IAEC) of Integral University (Lucknow, India).

Induction of Diabetes STZ (i.p) at a dose of 60 mg/kg, was used to induce diabetes by freshly dissolving it in cold buffer of citrate (4.5 pH, 0.1 M). Blood was taken from tail vein after 3 days and scrutinized for diabetes with the help of an Alere G1 glucometer, (Waltham, MA, USA). Rats with 250 mg/dL blood glucose level were considered as diabetic and used for further experiment. They were grouped into five cages of five rats each. The dose selection of different extracts was supported by our previous published in-vivo toxicity study [22]. Group-I was normal control (NC; 0.5% CMC), Group II was diabetic induced, Group-III, IV, and V diabetic treated with GMT-2.5, GMT-5, and pregabalin at a dose of 2.5, 5 and 10 mg/kg b.w., p.o. respectively [22].

2.7. Parameters

2.7.1. Blood Sugar Level and Body Mass At week zero and 2 weeks after of induction of diabetes, the body mass and blood sugar was measured.

2.7.2. Oral Glucose Tolerance Test (OGTT) Dextrose solution (40% w/v. p.o) was given to 12 h fasted animals. The level of glucose in blood was considered at specific intervals of 0, 1/2, 1 and 2 hr [22]. The OGTT measured the area under the curve (AUC) by the means of the formula below [23]: AreaUndertheCurve=(basalglycaemia+glycaemia30min) 0.25+(glycaemia 30 min + glycaemia 60 min) × 0.25 + (glycaemia 60 min + glycaemia 120 min) 0.5 × × Plants 2020, 9, 356 4 of 16

2.8. Behavioral Studies

2.8.1. Tail Immersion Test for the Assessment Hyperalgesia SD rat tail was sunken in a water bath at a temperature of water at 52.5 C 0.5 C until it flicked. ◦ ± ◦ This activity was observed for a period no more than 12 s. Hyperalgesia is a measure of reduction in time needed to withdrawal the tail [24].

2.8.2. Allodynia by Cold Water The procedure was same as above but in place of warm water we used cold water (10 0.5 C). ± ◦ 15 s was set as the cut off time. The shortening of tail immersion duration was recorded as allodynia. Three consecutive readings were recorded at a break of 30 min [25].

2.8.3. Motor Co-Ordination Test Rota rod (speed 25 rpm and time 2 min) was used for the estimation of motor coordination. The experiment was done at zero week and after two week of diabetes induction [26]. Time at which animal fall was recorded for the study.

2.8.4. Locomotor Activity The motor activity was done by using a photoactometer. The treated and normal control group animals were placed in the square closed field arena for a period of 5 min. The assembly was equipped with six photocells; the resulted were recorded [27].

2.9. Thio Barbituric Acid Reactive Substances (TBARS) Nerve homogenate (10%) was prepared; to that homogenate 1 mL was taken out in a tube and mixed with trichloroacetic acid (TCA, 0.5 mL; 30%) and thiobarbituric acid (TBA, 0.5 mL; 0.8%). Tubes are enclosed by means of aluminum foil and reserved on rotary water bath (30 min) and subsequently on the centrifuge (1080 g; 13 min). Absorbance of that supernatant was taken at 550 nm with reference to × the blank [28].

2.10. Reduced Glutathione-GSH Homogenized nerve tissue was mixed with ethylenediaminetetraacetic acid (EDTA, 0.02 M; 4 mL) along with distilled water (cold, 2 mL) and TCA (50%; 1 mL) and kept on vortex shaker for 10 min. Later on the content was transferred into EDTA tube for the centrifugation process at 1080 g for 12 min and supernatant was collected. Tris buffer (0.4 M, 4.0 mL, pH 8.9) was added to Supernatant (2 mL). The mixture was further put with 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB, 0.0.1 M; 0.1 mL) and processed for the absorbance at 412 nm against the blank at every 5 min [29].

2.11. Catalase One mL of tissue (nerve) was homogenized in 10 mL; 50 mM potassium phosphate buffer. The reaction mixture was then centrifuged at 3 C for less than 20 min at 3200 g. Approx 50 µL of ◦ × the supernatant was collected and mixed with hydrogen peroxide (2.95 mL). The absorbance at 240 nm was recorded at an interval of 1 min [30].

2.12. Superoxide Dismutase Assay The 100 µL supernatant was mixed with 2900 µL Tris HCl buffer (0.4 M, pH 8.9) making the final · volume to 3 mL. Pyrogallol in one forth ratio was used and absorbance was recorded at 420 nm at an interval of every minute for three minutes [31]. Plants 2020, 9, 356 5 of 16

2.13. Histopathology Animals were dissected and their respective sciatic nerves were removed and preserved in 10% formalin solution. Nerves were incised into segment and embedded in paraffin wax and further the sections were stained with the help of hematoxylin and eosin (H&E). The degenerative changes reflecting DN were observed with the help of a light microscope.

2.14. Development of Cell Culture and Treatment Protocol The human neuroblastoma SH-SY5Y cell line was procured from National Centre for Cell Sciences (NCCS, Pune, India. Cells were sustained in Eagle’s modified essential medium with L-glutamine (2.0 mM), NaHCO3 (1.5 g/L), antibiotic solutions (100 U/mL penicillin and 100 µg/mL streptomycin) and complemented with 10% (v/v) fetal bovine serum. The cells were incubated at a temperature of 37 ◦C in a 5% CO2 humidified atmosphere. Treatment of neuroblastoma cells with A. napellus extracts was carried out in following way: normal glucose (NG, 5.5 mM glucose), high glucose (HG, 50 mM glucose), 50 mM glucose along with A. napellus extracts at 10, 25 and 50 µg/mL respectively.

2.15. MTT Assay to Evaluate the Antiproliferative Activity of A. napellus Extracts on Neuroblastoma Cells The antiproliferative activity of A. napellus extracts on neuroblastoma cells was assessed by an MTT assay [32]. Neuroblastoma cells were seeded with a density of 1 105 cells/mL and treated with A. napellus × extracts at three different concentrations (10, 25 and 50 µg/mL) for 24 h. Later on, 10 µL of MTT reagent (5 mg/mL) was added, after that, 100 µL of dimethyl sulphoxide (DMSO) was added in the 96-well culture plate; absorbance was recorded by reader microplate (BIORAD-680, California, USA) at 540 nm. Effect of A. napellus extracts on cellular morphological changes was studied using contrast microscopy (ECLIPSETi-S, Nikon, Tokyo, Japan). In this experiment, glucose (50 mM) was used to create a high glucose concentration environment in the culture media comparable to that of human serum glucose concentrations. Further, to rationalize the neuroprotective effect of A. napellus extracts, neuroblastoma cells were incubated with NG (50 mM) and HG along with HG and different concentration of A. napellus extracts for 24 h.

2.16. Statistical Analysis Values acquired as mean SEM of five animals in every group. Statistical significance ± between groups was performed through Dunnett’s multiple comparisons using GraphPad Software (San Diego, CA, USA). All the parameters were compared at 5% level of significance.

3. Results

3.1. Yield of Plant Extract The percentage yield meant for the extracted material of A. napellus was 8.7% (w/w).

3.2. HPLC Analysis Reversed phase HPLC chromatography was performed to determine the levels of aconitine at 238 nm using acetonitrile: ammonium bicarbonate buffer as a mobile phase that may perhaps resolve aconitine from other constituents. We observed that the goat milk treated extract had less aconitine compared to the others. For HPLC chromatograms of standard aconitine, goat milk extract of A. napellus (GMT) and quantitative HPLC analysis please refer to the supplementary file.

3.3. Effect of the Chloroform Extract of Goat Milk Treated A. napellus on Body Mass: Group II animals confirmed significant (p < 0.05) body mass decline as compared with normal control. Treatment with A. napellus in group III, IV and V for two weeks created significant (p < 0.05) body mass gain as compared through DC (Table1). Plants 2020, 9, 356 6 of 16

Table 1. Effect of chloroform extract of goat milk treated A. napellus on body mass.

Change in Body Mass (g) Group(s) % Body Mass Variation At week 0 (g) At week 2 (g) % Gain % Loss NC 232.76 19.24 317.95 20.81 30.64 2.61 - ± ± ± DC 162.91 13.29 142.06 14.10 13.42 2.26 * ± ± ± GMT-2.5 238.28 17.69 260.10 16.27 10.23 3.40 # - ± ± ± GMT-5 203.48 7.53 264.75 6.54 30.04 6.71 # - ± ± ± Pregabalin 176.11 14.27 201.54 19.65 20.03 21.74 # - ± ± ± All the values were expressed as mean SEM; Where * denotes p < 0.05 compared to NC, while # denotes p < 0.05 compared to DC. NC = Normal Control± Group, DC = Diabetic Control Group, GMT-2.5 = Diabetic group receiving goat milk treated-chloroform extract of A. napellus (2.5 mg/kg), GMT-5 = Diabetic group receiving goat milk treated-chloroform extract of A. napellus (5 mg/kg).

3.4. Effect of the Extract of Goat Milk Treated A. napellus on Blood Sugar Level A significant (p < 0.05) rise in blood sugar level was observed in DC rats when compared with the NC group. Management of diabetic rats in groups GMT-2.5 and GMT-5 for 2 weeks showed significant (p < 0.05) decrease in blood sugar when compared with DC group while in the pregabalin treated group a non-significant decline in blood sugar level compared with the DC group was observed (Table2).

Table 2. Effect of chloroform extract of goat milk treated A. napellus on blood glucose level and oral glucose tolerance.

Blood Glucose Concentration (mg/dL) Oral Glucose Tolerance (h.mmol/L) Group(s) Initial Plasma Glucose Final Plasma Glucose Area Under Curve NC 82.40 3.47 85.60 2.94 12.94 0.75 ± ± ± DC 455.40 58.87 504.60 41.09 * 63.83 4.31 * ± ± ± GMT-2.5 514.00 67.62 204.80 14.02 # 30.85 1.72 # ± ± ± GMT-5 533.80 59.01 184.00 24.65 # 27.01 2.67 # ± ± ± Pregabalin 552.20 56.75 372.80 9.73 ns 51.33 1.61 ns ± ± ± All the values were expressed as mean SEM; where * denotes p < 0.05 compared to NC, while, # denotes p < 0.01 and ns p > ns compared to DC. NC = Normal± Control Group, DC = Diabetic Control Group, GMT-2.5 = Diabetic group receiving goat milk treated-chloroform extract of A. napellus (2.5 mg/kg), GMT-5 = Diabetic group receiving goat milk treated-chloroform extract of A. napellus (5 mg/kg)

3.5. Effect of the Extract of Goat Milk Treated A. napellus on Oral Glucose Tolerance: DC group animals had a significant (p < 0.01) increase in AUC while compared with NC group rats. Management of diabetic rats in groups GMT-2.5 and GMT-5 for 14 days produced significant (p < 0.05) decrease in AUC compared with DC animal group whereas pragabalin treated group produced non-significant decline in AUC compared with DC-treated animals (Table2).

3.6. Effect of the Extract of Goat Milk Treated A. napellus on Thermal Hyperalgesia: DC animals showed significant (p < 0.05) decline in withdrawal latency compared with control rats in tail immersion test. Treatment of diabetics rats in groups GMT-2.5, GMT-5 and pregabalin (10 mg/kg) significantly (p < 0.05) increased the latency of withdrawal compared with the DC group (Table3). Plants 2020, 9, 356 7 of 16

Table 3. Effect of chloroform extract of goat milk treated A. napellus on thermal hyperalgesia assessed by tail immersion (warm water) test.

Change in Thermal Hyperalgesia (in s) by Tail Immersion Group(s) (Warm Water) Test on Different Weeks 0 week 2 week NC 9.38 0.45 10.70 0.48 ± ± DC 5.94 0.36 3.62 0.20 * ± ± GMT-2.5 5.33 0.25 7.62 0.46 # ± ± GMT-5 5.25 0.32 8.97 0.50 # ± ± Pregabalin 7.34 0.66 11.75 0.07 # ± ± All the values were expressed as mean SEM; where * denotes p < 0.05 compared to NC, while, # denotes p < 0.05 compared to DC. NC = Normal Control± Group, DC = Diabetic Control Group, GMT-2.5 = Diabetic group receiving goat milk treated-chloroform extract of A. napellus (2.5 mg/kg), GMT-5 = Diabetic group receiving goat milk treated-chloroform extract of A. napellus (5 mg/kg)

3.7. Effect of the Chloroform Extract of Goat Milk Treated A. napellus on Cold Allodynia Diabetic animals in cold water showed (p < 0.05) a diminution in tail flick latency while compared to NC rats group, whereas treatment of diabetic animal with GMT (2.5 mg/kg and 5 mg/kg) as well as pregabalin (10 mg/kg) showed a significant reverse in allodynia due to cold as compared with DC group (Table4).

Table 4. Effect of chloroform extract of goat milk treated A. napellus on cold allodynia assessed by tail immersion (cold water) test.

Change in Cold Allodynia (s) on Different Weeks Groups(s) 0 week 2 week NC 13.00 0.30 13.08 0.45 ± ± DC 6.46 0.17 3.23 0.42 * ± ± GMT-2.5 6.96 0.41 12.08 0.37 # ± ± GMT-5 7.66 0.50 12.43 0.43 # ± ± Pregabalin 7.06 0.25 13.81 0.24 # ± ± All the values were expressed as mean SEM; where, * denotes p < 0.05 compared to NC, while # denotes p < 0.05 compared to DC. NC = Normal Control± Group, DC = Diabetic Control Group, GMT-2.5 = Diabetic group receiving goat milk treated-chloroform extract of A. napellus (2.5 mg/kg), GMT-5 = Diabetic group receiving goat milk treated chloroform extract of A. napellus (5 mg/kg)

3.8. Effect of the Chloroform Extract of Goat Milk Treated A. napellus on Motor Co-Ordination: A significant decrease (p < 0.05) in motor co-ordination was observed in DC compared to NC. Treatment with GMT produces observable change throughout the experimentation. Treatment of diabetic rats with GMT and pregabalin (2.5, 5 and 10 mg/kg respectively) considerably improved (p < 0.01) motor co-ordination as compared to DC group (Table5).

3.9. Effect of Chloroform Extract of Goat Milk Treated A. napellus on Locomotors Activity DC control group showed marked decrease in locomotor activity compared to NC. Treatment groups showed a significant improvement (p < 0.05) in locomotor activity as compared to DC group after two week of treatment (Table5). Plants 2020, 9, 356 8 of 16

Table 5. Effect of chloroform extract of goat milk treated A. napellus on motor co- ordination and locomotor activity.

Motor Coordination (s) Locomotor Activity (Count in 5 min) Group(s) 0 week 2 week 0 week 2 week NC 32.65 1.57 33.10 0.94 140.80 1.24 143.80 4.20 ± ± ± ± DC 10.82 0.44 10.94 0.49 * 65.00 3.39 36.00 2.77 * ± ± ± ± GMT-2.5 10.90 0.38 16.56 0.66 # 66.60 4.09 128.8 8.49 # ± ± ± ± GMT-5 11.00 0.83 21.68 0.66 # 64.40 4.35 133.6 6.13 # ± ± ± ± Pregabalin 11.38 0.41 28.20 0.87 # 63.80 5.73 140 5.32 # ± ± ± ± All the values were expressed as mean SEM; where, * p < 0.05 compared to NC, while, # denotes p < 0.05 compared to DC. NC = Normal Control Group, DC± = Diabetic Control Group, GMT-2.5 = Diabetic group receiving goat milk treated-chloroform extract of A. napellus (2.5 mg/kg), GMT-5 = Diabetic group receiving goat milk treated chloroform extract of A. napellus (5 mg/kg)

3.10. Effect of the Chloroform Extract of Goat Milk Treated A. napellus on Oxidative Biomarkers The nerve homogenate of DC group animals shows an elevation in TBARS and decline in SOD, GSH and catalase activity as compared to NC group. Whereas in GMT and Pregabalin treated group a significant decrease in dose dependent manner (p < 0.05) TBARS level and increase in SOD, catalase and GSH level was observed as compared with DC (Table6).

Table 6. Effect of chloroform extract of goat milk treated A. napellus on oxidative biomarkers.

Oxidative Parameters Group(s) TBARS SOD CATALASE GSH (m mole/mg Protein) (units/mg of Protein) (units/mg of Protein) (m mole/mg Protein) CONTROL 1.12 0.05 1.83 0.05 2.78 0.05 3.26 0.16 ± ± ± ± DC 2.85 0.22 * 0.64 0.08 * 0.56 0.02 * 1.564 0.10 * ± ± ± ± GMT-2.5 1.63 0.11 # 1.44 0.07 # 1.19 0.02 # 1.934 0.06 # ± ± ± ± GMT-5 1.22 0.09 # 1.72 0.04 # 2.18 0.05 # 2.194 0.14 # ± ± ± ± Pregabalin 2.09 0.07 # 1.95 0.01 # 2.71 0.03 # 2.468 0.05 # ± ± ± ± All the values were expressed as mean SEM; where, * denotes p < 0.05 compared to NC, while, # denotes p < 0.05 compared to DC. NC = Normal Control± Group, DC = Diabetic Control Group, GMT-2.5 = Diabetic group receiving goat milk treated-chloroform extract of A. napellus (2.5 mg/kg), GMT-5 = Diabetic group receiving goat milk treated-chloroform extract of A. napellus (5 mg/kg)

3.11. Histopathology of the Sciatic Nerve Histopathological examination of the sciatic nerve showed that H&E staining of the nerve tissue in NC shows no inflammatory and degenerative changes with regular collusion of nerve fibers along with stretched Schwann cells. The section of DC group shows nerve deranged cells and swelling in axons. The animals treated with GMT extract at a dose of 2.5 mg/kg b.w., shows constriction injury of the sciatic nerve and the nerve fibers shows deranged axonal swelling. The GMT-5 diabetic rats treated with GMT extract 5 mg/kg b.w., p.o shows improvement in the myelination and degenerative changes of the nerve fibers with mild derangement of nerve fibers. Complete restoration of neuronal integrity was not achieved. While the diabetic animals treated with pregabalin shows uniformly arranged fibres and decreased axonal swelling (Figure1).

3.12. Effect of A. napellus Extract on Morphological Variation and Cell Viability on Neuroblastoma Cells The neuroblastoma cells were incubated for 24 h after the treatment of NG, HG and HG plus A. napellus extracts with three different concentrations (10, 25 and 50 µg/mL) respectively. When compared with normal control, cells treated with HG showed cellular shrinkage and disintegrated bodies. Plants 2020, 9, x FOR PEER REVIEW 8 of 16

Diabetic group receiving goat milk treated-chloroform extract of A. napellus (2.5 mg/kg), GMT-5 = Diabetic group receiving goat milk treated chloroform extract of A. napellus (5 mg/kg)

3.9. Effect of Chloroform Extract of Goat Milk Treated A. napellus on Locomotors Activity DC control group showed marked decrease in locomotor activity compared to NC. Treatment groups showed a significant improvement (p < 0.05) in locomotor activity as compared to DC group after two week of treatment (Table 5).

3.10. Effect of the Chloroform Extract of Goat Milk Treated A. napellus on Oxidative Biomarkers The nerve homogenate of DC group animals shows an elevation in TBARS and decline in SOD, GSH and catalase activity as compared to NC group. Whereas in GMT and Pregabalin treated group a significant decrease in dose dependent manner (p < 0.05) TBARS level and increase in SOD, catalase and GSH level was observed as compared with DC (Table 6).

Table 6. Effect of chloroform extract of goat milk treated A. napellus on oxidative biomarkers.

Oxidative Parameters Group(s) TBARS SOD CATALASE GSH (m mole/mg protein) (units/mg of protein) (units/mg of protein) (m mole/mg protein) CONTROL 1.12 ± 0.05 1.83 ± 0.05 2.78 ± 0.05 3.26 ± 0.16 DC 2.85 ± 0.22* 0.64 ± 0.08* 0.56 ± 0.02* 1.564 ± 0.10* GMT-2.5 1.63 ± 0.11# 1.44 ± 0.07# 1.19 ± 0.02# 1.934 ± 0.06# GMT-5 1.22 ± 0.09# 1.72 ± 0.04# 2.18 ± 0.05# 2.194 ± 0.14# Pregabalin 2.09 ± 0.07# 1.95 ± 0.01 # 2.71 ± 0.03# 2.468 ± 0.05# All the values were expressed as mean ± SEM; where, * denotes p < 0.05 compared to NC, while, # denotes p < 0.05 compared to DC. NC = Normal Control Group, DC = Diabetic Control Group, GMT- 2.5 = Diabetic group receiving goat milk treated-chloroform extract of A. napellus (2.5 mg/kg), GMT- 5 = Diabetic group receiving goat milk treated-chloroform extract of A. napellus (5 mg/kg)

3.11. Histopathology of the Sciatic Nerve Histopathological examination of the sciatic nerve showed that H&E staining of the nerve tissue in NC shows no inflammatory and degenerative changes with regular collusion of nerve fibers along with stretched Schwann cells. The section of DC group shows nerve deranged cells and swelling in axons. The animals treated with GMT extract at a dose of 2.5 mg/kg b.w., shows constriction injury of the sciatic nerve and the nerve fibers shows deranged axonal swelling. The GMT-5 diabetic rats treated with GMT extract 5 mg/kg b.w., p.o shows improvement in the myelination and degenerative changes of the nerve fibers with mild derangement of nerve fibers. Plants 2020, 9, 356 9 of 16 Complete restoration of neuronal integrity was not achieved. While the diabetic animals treated with pregabalin shows uniformly arranged fibres and decreased axonal swelling (Figure 1).

Plants 2020, 9, x FOR PEER REVIEW 9 of 16

control group shows axonal swelling. (C) Treated with GMT-2.5 shows axonal swelling of nerve Figure 1. fibers.FigureAhematoxylin (1.D )A This hematoxylin group andtreated and eosineosin with section sectionGMT-5 of showingsciatic of sciatic nerve the showsmild nerve derangements (A) shows Control (animalA of) nerve Control shows fibers. treated animal (E) shows treated withpregabalinwith 0.5%0.5% CMC treated CMC with group with normal shows normal arrangement uniformly arrangement ofarranged nerve fibers nerve of nervewith fibers. elongated fibers Schwann with elongated cells. (B) Diabetic Schwann cells. (B) Diabetic control group shows axonal swelling. (C) Treated with GMT-2.5 shows axonal swelling 3.12. Effect of A. napellus Extract on Morphological Variation and Cell Viability on Neuroblastoma Cells of nerve fibers. (D) This group treated with GMT-5 showing the mild derangements of nerve fibers. (E) pregabalinThe neuroblastoma treated group cells shows were uniformly incubated arrangedfor 24 h after nerve the fibers.treatment of NG, HG and HG plus A. napellus extracts with three different concentrations (10, 25 and 50 µg/mL) respectively. When compared with normal control, cells treated with HG showed cellular shrinkage and disintegrated The resultsbodies. as found in Figure2A showed that the unexposed cells, in addition as NG, were smooth and in good physicalThe results shape. as found Nevertheless, in Figure 2A morphologicalshowed that the unexposed transformation cells, in addition was observed as NG, were in HG-treated cells as revealedsmooth and by thein good photomicrographs. physical shape. Nevertheless, morphological transformation was observed in HG-treated cells as revealed by the photomicrographs.

Figure 2. FigureIn vitro 2. Inneuroprotective vitro neuroprotective eff ectseffects of ofA. A. napellusnapellus extractextract against against mammalian mammalian neuroblastoma neuroblastoma SHSY5Y cellsSHSY5Y (A )cells Photomicrograph (A) Photomicrograph of of SHSY5Y SHSY5Y cell line line treated treated with with NG, HG NG, and HG 10 to and 50 µg/mL 10 to 50 µg/mL concentrationconcentration of ACON of extractACON extract (B) Dose-response (B) Dose-response eff ectseffects of of NG, NG, HG HG and andA. A. napellusnapellus extract at at different different concentrations on SHSY5Y cell line for 24 h. All values were expressed as Mean ± SEM. *p < concentrations on SHSY5Y cell line for 24 h. All values were expressed as Mean SEM. *p < 0.05, 0.05, high glucose vs normal glucose group; #p < 0.05, as compared to high glucose. ± high glucose vs normal glucose group; #p < 0.05, as compared to high glucose. The cell viability was reduced to 31.34% (p < 0.05) in MTT assay when compared against The cellcontrol viability and NG was treated reduced cells in to MTT 31.34% assay. (p Diminished< 0.05) in cell MTT viability assay was when noticeably compared improved against to control and NG treatedaround 56.01, cells 73.55 in MTT and 97.23 assay. % ( Diminishedp < 0.05) at 10, cell25 and viability 50 µg/mL was concentration noticeably of GMT improved extract, to around respectively. The data showed a significant rise in cell viability equal to normal level (Figure 2B). 56.01, 73.55 and 97.23 % (p < 0.05) at 10, 25 and 50 µg/mL concentration of GMT extract, respectively. The data showed3.13. Effect a of significant Cow Milk Treated rise Extract in cell of viability A. napellus equal (CMT) toon normalMorphological level Variation (Figure and2 B).Cell Viability on Neuroblastoma Cells Normal control group and NG treated cells remained even and well, as compared with CMT treated groups (10, 25 and 50 µg/mL). The photomicrographs revealed that macroscopic diversity Plants 2020, 9, 356 10 of 16

3.13. Effect of Cow Milk Treated Extract of A. napellus (CMT) on Morphological Variation and Cell Viability on Neuroblastoma Cells Normal control group and NG treated cells remained even and well, as compared with CMT treated groups (10, 25 and 50 µg/mL). The photomicrographs revealed that macroscopic diversity was lost in cells treatedPlants with 2020, HG.9, x FOR The PEER high REVIEW glucose-treated cells show shrinkage to that of normal10 cells. of 16 Whereas µ the CMT treatedwas lost in group cells treated shows with results HG. The similar high glucose-treated to normal control cells show at shrinkage a dose of to 50that ofg/ mLnormal (Figure 3A). MTT datacells. revealed Whereas that the CMT HG treated reduced group viability shows results of cells similar to aboutto normal 31.34 control % at (p a

Figure 3. InFigure vitro 3.neuroprotective In vitro neuroprotective effects effects of CMT of extractCMT extract against against mammalian mammalian neuroblastoma neuroblastoma SHSY5Y cells (A) PhotomicrographSHSY5Y cells (A) Photomicrograph of SHSY5Y cell of SHSY5Y line treated cell line with treated NG, with HG NG, and HG 10 and to 5010 µtog 50/mL µg/mL concentration concentration of CMT extract (B) Dose-response effects of NG, HG and CMT extract at different of CMT extract (B) Dose-response effects of NG, HG and CMT extract at different concentrations on concentrations on SHSY5Y cell line for 24 h. All values were expressed as Mean ± SEM *p < 0.05, HG SHSY5Y cell line for 24 h. All values were expressed as Mean SEM *p < 0.05, HG vs NG; #p < 0.05, vs NG; #p < 0.05, as compared to high glucose. ± as compared to high glucose. 3.14. Effect of Goat Milk Treated Extract of A. napellus (GMT) on Morphological Variation on 3.14. EffectNeuroblastoma of Goat Milk Cells Treated Extract of A. napellus (GMT) on Morphological Variation on Neuroblastoma Cells The SHSY5YThe SHSY5Y cells were cells were incubated incubated for for 24 24 hh with HG, HG, NG NG and and different diff erentconcentrations concentrations of GMT of GMT (10, 25 and 50 µg/mL). The morphology of cells treated with NG was similar to control, showing in µ (10, 25 andboth 50 casesg/mL). a healthyThe morphologyand smooth texture. of cells Opposite treated, the with cells NG treated was similarwith HGto showed control, shrinkage showing in both cases a healthy(Figure and 4 A). smooth The cell texture.viability results Opposite, are shown the cells of Figure treated 4B. The with MTT HG assay showed showshrinkage a decrease in (Figure 4A). The cell viabilitycell viability results to 31.34% are shown in HG-treated of Figure cells4B. compar The MTTed to assaycontrol show(100%) a and decrease NG cells in was cell significant viability to 31.34% in HG-treated(p < 0.05) cells (Figure compared 5B). The to controlcell viability (100%) had a and signif NGicant cells recovery was significant (56.01, 73.55 (andp < 0.05)97.23% (Figure for 10, 255B). The cell and 50 µg/mL, respectively) when the cells were treated with GMT extract. viability had a significant recovery (56.01, 73.55 and 97.23% for 10, 25 and 50 µg/mL, respectively) when the cells were treated with GMT extract.

3.15. Effect of Water Treated Extract of A. napellus (WT) on Cell Viability and Morphological Variation in Neuroblastoma Cells Normal cells and cells treated with normal glucose remained smooth and healthy. The cells treated with HG revealed morphological changes and cellular shrinkage and fragmented body as compared to normal cells. MTT data revealed that treatment with high glucose showed a decline in cell Plants 2020, 9, 356 11 of 16 viability approximately to 31.34% (p < 0.05) when compared with the normal treated cells. Nonetheless, diminished cell viability was noticeably improved to approximately 51.45, 68.55 and 95.02% (p < 0.05) (Figure5A,B). Plants 2020, 9, x FOR PEER REVIEW 11 of 16

Figure 4. In vitroFigure neuroprotective 4. In vitro neuroprotective effects ofeffects GMT of GMT extract extract against againstmammalian mammalian neuroblastoma neuroblastoma SHSY5Y cells (A) PhotomicrographSHSY5Y cells (A) of Photomicrograph SHSY5Y cell of lineSHSY5Y treated cell line with treated NG, with HG NG, andHG and 10 10 to to 50 50 µµg/mLg/mL concentration concentration of GMT extract (B) Dose-response effects of NG, HG and GMT extract at different of GMT extractconcentrations (B) Dose-response on SHSY5Y cell eff lineects for of24 h. NG, All values HG andwere expressed GMT extract as Mean ± at SEM, diff *perent < 0.05, concentrationsHG on SHSY5Y cell linevs NG; for # 24p < 0.05, h.All as compared values to were high glucose. expressed as Mean SEM, *p < 0.05, HG vs NG; #p < 0.05, ± as compared3.15.Plants to Effect 2020 high, 9of, x glucose.Water FOR PEER Treated REVIEW Extract of A. napellus (WT) on Cell Viability and Morphological Variation12 in of 16 Neuroblastoma Cells Normal cells and cells treated with normal glucose remained smooth and healthy. The cells treated with HG revealed morphological changes and cellular shrinkage and fragmented body as compared to normal cells. MTT data revealed that treatment with high glucose showed a decline in cell viability approximately to 31.34% (p < 0.05) when compared with the normal treated cells. Nonetheless, diminished cell viability was noticeably improved to approximately 51.45, 68.55 and 95.02 % (p < 0.05) (Figures 5A and B).

Figure 5. In vitroFigureneuroprotective 5. In vitro neuroprotective effects effects ofWT of WT extract extract against against mammalian mammalian neuroblastoma neuroblastoma SHSY5Y SHSY5Y cells (A) Photomicrographcells (A) Photomicrograph of SHSY5Y of cellSHSY5Y line cell treated line treated with with NG, NG, HG HG and and 10 10 to to 50 50µ µg/mLg/mL concentration concentration of WT extract (B) Dose-response effects of NG, HG and WT extract at different of WT extract (concentrationsB) Dose-response on SHSY5Y ecellffects line for of 24 NG, h. Phot HGomicrographs and WT were extract taken with at dian ffinvertederent phase concentrations on SHSY5Y cell linecontrast for 24 microscope. h. Photomicrographs Scale bar = 100 µm. All were the alues taken are expressed with an as invertedmean ± SEM. phase *p < 0.05, contrast HG vs microscope. NG; #p < 0.05, as compared to high glucose. Scale bar = 100 µm. All the alues are expressed as mean SEM. *p < 0.05, HG vs NG; #p < 0.05, ± as compared4. toDiscussion high glucose. There is no single treatment available which can treat or reverse neuropathy caused due to diabetes [33]. The naturally occurring flora is the largest and striking resource of novel drugs. A. napellus is a medicinal plant extensively used for the management of sciatica and rheumatic algesia [34,35]. Aconite is recognized as a valuable natural product for countering the symptoms of diabetic neuropathy e.g., sensation, cold and algesia [36]. Moreover, there are very few reports available that confirms its use as a neuroprotective agent [37]. The present research work was performed to confirm its use as a neuroprotective agent in STZ induced diabetic neuropathy. Streptozotocin (STZ) is an agent which induces diabetes in experimental laboratory animals. STZ inhibit hypoglycaemia and cause injury of exocrine gland of β-cells of pancrease. STZ inhibit the biogenesis and secretion of hypoglycaemic agent especially insulin by interfering with aldose hexose and glucose metabolism and consumption of oxygen. In animal models STZ induces diabetes mellitus as a result of destruction of the exocrine gland i.e. β cell of the pancreas in a similar manner as that of insulin dependent diabetes mellitus in humans. Diabetes mellitus has been characterized by the symptoms like excess of sugar in the urine, excessive appetite, decrees levels of insulin in the plasma, lipemia, and alteration in body mass [38,39]. Therefore within the DC group, we tend to additionally discover that there is a significant (p < 0.01) increase in blood sugar when contrast with normal control animals. As per Nasiry, et al. [40] the body weight of animals treated with STZ was significantly declined (p < 0.001) as compared to NC; in our study, we also observed identical trend as in above study; it demonstrate significant (p < 0.01) decline in body Plants 2020, 9, 356 12 of 16

4. Discussion There is no single treatment available which can treat or reverse neuropathy caused due to diabetes [33]. The naturally occurring flora is the largest and striking resource of novel drugs. A. napellus is a medicinal plant extensively used for the management of sciatica and rheumatic algesia [34,35]. Aconite is recognized as a valuable natural product for countering the symptoms of diabetic neuropathy e.g., sensation, cold and algesia [36]. Moreover, there are very few reports available that confirms its use as a neuroprotective agent [37]. The present research work was performed to confirm its use as a neuroprotective agent in STZ induced diabetic neuropathy. Streptozotocin (STZ) is an agent which induces diabetes in experimental laboratory animals. STZ inhibit hypoglycaemia and cause injury of exocrine gland of β-cells of pancrease. STZ inhibit the biogenesis and secretion of hypoglycaemic agent especially insulin by interfering with aldose hexose and glucose metabolism and consumption of oxygen. In animal models STZ induces diabetes mellitus as a result of destruction of the exocrine gland i.e. β cell of the pancreas in a similar manner as that of insulin dependent diabetes mellitus in humans. Diabetes mellitus has been characterized by the symptoms like excess of sugar in the urine, excessive appetite, decrees levels of insulin in the plasma, lipemia, and alteration in body mass [38,39]. Therefore within the DC group, we tend to additionally discover that there is a significant (p < 0.01) increase in blood sugar when contrast with normal control animals. As per Nasiry, et al. [40] the body weight of animals treated with STZ was significantly declined (p < 0.001) as compared to NC; in our study, we also observed identical trend as in above study; it demonstrate significant (p < 0.01) decline in body weight compared to animals in NC group [40]. Whereas the GMT and pregabaline treated animals showed marked increase (p < 0.01) in body mass compared to diabetic animals. Chronic form of diabetes mellitus induced change in perception of pain and harm along with loss of motor co-ordination [41]. We observed a decrease in tail flick latency in the animal treated with STZ during the experimentation in warm and icy water immersion test. The current experiment demonstrates the neuroprotective outcomes of A. napellus extracts owing to considerable improvement in locomotor activity, motor coordination, biochemical and histological examination. A decrease in blood sugar concentration was observed in A. napellus treated group as compared with diabetes induced group, which indicates that protective property of GMT on neuropathy associated with diabetes was independent of spectacular interventions with blood sugar concentration [42]. Pregabaline is the drug of choice for the treatment of diabetic neuropathy [43]. It acts as an antagonist of α2 delta subunit of calcium channel, pregabalin interacts with the voltage-gated calcium channels and shows antiallodynic effects [44]. For that reason pregabalin is selected as a drug for standard control. A change in sensitivity to touch and tenderness was reported in the animals after six week of inducing diabetes, these changes may occur due to significant hyperalgesia resulted by change in neurotransmitters [45]. The same was observed in our research that after two weeks of treatment animals showed a significant elevation (p < 0.001) in hyperalgesia. Treatment with GMT and pregabilin control treated animals with extract and standard markedly reversed the cold allodynia. Behavioral markers (e.g., thermal hyperalgesia, cold allodynia, motor coordination and locomotor activity) were observed as evaluation parameters for diabetic neuropathy [46]. The treatment with GMT and pregabalin showed clear decline in withdrawal latency while compared with control animals (p < 0.01), which illustrate that there is deterioration of sensory motor reflux. It has already been reported previously that pathogenicity of diabetic neuropathy is associated with loss of myelin sheath in nerve fibers, atrophy and deterioration of nerve fibers [47]. We observed that treatment with GMT in a dose dependent manner prevents the detoriation of nerve fibres. Fuzi, a Chinese folk medicine has been shown to efficiently diminish the elevated levels of ROS induced by glucose in Schwann cells [36]. ROS has been projected as potential mechanisms for HG-induced Schwann cell dysfunction in diabetic neuropathy [48,49]. The current study shows that in HG-induced neuropathy the SHSY-5Y cells shows a significant elevation in reaction oxygen level which Plants 2020, 9, 356 13 of 16 ultimately leads to apoptosis and death as compared with normal glucose cells, our observation was in agreement with earlier studies [36]. To promote this research we worked on the beneficial consequence of A. napellus extract, GMT, CMT and WT on HG-induced toxic effect, and observed that all these efficiently diminished the level of ROS, provoked by HG in a dose reliant manner. We have also observed that percentage of apoptosis or cell death significantly decreases in HG cells compared to NG cells.

5. Conclusions GMT-treated groups have indicated a neuroprotective role by improving the nerve dysfunctioning in STZ induced diabetic neuropathy in an experimental animal model. Processed and unprocessed extract of A. napellus tubers showed improvement in cell viability but the best results were obtained from GMT treated groups. Our results give supportive information to already existing research concerning the neuroprotective role of A. napellus extract. From our in vitro data, it can be concluded that GMT could be selected further for clinical analysis and its use in diabetic pathology. Also, more studies are warranted to explore the role of active principles isolated from A. napellus for their additional clinical use.

Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/3/356/s1, Figure S1: Calibration curve of mean area against concentration of standard aconitine, Table S1: Peak area and percentages of different components with specific retention time (Rt) in HPLC chromatograms of standard aconitine and Goat milk treated chloroform extract of Aconitum napellus. Author Contributions: Conceptualization, A.S., H.H.S. and B.D.; Data curation, A.S., H.H.S. and R.K.D.; Formal analysis, S.H.A., H.A.K., and P.A.; Funding acquisition, S.H.A, H.A.K., P.A.; Investigation, A.S., R.K.D., and S.S.; Methodology, A.S.; Project administration, A.S., and B.D.; Resources, S.H.A., H.A.K., B.D. and A.K.; Software, S.H.A., H.A.K.; Supervision, B.D.; Validation, H.H.S.; Writing—original draft, A.S., and B.D.; Writing—review & editing, R.K.D., S.S., A.K., S.H.A. and P.A. All authors have read and agreed to the published version of the manuscript Funding: This research was funded by RGP-1435-066 and 201213-MANF-2012-13-MUS-UTT-14306. Acknowledgments: Ambreen Shoaib acknowledges to University Grant Commission, Government of India for providing Fellowship (201213-MANF-2012-13-MUS-UTT-14306), Era University and Integral University (IU/R&D/2018-MCN000500), Lucknow for providing research facilities to carry out a part of research work. The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding the Research Group No. RGP-1435-066. Conflicts of Interest: We declare no conflict of interest.

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