Pushpraj S Gupta ¹, Sunil K Singh ², Abhishek K Tripathi ^{1 *}

The objective of the study was to prepare Bauhinia variegata loaded nanoemulsion(formulation and determine the efficacy of herbal drug formulation against diabetic peripheral neuropathic pain through acupuncture technique.

Nine different ba tches of nanoemulsion (NE1 NE9) of BVN was prepared by varying the S mix ratio and the concentration of oil. BVN was characterized to determine particle size, shape, zeta potential, polydispersity index, optical transmittance, drug release profile and stora ge stability. The optimized formulation was subjected to plantar test, behavioral tests of neuropathic pain and Von Frey filament stimulation test. Diabetes was induced by intraperitoneal injection of freshly prepared solution of Streptozotocin (60 mg/kg) to the experimental rats. Animals were made diabetic divided into four groups, Group I was untreated normal control group, Group II was diabetic control group, Group III was Bauhinia variegata extract ( treated group (100 mg/kg/day, p.o) and Group IV was BVN treated groups (100 mg/kg/day, p.o) acute and chronically.

The prepared B. variegata loaded nanoemulsion was nanosized (124 nm), spherical, uniform and stable over the period of 180 days with no change in physiochemical properties. The bl ood glucose and body weight of animals was normalizing after four weeks of treatment that was significant with BVN in comparison to diabetic control group. The chronic administration of BVN significantly (P<0.001) decreased hind paw withdrawal latency an d attenuated mechanical allodynia as compared with diabetic rats.

Thus, BVN may be an effective drug formulation against diabetic peripheral neuropathic pain.

Neuropathic pain, plantar test, Von Frey filament stimulation, Bauhinia variegata, diabetes, nanoemulsion

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Diabetes mellitus (DM) is a metabolic syndrome that have been affected people life around the globe, it is due to the body’s inability to either produce or use insulin [1]. In DM, chronic micro and macro vascular effects such as neuropathy, cardiovascular disease and stroke due to constant hyperglycemia [2, 3]. Neuropathic pain is most crucial complications of diabetes mellitus, it is due to severe muscle aches and a continuous burning or tinglin g symptoms mostly in lower extremities. Allodynia and hyperalgesia may be appeared in patients having diabetic peripheral neuropathic pain [4]. Conventional drug delivery systems for treatment of various metabolic disorder often face some limitations, lack o f efficacy or availability of drug in body due to improper or ineffective dosage, as well as reduced potency due to first pass metabolism. Nanotechnology has been diverged class of science and its application in several fields with nanometric range of obje cts to overcome trouble related to drug administration system. Application of nanocarriers, nanoemulsion and other nanotechnological approaches leads to enhance activity against the threating diseases, fewer side effects, proper drug accumulation and adsor ption in target. Nanoemulsions are the advanced nanocarriers for targeted drug delivery of several oral hypoglycemic drugs in comparison to other conventional oral hypoglycemic drugs. Now a days these modern approaches are using frequently to control hyper glycemia and becoming the most promising technology. Lipid nanoparticles and nano emulsion are an effective and novel colloidal delivery system for drug targeting, cosmetic and pharmaceutical products [5]. Bauhinia variegata belongs to family Leguminosae, it belongs to genus Bauhinia that comprises more than 300 species of plants all over the world [6] Bauhinia is also known as Cow's hoof because of its leaves shape. This species of Bahuinia is found in throughout the tropical zones, especially in Asian, A frican and South American countries. Bauhinia is used as a folk medicine in some parts of these zones by local people [7] Biological studies of Bauhinia extracts have affirmed the free radical reducing and blood lowering activities by its polyphenol and f lavonoid [8] Several glycosylated and aglycone flavonoids have been also reported for Bauhinia variegata [9] The present study was designed to investigate the effect of Bauhinia based nanoemulsion for treatment of diabetic peripheral neuropathic pain.

Streptozotocin, neem oil, coconut oil, cinnamon oil, black cumin seed oil, soyabean oil, span 80, tween 80, tween 20, ethanol, acetone and methanol were procured from Sigma Aldrich Co., USA.

The flowers of Bauhinia variegata were collected from the local surroundings of Allahabad, Uttar Pradesh. The air dried and powdered flowers (1.2 kg) of B. variegata were extracted with petroleum ether to remove fatty s ubstances. The mark was further extracted with 95% ethanol by hot percolation method. The extract was filtered and concentrated under vacuum at 40 and stored in desiccator. The percentage yield of ethanolic extract (BVE) was found to be 20.8% w/w.

Selection of components ( surfactants and co surfactants) is very importance for preparing a stable nanoemulsion system. It was selected by determining the solubility of B. variegata extrac tin different oils like neem oil, coconut oil, cinnamon oil, black cumin seed oil, soyabean oil; surfactants like span 80, tween 80, tween 20 and co surfactants like ethanol, acetone and methanol. The physical stability of the excipient and the plant extr act was evaluated by dissolving 1mg of the extract in 1ml of the above mentioned excipients at room temperature 25±1℃ and accelerated temperatures 40 ± 1℃ for 8 weeks and visually observed with eyes [10].

Ultrasonic emulsification method was used for preparation of nanoemulsion. Based on the solubility study, it was found that B. variegata extract was completely soluble in cinnamon oil, ethanol and Tween 80. Briefly, accurately weighed quantity of B. variegata dried extract was dissolved in measured quantity of ethanol (co surfactant) followed by addition of measured amount of Tween 80 (sur factant). Smix ratios were prepared by addition of varying concentration of surfactant and co surfactant. The mixture was homogenized with the help of a magnetic stirrer. Cinnamon oil was added to this mixture followed by addition of distilled water and a llowed to get a uniform, homogenized emulsion. The prepared emulsion was sonicated for 30 min to get. nanoemulsion. Nine different batches (NE1 - NE9) of B. variegata nano emulsion was prepared by varying the Smix ratio and the concentration of oil as sh own in (Table 1) [11].

Particle size of BVN was determined using optical microscope. The size distribution analysis was performed using particle size analyzer. The microstructure of BVN was characterized using TEM Hitachi (H-7500) at room tempe rature. The photo micrographs of BVN were obtained by drying on a microscopic carbon coated grid and viewed under microscope after staining at suitable magnification.

Zeta potential of BVN was determined using zeta sizer version 6.2. Sample (1 ml) was placed in a disposable zeta cell. Analysis was performed by inserting palladium electrode in distilled water in the cuvette at 25°C. All values were determined in triplicate.

In vitro release study of BVN was evaluated using Franz diffusion cell an artificial cellulose acetate membrane. Franz diffusion cell was maintained at 37 ± 1°C The membrane was equilibrated before application of the nanoemulsion equivalent to 10 mg of drug onto t he donor side. Aliquots of 1.0 ml were removed at suitable time interval and observed for drug content using UV visible spectrophotometer after appropriate dilutions [12].

Stability of BVN was determined for a period of 6 months. The formulation was placed at room temperature and studied for physical stability such as creaming, phase separation, or flocculation, accelerated centrifugation cycle and chemical stability such as particle size, drug content, and zeta potential determinati ons after 6 months of storage.

Male albino Wistar rats weighing 220 - 280 g were obtained from inbred animal house of C.D.R.I., Lucknow and maintained at standard environmental conditions such as temperature 25± 2℃ and relative humidity 45 - 55%. Animals were free access to standard pelleted diet and water ad libitum. The study was approved by IAEC (UIP/IAEC/ April 2015/03).

Stability of Streptozotocin (STZ) was given in the dose of 60 mg/kg, i/p to induce diabetes in animals. Hyperglycemia was produced within 2 days of STZ injection. The animals were included in the study showing blood glucose level more than 250 mg/dL (Fox et al., 1999)

Animals were selected randomly and divided into six groups each containing 10 animals after assessment of neuropathic pain at 7 th day of diabetes induction. Group I was untreated normal control group, Group II was diabetic control group, Group III was Bauhinia variegata extract (BVE) treated group (100 mg/kg/day, p.o) and Group IV was BVN treated groups (100 mg/kg/day, p.o); acute and chronically. Group V and VI received naloxone (1 mg/kg, i.p.) 30 min before BVN administration. The treatment schedule was single dose of BVN 30 min before pain assessment for acute condition; however, for chronic treatment administration of BVN was started from 7 th day till 21 st day injected once a day.

Thermal stimulus and mechanical stimuli causing hyperalgesia and allodynia, respectively was measured by radiant heat plantar and von Frey test, respectively. The behavioral tests were performed during day time after cage exploration and m ajor grooming activities ceased. The measurement of neuropathic pain, plasma glucose level and body weight was performed before the experiment and on 7, 14 and 21 day after induction of diabetes [13].

Rats were placed in a plexiglass enclosure with transparent glass floor. Infrared beam was passed as a heat source at the hind paw surface. Thermal withdrawal latency was determined to calculate the latency between heat stimulus and withdrawal of paw. It w as measured in seconds and paw latency was measured in every 5 min. Tissue damage was avoided with a cut off time of 22 sec. Hyperalgesia was scored by measuring mean latency of the withdrawal responses [14].

The response of hind paw withdrawal was measured using von Frey filament to quantify mechanical allodynia. VFFS was studied with the bending forces ranging from 2 to 60 g (Stoling Inc. Wood Dale, IL). The animals were allowed for 15 min to adapt or to cease exploratory behavior by placing on mesh floor enclosed with plastic chamber. The stimulation was provided at the plantar surface of paw and it was continued till the rat removed its paw. The withdrawal threshold was determined by change in filamen t size in three consecutive applications [15].

Data are represented as mean SEM. Statistical analysis was performed using MS excel and one way ANOVA by Bonferroni statistical hypothesis using Graph Pad Prism 8.0.2, statistically different at *P<0.05, P< 0.01, P < 0.001 in comparison to diabetic control group.

The characterizat ion of prepared nine nanoemulsion formulations of B. variegata is shown in (Table 2) The globule size was decreasing with increase in surfactant concentration upto certain extent i.e. NE1 - NE6 (311.00 - 124.33 nm); after that, increase in globule size was ob served. The nanoemulsion preparations NE5, NE6, NE7 and NE8 were showing small globule size, in which globule size of NE6 (124.33 nm) was smallest among all formulations. NE6 was also suitable with PDI, zeta potential and transmit- tance. Thus, NE6 was selected for further evaluation due to its suitability with other physiochemical characters. Initial observation of developed nanoemulsion under optical microscope showed uniform small size vesicles. It was also confirmed by TEM that reveals unilamellar and sph erical structure (Fig. 1) The formulation NE6 has 0.252 PDI that was indication of uniformity of globule size (Table 2) The zeta potential is a key indicator of the stability of the nanoemulsion formulation. The higher range of zeta potential showed nanosize globules and conferred stability. The zeta potential of NE6 was more negative compared to the other nanoemulsions. The percentage transmittance of 99.66 for NE6 indicated clear dispersion (Table 2) The batch NE6 was selected as the optimized batch as it displayed optimum response variables of 99.66% optical transparency, low globule size (124.33 nm), polydispersity of 0.252 and zeta potential of 34.48 mV. Hence, NE6 was selected for further processing.

It was observed that maximum drug release from nanoemulsion was achieved within 8h(92.19%). Tween 80 as a surfactant also contributed to release maximum percentage of drug from NE. It was observed that percent drug release was respective with time.

In stability studies, the NE6 was devoid of precipitation, phase separation, creaming and flocculation. In addition, the formulation was also stable after centrifugation (2000 rpm for 15min) at ambient temperature. The results of stabilit y testing represented in (Table 3), revealed that there were negligible changes in the parameters of CUN after 6 months of storage.

Blood glucose level was raised significantly higher with the injection of Streptozotocin (STZ). Treatment with BVE and BVN did not reduce hyperglycemia significantly in diabetic rats at first week (Fig. 2) Chronic administration of BVN from second to forth week significantly (P < 0.001) reduced raised blood glucose level in dia betic rats. However, BVE also decrease d blood glucose level but not much significant as BVN. Results showed that there was a significant decrease in the body weight of STZ-induced diabetic rats in comparison to aged matched control animals. Treatment with chronic administration of BVE and BVN caused raised in body weight, which was more significant with BVN.

Administration of STZ results into increased hind paw withdrawal latency to the given stimuli in rats after one week as compared with control animals. No withdrawal latency was achieved after acute treatment of BVN. However, chronic administration of BVN s ignificantly(P < 0.001) reduced withdrawal latency as compared with diabetic rats. However, BVE also reduced latency but not much as significant as BVN (Fig. 3)

STZ caused reduction in withdrawal threshold of von Frey filament diabetic rats as shown in (Fig. 4) The mechanical allodynia was started at first week due to STZ injection and it was continued for the three weeks. Administration of BVE and BVN significantly (P<0.001) attenuated mechanical allodynia after 7 days but acute dose of BVN did not change mechanical withdrawal threshold in STZinduced diabetic rats.

Diabetes is a disorder of carbohydrate, fat and protein metabolism attributed to diminished production of insulin or mounting resistance to its action [16, 17]. Phytotherapeutics are extracted from medicinal plants and their active constituents used either in whole food form or in the form of standardized extracts and su pplements, for healing purposes [18]. Diabetic neuropathy is a common disorder causes peripheral nerve dysfunction commonly in diabetic patient [19]. Inaddition, there is a sensory loss of pain because of nerve damage [20]. The major risk factor for diabetes neuropathy is hyperglycemia. Painful diabetic neuropathy cause increased blood glucose level condition called Glucojasinogen [21]. Diabetic neuropathy affects specific nerves, it may cause vision problem and nerve damage to the affected individual [22]. Chronic ad-ministration (4 weeks) of B. variegata loaded nanoemul-sion, 100 mg/kg (p.o.) decreased blood glucose level and progression neuropathy in STZ induced diabetic rats. Re-duction in blood glucose level due to herbal medicine was in accordance with Kumar et al., 2016 [23]. It has reported that STZ cause mechanical allodynia in rats [24, 25] Di-abetic neuropathy caused reduction of antinociceptive effect in diabetes, it is characterized by neurodegener-ation; it results in decreased pain perception [26, 27]. In this study the pain threshold was measured by plantar test apparatus, indicated to decrease in the foot with drawal in diabetic rats (Fig. 3) The tactile stimulus is transmitted throughmyelinated Aβ fibers [28]. Herbal drug loaded nanotechnology is effective in treatment of diabetic neuropathic pain as reported in earlier studies [29, 30]

Nanotechnology has emerged as an attractive field having several applications in the advancement of drugs. Results indicated that BVN was stable nanoemulsion formulation and its chronic treatment result in reduction of blood glucose level and prevention in progression of diabetic neuropathy. Thus, BVN may be an effective formulation for diabetic neuropathy. In conclusion, the present study demonstrates that chronic prevents weight loss and attenuates mechanical allodynia in STZ-induced diabetic rats. Althou gh further studies are required to clarify the exact mechanisms of BVN for therapeutic effect.

The authors declare no conflict of interest.

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