In vivo Study of Insulin-loaded Microemulsion Topical gel with Aloe vera for the Treatment of Dermatologic Manifestation of Diabetes

 

Chakraborty. T¹, Gupta. S^1*, Saini. V²

¹Department of Pharmaceutical Science, M M College of Pharmacy, , Maharishi Markandeshwar University

(Deemed to be University), Mullana, Ambala, Haryana 133207, India,

2Department of Pharmaceutical and Medical Sciences, Maharishi Markandeshwar University,

Solan, Laddo, Himachal Pradesh 173229, India,

 

ABSTRACT:

Insulin is useful for the treatment of diabetes mellitus. Because of its high molecular weight, it is difficult to permeate through transdermal routes. There is no existing formulation available for the dermatologic manifestation of diabetes. Thus an attempt was made to prepare insulin-loaded microemulsion topical gel with aloe vera. The insulin-loaded microemulsion was prepared using 3 square factorial design, using oleic acid as oil phase, tween-80 as the surfactant and poly ethylene glycol-400 as co-surfactant. Physical stability studies performed and amongst all of them, the best three formulations selected for ex vivo insulin permeation study. The batch with the best permeation was selected to prepare the topical gel with and without aloe vera. Post evaluation parameters of topical gel with skin irritation study and in vivo study performed. In vivo study results revealed that the groups of diabetes Wistar albino rats with skin infections; by the treatment of topical gel of insulin-loaded microemulsion with aloe vera gel was found to reduce the blood glucose level and skin infection as compared to one without aloe vera gel.  It was concluded that insulin loaded microemulsion topical gel with aloe vera can be used as dermatologic manifestation of diabetes significantly.

 

 

INTRODUCTION:

Diabetes mellitus is a heterogeneous disease which leads to an abnormal increase in blood glucose levels due to abnormal functioning of pancreatic beta cells. Increase in blood glucose level causes damage to a wide range of cell types includes endothelial cells, neurons, renal cells, keratinocytes and fibroblasts^1,2. Chronic diabetes mellitus can cause several diabetic manifestations like diabetic neuropathy, diabetic nephropathy, diabetic myopathy, diabetic dermopathy³. Globally, various clinical studies were conducted on a different population of diabetic patients and reported in the literature.

 

One of the studies conducted among Sweden population and the results exhibited that 33% of the patients had diabetic dermopathy in type 1diabetes mellitus and 39% of the patients had diabetic dermopathy in type 2 diabetes mellitus³. In Singapore, two clinical studies reported that 24% had diabetic dermopathy out of 135 hospitalized diabetic patients and 16% had diabetic dermopathy out of 100 outdoor diabetic patients⁴. In Iran, a study conducted on 181 outdoor diabetic patients and the results exhibited that 26% of the patient had diabetic dermopathy, 37.4% of the patient had diabetic retinopathy and 16.6% of the patients had both complications². In Helsinki, Finland a clinical study conducted on 750 outdoor diabetic patients and the result was reported that 6.9% of the patient had type 1 diabetes mellitus and 93% of the patient had type 2 diabetes mellitus⁵. Among them 38% of the patient had diabetic neuropathy, 23.3% of the patient had diabetic nephropathy, 22.9% of the patient had diabetic retinopathy and 79.2% of the patient had diabetic dermopathy⁵. Diabetic dermopathy can be recognized as a skin problem having patches due to a high level of blood glucose in chronic condition. It also recognized as little flaky red or purple macule (0.5 to 1.2cm) on the upper extremities, thigh region and stomach area due to alteration of skin blood flow^1,2,4,6,7,8. Extravasation of erythrocytes and invasion of lymphocytes occurred in a chronic condition of diabetes mellitus^1,6. Existing well established hypoglycemic agents exist for the treatment of diabetes mellitus in the form of parenteral and orally but among this insulin therapy is one of the best useful treatments for type 1 and type 2 and its diabetes complications^{9,10,11,12,13,14,15}. A drug therapy is still missing which can be used for both the treatment diabetes mellitus and diabetes dermopathy. A regular topical formulation which can be used for the treatment of dermopathy but these have limited use because most of them are not compatible with each other, show serious adverse effects and high cost^16,17. The microemulsion is one of the best techniques which are helpful for the preparation of the topical formulation^{18,19,20,21,22}. The topical formulation is specifically applicable for skin disorders. Mostly medicinal plants are widely used for a skin disorder^16,17. Aloe vera (Aloe barbadensis miller) is a natural medicinal plant, inner gel of aloe vera is safe and non-toxic according to the cosmetic ingredient review expert panel committee, aloe vera gel and the extract is in the list of FDA approved the ingredient^23,24, it has several therapeutic effects such as immune stimulation, wound healing,  anti-inflammatory, antibacterial, antiviral, anti-fungal, anti-diabetic, anti-oxidant, moisturizing, anti-ageing effects, anti-neoplastic activities and also significantly found to reduce the fasting blood glucose level^{25,26,27,28,29,30,31}. As it is now insulin therapy is only drug therapy which is fast relieving treatment but it is quite unstable for long term storage. Metacresol and/or phenol is used to enhance the stability of injectable insulin formulation but these causes serious adverse effects (allergic reaction, cell death and severe cutaneous reaction)^32,33,34. Based on the literature our study is aimed to attempt preparing insulin-loaded microemulsion topical gel with aloe vera for the treatment of dermatological manifestation of diabetes.

 

MATERIAL AND METHODS:

MATERIALS:

Insulin human recombinant dry powder and alloxan monohydrates were purchased from Sigma-Aldrich, Oleic acid, tween-80, Carbopol-934 Guar gum and Polyethylene glycol-400 were purchased from Qualikems Fine Chem. Pvt. Ltd.  Ethyl cellulose was purchased from Central drug house (P) Ltd. All other chemicals and reagents used in the study were of analytical grade.            

 

Aloe vera gel:

Aloe vera mucilaginous inner gel obtained from the plant leaf of aloe vera (Aloe barbadensis miller), the plant was obtained from the medicinal garden, M.M (DU) Mullana, Haryana, India and it was identified as Aloe barbadensis miller by the botanical survey of India, Dehradun. (BSI/NRC/Tech./Herb.(Ident.)/2019-20/528). The thick succulent leaves of aloe vera were obtained, washed with water and a mild chlorine solution and cut transversely. The thick epidermis selectively removed and the centre of the leaf, the inner gel-like pulp was separated, minced, homogenized by mechanical stirrer and pass through sieve #100³⁵.

 

Preparation methods of insulin loaded microemulsion:

S4, S5 and S6 batches (S1 to S9) insulin loaded microemulsion was prepared using 3square factorial designs by oleic acid as the oil phase, tween-80 as the surfactant and polyethylene glycol-400 as co-surfactant. At first dry human recombinant insulin dissolved in distilled sterile water (3.636mg/mL i.e.100IU/mL) and added drop wise with a mixture of oleic acid, tween-80 and polyethylene glycol-400 with vigorous stirring on ice bath maintain temperature below 25°C until the milky microemulsion has prepared. The composition was shown as (Table 1).

 

Physical Stability of insulin loaded microemulsion:

Physical stability study conducted of insulin-loaded microemulsion with the help of the instrument centrifugation (Remi Instruments, Remi c-24 bl, India) and it was rotated at 5000rpm for a period of 1h after that the microemulsion was observed that there have any phase separation or not³⁶.

 

Percentage insulin content uniformity of best three physical stability batches insulin loaded microemulsion:

Separately 0.5mL insulin loaded microemulsion was taken from the three physical stability batches formulation and separately dissolved in 10ml of 0.01 M hydrochloric acid. The solutions were vigorously shaken with the help of mechanical gyratory shaker at 100rpm for 30min maintain the temperature 25°C. Then the samples were filtered through a 0.45µm syringe filter and centrifuged at 1500rpm for 30 min at 25°C temperature. The supernatant centrifuged samples were filtered, suitably diluted and estimated the amount of insulin by previously reported High-Performance Liquid Chromatography (HPLC, LC-100, Cyberlab, USA) method³⁷. The mobile phase consists of 1 mmol sodium sulphate and 0.2% triethylamine in HPLC grade water, pH adjusted to 3.2 by phosphoric acid and acetonitrile (60:40). The flow rate was 1mL/min and selected the UV absorption at 214nm^37,38. Each sample solution analyzed in triplicate and the resulted data as summarized with mean±SD, n=3.

 

Table 1: Composition of nine batches insulin loaded micro emulsion

 

Ex vivo permeation studies of best three physical stability batches insulin-loaded microemulsion:

Ex vivo permeation study conducted of three physical stability batches insulin-loaded microemulsion with the help of modified Franz diffusion (FD) cell. The rats were anaesthetized, sacrificed and removed the abdominal hairs with the help of hair removal cream (Veet hair removal cream for sensitive skin). (3cmx3cm) the skin was isolated from the abdominal region and separated the connective tissue with the help of surgical scalpel and rinsed with double distilled water. The prepared skin mounted over the donor compartment of the diffusion cell (0.784 cm2). The dermal side overlaid in direct contact with the receptor chamber (5mL). Saline phosphate buffer pH 7.4 was used as a diffusion medium maintaining the temperature at 37±0.5°C by circulating water jacket with constant stirring using a magnetic bead (400-420rpm). Separately 0.5mL insulin-loaded microemulsion of each batch was poured in donor compartment of the FD cell. The study was carried out for 8h and in each 1h time interval, the 1mL sample solution was withdrawn from the receptor compartment and replaced with an equal volume of fresh saline phosphate buffer pH 7.4 as maintaining sink condition. Withdrawn sample solutions diluted by 0.01M hydrochloric acid and estimated the amount of insulin according to the previous method^36,37,38. Each sample analyzed in triplicate and the resulted data summarized with mean±SD, n=3.

 

Preparation methods of insulin-loaded microemulsion topical gel with and without aloe vera:

Insulin-loaded microemulsion topical gel (0.2 IU/g) was prepared using best insulin permeation batch (S5 batch) with and without aloe vera along with other excipients carbopol-934 as a viscoelastic gelling agent, ethyl cellulose as linear viscoelastic and thermomechanical stability polymer, guar gum as hydrocolloid thickening and binding agents^39,40.

Procedure:

Firstly carbopol-934 and guar gum were separately mixed with the required quantity of sterile distilled water and kept for overnight to prepare a colloidal solution. Simultaneously ethyl cellulose was also dissolved in ethanol in a separate beaker. Next both the solutions were mixed together and continuously stirred by mechanical stirrer at 5000rpm for 30min and sterilized by autoclave at 121°C for 15min. On the other hand, the required quantity of optimized batch’s (S5) insulin-loaded microemulsion and aloe vera and in another without aloe vera mixed with the above sterilized colloidal solution with the help of mechanical stirrer at 1000rpm for 30min maintaining the temperature below 25°C on an ice bath. Suitable and desired insulin-loaded microemulsion topical gel with and without aloe vera was prepared (composition as shown Table 2) and these stored in an amber colour airtight container for further evaluations.

 

Percentage insulin content uniformity test of insulin-loaded microemulsion topical gel with and without aloe vera:

Separately 0.5g insulin-loaded microemulsion topical gel with and without aloe vera formulations were weighed and dissolved in 10mL of 0.01(M) hydrochloric acid. The solutions were vigorously shaken with the help of a mechanical gyratory shaker at 100rpm for 30min and filtered through a 0.45µm syringe filter. Further, the sample solutions were centrifuged at 1500rpm for 30 min maintaining the temperature at 25°C. The supernatant centrifuge samples were filtered, suitably diluted and estimated the insulin content according to the previous method^37,38. Each sample solution analyzed in triplicate and the resulted data summarized with mean±SD, n=3.

 

Table 2: Compositions of insulin-loaded microemulsion topical gel with and without aloe vera

 

 

 

FTIR-ATR Spectrum of pure dry human recombinant insulin and prepared insulin formulations:

Fourier-Transform Infrared Spectroscopy, Attenuated Total Reflection (FTIR-ATR) spectrum of pure dry human recombinant insulin and insulin loaded microemulsion topical gel with and without aloe vera were recorded using FTIR-ATR spectra (Perkin Elmer, synthesis monitoring system, NIPER, Punjab) according to the previously reported FTIR-ATR spectra recorded method^41,42.

 

Differential Scanning Calorimetry (DSC) of pure dry human insulin and insulin formulations:

DSC thermogram of pure insulin and insulin loaded microemulsion topical gel with and without aloe vera formulations were measured using Differential Scanning Calorimetry (Mettler Toledo 823e). The instrument equipped with intra-cooler and its temperature and enthalpy was calibrated using the standard indium zinc. The samples were hermetically sealed in aluminium pans and heated over the temperature range -30°C to 40°C with a heating rate of 10°C/min. The inert atmosphere was provided by purging nitrogen gas flowing at 40 mL/min^42,43.

 

Mean particle size and zeta potential of insulin-loaded micro emulsion topical gel with and without aloe vera formulations:

Mean particle size and zeta potential were measured by diluting (0.1mg/mL) the insulin-loaded microemulsion topical gel with and without aloe vera formulations with distilled water at 25°C by photon correlation spectroscopy (Malvern zeta seizer).

 

Animals study:

Wistar albino rats of either sex 160-180g were used for all ex vivo and in vivo experiments. The animals were housed in polypropylene cages at 24±2°C and fed with commercial pellet diet and water.

 

The experiment was in accordance with the guidelines of Committee for the Purpose of Care and Supervision on Experimental Animals (CPCSEA) and the study approved by the Institutional Animal Ethics Committee (IAEC) (Reg. No MMCP/IAEC/17-14).

 

Assessment of drying time of the formulation on rat skin, skin irritation study and for in vivo study, dorsal side (3cm×3cm) hairs of the Wistar albino rats were removed by hair removal cream (Veet hair removal cream for sensitive skin) on the previous day of the experiment.

 

Assessment of drying time:

On rat skin the drying time of the prepared final topical formulations measured by previously reported literature method. For the study 500mg, insulin-loaded microemulsion topical gel with and without aloe vera formulations separately applied to the hairless area of the dorsal side of the two groups of Wistar albino rats (n=6) and after the 90s of application 15s time interval, a glass slide placed on the gel application area without pressure.  Drying time counted of the formulation in which no liquid was visible on the glass slide after removal⁴⁴, the resulted data as summarized with mean±SD, n=6.

 

Skin irritation study:

Skin irritation study conducted on Wistar albino rats (n=24). The animals randomly divided into 4 equal groups like normal control, standard irritant and insulin-loaded microemulsion topical gel with aloe vera and without aloe vera formulations. 0.8% v/v aqueous solution of formalin applied as a standard irritant group of rats and insulin-loaded microemulsion topical gel with and without aloe vera formulations applied to two groups of rats for assessing any kind of irritation at specified sites. Formulations removed after 24 h and skin examined for any sign of erythema and oedema and the test procedure repeated for another 6days. The resulting reactions compared against a normal control group⁴⁴.

 

In vivo study[42]:

In vivo study was conducted on Wistar albino rats (n=24). The animals randomly divided into 4 equal groups like normal control, diabetic positive and diabetic control with insulin-loaded microemulsion topical gel with aloe vera and without aloe vera.

 

Group A: Normal control (without formulation)

Group B: Diabetic positive (125mg/kg, intra peritoneal alloxan monohydrate)

Group C: Diabetic control with insulin loaded micro emulsion topical gel with aloe vera formulation.

Group D: Diabetic control with insulin loaded micro emulsion topical gel without aloe vera formulation.

 

In the in vivo study, Group A; six animals were taken as normal control groups and observed for 22 days without formulation to compare with the diabetic positive and diabetic control groups of animals. Six animals of each Group B, Group C and Group D have injected a single dose of 125mg/kg, intra-peritoneal alloxan monohydrate to prepare diabetic positive and taken care and observed for 22 days. To prevent hyperglycemia of Group B, Group C and Group D animals 10% glucose solution was provided in their cages at first day of alloxan monohydrate administered. On the other hand, insulin-loaded microemulsion topical gel with and without aloe vera formulations applied on Group C and Group D animals from 7th day to control diabetic positive. Fasting blood samples collected at 3rd day, 7th day, 12th day, 17th day and 22nd day from each group of animals by puncturing the retro-orbital sinus, under mild ether anaesthesia and measured the blood serum glucose by Erba glucose kit, Erba Mannheim, Mumbai and observed the dermatologic manifestation of diabetes rats. The resulted data as summarized with mean±SD, n=6.

 

RESULTS:

Physical Stability of insulin-loaded microemulsion:

Physical stability of nine batches (S1 to S9) insulin-loaded microemulsions were evaluated, among them S4, S5 and S6 batches insulin-loaded microemulsions were stable.

 

Percentage insulin content uniformity of best three physical stability batches insulin-loaded microemulsion:

Insulin content of S4, S5 and S6 batches insulin-loaded microemulsion was found to be in the range of 99.67±0.21% to 101±0.26%.

 

Ex vivo permeation study:

Through Wistar albino rat skin, insulin permeation from S4, S5 and S6 batches insulin loaded microemulsion were found to be 5.82±0.05037IUcm^-2h^-1, 6.4473±0.05037IUcm^-2h^-1 and  5.589±0.037IUcm^-2h^-1.

 

Percentage insulin content uniformity of insulin-loaded microemulsion topical gel with and without aloe vera:

Insulin content of insulin loaded microemulsion topical gel with and without aloe vera were found to be 99.86±0.22% and 100±0.32%.

 

FTIR-ATR Spectrum of pure dry human recombinant insulin and insulin formulations:

The Fourier-Transform Infrared Spectroscopy, Attenuated Total Reflection (FTIR-ATR) spectrum of pure dry human recombinant insulin was found to be a stronger peak at 1637.96 cm⁻¹ which denotes amide peak was present and previously reported (Amenabar et al.2013 and Prusty and Sahu 2013) insulin absorption peak at 1644 cm⁻¹ and 1664 cm⁻¹. On the other hand, FTIR-ATR spectrum of insulin loaded microemulsion topical gel with and without aloe vera formulations were found to be a stronger band at 1637.84 cm⁻¹ and1637.94 cm⁻¹. FTIR-ATR Spectrum of pure dry human recombinant insulin and insulin loaded microemulsion topical gel with and without aloe vera formulations were shown in (Fig. 1 (1(a), 1(b) and 1(c)).

 

Figure 1: FTIR-ATR Spectrum 1(a) pure dry human recombinant insulin 1(b) and 1(c) insulin-loaded microemulsion topical gel with and without aloe vera formulations

 

Figure 2: Differential Scanning Calorimetry peak, 2(a) endothermic peak of pure insulin at -2.48°, 2(b) and 2(c) endothermic peak of insulin loaded micro emulsion topical gel with and without aloe vera formulations at -2.89°C and -2.74°C and 2(d) Overlain of 2(a), 2(b) and 2(c).

 

Mean particle size and zeta potential:

Mean particle size and zeta potential of insulin-loaded microemulsion topical gel with and without aloe vera formulation was found to be 899nm and -10mV and 1340nm and -0.433mV.

 

Assessment of drying time:

After application, 215±20s and 120±13s of insulin-loaded microemulsion topical gel with and without aloe vera formulations on the hairless area of the dorsal side of the Wistar albino rats, it was observed that no remains of liquid were visible on the glass slide.

 

Skin irritation study:

The normal control group of animals and insulin-loaded microemulsion topical gel with and without aloe vera formulations in six days did not show any erythema and oedema but a standard irritant group of animals after six days were found to develop erythema and oedema.

 

In vivo study:

The results of in vivo study showed that in Group-A normal control rats the blood glucose levels not significantly changed at an interval twenty two days and skin infections were not observed at hair clean area of dorsal side or other body parts of Wistar albino rats. But in Group-B alloxan monohydrate induced diabetes positive Wistar albino rats the blood glucose levels were significantly changed in twenty two days and it was found to be 74.58±1.02 mg/dL to 323.33±1.96 mg/dL still skin infection observed at dorsal side of hair removal area, foot, eyelids and orbits as shown in (Fig. 3 (3(a), 3(b), 3(c) and 3(d)). Alloxan monohydrate induced Group-C animals the blood glucose levels were significantly increased in seven days 74.03±1.24 mg/dL to 263.81±1.48 mg/dL as well as dorsal side infections was observed as shown in (Fig. 4(a)). After fifteen days treatment with insulin loaded microemulsion topical gel with aloe vera formulation (0.5g applied on hair clean area of dorsal side) the blood glucose levels significantly reduced was 263.81±1.48 mg/dL to 74.±1.41 mg/dL and skin infection disappeared as shown in (Fig.4(b)). On the other hand, alloxan monohydrate induced Group-D animals the blood glucose levels were significantly increased in seven days 73.33±1.08 mg/dL to 262.63±1.47mg/dL as well as dorsal side infections was observed as shown in (Fig. 5(a)) and after fifteen days treatment with insulin-loaded microemulsion topical gel without aloe vera formulation (0.5g applied on hair clean area of dorsal side) the blood glucose levels significantly reduced  262.63±1.47mg/dL to 84.83±2.31mg/dL and skin, the infection was still observed as shown in (Fig. 5(b)). Before and after treatment mean fasting blood glucose levels of normal control, diabetic positive and diabetic control groups as shown in (Table 3) and (Fig. 6).

 

Figure 3: Group-B alloxan monohydrate induced diabetes positive Wistar albino rats in twenty two days (Fasting blood glucose level 323.33±1.96 mg/dL) 3(a) dorsal side skin infection, 3(b) foot infection, 3(c) and 3(d) eyelids and orbits infection.

 

 

Figure 4(a): Before treatment of Group-C diabetes Wistar albino rats (fasting blood glucose level 263.81±1.48 mg/dL) observed dorsal side skin infection. Figure 4(b): After 15 days treatment with insulin-loaded microemulsion topical gel with aloe vera, skin infection was disappeared (fasting blood glucose level 74.±1.41 mg/dL).

 

Figure 5(a): Before treatment of Group-D diabetes Wistar albino rats (fasting blood glucose level 262.63±1.47mg/dL) observed dorsal side skin infection. Figure 5(b): After 15 days treatment with insulin loaded micro emulsion topical gel without aloe vera; skin infection was still observed (fasting blood glucose level 84.83±2.31mg/dL).

Table 3: Fasting mean blood glucose levels of normal control, diabetic positive and diabetic control groups with before and after treatment

 

DISCUSSION:

Nine batches(S1 to S9) insulin-loaded microemulsions prepared using 3 square factorial design methods,  among this S4, S5 and S6 batches formulation were found to be milky in colour these were observed no phase separation after centrifugation (for 1h at 5000rpm). So it concluded that S4, S5 and S6 batches formulation were stable among nine batches formulation because of desirable concentrations oil phase oleic acid, surfactant tween-80 and co-surfactant polyethylene glycol-400. Ex vivo insulin permeation studied results revealed that among S4, S5 and S6 batches insulin-loaded microemulsion, S5 batch was found to be maximum (6.4473±0.05 IU cm^-2 h^-1) because of its composition consisting of oleic acid, tween-80 and polyethylene glycol-400. It observed that increasing and decreasing anyone excipient decreased the permeation of insulin. Insulin-loaded microemulsion topical gel was prepared by best insulin permeation batch (S5 batch) with and without aloe vera along with other excipients shown in Table 2. For pharmacological activity, the final prepared topical formulation was selected 2.5g/kg having different concentration insulin 0.2IU/g, aloe vera inner gel16mg/g, oleic acid 0.2µL/g, tween-80 0.7µL/g, polyethylene glycol-400 0.3µL/g, etc. The dose was selected on the basis of previous studies results in the literature and it was insulin dose 0.4 to 0.6 IU/kg/day; lethal dose (LD₅₀) on rats was aloe vera (ip) >50mg/kg, oleic acid (iv) 2.4mg/kg, oleic acid (oral) 74g/kg, Tween-80 (ip) 6.8g/kg, Tween-80 (iv) 1.8g/kg, Polyethylene glycol-400 (ip) 9.7g/kg, Polyethylene glycol-400 (iv) 7.3g/kg^11,12,24,45. Percentage insulin content in insulin-loaded microemulsion topical gel with and without aloe vera was found to be 99.86±0.22 and 100±0.32 which depicted that insulin formulations prepared were homogeneous. The FTIR-ATR spectrum of pure dry human insulin and insulin loaded microemulsion topical gel with and without aloe vera formulations were found to give strong peak at 1637.96 cm⁻¹, 1637.84 cm⁻¹ and1637.94 cm⁻¹ and previously reported (Amenabar et al.2013 and Prusty and Sahu 2013) insulin absorption peak at 1644 cm⁻¹ and 1664 cm⁻¹.^41,42 So the FTIR-ATR spectrum results revealed that in the formulations insulin was pure recombinant human insulin and insulin were present in insulin-loaded microemulsion topical gel with and without aloe vera formulations were shown in (Fig. 1 (1(a), 1(b) and 1(c)). Differential Scanning Calorimetry thermogram peak of insulin and insulin loaded microemulsion topical gel with and without aloe vera formulations revealed that the insulin was stable in both the formulations as shown in (Fig. 2(2(a), 2(b), 2(c) and 2(d)). The particle size and zeta potential results of insulin-loaded microemulsion topical gel with aloe vera are smaller and negative than without aloe vera gel, as the aloe vera gel reduced the particle size and decrease the negative charge of zeta potential⁴⁶. Formulations contain with aloe vera the drying time is comparatively more than without aloe vera also it has a moisturizing effect. Skin irritation studied results revealed that both the insulin-loaded microemulsion topical gel with and without aloe vera is not skin irritant formulations.

 

Group-A normal control Wistar albino rats in 22 days the blood glucose levels were not significantly changed and skin infections not found in any part of the body. But Group-B alloxan monohydrate induced diabetes positive Wistar albino rats in 22 days the fasting blood glucose levels were significantly changed (74.58±1.02mg/dL to 323.33±1.96mg/dL) and skin infection and brownish hyperpigmentation observed at the dorsal side of hair removal areas, foots, eyelids and orbits due to alterations in skin blood flow, isoneofedema of papillary dermis, shallow veins and gentle lymphocyte invade and extravasated erythrocytes released hemosiderin stores of diabetes Wistar albino rats^1,8. In Group-C alloxan monohydrate induced diabetes Wistar albino rats in 15 days treatment with insulin-loaded microemulsion topical gel with aloe vera, the fasting blood glucose level significantly reduced 263.81±1.48mg/dL to 74±1.41mg/dL due to higher molecular weight insulin, molecularly dispersed into the microemulsion; smaller particle size and more negative zeta potential which enhanced the permeation of insulin through topical skin, because epidermal tissue of diabetes rats were much thinner, which particularly altered the microvascular network and stratified arrangements of epidermis cell almost disappeared^47,48. The active potential compound aloe vera gel contained phytosterols, lophenol, cycloartenol and their alkylated derivatives normalizing membrane-bound enzyme activities of phosphatases and hydrolases which increased glucose metabolism and promoted hypoglycemic effect. Even skin infections were disappeared because aloe vera gel has varied active potential chemical constituents Glucomannan, a mannose-rich polysaccharide, and gibberellin, a growth hormone, interacted with growth factor receptors on the fibroblast, which stimulated its activity, multiplication, expanded and changed the collagen synthesis (more type III), increased level of collagen cross-linking due to increased contraction of wound and breaking strength of scar tissue and hyaluronic acid in the granulation tissue of a healing wound. Mucopolysaccharides help in binding moisture into the skin likewise it stimulates fibroblast which generates the collagen and elastin fibres making the skin progressively flexible and less wrinkled, it also have cohesive effects on the shallow epidermal cells by sticking them together, which softens the skin. Lupeol, salicylic acid, urea, nitrogen, cinnamonic acid and sulphur have inhibitory activity on parasites, microorganisms and infections. The amino acids additionally diminish solidified skin cells and zinc tightens skin pores^{26,27,28,29,30,31}. In the case of Group-D alloxan monohydrate induced diabetes Wistar albino rats in 15 days treatment with insulin-loaded microemulsion topical gel without aloe vera, the fasting blood glucose level significantly reduced 262.63±1.47mg/dL to 84.83±2.31mg/dL due to same mechanisms as Group-C animals, but comparatively insulin permeation is lower due to larger particle size and low negative zeta potential. Even skin infections observed due to the absence of multi anti-infective agents for various skin diseases. So from the in vivo studied results it concluded that without treatment of diabetes, blood sugar level, as well as skin infection ratios, were increased; on the other hand skin infections with diabetes, insulin therapy is not only effective treatment, is also required multi anti-infective agent for various skin disease topical drug therapy, in this study aloe vera used as multi anti-infective agent for various skin disease.

 

CONCLUSION:

On the basis of all evaluation parameters and in vivo animal studied results it was concluded that, the process or technology of insulin-loaded microemulsion topical gel with aloe vera gel can be used as dermatologic manifestation of diabetes as well as to penetrate the higher molecular weight insulin through topical skin.

 

ACKNOWLEDGMENT:

This study was supported by the M.M College of Pharmacy, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana-133207, India.

 

CONFLICT OF INTEREST:

Nil.

 

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Accepted on 27.01.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(9):4115-4124.