FROSTBITE WOUND HEALING ACTIVITY OF IBUPROFEN NANOEMULSION BASED GEL WITH THE FRACTIONAL DOSE FOR EXTRACTION OF MIMOSA PUDICA LEAVES

Akkas Ali Ahmed and Dr. Suvakanta Dash. Department of Pharmaceutics, Girijanadha Chowdhury Institute of Pharmaceutical Science, Azara, Guwahati, Assam, India, 781017. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 5 (8), 69-86 70 of dry skin and emollient applications. Gels for dermatological use have several favourable properties such as being thixotropic, greaseless, easily spreadable, easily removable, emollient, non-staining, compatible with several excipients and water-soluble or miscible. Emulgels are emulsions, either of the oil-in-water or water-in-oil type, which are gelled by mixing with a gelling agent. They have a high patient acceptability since they possess the previously mentioned advantages of both emulsions and gels. Therefore, they have been recently used as vehicles.
Types of Nanoemulsion:-Nanoemulsion are classified into mainly three types on the basis of their composition 1.
Oil in water Nanoemulsions: In oil in water nanoemulsion oil dispersed in water. Hence dispersion medium is water and this kind of nanoemulsion can be found in application which has small quantity of fatty material like moisturizing, creams. Example: milk which is also like these types of emulsion.

2.
Water in oil Nanoemulsions: In case of the Water in Oil Nanoemulsion water droplets are found to be dispersed in continuous oil phase.

3.
Bi-continuous Nanoemulsions: In case of bi-continuous nanoemulsion oil and water are interdispersed within the system.

Skin:-
The human skin is the outer covering of the body. In humans, it is the largest organ of the integumentary system. The skin has up to seven layers of ectodermal tissue and guards the underlying muscles, bones, ligaments and internal organs. Human skin is similar to that of most other mammals. Though nearly all human skin is covered with hair follicles, it can appear hairless. There are two general types of skin, hairy and glabrous skin. The adjective cutaneous literally means "of the skin" (from Latin cutis, skin) In humans, skin pigmentation varies among populations, and skin type can range from dry to oily. Such skin variety provides a rich and diverse habitat for bacteria that number roughly 1000 species from 19 phyla, present on the human skin. 4,5 Skin is composed of three primary layers: the epidermis, the dermis and the hypodermis. Different layer of skin is shown in Fig 1.   Fig 1:-A) Skin's layer; B) Skin layer without hair and with hair Frostbite:-Frostbite is an injury that is caused by exposure of parts of the body to the cold. The cold causes freezing of your skin and underlying tissues. Your fingers, toes and feet are most commonly affected. There are different degrees of frostbite. In superficial frostbite, the skin can recover fully with prompt treatment. However, if frostbite is deep, tissue damage can be permanent and tissue loss can occur. For example, the end of a finger or toe can gradually separate off. The most important way of preventing frostbite is to get out of the cold. If you are exposed to the cold, make sure that you have adequate protective clothing. Frostbite is an injury that is caused by exposure of parts of your body to temperatures below freezing point. The cold causes freezing of your skin and underlying tissues. The fingers, toes and feet are most commonly affected but other extremities including the nose, ears, and the cheeks can also develop frostbite 6 .

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Degrees of frostbite:-Rather like burns, frostbite injuries are classified by the degree of injury. The degree of frostbite basically refers to how deep the frostbite injury goes. Your skin has two layers -the outer layer (epidermis) and the dermis. The dermis sits just under the epidermis. Beneath the dermis is a layer of fat, and then the deeper structures such as muscles and tendons.  First-degree frostbite just affects the epidermis.  Second-degree frostbite may affect the epidermis and part of the dermis.  Third-degree frostbite affects the epidermis, the dermis and the fatty tissue beneath the dermis.  Fourth-degree frostbite affects the full thickness of the skin, the tissues that lie underneath the skin, and also deeper structures such as muscles, tendons and bone. Different type of frostbite injury indicated in Fig.2.

Fig.2:-Degree of frostbite in epidermis dermis and subcutaneous tissues of skin
Extraction of Mimosa pudica leaves:-Extraction in chemistry is a separation process consisting in the separation of a substance from a matrix. It includes Liquid-liquid extraction, and Solid phase extraction. A Soxhlet extractor is a piece of laboratory apparatus 7 invented in 1879 by Franz von Soxhlet 8 .It was originally designed for the extraction of a lipid from a solid material. Typically, a Soxhlet extraction is used when the desired compound has a limited solubility in a solvent, and the impurity is insoluble in that solvent. It allows for unmonitored and unmanaged operation while efficiently recycling a small amount of solvent to dissolve a larger amount of material. Mimosa pudica leaves. is a creeping annual or perennial herb. It has been identified as lajjalu in Ayurveda and has been found to have antiasthmatic, aphrodisiac, analgesic, and antidepressant properties. M. pudica is known to possess sedative, emetic, and tonic properties, and has been used traditionally in the treatment of various ailments including alopecia, diarrhea, dysentery, insomnia, tumor, and various urogenital infections. Phytochemical studies on M. pudica have revealed the presence of alkaloids, non-protein amino acid (mimosine), flavonoids C-glycosides, sterols, terpenoids, tannins, and fatty acids 9 . Mimosa pudica leaves was shown in

Material And Method:-
Materials:-Materials Ibuprofen as active ingredient was collected from the Merck Specialities private Ltd. Oleic acid and glycerol, and PEG 400, was collected from the Merck Specialities Private Ltd, Ethanol was collected from China, (Changshu Yangyuan Chemicals), Petroleum ether parches from the Merck Specialities private Ltd, Extract Raw material Mimosa pudica was collected from the Azara, Guwahati local area and air dried and powered the leaves.

Solubility Study by Different Solvents:-
The most important criteria for screening of excipients is the solubility of the poorly soluble drug in oil, surfactants, and co-surfactants. Solubility of the Drug Ibuprofen was carried out with different solvent by taking small amount of the sample to the test tube and shake well to observe if any crystal or precipitation form.
Melting points:-Small amount of sample inserted to the capillary tube, attaching to the stem to the Thermometer to the centre in a heating bath, heating the bath slowly and observed the temperature at which the sample started melting is pointed, pure sample usually have sharp melting point. The melting point of Ibuprofen was found to 76°C. Differential Scanning Calorimetric:-Drug and excipients weighted between 5 -10 mg and kept in the desiccators for 24 hrs. The sample was than kept in the sample holder and analysed was perform.

FTIR (Fourier transform infrared spectroscopy):-
Drug excipients and their physical mixture of smal amount were taken in the mortor and mixed well to form uniform mixture and placed to the deccicator if required and perform FTIR at scanning with 400 cm -1 to 4000cm -1 .

Thin Layer Chromatography [IP]
:-Silica gel plate:-Silica gel plate was prepared by Pouring method where the powder silica gel of approximately 30 gm dissolved in 60ml of water and forms the slurry which is poured to the glass plate and kept for drying at 120°c for 30 min for activation of the plate to remove the water. Punjabi name of this plant is lajwanti Mimosa pudica posses a wide area of therapeutic activity likes vulnerary, diuretic, antispasmodic, emetic, constipating and febrifuge. They used in haemorrhage, dysentery, inflammation, burning sensation & wounds. It is also used in jaundice, asthma, conjunctivitis, cut wounds, and glandular swelling 13,14,15,16,17 . The present study has been undertaken to examine the wound healing activity of the leaves of Mimosa pudica in excision and burn wounds models Preparation of leaves extract-The leaves of Mimosa pudica Linn were collected from local market, area. Powdered leaves were charged into soxhlet apparatus and successive hot continuous extraction was carried out using solvents such as Petroleum ether (600-800 0 C), and ethanol. The drug was extracted with each solvent. Each time before extraction with the next solvent, the powdered material was air dried. Each extract was concentrated by distilling the excess solvent to obtain the crude extract.The Petroleum Ether was used to remove the fat and tannins of the Mimosa pudica leaves than mark was kept for drying over night and carried out with solvent ethanol. Concentrated the ethanol and collected the extract. Process of Mimosa pudica leaves was have been shown in Fig.4.  74

Preparation of Nanoemulsion:-
The formulations were prepared by mixing appropriate amount of surfactant and co-surfactant and then oily part added, mix the formulation until completely dispersion occurs at room temperature. Then appropriate amount of drug was added and the Mimosa pudica extraction was dissolved with ethanol, transfer to the final mixture and mixed by Homogenizer for 5 hrs continuously. 21 After swellings of whole night the carbopol gel withdrawn and ibuprofen with extraction of Mimosa pudica was slowly added to the nanoemulsion with magnetic stirred and nanoemulsion gel was placed in well closed container for further studies.  Instruments, UK). The formulation (0.1 ml) was dispersed in 50 ml of water, mixed thoroughly and light scattering was carried out at an angle of 90 at 25˚C.
Drug Content:-Certain amounts of nanoemulsion was taken dissolved using 100ml of ethanol and sonicated for the period of 15 min filtered it by whatman filter paper. Further dilutions were made by using ethanol prepared concentration within Beer's range. The absorbance was measured by UV-Visible spectrophotometer and drug content was determined. Apart from this Western Blot method is used to measured amount of drug present.
Viscosity Determination:-Viscosity of nanoemulsion should be measured by using the rotary viscometer at different rate and temperature. Nanoemulsions have very low viscosity 21 . Brookfield viscosity usually refers to a viscosity measurement performed with a Brookfield Viscometer. There are several models of viscometer available from Brookfield but the majority operate in the same manner: the viscometer motor rotates the spindle at a defined speed (measured in rpm) or shear rate and the viscometer measures the resistance to rotation and reports a viscosity value. Various spindle designs can be employed, depending on the nature of the sample and the requirements.
pH Determination:-pH of the nanoemulsion was determined by litmus paper and pH meter which is maintained at 5 to 6 which is the skin pH due to that there is no skin irritation.

Zeta Potential:-
In most cases, colloidal particles possess a positive or negative electrostatic charge. As electrical fields are applied to the particle dispersion, the particles migrate in oppositely charged directions. As particles are irradiated in migration, scattering light causes Doppler shift depending on electrophoresis mobility. NanoPlus software calculates the amount of Doppler shift followed by electrophoretic mobility and zeta potential by combining a heterodyne system and photon correlation method to perform Fourier transforms (FFT) Slipping level Major part of medium of obtained correlation function.

Transmission Electron Microscopy:-
Morphology and structure of the nanoemulsion were studied using transmission electron microscopy TOPCON 002B operating at 200 KV (Topcon, USA) and capable of point to point resolution. Combination of bright field imaging at increasing magnification and of diffraction modes was used to reveal the form and size of nanoemulsion droplets. In order to perform the TEM observations, a drop of the nanoemulsion was directly deposited on the holey film grid and observed after.

Scanning electron microscopy:-
Scanning Electron Microscopy uses a focused beam of high-energy electrons to generate a variety of signals at the surface of solid specimens. In most SEM microscopy applications, data is collected over a selected area of the surface of the sample and a two-dimensional image is generated that displays spatial variations in properties including chemical characterization, texture and orientation of materials. The SEM is also capable of performing analyses of selected point locations on the sample. This approach is especially useful in qualitatively or semiquantitatively determining chemical compositions, crystalline structure and crystal orientations.

In-vitro drug Release:-
The in-vitro drug release studies were carried out using a Franz diffusion cell. The formulation was applied the surface of egg membrane which was placed between donor and receptor compartment of the Franz diffusion cell. Phosphate buffer pH 5.5 was used as a dissolution media. The temperature of the cell was maintained at 37 0 C by circulating water jacket. This whole assemble was kept on a magnetic stirrer and the solution was stirred continuously using a magnetic bead. Sample (1 ml) was withdrawn at suitable time intervals and dilute up to 10ml with same solvent and replaced with equal amounts of fresh dissolution media. Samples were analyzed spectrophotometrically at 263 nm and the cumulative % drug release was calculated 22 .

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Before day 0, Rats were individually anesthetized By using chloroform The skin surface from the base of the neck to the top of the rear haunches was shaved with an electric clipper, after which a depilatory cream (Nair, Church & Dwight, Princeton, NJ) was applied for 2 minutes to remove any remaining hair. After depilation, the skin was cleansed with a 70% isopropyl alcohol swab.

Continuously freezing method:-
Four Rats were studied using the CF method. Twenty four hours after skin preparation, ceramic (ferrite) magnets (diameter 0.5 inches, thickness 0.219 inches, weight 3.5 g) were placed in crushed dry ice (-78.5°C) and allowed to cool for 15 minutes. The centre of the tattooed circle was marked to determine precisely the placement of magnets. Using fingers, the back skin of the rats was lifted into a skin fold, and then, 2 frozen magnets were placed so that they adhered from opposite sides of the intervening skin fold with the mark at the fold's apex centre location. A silicone barrier was then slid underneath the magnets as a thermal shield to limit the rat's body temperature decline. Sets of 2 cooled magnets were left in place for 1 minute, and then removed to allow new magnets to be immediately placed in the same location against the frozen tissue. The magnet exchange occurred in less than 5 seconds, which did not allow any thaw to occur. The magnet exchange was repeated for a total of 5 applications, resulting in a freeze time slightly longer than 5 minutes. Injury was intentionally inflicted on a lifted skin fold rather than by applying topical pressure and freezing the tissue in its normal position directly underneath the magnet to simplify the method and avoid between-rat variability, confine the injury to the skin, create a precise injury, limit systemic hypothermia, and maximize survival. It was not the intention to create a wound below the dermis. Core temperature was monitored using an infrared thermometer (ThermoWorks, Alpine, UT) applied to the abdomen of therat. After 5 magnet placements and removals, the skin was allowed to completely thaw; Rats were given subcutaneous injections of buprenorphine (0.05 mg/kg) for analgesia after the thaw. No dressings were applied to the wounds 22, 23 .

Results and Discussion:-Preformulation Studies:-Organoleptic Properties:-
The result of the visual observation of the pure drug ibuprofen was found to be white crystalline and bitter in taste.

Solubility Study by Different Solvents:-
The solubility of Ibuprofen was found as insoluble with water but soluble with some other organic solvents like ethanol, acetone, at the same time ibuprofen was partially in soluble with pH solution of 6.8 and 7.4 respectively.     F3 Yes yes yes Stable From the above study, it was concluded that formulation 1, 2 and 3 found to be thermodynamically stable. It was concluded that peak was shown at the particle size range of 203.9 to 229.9 nm and the graph depict that it has a homogeneous distribution of particles. Thus the result showed that the particle size of formed nanoemulsion was in the required range, therefore nanoemulsion formulated successfully. Drug Contain determination:-Drug contain found as in the range of 88 % to 98%. pH Determination:-The pH of gel in between 5 to 6 which lies in between normal pH range of skin which does not produce any skin irritation.   After performed Transmission electron microscopy particle was determined in 211.15 nm,148.63 nm and apart from this more smaller number of particle also detected which is 3.97 and 46.65 nm. Particle was disributed well and found as spherical shape, which is shown in figure no.9 In case of the SEM particle are nano size and distributed. Particle are found as spherical in size.

In-vitro drug released:-
The in-vitro release of Ibuprofen from the Nanoemulsion gel was varied in amount according to concentration of emulsifying agents used on formulations. It is shown that the drug released in 76.93 to 91.07 % after the intervals of 300 minute for formulation F1 F2 F3 and F4 it found as 91.07%, which state as good drug released in ibuprofen nanoemulsion based gel. . Fig 11:-% cummulative drug release from different formulations In-vitro release of flavanoid:   Anima was treated with frozen condition and frostbite was developed over the skin of the rat as mentioned earlier. In initial stages of frostbite skin causes inflammation which is treated with nanoemulsion gel after intervals of 6 hours topically for couple of weeks and kept for observation. When Nanoemulsion used over skin Inflammation is showing decreasing after few applications. Due to the small particle size nanoemulsion formulation permeated through skin and shows good drug release.

Conclusion:-
Nanoemulsions are attractive system for the use in the cosmetic, pharmaceuticals foods and pharmaceutical industries because of amount of very less surfactant, high stability against the coalescence, lower of toxicity or irritants characteristic. Due to their better permeability characteristic through skin the colloidal dispersions of solid nanoscale particulates have received the considerable attention. Moreover, the development of high throughout production make the potential for wide spread commercial used of nanoemulsion in consumer products and medical applications. In future, we can predict that nanoemulsion will become as ubiquitous as many polymer solution and solid particulate dispersions are today.