1. INTRODUCTION:

Most of the non-steroidal anti-inflammatory medicine and drugs used for the management of pain and inflammation have number of side effects when used for the longer time in higher doses. At the same time some medicine related to this segment have poor bioavailability issue. Such problems of these medications can be eliminated with the use of novel drug delivery systems.

Lornoxicam is one of the examples, which is a potent non-steroidal anti-inflammatory drug (NSAID) and possess relatively short plasma half-life (3 to 5 hours) as compared to other oxicams¹. Data from preliminary clinical trials suggest that Lornoxicam is as effective as the opioid analgesics morphine, pethidine (meperidine) and tramadol in relieving postoperative pain following gynaecological or orthopaedic surgery, and as effective as other NSAIDs used postoperatively. Substantial concentrations of Lornoxicam are attained in synovial fluid, the proposed site of action in chronic inflammatory arthropathies. So it is considered equally effective as other NSAIDs in relieving symptoms of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, acute sciatica and low back pain and demonstrated as potential alternative to other NSAIDs for the management of arthritis and other painful and inflammatory conditions². Lornoxicam is available in market in the form tablet and parenteral dosage form with 4 mg, 8mg which suffers from the drawback of producing gastritis and also the daily dose of the Lornoxicam should not increase more than 16 mg. Considering the dose, plasma half life and its use, it is a potent candidate which can be developed in novel drug delivery formulations leading to reduction in associated side effects of the drug and increase in patient compliance. Hence novel drug delivery systems of the drug (lornoxicam) will be developed in this project¹. On the other hand herbal drugs like curcumin, quercetin etc has also been reported to be a potent candidate for anti-inflammatory property. Therefore drug of herbal origin will also be formulated separately and compared with that of the formulations developed with the synthetic drug² ³.

Novel drug delivery system is the advance drug delivery system which improve drug potency, control drug release to give a sustained therapeutic effect, provide greater safety, finally it is to target a drug specifically to a desired tissue⁴. Ethosomes are the modified forms of liposomes that are high in ethanol content. The ethosomal system is composed of phospholipid, ethanol and water. They can penetrate the skin and enhance compound delivery both to deep skin stratum corneum and systemically. This ethanol fluidizes both ethosomal lipids and bilayers of the stratum corneum intercellular lipid. The soft, malleable vesicles then penetrate the disorganized lipid bilayers. Ethosomes are soft, malleable vesicles composed mainly of phospholipids, ethanol (relatively high concentration) and water. These “soft vesicles” represents novel vesicular carriers for enhanced delivery to/through skin⁵. The vesicles have been well known for their importance in cellular communication and particle transportation for many years⁶. Researchers have understood the properties of vesicles structure for use in better drug delivery within their cavities. In contrast to liposomes, ethosomes have been shown to exhibit high encapsulation efficiency for a wide range of molecules including lipophilic drugs, and are selective at delivering molecules to through the skin as discussed⁷ ⁸ ⁹

2. MATERIALS AND METHODS:

2.1 Materials:

Lornoxicam was received from Sun pharmaindustry Ltd, Vrodara, India as a gift sample. Curcumin was received as a generous gift from Himedia Lab Mumbai, India. phospholipids and ethanol purchased from an SD fine chum, India.

2.2 Preparation of Lornoxicam and Curcumin loaded ethosomes:

Ethosomal formulations were prepared by using the cold method. This is the most common and widely used method for the ethosomal preparation.Phospholipid, drug and other lipid materials were dissolved in ethanol in a covered vessel at room temperature with vigorous stirring.The mixture was heated at 300c in a water bath. Water was heated up-to 30^oc in a separate vessel and was added to the mixture and then stirred for 5 min.The vesicle size of ethosomal formulation was decreased to desire extent using sonication. Finally, the formulation was properly stored. In the ethosomal formulation, the lipid: ethanol ratio was optimized by taking their different ratio such as 5:1, 10:1, 15:1 and 5:2, 10:2, 15:2 ratio and all other parameters were kept remain constant. The prepared formulations were optimized on the basis of average particle size and % entrapment efficiency¹⁰ ¹¹.

Table 1: Different composition of Lornoxicam Ethosomes Formulation

F. Code	Drug (mg)	Phospholipid (mg)	Ethanol (ml)	PEG (mg)	Water (ml)
LEF1	10	50	10	10	100
LEF2	10	100	10	10	100
LEF3	10	150	10	10	100
LEF4	10	50	20	10	100
LEF5	10	100	20	10	100
LEF6	10	150	20	10	100

Table 2: Different composition of Curcumin ethosomes formulation

F. Code	Drug (mg)	Phospholipid (mg)	Ethanol (ml)	PEG (mg)	Water (ml)
CEF1	10	50	10	10	100
CEF2	10	100	10	10	100
CEF3	10	150	10	10	100
CEF4	10	50	20	10	100
CEF5	10	100	20	10	100
CEF6	10	150	20	10	100

3. Characterization Lornoxicam and Curcumin loaded ethosomes:

3.1 Solubility:

Solubility of the drug was determined by taking a quantity of drug (about 1-2 mg) in the test tube separately and added the 5 ml of the solvent (water, ethanol, methanol, 0.1 N HCl, 0.1 N NaOH, 7.2 pH phosphate buffer and Chloroform) shake vigorously and kept for some time. The solubility of the drug in various solvents (at room temperature) was noted.

3.2 Melting point:

A small quantity of powder was placed into a fusion tube. That tube was placed in the melting point determining apparatus (Chemline CL-725) containing castor oil. The temperature of the castor oil was gradual increased automatically and read the temperature at which powder started to melt and the temperature when all the powder gets melted. Melting point of Lornoxicam and Curcumin were found180-182°C and 190-192°C respectively.

3.3 FTIR:

Spectroscopy Infra- red spectrum is an important record which gives sufficient information about the structure of a compound. This technique provides a spectrum containing a large number of absorption band from which a wealth of information can be derived about the structure of an organic compound. The region from 0.8 µ to 2.5 µ is called Near Infra-red and that from 15 µ to 200 µ is called Far infra-red region Identification of Lornoxicam and Curcumin was done by FT-IR Spectroscopy with respect to marker compound. Lornoxicam and Curcumin were obtained as yellow powder. It was identified from the result of IR spectrum as per specification show in fig 1 and 2.

3.4 Loss on drying:

Loss on drying is directly measured by Hot Air Oven. Firstly calibrated the instrument then 5 gm of sample (powder) was taken and the temp was set at 100°C to 105°C for 15 minutes. Set the knob and check % moisture content. Average of three determination was determined by n=3 (mean ± SD) Avg. % loss on drying of Lornoxicam Curcumin was obtain 2.33±0.41 1.93±0.61⁵

3.5 Shape and Surface Morphology:

The shape and surface morphology of the microspheres were investigated using scanning electron microscopy (IISER, Bhopal). The microspheres were fixed on supports with carbon- glue, and coated with gold using a gold sputter module in a high-vacuum evaporator. Samples were then observed with the Scanning Electron Microscope at 10 kV show in fig 3 and 4³.

3.6 Entrapment efficiency:

Entrapment efficiency was determined by measuring the concentration of unentrapped free drug in aqueous medium. About 1 ml of the drug loaded ethosomes dispersion was placed in the Ependorf tubes and centrifuged at 17000 rpm for 30 min. The ethosomesalong with encapsulated drug were separated at the bottom of the tubes. In order to measure the free drug concentration, the UV absorbance of the supernatant was determined at λ max378nm and 428nm for Lornoxicam and Curcumin respectively⁸ ¹² ¹³.

3.7 Determination of pH:

The pH of the gel was determined by digital pH meter. Tengram of gel was was taken in a clean beaker and the electrode was then dipped in to gel formulation until constant reading obtained. And constant reading was noted. The measurements of pH of each formulation were replicated two times

3.8 Spreadability:

An important criteriafor gels is that it must possess good spreadability. Spreadability is a term expressed to denote the extent of area to which the gel readily spreads on application to skin. The therapeutic efficacy of a formulation also depends on its spreading value. A special apparatus has been designed to study the spreadability of the formulations. Spreadability is expressed in terms of time in seconds taken by two slides to slip of from formulation, placed between, under the application of a certain load. Lesser the time taken for the separation of two slides, better the spreadability show in fig 5.

3.9 Viscosity:

The measurement of viscosity of the prepared gel was done using Brookfield digital Viscometer. The viscosity was measured using spindle no. 6 at 10 rpm and 25 °C. The sufficient quantity of gel was filled in appropriate wide mouth container. The gel was filled in the wide mouth container in such way that it should sufficiently allow to dip the spindle of the Viscometer. Samples of the gels were allowed to settle over 30 min at the constant temperature (25±/1 °C) before themeasurements¹⁰.

3.10 In-vitro drug release studies using the prehydrated cellophane membrane Preparation of cellophane membrane for the diffusion studies:

The cellophane membrane approximately 25 cm x 2cm was taken and washed in the running water. It was then soaked in distilled water for 24 hours, before used for diffusion studies to remove glycerin present on it and was mounted on the diffusion cell for further studies. The prepared Ethosomal gel was evaluated for in vitro drug release. In vitro diffusion study was carried out in a Franz diffusion cell using cellophane membrane. The cellophane membrane was mounted on the Franz diffusion cell. Formulation was applied through donor compartment on the dialysis membrane. Reservoir compartment was filled with 25 ml phosphate buffer of pH 7.4 The study was carried out at 37 ± 1°C and at a speed of 100 rpm for 8 h. Samples were withdrawn from reservoir compartment at 1 h interval and absorbance was measured spectrophotometrically at 378.0 nm and 428 nm for Lornoxicam and Curcumin respectively. . Each time the reservoir compartment was replenished with the same quantity of 6.8 pH phosphate buffer⁷ ¹⁴.

4. RESULTS AND DISCUSSION:

4.1 Solubility:

Lornoxicam was sparingly soluble in 0.1 N Hydrochloric acid and 7.2 pH Phosphate Buffer, soluble in ethanol, methanol and chloroform, freely soluble in 0.1 N NaOH. It was found that Curcumin was practically insoluble in water, freely soluble in ethanol and methanol, Soluble in 0.1 N NaOH and 7.2 pH Phosphate Buffer.

4.2 Entrapment efficiency:

Entrapment efficiency was best formulation of lornoxicam loaded Ethosomes Vesicle size was 356.45 and Entrapment efficiency of drug was 71.56, curcumin loaded Ethosomes Vesicle size of was 312.25 and Entrapment efficiency of drug was 73.23

4.5 Physical Characteristic of gel:

The Physical Characteristic was checked for gel formulations (homogeneity and texture) and observations were shown in Table.

Table 3: Results of evaluation of gel formulation

Formulation Code	Homogeneity and texture
LG1	Particles present
LG2	Smooth
LG3	Smooth
CG1	Particles present
CG2	Smooth
CG3	Smooth

4.6 Determination of pH:

The pH of the gel was determined by digital pH meter. Tengram of gel was was taken in a clean beaker and the electrode was then dipped in to gel formulation until constant reading obtained. and constant reading was noted. The measurements of pH of each formulation Lornoxicam loaded ethosomal gel were replicated two times pH was 6.98, 7.04, 6.95 and Curcumin loaded ethosomal gel was 6.95, 6.98, 7.56.

4.7 Washability:

Formulations were applied on the skin and then ease and extent of washing with water were checked manually and observations of Lornoxicam and curcumin loaded ethosomal gel were yes.

4.8 Method:

Two glass slides of standard dimensions (6×2) were selected. The gel formulation whose spreadability had to be determined was placed over one of the slides. The second slide was placed over the slide in such a way that the formulation was sandwiched between them across a length of 6 cms along the slide. 100 grams of weight was placed up on the upper slide so that the gel formulation between the two slides was traced uniformly to form a thin layer.

The weight was removed and the excess of the gel formulation adhering to the slides was scrapped off. The lower slide was fixed on the board of the apparatus and one end of the upper slide was tied to a string to which 20 gram load could be applied with the help of a simple pulley. The time taken for the upper slide to travel the distance of 6 cms and separate away from lower slide under the direction of the weight was noted. The experiment was repeated and the average of 6 such determinations was calculated for each gel formulation. Spreadability(gm.cm/sec.) ofLornoxicam loaded ethosomal gel was abstained 13.25, 12.32, 11.51 and Curcumin loaded ethosomal gel 14.56, 13.65, 12.4515.

Figure 5: Photographs taken during spreadability

4.9 Viscosity:

The measurement of viscosity of the prepared gel was done using Brookfield digital Viscometer. The viscosity was obtained all formulation was Lornoxicam loaded ethosomal gel 2565, 2315, 2215 and Curcumin loaded ethosomal gel 2654, 2512, 2452. % Assay Lornoxicam loaded ethosomal gel 98.89, 99.85, 99.75 and Curcumin loaded ethosomal gel 98.17, 98.98, 97.25

4.10 Preparation of cellophane membrane for the diffusion studies:

The prepared Ethosomalgelwas evaluated for in vitro drug release. In vitro diffusion study was carried out in a Franz diffusion cell using cellophane membrane. The cellophane membrane was mounted on the Franz diffusion cell. Formulation was applied through donor compartment on the dialysis membrane. Reservoir compartment was filled with 25 ml phosphate buffer of pH 7.4 The study was carried out at 37 ± 1°C and at a speed of 100 rpm for 8 h. Samples were withdrawn from reservoir compartment at 1 h interval and absorbance was measured spectrophotometrically at 378.0 nm and 428 nm for Lornoxicam and Curcuminrespectively. . Each time the reservoir compartment was replenished with the same quantity of 6.8 pH phosphatebuffer.

Table 4: Cumulative % drug release of Lornoxicam and Curcumin from optimized ethosomal gel formulation

Time (hrs)	% Cumulative Drug Release
Time (hrs)	LornoxicamLG2	CurcuminCG3
0.5	27.85	19.98
1	35.92	33.36
2	45.95	40.25
4	56.65	55.89
6	69.98	63.36
8	78.98	71.56

4.11 Release kinetics:

4.11.1 In-vitro:

diffusion has been recognized as an important element in drug development. Under certain conditions it can be used as a surrogate for the assessment of bioequivalence. Several theories/kinetic models describe drug dissolution from immediate and modified release dosage forms. There are several models to represent the drug dissolution profiles where ft is the function of t (time) related to the amount of drug dissolved from the pharmaceutical dosage system. To compare dissolution profiles between two drug products model dependent (curve fitting), statistic analysis and model independent methods can beused.

In order to elucidate mode and mechanism of drug release, the invitro data was transformed and interpreted at graphical interface constructed using various kinetic models. The zero order release Eq. (1) describes the drug dissolution of several types of modified release pharmaceutical dosage forms, as in the case of transdermal systems, matrix tablets with low soluble drugs, coated forms, osmotic systems etc., where the drug release is independent ofconcentration⁶.

Qt = Qo + Kot (1)

Where, Qt is the amount of drug released in time t, Qo is the initial amount of the drug in the solution and Ko is the zero order release constant.

The first order Eq. (2) describes the release from the system where release is concentration dependent e.g. pharmaceutical dosage forms containing water soluble drugs in porous matrices.

log Qt = log Qo + K1 t /2.303 (2)

Where Qt is the amount of drug released in time t, Q is the initial amount of drug in the solution and K1 is the first order release constant.

Higuchi described the release of drug from insoluble matrix as a square root of time as given in Eq. (3)

Qt = KH √t (3)

Where, Qt is the amount of drug released in time t, KH is Higuchi’s dissolution constant.

The following plots were made: cumulative % drug release vs. time (zero order kinetic models); log cumulative of % drug remaining vs. time (first order kinetic model); cumulative % drug release vs. square root of time (Higuchi model)

Table 5: In Vitrodrug release data for LG2

S. No.	Time (H)	Square Root of Time	Log Time	*Cumulative Percentage Drug Release±SD**	Log Cumulative PercentageDrug Release	Cumulative Percent Drug Remaining	Log cumulative Percent Drug Remaining
1	0.5	0.707	-0.301	27.85±0.45	1.444825	72.15	1.85823634
2	1	1	0	35.92±0.25	1.555336	64.08	1.8067225
3	2	1.414	0.301	45.95±0.36	1.662286	54.05	1.7327957
4	4	2	0.602	56.65±0.21	1.7532	43.35	1.6369891
5	6	2.449	0.778	69.98±0.45	1.844974	30.02	1.47741069
6	8	2.828	0.903	78.98±0.58	1.897517	21.02	1.32263271

Fig. 6: Cumulative Percent Drug Released Vs Time (Zero OrderPlots) and (First Order Plots)

Table 6: Regression analysis data of ethosomal gelformulation (lornoxicam)

Formulation	Zero order	First order
LG2	R² = 0.976	R² = 0.994

Table 7: In Vitro drug release data for CG3

S. No.	Time (H)	Square Rootof Time	Log Time	*Cumulative PercentageDrug Release ± SD**	Log Cumulative PercentageDrug Release	Cumulative PercentDrug Remaining	Log cumulative Percent Drug Remaining
1	0.5	0.707	-0.301	19.98±0.48	1.300595	80.02	1.90319855
2	1	1	0	33.36±0.52	1.523226	66.64	1.82373499
3	2	1.414	0.301	40.25±0.32	1.604766	59.75	1.77633791
4	4	2	0.602	55.89±0.14	1.747334	44.11	1.64453706
5	6	2.449	0.778	63.36±0.74	1.801815	36.64	1.56395546
6	8	2.828	0.903	71.56±0.23	1.85467	28.44	1.45392959

Figure 7: Cumulative Percent Drug Released Vs Time (Zero Order Plots and First Order Plots)

Table 8: Regression analysis data of ethosomal gel formulation(curcumin)

Formulation	Zero order	First order
CG3	R² = 0.934	R² = 0.983

4.11.2 Result of in vitro study:

The In vitro drug release data of the formulation was subjected to goodness of fit test by linear regression analysis according to zero order and first order kinetic in order to determine the mechanism of drug release. When the regression coefficient values of were compared, it was observed that ‘r’ values of formulation was maximum in first order i.e0.994 and 0.983 for LG2 and CG3 respectively hence indicating drug release from formulations was found to follow first order of drug release kinetics in both drug optimized formulation.

5. CONCLUSION:

In the present study, lornoxicam and curcumin entrapped ethosomal gel for transdermal drug delivery was prepared by using various concentrations of phospholipids and ethanol. The prepared formulation showed good entrapment efficiency, vesicle size and drug release. The result advocates the superiority of ethosomal formulation with respect to skin permeation. In conclusion, it can be suggested that ethosomes could be superior drug carrier for topical delivery of lornoxicam and curcumin in the treatment of skin infection.

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Received on 18.11.2020 Accepted on 24.11.2020

Research J. Topical and Cosmetic Sci. 2020; 11(2):70-76.

DOI: 10.5958/2321-5844.2020.00013.8