1. INTRODUCTION:

The solubility of BCS II drugs is a major challenge to the formulation scientist. Novel solubilization technologies have been developed from past two decades. The majority of the processes are not commercially viable due to the cost, sophisticated equipment, time and manpower. Use of synthetic surfactants of different classes, anionic, cationic, nonionic and amphiphilic in different dosage forms are available in the market.

Nonionic surfactants are the major role in solubilization poorly water soluble drug.

The average concentration is used in the different formulation is ranging from 2% to 20%. It is reported that synthetic surfactant supper from biodegradability, GIT irritation and skin sensitization [1]

Biosurfactants are a new class of surface active agents’ biosynthesized from yeast, bacteria and filamentous fungi using carbon and nitrogen source such as waste fried oil, molasses, expressed oil nuts and peptone, ammonium chloride respectively. Biosurfactants are classified into Glycolipids, Rhamnolipids, Sophorolipids, Trehalolipids, Lipoproteins and Lipopeptides etc. The advantages of biosurfactant are biodegradability, low toxicity, biocompatible, easy production with economical raw materials, stability in all pH and specificity. Biosurfactant have numerous uses in pharmaceuticals, for solubilization of lipophilic drugs in aqueous media, as components of emulsion or surfactant self-assembly vehicles for oral and transdermal drug delivery agents to improve drug absorption and as penetration enhancer, due to its very low critical micelle concentration (CMC). [2] In the present research, an attempt was made to produce biosurfactant from the flavno bacterium species using economical substrates and purified [2]. Enhancements of Solubility and permeation properties of biosurfactant were evaluated by selecting model the drug Lornoxicam.

Lornoxicam is a potent NSAID drug. It is practically water insoluble drug classified under the BCS II. The oral dose is 8 to 16mg daily divided doses and topically 0.5% w/w act as anti-inflammatory through Cox I & II inhibitor. Common side effect of the drug is GIT irritation hence the topical application of the drug in the gel form is preferred.[3] Solubilization of Lornoxicam is enhanced by biosurfactant and incorporated into gel base using sodium alginate as the gelling agent to achieve the uniform distribution of the drug. Gels were evaluated for its physicochemical properties and in vitro permeation study. Ex vivo permeation and in vivo anti-inflammatory activities were also studied for the effectiveness of biosurfactant as a permeation enhancer.

2 MATERIAL AND METHODS:

Lornoxicam procured from Optimus Drug Pvt Ltd Hyderabad gift sample. Sodium alginate, methyl parbens, Propyl paraben and tween 80 purchased from Hi-media Lab Ltd Mumbai. Solutol HS 15 USP BASF Germany. Calcium gluconate from Forbes Pharmaceutical Pvt Ltd Mumbai. All other chemicals were AR grades.

In- house biosynthesis of biosurfactant:[ 4,5]

Biosurfactant was produced from the biotechnology processes in the fermentor (SaraTousLite) using Flavobacterium Sp. 2495 in a modified mineral salt media. Molasses 2%, waste fried oil 5%, and 1% peptone, 0.5% ammonium chloride as carbon and nitrogen source respectively. Environmental conditions, pH at 7.4, temperature 35±1^oc aeration 40% and rpm 450.Duration of fermentation was 90 hrs. The fermented fluid was separated by solvent extraction using methanol-water mixture (1:2). Purification was carried out using 5 N HCl by precipitation method. The product was dried at 40^oc for 5hrs and passed through 60 mesh. Stored in the airtight container at room temperature. Skin irritation test was performed on the albumin rat as per OECD guidelines. It was found nonirritant.

Solubilization of Lornaxicam [6,7]

Excesses of Lornoxicam was added into 0.1%w/v to 2.5%w/v of biosurfactant and 2%w/v to 10%w/v Polysorbate 80 in 20ml conical flask respectively and stirred in the mechanical shaker for 24 hr at 10 rpm. Sonicated for 10 minutes, filtered, the filtrate is diluted with phosphate buffer in the Beer's range, drug content was determined using UV-Visible spectroscopy (Shemazid)at λmax 307 nm. Solubilization was carried in triplicate.

Drug compatibility study:

Drug excipients compatibility studies were performed to identify an interaction between drug and excipients used.IR spectra of drug and excipients mixture were carried out from 4000cm^-1 to 400cm^-1. (Shimadzu FTIR spectra)

Preparation of gel:[8]

Sodium alginate was used as the gelling agent in different concentrations. Polymer was dispersed in distilled water without lumps, kept for 24hrs for hydration. Lornoxicam was dissolved in of Solutol HS15, Biosurfactant and Tween 80 was added with constant stirring using propeller stirrer. Calcium gluconate was dissolved in hot water (50^oc) and added to the gelling mixture and stirred for 30 minutes. Aqueous solutions of Preservatives were added to the gel [Table no2]. The final weight of gel was adjusted using phosphate buffer pH 6.8 stirred for 20 minutes and stored in airtight container at room temperature until further analysis.

Evaluation of gels:

pH:

pH of all formulated gels was determined using calibrated pH meter ( Systroinic MK 6) All the reading were recorded in triplicate for three days and average reading was calculated.

Drug content:

Gels ( 1gm) were dissolved in the mixture of phosphate buffer pH 6.8 and methanol ( 1:1), stirred for 10min. one ml sample was diluted to 10ml using the same solution and filter through What man filter paper(No 40 ). Absorbance was measured at λmax 307 nm using UV-Visible spectroscopy ( Shemazid)

Rheological and apparent viscosity determination: [9]

The rheological determination was carried out using Brookfield Capcalc V3.0Build20-0.The system was equipped with cone and plate with plate diameter 40mm. About 0.5 gms of the sample was placed in the plate, allowed to set for constant temperature (25.0±0.1⁰c) for 5 minutes. Shear stress was determined by the gradual increase in rpm and recorded. Rheogram was plotted by the shear rate on the y axis and shear stress on the x axis. Yield stress was calculated by fitting the into the mathematical model Bingham.

Spreadability: [10]

The spreadability was performed on two glass plates (12cmx12cm).500mg of the gel was placed on one slide over it another slide was placed. 10grms of weight was placed on an upper slide for 5 min. weight was removed and time taken for separation of the slide was noted. Following formula was applied for calculation of spreadability.

S = ML/T

S = spread ability gm.cm/sec.

M = Weight tied on the upper slide.

L = Length of glass slide, cm.

T= Time taken to separate the two slides in sec

Extrudability:[11]

Formulated gel was filled in an aluminum tube (10gms) and the bottom is sealed. The Extrudability of the formulations were determined in terms of weights in grams required to extrude a 0.5 cm. ribbon of gel in 10 seconds. The measurement was done in triplicate.

Applied weighrt to extrude emugel from tube (g)

Extrudability= -------------------------------------------------------

Area (in cm²)

Gel strength: [12]

It was measured by the method reported by Jaiswal et al. An in-house modified apparatus was fabricated (Fig no4). The 50gms formulation was placed in 100ml graduated cylinder at ambient temperature. 35 grams weight was placed on the T-bar, allowed to penetrate 5cm. The time required to penetration was noted and reported as gel strength.

Fig: 2 In-house fabrication for gel strength measurement.

In vitro diffusion study: [13]

The experiment was conducted in Franz diffusion cell of 40ml capacity. Pretreated egg membrane was fixed in between the donor and receptor cells. The receiver contains phosphate buffer pH 6.8 and methanol [1:1] solution. Lornoxicam gel (1gm) was applied on the egg membrane and donor compartment was clamped. Water circulation was maintained at 37±1^oc with 20 rpm on the magnetic stirrer.1ml sample was withdrawn with the syringe and replace with fresh PBS solution to maintain the sink condition. Samples were analyzed by UV spectroscopy at λmax 307 nm. The study was conducted for 6 hrs in triplicate.

Ex-vivo permeation study:[10]

The ex vivo drug permeation study was carried out in Franz diffusion cell using wistar rat abdomen skin (IAEC, CPCSEA NO: 221) Clean, shaven, washed with saline abdomen rat skin was clamped between the donor and receiver compartment. Receiver compartment contains PBS buffer pH 6.8 and methanol (1:1) as dissolution medium. Lornoxicam 1 gram gel was spread on the upper skin layer. The temperature of the dissolution medium was maintained at 37±1^oc with 20 rpm on the magnetic stirrer.1ml sample was withdrawn with the syringe and replace with fresh PBS solution to maintain the sink condition. Samples were analyzed by UV spectroscopy at λmax 307 nm. The study was conducted for 6 hrs in triplicate.

Permeation Data Analysis:

The cumulative % drug release was plotted against time for all the formulations. Permeation rate (drug flux) at steady state (Jss)was determined by dividing the slope of the linear portion of the graph by the area of the diffusion cell. The permeation coefficient (Kp) was calculated by dividing Jss by the initial concentration of drug in the donor cell (Co)

Kp= ^Jss/_co

Jss of rat skin

Enhancement ratio was = -------------------------

determined by Jss of egg membrane

Drug release kinetics:

To understand the drug release kinetics of Lornoxicam gel, drug release data were fitted in different models of release kinetic using PCP Disso V3 software (Pune. India). To know which mathematical model best fits the obtained release profile

Skin irritation test:[10]

Skin irritation test was performed in six male Wistar rats. The animals were divided into two groups standard and test respectively containing three each. The dorsal side of the skin was shaven (6cm²) one day before the commencement of the experiment. The standard group was applied with 0.8% formalin. The test group was applied with optimized gel. All group animals were observed for erythrema, swelling or reddening at the end of 24 hrs. The score was given accordingly and analyzed for as per Draize table.

In vivo anti-inflammatory study:[14]

The experiment was conducted on male albino rats (150 to 200gms 10 weeks) by carrageenan induced paw edema model. Nine rats were taken, divided into three groups. Group, I control receiving 1% carrageenan in saline. Group II received standard marketed formulation (Piroxy gel). Group III optimized formulation with 1% carrageenan saline. Carrageenan saline injection of 0.1 ml was given to the plantar site of the right-hand paw to all groups, but group II and group III received the injection after 30 minutes of application of marked gel and optimized gel respectively. The volume of paw edema was measured using plethysmography immediately after injection and taken as zero reading. The volume was measured at different intervals up to 6 hrs and % inhibition of paw edema in the drug-treated group was compared with carrageen control group and calculated according to the formula.

(C-T)

% Inhibition edema= -------------- X 100

C= mean volume of edema for control

T= mean volume of edema for test.

Stability study:

Stability studies were conducted at 25^oc ±1^oc and RH 40%and 40^oc ±1oc at RH 65% in stability chamber ( Lab control ) Duration of stability study was for 3 months. Lornoxicam gel was filled in 10gms aluminum tubes with cap stored in the stability chamber. Every month gels were analyzed for the pH, Viscosity, drug content, and in vitro release.

Statistical analysis:

Data were expressed as mean ±SD. Differences were considered statistically significant at p < 0.05. Statistical analyses were performed using GraphPad Prism 4.01 software.

3 RESULTS AND DISCUSSION:

Solubility analysis:

Solubility studies of Lornoxicam with 0.1% to 2.5%w/v biosurfactant were in the range of 0.68mg/ml to 14.68mg/ml and with 2% to 10%w/v Polysorbate 80 were in the range of 0.44mg/ml to 2.25mg/ml. There were 7 fold increases in the solubility with the difference of 5% concentration of polysorbate 80. This could be due to the amphiphilic nature of the biosurfactant.SolutolHS15 solubility was 56mg/ml which was added to compensate the fraction of undissolved Lornoxicam. (Table no 1)

Table 1: Solubility of Lornoxicam

Biosurfactant %w/v	Concentration of drug dissolved mg/ml	Polysorbate80 %w/v	Concentration of drug dissolved mg/ml
0.1	0.68±0.01	2	0.44±0.07
1	1.81±0.03	4	0.76±0.01
1.5	6.41±0.03	6	1.45±.02
2	9.76±0.09	8	1.65±0.05
2.5	14.68±0.02	10	2.25±0.08

Mean ± SD. n=3

Drug compatibility study:

Results of IR spectra of pure drug Lornoxicam and with the physical mixture of excipients were interpreted for prominent functional groups of Lornoxicam structure. It was observed that presence of absorption peak at 3068 cm^-1 due to the presence of C=C stretching, 1592 cm^-1 for primary and secondary amines and amides, 831.7 cm^-1 for aromatic group, 1041.3 cm^-1 for S=O stretching. It concludes that drug is present in free form than the reacting form.(Fig no1)

Preparation of gel:

The concentration of Lornoxicam was 0.5%w/v in the gel. Solutol HS 15 was added in different proportions to solubilises the fraction of undissolved drug in the gel. Calcium gluconate is used as the complexing agent for sodium alginate and moisturizing agent. Methyl and Propyl Parabens were preservatives. All the formulations are shown in the table no 2.

Evaluation of gels:

Physical examination and pH:

The prepared Lornoxicam gels were viscous, yellow colour preparation with smooth and homogeneous appearance.

All the prepared formulations pHs were in the range of 6.62 to 6.72.Compatible with skin components. [Table no 3]

Table 2: Formulations of gels.

Sno	Ingredients	Unit	G1	G2	G3	G4
1	Lornoxicam	gm	0.5	0.5	0.5	0.5
2	Sodium alginate	gm	5	5	6	6
3	Calcium gluconate	gm	0.05	0.05	0.08	0.08
4	2.5 % w/v Biosurfactant	ml	10	---	10	---
5	10% w/v Polysorbate 80	ml	---	40	---	40
6	Solutol HS 15	ml	5	10	5	10

Drug content:

Formulations G1 to G4 drug contents were to be in the range of 97.89% to 98.50 % indicative of uniform distribution and within the compendial limits. [Table no 3]

Spreadability:

Topical gel formulations of lornoxicam were studied for its Spreadability property in order to determine the ease of application of gel onto the skin surface

The values of spreadability of all the formulations were in the range of 17.1 to 23.02g cm/sec High value indicate the spreadability is more with less shearing.[Table no 3]

Extrudability:

The Extrudability of the prepared gel formulations were found to be between 14.43gm to 17.51gm. For formulations containing the high concentration of Solutol HS 15, the extrudability was found to be more. [Table no 3]

Gel strength:

Gel strength of all the prepared formulations was in the ranges of 65.66sec to 88.33 Sec. Results indicate that all gels are having good integrity and homogeneity. [Table no 3]

Table no 3: Physicochemical evaluations.

Formulation code

% Drug content

Spreadability

(gm./cm²)

Extrudability

g.cm

Gel strength

Sec

6.71±

98.34±

2.31

21.91±

0.25

16.15±

0.27

73±

3.46

6.62±

97.84±2.45

26.02±

0.12

17.51±

0.34

65.66±

3.51

6.71±

98.13±1.54

17.10±0.21

14.43±

0.26

86±.

3.21

6.60±

98.50±1.89

22.03± 0.31

15.21±

0.39

88.33±

3.05

Mean ± SD. n=3

Rheological and apparent viscosity determination:

The rheological characteristics of prepared gels were essential in the technical application including manufacturing, material pumping, filling, and storage. Yield value is defined as minimum shear stress required to flow and below this point, the material will behave as solid. The pharmaceutical and cosmetic preparations should have high yield value so that they do not flow out of the container when placed up down position or in transport. The apparent viscosity and the yield values of G1 to G4 formulations are shown in the table no 4 and fig no 3. G1 formulation showed the highest yield value with optimum apparent viscosity as compared to the G2, G3, G4 formulations. The attributing factors might be the Solutol HS 15. G2 and G4formulation contain 10% of Solutol HS15 as compared to the G1 and G3 (5%). The higher value of viscosity showed in G3 and G4 as compared to the G1 and G2 due to the increase in the concentration of gelling polymer

Table: 4 Viscosity and Yield value

Formulations	Apparent Viscosity mPa.s	Yield stress Pa
G1	3563 ± 43.89	220 ±20
G2	3100 ±46.22	205 ±18
G3	3895 ±45.71	103 ±12
G4	3656±67.35	125 ±10

Mean ± SD. n=3

Fig; 3 Rheogramm of gels

In vitro and Ex-vivo diffusion study:

In vitro release of drug from the semisolid dosage form gives an idea of a number of free drug molecules available for partitioning into stratum corneum. A study conducted by two methods, results revealed that higher percentage of drug release through the egg membrane was found as that of abdomen rat skin, it could be due to the absence of stratum corneum layer in egg membrane. Permeation of drug molecules through the animal skin model of all gels was dependent on apparent viscosity, permeation enhancers and concentration of gelling polymers. All these three parameters have been studied results are tabulated in table no5.There was an increase in the percentage of drug release for low viscous gels (G1 and G2) as compared to the high viscous gels (G3 and G4). Lauffer's molecular diffusion theory states that diffusion coefficient of solute is inversely proportional to the volume occupied by the gel-forming agent. This statement is true with G3 and G4 formulations results. Ex Vivo study results revealed that biosurfactant act as good permeation enhancers as compared to the nonionic surfactant. The biosurfactant containing formulations ( G1 and G3) showed the increase in the transdermal flux as compared to the nonionic surfactant (G3 and G4) A possible explanation for this finding could be due to the surface active agents can increase the membrane fluidity increasing the drug diffusion through the membrane. All formulated gels were fit into kinetic modeling and followed the diffusion model.

Fig 4: In vitro egg membrane permeation studies.

Fig:5 Ex-vivo permeation study.

Skin irritation test:

The test group showed no irritation but standard group moderate erythema and mild edema were observed. The score given for test groups zero and the standard group was 2.6.From the results obtained from the skin irritation test optimized formulation G1 containing biosurfactant was nonirritant.

In vivo anti-inflammatory study:

Test and standard formulations exhibited the anti-inflammatory effect for 2.5 hrs and percentage inhibition of standard and test groups were 34.6% and 49.48% respectively. It concludes that the optimized formulation G1 has the better therapeutic effect of decreasing paw edema volume of the Wistar rat as compared to standard formulation. This could be due the effectiveness of biosurfactant as permeation enhancer.

Fig: 6 In vivo anti-inflammatory paw edema studies

Stability study:

Formulation G1 showed no changes in colour and appearance after three months of storage at room temperature and elevated the temperature. The pH, apparent viscosity, drug content and percentage of drug release showed no much variation in all the mentioned parameter at room temperature. But at elevated temperature, there was an average 10 percentage deviation in all parameters from the initial values. The results are tabulated in the table no 6 and 7 indicative of G1 formulation is stable at room temperature for three months

Table:6 For optimized formulation G1 at 25^oc ±1^oc and RH 40%

Duration	pH	Viscosity Pa.S	Drug content %	*In vitro* release studies %CDR
0 month	6.7±02	3565 ±45.71	99.5±2.31	82.23± 2.15
1^st month	6.6±0.3	3465±38.61	98.6±2.54	79.64±2.38
2^nd month	6.5±0.3	3478±41.2	98.5±2.98	79.52±2.48
3^rd month	6.5±0.2	3465±38.3	98.9±3.01	75.01±2.57

Mean ± SD n=3

Table: 7 for optimized formulation G1 at 40^oc ±1oc at RH 65%

Duration	pH	Viscosity Pa.S	Drug content %	*In vitro* release studies %CDR
0 month	6.7±02	3565±45.71	99.5±2.31	82.23± 3.15
1^st month	6.6±0.3	3265±38.61	98.6±2.54	76.64±1.08
2^nd month	6.3±0.3	3228±41.2	97.5±2.98	75.52±2.98
3^rd month	6.3±0.2	3215±38.3	96.9±3.01	74.01±2.12

Mean ± SD n=3

4 CONCLUSION:

Solubility studies of lornoxicam using the in-house biosynthesized biosurfactant showed maximum solubility with the lowest concentration (2.5%). There were no interactions between the ingredients selected for the formulation of Lornoxicam hydrogel by FTIR. Good consistency, uniform drug distribution, easily applicable with minimum shear and compatible skin pH were achieved for the optimized formulation G1.In vitro and ex vivo permeation studies proved that the incorporation of biosurfactant in the Lornoxicam hydrogel act as solublising agent and permeation enhancer with enhancement ratio 0.85 for the G1 formulation. Carrageenan-induced paw edema test revealed the anti-inflammatory activity of G1 formulation was 49%inhibition as compared with 34% inhibition of marketed formulation. The ideal temperature for storage of G1 formulation was at room temperature as compared to the elevated (40/^OC±1/65 RH). It can be concluded that biosurfactant is the good solublising agent as well as the permeation enhancer for the topical drug delivery containing the water in- soluble drugs. However, further preclinical and clinical studies are required.

5. ACKNOWLEDGMENTS:

Authors are very much thankful to M/s Optimus Drug Pvt Ltd Hyderabad for gift sample of Lornoxicam. We also thankful to the principal of KLEU College of Pharmacy Belagavi for providing the facilities to carry out the research work.

6. LIST OF ABBREVIATIONS AND SYMBOLS:

Symbols
w	Weight
v	Volume
λmax	Lambda max
nm	Nanometer
ml	Milliliter
gr	Gram
mg	Milligram
%	percentage
pH	Power of hydrogen

Abbreviations
BCS	Biopharmaceutical classification system
FTIR	Fourier tramission infrared spectroscopy
Pa S	Pascal second
GIT	Gastro intestinal trac
CMC	Critical micelle concentration
COX	Cyclooxygenase enzyme
OECD	Organisation for Economic Co-operation and Development
Jss	Transdermal flux
SD	Standard deviation
RH	Relative humidity
Max	Maximum

7. CONFLICT OF INTEREST:

There is no conflict of interest.

8. REFERENCES:

1. Van Bogaert INA, Saerens K, Muynck CD, Develter D, Soetaert W, Vandamme EJ. Microbial production and application of sophorolipids, Appl. Microbiol.Biotechnol.76; 2007: 23-34.

2. Sandeep Singh Bhadoriya1 , Narendra Madoriya , Karunakar Shukla , Parihar MS. Biosurfactants a new pharmaceutical Additive for solubility enhancement and pharmaceutical development, Biochemistry & Pharmacology.3;2013:1-5. Skjodt NM, Davies NM. Clinical pharmacokinetic of lornoxicam: short half-life oxicam. Clinical pharmacokinetic. 34(6); 1998:421-428

3. Krishnaswamy Muthusamy, Subbuchettiar G, Tiiengungal K, Ravi, Panchakarma S. Biosurfactants: Properties, commercial production, and application. Current Science. 94;2008:736- 748.

4. Krespky N, Da Silva FS, Fontana LF, Crapez MA. An alternative methodology for isolation of biosurfactant producing bacteria.Braz J Biol.67; 2007:117-124

5. Vishal J, Parekh, Vandana B Patravale, Aniruddha B Pandita. Mango kernel fat: A novel lipid source for the fermentative production of sophorolipid biosurfactant using Starmerella Bombicola NRRL-Y 17069. Annals of Biological Research.3 (4);2012:1798-1803

6. Higuchi K A, Connors, Phase-solubility techniques. Adv. Anal. Chem. Instr. , 4, 1965:117-212.

7. Desai KGH, Kulkarni AR., Aminabhavi TM. The solubility of rofecoxib in the presence of methanol, ethanol and sodium lauryl sulfate at (298.15, 303.15 and 308.15 K). J. Chem. Eng. Data. 48;2003:942–945.

8. Japan Patel, Brijesh Patel, Hardeep Singh banwait, Kaushal Parmar, Manish Patel. Formulation and Evaluation of Topical Aceclofenac Gel Using Different Gelling Agent. International Journal of Drug Development & Research .3; 2011: 156-164

9. Zaniol NA, MingTS, Darwis Y, Development of and characterization of cinnamon leaf oil nano cream for topical application, Indian Journal of Pharmaceutical science77(4);.2015: 422-433.

10. Rachit Khullar, Deepinder K, Namrata S, Seema S. formulation and evaluation of mefenamic acid emugel for topical delivery. Saudi Pharmaceutical Journal.20; 2012: 63-67.

11. Naga Sravan Kumar Varma et al., Calcipotriol delivery into the skin as emulgel for effective permeation. Saudi Pharmaceutical Journal. , 22(4) ;2014:591-599

12. Jyotivardhan Jaiswal, Anantvar SP, NarkhedE MR, Gore SV, Mehta K. Formulation and evaluation of thermoreversible in-situ nasal gel of Metoprolol succinate,.International Journal of Pharmacy and Pharmaceutical Sciences,.4(3); 2012:96-102

13. Mehandi Ansari, Maryam Kazemipour, Mohireh Aklami., The study of drug permeation through the natural membrane. Internation Journal of Pharmaceutics. , 32; 2006: 6-11

14. Tanaji Nandgude, et al. Formulation and evaluation of pH-induced in vitro nasal gel of salbutamol sulphate, International Journal f Pharmaceutical Science and Nanotechnology.1(2); 2008:177-183.

Received on 21.10.2017 Modified on 17.11.2017

Research J. Pharm. and Tech 2018; 11(7): 2848-2854.

DOI: 10.5958/0974-360X.2018.00525.5

Formulation	Flux Egg membrane Jss µm/cm².h	Permeability coefficient Egg membrane Kp	*Flux Ex-vivo* rat membrane Jss µm/cm².h**	Permeability coefficient *Ex vivo* rat membrane Kp	Enhancement ratio
F1	4.49±2.34	8.98±1.86	3.82±1.41	7.64±1.25	0.85
F2	3.61±0.98	7.22±1.25	2.73±2.11	5.46±1.65	0.75
F3	4.08±1.45	8.16±0.75	3.23±1.65	7.05±2.84	0.79
F4	3.39±0.85	6.78±0.89	2.41±2.74	4.80±2.22	0.72