Microemulsion formulation design and evaluation for hydrophobic compound: Catechin topical application

Accepted Manuscript Title: Microemulsion formulation design and evaluation for hydrophobic compound: catechin topical application Authors: Yu-Hsiang Lin, Ming-Jun Tsai, Yi-Ping Fang, Yaw-Syan Fu, Yaw-Bin Huang, Pao-Chu Wu PII: DOI: Reference:

S0927-7765(17)30664-1 https://doi.org/10.1016/j.colsurfb.2017.10.015 COLSUB 8896

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

5-6-2017 22-9-2017 4-10-2017

Please cite this article as: Yu-Hsiang Lin, Ming-Jun Tsai, Yi-Ping Fang, Yaw-Syan Fu, Yaw-Bin Huang, Pao-Chu Wu, Microemulsion formulation design and evaluation for hydrophobic compound: catechin topical application, Colloids and Surfaces B: Biointerfaces https://doi.org/10.1016/j.colsurfb.2017.10.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ms. Ref. No.: COLSUB-D-17-01032 Microemulsion formulation design and evaluation for hydrophobic compound: catechin topical application Yu-Hsiang Lin1#, Ming-Jun Tsai2,3#, Yi-Ping Fang1, Yaw-Syan Fu4, Yaw-Bin Huang1, Pao-Chu Wu*1 1

School of Pharmacy, 4Department of Biomedical Science and Environmental Biology, Kaohsiung Medical

University, 100 Shih-Chuan 1st Road, Kaohsiung city 807, Taiwan. ROC. 2

Department of Neurology, China Medical University Hospital, 3School of Medicine, Medical College, China

Medical University, 2Yuh-Der Road, Taichung city 404, Taiwan, ROC. *Correspondence Author: Pao-Chu Wu, Ph.D. School of Pharmacy Kaohsiung Medical University 100 Shih-Chuan 1st Road, Kaohsiung City 807, Taiwan, ROC. TEL: 886-7-3121101 ext 2660 FAX: 886-7-3210683 E-mail: [email protected] #

Ming-Jun Tsai and #Yu-Hsiang Lin had equal contribution.

Graphical Abstract

Highlights 

Response surface methodology was used in formulation optimization.



The percutaneous capacity of formulations was evaluated by permeation study.



The transdermal amount was remarkably increased by using microemulsion as carrier.

ABSTRACT The aim of the present study was to design a microemulsion for catechin topical application. A mixture experimental design with five independent variables (X1: oil, X2: surfactant, X3: catechin, X4: cosurfactant and X5: water) was developed, and the response surface methodology was used to study the effect of formulation components on physiochemical characteristics and penetration capacity of a catechin-loaded microemulsion, and to obtain an optimal microemulsion formulation. The results showed that the drug-loaded microemulsion formation and characteristics were related to many parameters of the components. The transdermal amounts in receiver cells and skin deposition amount remarkably increased about 4.1~111.6-fold and 0.6~7.6-fold respectively. The lag time was significantly shortened from 10 h to 1.0~6.7 h. The optimal formulation with 20% surfactant,

30% cosurfactant and 2.6% Catechin was subjected to stability and irritation tests. The results showed that the physicochemical characteristics and catechin level of the drug-loaded microemulsion did not show significant degradation after 3 months of storage at 25℃.The catechin-loaded microemulsion did not cause significant irritation compared to the watertreated group. Keywords: Catechin; Microemulsion; Mixture design; Stability; Skin irritation.

1. Introduction The transdermal drug delivery system is a much less invasive, more comfortable and convenient method for drug delivery. It can provide many advantages, including decreased first-pass metabolism effect of drug, avoidance of gastrointestinal irritations, sustained delivery of drug to provide steady plasma pattern, diminished systemic side effects, and enhanced patient compliance. Catechin, a polyphenol, rich in green tea, apple, red wine, and numerous nutritional products, has been reported to possess significant antioxidant potential, antiaging, antidiabetic, anti-obesity, antibacterial, neuroprotective, anti-HIV, hypolipidemic, and antiinflammatory effect properties [1-5]. Some studies [6, 7] also reported that catechin had significant skin photoprotection effect against UV-mediated oxidative stress, melanoma, basal cell carcinoma, and sunburn. After oral administration, the pharmacokinetic parameters showed that catechin possesses a short half-life (t1/2, 1.25 h), higher first-pass effect and low oral bioavailability at less than 5% [8-10]. Moreover, intestinal uptake is poor [11]. Hence, the topical application of catechin should offer benefits on skin photo-protective effects and bioavailability improvement, including used chemical enhancer, liposome, nanoemulsion gel and electrically methods to enhance skin drug delivery [6, 12-15] . With respect to the skin topical application, the therapeutic compounds drug can only act when it permeates at least the outermost layer (stratum corneum) of the skin, and its efficacy of topical application is often impeded, because transportation is slow due to the resistance of the stratum corneum of the skin. In past years, many strategies have been applied to improve the poor permeability of drugs through the skin, including the incorporation of penetration enhancers, drug carriers e.g. liposomes and microemulsions, and physical methods e.g. electroporation, iontophoresis, sonication, and microneedle technologies, either alone or in combination [16, 17]. The enhancement mechanism is included to increase

the solubility of drugs in formulations and to modify the structure of lipophilic and keratinized regions in skin layers [16, 17]. In recent years, the nanoscale carrier has attracted more attention to improve therapeutic efficacy of therapeutic compounds. Microemulsions are low viscosity, optically isotropic and thermodynamically stable colloid systems, and have been widely and extensively utilized for a variety of pharmaceutical products including oral, parental, transdermal and dermal drug delivery modalities [18-23]. They can provide several significant benefits such as ease of manufacturing, increased drug solubility in pharmaceutical formulation, and improved membrane permeability [22-25]. Furthermore, previous studies [26-29] have demonstrated that microemulsions do not induce barrier perturbation of the skin and can even decrease the skin irritation caused by drugs, even though the microemulsion system topically contains large amounts of oil and surfactants. Hence, the microemulsion system was used as a drug carrier to improve catechin penetration capacity in this present study. It is well known that the microemulsion formation and characteristics are related to many parameters of the components. In order to realize the influence of formulation components and to quickly obtain an optimal catechin-loaded microemulsion formulation with appropriate penetration rate and skin deposition amount of drug,

a statistical

experimental design with a constrained mixture design was used in this study [30-32]. Finally, the experimental formulation was also applied in conducting both skin irritant and stability tests to further confirm its utility. 2. Materials and methods 2.1. Materials

Catechin, acetaminophen (internal standard), carbamic acid ethyl ester (urethane), and paraformaldehyde were from Sigma-Aldrich (St. Louis, Missouri, USA). Polyoxyl 4-lauryl ether (Brij 35) and polyoxyl 23-lauryl ether (Brij 30) were purchased from Acros Organic

(Pennsylvania, USA). Isopropyl myristate (IPM) was obtained from Tokyo Chemical Industry (Tokyo, Japan). Ethanol was obtained from ECHO (Taiwan). All other chemicals were of analytical reagent grade.

2.2. Catechin-loaded microemulsion preparations After our preliminary study (data not shown), the IPM (3~10%), mixture surfactant of Brij35/Brij 30 (15~20%), catechin (0.5~3%), cosurfactant of ethanol (21.7~30%) and distilled water (40~50%) were utilized to prepare catechin-loaded microemulsions. The mixture experimental design was used to arrange a set of seventeen model formulations with different amounts of formulation ingredients. Then, the influencing degree of each dependent variable factor and the interaction variable factors on the physicochemical characteristics including viscosity and droplet size of formulations and permeation capacity of drug including penetration amount and skin deposition amount were investigated by response surface methodology (RSM) [30-32] by Design Experts software (state-Ease Inc, Mineapolis, USA). The model microemulsion formulations were randomly arranged and are listed in Table 1. The spontaneous emulsion method was used to prepared catechin-loaded microemulsions. The mixture surfactant of Polyoxyl 4-lauryl ether and polyoxyl 23-lauryl ether was prepared in advance. Accurately weighed quantities of catechin, oil phase, mixed surfactants and cosurfactant were mixed thoroughly by vortex for 1 min at ambient temperature, and then, distilled water was incorporated into the mixture by vortex for 5 mins to obtain a homogeneous mixture. The formulations were recorded for any change on turbidity or phase separation. The catechin-loaded microemulsions were stored in brown glass bottles at ambient temperature until use. 2.3. Physicochemical properties determination Viscosities of catechin-loaded formulations were measured by a Brookfield, Model LVDVII, cone-plate of viscometer (USA). A microemulsion of 0.5 mL was loaded into the cone-plate,

the cone-plate was then heated by a thermostatic pump, and maintained at 37℃ for 3 mins. The viscosity of sample was recorded after 30 s measurement made at the rotation rate of 120.0 rpm. The average droplet size of drug-loaded formulations was measured by Malvern particle size analyzer (Zetasizer 3000HSA, UK) with wavelength of 658 nm, scan angle of 90º, and temperature of 25 ºC. Three milliliters of sample were placed in a standard quartz cuvette and then put in the scattering chamber to determine the average droplet size and polydispersity index (PI). Each test formulation was measured in triplicate and the mean value was presented. 2.4. Skin permeation investigation The experimental protocol was reviewed and approved (# 104144) by the Institutional Animal Care and Use Committee of Kaohsiung Medical University (Kaohsiung, Taiwan). The committee confirmed that the experiment in this study followed the guidelines as set forth by the Guide for Laboratory Fact Lines and Care. The skin permeation capacity of tested catechin-loaded microemulsions and catechin saturated aqueous solution of 3941.62 μg/mL (control group) were measured by using modified Franz diffusion equipment with diffusion area of 3.46 cm2. The shaven abdomen skin samples of SD rats were set on the receptor cell with the stratum corneum side facing upward, and the donor cell was then clamped in place. The pH 5.5 phosphate-citric acid buffer of 20 mL [15] was used as receiver medium, and was maintained at 370.5 ℃ throughout the experiment by a thermostatic pump. Samples of 1 mL were withdrawn from the receiver cell at predetermined time intervals e.g. 1, 2, 3, 4, 6, 8, 10, and 12h, and then the same volume of fresh medium was refilled. The concentration of catechin was determined by a modified HPLC method [15].

At the end of the 12-h permeation experimental, the applied skin was carefully removed from the diffusion cells and washed with deionized water three times. The drug deposition amount in the skin was extracted with methanol by horizontal shake for 15h. The resulting methanol solution was filtered through a membrane of 0.45 mm, and the catechin content was then analyzed [15]. 2.5. Chromatographic condition A HPLC equipped with a Hitachi model L-7100 pump, a Spark Holland basic Marathon autosampler a Hitachi model L-4000H detector, and Merck Lichrocart® C18 column with 250×4 mm i.d. and particle size of 5 μm was used for catechin analysis. The mobile phase consisted of phosphoric acid aqueous solution (1 in 2000, pH 2.5) and acetonitrile at ratios of 88 and 12. The flow rate was at 1.0 mL/min and the UV detection was at 280 nm. Acetaminophen solution of 100 µg/mL was used as internal standard in drug analysis. The analytical method was successfully validated for linearity (3-100 ug/mL) with a determination coefficient (r) of 0.9993, coefficient of variation of 4.94%, and relative error of 4.19%. 2.6. Skin irritation study

Male Sprague Dawley rats weighted at 275-300 g were anesthetized with a urethane aqueous solution of 0.75 g/kg by intraperitoneal. Five hundred milliliters of aqueous water (negative control), 0.8% paraformaldehyde (positive control) and experimental formulation with and without catechin were evenly spread on the shaven abdomen skin of 2.54 cm 2 and occluded by parafilm [27, 33]. After 24 h application, the treated skin was excised for histological examination. In brief, the tissue was fixed in 10% neutral carbonated-buffered formalin for at least 24 h. Following fixation, tissue sample was rinsed with water, dehydrated with a series of ethanol solution and embedded in paraffin. The tissue block was sectioned at 10-μm thickness, rehydrated, and stained with hematoxylin and eosin (H&E). The stained

slides were examined under a light microscope (Nikon Eclipse Ci, Tokyo, Japan) and evaluated for histopathological changes associated with microemulsion exposure.

2.7. Stability study The centrifugation method at 3500 rpm for 30 min and 10000 rpm for 10 min, and three heating-cooling cycle (4℃/45℃ and 48 hr storage for each temperature) tests [34] were used to evaluate the thermodynamic stability of the experimental catechin-loaded microemulsion. The formulation was recorded for any changes in turbidity, phase separation, creaming or cracking. The optimal formulation was stored at 25±2℃ and 60±5% RH for 3 months. Samples were withdrawn at defined time intervals for appearance and drug content analysis. 2.8. Data analysis The permeability parameters such as cumulative amount in receiver cell, drug deposition amount in skin, and penetration retention time (lag time, the first detected time of drug) were used to evaluate the effective of microemulsions. The data was expressed as mean±standard deviation of triplicate determinations. The differences between the experimental formulations were assessed using analysis of variance, and then by Tukey multiple comparison test using Winks SDA 6.0 software. Mean differences with p value <0.05 were considered significant. The relationship between the dependent and independent variables was enlightened using RSM containing polynomial mathematical equation models such as linear, quadratic, and cubic models, provided by Design-Expert software. The best fitting mathematical equation model was selected based on the comparisons of the p value of model, p value of lack of fit multiple correlation coefficient (R2), adjusted multiple correlation coefficient (adjusted R2), and (Predicted Residual Sum of Squares (PRESS). The p value for the models

should be less than 0.05 and the p value for lack of fit should be larger than 0.05. The coefficients for the X term represented the intensity of effect of the independent variables [35-37]. 3. RESULTS AND DISCUSSION 3.1. Physicochemical characteristics Seventeen formulations were prepared as per the experimental design. As shown in Table 1, only fifteen model formulations were formed. The microemulsion formulation of F08 and F18 with lower surfactant and cosurfactant could not be formed. The mean droplet size and viscosity of all model drug-loaded nanostructured emulsions are measured and listed in Table 1. The mean droplets were in the nanosize range from 179.0 to 602.8 nm. Previous studies pointed out that only sizes ranging from 50 to 500 nm particles were possible to permeate the skin [38], demonstrating that the most current formulations were well-suited for dermal/ transdermal delivery. The result of multiple regression analysis for the response surface methodology is summarized in Table 2. It can be seen that the droplet size shows a good relationship with the independent variables (Table 2). The response surface plot (Fig. 1A) shows that the droplet size was significantly affected by the proportion of formulation components. The drug level (X3) and the interaction variables of (X3X5) were the crucial factors. The viscosity of catechin-loaded nanostructured emulsions was measured at 37℃ by a viscometer with rotation rate of 120.0 rpm. The viscosity ranged from 10.57 to 14.20 cps, indicating microemulsions with low viscosity. The response surface plot (Fig. 1B) depicts a linear relationship between the viscosity and formulation variables. It was found that the viscosity had incentive tendency with increase in drug level because the hydrophobic drug dissolved in the internal phase, then increased the size and viscosity.

The results showed that the microemulsion formation and characteristics were related to many parameters of the components. Physicochemical properties of formulations were significantly affected by the component of formulations [23, 25, 26, 29, 39] 3.2. In vitro skin permeation and drug deposition study The permeation parameters including cumulative amount of drug in receptor cell (Q12h), and deposition amount in skin (D12h) and lag time of catechin-loaded formulations after 12 h application are presented in Fig 2. The saturated aqueous solution of catechin was used as the control group to depict the enhancement effect by using microemulsion as carriers. The Q12h, D12h and LT were 13.46± 8.29 μg/cm3, 12.75±4.02 μg/cm3 and 10±2.83 h respectively, indicating that the catechin had difficulty penetrating the skin [14, 15]. When using microemulsion as the carrier, Q12h and D12h were significantly increased about 4.1~111.6-fold and 0.6~7.6-fold respectively. The LT was remarkably shortened from 10 h to 1.0~6.7 h. The result was consistent with previous studies reporting that microemulsion emulsions could enhance the permeability capacity of therapeutic compounds [20, 40-46]. From Table 2, the RSM analysis showed that the catechin concentration (X3) had greatest effect on Q12h and D12h, followed by cosurfactant, oil and surfactant. The response surface plots of Q12h (Fig. 3A) and D12h (Fig. 3B) were similar, showing that the skin deposition amount tended to increase in total transdermal amount, although a nonsignificant linear relationship (p > 0.05 ) between Q12h and D12h was found. From Fig. 3A and Fig. 3B, it was found that higher Q12h and D12h would be observed when the formulation incorporated higher amounts of catechin and cosurfactant, paired with the microemulsion with smaller particle size (Fig. 2A). The enhancement mechanism of the present microemulsion might be attributed to 1) higher level catechin resulted in higher thermodynamic activity and lower permeability capacity of drug. 2) the cosurfactant could act as permeation enhancer and increase the diffusivity of drug in the microemulsion. 3) nano-scale droplet size could offer high and robust skin contact [26].

In order to obtain an optimal catechin-loaded microemulsion, the software optimization process was used, with code selected for X1, X2, X3, X4, and X5 being 0.278, 0.334, 0.0078, 0.0019 and 0.385 respectively, which gives theoretical values of 333.05 nm, 10.25 cps, 868.81 µg/cm2, and 84.09 µg/cm2 for droplet size, viscosity, Q12h and D12h respectively. Fresh formulation was prepared using the optimum levels of independent variables. The observed values were found to be 368.63±3.96, 9.30±0.12, 994.27±150.15 µg/cm2 and 71.29±12.71 µg/cm2 84.09 µg/cm2 respectively, which were in close agreement with the theoretical values, indicating that the RSM with mixture design could be used in catechin-loaded microemulsion design.

3.3. Skin irritation The skin irritation test was conducted to assess the safety of tested formulations. The distilled water-treated group and 0.8% formalin solution-treated group were used as negative control and standard irritant group respectively [47]. The positive group (Fig. 4B) showed collagen fiber swelling in the dermis layer, small edema in the hypodermis layer, and slight damage and exfoliation of the stratum corneum in the epidermis layer when compared with the negative control group (Fig. 4A). In the tested formulation without and with drug (Figs. 4C and 4D), non-significant edema and erythema was observed when compared to the watertreated group (Fig. 4A), showing that the microemulsion formulation possessed good biocompatibility with skin, which might be used for other therapeutic compounds, and therefore drug-loaded formulations might be acceptable for human use. 3.4. Stability The catechin-loaded microemulsion was subject to centrifugation tests (3500 rpm for 30 min and 10000 rpm for 10 min) and three heating-cooling (4℃ and 45℃) cycles for quickly testing its thermodynamic stability. After these tests, there was no liquefaction, phase

separation, or precipitation observed, indicating that the catechin-loaded microemulsion possessed excellent physical stability. The result might be because the microemulsion system had low interfacial tension between oily and water phases, and was of nanoscale droplet size, which made it thermodynamically stable [48, 49]. The appearance, viscosity and droplet size of the catechin-loaded formulation showed no obvious change, and no drug crystal was observed after 3 months of storage at 25±2℃ and 60±5% RH. The residual drug content was 98.44±0.10%, indicating that the experimental catechin-loaded formulation was stable. 4. Conclusions

The optimization of microemulsion formulation is a complex process, which requires the consideration of quite a few variable factors. Response surface methodology with mixture design is a useful mathematical tool for catechin-loaded microemulsions design. It can identify the influence degree of variable factors, and interactions with each other, and obtain an optimal formulation. The drug permeability of experimental microemulsion formulations through rat skin in this study were significantly increased, including increase of Q12h and D12h about 4.1~111.6-fold and 0.6~7.6-fold respectively, and the lag time was remarkably shortened from 10 h to 1.0~6.7 h. The drug-loaded microemulsion formulation was stable for at least 3 months of storage at 25℃. Moreover, the microemulsion with and without catechin showed less irritant properties when compared with the standard irritant group. The result showed that the microemulsion is a suitable carrier for catechin topical application. Conflict of Interests The authors declare that there is no conflict of interest regarding the publication of this paper. Acknowledgment

This work was supported by Grants from the National Science Council of Taiwan (MOST 105-2320-B-037-010 and MOST 105-2632-B-037-003). References

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penetration/irritation profiles and pharmacokinetic performance, International journal of pharmaceutics, 496 (2015) 931-941. [44] D.S. Mahrhauser, H. Kahlig, E. Partyka-Jankowska, H. Peterlik, L. Binder, K. Kwizda, C. Valenta, Investigation of microemulsion microstructure and its impact on skin delivery of flufenamic acid, International journal of pharmaceutics, 490 (2015) 292-297. [45] M. Nasr, S. Abdel-Hamid, Optimizing the dermal accumulation of a tazarotene microemulsion using skin deposition modeling, Drug development and industrial

pharmacy, 42 (2016) 636-643. [46] P. Patel, A. Pol, S. More, D.R. Kalaria, Y.N. Kalia, V.B. Patravale, Colloidal soft nanocarrier for transdermal delivery of dopamine agonist: ex vivo and in vivo evaluation, Journal of biomedical nanotechnology, 10 (2014) 3291-3303. [47] A. Azeem, S. Talegaonkar, L.M. Negi, F.J. Ahmad, R.K. Khar, Z. Iqbal, Oil based nanocarrier system for transdermal delivery of ropinirole: a mechanistic, pharmacokinetic and biochemical investigation, International journal of pharmaceutics, 422 (2012) 436-444. [48] A. Azeem, F.J. Ahmad, R.K. Khar, S. Talegaonkar, Nanocarrier for the transdermal delivery of an antiparkinsonian drug, AAPS PharmSciTech, 10 (2009) 1093-1103. [49] M.J. Lawrence, G.D. Rees, Microemulsion-based media as novel drug delivery systems, Advanced drug delivery reviews, 45 (2000) 89-121.

Table legends Table 1: Factors investigated using constraint mixture design experimental design, and physical characterization of model catechin-loaded formulations. Table 2. Regression coefficients and statistical analysis of dependent variables.

Figure legends Fig. 1. Three-dimensional response surface plots demonstrating the effect of dependent variables on the independent variables including droplet size (A), viscosity (B). The dependent variable was fixed at oil phase (X1) of 0.184 and cosurfactant (X2) of 0.278.

Fig. 2. The permeability parameters of model catechin loaded formulations and control group (catechin-saturated solution).

Fig. 3. Three-dimensional response surface plots demonstrating the effect of dependent variables on the independent variables including transdermal amount (Q12h, C), and deposition amount in skin (D12h, D) after 12h application of formulations. The dependent variable was fixed at oil phase (X1) of 0.184 and cosurfactant (X2) of 0.278.

Fig. 4. Microscopic photos of rat skin after application of distilled water (A), 0.8% paraformaldehyde (B), nanostructured emulsion without drug (C), and nanostructured emulsion with catechin (D). (Original magnification 40)

Figr-1Fig. 1. Three-dimensional response surface plots demonstrating the effect of dependent variables on the independent variables including droplet size (A), viscosity (B). The dependent variable was fixed at oil phase (X1) of 0.184 and cosurfactant (X2) of 0.278.

rt?Software Coding: U_Pseudo nverted by U_Pseudo coding

700 600

A EtOH er

400

s iz e

omponents 84 8

500

300 200 100

X31(0.538288) (0.5) C

DX (0) 4(0.0)

5

X5(0.0) E (0)

XC3(0) (0.0) D X (0.538288) (0.5)

re _Pseudo y U_Pseudo coding

4

20.0

15.0

V is c o s ity

ts

EX(0.538288) (0.5)

10.0

5.0

0.0

C (0.5) X3(0.5)

X4(0.5) D (0.0) (0.5) XE5(0.5)

E (0.0) X5(0.5)

C (0.0) X3(0.0) D (0.5)

X4(0.5)

Fig. 2. The permeability parameters of model catechin loaded formulations and control group (catechin-saturated solution).

Q12h :Cumulative amount (g/cm2)

Control F17 F16 F15 F14 F13 F12 F11 F10 F09 F08 F07 F06 F05 F04 F03 F02 F01 0

600

400

200

800

1000

1400

1200

1600

1800

Cumulative amount (g/cm2)

Control F17 F16 F15 F14 F13 F12 F11 F10 F09 F08 F07 F06 F05 F04 F03 F02 F01

Lag time (h)

0

2

4

6

Control F17 F16 F15 F14 F13 F12 F11 F10 F09 F08 F07 F06 F05 F04 F03 F02 F01

8

10

12

14

D12h :Deposition amount (g/cm2)

0

20

40

60

80

100

120

140

Fig. 3. Three dimensional response surface plots demonstrating the effect of dependent variables on the independent variables including transdermal amount (Q12h, C), and deposition amount in skin (D12h, D) after 12h application of formulations. The dependent variable was fixed at oil phase (X1) of 0.184 and cosurfactant (X2) of 0.278.

Software ding: U_Pseudo erted by U_Pseudo coding

2000

1500

EtOH 1000

Q 12h

mponents 500

0

-500

X3(0.5) C (0.5) (0.0) X4D(0.0)

E (0.0) X5(0.0)

C (0.0)

X3(0.0)

D (0.5) X4(0.5)

ign-Expert?Software mponent Coding: U_Pseudo hs/Lows inverted by U_Pseudo coding h 12.592

120

.56363

100

= C: Drug = D: CoSA EtOH = E: Water

80

60

D 12h

seudo Components PM = 0.184 MS = 0.278

XE 5(0.5) (0.5)

40

20

0

XX3(0.5) C (0.5) 1(0.5)

X4

D (0.0) (0.5) XE5(0.5)

E (0.0) X5(0.0)

C (0.0)

X3(0.0)

D (0.5) X4(0.5)

Fig. 4. Microscopic photos of rat skin after application of distilled water (A), 0.8% paraformaldehyde (B), nanostructured emulsion without drug (C), and nanostructured emulsion with catechin (D). (Original magnification 40) (A)

(C)

(B)

(D)

Table 1: Factors investigated using constraint mixture design experimental design. Independent variables

Code value

X1: Amount of oil (3~10%)

0~0.389

X2: Amount of surfactant (15~25%)

0~0.556

X3: Amount of drug ( 1~3%)

0~0.5

X4: Amount of cosurfactant (21.7~30%)

0~0.461

X5: Amount of aqueous (40~50%) Code

0~556

X1

X2

X3

X4

X5

F01

0.000

0.020

0.139

0.286

0.556

349.50 ±

7.28

14.20 ±

0.45

F02

0.000

0.402

0.060

0.242

0.296

602.83 ±

17.04

12.83 ±

0.33

F03

0.000

0.174

0.098

0.366

0.362

561.87 ±

37.17

11.83 ±

0.48

F04

0.080

0.556

0.139

0.000

0.225

119.40 ±

1.45

12.73 ±

0.24

F05

0.309

0.421

0.039

0.000

0.231

488.37 ±

29.48

11.40 ±

0.73

F06

0.000

0.556

0.000

0.000

0.444

388.20 ±

6.48

11.80 ±

0.78

F07

0.100

0.248

0.083

0.014

0.556

542.77 ±

27.52

12.07 ±

0.31

F08

0.389

0.066

0.139

0.001

0.405

ND

F09

0.389

0.191

0.000

0.258

0.162

481.37 ±

34.46

11.13 ±

0.70

F10

0.142

0.214

0.000

0.182

0.462

496.23 ±

25.61

11.93 ±

0.70

F11

0.000

0.000

0.000

0.461

0.539

216.67 ±

20.21

12.73 ±

0.54

F12

0.389

0.050

0.000

0.006

0.556

179.00 ±

0.95

12.50 ±

1.48

F13

0.186

0.205

0.139

0.276

0.195

289.50 ±

8.21

10.57 ±

0.33

F14

0.389

0.427

0.117

0.067

0.000

379.17 ±

13.65

13.40 ±

1.93

F15

0.284

0.000

0.069

0.253

0.395

±

F16

0.000

0.020

0.139

0.286

0.556

488.37 ±

29.48

11.40 ±

0.73

F17

0.000

0.402

0.060

0.242

0.296

372.67 ±

9.95

11.13 ±

0.19

F18 ND: non-detected

Size (nm)

ND

Viscosity (Cps)

ND

±

ND

Table 2. Regression coefficients and statistical analysis of dependent variables. Q12h Regression

coefficient

D12h

Droplet size

Viscosity

Coefficient

Coefficient

Coefficient

Coefficient

Estimate

Estimate

Estimate

Estimate

b1 (X1)

970.08

88.28

1725.86

7.11

b2 (X2)

-505.55

-164.00

288.04

13.02

b3 (X3)

25859.86

+4431.10

-30491.57

21.73

b4 (X4)

-2083.02

-616.17

2272.84

15.59

b5 (X5)

2023.32

-16.12

514.95

10.06

b12 (X1X2) b13 (X1X3)

-1322.09 -4074.72

34634.36

b14 (X1X4)

432.64

-4181.33

b15 (X1X5)

269.65

-2995.99

b23 (X2X3)

-4634.04

32002.85

b23 (X2X4)

-24980.24

-24534.25

664.22

b23 (X2X5)

714.30

b34 (X3X4)

-2762.38

33473.57

-6518.15

38341.02

1222.81

-4624.51

< 0.0001

< 0.0001

< 0.0001

0.0021

Lack of Fit (P value)

0.5018

0.0915

0.4916

0.6753

R-Squared

0.9121

0.9246

0.9810

0.3574

Adj R-Squared

0.8940

0.8896

0.9732

0.2879

PRESS

907500

7122

28755

158

b35 (X3X5)

-50410.83

b45 (X4X5) Model (P value)

Q12h and D12h: Transdermal amount and deposition amount in skin after 12h application of formulation.