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657 Journal of Oleo Science Copyright ©2013 by Japan Oil Chemists’ Society J. Oleo Sci. 62, (9) 657-664 (2013) Penetration effect of Ostrich Oil as a Promising Vehicle on Transdermal Delivery of Sinomenine Xin Liu, Teng Chen, Xuesong Liu, Yong Chen and Longhu Wang＊ College of Pharmaceutical Sciences, Zhejiang University (310058, Hangzhou, China) 1 INTRODUCTION Sinomenine（Fig. 1）is a bioactive alkaloid derived from the Chinese medicinal plant, Sinomenium acutum, which has been widely used to treat inflammatory and arthritic diseases for one thousand years. However, oral administra- tion of sinomenine is often limited on account of gastroin- testinal adverse side effects, including nausea, diarrhea, constipation, gastralgia, and occasionally vomiting. In addi- tion, the oral route of drug delivery is not preferable for patients with low oral bioavailability; also, the short elimi- nation half-life of sinomenine requires frequent dosing1）. Hence, the transdermal route could be a better alternative route, because it bypasses first-pass metabolism, minimizes the gastrointestinal side effects, increases patient compli- ance, maintains a constant drug level in plasma and physi- ological effects. An essential prerequisite for transdermal absorption of drugs is that the drug must be capable of passing through the skin at a sufficiently high rate to achieve therapeutic ＊Correspondence to: Longhu Wang, College of Pharmaceutical Sciences, Zhejiang University 310058, Hangzhou, China E-mail: wang2000@zju.edu.cn Accepted March 31, 2013 (received for review February 7, 2013) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/　　http://mc.manusriptcentral.com/jjocs Abstract: The present study investigated the feasibility of ostrich oil utilizing as a promising vehicle for improved skin permeation of sinomenine with reference to vaseline matrix containing different content of chemical enhancers. The fatty acid composition of ostrich oil was analyzed by GC-MS. Penetration enhancing potential of ostrich oil on permeation of sinomenine across rat abdominal skin in vitro was studied using an automatic diffusion cell apparatus. The content of sinomenine percutaneous absorbed was determined by HPLC. Various parameters viz. steady-state skin flux (Jss), permeability coefficient (kP), cumulative amount of sinomenine (Q) and enhancement ratios (ER) were calculated from the permeation data. Fick’s law of diffusion and Scheuplein kinetic were used to evaluate the transdermal absorbent enhancement of ostrich oil to sinomenine. Ostrich oil showed significant penetration effect on sinomenine compared with vaseline matrix containing different content of chemical enhancers, the density sequence as follow: 2% Azone > ostrich oil > 1% Azone plus 1% propylene glycol > 1% Azone > 3% Azone > 1% propylene glycol. The percutaneous endosmic rate constant (Jss) and permeability coefficient (kP) of sinomenine in ostrich oil through rat skin were 10.01 µg/cm2/h and 0.087, respectively. Ostrich oil produced stronger enhancement (ER = 24.31) with greater cumulative amount of drug permeated (255.53 µg/cm2) up to 24 h and caused no skin irritation. The drug release of sinomenine was coincided with Fick’s equation. In summary, ostrich oil containing fatty acids is proposed as a promising adjuvant for use in cosmetics and pharmaceuticals for improved permeation of drug. Key words: ostrich oil, sinomenine, transdermal, penetration vehicle, fatty acid Fig. 1　The chemical structures of sinomenine. X. Liu, T. Chen, X. Liu et al. J. Oleo Sci. 62, (9) 657-664 (2013) 658 plasma concentrations. However, the major barrier to the percutaneous transport of drugs across the skin is stratum corneum（SC）2）. One of the most widely used strategies to deliver an effective dose of drug through skin is to revers- ibly reduce the barrier function of the skin with the aid of penetration enhancers3）. Fatty acids have been shown to interact with the stratum corneum lipids, and a number of fatty acids have been identified as skin permeation enhanc- ers4, 5）. Ostrich oil, a natural bird fat oil containing fatty acids, has been used for alleviating various dull pains caused by arthritis and cervical spondylosis since ancient times, exhibiting the remarkable curative effect on aceso- dyne and diminishing inflammation6）. Meanwhile, it has been used as an ideal adjuvant in cosmetics and pharma- ceuticals owing to its remarkable permeability, excellent lubrication and emulsion properties7）. In this study, ostrich oil was investigated as the test vehicle as it is an inexpensive and rich source of natural fat oil. Sinomenine was selected as the model drug, and rat abdominal skin was removed hairs and used in vitro pene- tration experiment. Various contents of Azone, propylene glycol（PG）and the combination of Azone and PG have been used as reference enhancers in vaseline matrix. The fatty acid composition of ostrich oil was analyzed by GC-MS. The influence and mechanism of ostrich oil on the transdermal delivery of sinomenine were also evaluated. 2 EXPERIMENTAL 2.1 Materials Standard of sinomenine（purity＞99％）was purchased from the Chinese Authenticating Institute of Material and Biological Products（Beijing, China）. Acutum alkaloid ex- tracts that contain 98％ sinomenine（w/w）was kindly pro- vided by Zelang Medical Technology Co., Ltd（Nanjing, China）. Ostrich oil was extracted from ostrich fat by super- critical fluids extraction（SFE）based on previously de- scribed procedures4）. Azone, propylene glycol, stearic acid, triethanolamine and lanolin were purchased from Aladdin Chemistry Co., Ltd.（Shanghai, China）. Methanol of HPLC grade was obtained from Amethyst Chemicals J & K Scien- tific Ltd. All other reagents used were of analytical grade. Deionized water purified by a Milli-Q water-purification system was used in all experiments. 2.2 Animals Male Sprague-Dawley rats（8 weeks old, 200-220 g）were supplied by Central Animal House Facility of Zhejiang Uni- versity and kept under standard laboratory conditions in 12 hr light/dark cycle at temperature 25±2℃ and humidity 70-75％. Animals were provided with standard rodent chow and water ad libitum. 2.3 Analytical Methodology Samples in solubility and permeation studies were ana- lyzed by Agilent 1200 series HPLC system（Agilent Tech- nologies, USA）with PAD detector. HPLC analysis was per- formed according to the method of China Pharmacopoeia 2010 edition part I. The mobile phase was methanol: 0.005 M phosphate buffered（55:45, v/v）adjusted to pH 9.0 with triethylamine. Volume of sample injected was 20 µL. Mobile phase was run at a flow rate 1ml/min through a reverse phase C-18 column（250 mm×4.6 mm, 5 µm, Agilent Tech- nologies, USA）. Detection wavelength was set at 265 nm, and the retention time of sinomenine was 6.439 min. The calibration curve was made by standard of sinomenine for sample analysis. For the identification of fatty acids composition in ostrich oil, the GC method described in a previous study was used4）. The GC-MS system consists of an Agilent 6890N GC equipped with a split-splitless injector coupled with an Agilent 5975I MS equipped with an EI ion source and a quadrupole array detector. In the GC assay, the column oven temperature was programmed from 130 to 170℃ at 5℃/min, holding for 30 min, and then heated to 270℃ at 30℃/min, holding for 7 min. The temperatures of quadrupole, ion source chamber, injector and additional channel were 150, 230, 250 and 280℃, respectively. The carrier gas, helium（99.99％）was adjusted to a linear veloc- ity of 1.0 mL/min. The ionization energy was 70 eV with the total scan mode and the MS scan interval was 40-500 amu. 2.4 Solubility Studies Solubility study was performed according to the method of Rahul Jain et al8）. Briefly, an excess amount of sinome- nine was added to water and PG and shaken at 37±0.5℃ for more than 48 hours until equilibration. The suspensions were centrifuged at 8,000 rpm for 15 min. The supernatant was then filtered through 0.45 µm membrane filters and the concentration of drug was measured by HPLC after ap- propriate dilution. The experiments were performed in quadruplicate and the results were shown in Table 1. 2.5 Partition Coefficient determination n-Octanol and water were mutually saturated for 24 hours before the experiment. Octanol/water partition coef- ficient（Po/w,）of sinomenine was determined by shake flask method that was based on thorough mixing of the two phases to reach the equilibrium9, 10）. Stock solutions of si- nomenine were made in aqueous solution with pH of 2, 3, 4, 5, 6, 7, 8, 9, 10 adjusted with hydrochloride solution or sodium hydroxide solution, and then presaturated with the analytic grade n-octanol. Ten milliliters of stock solutions were merged with 10 ml of n-octanol phase presaturated with water, and the phases of the solvent system were mu- tually saturated by shaking for 48 h on a mechanical shaker at the temperature of 25±0.5. After centrifugation at 6,000 Penetration effect of Ostrich Oil as a Promising Vehicle on Transdermal Delivery of Sinomenine J. Oleo Sci. 62, (9) 657-664 (2013) 659 rpm for 15 min, sinomenine concentrations in each phase were determined by HPLC method. Three independent measurements were conducted with all solutions. Apparent partition coefficient P was calculated according to the lit- erature11）. 2.6 Preparation of formulations The compositions of blank control formulation were vaseline as vehicle and sinomenine（5 mg/g）without either penetration enhancers and ostrich oil. Control formulations were prepared utilizing vaseline as the matrix with differ- ent penetration enhancers such as 1％ Azone, 2％ Azone, 3％ Azone, 1％ Azone＋1％ PG and 1％ PG, respectively. Sinomenine（5 mg/g）was then added to control formula- tions. Test formulation was prepared by the same method except that ostrich oil was used as vehicle without penetra- tion enhancers. 2.7 In vitro Skin Permeation studies 2.7.1 Preparation of Isolated Rat Abdominal Skin The rats were sacrificed by giving excess ether anesthe- sia. The hairs from the abdominal surface of the rat were removed by a clipper and isolated skin was surgically removed from the rats12）. The subcutaneous tissue adher- ing to the skin was separated with help of scalpels and the dermis side was wiped with isopropyl alcohol to remove the residual adhering fat. The skin was washed with normal saline and stored in a deep freezer at －20℃ until use. The skin was examined under light microscope before the per- meation experiments to ensure the integrity of the skin13, 14）. 2.7.2 Diffusion of sinomenine across rat skin in automatic diffusion apparatus The permeability of sinomenine across rat skin in vitro was studied using automatic diffusion apparatus（Pharma- copoeia Standard Instrument Factory of Tianjin, China） fitted with modified Franz diffusion cells at 37±0.5℃ through the use of a circulating water bath. The diffusion cell consisted of donor and receiver chambers each of ca- pacity 7 mL with a diffusional area of 1.65 cm2. The treated skin pieces were mounted over diffusion cells with the dermal side in contact with the receptor phase. The recep- tor compartments were filled with phosphate buffered saline（PBS, pH 6.8）containing 0.02％ w/v of sodium azide to retard microbial growth. The receptor phase was stirred at 500 rpm with a small magnetic bar to mix the concentra- tions uniformly. Skins were allowed to equilibrate for 1 h before experimentation. The stratum corneum faced the donor chamber filled with 2.0 g test cream containing sinomenine（5 mg/g）. Samples（2 mL）were withdrawn from the receiver solution at predetermined time intervals（1, 2, 4, 6, 8, 10 and 24 h）. Sinomenine concentrations were determined by the HPLC method. Sample volume was immediately replaced to their marked volumes with fresh receptor medium（maintained at 37±0.5℃）after each sampling. Addition of solution to the receiver compartment was performed with great care to avoid trapping air beneath the dermis samples. All ex- periments were performed in quadruplicate. 2.8 Data Analysis The cumulative amount of drug（Q, µg/cm2）permeated though a unit area was plotted as a function of time. The steady state flux（Jss, µg/cm 2/h）was determined as the slope obtained from the linear regression portion of the plot. The lag time（T, h）was determined by extrapolating the linear portion of the curve to the abscissa. Permeability coeffi- cient（Kp, cm/h）was calculated from the ratio of flux to drug concentration in the donor chamber. In addition, per- meation profiles were also analyzed by the skin concentra- tion of drugs（µg/g）. The ER was calculated from the Kp and skin concentration of drugs with enhancer divided by the same parameter without enhancer（control）. All percu- taneous permeation data are mean±S.D. Statistical signifi- cance was checked by student’s t test and considered to be granted at p＜0.05. 3 RESULTS AND DISCUSSION 3.1 Physicochemical Properties The physicochemical properties of the permeants, the molecular weight, solubility and log Po/w of drugs, are thought to play an important role in determining the pro- moting activity of penetration enhancers on the permeation of the drug across the skin3, 15）. In this study, the molecular weight of sinomenine is 329.38 g/mol（Table 1）, which is in the relatively range of 200-500 g/mol. The drug in this mo- lecular weight range has a potential in transdermal deliv- ery16）. The solubility of the drug, which depends on the chemical structure of itself and the vehicle, is important in determining the rate of delivery into the skin. As can be seen from Table 1, sinomenine has higher solubility in water at 87.24±8.93 mg/mL and moderately solubility in PG at 61.36±5.16 mg/mL, respectively. Contrarily, it pos- sesses low solubility in n-octanol at 3.71±0.24 mg/mL. The partition coefficient, Po/w, was defined as the ratio of Table 1　‌‌Physicochemical Properties of sinome- nine. Properties Results Molecular weight (g/mol) 329.38 Water solubility (mg/mL) 87.24±8.93 1, 2-PG solubility (mg/mL) 61.36±5.16 PBS (mg/mL) 114.97±9.61 Partition coefficient （Po/w） 0.043 Solubility parameter (MPa1/2) 24.42 X. Liu, T. Chen, X. Liu et al. J. Oleo Sci. 62, (9) 657-664 (2013) 660 the concentration of the compound in two immiscible liquid phases such as n-octanol and water. Po/w obtained from above calculation method, was 0.043. For practical purposes, the logarithm of the partition coefficient（log Po/w） which is a measure of how well a substance partitions between a lipid and water, determines the route of drug penetration through the skin. By comparison, log Po/w （－1.37）was not in the specified scope（2～3）. The possi- ble reason was that sinomenine molecule had considerable water solubility（87.24±8.93 mg/mL, Table 1）. In summary, sinomenine had a low potential of permeat- ing through the skin to the target, therefore, some addi- tives such as permeation enhancer should be used in order to increase the permeation ability of drug. 3.2 Fatty acid composition of ostrich oil Detailed identification and quantization of the fatty acid composition in the ostrich oil produced by SFE were per- formed by GC-MS. As shown in Fig. 2 and Table 2, 18 com- pounds were detected in the ostrich fat, among which 15 compounds were identified as fatty acids（99.90±0.92％）. The fatty acid composition of ostrich fat was characterized by 9-octadecenoic acid and hexadecanoic acid as the most major fatty acids. 9-octadecenoic acid（40.7±0.3％）was the principal unsaturated fatty acid, followed by 9, 12-octa- decadience acid（7.38±0.02％）and 9-hexadecenoic acid （7.13±0.15％）. Hexadecanoic acid（32.5±0.3％）was the predominant saturated fatty acid, followed by octadecanoic acid（7.43±0.05％）. These five fatty acids contributed to about 95.12％, and other ten minor fatty acids only made up 4.78％ approximately. Many fatty acids are generally recognized as safe and are approved by the FDA as inactive ingredients in cosmetic products. The effects of fatty acids as permeation enhancers have been shown to be depen- dent on their structure, alkyl chain length, and degree of saturation17）. Unsaturated fatty acids have been shown to promote higher magnitudes of permeation enhancement across skin when compared to saturated fatty acids of the same chain length. This has been attributed to the higher disrupting nature of the kinked chain of these fatty acids that would result in a higher magnitude of lipid disruption18－20）. 3.3 In vitro Percutaneous Permeation of sinomenine To further evaluate the penetration effect of ostrich oil as a vehicle in vitro, the classic permeation enhancers such as Azone and propylene glycol（PG）were chosen as positive reference-enhancing promoter in the control for- mulations. Previous studies demonstrated that Azone is generally used at low concentrations（1-5％ w/v）, which can increase percutaneous permeation of various drugs21）. PG has been extensively used as a solvent for transdermal formulation, which acts as a solvent for various compounds leading to an increased thermodynamic activity and en- hanced “solvent drag” of the drug molecule. Fig. 3（A）and（B）are examples of HPLC chromatograms resulting from analysis of the effect of ostrich oil on perme- ation of sinomenine across rat skin after 1 h and 24 h. A significant increasing height of the peak in sample collected from 1 to 24 h indicated that ostrich oil has higher perme- ation enhancement on sinomenine. Permeation profiles for sinomenine across rat abdominal skin in ostrich oil and control formulations containing other permeation enhanc- Fig. 2　GC chromatograms of ostrich oil obtained by SFE. Penetration effect of Ostrich Oil as a Promising Vehicle on Transdermal Delivery of Sinomenine J. Oleo Sci. 62, (9) 657-664 (2013) 661 ers are shown in Fig. 4. All results are expressed as means ±standard deviations. The results revealed that ostrich oil as vehicle significantly enhanced transdermal drug delivery of sinomenine. Among all the permeation enhancers evalu- ated, 2％ Azone had the highest enhancing effect on the permeation of sinomenine through the rat skin. The pene- tration effect of ostrich oil on sinomenine was higher than other enhancers except 2％ Azone. When 1％ PG used as an enhancer in the control formulation, there was no pro- moting effect on the permeation of sinomenine. The result of blank control experiment had no difference compared with that of the control formulation containing 1％ PG as permeation enhancer. The control formulation containing only 1％ Azone existed lower enhancing effect on the per- meation of sinomenine. Moreover, the combination of 1％ Azone plus 1％ PG used in the control formulation, the percutaneous permeation of sinomenine was obviously in- creased. The finding suggested that the enhancer activity of Azone may be increased by using cosolvents including PG22）. As shown in Fig. 4, with the increase of Azone con- centration from 1％ to 2％（w/v）, accumulative permeated amount of sinomenine increased, but decreased when Azone concentration reached to 3％（w/v）. This result indi- cated that there was an optimum concentration for Azone to achieve the best permeation-enhancing effect23）. Beyond the apparent “optimum” Azone concentration（2％ w/v）, the permeation of sinomenine did not increase further. The steady state flux of sinomenine obtained after per- meation enhancers treatment is in the following decreasing order: 2％ Azone＞ostrich oil＞1％ Azone＋1％ PG＞1％ Azone＞3％ Azone（p＜0.05, Table 3）. Moreover, it can also be clearly seen that ostrich oil exhibits better enhance- ment activity（ER＝24.31, p＜0.05）. Ostrich oil h