Materials
Rhizoma coptidis produced in Sichuan, evodia rutaecarpa produced in Henan were supplied by Jinan hebao traditional Chinese medicine co. LTD (China). Palmatine hydrochloride, berberine hydrochloride, evodiamine, rutacarpine were provided by Beijing solabao technology co. LTD (China). Fat-soluble azone, water-soluble azone of medical grade were obtained from Zhengzhou jinbei chemical industry co. LTD. Dimethyl sulfoxides of analytic grade was obtained from Tianjin kermiou chemical reagent co. LTD (China). Acetic acid of analytic grade was obtained from Tianjin guangfu technology development co. LTD (China). Acetonitrile, methanol were of high-performance liquid chromatography (HPLC) grade. Sodium dihydrogen phosphate, anhydrous formic acid were of analytic grade. 0.9% sodium chloride injection was of medical grade. The vinegar was of food grade.
Paste Preparation
Rhizoma coptidis and evodia rutaecarpa were crushed into powder and sifted through 100 mesh sieve. The main ingredients are rhizoma coptidis and evodia rutaecarpa powder with the mass ratio of 1:1. Then, the powder was made with 30-50% w/w dimethyl sulfoxides, 30-50% w/w acetic acid, 1-8% w/w water-soluble azone, 1-8% w/wfat-soluble azone, 100% vinegar surfactants, excluding promoter to prepare the preparation. 28 kinds of preparation with different transdermal absorbents were made for external use only.
The effects of different transdermal absorbents on percutaneous administration of external preparations of rhizoma coptidis and evodia rutaecarpa were investigated by using cumulative penetration ratio and cumulative retention ratio as indexes.
Skin Membrane Preparation
Male SD mice, (Jinan, China), initial weight 140±10 g, were used. The animals were housed in a conditioned environment (21±2°C, 40-70% relative humidity, 12-h light/12-h dark cycle), with free access to standard laboratory chow and tap water. The animal experiments are approved by Ethics Committee of the Hospital as Animal use project no. KYLL-201902. All animal experiments complied with the ARRIVE guidelines and were carried out in accordance the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). The mouse were sacrificed by dislocated neck. The full-thickness skin was excised from the abdominal site after removal of the fat and the subdermal tissues by surgical scissor. The skin was washed with saline solution, its integrity verified by means of visual inspection and then it was frozen at −80°C until use.
In Vitro Skin Permeation Study
The SD mouse skin was carefully mounted on the lower half of the Franz cell with the dermis facing downwards and the stratum corneum side in contact with the preparation. The upper and lower parts of the Franz cell were fastened together by means of a clamp, with the membrane acting as a barrier between the donor and receptor compartments. The diffusion area and the volume of the receptor compartment are 1.131 cm2 and about 15 mL, respectively. The receiver compartment was filled with a freshly prepared degassed saline. Special care was taken to avoid the formation of air bubbles between the solution and the membrane in the receptor compartment. The Franz cells were kept at 37±1°C throughout the experiment. Only the receptor compartment was in contact with the circulating water at 37°C and each Franz cell was equipped with a stirring magnet. At predetermined times, 5 mL samples were withdrawn at 2, 4, 6, 8, 10h from the receiver compartment and immediately replaced with fresh receiver medium. Sink conditions were maintained throughout the experiment. The withdrawn samples were assayed directly by an HPLC method to determine the concentrations of the compound.
In Vitro Skin Retention Study
At the end of skin permeation experiments, the full-thickness hairless mouse skin was removed from the Franz cell and the plaster specimens were carefully eliminated. The skin was cut into pieces and placed in a 5mL centrifuge tube. After 10 min of ultrasonic shaking with appropriate amount of methanol, methanol was transferred to a 5mL volumetric flask, cleaned repeatedly with methanol and transferred to the volumetric flask. Methanol was then stabilized to the scale, shaken well, and filtered with 0.45 μm microporous membrane.
Permeation and Retention Parameters
Permeation parameters were interpreted plotting cumulative penetration ratio and cumulative retention ratio.
MR2, the weight of the second reference (mg). LR, the labeled content of the reference (%). VS, the volume of the receptor compartment (15 mL) , the volume of constant volume (5 mL). AS, the peak area of the permeable or remaining active component in the sample. VR, dilution volume of the reference (5000 mL or 1000 mL). MS, sample weight (mg). AR2, mean peak area of the second reference.
HPLC Analysis
The concentrations of the main components palmatine hydrochloride, berberine hydrochloride, evodiamine, rutacarpine in the preparation were determined by an HPLC method. The HPLC method was developed for the simultaneous determination of the four main components. The HPLC system used throughout this study was a Agilent 1260 HPLC, equipped with a Diode Array Detector.
The column was C18, 250×4.6 mm, 5 μm (Kromasil, Sweden) which was kept at 40°C, with a specific precolumn. The mobile phase was 0.05 mol/L sodium dihydrogen phosphate (pH=3.00)/acetonitrile (49:51%, v/v).
The flow rate was set to 1 mL/min. Six concentrations of the four standard compound were injected at 10 μL (the ranges for UV 261 nm detection are 0.05-10 μg/mL). The linear regression equation of the standard curve, determinedin duplicate, was obtained by plotting the amount of the standard compound injected against the peak area.