Vol. 40, No. 12 2081Biol. Pharm. Bull. 40, 2081–2087 (2017)

© 2017 The Pharmaceutical Society of Japan

Regular Article

Prescription Optimization and Oral Bioavailability Study of Salvianolic Acid Extracts W/O/W Multiple EmulsionYanqing Song,a,b Huan Gao,a,b Sixi Zhang,a,b Yue Zhang,a,b Xiangqun Jin,*,a and Jingmeng Sun*,c

a Department of Pharmaceutics, School of Pharmaceutical Sciences, Jilin University; 1163 Xinmin Street, Changchun, Jilin 130021, P. R. China: b Department of Pharmacy, The First Hospital of Jilin University; 71 Xinmin Street, Changchun, Jilin 130021, P. R. China: and c Changchun University of Chinese Medicine; 1035 Boshuo Road, Changchun, Jilin 130117, P. R. China.Received February 21, 2017; accepted September 13, 2017; advance publication released online September 23, 2017

The purpose of this study is to develop a new method of preparing salvianolic acid extracts (SAE) water-in-oil-in-water (W/O/W) multiple emulsion (ME). SAE injection is used in the treatment of brain infarct and promotion of blood circulation in China. However, the injection is not convenient, and the oral preparation has poor bioavailability. Hence, a new preparation that is convenient and stable with good biological avail-ability is required. SAE ME was prepared by two-step emulsification method. Combined with single-factor investigation and orthogonal test, the embedding rate and centrifugal retention rate were taken as the com-prehensive indexes to optimize the formulation of SAE ME. The ME size was tested by laser particle size analyzer. The pharmacokinetic studies were conducted in Sprague-Dawley rats with HPLC-MS/MS method. The blood coagulation and hemorheology tests were conducted to assess the effect of preparation in rats. The best preparation technique for SAE ME is by the use of trospium chloride; SAE represent 12% of water in the phase, lipophilic emulsifier hydrophilic lipophilic balance value=4.3, lipophilic emulsifier is 20% of the oil phase. The median diameter of particle is (0.608±0.05) µm and the Cmax of ME is 3-fold higher compared to Cmax of free drug. The oral biavailability of ME is 26.71-fold higher than that of free drug with good effect on blood circulation. SAE ME is stable hence, improves the biological availability and slows down drug re-lease.

Key words salvianolic acid extract; multiple emulsion; encapsulation efficiency; bioavailability

Multiple emulsion (ME) was found by Seifritz in 1925, but people began to conduct in-depth research of its preparation in 1965.1) ME has a two interfaces structure, and there is a smaller dispersed phase in the dispersed layer.2) Water/oil/water (W/O/W) and oil/water/oil (O/W/O) are the most com-mon types of W/O/W. Inland water phase, oil phase and the external water phase, phase boundary play the role of “drug storehouse” to achieve sustained release and embedding of the drug.1)

ME carries water-soluble drug, lipid-soluble drugs and also has a certain carrying capacity of the water solubility of certain drugs. It has two or more layers of liquid emulsion membrane structure. The parcel to “pharmacy store” role in the complex drugs can effectively control the drug release rate. The drug can be embedded in the water phase and oil phase to avoid inactivation of drugs in the gastrointestinal tract which increases drug stability. ME is absorbed almost entirely through lymphatic pathway in association with intes-ninal lipoproteins namely chylomicron after oral administra-tion. They may directly be absorbed through the intestinal macrophage system and Peyer’s patches to gain access into mesenteric lymph from where they are drained into circula-tion through thoracic lymph duct. With the orientation of the lymphatic system, the emulsion droplet can be absorbed by the lymphatic system to distribute in the liver, kidney, spleen, rich organs.3) Oil droplets in the ME and the cell membrane has a strong affinity, it can be used as anti-cancer drugs and anti-cancer drug delivery systems. The drug concentration on target organs can improve the efficacy and reduce adverse reactions. In the study of pharmaceutical preparations, ME as an intermediate is widely used for the preparation of micro-

spheres, microcapsules, nanoparticles and many micron and nanometer grade solid or semi-solid drug delivery systems. For example, polycystic liposomes food and cosmetics.2,4)

Water-soluble components are the main active ingredients of traditional Chinese medicine. However, many of them are found to be poor membrane permeable through the gastro-intestinal wall after oral administration, which leads to low bioavailability and efficacy.5) Salvia miltiorrhiza has cured stasis pain which is the effect of invigorating circulation of menstruation. Water-soluble phenolic acid compound is one of the effective components. Salvia miltiorrhiza, is currently the most clinically used traditional Chinese medicine for acti-vating blood circulation. It is widely used in the treatment of disease of heart head blood-vessel.6–8) Nowadays, Salvianolic acid extracts (SAE), which is the water-soluble part from Radix Salvia miltiorrhiza, is an effective drug used in Chinese hospitals. Salvianolic acid B (Sal B) is one of the main ele-ments and the strongest pharmacologically active salvianolic acids.9) SAE can inhibit thrombosis, reduce plasma endothelin and thromboxane B2 levels. Sal B has a strong anticoagulant effect. However, it was reported that the bioavailability of Sal B is extremely low as a compound and has been made into phytosome to enhance the biomembrane permeability and im-prove its oral bioavailability.10,11)

This study provides a new method for developing new preparations of SAE with good stability, higher bioavailability and long-time effect. Furthermore, this study is an exploration of new drug delivery system for traditional Chinese medicine. SAE injections activate blood circulation, dredge collater-als, and help in recovery from blood stasis syndrome in mild cerebral infarctions. To verify these effects, we established

* To whom correspondence should be addressed. e-mail: jinxq@jlu.edu.cn; 350740439@qq.com

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an acute blood stasis model and observed changes in blood coagulation and hemorheology in rats. Oral administration of SAE ME and intravenous injection of SAE injections used in clinical settings were compared. In the present study, W/O/W ME loaded with SAE can be further made into oral solid drugs for patients convenience.

MATERIALS AND METHODS

Materials SAE (Lot. 20140604), was a gift from Tasly Pharmaceutical Co., Ltd., China. The chemicals used in the present study were of analytical grade. Tween-80 (polyoxy-ethylene sorbitol fatty acid esters) was obtained from Hunan Kang Pharmaceutical Limited by Share Ltd. (Hunan, China). Monooleate (Span 80) was purchased from Tianjin kwangfu Fine Chemical Industry Research Institute. Unless otherwise indicated, all other chemicals were of analytic or HPLC grade.

HPLC-MS/MS Analysis of Sal B The analysis was car-ried out by a model 1200 HPLC instrument (Agilent Tech-nologies, CA, U.S.A.) with a model API4000 tandem mass spectrometer (Applied Biosystems, U.S.A.). Chromatographic separation was done by SB C-18column (100×2.1 mm, 3.5 µm, Agilent, U.S.A.). The mobile phase consisted of 70% methanol (v/v) and 0.1% formic acid (v/v) at a flow rate of 0.2 mL/min. Analysis was performed under multiple reaction monitor-ing (MRM) conditions and the parameters of Sal B used for the mass spectrometer with the ESI− mode were as follows: the capillary voltage at −80V and the source temperature at 550°C MS/MS parameters of Sal B detection were shown in

Fig. 1 and quantitative daughter ion was shown in Table 1. The samples of ME and rat serum were diluted with 3 times methanol to destroy the structure of ME and remove pro-tein in serum, and then filtered by 0.22 µm filter membrane before injection. We validated the determination method for Sal B with the data of linearity, recovery and repeatability in ME and rat serum. To assess the linearity, five working solutions of Sal B were added into blank ME and rat serum and the concentraions of Sal B were 0.11, 1.10, 5.50, 27.50, 137.50 µg/mL in ME and rat serum respectively. The extrc-tion recovery was performed by comparing the peak areas of Sal B from extracted ME and rat serum samples with the working solution at the same concentration levels. We chose the 0.11, 5.50 and 137.50 µg/mL as the low-, middle- and high-concentration for recovery assessment and each sample analysis three times. For intra-day repeatability, the samples were determined on a single day for five times. The inter-day repeatability was determined for five consecutive days. The data in Table 2 demonstrated that the method for determining Sal B using HPLC-MS/MS was valid.

Preparation of SAE ME W/O/W ME was prepared by two-step emulsification method. The prescribed amount of lipophilic emulsifier, isopropyl myristate (IPM) was mixed evenly with a magnetic stirrer (30±1°C) at 500 rpm for 5 min; another SAE was dissolved in deionized water. The water was added to the oil phase, dispersed in 3 min to form W/O colostrum. Deionized water was added to another prescribed amount of hydrophilic emulsifier while stirring with a mag-netic stirrer (30±1°C) to mix evenly. The W/O colostrum was added to a water phase, dispersed in 5 min to form W/O/W ME.

Optimization of SAE W/O/W Composite Emulsion System optimization program uses the statistical method of L9(34) orthogonal test method, which fits the data by the math-ematical method. The orthogonal experimental design is one of the best experimental designs.12) By using the standard or-thogonal test scheme analysis, the results of calculation quick-ly find the optimization scheme which is an efficient scientific calculation method with multi-factor optimization problems.12)

Fig. 1. MS/MS Parameters of Sal BThe parent ion (m/z) is 717.9 and the daughter ions (m/z) are 520.1, 321.8 and

339.0.

Table 1. MS/MS Parameters of Salvianolic Acid B on the Parent and Quantitative Daughter Ion (m/z) and Collision Energy

Compound Parent ion (m/z)

Daughter ion (m/z)

Collision energy (eV)

Salvianolic acid B

717.9 520.1* −28321.8 −46339.0 −50

* Quantitative daughter ion.

Table 2. Method Validation of HPLC-MS/MS Detection for Salvianolic Acid B in Multiple Emulsion and Rat Serum

Sample Linearity equation, r value Linearity range (µg/mL)

Concentration (µg/mL) Recovery (%)

Repeatabiity (RSD%, n=5)

Intra-day Inter-day

SAE ME Y=3.32×106X+4380, r=0.9999 0.05–137.50 0.11 (97.15±3.41) 2.01% 1.84%5.50 (98.24±2.56) 1.29% 1.52%

137.50 (98.87±1.58) 1.10% 1.21%Rat serum Y=5.69×105X+3240, r=0.9994 0.05–137.50 0.11 (91.57±8.35) 6.98% 8.45%

5.50 (93.14±6.21) 5.87% 7.32%137.50 (94.21±5.20) 4.98% 5.84%

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Ln(Pγ)L is termed Mark orthogonal table, n denotes orthogonal table rows, γ denotes column number of the orthogonal table, including interaction and error; P denotes the number of levels of each factor, the column numerically represents the level code of each factor, and the actual level of the corresponding factor.13)

Surface Morphology Study by Optical Microscope Mi-croscopic analysis was conducted in order to explore the char-acters of the ME and their droplet size. A optical microscope (BA310 Motic, Motic China group Co., Ltd.) conducted with a camera was used to observe the particle sized and spheri-cal shaped. One drop of none diluted formulation was placed onto a system slide, covered with glass, and then observed at 1000× magnification microscope. The images of ME particle morphology were captured using a Nikon digital camera (D90, Nikon, Japan).

The Determination of SAE ME Embedding Rate Was Measured by Centrifugation Method14) Equal volume of SAE ME and deionized water was placed in a centrifuge tube (Shanghai Anting Scientific Instrument Factory) with an equal and centrifuged at the speed of 16000 rpm for 20 min. The supernatant was vaporized to dryness. The residue was dis-solved in methyl alcohol: water in the ratio of 8 : 2, placed in a flask, filtered and analyzed by HPLC-MS/MS. The formula for the SAE ME embedding rate is as follows:

total dissociate

dissociateEmbedding rate 100%= ×

−C CC

The Centrifugal Retention Rate of SAE ME15,16) A vol-ume of the freshly prepared composite emulsion was loaded to centrifuge tube using its scale and placed in a centrifuge (Shanghai Anting Scientific Instrument Factory) at the speed of 1800 rpm for 10 min. The centrifugal double emulsion layer was at the bottom of the precipitated external water. The vol-umes of the emulsion in centrifuge tubes, the total volume of the liquid phase and the precipitated aqueous phase volume were read.

t c

cCentrifuge retention capacity 100%

−= ×V VV

Vt is the total volume of the centrifuge tube liquid layer, Vc represents the volume of the continuous phase precipitated after stratification.

The Determination of Particle Size of SAE ME Deter-mination of SAE ME particle size and size distributions were used with a LS13320 Laser Particle Size Analyzer (Rong Hua Instrument Manufacturing Co., Ltd., Jiangsu, China). The ME was diluted with distilled water for 10 times before injecting into the analyzer. Then, the midian diametre and size distribu-tion were shown.

Drug Release Study in VitroRelease study of SAE from the ME was detected by dialysis

method.3) The dialysis bag (Solarbio, MD77, Beijing, China) was washed three times with distilled water and soaked in phosphate buffer saline (PBS) pH 7.4 overnight before use. Five gram SAE ME (contain 1.17 g Sal B) and same amount of free drugs were filled into the dialysis bag and seal at each end. The dialysis bag was placed into 200 mL of PBS (pH 7.4) diffusion medium at (37±1) °C and 75 rpm. At 0.5, 2, 4, 6, 8 and 12 h intervals, 5 mL diffusion medium was withdrawn and replenished with the same volume of fresh PBS solution. The

content of Sal B was detected by HPLC-MS/MS.The Preliminary Study on Stability SAE ME acceler-

ated test was carried out to assess the stability of formulation. The samples of we prepared with Lot. numbers 20150801, 20150802, 20150803 SAE was storaged at 40°C under the con-dition of relative humidity (RH) 75% for 6 months. And then the appearance, the embedding rate, centrifugal retention rate, particle size and morphology of ME was assessed in the 0, 1, 2, 3, 6 months.17,18)

Pharmacokinetic Experiments For the pharmacokinetic study, male Sprague-Dawley (SD) rats were provided by the Experimental Animal Center of Jilin University (Changchun, People’s Republic of China, permit number SCXK 2013-0001). This study was supported by the ethical committee of Jilin University. All animals were housed under controlled condi-tions (23±2°C, RH 50±5%) with natural light–dark cycle for at least one week before starting the experiments. All the rats were given free access to water and standard rodent food for one week. Twenty-four SD male rats were randomly assigned to three groups. All the rats fasted but had free access to water for 12 h. One group was treated with oral SAE ME, at a equal dose of 20 mg/kg Sal B, while the second group was orally administered SAE free drug with equal dose of Sal B. The third group had an intravenous injection of the same dose of Sal B.

The 3 mL blood samples were collected from the jugular vein at specified intervals (3, 5, 10, 20 min, 0.5, 1, 1.5, 2, 3, 5, 7, 10 and 24 h). Then the heparinized blood was immediately centrifuged at 3000 rpm for 10 min, and the serum was re-served after treating with three times volume of methanol and stored at −20°C.

Blood Coagulation and Hemorheology Experiments After 1 week of adaptive feeding, rats were randomly divided into five groups, each with six rats, including a blank group, model group, positive group (injection of salvianolic acid 0.01 g/kg), and high- and low-dose groups (0.1 and 0.05 g/kg, respectively). The positive group was treated by intraperito-neal injection, and the high- and low-dose groups were ad-ministered the drug orally. The blank and model groups were administered the same volume of distilled water, once per day, for a period of seven days.

Rats in each group (excepting the blank group) were inject-ed subcutaneously with 0.8 mL/kg 0.1% adrenaline hydrochlo-ride on the seventh day after administration of salvianolic acid for 30 min. After 6 h, the same process was repeated. Between these two injections, rats were placed in a swimming pool for 5 min at 2°C.19) Eight days after administration of SAE ME for 60 min, rats were anesthetized with 10% chloral hydrate and blood was collected from the abdominal aorta using a 3.8% sodium citrate anticoagulant solution. A LBY-N6K Blood Rheometer (Precil, Beijing, China) was used to determine hemorheology indicators. In addition, serum was obtained after blood centrifugation at 4°C and used to investigate pro-thrombin time (PT) and activated partial thromboplastin time (APTT) using a COATRON M4 Blood Coagulation Analyzer (ThermoFisher, Shanghai, China).

Statistical Analysis Data was presented as the mean±standard deviation (S.D.) and analyzed by SPSS 18.0 software (SPSS Inc., Chicago, IL, U.S.A.). The difference between groups was analyzed with one-way ANOVA test followed by Tukey test when equal variances were assumed.

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Results were considered statistically significant when p<0.05. Pharmacokinetic parameters were calculated using DAS 2.0 software (Shanghai Traditional Chinese Medicine Univer-sity, Shanghai, People’s Republic of China). Analysis of vari-ance of maximum concentration (Cmax) and area under the curve (AUC)(0–t), time of maximum concentration (tmax) and t1/2 was obtained, and the bioavailability was evaluated by BA(%)=(AUCp.o.×Dosei.v.)/(AUCi.v.×Dosep.o.).

RESULTS

The Prescription Optimization of SAE ME Several prescriptions were obtained by L9(34) orthogonal test, the rate of the maximum value of the embedding rate and centrifugal retention, the minimum particle size of indexes were reference criterion: SAE represent 12% of water in the phase, lipophilic emulsifier hydrophilic lipophilic balance (HLB) value=4.3, lipophilic emulsifier in an amount of 20% of the oil phase, oil water mass ratio=1 : 0.8, colostrum and outer aqueous phase mass ratio=1 : 1, hydrophilic emulsifier HLB value=8.1, hydrophilic emulsifier for 10% of the external phase. The em-bedding rate was 81.19%, the retention rate was 92.67%, and the particle size was 0.622 µm.

Based on the results of single factor determining factors and levels, Salvia miltiorrhiza polyphenols acid amount (A), emulsifying agent (B), fatty oil and inland water facies pro-portion (C) and the proportion of colostrum with web (D) to examine factors, the embedding rate and centrifugal retention as a comprehensive index, using L9(34) orthogonal test opti-mization of Salvia miltiorrhiza polyphenols acid W/O/W type emulsion process prescription, according to the factor level in Table 3 to design conditions of the experiment.

The results in Tables 4 and 5 show that the factors affecting

the preparation process are: A>C>D>B, that is, the amount of salvianolate> lipophilic emulsifier> the oil phase and the internal water phase (p<0.05). Therefore, the optimal process was A1B2C2D1. The repeatability test was performed for the total amount of polyphenolic acid in Salvia miltiorrhiza. The proportion of polyphenolic acid in Salvia miltiorrhiza was significantly higher than that in water; 12% of lipophilic emul-sifier, 20% of lipophilic emulsifier, 1 : 0.8 ratio of oil phase to the water phase, and 1 : 1 ratio of colostrum to the external water phase.

Surface Morphology Study by Optical Microscope SAE ME was observed as circular droplets, spherical, uni-formly sized, round in appearance and decentralized. It was also observed that many small droplets were present in the in-ternal phase of ME. The multiple nature of ME was confirmed by the optical images as shown in Fig. 2.

The Measurement Results of Embedding Rate, Centrifu-gal Retention Rate and Particle Diameter According to SAE ME optimized prescription, the rate of average value of three batches of SAE ME encapsulation was (81.59±1.34)%, the rate of the average value of centrifuge retention was (93.36±1.89)%. Particle size distribution is one of the stan-dards used to measure the quality of ME. The median particle diameter of SAE ME is (0.608±0.05) µm and associated with a narrow span of the particle diametre distribution (Fig. 3).

Release Study of SAE ME in VitroThe release profile of SAE from ME and free drugs through

dialysis bag was found in Table 6. Compared with free drugs, release rate of the ME was significantly lower. Sal B in ME was advantage with delay and control released that could be released at a ratio of (90.56±11.12)% of total Sal B in 12 h.

The Stability of SAE ME With sampling 0, 1, 2, 3, 6 months, the appearance is off-white–light yellow emulsion and no special smell buried rate. The centrifuge retention rate, the grain size and surface morphology had no obvious change. Therefore the product placed at 40°C and RH 75% conditions for 6 months have good stability. The experimental results were listed in Supplementary Table S1.

Pharmacokinetic Experiments Accordign to the DAS 2.0 software, the two compartment model was the optimal pharmacokinetic analysis model in SAE ME kinetic process. All the concentrations of Sal B in rats serum were between

Table 3. L9(34) Orthogonal Experimental Table

LevelFactors

A B C D

1 12 18 1 : 0.7 1 : 12 15 20 1 : 0.8 1 : 1.23 18 22 1 : 0.9 1 : 1.4

Table 4. L9(34) Orthogonal Experimental Table and Experimental Results

LevelFactors Embedding rate

(%)Retention rate

(%)Comprehensive score

(%)A B C D

1 1 1 1 1 83.43 90.61 87.022 1 2 2 2 86.04 92.28 89.163 1 3 3 3 81.04 91.32 86.184 2 1 2 3 80.37 83.45 81.915 2 2 3 1 81.40 83.64 82.526 2 3 1 2 75.25 80.97 78.117 3 1 3 2 69.78 70.16 69.978 3 2 1 3 73.08 73.02 73.059 3 3 2 1 73.80 75.86 74.83

Mean1 87.453 79.633 79.393 81.457Mean2 80.847 81.577 81.967 79.080Mean3 72.617 79.707 79.557 80.380Range 14.836 1.944 2.574 2.377

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linearity range. The mean plasma concentration versus time is shown in Fig. 4. The pharmacokinetic parameters of Sal B obtained from the study are shown in Table 7. The results indicate that Cmax of ME was 3-fold higher compared to Cmax of free drug. Meanwhile, both t1/2α and t1/2β of SAE ME were much longer than those of free drug solution for oral administration. And oral administrated SAE ME also gave a much greater AUC than that of free drug solution too. BA% of SAE ME was 30.98%, which was significantly higher than free drug with 1.16%. The BA% of Sal B in SAE ME was increased 26.71-fold than in free drug. Morevoer, there was another peak in SAE ME group which indicated enterohepatic

circulation might be taken place in the excretion. The Phar-macokinetic data showed that the SAE ME could improve the poor bioavailability of oral administrated free SAE.

Blood Coagulation and Hemorheology Experiments High-, medium-, and low-shear blood viscosities were sig-nificantly increased in the model group, indicating that the model of acute blood stasis in rats was successfully estab-lished. However, compared with model group, the viscosities were significantly decreased in the positive and high-dose ME groups. Additionally, high-shear blood viscosity was sig-nificantly decreased in the low-dose ME group. These results indicate that SAE ME could reduce blood viscosity in rats with acute blood stasis and improve hemorheology indicators (Table 8).

Compared with the blank group, PT and APTT were sig-nificantly decreased in the model group, but were significantly increased in the positive and high-dose SAE ME groups, which indicates an improvement in blood coagulation in acute

Table 5. The Comprehensive Score for the Assessment Indicators of Variance Analysis Table

Source of variance

Square of deviance Freedom F ratio F critical-

value Significant

A 331.508 2 45.543 19.000 *B 7.279 2 1.000 19.000C 12.457 2 1.711 19.000D 8.498 2 1.167 19.000Error 7.28 2

F0.05 (2, 2)=19.00; F0.01 (2, 2)=99.00

Fig. 2. SAE ME Showed in Microscope Observation at 1000× Magni-fication

ME was diluted at 1 : 10. The photogragh of ME was spherical, uniformly.

Fig. 3. Diagram of Polyphenol Acid of Salvia miltiorrhiza Multiple Emulsion Particle Size Distributions

The median particle diameter of SAE ME is (0.608±0.05) µm.

Table 6. The Data on the Releasing Profile of SAE ME in Vitro

Time (h)Ratio of accumulated release (%)

Multiple emulsions Free drugs

0.5 15.01±2.65 40.21±2.562 28.99±5.27 74.69±3.216 66.87±8.62 90.25±5.208 80.79±10.54 92.32±5.36

12 90.56±11.12 93.25±6.02

Fig. 4. Plasma Concentration–Time Profile of Sal B after Oral Admin-istrated SAE ME, Orally Administered SAE Free Drug and Intravenous Injection Administrated SAE Free Drug at a Dose of Equal 20 mg/kg Sal B in Rats

Table 7. Pharmacokinetic Parameters of Sal B

Parameters SAE free drug SAE ME SAE i.v.

t1/2α (min) 1.198 25.086 0.805t1/2β (min) 27.791 69.315 67.621AUC0–t (µg/mL·min) 4.27 114.425 368.76AUC0–∞ (µg/mL·min) 5.258 119.778 614.871MRT0–t (min) 31.456 170.068 421.259MRT0–∞ (min) 51.506 198.206 1677.876V (L/kg) 18.944 29.701 0.761CL (mL/min·kg) 3844 172 96

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blood stasis model rats (Table 9).

DISCUSSION

In this study, a new preparation of SAE with an excel-lent character was investigated. The productive technology, characteristic, stability and bioavailability of preparation were tested. Sal B, the main active chemical and major content, was determined by HPLC-MS/MS as a quantified method for SAE.

During this experiment, the liposome, nano-emulsions, ME and other new drug delivery system were chosen. Because of the strong hydrophilicity of SAE, the encapsulation effi-ciency was low, and the film-forming property was poor in the preparation of liposomes. Liposomes were prepared by film dispersion method, but the highest of encapsulation rate was 20%. The prepared nano-emulsions could not form a good shape and was not in good condition. In the W/O/W ME, the drug embedded in the water phase state is better. Hence the embedding rate is qualified to protect the drug from the dam-age in digestive tract and liver metabolism, while ME also possesses the character of slow controlled release.15,16) The re-search shows that W/O/W ME of SAE is associated with high embedding rate, centrifugal retention rate and uniform and narrow distributive particle diameter, which displayed a stabil-ity characteristic for 6 months. Moreover, the bioavailability of Sal B 26.80-fold was significantly higher than that of free drug and the t1/2 was also prolonged significantly.

Common preparation methods of ME are one step emulsifi-cation method, two step emulsification method and membrane emulsification method.18) Membrane emulsification method can be used to control the size and distribution of the particle size and distribution, but the preparation method and equipment are more complicated. Two-step emulsification is presently the most widely used complex system of preparation. This method has the advantages of simple operation and high preparation efficiency. The mechanical equipment is mainly used for the

magnetic mixing which can be used to optimize the process parameters and to prepare a more stable complex emulsion.20) Through the orthogonal test of the preparation process, it can be seen that the dosage of SAE has a significant effect on the preparation process, so the dosage of the drug is the most influential factor in the preparation. Pharmacodynamic results showed that SAE ME could reduce whole blood viscosity and improve blood coagulation in rats with acute blood stasis. Results indicate that the preparation of SAE ME was effective and could be used to replace injections.

In this research, Sal B as a detected component, biovaibil-ity was increased much significantly by loading to ME. Drug loaded-multiple emusions could avoid degradation in intestine as well as liver, and that might be the reason for enhancing absorption of poorly absorbable drugs. It also could prolong the exposure time and play a control release role. These ad-vantages indicated that SAE ME meet a good characteristic for further investigation.

It is well known that SAE injection has been extensively used in China for the treatment of cardiovascular and cerebro-vascular diseases. But the safety of traditional Chinese herbs injection is a hidden trouble since they are used in large. It usually brings infusion reaction, hence should be produced with most strict standard, leading to a high price in clini-cal practice. In addition, this drug needs to be applied for a long time, so the injective administration is inconvenient for patients. Therefore, an orally administered preparation is needed. In this research, there is a limitation that SAE ME is a liquid intermediate product. For further research, a terminal formulation such as the dripping pill, micropic capsule, clath-rate and so on should be used.

The present study reports the preparation of SAE ME. The technological research shows a high embedding rate, good stability and simple production. The in vivo pharmacokinetic results demonstrates that the formulation improves bioavail-ability and slows down the drug release more than free drug.

Acknowledgments This work was supported by the Na-tional Natural Science Foundation of China under Grant num-ber 81503168; Graduate Innovation Fund of Jilin University: 2017049.

Conflict of Interest The authors declare no conflict of interest.

Supplementary Materials The online version of this ar-ticle contains supplementary materials.

Table 8. Effect on Blood Viscosity in Acute Blood Stasis Rats with SAE ME (χ̄±s)

Group Dose (g/kg)Blood stasis (mpa/s)

High shear 120/s Medium shear 90/s Low shear 30/s

Blank group — 7.1±1.80 12.8±1.40 37.8±8.84Model group — 10.0±2.06Δ 16.1±2.49Δ 52.3±8.83Δ

Positive group 0.01 6.6±1.43** 13.0±1.47* 36.5±15.30*High dose group 0.1 6.5±2.06* 12.8±2.06* 38.6±8.65*Low dose group 0.05 5.6±0.47** 18.3±1.85 42.9±9.38

Compared with blank group, Δ p<0.05, ΔΔ p<0.01; compared with model group, * p<0.05, ** p<0.01; n=6.

Table 9. Effect on Prothrombin Time (PT) and Activated Partial Throm-boplastin Time (APTT) in Acute Blood Stasis Rats Treated with SAE ME (χ̄±s)

Group Dose (g/kg) PT (s) APTT (s)

Blank group — 19.80±2.42 25.90±3.27Model group — 16.76±1.92Δ 18.91±3.37ΔΔ

Positive group 0.01 20.43±3.07* 24.08±3.38*High dose group 0.1 19.21±1.52* 23.52±3.19*Low dose group 0.05 17.89±3.03 22.92±3.04*

Compared with blank group, Δ p<0.05, ΔΔ p<0.01; compared with model group, * p<0.05; n=6.

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