Solid self-microemulsifying drug delivery system of Sophoraflavanone G: Prescription optimization and pharmacokinetic evaluation

European Journal of Pharmaceutical Sciences 136 (2019) 104953

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Solid self-microemulsifying drug delivery system of Sophoraflavanone G: Prescription optimization and pharmacokinetic evaluation

T

Zhixin Yanga, , Ying Wanga, Jing Chenga, Baisong Shana, Yanhong Wanga, Rui Wanga, ⁎⁎ Liqiang Houb, ⁎

a b

College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, China The Second Affiliated Hospital of Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150001, China

ARTICLE INFO

ABSTRACT

Keywords: Sophoraflavanone G (SFG) Solid Self-microemulsifying Drug Delivery System (S-SMEDDS) Pharmacokinetic Bioavailability

Sophoraflavanone G (SFG) is promising component in clinical treatment. The purpose of this study was to develop a drug delivery system in order to improve oral bioavailability of SFG. The optimum formulation of Selfmicroemulsifying Drug Delivery System with SFG (SFG-SMEDDS) was selected by the solubility test, selfemulsifying grading test and ternary phase diagram test. The optimized formulation of SFG-S-SMEDDS was composed of Ethyl Oleate (38.5%, w/w), Cremophor RH40 (47.5%, w/w), PEG 400 (14.0%, w/w), and drug loading (20 mg/g). Mannitol as a solid absorbent was added to SFG-SMEDDS formulation with the mass adsorption ratio of 2:1 (w/w). The vitro release rate of SFG-S-SMEDDS reached 60% in 10 min and 80% in 30 min. After SD rats were given SFG and SFG-S-SMEDDS by oral administration, it was found that the area under the curve of SFG-S-SMEDDS was significantly larger than that of SFG suspension and the relative bioavailability of SFG in rats was 343.84%. In addition, the SFG-S-SMEDDS did not change greatly within 3 months. Therefore, the results show that SFG-S-SMEDDS can significantly improve the oral bioavailability of SFG so as to lay a foundation of further research on the new dosage form of SFG.

1. Introduction Sophora flavescens (S. flavescens) is a commonly used traditional Chinese medicine for treating dermatosis (Chen et al., 2017), hepatitis (Yang et al., 2018) and arrhythmia in orient countries (Li et al., 2015). Flavonoids and alkaloids are the main chemical constituents of S. flavescens (Ma et al., 2018). SFG is one of the most dazzling flavonoids and has been paid much attention nowadays (Ma et al., 2013; Guo et al., 2016a, 2016b; Yang et al., 2016), due to anti-bacterial (Fakhimi et al., 2006; Cha et al., 2009; Cha et al., 2016), anti-cancer (Kim et al., 2013; Li et al., 2016), and anti-inflammation (Guo et al., 2016a, 2016b). SFG has a molecular weight of 424.47 with a molecular formula C25H28O6. Its chemical name is (2S)-8-lavandulyl-5, 7, 2′, 4′-tetrahydroxydihydroflavone (see Fig. 1(A)) (Ohmoto et al., 1986). That lavandulyl is connected to C-8 position of the dihydroflavone A ring (see Fig. 1(B)) and there are hydroxyl groups in the mother nucleus C-5, C-7, C-2′, C-4′, respectively, of the dihydroflavone structure (Ryu et al., 1997), are the main prominent structural feature of SFG. It is

noteworthy that the above characteristics make SFG have unique pharmacological activities, but also bring very poor solubility. Preliminary experiment showed that SFG had good permeability (lgPapp ≧ 1) and slightly poor solubility (100 μg/mL–1000 μg/mL) resulted in low oral bioavailability. The absolute bioavailability of SFG was calculated to be only about 10.3%. Therefore, it is necessary to enhance SFG dissolution and improve the oral bioavailability through suitable dosage form. Oral administration is the most commonly route of administration, with the advantages of enhancing patient compliance and avoiding the risk of in vitro administration. The main absorption site of most drugs is the small intestine. However, after taking the oral medicine, it resulted in low bioavailability of the drug that the effect of actually reaching the lesion site is much lower than that before the actual administration due to the slow and incomplete absorption, fat-soluble and first-pass elimination of the drug. Since the first discovery of microemulsion structures in 1943 (Hoar and Schulman, 1943), it as a fat-soluble and watersoluble drug carrier is gradually attracting people's attention. SMEDDS

⁎ Correspondence to: Z. Yang, College of Pharmacy, Heilongjiang University of Chinese Medicine, No. 24, Peace Road, Xiangfang District, Harbin, Heilongjiang 150040, China. ⁎⁎ Correspondence to: L. Hou, The Second Affiliated Hospital of Heilongjiang University of Chinese Medicine, 411 Gogoli Street, Nangang District, Harbin, Heilongjiang 150001, China. E-mail addresses: [email protected] (Z. Yang), [email protected] (L. Hou).

https://doi.org/10.1016/j.ejps.2019.06.007 Received 27 September 2018; Received in revised form 8 May 2019; Accepted 4 June 2019 Available online 05 June 2019 0928-0987/ © 2019 Published by Elsevier B.V.

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(A)

MA, USA). Rutin (purity of 99.5%) was from the Chinse Food and Drug Inspection Institute (Beijing, China). Kolliphor HS15 and Cremphor RH40 were from BASF Corporation (Ludwigshafen, Germany). LABRAFIL M1944CS, Capyrol 90, Transcutol HP and Labrasol® were from GATTEFOSSE (Shanghai, China). Tween 20, Tween 80, PEG 400, PEG 4000, PEG 6000 and 1, 2-propylene carbonate were from Bodhi Chemical Co., Ltd (Tianjin, China). Sodium carboxy methyl cellulose, poloxamer, micropowder silica gel, isopropyl myristate and soybean oil were from China National Pharmaceutical Group Chemical Testing Co., Ltd (Shenyang, China). Isopropanol was from Commio Chemical Testing Co., Ltd (Tianjin, China). Miglyol 812N was from Feng Li Jing Business Co., Ltd (Beijing, China). Glycerol and castor oil were from Fuyu Fine Chemical Co., Ltd (Tianjin, China). Ethyl Oleate, mannitol and starch were from Guangfu Fine Chemical Research Institute (Tianjin, China). Citric acid was from Tongri Tianda Chemical Testing Factory (Tianjin, China). Soluble starch was from Ort Biotechnology Ltd (Beijing, China). Microcrystalline cellulose was from Hengxin Chemical Testing Co., Ltd (Shanghai, China). The solvent used were the analytical reagent grade. All of the liquid formulations were kept at −4 °C in airtight glass containers until analysis.

(B) 7′′

6′′ 5′′ 4′′

3′′ 2′′

9′′ 10′′ OH

OH

1′′

8′′

2′

8 9

O

7 6 5 OH

10

4

3′

1′ 2 3

4′ 6′

OH

5′

O

Fig. 1. Structure of SFG (A) and lavandulyl (B).

is a solid or liquid formulation formed with a drug, an oil phase, a surfactant and a co-surfactant in the absence of a water phase, which is spontaneously emulsified to form a particle size of < 100 nm under gastrointestinal motility. The formation of self-microemulsion depends on the type of surfactant selected and the ratio of oil to water. And the microemulsion as a drug carrier, the selection of components is more stringent. It requires that the selected components must be non-toxic, the bioavailability of drugs in microemulsion is high, the formed microemulsion area is large, the solubilization ability of drugs is good, and the stability is fine. Due to its thermodynamic stability, the advantages of preparation and preservation, enhancing the solubility of poorly water soluble, good drug dispersion, the prolonged release of watersoluble drugs, it has become a new research direction in the field of medicine to promote the absorption of poorly water-soluble drugs in vivo (Kamal and Nazzal, 2018). A large number of reports demonstrated SMEDDS could significantly improve the oral bioavailability of many insoluble natural components, such as curcumin (Cui et al., 2009), CKD-519 (Park et al., 2018), resveratrol (Bolko et al., 2018) and Emodin (Huang et al., 2017). However, liquid SMEDDS has the disadvantages of high production costs, low stability and portability, low drug loading and few choices of dosage forms. The new formulations, such as S-SMEDDS, supersaturated self-microemulsion delivery system (S-SEDDS), positive charge self-microemulsion drug delivery system (PO-SEDDS), self-double-emulsifying drug delivery system (SDEDDS), and self-microemulsifying drug delivery system of phospholipid complex (PC-SMEDDS), developed on the basis of SMEDDS not only have the advantages of SMEDDS to promote intestinal absorption, enhance oral availability of low permeability drugs and improve formulation stability, but also improve the above defects of SMEDDS. In particular, S-SMEDDS which refers to solid preparations made by mixing self-microemulsifying liquid with appropriate solid materials, reduces the dosage of surfactant, improves safety and reduces adverse reactions. It may be a great solution by combining the advantages of SMEDDS and solid preparations (Qi et al., 2014; Silva et al., 2018). In this experiment, SFG-S-SMEDDS were produced by SFG, oils, surfactants, co-surfactants, and mannitol. Ternary phase diagrams and central composite design (CCD) response surface methodology were used to optimize the formulation. Then, SFG-S-SMEDDS was further evaluated by particle size, polydispersity index (PDI), self-microemulsifying time, zeta potential, transmission electron microscopy (TEM), drug release in vitro and pharmacokinetic in vivo.

2.2. Solubility studies To find out suitable the oil phase, the surfactant and the co-surfactant as compositions of SMEDDS, the solubility of SFG in various oils (Capyrol 90, isopropyl myristate, Ethyl Oleate, Labrafil M1944CS, Miglyol 812N, castor oil and soybean oil), various surfactants (similarly, Labrasol®, Cremphor RH40, Kalliphor HS15, Tween 80 and Tween 20) and various co-surfactants (1, 2-propylene carbonate, PEG 400, Transcutol HP, isopropanol and glycerol) were identified. An excess amount of SFG was added to 1.0 mL of the dissolution medium, which comprises an oil phase, surfactant and co-surfactant in the 37 °C constant-temperature magnetic agitator (100 rpm, 10 min), and shaken at 37 °C for 24 h in a water-bath (50 rpm) to reach equilibrium. After that, the solution was centrifuged at 10,000 rpm for 10 min, the supernatant was diluted with methanol and filtered through a membrane filter (0.22 μm, organic) for analysis by 2996-2695 HPLC (Waters, USA) system with a DIKMA C18 column (250 mm × 4.6 mm, 5 μm), a column heater set at 30 °C and a UV detector set at a wave-length of 296 nm. The flow rate of mobile phase (methanol:water = 80:20) was 1.0 mL/ min. HPLC methods were validated for various parameters, including linearity (R2 = 0.9998) and the inter- and intra-day variance of this HPLC method was within the acceptable range of < 2.0%. Specificity was verified based on the absence of an interference peak at the retention time of SFG with blank self-microemulsion solution. The recovery of SFG was in the range of 99.67%–101.01% (RSD < 2.0%), and good stability was obtained within 24 h (RSD < 2.0%). Each solubility study was taken in triplicate. 2.3. Self-emulsifying grading test In order to evaluate self-emulsifying grading test between oil phases, surfactants and co-surfactants, oil phases and surfactants with high solubility of SFG were mixed under the mass ratio of 4:6 and a total mass of 1.0 g. The mixture was stirred for 10 min in a constant temperature water-bath at 37 °C (100 rpm). The emulsification process was observed and divided into five grades (Khoo et al., 1998). Among them, the A level is a phenomenon of the rapidly forming (within 1 min) microemulsion that was clear or slightly bluish in appearance, the B is a phenomenon of the rapidly forming, slightly less clear emulsion which had a bluish white appearance, the C is a phenomenon of the bright white emulsion (similar in appearance to milk) that formed within 2 min, D is a phenomenon of a dull and grayish white emulsion with a slightly oily appearance that was slow to emulsify (longer than 2 min), and E is a phenomenon of exhibited either poor or

2. Materials and methods 2.1. Materials and chemicals SFG (> 98%, HPLC grade) was separated by Heilongjiang University of Chinese Medicine (Harbin, China). Methanol was from DIKMA Technology Co., Ltd (Beijing, China). Formic acid, acetonitrile and methanol (analytical grade) were from Fisher Co., Ltd (Waltham, 2

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minimal emulsification with large oil droplets present on the surface. Similarly, the oils and surfactants with good emulsifying ability were mixed with the co-surfactants in the mass ratio of 2:4:4 (w/w). Compare the microemulsion effect of each combination and preliminary screening of SMEDDS consisting of the oil phases, surfactants and co-surfactants.

was adsorbed, 0.5 g of formulation was added to 50 mL distilled water and stirred at 50 rpm for 10 min in a constant temperature water-bath at 37 °C for 10 min. The microemulsifying time and particle size of the solution were recorded.

2.4. Preparation of SMEDDS

2.6.1. Determination of microemulsifying time 0.5 mL of each microemulsion preparation was dripped into 37 °C distilled water to 100 mL (using the Chinese Pharmacopoeia 2015 General Principles 0931 third method, the rotation speed is 50 rpm). The time of the formation of the light blue emulsion solutions was recorded and each sample was repeated three times to give the average.

2.6. Characterization of SFG-S-SMEDDS

2.4.1. Ternary phase diagram According to the results of 2.3, the oils (Ethyl Oleate, Capyrol 90, Mig 812N), surfactants (Tween 80 and Cremophor RH40) and co-surfactants (PEG 400, 1, 2-propylene carbonate, Transcutol HP) were selected as the three sides of phase diagram. 1.0 g of the oil and Km (Km = surfactant: co-surfactant = 1:9, 2:8 … 8:2 and 9:1 (w/w)) was added to the beaker in ratios of 1:9, 2:8 … 8:2 and 9:1. The mixtures were stirred in a constant temperature water-bath at 37 °C for 10 min, diluted with 50 times distilled water and recorded prescriptions and ratios that can form clear or light blue opalescent liquids. The point in the ternary phase diagram was composed of the weight percentage (w/ w) of oil, surfactant and co-surfactant. Origin 8.0 software was used to draw ternary phase diagrams (Cui et al., 2009; Qi et al., 2014).

2.6.2. Particle size distribution and zeta potential A zeta potential for each microemulsion was established using an Electron microscopy (TECNAI G2, The Netherlands). 0.5 g of SFG-SSMEDDS was dispersed 50 times with distilled water to form microemulsion. The particle size, potential and PDI of microemulsion were measured. 2.6.3. Transmission electron microscopy (TEM) The morphology of SFG-S-SMEDDS was observed by Transmission electron microscope (TECNAI G2, The Netherlands). The SFG-SSMEDDS was diluted 100 times with distilled water, 5 μL of sample was added to a copper wire and stained for 30s with a phosphotungstic acid solution with a mass fraction of 1%. After natural drying, the morphology of the microemulsion was observed and photographed under transmission electron microscopy.

2.4.2. Investigation of the dosage The particle size is an important indicator to measure the efficiency of self-microemulsion. Therefore, our study used particle size as an indicator to initially study the effect of the amount of excipients. Depending on the ternary phase diagram experiment, the oil was identified as Ethyl Oleate, the surfactant was Cremophor RH40, and the co-surfactant was PEG 400. Investigate the effect of adding different amounts of oil phase, surfactant, co-surfactant and SFG on particle size.

2.6.4. Stability of SFG-S-SMEDDS Three batches of SFG-S-SMEDDS were set at room temperature for 1, 2 and 3 months, and their stability were checked by traits, particle size after microemulsion, potential, and PDI (dispersion coefficient).

2.4.3. Optimization of formulation On the basis of dose studies experiments, oil (X1), surfactant (X2) and co-surfactant (X3) that have a significant effect on the formation of self-microemulsion as independent variables, and the scope of three factors was decided: oil percentage (X1): 20%–40% (w/w); surfactant percentage (X2): 25%–60% (w/w); co-surfactant percentage (X3): 5%–40% (w/w). And the particle size (Y1), self-microemulsifying time (Y2) and drug loading (Y3) as dependent variables. The three-factor, two-level D-optimal design was carried out. Each response of experiments was fit into linear, quadratic, cubic, and quadruple model respectively. After the best effect value was chose, the optimal prescription proportion was optimized by the Design-expert.V8.0.6.1.

2.7. Dissolution studies In order to find out if changing the formulation would affect the drug, in vitro release experiments were carried out. Depending on the dissolution method of the Chinese Pharmacopoeia (2015, General Principles 0931 Second Method), the cumulative dissolution was calculated and the dissolution curve was plotted. The suspensions of SFG and SFG-S-SMEDDS were diluted with 900 mL distilled water at (37 ± 0.5) °C. Samples were collected respectively at 5, 10, 20, 30, 45, 60 and 120 min to determine the content of SFG.

2.4.4. Preparation of liquid SMEDDS The optimal formulation was prepared by the results of D-optimal design and ternary phase diagram. In order to study the effect of temperature, stirring speed and microemulsifying medium, single-factor experiments were carried out. The microemulsifying medium of SFGSMEDDS was selected from 50 times distilled water, artificial gastric juice and artificial intestinal fluid at different temperature (20 °C, 37 °C, 50 °C) with speed of 50 rpm, 100 rpm or 200 rpm.

2.8. In vivo pharmacokinetic study All animal studies were conducted in accordance with the principles of Laboratory Animal Care and were approved by the Department of Laboratory Animal Research at Heilongjiang University of Chinese Medicine. Male Sprague Dawley (SD) rats (240–260 g) were supplied by the Animal Centre of the Heilongjiang University of Chinese Medicine. All the SD rats were clinically beneficial during the period of the experiment. SD Rats were fasted for 24 h prior at the beginning of experiments. Twelve SD rats were randomly divided into two groups and given 100 mg/kg of suspension of SFG-S-SMEDDS and SFG respectively. At 15, 30, 45, 60, 120, 240, 360, 480, 600, 720 and 1440 min, blood samples which were collected from the Retinal venous plexus and contained in the heparin tube, were centrifuged at 4000 rpm for 10 min. The obtained plasma was kept at −40 °C until analyzed. 10 μL of rutin (365 ng/mL, internal standard, IS) was added to 100 μL plasma in a 2.0 mL centrifuge tube and mixed for 3 min. Then, after 1.0 mL of Ethyl acetate was added slowly and the mixture was swirled for 3 min, 800 μL of the supernatant by centrifugal fluid (12,000 rpm, 10 min) was dehydrated at 40 °C nitrogen. The residue was dissolved in100 μL

2.5. Formulation optimization of S-SMEDDS Adsorption capacity of soluble adsorbent materials (PEG 4000, PEG 6000, Soluble starch, Mannitol, Citric acid) and insoluble adsorbent materials (poloxamer, Starch, Microsilica, Microcrystalline cellulose) on SFG liquid self-microemulsion was investigated. The above adsorbent materials were added respectively 1.0 g to SFG liquid SMEDDS and stir evenly so that the liquid was fully absorbed into the dry powder. When the powder was slightly wet, it meant that the adsorption material had been saturated and each adsorption material was repeated three times. The amount of SFG self-microemulsion adsorption was the maximum amount of adsorbed material. After solid adsorbent 3

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methanol, swirled for 3 min and centrifuged (12,000 rpm) for 10 min. The plasma sample was analyzed by UPLC-MS/MS. The UPLC system had an ACQUITY UPLC® HSS T3 C18 column (2.1 mm × 100 mm, 1.8 μm). The mobile phase was A (acetonitrile)-B (water with 0.1% formic acid), and the column heater was set for 35 °C and a UV detector with a wave-length of 296 nm. The flow rate was 0.2 mL/min and the injection volume was 5 μL. The MS/MS system uses the electrosurgery ionization as the ion source and the detection mode as the negative ion detection. The ionic charge ratios of SFG and rutin were form 423.2 m/z to 161.2 m/z and 609.3 m/z to 300.3 m/z respectively. The calibration curve had good linearity (R2 = 0.9975) over the concentration range of 0.08–160 ng/mL. The intra- and inter-day precisions and accuracy were no > 10% at each QC level. The mean extraction recovery was 79.6%, 83.5% and 82.7% at the three QC levels (low, medium and high) for SFG, respectively. The method was fully validated and applied to a pharmacokinetic study of SFG in rat plasma.

were used as the oil, Tween 80 and Cremophor RH40 were chosen as the surfactant. Based on the results, by comparing emulsion effect of each combination, show that 1, 2-propylene carbonate, PEG 400 and Transcutol HP were initially selected as the co-surfactant with level of A. 3.3. SMEDDS preparations 3.3.1. Ternary phase diagrams Consequences by establishing ternary phase diagrams are shown in Figs. 3(A, B, C), 4(A, B), 5(A, B, C). The oil of SFG self-microemulsion was identified as Ethyl Oleate, the surfactant was Cremophor RH40, and the co-surfactant was PEG 400. As the oil, Ethyl Oleate formed a larger self-emulsifying area and the solubility of SFG in Ethyl Oleate was (57.7 ± 3.03) mg/mL, which was the larger of the seven oil phases investigated. As the surfactant, the formation region of the Cremophor RH40 system was significantly larger than that of Tween 80. In addition, Cremophor RH40 has the optimum dissolution with high HLB, strong microemulsifying ability and high microemulsifying efficiency (Singh et al., 2012). It is likewise a very safe surfactant and has a wide range of applications even in injections. Cremophor RH40 can enhance the absorption rate of oral drugs by inhibiting P-glycoprotein drug leakage pump (Gursoy and Benita, 2004). As the co-surfactant, the selfemulsification system of the PEG 400 was much better than other cosurfactants. The reason is that low molecular weight of polar organics with water-solubility has the ability to destroy the structure of water by the intense interaction of molecules.

2.9. Statistical analyses All results were expressed as mean ± SD. The data from different formulations were comparable for statistical significance by a one-way analysis of variance (ANOVA). 3. Results and discussion 3.1. Solubility of SFG The solubility of SFG plays an important role in the formation of self-microemulsion. Fig. 2 shows the experimental data on solubility of SFG in oils, surfactants and co-surfactants. Capyrol 90, Isopropyl myristate, Ethyl Oleate, Labrafil M1944CS, Miglyol 812N and Soybean oil had high solubility so that could be chosen as the oil. Similarly, Labrasol®, Cremphor RH40, Kalliphor HS15, Tween 80 and Tween 20 were voted as the surfactant. 1, 2-Propylene carbonate, PEG 400, Transcutol HP, isopropanol, glycerol were selected as the co-surfactant. On this basis, we further carry out the experimental study of self-emulsifying grading test.

3.3.2. Investigation of the dosage First of all, the amount of fixed surfactant (Cremophor RH40) and co-surfactant (PEG 400) is added to different amounts of Ethyl Oleate. The results show that with the increase in oil content (22%~38%, w/ w), the particle size of the microemulsion formed by self-microemulsion gradually increased. But, when the proportion of Ethyl Oleate reaches 38% (w/w), the system cannot form microemulsion. Second, the amount of fixed oil phase (Ethyl Oleate) and co-surfactant (PEG400) was added to different amounts of Cremophor RH40. It was found about the amount of surfactant that as the mass fraction of the surfactant increased (42%–65%, w/w). The particle size of the microemulsion first decreased and then increased. When the surfactant concentration exceeded 60% (w/w), the particle size increased. This phenomenon may be caused by a large amount of surfactant causing water to penetrate into the oil droplets, and the oil droplets break into the water phase, and the surface membrane is destroyed (Kommuru et al., 2001; Rang and Miller, 1999). Then, the amount of fixed oil phase (Ethyl Oleate) and surfactant (Cremophor RH40) was added to different quality of PEG-400. The effect of the amount of co-surfactant on the self-emulsification efficiency was investigated. It found that as the co-surfactant concentration on the self-microemulsion increased, the particle size decreased. Finally, a blank self-microemulsion formulation was used to examine the drug dosage (0.5%–2.5%, w/w). It was demonstrated that the drug concentration increased and the particle size of the microemulsion increased slightly, but the increase was not significant. It shows that the addition of drugs has no obvious influence on the selfmicroemulsifying ability of the prescription.

3.2. Self-emulsifying grading test Grading results in different SMEDDS mixtures are recorded in Table 1, the self-emulsifying system grade was A with Ethyl OleateTween 80, Ethyl Oleate-Cremophor RH 40, Capyrol 90-Tween 80, Capyrol 90-Cremophor RH 40, Miglyol 812N-Tween 80 and Miglyol 812N-Tween 80. Therefore, Ethyl Oleate, Capyrol 90 and Miglyol 812N

3.3.3. Optimization of formulation Response plane equations were obtained by analyzing fitting results of the variance of the model. The resulting equation is reproduced bellow. Y1 and Y2 were minimized, Y3 was maximized as the optimization index, and the overall satisfaction D was used to optimize the experimental factors and designed the predicted value of the optimized prescription response.

Fig. 2. Saturated solubility of SFG in various oils, surfactants and co-surfactants (n = 3). 4

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Table. 1 The experimental results of oil surfactant with compatibility.

Labrasol Tween 80 Tween 20 Cremophor RH 40 Kolliphor HS 15

Isopropyl myristate

Ethyl Oleate

LabM1944CS

Capy 90

Soybean oil

Mig812N

D C D B D

D A C A C

D C D B D

D A D A D

D C C C C

D A C A C

Fig. 4. Three phase diagram of surfactant (A: Ethyl Oleate-Cremophor RH40PEG 400; B: Ethyl Oleate-Tween 80-PEG 400) (the gray area represent microemulsion range).

Y1 = + 19.06 X1 + 6.00 X2 + 12.36 X3 X2 X3 + 113.98 X1 X2 X3

Y2 = + 39.00 X1 + 76.82 X2

22.85 X1 X2

151.21 X12 X2 X3

X3 + 105.73 X1 X2 X3 + 2153.00 X1 X2 X3 467.91 X1 X3

0.62 X2

2

3454.47 X1 X2 X3

26.12 X2 + 79.48 X3 + 1072.91 X1 X2 + 915.04 X1

25.71 X2 X3

X2 (X1

21.63

2.30 X3 + 4.29 X1 X2 + 53.38 X1 X3 2

Y3 =

29.04 X1 X3 77.16 X1 X2 2 X3

1732.41 X1 X2 X3 + 820.03 X1

X2) + 944.15 X1 X3 (X1

X3 ) + 349.87 X2 X3 (X2

X3)

The consequences of formulation are Ethyl Oleate (38.5%, w/w), Cremophor RH40 (47.5%, w/w), and PEG 400 (14.0%, w/w). That the results of the best prescription and the measured value show in Table 2 illustrated the measured value is in good agreement with the predicted value, and that the prescription of experimental design has practical significance. SO, there was no significant difference between the predicted value and the real value. In order to facilitate the calculation at

Fig. 3. Three phase diagram of oil phase (A: Ethyl Oleate-Tween 80-PEG 400; B: Capy90-Tween 80-PEG 400; C: Mig 812N-Tween 80-PEG 400) (the gray area represent microemulsion range).

5

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the time of preparation, the 20.00 mg/g of drug loading was determined. 3.3.4. Preparation of SMEDDS The particle size declined with increasing temperature and the selfmicroemulsifying preparation was mainly passed through the oral microemulsion in the gastrointestinal tract. Therefore, 37 °C was chosen as self-microemulsion temperature (Song et al., 2009) and the particle size is (36.63 ± 1.38) nm. The stirring speed has little effect on the particle size, so we chose 50 rpm that was not hard to control. The particle size is (38.66 ± 1.54) nm. Comparing with different microemulsifying medium, we found no significant effect on the size of the SFG microemulsion. The preparation method of SFG-SMEDDS was as follows: 0.77 g Ethyl Oleate, 0.95 g RH40, 0.28 g PEG 400 uniformly stirred for 5 min, gradually add 40 mg SFG and magnetic stir 50 rpm for 10 min at 37 °C constant temperature. 3.4. Formulation optimization of S-SMEDDS In order to improve stability and the shortcomings of SFG-SMEDDS, solid materials were added to the microemulsion. It was found that the best adsorbent material was mannitol by comparing the adsorption force of solid adsorbents, the results are shown in Table 3. By studying the mass adsorption rate of SFG self microemulsion and mannitol, it was found that when the ratio was 1:1 (w/w), mannitol was difficult to dry because it was exudation from microemulsion in the drying process. Therefore, we further investigated the ratios of 1:2, 1:3, 1:4 and 1:5 (w/ w), and determined that the mass adsorption ratio was 1:2 (w/w). The preparation process of SFG-S-SMEDDS was carried out by adding 4 g mannitol to the SFG-SMEDDS, forming soft material through a 14 mesh sieves and drying. 3.5. Characterization of SFG-S-SMEDDS The appearance of SFG-S-SMEDDS is light yellow particles, the microemulsifying time was (34.6 ± 1.4) s, and the solution was light blue-opalescent. Particle size is (38.5 ± 1.0) nm, which is approximately the same as that of the SFG liquid self-microemulsion. Surface solid adsorbent has no impact on the formation of self-microemulsion. PDI was (0.15 ± 0.02), indicating that the particle distribution was uniform. The potential for the SFG microemulsion was (−39.1 ± 1.8) mV, indicating that the system was stable. The results obtained by TEM that the SFG-S-SMEDDS is a spherical particle of a more uniform appearance and a more homogeneous distribution. The preliminary stability results about the shape, particle size, potential and PDI, show that the SFG-S-SMEDDS did not change greatly within 3 months, and the properties were in accordance. The specific results are shown in Table 4. 3.6. Dissolution studies Fig. 6(A) displays the results obtained from the preliminary analysis of the dissolution study. The rate of SFG-S-SMEDDS reached 60% at 10 min and 80% at 30 min, which was significantly higher than that of SFG. It meant that dissolution rate effectively of SFG was increased.

Fig. 5. Three phase diagram of co-surfactant (A: Ethyl Oleate-Cremophor RH40-PEG 400; B: Ethyl Oleate-Cremophor RH40-1,2-propylene carbonate; C: Ethyl Oleate-Cremophor RH40-Transcutol HP) (the gray area represent microemulsion range).

3.7. In vivo pharmacokinetic study

Table. 2 The response value and predictive value of the optimized prescription. Response

Expected

Observed (n = 3)

Y1: Particle size (nm) Y2: Self-miciroemulsifying time (s) Y3: drug loading (mg/g)

39.16 32.75 23.69

38.40 ± 1.03 34.5 ± 1.40 23.3 ± 0.89

The methodological study shows that the UPLC-MS/MS method was suitable for the determination of plasma concentration. The average plasma concentration of SFG and SFG-SMEDDS was simulated by onechamber, two-chamber and three-chamber model respectively to obtain AIC value and r2. Depending on the minimum of AIC and the maximum value of r2, it was determined that the drug-time process of SFG and SFG-S-SMEDDS in rats was equal to two-compartment open model 6

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Table. 3 Effect of solid adsorption materials on particle size and emulsifying time of micro-emulsion (n = 3). Adsorption material

Emulsifying time (s)

PEG 4000 PEG 6000 Mannitol Citric acid Soluble starch Poloxamer Starch Micropowder silica gel Microcrystalline cellulose

37.5 37.2 35.5 36.9 38.3 43.1 45.4 44.9 47.2

± ± ± ± ± ± ± ± ±

1.5 0.8 0.5 1.4 1.2 0.9 1.0 1.4 1.6

particle size (nm)

Clarity

38.9 ± 1.0 39.7 ± 0.8 38.2 ± 1.4 40.2 ± 0.5 39.7 ± 1.2 104.5 ± 0.9 107.2 ± 1.4 106.5 ± 1.9 104.2 ± 1.8

With or without light blue-opalescent

Clarify Clarify Clarify Clarify Clarify Don't clarify Don't clarify Don't clarify Don't clarify

Yes Yes Yes Yes Yes No No No No

(weight 1/CC). The pharmacokinetic parameters are shown in Table 5. The relative bioavailability of SFG-S-SMEDDS was calculated based on Eq. (1): F = AUC 0

homemade preparation/AUC 0

reference preparation × 100%

(1) The relative bioavailability of SFG-S-SMEDDS was 343.84%. The plasma concentration-time curves about suspensions of SFG and SFG-SSMEDDS are obviously seen in Fig. 6(B). The results showed that the pharmacokinetic parameters of SFG-S-SMEDDS, especially Cmax and AUC, had been significantly changed. The absorption and peak time of SFG in vivo were increased and the absorption of drugs in the body were accelerated. 4. Conclusion In traditional Chinese medicine, many effective components and effective parts are water-insoluble substances. SMEDDS not only increases the oral bioavailability of the drug, but also reduces drug irritation to the gastrointestinal tract and mask the unpleasant smell of some drugs. Therefore, the introduction of SMEDDS preparation technology into traditional Chinese medicine preparation is of great significance for improving the efficacy of traditional Chinese medicine preparation. In this study, the prescription optimization and pharmacokinetic evaluation of SFG-S-SMEDDS with uniform spherical particles were studied. The relative bioavailability of SFG-SMEDDS was significantly improved. It may be that surfactants increase drug permeability by increasing the ability of drugs to penetrate the bilayer lipid of intestinal cells. And its particle size is generally < 50 nm, providing a relatively large absorption surface area, which is conducive to the absorption of drugs in the gastrointestinal tract. With the in-depth study of SFG-SSMEDDS, SFG will have a broad application prospect and become a good way to serve human beings. Since SFG is still in the early stage of the pre-clinical study and there is thus no clinical gold standard dose/ specifications for SFG. SMEDDS was adopted in this paper to provide SFG with an effective oral dosage form to enhance oral bioavailability. So, the result of this paper will lay a foundation for new drug RD based on SFG.

Fig. 6. In vitro cumulative release rate (A) and in vivo concentration time curve (B) of SFG and SFG-SMEDDS (superposition).

Table. 5 Statistical parameters of atrioventricular model of SFG and SFG-S-SMEDDS. Parameter

Unit

SFG

SFG-S-SMEDDS

Average (ng/mL) t1/2α h h t1/2β AUC(0→t) μg/L h AUC(0→∞) μg/L h – AUMC(0→t) AUMC(0→∞) – MRT(0→t) h h MRT(0→∞) Tmax h μg/L Cmax CLz L/h/kg

1.903 39.422 425.607 492.909 3474.532 5866.237 7.754 11.417 2.167 56.682 217.678

SD (%) 1.580 32.791 152.110 189.559 1358.437 3000.943 0.754 3.133 2.284 16.070 59.597

Average (ng/mL) 3.200 37.315 563.166 652.977 3833.022 8478.344 6.785 11.278 0.792 88.642 163.949

SD (%) 1.209 35.088 89.036 209.910 809.604 8836.086 0.769 6.965 0.102 23.827 41.715

Table. 4 Character, particle size and potential changes in 3 months of SFG-S-SMEDDS (n = 3). Placement time (month) 0 1 2 3

Character (character) Light Light Light Light

yellow yellow yellow yellow

Particle size (nm)

granule granule granule granule

38.5 37.1 38.9 37.9

7

± ± ± ±

1.0 0.9 1.1 1.4

Potential (mV)

PDI

−39.1 −40.4 −38.2 −35.2

0.15 0.16 0.15 0.18

± ± ± ±

1.8 1.3 2.1 1.7

± ± ± ±

0.02 0.02 0.03 0.03

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Declaration of Competing Interest

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There is no conflict of interest. Acknowledgments The authors are thankful to the Natural Science Foundation of Heilongjiang Province, China (No. H2016057, H2017066), financial support of outstanding innovative talents support program project from the Heilongjiang University of Chinese Medicine, China (No. 2018), Chunhui fund of Ministry of Education of China plans (No. Z2008-115016). The science and technology project of Department of Education, Heilongjiang Province, China (No. 12541198). References Bolko, Seljak. K., Ilić, I. G., Gašperlin, M., Zvonar, Pobirk, A., 2018. Self-microemulsifying tablets prepared by direct compression for improved resveratrol delivery. Int. J. Pharm. Sep 5; 548(1):263–275. Cha, J.D., Moon, S.E., Kim, J.Y., Jung, E.K., Lee, Y.S., 2009. Antibacterial activity of sophoraflavanone G isolated from the roots of Sophora flavescens against methicillinresistant Staphylococcus aureus. Phytother. Res. 23 (9), 1326–1331 Sep. Cha, S.M., Cha, J.D., Jang, E.J., Kim, G.U., Lee, K.Y., 2016. Sophoraflavanone G prevents Streptococcus mutans surface antigen I/II-induced production of NO and PGE2 by inhibiting MAPK-mediated pathways in RAW 264.7 macrophages. Arch. Oral Biol. 68, 97–104 Aug. Chen, Q., Zhou, H., Yang, Y., Chi, M., Xie, N., Zhang, H., Deng, X., Leavesley, D., Shi, H., Xie, Y., 2017. 2017. Investigating the potential of Oxymatrine as a psoriasis therapy. Chem. Biol. Interact. 271, 59–66 Jun 1. Cui, J., Yu, B., Zhao, Y., Zhu, W., Li, H., Lou, H., Zhai, G., 2009. Enhancement of oral absorption of curcumin by self-microemulsifying drug delivery systems. Int. J. Pharm. 371 (1–2), 148–155 Apr 17. Fakhimi, A., Iranshahi, M., Emami, S.A., Amin-Ar-Ramimeh, E., Zarrini, G., Shahverdi, A.R., 2006. Sophoraflavanone G from sophora pachycarpa enhanced the antibacterial activity of gentamycin against Staphylococcus aureus. Z. Naturforsch. C 61 (9–10), 769–772 Sep-Oct. Guo, C., Yang, L., Luo, J., Zhang, C., Xia, Y., Ma, T., Kong, L., 2016a. Sophoraflavanone G from Sophora alopecuroides inhibits lipopolysaccharide-induced inflammation in RAW264.7 cells by targeting PI3K/Akt, JAK/STAT and Nrf2/HO-1 pathways. Int. Immunopharmacol. 38, 349–356 Sep. Guo, C., Yang, L., Wan, C.X., Xia, Y.Z., Zhang, C., Chen, M.H., Wang, Z.D., Li, Z.R., Li, X.M., Geng, Y.D., Kong, L.Y., 2016b. Anti-neuroinflammatory effect of Sophoraflavanone G from Sophora alopecuroides in LPS-activated BV2 microglia by MAPK, JAK/STAT and Nrf2/HO-1 signaling pathways. Phytomedicine 23 (13), 1629–1637 Dec 1. Gursoy, R.N., Benita, S., 2004. Self-emulsifying drug delivery systems (SEDDS) for improved oral delivery of lipophilic drugs. Biomed. Pharmacother. 58 (3), 173–182 Apr. Hoar, T.P., Schulman, J.H., 1943. Transparent water-in-oil dispersions: the oleopathic hydro-micelle. Nature 152 (3847), 102–103. Huang, J., Gong, W., Chen, Z., Huang, J., Chen, Q., Huang, H., Zhao, C., 2017. Emodin self-emulsifying platform ameliorates the expression of FN, ICAM-1 and TGF-β1 in AGEs-induced glomerular mesangial cells by promoting absorption. Eur. J. Pharm. Sci. 1 (99), 128–136 Mar.

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