Pharmacokinetics of Jiaotai pill self-microemulsion in insomnia rats

BAI Dan-ni, WEI Xi-hong, SHEN Meng-ting, WANG Qing-wei, SU Jin

1. Heilongjiang Provincial Key Laboratory of New Drug Development and Pharmacotoxicological Evaluation, Department of Pharmacy,Jiamusi University,Heilongjiang Province,Jiamusi 154007,China

Keywords:

ABSTRACT

1. Introduction

Insomnia is one of the common sleep disorders in clinic.Epidemiological studies in China show that 45.4% of the respondents have experienced insomnia to varying degrees.Insomnia not only affects the quality of life, but also often accompanies other health problems[1-4]. Research shows that insomnia is closely related to a variety of mental diseases, metabolic syndrome, hypertension, coronary heart disease and other chronic diseases[5-9]. Jiaotai Pills is composed of Coptis chinensis and cinnamon herbs, which are found in the Han Shi Yi Tong[10], In the prescription, Coptis chinensis bitter in tastes, cold in nature, while cinnamon acrid in taste, hot in nature. The combination of the two drugs makes the heart and kidney intersect, and water and fire help each other. It is a classic prescription for the treatment of insomnia with heart kidney disharmony[11,12]. However, due to the poor water solubility of coptis alkaloid, the susceptibility to intestinal P-glycoprotein efflux, and the unstable nature of cinnamaldehyde,the main component of cinnamon, the oral bioavailability of Jiaotai Pill is low, which affects its efficacy[13,14]. Self microemulsifying drug delivery system (SMEDDS) can effectively improve the bioavailability of drugs by wrapping drugs in the oil phase, so that they can spontaneously disperse into o/w microemulsions after oral administration. SMEDDS is an excellent carrier of insoluble drugs[15,16]. In this paper, we compared the pharmacokinetic parameters of Jiaotai Pill self microemulsion and suspension in insomnia rats by intragastric administration, and investigated the effect of dosage form optimization on bioavailability, so as to provide reference for its pharmacodynamic study and clinical application.

2. Materials and methods

2.1 Drugs and reagents

Coptis chinensis(Hebei Meikang Pharmaceutical Co., Ltd., batch NO. 1901097); Coptis alkaloid(Self made in laboratory); Lotus leaf alkaloid reference substance (batch NO. MUST-17035501),Berberine(batch NO. MUST-16111115), Coptisine(batch NO.MUST-19052111), Palmatine(batch NO. MUST-17022604) and Jatrorrhizine(batch NO. MUST-19072609)(Chengdu mansite Biotechnology Co., Ltd., purity 98%); Cinnamon oil (Jiangxi Hengcheng natural flavor oil Co., Ltd., batch No. 20170301);Polyoxyethylene hydrogenated castor oil (RH40, BASF, Germany);P-chlorophenylalanine (PCPA, Shanghai Yuanye Biotechnology Co.,Ltd., batch NO. J01S7E20295); Anhydrous ethanol, concentrated sulfuric acid, sodium chloride, anhydrous calcium chloride (Tianjin kemio Chemical Reagent Co., Ltd., analytical reagent); Formic acid,methanol, acetonitrile (thermo company, chromatography pure).

2.2 Instrument

U3000-Q Exactive UPLC-MS/MS(thermo, USA); Sartorius XP105 Electronic analytical balance (saidoris AG, Germany); TGL-16G Desktop centrifuge (Shanghai Anting Scientific Instrument Factory);D10-12 nitrogen blowing instrument (Hangzhou Aosheng Instrument Co., Ltd.).

2.3 Animals

There are 16 healthy male SD rats, 8 in each group, weighing(200±20)g, provided by Changchun Yisi Experimental Animal Technology Co., Ltd., animal license No. SCXK (Ji) -2018-0007.Animal experiments have been approved by the experimental animal ethics committee of Jiamusi University.

Feeding conditions: adaptive feeding for 1 week, sub pack every 4 cages, maintain a 12 h circadian rhythm, room temperature 18℃～25 ℃, and eat and drink freely.

2.4 Methods

2.4.1 Preparation of Jiaotai Pill self microemulsion

Take 10 g of Rhizoma Coptidis powder and put it into a round bottom flask, add 100 mL of dilute sulfuric acid, reflux and extract for 2 h in a 70 ℃ water bath, filter by suction, add an appropriate amount of anhydrous CaCl2to the filtrate, stand for 1 hour, filter by suction, add an appropriate amount of NaCl to the filtrate, and store for 12 h at 4 ℃. Filter by suction and dry the filter residue(50 ℃, 2 h) to obtain Coptis alkaloids, collect and reserve.According to the optimized formula of the previous experiment of the research group, accurately weigh 0.28, 1.03 and 0.67 g of cinnamon oil, RH-40 and absolute ethanol respectively, add an appropriate amount of total alkaloid extract of Coptis chinensis,vortex evenly for 5 minutes, store at 4 ℃, and standby. Using UPLC-MS/MS, the contents of four alkaloids in Jiaotai Pill self microemulsion were berberine 7.67 mgbamatine 1.0/g, coptidine 2.13 mg/g, 9 mg/g, and jatrorrhizine 1.35 mg/g[17].

2.4.2 Preparation of Jiaotai Pill suspension

Accurately weigh the same amount of Rhizoma Coptidis alkaloid extract, mix it with cinnamon oil according to the prescription proportion, add an appropriate amount of normal saline and mix it,store it at 4 ℃ for standby.

2.4.3 Animal model and Administration

Sixteen rats were randomly divided into Jiaotai Pill suspension group and Jiaotai Pill self microemulsion group, with 8 rats in each group. Before the test, the rats were placed in a clean feeding cage for 3 minutes, and the spontaneous activity time and the lifting times of both forelimbs within 2 minutes were observed, taking this as the behavioral baseline[18]. The insomnia model was prepared by intraperitoneal injection of PCPA, with the dosage of 350 mg/kg and continuous injection for 4 days[19]. After confirming the success of the model, fasting and drinking for 12 h, the two groups were given Jiaotai Pill self microemulsion or suspension at the dose of 80 mg/kg of Coptis alkaloids, respectively[20]. Before and after administration,0.083, 0.167, 0.333, 0.667, 1, 1.5, 2, 4, 6, 8, 10, 12 and 24 h, orbital blood was taken from 0.30 mL to 0.50 mL in a heparin sodium centrifuge tube, centrifuged (4 ℃, 3500 r/min, 15 min), plasma was separated, and stored at ?20 ℃ for testing[21].

2.4.4 Preparation of solutionAccurately weigh 5.22 mg Berberine, 4.99 mg Coptisine, 5.50 mg Palmatine, 5.02 mg Jatrorrhizine, and fix the volume with methanol to 10 mL to prepare standard stock solutions with concentrations of 0.52, 0.20, 0.20, and 0.20 mg/mL respectively. Dilute each stock solution with methanol to 6 different concentrations (HB1-6), and store at 4 ℃. See Table 1 for concentration.

2.4.5 Determination of four alkaloids in plasma

2.4.5.1 Chromatographic conditions

Chromatographic column: Hypersil GOLD C18(100 mm×2.1 mm,1.9 μm); Mobile phase: 0.1% formic acid purified water (A) -0.1%formic acid acetonitrile (B); Volume flow: 0.3 mL/min; Column temperature: 20 ℃; injection volume: 5 μL, The linear gradient elution procedure is shown in Table 2.

Tab2 Linear gradient elution procedure

2.4.5.2 Mass spectrum conditions

ESI positive ion detection, single ion detection scanning (SIM)and parallel reaction monitoring scanning (PRM) were used;Test objects: Berberine m/z=336.12 26, Coptisine m/z=320.09 16,Palmatine m/z=352.15 42, Jatrorrhizine m/z=338.13 82, lotus leaf alkaloid (internal standard) m/z=296.13 19; Sheath gas pressure 35 bar, capillary voltage 3.5 kV, auxiliary gas pressure 10 bar; Heating(evaporation) temperature 350 ℃; The temperature of ion transfer tube is 320 ℃.

2.4.5.3 Plasma sample pretreatment

To 100 μL add 4 times the amount of methanol to the plasma,vortex for 1 min, stand for 15 min, centrifuge (4 ℃, 13000 r/min, 15 min), take 400 μL supernatant, blow dry with nitrogen (40 ℃) and add 100 μL methanol is dissolved by ultrasonic for 30 minutes, and the supernatant is taken for injection analysis.

2.4.5.4 Specificity inspectionBlank plasma, (mixed standard+blank plasma) and plasma samples after intragastric administration were analyzed respectively to investigate whether there was endogenous interference.

2.4.5.5 Linear relationship and limit of quantitation

Take 10 μL mixed standard solution of different concentrations is added to 90 μL blank rat plasma, mix well, treat the sample according to the method under "1.4.5.3", and conduct injection analysis to obtain the regression equation and linear range.

2.4.5.6 Precision

Prepare quality control samples of low, medium and high concentrations with rat blank plasma, operate according to the method under "1.4.5.3", measure continuously for 5 times to calculate the intra day precision, and measure for 5 d to calculate the intra day precision.

2.4.5.7 Accuracy

Prepare quality control samples (n=6) of low, medium and high concentrations with rat blank plasma, operate the injection according to the method under "1.4.5.3", calculate the measured concentrations of Berberine, Coptisine, Palmatine and Jatrorrhizine in the samples,express the accuracy as the percentage of the measured concentration and the marked concentration, and calculate the relative standard deviation (RSD%).

2.4.5.8 Stability

Prepare quality control samples of low, medium and high concentrations (n=6) with rat blank plasma, place the samples at room temperature for 24 hours, inject samples for detection, and investigate the stability at room temperature; After repeatedly freezing and thawing the sample for 3 times (freezing and thawing once for at least 1 day), inject samples for testing to investigate the freeze-thaw stability.

2.5 Statistical Analysis

The pharmacokinetic parameters were calculated by DAS 2.0 and analyzed by SPSS 17.0 independent sample t-test. The difference was statistically significant by P ＜ 0.05.

3. Results

3.1 Spontaneous activity experiment in rats

The spontaneous activity time and the lifting times of both forelimbs in the two groups increased significantly within 2 minutes after modeling (P ＜ 0.05), indicating that the establishment of insomnia model was successful. The results are shown in Table 3.

Tab3 Autonomous activity of rats in each group(n=8,x±s)

3.2 Mass spectrometric analysis of four alkaloids

Under the experimental conditions, the secondary mass spectra of Berberine, Coptisine, Palmatine and Jatrorrhizine are shown in Figure 1.According to Figure 1, the m/z of Berberine, Coptisine,Palmatine and Jatrorrhizine are 336, 320, 352 and 338 respectively.

Fig1 The mass spectra of Berberine (A),Coptisine (B),Palmatine (C) and Jatrorrhizine (D)

3.3 Specificity inspection

The results of UPLC-MS/MS showed that the Rt of the four alkaloids were Berberine 1.40 min, Coptisine 1.24 min, Palmatine 1.14 min, Jatrorrhizine 1.30 min. the separation was good, and there was no interference at the retention time of each component,indicating that the method was highly exclusive, as shown in Figure 2.

A. blank plasma B. plasma samples after intragastric administration C. Standard + blank plasma 1. Berberine 2 Coptidine 3 Palmatine 4 JatrorrhizineFig2 The UPLC-MS/MS chromatograms of four alkaloids

3.4 Linear relationship and limit of quantitation

The regression equation and limit of quantitation are shown in Table 4. The results show that the four alkaloids have a good linear relationship within the set concentration range, r ＞ 0.9995, and the limit of quantitation is 0.06 ng/mL.

Tab4 Regression equations and quantitative limits of four alkaloids

Tab5 Precision of four alkaloids in rat plasma(n=5,x±s)

3.5 Precision

The precision results are shown in Table 5. The results show that the RSD of the daytime and intraday precision of the four alkaloids is less than 10%, which meets the requirements for the determination of biological samples.

3.6 Accuracy

The accuracy results are shown in Table 6. The results show that the recoveries of the four alkaloids are all in the range of 85% - 115%,which meets the requirements for the determination of biological samples.

Tab6 Accuracy of four alkaloids in rat plasma(n=6,x±s)

3.7 Stability

The stability results are shown in Table 7. The results show that the four alkaloid samples have good stability under two storage conditions, RSD＜10%, meeting the requirements for the determination of biological samples.

3.8 Drug time curve

The drug time curve of Jiaotai Pill suspension and its self microemulsion in Berberine, Coptisine, Palmatine, Jatrorrhizine in insomnia rats is shown in Figure 3.

3.9 Pharmacokinetic parameters

The pharmacokinetic parameters of the four alkaloids are shown in Table 8. Compared with the suspension group, the Cmax、AUC0-tand AUC0-∞of Berberine, Coptisine, Palmatine and Jatrorrhizine in the self microemulsion group were significantly increased (p ＜0.01). In insomnia rats, the Cmaxof Berberine, Coptisine, Palmatine and Jatrorrhizine in Jiaotai Pill self microemulsion group were 1.46,1.18, 1.83 and 1.38 times that of suspension group, AUC0-twere 2.39,2.22, 2.04 and 2.27 times, and AUC0-∞were 2.25, 2.14, 2.05 and 2.11 times that of suspension group, respectively. The bioavailability was significantly improved.

Fig3 Mean plasma concentration-time curves of four alkaloids(n=6,x±s)

Tab7 Stability ( in room temperature for 24 h, three freeze-thaw cycles) of four alkaloids in rat plasma(n=6,x±s)

Tab8 Pharmacokinetic parameters of four alkaloids(n=6,x±s)

Conflict of interest statement:

4. Discuss

The main methods of making insomnia animal models are physical method and chemical stimulation method. In this study, the insomnia rat model was prepared by intraperitoneal injection of PCPA.Compared with other methods (selenides, 5-hydroxytryptamine receptor antagonists, etc.)[22,23], the PCPA method is simple and fast.The mechanism of insomnia is to block the synthesis of 5-HT by inhibiting tryptophan hydroxylation, reducing the concentration of 5-HT in the brain and peripheral blood of rats[24]. In this experiment,it was observed that the autonomous activity time and the number of forelimb lifts of the model rats increased significantly, which showed that they were constantly active during the day and were prone to hissing, attack and shock, which was consistent with the literature[25].In the study of TCM symptoms, it is often not caused by a single factor. In order to achieve the "heart kidney disharmony", it is reported that PCPA intraperitoneal injection can be supplemented by different anxiety stimuli. Therefore, the modeling method that is more in line with TCM symptoms and signs needs to be constantly explored and improved[26].

In this paper, male rats were selected to carry out the pharmacokinetic study of Jiaotai Pill self microemulsion in insomnia rats. Reference to the methods reported in the literature and through preliminary experiments, it was determined that the state of male rats was more stable after modeling. Jiaotai Pill is made of Coptis chinensis and cinnamon according to the ratio of 10:1. It is a classic traditional Chinese medicine prescription for insomnia[27].Coptis chinensis contains a variety of chemical components, such as flavonoids, phenylpropanoids, steroids, volatile oils, terpenoids,alkaloids, etc., and has a wide range of pharmacological activities such as antibacterial, blood lipid regulation, anti arrhythmia and hypnosis[28,29]. The results of pharmacokinetics study in vivo showed that the relative bioavailability of berberine, coptidine, bamatine and jatrorrhizine in Jiaotai pills in rats was significantly improved, t1/2 was prolonged, and the retention time of components in vivo was also increased. In addition to its solubility, the absorption of drugs in vivo is also related to its membrane permeability and gastrointestinal environment. The particle size of self microemulsion is smaller,which is easier to penetrate the hydration layer of gastrointestinal wall and increase the permeability of gastrointestinal epithelial cell membrane[30], so as to promote the absorption of drugs in the gastrointestinal tract and improve the dissolution of drugs. At the same time, the emulsifier and co emulsifier in the self microemulsion prescription can not only improve the fluidity of cell membrane and increase the permeability of membrane, but also inhibit the efflux of intestinal P-glycoprotein to Coptis alkaloids, Effectively improve the absorption of drug components[31].

All authors have no conflict of interest.

Description of author's contribution:

Associate Professor Su Jin provided the topic selection and experimental technical guidance of this paper, and put forward suggestions for revision of the paper; Bai Danni is responsible for data analysis and thesis writing; Wei Xihong was responsible for the analysis of samples in vivo and animal experiments, while Shen Mengting was responsible for assisting in the completion of the experiment; Wang Qingwei assisted in data processing and analysis.