Effects of Different Material Ratios on the Dissolution of Tongmai Pills

Xinhong ZHAO, Chao SUN, Yanwu ZHAO, Ying JIN, Bingya KANG, Tianchao CHEN*

1. Department of Pharmacy, The Affiliated Hospital of Chengde Medical College, Chengde 067000, China; 2. Department of Pharmacy, The First Affiliated Hospital of Henan University of Traditional Chinese Medicine, Zhengzhou 450000, China

Abstract [Objectives] To study the effects of different material ratios on the dissolution of Tongmai pills. [Methods] Based on mixture uniform experiment design, the fiber, starch and grease-resin materials in Tongmai pills were proportioned. The contents of strychnine, brucine, paeoniflorin, calycosin, ferulic acid, hesperidin and salvianolic acid B in the solution were determined by HPLC, and the content of total flavonoids was determined by ultraviolet spectrophotometry. The weight coefficient of index components was determined by analytic hierarchy process, and the SAS software was used to optimize the dissolution model and calculate the dissolution parameter T40. MATLAB was used to establish a mathematical model and realize the data visualization between material ratio and dissolution parameter T40. [Results] The mathematical model between material ratio and dissolution parameter was T40=8.93-5.31X1-5.83X2+17.67X1X3 (X1, X2, X3 are fiber, starch and grease-resin materials respectively, r2=0.970 6, P=0.01, RMSE=0.569 6). [Conclusions] Fiber can effectively promote the dissolution (P<0.05) .Starch can promote the dissolution, but the effect is not obvious (P=0.05). The interaction between fiber and grease-resin can inhibit the dissolution, but was not significant (P=0.07).

Key words Material ratio, Mixture uniform experiment design, Tongmai pills, Analytic hierarchy process, Mathematical model

1 Introduction

At present, traditional Chinese medicine pills are still one of the leading clinical forms among hospital preparations. Traditional Chinese medicine pills are the unity of medicines and excipients. Most pills contain fine powder of raw materials for botanicals, animal medicines, and mineral medicines. Materials with different properties often affect the dissolution of effective ingredients in the pills, thereby affecting their clinical efficacy. Presently, the research on the influence of materials with different properties on dissolution is mainly concentrated on the study of model drugs[1-6]. In this experiment, Tongmai pills containing 4 toxic drugs were selected as the research object, and the influence of material ratio on the dissolution was initially discussed.

2 Materials

2.1 ReagentsStrychnine reference substance (No.181027) was purchased from Shanghai Ronghe Pharmaceutical Technology Co., Ltd., with a mass fraction of ≥ 98%; brucine (No.201805), paeoniflorin (No.201804), calycosin (No.201801), and hesperidin (No.201804) reference substances were all purchased from Sichuan Weikeqi Biotechnology Co., Ltd., with a mass fraction of ≥ 98%; ferulic acid (No.110773-201811), salvianolic acid B (No.111562-201806), and rutin (No.100080-201806) reference substance were all purchased from National Institutes for Food and Drug Control, and the purity is 100%. Tongmai pills (No.180811, specification: 5 g per bag) were prepared by the preparation room of the First Affiliated Hospital of Henan University of Chinese Medicine. Methanol, acetonitrile, and phosphoric acid were chromatographically pure (Dima); water was self-made ultrapure water, and the rest of the reagents were analytically pure.

2.2 InstrumentsMain instruments included Waters e2695 high performance liquid chromatograph (Waters Company, USA), TU-1800PC UV-visible spectrophotometer (Beijing Puxi General Instrument Co., Ltd.), ZRS-8C intelligent dissolution tester (Tianjin University Radio Factory), GP225D one hundred thousandth analytical balance (Sartorius, Germany), and HK250 ultrasonic filter (Shanghai Kedao Ultrasonic Instrument Co., Ltd.).

3 Methods and results

3.1 Material classificationUnder the guidance of the theory of "unification of medicine and adjuvant" (Chinese herbal medicine is not only a medicine for curing diseases, but also a functional excipient for the molding of pharmaceutical preparations)[7], according to the content of primary metabolites in Chinese herbal medicine, traditional Chinese medicine was divided into seven types of materials, namely fiber, starch, protein, polysaccharide, grease, resin, and mineral according to the classification method of material science properties[8]. In this experiment, based on the previous research[1-7], various decoction pieces in the prescription of Tongmai pills were divided into three types of Chinese medicine materials: fiber, starch, and grease-resin according to the classification method of the seven types of materials.

3.2 Uniform design of the mixture for use in the compositionDPS software was used to arrange the experiment according to the uniform design tableUn(ns-1), wherenis the number of experimental levels and the frequency of experiments, andsis the number of factors. 7 levels for each of the 3 factors in Tongmai pills were taken to design a total of 7 ratios for investigation[9]. At the same time, a uniform design table with a small deviation (D=0.158 2) was chosen to arrange the test (Table 1).

Table 1 Uniform design table of the mixture %

3.3 Determination of the content of multiple index components in the dissolution liquid by HPLC

3.3.1Chromatographic conditions and system adaptability test. Chromatographic column Agilent SB-Aq C18(4.6 mm×250 mm, 5 m) and Agilent SB-Aq C18guard column (1.0 mm×20 mm, 5 m) were used; mobile phase was acetonitrile (A)-0.1% phosphoric acid solution (B), and gradient elution is as follows: A: 0-15 min, 16%-20%; 15-28 min, 20%-25%; 28-54 min, 25%-58%. The detection wavelength was 260 nm; the column temperature was 35 ℃; the flow rate was 0.8 mL/min, and the injection volume was 20 L. The theoretical number of plates was not less than 4 000, and the HPLC chromatogram is shown in Fig.1.

Note: 1. Strychnine; 2. Brucine; 3. Paeoniflorin; 4. Calycosin; 5. Ferulic acid; 6. Hesperidin; 7. Salvianolic acid B.

3.3.2Preparation of reference solution. Strychnine, brucine, paeoniflorin, calycosin, ferulic acid, hesperidin, salvianolic acid B reference substances were weighed accurately in appropriate amounts, to which methanol was added until constant volume, thereby making prepare mixed reference solutions with mass fractions of 2.69, 1.93, 4.30, 1.12, 0.78, 5.38, 1.58 mg/mL.

3.3.3Preparation of testing sample solution. The dosage of medicine in each test was 4.0 kg, and Tongmai pills were prepared according to the process parameters of preparing water pills in the preparation room of the hospital. By using the paddle method, 900 mL ultrapure water treated with ultrasound for 30 min was used as the dissolution medium; the temperature was (37±0.5) ℃, and the speed was 100 r/min. After the dissolution medium was kept warm for 30 min, about 15 g of Tongmai pills were weighed and placed in a dissolution cup. Afterwards,10 mL of the sample was taken at 2, 5, 10, 20, 30, 40, 60, 120, 240, and 1 440 min after administration, and the same volume of dissolution medium at the same temperature was replenished immediately. After filtering, the filtered residue was placed in a dissolution cup, and the filtrate was stored for later use. 3 mL of the dissolved solution was taken precisely and placed in a 10 mL measuring flask, to which methanol was added until constant volume. After filtering, it was detected.

3.3.4Linear relationship investigation. Methanol was added to the mixed reference solution in Section3.3.2to prepare the mixed reference solutions with different mass concentrations. 20 μL of each of the solutions was accurately taken and detected according to the chromatographic conditions in Section3.3.1. Linear regression with reference concentrationX(μg/mL) and peak areaYwas performed, and the results are shown in Table 2. The 7 compounds had a good linear relationship within their concentration ranges.

Table 2 Linear relationship of the seven index components

3.3.5Precision test. The solution dissolving for 4 h was injected 6 times under the chromatographic conditions in Section3.3.1, and theRSDvalues of the peak area of strychnine, brucine, paeoniflorin, calycosin, ferulic acid, hesperidin and salvianolic acid B were calculated as follows: 1.7%, 1.8%, 1.1%, 1.7%, 1.9%, 2.0%, and 1.7%. The result shows that the precision was good.

3.3.6Stability test. The solution dissolving for 4 h was injected under the chromatographic conditions in Section3.3.1after 0, 2, 4, 6, 12, and 24 h. TheRSDvalues of the peak area of strychnine, brucine, paeoniflorin, calycosin, ferulic acid, hesperidin and salvianolic acid B were 1.8%, 1.9%, 1.7%, 1.5%, 1.8%, 1.0%, and 1.7%. The result shows that the 7 components were stable within 24 h.

3.3.7Repeatability test. Firstly, 6 parts of Tongmai pills were weighed, and dissolution solutions were prepared according to the method in Section3.3.3. Afterwards, the samples were detected under the chromatographic conditions in Section3.3.1. TheRSDvalues of the peak area of strychnine, brucine, paeoniflorin, calycosin, ferulic acid, hesperidin and salvianolic acid B were 1.5%, 1.2%, 1.2%, 1.4%, 1.3%, 1.3%, and 1.5%, respectively. The result shows that the method had good repeatability.

3.3.8Sample recovery test. Firstly, 6 parts of the sample dissolution solution with known content were added to the mixed reference solution corresponding to the sample content, and they were measured under the chromatographic conditions in Section3.3.1. The results show that the average recovery rate of strychnine, brucine, paeoniflorin, calycosin, ferulic acid, hesperidin and salvianolic acid B was 101.4%, 100.2%, 100.1%, 100.8%, 99.1%, 101.3%, and 100.8%, respectively, and theRSDvalues were 1.6%, 1.6%, 1.8%, 1.8%, 1.6%, 2.0%, and 1.2%, respectively.

3.4 Determination of total flavonoids in the dissolution solution by ultraviolet spectrophotometry

3.4.1Preparation of reference solution. At first, 11.45 mg of the rutin reference substance was accurately weighed and placed in a 50 mL volumetric flask, to which an appropriate amount of methanol was added. Afterwards, the flask was heated in a water bath to dissolve the reference substance. After it was cool, methanol was added to the mark, and it was shaken well to get 0.229 mg/mL rutin reference substance solution.

3.4.2Linear relationship investigation. Firstly, 0.25, 1.0, 2.0, 4.0, 8.0, 12.0 mL of the rutin reference substance solution were taken precisely and placed in a 25 mL volumetric flask, to which 2 mL of 5% sodium nitrite solution was added. Aftering shaking well, it was placed for 6 min, to which 2 mL of 10% aluminum nitrate solution. Aftering shaking well, it was placed for 6 min again, to which 10 mL of 4% sodium hydroxide solution was added. Afterwards, water was added to it until reaching the mark. It was shaken well and then placed for 15 min. Corresponding reagent was as the blank, and absorbance A was detected at 500 nm by ultraviolet spectrophotometry. Matrix Laboratory Software (Matlab) curve fitting toolbox was used to perform linear regression of absorbance A and reference substance concentrationC(μg/mL), and the linear equation was obtained as follows:A=0.011 3C+0.010 4 (r2=0.999 6). When the concentration of the rutin reference substance was 2.29-109.92 μg/mL, it had a good linear relationship with absorbance.

3.4.3Methodology review. The same test solution was taken to perform precision, stability, repeatability and sample recovery tests according to the method in Section3.4.2. The results show that theRSDvalues of total flavonoid absorbance in the precision, stability, and repeatability tests were 1.5%, 1.6%, and 1.8%, respectively; the average recovery rate was 101.1%, and theRSDvalue was 1.7%. The results show that the methodological investigation met the requirements of the pharmacopoeia.

3.5 Evaluation of the influence of material ratio on the dissolution of Tongmai pills

3.5.1Analysis of the solubility of test indicators. Since the detected components were all the components of the material itself, its dissolution consisted of several processes of pill disintegration-penetration-dissolution-diffusion. The solubility of index components in water has become one of the main factors limiting the dissolution. Therefore, in accordance with the classification criteria for solubility in the 2015 edition of theChinesePharmacopoeia, the solubility of detected indicators was classified. The results show that paeoniflorin, salvianolic acid B, and calycosin were soluble components, and ferulic acid was classified as a slightly soluble ingredient; total flavonoids, strychnine, and brucine were very slightly soluble components, and hesperidin is almost insoluble.

3.5.2Determination of weight coefficient by analytic hierarchy process. According to the solubility classification of the detection index components, Analytic Hierarchy Process (AHP) was applied to the determination of the weight coefficient of index components[10]. Firstly, the judgment priority matrix for pairwise comparison was constructed, and the weight coefficient was calculated with the help of Matlab software programming. According to the results, the weighting coefficient of salvianolic acid B, calycosin and paeoniflorin were 0.212 1, and the weighting coefficient of ferulic acid was 0.121 1; the weighting coefficient of strychnine, brucine, and total flavonoids was 0.063 9; the weighting coefficient of hesperidin was 0.050 9. The random consistency ratio CR was calculated,CR=0.005 7<0.10, indicating that the weight calculation was correct and reasonable.

3.5.3Optimization of dissolution models and parameter estimation. The 24-h dissolution rate was 100%, and the cumulative dissolution rate after comprehensive evaluation was calculated according to the weight coefficient determined by the analytic hierarchy process. By using the statistical analysis system software (SAS) to write a program, 6 kinds of dissolution models were fitted, and determining coefficient (r2) and Akaike’s information criterion (AIC) were used to evaluate the fitting effect of dissolution models[11]. The results show that the Weibull distribution model was the optimal dissolution model. The timeT40required for 40% dissolution was calculated at the same time (Table 3).

Table 3 Estimated parameters of the optimal dissolution model and dissolution parameters (n=6)

3.5.4Quantitative characterization of the influence of material properties on dissolution. In order to quantificationally characterize the effect of three Chinese medicine material ratio on the dissolution of Tongmai pills, the stepwise multiple regression method (SMR) in Matlab software and the Stepwise (X,Y, inmodel, penter, premove) command were used to establish the following mathematical model between dissolution parameterT40and material ratioXn:T40=8.93-5.31X1-5.83X2+17.67X1X3, whereX1,X2, andX3are the proportion of fiber, starch, grease-resin materials in the prescription of Tongmai pills. The results show thatr2=0.970 6,P=0.01, and root mean square error (RMSE) was 0.569 6.

From the mathematical model, it can be seen that fiber materials could significantly promot the dissolution (P<0.05), and starch materials could also promote the dissolution, but it was not significant (P=0.05); the interaction term between fiber and grease-resin inhibited the dissolution, but it was not significant (P>0.05).

3.5.5Quantitative characterization between two types of materials and dissolution parameter. In order to investigate the influence of two types of materials on dissolution parameterT40, the SMR method in the Matlab software was used for stepwise regression, and the following mathematical model was obtained:T40=12.02-5.43X1-10.22X2(r2=0.893 2，P=0.01，RMSE=0.940 2)；T40=3.29+5.38X3+19.20X1X3(r2=0.969 8，P=0.000 9，RMSE=0.500 2);T40=3.03+26.50X3-21.89-18.65X2X3(r2=0.973 1,P=0.01,RMSE=0.544 9). At the same time, a three-dimensional graph between the two types of materials and the dissolution parameter was drawn (Fig.2).

Fig.2 Three dimensional relationship between fiber, starch, grease-resin and T40

From the analysis of the mathematical model and the three-dimensional relationship diagram between the two types of materials andT40, it can be seen that fiber and starch materials had the effect of promoting dissolution (P<0.05), and grease-resin had a significant effect of inhibiting dissolution (P=0.005 2< 0.01).

4 Discussion

4.1 Model drug selectionThe prescription of Tongmai pills contains 12 medicines such as Huangqi (sovereign drug), Zhifuzi (sovereign drug), Danggui (ministerial drug), Danshen (ministerial drug), and Chishao (ministerial drug). In this experiment, Tongmai pills were selected as the model drugs, and the dissolution differences of components in Huangqi, Danshen, Chishao, Danggui and toxic drug Maqianzi in the prescription were compared to discuss the effect of different material properties on dissolution.

4.2 Analysis of the influence of material ratio on dissolution time limitMaterial ratioXnwas used as the independent variable, and the disintegration time limitYwas the dependent variable to establish a mathematical modelY=5.57+2.53X1+20.47X3(X1is the proportion of fiber materials, andX3is the proportion of grease-resin materials,r2=0.806 5,P=0.04) by Matlab software. Analysis of the linear model shows that fiber and grease-resin materials inhibited disintegration; starch materials were not significantly correlated with the disintegration time limit and were eliminated. Grease-resin materials were relatively viscous and often inhibited the disintegration of the pellets, thus inhibiting their dissolution. For the starch and fiber contained in the prescription, countless irregular capillaries and pores can be formed inside the pills. These capillaries and pores were not only the main channels for water to enter the pills, but also exhibited low interfacial tension in the aqueous medium. The fiber, starch,etc.in the pills absorbed water to swell, so that the inside of the pills was loose and dissolved. After absorbing water and fully swelling, the drug components dissolved through these capillaries and pores[12]. The process was more "longer" than disintegration, and there were more influencing factors.

4.3 Choice of experimental design methodIn order to investigate the influence of material properties on dissolution, it is necessary to use mathematical and statistical methods to design experiments and find the quantitative relationship between the two. In the selection of experimental design, different experimental design methods such as orthogonal experimental design, uniform experimental design, effect surface design, and uniform design of mixture were compared. The uniform design of mixture has fewer experiments, and can avoid the phenomenon that the total dosage of each drug determined by the orthogonal design method and the uniform design method in the formulation optimization was not constant, so it was used to arrange the experiment.

There are many factors affecting the dissolution of pills[13], and material parameters will also affect the dissolution to a certain extent[3]. Therefore, the influence of three different material ratios on the dissolution of Tongmai pills were studied in this experiment, and the experimental results had certain limitations. In the later research, research on the influence of material parameters on dissolution should be carried out, and the evaluation of material parameters and material ratios will be stuided to realize the transformation of research from model drugs to specific drugs, further promoting the in-depth research on the influence of Chinese medicine materials oninvivoandinvitrodissolution.