An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

요약

Here, we test the dissolution of Rhodiola granules (RG) in vitro, draw dissolution curves of salidroside, gallic acid, and ethyl gallate in ultrapure water, and fit the curves to different mathematical models. This protocol provides information and guidance for in vivo bioequivalence and in vivo-in vitro correlation studies of RG.

초록

The composition of the Tibetan medicine Rhodiola granules (RG) is complex, and the overall quality of RG is difficult to determine. Therefore, establishing a method to determine the multi-component in vitro dissolution of RG is of great significance for quality control. This study uses the second paddle method of the fourth general rule 0931 from the Chinese Pharmacopoeia (2020 edition), compliant with apparatus 2 of the United States Pharmacopeia (USP). The dissolution apparatus was set to a rotation speed of 100 rpm with ultrapure water as the dissolution medium. A sample volume of 1 mL was collected at each timepoint. Furthermore, the cumulative dissolution of gallic acid, salidroside, and ethyl gallic acid in RG at different time points was determined by high-performance liquid chromatography (HPLC). Finally, the dissolution curves were drawn, and the curves were fitted to the GompertzMod, the Gompertz, the Logistic, and the Weibull equations. The results showed that the cumulative dissolution of gallic acid in RG was over 80% at 1 min, the cumulative dissolution of salidroside and ethyl gallic acid was over 65% at 5 min, and the cumulative dissolution of each index component decreased after 30 min. The curve fitting demonstrated that the GompertzMod equation was the best-fitting model for each index component of RG. In conclusion, the dissolution test method described in this protocol is simple, accurate, and reliable. It can characterize the dissolution behavior of the index components in RG in vitro, which provides a methodological reference for quality control of RG and quality evaluation of other ethnic compounds.

서문

In China, the prevalence of cardiovascular diseases continues to rise, and the morbidity and mortality rates of cardiovascular diseases rank first among Chinese residents¹. Angina pectoris of coronary heart disease is caused by luminal stenosis due to coronary atherosclerosis, which leads to relatively insufficient coronary blood supply and myocardial ischemia and hypoxia². In recent years, the curative effect of traditional Chinese medicine in the treatment of coronary heart disease has been recognized by many doctors³.

Traditional Chinese medicine plays an important role in alleviating clinical symptoms and improving the quality of life of patients⁴. Rhodiola granules (RG) are extracted and refined from the Tibetan Plateau medicinal plant Rhodiola rosea L. The main components of RG are salidroside, rhodiosin, and flavonoids^5,6. RG has the effect of supplementing Qi⁷ and activating and promoting blood circulation to relieve pain. Clinically, it is used to treat chest obstructions caused by Qi deficiency and blood stasis, coronary heart disease, angina pectoris⁸. Content determination alone does not fully reflect the intrinsic quality of the drugs, as both the disintegration and dissolution in vitro can affect the bioavailability and efficacy of the drugs^9,10. The inspection methods for the dissolution of Chinese medicine include the rotating basket method, the paddle method, and the small cup method. The disadvantage of the rotating basket method is that only the outer part of the rotating basket comes in contact with the dissolution medium during rotation, which does not reflect the real-world dissolution behavior. The paddle method can overcome the above shortcoming, which makes it more suitable than the basket method for some solid Chinese medicine preparations¹¹. At present, there is no report on the in vitro dissolution analysis of RG. In order to control the quality of RG more comprehensively, the dissolution behavior of the three index components (gallic acid, salidroside, and ethyl gallate) in RG was investigated. This study provides data for the quality control of RG and a methodological reference for quality evaluation of other ethnic compound preparations.

프로토콜

1. Solution preparation

1. Prepare the reference substance stock solution: Weigh 10.6 mg of salidroside, 5.24 mg of gallic acid, and 5.21 mg of ethyl gallic acid separately on an electronic analytical balance and add them individually into a 5 mL volumetric flask. Then, add HPLC-grade methanol to dissolve and dilute to 5 mL. Finally, shake well to obtain the reference substance stock solution with mass concentrations of 2.120 mg/mL, 1.048 mg/mL, and 1.042 mg/mL, respectively.
   NOTE: The reference substance stock solution contains 2.120 mg/mL salidroside, 1.048 mg/mL gallic acid, and 1.042 mg/mL ethyl gallate as the stock solution of each solution in the subsequent standard curve.
2. Prepare the test sample solution. Extract 2.8 g of RG (Table of Materials) with 10 mL of HPLC-grade methanol using an ultrasonic cleansing machine (Power: 200 W, frequency: 40 kHz) for 30 min, and then filter it with a 0.22 µm filter for the system adaptability test.
3. Prepare a mixed reference solution that contains 0.590 mg/mL salidroside, 2.030 mg/mL gallic acid, and 1.930 mg/mL ethyl gallate.
   NOTE: Each standard (2.950 mg of salidroside, 10.150 mg of gallic acid, and 9.650 mg of ethyl gallic acid) is dissolved in a 5 mL volumetric flask in HPLC-grade methanol as the dissolution medium.
4. Obtain the theoretical content of each characteristic component of RG for ultrapure water extraction.
   1. Place 2.8 g of RG in a 500 mL conical flask, add 200 mL of ultrapure water, and ultrasonically extract (Power: 200 W, frequency: 40 kHz) for 60 min. Then, filter it with a 0.22 µm filter.
   2. Determine the content of the test solution according to the linear equation obtained in the following experiment.

2. Chromatographic condition

1. Set the chromatographic conditions as shown in Table 1 for high-performance liquid chromatography. For details about the instrument used, refer to the Table of Materials.

3. System adaptability test

1. Investigate the linear relationship.
   1. Dilute the reference stock solutions of gallic acid and ethyl gallate by 5, 10, 25, 50, and 125 times, and the reference stock solutions of salidroside by 2, 4, 8, 16, and 32 times to obtain the gradient concentration solution for drawing a standard curve.
      NOTE: Adjust the dilution ratio of the standard curve according to the preliminary experiment of the sample treatment. In the preliminary experiment, the stock solutions of the three standards were first diluted 5, 10, 25, 50, and 125 times, and then the first standard curve was plotted. However, when the concentration of the test sample was detected, it was found that the concentrations of salidroside did not fall within the linear range of this standard curve and, therefore, the concentrations were adjusted to include them in the curve. In summary, the above preliminary experiments were used to determine the final dilution concentrations of the three test samples for subsequent experimental studies.
2. Precision testing: Inject 10 µL of the mixed reference solution into the HPLC system six times daily and run the samples with the same HPLC conditions described in step 2.1. Record the peak area of each feature component.
3. Stability testing experiments: Inject 10 µL of the prepared sample solution and determine the peak areas of the HPLC according to the chromatographic conditions after 0 h, 6 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, and 24 h, respectively.
   NOTE: The peak areas are recorded automatically by the HPLC system.
4. Reproducibility test: Take six samples of the same batch of RG to prepare the test sample solution according to the method in step 1.2. Inject 10 µL of each sample into the HPLC system. Run the samples as described in step 2.1 and determine reproducibility.
   NOTE: Repeatability was evaluated by comparing the concentration differences between the six samples.
5. Recovery experiment
   1. Prepare six portions of the same batch of RG for the test solution. Then, add about 50% of the reference substance of each index component in the test solution to calculate the recovery rate. Run these samples in the HPLC system with the same conditions described in step 2.1.
   2. Calculate the recovery rate.
      ​NOTE: Recovery rate = (C - A) / B x 100, where A is the amount of component to be measured in the test solution, B is the amount of reference substance added, and C is the measured value of the solution containing the reference substance and the RG sample. Refer to step 2.1 for the chromatographic conditions to perform the above steps (i.e., steps 3.1-3.5).

4. In vitro dissolution test

1. Carry out the dissolution test using the paddle method of the second method of general rule 0931 from Chinese Pharmacopoeia (2020 edition)¹².
   NOTE: Sampling technique and equipment: The drug dissolution apparatus (Table of Materials) has a dissolution cup, a paddle, a temperature control system, and a speed adjustment system. Before starting the dissolution experiment, the water is preheated to a set temperature, and then the corresponding speed is set. Start recording the time immediately after adding RG.
2. Add 100 mL of ultrapure water into the dissolution cup of the drug dissolution apparatus and maintain the temperature at 37 °C ± 0.5 °C. Set the rotation speed to 100 rpm.
   NOTE: The dissolution apparatus has a heating device that allows the temperature to be set within the system. There was no significant difference in the dissolution rate of salidroside in water, artificial gastric juice (16.4 mL of diluted hydrochloric acid [234 mL of concentrated hydrochloric acid diluted to 1000 mL with water] with about 800 mL of water and 10 g of pepsin, well shaken, and diluted with water to 1,000 mL), and artificial intestinal juice (phosphate buffer [pH 6.8] containing trypsin)¹³. The most readily available water (ultrapure) was selected as the dissolution medium.
3. Add 2.8 g of RG into a dissolution cup and start recording the duration of dissolution immediately. Collect a total of 1 mL of the sample with an injector (see Table of Materials) at 1 min, 5 min, 10 min, 20 min, 30 min, and 60 min, and make up the volume in the dissolution cup with the dissolution medium at the same temperature immediately.
   NOTE: The sampling tube in the dissolution cup cannot collect small sample volumes, so the injector is used to collect the sample. Samples must be collected quickly to avoid missing specified collection time points.
4. Immediately filter the collected samples through a 0.22 µm microporous membrane and take the subsequent filtrate. Determine the content of each component at each time point by HPLC (as per step 2.1) and calculate the cumulative dissolution.
   1. To calculate the cumulative dissolution, calculate the dissolution of each time point (X_n):
      X_n = A / B x 100, where A is the quantity of components measured at each time point and B is the theoretical content of each component.
   2. Then, calculate the cumulative dissolution (Y):
      Y = X_n + (X₁ + ... + X_n-1) x V₂ / V₁, where V₁ is the total volume of the dissolution medium and V₂ is the volume of solute added after each sampling.
      ​NOTE: Due to the low response values of salidroside and gallic acid in the chromatogram, the cumulative dissolution of salidroside and ethyl gallate at the 1 min time point was not plotted in the dissolution curve.

5. Fitting the dissolution model

1. Import the cumulative dissolution data at each time point into the data analysis software.
2. Use the drug dissolution analysis plug-in in the data analysis software to fit the GompertzMod equation, the Gompertz equation, the Logistic equation, and the Weibull equation¹⁴. The larger the value of R², the better the curve-fitting effect is.
   1. Start the software, select the Book1 window to enter the Origin Data Editing window.
   2. In the first column A(X)-Long Name input Time, define Time as time, and input each dissolution determination time. Input Data in the second column B(Y)-Long Name, define Data as the cumulative dissolution, input the cumulative dissolution percentage of each dissolution determination time.
   3. After data input, select the column A(X) and B(Y), and select the Drug Dissolution Analysis plug-in in the software menu bar and click on Fit Dissolution Data > Concatenate Fit > OK. The software generates the fitting results of each model.

결과

In this study, the precision, stability, repeatability, and sample recovery of RG were all within the methodological range specified in Chinese Pharmacopoeia (Volume 4, 2020)¹², indicating that the method was feasible. After repeated debugging, it was determined that the elution gradient used in this study had good resolution (Figure 1) for the three index components in RG. The three index components in RG had a good linear relationship within a specific concentration...

토론

The dissolution test is an ideal in vitro method to simulate the disintegration and dissolution of solid oral preparations in the gastrointestinal tract¹⁵. It is an important index for evaluating and controlling the quality of solid oral preparations. Therefore, the dissolution test plays an essential role in the development of solid drug oral preparations¹⁶. In particular, with the development of traditional Chinese medicine (TCM) quality control technology, the d...

자료

Name	Company	Catalog Number	Comments
Chromatographic column	ZORBAX Eclipse	XDB-C18	4.6 mm x 250 mm, 5 µm
Drug dissolution tester	Shanghai Huanghai Pharmaceutical Inspection Instrument Co., Ltd.	RCZ-6B3
Electronic analytical balance	Shanghai Liangping Instruments Co., Ltd.	FA1004
Ethyl gallate (HPLC, ≥98%)	Chengdu Desite Biotechnology Co., Ltd.	DSTDM006301
Function drawing software	OriginLab Corporation, Northampton, MA, USA	2022
Gallic acid (HPLC, ≥98%)	Chengdu Desite Biotechnology Co., Ltd.	DSTDM000802
High performance liquid chromatography	Agilent Technologies Singapore (International) Pte. Ltd.	Agilent 1260 Infinity
HPLC grade methanol	Thermo Fisher Scientific (China) Co., Ltd.	216565
Injector	Chengdu Xinjin Shifeng Medical Apparatus & Instrument Co., Ltd.	0.7 (22 G)
Millipore filter	Tianjin Jinteng Experimental Equipment Co., Ltd	φ13 0.22 Nylon66
Rhodiola granules	Tibet Nodikang Pharmaceutical Co., Ltd.	210501
Salidroside (HPLC, ≥98%)	Chengdu Desite Biotechnology Co., Ltd.	DST200425-037
Ultra pure water systemic	Merck Millipore Ltd.	Milli-Q
Ultrasonic cleansing machine	Ningbo Xinyi Ultrasonic Equipment Co., Ltd	SB-8200 DTS