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.
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.
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 flavonoids5,6. 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 drugs9,10. 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.
1. Solution preparation
2. Chromatographic condition
3. System adaptability test
4. In vitro dissolution test
5. Fitting the dissolution model
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 range (Table 2). The precision test results (Table 3) showed that the relative standard deviation (RSD) of the peak areas of salidroside, gallic acid, and ethyl gallate were 1.95%, 2.83%, and 1.42%, respectively, indicating that the precision of the instrument was good. The stability test results (Table 4) showed that the RSD of the peak areas of salidroside, gallic acid, and ethyl gallate were 2.37%, 2.47%, and 2.82%, respectively, suggesting that the sample solution was stable within 24 h. The repeatability test results (Table 5) showed that the RSDs of the peak areas of salidroside, gallic acid, and ethyl gallate were 2.79%, 2.67%, and 1.55%, respectively, showing that the repeatability of this method was good. The recovery experiment results indicated the average recoveries of salidroside, gallic acid, and ethyl gallate were 99.91%, 100.40%, and 102.80%, respectively (Table 6).
The in vitro dissolution experiment in this study was to determine the content of three characteristic components (salidroside, gallic acid, and ethyl gallate) in RG samples at each time point by HPLC, and then calculate the cumulative dissolution. The dissolution curves of each component are shown in Figure 2. After the sample was put into the dissolution cup, the cumulative dissolution of gallic acid in RG was over 80% after 1 min. The cumulative dissolution of salidroside and ethyl gallic acid was over 65% after 5 min, which was reflected in the data that each index component could dissolve over 60% after 5 min. However, the cumulative dissolution of each index component decreased after 30 min. Further, the dissolution curves were fitted to the GompertzMod equation, the Gompertz equation, the Logistic equation, and the Weibull equation. The results showed that the GompertzMod equation was the best-fitting model for the three index components (salidroside, gallic acid, and ethyl gallate) in RG. The dissolution model fitting results of three index components in RG are shown in Table 7.
Figure 1: Representative chromatograms of the three characteristic components after setting the chromatographic conditions mentioned in step 2.1 (n = 1). (A) The chromatogram of the sample solution. Peak 1 is gallic acid, peak 2 is salidroside, and peak 3 is ethyl gallate. (B) The chromatogram of the reference solution. Peak 1 is gallic acid, peak 2 is salidroside, and peak 3 is ethyl gallate. Please click here to view a larger version of this figure.
Figure 2: Dissolution curve of characteristic components (n = 4). (A) Cumulative dissolution of gallic acid at 1 min, 5 min, 10 min, 20 min, 30 min, and 60 min after administration. (B) Cumulative dissolution of salidroside at 5 min, 10 min, 20 min, 30 min, and 60 min after administration. (C) Cumulative dissolution of ethyl gallate at 5 min, 10 min, 20 min, 30 min, and 60 min after administration. Please click here to view a larger version of this figure.
Condition | Parameter |
Chromatographic column | C18 (4.6 mm x 250 mm, 5 µm) |
Mobile phase | Acetonitrile (A)-0.2% Acetic acid (B) |
Gradient elution | 0–5min, 0%–4%A; 5–15min, 4%–5%A; 15–20min, 5%–7%A; 20–30min, 7%–14%A; 30–40min, 14%–13%A; 40–45min, 13%–4%A |
Flow rate | 1.0 mL/min |
Column temperature | 30 °C |
Detecting wavelength | 275 nm |
Sample volume | 10 μL |
Table 1: The chromatographic conditions set in this experiment. The table lists the details of the chromatographic column, the mobile phase, the gradient elution, the flow rate, the column temperature, the detection wavelength, and the sample volume.
Index components | Linear equation | R2 | Range of linearity (mg/mL) |
Salidroside | Y = 2221X – 19.742 | 0.9996 | 0.06625–2.12 |
Gallic acid | Y = 29497X – 224 | 0.9997 | 0.008384–1.048 |
Ethyl gallate | Y = 28902X – 86.171 | 0.9999 | 0.008336–1.042 |
Table 2: The linear relationship of the index components in RG. The three index components in RG had a good linear relationship in a specific concentration range.
Peak area of index components | 1 | 2 | 3 | 4 | 5 | 6 | RSD % |
Salidroside | 900.6 | 917.4 | 899.8 | 917.4 | 940.1 | 890.5 | 1.95 |
Gallic acid | 6430.2 | 6544.2 | 6281.2 | 6327.7 | 6142.5 | 6636.9 | 2.83 |
Ethyl gallate | 12748.9 | 12833.1 | 13190.4 | 13152.3 | 13128.3 | 13090.5 | 1.42 |
Table 3: The results of the precision measurement. The RSD of the peak areas of salidroside, gallic acid, and ethyl gallate were 1.95%, 2.83%, and 1.42% (n = 6).
Peak area of index components | 0 h | 6 h | 12 h | 18 h | 21 h | 24 h | RSD % |
Salidroside | 486.6 | 509 | 479 | 505.1 | 502.8 | 492 | 2.37 |
Gallic acid | 3236.5 | 3359.8 | 3152.2 | 3347.6 | 3337 | 3319.9 | 2.47 |
Ethyl gallate | 442 | 413 | 421 | 429 | 443.8 | 436 | 2.82 |
Table 4: The results of the stability test. The RSD of the peak areas of salidroside, gallic acid, and ethyl gallate were 2.37%, 2.47%, and 2.82% (n = 6).
Peak area of index components | 1 | 2 | 3 | 4 | 5 | 6 | RSD % |
Salidroside | 1337.3 | 1276.5 | 1283.7 | 1286.8 | 1242.6 | 1237.2 | 2.83 |
Gallic acid | 8432.1 | 8976.1 | 8792 | 9083.1 | 9040.2 | 8751.4 | 2.74 |
Ethyl gallate | 422.8 | 415.3 | 421.9 | 416.3 | 428.9 | 406.1 | 1.87 |
Table 5: The results of the reproducibility test. The RSD of the peak areas of salidroside, gallic acid, and ethyl gallate were 2.83%, 2.74%, and 1.87% (n = 6).
Known content (mg) | Adding quantity (mg) | Measuring quantity (mg) | Recoveries (%) | Average recoveries (%) | RSD (%) |
0.5838 | 0.406 | 0.9783 | 97.18 | 99.91 | 2.70 |
0.5743 | 0.406 | 0.9984 | 104.47 | ||
0.5751 | 0.406 | 0.9755 | 98.63 | ||
0.5764 | 0.406 | 0.9776 | 98.81 | ||
0.5906 | 0.406 | 0.991 | 98.6 | ||
0.5802 | 0.406 | 0.9934 | 101.77 | ||
0.1234 | 0.118 | 0.2424 | 100.87 | 100.4 | 1.67 |
0.1214 | 0.118 | 0.2428 | 102.85 | ||
0.1216 | 0.118 | 0.2396 | 100 | ||
0.1218 | 0.118 | 0.2389 | 99.19 | ||
0.1249 | 0.118 | 0.2406 | 98.09 | ||
0.1226 | 0.118 | 0.2423 | 101.4 | ||
0.0221 | 0.386 | 0.4232 | 103.91 | 103.8 | 2.02 |
0.0218 | 0.386 | 0.4115 | 100.97 | ||
0.0218 | 0.386 | 0.4176 | 102.55 | ||
0.0218 | 0.386 | 0.4337 | 106.7 | ||
0.0224 | 0.386 | 0.4302 | 105.65 | ||
0.022 | 0.386 | 0.4198 | 103.05 |
Table 6: The results of the sample recovery rate measurement. The RSD of the recovery rate of salidroside, gallic acid, and ethyl gallate were 2.70%, 1.67%, and 2.02%, respectively.
Index components | Dissolution equation | R2 |
Gallic acid | GompertzMod | 0.4978 |
Gompertz | 0.3740 | |
Logistic | 0.3739 | |
Weibull | 0.3739 | |
Salidroside | GompertzMod | 0.9894 |
Gompertz | 0.9783 | |
Logistic | 0.9781 | |
Weibull | 0.9781 | |
Ethyl gallate | GompertzMod | 0.9895 |
Gompertz | 0.9852 | |
Logistic | 0.9853 | |
Weibull | 0.9853 |
Table 7: Curve-fitting results of the dissolution model of three index components in ultrapure water. The fitting results of each index component of RG were the best with the GompertzMod equation.
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 determination of dissolution has been gradually applied to the screening studies of Chinese and ethnic medicine compound preparations17,18.
Currently, the determination of the dissolution of TCM and ethnic medicine in vitro is mainly based on detecting a single index component. However, the solid preparation of traditional Chinese medicine and ethnic medicine is a complex, and their dissolution is affected by many factors (e.g., temperature, dissolution medium, etc.) and their complex chemical composition19,20. Therefore, detection of multi-index components can better reflect the mutual influence and dissolution difference of different components. In this paper, the in vitro dissolution test of the three index components (gallic acid, salidroside, and ethyl gallate) in RG was measured, and the dissolution curves of these three characteristic components were plotted, which provided a reference for the quality control of RG.
During the experiment, the following two points should be particularly noted. Firstly, when sampling for the dissolution test according to the Chinese Pharmacopoeia 2020 edition12, an equal volume of dissolution medium at a temperature of 37 °C ± 0.5 °C should be replenished immediately after sample collection, which is the key step in the experimental process. Secondly, the samples should be collected from an area midway between the top of the blade and the surface of the dissolution medium, ~10 mm from the inner wall of the dissolution cup. This is because there is a concentration gradient from the start of the dissolution of the drug to the time of complete dissolution. The concentration gradient is inversely proportional to the stirring speed, so the dissolved drug concentration is highest near the undissolved drug and the lowest where the stirring is weak. Therefore, sampling at these two extremes should be avoided21.
Although the detection of multi-index components can better reflect the dissolution variation of different components of TCM/ethnic medicine compounded formulations compared to the detection of single-index components, it has certain limitations. There is the potential of human error when using a syringe to collect the samples. The precision and accuracy of the measurement can be improved if automatic drug dissolution measurements can be implemented22.
In summary, we have established an in vitro dissolution method for determining multi-index components in RG, which provides a basis for further studies of RG. This experiment can provide information and guidance for in vivo bioequivalence studies and in vivo-in vitro correlation studies of other ethnic medicines23.
The authors have nothing to disclose.
This work was funded by the National Key Research and Development Program of China (2017YFC1703904), the University (Chengdu University of TCM) – enterprise (Tibet Rhodiola Pharmaceutical Holding Co. LTD) cooperation project (1052022040101); the Regional Innovation and Cooperation Project of the Science & Technology Department of Sichuan Province (2020YFQ0032); and the Key R&D and Transformation Program of the Science & Technology Department of Qinghai Province (2020-SF-C33).
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 |