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 &#38; Technology Department of Sichuan Province (2020YFQ0032); and the Key R&#38;D and Transformation Program of the Science &#38; Technology Department of Qinghai Province (2020-SF-C33).

1. Solution preparation
<ol>
	<li>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.</li>
	<li>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 &#181;m filter for the system adaptability test.</li>
	<li>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.</li>
	<li>Obtain the theoretical content of each characteristic component of RG for ultrapure water extraction.
	<ol>
		<li>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 &#181;m filter.</li>
		<li>Determine the content of the test solution according to the linear equation obtained in the following experiment.</li>
	</ol>
	</li>
</ol>
2. Chromatographic condition
<ol>
	<li>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.</li>
</ol>
3. System adaptability test
<ol>
	<li>Investigate the linear relationship.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Precision testing: Inject 10 &#181;L 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.</li>
	<li>Stability testing experiments: Inject 10 &#181;L 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.</li>
	<li>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 &#181;L 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.</li>
	<li>Recovery experiment
	<ol>
		<li>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.</li>
		<li>Calculate the recovery rate. 
		&#8203;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).</li>
	</ol>
	</li>
</ol>
4. In vitro dissolution test
<ol>
	<li>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.</li>
	<li>Add 100 mL of ultrapure water into the dissolution cup of the drug dissolution apparatus and maintain the temperature at 37 &#176;C &#177; 0.5 &#176;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.</li>
	<li>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.</li>
	<li>Immediately filter the collected samples through a 0.22 &#181;m 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.
	<ol>
		<li>To calculate the cumulative dissolution, calculate the dissolution of each time point (Xn): 
		Xn = 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.</li>
		<li>Then, calculate the cumulative dissolution (Y): 
		Y = Xn + (X1 + ... + Xn-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. 
		&#8203;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.</li>
	</ol>
	</li>
</ol>
5. Fitting the dissolution model
<ol>
	<li>Import the cumulative dissolution data at each time point into the data analysis software.</li>
	<li>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.
	<ol>
		<li>Start the software, select the Book1 window to enter the Origin Data Editing window.</li>
		<li>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.</li>
		<li>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 &#62; Concatenate Fit &#62; OK. The software generates the fitting results of each model.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 &#176;C &plusmn; 0.5 &#176;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.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chromatographic column</td>
			<td>ZORBAX Eclipse&#160;&#160;</td>
			<td>XDB-C18</td>
			<td>4.6 mm x 250 mm, 5 &#181;m</td>
		</tr>
		<tr>
			<td>Drug dissolution tester</td>
			<td>Shanghai Huanghai Pharmaceutical Inspection Instrument Co., Ltd.</td>
			<td>RCZ-6B3</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic analytical balance</td>
			<td>Shanghai Liangping Instruments Co., Ltd.</td>
			<td>FA1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl gallate (HPLC, &#8805;98%)</td>
			<td>Chengdu Desite Biotechnology Co., Ltd.</td>
			<td>DSTDM006301</td>
			<td></td>
		</tr>
		<tr>
			<td>Function drawing software</td>
			<td>OriginLab Corporation, Northampton, MA, USA</td>
			<td>2022</td>
			<td></td>
		</tr>
		<tr>
			<td>Gallic acid (HPLC, &#8805;98%)</td>
			<td>Chengdu Desite Biotechnology Co., Ltd.</td>
			<td>DSTDM000802</td>
			<td></td>
		</tr>
		<tr>
			<td>High performance liquid chromatography</td>
			<td>Agilent Technologies Singapore (International) Pte. Ltd.</td>
			<td>Agilent 1260 Infinity <img alt="Equation 1" src="/files/ftp_upload/64670/64670eq01.jpg" style="margin: auto;" /></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC grade methanol</td>
			<td>Thermo Fisher Scientific (China) Co., Ltd.</td>
			<td>216565</td>
			<td></td>
		</tr>
		<tr>
			<td>Injector</td>
			<td>Chengdu Xinjin Shifeng Medical Apparatus &#38; Instrument Co., Ltd.</td>
			<td>0.7 (22 G)</td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore filter</td>
			<td>Tianjin Jinteng Experimental Equipment Co., Ltd</td>
			<td>&#966;13 0.22 Nylon66</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodiola granules</td>
			<td>Tibet Nodikang Pharmaceutical Co., Ltd.</td>
			<td>210501</td>
			<td></td>
		</tr>
		<tr>
			<td>Salidroside (HPLC, &#8805;98%)</td>
			<td>Chengdu Desite Biotechnology Co., Ltd.</td>
			<td>DST200425-037</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra pure water systemic</td>
			<td>Merck Millipore Ltd.</td>
			<td>Milli-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleansing machine</td>
			<td>Ningbo Xinyi Ultrasonic Equipment Co., Ltd</td>
			<td>SB-8200 DTS</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro dissolution determination of multi-index components in tibetan medicine rhodiola granules

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 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64670/64670fig01.jpg" /> 
Figure 1: Representative chromatograms of the three characteristic components after setting the chromatographic conditions mentioned in step 2.1 (n = 1). (A)&#160;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. <a href="https://www.jove.com/files/ftp_upload/64670/64670fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64670/64670fig02.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/64670/64670fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Condition</td>
			<td>Parameter</td>
		</tr>
		<tr>
			<td>Chromatographic column</td>
			<td>C18 (4.6 mm x 250 mm, 5 &#181;m)</td>
		</tr>
		<tr>
			<td>Mobile phase</td>
			<td>Acetonitrile (A)-0.2% Acetic acid (B)</td>
		</tr>
		<tr>
			<td>Gradient elution</td>
			<td>0&#8211;5min, 0%&#8211;4%A; 5&#8211;15min, 4%&#8211;5%A; 15&#8211;20min, 5%&#8211;7%A; 20&#8211;30min, 7%&#8211;14%A; 30&#8211;40min, 14%&#8211;13%A; 40&#8211;45min, 13%&#8211;4%A</td>
		</tr>
		<tr>
			<td>Flow rate</td>
			<td>1.0 mL/min</td>
		</tr>
		<tr>
			<td>Column temperature</td>
			<td>30 &#176;C</td>
		</tr>
		<tr>
			<td>Detecting wavelength</td>
			<td>275 nm</td>
		</tr>
		<tr>
			<td>Sample volume</td>
			<td>10 &#956;L</td>
		</tr>
	</tbody>
</table>
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.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Index components</td>
			<td>Linear equation</td>
			<td>R2</td>
			<td>Range of linearity (mg/mL)</td>
		</tr>
		<tr>
			<td>Salidroside</td>
			<td>Y = 2221X - 19.742</td>
			<td>0.9996</td>
			<td>0.06625&#8211;2.12</td>
		</tr>
		<tr>
			<td>Gallic acid</td>
			<td>Y = 29497X - 224</td>
			<td>0.9997</td>
			<td>0.008384&#8211;1.048</td>
		</tr>
		<tr>
			<td>Ethyl gallate</td>
			<td>Y = 28902X - 86.171</td>
			<td>0.9999</td>
			<td>0.008336&#8211;1.042</td>
		</tr>
	</tbody>
</table>
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.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Peak area of index components</td>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>4</td>
			<td>5</td>
			<td>6</td>
			<td>RSD %</td>
		</tr>
		<tr>
			<td>Salidroside</td>
			<td>900.6</td>
			<td>917.4</td>
			<td>899.8</td>
			<td>917.4</td>
			<td>940.1</td>
			<td>890.5</td>
			<td>1.95</td>
		</tr>
		<tr>
			<td>Gallic acid</td>
			<td>6430.2</td>
			<td>6544.2</td>
			<td>6281.2</td>
			<td>6327.7</td>
			<td>6142.5</td>
			<td>6636.9</td>
			<td>2.83</td>
		</tr>
		<tr>
			<td>Ethyl gallate</td>
			<td>12748.9</td>
			<td>12833.1</td>
			<td>13190.4</td>
			<td>13152.3</td>
			<td>13128.3</td>
			<td>13090.5</td>
			<td>1.42</td>
		</tr>
	</tbody>
</table>
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).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Peak area of index components</td>
			<td>0 h</td>
			<td>6 h</td>
			<td>12 h</td>
			<td>18 h</td>
			<td>21 h</td>
			<td>24 h</td>
			<td>RSD %</td>
		</tr>
		<tr>
			<td>Salidroside</td>
			<td>486.6</td>
			<td>509</td>
			<td>479</td>
			<td>505.1</td>
			<td>502.8</td>
			<td>492</td>
			<td>2.37</td>
		</tr>
		<tr>
			<td>Gallic acid</td>
			<td>3236.5</td>
			<td>3359.8</td>
			<td>3152.2</td>
			<td>3347.6</td>
			<td>3337</td>
			<td>3319.9</td>
			<td>2.47</td>
		</tr>
		<tr>
			<td>Ethyl gallate</td>
			<td>442</td>
			<td>413</td>
			<td>421</td>
			<td>429</td>
			<td>443.8</td>
			<td>436</td>
			<td>2.82</td>
		</tr>
	</tbody>
</table>
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).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Peak area of index components</td>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>4</td>
			<td>5</td>
			<td>6</td>
			<td>RSD %</td>
		</tr>
		<tr>
			<td>Salidroside</td>
			<td>1337.3</td>
			<td>1276.5</td>
			<td>1283.7</td>
			<td>1286.8</td>
			<td>1242.6</td>
			<td>1237.2</td>
			<td>2.83</td>
		</tr>
		<tr>
			<td>Gallic acid</td>
			<td>8432.1</td>
			<td>8976.1</td>
			<td>8792</td>
			<td>9083.1</td>
			<td>9040.2</td>
			<td>8751.4</td>
			<td>2.74</td>
		</tr>
		<tr>
			<td>Ethyl gallate</td>
			<td>422.8</td>
			<td>415.3</td>
			<td>421.9</td>
			<td>416.3</td>
			<td>428.9</td>
			<td>406.1</td>
			<td>1.87</td>
		</tr>
	</tbody>
</table>
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).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Known content (mg)</td>
			<td>Adding quantity (mg)</td>
			<td>Measuring quantity (mg)</td>
			<td>Recoveries (%)</td>
			<td>Average recoveries (%)</td>
			<td>RSD (%)</td>
		</tr>
		<tr>
			<td>0.5838</td>
			<td>0.406</td>
			<td>0.9783</td>
			<td>97.18</td>
			<td>99.91</td>
			<td>2.70</td>
		</tr>
		<tr>
			<td>0.5743</td>
			<td>0.406</td>
			<td>0.9984</td>
			<td>104.47</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.5751</td>
			<td>0.406</td>
			<td>0.9755</td>
			<td>98.63</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.5764</td>
			<td>0.406</td>
			<td>0.9776</td>
			<td>98.81</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.5906</td>
			<td>0.406</td>
			<td>0.991</td>
			<td>98.6</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.5802</td>
			<td>0.406</td>
			<td>0.9934</td>
			<td>101.77</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.1234</td>
			<td>0.118</td>
			<td>0.2424</td>
			<td>100.87</td>
			<td>100.4</td>
			<td>1.67</td>
		</tr>
		<tr>
			<td>0.1214</td>
			<td>0.118</td>
			<td>0.2428</td>
			<td>102.85</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.1216</td>
			<td>0.118</td>
			<td>0.2396</td>
			<td>100</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.1218</td>
			<td>0.118</td>
			<td>0.2389</td>
			<td>99.19</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.1249</td>
			<td>0.118</td>
			<td>0.2406</td>
			<td>98.09</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.1226</td>
			<td>0.118</td>
			<td>0.2423</td>
			<td>101.4</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.0221</td>
			<td>0.386</td>
			<td>0.4232</td>
			<td>103.91</td>
			<td>103.8</td>
			<td>2.02</td>
		</tr>
		<tr>
			<td>0.0218</td>
			<td>0.386</td>
			<td>0.4115</td>
			<td>100.97</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.0218</td>
			<td>0.386</td>
			<td>0.4176</td>
			<td>102.55</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.0218</td>
			<td>0.386</td>
			<td>0.4337</td>
			<td>106.7</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.0224</td>
			<td>0.386</td>
			<td>0.4302</td>
			<td>105.65</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.022</td>
			<td>0.386</td>
			<td>0.4198</td>
			<td>103.05</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
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.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Index components</td>
			<td>Dissolution equation</td>
			<td>R2</td>
		</tr>
		<tr>
			<td>Gallic acid</td>
			<td>GompertzMod</td>
			<td>0.4978</td>
		</tr>
		<tr>
			<td></td>
			<td>Gompertz</td>
			<td>0.3740</td>
		</tr>
		<tr>
			<td></td>
			<td>Logistic</td>
			<td>0.3739</td>
		</tr>
		<tr>
			<td></td>
			<td>Weibull</td>
			<td>0.3739</td>
		</tr>
		<tr>
			<td>Salidroside</td>
			<td>GompertzMod</td>
			<td>0.9894</td>
		</tr>
		<tr>
			<td></td>
			<td>Gompertz</td>
			<td>0.9783</td>
		</tr>
		<tr>
			<td></td>
			<td>Logistic</td>
			<td>0.9781</td>
		</tr>
		<tr>
			<td></td>
			<td>Weibull</td>
			<td>0.9781</td>
		</tr>
		<tr>
			<td>Ethyl gallate</td>
			<td>GompertzMod</td>
			<td>0.9895</td>
		</tr>
		<tr>
			<td></td>
			<td>Gompertz</td>
			<td>0.9852</td>
		</tr>
		<tr>
			<td></td>
			<td>Logistic</td>
			<td>0.9853</td>
		</tr>
		<tr>
			<td></td>
			<td>Weibull</td>
			<td>0.9853</td>
		</tr>
	</tbody>
</table>
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.

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

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.

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

The composition of the Tibetan medicine Rhodiola granules (RG) is complex, and the overall quality of RG is difficult to determine. Therefore, ...

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1.6K 조회수.  Chengdu University of Traditional Chinese Medicine. 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.

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

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Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody mAb by Neutralizing TNF Using an In Vitro Bioanalytical Method

A protocol for the determination of the relative anti-apoptotic activity of an anti-TNF&#945; mAb using a neutralization mechanism with WEHI 164 cells is presented here. This protocol is useful for comparing the neutralization strength of different molecules with the same biological functionality.

상대적인 힘의 안티-TNF 단 클론 항 체 (mAb) 중화 방 청 TNF는 시험관에서 생물 메서드를 사용 하 여의 결정

This protocol shows the measurement of the apoptotic activity neutralization of TNF&#945; in a mouse fibroblast cell model (WEHI 164) using an ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

신생아 쥐의 달팽이관 Explant 기법으로<em&gt; 체외에서</em&gt; 청력 연구의 스크리닝 도구

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

Photodynamic 치료 체 외에서 접근

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

구슈강(GSK) 생체내 및 체외 실험을 위한 과립 준비

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Optimized Griess Reaction for UV-Vis and Naked-eye Determination of Anti-malarial Primaquine

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening infections caused by P. vivax and ovale. However, PMQ has unwanted adverse effects that lead&#160;to acute hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. There is a need to develop simple and reliable methods for PMQ determination with the purpose of dosage monitoring. In early 2019, we have reported an UV-Vis and naked-eye based approach for PMQ colorimetric quantification. The detection was based on a Griess-like reaction between PMQ and anilines, which can generate colored azo products. The detection limit for direct measurement of PMQ in synthetic urine is in the nanomolar range. Moreover, this method has shown great potential for PMQ quantification from human serum samples at clinically relevant concentrations. In this protocol, we will describe the technical details regarding the syntheses and characterization of colored azo products, the reagent preparation, and the procedures for PMQ determination.

This protocol describes a novel colorimetric method for antimalarial primaquine (PMQ) detection in synthetic urines and human serums.

UV-Vis및 말라리아 방지 프리마퀸의 육안 측정을 위한 최적화된 Griess 반응

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening ...

A Surgical Model of Heart Failure with Preserved Ejection Fraction in Tibetan Minipigs

More than half of heart failure (HF) cases are classified as heart failure with preserved ejection fraction (HFpEF) worldwide. Large animal models are limited for investigating the fundamental mechanisms of HFpEF and identifying potential therapeutic targets. This work provides a detailed description of the surgical procedure of descending aortic constriction (DAC) in Tibetan minipigs to establish a large animal model of HFpEF. This model used a precisely controlled constriction of the descending aorta to induce chronic pressure overload in the left ventricle. Echocardiography was used to evaluate the morphological and functional changes in the heart. After 12 weeks of DAC stress, the ventricular septum was hypertrophic, but the thickness of the posterior wall was significantly reduced, accompanied by dilation of the left ventricle. However, the LV ejection fraction of the model hearts was maintained at &gt;50% during the 12-week period. Furthermore, the DAC model displayed cardiac damage, including fibrosis, inflammation, and cardiomyocyte hypertrophy. Heart failure marker levels were significantly elevated in the DAC group. This DAC-induced HFpEF in minipigs is a powerful tool for investigating molecular mechanisms of this disease and for preclinical testing.

The present protocol describes a step-by-step procedure to establish a minipig model of heart failure with preserved ejection fraction using descending aortic constriction. The methods for evaluating cardiac morphology, histology, and function of this disease model are also presented.

티베트 Minipigs에서 박출률이 보존된 심부전의 수술 모델

More than half of heart failure (HF) cases are classified as heart failure with preserved ejection fraction (HFpEF) worldwide. Large animal models are ...

In Vitro Application of a Wireless Sensor in Flexion-Extension Gap Balance of Unicompartmental Knee Arthroplasty

Unicompartmental knee arthroplasty (UKA) is an effective treatment for end-stage anteromedial osteoarthritis (AMOA). The key to UKA is the flexion-extension gap balance, which is closely related to postoperative complications such as bearing dislocation, bearing wear, and arthritis progression. The traditional gap balance assessment is performed by indirectly sensing the tension of the medial collateral ligament by a gap gauge. It relies on the surgeon's feel and experience, which is imprecise and difficult for beginners. To accurately assess the flexion-extension gap balance of UKA, we developed a wireless sensor combination consisting of a metal base, a pressure sensor, and a cushion block. After osteotomy, the insertion of a wireless sensor combination allows the real-time measurement of intra-articular pressure. It accurately quantifies the flexion-extension gap balance parameters to guide further femur grinding and tibia osteotomy, to improve the accuracy of gap balance. We conducted an in vitro experiment with the wireless sensor combination. the results showed that there was a difference of 11.3 N after applying the traditional method of flexion-extension gap balance performed by an experienced expert.

In Vitro Application of a Wireless Sensor in Flexion-Extension Gap Balance of Unicompartmental Knee Arthroplasty

This protocol presents a cadaveric study of a wireless sensor used in medial unicompartmental knee arthroplasty. The protocol includes the installation of an angle measuring device, standardized Oxford unicompartmental knee arthroplasty osteotomy, preliminary assessment of flexion-extension balance, and application of the sensor to measure flexion-extension gap pressure.

체외 Unicompartmental Knee Arthroplasty의 Flexion-Extension Gap Balance에 무선 센서 적용

Unicompartmental knee arthroplasty (UKA) is an effective treatment for end-stage anteromedial osteoarthritis (AMOA). The key to UKA is the ...

EV-Rich Plasma의 혈액 과응고 평가

Determination of the Procoagulant Activity of Extracellular Vesicle (EV) Using EV-Activated Clotting Time (EV-ACT)

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. However, there is an urgent need for a bedside test to assess the procoagulant activity of EV in clinical settings. This study proposes the use of thrombin activation time of EV-rich plasma as a measure of EV's procoagulant activity. Standardized procedures were employed to obtain sodium-citrated whole blood, followed by differential centrifugation to obtain EV-rich plasma. The EV-rich plasma and calcium chloride were added to the test cup, and the changes in viscoelasticity were monitored in real-time using an analyzer. The natural coagulation time of EV-rich plasma, referred to as EV-ACT, was determined. The results revealed a significant increase in EV-ACT when EV was removed from plasma obtained from healthy volunteers, while it significantly decreased when EV was enriched. Furthermore, EV-ACT was considerably shortened in human samples from preeclampsia, hip fracture, and lung cancer, indicating elevated levels of plasma EV and promotion of blood hypercoagulation. With its simple and rapid procedure, EV-ACT shows promise as a bedside test for evaluating coagulation function in patients with high plasma EV levels.

저자 스포트라이트: 외상성 뇌 손상 환자의 응고 장애 해독 

This protocol investigates the use of extracellular vesicle (EV)-rich plasma as an indicator of the coagulative ability of EV. EV-rich plasma is obtained through a process of differential centrifugation and subsequent recalcification.

EV-ACT(Activated Clotting Time)를 사용한 세포외 소포(EV)의 응고 활성 측정

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. ...