Name: an in vitro dissolution determination of multi-index components in tibetan medicine rhodiola granules
Uploaded: 2022-11-04T15:00:00
Description: Watch this Scientific Journal Video about An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules at JoVE.com

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 re...

<ol>
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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...

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...

Watch this Scientific Journal Video about An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules at JoVE.com

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, ...

In vitro measure is a suitable measure for inspecting the dissolution of solutions of solid Chinese medicine preparation which take into account the effects of drug disintegration, and the dissolution behavior, and the content of active ingredient. The detection of multi-index component can better reflect the neutral influence, and the dissolution difference of different components. Demonstrating the procedure will be Quinyun Du a extraordinary student from my laboratory.To begin, prepare the reference substance stock solution by weighing 10.6 milligrams of salidrocide, 5.24 milligrams of gallic acid, and 5.21 milligrams of ethyl gallic acid separately on an electronic analytical balance. Then add them individually into a five milliliter volumetric flask. Then add HPLC grade methanol to dissolve, and dilute to five milliliters.And finally, shake well to obtain the reference substance stock solution with mass concentrations of 2.120, 1.048, and 1.042 milligrams per milliliter, respectively. To prepare the test sample solution, extract 2.8 grams of rhodiola granules with 10 milliliters of HPLC grade methanol, using an ultrasonic cleansing machine for 30 minutes. Then filter it with a 0.22 micrometer filter for the system adaptability test.Then prepare a mixed reference solution that contains 0.590 milligrams per milliliter salidrocide, 2.030 milligrams per milliliter gallic acid, and 1.930 milligrams per milliliter ethyl gallate. To obtain the theoretical content of each characteristic component of rhodiola granules, place 2.8 grams of rhodiola granules in 500 milliliter conical flask. Add 200 milliliters of ultrapure water, and ultrasonically extract for 60 minutes.Then filter it with a 0.22 micrometer filter. After determining the content of the test solution, set the chromatographic conditions for high performance liquid chromatography as described in the manuscript. To investigate the linear relationship, dilute the reference stock solutions of gallic acid and ethyl gallate, and the reference stock solutions of salidrocide to obtain the gradient concentration solution for drawing a standard curve.Then inject 10 microliters of the mixed reference solution into the HPLC system six times daily, and run the samples with the same HPLC conditions, while recording the peak area of each feature component. Next inject 10 microliters of the prepared sample solution, and determine the peak areas of the HPLC according to the chromatographic conditions at different time intervals. Take six samples of the same batch of rhodiola granules to prepare the test sample solution as demonstrated previously, and inject 10 microliters of each sample into the HPLC system.Run the samples as demonstrated previously to determine reproducibility. After preparing six portions of the same batch of rhodiola granules for the test solution, add about 50%of the reference substrate of each index component. Run these samples in the HPLC system, as demonstrated previously, to calculate the recovery rate as described in the manuscript.To carry out the dissolution test, add 100 milliliters of ultrapure water into the dissolution cup of the drug dissolution apparatus, and maintain the temperature at 37 degrees Celsius with a rotation speed of 100 rpm. Next add 2.8 grams of rhodiola granules into a dissolution cup, and start recording the duration of dissolution immediately. Collect a total of one milliliter of the sample with an injector at different time intervals, and make up the volume in the dissolution cup with a dissolution medium at the same temperature immediately.Immediately fill the collected samples through a 0.22 micrometer microporous membrane, and take the subsequent filtrate. After determining the content of each component, at each time point by HPLC, calculate the dissolution of each time point, and cumulative dissolution as described in the manuscript. The chromatogram of the sample, and the reference solution were obtained.Three different peaks corresponding to gallic acid, salidrocide, and ethyl gallate shows that the elution gradient had a good resolution for the three index components in rhodiola granules. The dissolution curves of each component show that the cumulative dissolution of gallic acid was over 80%after one minute. Whereas for salidrocide and ethyl gallic acid, it was over 65%after five minutes.However, the cumulative dissolution of each index component decreased after 30 minutes. Adjusting the dilution issue of the standard curve, according to the preliminary experiment of the sample treatment, samples must be collected quickly to avoid them missing special collection time point.

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