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

<ol>
	<li>Smith, S. C., Zheng, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impending+cardiovascular+pandemic+in+China.">The impending cardiovascular pandemic in China.</a> Circulation Cardiovascular Quality and Outcomes. 3 (3), 226-227 (2010).</li><li>Wang, D., Wang, P., Zhang, R., Xi, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+Xuefu+Zhuyu+decoction+combined+with+Western+medicine+for+angina+pectoris+in+coronary+heart+disease:+A+protocol+for+systematic+review+and+meta-analysis.">Efficacy and safety of Xuefu Zhuyu decoction combined with Western medicine for angina pectoris in coronary heart disease: A protocol for systematic review and meta-analysis.</a> Medicine. 99 (50), 23195 (2020).</li><li>Yang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+traditional+Chinese+medicine+in+the+regulation+of+stress+in+treating+coronary+heart+disease.">The role of traditional Chinese medicine in the regulation of stress in treating coronary heart disease.</a> Oxidative Medicine and Cellular Longevity. 2019, 3231424 (2019).</li><li>Yang, J., Tian, S., Zhao, J., Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+mechanism+of+TCM+formulae+in+the+treatment+of+different+types+of+coronary+heart+disease+by+network+pharmacology+and+machining+learning.">Exploring the mechanism of TCM formulae in the treatment of different types of coronary heart disease by network pharmacology and machining learning.</a> Pharmacological Research. 159, 105034 (2020).</li><li>Pu, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+effects+of+Rhodiola+rosea+L.:+A+review.">Anti-inflammatory effects of Rhodiola rosea L.: A review.</a> Biomedicine &amp; Pharmacotherapy. 121, 109552 (2020).</li><li>Tao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodiola+species:+A+comprehensive+review+of+traditional+use,+phytochemistry,+pharmacology,+toxicity,+and+clinical+study.">Rhodiola species: A comprehensive review of traditional use, phytochemistry, pharmacology, toxicity, and clinical study.</a> Medicinal Research Reviews. 39 (5), 1779-1850 (2019).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+biochemical+basis+of+the+meridian+tropism+theory+for+the+qi-invigorating+traditional+Chinese+medicine+herb+Panax+ginseng.">Exploring the biochemical basis of the meridian tropism theory for the qi-invigorating traditional Chinese medicine herb Panax ginseng.</a> Journal of Evidence-Based Integrative Medicine. 26, 2515690 (2021).</li><li>Pang, Y., Liang, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Nordicam+on+hemodynamics+in+rats+with+myocardial+ischemia-reperfusion+injury.">Effect of Nordicam on hemodynamics in rats with myocardial ischemia-reperfusion injury.</a> Journal of Chinese Medicinal Materials. 36 (2), 276-279 (2013).</li><li>Nickerson, B., Kong, A., Gerst, P., Kao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+dissolution+and+disintegration+results+for+an+immediate-release+tablet.">Correlation of dissolution and disintegration results for an immediate-release tablet.</a> Journal of Pharmaceutical and Biomedical Analysis. 150, 333-340 (2018).</li><li>Kambayashi, A., Yomota, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+clinically+relevant+dissolution+specifications+for+oral+solid+dosage+forms+of+weak+acid+drugs+using+an+in+silico+modeling+and+simulation+approach.">Exploring clinically relevant dissolution specifications for oral solid dosage forms of weak acid drugs using an in silico modeling and simulation approach.</a> European Journal of Pharmaceutical Sciences. 159, 105728 (2021).</li><li>Meng, S., Jiang, T. Y., Bu, C. J., Liu, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+progress+on+the+dissolution+of+traditional+Chinese+medicine+preparations.">Research progress on the dissolution of traditional Chinese medicine preparations.</a> Chinese Journal of Clinical Rational Drug Use. 8 (32), 180-181 (2015).</li><li>Chinese Pharmacopoeia Committee. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pharmacopoeia of the People's Republic of China. 4, (2020).</li><li>Lin, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Evaluation on Pre-Preparation of Rhodiola Extract. , (2013).</li><li>Zhou, Y. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+drug+solubility+Weibull+distribution+parameters+by+Origin+software.">Calculation of drug solubility Weibull distribution parameters by Origin software.</a> Herald of Medicine. 30 (06), 721-723 (2011).</li><li>Hu, C. Q., Pan, R. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+evaluation/prediction+of+bioequivalence+of+solid+oral+preparations+by+dissolution+test.">Progress in evaluation/prediction of bioequivalence of solid oral preparations by dissolution test.</a> Chinese Journal of New Drugs. 23 (01), 44-51 (2014).</li><li>Zhang, H., Yu, L. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissolution+testing+for+solid+oral+drug+products:+Theoretical+considerations.">Dissolution testing for solid oral drug products: Theoretical considerations.</a> American Pharmaceutical Review. 7 (5), 26-30 (2004).</li><li>Ren, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+strategies+for+the+discovery+and+validation+of+quality-markers+of+traditional+Chinese+medicine.">Analytical strategies for the discovery and validation of quality-markers of traditional Chinese medicine.</a> Phytomedicine. 67, 153165 (2020).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+modified+biopharmaceutics+classification+system+absorption+model+for+oral+Traditional+Chinese+Medicine+(Sanye+Tablet).">Establishment of modified biopharmaceutics classification system absorption model for oral Traditional Chinese Medicine (Sanye Tablet).</a> Journal of Ethnopharmacology. 244, 112148 (2019).</li><li>Song, X. Y., Li, Y. D., Shi, Y. P., Jin, L., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+control+of+traditional+Chinese+medicines:+a+review.">Quality control of traditional Chinese medicines: a review.</a> Chinese Journal of Natural Medicines. 11 (6), 596-607 (2013).</li><li>Wu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+markers+based+on+biological+activity:+A+new+strategy+for+the+quality+control+of+traditional+Chinese+medicine.">Quality markers based on biological activity: A new strategy for the quality control of traditional Chinese medicine.</a> Phytomedicine. 44, 103-108 (2018).</li><li>Wei, N. -. N., Wang, X., Su, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+of+dissolution+test+methodologies.">Progress of dissolution test methodologies.</a> Chinese Journal of New Drugs. 22 (10), 1119-1124 (2013).</li><li>Chi, Z., Azhar, I., Khan, H., Yang, L., Feng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+dissolution+testing+with+high-temporal+resolution+for+both+immediate-release+and+fixed-combination+drug+tablets.">Automatic dissolution testing with high-temporal resolution for both immediate-release and fixed-combination drug tablets.</a> Scientific Reports. 9 (1), 17114 (2019).</li><li>Haidar, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioequivalence+approaches+for+highly+variable+drugs+and+drug+products.">Bioequivalence approaches for highly variable drugs and drug products.</a> Pharmaceutical Research. 25 (1), 237-241 (2008).</li></ol>

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 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><tbody><tr><td>Chromatographic column</td><td>ZORBAX Eclipse&amp;nbsp;&amp;nbsp;</td><td>XDB-C18</td><td>4.6 mm x 250 mm, 5 &amp;micro;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, &amp;ge;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, &amp;ge;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 &lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/64670/64670eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;</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 &amp;amp; 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>&amp;phi;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, &amp;ge;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...

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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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1.7K Views.  Chengdu University of Traditional Chinese Medicine. In vitro maatregel is een geschikte maatregel voor het inspecteren van het oplossen van oplossingen van vast Chinees medicijnpreparaat die rekening houden met de effecten van desintegratie van geneesmiddelen, en het oplosgedrag en de inhoud van het actieve ingrediënt. De detectie van multi-index componenten kan de neutrale invloed en het oplossingsverschil van verschillende componenten beter weerspiegelen. De procedure wordt gedemonstreerd door Quinyun Du, een buitengewone student uit mijn l...

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

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis

A method for identifying Chinese medicinal materials and their related adulterants was constructed by taking Clematidis Armandii Caulis (Chuanmutong, a universally used traditional Chinese medicine) as an example. Ten batches of genuine Chuanmutong varieties and five batches of related adulterants were analyzed and compared based on the high-performance liquid chromatography (HPLC) fingerprints combined with chemometrics, including cluster analysis (CA), principal component analysis (PCA), and orthogonal partial least-squares discrimination analysis (OPLS-DA). In addition, the content of &#946;-sitosterol was determined. The control chemical fingerprint of Chuanmutong was established, and 12 common peaks were identified. The similarity between the fingerprint of 10 batches of genuine Chuanmutong varieties and the control fingerprint was 0.910-0.989, while the similarity of five batches of adulterants was only 0.133-0.720. Based on the common peaks in the chromatogram, 15 batches of samples were classified into three content levels by PCA, and were aggregated into four categories by CA, achieving a clear distinction between authentic Chuanmutong and adulterants of Chuanmutong. Further, seven differential components that can effectively identify authentic Chuanmutong and adulterants of Chuanmutong were found through OPLS-DA. The &#946;-sitosterol content of 10 batches of genuine Chuanmutong varieties was 97.53-161.56 &#181;g/g, while the &#946;-sitosterol content of the five batches of adulterants varied greatly, among which the &#946;-sitosterol content of Clematis peterae Hand.-Mazz. and Clematis gouriana Roxb. Var. finetii Rehd. et Wils. was significantly lower than that of authentic varieties of Chuanmutong. The HPLC index component content and chemical fingerprint multi-pattern recognition method established in this study provide a new strategy for effectively identifying authentic Chinese medicinal materials and related adulterants.

HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis

Here, we present a protocol to establish high-performance liquid chromatography (HPLC), coupled with chemical fingerprint multi-pattern recognition, which provides a new strategy for effectively identifying the genuine varieties of Clematidis Armandii Caulis and its adulterants.

A method for identifying Chinese medicinal materials and their related adulterants was constructed by taking Clematidis Armandii Caulis (Chuanmutong, a ...

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Chemistry

Standardized Identification of Compound Structure in Tibetan Medicine Using Ion Trap Mass Spectrometry and Multiple-Stage Fragmentation Analysis

Tibetan medicines are complex and contain numerous unknown compounds, making in-depth research on their molecular structures crucial. Liquid chromatography-electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) is commonly used to extract Tibetan medicine; however, many unpredictable unknown compounds remain after using the spectrum database. The present article developed a universal method for identifying components in Tibetan medicine using ion trap mass spectrometry (IT-MS). The method includes standardized and programmed protocols for sample preparation, MS setting, LC prerun, method establishment, MS acquisition, multiple-stage MS operation, and manual data analysis. Two representative compounds in the Tibetan medicine Abelmoschus manihot seeds were identified using multiple-stage fragmentation, with a detailed analysis of typical compound structures. In addition, the article discusses aspects such as ion mode selection, mobile phase adjustment, scanning range optimization, collision energy control, collision mode switchover, fragmentation factors, and limitations of the method. The developed standardized analysis method is universal and can be applied to unknown compounds in Tibetan medicine.

Here, we describe a general protocol and design that could be applied to identify trace amounts and minor constituents in the complex natural product formulations (matrixes) in Tibetan medicine.

Tibetan medicines are complex and contain numerous unknown compounds, making in-depth research on their molecular structures crucial. Liquid ...

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is the most widely used method in qualitative analysis of compounds. The method includes standardized and programmed protocols for sample pretreatment, MS tune, MS acquisition, and data processing. The sample pretreatments include collection, pulverization, solvent extraction, ultrasound, centrifugation, and filtration. Data post-processing was described in detail and includes data importing, self-established database construction, method establishment, data processing, and other manual operations. The above-ground part of the alpine yarrow herb, Achillea millefolium L., is used to treat inflammation, gastrointestinal disturbances, and pain and its 3-oxa-guaianolides could be useful leads for anti-inflammatory drug development. Three representative compounds in AML were identified, combining TOF-MS with a self-established database. Moreover, the differences from existing literature, liquid-phase parameter optimization, scan mode selection, ion source suitability, collision energy adjustment, isomer screening, method limitation, and possible solutions were discussed. This standardized analysis method is universal and can be applied to identify complex compounds in Chinese herbal medicine.

We describe a general protocol and systematic design that could be applied to separate and recognize complex components in alpine yarrow herb, Achillea millefolium L., a Chinese herbal medicine.

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography ...

Identifying Chemical Composition of Gynura bicolor by UPLC-TOF Mass Spectrometry

High-resolution Tandem Mass Spectrometry for Studying Chemical Constituents of Gynura bicolor DC

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). Ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) has gradually become a leading technology for the identification of TCM ingredients. Gynura bicolor DC. (BFH), a perennial stemless herb used for medicine and food in China has medicinal effects such as clearing heat, moistening the lung, relieving cough, dispersing stasis, and relieving swelling. Polyphenols and flavonoids contain numerous isomers, which hinder the identification of the complex compounds in BFH. This paper presents a systematic protocol for studying chemical constituents of BFH based on solvent extraction and integrated data via UPLC-Q-TOF-MS.
The method described here includes systematic protocols for sample pretreatment, MS calibration, MS acquisition, data processing, and analysis of results. Sample pretreatment includes collection, cleaning, drying, crushing, and extraction. MS calibration consists of multipoint and single-point correction. Data processing includes data importing, method establishment, analysis processing, and result presentation. Representative results of the typical fragmentation pattern of phenolic acids, esters, and glycosides in Gynura bicolor DC. (BFH) are presented in this paper. In addition, organic solvent selection, extraction, data integration, collision energy selection, and method improvement are discussed in detail. This universal protocol can be widely used to identify complex compounds in TCM.

Preparation of Gynura bicolor DC samples for High-Resolution Tandem Mass Spectrometry

We describe a general protocol that integrates high-resolution mass spectrometry analysis and molecular docking in traditional Chinese medicine research.

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). ...

Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is enhanced after processing with vinegar, CR processed with vinegar (CRV) is generally used clinically. However, the mechanism by which the analgesic effect is enhanced by vinegar processing is unclear. In this study, the ultra-high pressure liquid chromatographytandem mass spectrometry (UPLC-MS/MS) technique was used to examine changes in the blood levels of the exogenous constituents and metabolites between CR-treated and CRV-treated rats with dysmenorrhea. The results revealed differing levels of 15 constituents and two metabolites in the blood of these rats. Among them, the levels of (-)-myrtenol and [(1R,2S,3R,4R)-3-hydroxy-1,4,7,7-tetramethylbicyclo[2.2.1]hept-2-yl]acetic acid in the CRV group were considerably higher than in the CR group. CRV reduced the level of 2-series prostanoids and 4-series leukotrienes with proinflammatory, platelet aggregation, and vasoconstriction activities and provided analgesic effects by modulating arachidonic acid and linoleic acid metabolism and the biosynthesis of unsaturated fatty acids. This study revealed that vinegar processing enhances the analgesic effect of CR and contributes to our understanding of the mechanism of action of CRV.

Here, a comparative analysis of raw and processed Cyperi rhizoma (CR) samples is presented using ultra-high performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-MS/MS) in rats with primary dysmenorrhea. The changes in blood levels of the metabolites and the sample constituents were examined between rats treated with CR and CR processed with vinegar (CRV).

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is ...

Formation of Dispersible Taohong Siwu Tablets

Here, we optimize the process used to formulate and prepare dispersible Taohong Siwu tablets and provide a foundation for expanding their clinical application. Taking dispersion uniformity and disintegration time as the indices for investigation, we used a single-factor test to match and filter the excipient categories for Taohong Siwu tablets. The formulation was optimized by an orthogonal test design. The content and dissolution rates of the effective substances in dispersible Taohong Siwu tablets when prepared with optimized prescriptions were determined by ultra-high performance liquid chromatography (UPLC), and the optimal preparation process was determined.
The optimal composition for dispersible Taohong Siwu tablets was 17% Taohong Siwu extract powder, 1% magnesium stearate, 49% microcrystalline cellulose, 20% cross-linked polyvinylpyrrolidone, and 13% sodium carboxymethyl starch. When dispersible Taohong Siwu tablets were prepared by direct compression and the optimized prescription powder was uniformly dispersed within 3 min, the dissolution rate reached more than 90% within 50 min.When prepared according to the optimized methods,dispersible Taohong Siwu tablets disintegrate rapidly in water with good dispersion uniformity and controllable quality.

This article discusses the process used to formulate and prepare Taohong Siwu, a traditional Chinese medicine, as a dispersible tablet. We performed single-factor and orthogonal experiments to screen the prescription excipients, optimize the formulation process, and produce dispersible Taohong Siwu tablets.

Here, we optimize the process used to formulate and prepare dispersible Taohong Siwu tablets and provide a foundation for expanding their clinical ...

A Method for Zanba-Stir-Fried Tiebangchui Process Optimization

Optimization of Processing Technology for Tiebangchui with Zanba Based on CRITIC Combined with Box-Behnken Response Surface Method

The dried root of Aconitum pendulum Busch., called Tiebangchui (TBC) in Chinese, is one of the most famous Tibetan medicines. It is a widely used herb in northwest China. However, many cases of poisoning have occurred because of TBC's intense toxicity and because its therapeutic and toxic doses are similar. Therefore, finding a safe and effective method to reduce its toxicity is an urgent task. A search through the Tibetan medicine classics shows that the processing method of TBC stir-fried with Zanba was recorded in the "Processing specification of Tibetan medicine of Qinghai Province (2010)". However, the specific processing parameters are not yet clear. Thus, this study aims to optimize and standardize the processing technology of Zanba-stir-fried TBC.
First, a single-factor experiment was conducted on four factors: the slice thickness of TBC, amount of Zanba, processing temperature, and time. With monoester and diester alkaloid contents in Zanba-stir-fried TBC as indexes, CRITIC combined with the Box-Behnken response surface method was used to optimize the processing technology of Zanba-stir-fried TBC. The optimized processing conditions of Zanba-stir-fried TBC were a TBC slice thickness of 2 cm, three times more Zanba than TBC, a processing temperature of 125 &#176;C, and 60 min of stir-frying. This study determined the optimized and standard processing conditions for the usage of Zanba-stir-fried TBC, thus providing an experimental basis for the safe clinical use and industrial production of Zanba-stir-fried TBC.

Author Spotlight: Optimization of Processing Technology for Tiebangchui with Zanba Based on CRITIC Combined with Box-Behnken Response Surface Method  

The present protocol describes an efficient and standard detoxification processing method for Zanba-stir-fried Tiebangchui using CRITIC combined with the Box-Behnken response surface method.

The dried root of Aconitum pendulum Busch., called Tiebangchui (TBC) in Chinese, is one of the most famous Tibetan medicines. It is a widely used herb in ...

Microscopic Characterization and TLC-Based Identification of Rhodiola crenulate

Field Collection and Laboratory Routine Identification of Rhodiola crenulata

The identification of medicinal materials is the premise and guarantee of drug safety. The majority of scientific researchers are bound to favor the simple, fast, effective, and inexpensive identification process of herbals. Rhodiola crenulata is a traditional Tibetan medicine grown at high altitudes, mainly distributed in Tibet, Yunnan, and Sichuan regions of China. Rhodiola crenulate possesses multiple bioactivities, such as anti-inflammatory, anti-hypoxia, and antioxidant properties, and has great potential for development. With the increasing market demand and a rapid decrease in resource content, a large number of confused products of Rhodiola crenulata have been troubling people. Therefore, this protocol introduces a standard process for the identification of Rhodiola crenulata in the field combined with routine laboratory testing. The combination of habitat, microscopic features, and thin-layer chromatography will undoubtedly identify Rhodiola crenulata quickly, efficiently, and economically, contributing to the continuous development of Tibetan medicine and the quality control of medicinal materials.

Author Spotlight: Developing Identification Methods for Rhodiola crenulata and Investigating Its Resource Distribution and Pharmacological Effects

Here, we describe the identification of Rhodiola Crenulata from habitat, plant morphology, medicinal properties, microscopic features, and thin-layer chromatography.

The identification of medicinal materials is the premise and guarantee of drug safety. The majority of scientific researchers are bound to favor the ...

Analyzing Tibetan Medicinal Plant APB Using 2D-HPLC-MS with Molecular Networking

2D-HPLC-MS Technology Combined with Molecular Network for the Identification of Components in Tibetan Medicine Aconitum pendulum

In this study, a comprehensive approach was employed, utilizing 2D-HPLC-MS technology in conjunction with the molecular network to unravel the intricate chemical composition of the Tibetan medicinal plant APB. Through the implementation of 2D-HPLC, enhanced separation of complex mixtures was achieved, enabling the isolation of individual compounds for subsequent analysis. The molecular network approach further aided in elucidating structural relationships among these compounds, contributing to the determination of potential bioactive molecules. This integrated strategy efficiently identified a wide array of chemical components present within the plant. The findings revealed a diverse spectrum of chemical constituents within APB, including alkaloids, among others. This research not only advances understanding of the phytochemical profile of this traditional Tibetan medicine but also provides valuable insights into its potential therapeutic properties. The integration of 2D-HPLC-MS and molecular network proves to be a powerful tool for systematically exploring and identifying complex chemical compositions in herbal medicines, paving the way for further research and development in the field of natural product discovery.

Author Spotlight: Integrating 2D-HPLC-MS and Molecular Networking in Natural Medicine Analysis

This study utilizes two-dimensional high-performance liquid chromatography-mass spectrometry (2D-HPLC-MS) technology in conjunction with molecular networking to unravel the intricate chemical composition of the Tibetan medicinal plant Aconitum pendulum Busch (APB). The article provides a detailed protocol for the systematic exploration and identification of complex chemical components of herbal medicines.

In this study, a comprehensive approach was employed, utilizing 2D-HPLC-MS technology in conjunction with the molecular network to unravel the intricate ...

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

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