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>
	<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 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, ...

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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.

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.

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.

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

Microscopic Characterization and TLC-Based Identification of &lt;em&gt;Rhodiola crenulate&lt;/em&gt;

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 &lt;em&gt;Rhodiola crenulata&lt;/em&gt; 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 ...