The work wishes to acknowledge support from the National Natural Science Foundation of China (No. 82173976), National Key Research and Development Program (No. 2018YFC1707205), and State Key Laboratory of Innovative Drugs and High Energy Saving Pharmaceutical Equipment, Jiangxi University of Traditional Chinese Medicine (No. GZSYS202003).

1. Preparation of test sample
<ol>
	<li>Set up four different batches of Indigo Naturalis herbal powder for the test. Pass each sample through the seventh sieve and ninth sieve in turn and collect the sample between the seventh and ninth sieve5. 
	NOTE: The average aperture size of the seventh sieve is 125 &#181;m &#177; 5.8 &#181;m. The average aperture size of the ninth sieve is 75 &#181;m &#177; 4.1 &#181;m.</li>
	<li>Accurately weigh 0.2 g of the sample (Indigo Naturali...

<ol>
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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=Treatment+of+non-high-risk+acute+promyelocytic+leukemia+with+realgar-indigo+naturalis+formula+(RIF)+and+all-trans+retinoid+acid+(ATRA):+study+protocol+for+a+randomized+controlled+trial.">Treatment of non-high-risk acute promyelocytic leukemia with realgar-indigo naturalis formula (RIF) and all-trans retinoid acid (ATRA): study protocol for a randomized controlled trial.</a> Trials. 21 (1), 7 (2020).</li><li>Sugimoto, S., 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=Clinical+efficacy+and+safety+of+oral+Qing-Dai+in+patients+with+ulcerative+colitis:+a+single-center+open-label+prospective+study.">Clinical efficacy and safety of oral Qing-Dai in patients with ulcerative colitis: a single-center open-label prospective study.</a> Digestion. 93 (3), 193-201 (2016).</li><li>Lin, Y. K., 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=Clinical+assessment+of+patients+with+recalcitrant+psoriasis+in+a+randomized,+observer-blind,+vehicle-controlled+trial+using+indigo+naturalis.">Clinical assessment of patients with recalcitrant psoriasis in a randomized, observer-blind, vehicle-controlled trial using indigo naturalis.</a> Archives of Dermatology. 144 (11), 1457-1464 (2008).</li><li>Sun, Q., Leng, J., Tang, L., Wang, L., Fu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comprehensive+review+of+the+chemistry,+pharmacokinetics,+pharmacology,+clinical+applications,+adverse+events,+and+quality+control+of+indigo+naturalis.">A Comprehensive review of the chemistry, pharmacokinetics, pharmacology, clinical applications, adverse events, and quality control of indigo naturalis.</a> Frontiers in Pharmacology. 12, 664022 (2021).</li><li>Yang, Y. 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The authors have no conflicts of interest to disclose.

According to traditional Chinese medicine, Indigo Naturalis has the power to clear heat and detoxification, cool blood, eliminate spots, purge fire, and arrest convulsion. Based on randomized double-blind controlled clinical trials11,12,13, Indigo Naturalis is effective in the treatment of psoriasis, ulcerative colitis, and acute promyelocytic leukemia in addition to its traditional treatment of cough and phlegm, hemorrhagic sym...

In traditional Chinese medicine, during the course of disease treatment, the clinical effectiveness of medications and the safety of the course of treatment are directly impacted by the quality of Chinese herbal medicine. Utilizing cutting-edge identification technology, one can assess the efficacy of Chinese herbal medicine and guarantee user safety. The Chinese herbal medicine water test method refers to immersing the herbs in water or solvent, then quickly and precisely determining the medicine&#39;s authenticity by observing the changes in color, size, and shape1.
It was originally a good choice for the identification of Chinese medicine. However, the disadvantage of the traditional water test method is that the accuracy and sensitivity of distinguishing the authenticity of Chinese medicine are low because of the subjectivity of observation by the naked eye2. One of the key medicinal materials used in the water test method is Indigo Naturalis, considered an effective remedy for pediatric hyperthermia, psoriasis, leukemia, and ulcerative colitis3. The genuine Indigo Naturalis floats on the surface of water, and the water does not turn dark blue after shaking. However, the fake Indigo Naturalis has particles that sink, and the water will turn dark blue after shaking4. Its principle is due to hydrophobic and easily floatable indigo, indirubin, and other organic components of high-quality Indigo Naturalis. On the contrary, due to low organic matter, a large amount of lime, and heavy texture, some particles doped with fake Indigo Naturalis will sink quickly5. However, this method is only a simple qualitative identification, and it limits the rapid identification of the authenticity of Chinese herbal medicine and fails to reveal the changes of Indigo Naturalis in water.
Multiple light scattering technology is a technology that can measure multi-angle light signal scanning based on a laser passing through the sample. The incident light will be scattered when it penetrates the sample or encounters particles. If the scattered light penetrates through the sample, a transmission light signal is formed; If the sample concentration is high, the light will be reflected by the particles, forming a backscattering light signal. The changes in light intensity reflect the changes in particle concentration and particle size in liquid preparation6. The multiple light scattering instruments can quickly and accurately analyze phenomena such as emulsification, flocculation, precipitation, and rupture of suspension, emulsion, and foam liquid by multiple light scattering technology, as well as quantitatively analyze characteristics such as the rate of occurrence of the above phenomena.
Multiple light scattering technology has demonstrated significant benefits in particle stability monitoring7, red wine clarification8, and milk fermentation quality control9. Using this technology, the traditional water test method of Indigo Naturalis may be intuitive, quantitative, and scientific. Therefore, based on the principle of multiple light scattering technology, this study established a rapid evaluation method for the quality of Indigo Naturalis, taking the Turbiscan stability index (TSI) of the sample as the index of quality control (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Analytical balance (1/10,000)</td>
			<td>Sartorious, Germany</td>
			<td>BSA224S&#160;</td>
			<td>www.sartorius.com.cn</td>
		</tr>
		<tr>
			<td>Funnel</td>
			<td>Chengdu Kelong Chemical Co. LTD</td>
			<td>Diameter 5 cm</td>
			<td>www.cdkelongchem.com</td>
		</tr>
		<tr>
			<td>Indigo Naturalis S1</td>
			<td>Xianyou, Fujian</td>
			<td>20210501</td>
			<td></td>
		</tr>
		<tr>
			<td>Indigo Naturalis S2</td>
			<td>Yaan, Sichuan</td>
			<td>20201102</td>
			<td></td>
		</tr>
		<tr>
			<td>Indigo Naturalis S3</td>
			<td>Xianyou, Fujian</td>
			<td>20161012</td>
			<td></td>
		</tr>
		<tr>
			<td>Indigo Naturalis S4</td>
			<td>Xianyou, Fujian</td>
			<td>20180305</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Iron ring</td>
			<td>Chengdu Kelong Chemical Co. LTD</td>
			<td>/</td>
			<td>www.cdkelongchem.com</td>
		</tr>
		<tr>
			<td>Iron stand</td>
			<td>Chengdu Kelong Chemical Co. LTD</td>
			<td>/</td>
			<td>www.cdkelongchem.com</td>
		</tr>
		<tr>
			<td>Mili-Q ultra-pure water meter</td>
			<td>Milipore, USA</td>
			<td>Mili-Q</td>
			<td>www.merckmillipore.com</td>
		</tr>
		<tr>
			<td>Ninth sieve</td>
			<td>Chengdu Kelong Chemical Co. LTD</td>
			<td>Average aperture size 75 &#181;m</td>
			<td>www.cdkelongchem.com</td>
		</tr>
		<tr>
			<td>Sample bottle</td>
			<td>French Formulaction Company</td>
			<td>Bottom diameter 2.6 cm, height 6 cm</td>
			<td>www.formulaction.com</td>
		</tr>
		<tr>
			<td>Seventh sieve</td>
			<td>Chengdu Kelong Chemical Co. LTD</td>
			<td>Average aperture size 125 &#181;m</td>
			<td>www.cdkelongchem.com</td>
		</tr>
		<tr>
			<td>Turbisoft Lab multiple light scattering instrument</td>
			<td>French Formulaction Company</td>
			<td>Turbisoft Lab 2.3.1.125 Fanalyser 1.3.5</td>
			<td>www.formulaction.com</td>
		</tr>
		<tr>
			<td>Weighing paper</td>
			<td>Chengdu Kelong Chemical Co. LTD</td>
			<td>/</td>
			<td>www.cdkelongchem.com</td>
		</tr>
	</tbody>
</table>

fast inspection of quality of indigo naturalis by multiple light scattering

Quality control of Chinese herbal medicine is a crucial component of Chinese herbal medicine research and development. Faced with the challenges of modernization and internationalization of Chinese herbal medicine, it is urgent to establish thorough and effective procedures for quality identification of Chinese herbal medicine, and there is an urgent need for new analytical and testing techniques that are efficient, accurate, and environmentally friendly. 
Multiple light scattering is a cutting-edge and analytical method that can accurately and rapidly assess the quality of Chinese herbal medicine without altering the nature or state of the sample or using organic reagents. Indigo Naturalis is considered a good remedy for pediatric hyperthermia, psoriasis, leukemia, and ulcerative colitis. In this study, the process of addition of Indigo Naturalis powder in water was recorded precisely using a multiple light scattering instrument. 
The qualitative and quantitative measurements of the instrument can be used to accurately capture the overall trajectory and sinking behavior of Indigo Naturalis powder into water and to establish a rapid evaluation method for the quality of Indigo Naturalis with the transmission and backscattering spectrograms of the sample as qualitative indicators and stability index as a quantitative indicator. The analytical technique based on multiple light scattering provides a fast, accurate, green, and environmentally friendly method for the quality evaluation of Indigo Naturalis and supports the development and transformation of high-quality Indigo Naturalis.

Figure 2A-D correspond to S1, S2, S3, and S4 of Indigo Naturalis, respectively. A is of high-quality Indigo Naturalis, showing the same light transmittance at any height within 0-10 min, which is very stable. B is the common Indigo Naturalis, and its light transmittance fluctuates slightly with the change of time and is generally stable. C and D are fake and inferior products. There may be two conditions in the transmission spectrograms of fake Indigo Natura...

Watch this Scientific Journal Video about Fast Inspection of Quality of Indigo Naturalis by Multiple Light Scattering at JoVE.com

Fast Inspection of Quality of Indigo Naturalis by Multiple Light Scattering

Here, we present a quality evaluation method based on a multiple light scattering technique to evaluate the quality of Indigo Naturalis.

Quality control of Chinese herbal medicine is a crucial component of Chinese herbal medicine research and development. Faced with the challenges of ...

The protocol can accurately and rapidly assess the quality of Chinese medicine without altering the nature of the sample or using organic reagents. They main advantage of this technique provides a fast, accurate, and method for quantity evaluation and supports the development and the transformation of high quality Chinese medicine. We believe that this method will be helpful in the field of traditional Chinese medicine quality control, particularly in the assessment of authenticity.Begin by selecting four distinct batches of Indigo naturalis herbal powder for testing. For each sample, pass it successively through the seventh and ninth sieves and collect the powder that remains between these two sieves. Now accurately weigh 0.2 grams of the collected powder on a piece of weighing paper and set aside this measured quantity for later use.Next, set up an iron support stand and place an iron ring with a five centimeter diameter funnel on top of it. Pipette 20 milliliters of pure water to the sample glass bottle, then place the sample glass bottle directly under the funnel, ensuring the lower edge of the funnel aligns with the mouth of the bottle. Release the sample at a height of 80 centimeters from the lower edge of the funnel, allowing it to slide freely along the funnel and into the sample bottle.Turn on the Turbiscan lab instrument and allow it to warm up for 30 minutes. Click on the New File button at the top menu to create a new empty measurement file. Click on the Turbiscan lab temperature icon in the top menu to set the instrument target temperature to 25 degrees Celsius.Next, click on Programmed Analysis to enter the setup analysis program. Add the program to the list. Then input a 10 minute task duration with 30 second task periods as a cycle and 21 scans into the analysis sequence within the taskbar.Place the prepared sample vial into the measurement system. Once the program has been set up, click on Start to begin measurement. After data acquisition, click on the list of calculated parameters to automatically calculate the Turbiscan stability index.High quality Indigo naturalis showed the same light transmittance at all heights within 0 to 10 minutes. The transmittance of common Indigo naturalis showed slight fluctuations over time. Fake Indigo naturalis products showed a rapid decrease in transmittance with significantly lower light at the bottom of the sample bottle.Inferior samples showed stable transmission at zero time, which decreased over time. The back scatter of stable samples one and two showed little to no fluctuations, whereas unstable samples three and four showed sedimentation-derived turbidity. High Turbiscan stability index values were observed in samples three and four, indicative of instability.However, the slopes of samples one and two were close to zero. A key step in the process is that the glass bottle containing the sample should be moved inside the sample tank as quickly as possible while avoiding shaking

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294 vistas.  Chengdu University of Traditional Chinese Medicine. El protocolo puede evaluar de manera precisa y rápida la calidad de la medicina china sin alterar la naturaleza de la muestra ni utilizar reactivos orgánicos. La principal ventaja de esta técnica proporciona un método rápido, preciso y para la evaluación de cantidades y apoya el desarrollo y la transformación de la medicina china de alta calidad. Creemos que este método será útil en el campo del control de calidad de la medicina tradicional china, particularmente en la evaluación d...