Summary
Here, we present a quality evaluation method based on a multiple light scattering technique to evaluate the quality of Indigo Naturalis.
Abstract
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.
Introduction
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'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).
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Protocol
1. Preparation of test sample
- 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 µm ± 5.8 µm. The average aperture size of the ninth sieve is 75 µm ± 4.1 µm. - Accurately weigh 0.2 g of the sample (Indigo Naturalis powder) on a weighing paper and set it aside.
2. Sample addition
- Build an iron support stand and put an iron ring with a 5 cm diameter funnel on it.
- Use a pipette to add 20 mL of pure water to the sample glass bottle (bottom diameter 2.6 cm, height 6 cm). Place the sample glass bottle directly under the funnel so that the lower edge of the funnel is flush with the mouth of the bottle.
NOTE: Clean the exterior of the sample glass vial with a clean, non-abrasive paper towel and inspect the glass surface for visible marks. If there are, change the glass bottle. Be careful not to spill when adding liquid. - Release the sample at a height of 80 cm from the lower edge of the funnel so that it can slide freely along the funnel into the sample bottle.
3. Instrument operation
- Turn on the Turbiscan Lab instrument and warm it up for 30 min.
- Create the file. Click the Create file button in the top menu (or the New file function in the File menu) to create a new empty measurement file. Define its name and save location (by default, the data folder is located at: "C:/users/admin/Formulaction/FAnalyser/Data".
- Click the Show Turbiscan Lab Temperature button in the top menu to set the instrument target temperature to 25 °C.
NOTE: The instrument temperature is influenced by the room temperature, so be careful to adjust the ambient temperature. - Click Program Scan in the top menu to enter the setup analysis program. Add the program to the list, and in the taskbar, add 30 s as a cycle and 21 scans to the analysis sequence. Select this analysis program for all subsequent measurements.
- Move the prepared sample vial into the measurement system. After setting up the program, click Start to start the measurement.
NOTE: Be careful not to shake the glass bottle when moving and only move it slightly. - After data acquisition, click on the list of calculated parameters to automatically calculate TSI.
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Representative Results
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 Naturalis, namely the transmission light in C decreased rapidly at the first moment of measurement, and the transmission light at the bottom of the sample bottle was significantly lower than that at the top, indicating that the deposition occurred in the sample bottle at the first time, and the deposition was very fast. However, the transmission light in D is stable at zero time and then decreases slowly with the prolongation of time. Compared with C, it indicates that there is slow sedimentation in the sample bottle.
Figure 3A-D correspond to S1, S2, S3, and S4 of Indigo Naturalis, respectively. From the spectral data of backscattering light, it can be roughly inferred that the sample data are consistent with the transmission light. No or small fluctuations (Figure 3A and Figure 3B, respectively) indicate that the sample is stable. Figure 3C, D may be turbid due to the sedimentation of the sample, and the fluctuation increases, which leads to the instability of the sample.
By comparing the transmission spectrogram (Figure 2) and backscattering spectrograms (Figure 3) of the four Indigo Naturalis with the start and end snapshots of the videos (Figure 4) and Supplemental Video S1, Supplemental Video S2, Supplemental Video S3, and Supplemental Video S4 captured by the multiple light scattering instrument, the authenticity of the Indigo Naturalis can be quickly and roughly identified.
With the extension of the measurement time, the transmission spectrograms and backscattering spectrograms of high-quality Indigo Naturalis should fluctuate little or not at all, and the transmission spectrograms and backscattering spectrograms of pseudo or inferior Indigo Naturalis may gradually or sharply decrease. Supplemental Video S1, Supplemental Video S2, Supplemental Video S3, and Supplemental Video S4 also clearly reflected this result. The TSI values reflect the accumulation of the intensity changes of transmission light or backscattering light compared with the previous measurement during the measurement time, and it is also the comprehensive change of the volume concentration and particle size of the sample during the whole scan period. The quality of the four types of Indigo Naturalis can be accurately distinguished by contrasting their TSI at 10 min (Figure 5 and Table 1). The higher the TSI value, the more unstable the system will be, and the greater the change range of the sample will be10. If the TSI value is <10 during the scan period, the sample will be considered stable. Therefore, the current protocol shows a method for quick identification of good quality Indigo Naturalis based on TSI in a multiple light scattering instrument.
Figure 1: Principle of evaluating the quality of Indigo Naturalis by multiple light scattering. Indigo and indirubin are the main reasons for the strong hydrophobicity of Indigo Naturalis. The content of indigo and indirubin determines the sedimentation speed of particles. With this characteristic, a multiple light scattering instrument can distinguish different qualities of Indigo Naturalis. The multiple light scattering instrument has multiple light scattering technology, and its measuring probe consists of a pulsed near-infrared light source (λ = 880 nm) and two synchronous detectors. One of them is a transmission light detector, which is used to receive the light passing through the sample bottle (0° with the incident light) and to determine the clear sample. The other is a backscattering light detector, which is used to receive the backscattering light of the sample (45° from the incident light) and to determine the high-concentration sample. The measuring probe scans the whole sample cell from bottom to top, once every 40 µm, and collects the transmission light (T) and backscattering light (BS) data. By setting the measurement times and scanning time, the sample will be scanned repeatedly, and the signal and data acquisition will be processed by the current-voltage converter to obtain an atlas representing the stability characteristics of the sample. Please click here to view a larger version of this figure.
Figure 2: Transmission spectrograms of four Indigo Naturalis in the multiple light scattering instrument. A-D correspond to S1, S2, S3, and S4 of Indigo Naturalis, respectively. (A) High-quality, stable Indigo Naturalis, showing the same light transmittance at any height within 0-10 min. (B) The common Indigo Naturalis and its light transmittance fluctuates slightly with the change of time and is generally stable. (C, D) Fake and inferior products. (C) The transmission light at the bottom of the sample bottle was significantly lower than that at the top, indicating that the deposition occurred in the sample bottle at an early stage, and the deposition was very fast. (D) However, the transmission light is stable at zero time and then decreases slowly with time. Compared with C, it indicates that there is slow sedimentation in the sample bottle. Please click here to view a larger version of this figure.
Figure 3: Backscattering spectrograms of four Indigo Naturalis in the multiple light scattering instrument. A-D correspond to S1, S2, S3, and S4 of Indigo Naturalis, respectively. From the spectral data of backscattering light, it can be roughly inferred that the sample data are consistent with the transmission light. (A) No fluctuation, indicating that the sample is very stable. (B) The fluctuation is small, indicating that the sample is relatively stable. (C, D) Turbidity is due to the sedimentation of the sample, and the fluctuation increases, which leads to the instability of the sample. Please click here to view a larger version of this figure.
Figure 4: Start and end snapshots of the videos of the settling process of four Indigo Naturalis in the multiple light scattering instrument. A-D correspond to S1, S2, S3, and S4 of Indigo Naturalis, respectively. In the results, by comparing the scanning pictures of 0 min and 10 min, it can be seen that A (Supplemental Video S1) and B (Supplemental Video S2) are very clear in the whole process. C (Supplemental Video S3) is partially turbid at first, and then completely turbid at last. D (Supplemental Video S4) gradually changes from clarification to turbidity. Please click here to view a larger version of this figure.
Figure 5: TSI of four Indigo Naturalis from 0 min to 10 min. The graph shows the curve of TSI with scan time. According to the variation of the TSI curve, S4 has the highest slope and the TSI value changes particularly significantly. Next, the slope of S3 is also relatively large, and the TSI value has been in a slow increase. However, the slopes of S1 and S2 are close to zero, and the TSI values change less. Please click here to view a larger version of this figure.
| No. | Indigo content | Indirubin content | TSI of 10 min |
| S1 | 9.00% ± 0.38% | 0.60% ± 0.00% | 0.61 ± 0.06 |
| S2 | 2.07% ± 0.01% | 0.20% ± 0.00% | 2.74 ± 0.14 |
| S3 | 1.40% ± 0.02% | 0.00% ± 0.00% | 28.46 ± 3.51 |
| S4 | 0.00% ± 0.00% | 0.00% ± 0.00% | 68.75 ± 1.28 |
Table 1: TSI of four Indigo Naturalis at 10 min (n=3). According to the strong hydrophobicity of Indigo Naturalis, it can be inferred that the indigo and indirubin content of Indigo Naturalis determines its quality. When the content of indigo and indirubin is high, the sample floats almost completely on the water surface, resulting in small TSI values. At 10 min, the TSI sequence of each batch is S4 > S3 > S2 > S1. For S1 and S2, the TSI value is quite small, which reflects that the samples are relatively stable and of good quality. For S3 and S4, the TSI value is extremely large, which also reflects the instability of the sample and the quality is inferior.
Supplemental Video S1: Animation video of the settling process of a good-quality Indigo Naturalis in the multiple light scattering instrument. In the whole animation video, it can be seen that S1 is almost unchanged, indicating that it is relatively stable. Please click here to download this File.
Supplemental Video S2: Animation video of the settling process of common Indigo Naturalis in the multiple light scattering instrument. In the whole animation video, it can be seen that S2 is almost unchanged, indicating that it is relatively stable. Please click here to download this File.
Supplemental Video S3: Animation video of the settling process of fake Indigo Naturalis in the multiple light scattering instrument. In the whole animation video, it can be seen that S3 is turbid at the third minute of scanning, indicating instability. Please click here to download this File.
Supplemental Video S4: Animation video of the settling process of fake Indigo Naturalis in the multiple light scattering instrument. In the whole animation video, it can be seen that S4 is turbid at the third minute of scanning, indicating instability. Please click here to download this File.
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Discussion
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 symptoms, sores and swellings, liver heat and epilepsy. Due to the variety of Indigo Naturalis, the quality difference is large, and the content detection process is complicated. On the one hand, the sources of Indigo Naturalis include Strobilanthes cusia (Nees) Kuntze, Persicaria tinctoria (Aiton) Spach, and Isatis tinctoria L., the geographical environment, and various harvesting times lead to the inherent quality differences14. On the other hand, the preparation process of Indigo Naturalis requires steps like soaking fermentation, lime-beating indigo, water fly refining, etc. However, this process is likely to produce different qualities of Indigo Naturalis, and the content of Indigo Naturalis varies from batch to batch. At present, several studies have shown that the qualifying rate of the index components content of Indigo Naturalis is low. Fake and inferior products cause major quality issues, which make it extremely difficult to use Indigo Naturalis clinically15,16,17. Therefore, it is urgent and indispensable for Indigo Naturalis to develop a standardized quality control method.
A key step in the process described is that the glass bottle containing the sample should be moved inside the sample tank as quickly as possible while avoiding shaking the bottle. Otherwise, inconsistent handling may produce misleading results. Second, the ambient temperature will have an impact on the instrument's set temperature. When the room temperature exceeds 30 °C, and the instrument temperature is lower than the room temperature, the instrument temperature will rise. It is worth noting that the room temperature should be controlled below the instrument temperature.
Although multiple light scattering has unique advantages over traditional methods, it also has its limitations. First, multiplex light scattering cannot definitively yield the exact content of a sample, but can only identify the authenticity and a roughly given range. Second, without further identification of Chinese herbal medicine, it is currently only relevant to the quality rapid assessment of Indigo Naturalis. Thirdly, the criteria of contemporary quality evaluation research are far from being met by depending on multiple light scattering methodologies and multiple light scattering instruments.
Compared with the existing water test method, the significance of the multiple light scattering method lies in the following points. First, it has high sensitivity and reliability. The sensitivity and resolution are much higher than that of naked-eye observation. The multiple light scattering device can capture how the solution changes over time and create an animated video of the entire process. Second, it can be analyzed qualitatively and quantitatively. Through non-contact measurement, the instrument may automatically determine the stability of the sample using its optical characteristics (transmission light, backscattering light, TSI, and particle size).
In the future, we believe that this method will eventually be helpful in the field of traditional Chinese medicine quality control, particularly in the assessment of authenticity. This study supported the validity and accuracy of the multiple light scattering approach in the quick assessment of Chinese medicine quality using Indigo Naturalis as an example. Consequently, as equipment and application technology advance continuously, multiple light scattering technologies will be combined with other detection techniques to complement one another, which have a greater impact on the quality control of Chinese herbal medicines in the future.
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Disclosures
The authors have no conflicts of interest to disclose.
Acknowledgments
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).
Materials
| Name | Company | Catalog Number | Comments |
| Analytical balance (1/10,000) | Sartorious, Germany | BSA224S | www.sartorius.com.cn |
| Funnel | Chengdu Kelong Chemical Co. LTD | Diameter 5 cm | www.cdkelongchem.com |
| Indigo Naturalis S1 | Xianyou, Fujian | 20210501 | |
| Indigo Naturalis S2 | Yaan, Sichuan | 20201102 | |
| Indigo Naturalis S3 | Xianyou, Fujian | 20161012 | |
| Indigo Naturalis S4 | Xianyou, Fujian | 20180305 | |
| Iron ring | Chengdu Kelong Chemical Co. LTD | / | www.cdkelongchem.com |
| Iron stand | Chengdu Kelong Chemical Co. LTD | / | www.cdkelongchem.com |
| Mili-Q ultra-pure water meter | Milipore, USA | Mili-Q | www.merckmillipore.com |
| Ninth sieve | Chengdu Kelong Chemical Co. LTD | Average aperture size 75 µm | www.cdkelongchem.com |
| Sample bottle | French Formulaction Company | Bottom diameter 2.6 cm, height 6 cm | www.formulaction.com |
| Seventh sieve | Chengdu Kelong Chemical Co. LTD | Average aperture size 125 µm | www.cdkelongchem.com |
| Turbisoft Lab multiple light scattering instrument | French Formulaction Company | Turbisoft Lab 2.3.1.125 Fanalyser 1.3.5 | www.formulaction.com |
| Weighing paper | Chengdu Kelong Chemical Co. LTD | / | www.cdkelongchem.com |
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