Using Multiple Light Scattering to Examine the Stability of Phyllanthus emblica L. Extracts Obtained with Different Extraction Methods

Podsumowanie

Here, we introduce a stability evaluation method based on multiple light scattering technology to evaluate the stability of traditional Chinese medicine extracts.

Streszczenie

The extraction intermediate of traditional Chinese medicine is the key intermediate in the preparation process, and its stability has an important impact on the effectiveness and quality of the final product. However, existing stability evaluation methods are often time-consuming and labor-intensive, requiring long-term observation and the operation of complex equipment (such as high-performance liquid chromatography), and it is difficult to obtain more physical information about the instability of the system. Therefore, there is an urgent need to establish a fast and accurate stability analysis technology for traditional Chinese medicine. Multiple light scattering is a cutting-edge analytical method that can accurately and rapidly evaluate the stability of traditional Chinese medicines in an environment-friendly manner without changing the nature or state of the sample or using organic reagents.

In this work, using the precise scanning data of multiple light scattering, the present protocol rapidly acquired the variation curves for layer thickness, particle migration speed, and average particle size over time. This enabled the precise identification of the mechanism and crucial characteristics causing the system's instability in its early stages. Of note, the research period for the extraction process can be considerably shortened by the detailed quantification of the system stability, which also allows for a quick, accurate, and in-depth analysis of the effects of various extraction processes on the stability of Phyllanthus emblica L.

Wprowadzenie

In the manufacturing of traditional Chinese medicine (TCM), the stability of the TCM extraction intermediates and related liquid preparations has always been the focus of inspection¹. The clinical efficacy of medicinal products, especially with polyphenols as the primary active ingredient, suffers due to significant stability issues^2,3. Sanajon oral liquid and Nuodikang oral liquid are examples of typical cases of this issue⁴. Therefore, it is crucial to learn how to use efficient tools to rapidly and accurately evaluate and optimize the stability of liquid intermediates in the TCM production process. Phyllanthus emblica L. (PE), a widespread medicinal plant in Southeast Asia, is thought to have good antioxidant properties⁵, as well as anti-inflammatory⁶, antibacterial⁷, and antitumor actions⁸. During the thermal extraction procedure, the tannins in PE transform violently⁹. Under catalysis with high temperatures, these tannins hydrolyze quickly to produce molecules such as gallic acid and ellagic acid, which lead to instability or precipitation due to their poor solubility¹. Current methods for evaluating TCM stability, such as accelerated testing or centrifugation, are usually cumbersome⁴, which limits the further development of pertinent preparation processes.

Based on the principle of multiple light scattering (MLS), we established a fast stability evaluation method for PEF extracts and analyzed the instability mechanism. MLS is a measurement method based on the scanning of near-infrared light sources. Any solution system change results in a change in the light intensity. The incident light is scattered when it is absorbed or penetrated by the particles of the sample. The system records the transmission light signal when it passes through the sample; if the light transmittance of the sample is poor, the system records the backscattering light signal. Compared with visual observation, this can save a lot of time¹ and can quickly and accurately analyze the instability phenomenon in detail, thus providing more useful information for guiding the optimization of the extraction process.

Protokół

1. Extract preparation

1. Accurately weigh an appropriate amount of PE, and add 10x (weight) of deionized water for reflux extraction.
2. Set five samples for reflux extraction for 0 h (E1), 0.5 h (E2), 1 h (E3), 1.5 h (E4), and 2 h (E5) after weighing.
3. After extraction, cool the samples to room temperature, and weigh to make up for the lost weight to ensure consistency with the pre-extraction weights.
4. Centrifuge the samples at 8,581 × g for 10 min to ensure the removal of insoluble material and herbal residues from the sample solution.
5. Use a pipette to add 20 mL of sample solution into the sample bottle to ensure that the solution added each time is at the same height.
   ​NOTE: Avoid contamination, such as fingerprints, on the scanning part of the sample bottle, ensure that the sample bottle is clean, and check whether there are visible scratches on the bottle surface. When adding the sample solution, be careful not to spill or splash on the sample bottle, and ensure that the liquid level is at the same height in each bottle.

2. Instrument operation

1. Turn on the MLS detection instrument, and warm it up for 30 min.
2. Click on the Create file button in the top menu (or click on the File | New file function) to create a new test file.
3. Click on the Show Turbiscan Lab Temperature button in the top menu to set the instrument target temperature to 25 °C.
   NOTE: The set temperature of the instrument must be higher than the room temperature; otherwise, the sample temperature will be affected by the room temperature.
4. Click on Program Scan in the top menu to enter the setup analysis program. Add the program to the list, and in the taskbar, add 5 min as a cycle, scan for 48 h to the analysis sequence, and set the balance time to 20 min. Select this analysis program for all the subsequent measurements.
5. Move the prepared sample bottle into the MLS detection system. After setting up the program, click on Start to start the measurement.
   ​NOTE: Be careful not to shake the glass bottle when loading the sample. The measurement can only be started after the sample temperature and setting temperature are balanced.

3. Multiple light scattering analysis program setting

1. After the data collection, click on the calculation parameters list to set the optical parameters to calculate the stability index (SI), particle size, and particle migration speed.
2. Set the optical parameters as follows: the continuous phase light transmission intensity (T₀) as 99.99% (water), the dispersed phase refractive index (np) as 1.36, and the continuous phase refractive index (nf) as 1.33.

Wyniki

Figure 1 shows the principle of multiple light measurement and the meaning of the collected results. In the MLS spectra results (Figure 2), the abscissa was the height of the sample cell, and the ordinate was the transmission (T%) and backscattering (BS%) intensity. By calculating the MLS spectra results, the system can obtain the changes in the key physical parameters of the sample during the measurement period, including the delta transm...

Dyskusje

The rapid and accurate assessment of TCM stability has always been a focus of TCM research. To provide more useful information for directing the improvement of the extraction process, this study analyzed the stability and instability mechanisms of a sample using a near-infrared non-destructive technology.

In this protocol, the important stability parameters are calculated based on accurate MLS scan data. MLS scans can collect the transmission (T%) and backscattering (BS%) of ...

Materiały

Name	Company	Catalog Number	Comments
Adjustable electric heating jacket	Beijing Kewei Yongxing Instrument Co., Ltd	MH-1000	www.keweiyq.com
Analytical balance(1/10000)	Sartorious, Germany	BSA224S	www.sartorius.com.cn
CNC ultrasonic instrument	Kunshan Ultrasonic Instrument Co., Ltd	KQ-500DE	www.ks-csyq.com
GL-16 high-speed centrifuge	Sichuan Shuke Instrument Co., Ltd	18091403	www.sklxj.com
Phyllanthus emblica L.	Hehuachi medicinal materials market	YJL2004	Produced in Yunnan
Turbisoft Lab multiple light scattering instrument	French Formulaction Company	Turbisoft Lab 2.3.1.125 Fanalyser 1.3.5	www.formulaction.com
UPR-II-5T ultra-pure water device	Sichuan ULUPURE  Ultrapure Technology Co., Ltd	Z16030559	www.ccdup.com