Name: optimization of processing of tiebangchui with highland barley wine based on the box-behnken design combined with the entropy method
Uploaded: 2023-05-19T14:40:00.000+00:00
Duration: 9 min 12 s
Description: Watch this Scientific Journal Video about Optimization of Processing of Tiebangchui with Highland Barley Wine Based on the Box-Behnken Design Combined with the Entropy Method at JoVE.com

This work was financially supported by the National Natural Science Foundation of China (No. 82130113), the China Postdoctoral Science Foundation (No. 2021MD703800), the Science Foundation for Youths of Science &#38; Technology Department of Sichuan Province (No. 2022NSFSC1449), and the &#34;Xinglin Scholars&#34; Research Promotion Program of Chengdu University of Traditional Chinese Medicine (No. BSH2021009).

In this study, the processing technology of TBC processed with highland barley wine was optimized with a Box-Behnken design combined with the entropy method. DDA and MDA content were used as evaluation indicators, and the weight coefficient of each evaluation index was determined by the entropy method.
1. Experimental preparation
<ol>
	<li>Prepare highland barley wine15.
	<ol>
		<li>Take 500.00 g of black highland barley rice and add five times the amount of water. Cook the rice until the remaining water is absorbed (~2 h). Pour it out, wait until the temperature falls to 37 &#176;C, add 4 g of Jiuqu (see Table of Materials), mix well, seal the can, wrap the container with cotton wool, and let it stew for 7 days.</li>
		<li>Add 300 mL of water on the 7th day and seal again. On the 8th day, begin removing the wine and replace with 300 mL of water afterward. Seal and ferment for 1 day, take the wine, and add 300 mL of water again. Repeat this procedure three times and combine the liquors.</li>
		<li>Bring to a boil, then reduce the heat to a simmer, and continue cooking until the remaining water is absorbed.</li>
	</ol>
	</li>
	<li>To prepare processed products, accurately weigh the TBC in a container, add highland barley wine, and soak for 1 day. Then, dry in a constant temperature electric drying oven. 
	NOTE: The drying temperature should be less than 40 &#176;C to avoid changes in the alkaloid composition.</li>
	<li>Prepare test sample solution.
	<ol>
		<li>Accurately weigh TBC processed product powder (2 g) in a conical flask, add 40% ammonia solution, and perform ultrasound-assisted extraction with isopropanol-ethyl acetate (1:1) mixed solvents (50 mL) (power: 200 W; frequency: 40 kHz; temperature: 40 &#176;C) for 30 min. 
		NOTE: To prepare 40% ammonia solution, transfer 40 mL of ammonia to a 100 mL volumetric flask and then dilute with pure water.</li>
		<li>Adjust the extracted solution to the original weight by adding an isopropanol-ethyl acetate mixture (1:1 v/v).</li>
		<li>Accurately transfer the extracted solution (25 mL) to a round-bottom flask for the recovery of the solvent under reduced pressure until dry.</li>
		<li>Finally, transfer 0.05% hydrochloric acid-methanol solution to dissolve the residue from step 1.3.3 in a 5 mL volumetric flask and dilute with 0.05% methanol hydrochloride solution. Filter the solution through a 0.22 &#181;m microporous membrane filter prior to injection into the high-performance liquid chromatography (HPLC) systems. 
		NOTE: Prepare 0.05% methanol hydrochloride acid by adding 0.05 mL of hydrochloric acid to a 100 mL volumetric flask, then dilute with methanol.</li>
	</ol>
	</li>
	<li>Prepare a standard solution by weighing 5.18 mg of benzoylaconine, 13.13 mg of aconitine, 10.05 mg of 3-deoxyaconitine, and 10.09 mg of 3-acetylaconitine accurately, and then place the solids in a 5 mL volumetric flask individually. Dilute with 0.05% methanol hydrochloride solution.</li>
</ol>
2. Chromatographic condition
<ol>
	<li>Set up the chromatographic conditions as shown in Table 1 for HPLC. Details of the instruments used are provided in the Table of Materials.</li>
</ol>
3. System adaptability test
<ol>
	<li>Range of linearity 
	NOTE: First, we used HPLC to determine the peak areas of benzoylaconitine, aconitine, 3-deoxyaconitine, and 3-acetylaconitine in the sample, and then randomly determined the peak area of one known concentration of standard solution. Next, we compared the difference between two peak areas (sample solution and standard solution) to estimate the concentration of benzoylaconitine, aconitine, 3-deoxyaconitine, and 3-acetylaconitine in different samples, and then adjusted the standard solution into a linear range to include the concentration of the sample in the curve. The standard curve concentrations are shown in Table 2.
	<ol>
		<li>Prepare benzoylaconitine reference solutions containing 1.036 mg/mL, 0.518 mg/mL, 0.2072 mg/mL, 0.1036 mg/mL, and 0.0518 mg/mL.</li>
		<li>Prepare aconitine reference solutions containing 1.313 mg/mL, 0.5252 mg/mL, 0.2626 mg/mL, 0.1313 mg/mL, and 0.05252 mg/mL.</li>
		<li>Prepare 3-deoxyaconitine reference solutions containing 1.005 mg/mL, 0.5025 mg/mL, 0.201 mg/mL, 0.1005 mg/mL, and 0.402 mg/mL.</li>
		<li>Prepare 3-acetylaconitine reference solutions containing 0.2018 mg/mL, 0.1009 mg/mL, 0.04036 mg/mL, 0.02018 mg/mL, and 0.01009 mg/mL.</li>
		<li>Investigate the linearity of each compound by plotting the peak area versus injection concentration.</li>
	</ol>
	</li>
	<li>To perform the precision test, inject 10 &#181;L of each reference solution into the HPLC system six times daily and employ the same HPLC conditions described in step 2.1 to run the samples Record the peak area of each component.</li>
	<li>Perform intraday stability testing by injecting 10 &#181;L of the prepared sample solution via step 1.3 and determine the peak areas after 0 h, 2 h, 4 h, 8 h, 14 h, 12 h, and 24 h16.</li>
	<li>Perform a reproducibility test by taking six samples of the same batch of TBC to prepare the test sample solution, according to step 1.3. Inject 10 &#181;L of each sample into the HPLC system and run the samples as described in step 2.1.</li>
	<li>Perform the recovery test to evaluate the accuracy of the method. Add 100% of the standard solution of each index component (benzoylaconitine, aconitine, 3-deoxyaconitine, and 3-acetylaconitine) in the test solution to calculate the recovery rate, respectively. For example, as the content of benzoylaconitine is 0.1524 mg/mL in the TBC sample, accurately weigh 0.1524 mg of benzoylaconitine standards and add to the TBC sample, then prepare the test sample solution according to step 1.3. Run these samples with the same HPLC conditions described in step 2.1. Calculate the recovery rate using Equation (1): 
	<img alt="Equation 1" src="/files/ftp_upload/65154/65154eq01.jpg" style="margin: auto;" />&#160; &#160; (1) 
	Here, A is the amount of component (benzoylaconitine, aconitine, 3-deoxyaconitine, or 3-acetylaconitine) to be measured in the sample solution, B is the amount of standard added (benzoylaconitine, aconitine, 3-deoxyaconitine, or 3-acetylaconitine), and C is the measured value of the solution containing the standard solution and the sample solution (see Table 3). Refer to step 2.1 for the chromatographic conditions to perform the above steps. The recovery rate reflects the degree of loss of the target component (benzoylaconitine, aconitine, 3-deoxyaconitine, or 3-acetylaconitine) during the sample analysis; the higher the recovery rate, the lower the loss of the target component.</li>
</ol>
4. Single factor test of TBC processed with highland barley wine
NOTE: The ratio between highland barley wine and TBC, slice thickness of TBC, and soaking time will affect the dissolution of more toxic components (aconitine, 3-deoxyaconitine, and 3-acetylaconitine) in TBC during the TBC processed with highland barley wine17. The single factor test and Box-Behnken design were used to investigate the influence of the ratio of highland barley wine to TBC, slice thickness of TBC, and soaking time.
<ol>
	<li>Perform the highland barley wine addition test (A) by setting up five groups of tests, each with 30 g of TBC, where the amount of highland barley wine is two, three, four, five, and six times the amount of TBC in the recipe. The soaking time is 12 h, and the slices are 1.0 cm thick18. 
	NOTE: Each group of the same condition test should be processed in three parallel groups.</li>
	<li>Perform the soaking time test (B) by setting up five groups of tests, each with 30 g of TBC. The soaking times are 12 h, 24 h, 36 h, and 48 h. The amount of highland barley wine is five times that of TBC, and the slices are 1.0 cm thick19. 
	NOTE: Each group of the same condition experiment should be processed in three parallel groups.</li>
	<li>Perform the slicing thickness test (C) by setting up five groups of tests, each with 30 g of TBC. The slices are 0.5, 1.0, 1.5, 2.0, and 2.5 cm thick, the soaking time is 24 h, and the amount of highland barley wine is five times that of TBC20. 
	NOTE: Each group of the same condition experiment should be processed in three parallel groups.</li>
	<li>Accurately weigh processed products for each test group to prepare test sample solution according to step 1.3. Determine the peak area of each sample by HPLC and use the standard curve to estimate the amounts of MDAs and DDAs. In the standard curve, y is the peak area and x is the content. The content of MDAs is benzoyl aconitine, and the content of DDAs is the sum of aconitine, 3-deoxyaconitine, and 3-acetylaconitine.</li>
	<li>Use the total content of DDAs and the content of MDAs as evaluation indicators, and determine the weight coefficient of each evaluation index and the comprehensive scoring via the entropy method (section 5). 
	CAUTION: TBC is toxic, and thus protective measures should be taken during processing.</li>
</ol>
5. Entropy method to calculate the comprehensive scoring
NOTE: We use the experimental data of the slicing thickness test in the single factor test as an example to illustrate the calculation process in detail. We use the peak area of the components in each sample in Supplementary Table S1 and the standard curve in Table 2 to calculate the content of MDAs and DDAs (see Supplementary Table S2). In the linear equation, y is the peak area and x is the content. In this study, the moderately toxic MDA (benzoylaconitine) was used as the positive indicator, and the total content of DDAs (aconitine, 3-deoxyaconitine, and 3-acetylaconitine) with high toxicity was used as the negative indicator. The content of MDAs is benzoyl aconitine, and the content of DDAs is the sum of aconitine, 3-deoxyaconitine, and 3-acetylaconitine. Each sample has two evaluation indicators: i = 1,2,&#8230;,n, and j = 1,2,&#8230;m21.
<ol>
	<li>Use Equation (2) to standardize the content of MDAs22. 
	<img alt="Equation 2" src="/files/ftp_upload/65154/65154eq02.jpg" style="margin: auto;" />&#160; &#160; &#160;(2) 
	Thus, <img alt="Equation 3" src="/files/ftp_upload/65154/65154eq03.jpg" style="margin: auto;" /> 
	NOTE: Xij is the value of the j-th indicator of the i-th sample. Xij* is the standardized value of Xij. For example, i = 3 and j = 1, X31 represents the value of the first indicator of the third sample, and <img alt="Equation 4" src="/files/ftp_upload/65154/65154eq04.jpg" style="margin: auto;" /> is the standardized value of the first indicator in the third sample. <img alt="Equation 5" src="/files/ftp_upload/65154/65154eq05.jpg" style="margin: auto;" /> are shown in Supplementary Table S3.</li>
	<li>Use Equation (3) to standardize the total content of the DDAs23. 
	<img alt="Equation 6" src="/files/ftp_upload/65154/65154eq06.jpg" style="margin: auto;" />&#160; &#160; &#160;(3) 
	<img alt="Equation 7" src="/files/ftp_upload/65154/65154eq07.jpg" style="margin: auto;" /> 
	NOTE: Here, i = 3, j = 2, represents the second indicator of the third sample. <img alt="Equation 8" src="/files/ftp_upload/65154/65154eq08.jpg" style="margin: auto;" /> is the standardized value of the second indicator in the third sample. <img alt="Equation 9" src="/files/ftp_upload/65154/65154eq09.jpg" style="margin: auto;" /> are shown in Supplementary Table S3.</li>
	<li>Use Equations (4) and (5) to define the entropy value (Hj) of each indicator23.
	<ol>
		<li>Calculate the probability of the j-th trial under the i-th evaluation indicator Pij using equation (4). 
		<img alt="Equation 10" src="/files/ftp_upload/65154/65154eq10.jpg" style="margin: auto;" />&#160; &#160; &#160;(4) 
		For number 3, 
		<img alt="Equation 11" src="/files/ftp_upload/65154/65154eq11.jpg" style="margin: auto;" /> 
		<img alt="Equation 12" src="/files/ftp_upload/65154/65154eq12.jpg" style="margin: auto;" /> 
		&#8203;NOTE: The probability values for the first indicator and second indicator of the third sample are 0.2374 and 0.2812, respectively. <img alt="Equation 13" src="/files/ftp_upload/65154/65154eq13.jpg" style="margin: auto;" /> are shown in Supplementary Table S3.</li>
		<li>Calculate the information entropy Hj. 
		<img alt="Equation 14" src="/files/ftp_upload/65154/65154eq14.jpg" style="margin: auto;" />&#160; &#160; &#160;(5) 
		<img alt="Equation 15" src="/files/ftp_upload/65154/65154eq15.jpg" style="margin: auto;" /> 
		<img alt="Equation 16" src="/files/ftp_upload/65154/65154eq16.jpg" style="margin: auto;" /> 
		NOTE: H1 is the entropy of the first indicator (MDAs) and H2 is the entropy of the second indicator (DDAs) in the slicing thickness test.</li>
	</ol>
	</li>
	<li>Use Equation (6) to calculate the indicator weights (Wj)23. 
	<img alt="Equation 17" src="/files/ftp_upload/65154/65154eq17.jpg" style="margin: auto;" /> (6) 
	<img alt="Equation 18" src="/files/ftp_upload/65154/65154eq18.jpg" style="margin: auto;" />&#160; &#160;= 33.3% 
	<img alt="Equation 19" src="/files/ftp_upload/65154/65154eq19.jpg" style="margin: auto;" />&#160; &#160;= 66.7% 
	NOTE: Wj is the weight coefficient of each indicator. In the slicing thickness test, the weight coefficient of the positive indicator (MDAs) and negative indicator (DDAs) are 33.3% and 66.7%, respectively.</li>
	<li>Use Equation (7) to calculate the comprehensive scoring of the indicators23. 
	<img alt="Equation 20" src="/files/ftp_upload/65154/65451eq20v2.jpg" style="margin: auto;" /> (7) 
	For number 3, <img alt="Equation 21" src="/files/ftp_upload/65154/65154eq21.jpg" style="margin: auto;" /> 
	<img alt="Equation 22" src="/files/ftp_upload/65154/65154eq22.jpg" style="margin: auto;" /> 
	NOTE: Si is the comprehensive scoring of each sample. We need to obtain the highest score as the central point in the Box-Behnken design. S1, S2, S3, S4, and S5 are show in Supplementary Table S3.</li>
</ol>
6. Box-Behnken design
<ol>
	<li>Through the single factor test, use the condition with the highest comprehensive scoring (see Table 4, Table 5, Table 6, and Figure 2) as the center point of the response surface. Use the amount of highland barley wine (A), soaking time (B), and slice thickness of TBC (C) as the influencing factors and the comprehensive scoring as the response value24. 
	NOTE: Based on the single factor data in Table 4, Table 5, and Table 6, the highest comprehensive scoring is calculated by equations (2), (3), (4), (5), (6), and (7) in section 5, and the best point is obtained. The amount of highland barley wine was five times that of TBC, the soaking time was 36 h, and the slicing thickness was 1.0 cm.</li>
</ol>
7. Box-Behnken design software operation steps
<ol>
	<li>Open the software (see Table of Materials) and select New Design | Box-Behnken Design (see step 5.1; Supplementary File 1).
	<ol>
		<li>Input the number of influencing factors and input the level information (three-level-three-factor; see Table 7). The Box-Behnken design is composed of 17 experiments in this study. Finally, click Continue (see step 5.2; Supplementary File 1).</li>
		<li>Set the comprehensive scoring (Y) by equations (2), (3), (4), (5), (6), and (7) in section 5 as the response. Input the number of response values (image shows only one response value) and click Finish (see step 5.3; Supplementary File 1).</li>
		<li>Process the TBC with highland barley wine according to the design results and complete the experiment based on the 17 scenarios designed for the response surface.</li>
		<li>Prepare the sample solutions by following step 1.3 and calculate the total content of the MDAs and DDAs by the HPLC system.</li>
		<li>Calculate the comprehensive scoring for each group by equations (2), (3), (4), (5), (6), and (7) in step 5, and input the score results (see step 5.4; Supplementary File 1).</li>
	</ol>
	</li>
	<li>Click analyze to analyze the date and model information (see step 5.4.1; Supplementary File 1).
	<ol>
		<li>Perform statistical validation of polynomial equations and response surface analysis plotted in 3D model plots obtained by the software.</li>
		<li>Click on ANOVA in the top menu and observe the results table.</li>
	</ol>
	</li>
	<li>Click Optimization to view the predicted optimal process conditions (see step 5.4.2; Supplementary File 1).</li>
</ol>
8. Validation test
<ol>
	<li>According to the results predicted from the Box-Behnken response surface design, in step 7.3, identify the optimal processing condition of TBC. Here, it is as follows: TBC is soaked for 24 h in five times the amount of highland barley wine, and the thickness of the TBC is 1.5 cm. Take the optimal level of influencing factors as processing conditions and set up three parallel sets of experiments to verify the stability of the processing technology.</li>
</ol>

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C., 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=Box-Behnken+design:+an+alternative+for+the+optimization+of+analytical+methods.">Box-Behnken design: an alternative for the optimization of analytical methods.</a> Analytica Chimica Acta. 597 (2), 179-186 (2007).</li><li>Dong, R., Lu, Y., Wang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+process+optimization+of+vinegar+roasting+of+Bupleurum+chinense+by+entropy+weight+method+combined+with+Box-Behnken+response+surface+method+and+its+protective+effect+on+mice+liver+injury.">The process optimization of vinegar roasting of Bupleurum chinense by entropy weight method combined with Box-Behnken response surface method and its protective effect on mice liver injury.</a> Science and Technology of Food Industry. 42 (23), 209-217 (2021).</li><li>Li, W. J., 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=Analysis+on+the+times+of+Polygonati+Rhizoma+steamed+by+multiple+times+based+on+entropy+weight+and+gray+relative+analysis+method.">Analysis on the times of Polygonati Rhizoma steamed by multiple times based on entropy weight and gray relative analysis method.</a> China Journal of Traditional Chinese Medicine and Pharmacy. 36 (11), 6764-6769 (2021).</li><li>Huang, B. J., Liu, X. T., Mao, Y. M., Qi, B., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+surface+methodology+combined+with+analytic+hierarchy+process+to+optimize+the+processing+technology+of+Custutae+semen+with+wine.">Response surface methodology combined with analytic hierarchy process to optimize the processing technology of Custutae semen with wine.</a> Lishizhen Medicine and Materia Medica Research. 33 (8), 1890-1894 (2022).</li><li>Wang, J., Meng, X. H., Chai, T., Yang, J. L., Shi, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diterpenoid+alkaloids+and+one+lignan+from+the+roots+of+Aconitum+pendulum+Busch.">Diterpenoid alkaloids and one lignan from the roots of Aconitum pendulum Busch.</a> Natural Products and Bioprospecting. 9 (6), 419-423 (2019).</li><li>Xie, H. 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=Metabolomics+study+of+aconitine+and+benzoylaconine+induced+reproductive+toxicity+in+Be+Wo+cell.">Metabolomics study of aconitine and benzoylaconine induced reproductive toxicity in Be Wo cell.</a> Chinese Journal of Analytical Chemistry. 43 (12), 1808-1813 (2015).</li><li>Han, Y. F., 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=Optimization+of+extraction+process+for+Yangyin+Runmu+granules+by+Box-Behnken+design+based+on+entropy+weight+method-analytic+hierarchy+process+method.">Optimization of extraction process for Yangyin Runmu granules by Box-Behnken design based on entropy weight method-analytic hierarchy process method.</a> Chinese Journal of Modern Applied Pharmacy. 39 (7), 896-903 (2022).</li><li>Chen, F. G., 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=Optimization+of+the+baked+drying+technology+of+Clinamomi+Ramulus+based+on+CRITIC+combined+with+Box-Behnken+response+surface+method.">Optimization of the baked drying technology of Clinamomi Ramulus based on CRITIC combined with Box-Behnken response surface method.</a> Journal of Chinese Medicinal Materials. 2022 (8), 1838-1842 (2022).</li><li>Pan, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+stir-baking+process+of+Coix+lacryma-Jobi+Var.Mayuen+Kernel+by+Box-Behnken+response+surface+methodology.">Optimization of stir-baking process of Coix lacryma-Jobi Var.Mayuen Kernel by Box-Behnken response surface methodology.</a> Shandong Chemical Industry. 51 (14), 73-75 (2022).</li></ol>

The authors have no conflicts of interest to disclose.

As a commonly used Tibetan medicine with toxic effects, the toxicity-attenuating effect of processing is extremely important for TBC&#39;s clinical application25. In this study, the processing technology of TBC processed with highland barley wine was optimized. By reviewing the main active ingredients and relating the pharmacological effects of TBC, we found that TBC alkaloids have anti-inflammatory and analgesic effects and can be used to treat rheumatoid arthritis. In this study, a moderate toxi...

Tiebangchui (TBC), the dried root of Aconitum pendulum Busch, is a well-known Tibetan medicine and was initially recorded in the classic Tibetan medical book &#34;Four Medical Tantra&#34;1,2. According to &#34;Drug Standards of the Ministry of Health of the People&#39;s Republic of China (Tibetan Medicine)&#34;, TBC is effective in expelling cold, relieving pain, dispelling wind, and calming shock, and is commonly used to treat rheumatoid arthritis in clinics3,4,5.
TBC mainly contains alkaloids, including highly toxic diester-diterpenoid alkaloids (DDAs), and the moderately toxic monoester-diterpenoid alkaloids (MDAs)6,7,8. These chemical components are active ingredients with medicinal effects but are toxic. One of the most famous active and toxic ingredients, aconitine, causes poisoning when it exceeds 1 mg9. Therefore, improper or excessive use of TBC might result in poisoning and even death, and the toxicity attenuation and efficacy reservation of TBC is crucial for its safe clinical application10,11.
Processing is an effective method for detoxifying TBC. According to ancient Tibetan medicine books, processing with highland barley wine is an efficient way to attenuate toxicity and preserve the efficacy of TBC. TBC is soaked in highland barley wine, stored for one night, dried, and added to medicines12. However, the specific processing technology and potential influencing factors are rarely reported, and the traditional processing process often relies on experience and lacks standardized methods. Hence, modern scientific and technological methods for optimizing and standardizing the processing process are needed.
The Box-Behnken design method is used in investigating interactions among different factors and their influence on comprehensive scoring through quadratic polynomial fitting. This design allows the intuitive observation of optimal conditions and has been widely used in the field of pharmacy13. For example, the Box-Behnken design method, based on the entropy method, successfully optimized the processing technology of stir-frying with vinegar of Curcuma Longa Radix14. In this study, the Box-Behnken response surface experimental design combined with the entropy method was used in optimizing the processing technology of TBC processed with highland barley wine. The optimized processing technology is expected to ensure quality control and safe clinical use.

<table><tbody><tr><td>Aconitine</td><td>Chengdu Push Biotechnology Co.,Ltd</td><td>PS000905</td><td></td></tr><tr><td>3-Acetylaconitine</td><td>Chengdu Push Biotechnology Co.,Ltd</td><td>PS010552</td><td></td></tr><tr><td>3-Deoxyaconitine</td><td>Chengdu Push Biotechnology Co.,Ltd</td><td>PS011258</td><td></td></tr><tr><td>Benzoylaconine</td><td>Chengdu Push Biotechnology Co.,Ltd</td><td>PS010300</td><td></td></tr><tr><td>Circulating water vacuum pump</td><td>Gongyi City Yuhua Instrument Co., Ltd</td><td>SHZ-DIII</td><td></td></tr><tr><td>Design-Expert&amp;nbsp;</td><td>State-East Corporation</td><td>8.0.6</td><td></td></tr><tr><td>Electric constant temperature drying oven</td><td>Shanghai Yuejin Medical Equipment Co., Ltd</td><td>101-3-BS</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>High performance liquid chromatography</td><td>Shimadzu Enterprise Management (China) Co., Ltd</td><td>shimadzu 2030</td><td></td></tr><tr><td>Highland barley rice</td><td>Kangding City, Ganzi Tibetan Autonomous Prefecture, Sichuan Province</td><td>20221015</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>Rotary evaporator</td><td>Shanghai Yarong Biochemical Instrument Factory</td><td>RE-2000A</td><td></td></tr><tr><td>Starter of liquor-making</td><td>Angel Yeast CO., Ltd</td><td>BJ22-104</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>

optimization of processing of tiebangchui with highland barley wine based on the box-behnken design combined with the entropy method

The processing of toxic ethnomedicines is of great significance for their safe clinical application. Thus, the limitations of traditional processing should be addressed, and the processing method of ethnomedicines should be standardized using modern research methods. In this study, the processing technology of a commonly used Tibetan medicine Tiebangchui (TBC), the dried root of Aconitum pendulum&#160;Busch, processed with highland barley wine was optimized. Diester-diterpenoid alkaloid (DDA) (aconitine, 3-deoxyaconitine, 3-acetylaconitine) and monoester-diterpenoid alkaloid (MDA) (benzoylaconine) content were used as evaluation indicators, and the weight coefficient of each evaluation index was determined by the entropy method.
The single factor test and Box-Behnken design were used in investigating the influence of the ratio between highland barley wine and TBC, slice thickness of TBC, and processing time. Comprehensive scoring was performed according to the objective weight of each index determined by the entropy method. The optimal processing conditions of TBC with highland barley wine were as follows: the amount of highland barley wine is five times that of TBC, a soaking time of 24 h, and a TBC thickness of 1.5 cm. The results showed that the relative standard deviation between the verification test and predicted value was less than 2.55% and the optimized processing technology of TBC processed with highland barley wine is simple, feasible, and stable, and so can provide a reference for industrial production.

In this study, the precision, stability, repeatability, and sample recovery of TBC indicated that the method is feasible. The four index components in TBC had a good linear relationship within a specific concentration range. Typical chromatograms are shown in Figure 1. The precision test results (Table 8) showed that the relative standard deviation (RSD) of the peak areas were 2.56%, 1.49%, and 2.03% for benzoylaconine, aconitine, and 3-deoxyaconitine, respectively, and 0.21...

Watch this Scientific Journal Video about Optimization of Processing of Tiebangchui with Highland Barley Wine Based on the Box-Behnken Design Combined with the Entropy Method at JoVE.com

Optimization of Processing of Tiebangchui with Highland Barley Wine Based on the Box-Behnken Design Combined with the Entropy Method

The present protocol describes an efficient method for optimization of the processing technology of Tiebangchui processed with highland barley wine based on a Box-Behnken design response surface combined with the entropy method.

The processing of toxic ethnomedicines is of great significance for their safe clinical application. Thus, the limitations of traditional processing ...

optimization-processing-tiebangchui-with-highland-barley-wine-based

Research

JoVE Journal

Medicine

561 Views.  Chengdu University of Traditional Chinese Medicine. Zoals het bedrijf vroeger debatteerde over medicijnen met toxisch effect, is Tiebangchui voor toxiciteitsverzwakking uiterst belangrijk voor klinische toepassing. De optimalisatietechnologie van de Tiebangchui met een hooglandgerstwijn is eenvoudig, zichtbaar en stabiel. die deze variantie kan bieden voor industriële productie.Box-Behnken-ontwerp in combinatie met de entropiemethode maakt de observatie tussen tafels van geoptimaliseerde omstandigheden mogelijk en is op grote schaal ge...

Video: Optimization of Processing of Tiebangchui with Highland Barley Wine Based on the Box-Behnken Design Combined with the Entropy Method

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (&#946;-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

Genetics

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (Zea mays L.)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole grain composition and processing characteristics vary among maize hybrids and can impact the quality of the final processed product. Therefore, in order to produce healthier processed food products from maize, it is necessary to know how to optimize processing parameters for particular sets of germplasm to account for these differences in grain composition and processing characteristics. This includes a better understanding of how current processing techniques impact the nutritional quality of the final processed food product. Here, we describe a microscale protocol that both simulates the processing pipeline to produce cornflakes from large flaking grits and allows for the processing of multiple grain samples simultaneously. The flaking grits, the intermediate processed products, or final processed product, as well as the corn grain itself, can be analyzed for nutritional content as part of a high-throughput analytical pipeline. This procedure was developed specifically for incorporation into a maize breeding research program, and it can be modified for other grain crops. We provide an example of the analysis of insoluble-bound ferulic acid and p-coumaric acid content in maize. Samples were taken at five different processing stages. We demonstrate that sampling can take place at multiple stages during microscale processing, that the processing technique can be utilized in the context of a specialized maize breeding program, and that, in our example, most of the nutritional content was lost during food product processing.

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize Zea mays L.

Here, we present a microscale protocol for processing grain samples and for incorporating this microscale approach into a high-throughput analytical pipeline. This is a higher throughput adaptation of currently available protocols.

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole ...

Environment

Derivatization and Separation of Barley Flour Metabolites into Four Fractions

Metabolomic Analysis of Barley by Gas Chromatography/Mass Spectrometry

Climate change increases drought risk to agriculture and impacts both food nutrient content and overall food security. Metabolomics is one way to observe and quantify the impacts of drought on grain and other agricultural products. The identified metabolites may allow for the identification of the biochemical response that allows the plant to tolerate stressful environments. The methodology presented herein allowed for the total metabolomic analysis of barley flour using gas chromatography/mass spectrometry (GC/MS). 
Barley flour metabolite extracts were fractionated into four fractions based on polarity. To allow for analysis by GC/MS, metabolites were derivatized to increase volatility and metabolite separation: fatty acids esters were derivatized into fatty acid methyl esters; sugars were oximated into their straight chain form; and metabolites with hydroxyl groups were converted to their corresponding silyl ethers. The derivatized samples were injected into the GC/MS and the generated mass spectra were used for metabolite identification by comparing the generated spectra to the National Institute of Standards and Technology (NIST) Tandem Mass Spectra library. The method described here can also be used to examine the total metabolome for other plants, furthering our understanding of the biochemical responses of stressed plants.

A method for metabolomic analysis of barley is presented. The method entails fractionation and derivatization of metabolites and analysis thereof by gas chromatography/mass spectrometry (GC/MS). Metabolomic analysis can be used to determine the effect of intercropping and drought stress on the grain.

Climate change increases drought risk to agriculture and impacts both food nutrient content and overall food security. Metabolomics is one way to observe ...

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices currently entering the market. In order to evaluate the safety of these devices, in preclinical studies, reliable techniques are needed. However, the hard metal components utilised in some retinal implants are not compatible with traditional histological processes, particularly in consideration for the delicate nature of the surrounding tissue. Here we describe techniques for assessing the health of the eye directly adjacent to a retinal implant secured epiretinally with a metal tack.
Retinal prostheses feature electrode arrays in contact with eye tissue. The most commonly used location for implantation is the epiretinal location (posterior chamber of the eye), where the implant is secured to the retina with a metal tack that penetrates all the layers of the eye. Previous methods have not been able to assess the proximal ocular tissue with the tack in situ, due to the inability of traditional histological techniques to cut metal objects. Consequently, it has been difficult to assess localized damage, if present, caused by tack insertion.
Therefore, we developed a technique for visualizing the tissue around a retinal tack and implant. We have modified an established technique, used for processing and visualizing hard bony tissue around a cochlear implant, for the soft delicate tissues of the eye. We orientated and embedded the fixed eye tissue, including the implant and retinal tack, in epoxy resin, to stabilise and protect the structure of the sample. Embedded samples were then ground, polished, stained, and imaged under various magnifications at incremental depths through the sample. This technique allowed the reliable assessment of eye tissue integrity and cytoarchitecture adjacent to the metal tack.

Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

Design and Optimization Strategies of a High-Performance Vented Box

This study aims to solve the problems of air flow chaos and poor performance in a vented box caused by the heterogeneous distribution of airflow through the design of the internal structure of the vented box with constant energy consumption. The final goal is to evenly distribute the airflow inside the vented box. Sensitivity analysis was performed for three structural parameters: the number of pipes, the number of holes in the middle pipe, and the number of each increment from the inside to the outside pipe. A total of 16 random array sets of three structural parameters with four levels were determined using the orthogonal experimental design. Commercial software was used for the construction of a 3D model for the selected experimental points, and this data was used to obtain the airflow velocities, which were then used to obtain the standard deviation of each experimental point. According to the range analysis, the combination of the three structural parameters was optimized. In other words, an efficient and economical optimization method considering the performance of the vented box was established, and it could be widely used to extend the storage time of fresh food.

Here, we present the range analysis method to optimize the sample points generated by an orthogonal experimental design to ensure that fresh food can be stored in a vented box for a long time by regulating the airflow pattern.

This study aims to solve the problems of air flow chaos and poor performance in a vented box caused by the heterogeneous distribution of airflow through ...

Engineering

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Bioengineering

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

Process Evaluation and Kinetics of Recombinant Chitin Deacetylase Expression in E. coli Rosetta pLysS Cells Using a Statistical Technique

In recent years, the greener route of the deacetylation of chitin to chitosan using the enzyme chitin deacetylase has gained importance. Enzymatically converted chitosan with emulating characteristics has a broad range of applications, particularly in the biomedical field. Several recombinant chitin deacetylases from various environmental sources have been reported, but there are no studies on process optimization for the production of these recombinant chitin deacetylases. The present study used the central composite design of response surface methodology to maximize the recombinant bacterial chitin deacetylase (BaCDA) production in E. coli Rosetta pLysS. The optimized process conditions were 0.061% glucose concentration, 1% lactose concentration, an incubation temperature of 22 &#176;C, an agitation speed at 128 rpm, and 30 h of fermentation. At optimized conditions, the expression due to lactose induction was initiated after 16 h of fermentation. The maximum expression, biomass, and BaCDA activity were recorded 14 h post-induction. At the optimized condition, the BaCDA activity of expressed BaCDA was increased ~2.39-fold. The process optimization reduced the total fermentation cycle by 22 h and expression time by 10 h post-induction. This is the first study to report the process optimization of recombinant chitin deacetylase expression using a central composite design and its kinetic profiling. Adapting these optimal growth conditions could result in cost-effective, large-scale production of the lesser-explored moneran deacetylase, embarking on a greener route for biomedical-grade chitosan production.

Process Evaluation and Kinetics of Recombinant Chitin Deacetylase Expression in E. coli Rosetta pLysS Cells Using a Statistical Technique

In the current protocol, a statistical technique, central composite design (CCD), was applied to optimize the process conditions for the expression of recombinant bacterial chitin deacetylase (BaCDA) in E. coli Rosetta pLysS cells. The employment of CCD resulted in a ~2.39-fold increase in the expression and activity of BaCDA.

In recent years, the greener route of the deacetylation of chitin to chitosan using the enzyme chitin deacetylase has gained importance. Enzymatically ...

Optimization of the Epimedii Folium Mutton-Oil Processing Technology and Testing Its Effect on Zebrafish Embryonic Development

As a traditional Chinese medicine (TCM), Epimedii folium (EF) has a history in medicine and food that is &#62; 2,000 years old. Clinically, EF processed with mutton oil is often used as a medicine. In recent years, reports of safety risks and adverse reactions of products that use EF as a raw material have gradually increased. Processing can effectively improve the safety of TCM. According to TCM theory, mutton-oil processing can reduce the toxicity of EF and enhance its tonifying effect on the kidneys. However, there is a lack of systematic research and evaluation of EF mutton-oil processing technology. In this study, we used the Box-Behnken experimental design-response surface methodology to optimize the key parameters of the processing technology by assessing the contents of multiple components. The results showed that the optimal mutton-oil processing technology of EF was as follows: heating the mutton oil at 120 &#176;C &#177; 10 &#176;C, adding the crude EF, stir-frying it gently to 189 &#176;C &#177; 10 &#176;C until it is evenly shiny, and then removing it and cool. For every 100 kg of EF, 15 kg of mutton oil should be used. The toxicities and teratogenicities of an aqueous extract of crude and mutton-oil processed EF were compared in a zebrafish embryo developmental model. The results showed that the crude herb group was more likely to cause zebrafish deformities, and its half-maximal lethal EF concentration was lower. In conclusion, the optimized mutton-oil processing technology was stable and reliable, with good repeatability. At a certain dose, the aqueous extract of EF was toxic to the development of zebrafish embryos, and the toxicity was stronger for the crude drug than for the processed drug. The results showed that mutton-oil processing reduced the toxicity of crude EF. These findings can be used to improve the quality, uniformity, and clinical safety of mutton oil-processed EF.

In this protocol, the mutton-oil processing technology of Epimedii folium (EF) was optimized by applying a Box-Behnken experimental design-response surface methodology, and the effect of crude and optimized water-extracted EF on zebrafish embryonic development was preliminarily investigated.

As a traditional Chinese medicine (TCM), Epimedii folium (EF) has a history in medicine and food that is &#62; 2,000 years old. Clinically, EF processed ...

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 ...

Optimization of Processing of Tiebangchui with Highland Barley Wine Based on the Box-Behnken Design Combined with the Entropy Method

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