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 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 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.
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 "Four Medical Tantra"1,2. According to "Drug Standards of the Ministry of Health of the People's Republic of China (Tibetan Medicine)", 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.
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
2. Chromatographic condition
3. System adaptability test
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.
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,…,n, and j = 1,2,…m21.
6. Box-Behnken design
7. Box-Behnken design software operation steps
8. Validation test
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% for 3-acetylaconitine, indicating that the precision of the instrument was good. The study of stability performed for 24 h (n = 6) indicated relative standard deviation values of 2.76%, 2.21%, 2.98%, and 2.31% for benzoylaconine, aconitine, 3-deoxyaconitine, and 3-acetylaconitine, respectively (Table 9), suggesting that the sample solution was stable for 24 h. The repeatability test results (Table 10) showed that the RSDs of the peak areas of benzoylaconine, aconitine, 3-deoxyaconitine, and 3-deoxyaconitine were 2.80%, 2.92%, 2.92%, and 2.07%, respectively, showing that the repeatability of this method was good. The recovery experiment results indicated that the average recovery rates of benzoylaconine, aconitine, 3-deoxyaconitine, and 3-deoxyaconitine were 99.7%, 100.84%, 103.27%, and 100.92%, respectively.
The single factor test of TBC processed with highland barley wine revealed that 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 (Figure 2). The experimental design and results of the response surface model are shown in Table 11. The results of the experimental ANOVA are shown in Table 12. The factors are fitted by regression to obtain a quadratic multinomial regression equation (8). A: Highland barley wine addition; B: soaking time; C: slicing thickness. The results showed that the model was well fitted and was able to predict the relationship between the comprehensive scoring of highland barley wine addition, soaking time, and slice thickness. The order of the factors by the strength of the effects was highland barley wine addition > slice thickness of medicinal herbs > soaking time.
(8)
According to Equation (8), Design-Expert 8.0.6 analysis software is used to plot a 3D curve via step 7.2.1 (Figure 3). A steeper slope of the response surface indicates a stronger horizontal interaction of factors, and a gentler slope is the opposite. The p-value (p < 0.0001) of the model in Table 12 shows that the model is significant, with an R2 of 0.9754 and a non-significant misfit term (p = 0.7253), indicating that the model is a good fit and better reflects the relationship among the highland barley wine addition, soaking time, slice thickness of medicinal herbs, and overall score.
According to the objective weight of each index determined by the analytic entropy method, comprehensive scoring was performed, and the optimal processing condition of TBC was determined 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. The validation test results showed that the total DDAs were 0.6963, 0.6793, and 0.7023 mg/g, respectively, and the MDA content in three sets of parallel tests was 0.2096, 0.2237, and 0.2109 mg/g. The average comprehensive scoring was 83. The RSD between the verification test and the predicted value was less than 1.8%, indicating that the optimized processing technology of TBC processed with highland barley wine is simple, feasible, and stable, providing a reference for industrial production.
Figure 1: Representative chromatograms of the four characteristic components after setting the chromatographic conditions mentioned in step 2.1 (n = 1). (A) Typical chromatograms of the reference solution. Peak 1 is benzoylaconine, peak 2 is aconitine, peak 3 is 3-deoxyaconitine, and peak 4 is 3-acetylaconitine. (B) Typical chromatograms of the sample solution. Peak 1 is benzoylaconine, peak 2 is aconitine, peak 3 is 3-deoxyaconitine, and peak 4 is 3-acetylaconitine. Please click here to view a larger version of this figure.
Figure 2: The comprehensive scoring of the single factor test of TBC processed with highland barley wine (n = 3). (A) Amount of highland barley wine (times); (B) Soaking time (h); (C) Slice thickness (cm). Please click here to view a larger version of this figure.
Figure 3: 3D response surface map of the effect of interaction of various factors on comprehensive scoring. Please click here to view a larger version of this figure.
Condition | Parameter |
Chromatographic column | Ultimate ODS-3 C18 (4.6 mm x 250 mm, 5 μm) |
Mobile phase | Acetonitrile (A) – 0.04 mol/L ammonium acetate solution (B) pH= 8.5 ± 0.5 |
Gradient elution | 0–10 min, 0%-70% A; 10–15 min, 70–50% A; 15–30 min, 50–40% A; 30–38min, 40–15% A; 38–45 min, 15–15% A; 45–55 min, 15–70% A |
Flow rate | 1 mL/min |
Column temperature | 30 °C |
Detecting wavelength | 235 nm |
Sample volume | 10 μL |
Table 1: The chromatographic conditions set in this experiment. Details about the chromatographic column, the mobile phase, the gradient elution, the flow rate, the column temperature, the detection wavelength, and the sample volume.
Index components | Linear equation | Range of linearity (mg/mL) | R2 |
Benzoylaconine | y=11,658,706.1677x +19,717.0872 | 1.036-0.0518 | 0.9995 |
Aconitine | y=11,199,784.3030x -67,641.2429 | 1.313-0.05252 | 0.9999 |
3-Deoxyaconitine | y=11,214,550.3140x +59,795.9119 | 1.005-0.0402 | 0.9999 |
3-Acetylaconitine | y=9,887,511.9074x +26,713.6359 | 0.2018-0.01009 | 0.9994 |
Table 2: The linear relationship of the index components in TBC. The four index components in TBC had a good linear relationship in a specific concentration range.
Index components | Known content (mg) | Adding quantity (mg) | Measuring quantity (mg) | Recoveries (%) | Average recoveries (%) | RSD (%) |
Benzoylaconine | 0.1558 | 0.1295 | 0.2901 | 96.4 | 99.7 | 3.14 |
0.1574 | 0.1295 | 0.2849 | 98.46 | |||
0.156 | 0.1295 | 0.2871 | 101.24 | |||
0.1574 | 0.1295 | 0.2923 | 104.95 | |||
0.1449 | 0.1295 | 0.2736 | 99.38 | |||
0.1566 | 0.1295 | 0.2839 | 98.3 | |||
Aconitine | 0.3099 | 0.3283 | 0.645 | 102.07 | 100.84 | 2.02 |
0.3153 | 0.3283 | 0.6371 | 98.02 | |||
0.2928 | 0.3283 | 0.6314 | 103.14 | |||
0.2969 | 0.3283 | 0.6325 | 102.23 | |||
0.3035 | 0.3283 | 0.6343 | 100.76 | |||
0.3094 | 0.3283 | 0.6339 | 98.84 | |||
3-Deoxyaconitine | 0.1789 | 0.201 | 0.3788 | 99.45 | 103.27 | 2.65 |
0.1793 | 0.201 | 0.3845 | 102.09 | |||
0.1741 | 0.201 | 0.3774 | 101.14 | |||
0.1635 | 0.201 | 0.3753 | 105.37 | |||
0.1708 | 0.201 | 0.383 | 105.57 | |||
0.1653 | 0.201 | 0.3783 | 105.97 | |||
3-Acetylaconitine | 0.0169 | 0.02 | 0.0374 | 102.5 | 100.92 | 1.15 |
0.0168 | 0.02 | 0.037 | 101 | |||
0.0166 | 0.02 | 0.0366 | 100 | |||
0.0161 | 0.02 | 0.0365 | 102 | |||
0.017 | 0.02 | 0.0369 | 99.5 | |||
0.0171 | 0.02 | 0.0372 | 100.5 |
Table 3: The results of the sample recovery rate measurement. The RSD of the recovery rate of benzoylaconine, aconitine, 3-deoxyaconitine, and 3-acetylaconitine were 3.14%, 2.02%, 2.65%, and 1.15%, respectively.
Number | Highland barley wine addition test (times) | Content of MDAs (mg/g) | Content of DDAs (mg/g) | Comprehensive scoring/points |
1 | 2 | 0.1875 | 0.8254 | 58.98421777 |
2 | 3 | 0.1099 | 0.9847 | 0.056898711 |
3 | 4 | 0.2296 | 0.8487 | 71.12048666 |
4 | 5 | 0.2161 | 0.6894 | 94.6966946 |
5 | 6 | 0.2006 | 0.7472 | 78.22537224 |
Table 4: The results of the single factor test of the ratio between highland barley wine and TBC.
Number | Soaking time test (h) | Content of MDAs (mg/g) | Content of DDAs (mg/g) | Comprehensive scoring/points |
1 | 6 | 0.236292609 | 1.047811476 | 59.67501032 |
2 | 12 | 0.193880685 | 1.164420534 | 23.10718817 |
3 | 24 | 0.229606225 | 0.848736346 | 53.86313899 |
4 | 36 | 0.151447388 | 0.701045217 | 79.15664943 |
5 | 48 | 0.193311963 | 0.767427412 | 68.88872066 |
Table 5: The results of the single factor test of the soaking time.
Number | slicing thickness test (cm) | Content of MDAs (mg/g) | Content of DDAs (mg/g) | Comprehensive scoring/points |
1 | 0.5 | 0.1043 | 0.6190 | 66.96 |
2 | 1 | 0.1709 | 0.6992 | 75.05 |
3 | 1.5 | 0.1507 | 0.6954 | 66.23 |
4 | 2 | 0.1459 | 0.8347 | 20.66 |
5 | 2.5 | 0.1451 | 0.8298 | 21.79 |
Table 6: The results of the single factor test of the slice thickness of TBC.
Level | Factor | ||
A (amount of highland barley wine, times) | B (soaking time, h) | C (slice thickness, cm) | |
1.0000 | 4.0000 | 24.0000 | 0.5000 |
2.0000 | 5.0000 | 36.0000 | 1.0000 |
3.0000 | 6.0000 | 48.0000 | 1.5000 |
Table 7: Box-Behnken design response surface level factor table.
Peak area in index components | 1 | 2 | 3 | 4 | 5 | 6 | RSD (%) |
Benzoylaconine | 1281252 | 1290912 | 1198912 | 1256056 | 1256704 | 1266738 | 2.56% |
Aconitine | 2861208 | 2881686 | 2785022 | 2790990 | 2859024 | 2799395 | 1.50% |
3-Deoxyaconitine | 2356317 | 2328383 | 2429059 | 2350987 | 2406114 | 2450374 | 2.04% |
3-Acetylaconitine | 2008110 | 2021560 | 2014519 | 2015881 | 2015209 | 2012529 | 0.22% |
Table 8: The results of the precision measurement. The RSD of the peak areas of benzoylaconine, aconitine, 3-deoxyaconitine, and 3-acetylaconitine were 2.56%, 1.49%, 2.03%, and 0.22%, respectively (n = 6).
Peak area in index components | 0 | 2 | 4 | 8 | 12 | 24 | RSD (%) |
Benzoylaconine | 191657 | 189590 | 193934 | 205135 | 196159 | 195954 | 2.76 |
Aconitine | 312259 | 310240 | 294331 | 309104 | 312199 | 305360 | 2.22 |
3-Deoxyaconitine | 230174 | 246787 | 239760 | 249302 | 248806 | 243396 | 2.98 |
3-Acetylaconitine | 17086 | 16953 | 16826 | 16914 | 16979 | 17896 | 2.31 |
Table 9: The results of the stability test. The RSD of the peak areas of benzoylaconine, aconitine, 3-deoxyaconitine and 3-acetylaconitine were 2.76%, 2.21%, 2.98%, and 2.31%, respectively (n = 6).
Peak area of index components | 1 | 2 | 3 | 4 | 5 | 6 | RSD (%) |
Benzoylaconine | 191067 | 192795 | 191058 | 192907 | 179103 | 192008 | 2.79 |
Aconitine | 308142 | 313754 | 290487 | 294740 | 301515 | 307654 | 2.92 |
3-Deoxyaconitine | 249021 | 249456 | 243963 | 232781 | 240524 | 234661 | 2.92 |
3-Acetylaconitine | 17465 | 17451 | 17247 | 16691 | 17608 | 17686 | 2.07 |
Table 10: The results of the reproducibility test. The RSD of the peak areas of benzoylaconine, aconitine, 3-deoxyaconitine, and 3-acetylaconitine were 2.79%, 2.92%, 2.92%, and 2.07%, respectively (n = 6).
Number | A (Highland barley wine addition, times) | B (Soaking time, h) | C (Slicing thickness, cm) | Content of MDAs (mg/g) | Content of DDAs (mg/g) | Comprehensive scoring/points |
1 | 4 | 36 | 0.5 | 0.1032 | 0.6882 | 28.2 |
2 | 5 | 48 | 1.5 | 0.1688 | 0.6588 | 56.49 |
3 | 6 | 24 | 1 | 0.1236 | 0.6535 | 33.02 |
4 | 5 | 24 | 1.5 | 0.2201 | 0.692 | 87.23 |
5 | 5 | 36 | 1 | 0.2094 | 0.6199 | 70.71 |
6 | 5 | 24 | 0.5 | 0.1809 | 0.5689 | 48.56 |
7 | 4 | 24 | 1 | 0.2016 | 0.7744 | 90.74 |
8 | 5 | 36 | 1 | 0.2169 | 0.6889 | 85.15 |
9 | 5 | 36 | 1 | 0.2103 | 0.6802 | 80.5 |
10 | 6 | 36 | 0.5 | 0.1036 | 0.5072 | 0.36 |
11 | 6 | 36 | 1.5 | 0.1089 | 0.5062 | 2.86 |
12 | 4 | 48 | 1 | 0.1789 | 0.6789 | 64.6 |
13 | 6 | 48 | 1 | 0.1036 | 0.5536 | 7.55 |
14 | 5 | 36 | 1 | 0.2062 | 0.6084 | 67.33 |
15 | 4 | 36 | 1.5 | 0.1832 | 0.6954 | 69.31 |
16 | 5 | 48 | 0.5 | 0.1759 | 0.5569 | 44.21 |
17 | 5 | 36 | 1 | 0.2161 | 0.6894 | 84.82 |
Table 11: Design and results of the response surface design test.
Source | Sum of Squares | df | Mean Square | F -Value | P-Value |
Model | 14403.27 | 9 | 1600.36 | 30.8 | <0.0001 |
A | 5463.26 | 1 | 5463.26 | 105.15 | <0.0001 |
B | 939.61 | 1 | 939.61 | 18.08 | 0.0038 |
C | 1117.7 | 1 | 1117.7 | 21.51 | 0.0024 |
AB | 0.11 | 1 | 0.11 | 0.00216 | 0.9642 |
AC | 372.68 | 1 | 372.68 | 7.17 | 0.0316 |
BC | 174.11 | 1 | 174.11 | 3.35 | 0.1099 |
A2 | 4133.52 | 1 | 4133.52 | 79.55 | <0.0001 |
B2 | 28.63 | 1 | 28.63 | 0.55 | 0.482 |
C2 | 1890.1 | 1 | 1890.1 | 36.38 | 0.0005 |
Residual | 363.71 | 7 | 51.96 | ||
Lack of Fit | 93.28 | 3 | 31.09 | 0.46 | 0.7253 |
Pure Error | 270.43 | 4 | 67.61 | ||
Cor Total | 14766.99 | 16 |
Table 12: ANOVA for the regression model.
Supplementary File 1: A detailed guide of the Box-Behnken design software. Please click here to download this File.
Supplementary Table S1: Sample peak area of the slicing thickness test by HPLC. Please click here to download this File.
Supplementary Table S2: Content of MDAs (benzoylaconine) and DDAs (aconitine, 3-deoxyaconitine, and 3-acetylaconitine) in the slicing thickness test. Please click here to download this File.
Supplementary Table S3: Comprehensive scoring of the slicing thickness test. Please click here to download this File.
As a commonly used Tibetan medicine with toxic effects, the toxicity-attenuating effect of processing is extremely important for TBC'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 toxic MDA was used as the positive indicator. The total content of DDAs was used as the negative indicator. The entropy method was used in calculating the index weights and optimizing the processing technology26.
During the experiment, two points should be particularly noted. First, three parallel sets of experiments are required for each condition of a single factor to improve the accuracy of subsequent results. Second, in the calculation of the comprehensive scores, the standardized values of each indicator are multiplied by the weighting factor instead of the raw data. This procedure ensures the accuracy of calculation results. In addition, the R2 value in ANOVA should be as close as possible to one; otherwise, the levels of factors are extremely close, having little impact on results; the difference between the levels should be moderately wide.
Although the use of multi-index comprehensive scoring combined with the response surface method ensures the precise prediction of the processing process of TBC, it does have limitations. First, when laboratories use HPLC to measure indicated components in medicinal materials, human error may occur due to the small scale of experiments. More convincing results can be obtained if pilot evaluation is carried out in a large-size herb processing factory. Second, the Box-Behnken design method is not suitable for optimization of the whole process. After importing the experimental data into the response surface software, the model term needs to be significant (p < 0.05), and the lack of fit needs to be nonsignificant (p > 0.05) in the ANOVA data. If the result is reversed, the process is unsuitable for optimization by this method.
In conclusion, compared with the commonly used single factor test, uniform design, orthogonal design, and star point design, the Box-Behnken response surface approach is an experimental optimization design method that uses multivariate linear and quadratic term model fitting27. The model predicted by the Behnken response surface method has continuity and high experimental accuracy, and predicts optimal points28,29. In the present experiment, the preferred process of TBC was determined by comprehensive scoring based on the single factor test, and the final validation test did not deviate much from the predicted value, indicating that the selected model is reasonable and can provide references for the processing technology of TBC processed with highland barley wine. The optimized processing technology can provide information and guidance for the study of the toxicity-attenuating effect of processing other toxic ethnic medicines.
The authors have nothing to disclose.
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 & Technology Department of Sichuan Province (No. 2022NSFSC1449), and the "Xinglin Scholars" Research Promotion Program of Chengdu University of Traditional Chinese Medicine (No. BSH2021009).
Aconitine | Chengdu Push Biotechnology Co.,Ltd | PS000905 | |
3-Acetylaconitine | Chengdu Push Biotechnology Co.,Ltd | PS010552 | |
3-Deoxyaconitine | Chengdu Push Biotechnology Co.,Ltd | PS011258 | |
Benzoylaconine | Chengdu Push Biotechnology Co.,Ltd | PS010300 | |
Circulating water vacuum pump | Gongyi City Yuhua Instrument Co., Ltd | SHZ-DIII | |
Design-Expert | State-East Corporation | 8.0.6 | |
Electric constant temperature drying oven | Shanghai Yuejin Medical Equipment Co., Ltd | 101-3-BS | |
Electronic analytical balance | Shanghai Liangping Instruments Co., Ltd. | FA1004 | |
High performance liquid chromatography | Shimadzu Enterprise Management (China) Co., Ltd | shimadzu 2030 | |
Highland barley rice | Kangding City, Ganzi Tibetan Autonomous Prefecture, Sichuan Province | 20221015 | |
Millipore filter | Tianjin Jinteng Experimental Equipment Co., Ltd | φ13 0.22 Nylon66 | |
Rotary evaporator | Shanghai Yarong Biochemical Instrument Factory | RE-2000A | |
Starter of liquor-making | Angel Yeast CO., Ltd | BJ22-104 | |
Ultra pure water systemic | Merck Millipore Ltd. | Milli-Q | |
Ultrasonic cleansing machine | Ningbo Xinyi Ultrasonic Equipment Co., Ltd | SB-8200 DTS |