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 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 °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.
The dried root of Aconitum pendulum Busch and A. flavum Hand.-Mazz., one of the most famous Tibetan medicines, is called Tiebangchui (TBC) in Chinese1,2. The dried roots of TBC are helpful in dispelling cold and wind, reducing pain, and calming shock. It was recorded in the first volume of “Drug Standards (Tibetan Medicine) of the Ministry of Health of the People’s Republic of China,” which states that the dried roots of TBC are commonly used to treat rheumatoid arthritis, bruises, and other cold diseases3. However, the clinical therapeutic dose of TBC is similar to its toxic dose, and incidents of poisoning or death have been frequently reported due to improper use4. Therefore, reducing the toxicity and preserving the efficacy of TBC has become a research hot spot over the years.
In Tibetan medicine, processing is one of the most effective methods to attenuate the toxicity of TBC. According to “Processing specification of Tibetan medicine of Qinghai Province (2010)”, the original herbs (TBC) should be placed in an iron pot and stir-fried with Zanba until the Zanba turns yellow, after which Zanba is removed and the herbs are dried in air5,6. However, no specific process parameters have been documented, which makes controlling the processing technology and the quality of Zanba-stir-fried TBC difficult. The CRITIC method is an objective weight method that can avoid fuzzification and subjectivity, and enhance the objectivity of weighing7. The Box-Behnken response surface method can directly reflect the interaction between each factor through polynomial fitting8. The combination of the Box-Behnken response surface and CRITIC method is commonly used to optimize processing technology to acquire the optimized processing protocol9,10. In this paper, a monoester-diterpenoid alkaloid (MDA) (benzoylaconitine) and two diester-diterpenoid alkaloids (DDAs) (aconitine, 3-deoxyaconitine) were used as evaluation indexes. CRITIC combined with the Box-Behnken response surface method was applied to optimize the processing technology of Zanba-stir-fried TBC and establish a standard processing method for clinical safe use.
The Zanba-stir-fried TBC processing method was optimized and standardized by CRITIC combined with the Box-Behnken response surface method. Benzoylaconitine, aconitine, and 3-deoxyaconitine were used as evaluation indexes during this procedure.
1. Sample solution preparation
2. Chromatographic condition
3. System adaptability test
NOTE: Refer to section 2 for the chromatographic conditions to perform steps 3.1-3.5.
4. Single-factor experiments
5. Processing technology optimization of Zanba-stir-fried TBC using response surface methodology (RSM)
6. Model evaluation
NOTE: This step is to be performed after each single-factor experiment or response surface experiment has been completed. After each experiment (e.g., comparison of slice thickness) is completed, the content of the MDA and DDAs in the different samples are measured to obtain five datasets, according to step 1.2 and section 2. The data are shown in Supplementary Table S1.
In this study, the elution gradient used had a good resolution (Figure 1) for the three index components in Zanba-stir-fried TBC, as determined after repeated debugging. The three index components in Zanba-stir-fried TBC had a good linear relationship within a specific concentration range (Table 2). The precision (Table 5), stability (Table 6), repeatability (Table 7), and sample recovery (Table 8) of Zanba-stir-fried TBC were all within the methodological range specified in the Chinese Pharmacopoeia (Volume 4, 2020)22, indicating that the method was feasible. Therefore, the HPLC method was reliable to conduct the analysis of Zanba-stir-fried TBC.
The effect of each factor on processing technology was elucidated using single-factor tests, the results of which are shown in Figure 2. The trend of the comprehensive score of Zanba-stir-fried TBC under different conditions was visualized. The range of slice thickness (A, 1-3 cm), amount of Zanba (B, 2-4x), processing temperature (C, 100-140 °C), and processing time (D, 40-80 min) were determined using single-factor tests (Figure 2).
The CRITIC method is an objective evaluation method that takes advantage of measured data19,20. When each index has very different levels, using the original index value directly for analysis results in a larger role of the index with a higher value in the comprehensive analysis and a smaller role of the index with a lower value. Therefore, the original indicator data must be standardized to ensure the reliability of the results, as applied to the experimental values in this study. According to the response surface test results and CRITIC method, the weight coefficients of the MDA and DDAs in the response surface experiment were established as 0.5295 and 0.4705, respectively. The comprehensive score (Y) could be calculated according to Eq. (8).
(8)
The results of the Box-Behnken experimental design are shown in Table 4, while Table 9 presents the results of the ANOVA and regression coefficients. The polynomial equations of comprehensive scores were also obtained after the software analysis. Values of probability less than 0.05 suggested that model was significant (p < 0.0001)23; The equation in terms of actual factors was obtained in Eq. (9) (Y: comprehensive score; A: slicing thickness; B: amount of Zanba; C: processing temperature; and D: processing time). The equation indicated that the intensity of the influence on comprehensive scores follows this order: processing time > processing temperature > slice thickness > amount of Zanba for four different factors.
Y = 89.05 + 4.57A + 2.88B + 4.63C – 4.83D + 5.19AB + 4.91AC + 6.97AD + 6.69BC – 7.05BD – 1.17CD – 22.80A2 – 21.93B2 – 19.58C2 – 27.19D2 (9)
The response surfaces and contour plots are shown in Figure 3, demonstrating the changes in synthetic scores as a function of four variables. On the basis of the experimental results, the optimal processing parameters of Zanba-stir-fried TBC were determined to be as follows: slice thickness of 2.117 cm, 3.118 times more Zanba than TBC, processing temperature of 123.106 °C, and processing time of 58.156 min. Depending on the feasibility of the operation, the optimal processing technology of Zanba was adjusted – the optimal slice thickness of TBC was 2 cm, the amount of Zanba was three times, the processing temperature was 125 °C, and the processing time was 60 min. The reliability of the model was proven through three tests that were conducted according to the obtained processing parameters (Table 10).
Figure 1: Chromatograms. The chromatogram of the sample solution (A) and the mixed standard solution (B) (1: benzoylaconitine; 2: aconitine; 3: 3-deoxyaconitine). Please click here to view a larger version of this figure.
Figure 2: The synthetic scores of all single factors. (A) Slice thickness; (B) the amount of Zanba; (C) processing temperature; and (D) processing time. The results showed that the comprehensive scores of Zanba-stir-fried TBC are highest when the slice thickness is 2 cm, the amount of Zanba is three times, the processing temperature is 120 °C, and the processing time is 60 min. So, the results showed the range of slicing thickness (A, 1-3 cm), amount of Zanba (B, 2-4x), processing temperature (C, 100-140 °C), and processing time (D, 40-80 min) to be used to design the next experiment. Please click here to view a larger version of this figure.
Figure 3: Response surface plots (3D) reflecting the effects of processing parameters on comprehensive scores. Please click here to view a larger version of this figure.
Table 1: The HPLC gradient. Please click here to download this Table.
Table 2: The linear relationship of the index components in Zanba-stir-fried TBC. The results suggested that the three index components in Zanba-stir-fried TBC had a good linear relationship within a certain concentration range. Please click here to download this Table.
Table 3: Levels of variables for the experimental design. Please click here to download this Table.
Table 4: The Box-Behnken experimental design with responses. Please click here to download this Table.
Table 5: The results of the precision measurement. The relative standard deviation (RSD) values of the peak areas of benzoylaconitine, aconitine, and 3-deoxyaconitine were 0.42%, 0.71%, and 2.95%, respectively (n = 6). Abbreviation: RSD = relative standard deviation. Please click here to download this Table.
Table 6: The results of the stability test. The RSD values of the peak areas of benzoylaconitine, aconitine, and 3-deoxyaconitine were 1.86%, 0.54%, and 2.81%, respectively (n = 6). Abbreviation: RSD = relative standard deviation. Please click here to download this Table.
Table 7: The results of the reproducibility test. The RSD values of the peak areas of benzoylaconitine, aconitine, and 3-deoxyaconitine were 1.99%, 1.84%, and 2.41%, respectively (n = 6). Abbreviation: RSD = relative standard deviation. Please click here to download this Table.
Table 8: Sample recovery rate measurements. The RSD values of the recovery rate of benzoylaconitine, aconitine, and 3-deoxyaconitine were 2.47%, 1.88%, and 2.33%, respectively. Abbreviation: RSD = relative standard deviation. Please click here to download this Table.
Table 9: Analysis of variance (ANOVA) results of the experiment model. Please click here to download this Table.
Table 10: The results of verifying tests. Please click here to download this Table.
Supplementary File 1: The instructions of the Box-Behnken design software Please click here to download this File.
Supplementary Table S1: The calculation result of slice thickness. Please click here to download this File.
TBC is an important Tibetan medicine with the effects of dispelling cold and relieving pain. It has been mostly used to treat traumatic injury and rheumatic arthralgia in China for thousands of years24,25,26. Diterpenoid alkaloids are both active and toxic ingredients of TBC27,28,29. The main toxic effects of the aconitum alkaloids of TBC are neurotoxicity, cardiotoxicity, and gastrointestinal toxicity30,31. TBC is generally processed before oral use to mitigate the risk of toxicity. Various processing methods, such as steaming, decocting, and sand-frying, as well as processing with Hezi decoction, Qingke wine, and Zanba, have been useful in reducing the toxicity of TBC while preserving its efficacy1. Among them, Zanba-stir-frying is an important processing method. Zanba is produced from highland barley (Hordeum vulgare L. var. nudum Hook. f), which is an important grain for people living in the Qinghai-Tibet Plateau32,33. However, the precise parameters of formulating Zanba-stir-fried TBC are still unclear, which is why this processing technology needs to be standardized to ensure its quality control and safe application.
The most crucial aspect of the method is that the evaluation index was determined with the CRITIC method. According to recent studies, highly toxic DDAs can be hydrolyzed or pyrolyzed into an MDA with moderate toxicity during the heating process34,35. Studies have showed that aconitine hydrolysis to benzoylaconine is the typical example36. Therefore, the composition changes in the processing process were taken as the evaluation index in the process technology optimization. The CRITIC method is an objective weight method that mainly considers the variation of indicators and the conflict among indicators, which are expressed by the standard deviation and correlation coefficient, respectively. It has been widely applied in the processing of traditional Chinese medicine37,38. In this protocol, the weight of the main components of Zanba-stir-fried TBC, including benzoylaconitine, aconitine, and 3-deoxyaconitine, were calculated using the CRITIC weighing method of objective assignment, which was used as the evaluation standard of Zanba-stir-fried TBC.
One of the key experimental procedures is ensuring a constant processing temperature during processing, as the processing temperature greatly affects the decomposition of DDAs. Therefore, the pre-experiment involved the use of many kinds of heating devices, such as an induction cooker, electric ceramic stove, and multifunctional stir-fry machine. The multifunctional stir-fry machine could maintain a constant temperature and stabilize the quality of the processed product.
Although the optimized processing technology could reduce TBC's toxicity effectively, limitations still exist. First, some of the active ingredients in Zanba-stir-fried TBC remain unknown. Therefore, qualitative and quantitative analysis cannot be conducted as the relevant reference product is not available. More attention should be paid to phytochemical investigations to obtain the target quality control components. In addition, the pharmacological comparison of raw and Zanba-stir-fried TBC is unclear. Detoxification and evaluation of the efficacy reservation effects in animal models will be the next objectives.
Traditional Chinese medicine processing culture is mainly passed down from master to apprentice, and the end point of processing is generally judged by people's subjective consciousness, which is not conducive to the establishment of a standardized processing method. In this study, digital process parameters were used to specify the processing endpoint, which can realize the combination of modern technology to a certain extent. In summary, this study standardized Zanba-stir-fried processing technology for the toxic attenuation and efficacy reservation of TBC. This approach can provide useful information and guidance for processing technology of other poisonous 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).
3-Deoxyaconitine | Chengdu Desite Biotechnology Co., Ltd. | DST221109-033 | |
Aconitine | Chengdu Desite Biotechnology Co., Ltd. | DSTDW000602 | |
Ammonium acetate | Tianjin Kermel Chemical Reagent Co., Ltd | Chromatographic grade | |
Benzoylaconitine | Chengdu Desite Biotechnology Co., Ltd. | DSTDB005502 | |
Design-Expert software | Stat-Ease, Inc., Minneapolis, MN, USA | version 13.0 | |
Electronic analytical balance | Shanghai Liangping Instruments Co., Ltd. | FA1004 | |
High performance liquid chromatography | SHIMADZU Co., Ltd. | LC-20A | |
High-speed smashing machine | Beijing Zhongxing Weiye Instrument Co., Ltd. | FW-100 | |
Millipore filter | Tianjin Jinteng Experimental Equipment Co., Ltd | φ13 0.22 Nylon66 | |
stir-Fry machine | Changzhou Maisi Machinery Co., Ltd | Type 5 | |
Tiebangchui | Gannan Baicao Biotechnology Development Co., Ltd | 20211012 | |
Ultra pure water systemic | RephiLe Bioscience, Ltd. | Genie G | |
Ultrasonic cleansing machine | Ningbo Xinyi Ultrasonic Equipment Co., Ltd | SB2200 | |
Zanba | 27 Chuanzang Road, Ganzi County | – |