Chemical composition, pharmacodynamic activity of processed Aconitum brachypodum Diels., and molecular docking analysis of its active target

Aconitum brachypodum Diels. (AB.) is a plant of Aconitum L. The dried roots of AB have analgesic and anti-inflammatory activity. However, the processing is required to reduce toxicity before use because of its high toxicity. Studies on the toxicity, pharmacodynamics, and chemical composition of processed Aconitum brachypodum Diels. PAB. are still lacking at present. In this study, the composition changes of AB and PAB were determined by ultra-performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry (UPLC-QE-Orbitrap-MS). The intensity of diester alkaloids was greatly reduced, while the monoester alkaloids were significantly increased. An acute toxicity experiment was used to evaluate the toxicity differences between AB and PAB, while the acetic acid-induced writhing pain experiment and croton oil-induced ear edema experiment were applied to evaluate the analgesic and anti-inflammatory properties. The acute toxicity test of AB showed that the median lethal dose (LD50) was 1.37 g/kg, while the maximal feasible dose (MFD) of PAB was 30.0 g/kg. It was apparent that the toxicity of PAB was significantly reduced. The alkaloid component of PAB could significantly inhibit the mice’s ear edema and significantly reduce the writhing times of mice. Based on the above findings, ten compounds, including songoramine (1), neoline (2), bullatine C (3), dihydroatisine (4), bullatineA (5), maltol (6), 15-O-acetylsongorine (7), 15-O-acetylsongoramine (8), songorine (9), and aldohypaconitine (10) were isolated and identified from the alkaloid component of PAB. Compounds 4, 6, 8, and 10 were firstly separate from Aconitum brachypodum Diels. Finally, molecular docking to anti-inflammatory analgesic target protein was carried out. The results indicated that 15-O-acetylsongoramine has a strong binding ability with target proteins, which may interact with the target protein Akt1 of phosphatidylinositol-3-kinase/serine-threonine kinase (Pi3k/Akt) pathway, while, adjust the downstream nuclear factor kappa B (NF-κB) signaling pathway to play an anti-inflammatory analgesic effect. UPLC-QE-Orbitrap-MS, acute toxicity, analgesia, anti-inflammatory experiment, and molecular docking analysis were made to speculate the toxicity and efficacy of processed Aconitum brachypodum

Aconitine is the representative highly toxic substance in AB, which is known to have strong cardiotoxicity and neurotoxicity [35][36][37][38]. According to literature research, aconitine can be hydrolyzed into aconine with very little toxicity during decoction. Generally, aconitine in AB is almost completely destroyed after decocting for 3 to 4 h. Therefore, when using AB as medicine, it must be processed first. Currently, most research has focused on the separation and identification of active components of AB. So far, the research about processed Aconitum brachypodum Diels. (P.A.B.) are lacking.
With the development of the traditional Chinese medicine industry and material technology [3,[39][40][41][42][43][44], traditional Chinese medicine has been developed and utilized in different application fields. For example, combining traditional Chinese medicine and material science has expanded the application direction of traditional Chinese medicine. In recent years, using cheap agricultural and forestry waste and other biomass as raw materials, and using simple and clean processes to prepare carbon materials such as activated carbon with good performance, has become a research hotspot. Some scholars have processed Chinese herbal medicine into nanoscale biological functional materials to enhance and improve the therapeutic effect of traditional Chinese medicine in various diseases [45,46]. Some scholars have studied the applications of Chinese medicine residue in the field of composite materials, which do not pollute the environment and become a high-value-added product [47,48]. The main active component of AB is diterpene alkaloids, which have analgesic, anti-inflammatory, local anesthetic, anti-tumor, and other pharmacological effects. The study of chemical components of Aconitum brachypodum Diels. (AB.) and processed Aconitum brachypodum Diels. (PAB.) can provide basic research data for the new application of this Chinese herbal medicine.
This study first reported the composition and acute toxicity of PAB. Then, alkaloid, non-alkaloid, alcohol extraction, and water extraction components were prepared by different separation and extraction methods, and their pharmacological activities were determined. The structures of the compounds, which were isolated from the alkaloids component of PAB, were further identified. Finally, molecular docking technology was applied. The analgesic and anti-inflammatory protein targets were selected as a receptor to carry out molecular docking with isolated compounds, and the contribution of isolated compounds to inflammation and analgesia pharmacological activity was analyzed. Based on the research results of this paper are expected to guide the scientific processing of AB and provide data support for clinically safe drug use.

Processing of Aconitum brachypodum
AB was processed by the traditional steaming and boiling method in this work [49,50]. Firstly, the roots of AB were washed, soaked in water for 20 h, then taken out, sliced, and steamed at 90-100 °C for 2 to 3 h. After that, AB was taken out to dry at 130 °C for 4 h, the processed Aconitum brachypodum Diels. (PAB.) were thus prepared.

Acute toxicity experiment of AB and PAB
The acute toxicity experiment was used to assess the acute toxicity of AB, and PAB LD 50 was calculated according to the previously described methods [51]. According to the result of preparation, the initial dose of AB was 2.0 g/kg, then diluted eight dose groups down based on 0.9-fold, that was 1.80, 1.62, 1.46, 1.31, 1.18, 1.06, 0.96, 0.86 g/kg, and the solvent control group which gave equal volume pure water was established. Different doses of samples were provided by single gavage administration in 24 h, and the potential toxicity reaction and death of animals were observed.
In the pre-experiment, the PAB sample was given to the mice in the highest dose (10.0 g/kg) 3 times in 24 h. There were no toxic reactions or deaths in the animals. Therefore, in the formal experiment, the maximum dose of PAB was given three times in 24 h (the time interval between 3 times gavage administrations was 4 h), and the possible toxicity reaction and death of animals were observed.

Pharmacological investigation
The alkaloid, non-alkaloid, water extract, and alcohol extract of PAB were prepared by scientific extraction methods (see Supporting Information for full details).

In vitro anti-inflammatory experiment
Raw 264.7 cells were cultured with DMEM (see Supporting Information for full details).
The extracts and indomethacin were prepared into corresponding working solutions with the culture medium containing 100 μg/L LPS RAW264.7 cells (1.0 × 10 5 cells/mL, 100 μL/well) were inoculated for 24 h (37 °C, 5% CO 2 ). The blank control group was added to the fresh culture medium, the LPS model group was added to a culture solution containing 100 μg/L LPS, the positive control group, and the test sample group were added with the corresponding concentrations of prepared solutions, and continued to cultivate for 24 h. The ELISA kit was used to determine the levels of IL-6 and TNF-α in the supernatant.

In vivo anti-inflammatory experiment
Croton oil-induced ear edema was applied to evaluate the antiinflammatory activity [52]. One hundred twenty mice were randomly assigned, including a blank control group, a positive control group (Voltaren 40 mg), and two dose groups (1 mg and 4 mg) of alkaloid, non-alkaloid, water, and alcohol extract. Each trial group was smeared to the right ear at a volume of 0.04 mL for three consecutive days.
One hour after the last operation, cotton dipped in pure water was used to gently wipe the residual subject on both sides of the right ear, then 2% croton oil (20 μL) was applied to the right ear of mice to make inflammation model. After 4 h of modeling, the mice were sacrificed, then the disk of the same part was taken from both ears and weighed. The ear edema was calculated based on Eq. (1): where W t is the weight of the right ear, and W 0 is the weight of the left ear.

Mice pain resistant (writhing times) experiment
Mice were divided randomly, which included a blank control group, a positive control group (Voltaren 100 mg), and two dose groups (2.5 mg and 10 mg) of alkaloid, non-alkaloid, water extract, and alcohol extract. Each day for 3 days, 0.1 mL of the respective group's solution was applied to the abdomen of the mice, while the solvent group received 0.1 mL of pure water.
After the last operation of 1 h, 0.62% of the acetic acid solution was injected (10 mL/kg). The writhing times between 5 to 20 min after injection were recorded.

Isolation and identification of compounds from PAB
Sixty kilograms of PAB were ground into powders and extracted with 70% ethanol (three times, reflux for 2 h). Under reduced pressure, the extracting solution was concentrated at 68 L. Then, about 6 L of 7% hydrochloric acid solution was added under constant stirring to adjust the pH to about 3. After extracting with EtOAc (3 × 90 L), the acidified solution was treated with ammonia water to bring them to pH 10. EtOAc (380 L) was used to extract the solutions, and the resulting extract was concentrated to create the crude alkaloid fraction (475 g). After separation and purification, compounds were isolated by classical methods, such as column chromatography and preparative liquid phase (Agilent 1100). Compounds were identified by classical methods, such as Bruker DRX-500 spectrometers ( 1 H and 13 C NMR spectra).

Molecular docking analysis of active target
Chemdraw2019 software, target Prediction database, and string database were used to evaluate the interactions between compound targets and anti-inflammatory analgesic interactions. The interactions between targets and proteins were displayed using the Cytoscape 3.7.0 software to create PPI networks [53,54]. The program Autodock-vina (1.1.2) was used for docking

Statistical analysis
The data were analyzed using SPSS 17.0 statistical software, and the experimental results were represented by x ± s. The level of the inspection was α = 0.05. The acute toxicity experimental LD 50 and 95% were calculated using Bliss [56].

UPLC-QE-Orbitrap-MS qualitative analysis of AB and PAB
AB and PAB's positive total ion chromatogram ( Fig. 1) showed that the chemical composition changed significantly, especially in 5-7 min. It was identified that the differences between 5 and 7 min were mainly due to the change in alkaloid composition. Using OPLS-DA method, eight different alkaloid components were identified (VIP > 1 and P < 0.05). The variation trend of the intensity of the alkaloid component in AB and PAB (Fig. 2) showed that the diester alkaloids (aconitine, hypaconitine, 3-acetylaconitine, and 3-deoxyaconitine) were significantly reduced, while the monoester alkaloids (14-benzoylaconine, benzoylhypaconine, benzoylmesaconine) and aconine were significantly increased. Retrieved the NiH Chemid Plus chemical toxic database, the LD 50 of aconitine, hypaconitine and 3-acetylaconitine, 14-benzoylaconine, benzoylhypaconine, benzoylmesaconine, and aconine were 0.10, 0.47, 0.4, 10.1, 23, 21, and 117 mg/kg (intravenous injection). The LD 50 of 3-deoxy aconitine was 1.9 mg/kg (intraperitoneal injection). The reciprocal of the above LD 50 was normalized and then multiplied by the intensity of response to obtain the virulence values in the form of a heatmap (Fig. 3). From the perspective of total virulence, the toxicity of AB was significantly higher than that of PAB, suggesting that AB can reduce toxicity after processing. From the perspective of component toxicity contribution, aconitine, 3-acetylaconitine, and 3-deoxyaconitine accounted for a large proportion of the toxicity contribution, suggesting that the mechanism of toxicity reduction was related to the reduction of the content of diester alkaloids, which was consistent with the literature reports [57][58][59].

Acute toxicity experiment
As shown in Table 1, all animals were generally good in the control group, and there was no obvious abnormal reaction or death. In the AB group, the maximal tolerance dose was 0.86 g/kg with no animal death. The mice's minimal lethal dose of the AB was 0.96 g/kg, with the animal mortality of 10%. Additionally, the mortality increased in a gradient with increasing AB dose. All mice died when the AB dose was 2 g/kg. The LD 50 of AB was 1.37 g/kg, and the 95% confidence interval was 1.28-1.46 g/kg.
Anatomical observation of animal death during the observation period did not reveal significant abnormalities. In the PAB group, some mice showed a mild toxic reaction, mainly diarrhea, and no significant toxic reactions or death were observed throughout the observation period. Therefore, we concluded that the MFD of PAB was 30.0 g/kg. The MFD of PAB was 30.0 g/kg while the LD 50 of AB was 1.37 g/kg, indicating that the toxicity of PAB was less than that of AB and also indicating that the toxicity of processed Aconitum brachypodum Diels. was significantly reduced.

In vitro anti-inflammatory experiment
As shown in Figs. 4 and 5, the alkaloid components of PAB at concentrations of 2.5 μg/mL, 5 μg/mL, and 10 μg/mL at a concentration of 2 μg/mL (P < 0.05). However, there was no effect on the content of TNF-α significantly. These results suggest that some components of PAB might substantially alleviate the inflammatory response of cells induced by LPS.

In vivo anti-inflammatory experiment (croton oil-induced ear edema)
As shown in Fig. 6, the Voltaren (positive control, 40 mg) and the alkaloid component of PAB (4 mg) significantly decreased the mice's ear edema (P < 0.05) after three continuous days. In contrast, the non-alkaloid component of PAB, water extract component of PAB, and alcoholic extract component of PAB did not decrease the mice's ear edema. Therefore, we speculate that PAB plays an anti-inflammatory role mainly through the alkaloid component.

Mice pain resistant (writhing times) experiment
To detect the analgesic effect of PAB component, we conducted the mice pain-resistant experiment. As shown in Table 2 and Fig. 7, there were no significant effects on the body weight of mice compared to the control group (P > 0.05). After 3 days, the Voltaren (positive control, 100 mg) and alkaloid component (10 mg) significantly reduced the writhing times in mice (P < 0.05). The nonalkaloid component of PAB, the water extract component of PAB and the alcohol extract component of PAB did not significantly affect the writhing of mice. The acetic acid writhing method in mice is a classic model of chemical injury causing pain, which can effectively respond to the test substance and reduce the pain reaction through anti-inflammatory. This study confirmed that the alkaloid extract component of PAB could reduce the writhing times in mice, and we preliminarily concluded that the alkaloid extract component of PAB has the analgesic effect while anti-inflammatory.

Isolation and identification of compounds from PAB
Overall, N.M.R. and MS were used to determine the structures. Firstly, ten compounds ( Fig. 8) were isolated and purified from PAB using chromatographic techniques such as HPD 100 and preparative HPLC. Subsequently, physicochemical   (9), and aldohypaconitine (10). For the first time, compounds 4, 6, 8, and 10 were isolated from this plant. The isolation and identification data of these compounds see Supporting Information.

Molecular docking analysis of active target
The top six target proteins in the PPI networks were selected as receptors (AKT1, JUN, PTGS2, PIK3CA, SLC6A2, and SLC6A3, see Supporting Information), semi-flexible molecular docking was used to predict the binding energy (kcal/mol) of the 10 isolated compounds ( Table 3). The binding energy lower than − 5.0 kcal/mol indicates good interactions [60,61], which indicates that 15-O-acetylsongoramine had the better affinity with AKT1, JUN, PTGS2, PIK3CA, and SLC6A3 target proteins, with binding energies of − 9.5, − 7.8, − 7.6, − 7.8, and − 7.6 kcal/mol, respectively. The binding energy of 15-O-acetylsongoramine with target SLC6A2 was − 5.3 kcal/ mol, which showed that 15-O-acetylsongoramine could interact with the target SLC6A2. The molecular docking visualization of 15-O-acetylsongoramine with six target proteins was also carried out (Fig. 9) These interactions promote 15-O-Acetylsongoramine to be well bonded to the active cavity of the AKT1, JUN, PTGS2, PIK3CA, SLC6A2, and SLC6A3, which may interact with the target protein Akt1 of Pi3k/Akt pathway and adjust the downstream nuclear factor kappa B (NF-κB) signaling pathway to play an anti-inflammatory analgesic effect. In the future, our research team will conduct in-depth research on this mechanism to clarify PAB's anti-inflammatory and analgesic mechanisms.

Conclusions
In this study, AB was processed to PAB, and the alkaloid component, non-alkaloid component, water extract component, and alcohol extract component were prepared. The chemical composition, acute toxicity, and pharmacological activity were measured. There were mainly eight different alkaloid components in both AB and PAB, with PAB including lower diester alkaloids and higher monoester alkaloids, which suggests that the toxicity components of PAB were reduced. And the acute toxicity experiment also showed that LD 50 to mice of AB was 1.37 g/kg, but PAB only showed low toxicity to mice. Analgesia, anti-inflammatory experimental results showed that the alkaloid component, non-alkaloid component, water extract component, and alcohol extract component of PAB have in vitro anti-inflammatory effects. The alkaloid component of PAB had good anti-inflammatory and analgesic effects in vivo. Finally, ten compounds were isolated from the alkaloid component of PAB and then docked to analgesic, active anti-inflammatory targets through molecular docking techniques. The results indicated that 15-O-acetylsongoramine had a strong binding ability with target proteins, which may affect the effect of anti-inflammatory analgesia by participating in the adjustment of the Pi3k/Akt signaling pathway. Data availability All data used are given in the paper or are taken from references that are properly cited.