1. Introduction
Decoction is one of the oldest and most common forms of administration of herbal medicine in China. Chinese herbs with medicinal properties, such as
Aconitum (
Aconitum carmichaeli Debx L.) [
1,
2] and
Nux vomica (
Strychnos nux-vomica L.) [
3,
4], are often combined to prepare tonics to reduce toxicity and increase effectiveness [
5,
6,
7]. Modern science has found that the high temperature and complex chemistry of the decoction process, together with the dissolved secondary metabolites, including small molecules, biological macromolecules and inorganic elements, alter the physicochemical properties of herbal preparations. These natural substances, which act as suspension aids, flocculants and antiflocculants, turn the decoction into a multiphase integrated system of true solution, colloid solution, mixed suspension and sedimentary at the same time [
8]. For example, a nanophase state of Baihu Tang was reported [
9]. The study confirmed that herbs containing phenolic and carboxylic acid components, when combined with herbs containing alkaloid components, generate new compounds, which are obtained as precipitates and deposits during decoction [
10]. Jinming et al. found that the sedimentary phase was generated by combinations of
Glycyrrhiza glabra (
Glycyrrhiza glabra L.) and
Aconitum, which decreased its toxicity [
11]. Despite the progress achieved, the composition, drug release behavior and gastrointestinal absorption of drugs in different phase states of herbal medicine decoctions show variation. Further studies analyzing the distribution and release of toxins (potent) in each phase are needed to determine the phase characteristics, but also provide a standard of reference for further exploration of the mechanism of toxicity reduction and potency enhancement of traditional Chinese medicines (TCM).
As a typical toxic TCM,
Nux vomica (NV) has been recommended by physicians because of its remarkable pharmacological activity [
12,
13,
14]. However, due to their inherent toxicity associated with brucine and strychnine, careless use of NV can result in poisoning and even death. Excessive use of NV can overstimulate medulla oblongata and result in neurotoxicity [
15]. In order to effectively reduce the toxicity of NV, it is combined with
Glycyrrhiza glabra (GG). GG is an ancient TCM and is an antidote against poisoning [
16,
17,
18,
19]. GG can be used to ameliorate the toxicity of NV. Many ancient books recorded the use of “raw NV plus GG immersion”, administered orally as a prescription of NV combined with GG to hydrolyze NV. The combined intake of NV and GG can reduce toxicity. However, the phase characteristics of “
Glycyrrhiza glabra-
Nux vomica” decoction (NGD) have yet to be analyzed comprehensively. The correlation between the content distribution in different phases and the reduction of NV toxicity using GG has yet to be explored.
It has been found that TCM prescriptions containing flavonoids and alkaloids precipitate significantly in the process of decocting [
20]. For example, liquiritin and glycyrrhizic acid in Sini decoction and Mahuang-Fuzi-Gancao decoction can combine with alkaloids in Aconitum aconite to form insoluble salts. Some studies [
21] have also shown that alkaloids in GG and Aconitum can form molecular complexes, which can release toxic components slowly in the body. Further investigation revealed that the formation mechanism of composite deposition was the association of tertiary amine N in alkaloids with carboxylic acid C=O in GG, which avoided the toxic reaction caused by excessive alkaloids absorbed by the organisms in a short time. Based on the above, the main alkaloids that contribute more to the formation of phase state, brucine and strychnine, and the characteristic flavonoids and carboxylic acid components of GG, liquiritin, isoliquiritin, isoliquiritigenin and glycyrrhizic acid, were selected as indexes to investigate the content distribution and release behavior of phase states.
At present, the HPLC method is still the main method for the determination of the compatible components of GG and NV. However, it is difficult to sensitively determine the content of substances in the phase states with lower concentration. In this study, centrifugation and dialysis were used to separate the phase states of NGD. The particle size and potential of different phase states, including the sedimentary phase group (NG1), suspension phase group (NG2), colloid phase group (NG3) and solution phase group (NG4) were characterized. A simple, sensitive and selective UPLC-MS/MS method was developed to determine the concentration of six components, namely brucine, strychnine, liquiritin, isoliquiritin, isoliquiritigenin and glycyrrhizic acid, in different phase states of NGD, and to analyze the differences in the distribution of the key ingredients in different phase states. Further in-depth analysis of the release behavior of NG1 revealed a relatively high proportion of macromolecules. The study showed that the distribution of compounds in different phase states varied significantly, and NG1 was the key phase containing enriched active ingredients and manifested a slow release of soluble skeleton material. The results provide a standard of reference for the study of toxicity reduction by combining NV and GG.
3. Discussion
Chinese medicine is essentially based on the compatibility of the effective components. The complexity of the biocompatibility is reflected by the diversity of chemical components and body reactions. “Phase state” refers to the relatively stable structure formed by the interaction between different or the same components in a certain environment. The chemical components in different phase states of the decoction exhibit different chemical states, including free, combined and complex types. The distribution of active components in different phase states directly affects their physical and chemical properties and biological activities. A large amount of sediment will appear in TCM after decoction is allowed to settle, which is defined as the sedimentary phase. The NG1, NG2, NG3 and NG4 phases in the decoction, especially in the toxic TCM decoction, carry different components. The determination and analysis of the distribution of the principal components in different phase states and the release behavior of the main phases can provide a standard of reference for the elucidation of the synergistic mechanism of toxicity reduction based on the compatibility of TCM.
Now, the content determination methods of NGD are mostly used in the HPLC method [
22]. The report of liquid mass spectrometry is only limited to the research results of our research group [
23,
24,
25]. On the basis of previous research results, the UPLC-MS/MS method was established for the determination of principal components in the NGD and its different phases. The components with lower content in NG4 and NG2 could be accurately determined. The contents of brucine and strychnine in NV were decreased after the combination with GG, and the decrease in strychnine was more obvious. Further analysis of the content distribution of the six main components in different phase states showed that the content distributions of brucine and strychnine in different phase states were in the following order: NG1, NG3, NG2, NG4. Strychnine, the most toxic component in NV, accounted for 75.7% of the NG1. The distribution of the main components in GG from large to small was as follows: NG1, NG2, NG3, NG4. Similarly, the levels of glycyrrhizic acid and the other four components in the NG1 ranged from 51.4% to 75.5%. Thus, NG1 is the key phase for the enrichment of active substances among the different phases of NGD obtained.
An in-depth study of the release of each substance in the sedimentary phase in different dissolution media and fitting analysis revealed that brucine, strychnine and glycyrrhizic acid were released in large amounts in the artificial intestinal fluid. Brucine, strychnine and glycyrrhizic acid were released in descending order because brucine and strychnine are carbazole alkaloids, which are released easily due to the decreased interaction between the molecules in a weakly alkaline environment. Glycyrrhizic acid is a triterpenoid saponin with carboxyl group and acidic structure, which is easily dissociated in alkaline environment. Results of the model-fitting studies suggest that the release behavior of the most toxic strychnine fitted best with the Ritger–Peppas model, showing the slow release of the soluble components. Combined with previous reports of glycyrrhizic acid adsorption of strychnine, sediments reacted to form water-insoluble compounds, suggesting that the toxic (effective) components, such as strychnine and glycyrrhizic acid, existed in the form of sedimentary copolymers due to their inherent dissolution characteristics, which resulted in a slow-release behavior in the body. This phenomenon is also consistent with the large number of flocculents generated in NG, reported in our previous study.
4. Materials and Methods
4.1. Chemicals and Reagents
Brucine and strychnine (purity 98%, Y16A7S13272 and Z02A7S18869) were purchased from Shanghai Yuanye Biotechnology Co. Ltd. (Shanghai, China). Glycyrrhizic acid, liquiritin and isoliquiritin (purity 98%, HI042238196, HL04223898 and HI042234198) were purchased from Baoji Chenguang Technology Co., Ltd. (Baoji, China)
NV (number 20201102) and GG (number 20200601) were purchased from Harbin Tongren Medicine Market and identified by Mr. Wang Zhenyue from Heilongjiang University of TCM. HPLC-grade methanol, acetonitrile and formic acid were purchased from Dikama (Dikama, USA). PBS solution was purchased from Beijing Boao Tuoda Technology Co., Ltd. (Biotopped, Beijing). Deionized water was prepared using a Milli-Q ultrapure water preparation instrument (Millipore, Burlington, MA, USA). Other reagents were of analytical grade.
4.2. Preparation of NGD Phase States
Amounts of 100 g of NV and 300 g of GG were weighed and mixed with 5 volumes of water, heated and refluxed twice, initially for 60 min and then for 30 min, then mixed and refluxed twice and filtered. The filtrate was stored at −4 °C for 24 h. Phase separation was carried out via the following steps to obtain NG1, NG2, NG3 and NG4 phases (
Figure 5).
- (1)
Preparation of NG1: After standing, the extract was filtered with a dust-free filter paper and the solids precipitated on the filter medium were identified as NG1.
- (2)
Preparation of NG2: The supernatant obtained in step (1) was centrifuged at 12,000 r/min for 30 min at low temperature, and the precipitate obtained was designated as NG2.
- (3)
Preparation of NG3: The supernatant obtained in step (2) was transferred carefully into a dialysis bag. The extract was separated into two portions, according to the method described for dialysis. The portion inside the dialysis bag was considered as NG3.
- (4)
Preparation of NG4: The portion outside the dialysis bag in step (3) was designated as NG4.
All samples mentioned above were vacuum freeze-dried for backup. The yields in NG1, NG2, NG3 and NG4 groups were 41.78%, 2.57%, 48.51% and 7.10%, respectively. The obtained phases were analyzed using a Malvern laser particle sizer to determine the particle size and potential differences between the different samples (
Figure 6).
4.3. Chromatographic and Mass Spectrometric Determination Conditions
An ACQUlTY ultra-performance liquid chromatographic system (Waters, Milford, MA, USA) was used. The column temperature was set to 40 °C. The mobile phase consisted of solvent A (containing 0.1% aqueous formic acid) and solvent B (acetonitrile containing 0.1% formic acid). The gradient elution procedure was as follows: 0–5 min, 81% A; 5–9 min, 81–50% A; 9 min–12 min, 50–10% A; 12–13 min, 10–90% A; 13–19 min, 90% A. The flow rate was 0.3 mL/min. The sample injection volume was 3 μL.
Mass spectrometry was performed using an AB SCIEX 4500 QTRAP-triple quadrupole linear ion trap tandem mass spectrometer (QTrap–MS/MS, AB Sciex, Foster City, CA, USA) equipped with an electrospray ionization source (ESI). A multistage reaction monitoring (MRM) mode with a switch between positive and negative ions was used; the source injection voltage was set to 3.5 kV. The ion source temperature was set to 320 °C. The pressure of the nebulizer was 35 psi. Depolymerization potential (DP) and collision energy (CE) were optimized for all the analytes. The optimized MS conditions are shown in
Table 10.
4.4. Preparation of Standard Solutions, Samples and Negative Reference Solution
Brucine, strychnine, glycyrrhizic acid, liquiritin, isoliquiritin and isoliquiritigenin standards were precisely weighed and transferred to a 25 mL measuring bottle and dissolved in methanol to obtain a constant volume. The mixed control solution with brucine (0.276 mg/mL), strychnine (0.245 mg/mL), glycyrrhizic acid (0.222 mg/mL), liquiritin (0.193 mg/mL), isoliquiritin (0.290 mg/mL) and isoliquiritigenin (0.041 mg/mL) was obtained.
About 0.5 g each of NG1, NG2, NG3 and NG4 prepared as mentioned above were weighed accurately and mixed with 1 mL of concentrated ammonia water and 25 mL of chloroform in a conical bottle with a plug, and then sealed with ultrasound for 40 min to reduce the loss. After filtration, 10 mL of filtrate was removed accurately and dried in a water bath. Methanol was redissolved in a 10 mL flask, shaken well, and filtered through a microporous membrane (0.22 μm) to obtain the test solution. The negative reference solution without the test substance was prepared as described above.
4.5. Method Validation
The method was comprehensively validated for selectivity, calibration curve linearity, LLOQ, precision, matrix effect, recovery and stability according to the US FDA and Chinese Pharmacopoeia (2020) Guidelines for the Validation of Methods.
4.5.1. Selectivity
The selectivity of the method was assessed by comparing the chromatograms of the negative samples without the measured components with those of all analyte samples. Six analytes showed no peaks at the retention time, which indicated lack of interference with the test samples.
4.5.2. Precision
The intra- and interday precision were determined by testing the same standard solution in six replicates for three days in a row. Precision was shown by the coefficient of variation (RSD, %). The RSD values should not exceed 2%.
4.5.3. Calibration Curve and LOQs
Under the optimal chromatographic conditions, a detection signal-to-noise ratio of 10 was used as the limit of quantification of the analytes. The signal-to-noise ratio of 3:1 was used to determine the detection limit of each component. Linearity was investigated by plotting the peak areas of the analytes against the measured concentrations of the standards. The calibration equation was calculated using weighted least-squares regression (weighting factor of l/χ2) and the correlation coefficient r was greater than 0.999.
4.5.4. Repeatability
To evaluate the precision of the analytical method, six test samples were analyzed and the concentration of the components in each sample was calculated and expressed as RSD ≤ 2.0%, indicating good reproducibility.
4.5.5. Stability
To evaluate the stability of the analytes during sample preparation and storage, we monitored the concentrations of six analytes under different storage conditions and over 24 h. Expressed as the mean relative deviation (RSD, %), good stability was indicated when the RSD ≤ 2.0%.
4.5.6. Recovery and Matrix Effect
In order to evaluate the accuracy of the method, three test samples (high, medium and low concentrations) were prepared and the spiked recoveries were determined to ensure an accuracy greater than 95% and the RSD ≤ 2.0%. The matrix effect was determined by comparing the peak area ratio of the extracted NGD with the standard solution of the same concentration. Taking into account instrument fluctuations, matrix effects between 90% and 110% indicate that the analyte is hardly affected.
4.6. Content Determination
Four samples from 3 batches of NG1, NG2, NG3 and NG4, respectively, were selected, according to the optimal chromatographic conditions of sample analysis. The chromatographic peak area and the concentrations of six components in different phase states were calculated based on the composition of each phase in the raw herb.
4.7. In Vitro Release and Model Fitting
According to the Pharmacopoeia of the People’s Republic of China 2020 edition, 500 mL of degassed water, artificial gastric juice and artificial intestinal juice were injected into each operating container, and the temperature was adjusted to 37 ± 0.5 °C. The sedimentary phase was weighed to about 2.0 g in 3 portions, weighed accurately and transferred to containers containing water, artificial gastric juice and artificial intestinal juice. The centrifugation speed was adjusted to 100 r/min, and a 2 mL sample solution was drawn from the containers at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h and 12 h. Meanwhile, the same medium was added to the sample solution containing 0.22 μL of filtering agent, and the filtrate was obtained for later use. According to the selected method, the total content of brucine, strychnine, glycyrrhizic acid, liquiritin, isoliquiritin and isoliquiritigenin in the sedimentary phase was 100%. The cumulative release percentage was calculated, the release curve was drawn and the release kinetics was determined.
The cumulative drug release of the original indicator components was analyzed according to zero-order and first-order kinetics, and Higuchi and Ritger–Peppas models. The correlation equation and correlation coefficient R2 were obtained, and the goodness of fit was judged by R2.
4.8. Statistical Analysis
The experimental data were analyzed using SPSS 26.0 software. One-way ANOVA was used for comparison of multiple groups, and LSD test was used for comparison of two groups.
5. Conclusions
A rapid, simple and accurate UPLC/MS method was established for the determination of six main components in different phase states of NGD, and the release characteristics of NG1 were studied. The specificity, precision, linearity, repeatability, recovery and stability of the method was validated via various tests. Based on the perspective of microscopic ingredients, the concentrations of different phase components in the decoction were determined and the release characteristics of NG1 were analyzed. It was found that the toxic (effective) components were concentrated in NG1 after NV was combined with GG, and large concentrations of the toxic (effective) components in NG1 were released in the artificial intestinal fluid, demonstrating a slow-release behavior. The results provide a scientific basis for toxicity reduction and survival using the combination of NV and GG.