Material Basis For Combining Euphrobia Kansui And Licorice Based On Rat Liver Microsomal

Background: The herbal-pair, Kansui and Licorice, belongs to the "eighteen incompatible medicaments" category of traditional Chinese medicine. Kansuiphorin C (KC) is the main toxic component of Kansui. The main component of licorice is glycyrrhizic acid, which is hydrolyzed to glycyrrhetinic acid. Currently, the synergistic mechanism between Kansui and Licorice is unclear. Methods: Rat liver microsomes were used in this experiment, HPLC was used to detect the contents of KC, glycyrrhizic acid, and glycyrrhetinic acid to determine whether these compounds are metabolic substrates of CYP450. A control group with isozyme inhibitors was also employed to reveal the isozyme subtypes involved in compound metabolism. To further explain the induction or inhibitory effect of the above compounds on liver microsomal enzymes, enzyme activity was indirectly revealed based on changes in the contents of known metabolites of CYP2E1, CYP2C9, and CYP3A4. Results: KC and glycyrrhetinic acid were metabolic substrates of CYP450. CYP2E1 and CYP2C9 are mainly involved in the partial metabolism of glycyrrhizic acid in the liver. CYP2E1 and CYP3A4 are mainly involved in the partial metabolism of glycyrrhetinic acid in the liver. CYP2E1, CYP2C9, and CYP3A4 did not play a major co-first authors. role in the metabolism of KC. KC had little effect on the metabolism of glycyrrhizic acid and glycyrrhetinic acid. Glycyrrhizic acid, glycyrrhetinic acid, and KC induced CYP3A4 and inhibit CYP2E1. Both glycyrrhizic acid and glycyrrhetinic acid could inhibit the induction of CYP3A4 after combination with KC. KC with glycyrrhizic acid could synergistically inhibit the activity of CYP2E1, while KC with glycyrrhetinic acid could synergistically induce the activity of CYP2E1 Conclusion: KC and glycyrrhetinic acid were metabolic substrates of CYP450. KC, glycyrrhizic acid and glycyrrhetinic acid have different inducing and inhibiting effects on CYP450 enzyme.


Background
The eighteen incompatible medicaments are important for explaining the properties of traditional Chinese medicine [1] . Based on the traditional Chinese medicine theory, the use of contraindicating drugs will lead to an increase in toxicity and side effects or a decrease in efficacy. As a result, these drugs belong to the incompatibility category of traditional Chinese medicine [2][3] . The herbal-pair, Kansui and licorice, belongs to the category of "18 incompatible medicaments" [4] . Kansui is the dried root of the euphorbiaceae plant, euphorbia kansui T. N. Liou ex S. B. Hois, a type of markedly purgative water drug [5] . The major component of this plant is giant euphorbia officinalis diterpenoids [6][7] , which is the main irritant and toxic ingredient.
Kansuiphorin C (KC) is also an ingredient found in kansui [8] . Licorice is the dried root and rhizome of the leguminous plant, Glycyrrhiza uralensis Fisch. One of the main components of licorice is glycyrrhizic acid [9][10] . Glycyrrhizic acid is hydrolyzed to produce glycyrrhetinic acid [11] . Further, it has a function similar to adrenocortical hormones and can lead to water-sodium retention [12] . Therefore, from the viewpoint of water-electrolyte metabolism, the co-administration of Kansui and Licorice should be prohibited owing to their synergistic induction of edema [13] . However, the mechanism of Euphorbia kansui and licorice has not been fully studied, thereby markedly hindering further development of these compounds.
Cytochrome P450 (CYP450) is the main component of the liver microsomal mixed function oxidase system [14] and is involved in the phase I biotransformation of many endogenous and exogenous compounds in vivo [15] . Thus, CYP450 not only reduces the long-term accumulation of drugs in the body and prevents drug accumulation poisoning, but also converts some protoxicants into toxicants, resulting in a toxic effect [16] . The P450 enzymes that are involved in the metabolism of most drugs in vivo are mainly composed of three gene families: CYP1, CYP2, and CYP3.
Each gene family is known to possess many subtypes [17] . Among them, CYP3A4/5, CYP2C9, and CYP2E1 are involved in approximately 96% of the biotransformation of clinical drugs metabolized by CYP450 enzymes. Further, each subtype has its own metabolic substrate and metabolic mode [18][19] .
In this study, HPLC combined with metabolic pathway analysis was used to determine whether KC, glycyrrhetinic acid, and glycyrrhizic acid are metabolic substrates of the CYP450 enzymes and isozyme substrates involved in compound metabolism. We also sought to determine whether kansui and licorice are synergistically metabolized by liver enzymes and play a role in the metabolism of toxic substances or glycyrrhetinic acid. Through metabonomics, studying the interaction between traditional Chinese medicine from the viewpoint of CYP450 enzymes can provide an experimental basis for exploring the mechanism of action of the 18 incompatible medicaments. (chromatography-grade, Pure, Pittsburgh, USA). All other reagents were of analytical grade.

Solution preparation
The enzyme inhibitors, amiodarone hydrochloride, diethyldithiocarbamic acid, and ketoconazole were prepared at a concentration of 1 g/L using PBS. Briefly, the application solution was placed in a hot water bath to maintain its solubility. Glycyrrhizic acid (1 mg) was mixed with 100 µL of ethanol. Thereafter, PBS was added to achieve a final volume of 1 mL and a concentration of 1 g/L.

Liver microsomal incubation system
The total volume of the incubation system was 200 μL and the final concentration of liver microsomal protein was 1 g/L [20] . For the NADPH regeneration system (containing 0.5 mmol/L NADP, 10 mmol/L G-6-P, 1 kU/L G-6-P-OH, 0.5 mmol/L MgCl 2 ), NADPH was prepared at a concentration of 1 mg·ml -1 . The liver microsome was used on ice, stored in liquid nitrogen at -26 ℃, and dissolved at 4 ℃ for use [21] . In the enzyme inhibitor groups, the enzyme inhibitors amiodarone hydrochloride, diethyldithiocarbamic acid, and ketoconazole were separately added to the liver microsomal incubation system and incubated at 37 °C for 5 min. Other controls were incubated at 37 °C for 5 min. The corresponding compounds were added to start the reaction. After incubation at 37 °C for 30 min, 50 μL of ice-cold acetonitrile was added to stop the reaction. The homogenate was centrifuged at 12000 rpm for 12 min at 4 °C, and the supernatant was injected into the system for HPLC analysis.

HPLC conditions
The Agilent 1100 HPLC system was employed. This system was equipped with a degasser, binary pump, auto-sampler, and Zorbax Eclipse Plus-C18 (250 × 4.6 mm, 5 µm). The analytical column was maintained at 38 ℃ and the mobile phase was mL/min, and the injection volume was 10 μL. Three parallel tubes were prepared for each isozyme. To ensure consistent performance of the analytical system, the reference substances 500 μg/mL KC, glycyrrhizic acid, and glycyrrhetinic acid were repeatedly injected six times to determine the precision during the operation of the instrument.

Grouping and Sample preparation
The following groups were employed: supernatant was retrieved to determine the content of 1-hydroxymidazolam.

6-hydroxychlorzoxazone HPLC conditions
The analytical column was maintained at 25 °C, and the mobile phase was composed of water (A) and acetonitrile (D). The following gradient program was employed : 0 min, 37%-75% D; 10 min, 75%-37% D; 12 min, 37% D; 13 min, 37% D; and return to the initial condition for 3 min. The flow rate was 1 mL/min and the injection volume was 10 μL. Detection was carried out at a wavelength of 280 nm.

4-hydroxytoluenesulfonbutylurea HPLC conditions
The analytical column was maintained at 40 °C, and the mobile phase was composed of water (A) and acetonitrile (D). The following gradient program was employed: 0 min, 5%-60% D; 20 min, 60%-5% D; 25min, 60%-5% D; return to the initial condition in 3 min. The flow rate was 1 mL/min and the injection volume was 10 μL.
Detection was carried out at a wavelength of 230 nm.

1-hydroxymidazolam HPLC conditions
The analytical column was maintained at 35 °C, and the mobile phase was composed of water (A) and methanol (D). The following gradient program was employed: 0 min, 40%-60% D; 11 min, 60%-40% D; 12 min, 40%-40% D; 16 min, 40%; return to the initial conditions for 3 min. The flow rate was 1 mL/min and the injection volume was 10 μL. Detection was carried out at a wavelength of 240 nm.

Effects of CYP450 isozymes on the contents of KC, glycyrrhetinic acid, and glycyrrhizic acid in rat liver microsomes
After the addition of liver microsomes, the content of KC and glycyrrhetinic acid decreased by 50%, while that of glycyrrhetinic acid decreased by only 16% under the same conditions.
To further explore the metabolic enzyme subtypes of the three compounds, the corresponding inhibitors of CYP2E1, CYP2C9, and CYP3A4 were added to the liver microsome system. The content of glycyrrhizic acid and glycyrrhetinic acid increased after the addition of enzyme inhibitors. However, the content of glycyrrhizic acid was close to that of the control group after the addition of the CYP2E1 inhibitor, suggesting that CYP2E1 is mainly involved in the partial metabolism of glycyrrhizic acid in the liver. Glycyrrhizic acid content increased after the addition of the CYP2C9 inhibitor, indicating that CYP2C9 was also involved in the metabolism of glycyrrhizic acid in the liver. Further, the content of glycyrrhizic acid increased slightly after the addition of CYP3A4 inhibitor, suggesting that CYP3A4 was less involved in the metabolism of glycyrrhizic acid in the liver. As the content of glycyrrhetinic acid increased significantly after the addition of the CYP3A4 and CYP2E1 inhibitors, these inhibitors were found to be mainly involved in the metabolism of glycyrrhetinic acid, while CYP2C9 had less effect on the metabolism of glycyrrhetinic acid. After the addition of KC, the content of glycyrrhizic acid increased slightly, while that of glycyrrhetinic acid decreased slightly; however, the change was not obvious, suggesting that KC had little effect on the metabolism of glycyrrhizic acid and glycyrrhetinic acid ( Table 1, Figures 1-4). The content of KC decreased after the addition of the inhibitors, glycyrrhizic acid, and glycyrrhetinic acid, suggesting that CYP2E1, CYP2C9, and CYP3A4 did not play a major role in the metabolism of KC. Further, no substance was detected after the addition of the inhibitors of CYP2C9. After three inhibitors were added to KC without liver microsomes, the content of KC decreased. KC reacted with three inhibitors, and glycyrrhizic acid and glycyrrhetinic acid promoted the metabolism of KC (Table 2).

DISCUSSION
The processes of metabolism and transformation of drugs in the body are complex.
Although the components of traditional Chinese medicine are complex, in the final analysis, the basic of its function is still the interaction between compounds of traditional Chinese medicine, and the metabolism of its effective components in the body still follows the basic law of general drug metabolism.

Function of subtypes of liver drug enzymes
CYP3A4 is one of the most important drug metabolic enzymes in the liver [22] that participates in the metabolism of approximately 45% to 60% of drugs commonly used in clinics. However, the metabolism of drugs markedly varies among individuals (i.e., approximately 10-100 times in the liver and 30 times in the small intestine) [23] .
CYP2E1 is a key member of the CYP450 enzyme system and a key enzyme involved in the metabolism and inactivation of drugs and some toxic substances (ethanol, acetone, benzene, carbon tetrachloride, etc.) [24][25] . CYP2E1 is genetically polymorphic, which can affect its expression and lead to differences in drug metabolism and toxicity among individuals; this is crucial for the active transformation of drugs and poisons [26] . Previous studies have shown that the combination of kansui and licorice can induce the gene and protein expression of CYP2E1 to a greater extent, and promote the transformation of its pre-carcinogens and pre-toxicants into carcinogens and toxicants [27] .
The CYP2C family is the largest subfamily of CYP450 enzymes in mammals.
CYP2C9 is the main member of the family, accounting for 20% of the total liver microparticle CYP450 enzymes, and approximately 16% of commonly used clinical drugs are catalyzed by this enzyme [28][29] . CYP2C9 gene polymorphisms are an important risk factor for hepatotoxicity. The high mutation rates of CYP2C9*2 and CYP2C9*3 mutants reduce the metabolic rate of CYP2C9 for drugs by tens of times, thereby markedly changing the clearance rate of drugs in the body, resulting in drug accumulation, and a 7.50-fold increase in the risk of liver injury [30] .
Many subtypes of liver drug enzymes have been demonstrated to play the role of drug metabolizer, promoting the transformation of inactive substances into biologically active substances, degrading biotoxicants, and promoting the transformation of pre-toxicants or pre-carcinogens into carcinogens or poisons.

Effect of CYP450 on the contents of glycyrrhizic acid, glycyrrhetinic acid, and KC
Based on the results presented in 3.1, when glycyrrhizic acid and glycyrrhetinic acid were combined with KC, the content of glycyrrhizic acid increased slightly, while that of glycyrrhetinic acid decreased slightly. However, such change was not obvious, suggesting that KC had little effect on the metabolism of glycyrrhizic acid and glycyrrhetinic acid. Based on these findings, including those of metabonomics, there may be other substances in Fructus kansui that promote the metabolism of glycyrrhetinic acid. KC and glycyrrhetinic acid are suggested to be the metabolic substrates of liver drug enzymes. Further, the main metabolic pathway of glycyrrhizic acid is not liver microsome enzymes.

Subtypes of liver drug enzymes involved in glycyrrhizic acid, glycyrrhetinic acid, and KC metabolism
Liver microsomal experiments in vitro showed that glycyrrhetinic acid was less metabolized in the liver, and glycyrrhetinic acid was mainly metabolized through CYP3A4 and CYP2E1. Although KC is the metabolic substrate of liver microsomal enzymes, it may not be metabolized by CYP2C9, CYP3A1, and CYP2E1, as well as other metabolic subtypes. Some researchers believe that CYP2C19 may be involved in the metabolism of KA and KB, which are toxic components of kansui. KA, KB, and KC are terpenoids of Euphorbia kansui; thus, KC may also be metabolized by CYP2C19 [31] . The combination of glycyrrhizic acid and glycyrrhetinic acid with KC enhanced the metabolism of KC, inhibited the metabolism of glycyrrhizic acid, and promoted the metabolism of glycyrrhetinic acid. However, studies have shown that glycyrrhizic acid and glycyrrhetinic acid can inhibit the metabolism of KA and KB [32] ; thus, it is necessary to combine the content, absorption, and distribution of three terpenoid esters in Fructus kansui to further reveal the synergistic mechanism.
The enzyme activity experiment revealed that glycyrrhizic acid, glycyrrhetinic acid, variety of processing, route of administration, administration time, etc. [33][34][35][36][37] . Among them, there have been many studies on compatible doses and proportion [38][39] . When the ratio of kansui and licorice is 1: 15 or 1: 10 based on the low dose (close to the clinical dosage), licorice not only plays a role in diuresis, but can also reduce toxicity to a certain extent [40] .
From the point of view of drug metabolic enzymes, to study the biological effects of the synergy between Fructus kansui and licorice, our findings not only provide a reference for the security of drug usage in clinical practice, but also serve as a useful reference for exploring the mechanism of compatibility of traditional Chinese medicine and expanding this research idea.

CONCLUSIONS
Through in vitro liver microsomal experiments, KC and glycyrrhetinic acid were identified as the metabolic substrates of liver microsomal enzymes.