Study on the mechanism of toxicity reduction and preservation of processed Ma Qian Zi based on metabolomics of inflammatory rats
Ma Qian Zi is an important and commonly used traditional Chinese medicine in China, first recorded in the Compendium of Materia Medica. It has the effects of unblocking collaterals, relieving pain, dispersing nodules, and reducing swelling; Mainly used for treating diseases such as swelling, breast abscess, and scrofula. The severe toxicity of raw horsehair greatly limits its clinical application. Traditional Chinese medicine believes that processing can achieve the effect of reducing toxicity and preserving efficacy. There are many methods for processing horseradish, including sand blanching, deep frying, stir frying, tofu making, etc. Modern research has shown that during the high-temperature frying process, the main toxic components of strychnine and strychnine are effectively reduced in the production of nitrogen oxides with lower toxicity and better therapeutic effects. At present, research still remains at the level of characteristic component analysis. Although the mechanism of reducing toxicity in the processing of Fructus Strychni has been preliminarily explained, the complex chemical composition of Fructus Strychni and the unclear mechanisms of absorption, transformation, metabolism, and regulation after entering the human body hinder the in-depth interpretation of its pharmacological effects and active substances.

In recent years, metabolomics has rapidly developed with the help of high-throughput and high-resolution instruments, especially in the field of traditional Chinese medicine, where it has been widely applied. By monitoring endogenous small molecule qualitative and quantitative changes, combined with changes in the body’s functional status, the molecular mechanisms of traditional Chinese medicine efficacy can be elucidated. This study preliminarily evaluated the toxicity changes of processed Semen Strychni before and after processing based on mortality rate; Evaluate the anti-inflammatory effect before and after processing based on swelling rate and inflammatory factor levels; Afterwards, the effective substances after entering the bloodstream were analyzed by plasma drug chemistry. Finally, the endogenous metabolites in plasma were analyzed and identified using an inflammation intervention model with Ma Qian Zi. The mechanism of Ma Qian Zi’s detoxification and efficacy before and after processing was explored at the chemical, molecular, and metabolic levels.

Preliminary toxicity experiments have shown that the mortality rate of the sand scalding group at high doses is significantly lower than that of the raw group, indicating that the toxicity of dried horse seeds is indeed significantly reduced after high-temperature processing. The experiment on plantar swelling rate showed that at high doses, the swelling rate of both the raw product group and the sandhot product group was significantly reduced, proving that the raw product and sandhot product do have certain anti-inflammatory effects. There was no statistically significant difference in swelling rate between the sand scalding group and the raw product group, indicating that there was no significant change in anti-inflammatory efficacy before and after processing. The detection results of inflammatory factors are roughly consistent with the swelling rate experiment. There are various processing methods for making horseradish, including sand blanching, deep frying, stir frying, explosive pressing, baking, microwaving, boiling, etc. Most of the processing steps involve heating, and it is generally believed that heating will reduce the content of the main toxic components, brucine and strychnine. However, the more effective treatments, such as isobrucine, isobrucine, strychnine nitrogen oxides, and brucine nitrogen oxides, will increase in content. Liu Jun et al. believed that heating processing is the result of alkaloid decomposition and oxidation reactions, and verified it using H2O2. This study further confirmed through preliminary toxicity experiments and pharmacological experiments that sand blanching processing can indeed reduce the toxicity of Semen Strychni and retain a certain anti-inflammatory therapeutic effect.

Through the study of plasma metabolites, it was found that alkaloids identified in the drug containing plasma accounted for 77% of the transitional components, indicating that alkaloids are mainly responsible for anti-inflammatory effects; The identification of nitrogen oxides with better therapeutic effects and lower toxicity in the mobile components is consistent with previous studies on the processing mechanism of Semen Strychni. Out of the 60 identified metabolites, only 7 underwent phase II reactions, indicating that these migratory components are easily cleared by the body after undergoing phase I reactions. Through differential analysis of drug containing plasma, it was found that alkaloids accounted for the majority of the identification results, and the substance quality of the sand scalding group was significantly higher than that of the raw product group; Nitrogen oxides of brucine and strychnine were also identified in the sand scalding group; In the differential analysis, only Shidianing was identified, and according to literature reports, the pharmacological strength of Shidianing is 40 times that of strychnine; The above results confirm that the anti-inflammatory therapeutic effect of processed products should not be lower than that of raw products. In the differential analysis, three organic acids were also identified, and the mass of the sand scalding group was lower than that of the raw group, which may be related to the easy decomposition of organic acid components at high temperatures. The above plasma medicinal chemistry research provides data reference for clarifying the in vivo functional substance basis of processed products of Fructus Strychni.
Through metabolomics research, it was found that, except for the dexamethasone group, the metabolic profile of the Ma Qian Zi sand scalding group was closest to that of the normal group, indicating that the processed product has a good regulatory effect on the metabolic disorders caused by inflammation in the body. The analysis of biomarkers proves that the anti-inflammatory mechanism of processed Ma Qian Zi products is mainly related to amino acid metabolism and lipid metabolism. The liver is the main organ for amino acid metabolism in animals, and liver damage can cause metabolic disorders and functional impairments. The decrease in amino acid metabolism within liver cells can lead to an increase in the concentration of amino acids in the blood, thereby affecting changes in the levels of amino acids in the blood, urine, and bile. Glutamate is an important excitatory neurotransmitter. If glutamate metabolism is disrupted, the “glutamate glutamine cycle” is disrupted. Excessive concentration of glutamate in extracellular fluid can activate too many excitatory amino acids, leading to toxic reactions. Glutamine is an important raw material for the synthesis of α – ketoglutarate. α – ketoglutarate can reduce the levels of inflammatory factors in stress to a certain extent, and after modeling inflammation, it can cause metabolic disorders of glutamate in tissues, resulting in toxic effects. The experimental results showed that the glutamate content in the plasma of the sand scalding group was significantly reduced, thereby reducing the excitability of the brain nerves and exerting a sedative effect. At the same time, the glutamine content was upregulated to achieve anti-inflammatory therapeutic effects. Phenylalanine is mainly metabolized into tyrosine in tissues such as the liver, and then synthesized in the nervous system and adrenal medulla, such as dopamine, norepinephrine, adrenaline, etc. The metabolic status of phenylalanine will directly affect the growth, development, and normal physiological functions of the body. The results of this study indicate that phenylalanine is significantly upregulated in the normal group, while it is slightly downregulated in the raw product group and sand scald group. The sand scald group is closer to the normal group, indicating that the sand scald group can better regulate the phenylalanine metabolism disorder caused by inflammation. Citrulline is produced in the metabolic pathways of arginine and proline, and under the action of corresponding enzymes, it generates arginine, ornithine, etc. It can effectively reduce blood ammonia levels, avoid organ damage, and alleviate pain reactions in the body. The content of citrulline in the model group was significantly downregulated compared to the normal group, while it was up-regulated in both the raw product group and the sand scald group, indicating that sand scald can effectively regulate arginine metabolism, dilate blood vessels, and alleviate pain caused by inflammation. Tyrosine is mainly metabolized by tyrosine hydrogenase to produce dihydroxyphenylalanine, which is then converted into dopa and other sympathetic neurotransmitters under the action of tyrosine hydroxylase. The rat egg white induced plantar swelling inflammation model can cause a rapid decrease in local blood flow, accompanied by inflammatory reactions and pain, and the pain sensitivity response will gradually increase. The content of dopamine was upregulated in the model group, while it was significantly downregulated in the sandhot group, indicating that sandhot products may reduce pain sensitivity by regulating dopamine metabolism. The metabolism of glycerophospholipids is a key metabolic pathway for the hepatotoxicity and processing attenuation of Semen Strychni. There are two key targets involved in glycerophospholipid metabolism, namely phosphatidylcholine and phosphatidylethanolamine, which can be converted into each other mainly in liver tissue. Previous studies have shown that liver damage caused by horsehair can lead to lipid metabolism disorders, involving changes in substances such as glycerides, acylglycerols, phosphatidylethanolamines, phosphatidylcholine, as well as related inflammatory factor levels. Phosphatidylcholine has anti-inflammatory and immune enhancing effects, and its anti-inflammatory and potential therapeutic effects may be related to reducing the microcirculatory inflammatory response induced by neutrophils; Lysophosphatidylcholine is a product or metabolite of phosphatidylcholine, distributed in the plasma pool of extrahepatic tissues, and can induce various pro-inflammatory reactions. According to the experimental results, the levels of phosphatidylcholine and lysophosphatidylcholine were upregulated in the model group, while they were decreased in the raw product group and the sand scalding group, and were close to the normal group. This indicates that the extract of Malvaceae can effectively regulate the levels of phosphatidylcholine and lysophosphatidylcholine to restore balance. The liver is the most active site for the synthesis of glycerophospholipids in the body, which to some extent confirms the objective existence of the hepatotoxicity of Malvaceae; At the same time, it also confirms that Ma Qian Zi Sha Hot Pot has good anti-inflammatory and detoxifying effects.
In summary, this study used metabolomics to confirm that the toxicity of processed Ma Qian Zi was reduced while still retaining good anti-inflammatory therapeutic effects. The active substances in its body should be mostly alkaloids; The anti-inflammatory and analgesic effects, as well as the liver and kidney toxicity, of Ma Qian Zi are closely related to the disruption of pathways such as glutamine glutamate metabolism, phenylalanine metabolism, thiometabolism, glycerophospholipid metabolism, arginine and proline metabolism. The research results provide useful scientific references for exploring the mechanisms of reducing toxicity, enhancing efficacy, or preserving efficacy in the processing of toxic traditional Chinese medicinal materials containing alkaloids.