Integrated virtual screening and in vitro studies for exploring the mechanism of triterpenoids in Chebulae Fructus alleviating mesaconitine-induced cardiotoxicity via TRPV1 channel.

Background: In traditional Mongolian or Tibetan medicine in China, Chebulae Fructus (CF) is widely used to process or combine with aconitums to decrease the severe toxicity of aconitums. Researches in this area have predominantly focused on tannins, with few research on other major CF components for cardiotoxicity mitigation. The present study aimed to clarify whether triterpenoids can attenuate the cardiotoxicity caused by mesaconitine (MA) and investigate the mechanism of cardiotoxicity attenuation. Methods: Firstly, the pharmacophore model, molecular docking, and 3D-QSAR model were used to explore the mechanism of CF components in reducing the toxicity of MA mediated by the TRPV1 channel. Then three triterpenoids were selected to verify whether the triterpenoids had the effect of lowering the cardiotoxicity of MA using H9c2 cells combined with MTT, Hoechst 33258, and JC-1. Finally, Western blot, Fluo-3AM, and MTT assays combined with capsazepine were used to verify whether the triterpenoids reduced H9c2 cardiomyocyte toxicity induced by MA was related to the TRPV1 channel. Results: Seven triterpenoids in CF have the potential to activate the TRPV1 channel. And they exhibited greater affinity for TRPV1 compared to other compounds and MA. However, their activity was relatively lower than that of MA. Cell experiments revealed that MA significantly reduced H9c2 cell viability, resulting in diminished mitochondrial membrane potential and nuclear pyknosis and damage. In contrast, the triterpenoids could improve the survival rate significantly and counteract the damage of MA to the cells. We found that MA, arjungenin (AR), and maslinic acid (MSA) except corosolic acid (CRA) upregulated the expression of TRPV1 protein. MA induced a significant influx of calcium, whereas all three triterpenoids alleviated this trend. Blocking the TRPV1 channel with capsazepine only increased the cell viability that had been simultaneously treated with MA, and AR, or MSA. However, there was no significant difference in the CRA groups treated with or without capsazepine. Conclusion: The triterpenoids in CF can reduce the cardiotoxicity caused by MA. The MSA and AR function as TRPV1 agonists with comparatively reduced activity but a greater capacity to bind to TRPV1 receptors, thus antagonizing the excessive activation of TRPV1 by MA.

Metabolomics-based investigation of the chemical composition changes in Mongolian medicinal plant Euphorbia pekinensis before and after processing with Chebulae Fructus.

Euphorbia pekinensis (EP), known for its diuretic properties, is clinically utilized for treating conditions such as edema and malignant tumors. However, in its raw form, Euphorbia pekinensis is toxic, and oral administration of this crude medicine can lead to gastrointestinal stimulation, resulting in abdominal pain and diarrhea. In Mongolian medicine's ethnomedicinal system, a distinctive processing method called "Chebulae Fructus processing" is employed. Chebulae Fructus is used to mitigate the toxicity of EP and alleviate its purgative effects. Nevertheless, the detoxification mechanism associated with this processing method remains unexplored. It is hypothesized that processing with Chebulae Fructus may alter the chemical composition of EP, and the residual components of Chebulae Fructus within processed Chinese medicine might exhibit pharmacological antagonistic effects, thereby achieving the purpose of processing and reducing toxicity. To investigate this further, a combination of UPLC-QTOF-MS-based metabolomics technology and multivariate statistical analysis was employed to analyze and compare the chemical composition of raw and processed EP. Differential variables contributing to group separation were identified based on specific criteria, including VIP (Variable Importance in Projection) values of ≥ 1 in PLS-DA models, p-values < 0.05, and fold changes (FC) > 1.2 or < 0.8. The resulting differentially expressed features were then identified through database matching, literature review, or manual annotation. In total, 47 components were identified from the PEP samples in both positive and negative ionization modes, primarily belonging to flavonoids, terpenoids, organic acids, glycosides, and fatty acids. Among the raw EP group and PEP S4 group, 10 differential compounds were identified. Notably, one toxic terpene and one phenylpropanoid from EP were downregulated, while two bioactive components from Chebulae Fructus were upregulated in the processed group. The possible conversion reactions of these two processing Q-markers were also elucidated. The characteristic processing with Chebulae Fructus resulted in a change in the composition of this Mongolian medicine EP. Furthermore, this study provides a scientific foundation for optimizing the processing technology of EP and offers insights into the processing of other ethnomedicines with toxic properties.

The mechanism of Chebulae Fructus Immaturus promote diabetic wound healing based on network pharmacology and experimental verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Diabetic ulcers (DUs) are commonly seen in the lower limbs, especially the feet. Long-term hyperglycaemia in diabetic patients may cause peripheral microvascular damage, which affects local blood flow reconstruction when the skin is ruptured. This results in delayed or even non-healing of skin wounds. Chebulae Fructus Immaturus (CFI) is a traditional Chinese medicine. According to traditional Chinese medicine theory, CFI belongs to the lung channel and large intestine channel. Clinical data confirm a significant clinical effect of CFI in the treatment of skin diseases. CFI can be safely used to treat wounds due to its natural active ingredients. AIM OF THE STUDY: This study utilised HPLC-ESI-QTOF-MS/MS combined with network pharmacology to investigate the mechanism of Chebulae Fructus Immaturus extract (CFIE) in the treatment of DU. Moreover, the efficacy of CFIE on DU was verified in vitro and in vivo by constructing cell models and mouse models. MATERIALS AND METHODS: The main ingredients of CFIE were identified by HPLC-ESI-QTOF-MS/MS. The targets of these ingredients were predicted by database analysis and intersected with the DU targets. Gene ontology (GO) was used for functional enrichment of differential genes, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) was used for enrichment of signalling pathways related to the differential genes. The network pharmacology findings were validated in vivo and in vitro, and the affinity of key targets and active components was assessed using molecular docking. RESULTS: Twenty-nine compounds of CFIE were identified by HPLC-ESI-QTOF-MS/MS, and their potential targets were predicted. Among these, 41 targets were associated with DU. KEGG enrichment analysis showed that the PI3K/AKT and HIF-1α signalling pathways were significantly enriched, which may be related to the promotion of wound angiogenesis. In vitro cell experiments showed that CFIE promoted the proliferation, migration and angiogenesis of HUVECs, and also affected the expression of pathway-related proteins. In vivo experiments showed that CFIE increased the expression of pathway-related proteins in wound tissue and promoted the formation of blood vessels. CONCLUSIONS: In summary, this study systematically demonstrated the possible therapeutic effects and mechanisms of CFIE on DU through network pharmacology analysis and experimental verification. The results revealed that CFIE can accelerate the angiogenesis of diabetic wounds through the PI3K/AKT and HIF-1α signalling pathways, ultimately promoting the healing of diabetic wounds.

Comparative analysis of chemical components in fruits of Chebulae Fructus and its pulp based on chromatographic technology coupled with multivariate chemometric methods.

Chebulae Fructus, was extensively used as a food supplement and medicinal herb, which contained two medicinal forms corresponding to the mature fruit of Chebulae Fructus (CF) and CF pulp. They were widely used in the Chinese clinical medicine and it played a significant role in the Mongolian and Tibetan medicine for the treatment of sore throat, asthma, diarrhea and other diseases. Both of them were recorded in the 2020 Edition (Volume I) of the Chinese Pharmacopoeia. However, the chemical components of CF and CF pulp have not been holistically explored, which seriously hindered its quality evaluation. This study investigated the overall chemical profile of the CF and CF pulp using ultra high-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UHPLC-Q-TOF/MS) and ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Sixty-four chemical components were tentatively identified, and 13 components were quantified in Chebulae Fructus. Furthermore, multivariate chemometric methods were applied to compare the differences among CF samples, and all samples were classified by orthogonal partial least squares-discriminant analysis (OPLS-DA) based on the 13 quantified compounds. The results showed that CF and CF pulp were clustered in two different areas. Ellagic acid, chebulagic acid, chebulinic acid, corilagin and pentagalloyl glucose were selected as the significant constituents to different of CF and CF pulp. LC-MS coupled with chemometrics strategy analysis could comprehensively evaluate the holistic quality of CF, which provided a necessary information for the rational development and utilization of CF and CF pulp resource.

The therapeutic mechanism of Chebulae Fructus in the treatment of immunosuppression in Chinese yellow quail on the basis of network pharmacology.

Introduction: Chebulae Fructus (Terminalia chebula Retz.) is a well-known traditional Chinese medicine (TCM), one of the family Combretaceae, whose immature fruit is called Fructus Chebulae Immaturus or Zangqingguo. This present study aimed at detecting the target and therapeutic mechanism of Chebulae Fructus against immunosuppression through network analysis and experimental validation. Methods: Effective components and potential targets of Chebulae Fructus were Search and filtered through the Chinese herbal medicine pharmacology data and analysis platform. A variety of known disease target databases were employed to screen the therapeutic target proteins against immunosuppression and thus constructing a protein-protein interaction network. Hub genes and key pathways in this study were identified by continuous project enrichment analysis. Further, the core targets and therapeutic mechanism of Chebulae Fructus against immunosuppression in Chinese yellow quail through animal experiment. Results: Seventy-five identifiable major candidate targets of Chebulae Fructus were found and thus constructing a drug-compound-target-disease network. Targets derived from gene enrichment analysis play pivotal roles in lipid and atherosclerosis, fluid shear stress and atherosclerosis, and the hepatitis B pathway. Height of plicate and areas of lymphoid follicle were both increased and the expression of GATA-3 and T-bet was upregulated in Chinese yellow quail fed with Chebulae Fructus in animal experiment. Conclusion: Chebulae Fructus may be a helpful Chinese medicine with immunosuppressive effect and prospective applications in future. Further research is also needed to understand the mechanisms of immunosuppression and the mechanism of action of immunomodulators.

Hepatotoxic Components Effect of Chebulae Fructus and Associated Molecular Mechanism by Integrated Transcriptome and Molecular Docking.

Chebulae Fructus (CF) is a natural medicinal plant widely used for its various pharmacological properties. Natural products used to cure several diseases have been considered safe thanks to their little or no side effects. However, in recent years, a hepatotoxic effect has been found due to the abuse of herbal medicine. CF has been reported to have hepatotoxicity, but the mechanism is unclear. In this experiment, the toxic aspect and mechanism of CF action were evaluated by transcriptome analysis. Components of toxic CF fractions were identified by LC-MS, and hepatotoxic toxic components in toxic CF fractions were predicted by molecular docking. The results showed that the ethyl acetate part of CF was the main toxic fraction, and transcriptome analysis found that the toxic mechanism was highly related to lipid metabolism-related pathways, and CFEA could inhibit the PPAR signaling pathway. Molecular docking results showed that 3'-O-methyl-4-O-(n″-O-galloyl-ß-d-xylopyranosyl) ellagic acid (n = 2, 3 or 4) and 4-O-(3″,4″-O-digalloyl-α-l-rhamnosyl) ellagic acid have better docking energies with PPARα protein and FABP protein than other components. In summary, 3'-O-methyl-4-O-(n″-O-galloyl-ß-d-xylopyranosyl) ellagic acid (n = 2, 3 or 4) and 4-O-(3″,4″-O-digalloyl-α-l-rhamnosyl) ellagic acid were the main toxic components, which may play a toxic role by inhibiting the PPAR signaling pathway and affect lipid metabolism.

The potential mechanism of Chebulae Fructus in the treatment of hepatocellular carcinoma on the basis of network pharmacology.

INTRODUCTION AND OBJECTIVES: Hepatocellular carcinoma (HCC) ranks third on the list of the leading cause for cancer death globally. The treatment of HCC patients is unsatisfactory. However, the traditional Chinese medicine Chebulae Fructus has potential efficacy in the treatment of HCC. MATERIALS AND METHODS: We mined the active ingredients of Chebulae Fructus and its main targets from the Traditional Chinese Medicine Systems Pharmacology database. HCC-related datasets were downloaded from The Cancer Genome Atlas database and differentially expressed genes (DEGs) in HCC were obtained by differential expression analysis. Top10 small molecule compounds capable of reversing HCC pathology were screened by the Connectivity Map database based on DEGs. Ellipticine, an extract of Chebulae Fructus, had the potential to reverse HCC pathology. Protein-Protein Interaction (PPI) networks of DEGs in HCC were constructed using STRING. Eighteen potential targets of Chebulae Fructus for the treatment of HCC were obtained by taking intersection of DEGs in HCC with targets corresponding to the active constituents of Chebulae Fructus. In addition, MTT assay was also employed to examine the effect of ellipticine on HCC cell viability. RESULTS: It has been shown that ellipticine and ellagic acid have antitumor activity. Random Walk with Restart analysis of PPI networks was performed using potential targets as seeds, and the genes with the top 50 affinity coefficients were selected to construct a drug-active constituent-gene interaction network. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of key genes involved in the treatment of HCC with Chebulae Fructus demonstrated that these genes were mainly enriched in signaling pathways related to tumor metabolism such as cAMP signaling pathway and Ras signaling pathway. Finally, it was verified by MTT assay that proliferation of HCC cells could be remarkably hindered. CONCLUSIONS: We excavated ellipticine, a key active constituent of Chebulae Fructus, by network pharmacology, and elucidated the signaling pathways involved in Chebulae Fructus, providing a theoretical basis for the use of Chebulae Fructus for HCC clinical application.

[Study on regulation of CYP450 enzyme system to reduce liver toxicity through compatibility of Aconiti Kusnezoffii Radix Cocta with Chebulae Fructus and Glycyrrhizae Radix et Rhizoma].

Aconiti Kusnezoffii Radix Cocta is one of the most commonly used medicinal materials in Mongolian medicine. Due to the strong toxicity of Aconiti Kusnezoffii Radix Cocta, Mongolian medicine often uses Chebulae Fructus, Glycyrrhizae Radix et Rhizoma to reduce the toxicity, so as to ensure the curative effect of Aconiti Kusnezoffii Radix Cocta while ensuring its clinical curative effect, but the mechanism is not clear. The aim of this study was to investigate the effects of Chebulae Fructus, Glycyrrhizae Radix et Rhizoma and Aconiti Kusnezoffii Radix Cocta on the mRNA transcription and protein translation of cytochrome P450(CYP450) in the liver of normal rats. Male SD rats were randomly divided into negative control(NC) group, phenobarbital(PB) group(0.08 g·kg~(-1)·d~(-1)), Chebulae Fructus group(0.254 2 g·kg~(-1)·d~(-1)), Glycyrrhizae Radix et Rhizoma group(0.254 2 g·kg~(-1)·d~(-1)), Aconiti Kusnezoffii Radix Cocta group(0.254 2 g·kg~(-1)·d~(-1))and compatibility group(0.254 2 g·kg~(-1)·d~(-1),taking Aconiti Kusnezoffii Radix Cocta as the standard). After continuous administration for 8 days, the activities of total bile acid(TBA), alkaline phosphatase(ALP), amino-transferase(ALT) and aspartate aminotransferase(AST)in serum were detected, the pathological changes of liver tissue were observed, and the mRNA and protein expression levels of CYP1 A2, CYP2 C11 and CYP3 A1 were observed. Compared with the NC group, the serum ALP, ALT and AST activities in the Aconiti Kusnezoffii Radix Cocta group were significantly increased, and the ALP, ALT and AST activities were decreased after compatibility. At the same time, compatibility could reduce the liver injury caused by Aconiti Kusnezoffii Radix Cocta. The results showed that Aconiti Kusnezoffii Radix Cocta could inhibit the expression of CYP1 A2, CYP2 C11 and CYP3 A1, and could up-regulate the expression of CYP1 A2, CYP2 C11 and CYP3 A1 when combined with Chebulae Fructus and Glycyrrhizae Radix et Rhizoma. The level of translation was consistent with that of transcription. The compatibility of Chebulae Fructus and Glycyrrhizae Radix et Rhizoma with Aconiti Kusnezoffii Radix Cocta could up-regulate the expression of CYP450 enzyme, reduce the accumulation time of aconitine in vivo, and play a role in reducing toxicity, and this effect may start from gene transcription.

Study on regulation of CYP450 enzyme system to reduce liver toxicity through compatibility of Aconiti Kusnezoffii Radix Cocta with Chebulae Fructus and Glycyrrhizae Radix et Rhizoma / 中国中药杂志

Aconiti Kusnezoffii Radix Cocta is one of the most commonly used medicinal materials in Mongolian medicine. Due to the strong toxicity of Aconiti Kusnezoffii Radix Cocta, Mongolian medicine often uses Chebulae Fructus, Glycyrrhizae Radix et Rhizoma to reduce the toxicity, so as to ensure the curative effect of Aconiti Kusnezoffii Radix Cocta while ensuring its clinical curative effect, but the mechanism is not clear. The aim of this study was to investigate the effects of Chebulae Fructus, Glycyrrhizae Radix et Rhizoma and Aconiti Kusnezoffii Radix Cocta on the mRNA transcription and protein translation of cytochrome P450(CYP450) in the liver of normal rats. Male SD rats were randomly divided into negative control(NC) group, phenobarbital(PB) group(0.08 g·kg~(-1)·d~(-1)), Chebulae Fructus group(0.254 2 g·kg~(-1)·d~(-1)), Glycyrrhizae Radix et Rhizoma group(0.254 2 g·kg~(-1)·d~(-1)), Aconiti Kusnezoffii Radix Cocta group(0.254 2 g·kg~(-1)·d~(-1))and compatibility group(0.254 2 g·kg~(-1)·d~(-1),taking Aconiti Kusnezoffii Radix Cocta as the standard). After continuous administration for 8 days, the activities of total bile acid(TBA), alkaline phosphatase(ALP), amino-transferase(ALT) and aspartate aminotransferase(AST)in serum were detected, the pathological changes of liver tissue were observed, and the mRNA and protein expression levels of CYP1 A2, CYP2 C11 and CYP3 A1 were observed. Compared with the NC group, the serum ALP, ALT and AST activities in the Aconiti Kusnezoffii Radix Cocta group were significantly increased, and the ALP, ALT and AST activities were decreased after compatibility. At the same time, compatibility could reduce the liver injury caused by Aconiti Kusnezoffii Radix Cocta. The results showed that Aconiti Kusnezoffii Radix Cocta could inhibit the expression of CYP1 A2, CYP2 C11 and CYP3 A1, and could up-regulate the expression of CYP1 A2, CYP2 C11 and CYP3 A1 when combined with Chebulae Fructus and Glycyrrhizae Radix et Rhizoma. The level of translation was consistent with that of transcription. The compatibility of Chebulae Fructus and Glycyrrhizae Radix et Rhizoma with Aconiti Kusnezoffii Radix Cocta could up-regulate the expression of CYP450 enzyme, reduce the accumulation time of aconitine in vivo, and play a role in reducing toxicity, and this effect may start from gene transcription.

[Chemical variation in Aconti Kusnezoffii Radix before and after processing based on UPLC-Orbitrap-MS].

Some Chinese herbal medicine needs to be processed before it can be used as medicine, especially toxic Chinese medicine. Highly toxic Aconti Kusnezoffii Radix(Caowu in Chinese) is widely used in traditional Chinese medicine and Mongolian medicine. In traditional Chinese medicine, Caowu is usually processed by boiling with water(CW) until no white part inside and being tasted without tongue-numbing. In Mongolian medicine, it is usually soaked in Chebulae Fructus(Hezi in Chinese) decoction for several days(CH). Both methods could reduce toxicity according to reports. The biggest difference between CW and CH is that CW needs to be heated for 4-6 h, while CH needs Hezi as processing adjuvants. To explore the toxicity reduction mechanism of CW and CH, we studied the contents of various compounds in Caowu processed by two methods by UPLC-Orbitrap-MS. The results indicated that CW had 14 new ingredients, such as 14-O-anisoylneoline and dehydro-mesaconitine, while N-demethyl-mesaconitine and aconitine disappeared. At the same time, it could significantly decrease the content of diester diterpenoid alkaloids and increase the contents of monoester diterpenoid alkaloids and amine-diterpenoid alkaloids. CH had 9 new ingredients from Hezi, like gallic acid, chebulic acid and shikimic acid. Neither the kinds nor the contents of compositions from Caowu in CH changed little. This suggested that the processing mechanism of CW reduced highly toxic components(diester diterpenoid alkaloids) and increased the content of lowly toxic components(monoester diterpenoid alkaloids and amine-diterpenoid alkaloids). Attenuated principle of CH may be related to the components of Hezi. In this experiment, the conclusion shows that the chemical constituents of CW and CH are essentially different, and the two methods have different toxicity reduction principles.

Protective effect of phenolic acids from Chebulae Fructus immaturus on carbon tetrachloride induced acute liver injury via suppressing oxidative stress, inflammation and apoptosis in mouse.

Chebulae Fructus immaturus, a traditional Tibetan medicine, originated from the immature fruit of Terminalia chebula Retz., has been proven to have antioxidat function. However, its protection to injury liver cell caused by carbon tetrachloride (CCl4) has not been clarified. This study evaluated the effect of phenolic acid from Chebulae Fructus immaturus (PATC) on CCl4-induced acute liver injury in mice and related molecular mechanisms. Our data showed that PATC had convincing protective effects on the CCl4-induced acute liver injury by enhancing the anti-oxidative defense system, ameliorating inflammation and inhibiting the hepatocyte apoptosis.