Optimal compatibility proportional screening of Trichosanthis Pericarpium- Trichosanthis Radix and its anti- Inflammatory components effect on experimental zebrafish and coughing mice

Ethnopharmacological relevance: The herbal pair of Trichosanthis Pericarpium (TP) -Trichosanthis Radix (TR) can be seen in the famous formula Beimu Gualou San. It is a commonly selected combination of medicinal herbs for the treatment of cough with lung heat. Both drugs are derived from Trichosanthes kirilowii Maxim, a medicinal plant known for its ability to clear heat, resolve phlegm, produce saliva, and alleviate dryness. However, the optimal combination ratio and active ingredients of TP-TR have yet to be determined. Aim of the study: This study aims to investigate the optimal combination ratio of TP-TR and its anti-inflammatory active ingredients in cough treatment. Materials and methods: A zebrafish (Danio rerio) inflammatory injury model and response surface method were applied in the present study to determine the appropriate proportion of TP-TR. Chemical constituents in TP-TR were identified using HPLC-ELSD and UPLC-MS/MS methods. Subsequently, a cough mouse model was created using an ammonia solution to evaluate the effectiveness of the optimal TP-TR ratio. Network pharmacology and intestinal flora sequencing were used to validate the anti-inflammatory components of TP-TR. Results: The herbal pair of TP -TR at the ratio of 1:2 showed an optimal anti-inflammatory effect, with a composite inflammatory factor score of 119.645 in the zebrafish experiment. TP-TR combination facilitated the dissolution of glutamine, inosine, cytosine, isoquercetin, and other substances. In the animal model, the TP-TR (1:2) treatment significantly reduced the frequency of coughs and prolonged cough latency compared to the model group. Results of the network pharmacology indicated that inflammatory-related factors such as TLR4, STAT3, EGFR, and AKT1 played crucial roles in cough treatment with TP-TR, consistent with the validation experiment. The 16s rDNA sequencing results revealed a significant increase in the abundance of Clostridia_UCG014, Lachnospiraceae, Christenella, Ruminococcus, and other species in the intestinal tract of mice after modelling. TP-TR (1:2) reduced the abundance of pro-inflammatory flora such as Clostridium_UCG-014 and Lachnospira, which were closely associated with L-lysine and trans-4-hydroxy-L-proline present in TP-TR according to correlation analysis. Conclusion: TP-TR may promote the dissolution of glutamine, thymidine, inosine, cytosine, isoquercetin, and other components through their combination, thereby regulating the abundance of Clostridium_UCG-014 and Lachnospira and exerting an antitussive effect. This study, for the first time, showed that TP-TR at a 1:2 ratio exhibits superior anti-inflammatory effects. In addition to inflammatory mediators like EGFR, TLR4, AKT1, and STAT3, gut microbes could also serve as potential regulatory targets of TP-TR in the treatment of cough. 2'- Deoxyguanosine monohydrate, L-lysine, L-leucine, gamma-aminobutyric acid, L-valine, L-tryptophan, L-proline, trans4-hydroxy-L-proline, L-methionine, uridine, 2'-deoxyinosine, guanosine, cucurbitacin B and cucurbitacin D were identified as its anti-inflammatory active ingredients.

Automated summary

The study aims to investigate the optimal combination ratio of Trichosanthis Pericarpium - Trichosanthis Radix (TP-TR) and its anti-inflammatory active ingredients in the treatment of cough. Through zebrafish and mouse models, the study found that TP-TR at a ratio of 1:2 exhibited superior anti-inflammatory effects and identified several anti-inflammatory active ingredients. Furthermore, network pharmacology and intestinal flora sequencing validated the anti-inflammatory components of TP-TR and revealed its potential regulatory effects on gut microbes.