Introduction

Herbal medicine decoction has been a crucial method for preventing and treating illnesses for millennia, and remains one of the most prevalent forms of medication used in clinical settings [8, 9]. Supramolecular chemistry was proposed by Nobel Prize winner Jean-Marie Len in 1973. It typically refers to the combine of two or more molecules through intermolecular interactions, resulting in intricate and well-organized aggregates [10, 11]. At the same time, with the popularization of microscopic morphology research techniques, the study of supramolecular aggregates in herb medicine decoction has made some breakthroughs. For example, our laboratory has identified supermolecules present in the decoction of Rhei Radix et Rhizoma and Coptidis Rhizoma, namely emodin and coptisine, have the ability to form nanoparticles (NPs) with a size of approximately 50 nm, nanofibers can be formed by rhein and coptidine [12]; the supramolecular components of Glycyrrhiza uralensis Fisch (GC) and Coptidis Rhizoma co-decoction were important substances to exert their antibacterial effects [13]; NPs with different sizes in the Huanglian Jiedu decoction showed good inhibitory effect on bacteria [14]. In addition, Zhou et al. [15] was able to effectively isolate colloidal nanoparticles from Maxing Shigan decoction and found that these particles were linked to ephedrine and pseudoephedrine. The supramolecules present in the herb medicine decoction serve as a significant material foundation for the therapeutic effects of the substance.

At present, due to the problems of inconveniently carrying decoction and the low utilization rate of herb medicines, modern medicine has changed traditional decoction into granules, extracts, and other new dosage forms [16]. Nevertheless, compared to the thousands of years of conventional decoction, they had been the subject of significant and contentious debate because of novel dosage forms that lack the method of co-decocting combination herb medicines. By comparing the difference between formula granules and combined decoction of Coptidis Rhizome and Evodiae Fructus, Quan [17] found that the formula granules could not reproduce the trend of influence of combined decoction of herb medicine on proportion and component content. Huang [18] found that the thermodynamic heating process promoted the assembly of berberine and baicalin to form supermolecule’ nanospheres, which produced a better antibacterial effect than the physical mixture. These results indicate that herb medicine co-decoction is one of the necessary conditions for the formation of herb medicine supermolecules, which affects the biological activity of clinical herb medicine. However, there are few reports on the effect of co-decocting on the supermolecules’ micromorphology and biological activity of herb medicine, and the effect of supermolecules’ micromorphology changes on its biological activity under decoction regulation is not clear.

Mahuang Fuzi decoction (MGF), as a classic prescription in the Synopsis of Prescriptions of the Golden Chamber used clinically for 1800 years, consists of three kinds of Chinese herbs, namely Ephedra sinica Stapf (MH), GC, Aconitum carmichaeli Debx (FZ). Among them, MH as a traditional medicine used in Oriental medicine for thousands of years, can effectively reduce inflammation, lower fever and relieve pain [19,20,21,22]. FZ is first recorded in Shennong Classic of Materia Medica and its main function is analgesic, anti-inflammatory, antibiotic, antipyretic, cardiotonic, etc. [23, 33], so the influence of MGF, MGF SA and MIX in activating the NF-κB pathway were investigated. In the normal group, Fig. 8 showed the green fluorescence of NF-κB p65 was mainly observed in the cytoplasm and not in the Hoechst-stained blue nucleus. There was intense green fluorescence in the nucleus, which showed that LPS had caused NF-κB p65 to move from the cytoplasm to the nucleus in the model group. By introducing MGF and MGF SA treatment at a concentration of 500 mg/L, significant inhibition of the LPS-induced NF-κB p65 nuclear migration was possible. Among them, the inhibition effect of MGF SA was more obvious. Then the expression of NF-κB signaling pathway related proteins were determined by western blotting.

Fig. 8
figure 8

Effects of MGF, MGF SA and MIX on nuclear translocation of NF-κB p65 in LPS-induced RAW 264.7 cells (n = 3)

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LPS stimulation could significantly induce the total protein expression of NF-κB p65 in RAW264.7 cells. MGF and MGF SA at the concentration of 500 mg/L could significantly reduce the total protein expression of NF-κB p65 in inflammatory cells, as shown in Fig. 9A, B. In the normal state of the cells, NF-κB p65 expression in the nucleus was minimal. However, upon LPS stimulation, NF-κB p65 translocated from the cytoplasm to the nucleus, leading to an increase in NF-κB p65 expression in the nucleus and a decrease in its expression in the cytoplasm, as depicted in Fig. 9C–F. Notably, Fig. 9E, F, showed that MGF SA could inhibit the transfer of NF-κB p65 from cytoplasm to nucleus and had a significant difference compared with the MGF group, which was consistent with immunofluorescence results.

Fig. 9
figure 9

Effects of MGF, MGF SA and MIX on expression of NF-κB p65 total protein, NF-κB p65 nuclear protein and cytoplasmic protein (n = 3). Note: *p < 0.05, **p < 0.01 compared with normal group; #p < 0.05 compared with model group; △p < 0.05 compared with MGF group

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Effect of MGF SA on metabolites in RAW264.7 cells induced by LPS

Metabolomics as a novel field “-omics” technology is a scientific approach for evaluating and researching the mechanisms of herbal medicine [34]. Previous studies had shown that MGF SA could effectively relieve the nuclear metastasis of NF-κB p65 and the release of inflammatory cytokines induced by LPS. Therefore, we further used metabolomics to explore the mechanism of MGF SA intervention on the inflammatory metabolic profile induced by LPS. As shown in Fig. 10A–D, PCA and PLS-DA models were established for statistical analysis of the two groups of metabolic data. It could be seen that PCA and PLS-DA data were separated on both sides of the Y axis, indicating significant differences. R2X = 0.729, Q2 = 0.347 in PCA model; R2Y and Q2 of PLS-DA model were 0.999 and 0.977, respectively. The results of permutation test showed that R2 and Q2 of the MGF SA group and the model group were 0.955 and 0.452. It could be shown that the fit degree and reliability of the two models meet the requirements.

Fig. 10
figure 10

Effects of MGF SA on RAW264.7 inflammatory cells metabolism. A, B PCA score plot and PLS-DA score plot; C, D 200-time permutations plots; E metabolic pathway enrichment analyses between M and MGF SA)

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The differential metabolites of the MGF SA group and model (M) group were then examined. A total of 29 differential metabolites were chosen as per the screening criteria (VIP > 1.2, p < 0.05), as shown in Table 4. In both healthy and diseased situations, arginine was essential for several metabolic functions, including the urea cycle, synthesis of polyamino acids and creatine, immunological control, and NO synthesis. Studies had shown that inflammation may affect the metabolism of arginine, and abnormal metabolism of arginine and proline may exacerbate inflammation [34,35,36]. Inflammatory responses stimulate metabolism and cells degrade AMP into hypoxanthine nucleotides, which in turn degrade into inosine, hypoxanthine, and urate, which could lead to arthritis [37]. The probable metabolic pathways were further examined based on the different metabolites. The results of metabolic pathway analysis in this study were of great significance for the study of the mechanism of MGF SA. As shown in Fig. 10D, the metabolism of arginine biosynthesis (p < 0.01), purine metabolism (p = 0.02), arginine and proline metabolism (p = 0.03) and terpenoid backbone biosynthesis (p = 0.04) were significantly affected in RAW264.7 inflammatory cells. Therefore, it was preliminary suggested that MGF SA acted as an anti-inflammatory by regulating the metabolic pathways of arginine, proline and purine.

Table 4 The MGF SA affect the differential metabolites of RAW264.7 inflammatory cells metabolism
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Conclusions

In this study, it was found that the morphology of MGF co-decoction was more uniform than that of the physical mixture of single decoction. Meanwhile, the MGF SA extracted from co-decoction displayed spherical nanoparticles. Interestingly, though the micromorphology was different, the comparison of phytochemicals of the co-decoction and physical mixing were roughly the same. Anti-inflammatory evaluation showed that MGF and MGF SA had better activity than MIX; and MGF SA had a significant effect on improving nuclear transfer of NF-κB p65 than other groups, suggesting that supermolecules were the key component of the pharmacodynamic contribution of MGF decoction. LPS caused a series of metabolic pathways of RAW264.7 cells to be disordered, and the disordered metabolic pathways were effectively regulated after MGF SA intervention. The mechanism might be connected to MGF SA’s control of arginine biosynthesis, purine metabolism, arginine and proline metabolism. In summary, MGF, MGF SA and MIX had the similar main phytochemicals’ composition and administered at the same dose, but diverse biological effects were produced by their varied molecular morphologies. This work exhibited that decoction could regulate phytochemicals’ micromorphology and anti-inflammation activity of supermolecules originated from MGF decoction. In addition, current study displayed that the supermolecules as one of the main pharmacodynamic site of herb medicine decoction would be a new hotspot in future research and co-decocting herb might be more beneficial to the treatment of diseases than the mixture of the single herbs’ extraction.