Untargeted Metabolomics Reveals Multiple Phytometabolites in the Agricultural Waste Materials and Medicinal Materials of Codonopsis pilosula

Codonopsis pilosula has been used in traditional Chinese medicine for hundreds of years, where it has been used to treat anaemia, fatigue, a weak spleen, and stomach problems, among other ailments. The roots of C. pilosula are considered medicinal, while the aerial parts are always directly discarded after harvest in autumn or winter. Some studies have shown that the stems and leaves of C. pilosula also contain a variety of active metabolites, including saponins, flavonoids, terpenoids, and polysaccharides. To efficiently utilise resources, waste products from C. pilosula leaves and stems were analysed by untargeted metabolomics and chemometrics. A total of 1508 metabolites were detected and annotated, of which 463 were identified as differentially expressed metabolites (DEMs). These DEMs were grouped into classes, such as carboxylic acids and derivatives, steroids, organic oxygen compounds, fatty acyls, prenol lipids, and flavonoids. Metabolic profiling of C. pilosula tissues showed that the contents of polyacetylenes, polyenes, flavonoids, some alkaloids, steroids, terpenoids, and organic acids were higher in stems and leaves, whereas the contents of the main lignans and some alkaloids were more enriched in roots. Moreover, C. pilosula stems and leaves also contained a lobetyolin, syringin and atractylenolide III, which were detected by LC-MS/MS and HPLC-UV. The extracts of C. pilosula aerial parts also showed stronger antioxidant properties than roots. C. pilosula stems and leaves were rich in active ingredients and might have great value for development and utilisation.

The Promotive Effect of the Active Ingredients of Atractylodes macrocephala on Intestinal Epithelial Repair Through Activating Ca2+ Pathway

Atractylodes macrocephala ( AM) is a famous traditional Chinese medicine for intestinal epithelial restitution through activating Ca2+ channels. However, the roles of specific AM compositions in intestinal epithelial restitution are sparse. Therefore, this study aimed to compare the concrete effects of the 4 active ingredients (atractylon, β-eudesmol, atractylenolide II, atractylenolide III) of AM and their combination on intestinal epithelial repair and the Ca2+ pathway in intestinal epithelial cell (IEC-6) cells. First, the best combination of the 4 ingredients with an optimal mixing ratio of atractylon: β-eudesmol: atractylenolide II: atractylenolide III = 1:2:2:2 was demonstrated by a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide orthogonal experiment. Subsequently, enzyme-linked immunosorbent assay was used to measure anti-inflammatory cytokine levels, the migratory ability was evaluated by cell scratch experiments, cell cycle analysis and [Ca2+]cyt concentration in cells were detected by flow cytometry, and the expression of the Ca2+ pathway-related genes was detected by immunofluorescence staining, quantitative polymerase chain reaction and whole blood assays. Our result showed that atractylon, β-Eudesmol, atractylenolide II, and atractylenolide III showed different abilities to promote the IEC-6 cells proliferation, migration, and the expression of anti-inflammatory cytokines interleukin (IL)-2, IL-10, and ornithine decarboxylase, as well as the intracellular [Ca2+]cyt concentration through stromal interaction molecule 1 transposition to activate Ca2+ pathway. Thereinto, atractylenolide III was the main active ingredient of AM for pro-proliferation and anti-inflammation, and the combination of 4 AM ingredients performed better beneficial effects on IEC-6 cells. Therefore, our study suggested that atractylenolide III was the active ingredient of AM for intestinal epithelial repair through activating the Ca2+ pathway, and the 4 ingredients of AM have a synergy in intestinal epithelial repair.

Effect of atractylenolide III on interstitial cells of Cajal and C-kit/SCF pathway of rats with loperamide-induced slow transit constipation

Purpose: To determine the effect of atractylenolide-III (ATL-III) on loperamide-induced slow transit constipation (STC) in a rat STC model, and to elucidate the mechanisms involved. Methods: Male Wistar rats were divided into five groups (n=6 per group): normal control group (NG), model group, and three STC rat groups treated with different doses of ATL-III, viz, 5, 10 and 15 mg/kg. The rats were treated for 15 days. Feed consumption, fecal excretion and intestinal transit rate were determined. Nitric oxide synthase (NOS), somatostatin (SS), serotonin (5-HT), and vasoactive intestinal peptide (VIP) were measured with enzyme-linked immunosorbent assay (ELISA). The protein and mRNA expressions of C-kit, SCF, PKC, and PI-3K were assayed using Western blot analysis and realtime reverse transcription polymerase chain reaction (RT-PCR), respectively. Results: The amount, weight, and moisture content of stool, and water consumption were significantly higher in ATL-III-treated groups than in the untreated (model) group (p < 0.05), whereas no difference was observed in feed intake. Intestinal transit rate was higher in the ATL-III-treated groups (p < 0.05). Decreased NOS, SS and VIP levels and increased 5-HT level were seen in the ATL-III-treated groups (p < 0.05). ATL-III treatment also induced increases in smooth muscle cells, neuronal cells, and mucous layer (p<0.05). Results from RT-PCR and Western blot revealed that ATL-III–treated groups had elevated c-kit, SCF, PKC, as well as PI-3K mRNA and protein expressions (p < 0.05). Conclusion: These results suggest that ATL-III mitigates loperamide-induced STC in rats via stimulation of NOS, SS, VIP, and 5-HT secretions. It also increases smooth muscle cells, neuronal cells, and mucous layer, and regulates the signaling pathways involving PKC, PI3K, SCF, and c-kit.

Selecting and Characterizing Tyrosinase Inhibitors from Atractylodis macrocephalae Rhizoma Based on Spectrum-Activity Relationship and Molecular Docking

Atractylodis macrocephalae Rhizoma (AMR) is a famous classical Chinese traditional medicine (CTM), which has been used as a tonic for many diseases for thousands of years. In ancient China, it was used as a supplementary food for beauty in the palace. In preliminary studies, the function of whitening skin and the significant inhibiting effect on tyrosinase (TYR) which is the reactive enzyme in the composition of melanin of AMR were discovered, and the relevant research was rarely reported. In this study, high-performance liquid chromatography (HPLC) along with partial least squares regression analysis (PLS) was applied to survey the coherence between the chemical constituents and the inhibiting activity of 11 batches of AMR on TYR activity. The results of PLS showed that the chromatographic peaks 11 (atractylenolide III) and 15 could be important effective ingredients of the inhibition TYR activity as ascertained by spectrum-activity relationships. Furthermore, TYR inhibitory activity of atractylenolide III was validated by in vitro test by β-arbutin served as a positive control drug. The results of the in vitro test and the molecular docking showed that atractylenolide III has high TYR inhibitory activity and could link to the residues in TYR catalytic pocket. Therefore, bioassay, molecular docking, and spectrum-activity relationships are appropriate for linking the quality of samples with pharmaceutical-related active ingredients. And our studying would lay a theoretical foundation for applying the water extracts of AMR in whitening cosmetics.