Berberine is an effective antimicrobial and antidiabetic alkaloid, primarily extracted from divergent botanical lineages, specifically Coptis (Ranunculales, early-diverging eudicot) and Phellodendron (Sapindales, core eudicot). In comparison with its known pathway in Coptis species, its biosynthesis in Phellodendron species remains elusive. Using chromosome-level genome assembly, coexpression matrix, and biochemical assays, we identified six key steps in berberine biosynthesis from Phellodendron amurense , including methylation, hydroxylation, and berberine bridge formation. Notably, we discovered a specific class of O -methyltransferases (NOMT) responsible for N -methylation. Structural analysis and mutagenesis of PaNOMT9 revealed its unique substrate-binding conformation. In addition, unlike the classical FAD-dependent berberine bridge formation in Ranunculales, Phellodendron uses a NAD(P)H-dependent monooxygenase (PaCYP71BG29) for berberine bridge formation, originating from the neofunctionalization of tryptamine 5-hydroxylase. Together, these findings reveal the convergence of berberine biosynthesis between Coptis and Phellodendron and signify the role of the convergent evolution in plant specialized metabolisms.

The activity of polysaccharides is usually related to molecular weight. The molecular weight of polysaccharides is critical to their immunological effect in cancer therapy. Herein, the Codonopsis polysaccharides of different molecular weights were isolated using ultrafiltration membranes of 60- and 100-wDa molecular weight cut-off to determine the relationship between molecular weight and antitumor activities. First, three water-soluble polysaccharides CPPS-I (100 wDa) from Codonopsis were isolated and purified using a combination of macroporous adsorption resin chromatography and ultrafiltration. Their structural characteristics were determined through chemical derivatization, GPC, HPLC, FT–IR, and NMR techniques. In vitro experiments indicated that all Codonopsis polysaccharides exhibited significant antitumor activities, with the tumor inhibition rate in the following order: CPPS-II > CPPS-I > CPPS-III. The treatment of CPPS-II exhibited the highest inhibition rate at a high concentration among all groups, which was almost as efficient as that of the DOX·HCL (10 μg/mL) group at 125 μg/mL concentration. Notably, CPPS-II demonstrated the ability to enhance NO secretion and the antitumor ability of macrophages relative to the other two groups of polysaccharides. Finally, in vivo experiments revealed that CPPS-II increased the M1/M2 ratio in immune system regulation and that the tumor inhibition effect of CPPS-II + DOX was superior to that of DOX monotherapy, implying that CPPS-II + DOX played a synergistic role in regulating the immune system function and the direct tumor-killing ability of DOX. Therefore, CPPS-II is expected to be applied as an effective cancer treatment or adjuvant therapy.

A rapid and sensitive method based on HPLC–DAD-MS was developed for quantitative analysis of two flavonoids and chemical fingerprint analysis to evaluate the quality of Receptaculum Nelumbinis. The analysis was conducted on a Poroshell 120 C₁₈ column (100 × 4.6 mm, 2.7 μm) with 0.2% formic acid buffer solution and methanol as mobile phases with gradient elution. This method displayed good linearity with R² at >0.9999 and limits of quantity <0.37 μg mL⁻¹. Relative standard deviation values for intra- and interday precision were <0.82 and 1.03%, respectively. The mean recovery of hyperoside was 95.54% and of isoquercitrin was 92.10%. Hyperoside and isoquercitrin were determined simultaneously, and 12 peaks in the chemical fingerprint were identified. The chemometric methods, including similarity analysis, hierarchical clustering analysis and principal component analysis, were applied to distinguish 11 batches of Receptaculum Nelumbinis samples. The above results could validate each other and successfully divide these samples into two groups. Moreover, hyperoside and isoquercitrin could be selected as chemical markers to evaluate the quality of Receptaculum Nelumbinis from different localities. This study demonstrated that the developed method was a powerful and beneficial tool to carry out the quality control of Receptaculum Nelumbinis.