Introduction: Hypertension is a major risk factor for vascular diseases. The roots and rhizomes of Veratrum nigrum L. have been used to treat hypertension with alkaloids (component A) from these plants showing potential as antihypertensive agents.

Objectives: This study aims to identify the key active ingredients in component A and investigate their mechanisms in treating hypertension and vascular remodeling.

Methods: The active ingredients of component A were predicted through blood-entry compounds analysis and validated its effects on hypertension and vascular remodeling in SHRs and HUVECs. Network pharmacology and molecular docking were employed to identify related targets and pathways, with further confirmation from experiments.

Results: The blood-entry compounds of component A consisted of 12 Veratrum alkaloids, including Rubijervine, Jervine, Cevine, etc. Component A ‌dose-dependently and effectively lowered blood pressure (BP), improved vascular remodeling and endothelial dysfunction, and regulated the renin-angiotensin system (RAS) in SHRs. Network pharmacology identified 10 hub genes associated with 12 blood-entry compounds for the treatment of hypertension. Subsequent experiments validated that component A modulates the expression of key targets such as ADRB2, AGTR1, MMP9, eNOS, TNF-α, and NNMT. When NNMT was knocked down in HUVEC, the levels of P-eNOS, eNOS, and NO were decreased simultaneously. These modulations inhibit renal sympathetic nerve activity and the Smad pathway, while activating vagal nerve activity, NNMT/MNA/eNOS/NO pathway and the cAMP-mediated inhibition of the RhoA/MLC pathway. Moreover, component A, specificably rubijervine, binds directly to ADRB2, AGTR1, MMP9, and eNOS.

Conclusions: Rubijervine may be the active ingredient of component A. Component A effectively alleviate hypertension and vascular remodeling. The underlying mechanism involves modulating ADRB2, AGTR1, MMP9, eNOS, TNF-α, and NNMT to inhibit TGF-β/Smad pathway, activate NNMT/MNA/ eNOS/NO pathway and cAMP-mediated inhibition of RhoA/MLC pathway, as well as balance RAS and nervous system.

Carotenoid cleavage dioxygenase 4 (CCD4) controls the rate-limiting step of β-ionone biosynthesis, making it a valuable target for healthcare and pharmaceutical applications. Nicotiana tabacum, a carotenoid-richd crop species, is a promising source for β-ionone production. This study aimed to modify CCD4 activity to increase β-ionone yield in tobacco. We identified two isoforms of CCD4 in N. tabacum, NtCCD4a and NtCCD4b, with NtCCD4a exhibiting significantly higher expression levels than NtCCD4b. Using solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC–MS), we demonstrated that NtCCD4a effectively catalyzes the cleavage of β-carotene to produce β-ionone. To improve its enzymatic activity, we applied structure-based rational design to reconstruct the active pocket of NtCCD4a, followed by high-throughput screening of mutant variants. Three single base mutants, F181G, F184L, and F337M, in NtCCD4a showed enhanced β-ionone production compared to the wild-type, with F337M yielding the highest amount. No synergistic effects were observed among the three mutants. Transgenic tobacco plants expressing the F181G, F184L, and F337M mutations had accelerated β-carotene cleavage and increased β-ionone production relative to the wild-type NtCCD4a. Our results establish a framework for the design of CCD4 in major crop species through genome editing technology.