Diterpenoid Alkaloids Research Articles

Study on the embryotoxic effects and potential mechanisms of Aconitum diterpenoid alkaloids in rat whole embryo culture through morphological and transcriptomic analysis

Ethnopharmacological relevanceThe lateral root of Aconitum carmichaelii Debeaux, or Fuzi, is recognized in Asia for its anti-inflammatory, analgesic, and cardiotonic effects. Its main active compounds are diester diterpenoid alkaloids (DDAs) such as aconitine (AC), mesoacitine (MA), and hypoaconitine (HA), which are also toxic and have a narrow therapeutic window, limiting their clinical use. Although Aconitum DDAs are known for cardiotoxic and neurotoxic effects, their impact on embryonic development remains unclear. Aim of the studyThe embryotoxicity of three representative Aconitum DDAs (AC, MA, and HA) and their metabolites were systematically assessed, and the mechanisms underlying AC-induced embryotoxicity was explored. Materials and methodsThe embryotoxicity of these DDAs was assessed by indicators such as morphological scores in a whole embryo culture (WEC) system. Immunofluorescence analysis was conducted to detect DNA damage and apoptosis in embryos, and transcriptomic analysis and western blotting were performed to explore the underlying mechanisms. ResultsDDAs, particularly AC, induced dose-dependent developmental retardation and malformation in rat embryos. Notably, the embryotoxicity of AC metabolites such as benzoyltrypine (BAC) and aconine, was significantly reduced. AC treatment caused substantial DNA damage and apoptosis in embryos. Transcriptomic analysis indicate that AC treatment may impair DNA replication and histone synthesis by activating the p53/p21/CDK2/NPAT pathway, ultimately affecting embryonic development. ConclusionAmong these Aconitum DDAs, AC exhibited the strongest embryotoxicity, mainly through DNA damage and regulation of histone genes via the p53/p21/CDK2/NPAT pathway.

A New Diterpenoid Alkaloid with Antimicrobial Activity from Aconitum brachypodum Diels.

A new C20-denudatine-type diterpenoid alkaloid (DA) 11S-aconicarnine D (1) and fifteen known DAs were isolated from the lateral roots of Aconitum brachypodum Diels. Their structures were identified on the basis of extensive spectroscopic analyses, NMR calculations and DP4+ analysis. Compounds 1 and 4 exhibited antimicrobial activity against Alternaria panax with MICs of 2.00 and 8.00 μg/mL (Nystatin, 1.00 μg/mL), respectively.

Seven new aconitine-type diterpenoid alkaloids from the roots of Aconitum apetalum.

Seven new aconitine-type C19-diterpenoid alkaloids, apetaldines K-Q (1-7), were isolated from the roots of Aconitum apetalum (Huth) B. Fedtsch. Their structures were established on the basis of extensive spectroscopic analyses (HRESIMS, 1D and 2D NMR). Among them, compounds 1 and 2 possess a unique 2-(E)-(2-methylbut-2-enamido)benzoate moiety at the C-18 position. Furthermore, cytotoxic activities of these diterpenoid alkaloids were also evaluated.

Anti-inflammatory oxazole-, nitro- and hexahydropyrrolo[2,1-b]oxazole-containing abietane diterpenoid alkaloids from Salvia miltiorrhiza

Nine undescibed abietane diterpenoid alkaloids (DAs), salviamines G‒H (1–2), isosalviamines G‒J (3–6), and miltiorramines A‒C (7–9) were isolated from the roots of Salvia miltiorrhiza. Their chemical structures including absolute configurations were elucidated by extensive spectroscopic analysis (including 1D and 2D NMR, and HRESIMS), combined with the calculated ECD method and single-crystal X-ray diffraction analysis. Among them, compounds 1–6 are unusual 20-nor- or 19,20-bisnor-abietane DAs with an oxazole ring. Compounds 7–8 are the first examples of DAs with a nitro group isolated from plant sources. Notably, compound 9 possesses a rare hexahydropyrrolo[2,1-b]oxazole unit that is fused in the ring B of the abietane skeleton. Bioactivity assay indicated that compound 3 showed significant anti-inflammatory activity by decreasing the gene expressions of IL-1β, IL-6, and TNF-α in LPS-induced RAW264.7 cells in a dose-dependent manner.

Inhibitory Effect of Aconitine on Colorectal Cancer Malignancy via Inducing Apoptosis and Suppression of Cell Motion.

The incidence of colorectal cancer (CRC) has been increasing in recent years worldwide. Aconitine is a diester diterpenoid alkaloid that exhibits an antitumor role in several cancers. Nevertheless, it remains unclear whether aconitine also has antitumor activity in CRC. This study aims to investigate the effects of aconitine on the malignant behaviors of CRC cells. 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay was utilized for cell viability assessment. Flow cytometry, western blotting, wound healing, and Transwell assays were implemented for examining the aconitine effect on CRC cell apoptosis, migration, and invasiveness. Animal experiments were performed to further elucidate aconitine's effect on CRC tumorigenesis. Aconitine time- and dose-dependently restrained CRC cell viability but was not cytotoxic to normal colorectal mucosa cells. Aconitine facilitated CRC cell apoptosis and hindered cell migration and invasiveness. Aconitine blocked tumor growth in xenograft mouse models. Aconitine exerts an anti-CRC effect by promoting cell apoptosis and blocking cell migration and invasiveness.

Biologically active franchetine-type diterpenoid alkaloids: Isolation, synthesis, anti-inflammatory, agalgesic activities, and molecular docking

In this study, four franchetine-type diterpenoid alkaloids (1–4) were isolated from Aconitum sinoaxillare, and fourteen diverse franchetine analogs (5–18) were synthesized. Compounds 1, 2, 7 and 16 exhibited stronger inhibitory effects on NO production when compared to celecoxib. Among them, compound 1 had the best inhibitory effect on iNOS and COX-2 inflammatory proteins. The in vitro studies displayed that the anti-inflammatory effect of the most active compound 1 was ascribed to the inhibition of the TLR4-MyD88/NF-κB/MAPKs signalling pathway. Consequently, this led to a inhibition in the expression of inflammatory factors or mediators including NO, ROS, TNF-α, IL-6, IL-1β, iNOS, and COX-2. Additionally, compound 1 had low toxicity (LD50 > 20 mg/kg) in mice, and it had notable analgesic effects on acetic acid-induced visceral pain (ED50 = 2.15 ± 0.07 mg/kg). Moreover, compound 1 exhibited a distinct reduction in the NaV1.7 and NaV1.8 channel currents during both resting and half-inactivated states at 50 μM. The present study enriches the pharmacological activities of franchetine derivatives and provides valuable insights for the development of novel anti-inflammatory and analgesic agents.

New insights into the chemotaxonomic implications of diterpenoid alkaloid composition in Aconitum sessiliflorum

Through phytochemical research, the composition of diterpenoid alkaloids in Aconitum sessiliflorum was analyzed, and sixteen compounds were identified. Among them, there are seven napelline type C20-diterpenoid alkaloids (1–4, 7–8, 15), three denudatine type C20-diterpenoid alkaloids (5–6, 16), and six aconitine type C19-diterpenoid alkaloids (9–14). On the basis of NMR spectroscopy, the structural analysis of the separated compounds was achieved by comparing with previous data. All sixteen compounds were isolated for the first time from the Aconitum sessiliflorum, of which five compounds (1, 6, 9, 13, 15) were isolated for the first time from series Brachypoda, where Aconitum sessiliflorum is classified. These diterpenoid alkaloids not only enrich the chemical diversity of Aconitum sessiliflorum, but also facilitate the analysis of their chemotaxonomic implications. Importantly, the diverse types and structural features of these diterpenoid alkaloids provide new insights into both the potential phylogenetic affiliations within the series Brachypoda and the varying degrees of evolutionary divergence among these related taxa.

UHPLC-PDA-ELSD-MS-based simultaneous determination of diterpenoid alkaloids in commercially important Himalayan medicinal plant Aconitum heterophyllum Wall. and network pharmacology-based target prediction

Aconitum heterophyllum Wall., a commercially important Himalayan medicinal plant, is used in various herbal formulations and has high market demand. Given its esteemed medicinal properties, considerable demand, and prioritized status, there’s a pressing requirement for thorough quality assessment methods to ensure high throughput. Genus Aconitum is also well known for its toxicity owing to the presence of diester diterpenoid alkaloids. Hence it is essential to develop a UHPLC-PDA-ELSD-MS based quantification method enabling simultaneous quantification of primary marker compounds alongside toxic diterpenoid alkaloids. The method showcased strong linearity, with detection and quantification limits covering a broad range. Precision was evident with consistently low relative standard deviations both within and across days. Furthermore, accuracy was demonstrated within acceptable bounds. Quantification results revealed the maximum content of atisinium and hetisine in hydroethanolic root extract [atisinium (≅78.88 mg/g) and hetisine (≅50.17 mg/g)] In addition, a network pharmacology-based approach has been applied to predict potential biological targets. The network pharmacology approach suggests the plant’s potential in treating neuralgia, renal fibrosis, diabetes mellitus, lung diseases, cancer, and more, emphasizing the need for scientific validation and indicating future research directions. Overall, the developed method represents a reliable and efficient quality assessment method for the simultaneous quantification of marker compounds and toxic diterpenoid alkaloids in the roots of A. heterophyllum. Network pharmacology predicts pharmacological potentials that require scientific validation highlighting future scope.

Two Novel Diterpenoid Alkaloids from Aconitum Pendulum.

Two previously uncharacterized compounds, an aconitine-type C19-diterpenoid alkaloid (1) and a napelline-type diterpenoid alkaloid C20-diterpenoid alkaloid (2), as well as ten known compounds (3-12), were isolated from Aconitum pendulum. Their structures were elucidated based on spectroscopic data, including 1D and 2D NMR, IR, HR-ESI-MS, and single-crystal X-ray diffraction analysis. The anti-insecticidal activities of these compounds were evaluated by contact toxicity tests against two-spotted spider mites, and compounds 1, 2, and 9 showed moderate contact toxicity, with LC50 values of 0.86±0.09, 0.95±0.23, and 0.89±0.19 mg/mL, respectively. This study highlights the potential use of diterpenoid alkaloids as natural plant-derived pesticides for the management of plant pests.

Diterpenoid alkaloids from Delphinium sherriffii

Three new diterpenoid alkaloids (1, 2, 3) and seventeen known (4–20) compounds were isolated from the whole plant of Delphinium sherriffii Munz (Ranunculaceae). Their structures were elucidated by various spectroscopic analyses, including IR, HR-ESI-MS, 1D and 2D NMR spectra. All compounds were evaluated for the inhibitory activity of Sf9 cells and compound 5 exhibited the strongest cytotoxicity (IC50 = 8.97 μM) against Sf9 cell line.

Two new diterpenoid alkaloids from the roots of Aconitum nagarum and their cytotoxic activity

A chemical investigation on the roots of Aconitum nagarum afforded two undescribed C19-diterpenoid alkaloids nagarumines D and E (1 and 2). The structures of the new compounds were elucidated by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy, as well as HR-ESI-MS. The two isolated alkaloids were tested in vitro for cytotoxic activity against five gastric tumor cell lines. Consequently, compound 2 exhibited some cytotoxicities against several human cancer cell lines with IC50 value less than 20.0 μM.

Diterpenoid alkaloids from Delphinium trichophorum

The ethanol extract of the whole plant of Delphinium trichophorum Franch was subjected to a phytochemical study, leading to the isolation of ten unprecedented diterpenoid alkaloids, including nine delnudine-type C20-diterpenoid alkaloids named trichophodines A–I and one kusnezoline-type C20-diterpenoid alkaloid named trichophozine A. Additionally, seven known compounds were also identified. Their structures were elucidated on the basis of extensive spectroscopic analysis, including HSQC, HMBC, 1H–1H COSY, NOESY and X-ray crystallographic analysis. Most isolated compounds were screened for inhibitory activities against LPS-induced NO production in RAW 264.7 macrophage cells and acetylcholinesterase inhibitory effects. Guan-fu base V exhibited potent inhibitory activity against acetylcholinesterase, demonstrating an inhibitory rate of 53.81% at a concentration of 40 μM.

Discovery of Epichloë as novel endophytes of Psathyrostachys lanuginosa in China and their alkaloid profiling

The Epichloë genus represents a significant group of above-ground endophytes extensively researched for their potential applications in agriculture and ecology. Additionally, Epichloë species synthesize bioactive alkaloids, which generally cause health problems in livestock and have detrimental effects on the performance of insect herbivores. Psathyrostachys lanuginosa serves as a valuable forage grass for livestock owing to its high nutritional value and resilience in adverse environmental conditions. Nevertheless, to date, no reports have documented Epichloë as endophytes of P. lanuginosa. In this study, four strains (PF5, PF9, QG2, and QG4) were isolated and identified through morphological, molecular, and phylogenetic analyses as endophytes of P. lanuginosa. Morphological analysis indicated colony characteristics and conidia features consistent with symbiotic Epichloë, with no significant differences observed in growth rates or conidia dimensions among the four strains. Phylogenetic analysis confirmed all strains as E. bromicola. Additionally, alkaloid biosynthetic genes were detected, revealing differences in the potential synthesis of peramine and indole diterpenoid alkaloids among strains from different geographic origins. However, all four E. bromicola strains exhibited similar potential for synthesizing ergot alkaloids, but not loline alkaloids. Overall, this study identified P. lanuginosa as a novel host for E. bromicola and provided insights into the alkaloid profiles of these strains, laying a solid foundation for the scientific and rational utilization of Epichloë resources.

Brunonianines D-F, three new C19-diterpenoid alkaloids from the Delphinium brunonianum, with therapeutic effect on ovarian cancer in vitro and in vivo

The current standard treatment for ovarian cancer consists of surgery to reduce the size of the tumor, followed by treatment with chemotherapeutic drugs, which have major side effects. Therefore, finding a new natural product drug with fewer side effects is a strategy. Delphinium brunonianum (D. brunonianum) is a traditional Tibetan medicine, mainly from southern Tibet, China, whereas the chemical constituents in this plant remain elusive. The major metabolites in the dichloromethane fraction of D. brunonianum were analyzed and purified by HPLC and various column chromatography techniques. Nine diterpenoid alkaloids (1–9) and one amide alkaloid (10) were isolated from D. brunonianum, including three novel C19-type diterpenoid alkaloids (Brunonianines D-F) (1–3). Their structures were elucidated by 1D/2D NMR, HR-ESI-MS and single-crystal X-ray diffraction analyses. All compounds were evaluated for toxicity in four tumor cell lines. Most of the compounds exhibited potent inhibitory effects on Skov-3 cell lines, with IC50 values ranging from 2.57 to 8.05 μM. The western blotting experiment was used to further analyze the expression levels of molecules in the Bax/Bcl-2/Caspase-3 signaling pathway for compound 1. Molecular docking was performed to predict the binding modes of Brunonianine D with target proteins. In vivo experiments were also performed and evaluated in real time by monitoring the size of the Skov-3 tumor. Additionally, tumor H&E staining and the TUNEL assay used to evaluate anti-tumor effects.

Norditerpenoid alkaloids from Aconitum refractum var. circinatum as autophagy inducers

A total of twenty-two diterpenoid alkaloids, including ten unprecedented ones, namely refractines C–L, were isolated from the roots of Aconitum refractum (Finet et Gagnep.) Hand.-Mazz. Refractine C was the first example of a natural diterpenoid alkaloid wherein C-19 is linked to N position by an oxaziridine ring. Refractine L was a rare glycosidic diterpenoid alkaloid with fructofuranoside. Most of the isolated compounds obtained from a previous study were screened for their anti-inflammatory and myocardial protective activities. The autophagy-inducing effects of some of these compounds on RAW 264.7 cells were evaluated by assessing the expression of microtubule-associated protein 1 light chain 3 (LC3-II/LC3-I). Results revealed that some compounds exerted varying levels of inhibitory effects on the proliferative activity of RAW 264.7 cells.

The expression of AcIDI1 reveals diterpenoid alkaloids’ allocation strategies in the roots of Aconitum carmichaelii Debx

Isopentenyl diphosphate isomerase (IDI), a key enzyme in the biosynthetic pathway of diterpenoid alkaloids (DAs), plays an essential regulatory role in the synthesis and accumulation of DAs. In this study, the coding sequence (CDS) of AcIDI1 was isolated from the mother roots of Aconitum carmichaelii Debx. (GeneBank accession number OR915879). Bioinformatics analysis showed that the CDS of AcIDI1 was 894 bp, encoding a protein with 297 amino acids and the putative protein localized in the chloroplast. AcIDI1 exhibited significant homology with sequences encoding IDI in other species, and was most closely related to Aconitum vilmorinianum. Furthermore, the fusion protein has been successfully expressed in Escherichia coli (E. coli), providing a basis for future functional studies of AcIDI1. The expression pattern of AcIDI1 was analyzed by real-time quantitative PCR (qPCR), which demonstrates that AcIDI1 is a tissue-specific gene in the roots of A. carmichaelii and exhibits high expression in both daughter and mother roots. By comparing the expression levels of AcIDI1 in three tissues of the roots of A. carmichaelii at different growth stages, we propose that the mother roots (MRs) are the centers of resources allocation. The roots of A. carmichaelii continuously absorb the energy from external environment, while resources transfer behavior from MRs to both daughter roots (DRs) and axillary buds (ABs) occurs as the plant grows. This study establishes a foundation for applying the IDI gene to regulate the biosynthesis and accumulation of DAs in A. carmichaelii.

A new illudane sesquiterpene from the edible fungus Pholiota nameko

Fungi have different genetic expression abilities and biosynthetic pathways under different cultivation conditions, which can produce various secondary metabolites. The “one strain many compounds” strategy is used to activate silent biosynthetic genes of fungi to produce various compounds, which is an effective method. In order to discover various new compounds in the edible fungus Pholiota nameko, a fermentation strategy involving precursor feeding and enzyme inhibitor addition has been employed. A new illudane sesquiterpene (1), along with one known indole diterpenoid alkaloid, cladosporine A (2) were isolated from the extracts of liquid culture of P. nameko. The new compound was identified by combination of 1D and 2D NMR, MS, optical rotation, and ECD calculations. We conducted experiments on the cytotoxicity of all isolated compounds on three cancer cell lines, but we did not observe any significant cytotoxicity (IC50 > 40 μM).

Synthesis and biological evaluation of lycoctonine derivatives with cardiotonic and calcium channels inhibitory activities

In our previous study, a screening of a variety of lycotonine-type diterpenoid alkaloids were screened for cardiotonic activity revealed that lycoctonine had moderate cardiac effect. In this study, a series of structurally diverse of lycoctonine were synthesized by modifying on B-ring, D-ring, E-ring, F-ring, N-atom or salt formation on lycoctonine skeleton. We evaluated the cardiotonic activity of the derivatives by isolated frog heart, aiming to identify some compounds with significantly enhanced cardiac effects, among which compound 27 with a N-isobutyl group emerged as the most promising cardiotonic candidate. Furthermore, the cardiotonic mechanism of compound 27 was preliminarily investigated. The result suggested that the cardiotonic effect of compound 27 is related to calcium channels. Patch clamp technique confirmed that the compound 27 had inhibitory effects on CaV1.2 and CaV3.2, with inhibition rates of 78.52 % ± 2.26 % and 79.05 % ± 1.59 % at the concentration of 50 μM, respectively. Subsequently, the protective effect of 27 on H9c2 cells injury induced by cobalt chloride was tested. In addition, compound 27 can alleviate CoCl2-induced myocardial injury by alleviating calcium overload. These findings suggest that compound 27 was a new structural derived from lycoctonine, which may serve as a new lead compound for the treatment of heart failure.

Two new cytotoxic diterpenoid alkaloids from Delphinium grandiflorum

Delphinium grandiflorum, one of the well-known Delphinium species, contains diverse bioactive diterpenoid alkaloids (DAs), which promoted a systematic phytochemical study on this plant. As a result, two new DAs, namely 16-demethyl tuguaconitine (1) and 6-O-acetyl-16-demethyldelsoline (2), together with one known analogue pseudophnine A (3) were obtained from the whole herbs of D. grandiflorum. Their structures were elucidated by use of extensive spectroscopic analysis (HR-ESIMS, IR, 1D and 2D NMR). Additionally, the inhibitory effects of these obtained individual compounds were also evaluated in vitro for lung cancer cell line (A549), liver cancer cell line (SMMC-7721), and breast cancer cell line (MCF-7). The results of cytotoxic activities showed that only compound 2 exhibit weak inhibitory effect on SMMC-7721 and MCF-7, and their IC50 values were 37.4 and 33.1 μM, respectively. However, compound 1 and 3 had no obvious cytotoxic activities (IC50>80 μM).

The acute toxic effect of Chinese medicine Fuzi is exacerbated in kidney yang deficiency mice due to metabolic difference

Ethnopharmacological relevanceThe proper application of toxic medicines is one of the characteristics of traditional Chinese medicines, and the use of traditional Chinese medicines follows the principle of dialectical treatment. It is necessary to combine different “syndrome” or “disease” states with the toxicity of traditional Chinese medicines to form a reliable toxicity evaluation system. Fuzi, the lateral root of Aconitum carmichaelii Debx, is recognized as a panacea for kidney yang deficiency syndrome, however, its toxic effects significantly limit its clinical application. Aim of the studyHerein, our research aimed to explore the toxic effects of Fuzi on syndrome models, and tried to reveal the underlying mechanisms. Materials and methodsFirstly, the mouse model of kidney yang deficiency syndrome was established through intramuscular injection of 25 mg/kg hydrocortisone per day for 10 consecutive days. Then, the acute toxicity of Fuzi in normal mice and kidney yang deficiency model mice was explored. Finally, the plasma metabolite concentrations and liver CYP3A4 enzyme activity were analyzed to reveal the possible mechanisms of the different pharmacological and toxicological effects of Fuzi in individuals with different physical constitutions. ResultsIt was found that the treatment with Fuzi (138 g/kg) had serious toxic effects on kidney yang deficiency mice, leading to the death of 80% of the mice, whereas it showed no lethal toxicity in normal mice. This indicates that Fuzi induced greater toxicity in kidney yang deficiency mice than in normal ones. The liver CYP3A4 enzyme activity in kidney yang deficiency mice was decreased by 20% compared to the controls, resulting in slower metabolism of the toxic diester diterpenoid alkaloids in Fuzi. ConclusionIn conclusion, our study showed that changes of the metabolic enzyme activity in individuals with different syndromes led to different toxic effects of Chinese medicines, emphasizing the crucial importance of considering individual physical syndromes in the clinical application of traditional Chinese medicine, and the significance of conducting safety evaluations and dose predictions on animal models with specific syndromes for traditional Chinese medicines.