Effects of Abiotic Elicitors on Expression and Accumulation of Three Candidate Benzophenanthridine Alkaloids in Cultured Greater Celandine Cells

Efforts to develop the necessary biotechnologies in Greater Celandine (Chelidonium majus L.), a leading plant resource for the development of plant-derived medicines, have been hampered by the lack of knowledge about transcriptome and metabolome regulations of its medicinal components. Therefore, this study aimed to examine the effect of abiotic elicitors, methyl jasmonate (MJ) and salicylic acid (SA), at different time courses (12, 24, 48, and 72 h), on expression and metabolome of key benzophenanthridine alkaloids (BPAs) in an optimized in vitro culture. Gene expression analysis indicated the upregulation of CFS (cheilanthifoline synthase) to 2.62, 4.85, and 7.28 times higher than the control at 12, 24, and 48 h respectively, under MJ elicitation. Besides, MJ upregulated the expression of TNMT (tetrahydroprotoberberine N-methyltransferase) to 2.79, 4.75, and 7.21 times at 12, 24, and 48 h respectively, compared to the control. Investigation of BPAs revealed a significant enhancement in the chelidonine content (9.86 µg/mg) after 72 h of MJ elicitation. Additionally, sanguinarine content increased to its highest level (3.42 µg/mg) after 24 h of MJ elicitation; however, no significant enhancement was detected in its content in shorter elicitation time courses. Generally, higher gene expression and BPAs’ level was observed through longer elicitation courses (48 and 72 h). Our findings take part in improving the understanding of transcription and metabolic regulation of BPAs in cultured Greater Celandine cells.

Synthetic Lethal Activity of Benzophenanthridine Alkaloids From Zanthoxylum coco Against BRCA1-Deficient Cancer Cells

Several plants from South America show strong antitumoral properties based on anti-proliferative and/or pro-apoptotic activities. In this work we aimed to identify selective cytotoxic compounds that target BRCA1-deficient cancer cells by Synthetic Lethality (SL) induction. Using a high-throughput screening technology developed in our laboratory, we analyzed a collection of extracts from 46 native plant species from Argentina using a wide dose-response scheme. A highly selective SL-induction capacity was found in an alkaloidal extract from Zanthoxylum coco (Fam. Rutaceae). Bio-guided fractionation coupled to HPLC led to the identification of active benzophenanthridine alkaloids. The most potent SL activity was found with the compound oxynitidine, which showed a remarkably low relative abundance in the active fractions. Further validation experiments were performed using the commercially available and closely related analog nitidine, which showed SL-induction activity against various BRCA1-deficient cell lines with different genetic backgrounds, even in the nanomolar range. Exploration of the underlying mechanism of action using BRCA1-KO cells revealed AKT and topoisomerases as the potential targets responsible of nitidine-triggered SL-induction. Taken together, our findings expose an unforeseen therapeutic activity of alkaloids from Zanthoxylum-spp. that position them as novel lead molecules for drug discovery.

Effects of Abiotic Elicitors on Expression and Accumulation of Three Candidate Benzophenanthridine Alkaloids in Cultured Greater Celandine Cells

Efforts to develop the necessary biotechnologies in Greater Celandine (Chelidonium majus L.), a leading plant resource for the development of plant-derived medicines, have been hampered by the lack of knowledge about transcriptome and metabolome regulations of its medicinal components. Therefore, this study aimed to examine the effect of abiotic elicitors, methyl jasmonate (MJ) and salicylic acid (SA), at different time courses (12, 24, 48, and 72 h), on expression and metabolome of key benzophenanthridine alkaloids (BPAs) in an optimized in vitro culture. Gene expression analysis indicated the up-regulation of CFS to 2.62, 4.85, and 7.28 times higher than the control at 12, 24, and 48 h, respectively, under MJ elicitation. Besides, MJ up-regulated the expression of TNMT to 2.79, 4.75, and 7.21 times at 12, 24, and 48 h, respectively, compared to the control. Investigation of BPAs revealed a significant enhancement in the chelidonine content (9.86 µg/mg) after 72 h of MJ elicitation. Additionally, sanguinarine content increased to its highest level (3.42 µg/mg) after 24 h of MJ elicitation; however, no significant enhancement was detected in its content in shorter elicitation time courses. Generally, higher gene expression and BPAs’ level was observed through longer elicitation courses (48 and 72 h). Our findings take part in improving the understanding of transcription and metabolic regulation of BPAs in cultured Greater Celandine cells.

Purification, identification, and characterization of two benzophenanthridine alkaloids from Corydalis longicalcarata rhizomes with anti-mitotic and polyploidy-inducing activities

ABSTRACTMitosis has long been a therapeutic target for the treatment of cancer. After conducting a phenotypic screen for anti-mitotic activity in a crude extract library of more than 2000 medicinal plants, we identified the rhizomes of Corydalis longicalcarata. Guided by the bioactivity-based assay, two benzophenanthridine alkaloids were purified and subsequently identified as corynoline and its close analog acetylcorynoline. This study uncovers a previously unknown antimitotic activity for these two phytochemicals, discovers their potential to be developed as anticancer drugs, and highlights their pleiotropic effects on cell division, including prevention of chromosome congression, compromise of spindle checkpoint response, and blockage of cytokinesis.

ISOLATION OF BENZOPHENANTHRIDINE ALKALOIDS FROM MACLEAYA LEAVES WITHOUT USING TOXIC SOLVENTS

This study aims to develop a new method for isolation of benzophenanthridine alkaloids from Macleaya leaves, which do not use toxic or ecologically dangerous solvents or reagents. According to this method, the extraction of alkaloids from the plant material is performed with 90% ethanol by percolation at 40±2 °C until 5–8 parts (V/M) of extract is obtained. The extract is acidified with sulphuric acid to pH 4.0–4.5 for sedimentation of ballast substances. After filtration or centrifugation, sulphuric acid is added to the extract to obtain the concentration of 0.1–0.12 M. Crystallisation of bisulphates of alkaloids occurs during 14 days at room temperature, then is finished in 7 days at 2–8 °C. The crude product is separated by filtration, washed with 96% ethanol and dried. Upon purification, alkaloids pass into a concentrated aqueous solution in the form of sulphates. A major part of impurities is removed by sedimentation, the remainder – by sorption on activated carbon. Than alkaloids are crystallised again in the form of bisulphates or other salts as required. The procedure has been created to obtain the sum of benzophenanthridine alkaloids of Macleaya in form of salicylates. This substance, named "Sanguirisal", is proposed as an active substance for preparation of pharmaceutical forms for topic administration, being much more lipophilic than sanguiritrine.