Identification of the cytochrome P450s responsible for the biosynthesis of two types of aporphine alkaloids and their de novo biosynthesis in yeast.

Aporphine alkaloids have diverse pharmacological activities; however, our understanding of their biosynthesis is relatively limited. Previous studies have classified aporphine alkaloids into two categories based on the configuration and number of substituents of the D-ring and have proposed preliminary biosynthetic pathways for each category. In this study, we identified two specific cytochrome P450 enzymes (CYP80G6 and CYP80Q5) with distinct activities toward (S)-configured and (R)-configured substrates from the herbaceous perennial vine Stephania tetrandra, shedding light on the biosynthetic mechanisms and stereochemical features of these two aporphine alkaloid categories. Additionally, we characterized two CYP719C enzymes (CYP719C3 and CYP719C4) that catalyzed the formation of the methylenedioxy bridge, an essential pharmacophoric group, on the A- and D-rings, respectively, of aporphine alkaloids. Leveraging the functional characterization of these crucial cytochrome P450 enzymes, we reconstructed the biosynthetic pathways for the two types of aporphine alkaloids in budding yeast (Saccharomyces cerevisiae) for the de novo production of compounds such as (R)-glaziovine, (S)-glaziovine, and magnoflorine. This study provides key insight into the biosynthesis of aporphine alkaloids and lays a foundation for producing these valuable compounds through synthetic biology.

Rare oxoisoaporphine alkaloids from Menispermum dauricum with potential anti-inflammatory activity.

Eleven alkaloids including four previously undescribed oxoisoaporphine alkaloids, menisoxoisoaporphines A-D (1-4), four known analogues (5-8), and three aporphine alkaloids (9-11), were isolated and identified from the rhizomes of Menispermum dauricum. Their structures were elucidated by extensive spectroscopic data and single-crystal X-ray diffraction analyses. Among them, compounds 1 and 4 were the first samples of oxoisoaporphine with C-6 isopentylamino moiety, and 2 was a rare C-4 methylation product of oxoisoaporphine alkaloid. The in vitro anti-inflammatory activity of compounds 1-11 was performed by evaluating the inhibition of NO level in LPS-induced RAW264.7 macrophages. Among them, compound 4 exhibited the most potent NO inhibition activity with an IC50 value of 1.95 ± 0.33 µM. The key structure-activity relationships of those oxoisoaporphine alkaloids for anti-inflammatory effects have been summarized.

Boldine prevents the inflammatory response of cardiac fibroblasts induced by SGK1-NFκB signaling pathway activation.

Cardiac fibroblasts (CF) are mesenchymal-type cells responsible for maintaining the homeostasis of the heart's extracellular matrix (ECM). Their dysfunction leads to excessive secretion of ECM proteins, tissue stiffening, impaired nutrient and oxygen exchange, and electrical abnormalities in the heart. Additionally, CF act as sentinel cells in the cardiac tissue microenvironment, responding to various stimuli that may affect heart function. Deleterious stimuli induce an inflammatory response in CF, increasing the secretion of cytokines such as IL-1ß and TNF-α and the expression of cell adhesion molecules like ICAM1 and VCAM1, initially promoting damage resolution by recruiting immune cells. However, constant harmful stimuli lead to a chronic inflammatory process and heart dysfunction. Therefore, it is necessary to study the mechanisms that govern CF inflammation. NFκB is a key regulator of the cardiac inflammatory process, making the search for mechanisms of NFκB regulation and CF inflammatory response crucial for developing new treatment options for cardiovascular diseases. SGK1, a serine-threonine protein kinase, is one of the regulators of NFκB and is involved in the fibrotic effects of angiotensin II and aldosterone, as well as in CF differentiation. However, its role in the CF inflammatory response is unknown. On the other hand, many bioactive natural products have demonstrated anti-inflammatory effects, but their role in CF inflammation is unknown. One such molecule is boldine, an alkaloid obtained from Boldo (Peumus boldus), a Chilean endemic tree with proven cytoprotective effects. However, its involvement in the regulation of SGK1 and CF inflammation is unknown. In this study, we evaluated the role of SGK1 and boldine in the inflammatory response in CF isolated from neonatal Sprague-Dawley rats. The involvement of SGK1 was analyzed using GSK650394, a specific SGK1 inhibitor. Our results demonstrate that SGK1 is crucial for LPS- and IFN-γ-induced inflammatory responses in CF (cytokine expression, cell adhesion molecule expression, and leukocyte adhesion). Furthermore, a conditioned medium (intracellular content of CF subject to freeze/thaw cycles) was used to simulate a sterile inflammation condition. The conditioned medium induced a potent inflammatory response in CF, which was completely prevented by the SGK1 inhibitor. Finally, our results indicate that boldine inhibits both SGK1 activation and the CF inflammatory response induced by LPS, IFN-γ, and CF-conditioned medium. Taken together, our results position SGK1 as an important regulator of the CF inflammatory response and boldine as a promising anti-inflammatory drug in the context of cardiovascular diseases.

Magnoflorine Ameliorates Chronic Kidney Disease in High-Fat and High-Fructose-Fed Mice by Promoting Parkin/PINK1-Dependent Mitophagy to Inhibit NLRP3/Caspase-1-Mediated Pyroptosis.

Excessive intake of fat and fructose in Western diets has been confirmed to induce renal lipotoxicity, thereby driving the progression of chronic kidney disease (CKD). This study was conducted to evaluate the efficacy of magnoflorine in a CKD mouse model subjected to high-fat and high-fructose diets. Our results demonstrated that magnoflorine treatment ameliorated abnormal renal function indices (serum creatinine, urea nitrogen, uric acid, and urine protein) in high-fat- and high-fructose-fed mice. Histologically, renal tubular cell steatosis, lipid deposition, tubular dilatation, and glomerular fibrosis were significantly reduced by the magnoflorine treatment in these mice. Mechanistically, magnoflorine promotes Parkin/PINK1-mediated mitophagy, thereby inhibiting NLRP3/Caspase-1-mediated pyroptosis. Consistent findings were observed in the palmitic acid-incubated HK-2 cell model. Notably, both silencing of Parkin and the use of a mitophagy inhibitor reversed the inhibitory effect of magnoflorine on NLRP3 inflammasome activation in vitro. Therefore, the present study provides compelling evidence that magnoflorine improves renal injury in high-fat- and high-fructose-fed mice by promoting Parkin/PINK1-dependent mitophagy to inhibit NLRP3 inflammasome activation and pyroptosis. Our findings suggest that dietary supplementation with magnoflorine and magnoflorine-rich foods (such as magnolia) might be an effective strategy for the prevention of CKD.

Cytotoxic effects of aporphine alkaloids from the stems and leaves of Stephania dielsiana Y.C.Wu.

Phytochemical studies of the stems and leaves of Stephania dielsiana Y.C.Wu yielded two new aporphine alkaloids (1 and 5), along with six known alkaloids (2-4 and 6-8). Their structures were characterised based on analyses of spectroscopic data, including one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS). The cytotoxic activities of the isolated compounds against a small panel of tumour cell lines were assessed by MTS assay. Interestingly, compound 2 exhibited particularly strong cytotoxic activities against HepG2, MCF7 and OVCAR8 cancer cell lines, with IC50 values of 3.20 ± 0.18, 3.10 ± 0.06 and 3.40 ± 0.007 µM, respectively. Furthermore, molecular docking simulations were carried out to explore the interactions and binding mechanisms of the most active compound (compound 2) with proteins. Our results contribute to understanding the secondary metabolites produced by S. dielsiana and provide a scientific rationale for further investigations of cytotoxicity of this valuable medicinal plant.

Synthetic aporphine alkaloids are potential therapeutics for Leigh syndrome.

Mitochondrial diseases are mainly caused by dysfunction of mitochondrial respiratory chain complexes and have a variety of genetic variants or phenotypes. There are only a few approved treatments, and fundamental therapies are yet to be developed. Leigh syndrome (LS) is the most severe type of progressive encephalopathy. We previously reported that apomorphine, an anti- "off" agent for Parkinson's disease, has cell-protective activity in patient-derived skin fibroblasts in addition to strong dopamine agonist effect. We obtained 26 apomorphine analogs, synthesized 20 apomorphine derivatives, and determined their anti-cell death effect, dopamine agonist activity, and effects on the mitochondrial function. We found three novel apomorphine derivatives with an active hydroxy group at position 11 of the aporphine framework, with a high anti-cell death effect without emetic dopamine agonist activity. These synthetic aporphine alkaloids are potent therapeutics for mitochondrial diseases without emetic side effects and have the potential to overcome the low bioavailability of apomorphine. Moreover, they have high anti-ferroptotic activity and therefore have potential as a therapeutic agent for diseases related to ferroptosis.

Nuciferine alleviates collagen-induced arthritic in rats by inhibiting the proliferation and invasion of human arthritis-derived fibroblast-like synoviocytes and rectifying Th17/Treg imbalance.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder marked by persistent synovial inflammation and joint degradation, posing challenges in the development of effective treatments. Nuciferine, an alkaloid found in lotus leaf, has shown promising anti-inflammatory and anti-tumor effects, yet its efficacy in RA treatment remains unexplored. This study investigated the antiproliferative effects of nuciferine on the MH7A cell line, a human RA-derived fibroblast-like synoviocyte, revealing its ability to inhibit cell proliferation, promote apoptosis, induce apoptosis, and cause G1/S phase arrest. Additionally, nuciferine significantly reduced the migration and invasion capabilities of MH7A cells. The therapeutic potential of nuciferine was further evaluated in a collagen-induced arthritis (CIA) rat model, where it markedly alleviated joint swelling, synovial hyperplasia, cartilage injury, and inflammatory infiltration. Nuciferine also improved collagen-induced bone erosion, decreased pro-inflammatory cytokines and serum immunoglobulins (IgG, IgG1, IgG2a), and restored the balance between T helper (Th) 17 and regulatory T cells in the spleen of CIA rats. These results indicate that nuciferine may offer therapeutic advantages for RA by decreasing the proliferation and invasiveness of FLS cells and correcting the Th17/Treg cell imbalance in CIA rats.

Advances in the pharmacological effects and mechanisms of Nelumbo nucifera gaertn. Extract nuciferine.

ETHNOPHARMACOLOGIC RELEVANCE: The leaves of Nelumbo nucifera Gaertn. Are recorded in the earliest written documentation of traditional Chinese medicinal as "Ben Cao Gang Mu", a medicinal herb for blood clotting, dysentery and dizziness. Nuciferine, one of N. nucifera Gaertn. leaf extracts, has been shown to possess several pharmacological properties, including but not limited to ameliorating hyperlipidemia, stimulating insulin secretion, inducing vasodilation, reducing blood pressure, and demonstrating anti-arrhythmic properties. AIM OF THE STUDY: In light of the latest research findings on nuciferine, this article provides a comprehensive overview of its chemical properties, pharmacological activities, and the underlying regulatory mechanisms. It aims to serve as a dependable reference for further investigations into the pharmacological effects and mechanisms of nuciferine. MATERIALS AND METHODS: Use Google Scholar, Scifinder, PubMed, Springer, Elsevier, Wiley, Web of Science and other online database search to collect the literature on extraction, separation, structural analysis and pharmacological activity of nuciferine published before November 2023. The key words are "extraction", "isolation", "purification" and "pharmacological action" and "nuciferine". RESULTS: Nuciferine has been widely used in the treatment of ameliorating hyperlipidemia and lose weight, Nuciferine is a monomeric aporphine alkaloid extracted from the leaves of the plant Nymphaea caerulea and Nelumbo nucifera Gaertn. Nuciferine has pharmacological activities such as relaxing smooth muscles, improving hyperlipidemia, stimulating insulin secretion, vasodilation, inducing hypotension, antiarrhythmic effects, and antimicrobial and anti-HIV activities. These pharmacological properties lay a foundation for the treatment of tumors, inflammation, hyperglycemia, lipid-lowering and weight-loss, oxidative stress and other diseases with nuciferine. CONCLUSION: Nuciferine has been clinically used to treat hyperlipidemia and aid in weight loss due to its effects on lipid levels, insulin secretion, vasodilation, blood pressure reduction, anti-tumor properties, and immune enhancement. However, other potential benefits of nuciferine have not yet been fully explored in clinical practice. Future research should delve deeper into its molecular structure, toxicity, side effects, and clinical pharmacology to uncover its full range of effects and pave the way for its safe and expanded clinical use.

Exploring the antileishmanial activity of dicentrine from Ocotea puberula (Lauraceae) using biomembrane models.

This study aimed to assess the antiprotozoal efficacy of dicentrine, an aporphine alkaloid isolated from Ocotea puberula, against amastigote forms of Leishmania (L.) infantum. Our findings reveal that dicentrine demonstrated a notable EC50 value of 10.3 µM, comparable to the positive control miltefosine (EC50 of 10.4 µM), while maintaining moderate toxicity to macrophages (CC50 of 51.9 µM). Utilizing an in silico methodology, dicentrine exhibited commendable adherence to various parameters, encompassing lipophilicity, water solubility, molecule size, polarity, and flexibility. Subsequently, we conducted additional investigations to unravel the mechanism of action, employing Langmuir monolayers as models for protozoan cell membranes. Tensiometry analyses unveiled that dicentrine disrupts the thermodynamic and mechanical properties of the monolayer by expanding it to higher areas and increasing the fluidity of the film. The molecular disorder was further corroborated through dilatational rheology and infrared spectroscopy. These results contribute insights into the role of dicentrine as a potential antiprotozoal drug in its interactions with cellular membranes. Beyond elucidating the mechanism of action at the plasma membrane's external surface, our study sheds light on drug-lipid interface interactions, offering implications for drug delivery and other pharmaceutical applications.

Synthesis, Stereochemical Resolution, and Analogue Synthesis of Variabiline, an Aporphine Alkaloid That Sensitizes Acinetobacter baumannii and Klebsiella pneumoniae to Colistin.

Increasing antimicrobial resistance, coupled with the absence of new antibiotics, has led physicians to rely on colistin, a polymyxin with known nephrotoxicity, as the antibiotic of last resort for the treatment of infections caused by Gram-negative bacteria. One approach to increasing antibiotic efficacy and thereby reducing dosage is the use of small-molecule potentiators that augment antibiotic activity. We recently identified the aporphine alkaloid (±)-variabiline, which lowers the minimum inhibitory concentration of colistin in Acinetobacter baumannii and Klebsiella pneumoniae. Herein, we report the first total synthesis of (±)-variabiline to confirm structure and activity, the resolution, and evaluation of both enantiomers as colistin potentiators, and a structure-activity relationship study that identifies more potent variabiline derivatives. Preliminary mechanistic studies indicate that (±)-variabiline and its derivatives potentiate colistin by targeting the Gram-negative outer membrane.

Nuciferine reduces vascular leakage and improves cardiac function in acute myocardial infarction by regulating the PI3K/AKT pathway.

The destruction of the microvascular structure and function can seriously affect the survival and prognosis of patients with acute myocardial infarction (AMI). Nuciferine has a potentially beneficial effect in the treatment of cardiovascular disease, albeit its role in microvascular structure and function during AMI remains unclear. This study aimed to investigate the protective effect and the related mechanisms of nuciferine in microvascular injury during AMI. Cardiac functions and pathological examination were conducted in vivo to investigate the effect of nuciferine on AMI. The effect of nuciferine on permeability and adherens junctions in endothelial cells was evaluated in vitro, and the phosphorylation level of the PI3K/AKT pathway (in the presence or absence of PI3K inhibitors) was also analyzed. In vivo results indicated that nuciferine inhibited ischemia-induced cardiomyocyte damage and vascular leakage and improved cardiac function. In addition, the in vitro results revealed that nuciferine could effectively inhibit oxygen-glucose deprivation (OGD) stimulated breakdown of the structure and function of human coronary microvascular endothelial cells (HCMECs). Moreover, nuciferine could significantly increase the phosphorylation level of the PI3K/AKT pathway. Finally, the inhibitor wortmannin could reverse the protective effect of nuciferine on HCMECs. Nuciferine inhibited AMI-induced microvascular injury by regulating the PI3K/AKT pathway and protecting the endothelial barrier function in mice.

Boldine protects against carbon tetrachloride-induced chronic liver injury by regulating NF-κB signaling pathway.

Sustained liver injuries predominantly promote oxidative stress and inflammation that lead to the progression of chronic liver disease (CLD), including fibrosis, cirrhosis, and hepatocellular carcinoma. Boldine, an alkaloid isolated from Peumus boldus, has been shown to have antioxidant and anti-inflammatory effects. Currently, there is no definitive treatment option available for CLD. Therefore, we investigated the hepatoprotective effect of boldine against carbon tetrachloride (CCl4 )-induced chronic liver injury in rats. CCl4 (2 mL/kg., b.w., i.p.) was administered twice weekly for 5 weeks to induce chronic liver injury in rats. Separate groups of rats were given boldine (20 mg/kg b.w., and 40 mg/kg b.w.) and silymarin (100 mg/kg b.w.) orally, daily. Serum transaminases, lipid peroxidation, and antioxidant levels were measured, and nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (cox-2), interleukin-1 ß (IL-1ß), and α-smooth muscle actin (α-SMA) gene and protein expressions were evaluated. CCl4 administration increased liver marker enzymes of hepatotoxicity in serum and oxidative stress markers, inflammatory genes and α-smooth muscle actin expression in liver tissue. Boldine concurrent treatment suppressed CCl4 -induced elevation of transaminase levels in serum, restored enzymic and non-enzymic antioxidants, and downregulated NF-κB, TNF-α, Cox-2 and IL-1ß expressions, thereby suppressing hepatic inflammation. Boldine administration also repressed α-SMA expression. The results of this study demonstrate the antioxidant, anti-inflammatory, and antifibrotic properties of boldine, and it can be a potential therapeutic candidate in the treatment of CLD.

Four unreported aporphine alkaloids with antifungal activities from Artabotrys hexapetalus.

In this study, the extract from Artabotrys hexapetalus showed strong antifungal activity against phytopathogenic fungi in vitro. Four unreported aporphine alkaloids, hexapetalusine A-D (1-4), were isolated from stems and roots of Artabotrys hexapetalus (L.f.) Bhandari, along with six known aporphine alkaloids (5-10). Their chemical structures were elucidated by extensive spectroscopic analysis. The absolute configurations of 1-3 were determined using single-crystal X-ray diffractions and ECD calculations. Hexapetalusine A-C (1-3) were special amidic isomers. Additionally, all isolated compounds were evaluated for their antifungal activity against four phytopathogenic fungi in vitro. Hexapetalusine D (4) exhibited weak antifungal activity against Curvularia lunata. Liriodenine (5) displayed significant antifungal activity against Fusarium proliferatum and Fusarium oxysporum f. sp. vasinfectum, which is obviously better than positive control nystatin, suggesting that it had great potential to be developed into an effective and eco-friendly fungicide.

Characterization of two CYP80 enzymes provides insights into aporphine alkaloid skeleton formation in Aristolochia contorta.

Aporphine alkaloids are a large group of natural compounds with extensive pharmaceutical application prospects. The biosynthesis of aporphine alkaloids has been paid attentions in the past decades. Here, we determined the contents of four 1-benzylisoquinoline alkaloids and five aporphine alkaloids in root, stem, leaf, and flower of Aristolochia contorta Bunge, which belongs to magnoliids. Two CYP80 enzymes were identified and characterized from A. contorta. Both of them catalyze the unusual C-C phenol coupling reactions and directly form the aporphine alkaloid skeleton. AcCYP80G7 catalyzed the formation of hexacyclic aporphine corytuberine. AcCYP80Q8 catalyzed the formation of pentacyclic proaporphine glaziovine. Kingdom-wide phylogenetic analysis of the CYP80 family suggested that CYP80 first appeared in Nymphaeales. The functional divergence of hydroxylation and C-C (or C-O) phenol coupling preceded the divergence of magnoliids and eudicots. Probable crucial residues of AcCYP80Q8 were selected through sequence alignment and molecular docking. Site-directed mutagenesis revealed two crucial residues E284 and Y106 for the catalytic reaction. Identification and characterization of two aporphine skeleton-forming enzymes provide insights into the biosynthesis of aporphine alkaloids.

A Combination of Magnoflorine and Spinosin Improves the Antidepressant effects on CUMS Mouse Model.

BACKGROUND: Depression is a common neuropsychiatric disease. As a famous traditional Chinese medicine with significant anti-depressive and sleep-promoting effects, Ziziphi Spinosae Semen (ZSS) has attracted the attention of many researchers. Although it is well known that Magnoflorine (MAG) and Spinosin (SPI) were the main active components isolated from ZSS, there is a lack of research on the combined treatment of depression with these two ingredients. METHODS: The shaking bottle method was used to simulate the human environment for detecting the changes in oil-water partition coefficient before and after the drug combination. Cell viability was evaluated by the MTT assay. To establish a mouse model of depression and insomnia by CUMS method, and then to explore the effect of combined administration of MAG and SPI on depression in CUMS model by observing behavior and analyzing pharmacokinetics. RESULTS: The change in LogP values affected the lipid solubility of MAG and increased the water solubility of SPI, allowing them to penetrate more easily through the blood-brain barrier into the brain. Compared with the model group, MAG-SPI with a concentration of 60 µM significantly increased cell survival rate. In both the TST and FST experiments, the mice showed a decrease in immobilization time. Pharmacokinetic results showed that the pharmacokinetic parameters, Cmax and AUC of MAG and SPI, were increased in the case of combination, which resulted in enhancement of their relative bioavailability and improvement of in vivo effects. CONCLUSIONS: The present study demonstrated that a combination of MAG and SPI had a synergistic antidepressant effect in CUMS mouse model.

Aporphine alkaloids and a naphthoquinone derivative from the leaves of Phaeanthus lucidus Oliv. and their α-glucosidase inhibitory activity.

Three previously undescribed aporphine alkaloids, phaeanthuslucidines E-G, one previously undescribed naphthoquinone derivative, phaeanthusnaphthoquinone, and three known compounds were isolated from an EtOAc extract of the leaves of Phaeanthus lucidus Oliv. The structures of all previously undescribed compounds were established through extensive spectroscopic investigations and high-resolution mass spectroscopy. The 6aR configuration of phaeanthuslucidines E-G was assigned by comparing their ECD spectra and specific rotation values with the reported known compounds. Some isolated compounds were evaluated for their α-glucosidase inhibitory activity. Among these compounds, phaeanthuslucidine E showed the highest α-glucosidase inhibitory activity with an IC50 value of 17.9 ± 0.4 µM. The molecular docking of phaeanthuslucidine E was further studied.

Bitter yet beneficial: The dual role of dietary alkaloids in managing diabetes and enhancing cognitive function.

With the rising prevalence of diabetes and its association with cognitive impairment, interest in the use of dietary alkaloids and other natural products has grown significantly. Understanding how these compounds manage diabetic cognitive dysfunction (DCD) is crucial. This comprehensive review explores the etiology of DCD and the effects of alkaloids in foods and dietary supplements that have been investigated as DCD therapies. Data on how dietary alkaloids like berberine, trigonelline, caffeine, capsaicin, 1-deoxynojirimycin, nuciferine, neferine, aegeline, tetramethylpyrazine, piperine, and others regulate cognition in diabetic disorders were collected from PubMed, Research Gate, Web of Science, Science Direct, and other relevant databases. Dietary alkaloids could improve memory in behavioral models and modulate the mechanisms underlying the cognitive benefits of these compounds, including their effects on glucose metabolism, gut microbiota, vasculopathy, neuroinflammation, and oxidative stress. Evidence suggests that dietary alkaloids hold promise for improving cognition in diabetic patients and could open exciting avenues for future research in diabetes management.

Nuciferine reduces inflammation induced by cerebral ischemia-reperfusion injury through the PI3K/Akt/NF-κB pathway.

BACKGROUND: Cerebral ischemia has the characteristics of high incidence, mortality, and disability, which seriously damages people's health. Cerebral ischemia-reperfusion injury is the key pathological injury of this disease. However, there is a lack of drugs that can reduce cerebral ischemia-reperfusion injury in clinical practice. At present, a few studies have provided some evidence that nuciferine can reduce cerebral ischemia-reperfusion injury, but its specific mechanism of action is still unclear, and further research is still needed. OBJECTIVE: In this study, PC12 cells and SD rats were used to construct OGD/R and MCAO/R models, respectively. Combined with bioinformatics methods and experimental verification methods, the purpose of this study was to conduct a systematic and comprehensive study on the effect and mechanism of nuciferine on reducing inflammation induced by cerebral ischemia-reperfusion injury. RESULTS: Nuciferine can improve the cell viability of PC12 cells induced by OGD/R, reduce apoptosis, and reduce the expression of inflammation-related proteins; it can also improve the cognitive and motor dysfunction of MCAO/R-induced rats by behavioral tests, reduce the area of cerebral infarction, reduce the release of inflammatory factors TNF-α and IL-6 in serum and the expression of inflammation-related proteins in brain tissue. CONCLUSION: Nuciferine can reduce the inflammatory level of cerebral ischemia-reperfusion injury in vivo and in vitro models by acting on the PI3K/Akt/NF-κB signaling pathway, and has the potential to be developed as a drug for the treatment of cerebral ischemia-reperfusion injury.

The CYP80A and CYP80G Are Involved in the Biosynthesis of Benzylisoquinoline Alkaloids in the Sacred Lotus (Nelumbo nucifera).

Bisbenzylisoquinoline and aporphine alkaloids are the two main pharmacological compounds in the ancient sacred lotus (Nelumbo nucifera). The biosynthesis of bisbenzylisoquinoline and aporphine alkaloids has attracted extensive attention because bisbenzylisoquinoline alkaloids have been reported as potential therapeutic agents for COVID-19. Our study showed that NnCYP80A can catalyze C-O coupling in both (R)-N-methylcoclaurine and (S)-N-methylcoclaurine to produce bisbenzylisoquinoline alkaloids with three different linkages. In addition, NnCYP80G catalyzed C-C coupling in aporphine alkaloids with extensive substrate selectivity, specifically using (R)-N-methylcoclaurine, (S)-N-methylcoclaurine, coclaurine and reticuline as substrates, but the synthesis of C-ring alkaloids without hydroxyl groups in the lotus remains to be elucidated. The key residues of NnCYP80G were also studied using the 3D structure of the protein predicted using Alphafold 2, and six key amino acids (G39, G69, A211, P288, R425 and C427) were identified. The R425A mutation significantly decreased the catalysis of (R)-N-methylcoclaurine and coclaurine inactivation, which might play important role in the biosynthesis of alkaloids with new configurations.

Nuciferine prevents obesity by activating brown adipose tissue.

Increasing evidence suggests that brown adipose tissue (BAT) plays an important role in obesity and related diseases. Increasing the amount or activity of BAT could prevent obesity. Therefore, a safe and effective method of activating BAT is urgently required. Here, we evaluated the potential effects of lotus leaf extract (LLE) on BAT function. We found that LLE substantially increased UCP1 mRNA and protein levels as well as thermogenic protein expression in primary brown adipocytes. Additionally, LLE treatment reduced diet-induced obesity and improved glucose homeostasis owing to BAT activation and increased energy expenditure. We found that nuciferine, an active ingredient of LLE, could dose-dependently activate BAT in vitro and in vivo, alleviate diet-induced obesity, and improve glucose homeostasis by increasing energy expenditure. Mechanistically, we found that nuciferine induced PPARG coactivator 1 alpha (PGC1-α) expression, which is a key gene involved in mitochondrial biogenesis promoter activity, by directly binding to RXRA. Furthermore, RXRA knockdown abolished expression of the nuciferine-induced mitochondrial and thermogenesis-related gene in primary brown adipocytes. In summary, we found that LLE and nuciferine have a notable effect on BAT activation and highlight the potential applications of the main component of LLE in preventing obesity and treating metabolic disorders.