Magnoflorine Research Articles

Magnoflorine attenuates Ang II-induced cardiac remodeling via promoting AMPK-regulated autophagy.

Heart failure (HF) remains one of the most common events in the progression of hypertension. Magnoflorine (MNF) has been shown beneficial effects on the cardiovascular system. However, the action of MNF on angiotensin (Ang) II-induced cardiac remodeling and its underlying mechanisms have not yet been characterised. Here, we assessed the action of MNF in the development of hypertension-related HF. C57BL/6 male mice were subjected to Ang II through a micro-osmotic pump infusion continuously for 4 weeks to induce hypertensive HF. MNF (10 and 20 mg/kg) was administered in the final 2 weeks. Ang II content was measured by enzyme-linked immunosorbent assay (ELISA) kit. Values of ejection fraction (EF) and fractional shortening (FS) were detected using an ultrasound diagnostic instrument. The mRNA levels of hypertrophic and fibrotic genes were determined by real-time quantitative polymerase chain reaction (RT-qPCR). Haematoxylin and eosin (H&E), wheat germ agglutinin (WGA), Masson trichrome, and Sirius Red staining were used to analyse pathologic changes in heart tissues. The expression levels of phosphorylated AMP-activated protein kinase (AMPK), light chain 3 microtubule associated protein II (LC3 II) to LC3 I, and p62 were detected by western blot assay. MNF significantly improved cardiac dysfunction and the content of creatine kinase-MB without altering blood pressure in Ang II-challenged mice. MNF obviously corrected the phenotypes of cardiac hypertrophy and fibrosis, including the high mRNA levels of atrial natriuretic peptide (Anp), brain natriuretic peptide (Bnp), collagen1a (Col1a1), transforming growth factor beta (Tgfb1), enlarged myocardial areas, and increased positive areas of Masson trichrome and Sirius Red staining. In addition, MNF alleviated oxidative injury, reflected by the upregulation of glutathione and the downregulation of reactive oxygen species and malondialdehyde. The activation of AMPK was elevated accompanied by an increased level of autophagy by MNF in hypertensive heart tissues. The therapeutic action of MNF was confirmed in Ang II-challenged H9c2 cells. Specifically, the AMPK inhibitor could eliminate the autophagy pathway in which MNF is involved. MNF has benefits in hypertension-induced cardiac remodeling, which was partially associated with the improvement of oxidative stress via the mediation of the AMPK/autophagy axis.

Structural characterization and biological activity evaluation of Magnoflorine alkaloid, a potential anticonvulsant agent

Magnoflorine (MGF), an isoquinoline alkaloid, emerges as a quaternary aporphine alkaloid derived from L-tyrosine amino acid, found in families Magnoliaceae plants and it presents important biological activity being noted for its effect on the nervous system. In this work, a deep study of structural, vibrational and electronic properties has been carried out for the MGF. From Potential Energy Surface (PES) scan the most stable conformer of alkaloid was identified and geometrical parameters were determined by Density Functional Theory (DFT) using B3LYP/6–311++G** method. A complete vibrational assignment of the normal modes was theoretical and experimentally achieved from FT-IR and Raman spectroscopies and scaled SQM methodology. Electronic transitions observed in UV–visible spectrum in aqueous medium were identified using TD-DFT methodology, with the π→π* transition being the most probable UV signal. The MEPs, the electric charge distribution along with the HOMO and LUMO frontier orbitals were analyzed and the GAP energy values justified the stability of the molecule in aqueous medium. The molecular electrostatic potential map reveals the most favourable region for electrophilic attack on MGF. Molecular docking was used to analyze the anxiolytic biological activity of the title molecule on the GABAA receptor. The results suggest an intermolecular binding capability of MFG toward both intracellular and extracellular domains of GABAA receptor, and the extracellular domain presents a higher affinity by alkaloid. Finally, the biological study infers that MGF could be used as a potential anxiolytic compound.

Natural alkaloids from Dicranostigma leptopodum (Maxim.) Fedde and their G5. NHAc-PBA dendrimer-alkaloid complexes for targeting chemotherapy in breast cancer MCF-7 cells.

Herein, we isolated five natural alkaloids, iso-corydine (iso-CORY), corydine (CORY), sanguinarine (SAN), chelerythrine (CHE) and magnoflorine (MAG), from traditional medicinal herb Dicranostigma leptopodum (Maxim.) Fedde (whole herb) and elucidated their structures. Then we synthesised G5. NHAc-PBA as targeting dendrimer platform to encapsulate the alkaloids into G5. NHAc-PBA-alkaloid complexes, which demonstrated alkaloid-dependent positive zeta potential and hydrodynamic particle size. G5. NHAc-PBA-alkaloid complexes demonstrated obvious breast cancer MCF-7 cell targeting effect. Among the G5. NHAc-PBA-alkaloid complexes, G5.NHAc-PBA-CHE (IC50=13.66 μM) demonstrated the highest MCF-7 cell inhibition capability and G5.NHAc-PBA-MAG (IC50=24.63 μM) had equivalent inhibitory effects on cell proliferation that comparable to the level of free MAG (IC50=23.74 μM), which made them the potential breast cancer targeting formulation for chemotherapeutic application. This work successfully demonstrated a pharmaceutical research model of 'natural bioactive product isolation-drug formulation preparation-breast cancer cell targeting inhibition'.

Green solvent selection and extractin protocol for selective recovery of anti-diabetic components from T. crispa

This study systematically explores a green extraction process of T. crispa stems to selectively isolate therapeutic compounds, borapetoside C (BPC) and magnoflorine (MGF), recognized for their anti-diabetic properties. In agreement with Hansen solubility parameters prediction, H2O and EtOH were found to be suitable solvents for extraction of BPC and MGF, respectively, resulting in 33 % and 45 % extractabilities after 60 min. Further investigations demonstrated considerable increase in BPC extractability using 20 % EtOH:H2O mixture at 40 °C, with complete extraction achieved after 60 min. While for MGF, pure-EtOH at 40 °C, was the most suitable solvent, showing the highest selectivity and complete extraction after 100 min. The BPC-rich extract from 20 % EtOH:H2O exhibited higher anti-diabatic activity (IC50 = 0.63 ± 0.03 and 0.72 ± 0.04 mg/mL, respectively, for α-glucosidase and α-amylase enzymes inhibition activity), and considerably lower cytotoxicity to L6 and HepG2 cells, with IC50 = 0.26 ± 0.16 and 0.24 ± 0.02 mg/mL, respectively, compared with the MGF-rich extract obtained with pure-EtOH. Based on these results, a sequential extraction scheme was proposed involving initial pure-EtOH extraction to selectively and completely remove MGF, followed by extraction with a 20 % EtOH:H2O mixture to recover the remaining BPC, which was approximately 80 % of the BPC originally present. The obtained MGF-free BPC-rich extract showed significantly lower cytotoxicity (IC50 = 0.31 ± 0.06 mg/mL against L6 cell) and higher enzyme inhibition activities (IC50 = 0.53 ± 0.32 and 0.52 ± 0.02 mg/mL for α-glucosidase and α-glucosidase enzymes inhibition activity), comparable to acarbose (IC50 = 0.43 ± 0.02 and 0.83 ± 0.03 mg/mL for α-glucosidase and α-glucosidase enzymes inhibition activity), the result that potentially leads to the development of a promising industrial process to harness T. crispa for diabetes prevention and treatment.

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

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. 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. 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. The present study demonstrated that a combination of MAG and SPI had a synergistic antidepressant effect in CUMS mouse model.

Analyzing Biomarkers in an Enhanced Polyherbal Formula with Antidiabetic Attributes through High-performance Thin-layer Chromatography and Liquid Chromatography–Mass Spectrometry/ Mass Spectrometry

ABSTRACT Context: In this research, we subjected a polyherbal formulation (PHF) prepared in-house, previously evaluated for its antidiabetic effects in streptozotocin-induced diabetic rats, to further analytical characterization using high-performance thin-layer chromatography (HPTLC) and Liquid Chromatography coupled with Tandem Mass Spectrometry (LC-MS/MS). Aim: The aim of this research was to measure the active components within a newly developed in-house PHF with antidiabetic properties, prepared using dried leaves of Tinospora cordifolia, dried bark of Cinnamomum zeylanicum, dried seeds of Trigonella foenum, and dried seeds of Nigella sativa, employing HPTLC and LC-MS/MS techniques. Materials and Methods: Magnoflorine (MAG), cinnamaldehyde (CIN), trigonelline (TRI), and thymoquinone (THY) were identified as the active components of the ethanolic extract of the PHF. We utilized HPTLC and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the characterization of these constituents. The gradient mobile phase consisted of water, acetonitrile, and 0.1% trifluoroacetic acid. Chromatographic separation was achieved using an XBridge C18, 250 × 4.6, 3 mm analytical column with a flow rate set at 1.0 mL/min, maintained at ambient temperature employing a gradient system that spanned 30 min. Mass scanning was conducted in positive mode, with a scan speed ranging from 100 to 2000 amu/s, covering a mass range from 20 to 1974 Da. Electron spray ionization mode was utilized, with a source temperature maintained at 150°C and a desolvation temperature at 350°C. For HPTLC separation, precoated silica gel 60 GF254 plates were employed, employing a mobile phase consisting of toluene/chloroform/ethyl acetate/methanol/formic acid in a ratio of 2.5:2.5:2.5:2.0:0.5 (v/v). The plates were developed at room temperature, extending to a distance of 9.0 cm. Each individual component (MAG, CIN, TRI, and THY) was scanned and quantified at its respective maximum absorption wavelength (237 nm, 259 nm, 305 nm, and 338 nm). Results: Experimental parameters such as band size, chamber saturation duration, solvent front migration, slit width, and others were carefully examined to determine the optimized chromatographic conditions. LC-MS analysis of the alcoholic extract of the PHF successfully identified the presence of all five bioactive chemical constituents, namely, MAG, CIN, TRI, and THY. In addition, the drug samples exhibited satisfactory resolution with retention factor values of 0.27 ± 0.01, 0.64 ± 0.01, 0.55 ± 0.01, and 0.92 ± 0.01 for CIN, MAG, TRI, and THY respectively, using HPTLC. Conclusion: The integration of Ayurvedic formulations with contemporary high-throughput screening techniques holds great promise in discovering more potent and biocompatible drug leads. The results of this study offer compelling scientific evidence that strengthens the case for the therapeutic utility of the PHF, further underscoring its significance in the field of health care.

Computational investigation of four isoquinoline alkaloids against polycystic ovarian syndrome

Hyperandrogenism, insulin resistance, and estrogen dominance are the prime defining traits of women with polycystic ovarian syndrome which disrupts hormonal, adrenal, or ovarian functions resulting in impaired folliculogenesis and excess androgen production. The purpose of this study is to identify an appropriate bioactive antagonistic ligand from isoquinoline alkaloids [palmatine (PAL), jatrorrhizine (JAT), magnoflorine (MAG) and berberine (BBR)] from stems of Tinospora cordifolia. Phytocomponents inhibit/prevent androgenic, estrogenic, and steroidogenic receptors, insulin binding, and resultant hyperandrogenism. Intending to develop new inhibitors for human androgen receptor (1E3G), insulin receptor (3EKK), estrogen receptor beta (1U3S), and human steroidogenic cytochromeP450 17A1 (6WR0), here we report the docking studies by employing a flexible ligand docking approach using AutodockVina 4.2.6. ADMET screened swissADME and toxicological predictions to identify novel and potent inhibitors against PCOS. Binding affinity was obtained using Schrodinger. Two ligands, mainly BER (−8.23) and PAL (−6.71) showed the best docking score against androgen receptors. A molecular docking study reveals that compounds BBR and PAL were found to be tight binder at the active site of IE3G. Molecular dynamics results suggest that BBR and PAL showed good binding stability of active site residues. The present study corroborates the molecular dynamics of the compound BBR and PAL, potent Inhibitors of IE3G, having therapeutic potential for PCOS. We project that this study’s findings will be helpful in drug development efforts targeting PCOS. Hence isoquinoline alkaloids (BER& PAL) have potential roles against androgen receptors, and in specific PCOS, scientific evaluation has been put forth based on virtual screening. Communicated by Ramaswamy H. Sarma

Magnoflorine from Berberis vulgaris Roots-Impact on Hippocampal Neurons in Mice after Short-Term Exposure.

In search of novel potential drug candidates that could be used as treatments or prophylactics for memory impairment, an aporphine alkaloid magnoflorine (MAG) isolated from the root of Berberis vulgaris was proven to exhibit beneficial anti-amnestic properties. Its effects on immunoreactivity to parvalbumin in the mouse hippocampus were assessed together with a study on its safety and concentration in the brain and plasma. For this purpose, four experimental groups were created: the MAG10 group-treated with 10 mg MAG/kg b.w. i.p., the MAG20 group-treated with 20 mg MAG/kg b.w. i.p., the MAG50 group-treated with 50 mg MAG/kg b.w. i.p., and a control group-injected with saline i.p. at a volume corresponding to their weight. Our results indicated that the hippocampal fields CA1-CA3 were characterized by an elevated number of parvalbumin-immunoreactive neurons (PV-IR) and nerve fibers in mice at the doses of 10 and 20 mg/kg b.w. (i.p.). No significant changes to the levels of IL-1β, IL-6 or TNF-α were observed for the above two doses; however, the administration of 50 mg/kg b.w. i.p. caused a statistically significant elevation of IL-6, IL-1beta plasma levels and an insignificant raise in the TNF-alpha value. The HPLC-MS analysis showed that the alkaloid's content in the brain structures in the group treated with 50 mg/kg b.w. did not increase proportionally with the administered dose. The obtained results show that MAG is able to influence the immunoreactivity to PV-IR in hippocampal neurons and might act as a neuroprotective compound.

Co-existing polysaccharides affect the systemic exposure of major bioactive ingredients in Chang-Kang-Fang, a multi-herb prescription for treatment of irritable bowel syndrome

Ethnopharmacological relevanceChang-Kang-Fang (CKF) is a traditional Chinese herbal formula used for treatment of irritable bowel syndrome (IBS) in China. Decoction is the administration form of CKF in clinical practice. Previously, CKF has been confirmed with activities of releasing pain and reversing disorders of intestinal propulsion. And alkaloids, monoglycosides, chromones were found as the main bioactive components potentially contributing to the efficacy of CKF. Polysaccharide was also a major constituent in CKF. But if and how polysaccharides influence the systemic exposure of bioactive components in CKF is unknown. Aim of the studyIn this study, we aimed to demonstrate the contribution of the co-existed polysaccharides on the systemic exposure of the major bioactive components from CKF in normal and IBS model rats. Materials and methodsAn UPLC-TQ-MS with multiple reaction monitoring (MRM) scan method was developed and validated for quantifying six major small molecular bioactive ingredients of CKF in the plasma samples, including magnoflorine (MAG), berberine (BBR), albiflorin (ALB), paeoniflorin (PAE), 5-O-methylvisamminol (5-OM) and prim-O-glucosylcimifugin (POG). The rats received CKF decoction (CKF) and CKF small molecule portion (knockout of polysaccharides, CKFSM), respectively. IBS model rats were induced by daily bondage and gavage of Sennae Folium decoction (derived from the leaf of Cassia angustifolia Vahl). The effects of the co-existing polysaccharides on the pharmacokinetic parameters of six small molecular bioactive components in normal and IBS model rats were systematically evaluated. The potential gut microbiota involved mechanisms of the effects was validated by broad-spectrum antibiotic (ABX) treatment. ResultsThe selectivity, precision, accuracy, recovery and matrix effect of the established quantification method were all within acceptable limits of biological sample. In normal rats, the co-existing polysaccharides significantly reduced the AUC(0-t) of MAG and PAE compared with CKFSM group. The Cmax and AUC(0-t) of other four compound were not influenced by co-existing polysaccharides. However, in IBS model rats, compared with CKFSM group, the Cmax and AUC(0-t) of the six ingredients significantly increased in CKF group. For CKF + ABX group, the Cmax of six ingredients decreased significantly when compared with CKF group, and the AUC(0-t) of MAG, BBR, ALB, PAE also reduced with significant differences. ConclusionsA reliable and sensitive UPLC-TQ-MS method was successfully developed and validated for evaluating influence of co-existing polysaccharides on pharmacokinetic behavior of six major small molecules components in CKF. The co-existing polysaccharides enhanced the systemic exposure of six bioactive small molecules in CKF under IBS pathological state potentially via gut microbiota involvement.

Direct infusion electrospray ionization-ion mobility-mass spectrometry for rapid metabolite marker discovery of medicinal Phellodendron Bark

Ion-mobility mass spectrometry (IM-MS) currently serves as a powerful tool for the structural identification of numerous biological compounds and small molecules. In this work, rapid metabolomic analysis of closely-related herbal medicines by direct injection ion mobility-quadrupole time-of-flight mass spectrometry (DI-IM-QTOF MS) was established. Phellodendron chinense Bark (PC) and Phellodendron amurense Bark (PA) were studied as a case. Thirty-three batches of PC and twenty-two batches of PA have been directly injected in electrospray ionization-IM-QTOF MS in positive mode. Without chromatographic separation, each run was completed within 3 min. After data alignment and statistical analysis, a total of seven chemical markers were found (p-value < 0.05, VIP > 1.00). Among them, the ion m/z 342.17 and m/z 356.18 present a single peak in the drift spectrum, respectively, but their drift time has a certain deviation compared with the pure substance of known compounds. In addition, the MS/MS spectra also confirmed that the single peak includes two chemical isomers. To investigate the composition ratio of individual isomers, the calibration curves of relative drift time (rDT) based on the standard superposition method were established, which were found to fit the least square regression. The ion [M]+m/z 342.17 was recognized consisting of magnoflorine (MAG) and phellodendrine (PHE), and their composition ratio in PA and PC samples was calculated. The results were compared with those obtained by the HPLC quantitative method, which produced equivalent quantification results. Our DI-IM-QTOF MS methodology provides an additional methodology for the relative quantification of unresolved isomers in drift tube IM-MS and offers DI-IM-QTOF MS based metabolomics.

Magnoflorine attenuates inflammatory responses in RA by regulating the PI3K/Akt/NF-κB and Keap1-Nrf2/HO-1 signalling pathways in vivo and in vitro

BackgroundAs a prolonged autoimmune disorder, rheumatoid arthritis (RA) is characterised by synovial hyperplasia and the erosion of bone and cartilage. Magnoflorine (MAG) is the main component purified from Clematis manshurica Rupr. Recent studies have shown that MAG has anti-inflammatory, antioxidant, and immunosuppressive effects, which are relevant to anti-RA activities. ObjectiveThe current investigation was conducted to explore the anti-RA effects of MAG and to discover the possible molecular mechanisms. MethodsIn vitro experiments, CCK-8, wound healing, and transwell assays were utilized to evaluate the anti-proliferative, anti-migratory, and anti-invasive activities of MAG, respectively. The rate of cell distribution and cell apoptosis were evaluated by flow cytometry. ROS generation was detected by DCFH-DA staining. Western blotting, quantitative real-time polymerase chain reaction assay, and immunofluorescent staining were employed to test the anti-RA effect of MAG as well as to explore the potential mechanisms by evaluating related gene and protein expression. For in vivo experiments, an adjuvant-induced arthritis (AIA) rat model was established. The related parameters were measured in rats. Then, rats were sacrificed, and ankle joints were collected for histopathological analysis and observation. ResultsMAG significantly decreased the proliferation, migration, invasion, and reactive oxygen species levels in IL-1β-treated MH7A cells. Furthermore, MAG promoted cell apoptosis by increasing Bax levels and decreasing Bcl-2 levels. MAG also induced cell cycle arrest. Inflammatory cytokines (iNOS, COX-2, IL-6, and IL-8) and MMPs (MMP-1, 2, 3, 9, and 13) were reduced by MAG treatment. Molecular analysis revealed that MAG exerted anti-RA effects by partly inhibiting the PI3K/Akt/NF-κB signalling axis and activating the Keap1-Nrf2/HO-1 signalling pathway. In vivo studies have revealed that MAG treatment substantially improved severe symptoms in AIA rats, and these curative effects were linked to the attenuation of inflammatory responses. ConclusionThese results first suggested that MAG exhibits anti-arthritic effects in IL-1β-treated MH7A cells and AIA rat models. Thus, MAG may be used as a new drug to treat RA clinically.

Nanonization of Magnoflorine-Encapsulated Novel Chitosan-Collagen Nanocapsules for Neurodegenerative Diseases: In Vitro Evaluation.

Neurodegeneration is one of the most common diseases in the aged population, characterized by the loss in the function of neuronal cells and their ultimate death. One of the common features in the progression of this type of diseases is the oxidative stress. Drugs which are currently being used have been found to show lateral side effects, which is partly due to their inefficiency to cross blood–brain barrier. Nanoencapsulation of bioactive compounds is a profound approach in this direction and has become a method of choice nowadays. This study involved the evaluation of the anti-oxidative properties of magnoflorine (MF), which is an aporphine quaternary alkaloid, and synthesis of MF-loaded chitosan–collagen nanocapsules (MF-CCNc) for its better efficacy as a potent anti-oxidant. Physiochemical characterization of the synthesized nanocapsules was done by using dynamic light scattering and transmission electron microscopy. It revealed that the synthesized nanocapsules are of small size range, as small as 12 ± 2 nm, and are more or less of spherical shape. Sustained release was shown by MF in the in vitro drug release studies. Both MF and MF-CCNc were found to have good anti-oxidant potential with IC50 < 25 μg/mL. No major cytotoxicity was shown by the synthesized nanocapsules on SH-SY5Y cells. In silico anti-acetylcholinesterase (AChE) studies were also done, and they revealed that MF can be a potent inhibitor of AChE.

Magnoflorine promotes Huh-7 cell apoptosis and autophagy by regulating PI3K/Akt/mTOR pathway

Hepatoma is a malignant tumor with high rates of heterogeneity, metastasis, and mortality. Currently, there is no effective treatment available for hepatoma. In order to treat advanced hepatoma in a better manner, new and more effective therapeutic targets still need to be developed. Magnoflorine (MGN) is a quaternary ammonium alkaloid with a variety of therapeutic properties. MGN inhibited the proliferation of lung cancer, breast cancer, glioma, and rhabdomyosarcoma cells, induced apoptosis, and blocked cell cycle. However, its possible effects on the progression of hepatoma are still indefinite. In this study, the effects of MGN on the progression of hepatoma in vitro and the underlying mechanisms were determined. MGN suppressed the proliferation, induced the autophagy, and stimulated the apoptosis of human hepatoma Huh-7 cells. Mechanically, MGN could regulate PI3K/AKT/mTOR pathway, which therefore affects the progression of hepatoma in vitro. Taken together, MGN affected Huh-7 cell proliferation, autophagy, and apoptosis, and might act as a promising therapeutic drug for treating hepatoma.

Super-resolution image-based tracking of drug distribution in mitochondria of a label-free naturally derived drug molecules

In current practice, drug visualization strategies mainly include 3H-or 14C-modification, or dye-labeling steps. Compared to current drug labeling strategies, drug molecules with autofluorescence can achieve accurate visualization of the drug's subcellular distribution. To this end, we screened various compounds in the traditional Chinese medicine compound library and selected a natural, label-free, fluorescent drug molecule named magnoflorine (MF). MF has fluorescent properties and does not require external intervention by the current labeling strategies, thereby proving to be suitable for reporting its distribution in living cells using structured illumination microscopy (SIM). In addition, using SIM, we found that MF not only had a high quantum yield but could also be well localized to the mitochondria. More importantly, the binding target of MF in mitochondria, namely hypochlorite (ClO-), was also revealed for the first time at the nanoscale visualization level. Finally, we also found that MF can play a role in binding to the ClO- as a target during ferroptosis, hence indicating that MF is a possible intervention drug for this process. In conclusion, we have identified for the first time a new fluorescent molecule, MF, that allows visualizing accurate drug distribution in organelles without additional labeling strategies with SIM. Furthermore, we have discovered the binding target of MF, which is beneficial for understanding of regulatory mechanism of drugs in various diseases.

Magnoflorine Alleviates "M1" Polarized Macrophage-Induced Intervertebral Disc Degeneration Through Repressing the HMGB1/Myd88/NF-κB Pathway and NLRP3 Inflammasome.

Intervertebral disc degeneration (IDD) is related to the deterioration of nucleus pulposus (NP) cells due to hypertrophic differentiation and calcification. The imbalance of pro-inflammatory (M1 type) and anti-inflammatory (M2 type) macrophages contributes to maintaining tissue integrity. Here, we aimed to probe the effect of Magnoflorine (MAG) on NP cell apoptosis mediated by “M1” polarized macrophages. THP-1 cells were treated with lipopolysaccharide (LPS) to induce “M1” polarized macrophages. Under the treatment with increasing concentrations of MAG, the expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, IL-18), high mobility group box protein 1 (HMGB1), as well as myeloid differentiation factor 88 (MyD88), nuclear factor kappa B (NF-κB) and NOD-like receptor 3 (NLRP3) inflammasomes in THP-1 cells were determined. What’s more, human NP cells were treated with the conditioned medium (CM) from THP-1 cells. The NP cell viability and apoptosis were evaluated. Western blot (WB) was adopted to monitor the expression of apoptosis-related proteins (Bax, Caspase3, and Caspase9), catabolic enzymes (MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5), and extracellular matrix (ECM) compositions (collagen II and aggrecan) in NP cells. As a result, LPS evidently promoted the expression of pro-inflammatory cytokines and HMGB1, the MyD88-NF-κB activation, and the NLRP3 inflammasome profile in THP-1 cells, while MAG obviously inhibited the "M1″ polarization of THP-1 cells. After treatment with “M1” polarized THP-1 cell CM, NP cell viability was decreased, while cell apoptosis, the pro-inflammatory cytokines, apoptosis-related proteins, and catabolic enzymes were distinctly up-regulated, and ECM compositions were reduced. After treatment with MAG, NP cell damages were dramatically eased. Furthermore, MAG dampened the HMGB1 expression and inactivated the MyD88/NF-κB pathway and NLRP3 inflammasome in NP cells. In conclusion, this study confirmed that MAG alleviates “M1” polarized macrophage-mediated NP cell damage by inactivating the HMGB1-MyD88-NF-κB pathway and NLRP3 inflammasome, which provides a new reference for IDD treatment.

Magnoflorine Ameliorates Inflammation and Fibrosis in Rats With Diabetic Nephropathy by Mediating the Stability of Lysine-Specific Demethylase 3A.

Diabetic nephropathy (DN) represents one of the most devastating complications for patients with diabetes. The anti-diabetic activities of Magnoflorine (MF) were reported, with underlying mechanism unknown. Lysine-specific demethylase 3A (KDM3A) was identified in the renal injuries. In the current study, we investigated the functional role of MF in DN progression with the involvement of KDM3A. We reported that in the animal model of DN induced by streptozotocin (STZ) injection, MF attenuated inflammatory response and fibrosis in the kidneys. In cultured mesangial cells, MF similarly ameliorated abnormal proliferation and lowered the expression of inflammation- and fibrosis-related factors stimulated by high glucose (HG) treatment. Upon MF treatment, there was a decline in KDM3A-positive cells in renal tissues of rats, accompanying an augment in KDM3A ubiquitination. KDM3A upregulation in vitro by a proteasome inhibitor MG132 comparably dampened the inhibitory role of MF in inflammatory response and fibrosis. Further analyses revealed that MF increased transforming growth factor β-induced factor 1 (TGIF1) transcriptional activity by promoting ubiquitination and degradation of KDM3A, thus inhibiting the activation of TGF-β1/Smad2/3 signaling pathway. TGIF1 silencing weakened the repressive role of MF in mesangial cells as well. In conclusion, MF contributes to TGIF1 transcription via an epigenetic mechanism.

Magnoflorine-Isolation and the Anticancer Potential against NCI-H1299 Lung, MDA-MB-468 Breast, T98G Glioma, and TE671 Rhabdomyosarcoma Cancer Cells.

Magnoflorine (MGN) is a quaternary aporphine alkaloid that exhibits numerous therapeutic properties, including neuropsychopharmacological, anti-anxiety, immunomodulatory, anti-inflammatory, antioxidant, or antifungal activities. The aim of the present study was an investigation of the influence of MGN on viability, proliferation, induction of apoptosis, and cell cycle arrest in NCI-H1299 lung, MDA-MB-468 breast, T98G glioma, and TE671 rhabdomyosarcoma cancer cells. MGN was isolated from the roots of Berberis cretica L. by counter-current partition chromatography (CPC). Cell viability and proliferation assessments were performed by means of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and 5-bromo-2ʹ-deoxyuridine (BrDU) assays, respectively. The induction of apoptosis and cell cycle progression was measured using fluorescence-activated cell sorting analysis. MGN in high doses inhibits proliferation, induces apoptosis, and inhibits cell cycle in S/G2 phases in a dose-dependent manner. MGN seems to be a promising anti-cancer compound in therapy of some types of lung, breast, glioma, and rhabdomyosarcoma cancers, for which current standard therapies are limited or have severe strong side effects.

A rapid method for simultaneous quantification of berberine, berbamine, magnoflorine and berberrubine in mouse serum using UPLC-MS/MS

Berberidis cortex, the dry bark of Berberis L., is used to treat diabetes and contains at least three bioactive components: berberine (BBR), berbamine (BBM) and magnoflorine (MGF). BBR in turn is metabolized into berberrubine (BRB). Although it is possible to quantify each of these components individually in serum, there are currently no methods for simultaneously quantifying all four. Here, we developed a specific and rapid method for simultaneously quantifying BBR, BBM, MGF and BRB in mouse serum using ultra high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Samples were pretreated by protein precipitation, separated using an ACQUITY UPLC® BEH C18 column and detected by a triple quadrupole mass spectrometer with electrospray ionization. The compound [9,10-(OC2H3)2]-BBR (d6-BBR) was used as internal standard for BBR and BRB, boldine (BOL) for MGF and tetrandrine (TET) for BBM. The m/z transitions for precursor/product ion pairs were 336.1/320.2 for BBR, 305.2/566.3 for BBM, 342.0/297.1 for MGF, 322.1/307.2 for BRB, 342.2/294.3 for d6-BBR, 312.2/580.3 for TET and 328.1/265.2 for BOL. We validated our method in terms of selectivity, linearity and lower limit of quantification, accuracy, precision, matrix effect and recovery, dilution integrity and stability. This method showed good linearity from 0.1 to 40 ng/mL for BBR, 8 to 3200 ng/mL for BBM, 5 to 2000 ng/mL for MGF and 0.2 to 80 ng/mL for BRB. The chromatographic run time was 3.9 min, and sample preparation took approximately 15 min per batch. Finally, we used our method to measure BBR, BBM, MGF and BRB in serum from diabetic mice after gavage administration of BBR hydrochloride, BBM hydrochloride, and MGF. This method is precise, accurate and suitable for high-throughput sample analysis.

Variation in the microbial community contributes to the improvement of the main active compounds of Magnolia officinalis Rehd. et Wils in the process of sweating

BackgroundMagnolia officinalis Rehd. et Wils, commonly called Houpo, has been used for thousands of years in China as a traditional herbal medicine. The primary processing of Houpo requires sweating treatment, which is a special drying process and is considered to be an essential embodiment of high quality and genuine medicinal materials. The sweating of Houpo leads to peculiar changes in the microbial community structure and the content of main active substances (magnolol, honokiol, syringin and magnoflorine). Variation in the microbial community was considered the cause of the change in content of active substances of Houpo, although the microbial taxa responsible for the improvement of content remain unidentified.MethodsIn this study, we used MiSeq high-throughput sequencing methods for partial bacterial 16S rRNA and 18S rRNA gene sequences to compare the bacterial and fungal community structures at different timepoints in the process of sweating. The content of the main active substances (magnolol, honokiol, syringin and magnoflorine) were determined by high-performance liquid chromatography analysis to evaluate the effects of sweating. UPLC-Q-Extractive Orbitrap mass spectrometry (UPLC-QE Orbitrap MS) was used to detection of differential metabolites of unsweated Houpo before and after co-culture with core bacterial solutions.ResultsIn this study, the total contents of magnolol (MG) and honokiol (HK) were significantly increased at 4 dp (dp for day PM sample), up to 3.75%, and the contents of syringin (SG) and magnoflorine (MF) were as high as 0.12% and 0.06%, respectively. Bacterial abundance and diversity were higher in the early stage (0 day–2 da; da for day AM sample) than in the later stage (4–5 dp), while fungal abundance was more obvious in the later stage than in the early stage. Positive correlation coefficients revealed that the relative abundance of Enterobacter (P < 0.05), Klebsiella (P < 0.05), Weissella (P < 0.05), Bacillus (P < 0.05) and Candida (P < 0.05) would be conducive to improving the quality of Houpo. Negative correlation coefficients revealed that the relative abundance of Actinomycetospora, Singulisphaera, Mucilaginibacter, Deinococcus, Gemmatirosa, Methylobacterium, Sphingomonas, Hymenobacter, Halomonas and Capnobotryella could be a potential antagonist for the decrease in the quality of Houpo. After co-culture of single core strain and unsweated Houpo, there was no significant difference in the four main active components, but there were other metabolites with significant difference.ConclusionsOur findings reveal that sweating increased the content of the main active compounds, promoted the relative abundance of potentially beneficial microbes, decreased the abundance of potentially harmful microbes, the core functional genera group together, forming a core microbiome, these genera are dominant across the different stages of the sweating process and contribute to the quality development of the characteristics of Houpo. Meanwhile, this study presented a clear scope for potential beneficial microbes that improve the quality of Houpo.

Simultaneous determination of multiple components in rat plasma and pharmacokinetic studies at a pharmacodynamic dose of Xian-Ling-Gu-Bao capsule by UPLC-MS/MS

Xian-Ling-Gu-Bao capsule (XLGB) is an effective traditional Chinese medicine prescription (TCMP) that is used for the prevention and treatment of osteoporosis in China. A rapid, simple, efficient and stable method based on UPLC-MS/MS technology was developed for simultaneous determination of multiple components of XLGB in rat plasma. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode with electrospray ionization (ESI). For twenty-one selected quantitative prototypes, all calibration curves showed favourable linearity (r>0.9932) in linear ranges. The lower limits of quantification (LLOQs) were 2 ng/mL for psoralen (PL), 2.5 ng/mL for asperosaponin VI (AS), 1 ng/mL for isopsoralen (IPS) and sweroside (SW), 0.5 ng/mL for magnoflorine (MA), bavachinin (BVN), tanshinone IIA (TA), timosaponin BII (TBII) and icaritin (ICT), 0.1 ng/mL for epimedin B (EB) and epimedin C (EC), 0.05 ng/mL for icariin (IC), isobavachalcone (IBC), psoralidin (PD), bavachin (BV), bavachalcone (BC), epimedin A (EA) and isobavachin (IBV), 0.02 ng/mL for neobavaisoflavone (NEO) and icariside I (ICI) and 0.01 ng/mL for icariside II (ICII). The intra-day and inter-day (low, medium, high) precision (relative standard deviation) for all analytes was less than 8.63%, and the accuracies (as relative error) were in the range of -12.45% to 8.91%. Extraction recoveries and matrix effects of analytes and IS were acceptable. All analytes were stable during the assay and storage in plasma samples. The validated method was successfully applied to the pharmacokinetics (PK) studies of the twenty-one prototypes at pharmacodynamic doses (0.3 and 1 g/kg/day). In addition, dynamic profiles of 28 metabolites (phase II conjugates: 23 glucuronide conjugates, 2 sulfate conjugates and 3 glucuronide or sulfate conjugates) were also monitored by their area/IS area-time curves. As a result, coumarins, prenylated flavonoids from Psoraleae Fructus, alkaloids and prenylated flavonol glycosides from Epimedii Herba, and iridoid glycosides, triterpenoid saponins from Dipsaci Asperoidis Radix were considered to be the key effective substances of XLGB due to their high exposure and appropriate pharmacokinetic features. This is the first report to reveal pharmacodynamic ingredients by a reversed pharmacodynamic (PD) - pharmacokinetics (PK) study.