Study on quality control methods and pharmacodynamic material basis of different specifications of Corydalis Rhizoma produced in Zhejiang Province.

ETHNOPHARMACOLOGICAL RELEVANCE: The quality control methods of different specifications of Corydalis Rhizoma in Zhejiang China (ZJ CR) are the same, so the quality of each specification couldnot be guaranteed. To clarify the quality control methods and pharmacodynamic material basis of ZJ CR with different specifications could provide reference for the quality control of ZJ CR. AIM OF THE STUDY: The purpose of this study was to establish a quality control method for ZJ CR with different specifications and to screen out the pharmacodynamic material basis of ZJ CR with different specifications. MATERIALS AND METHODS: Firstly, according to the existing grading standards, the medicinal materials were divided into specifications, and the character indexes of ZJ CR with different specifications were established. The quality indexes were established by HPLC, network pharmacology and literature retrieval. The correlation between the trait indexes and quality indexes of ZJ CR with different specifications was analyzed, and the best quality control method was established. Further combined with the pharmacodynamic indexes of ZJ CR with different specifications, the pharmacodynamic material basis of ZJ CR with different specifications was screened out by spectrum-effect analysis. The correlation between trait indexes and pharmacodynamic indexes was analyzed to verify the rationality of grade standard. RESULTS: The three specifications of ZJ CR were CR (Diameter ≥1.1 cm), CR (Diameter <1.1 cm), and CR (No size distinction). Diameter, width, thickness, grain weight, volume and 50 g grain number could be used as the trait indexes of ZJ CR. Protopine (CR1), palmatine hydrochloride (CR2), berberine hydrochloride (CR3), dehydrocorydaline (CR4), tetrahydropalmatine (CR5), tetrahydroberberine (CR6), corydaline (CR7), stylopine (CR8) and isoimperatorin (CR9) were identified. Total components, core components (CR5, CR6, CR7 and CR8), alcohol-soluble extracts (ASE) could be used as quality indexes. The best quality control methods of the three specifications respectively were: the larger the diameter and grain weight, the smaller the number of 50 g grains; The larger the diameter, the smaller the volume, thickness, width and number of 50 g particles; The larger the grain weight and volume, the smaller the number of 50 g grains. The main analgesic components of the three specifications respectively were: CR1, CR2 and core components; CR2, CR4; CR8, CR9. The larger the diameter and the less the number of 50 g grains, the better the analgesic effect of ZJ CR, and the grade standard was reasonable. CONCLUSIONS: This study showed that the quality control methods and pharmacodynamic material basis of ZJ CR with different specifications were different, which may be caused by the differences in traits and the contribution of main active ingredients. This study constructed an evaluation model combining external traits, internal quality and overall efficacy, and provided theoretical support for the rationality of ZJ CR grade standard.

Piperine Enhances Antimalarial Activity of Methyl Gallate and Palmatine Combination.

PURPOSE: Artemisinin combination therapies, the first-line antimalarials in Nigeria, have reportedly suffered multiple failures in malaria treatment, hence the search for novel combination of other compounds. Methyl gallate and palmatine have been reported to exhibit antiplasmodial activities but the antimalarial activity of their combination has not been evaluated. Therefore, the evaluation of the combination of methyl gallate and palmatine for antimalarial activity in vitro and in vivo in the presence of piperine was carried out. MATERIALS AND METHODS: The inhibitory potential of methyl gallate and palmatine combination on ß-hematin (hemozoin) formation was studied in vitro. Also, the antimalarial activity of methyl gallate and palmatine combination with/without a bioenhancer (piperine) was evaluated in Plasmodium berghei NK65-infected mice. RESULTS: Methyl gallate and palmatine in the ratio 3:2 acted synergistically in vitro and had the highest inhibitory effect (IC50 = 0.73 µg/mL) on ß-hematin (hemozoin) formation. The 3:2 combination of methyl gallate and palmatine exhibited no antimalarial activity in vivo in the absence of piperine but caused reduction in parasitemia that exceeded 40% in the presence of piperine at the dose of 25 mg/kg body weight on days 6 and 8 post-inoculation in mice. CONCLUSION: The 3:2 combination of methyl gallate and palmatine in the presence of piperine exhibited antimalarial activity in vivo, possibly by synergistic inhibition of hemozoin formation which may cause accumulation of haem within the food vacuole of Plasmodium spp. and its death.

Metabolomics and transcriptomics reveal the mechanism of alkaloid synthesis in Corydalis yanhusuo bulbs.

Corydalis yanhusuo W.T. Wang is a traditional herb. Benzylisoquinoline alkaloids (BIAs) are the main pharmacological active ingredients that play an important role in sedation, relieving pain, promoting blood circulation, and inhibiting cancer cells. However, there are few studies on the biosynthetic pathway of benzylisoquinoline alkaloids in Corydalis yanhusuo, especially on some specific components, such as tetrahydropalmatine. We carried out widely targeted metabolome and transcriptomic analyses to construct the biosynthetic pathway of benzylisoquinoline alkaloids and identified candidate genes. In this study, 702 metabolites were detected, including 216 alkaloids. Protoberberine-type and aporphine-type alkaloids are the main chemical components in C. yanhusuo bulbs. Key genes for benzylisoquinoline alkaloids biosynthesis, including 6-OMT, CNMT, NMCH, BBE, SOMT1, CFS, SPS, STOX, MSH, TNMT and P6H, were successfully identified. There was no significant difference in the content of benzylisoquinoline alkaloids and the expression level of genes between the two suborgans (mother-bulb and son-bulb). The expression levels of BIA genes in the expansion stage (MB-A and SB-A) were significantly higher than those in the maturity stage (MB-C and SB-C), and the content of benzylisoquinoline alkaloids was consistent with the pattern of gene regulation. Five complete single genes were likely to encode the functional enzyme of CoOMT, which participated in tetrahydropalmatine biosynthesis in C. yanhusuo bulbs. These studies provide a strong theoretical basis for the subsequent development of metabolic engineering of benzylisoquinoline alkaloids (especially tetrahydropalmatine) of C. yanhusuo.

Protopine and Allocryptopine Interactions with Plasma Proteins.

A comprehensive study of the interactions of human serum albumin (HSA) and α-1-acid glycoprotein (AAG) with two isoquinoline alkaloids, i.e., allocryptopine (ACP) and protopine (PP), was performed. The UV-Vis spectroscopy, molecular docking, competitive binding assays, and circular dichroism (CD) spectroscopy were used for the investigations. The results showed that ACP and PP form spontaneous and stable complexes with HSA and AAG, with ACP displaying a stronger affinity towards both proteins. Molecular docking studies revealed the preferential binding of ACP and PP to specific sites within HSA, with site 2 (IIIA) being identified as the favored location for both alkaloids. This was supported by competitive binding assays using markers specific to HSA's drug binding sites. Similarly, for AAG, a decrease in fluorescence intensity upon addition of the alkaloids to AAG/quinaldine red (QR) complexes indicated the replacement of the marker by the alkaloids, with ACP showing a greater extent of replacement than PP. CD spectroscopy showed that the proteins' structures remained largely unchanged, suggesting that the formation of complexes did not significantly perturb the overall spatial configuration of these macromolecules. These findings are crucial for advancing the knowledge on the natural product-protein interactions and the future design of isoquinoline alkaloid-based therapeutics.

Discovery of palmatine derivatives as potent neuroprotective agents.

Alzheimer's disease is a neurodegenerative disorder characterized by the presence of neurodegenerative lesions and cognitive impairment. In this study, a series of novel palmatine derivatives were designed and synthesized through the introduction of a heteroatom using carbodiimide-mediated condensation. The synthesized compounds were then screened for toxicity and potency, leading to the identification of compound 2q, which exhibited low toxicity and high potency. Our findings demonstrated that compound 2q displayed significant neuroprotective activity in vitro, emerging as a promising candidate for Alzheimer's disease treatment.

[Tetrahydropalmatine inhibiting mitophagy through ULK1/FUNDC1 pathway to alleviate hypoxia/reoxygenation injury in H9c2 cells].

This study explored the specific mechanism by which tetrahydropalmatine(THP) inhibited mitophagy through the UNC-51-like kinase 1(ULK1)/FUN14 domain containing 1(FUNDC1) pathway to reduce hypoxia/reoxygenation(H/R) injury in H9c2 cells. This study used H9c2 cells as the research object to construct a cardiomyocyte H/R injury model. First, a cell viability detection kit was used to detect cell viability, and a micro-method was used to detect lactate dehydrogenase(LDH) leakage to evaluate the protective effect of THP on H/R injury of H9c2 cells. In order to evaluate the protective effect of THP on mitochondria, the chemical fluorescence method was used to detect intracellular reactive oxygen species, intramitochondrial reactive oxygen species, mitochondrial membrane potential, and autophagosomes, and the luciferin method was used to detect intracellular adenosine 5'-triphosphate(ATP) content. Western blot was further used to detect the ratio of microtubule-associated protein 1 light chain 3(LC3) membrane type(LC3-Ⅱ) and slurry type(LC3-Ⅰ) and activated cleaved caspase-3 expression level. In addition, ULK1 expression level and its phosphorylation degree at Ser555 site, as well as the FUNDC1 expression level and its phosphorylation degree of Ser17 site were detected to explore its specific mechanism. The results showed that THP effectively reduced mitochondrial damage in H9c2 cells after H/R. THP protected mitochondria by reducing the level of reactive oxygen species in cells and mitochondria, increasing mitochondrial membrane potential, thereby increasing cellular ATP production, enhancing cellular activity, reducing cellular LDH leakage, and finally alleviating H/R damage in H9c2 cells. Further studies have found that THP could reduce the production of autophagosomes, reduce the LC3-Ⅱ/LC3-Ⅰ ratio, and lower the expression of the apoptosis-related protein, namely cleaved caspase-3, indicating that THP could reduce apoptosis by inhibiting autophagy. In-depth studies have found that THP could inhibit the activation of the ULK1/FUNDC1 pathway of mitophagy and the occurrence of mitophagy by reducing the phosphorylation degree of ULK1 at Ser555 and FUNDC1 at Ser17. The application of ULK1 agonist BL-918 reversely verified the effect of THP on reducing the phosphorylation of ULK1 and FUNDC1. In summary, THP inhibited mitophagy through the ULK1/FUNDC1 pathway to reduce H/R injury in H9c2 cells.

Compound-compound interaction analysis of baicalin and berberine derivatives in aqueous solution.

Baicalin and berberine are biologically active constituents of the crude drugs Scutellaria root and Coptis rhizome/Phellodendron bark, respectively. Baicalin and berberine are reported to combine together as a 1:1 complex that forms yellow precipitates by electrostatic interaction in decoctions of Kampo formulae containing these crude drugs. However, the structural basis and mechanism for the precipitate formation of this compound-compound interaction in aqueous solution remains unclarified. Herein, we searched for berberine derivatives in the Coptis rhizome that interact with baicalin and identified the chemical structures involved in the precipitation formation. Precipitation assays showed that baicalin formed precipitates with berberine and coptisine but not with palmatine and epiberberine. Thus, the 2,3-methylenedioxy structure may be crucial to the formation of the precipitates, and electrostatic interaction is necessary but is not sufficient. In this multicomponent system experiment, palmatine formed a dissociable complex with baicalin and may competitively inhibit the formation of berberine and coptisine precipitation with baicalin. Therefore, the precipitation formed by berberine and baicalin was considered to be caused by the aggregation of the berberine-baicalin complex, and the 2,3-methylenedioxy structure is likely crucial to the aggregation of the complex.

Palmatine inhibits expression fat mass and obesity associated protein (FTO) and exhibits a curative effect in dextran sulfate sodium (DSS)-induced experimental colitis.

BACKGROUND: Ulcerative colitis (UC) is an inflammatory disease whose pathogenesis and mechanisms have not been fully described. The m6A methylation modification is a general mRNA modification in mammalian cells and is closely associated with the onset and progression of inflammatory bowel disease (IBD). Palmatine (PAL) is a biologically active alkaloid with anti-inflammatory and protective effects in animal models of colitis. Accordingly, we examined the role of PAL on colitis by regulating N6-methyladenosine (m6A) methylation. METHODS: A rat experimental colitis model was established by 5 % dextran sulfate sodium (DSS) in drinking water for seven days, then PAL treatment was administered for seven days. The colonic tissue pathology was assessed using hematoxylin-eosin (HE) and disease activity index (DAI). In in vitro studies, a human, spontaneously immortalized non-cancerous colon mucosal epithelial cell line (NCM460) was exposed to 2 % DSS and treated with PAL and cell viability was assayed using Cell Counting Kit-8 (CCK-8). The levels of tumor necrosis factor α (TNF-α), interleukin (IL)-1ß, IL-6, and IL-8 were detected by enzyme-linked immunosorbent assay (ELISA) kits. The level of Zonula occludens-1 (ZO-1) was dectected by immunofluorescence. Transepithelial electrical resistance (TEER) of cells was also assessed. The methyltransferase-like 3 (METTL3), METTL14, AlkB homologate 5 (ALKBH5), and fat mass and obesity-associated protein (FTO) expression levels were assessed by western blotting. The localized expression of m6A was measured by immunofluorescence. RESULTS: PAL significantly prevented bodyweight loss and shortening of the colon in experimental colitis rats, as well as decreasing the DAI and histological damage scores. Furthermore, PAL inhibited the levels of inflammatory factors (TNF-α, IL-6, IL-8, and IL-1ß) in both DSS treated rats and NCM460 cells. In addition, PAL enhanced the expression level of ZO-1, and increased the transepithelial electrical resistance to repaire intestinal barrier dysfunction. Colitis occurred due to decreased m6A levels, and the increased FTO expression led to a colitis phenotype. PAL markedly enhanced the METTL3 and METTL14 expression levels while decreasing ALKBH5 and FTO expression levels. CONCLUSIONS: The findings demonstrated that PAL improved DSS-induced experimental colitis. This effect was associated with inhibiting FTO expression and regulating m6A methylation.

Protopine protects chondrocytes from undergoing ferroptosis by activating Nrf2 pathway.

Osteoarthritis is a highly prevalent joint disease; however, effective treatments are lacking. Protopine (PTP) is an isoquinoline alkaloid with potent anti-inflammatory and antioxidant properties; however, it has not been studied in osteoarthritis. This study aimed to investigate whether PTP can effectively protect chondrocytes from ferroptosis. Primary mouse chondrocytes were treated with tert-butyl hydroperoxide (TBHP) to simulate oxidative stress in an in vitro model of osteoarthritis. Two concentrations of PTP (10 and 20 µg/mL) were validated for in vitro experiments. Cellular inflammation and metabolism were detected using RT-qPCR and western blotting (WB). Ferroptosis was assessed via WB, qPCR, reactive oxygen species (ROS) levels, lipid ROS, and immunofluorescence staining. In vitro, PTP significantly ameliorated chondrocyte inflammation and cytolytic metabolism and significantly suppressed chondrocyte ferroptosis through the activation of the Nrf2 pathway. The anterior cruciate ligament transection (ACLT) mouse model was used to validate the in vivo effects of PTP. The joint cartilage was assessed using the Osteoarthritis Research Society International (OARSI) score, Safranin O staining, and immunohistochemistry. The intra-articular administration of PTP alleviated cartilage inflammation and ferroptosis, as evidenced by the expression of MMP3, MMP13, COL2A1, GPX4, and Nrf2. Overall, we find that PTP exerted anti-ferroptosis and anti-inflammatory effects on chondrocytes to protect the articular cartilage.

Thrombosis inhibited by Corydalis decumbens through regulating PI3K-Akt pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Corydalis decumbens (Thunb.) Pers. was used as stasis-eliminating medicine traditionally to treat cardiovascular disease potentially attributed to its antithrombotic effect, but lack of pharmacological research on it. AIM OF THE STUDY: To investigate the antithrombotic effect of C. decumbens and its preliminary mechanism. MATERIALS AND METHODS: A carrageenan-induced mouse thrombus model and adenosine diphosphate stimulated platelet aggregation of rabbits were used to confirm the inhibitory effect of C. decumbens extract and compounds on thrombosis in vivo. Then, H2O2-induced human umbilical vein endothelial cells (HUVECs) injury model was further adopted to verify the effects of bioactive compounds in vitro. Moreover, in silico network pharmacology analyses and molecular docking were performed to predict the underlying mechanisms, targets, and pathways, and which were further confirmed through western blotting assay. RESULTS: The administration of total extract (TE), total alkaloids (TA) and tetrahydropalmatine (TET) resulted in a significant reduction in black tail thrombus and congestion, along with a decreasing in platelet aggregation of rabbits. A superior antithrombotic effect indicated the bioactive fraction, and then the isolated bioactive compounds, TET and protopine (PRO) increased cell survival, and decreased reactive oxygen species (ROS) and lactate dehydrogenase (LDH) release in H2O2-induced HUVECs injury model. Moreover, the two alkaloids targeted 33 major proteins and influenced 153 pathways in network pharmacology prediction. Among these, HSP90AA1, COX-2, NF-κB/p65, MMP1 and HIF-1α were the key proteins and PI3K-Akt emerged as the major signaling pathway. Further western blotting results supported that five key proteins were downregulated by the two bioactive compounds in H2O2-stimulated HUVECs model. CONCLUSION: C. decumbens exerted protective effect on thrombosis through inhibiting PI3K-Akt pathway and related key proteins, which supported the traditional use and presented potential antithrombotic alkaloids for further investigation.

Palmatine improves cognitive dysfunction in Alzheimer's disease model rats through autophagy pathway and regulation of gut microbiota.

Alzheimer's disease (AD) is a primary degenerative encephalopathy that first appeared as a decline in memory and learning skills. Over time, the condition's severity grew. Palmatine (Pal) alleviates Alzheimer's disease symptoms, which has neuroprotective benefits. Numerous investigations have demonstrated a close relationship among AD and gut structure changes. The aim of the research was investigating whether the improvement of Pal on AD is linked to regulating gut flora and autophagy. First, we used Aß1-40 to induce apoptosis in HT22 cells. After Pal treatment, apoptosis can be improved. Then, We used bilateral intracranial hippocampal injection of Aß1-40 for establishing the AD model, after treatment with Pal, the morris water maze experiment and eight-arm maze test demonstrated that Pal enhanced the AD rats' capacity for learning and memory, HE staining illustrated that Pal improved the morphological abnormalities of brain cells and gut tissue damage. Pal reduced the death of hippocampus neurons, as shown by Nissl staining. Pal substantially reduced Tau hyperphosphorylation and Aß accumulation in the brain, according to immunohistochemical labelling. Pal improved the expression of LC3, Beclin 1, AMPK, and suppressed the expression of mTOR and P62, as validated by RT-qPCR and immunofluorescence labelling. This suggests that Pal's treatment of AD may be associated with the control of the AMPK/mTOR autophagy signalling system. 16S rRNA sequencing and short-chain fatty acids (SCFAs) content detection analysis illustrated that Pal has the potential to enhance the content of SCFAs, reverse the alterations in gut microorganisms. It has been showed by the study that Pal could improve AD by activating autophagy signaling pathway and improving gut barrier changes.

Insights into the interaction and inhibitory action of palmatine on lysozyme fibrillogenesis: Spectroscopic and computational studies.

Interaction under amyloidogenic condition between naturally occurring protoberberine alkaloid palmatine and hen egg white lysozyme was executed by adopting spectrofluorometric and theoretical molecular docking and dynamic simulation analysis. In spetrofluorometric method, different types of experiments were performed to explore the overall mode and mechanism of interaction. Intrinsic fluorescence quenching of lysozyme (Trp residues) by palmatine showed effective binding interaction and also yielded different binding parameters like binding constant, quenching constant and number of binding sites. Synchronous fluorescence quenching and 3D fluorescence map revealed that palmatine was able to change the microenvironment of the interacting site. Fluorescence life time measurements strongly suggested that this interaction was basically static in nature. Molecular docking result matched with fluorimetric experimental data. Efficient drug like interaction of palmatine with lysozyme at low pH and high salt concentration prompted us to analyze its antifibrillation potential. Different assays and microscopic techniques were employed for detailed analysis of lysozyme amyloidosis.Thioflavin T(ThT) assay, Congo Red (CR) assay, 8-anilino-1-naphthalenesulfonic acid (ANS) assay, Nile Red (NR) assay, anisotropy and intrinsic fluorescence measurements confirmed that palmatine successfully retarded and reduced lysozyme fibrillation. Dynamic light scattering (DLS) and atomic force microscopy (AFM) further reiterated the excellent antiamyloidogenic potency of palmatine.

Corynoline Suppresses Osteoclastogenesis and Attenuates ROS Activities by Regulating NF-κB/MAPKs and Nrf2 Signaling Pathways.

Declining estrogen production in postmenopausal females causes osteoporosis in which the resorption of bone exceeds the increase in bone formation. Although clinical drugs are currently available for the treatment of osteoporosis, sustained medication use is accompanied by serious side effects. Corydalis bungeana Herba, a famous traditional Chinese herb listed in the Chinese Pharmacopoeia Commission, constitutes various traditional Chinese Medicine prescriptions, which date back to thousands of years. One of the primary active components of C. bungeana Turcz. is Corynoline (Cor), a plant isoquinoline alkaloid derived from the Corydalis species, which possesses bone metabolism disease therapeutic potential. The study aimed at exploring the effects as well as mechanisms of Cor on osteoclast formation and bone resorption. TRAcP staining, F-actin belt formation, and pit formation were employed for assessing the osteoclast function. Western blot, qPCR, network pharmacology, and docking analyses were used for analyzing the expression of osteoclast-associated genes and related signaling pathways. The study focused on investigating how Cor affected OVX-induced trabecular bone loss by using a mouse model. Cor could weaken osteoclast formation and function by affecting the biological receptor activators of NF-κB and its ligand at various concentrations. Mechanistically, Cor inhibited the NF-κB activation, and the MAPKs pathway stimulated by RANKL. Besides, Cor enhanced the protein stability of the Nrf2, which effectively abolished the RANKL-stimulated ROS generation. According to an OVX mouse model, Cor functions in restoring bone mass, improving microarchitecture, and reducing the ROS levels in the distal femurs, which corroborated with its in vitro antiosteoclastogenic effect. The present study indicates that Cor may restrain osteoclast formation and bone loss by modulating NF-κB/MAPKs and Nrf2 signaling pathways. Cor was shown to be a potential drug candidate that can be utilized for the treatment of osteoporosis.

Antifungal Activity and Putative Mechanism of HWY-289, a Semisynthetic Protoberberine Derivative, against Botrytis cinerea.

The emergence of resistant pathogens has increased the demand for alternative fungicides. The use of natural products as chemical scaffolds is a potential method for developing fungicides. HWY-289, a semisynthetic protoberberine derivative, demonstrated broad-spectrum and potent activities against phytopathogenic fungi, particularly Botrytis cinerea (with EC50 values of 1.34 µg/mL). SEM and TEM imaging indicated that HWY-289 altered the morphology of the mycelium and the internal structure of cells. Transcriptomics revealed that it could break down cellular walls through amino acid sugar and nucleotide sugar metabolism. In addition, it substantially decreased chitinase activity and chitin synthase gene (BcCHSV) expression by 53.03 and 82.18% at 1.5 µg/mL, respectively. Moreover, this impacted the permeability and integrity of cell membranes. Finally, HWY-289 also hindered energy metabolism, resulting in a significant reduction of ATP content, ATPase activities, and key enzyme activities in the TCA cycle. Therefore, HWY-289 may be a potential candidate for the development of plant fungicides.

Programming the Assembly of Oligo-Adenine with Coralyne into a pH-Responsive DNA Hydrogel.

External stimuli-responsive DNA hydrogels present interesting platforms for drug loading and triggered release. Typically, drug molecules are encapsulated within three-dimensionally hybridized DNA networks. However, the utilization of drug molecules as cofactors to facilitate the directed assembly of DNA strands into hydrogel frameworks and their subsequent controlled release remains to be explored. Herein, we introduce the guided assembly of oligo-adenine (A-strand) into an acidic pH-responsive DNA hydrogel using an anticancer drug, coralyne (COR), as a low-molecular-weight cofactor. At pH 7, COR orchestrates the assembly of A-strand into an antiparallel duplex configuration cross-linked by A-COR-A units at a stoichiometric ratio of one COR cofactor per four adenine bases, resulting in a DNA hydrogel characterized by A-COR-A duplex bridges. At pH 4-5, the instability of A-COR-A units results in the disintegration of the duplex into its constituent components, leading to the release of COR and simultaneous dissociation of the DNA hydrogel matrix. This study introduces a method by which drug molecules, exemplified here by COR, facilitate the direct formation of a supramolecular cofactor-DNA complex, subsequently leading to the creation of a stimuli-responsive DNA hydrogel. This approach may inspire future investigations into DNA hydrogels tailored for controlled drug encapsulation and release applications.

A comprehensive assessment of palmatine as anticonvulsant agent - In vivo and in silico studies.

Previously, we demonstrated that palmatine (PALM) - an isoquinoline alkaloid from Berberis sibrica radix, exerted antiseizure activity in the pentylenetetrazole (PTZ)-induced seizure assay in larval zebrafish. The aim of the present study was to more precisely characterize PALM as a potential anticonvulsant drug candidate. A range of zebrafish and mouse seizure/epilepsy models were applied in the investigation. Immunostaining analysis was conducted to assess the changes in mouse brains, while in silico molecular modelling was performed to determine potential targets for PALM. Accordingly, PALM had anticonvulsant effect in ethyl 2-ketopent-4-enoate (EKP)-induced seizure assay in zebrafish larvae as well as in the 6 Hz-induced psychomotor seizure threshold and timed infusion PTZ tests in mice. The protective effect in the EKP-induced seizure assay was confirmed in the local field potential recordings. PALM did not affect seizures in the gabra1a knockout line of zebrafish larvae. In the scn1Lab-/- zebrafish line, pretreatment with PALM potentiated seizure-like behaviour of larvae. Repetitive treatment with PALM, however, did not reduce development of PTZ-induced seizure activity nor prevent the loss of parvalbumin-interneurons in the hippocampus of the PTZ kindled mice. In silico molecular modelling revealed that the noted anticonvulsant effect of PALM in EKP-induced seizure assay might result from its interactions with glutamic acid decarboxylase and/or via AMPA receptor non-competitive antagonism. Our study has demonstrated the anticonvulsant activity of PALM in some experimental models of seizures, including a model of pharmacoresistant seizures induced by EKP. These results indicate that PALM might be a suitable new drug candidate but the precise mechanism of its anticonvulsant activity has to be determined.

Palmatine alleviates cardiac fibrosis by inhibiting fibroblast activation through the STAT3 pathway.

Cardiac fibrosis, the hallmark of cardiovascular disease, is characterized by excessive deposition of extracellular matrix in the heart. Emerging evidence indicates that cardiac fibroblasts (CFs) play pivotal roles in driving cardiac fibrosis. However, due to incomplete insights into CFs, there are limited effective approaches to prevent or reverse cardiac fibrosis currently. Palmatine, a protoberberine alkaloid extracted from traditional Chinese botanical remedies, possesses diverse biological effects. This study investigated the potential therapeutic value and mechanism of palmatine against cardiac fibrosis. Adult male C57BL/6 mice were treated with vehicle, isoproterenol (ISO), or ISO plus palmatine for one week. After echocardiography assessment, mice hearts were collected for histopathology, real-time polymerase chain reaction, and Western blot analyses. Primary rat CFs were utilized in vitro. Compared to control, ISO-treated mice exhibited cardiac hypertrophy and structural abnormalities; however, treatment with palmatine ameliorated these effects of ISO. Moreover, palmatine treatment mitigated ISO-induced cardiac fibrosis. Network pharmacology and molecular docking analysis showed that palmatine strongly binds the regulators of cardiac fibrosis including signal transducer and activator of transcription 3 (STAT3) and mammalian target of rapamycin. Furthermore, palmatine reduced the elevated fibrotic factor expressions and overactivated STAT3 induced by ISO, Transformed growth factor ß1 (TGF-ß1), or interleukin-6 both in vivo and in vitro. Additionally, blocking STAT3 suppressed the TGF-ß1-induced CF activation. Collectively, these data demonstrated that palmatine attenuated cardiac fibrosis partly by inhibiting fibroblast activation through the STAT3 pathway. This provides an experimental basis for the clinical treatment of cardiac fibrosis with palmatine.

Protopine ameliorates OVA-induced asthma through modulatingTLR4/MyD88/NF-κB pathway and NLRP3 inflammasome-mediated pyroptosis.

BACKGROUND: Chronic airway inflammation and hyperresponsiveness are characteristics of asthma. The isoquinoline alkaloid protopine (PRO) has been shown to exert anti-inflammatory effects, but its mechanism of action in asthma is not known. PURPOSE: Investigate the protective properties of PRO upon asthma and elucidate its mechanism. STUDY DESIGN AND METHODS: The effects of PRO in asthma treatment were assessed by histology, biochemical analysis, and real-time reverse transcription-quantitative polymerase chain reaction. Then, we integrated molecular docking, western blotting, cellular experiments, immunohistochemistry, immunofluorescence analysis, flow cytometry, and metabolomics analysis to reveal its mechanism. RESULTS: In vivo, PRO therapy reduced the number of inflammatory cells (eosinophils, leukocytes, monocytes) in bronchoalveolar lavage fluid (BALF), ameliorated pathologic alterations in lung tissues, and inhibited secretion of IgG and histamine. Molecular docking showed that PRO could dock with the proteins of TLR4, MyD88, TRAF6, TAK1, IKKα, and TNF-α. Western blotting displayed that PRO inhibited the TLR4/NF-κB signaling pathway. PRO regulated expression of the pyroptosis-related proteins NLR family pyrin domain containing 3 (NLRP3) inflammasome, gasdermin D, caspase-1, and drove caspase-1 inactivation to affect inflammatory responses by inhibiting the NLRP3 inflammasome. In vitro, 24 h after treatment with PRO, cell activity, as well as levels of reactive oxygen species (ROS) and interleukin (IL)-1ß and IL-18, decreased significantly. Immunofluorescence staining showed that PRO decreased expression of TLR4 and MyD88 in vitro. PRO decreased nuclear translocation of NF-κB p65. Twenty-one potential biomarkers in serum were identified using metabolomics analysis, and they predominantly controlled the metabolism of phenylalanine, tryptophan, glucose, and sphingolipids. CONCLUSION: PRO reduced OVA-induced asthma. The underlying mechanism was associated with the TLR4/MyD88/NF-κB pathway and NLRP3 inflammasome-mediated pyroptosis.

Protoberberine alkaloids: A review of the gastroprotective effects, pharmacokinetics, and toxicity.

BACKGROUND: Stomach diseases have become global health concerns. Protoberberine alkaloids (PBAs) are a group of quaternary isoquinoline alkaloids from abundant natural sources and have been shown to improve gastric disorders in preclinical and clinical studies. The finding that PBAs exhibit low oral bioavailability but potent pharmacological activity has attracted great interest. PURPOSE: This review aims to provide a systematic review of the molecular mechanisms of PBAs in the treatment of gastric disorders and to discuss the current understanding of the pharmacokinetics and toxicity of PBAs. METHODS: The articles related to PBAs were collected from the Web of Science, Pubmed, and China National Knowledge Infrastructure databases using relevant keywords. The collected articles were screened and categorized according to their research content to focus on the gastroprotective effects, pharmacokinetics, and toxicity of PBAs. RESULTS: Based on the results of preclinical studies, PBAs have demonstrated therapeutic effects on chronic atrophic gastritis and gastric cancer by activating interleukin-4 (IL-4)/signal transducer and activator of transcription 6 (STAT6) pathway and suppressing transforming growth factor-beta 1 (TGF-ß1)/phosphoinositide 3-kinase (PI3K), Janus kinase-2 (JAK2)/signal transducers and activators of transcription 3 (STAT3), and mitogen-activated protein kinase (MAPK) pathways. The major PBAs exhibit similar pharmacokinetic properties, including rapid absorption, slow elimination, and low bioavailability. Notably, the natural organ-targeting property of PBAs may account for the finding of their low blood levels and high pharmacological activity. PBAs interact with other compounds, including conventional drugs and natural products, by modulation of metabolic enzymes and transporters. The potential tissue toxicity of PBAs should be emphasized due to their high tissue accumulation. CONCLUSION: This review highlights the gastroprotective effects, pharmacokinetics, and toxicity of PBAs and will contribute to the evaluation of drug properties and clinical translational studies of PBAs, accelerating their transfer from the laboratory to the bedside.

Tetrahydropalmatine promotes random skin flap survival in rats via the PI3K/AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Flap necrosis is the most common complication after flap transplantation, but its prevention remains challenging. Tetrahydropalmatine (THP) is the main bioactive component of the traditional Chinese medicine Corydalis yanhusuo, with effects that include the activation of blood circulation, the promotion of qi, and pain relief. Although THP is widely used to treat various pain conditions, its impact on flap survival is unknown. AIM OF THE STUDY: To explore the effect and mechanism of THP on skin flap survival. MATERIALS AND METHODS: In this study, we established a modified McFarlane flap model, and the flap survival rate was calculated after 7 days of THP treatment. Angiogenesis and blood perfusion were evaluated using lead oxide/gelatin angiography and laser Doppler, respectively. Flap tissue obtained from zone II was evaluated histopathologically, by hematoxylin and eosin staining, and in assays for malondialdehyde content and superoxide dismutase activity. Immunofluorescence was performed to detect interleukin (IL)-6, tumor necrosis factor (TNF)-α, hypoxia-inducible factor (HIF)-1α, Bcl-2, Bax, caspase-3, caspase-9, SQSTM1/P62, Beclin-1, and LC3 expression, and Western blot to assess PI3K/AKT signaling pathway activation and Vascular endothelial growth factor (VEGF) expression. The role played by the autophagy pathway in flap necrosis was examined using rapamycin, a specific inhibitor of mTOR. RESULTS: Experimentally, THP improved the survival rate of skin flaps, promoted angiogenesis, and improved blood perfusion. THP administration reduced the inflammatory response, oxidative stress, and apoptosis in addition to inhibiting autophagy via the PI3K/AKT/mTOR pathway. Rapamycin partially reversed these effects. CONCLUSION: THP promotes skin flap survival via the PI3K/AKT signaling pathway.