[Mechanism of astragaloside â £ combined with Panax notoginseng saponins in regulating angiogenesis to treat cerebral ischemia based on network pharmacology and experimental verification].

Network pharmacology and animal and cell experiments were employed to explore the mechanism of astragaloside Ⅳ(AST Ⅳ) combined with Panax notoginseng saponins(PNS) in regulating angiogenesis to treat cerebral ischemia. The method of network pharmacology was used to predict the possible mechanisms of AST Ⅳ and PNS in treating cerebral ischemia by mediating angiogenesis. In vivo experiment: SD rats were randomized into sham, model, and AST Ⅳ(10 mg·kg~(-1)) + PNS(25 mg·kg~(-1)) groups, and the model of cerebral ischemia was established with middle cerebral artery occlusion(MCAO) method. AST Ⅳ and PNS were administered by gavage twice a day. the Longa method was employed to measure the neurological deficits. The brain tissue was stained with hematoxylin-eosin(HE) to reveal the pathological damage. Immunohistochemical assay was employed to measure the expression of von Willebrand factor(vWF), and immunofluorescence assay to measure the expression of vascular endothelial growth factor A(VEGFA). Western blot was employed to determine the protein levels of vascular endothelial growth factor receptor 2(VEGFR2), VEGFA, phosphorylated phosphatidylinositol 3-kinase(p-PI3K), and phosphorylated protein kinase B(p-AKT) in the brain tissue. In vitro experiment: the primary generation of rat brain microvascular endothelial cells(rBEMCs) was cultured and identified. The third-generation rBMECs were assigned into control, model, AST Ⅳ(50 µmol·L~(-1)) + PNS(30 µmol·L~(-1)), LY294002(PI3K/AKT signaling pathway inhibitor), 740Y-P(PI3K/AKT signaling pathway agonist), AST Ⅳ + PNS + LY294002, and AST Ⅳ + PNS + 740Y-P groups. Oxygen glucose deprivation/re-oxygenation(OGD/R) was employed to establish the cell model of cerebral ischemia-reperfusion injury. The cell counting kit-8(CCK-8) and scratch assay were employed to examine the survival and migration of rBEMCs, respectively. Matrigel was used to evaluate the tube formation from rBEMCs. The Transwell assay was employed to examine endothelial cell permeability. Western blot was employed to determine the expression of VEGFR2, VEGFA, p-PI3K, and p-AKT in rBEMCs. The results of network pharmacology analysis showed that AST Ⅳ and PNS regulated 21 targets including VEGFA and AKT1 of angiogenesis in cerebral infarction. Most of these 21 targets were involved in the PI3K/AKT signaling pathway. The in vivo experiments showed that compared with the model group, AST Ⅳ + PNS reduced the neurological deficit score(P<0.05) and the cell damage rate in the brain tissue(P<0.05), promoted the expression of vWF and VEGFA(P<0.01) and angiogenesis, and up-regulated the expression of proteins in the PI3K/AKT pathway(P<0.05, P<0.01). The in vitro experiments showed that compared with the model group, the AST Ⅳ + PNS, 740Y-P, AST Ⅳ + PNS + LY294002, and AST Ⅳ + PNS + 740Y-P improved the survival of rBEMCs after OGD/R, enhanced the migration of rBEMCs, increased the tubes formed by rBEMCs, up-regulated the expression of proteins in the PI3K/AKT pathway, and reduced endothelial cell permeability(P<0.05, P<0.01). Compared with the LY294002 group, the AST Ⅳ + PNS + LY294002 group showed increased survival rate, migration rate, and number of tubes, up-regulated expression of proteins in the PI3K/AKT pathway, and decreased endothelial cell permeability(P<0.05,P<0.01). Compared with the AST Ⅳ + PNS and 740Y-P groups, the AST Ⅳ + PNS + 740Y-P group presented increased survival rate, migration rate, and number of tubes and up-regulated expression of proteins in the PI3K/AKT pathway, and reduced endothelial cell permeability(P<0.01). This study indicates that AST Ⅳ and PNS can promote angiogenesis after cerebral ischemia by activating the PI3K/AKT signaling pathway.

Astragaloside IV-PESV inhibits prostate cancer tumor growth by restoring gut microbiota and microbial metabolic homeostasis via the AGE-RAGE pathway.

BACKGROUND: Prostate cancer (PCa) is becoming the most common malignancy in men worldwide. We investigated the effect of astragaloside IV combined with PESV on the gut microbiota and metabolite of PCa mice and the process of treating PCa. METHODS: Nude mice were genetically modified to develop tumors characteristic of PCa. The treatment of PCa mice involved the administration of a combination of astragaloside IV and peptides derived from scorpion venom (PESV). Feces were collected for both 16 S rDNA and metabolic analysis. Fecal supernatant was extracted and used for fecal transplantation in PCa mice. Tumor development was observed in both PCa mice and nude mice. Tumor histopathology was examined, and the expression of inflammatory factors and the AGE-RAGE axis in PCa tissues were analyzed. RESULTS: PCa mice treated with Astragaloside IV in combination with PESV showed a significant reduction in tumor volume and weight, and stabilization of gut microbiota and metabolites. At the Genus level, significant differences were observed in Porphyromonas, Corynebacterium, Arthromitus and Blautia, and the differential metabolites were PA16_016_0, Astragaloside+, Vitamin A acid, Nardosinone, a-Nortestoster, D-Pantethine, Hypoxanthine, Pregnenolone, cinnamic acid, Pyridoxa, Cirtruline and Xanthurenate. There was a correlation between gut microbiota and metabolites. After the fecal transplantation, tumor growth was effectively suppressed in the PCa mice. Notably, both the mRNA and protein levels of the receptor for advanced glycation end products (RAGE) were significantly decreased. Furthermore, the expression of inflammatory factors, namely NF-κB, TNF-α, and IL-6, in the tumor tissues was significantly attenuated. Conversely, upregulation of RAGE led to increased inflammation and reversed tumor growth in the mice. CONCLUSION: Astragaloside IV combined with PESV could treat PCa by intervening in gut microbiota composition and metabolite by targeting RAGE.

Alleviatory Role of Panax Notoginseng Saponins in Modulating Inflammation and Pulmonary Vascular Remodeling in Chronic Obstructive Pulmonary Disease: mechanisms and Implications.

COPD is an inflammatory lung disease that limits airflow and remodels the pulmonary vascular system. This study delves into the therapeutic potential and mechanistic underpinnings of Panax notoginseng Saponins (PNS) in alleviating inflammation and pulmonary vascular remodeling in a COPD rat model. Symmap and ETCM databases provided Panax notoginseng-related target genes, and the CTD and DisGeNET databases provided COPD-related genes. Intersection genes were subjected to protein-protein interaction analysis and pathway enrichment to identify downstream pathways. A COPD rat model was established, with groups receiving varying doses of PNS and a Roxithromycin control. The pathological changes in lung tissue and vasculature were examined using histological staining, while molecular alterations were explored through ELISA, RT-PCR, and Western blot. Network pharmacology research suggested PNS may affect the TLR4/NF-κB pathway linked to COPD development. The study revealed that, in contrast to the control group, the COPD model exhibited a significant increase in inflammatory markers and pathway components such as TLR4, NF-κB, HIF-1α, VEGF, ICAM-1, SELE mRNA, and serum TNF-α, IL-8, and IL-1ß. Treatment with PNS notably decreased these markers and mitigated inflammation around the bronchi and vessels. Taken together, the study underscores the potential of PNS in reducing lung inflammation and vascular remodeling in COPD rats, primarily via modulation of the TLR4/NF-κB/HIF-1α/VEGF pathway. This research offers valuable insights for developing new therapeutic strategies for managing and preventing COPD.

Studies Related to the Involvement of EsA in Improving Intestinal Inflammation in Acute Pancreatitis via the NF-κB Pathway.

Background: Acute pancreatitis (AP) is a clinically frequent acute abdominal condition, which refers to an inflammatory response syndrome of edema, bleeding, and even necrosis caused by abnormal activation of the pancreas's own digestive enzymes. Intestinal damage can occur early in the course of AP and is manifested by impaired intestinal mucosal barrier function, and inflammatory reactions of the intestinal mucosa, among other factors. It can cause translocation of intestinal bacteria and endotoxins, further aggravating the condition of AP. Therefore, actively protecting the intestinal mucosal barrier, controlling the progression of intestinal inflammation, and improving intestinal dynamics in the early stages of AP play an important role in enhancing the prognosis of AP. Methods: The viability and apoptosis of RAW264.7 cells treated with Esculentoside A (EsA) and/or lipopolysaccharide were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry, respectively. The expression of apoptosis-related proteins and NF-κB signaling pathway-related proteins were detected by western blot (WB). An enzyme-linked immunosorbent assay was used to measure TNF-α and IL-6 secretion. Results: In vitro experiments demonstrated that EsA not only promoted the apoptosis of inflammatory cells but also reduced the secretion of TNF-α and IL-6 in a dose-dependent manner. Additionally, it inhibited the activation of the NF-κB signaling pathway by decreasing the expression of phosphorylated-p65(p-p65) and elevating the expression of IκBα. Similarly, in vivo experiments using a rat AP model showed that EsA inhibited the expression of p-p65 elevating the expression of IκBα in the intestinal tissues of the rat AP model and promoting the apoptosis of inflammatory cells in the intestinal mucosa in vivo experiments, while improving the pathological outcome of the pancreatic and intestinal tissues. Conclusion: Our results suggest that EsA can reduce intestinal inflammation in the rat AP model and that EsA may be a candidate for treating intestinal inflammation in AP and further arresting AP progression.

Panax notoginseng saponins prevent dementia and oxidative stress in brains of SAMP8 mice by enhancing mitophagy.

BACKGROUND: Mitochondrial dysfunction is one of the distinctive features of neurons in patients with Alzheimer's disease (AD). Intraneuronal autophagosomes selectively phagocytose and degrade the damaged mitochondria, mitigating neuronal damage in AD. Panax notoginseng saponins (PNS) can effectively reduce oxidative stress and mitochondrial damage in the brain of animals with AD, but their exact mechanism of action is unknown. METHODS: Senescence-accelerated mouse prone 8 (SAMP8) mice with age-related AD were treated with PNS for 8 weeks. The effects of PNS on learning and memory abilities, cerebral oxidative stress status, and hippocampus ultrastructure of mice were observed. Moreover, changes of the PTEN-induced putative kinase 1 (PINK1)-Parkin, which regulates ubiquitin-dependent mitophagy, and the recruit of downstream autophagy receptors were investigated. RESULTS: PNS attenuated cognitive dysfunction in SAMP8 mice in the Morris water maze test. PNS also enhanced glutathione peroxidase and superoxide dismutase activities, and increased glutathione levels by 25.92% and 45.55% while inhibiting 8-hydroxydeoxyguanosine by 27.74% and the malondialdehyde production by 34.02% in the brains of SAMP8 mice. Our observation revealed the promotion of mitophagy, which was accompanied by an increase in microtubule-associated protein 1 light chain 3 (LC3) mRNA and 70.00% increase of LC3-II/I protein ratio in the brain tissues of PNS-treated mice. PNS treatment increased Parkin mRNA and protein expression by 62.80% and 43.80%, while increasing the mRNA transcription and protein expression of mitophagic receptors such as optineurin, and nuclear dot protein 52. CONCLUSION: PNS enhanced the PINK1/Parkin pathway and facilitated mitophagy in the hippocampus, thereby preventing cerebral oxidative stress in SAMP8 mice. This may be a mechanism contributing to the cognition-improvement effect of PNS.

Response Surface Optimization for Water-Assisted Extraction of Two Saponins from Paris polyphylla var. yunnanensis Leaves.

The process of extracting polyphyllin II and polyphyllin VII by water-assisted extraction was established and optimized in this study. Response surface methodology was used to establish a prediction model to optimize the extraction conditions. Based on the one-way test, the Box-Behnken design with three factors and three levels was used for the experimental program, and the composition analysis was carried out by high-performance liquid chromatography (HPLC). The optimal extraction conditions for polyphyllin II and polyphyllin VII were as follows: extraction time of 57 and 21 min, extraction temperature of 36 and 32 °C, solid-to-liquid ratio of 1:10 and 1:5 g/mL, respectively, and the yields of polyphyllin II and polyphyllin VII were 1.895 and 5.010%, which was similar to the predicted value of 1.835 and 4.979%. The results of the ANOVA showed that the model fit was good, and the Box-Behnken response surface method could optimize the water-assisted extraction of saponins from the leaves of Paris polyphylla var. yunnanensis. This study provides a theoretical basis for the application of polyphyllin II and polyphyllin VII in pharmaceutical production.

Anti-Biofilm Activity of Assamsaponin A, Theasaponin E1, and Theasaponin E2 against Candida albicans.

Biofilm formation plays a crucial role in the pathogenesis of Candida albicans and is significantly associated with resistance to antifungal agents. Tea seed saponins, a class of non-ionic triterpenes, have been proven to have fungicidal effects on planktonic C. albicans. However, their anti-biofilm activity and mechanism of action against C. albicans remain unclear. In this study, the effects of three Camellia sinensis seed saponin monomers, namely, theasaponin E1 (TE1), theasaponin E2 (TE2), and assamsaponin A (ASA), on the metabolism, biofilm development, and expression of the virulence genes of C. albicans were evaluated. The results of the XTT reduction assay and crystal violet (CV) staining assay demonstrated that tea seed saponin monomers concentration-dependently suppressed the adhesion and biofilm formation of C. albicans and were able to eradicate mature biofilms. The compounds were in the following order in terms of their inhibitory effects: ASA > TE1 > TE2. The mechanisms were associated with reductions in multiple crucial virulence factors, including cell surface hydrophobicity (CSH), adhesion ability, hyphal morphology conversion, and phospholipase activity. It was further demonstrated through qRT-PCR analysis that the anti-biofilm activity of ASA and TE1 against C. albicans was attributed to the inhibition of RAS1 activation, which consequently suppressed the cAMP-PKA and MAPK signaling pathways. Conversely, TE2 appeared to regulate the morphological turnover and hyphal growth of C. albicans via a pathway that was independent of RAS1. These findings suggest that tea seed saponin monomers are promising innovative agents against C. albicans.

Astragaloside IV as a Memory-Enhancing Agent: In Silico Studies with In Vivo Analysis and Post Mortem ADME-Tox Profiling in Mice.

Many people around the world suffer from neurodegenerative diseases associated with cognitive impairment. As life expectancy increases, this number is steadily rising. Therefore, it is extremely important to search for new treatment strategies and to discover new substances with potential neuroprotective and/or cognition-enhancing effects. This study focuses on investigating the potential of astragaloside IV (AIV), a triterpenoid saponin with proven acetylcholinesterase (AChE)-inhibiting activity naturally occurring in the root of Astragalus mongholicus, to attenuate memory impairment. Scopolamine (SCOP), an antagonist of muscarinic cholinergic receptors, and lipopolysaccharide (LPS), a trigger of neuroinflammation, were used to impair memory processes in the passive avoidance (PA) test in mice. This memory impairment in SCOP-treated mice was attenuated by prior intraperitoneal (ip) administration of AIV at a dose of 25 mg/kg. The attenuation of memory impairment by LPS was not observed. It can therefore be assumed that AIV does not reverse memory impairment by anti-inflammatory mechanisms, although this needs to be further verified. All doses of AIV tested did not affect baseline locomotor activity in mice. In the post mortem analysis by mass spectrometry of the body tissue of the mice, the highest content of AIV was found in the kidneys, then in the spleen and liver, and the lowest in the brain.

Functional Identification of HhUGT74AG11-A Key Glycosyltransferase Involved in Biosynthesis of Oleanane-Type Saponins in Hedera helix.

Hedera helix is a traditional medicinal plant. Its primary active ingredients are oleanane-type saponins, which have extensive pharmacological effects such as gastric mucosal protection, autophagy regulation actions, and antiviral properties. However, the glycosylation-modifying enzymes responsible for catalyzing oleanane-type saponin biosynthesis remain unidentified. Through transcriptome, cluster analysis, and PSPG structural domain, this study preliminarily screened four candidate UDP-glycosyltransferases (UGTs), including Unigene26859, Unigene31717, CL11391.Contig2, and CL144.Contig9. In in vitro enzymatic reactions, it has been observed that Unigene26859 (HhUGT74AG11) has the ability to facilitate the conversion of oleanolic acid, resulting in the production of oleanolic acid 28-O-glucopyranosyl ester. Moreover, HhUGT74AG11 exhibits extensive substrate hybridity and specific stereoselectivity and can transfer glycosyl donors to the C-28 site of various oleanane-type triterpenoids (hederagenin and calenduloside E) and the C-7 site of flavonoids (tectorigenin). Cluster analysis found that HhUGT74AG11 is clustered together with functionally identified genes AeUGT74AG6, CaUGT74AG2, and PgUGT74AE2, further verifying the possible reason for HhUGT74AG11 catalyzing substrate generalization. In this study, a novel glycosyltransferase, HhUGT74AG11, was characterized that plays a role in oleanane-type saponins biosynthesis in H. helix, providing a theoretical basis for the production of rare and valuable triterpenoid saponins.

The Effect of Composition on the Properties and Application of CuO-NiO Nanocomposites Synthesized Using a Saponin-Green/Microwave-Assisted Hydrothermal Method.

In this study, we explored the formation of CuO nanoparticles, NiO nanoflakes, and CuO-NiO nanocomposites using saponin extract and a microwave-assisted hydrothermal method. Five green synthetic samples were prepared using aqueous saponin extract and a microwave-assisted hydrothermal procedure at 200 °C for 30 min. The samples were pristine copper oxide (100C), 75% copper oxide-25% nickel oxide (75C25N), 50% copper oxide-50% nickel oxide (50C50N), 25% copper oxide-75% nickel oxide (25C75N), and pristine nickel oxide (100N). The samples were characterized using FT-IR, XRD, XPS, SEM, and TEM. The XRD results showed that copper oxide and nickel oxide formed monoclinic and cubic phases, respectively. The morphology of the samples was useful and consisted of copper oxide nanoparticles and nickel oxide nanoflakes. XPS confirmed the +2 oxidation state of both the copper and nickel ions. Moreover, the optical bandgaps of copper oxide and nickel oxide were determined to be in the range of 1.29-1.6 eV and 3.36-3.63 eV, respectively, and the magnetic property studies showed that the synthesized samples exhibited ferromagnetic and superparamagnetic properties. In addition, the catalytic activity was tested against para-nitrophenol, demonstrating that the catalyst efficiency gradually improved in the presence of CuO. The highest rate constants were obtained for the 100C and 75C25N samples, with catalytic efficiencies of 98.7% and 78.2%, respectively, after 45 min.

[Saikosaponin a alleviates pentylenetetrazol-induced acute epileptic seizures in mouse models of depression by suppressing microglia activation-mediated inflammation].

OBJECTIVE: To explore the inhibitory effect of saikosonin a (SSa) on pentylenetetrazol-induced acute epilepsy seizures in a mouse model of depression and explore the mechanism mediating this effect. METHODS: Male C57BL/6J mouse models of depression was established by oral administration of corticosterone via drinking water for 3 weeks, and acute epileptic seizures were induced by intraperitoneal injection of a single dose of pentylenetetrazole. The effect of intraperitoneal injection of SSa prior to the treatment on depressive symptoms and epileptic seizures were assessed using behavioral tests, epileptic seizure grading and hippocampal morphology observation. ELISA was used to detect blood corticosterone levels of the mice, and RTqPCR was performed to detect the pro- and anti-inflammatory factors. Microglia activation in the mice was observed using immunofluorescence staining. RESULTS: The mouse model of corticosterone-induced depression showed body weight loss and obvious depressive behaviors with significantly increased serum corticosterone level (all P < 0.05). Compared with those with pentylenetetrazole-induced epilepsy alone, the epileptic mice with comorbid depression showed significantly shorter latency of epileptic seizures, increased number, grade and duration of of seizures, reduced Nissl bodies in hippocampal CA1 and CA3 neurons, increased number of Iba1-positive cells, and significantly enhanced hippocampal expressions of IL-1ß, IL-10, TNF-α and IFN-γ. Pretreatment of the epileptic mice with SSa significantly prolonged the latency of epileptic seizures, reduced the number, duration, and severity of seizures, increased the number of Nissl bodies, decreased the number of Iba1-positive cells, and reduced the expression levels of IL-1ß, IL-10, TNF-α, and IFN-γ in the hippocampus (P < 0.05). CONCLUSION: Depressive state aggravates epileptic seizures, increases microglia activation, and elevates inflammation levels. SSA treatment can alleviate acute epileptic seizures in mouse models of depression possibly by suppressing microglia activation-mediated inflammation.

Astragaloside IV combined with ligustrazine ameliorates abnormal mitochondrial dynamics via Drp1 SUMO/deSUMOylation in cerebral ischemia-reperfusion injury.

OBJECTIVES: Astragaloside IV (AST IV) and ligustrazine (Lig), the main ingredients of Astragali Radix and Chuanxiong Rhizoma respectively, have demonstrated significant benefits in treatment of cerebral ischemia -reperfusion injury (CIRI); however, the mechanisms underlying its benificial effects remain unclear. SUMO-1ylation and deSUMO-2/3ylation of dynamin-related protein 1 (Drp1) results in mitochondrial homeostasis imbalance following CIRI, which subsequently aggravates cell damage. This study investigates the mechanisms by which AST IV combined with Lig protects against CIRI, focusing on the involvement of SUMOylation in mitochondrial dynamics. METHODS: Rats were administrated AST IV and Lig for 7 days, and middle cerebral artery occlusion was established to mimic CIRI. Neural function, cerebral infarction volume, cerebral blood flow, cognitive function, cortical pathological lesions, and mitochondrial morphology were measured. SH-SY5Y cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) injury. Mitochondrial membrane potential and lactic dehydrogenase (LDH), reactive oxygen species (ROS), and adenosine triphosphate (ATP) levels were assessed with commercial kits. Moreover, co-immunoprecipitation (Co-IP) was used to detect the binding of SUMO1 and SUMO2/3 to Drp1. The protein expressions of Drp1, Fis1, MFF, OPA1, Mfn1, Mfn2, SUMO1, SUMO2/3, SENP1, SENP2, SENP3, SENP5, and SENP6 were measured using western blot. RESULTS: In rats with CIRI, AST IV and Lig improved neurological and cognitive functions, restored CBF, reduced brain infarct volume, and alleviated cortical neuron and mitochondrial damage. Moreover, in SH-SY5Y cells, the combination of AST IV and Lig enhanced cellular viability, decreased release of LDH and ROS, increased ATP content, and improved mitochondrial membrane potential. Furthermore, AST IV combined with Lig reduced the binding of Drp1 with SUMO1, increased the binding of Drp1 with SUMO2/3, suppressed the expressions of Drp1, Fis1, MFF, and SENP3, and increased the expressions of OPA1, Mfn1, Mfn2, SENP1, SENP2, and SENP5. SUMO1 overexpression promoted mitochondrial fission and inhibited mitochondrial fusion, whereas SUMO2/3 overexpression suppressed mitochondrial fission. AST IV combined with Lig could reverse the effects of SUMO1 overexpression while enhancing those of SUMO2/3 overexpression. CONCLUSIONS: This study posits that the combination of AST IV and Lig has the potential to reduce the SUMO-1ylation of Drp1, augment the SUMO-2/3ylation of Drp1, and thereby exert a protective effect against CIRI.

Purification of triterpenoid saponins and 25R/25S-inokosterone from Achyranthes bidentata Bl. by high-speed countercurrent chromatography coupled silver nitrate coordination.

An effective method by high-speed countercurrent chromatography coordinated with silver nitrate for the preparative separation of sterones and triterpenoid saponins from Achyranthes bidentata Blume was developed. Methyl tert-butyl ether/n-butanol/acetonitrile/water (4:2:3:8, v/v/v/v) was selected for 20-hydroxyecdysone (compound 1), chikusetsusaponin IVa methyl ester (compound 4), 2'-glycan-11-keto-pigmented saponin V (compound 5), as well as a pair of isomers of 25S-inokosterone (compound 2) and 25R-inokosterone (compound 3), which were further purified by silver nitrate coordinated high-speed countercurrent chromatography. What is more, dichloromethane/methanol/isopropanol/water (6:6:1:4, v/v/v/v) was applied for calenduloside E (compound 6), 3ß-[(O-ß-d-glucuronopyranosyl)-oxy]-oleana-11,13-dien-28-oic acid (compound 7), zingibroside R1 (compound 8) and chikusetsusaponin IVa (compound 9). Adding Ag+ to the solvent system resulted in unique selectivity for 25R/25S isomers of inokosterone, which increased the complexing capability and stability of Ag+ coordinated 25S-inokosterone, as well as the α value between them. These results were further confirmed by the computational calculation of geometry optimization and frontier molecular orbitals assay. Comprehensive mass spectrometry and nuclear magnetic resonance analysis demonstrated the structures of the obtained compounds.

In vitro suppression of porcine epidemic diarrhea virus by Panax notoginseng saponins: assessing antiviral potential.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality in neonatal suckling piglets, leading to significant economic losses to the swine industry. Panax notoginseng saponins (PNS) are bioactive extracts derived from the P. notoginseng plant. In this study, we investigated the anti-PEDV effect of PNS by employing various methodologies to assess their impact on PEDV in Vero cells. Using a CCK-8 (Cell Counting Kit-8) assay, we found that PNS had no significant cytotoxicity below the concentration of 128 µg/mL in Vero cells. Using immunofluorescence assays (IFAs), an enzyme-linked immunosorbent assay (ELISA), and plaque formation assays, we observed a dose-dependent inhibition of PEDV infection by PNS within 24-48 hours postinfection. PNS exerts its anti-PEDV activity specifically at the genome replication stage, and mRNA-seq analysis demonstrated that treatment with PNS resulted in increased expression of various genes, including IFIT1 (interferon-induced protein with tetratricopeptide repeats 1), IFIT3 (interferon-induced protein with tetratricopeptide repeats 3), CFH (complement factor H), IGSF10 (immunoglobulin superfamily member 10), ID2 (inhibitor of DNA binding 2), SPP1 (secreted phosphoprotein 1), PLCB4 (phospholipase C beta 4), and FABP4 (fatty acid binding protein 4), but it resulted in decreased expression of IL1A (interleukin 1 alpha), TNFRSF19 (TNF receptor superfamily member 19), CDH8 (cadherin 8), DDIT3 (DNA damage inducible transcript 3), GADD45A (growth arrest and DNA damage inducible alpha), PTPRG (protein tyrosine phosphatase receptor type G), PCK2 (phosphoenolpyruvate carboxykinase 2), and ADGRA2 (adhesion G protein-coupled receptor A2). This study provides insights into the potential mechanisms underlying the antiviral effects of PNS. Taken together, the results suggest that the PNS might effectively regulate the defense response to the virus and have potential to be used in antiviral therapies.

Novel glycosidase from Paenibacillus lactis 154 hydrolyzing the 28-O-ß-D-glucopyranosyl ester bond of oleanane-type saponins.

Oleanane-type ginsenosides are a class of compounds with remarkable pharmacological activities. However, the lack of effective preparation methods for specific rare ginsenosides has hindered the exploration of their pharmacological properties. In this study, a novel glycoside hydrolase PlGH3 was cloned from Paenibacillus lactis 154 and heterologous expressed in Escherichia coli. Sequence analysis revealed that PlGH3 consists of 749 amino acids with a molecular weight of 89.5 kDa, exhibiting the characteristic features of the glycoside hydrolase 3 family. The enzymatic characterization results of PlGH3 showed that the optimal reaction pH and temperature was 8 and 50 °C by using p-nitrophenyl-ß-D-glucopyranoside as a substrate, respectively. The Km and kcat values towards ginsenoside Ro were 79.59 ± 3.42 µM and 18.52 s-1, respectively. PlGH3 exhibits a highly specific activity on hydrolyzing the 28-O-ß-D-glucopyranosyl ester bond of oleanane-type saponins. The mechanism of hydrolysis specificity was then presumably elucidated through molecular docking. Eventually, four kinds of rare oleanane-type ginsenosides (calenduloside E, pseudoginsenoside RP1, zingibroside R1, and tarasaponin VI) were successfully prepared by biotransforming total saponins extracted from Panax japonicus. This study contributes to understanding the mechanism of enzymatic hydrolysis of the GH3 family and provides a practical route for the preparation of rare oleanane-type ginsenosides through biotransformation. KEY POINTS: • The glucose at C-28 in oleanane-type saponins can be directionally hydrolyzed. • Mechanisms to interpret PlGH3 substrate specificity by molecular docking. • Case of preparation of low-sugar alternative saponins by directed hydrolysis.

DNA methylation regulates the secondary metabolism of saponins to improve the adaptability of Eleutherococcus senticosus during drought stress.

Plant growth and development can be significantly impacted by drought stress. Plants will adjust the synthesis and accumulation of secondary metabolites to improve survival in times of water constraint. Simultaneously, drought stress can lead to modifications in the DNA methylation status of plants, and these modifications can directly impact gene expression and product synthesis by changing the DNA methylation status of functional genes involved in secondary metabolite synthesis. However, further research is needed to fully understand the extent to which DNA methylation modifies the content of secondary metabolites to mediate plants' responses to drought stress, as well as the underlying mechanisms involved. Our study found that in Eleutherococcus senticosus (E. senticosus), moderate water deprivation significantly decreased DNA methylation levels throughout the genome and at the promoters of EsFPS, EsSS, and EsSE. Transcription factors like EsMYB-r1, previously inhibited by DNA methylation, can re-bind to the EsFPS promotor region following DNA demethylation. This process promotes gene expression and, ultimately, saponin synthesis and accumulation. The increased saponin levels in E. senticosus acted as antioxidants, enhancing the plant's adaptability to drought stress.

Effects of Saikosaponin-A on Insulin Resistance in Obesity: Computational and Animal Experimental Study.

Obesity is known to be associated with increased inflammation and dysregulated autophagy, both of which contribute to insulin resistance. Saikosaponin-A (SSA) has been reported to exhibit anti-inflammatory and lipid-lowering properties. In this research, we employed a combination of computational modeling and animal experiments to explore the effects of SSA. Male C57BL/6 mice were categorized into four groups: normal diet, high-fat diet (HFD), HFD + atorvastatin 10 mg/kg, and HFD + SSA 10 mg/kg. We conducted oral glucose and fat tolerance tests to assess metabolic parameters and histological changes. Furthermore, we evaluated the population of Kupffer cells (KCs) and examined gene expressions related to inflammation and autophagy. Computational analysis revealed that SSA displayed high binding affinity to tumor necrosis factor (TNF)-α, nuclear factor (NF)-κB, fibroblast growth factor 21 (FGF21), and autophagy-related 7 (ATG7). Animal study demonstrated that SSA administration improved fasting and postprandial glucose levels, homeostatic model assessment of insulin resistance (HOMA-IR) index, as well as triglyceride, free fatty acid, total cholesterol, low-density lipoprotein cholesterol (LDL-C)-cholesterol, and high-density lipoprotein cholesterol (HDL-C)-cholesterol levels in HFD-fed mice. Moreover, SSA significantly reduced liver weight and fat accumulation, while inhibiting the infiltration and M1 activation of KCs. At the mRNA level, SSA downregulated TNF-α and NF-κB expression, while upregulating FGF21 and ATG7 expression. In conclusion, our study suggests that SSA may serve as a therapeutic agent for addressing the metabolic complications associated with obesity. This potential therapeutic effect is attributed to the suppression of inflammatory cytokines and the upregulation of FGF21 and ATG7.

Methyl jasmonate inducible UGT79A18 is a novel glycosyltransferase involved in the bacoside biosynthetic pathway in Bacopa monnieri.

Bacosides are dammarane-type triterpenoidal saponins in Bacopa monnieri and have various pharmacological applications. All the bacosides are diversified from two isomers, i.e., jujubogenin and pseudojujubogenin. The biosynthetic pathway of bacoside is not well elucidated. In the present study, we characterized a UDP-glycosyltransferase, UGT79A18, involved in the glycosylation of pseudojujubogenin. UGT79A18 shows higher expression in response to 5 h of wounding, and 3 h of MeJA treatment. The recombinant UGT79A18 shows in vitro activity against a wide range of flavonoids and triterpenes and has a substrate preference for protopanaxadiol, a dammarane-type triterpene. Secondary metabolite analysis of overexpression and knockdown lines of UGT79A18 in B. monnieri identify bacopasaponin D, bacopaside II, bacopaside N2 and pseudojujubogenin glucosyl rhamnoside as the major bacosides that were differentially accumulated. In the overexpression lines of UGT79A18, we found 1.7-fold enhanced bacopaside II, 8-fold enhanced bacopasaponin D, 3-fold enhanced pseudojujubogenin glucosyl rhamnoside, and 1.6-fold enhanced bacopaside N2 content in comparison with vector control plant, whereas in the knockdown lines of UGT79A18, we found 1.4-fold reduction in bacopaside II content, 3-fold reduction in the bacopasaponin D content, 2-fold reduction in the pseudojujubogenin glucosyl rhamnoside content, and 1.5-fold reduction in bacopaside N2 content in comparison with vector control. These results suggest that UGT79A18 is a significant UDP glycosyltransferase involved in glycosylating pseudojujubogenin and enhancing the pseudojujubogenin-derived bacosides.

Saikosaponin D mitigate pilocarpine-induced astrocyte injury by regulating the NLRP3/caspase-1 signaling pathway.

Studies have shown that saikosaponin D (SSD) has favorable neurotherapeutic effects. Therefore, the objective of this study was to explore the efficacy and possible molecular mechanisms of SSD on pilocarpine (PP)-induced astrocyte injury. Primary astrocytes were isolated from juvenile rats and identified using immunofluorescence. The cells were treated with PP and/or SSD for 6 h and 12 h, respectively, followed by measurement of their viability through 3-(4,5-dimethylthiazol)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Next, quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression levels of Glial fibrillary acidic protein (GFAP), C3, S100 calcium binding protein A10 (S100a10), pentraxin 3 (Ptx3), toll-like receptor 4 (TLR4), and RAG in astrocytes after different treatments. Enzyme-linked immunosorbent assay and biochemical tests were utilized to evaluate the level of inflammatory factors [interleukin (IL)-1ß, IL-6, and tumor necrosis factor alpha (TNF-α)] secreted by cells and the content of oxidative stress-related factors (malondialdehyde [MDA] and glutathione [GSH]) or enzyme activity (catalase [CAT] and glutathione peroxidase [GPX]) in cells. The JC-1 mitochondrial membrane potential (MMP) fluorescence probe was used to measure the MMP in astrocytes. Additionally, western blot was applied to test the expression of proteins related to the nod-like receptor protein 3 (NLRP3)/caspase-1 signaling pathway. PP treatment (1 mM) induced cell injury by significantly reducing the viability of astrocytes and expression of cellular markers. SSD treatment (4 µM) had no toxicity to astrocytes. Besides, SSD (4 µM) treatment could significantly up-regulate the cell viability and marker expression of PP-induced astrocytes. Furthermore, SSD could be employed to inhibit inflammation (reduce IL-1ß, IL-6, and TNF-α levels) and oxidative stress (decrease MDA level, elevate GSH level, the activity of CAT and GPX), and ameliorate mitochondrial dysfunction (upregulate JC-1 ratio) in PP-induced astrocytes. Moreover, further mechanism exploration revealed that SSD treatment significantly reduced the activity of the NLRP3/caspase-1 signaling pathway activated by PP induction. SSD increased cell viability, inhibited inflammation and oxidative stress response, and ameliorated mitochondrial dysfunction in PP-induced astrocyte injury model, thus playing a neuroprotective role. The mechanism of SSD may be related to the inhibition of the NLRP3/caspase-1 inflammasome.

Astragaloside IV restrains pulmonary fibrosis progression via the circ_0008898/miR-211-5p/HMGB1 axis.

Pulmonary Fibrosis (PF) is a fatal lung disease with complicated pathogenesis. Astragaloside IV (ASV) has been discovered to alleviate PF progression, and the potential molecular mechanism of ASV in the development of PF need to be further clarified. Bleomycin (BLM) was used to construct PF in vivo model. Expression levels of circ_0008898, miR-211-5p, high mobility group protein B1 (HMGB1), alpha smooth muscle Actin (α-SMA) and Collagen I were examined by Quantitative real time polymerase chain reaction (qRT-PCR) and western blot. Cell survival was analyzed using Cell Counting Kit-8 (CCK-8) and EdU (5-ethynyl-2'-deoxyuridine) assay. The invasion abilities were investigated by transwell assay. The levels of inflammatory factors were tested via using Enzyme-linked immunosorbent assay (ELISA). The relationship between circ_0008898 or HMGB1 and miR-211-5p was identified by dual-luciferase reporter assay. The results showed that ASV attenuated BLM-induced pulmonary fibrosis in vivo. In vitro study, ASV alleviated TGF-ß1-induced fibrogenesis in HFL1 cells. Circ_0008898 was increased in TGF-ß1-induced HFL1 cells. ASV-induced impacts were abrogated by circ_0008898 overexpression in TGF-ß1-induced HFL1 cells. Mechanistically, circ_0008898 competitively bound to miR-211-5p to increase the expression of its target HMGB1. MiR-211-5p deficiency rescued ASV-mediated effects in TGF-ß1-induced HFL1 cells. In addition, HMGB1 overexpression partially overturned circ_0008898 interference-induced impacts in HFL1 cells upon TGF-ß1 treatment. In conclusion, our work manifested that ASV hindered PF process by mediating the circ_0008898/miR-211-5p/HMGB1 network.