RNA polymerase inhibitor（IC50=32 nM）

Cynaroside reduces liver inflammatory damage linked to sepsis and promotes the phenotypic shift of macrophages from pro-inflammatory M1 to anti-inflammatory M2.
By preventing PKM2 from translocating to the nucleus, encouraging the formation of PKM2 tetramers, and inhibiting PKM2 phosphorylation at Y105 both in vivo and in vitro, cynaroside decreases the binding of PKM2 to hypoxia-inducible factor-1α (HIF-1α).
In septic liver, cynaroside prevents the hyperacetylation of HMGB1 due to glycolysis, restores pyruvate kinase activity, and inhibits proteins related to glycolysis such as PFKFB3, HK2, and HIF-1α.
By reducing the production of reactive oxygen species and preventing caspase activation in the mitochondrial and death receptor pathways, cynaroside shields H9c2 cells from H2O2-induced apoptosis.
By controlling the expression of JNK, P53, and the Bcl-2 protein, cynaroside preserves mitochondrial function[2][3].

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Cynaroside regulates macrophage phenotype shift associated with PKM2 mediated pro-inflammatory pathways [2]
To further explore the effects of cynaroside on PKM2 in macrophage phenotype shift, we performed cellular phenotype identification using RAW264.7 cells under LPS stimulators. Cynaroside, as well as PKM2 inhibitor shikonin significantly reduced mRNA levels of M1 markers CXCL10, iNOS and TNF-α and protein expression levels of iNOS induced by LPS (Fig. 5A and C). In contrast, treatment with cynaroside, as well as shikonin increased expression levels of M2 markers Arg-1, IL-10 and CD206 (Fig. 5B and C). These findings show that Cynaroside inhibits pro-inflammatory M1-type macrophage polarization and increases anti-inflammatory and tissue repair M2-type macrophage polarization in vitro. Furthermore, cynaroside and shikonin decreased expression levels of glycolysis related genes HK2, PFKFB3 and PKM2, which were increased by LPS in RAW264.7 cells (Fig. 6A). PKM2 interacts with Hif-1α and stimulates Hif-1α transactivation domain function. Furthermore, PKM2 promotes recruitment of p300 into Hypoxia responsive elements (HREs) associated with the HIF-1 target genes. We measured HIF-1α levels to determine the effect of cynaroside on the binding of PKM2 with Hif-1α. Cynaroside treatment reduced HIF-1α expression levels in nucleus extracts or whole cell extracts of liver tissues in CLP surgical mice (Fig. 4A) but decreased PKM2 nuclear translocation in hepatic macrophages of septic mice (Fig. 4E). In addition, cynaroside inhibited PKM2 and HIF-1α expression levels in nucleus extracts and co-localization of PKM2 with HIF-1α in LPS-exposed RAW264.7 nuclei in vitro (Figs. 4B, 6B and F). Co-IP experiment results revealed that cynaroside reduced the level of a complex formed by PKM2 and HIF-1α in the nucleus in vitro and in vivo (Figs. 4D and 6E). Furthermore, we investigated the effects of cynaroside on PKM2 tetramerization. TEPP-46 promotes the conversion of PKM2 dimers to tetramers by functioning as an agonist. Cynaroside and TEPP-46 treatments significantly increased the ratio of tetrameric PKM2 in LPS-exposed macrophages (Fig. 6C). Phosphorylation of Y105 indicates monomer or dimer formation as it prevents PKM2 tetramer configuration, which further promotes the Warburg effect. CLP and LPS induced high phosphorylation of PKM2Y105 in vitro and in vivo. Cynaroside treatment inhibited phosphorylation of residue Y105 in PKM2 in vitro and in vivo (Figs. 4C and 6D). Molecular docking analysis using SwissDock (http://www.swissdock.ch/) and Autodock vina showed that cynaroside bound to PKM2 (PDB ID:1T5A) (Fig. 6H). The docking results predicted that cynaroside might bond to key amino acid residues LYS311 and ASN350, ALA388, TYE390 via hydrogen bond. Meanwhile, a hydrophobic interaction and π-π stacking formed between PHE306 of PKM2 and cynaroside. Therefore, the molecular docking information illustrated that PKM2 could be a potential druggable target of cynaroside. All the results above suggested cynaroside possibly targeted PKM2 to inhibit phosphorylation at the Y105 site and enhance PKM2 tetramer stability like TEPP-46, thereby blocking the PKM2-mediated pro-inflammatory pathways.

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Flavonoids with potent anti-oxidative effects are the major effective components in traditional herbal medicine used in treating cardiovascular diseases. Cynaroside is a flavonoid compound that exhibits anti-oxidative capabilities. However, little is known about its effect on oxidative injury to cardiac myocytes and the underlying mechanisms. This study was designed to investigate the protective effects of cynaroside against H(2) O(2) -induced apoptosis in H9c2 cardiomyoblasts. H9c2 cells were pretreated with cynaroside for 4 h before exposure to 150 µM H(2) O(2) for 6 h. H(2) O(2) treatment caused severe injury to the H9c2 cells, which was accompanied by apoptosis, as revealed by analysis of cell nuclear morphology, through Annexin V FITC/PI staining and caspase proteases activation. Cynaroside pretreatment significantly reduced the apoptotic rate by enhancing the endogenous anti-oxidative activity of superoxide dismutase, glutathione peroxidase, and catalase, thereby inhibiting intracellular reactive oxygen species (ROS) generation. Moreover, cynaroside moderated H(2) O(2) -induced disruption of mitochondrial membrane potential, increased the expression of anti-apoptotic protein Bcl-2 while decreased the expression of pro-apoptotic protein Bax, and thereby inhibited the release of apoptogenic factors (cytochrome c and smac/Diablo) from mitochondria in H9c2 cells. Our data also demonstrated that cynaroside pretreatment showed an inhibitory effect on the H(2) O(2) -induced increase in c-Jun N-terminal kinase (JNK) and P53 protein expression. These results suggest that cynaroside prevents H(2) O(2) -induced apoptosis in H9c2 cell by reducing the endogenous production of ROS, maintaining mitochondrial function, and modulating the JNK and P53 pathways. [3]

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Cynaroside, a flavonoid, has been shown to have antibacterial, antifungal and anticancer activities. Here, we evaluated its antileishmanial properties and its mechanism of action through different in silico and in vitro assays. Cynaroside exhibited antileishmanial activity in time- and dose-dependent manner with 50% of inhibitory concentration (IC50) value of 49.49 ± 3.515 µM in vitro. It inhibited the growth of parasite significantly at only 20 µM concentration when used in combination with miltefosine, a standard drug which has very high toxicity. It also inhibited the intra-macrophagic parasite significantly at low doses when used in combination with miltefosine. It showed less toxicity than the existing antileishmanial drug, miltefosine at similar doses. Propidium iodide staining showed that cynaroside inhibited the parasites in G0/G1 phase of cell cycle. 2,7-dichloro dihydro fluorescein diacetate (H2DCFDA) staining showed cynaroside induced antileishmanial activity through reactive oxygen species (ROS) generation in parasites. Molecular-docking studies with key drug targets of Leishmania donovani showed significant inhibition. Out of these targets, cynaroside showed strongest affinity with uridine diphosphate (UDP)-galactopyranose mutase with -10.4 kcal/mol which was further validated by molecular dynamics (MD) simulation [4].

Cynaroside (i.p.; 5 mg/kg) inhibits PKM2's binding to hypoxia-inducible factor-1α (HIF-1α) by preventing PKM2 from translocating to the nucleus, encouraging the formation of PKM2 tetramers, and preventing PKM2 from being phosphorylated at Y105[2].

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Cynaroside inhibits CLP-induced inflammation in the liver [2]
To investigate the role of Cynaroside in CLP-induced liver injury, mice were intraperitoneally injected with cynaroside or physiological saline solution after performing cecal ligation and puncture (CLP) of the liver to mimic clinical sepsis effects (Fig. 1A). Plasma ALT and AST levels in mice were determined 16 h after performing CLP. High plasma ALT and AST levels induced by CLP were significantly reduced by cynaroside treatment (Fig. 1C and D). To confirm effects of cynaroside on CLP-induced hepatic damage, histopathological changes in the liver of mice were determined by H&E staining. Results of H&E staining of liver tissues from CLP-induced sepsis in mice showed infiltration of lymphocytes and neutrophils in the liver. However, inflammatory cell infiltration in drug treated groups showed different degrees of recovery (Fig. 1B). We performed immunofluorescence of liver tissues to further explore the effects of cynaroside on immunological reaction in liver injury induced by CLP. IF staining of liver sections showed that cynaroside reduced infiltration of F4/80-positive macrophages in the liver (Fig. 2A). Pro-inflammatory cytokines play a key role in progression of CLP-induced liver injury [7]. Further, we compared expression of early pro-inflammatory factors IL-1β, IL-6, TNF-α and late HMGB1 in liver 16 h after CLP in different treatment groups through Western blot (Fig. 2B) and IHC staining (Fig. 2A). Expression levels of IL-1β, IL-6 and TNF-α, as well as HMGB1, were significantly higher in CLP mice whereas cynaroside pretreatment group showed significantly lower levels compared with levels in the control group. In summary, these findings imply that cynaroside alleviates liver inflammation caused by CLP.

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Cynaroside suppresses M1 polarized phenotype of macrophages in the liver of septic mice [2]
Macrophage polarization plays a key role in hepatic inflammation and tissue impairment during sepsis progression. Therefore, we explored the effect of cynaroside on hepatic macrophage polarization. F4/80 is a surface glycoprotein of macrophages and is used as a marker for macrophages. iNOS is a marker for M1 macrophages, therefore, it was used as a M1 marker in tissues. Further, CD206 was used as a surface receptor protein for labeling M2 markers. CLP surgery significantly increased levels of iNOS+ (shown in red), F4/80+ (shown in green) markers (Fig. 3A). Notably, cynaroside treatment significantly reduced levels of iNOS+ enzyme. Conversely, cynaroside treatment increased the number of CD206+ (red) and F4/80+ (green) markers (Fig. 3A). Western blot results revealed that cynaroside downregulated the high iNOS protein levels induced by CLP, and upregulated CD206 (Fig. 3B). The results suggest that cynaroside promotes M2 polarization and inhibits M1 polarization in the liver of septic mice.

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Cynaroside inhibits PKM2 expression in the liver of septic mice [2]
Excessive inflammatory response and polarization of M1 macrophages are controlled by aerobic glycolysis [8]. Therefore, we investigated liver glycolysis-associated signals by performing Western blot analysis. The results revealed that CLP upregulated key enzymes involved in glycolysis but induction of cynaroside significantly reversed upregulation of the enzymes, including HK2, PFKFB3 and PKM2 (Fig. 4A). PKM2 is a key protein for bridging immunity and metabolism, balancing glycolysis and OXPHOS and enabling activation of inflammatory response through nuclear translocation [27]. A series of experiments were performed to explore the relationship between cynaroside and PKM2, with an aim of elucidating the mechanisms of cynaroside in the regulation of inflammatory response and conversion of macrophage phenotypes by regulating PKM2. In addition, cynaroside treatment inhibited CLP-induced HMGB1 acetylation, which was activated by PKM2-mediated abnormal glycolysis in liver tissues (Fig. 2B). IF staining of liver sections showed that PKM2 (shown in red) is highly expressed in whole liver as well as in hepatic macrophages (F4/80+, shown in green) in sepsis mice (Fig. S1 and S2). Moreover, cynaroside reduced PKM2 expression level both in hepatic macrophages and the entire liver.

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Cynaroside inhibits PKM2 nuclear translocation in the liver of septic mice [2]
PKM2 can occur as a monomer or dimer, and as a tetramer. The high level of pyruvate kinase activity of PKM2 tetramer promotes normal cell metabolism. However, a low enzymatic activity of PKM2 dimer results in decreased cellular metabolism during immune activation, which also activates inflammatory genes following translocation into the nucleus. PKM2 dimer expression in the liver of septic mice was detected using Native-PAGE electrophoresis. CLP increased the expression levels of PKM2 dimers in the liver, which were reduced by cynaroside treatment (Fig. 4B) and the observation was consistent with the findings of a previous study. In addition, treatment with cynaroside restored pyruvate kinase activity in the livers of septic mice (Fig. 4F). Cynaroside reversed the upregulation of PKM2 in the nuclei of mice livers after subjecting mice to CLP treatment (Fig. 4A). Moreover, IF staining revealed that cynaroside reduced nuclear translocation of PKM2 in hepatic macrophages of septic mice (Fig. 4E).

Molecular docking and molecular dynamics simulation of protein–ligand complex [4]
The protein molecule was loaded in the PyRx Virtual Screening Tool and was converted into a macromolecule pdbqt format. The Cynaroside to be screened was imported and converted into the ligand pdbqt format. In the Vina wizard the pdbqt macromolecules and pdbqt ligands are selected and the grid box is set up with all the active site residues containing within the grid box. The docking is performed by running Vina. The output file was analyzed to find binding energy. The best orientation of the Cynaroside was selected and saved as a PDB file. The protein molecule and the best-oriented ligand molecule were loaded in Pymol and the protein–ligand complex was visualized. The protein–ligand complex was loaded in Ligplot software and the output 2D diagram was analyzed to find the number of hydrogen bonds and the binding site residues of the protein.
The most favourable binding poses of the Cynaroside were analyzed by choosing the lowest free energy of binding (ΔG) and the lowest inhibition constant (Ki) which is calculated using the following formula:

Cell Line: H9c2 cells
Concentration: 25, 50, 100 μg/mL
Incubation Time: 4 h
Result: Protected H9c2 cells from oxidative stress-induced cell injury.

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RAW264.7 cell line was cultured in DMEM supplemented with 10% FBS, and incubated with 5% CO2 at 37 °C. Cells were passaged every 2–3 days. RAW264.7 cells were seeded at a density of 1 × 105 cells/mL and cultured overnight in six-well plates to achieve a cover of 70% confluence. RAW264.7 macrophages were pretreated with Cynaroside or shikonin (specific inhibitor of PKM2) for 4 h and stimulated by LPS (0.5 μg/mL) for 8 h.[2]

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Analysis of Cell Viability and Morphological Changes [3]
Cell viability was determined colorimetrically by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay. Briefly, cells were seeded at 1 × 104 cells/well in 96-well plates. After 4 h of treatment with different concentrations of Cynaroside followed by incubation with 150 µM H2O2 for 6 h, 20 µl of 5 mg/ml MTT solution was added to each well (0.1 mg/well), and incubated for 4 h. The supernates were aspirated, and the formazan crystals in each well were dissolved in 100 µl of DMSO. The absorbance was measured at 570 nm on a microplate reader (BioTek, Vermont). The morphological changes in the cells were also observed and the images were captured under an inverted microscope connected to a digital camera.

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Detection of Intracellular ROS Production [3]
To investigate the effect of Cynaroside on the generation of intracellular ROS, the cells were pretreated with cynaroside for 4 h prior to the addition of 150 µM H2O2 for 6 h. Then, the level of intracellular ROS was monitored using the total ROS detection kit according to the manufacturer's brochures. The cells were harvested, placed into 5 ml round-bottom polystyrene tubes after treatment, and washed with 1× wash buffer. Then, the cells were centrifuged for 5 min at 400g at room temperature and the supernate was discarded. The cells were resuspended in 500 µl ROS detection solution, stained at 37°C in the dark for 30 min, and then analyzed by flow cytometry.

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Anti-promastigote evaluation and IC50 determination [4]
Log-phase promastigotes were enumerated and incubated at the density of 5 × 106 parasites in the absence and presence of Cynaroside at two-fold serial dilutions starting at 120 µM for 48 h at 22°C. The percentage parasite viability was calculated as (Mean parasite number of treated sample/Mean parasite of control) × 100. The 50% inhibitory concentration was determined by extrapolating the graph of % viability of parasites against concentration of the drug/compound. Morphological changes of the parasites were observed under 20× and 40× lenses using LMI U.K. vImage software.

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Anti-amastigote and cytotoxicity evaluation of Cynaroside [4]
A total of 2 × 106 THP-1 differentiated macrophages were plated in 96-well tissue culture-grade plates in RPMI 1640 complete media with 5% CO2 at 37°C to evaluate the cytotoxic activity of Cynaroside. Differentiated adherent cells were washed with plain RPMI 1640 media and exposed to two-fold serial dilution of cynaroside starting from 500 µM for 24 h. Cells were further incubated with 50 µM of 5 mg/ml of MTT for 3–4 h, thereafter the resulting formazan was dissolved in 150 µM of DMSO. The amount of formazan produced represented the relative number of viable cells which was recorded spectrophotometrically at 570 nm by ELISA plate reader. The cytotoxic concentration 50%, i.e. CC50 value was determined by extrapolation of the dose–response curve of percentage cell viability vs concentration of the compound. For parasite load calculation, THP-1-differentiated macrophages were plated on coverslip in six-well plates and infected by L. donovani with 1:10. Infected macrophages were treated with different concentrations of the drugs/compounds for 48 h and then cells were fixed by chilled methanol and stained by Giemsa to calculate the % parasites load.

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Study of cell cycle of promastigotes [4]
In brief, the promastigotes were cultured in the absence and presence of different concentrations of Cynaroside and miltefosine as the positive control. The promastigotes were harvested after 48 h of incubation and washed thrice with PBS followed by fixation with 80% chilled ethanol and kept at 4°C overnight. The fixed cells were washed twice with PBS and incubated with 200 μg/ml of RNase at 37°C for 1 h followed by staining with 50 µM of 1 mg/ml of propidium iodide (PI) for 20 min in dark. Cells were analyzed through flow cytometer.

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ROS estimation [4]
To assess the Cynaroside-induced ROS generation, 5 × 106 parasites were incubated with different concentrations of compounds/drugs at 22°C for 48 h. The treated parasites were washed with PBS and incubated with 10 µM of fluorescent dye, 2,7-dichloro dihydro fluorescein diacetate (H2DCFDA) for 20 min in dark and analyzed through BD FACS ARIA. Data were represented in the form of histograms.

Animal Model: Mice model of sepsis[2]
Dosage: 5mg/kg
Administration: Cynaroside (i.p.; 5mg/kg)
Result: Inhibited PKM2 dimer formation in liver of septic mice.

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Mice model of sepsis [2]
A mouse model of CLP-induced severe sepsis was developed following a method by Daniel Rittirsch et al. C57BL/6 mice were randomly divided into three groups: sham, CLP, CLP + Cynaroside (5 mg/kg). Animals received Cynaroside or vehicle treatment through intraperitoneal injection 30 min before CLP. Afterwards, anesthesia was induced using ketamine (80–100 mg/kg/i.p., 1867-66-9) and xylazine (5 mg/kg/i.p.). A small midline abdominal incision was made to externalize the cecum and ligate the distal aspect of ileocecal valve using a 4–0 silk wire without causing intestinal obstruction. Furthermore, a 21-gauge needle was used to puncture the appendix twice. The abdomen was closed using two-layer sutures. In the sham-treated group, mice underwent the same surgical procedures, although the ceca were neither ligated nor perforated. Finally, the mice were returned to the cages in a temperature-controlled room (22 °C) and observed every 6 h.

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Pharmacokinetics studies [4]
The selected ligand was evaluated for pharmacological profiles by analyzing for Lipinski’s rule of violation-5, which was analyzed by Molsoft L.L.C.: Drug-Likeness and molecular property prediction for drug-likeness (http://www.molsoft.com/mprop/). The bioactivity of Cynaroside was checked by Molinspiration (https://molinspiration.com/cgi-bin/properties). The Cynaroside was further evaluated for ADMET (absorption, distribution, metabolism, excretion and toxicity) properties by GUSAR and SwissADME database. SwissADME software helps us to identify the selected drug molecule by applying different virtual screening methods. Different components of lipophilicity (iLOGP, WLOGP, XLOGP3, MLOGP, Log Po/w), pharmacokinetics (GI absorption, BBB permeant, P-gp substrate, Log (Kp)), water solubility also helped in the preliminary testing of the suitable drug molecule. OSIRIS Property Explorer programme was used to evaluate the mutagenic, tumorigenic, irritant and reproductive risks, and which also provides information on the compound’s toxicity, solubility (LogS), hydrophilicity (LogP), molecular weight, drug-likeness and drug score

The pharmacological studies were done on Cynaroside for a good oral administration established through the Lipinski’s rule of five, which was evaluated by Molsoft L.L.C.: Drug-likeness and molecular property prediction. Cynaroside followed all the parameters of Lipinski’s rule of five, except HBD and HBA criteria which are exceeding by 2 and 1 atoms, respectively. The lipophilicity of cynaroside showed value of 0.47 that indicates moderate sublingual absorption as observed from Table 2. Lipinski’s ‘rule of five’is an analytical approach for predicting drug-likeness stating that molecules had molecular weight (M.W. ≤ 500 Da), high lipophilicity expressed as LogP (LogP ≤ 5), hydrogen bond donors (HBD ≤ 5) and hydrogen bond acceptors (HBA ≤ 10) have good absorption or permeation across the cell membrane. For choosing the selected drug molecules through virtual screening, sometimes we found violation of some selection rules like Ro5, Veber etc. At present, in drug industry there are several important drugs available in the market which violate some likeness rules. Among the very popular drugs, some like fosinapril, bromocriptine mesylate, dabigitranetexilate, olmesartanmedoxomil and reserpine etc revealed two Ro5 rule violations. The SwissADME was used for pharmacodynamic study of cynaroside to understand the action of drug inside a host’s body. Cynaroside possess low gastrointestinal absorption and good solubility with value −3.65, which is higher than −4 (≥ −4). Cynaroside is not permeable to the blood–brain barrier. The leadlikeness criteria is violated only for molecular mass (≥350) and possess a moderate bioavailability score. The results are summarized in Table 2. The ADMET study focused on the parameters that can define absorption, distribution, metabolism, excretion, toxicity, gastrointestinal absorption (GIA), solubility (LogS), P-glycoprotein substrate inhibition, cytochrome substrate/inhibitor. Cynaroside was evaluated as an active enzyme inhibitor with value 0.42. The predicted bioactivity by molinspiration is shown in Table 3. Molinspiration was used to evaluate the bioactivity of cynaroside by calculating the activity against GPCR ligand, kinase inhibitor, ion channel modulator, protease inhibitor, nuclear receptor ligand and enzyme inhibitor. The bioactivity values were interpreted as follows: inactive (bioactivity score ≤ −5.0), moderately active (bioactivity score: −5.0 to 0.0) and active (bioactivity score ≥ 0). As per the Organisation for Economic Co-operation and Development (OECD) chemical classification, cynaroside was found to be a non-toxic as mentioned in Table 4. The principal aim of predicting the acute toxicity is to evaluate undesirable side effects of a compound after single or multiple exposures to an organism via a known administration route (oral, inhalation, subcutaneous (sc), intravenous (iv) or intraperitoneal (ip)). GUSAR was used to determine the acute toxicity of the successfully docked cynaroside based on the Prediction of Activity Spectra for Substances algorithm and Quantitative Neighborhoods of Atoms descriptors. The obtained result was compared with Toxicity Database to categorise on the basis of OECD chemical classification manual. The criteria used for cynaroside to elicit toxicity based upon the administration route when the compound dose is more than 7000 mg/kg for intravenous route, more than 500000 mg/kg in case of the oral route, and more than 20000 mg/kg for intraperitoneal route and subcutaneous database as shown in Table 4. All the predicted toxicity risk factors for cynaroside were low and molecular weights less than 500, implied that it is likely to be absorbed and are capable to reach the place of action when administered as drugs. [4]

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The bioactivity, ADMET (absorption, distribution, metabolism, excretion and toxicity) properties, Organisation for Economic Co-operation and Development (OECD) chemical classification and toxicity risk prediction showed cynaroside as an enzyme inhibitor having sufficient solubility and non-toxic properties. In conclusion, cynaroside may be used alone or in combination with existing drug, miltefosine to control leishmaniasis with less cytotoxicity.

Pharmacodynamic studies showed non-toxic properties of Cynaroside. It was predicted that cynaroside possessed no mutagenic, tumorigenic, irritant and reproductive effective toxicity risks as shown in Table 5. [4]

[1]. Unraveling the Anti-Influenza Effect of Flavonoids: Experimental Validation of Luteolin and its Congeners as Potent Influenza Endonuclease Inhibitors. Eur J Med Chem. 22 August 2020, 112754.

[2]. Cynaroside prevents macrophage polarization into pro-inflammatory phenotype and alleviates cecal ligation and puncture-induced liver injury by targeting PKM2/HIF-1α axis. Fitoterapia. 2021 Jul;152:104922.

[3]. Protective effects of cynaroside against H₂O₂-induced apoptosis in H9c2 cardiomyoblasts. J Cell Biochem. 2011 Aug;112(8):2019-29.

[4]. Cynaroside inhibits Leishmania donovani UDP-galactopyranose mutase and induces reactive oxygen species to exert antileishmanial response. Biosci Rep. 2021 Jan 29;41(1):BSR20203857.

Luteolin 7-O-beta-D-glucoside is a glycosyloxyflavone that is luteolin substituted by a beta-D-glucopyranosyl moiety at position 7 via a glycosidic linkage. It has a role as an antioxidant and a plant metabolite. It is a beta-D-glucoside, a glycosyloxyflavone, a trihydroxyflavone and a monosaccharide derivative. It is functionally related to a luteolin. It is a conjugate acid of a luteolin 7-O-beta-D-glucoside(1-).
Cynaroside has been reported in Coreopsis lanceolata, Sonchus fruticosus, and other organisms with data available.
See also: Cynara scolymus leaf (part of); Lonicera japonica flower (part of); Chamaemelum nobile flower (part of).

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The biological effects of flavonoids on mammal cells are diverse, ranging from scavenging free radicals and anti-cancer activity to anti-influenza activity. Despite appreciable effort to understand the anti-influenza activity of flavonoids, there is no clear consensus about their precise mode-of-action at a cellular level. Here, we report the development and validation of a screening assay based on AlphaScreen technology and illustrate its application for determination of the inhibitory potency of a large set of polyols against PA N-terminal domain (PA-Nter) of influenza RNA-dependent RNA polymerase featuring endonuclease activity. The most potent inhibitors we identified were luteolin with an IC50 of 72 ± 2 nM and its 8-C-glucoside orientin with an IC50 of 43 ± 2 nM. Submicromolar inhibitors were also evaluated by an in vitro endonuclease activity assay using single-stranded DNA, and the results were in full agreement with data from the competitive AlphaScreen assay. Using X-ray crystallography, we analyzed structures of the PA-Nter in complex with luteolin at 2.0 Å resolution and quambalarine B at 2.5 Å resolution, which clearly revealed the binding pose of these polyols coordinated to two manganese ions in the endonuclease active site. Using two distinct assays along with the structural work, we have presumably identified and characterized the molecular mode-of-action of flavonoids in influenza-infected cells.[1]

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The treatment of sepsis is still challenging and the liver is an important target of sepsis-related injury. Macrophages are important innate immune cells in liver, and modulation of macrophages M1/M2 polarization may be a promising strategy for septic liver injury treatment. Macrophage polarization and inflammation of liver tissue has been shown regulated by pyruvate kinase M2 (PKM2)-mediated aerobic glycolysis and immune inflammatory pathways. Therefore, modulating PKM2-mediated immunometabolic reprogramming presents a novel strategy for inflammation-associated diseases. In this study, cynaroside, a flavonoid compound, promoted macrophage phenotypic transition from pro-inflammatory M1 to anti-inflammatory M2, and mitigated sepsis-associated liver inflammatory damage. We established that cynaroside reduced binding of PKM2 to hypoxia-inducible factor-1α (HIF-1α) by abolishing translocation of PKM2 to the nucleus and promoting PKM2 tetramer formation, as well as suppressing phosphorylation of PKM2 at Y105 in vivo and in vitro. Moreover, cynaroside restored pyruvate kinase activity, inhibited glycolysis-related proteins including PFKFB3, HK2 and HIF-1α, and inhibited glycolysis-related hyperacetylation of HMGB1 in septic liver. Therefore, this study reports a novel function of cynaroside in hepatic macrophage polarization, and cecum ligation and puncture-induced liver injury in septic mice. The findings provide crucial information with regard to therapeutic efficacy of cynaroside in the treatment of sepsis. [2]

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In conclusion, the results of this study demonstrated that cynaroside protected H9c2 cells against H2O2-induced apoptosis by decreasing ROS generation and inhibiting caspase activation in both the mitochondrial and death receptor pathways. Furthermore, cynaroside maintained mitochondrial function by regulating Bcl-2 protein expression, as well as JNK and P53 expression. Although cynaroside is a promising agent for the treatment of H2O2-induced apoptosis, further investigations are necessary to explore the underlying mechanisms of cynaroside cytoprotection against oxidative stress-induced cardiovascular diseases. [3]

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Here, it was observed that cynaroside has the potential antileishmanial activity. It showed better response when used in combination with low concentrations of miltefosine. Through in silico study, we have found that the binding energy and the binding site residues of the LdUGM exhibited best interaction with the inhibitory flavonoid, cynaroside. It was confirmed by MD simulation study also. Our results suggested that cynaroside may be used as food constituent after the detailed in vivo studies on experimental visceral leishmanaisis, to fight against leishmaniasis.[4]

C21H20O11

448.3769

448.1

5373-11-5

5280637

Light yellow to green yellow solid powder

1.7±0.1 g/cm3

838.1±65.0 °C at 760 mmHg

256 - 258 °C

296.8±27.8 °C

0.0±3.2 mmHg at 25°C

1.740

-0.09

7

11

4

32

714

5

C1=CC(=C(C=C1C2=CC(=O)C3=C(C=C(C=C3O2)O[C@H]4[C@@H]([C@H]([C@@H]([C@H](O4)CO)O)O)O)O)O)O

PEFNSGRTCBGNAN-QNDFHXLGSA-N

InChI=1S/C21H20O11/c22-7-16-18(27)19(28)20(29)21(32-16)30-9-4-12(25)17-13(26)6-14(31-15(17)5-9)8-1-2-10(23)11(24)3-8/h1-6,16,18-25,27-29H,7H2/t16-,18-,19+,20-,21-/m1/s1

2-(3,4-dihydroxyphenyl)-5-hydroxy-7-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one

Cynaroside;Luteolin 7-glucoside; Cinaroside; Luteolin-7-glucoside; Luteolin 7-O-glucoside; Luteolin-7-O-glucoside

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~83.33 mg/mL (~185.85 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.64 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.64 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 16.67 mg/mL (37.18 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)