Natural biflavonoid; matrix metalloproteinases (MMPs); pre-mRNA splicing
SENP1 (Sentrin-specific protease 1) – Hinokiflavone inhibits SENP1 catalytic activity in vitro (no IC50/Ki reported; tested at 500 μM shows clear inhibition). [1]

Hinokiflavone modulates splicing in cells. Hinokiflavone prevents assembly of the spliceosome B complex. Hinokiflavone blocks cell cycle progression. Hinokiflavone alters nuclear organization of a subset of splicing factors.Hinokiflavone promotes nuclear relocalization of SUMO.[1]

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Hinokiflavone reduced the proliferation, migration, and invasion and promoted the apoptosis in colorectal tumor cells in vitro. [2]

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Hinokiflavone inhibited the activity of ClpP of MRSA strain USA300 with an IC50 of 34.36 μg/mL. Further assays showed that hinokiflavone reduced the virulence of S. aureus by inhibiting multiple virulence factors expression. Results obtained from cellular thermal transfer assay (CETSA), thermal shift assay (TSA), local surface plasmon resonance (LSPR) and molecular docking (MD) assay enunciated that hinokiflavone directly bonded to ClpP with confirmed docking sites, including SER-22, LYS-26 and ARG-28. [3] Hinokiflavone increases levels of SUMOylated proteins. Hinokiflavone inhibits SENP1 activity. SUMOylated splicing factors accumulate in the insoluble fraction in cell extracts treated with hinokiflavone.

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Hinokiflavone inhibits splicing of Ad1 and HPV18 E6 pre-mRNAs in HeLa nuclear extract in a non-radioactive RT-PCR based in vitro splicing assay at 500 μM (strong inhibitory effect). The lowest concentration that inhibits splicing in vitro is 50 μM. [1]
Hinokiflavone prevents spliceosome assembly: in radioactive in vitro splicing reactions with Ad1 pre-mRNA, treatment with 500 μM Hinokiflavone blocks formation of B and C spliceosome complexes; only H/E and A complexes are detected on native agarose gels, indicating failure to transition from A to B complex. [1]
Hinokiflavone increases protein SUMOylation levels in vitro: HeLa nuclear extract incubated under splicing conditions with 100, 300, or 500 μM Hinokiflavone for 90 min at 30°C shows a concentration-dependent increase in high molecular weight SUMO1- and SUMO2/3-modified proteins compared to DMSO control (by immunoblotting). [1]
Hinokiflavone inhibits purified recombinant SENP1 (aa 415-643) activity: in a gel-based activity assay using SUMOylated YFP-RANGAP-ECFP-SUMO2 substrate, 500 μM Hinokiflavone clearly inhibits SENP1 isopeptidase activity compared to DMSO control. Other biflavones (amentoflavone, cupressuflavone, isoginkgetin, scidopitysin) tested at same concentration show varying degrees of inhibition, with amentoflavone also showing strong inhibition. [1]
Hinokiflavone binds to SENP1 as shown by DARTS (Drug Affinity Responsive Target Stability) assay: recombinant SENP1 fragment incubated with 500 μM Hinokiflavone or DMSO, then digested with pronase (16–200 μg/ml). In the presence of Hinokiflavone, SENP1 shows increased sensitivity to protease digestion, indicating direct interaction. [1]
Hinokiflavone binds to SENP1 as shown by CETSA (Cellular Thermal Shift Assay): HeLa nuclear extract treated with 500 μM Hinokiflavone or DMSO, heated to temperatures from 30°C to 50°C, then ultracentrifuged. In DMSO control, SENP1 remains soluble up to 43°C; in the presence of Hinokiflavone, no soluble SENP1 is detected at any temperature tested, indicating reduced thermal stability. [1]
Hinokiflavone alters alternative splicing in human cell lines (HeLa, HEK293, NB4): treatment with 10, 20, or 30 μM for 24 hr (NB4 also tested at 0.5, 1, 2.5, 5 μM) causes changes in MCL1, NOP56, EIF4A2, FAS, HSP40, RIOK3, ACTR1b, and DXO pre-mRNAs detected by semiquantitative RT-PCR. Effects include exon skipping, intron retention, and alternative splice site usage. In HeLa and HEK293, Hinokiflavone promotes MCL1 exon 2 skipping (pro-apoptotic isoform); in NB4, multiple aberrant MCL1 isoforms appear. [1]
Hinokiflavone blocks cell cycle progression and induces apoptosis: HeLa, HEK293, and NB4 cells treated with 10, 20, or 30 μM for 24 hr (NB4 also 0.5–5 μM) show cell cycle arrest and/or cell death in a concentration-dependent manner by propidium iodide staining and flow cytometry. NB4 cells become mostly apoptotic after 24 hr exposure to 10 μM Hinokiflavone. [1]
Hinokiflavone alters nuclear organization of splicing factors: HeLa cells treated with 20 μM for 24 hr show relocalization of early spliceosome factors (SRSF2, U1A, DDX46, U2AF65, SF3B1, SR proteins) into enlarged “mega speckles” (0.5–4 μm size, 10–30 per cell), while late-assembling factors (CDC5L, PLRG1, BCAS2, PRP19, CTNNBL1, snRNP200) remain diffuse. Cajal bodies are disrupted; coilin, SMN, TMG-cap, Y12, SNRPA1 relocalize to mega speckles; CDK and fibrillarin remain in nucleoli. [1]
Hinokiflavone causes loss of cytoplasmic polyadenylated RNA and accumulation in nuclear mega speckles: HeLa cells treated with 10–30 μM for 4–24 hr show by FISH with Cy3-Oligo dT that poly(A) RNA concentrates in mega speckles; after 24 hr at 30 μM, poly(A) RNA appears in small spots at periphery of mega speckles. [1]
Hinokiflavone promotes relocalization of SUMO1 and SUMO2/3 to mega speckles: HeLa cells treated with 20 μM for 2 hr or 24 hr show co-localization of SUMO1 and SUMO2/3 with SRSF2 in enlarged speckles by immunofluorescence. [1]
Hinokiflavone increases SUMOylation in cells: HEK293 cells treated with 10, 20, or 30 μM for 24 hr show increased high molecular weight SUMO1- and SUMO2/3-modified proteins by immunoblotting, while ubiquitin and NEDD8 conjugation levels show a modest decrease. Similar increase seen in HeLa and NB4 cells. [1]
Hinokiflavone increases SUMO2 modification on specific lysine residues identified by quantitative SILAC proteomics: HEK293 SUMO2 T90K cells treated with 20 μM Hinokiflavone for 8 hr show 924 SUMO2-modified lysines in 543 proteins; 22 lysines show >5-fold increase. Most increased sites are in U2 snRNP components: PRPF40A (K241: 145.8-fold, K375: 45.5-fold, K517: 47.5-fold, K707: 7.8-fold), SF3B2 (K680: 94.4-fold, K563: 56.5-fold), SF3A2 (K10: 22.6-fold), SNRPD2 (K8: 16.2-fold), U2SURP (K822: 10.0-fold), SF3B1 (K413: 5.0-fold). [1]
Hinokiflavone causes accumulation of SUMOylated PRPF40A: HeLa cells stably expressing YFP-SUMO2 treated with 20 μM for 8 hr show >15% of total PRPF40A is SUMO2-modified after immunoprecipitation. Immunofluorescence shows PRPF40A relocalizes to mega speckles. [1]
Hinokiflavone does not inhibit RNA polymerase II transcription significantly: HeLa cells treated with 10–30 μM for 4 or 8 hr show little or no change in EU-labeled newly synthesized RNA compared to DRB control. [1]

Treatment of hinokiflavone at a tolerable and safe dose (50 mg/kg) significantly suppressed tumor growth in mice bearing CT26 tumors by reducing tumor proliferation and metastasis and inducing apoptosis. Mechanically, treatment of hinokiflavone induced apoptosis by loss of mitochondrial transmembrane potential and increased reactive oxygen species generation. Conclusions: Hinokiflavone suppressed colorectal tumor cell proliferation, induced apoptosis via the reactive oxygen species-mitochondria-mediated apoptotic pathway, and inhibited tumor cell migration and invasion. Antitumor activity of hinokiflavone was also validated in mice model without observed toxicity. Our findings suggested that the plant-derived hinokiflavone could be used as an antitumor agent against colorectal cancer[2].

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In vivo, the evaluation of anti-infective activity showed that hinokiflavone in combination with vancomycin effectively protected mice from MRSA-induced fatal pneumonia, which was more potent than vancomycin alone. As mentioned above, hinokiflavone, as an inhibitor of ClpP, could be further developed into a promising adjuvant against S. aureus infections[3].

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Cellular thermal shift assay[1]
To 1 ml of HeLa NE, either 50 µl of DMSO, or 50 µl of 10 mM hinokiflavone, was added and incubated at RT for 20 min. DMSO as well as hinokiflavone treated NE samples were then split into seven 100 µl aliquots. Each sample was incubated at a specific temperature (30°C, 33°C, 37°C, 40°C, 43°C, 47°C or 50°C) for 3 min, which was followed by 3 min incubation at RT. After ultracentrifugation at 35,000 rpm, 4°C for 20 min using a Optima MAX ultracentrifuge and a TLA 120.2 rotor, the supernatant was transferred to a new tube containing 25 µl 4x LDS buffer and boiled for 10 min at 95°C. The samples were subjected to western blot analysis with the indicated primary antibodies.

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Effect of Hinokiflavone on the protease activity of ClpP. [3]
A fluorescence resonance energy transfer (FRET)-based assay was used to screen for ClpP inhibitors based on the ability of ClpP to specifically cleave the fluorescent peptide substrate Suc-LY-AMC. The difference in RFU was used to define the enzymatic activity of ClpP and calculated the relative inhibitory activity. The compound was considered a potential inhibitor when the inhibitory activity was greater than 60%. The 100 μL reaction system consisting of 1 μΜ ClpP protein, various concentrations of hinokiflavone (0.5 to 256 μg/mL) and ClpP buffer (100 mM HEPES, 100 mM NaCl, pH = 7.0) were added to 96-well plates and incubated for 30 min, followed by the addition of the fluorescent substrate Suc-LY-AMC. After continuing incubation for 20 min, the activity of ClpP was determined by measuring excitation at 360 nm and emission at 465 nm using a microplate reader. Then the IC50 value of hinokiflavone was calculated. The reaction system containing an equal volume of DMSO was used as a negative control.

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Thermal shift assay (TSA). [3]
To determine the interaction between hinokiflavone and ClpP, a TSA assay was performed. ClpP protein (final concentration, 2 μM), SYPRO Orange, hinokiflavone and TSA buffer (150 mM NaCl, 10 mM HEPES, pH = 7.5) were added to a 96-well optical PCR plate. The mixture was heated at the rate of 1°C/min from 25 to 90°C by a real-time PCR instrument. This interaction was verified by observing the Tm shift after drug treatment. Tm was estimated as the temperature corresponding to 50% denaturation of the protein.

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Cellular thermal shift assay (CETSA). [3]
E. coli BL21(DE3) containing pET28a-ClpP plasmid was incubated until OD600 of 0.8, and 0.5 mM IPTG was added to induce ClpP protein expression. The supernatant was incubated with hinokiflavone and an equal volume of DMSO at 37°C for 1 h through centrifugation at 18,000 g for 20 min at 4°C. The reaction mixture was heated for 5 min at a temperature gradient of 25.0, 45.0, 51.2, 56.2, 61.4, and 65.0°C, respectively. It is worth noting that the mixture should be placed into ice water for 3 min immediately. Then gently centrifuged at 18,000g for 20 min to yield a supernatant and analyzed by SDS-PAGE. The samples were incubated with Coomassie brilliant blue G-250 stain, and the relative intensities of the indicated proteins were visualized using ImageJ software.

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Localized surface plasmon resonance (LSPR). [3]
The interaction between hinokiflavone and ClpP was performed by OpenSPR instruments. After a stable baseline is reached, PBS containing 1% DMSO (pH = 7.4) is first applied to enable activation of the chip surface. Following this, the ClpP protein was used as an analyte on the sensor chip. After stabilizing the reaction signal, and infusion of each concentration of hinokiflavone, the time for association and dissociation was 240 s, respectively. The results were finally analyzed by Trace Drawer and One To One.

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Gel-based SENP activity assay: Recombinant SENP1 fragment (amino acids 415-643) expressed in E. coli is used. Assay reactions (20 μl) contain 2 μl 10x reaction buffer (200 mM Hepes, 500 mM NaCl, 30 mM MgCl2, pH 7.5), 186 nM SENP1 fragment, 5 μM SUMOylated substrate (YFP-RANGAP aa 418-587-ECFP-SUMO2), and either 1 μl DMSO (control) or 1 μl compound dissolved in DMSO (final concentration 500 μM). Reactions are incubated at 37°C for 15 min, stopped by adding 5 μl 4x LDS loading buffer, heated at 70°C for 10 min, separated on 4-12% Tris-Bis PAGE gel, and visualized with Coomassie blue. [1]
DARTS (Drug Affinity Responsive Target Stability) assay: Recombinant SENP1 fragment (186 nM) in 20 μl SENP buffer (50 mM Tris-HCl, 150 mM NaCl) is incubated with either 1 μl DMSO or 1 μl compound (500 μM) at 4°C for 1 hr. Samples are then treated with different concentrations of pronase (16–200 μg/ml) for 30 min at room temperature. Reactions are stopped by adding 5 μl 4x LDS loading buffer, heated at 70°C for 10 min, separated by SDS-PAGE, and visualized with Coomassie blue. [1]
CETSA (Cellular Thermal Shift Assay): 1 ml HeLa nuclear extract is mixed with either 50 μl DMSO or 50 μl 10 mM Hinokiflavone (final 500 μM) and incubated at room temperature for 20 min. Samples are split into seven 100 μl aliquots, each incubated at a specific temperature (30, 33, 37, 40, 43, 47, or 50°C) for 3 min, followed by 3 min at room temperature. After ultracentrifugation at 35,000 rpm, 4°C for 20 min, supernatants are transferred, mixed with 25 μl 4x LDS buffer, boiled for 10 min at 95°C, and analyzed by western blot with indicated primary antibodies. [1]

Cell viability and cell colony formation assay[2]
For MTT assay, cells were seeded in 96-well culture plates and treated with hinokiflavone and then incubated with MTT solution. Formazan crystal was dissolved, and absorbance was measured by Spectra MAX M5 microplate spectrophotometer at 570 nm. For cell colony formation assay, cells were seeded in six-well plates and incubated with hinokiflavone for additional 12 days. The colonies were stained and counted under microscope.

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Hoechst staining and flow cytometry[2]
For Hoechst 33258 staining, cells were seeded in a six-well plate and incubated with hinokiflavone for 24 h and then fixed and stained with Hoechst 33258 solution. The nuclear morphology was observed and imagined by fluorescence microscopy). For flow cytometry (FCM), cells were harvested after incubated with hinokiflavone for 24 h and stained with Annexin V–fluorescein isothiocyanate/propidium iodide detection kit and then detected by flow cytometer.

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Mitochondrial membrane potential (ΔΨm) assay and reactive oxygen species detection[2]
Rh123 was used to determine the changes of mitochondrial transmembrane potential by FCM.24 Cells were harvested after incubated with hinokiflavone for 24 h, incubated with Rh123 solution, and then measured by FCM. To detect ROS level, cells were seeded and incubated with different concentration of hinokiflavone for 24 h, incubated with DCFH-DA, and then measured by FCM.25

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Boyden chamber migration and invasion assay[2]
Modified Boyden chamber migration and invasion assay was conducted as previously described.26, 27 For migration assay, cells in serum-free medium with hinokiflavone were added into the upper chamber, and medium containing 10% FBS and hinokiflavone was added at the bottom. For invasion assay, the upper surface of transwell membrane was coated with serum-free medium diluted with Matrigel, and then cells in serum-free medium containing hinokiflavone were added into the upper layer of the chamber, and the lower compartment of the chamber was filled with medium containing 10% FBS. Migrating and invasion cells were counted and photographed under a light microscope.[2]

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Determination of growth curves. [3]
The strain is kept in −20°C environment. Before experimenting, a single colony of USA300 and USA300 ΔclpP was picked for recovery after overnight incubation. The experiments are then carried out to ensure the activity of strains. Then the cultures were added to 2 mL of TSB medium at a ratio of 1:100, respectively. Hinokiflavone (64 μg/mL) or DMSO was also added. One hundred microliters of culture were used to measure absorbance values (OD600) at different time points.

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Determination of MIC. [3]
The MIC of hinokiflavone against S. aureus USA300 was determined by following the Clinical and Laboratory Standard Institute guidelines (CLSI). Briefly, 96-well plates containing hinokiflavone (0 to 512 μg/mL) and S. aureus USA300 (1 × 105 CFU) in 100 μL fresh CAMHB medium were incubated at 37°C for 16 to 20 h. The MIC was the lowest concentration at which no bacterium were found on visual observation.

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Cytotoxicity experiments. [3]
HEK293T cells were seeded into 96-well plates at 100 μL per well at a density of 5 × 104 cells/mL and cultured for 16 h. After adhering, various concentrations of hinokiflavone (0 to 256 μg/mL) were added to the 96-well plate. Cells were continued to grow in a 5% CO2 incubator at 37°C for 24 h. Subsequently, MTT solution was mixed to the medium at 10 μL/well and incubated for 4 h at 37°C. The culture medium was discarded, followed by the addition of 100 μL of DMSO. The absorbance at 490 nm was calculated using a microplate reader.

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Cell culture and treatment: HeLa, HEK293, and NB4 cells are cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, and 100 μg/ml streptomycin. Cells are treated with Hinokiflavone at concentrations 10, 20, 30 μM (NB4 also 0.5, 1, 2.5, 5 μM) or DMSO (control) for 24 hr (or shorter times as indicated). [1]
RNA isolation and RT-PCR: Total RNA is extracted using NucleoSpin RNA II Kit. 200 ng total RNA is reverse transcribed and amplified using One Step RT-PCR kit with primer pairs for ACTR1b, DXO, EIF4A2, HSP40, MCL1, NOP56, FAS, RIOK3. PCR products are separated on 1% agarose gels containing SYBR safe DNA gel stain. [1]
In vitro splicing assay (non-radioactive): Standard splicing reactions contain 30% HeLa nuclear extract, DMSO or compound, and in vitro transcribed Ad1 or HPV18 E6 pre-mRNA, incubated at 30°C for 90 min. Reactions are heat-inactivated at 95°C for 5 min, digested with proteinase K at 55°C for 30 min, and heat-inactivated again. Spliced and unspliced RNA are amplified using One step RT-PCR kit, products separated on agarose gels. [1]
Radioactive in vitro splicing and native gel electrophoresis: 32P-labeled pBsAd1 pre-mRNA is used. Splicing complexes are analyzed on 1.5% low melting point agarose gels and visualized by phosphor imaging. [1]
Immunofluorescence: Cells grown on coverslips are treated with compound or DMSO, fixed with 4% paraformaldehyde in PHEM buffer for 10 min at RT, permeabilized with 0.5% Triton X100 in PBS, incubated with primary antibodies (anti-SRSF2, U1A, DDX46, U2AF65, SF3B1, SR proteins, CDC5L, PLRG1, BCAS2, PRP19, CTNNBL1, snRNP200, coilin, SMN, TMG-cap, Y12, SNRPA1, CDK, fibrillarin, PML, CSTF2, SUMO1, SUMO2/3, PRPF40A) for 1 hr at RT, then with dye-conjugated secondary antibody for 30 min, stained with DAPI, mounted in Vectashield, and visualized by fluorescence microscopy. [1]
Fluorescence in situ hybridization (FISH) for poly(A) RNA: Cells treated with compound or DMSO are fixed with 4% paraformaldehyde, incubated in ice-cold methanol for 10 min, then 70% ethanol for 15 min, washed with Tris-HCl pH 8.0, hybridized overnight at 37°C with Cy3-labeled Oligo-dT(30) (1 ng/μl) in hybridization buffer (1 mg/ml yeast tRNA, 0.005% BSA, 10% dextran sulphate, 25% deionized formamide), washed with 4x SSC and 2x SSC, stained with DAPI, mounted, and visualized. [1]
Flow cytometry for cell cycle analysis: Cells seeded in 12-well plates are treated for 24 hr, harvested, washed with PBS, resuspended in cold 70% ethanol, fixed for 30 min at RT, washed twice with PBS, resuspended in PI stain solution (50 μg/ml propidium iodide and 100 mg/ml ribonuclease A in PBS), incubated for 30 min, and analyzed on a BD FACScalibur using FlowJo software. [1]
Pulse labeling of newly synthesized RNA with EU: HeLa cells treated with compound or DMSO for 4 or 8 hr are pulse-labeled with 5-ethynyluridine (EU) for 20 min, fixed, and detected using Click-iT RNA imaging Kit according to manufacturer’s instructions. [1]
Western blotting: Treated cells are harvested, washed with PBS, lysed in 1x LDS buffer, proteins separated on 4-12% NuPAGE Bis-Tris gels, transferred to nitrocellulose membranes, and detected with primary antibodies (anti-SUMO1, SUMO2/3, ubiquitin, NEDD8, SRSF1, PRPF40A, etc.) using chemiluminescent kit. [1]
Stable cell lines and immunoprecipitation: HEK293 cells stably expressing GFP-PRPF40A are generated by transfecting pEGFP-C3-PRPF40A and selecting with G418 (0.5 mg/ml). Cells treated with 20 μM Hinokiflavone or DMSO for 8 hr are lysed in Co-IP buffer (1 mM EDTA, 100 μM Na3VO4, 0.5% Triton X-100, 20 mM beta-Glycerol P) with protease inhibitors, centrifuged, incubated with GFP-Trap magnetic beads overnight at 4°C, washed, eluted with 1x LDS buffer, boiled, and analyzed by western blot. For YFP-SUMO2 IP, HeLa stable cells are lysed in RIPA buffer, homogenized, cleared, incubated with GFP-Trap beads, and processed similarly. [1]
SILAC proteomics for SUMO2 modification sites: HEK293 6His-SUMO2-T90K cells are cultured in DMEM lacking L-lysine, L-arginine, L-glutamine with 10% dialysed FBS, 1 mg/ml puromycin, and either natural or heavy stable isotope-labeled lysine/arginine. Heavy-labeled cells are treated with 20 μM Hinokiflavone for 8 hr, light-labeled with DMSO. Cells are harvested, mixed, lysed in 6 M guanidine hydrochloride buffer, sonicated, cleared, and His-tagged SUMO2 conjugates are purified on Ni2+-NTA agarose, eluted with imidazole, digested with Lys-C and Glu-C, and diGly-Lys-containing peptides are immunoaffinity purified using K-ε-GG-specific antibody coupled to protein A agarose. Peptides are analyzed by LC-MS/MS on Q Exactive Orbitrap, and data processed with MaxQuant. [1]

Mice model and toxicity evaluation[2]
Female BALB/c mice (6–8 weeks of age) were maintained in a specific pathogen-free condition facility. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. CT26 cells (0.5 × 106) were injected into the right flank of mice. When the tumor volume reached ~60mm3, mice were randomly divided and received intraperitoneally injection of hinokiflavone 25 and 50 mg/kg or vehicle once daily. Tumor volumes were calculated by the formula 0.52 × length × width2. Tumor inhibition rates (TIs) are calculated by TI = (Vcontrol − Vexperiment)∕Vcontrol. Internal organs and blood samples were collected after final treatment, and tumors were also collected and weighed (on day 24 after tumor cell inoculation). For toxicity assessment, related indices including body weight, diarrhea, anorexia, and other clinical symptoms were monitored continuously. Tissue samples (tumor, heart, liver, spleen, lung, and kidney) were examined by hematoxylin and eosin (HE) staining, and blood samples were subjected to biochemical and blood routine analyses. Pneumonia model experiment.[3]
Hinokiflavone was evaluated in vivo using a mouse model of pneumonia infection. Liaoning Changsheng Biologicals provided the mice as 6 weeks old male SPF grade C57BL/6J mice, weight 18 to 22 g. The mouse model of S. aureus pneumonia was constructed as previously described.[3]
To assess survival rates, mice were randomly divided into 7 groups of 10 mice. Each group was infected with 2 × 108 CFU/30 μL S. aureus. One hour after inoculating, hinokiflavone (100 mg/kg/d) was injected subcutaneously every 12 h to assess the survival rate at 96 h. After 2 h of infection, a specific dosage of hinokiflavone (100 mg/kg·d−1), vancomycin (100 mg/kg·d−1), or the combination of hinokiflavone (100 mg/kg·d−1) and vancomycin (100 mg/kg·d−1) was administered through the subcutaneous injection route for hinokiflavone and the intraperitoneal route for the antibiotic.

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Hinokiflavone induces cell cycle arrest and apoptosis in human cell lines in a concentration-dependent manner. In HeLa and HEK293 cells, treatment with 10-30 μM for 24 hr causes cell cycle arrest; NB4 cells are most sensitive, becoming mostly apoptotic after 24 hr exposure to 10 μM Hinokiflavone. [1]

[1]. Characterisation of the biflavonoid hinokiflavone as a pre-mRNA splicing modulator that inhibits SENP. Elife. 2017 Sep 8;6. pii: e27402.

Juniper flavonoids are biflavonoid compounds with a structure in which apigenin is substituted at the 6-position with a 4-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)phenoxy group. It is a biflavonoid ether isolated from Rhus succedanea and has been found to possess significant cytotoxicity. It functions as a neuroprotective agent, antitumor agent, and metabolite. It is a biflavonoid compound, an aromatic ether, and a hydroxyflavonoid. Its function is related to apigenin. Juniper flavonoids have also been reported in Garcinia multiflora, Rhus punjabensis, and several other organisms with relevant data.

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Hinokiflavone is a biflavonoid (biapigenin) found in many plant families. It has been previously suggested by in silico screens to target prostaglandin D2 synthetase and matrix metalloproteinase-9, but this study shows it inhibits SUMO protease SENP1. Hinokiflavone does not significantly inhibit any of 120 different purified kinases tested in vitro (weak, non-specific effects only). The compound increases SUMOylation specifically (not ubiquitin or NEDD8). Chemically synthesized Hinokiflavone is spectroscopically identical to natural isolates and shows same splicing modulation activity, confirming it as the active molecule. Hinokiflavone treatment changes MCL1 alternative splicing to favor pro-apoptotic isoform MCL1-S, similar to SF3B1 inhibitors. The NB4 cell line (acute promyelocytic leukemia expressing PML-RARα fusion) shows extreme sensitivity to Hinokiflavone-induced apoptosis, possibly due to increased PML SUMOylation. The study suggests Hinokiflavone or its derivatives could be developed as novel cancer therapeutics. [1]

C30H18O10

538.4579

538.09

C, 66.92; H, 3.37; O, 29.71

19202-36-9

5281627

Light yellow to yellow solid

1.6±0.1 g/cm3

841.5±65.0 °C at 760 mmHg

353-355ºC

284.8±27.8 °C

0.0±3.2 mmHg at 25°C

1.771

4.33

5

10

4

40

1020

0

O1C(C2C([H])=C([H])C(=C([H])C=2[H])O[H])=C([H])C(C2C(=C(C(=C([H])C1=2)O[H])OC1C([H])=C([H])C(=C([H])C=1[H])C1=C([H])C(C2=C(C([H])=C(C([H])=C2O1)O[H])O[H])=O)O[H])=O

WTDHMFBJQJSTMH-UHFFFAOYSA-N

InChI=1S/C30H18O10/c31-16-5-1-14(2-6-16)24-12-21(35)28-26(40-24)13-22(36)30(29(28)37)38-18-7-3-15(4-8-18)23-11-20(34)27-19(33)9-17(32)10-25(27)39-23/h1-13,31-33,36-37H

6-[4-(5,7-dihydroxy-4-oxochromen-2-yl)phenoxy]-5,7-dihydroxy-2-(4-hydroxyphenyl)chromen-4-one

4',6''-O-Biapigenin; Hinokiflavone; 19202-36-9; 4',6''-O-Biapigenin; GFF5VYC4NB; 4H-1-benzopyran-4-one, 6-[4-(5,7-dihydroxy-4-oxo-4H-1-benzopyran-2-yl)phenoxy]-5,7-dihydroxy-2-(4-hydroxyphenyl)-; 6-[4-(5,7-dihydroxy-4-oxochromen-2-yl)phenoxy]-5,7-dihydroxy-2-(4-hydroxyphenyl)chromen-4-one; CHEBI:5721; 4H-1-Benzopyran-4-one, 6-(4-(5,7-dihydroxy-4-oxo-4H-1-benzopyran-2-yl)phenoxy)-5,7-dihydroxy-2-(4-hydroxyphenyl)-; Hinokiflavone

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~50 mg/mL (~92.86 mM)
Ethanol : ~2 mg/mL (~3.71 mM)

Solubility in Formulation 1: ≥ 0.83 mg/mL (1.54 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 + to the above solution and mix evenly; then add 450 μL of 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: 10 mg/mL (18.57 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.)