In pooled human liver microsomes, the reported CYP2C19 IC50 was 63.8 µM; IC50 values for CYP1A2, CYP2C8, CYP2C9, CYP2D6 and CYP3A4 were >100 µM. These findings describe the tested enzyme activities and do not establish clinical drug-interaction safety. [3]
Scutellarin at 4.1–1000 µM remained below the positive-inhibition threshold in a WM-266-4 calcein-AM P-glycoprotein screen. The threshold was 25% of the response to 100 µM verapamil. [3]
Scutellarin reduced IL-1β-induced inflammatory responses in primary mouse chondrocytes. COX-2 and iNOS protein reductions were nonsignificant at 15 µM but evident at 30–60 µM; the 15 µM IL-6 response was also nonsignificant. A separate viability assay showed reduced viability at 120 µM after 48 hours. [2]
Human liver microsomal CYP inhibition
Biochemical assay · Pooled human liver microsomes from 50 donors, lot 32556; 0.3 mg/mL protein. [3]
Scutellarin weakly inhibited CYP2C19; the other five tested human CYPs did not reach 50% inhibition within the tested range. These endpoints do not establish clinical interaction safety.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0.03, 0.1, 0.3, 1.0, 3.0, 10, 30 and 100 µM
- Exposure duration
- 15 min preincubation at 37°C; 30 min reaction after NADP+ addition.
- Method
- Probe-substrate cocktail assay; metabolite formation measured by LC-MS/MS.
- Co-treatment
- Probe substrates: phenacetin, paclitaxel, diclofenac, S-mephenytoin, dextromethorphan and midazolam. Isoform-specific inhibitors served as positive controls.
- Reported limits
- Negative-control solvent composition is not specified in the retrieved microsomal protocol.
Methods and source locations
Compound reported in the paper: Scutellarin; Compound label in the paper: scutellarin
Applies to the reported research material and experimental model; no Adooq lot validation or salt/form conversion.
Results 2.1; Figure 3; Methods 3.1, 3.3.1–3.3.2 and 3.5; Table 2
Inhibition of CYP2C19-mediated S-mephenytoin metabolism: LC-MS/MS metabolite assay; nonlinear regression. — Results 2.1; Figure 3; Methods 3.3 and 3.5
CYP1A2, CYP2C8, CYP2C9, CYP2D6 and CYP3A4 inhibition: LC-MS/MS probe-metabolite formation. — Results 2.1; Figure 3; Methods 3.3 and 3.5
Negative P-glycoprotein inhibition screen
Cell-based study · Human WM-266-4 melanoma cells. [3]
Scutellarin did not reach the assay positivity threshold for P-glycoprotein inhibition. This is a cell-based calcein-AM screening result, not a general exclusion of transporter interactions.
Literature-Reported Treatment Conditions
- Treatment concentration
- 4.1–1000 µM
- Exposure duration
- 30 min scutellarin incubation, then another 30 min with calcein-AM.
- Method
- Calcein-AM fluorescence assay.
- Vehicle
- 1% DMSO negative control.
- Co-treatment
- Calcein-AM 1 µM; verapamil 100 µM positive control.
- Reported limits
- Intermediate concentration labels differ slightly between Figure 2/caption and Methods 3.4; only the common range is retained.
Methods and source locations
Compound reported in the paper: Scutellarin; Compound label in the paper: scutellarin
Applies to the reported research material and experimental model; no Adooq lot validation or salt/form conversion.
Results 2.3; Figure 2; Methods 3.1, 3.4–3.5
Calcein-AM response relative to verapamil 100 µM: Fluorescence at excitation/emission 494/517 nm. Assay criterion: ≥25% is positive; positive-control signal increase >5-fold defines validity. — Results 2.3; Figure 2 caption; Methods 3.4–3.5
PDK2 kinase assay versus dichloroacetic acid
Biochemical assay · Full-length human GST-PDK2; mobility-shift/IMAP phosphorylation assay. [1]
Scutellarin produced greater PDK2 inhibition than dichloroacetic acid at the two concentrations plotted. This comparison supplies no IC50 and does not establish selectivity across the PDK family.
Literature-Reported Treatment Conditions
- Treatment concentration
- 10 and 100 µmol/L (Figure 8D labels).
- Method
- Mobility-shift/IMAP measurement of phosphorylated and nonphosphorylated substrate.
- Co-treatment
- Dichloroacetic acid (DCA) positive comparator at matched plotted concentrations.
- Reported limits
- Main-text assay description does not specify reaction duration, ATP/substrate concentrations or vehicle. SI was not retrieved. PDK4 is named in Methods, but no PDK4 result is adopted.
Methods and source locations
Compound reported in the paper: Scutellarin; Compound label in the paper: SG
Applies to the reported research material and experimental model; no Adooq lot validation or salt/form conversion.
Target-characterization Results section 2.7; Figure 8D; Experimental Section: Materials; Mobility Shift Assay for Kinase Activity Evaluation
PDK2 substrate phosphorylation: Mobility-shift/IMAP assay; figure caption describes mean ± SEM. — Figure 8D and caption; Results section 2.7; Mobility Shift Assay for Kinase Activity Evaluation
Inflammatory and pathway responses in primary chondrocytes
Cell-based study · Second-passage primary knee-articular chondrocytes from 2-week-old C57BL/6 mice. [2]
Scutellarin reduced IL-1β-associated inflammatory readouts and nuclear p65 accumulation, with increased nuclear Nrf2 and HO-1 at 60 µM. Some low-dose inflammatory effects were nonsignificant. These are pathway associations rather than demonstrated direct Nrf2 binding.
Literature-Reported Treatment Conditions
- Treatment concentration
- 15, 30 and 60 µM for inflammatory and NF-κB assays; 60 µM for Nrf2/HO-1 panels. Separate viability controls: 15–120 µM.
- Exposure duration
- Experimental Design specifies 24 h IL-1β exposure after SCU pretreatment; inflammatory Results also describe 24 h SCU pretreatment. NF-κB Results describe a 2 h IL-1β response. Viability assessed at 24 and 48 h.
- Method
- qRT-PCR, Western blot, immunofluorescence and soluble-mediator assays; CCK-8 viability controls.
- Co-treatment
- IL-1β 10 ng/mL; untreated and IL-1β-only controls; Figure 5 also includes SCU alone.
- Reported limits
- A unified pretreatment interval is not unambiguously specified across assays. Cell-treatment solvent/concentration not specified; reagent list mentions DMSO (printed DMOS).
Methods and source locations
Compound reported in the paper: Scutellarin; Compound label in the paper: SCU
Applies to the reported research material and experimental model; no Adooq lot validation or salt/form conversion.
Figures 1B–C, 2, 4 and 5; corresponding Results; Methods: Reagents, Primary Mouse Chondrocyte Culture, Experimental Design, CCK-8, immunofluorescence, Western blot, qRT-PCR and statistics
IL-1β-induced COX-2 and iNOS protein expression: Western blot; β-actin loading control. — Inflammatory-mediator Results; Figure 2B
IL-6 response at 15 µM scutellarin: Soluble-mediator assay described as ELISA in Figure 2D caption. — Inflammatory-mediator Results; Figure 2D
Nuclear p65, cytoplasmic IκBα, nuclear Nrf2 and cytoplasmic HO-1: Western blots with Lamin B1/β-actin controls and immunofluorescence. — Figures 4–5; NF-κB and Nrf2/HO-1 Results
Chondrocyte viability after scutellarin alone: CCK-8; caption n=5, mean ± SD; Methods says triplicate experiments. — Figure 1B–C and caption; CCK-8 Methods
