Cat's-claw (Uncaria tomentosa)
Identification of PXR activators from Uncaria tomentosa (Cat’s Claw) CSV JSON

DPX2 cells (~ 60,000 cells/well) were seeded on 96-well plate (clear bottom and white wall). After 24 h incubation in 37 °C incubator (5% CO2), cells were treated with herbal extracts or chemicals in dosing medium for another 24 h. Each chemical tested was dissolved in DMSO to prepare a stock solution (10 mM). The final concentration of DMSO was 1‰ in control and treatment groups. Cell viability was detected by replacing the medium with 100 µl of CellTiter-FluroTM reagent, incubating for 1 h and measuring fluorescence (RFU) with an excitation wavelength of 390 nm and emission wavelength of 505 nm. Afterward, 100 µl ONE-GloTM assay reagent was added into each well. Plate was agitated carefully, and then luminescence (RLU) was detected 5 min later. The efficacy of PXR activation was calculated by the ratio of RLU/RFU for each well. Both RLU and RFU were measured by BioTekTM Synergy TM H1 Hybrid Reader. The half maximal effective concentration (EC50) was determined from the dose-response curve using GraphPad prism.

For the analysis of CYP3A4 expression, ~500,000 DPX2 cells were seeded on a 12-well plate and cultured for 24 h. Cells were treated with herbal extracts or chemicals for another 24 h and then washed with PBS. Total RNA of the cells was extracted using TRIzolTM Reagent and 2 µg of them was used for reverse transcription. Quantitative RTPCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems by Thermo Fisher Scientific, Warrington, UK). mRNA levels of CYP3A4 were normalized to Gapdh.

DPX2 cells (~ 60,000 cells/well) were seeded on 96-well plate (clear bottom and white wall). After 24 h incubation in 37 °C incubator (5% CO2), cells were treated with herbal extracts or chemicals in dosing medium for another 24 h. Each chemical tested was dissolved in DMSO to prepare a stock solution (10 mM). The final concentration of DMSO was 1‰ in control and treatment groups. Cell viability was detected by replacing the medium with 100 µl of CellTiter-FluroTM reagent, incubating for 1 h and measuring fluorescence (RFU) with an excitation wavelength of 390 nm and emission wavelength of 505 nm. Afterward, 100 µl ONE-GloTM assay reagent was added into each well. Plate was agitated carefully, and then luminescence (RLU) was detected 5 min later. The efficacy of PXR activation was calculated by the ratio of RLU/RFU for each well. Both RLU and RFU were measured by BioTekTM Synergy TM H1 Hybrid Reader. The half maximal effective concentration (EC50) was determined from the dose-response curve using GraphPad prism.

DPX2 cells (~ 60,000 cells/well) were seeded on 96-well plate (clear bottom and white wall). After 24 h incubation in 37 °C incubator (5% CO2), cells were treated with herbal extracts or chemicals in dosing medium for another 24 h. Each chemical tested was dissolved in DMSO to prepare a stock solution (10 mM). The final concentration of DMSO was 1‰ in control and treatment groups. Cell viability was detected by replacing the medium with 100 µl of CellTiter-FluroTM reagent, incubating for 1 h and measuring fluorescence (RFU) with an excitation wavelength of 390 nm and emission wavelength of 505 nm. Afterward, 100 µl ONE-GloTM assay reagent was added into each well. Plate was agitated carefully, and then luminescence (RLU) was detected 5 min later. The efficacy of PXR activation was calculated by the ratio of RLU/RFU for each well. Both RLU and RFU were measured by BioTekTM Synergy TM H1 Hybrid Reader. The half maximal effective concentration (EC50) was determined from the dose-response curve using GraphPad prism.

DPX2 cells (~ 60,000 cells/well) were seeded on 96-well plate (clear bottom and white wall). After 24 h incubation in 37 °C incubator (5% CO2), cells were treated with herbal extracts or chemicals in dosing medium for another 24 h. Each chemical tested was dissolved in DMSO to prepare a stock solution (10 mM). The final concentration of DMSO was 1‰ in control and treatment groups. Cell viability was detected by replacing the medium with 100 µl of CellTiter-FluroTM reagent, incubating for 1 h and measuring fluorescence (RFU) with an excitation wavelength of 390 nm and emission wavelength of 505 nm. Afterward, 100 µl ONE-GloTM assay reagent was added into each well. Plate was agitated carefully, and then luminescence (RLU) was detected 5 min later. The efficacy of PXR activation was calculated by the ratio of RLU/RFU for each well. Both RLU and RFU were measured by BioTekTM Synergy TM H1 Hybrid Reader. The half maximal effective concentration (EC50) was determined from the dose-response curve using GraphPad prism.

DPX2 cells (~ 60,000 cells/well) were seeded on 96-well plate (clear bottom and white wall). After 24 h incubation in 37 °C incubator (5% CO2), cells were treated with herbal extracts or chemicals in dosing medium for another 24 h. Each chemical tested was dissolved in DMSO to prepare a stock solution (10 mM). The final concentration of DMSO was 1‰ in control and treatment groups. Cell viability was detected by replacing the medium with 100 µl of CellTiter-FluroTM reagent, incubating for 1 h and measuring fluorescence (RFU) with an excitation wavelength of 390 nm and emission wavelength of 505 nm. Afterward, 100 µl ONE-GloTM assay reagent was added into each well. Plate was agitated carefully, and then luminescence (RLU) was detected 5 min later. The efficacy of PXR activation was calculated by the ratio of RLU/RFU for each well. Both RLU and RFU were measured by BioTekTM Synergy TM H1 Hybrid Reader. The half maximal effective concentration (EC50) was determined from the dose-response curve using GraphPad prism.

DPX2 cells (~ 60,000 cells/well) were seeded on 96-well plate (clear bottom and white wall). After 24 h incubation in 37 °C incubator (5% CO2), cells were treated with herbal extracts or chemicals in dosing medium for another 24 h. Each chemical tested was dissolved in DMSO to prepare a stock solution (10 mM). The final concentration of DMSO was 1‰ in control and treatment groups. Cell viability was detected by replacing the medium with 100 µl of CellTiter-FluroTM reagent, incubating for 1 h and measuring fluorescence (RFU) with an excitation wavelength of 390 nm and emission wavelength of 505 nm. Afterward, 100 µl ONE-GloTM assay reagent was added into each well. Plate was agitated carefully, and then luminescence (RLU) was detected 5 min later. The efficacy of PXR activation was calculated by the ratio of RLU/RFU for each well. Both RLU and RFU were measured by BioTekTM Synergy TM H1 Hybrid Reader. The half maximal effective concentration (EC50) was determined from the dose-response curve using GraphPad prism.

For the analysis of CYP3A4 expression, ~500,000 DPX2 cells were seeded on a 12-well plate and cultured for 24 h. Cells were treated with herbal extracts or chemicals for another 24 h and then washed with PBS. Total RNA of the cells was extracted using TRIzolTM Reagent and 2 µg of them was used for reverse transcription. Quantitative RTPCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems by Thermo Fisher Scientific, Warrington, UK). mRNA levels of CYP3A4 were normalized to Gapdh.

CYP3A4 mRNA level (% Rifampicin): 175 ± 2 (mean ± SEM)
p-value < 0.0001

For the analysis of CYP3A4 expression, ~500,000 DPX2 cells were seeded on a 12-well plate and cultured for 24 h. Cells were treated with herbal extracts or chemicals for another 24 h and then washed with PBS. Total RNA of the cells was extracted using TRIzolTM Reagent and 2 µg of them was used for reverse transcription. Quantitative RTPCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems by Thermo Fisher Scientific, Warrington, UK). mRNA levels of CYP3A4 were normalized to Gapdh.

CYP3A4 mRNA level (% Rifampicin): 160 ± 30 (mean ± SEM) 
p-value < 0.0001

For the analysis of CYP3A4 expression, ~500,000 DPX2 cells were seeded on a 12-well plate and cultured for 24 h. Cells were treated with herbal extracts or chemicals for another 24 h and then washed with PBS. Total RNA of the cells was extracted using TRIzolTM Reagent and 2 µg of them was used for reverse transcription. Quantitative RTPCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems by Thermo Fisher Scientific, Warrington, UK). mRNA levels of CYP3A4 were normalized to Gapdh.

PXR activation (% Rifampicin): 110 ± 0.5 (mean ± SEM) 
p-value < 0.0001

DPX2 cells (~ 60,000 cells/well) were seeded on 96-well plate (clear bottom and white wall). After 24 h incubation in 37 °C incubator (5% CO2), cells were treated with herbal extracts or chemicals in dosing medium for another 24 h. Each chemical tested was dissolved in DMSO to prepare a stock solution (10 mM). The final concentration of DMSO was 1‰ in control and treatment groups. Cell viability was detected by replacing the medium with 100 µl of CellTiter-FluroTM reagent, incubating for 1 h and measuring fluorescence (RFU) with an excitation wavelength of 390 nm and emission wavelength of 505 nm. Afterward, 100 µl ONE-GloTM assay reagent was added into each well. Plate was agitated carefully, and then luminescence (RLU) was detected 5 min later. The efficacy of PXR activation was calculated by the ratio of RLU/RFU for each well. Both RLU and RFU were measured by BioTekTM Synergy TM H1 Hybrid Reader. The half maximal effective concentration (EC50) was determined from the dose-response curve using GraphPad prism.

For the analysis of CYP3A4 expression, ~500,000 DPX2 cells were seeded on a 12-well plate and cultured for 24 h. Cells were treated with herbal extracts or chemicals for another 24 h and then washed with PBS. Total RNA of the cells was extracted using TRIzolTM Reagent and 2 µg of them was used for reverse transcription. Quantitative RTPCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems by Thermo Fisher Scientific, Warrington, UK). mRNA levels of CYP3A4 were normalized to Gapdh.

DPX2 cells (~ 60,000 cells/well) were seeded on 96-well plate (clear bottom and white wall). After 24 h incubation in 37 °C incubator (5% CO2), cells were treated with herbal extracts or chemicals in dosing medium for another 24 h. Each chemical tested was dissolved in DMSO to prepare a stock solution (10 mM). The final concentration of DMSO was 1‰ in control and treatment groups. Cell viability was detected by replacing the medium with 100 µl of CellTiter-FluroTM reagent, incubating for 1 h and measuring fluorescence (RFU) with an excitation wavelength of 390 nm and emission wavelength of 505 nm. Afterward, 100 µl ONE-GloTM assay reagent was added into each well. Plate was agitated carefully, and then luminescence (RLU) was detected 5 min later. The efficacy of PXR activation was calculated by the ratio of RLU/RFU for each well. Both RLU and RFU were measured by BioTekTM Synergy TM H1 Hybrid Reader. The half maximal effective concentration (EC50) was determined from the dose-response curve using GraphPad prism.

For the analysis of CYP3A4 expression, ~500,000 DPX2 cells were seeded on a 12-well plate and cultured for 24 h. Cells were treated with herbal extracts or chemicals for another 24 h and then washed with PBS. Total RNA of the cells was extracted using TRIzolTM Reagent and 2 µg of them was used for reverse transcription. Quantitative RTPCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems by Thermo Fisher Scientific, Warrington, UK). mRNA levels of CYP3A4 were normalized to Gapdh.

DPX2 cells (~ 60,000 cells/well) were seeded on 96-well plate (clear bottom and white wall). After 24 h incubation in 37 °C incubator (5% CO2), cells were treated with herbal extracts or chemicals in dosing medium for another 24 h. Each chemical tested was dissolved in DMSO to prepare a stock solution (10 mM). The final concentration of DMSO was 1‰ in control and treatment groups. Cell viability was detected by replacing the medium with 100 µl of CellTiter-FluroTM reagent, incubating for 1 h and measuring fluorescence (RFU) with an excitation wavelength of 390 nm and emission wavelength of 505 nm. Afterward, 100 µl ONE-GloTM assay reagent was added into each well. Plate was agitated carefully, and then luminescence (RLU) was detected 5 min later. The efficacy of PXR activation was calculated by the ratio of RLU/RFU for each well. Both RLU and RFU were measured by BioTekTM Synergy TM H1 Hybrid Reader. The half maximal effective concentration (EC50) was determined from the dose-response curve using GraphPad prism.

For the analysis of CYP3A4 expression, ~500,000 DPX2 cells were seeded on a 12-well plate and cultured for 24 h. Cells were treated with herbal extracts or chemicals for another 24 h and then washed with PBS. Total RNA of the cells was extracted using TRIzolTM Reagent and 2 µg of them was used for reverse transcription. Quantitative RTPCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems by Thermo Fisher Scientific, Warrington, UK). mRNA levels of CYP3A4 were normalized to Gapdh.

Midazolam was utilized as a substrate for CYP3A4 activity assay (Olsen et al., 2016). Briefly, ~250,000 DPX2 cells were seeded on a 24-well plate and cultured for 24 h. After treatment with herbal extracts or chemicals for another 24 h, cells were washed with PBS. Midazolam (50 µM) in fresh medium was added and incubated for 2 h. Afterward, 100 µl medium was collected from each well, and mixed with 100 µl cold acetonitrile. The mixtures were centrifuged at 13,000 rpm for 15 min, and the supernatant was collected for analysis of 1'- OH-MDZ, a CYP3A4-mediated metabolite of midazolam.

Midazolam was utilized as a substrate for CYP3A4 activity assay (Olsen et al., 2016). Briefly, ~250,000 DPX2 cells were seeded on a 24-well plate and cultured for 24 h. After treatment with herbal extracts or chemicals for another 24 h, cells were washed with PBS. Midazolam (50 µM) in fresh medium was added and incubated for 2 h. Afterward, 100 µl medium was collected from each well, and mixed with 100 µl cold acetonitrile. The mixtures were centrifuged at 13,000 rpm for 15 min, and the supernatant was collected for analysis of 1'- OH-MDZ, a CYP3A4-mediated metabolite of midazolam.

Midazolam was utilized as a substrate for CYP3A4 activity assay (Olsen et al., 2016). Briefly, ~250,000 DPX2 cells were seeded on a 24-well plate and cultured for 24 h. After treatment with herbal extracts or chemicals for another 24 h, cells were washed with PBS. Midazolam (50 µM) in fresh medium was added and incubated for 2 h. Afterward, 100 µl medium was collected from each well, and mixed with 100 µl cold acetonitrile. The mixtures were centrifuged at 13,000 rpm for 15 min, and the supernatant was collected for analysis of 1'- OH-MDZ, a CYP3A4-mediated metabolite of midazolam.

Midazolam was utilized as a substrate for CYP3A4 activity assay (Olsen et al., 2016). Briefly, ~250,000 DPX2 cells were seeded on a 24-well plate and cultured for 24 h. After treatment with herbal extracts or chemicals for another 24 h, cells were washed with PBS. Midazolam (50 µM) in fresh medium was added and incubated for 2 h. Afterward, 100 µl medium was collected from each well, and mixed with 100 µl cold acetonitrile. The mixtures were centrifuged at 13,000 rpm for 15 min, and the supernatant was collected for analysis of 1'- OH-MDZ, a CYP3A4-mediated metabolite of midazolam.

Midazolam was utilized as a substrate for CYP3A4 activity assay (Olsen et al., 2016). Briefly, ~250,000 DPX2 cells were seeded on a 24-well plate and cultured for 24 h. After treatment with herbal extracts or chemicals for another 24 h, cells were washed with PBS. Midazolam (50 µM) in fresh medium was added and incubated for 2 h. Afterward, 100 µl medium was collected from each well, and mixed with 100 µl cold acetonitrile. The mixtures were centrifuged at 13,000 rpm for 15 min, and the supernatant was collected for analysis of 1'- OH-MDZ, a CYP3A4-mediated metabolite of midazolam.

Midazolam was utilized as a substrate for CYP3A4 activity assay (Olsen et al., 2016). Briefly, ~250,000 DPX2 cells were seeded on a 24-well plate and cultured for 24 h. After treatment with herbal extracts or chemicals for another 24 h, cells were washed with PBS. Midazolam (50 µM) in fresh medium was added and incubated for 2 h. Afterward, 100 µl medium was collected from each well, and mixed with 100 µl cold acetonitrile. The mixtures were centrifuged at 13,000 rpm for 15 min, and the supernatant was collected for analysis of 1'- OH-MDZ, a CYP3A4-mediated metabolite of midazolam.

Midazolam was utilized as a substrate for CYP3A4 activity assay (Olsen et al., 2016). Briefly, ~250,000 DPX2 cells were seeded on a 24-well plate and cultured for 24 h. After treatment with herbal extracts or chemicals for another 24 h, cells were washed with PBS. Midazolam (50 µM) in fresh medium was added and incubated for 2 h. Afterward, 100 µl medium was collected from each well, and mixed with 100 µl cold acetonitrile. The mixtures were centrifuged at 13,000 rpm for 15 min, and the supernatant was collected for analysis of 1'- OH-MDZ, a CYP3A4-mediated metabolite of midazolam.

The chemical profiling of Cat's claw extracts was conducted using ultra-performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS, Waters, Milford, MA). An UPLC BEH C-18 column was utilized for chemical separation. The mobile phase was aqueous acetonitrile containing 0.1% formic acid and was delivered in a gradient elution from 2% to 98% acetonitrile. QTOFMS was performed in a positive mode. 1'-OH-MDZ was also analyzed by UPLC-QTOFMS using the same column. The UPLC-QTOFMS data for herbal extracts were collected using MassLynx 4.1 (Waters Corp., Milford, MA) and further analyzed by orthogonal partial least-squares discriminant analysis using SIMCA-P (Umetrics, Kinnelon, NJ).