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  • 1 Northwest A&F University, Yangling, Shaanxi 712100, China
  • 2 Fourth Military Medical University, Xi'an, China
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A simple, sensitive, and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for determination of phloretin in dog plasma using darunavir as internal standard. The phloretin was separated by the Inertsil® ODS3 C18 column (150 mm × 4.6 mm, 5 μm) and determined by LC–MS/MS. The electrospray ionization (ESI) source was operated in negative ionization mode for phloretin and positive ionization mode for darunavir (internal standard, IS). The multiple reaction monitoring (MRM) transitions were chosen to be m/z 273.0 → m/z 148.9 for phloretin, m/z 443.2 → m/z 401.0 for 2′,4′,6′,4-tetra-acetylphloretin and m/z 548.1 → m/z 69.1 for IS. The method was validated for accuracy, precision, linearity, range, selectivity, lower limit of quantification (LLOQ), recovery, and matrix effect. All validation parameters met the acceptance criteria according to regulatory guidelines. 2′,4′,6′,4-Tetra-acetylphloretin, as a prodrug of phloretin, is more stable than phloretin (PH) in vitro, protecting phenolic hydroxy from being oxygenated. The method had been successfully applied to a pharmacokinetic study of administration of phloretin and 2′,4′,6′,4-tetra-acetylphloretin in beagle dogs. Significant differences of tmax, Cmax, and area under the plasma concentration curve (AUC) were observed between phloretin and 2′,4′,6′,4-tetra-acetylphloretin.

Abstract

A simple, sensitive, and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for determination of phloretin in dog plasma using darunavir as internal standard. The phloretin was separated by the Inertsil® ODS3 C18 column (150 mm × 4.6 mm, 5 μm) and determined by LC–MS/MS. The electrospray ionization (ESI) source was operated in negative ionization mode for phloretin and positive ionization mode for darunavir (internal standard, IS). The multiple reaction monitoring (MRM) transitions were chosen to be m/z 273.0 → m/z 148.9 for phloretin, m/z 443.2 → m/z 401.0 for 2′,4′,6′,4-tetra-acetylphloretin and m/z 548.1 → m/z 69.1 for IS. The method was validated for accuracy, precision, linearity, range, selectivity, lower limit of quantification (LLOQ), recovery, and matrix effect. All validation parameters met the acceptance criteria according to regulatory guidelines. 2′,4′,6′,4-Tetra-acetylphloretin, as a prodrug of phloretin, is more stable than phloretin (PH) in vitro, protecting phenolic hydroxy from being oxygenated. The method had been successfully applied to a pharmacokinetic study of administration of phloretin and 2′,4′,6′,4-tetra-acetylphloretin in beagle dogs. Significant differences of tmax, Cmax, and area under the plasma concentration curve (AUC) were observed between phloretin and 2′,4′,6′,4-tetra-acetylphloretin.

Introduction

Apple polyphenols are naturally occurring plant compounds that act as potent antioxidants [1, 2]. In addition, apple polyphenols also can provide cardiovascular protection [3, 4], prevent dental caries [5], and have anti-inflammatory effects [2, 6], with lots of developing potential [79]. Phloretin (2′,4′,6′-trihydroxy-3-(4-hydroxyphenyl)-propiophenone, PH, Figure 1A) is a dihydrochalcone, a main component of apple polyphenols. It can be found in apple tree leaves [10], pear, and strawberries [11].

Figure 1.
Figure 1.

Chemical structures of PH (A), TAPH (B), and darunavir (IS) (C)

Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2016.00097

The unique chemical structure of PH determines its strong antioxidant activity. PH displayed a potent antioxidant activity in peroxynitrite scavenging and the inhibition of lipid peroxidation. The research revealed that the antioxidant pharmacophore of PH is 2,6-dihydroxyacetophenone [12]. De Jonge et al. [13] found that PH was an oxidative phosphorylation uncoupler and inhibitor in 1983. Both NADH- and succinate-dependent respiration were inhibited by PH. Glutamic acid, succinic acid, and ascorbic acid oxidation in mitochondria were inhibited by PH. Studies have shown that agents with antioxidant activity can effectively protect the skin from damage which caused by free radicals. It was confirmed that PH has a strong activity of antioxidant and inhibition of tyrosinase, so the PH was widely used in the field of cosmetics to eliminate skin wrinkles, reduce the appearance of lines and pigmentation, prevent skin damage caused by UV rays, and whiten skin [1416].

Besides its potent antioxidant property, PH was known to be a competitive inhibitor of sodium d-glucose cotransporter [17]. Wilbrandt and Rosenberg [18] have found that transport of glucose in human erythrocytes containing a certain concentration of PH was inhibited. PH could also play a role in immune suppression by adjusting the level of pro-inflammatory cytokines [19, 20]. For PH, a lot of research suggested that PH may be useful for cancer chemotherapy and chemoprevention [2125]. PH has also some other biological functions. Fat cells are closely related to human health. The dysfunction of fat cells can lead to obesity, diabetes, cardiovascular disease, etc. However, PH has an effect on protein-coding genes including fat cytokine, adiponectin, and its receptors, thereby affecting the fat cells to generate and store triglycerides, to achieve the purpose of disease prevention and cure [26, 27].

Due to the therapeutic effects of PH, the pharmacokinetic studies of PH are essential. Some previous studies have shown that the poor bioavailability of apple polyphenols is mainly due to the wide range of phase II metabolism in vivo [2831]. Kahle et al. [32] assessed phloretin content in apple juices through high-performance liquid chromatography (HPLC) using photodiode array detection and gradient elution. Andrews et al. [33] reported an analytical method for the determination of phloretin in rat serum using HPLC. Lijia et al. [34] reported an analytical method for the determination of phlorizin and phloretin in human plasma using ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). In the present study, we established a simple, sensitive, and rapid liquid chromatography (LC)–MS/MS method and validated for determination of PH in dog plasma using darunavir as internal standard. It was successfully applied to the pharmacokinetic study of PH after oral administration to dogs. We found that its pharmacokinetic properties are not favorable since this compound has poor bioavailability being rapidly and extensively metabolized. Yang et al. [35] have found that acetylated hydroxyl groups did not undergo glucuronidation and the modified compound exhibited a better pharmacokinetic profile. In order to improve the bioavailability of PH, we protected the hydroxyl groups of PH from metabolism by acetylating them and synthesized 2′,4′,6′,4-tetra-acetylphloretin (TAPH, Figure 1B). TAPH as a prodrug of PH is more stable than PH in vitro, protecting phenolic hydroxy from being oxygenated. TAPH transformed into PH in vivo, and we only detected PH in dog plasma after oral administration of TAPH to dogs by LC–MS/MS. In this study, we examined and compared the pharmacokinetics of TAPH with those of PH in dogs by taking advantage of LC–MS/MS.

Method

Materials and Reagents.

PH (Lot No. XC20140403, purity >99%) was purchased from Xi'an Plants of Grass Technology Co. Ltd. (Shaanxi, China), TAPH and darunavir (internal standard, IS) were synthesized at the Department of Medicinal Chemistry, Fourth Military Medical University and identified by infrared (IR), proton nuclear magnetic resonance (1H NMR), and high-resolution mass spectrometry (HRMS) with purity greater than 99%. PH capsules (each capsule contains 100 mg PH) and TAPH capsules (each capsule contains 160 mg TAPH) were manufactured by the Department of Pharmacy, Tangdu Hospital, Fourth Military Medical University. Ethyl acetate was purchased from Tianli Chemical Reagent Co. Ltd. (Tianjin, China). Methanol and acetonitrile were of HPLC grade (Tedia Co. Inc., Ohio OH, USA). Milli-Q water was used throughout the whole experiments (Millipore, Bedford, MA, USA). Other solvents used for analysis were of analytical grade.

Animals.

All the pharmacokinetic studies on animals were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals. Eighteen healthy beagle male dogs weighing 12.0 ± 1.0 kg (Certificate No. 61001600000032) were purchased from Chengdu Dossy Biological Technology Co. Ltd. (Sichuan, China) and acclimated in the laboratory for 2 weeks prior to the experiments. All the animals were fasted for 12 h before the experiments with free access to water. Dogs were set free in their individual cages throughout the experiment after the capsules were administered. The experimental protocol (2014-0367-R) involving animals was reviewed and approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University.

Instrumentation and Analytical Conditions.

The liquid chromatography was performed on an Agilent 1260 Series liquid chromatography (Agilent Technologies, Palo Alto, CA, USA), which included an Agilent 1260 Quat pump VL (model G1311C), Agilent 1260 Autosampler (model G1329B), and temperature-controlled column compartment (model G1316A). The LC system was coupled with an Agilent 6460 triple quadrupole mass spectrometer equipped with an electrospray ionization source. Chromatographic separation was achieved on an Inertsil® ODS3 C18 column (150 mm × 4.6 mm, 5 μm) and maintained at 30 °C. The mobile phase consisted of methanol–0.1% ammonia in water (85:15, v/v) with fast gradient elution at a flow rate of 0.5 mL min−1 and run time of 5 min. The eluent flow was led into the MS/MS starting 0.5 min after injection by switching the MS inlet valve. The sample volume injected was 5 μL, and the temperature of autosampler was set at 4 °C.

The mass spectrometer was run in electrospray ionization (ESI) mode using multiple reaction monitoring (MRM) to monitor the mass transitions. The mass analysis and detection were conducted using an Agilent 6460 triple quadrupole mass spectrometer (USA) equipped with an ESI source. The ESI source was operated in negative ionization mode for PH and positive ionization mode for TAPH and darunavir (IS). The MRM transitions were chosen to be m/z 273.0 → m/z 148.9 for PH, m/z 443.2 → m/z 401.0 for TAPH, and m/z 548.1 → m/z 69.1 for IS. The fragmentor voltage values set for PH and IS were 120, 48, and 110 V. The collision energy values set for PH and the IS were 45, 17, and 22 eV. The mass resolution was set at 0.7 μFWHM (unit mass resolution) for both quadrupoles. Other parameters of the mass spectrometer were as follows: gas flow, 12.0 L min−1; gas temperature, 300 °C; sheath gas flow, 10 L min−1; sheath gas temperature, 250 °C; nebulizer, 45 psi; capillary, 3500 V; chamber current, 0.30 μA; and capillary current, 5641 nA. Data were acquired and analyzed with Agilent MassHunter software.

Preparation of Stock Solutions.

Stock solutions (1 mg mL−1) were prepared in methanol. These stock solutions were diluted with methanol to obtain calibration solutions (range, 20–50,000 ng mL−1). Internal standard solution was prepared by dilution of stock solution to a concentration of 10 ng mL−1. All the solutions were stored at −20 °C and were brought to room temperature before use.

Drug Administration and Plasma Sample Collection.

In the pharmacokinetic study, 18 dogs were randomly divided into three groups (n = 6 per group). The dogs of the 1st groups were orally administered with three PH capsules (300 mg), the dogs of the 2nd groups were orally administered with six PH capsules (600 mg), the dogs of the 3rd groups were orally administered with twelve PH capsules (1200 mg). Then, 2 mL of blood samples were taken into a pre-heparinized blood collection tube at predose, 10, 30, 60, 120, 240, 360, 480, 720, and 1440 min postdosing. After a washout period of 1 month, the dogs of the 1st groups were orally administered with three TAPH capsules (480 mg), the dogs of the second groups were orally administered with six TAPH capsules (960 mg), the dogs of the third groups were orally administered with twelve TAPH capsules (1920 mg). Then, 2 mL of blood samples were taken into a preheparinized blood collection tube at predose, 10, 30, 60, 120, 240, 360, 480, 720, 1440, and 2160 min postdosing. Each blood sample was immediately centrifuged at approximately 12,000g, 4 °C for 15 min, and a 400-μL aliquot of supernatant plasma layer was transferred into another tube and stored at −20 °C until analysis.

Sample Preparation.

An aliquot of 400 μL dog plasma sample was mixed with 10 μL of internal standard solution (10 ng mL−1), and 1 mL of ethyl acetate was added. The sample was vortex mixed for 3 min, which was followed by centrifugation at 12,000g for 15 min. Finally, 800 μL of upper organic phase was transferred to another tube and evaporated to dryness at 40 °C under nitrogen. The resulting extract was dissolved in 80 μL of methanol and vortex mixed for 3 min. After centrifugation at 12,000g for 15 min, an aliquot of 5 μL sample was injected into the LC–MS/MS.

Selectivity and Matrix Effect.

Selectivity was assessed by comparing chromatograms of six different batches of drug-free plasma. Blank samples were and analyzed by LC–MS/MS for potential interfering peaks within the range of the retention time of each analyte. To evaluate the matrix effect in the experiment, chromatographic peak areas of each analyte from the unextracted standard biological samples, at three different concentrations (high, medium, and low), were compared to those for the clean standard solutions at the same concentrations. Matrix effects for the IS were also investigated.

Linearity and Calibration Curves.

Linearity was analyzed by the weighted regression method (1/x2) of peak area ratios of PH to IS versus actual concentrations. The calibration curves (n = 7) were prepared by blank plasma with standard solution of PH. The lowest limit of quantification (LLOQ) was defined as the lowest concentration which could be determined with both accuracy and precision.

Accuracy and Precision.

Accuracy and precision were determined in six replicates at three concentration levels (high, medium, and low) on the same day and three analytical batches on three consecutive days using calibration curve, respectively. The accuracy was estimated by the relative error (expressed as relative error, %RE), and the precision, by relative standard deviation (%RSD) for the same samples by comparing concentration measured with the nominal value. The assay accuracy was calculated as (observed concentration − spiked concentration)/(spiked concentration) × 100%.

Stability.

The stability of PH in dog biological samples was evaluated by six replicates at three different concentrations (high, medium, and low). The short-term stability was examined by keeping the replicates of samples at room temperature for 12 h. The long-term stability was assessed by carrying out the experiment after 45 days of storage at −20 °C. Freeze–thaw stability was assessed over three freeze–thaw cycles. Postpreparation stability was assessed by analyzing the extracted samples kept in the autosampler at 4 °C for 24 h.

Extraction Recovery.

For the determination of recovery, blank plasma was processed according to the sample preparation procedure described above. The supernatant was evaporated to dryness, and dry extracts were reconstituted in the initial mobile phase with addition of appropriate standards at concentrations corresponding to the final concentration of the pretreated plasma samples. These spike-after-extraction samples represented 100% recovery. The extraction recoveries for PH were determined by comparing the mean peak areas of six extracted low, medium, and high QC samples to mean peak areas of six spike-after-extract samples at the same concentrations. The extraction recoveries for IS were determined at a single concentration of 1 ng mL−1.

Pharmacokinetic and Statistical Analysis.

The LC–MS/MS procedure developed here was used to investigate the plasma profiles of PH after oral administration. All data were subsequently processed by the Agilent MassHunter software and Statistics version 2.0 (DAS 2.0, Anhui Provincial Center for Drug Clinical Evaluation). Terminal elimination rate constant (Ke) was determined by linear regression of the terminal portion of plasma concentration–time data, and the elimination half-life (t1/2) was calculated as 0.693/Ke. The area under the plasma concentration curve (AUC) versus time (AUC0−t) from time zero to the time of last measured concentration (Clast) was calculated by the linear trapezoidal rule. The AUC from time zero to infinity (AUC0−) was obtained by the addition of AUC0−t, and the extrapolated area was determined by Clast/Ke. Data were shown as mean ± standard deviation (mean ± SD). All the data are expressed as mean ± SD and a P value < 0.05 was deemed to be statistically significant.

Results and Discussion

Method Validation

Specificity and matrix effect.

Selectivity was assessed by comparing chromatograms of six different batches of blank dog plasma with the corresponding spiked plasma. Figure 2 shows the typical chromatograms of a blank plasma sample, a blank plasma sample spiked with PH and IS, and a plasma sample from a healthy dog 0.5 h after an oral administration and IS. Under the given condition, PH and IS were eluted at retention time of 2.4 min and 4.3 min, respectively. The result demonstrated that there was no interference with the determination of the PH and IS, granting good method selectivity. Matrix effects on recovery of blank dog plasma samples spiked after the sample preparation with low, medium, and high concentration levels of all three analytes were found to be within the acceptable limits. The same evaluation was performed on the IS (10 ng mL−1). Table 1 indicates that the plasma matrix effect was negligible for the assay.

Figure 2.
Figure 2.

Representative MRM chromatograms for PH and IS in dog plasma samples: (A) a blank plasma sample; (B) a blank plasma sample spiked with PH (100 ng mL−1) and IS (1 ng mL−1); (C) a plasma sample from a healthy dog 30 min after an oral administration of PH (300 mg); (D) a plasma sample from a healthy dog 30 min after an oral administration of TAPH (480 mg)

Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2016.00097

Table 1.

Matrix effect for the assay of PH and IS in dog plasma (n = 5)

SampleCompoundConcentration (ng mL−1)Absolute matrix effect (mean ± SD)Relative matrix effect (RSD, %)
PlasmaPH2092.31 ± 4.155.4
100092.14 ± 3.874.2
30,00094.25 ± 3.514.1
IS1094.56 ± 3.043.9

Linearity and calibration curves.

The standard calibration curve for spiked dog plasma containing PH was linear over the range 20–50,000 ng mL−1 with a correlation coefficient r > 0.9975. The results indicated that the calibration curve was linear, accurate, and precise over the range of the method.

Accuracy and precision.

The results of intra- and inter-day precision and accuracy for the assay of PH in blank plasma of dogs were summarized in Table 2. The intra- and inter-day RSD (%) were both no more than 6.1%, while the RE (%) of assay accuracies ranged from 2.1 to 5.4%. This indicated that the method was accurate and precise over the range of the assay. The LLOQ for PH was 20 ng mL−1 at which both precision and accuracy were less than 20% and within ±20%, respectively.

Table 2.

Precision and accuracy for the assay of PH in dog plasma (intra-day: 6 replicates at each concentration; inter-day: 18 replicates at each concentration)

SampleConcentration (ng mL−1)Intra-day (n = 3)
Inter-day (n = 6 × 3)
RSD (%)RE (%)RSD (%)RE (%)
Plasma205.32.65.63.6
10004.32.13.63.4
30,0005.14.36.15.4

Stability.

Table 3 demonstrates that the dog plasma samples were stable at −20 °C for at least 4 weeks with no significant loss. Plasma samples were stable over at least three freeze–thaw cycles.

Table 3.

Stability of PH under a variety of storage conditions (n = 3)

SampleConcentration (ng mL−1)Frozen for 30 days (mean ± SD)Three freeze–thaw cycles (mean ± SD)Room temperature for 12 h (mean ± SD)Prepared samples in freezer (−20 °C) for 24 h (mean ± SD)
Plasma2020.03 ± 0.0120.04 ± 0.0020.03 ± 0.0020.02 ± 0.01
10001030 ± 341036 ± 561061 ± 421054 ± 53
30,00029,831 ± 17629,746 ± 16928,947 ± 18229,816 ± 146

Extraction recovery.

Table 4 illustrates the recoveries of PH at three concentration levels and the recovery of IS. The method was consistent, reproducible, and acceptable.

Table 4.

The mean recoveries of PH and IS in dog plasma (n = 6)

CompoundAdd concentration (10 ng mL−1)Recovery (%) (mean ± SD)RSD (%)
PH2093.15 ± 5.435.2
100094.87 ± 5.944.6
30,00098.17 ± 3.753.4
IS191.62 ± 5.416.1

Pharmacokinetic Study.

TAPH is a prodrug of PH by acetylating its four hydroxyl groups. To our knowledge, this study constituted the first report on the pharmacokinetics of TAPH in dogs after oral administration and compared its properties with those of its parent drug PH. A simple, specific, and sensitive LC–MS/MS method has been successfully validated and applied to quantification of PH in dog plasma.

Mean plasma concentration–time curves (n = 6) were presented in Figures 3 and 4. The main pharmacokinetic parameters calculated using noncompartmental analysis were shown in Tables 5 and 6.

Figure 3.
Figure 3.

Mean plasma concentration–time curves (mean ± SD, n = 6) of PH after oral administration of PH (300, 600, and 1200 mg) to dogs

Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2016.00097

Figure 4.
Figure 4.

Mean plasma concentration–time curves (mean ± SD, n = 6) of PH after oral administration of TAPH (480, 960, and 1920 mg) to dogs

Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2016.00097

Table 5.

Pharmacokinetic parameters of PH in dogs after oral administration of PH (n = 6)

ParameterUnitAdministration of PH
300 mg600 mg1200 mg
AUC(0–t)mg L−1*min296.2 ± 37.4929.3 ± 108.22002.3 ± 140.1
AUC(0–)mg L−1*min329.5 ± 53.71044.6 ± 199.72041.1 ± 116.3
t1/2min120.7 ± 14.1187.5 ± 53.7268.3 ± 123.1
tmaxmin180.0 ± 69.28240240
CL/FL min−1 kg−10.093 ± 0.0150.059 ± 0.0110.059 ± 0.003
Cmaxng L−11025.1 ± 189.23075.9 ± 242.35712.3 ± 600.8
Table 6.

Pharmacokinetic parameters of PH in dogs after oral administration of TAPH (n = 6)

ParameterUnitAdministration of TAPH
480 mg960 mg1920 mg
AUC(0–t)mg L−1*min677.1 ± 28.41291.8 ± 38.74440.7 ± 205.4
AUC(0–)mg L−1*min770.7 ± 131.21519.3 ± 200.55195.1 ± 1065.9
t1/2min677.54 ± 285.4762.7 ± 288.3745.7 ± 449.1
tmaxmin240.0 ± 75.89220.0 ± 48.99240.0 ± 75.9
CL/FL min−1 kg−10.063 ± 0.0090.064 ± 0.0080.038 ± 0.006
Cmaxng L−1631.7 ± 285.61274.5 ± 142.24121.5 ± 157.9

The concentration of PH was determined at different times after oral administration at the dosages of PH (300 mg, 600 mg, and 1200 mg) and TAPH (480 mg, 960 mg, and 1920 mg), respectively. The data were analyzed by DAS 2.0, and pharmacokinetic parameters were attained under different conditions. The results indicated that the concentration–time (C–T) curves of different dosages accorded with first-order linear equation. Three curves of TAPH all possess double peaks, which may be related to enterohepatic circulation, recirculation, and discordant absorption. PH was rapidly absorbed within 4 h, then reached the maximum plasma concentration (Cmax), and the concentration decreased sharply. After PH was transformed into TAPH, PH eliminated slowly after Cmax; meanwhile, AUC and t1/2 increased among the three dosages. However, there were no differences in other pharmacokinetic parameters such as tmax and CL/F among the three dosages. Table 5 shows that PH was absorbed and distributed rapidly after oral administration to dogs, and then eliminated from dog plasma with t1/2 of approximately 120 min (300 mg), 187 min (600 mg), and 268 min (1200 mg). The residence time of PH was very long which might have some relationships with its chemical structure and the physicochemical property. After oral administration of TAPH to dogs, there can be PH measured after 10 min. This shows that TAPH decomposed and was absorbed very quickly; meanwhile, the Cmax of the three dosages (480 mg, 960 mg, and 1920 mg) decreased, indicating that TAPH was absorbed and distributed and is more stable than PH. AUC and t1/2 of TAPH increased among the three dosages, showing that TAPH can effectively increase the bioavailability and extend the duration of action of PH.

Conclusion

To the best of our knowledge, this is the first report to evaluate the pharmacokinetics of TAPH in dogs after oral administration and compared its properties with those of its parent drug PH. A validated LC–MS/MS method for the determination of PH in dog plasma was developed and applied to investigate the pharmacokinetics of PH and TAPH. The validation was performed according to regulatory guidelines. After PH was transformed into TAPH, its pharmacokinetic properties were improved; for instance, AUC and t1/2 increased among the three dosages. The present in vivo pharmacokinetics studies of PH and TAPH in dogs will provide helpful information for the development of suitable dosage forms and clinical references on rational administration.

Conflict of interest

The authors report no conflict of interest.

Acknowledgments

This research was supported by grants from the National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (No. 2011ZX09401-308-23).

References

  • 1.

    Lu, Y.; Foo, L. Y. Food Chem. 2000 , 68 , 81 85.

  • 2.

    JA N. ; SK K. Arch. Dermatol. Res. 2010 , 302 , 71 83.

  • 3.

    Manach, C.; Mazur, A.; Scalbert, A. Curr. Opin. Lipidol 2005 , 16 , 77.

  • 4.

    Ruxton , C. H. S. ; Gardner, E. J.; Walker, D. Int. J. Food Sci. Nutr. 2006 , 57 , 249 272.

  • 5.

    Yanagida A. ; Kanda T.; Tanabe M.; Matsudaira F.; Oliveira C. J. J. Agric. Food Chem. 2000 , 48 , 5666 5671.

  • 6.

    Jung M. ; Triebel S.; Anke T.; et al. Mol. Nutr. Food Res. 2009 , 53 , 1263.

  • 7.

    Gerhauser C. Planta Med. 2008 , 74 , 1608 1624.

  • 8.

    Zessner H. ; Pan L.; Will F.; Klimo K.; Knauft J.; Niewohner R.; Hummer W.; Owen R.; Richling E.; Frank N.; Schreier P.; Becker H.; Gerhauser C. Mol. Nutr. Food Res. 2008 , 52 , S2 S44.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Sugiyama H. ; Akazome Y.; Shoji T.; Yamaguchi A.; Yasue M.; Kanda T.; Ohtake Y.; Agric J. Food Chem. 2007 , 55 , 4604 4609.

  • 10.

    Picinelli A. ; Dapena E.; Mangas J. J. J. Agric. Food Chem. 1995 , 43 , 2273 2278.

  • 11.

    Miksicek R. J. J. Steroid Biochem. Mol. Biol. 1994 , 49 , 153 160.

  • 12.

    Rezk B. M. ; Haenen G. R.; van der Vijgh W. J.; Bast A. Biochem. Biophys. Res. Commun. 2002 , 295 , 9 13.

  • 13.

    De Jonge P. C. ; Wieringa T.; Van Putten J. P.; Michiel H.; Krans J.; Van Dam K. Biochim. Biophy. Acta, Bioenerg. 1983 , 722 , 219 225.

  • 14.

    Gaudout D. ; Megard D.; Inisan C.; et al. Phloridzin-rich phenolic fraction and use thereof as a cosmetic, dietary or nutraceutical agent: U.S. Patent 7,427,418. 2008 , 9 23.

    • Search Google Scholar
    • Export Citation
  • 15.

    Oresajo C. ; Stephens T.; Hino P. D.; Law R. M.; Yatskayer M.; Foltis P.; Pillai S.; Pinnell S. R. J. Cosmet. Dermatol. 2008 , 7 , 290 297.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Gaudout D. ; Megard D.; Inisan C.; et al. Phloridzin-rich phenolic fraction and use thereof as a cosmetic, dietary or nutraceutical agent: U.S. Patent 7,041,322. 2006 , 5 9.

    • Search Google Scholar
    • Export Citation
  • 17.

    Kobori M. ; Shinmoto H.; Tsushida T.; Shinohara K. Cancer Lett. 1997 , 119 , 207 212.

  • 18.

    Rosenberg T. ; Wilbrandt W. Helv. Physiol. Pharmacol. Acta 1956 , 15 , 168 176.

  • 19.

    Jung M. ; Triebel S.; Anke T.; Richling E.; Erkel G. Mol. Nutr. Food Res. 2009 , 53 , 1263 1280.

  • 20.

    Devi M. A. ; Das N. P. Cancer Lett . 1993 , 69 , 191 196.

  • 21.

    Wu C. H. ; Ho Y. S.; Tsai C. Y.; Wang Y. J.; Tseng H.; Wei P. L.; Lee C. H.; Liu R. S.; Lin S. Y. Int. J. Cancer 2009 , 124 , 2210 2219.

  • 22.

    Yang K. C. ; Tsai C. Y.; Wang Y. J.; Wei P. L.; Lee C. H.; Chen J. H.; Wu C. H.; Ho Y. S. Mol. Carcinog. 2009 , 48 , 420 431.

  • 23.

    Sabzevari O. ; Galati G.; Moridani M. Y.; Siraki A.; O'Brien P. J. Chem.-Biol. Interact. 2004 , 148 , 57 67.

  • 24.

    Nelson J. A. ; Falk R. E. Anticancer Res. 1992 , 13 , 2293 2299.

  • 25.

    Ren W. ; Qiao Z.; Wang H.; Zhu L.; Zhang L. Med. Res. Rev. 2003 , 23 , 519 534.

  • 26.

    Hassan M. ; El Yazidi C.; Malezet-Desmoulins C.; Amiot M.; Margotat A. J. Nutr. Biochem. 2010 , 21 , 645 652.

  • 27.

    Hassan M. ; El Yazidi C.; Landrier J.; Lairon D.; Margotat A.; Amiot M. Biochem. Biophys. Res. Commun. 2007 , 361 , 208 213.

  • 28.

    Kahle K. ; Kempf M.; Schreier P.; Scheppach W.; Schrenk D.; Kautenburger T.; Hecker D.; Huemmer W.; Ackermann M.; Richling E. Eur. J. Nutr. 2011 , 50 , 507 522.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Petermann A. ; Miene C.; Schulz-Raffelt G.; et al. Mol. Nutr. Food Res. 2009 , 53 , 1245 1253.

  • 30.

    Yáñez J. A. ; Remsberg C. M.; Takemoto J. K.; et al. Polyphenols and Flavonoids: An Overview. Flavonoid Pharmacokinetics 2012 , 1 69.

  • 31.

    Yang H. ; Hao H. P.; Wang G. J.; et al. Prog. Pharm. Sci. 2009 , 33 , 498 502.

  • 32.

    Kahle K. ; Kraus M.; Richling E. , Mol. Nutr. Food Res. 2005 , 49 , 797 806.

  • 33.

    Remsberg C. M. ; Yáñez J. A.; Vega-Villa K. R.; et al. J. Chromatogr. Sep. Tech. 2010 , 1 , 2.

  • 34.

    Lijia X. ; Guo J.; Chen Q.; Baoping J.; Zhang W. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2014 , 960 , 67 72.

  • 35.

    Chan A. ; Chen S.; Li Y.; et al. Puerarin derivatives and their medical uses: U.S. Patent Application 10/969,571. 2004 , 10 20.

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  • 1.

    Lu, Y.; Foo, L. Y. Food Chem. 2000 , 68 , 81 85.

  • 2.

    JA N. ; SK K. Arch. Dermatol. Res. 2010 , 302 , 71 83.

  • 3.

    Manach, C.; Mazur, A.; Scalbert, A. Curr. Opin. Lipidol 2005 , 16 , 77.

  • 4.

    Ruxton , C. H. S. ; Gardner, E. J.; Walker, D. Int. J. Food Sci. Nutr. 2006 , 57 , 249 272.

  • 5.

    Yanagida A. ; Kanda T.; Tanabe M.; Matsudaira F.; Oliveira C. J. J. Agric. Food Chem. 2000 , 48 , 5666 5671.

  • 6.

    Jung M. ; Triebel S.; Anke T.; et al. Mol. Nutr. Food Res. 2009 , 53 , 1263.

  • 7.

    Gerhauser C. Planta Med. 2008 , 74 , 1608 1624.

  • 8.

    Zessner H. ; Pan L.; Will F.; Klimo K.; Knauft J.; Niewohner R.; Hummer W.; Owen R.; Richling E.; Frank N.; Schreier P.; Becker H.; Gerhauser C. Mol. Nutr. Food Res. 2008 , 52 , S2 S44.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Sugiyama H. ; Akazome Y.; Shoji T.; Yamaguchi A.; Yasue M.; Kanda T.; Ohtake Y.; Agric J. Food Chem. 2007 , 55 , 4604 4609.

  • 10.

    Picinelli A. ; Dapena E.; Mangas J. J. J. Agric. Food Chem. 1995 , 43 , 2273 2278.

  • 11.

    Miksicek R. J. J. Steroid Biochem. Mol. Biol. 1994 , 49 , 153 160.

  • 12.

    Rezk B. M. ; Haenen G. R.; van der Vijgh W. J.; Bast A. Biochem. Biophys. Res. Commun. 2002 , 295 , 9 13.

  • 13.

    De Jonge P. C. ; Wieringa T.; Van Putten J. P.; Michiel H.; Krans J.; Van Dam K. Biochim. Biophy. Acta, Bioenerg. 1983 , 722 , 219 225.

  • 14.

    Gaudout D. ; Megard D.; Inisan C.; et al. Phloridzin-rich phenolic fraction and use thereof as a cosmetic, dietary or nutraceutical agent: U.S. Patent 7,427,418. 2008 , 9 23.

    • Search Google Scholar
    • Export Citation
  • 15.

    Oresajo C. ; Stephens T.; Hino P. D.; Law R. M.; Yatskayer M.; Foltis P.; Pillai S.; Pinnell S. R. J. Cosmet. Dermatol. 2008 , 7 , 290 297.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Gaudout D. ; Megard D.; Inisan C.; et al. Phloridzin-rich phenolic fraction and use thereof as a cosmetic, dietary or nutraceutical agent: U.S. Patent 7,041,322. 2006 , 5 9.

    • Search Google Scholar
    • Export Citation
  • 17.

    Kobori M. ; Shinmoto H.; Tsushida T.; Shinohara K. Cancer Lett. 1997 , 119 , 207 212.

  • 18.

    Rosenberg T. ; Wilbrandt W. Helv. Physiol. Pharmacol. Acta 1956 , 15 , 168 176.

  • 19.

    Jung M. ; Triebel S.; Anke T.; Richling E.; Erkel G. Mol. Nutr. Food Res. 2009 , 53 , 1263 1280.

  • 20.

    Devi M. A. ; Das N. P. Cancer Lett . 1993 , 69 , 191 196.

  • 21.

    Wu C. H. ; Ho Y. S.; Tsai C. Y.; Wang Y. J.; Tseng H.; Wei P. L.; Lee C. H.; Liu R. S.; Lin S. Y. Int. J. Cancer 2009 , 124 , 2210 2219.

  • 22.

    Yang K. C. ; Tsai C. Y.; Wang Y. J.; Wei P. L.; Lee C. H.; Chen J. H.; Wu C. H.; Ho Y. S. Mol. Carcinog. 2009 , 48 , 420 431.

  • 23.

    Sabzevari O. ; Galati G.; Moridani M. Y.; Siraki A.; O'Brien P. J. Chem.-Biol. Interact. 2004 , 148 , 57 67.

  • 24.

    Nelson J. A. ; Falk R. E. Anticancer Res. 1992 , 13 , 2293 2299.

  • 25.

    Ren W. ; Qiao Z.; Wang H.; Zhu L.; Zhang L. Med. Res. Rev. 2003 , 23 , 519 534.

  • 26.

    Hassan M. ; El Yazidi C.; Malezet-Desmoulins C.; Amiot M.; Margotat A. J. Nutr. Biochem. 2010 , 21 , 645 652.

  • 27.

    Hassan M. ; El Yazidi C.; Landrier J.; Lairon D.; Margotat A.; Amiot M. Biochem. Biophys. Res. Commun. 2007 , 361 , 208 213.

  • 28.

    Kahle K. ; Kempf M.; Schreier P.; Scheppach W.; Schrenk D.; Kautenburger T.; Hecker D.; Huemmer W.; Ackermann M.; Richling E. Eur. J. Nutr. 2011 , 50 , 507 522.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Petermann A. ; Miene C.; Schulz-Raffelt G.; et al. Mol. Nutr. Food Res. 2009 , 53 , 1245 1253.

  • 30.

    Yáñez J. A. ; Remsberg C. M.; Takemoto J. K.; et al. Polyphenols and Flavonoids: An Overview. Flavonoid Pharmacokinetics 2012 , 1 69.

  • 31.

    Yang H. ; Hao H. P.; Wang G. J.; et al. Prog. Pharm. Sci. 2009 , 33 , 498 502.

  • 32.

    Kahle K. ; Kraus M.; Richling E. , Mol. Nutr. Food Res. 2005 , 49 , 797 806.

  • 33.

    Remsberg C. M. ; Yáñez J. A.; Vega-Villa K. R.; et al. J. Chromatogr. Sep. Tech. 2010 , 1 , 2.

  • 34.

    Lijia X. ; Guo J.; Chen Q.; Baoping J.; Zhang W. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2014 , 960 , 67 72.

  • 35.

    Chan A. ; Chen S.; Li Y.; et al. Puerarin derivatives and their medical uses: U.S. Patent Application 10/969,571. 2004 , 10 20.

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