| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Drug Monitoring and Toxicology |
1 State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, and 2 Key Laboratory of Analytical Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China.
aAddress correspondence to these authors at: School of Life Sciences, Nanjing University, 22 Han Kou Rd., Nanjing 210093, China. Fax 86-25-8359-7620; e-mail molpharm{at}163.com.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Methods: We incubated astilbin with rat liver microsomal/cytosolic fractions and isolated the metabolite of astilbin, which was fully characterized by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. We administered astilbin orally via a gastric tube to rats at 0.22 mmol/kg and collected whole blood samples after 30 min and urine samples after 0 to 12 h. We applied HPLC and liquid chromatography/MS to measure the metabolite in the samples, and we assayed cytokine expression by reverse-transcription PCR.
Results: After incubation of astilbin with rat liver microsomal/cytosolic fractions, we detected a new metabolite of astilbin and isolated it from the culture solution. We characterized this metabolite by MS and NMR techniques as 3'-O-methylated astilbin. We detected the metabolite in both blood and urine samples after oral administration of astilbin, and the metabolite inhibited picryl chloride–induced ear swelling in mice and suppressed the expression of tumor necrosis factor-
and interferon-
, similarly to astilbin.
Conclusion: This is the first identification of 3'-O-methylastilbin as a new flavonoid, as well as an active metabolite of astilbin in vivo, and is helpful for studying the kinetics of astilbin and its clinical applications.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-L-mannopyranoside)] (see Fig. 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue3). This flavonoid shows properties of coenzyme A reductase inhibition (3), aldose reductase inhibition (4), and antioxidation (5)(6). In our previous work (7)(8), astilbin significantly inhibited both ear contact dermatitis and liver injury induced by delayed-type hypersensitivity (DTH) at the effector phase. The mechanism of inhibition involved selective induction of apoptosis in liver-infiltrating T lymphocytes, activated spleen T cells, and activated Jurkat cells, without affecting hepatocytes, naive spleen T cells, nonactivated Jurkat cells, or other tissue cells (7)(8). Astilbin also prevented concanavalin A–induced liver injury by reducing tumor necrosis factor- (TNF-) production and T lymphocyte adhesion (9), and it significantly suppressed collagen-induced arthritis by causing the dysfunction of lymphocytes and inhibiting lymphocyte migration (10)(11). Such a selective activity of astilbin is quite different from the nonselective activity of other immunosuppressive drugs (12). For example, astilbin alleviated contact hypersensitivity by inhibiting Th1 cytokine production and stimulating endogenous interleukin-10, and it up-regulated the downstream proteins of interleukin-10, suppressor of cytokine signaling 1 (SOCS1), and SOCS3. This feature of astilbin is distinct from the immunosuppressant cyclosporin A, which strongly inhibits proinflammatory cytokines but does not influence interleukin-10, SOCS1, or SOCS3 (13). These characteristics of astilbin may lead to fewer side effects than other treatments. For example, astilbin does not show any influence on thymus and spleen weights, whereas dexamethasone significantly lowers organ weights (11). The selective immunosuppression by astilbin is also of significance for developing a new immunosuppressant from flavones, since with the exception of the antiinflammatory activity of rutin against rat paw edema (14), selective immunosuppressive ability of other flavones has not been reported. Therefore, astilbin shows promise for the treatment of immune diseases.
To fully understand astilbin’s mechanism of action, we studied its metabolic fate, as well as the role of any active metabolites. Here, we aim to elucidate the metabolic pathway of astilbin, and we describe the first identification of a new flavonoid as an active metabolite of astilbin in rats.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
animals and animal experiments
We purchased male Sprague–Dawley rats (6 weeks old) from the Experimental Animal Center of China Pharmaceutical University (Nanjing). Female Balb/c mice (6 to 8 weeks old, 18 to 22 g) were supplied by the Laboratory Animal Center of Shanghai. They were kept in plastic cages at mean (SD) temperatures of 21 (2) °C on a 12-h light-dark cycle, with free access to pellet food and water. Animal welfare and experimental procedures were strictly in accordance with the Guide for the Care and Use of Laboratory Animals (US National Research Council, 1996) and the related ethics regulations of Nanjing University. After the rats fasted overnight, we collected baseline (blank) plasma and urine samples, then administered 0.22 mmol/kg astilbin, orally in 1 mL water, via a gastric tube. Thirty minutes later, we collected whole blood samples (
1 mL) in heparin-containing polythene tubes, which we immediately centrifuged at 1500g for 10 min at 4 °C to obtain plasma. We collected urine samples 0 to 12 h after astilbin administration.
sample preparation
We extracted 200 µL plasma or urine with 5 mL ethyl acetate. After mixing for 1 min and centrifugation at 1200g for 10 min, we transferred the organic layer to a test tube and dried it under reduced pressure. We dissolved the dried extracts in 100 µL during mobile phase and injected 20 µL into the HPLC. To determine the glucuronide of astilbin, we mixed the samples with methanol, centrifuged them at 7000g for 5 min, and injected 20 µL supernatant into the HPLC.
hplc analysis
The HPLC system consisted of a Waters 600 pump, a Waters 2487 Dual 
Absorbance UV detector, an Empower workstation, and a Waters Nova-Pak C-18 column (4.6 µm, 150 x 3.9 mm). The solvent in mobile phase was methanol:water:acetic acid (36:63.7:0.3, vol/vol/vol) at a flow rate of 0.8 mL/min. The detector was operated at 291 nm and 0.2 absorbance units full scale. Under these conditions the retention time for astilbin was 9.6 min.
methylation of astilbin with rat liver microsomal/cytosolic fractions and metabolite isolation for nuclear magnetic resonance
To determine the structure of the astilbin metabolite by nuclear magnetic resonance (NMR) spectroscopy, we used rat liver microsomal/cytosolic fractions to produce sufficient amounts of the metabolite. Liver tissues (50 g) from 4 freshly killed rats were homogenized at 4 °C with 500 mL of 10 mmol/L sodium phosphate buffer (pH 7.4) containing 10 mmol/L MgCl2, according to the method of Okushio et al. (16). We centrifuged the homogenate at 12 000g for 15 min at 4 °C to remove the insoluble materials and further centrifuged the supernatant at 100 000 g for 90 min to obtain microsomal and cytosolic fractions, which were stored at –80 °C. We carried out protein determination according to Lowry et al. (17).
The incubation mixture contained 1.12 g/L protein, 5 mmol/L MgCl2, 2 mmol/L S-adenosyl-L-methionine, and 1 mmol/L astilbin in 10 mmol/L sodium phosphate buffer (pH 7.4). We incubated the mixture at 37 °C for 4 h in a shaking water bath. We separated the products by silica gel flash column chromatography (2 x 50 cm), eluted with CH2Cl2:methanol (19:1). We carried out repeated column purifications by use of Sephadex LH-20 column chromatography, eluted with methanol:H2O (30:70), to obtain the major metabolite of astilbin. We characterized the isolated metabolite by use of 1D and 2D NMR techniques and mass spectrometry (MS) analysis.
identification of metabolite
We performed negative-ion electron spectroscopic imaging–MS analyses, with direct loop injection, by use of a Finnigan TSQ 7000 mass spectrometer. We recorded NMR spectra on a Bruker DPX-300 spectrometer. We recorded the 13C-1H correlation spectroscopy (COSY) and 13C-1H long-range COSY NMR spectra using generally accepted pulse sequences. We measured circular dichroism spectra, in methanol solution, at room temperature by use of a Jasco model 810 spectropolarimeter. Circular dichroism spectra were expressed by molar ellipticity in units of millidegrees (m°).
liquid chromatography/ms analysis of methylated astilbin in rat urine and plasma
We carried out the liquid chromatography (LC)/MS analysis by use of an Agilent 1100 LC separation module with HPLC conditions as described above. We performed the mass spectral analysis using electrospray ionization in the positive ion mode. We used full-scan MS and selected ion monitoring techniques to identify the enzymatically synthesized methylated astilbin and the corresponding metabolite in the urine and plasma.
picryl chloride–induced contact hypersensitivity
To sensitize mice, we painted 0.1 mL of 1% picryl chloride in ethanol onto the shaved skin of their abdomens. Five days after sensitization, we applied 30 µL of 1% picryl chloride in olive oil on the right ear. Twenty-four hours later, we evaluated ear swelling (difference in thickness between right and left ears) by use of an engineer’s micrometer (0.001 mm; Mitutoyo).
quantification of MRNA expression by reverse-transcription pcr
We performed reverse-transcription PCR as previously described (13). Briefly, we cultured lymph node cells (5 x 106 cells/well) from mice, 5 days after sensitization, at 37 °C in the presence of drugs. PCR was performed under the following conditions: 94 °C for 30 s, 60 °C for 1 min, and 72 °C for 1 min. Cycle numbers were 28, 35, and 33 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), interferon- (IFN-), and TNF-, respectively. The PCR primer sequences (Sangon) were as follows: GAPDH, forward: 5'-AAC GAC CCC TTC ATT GAC; GAPDH, reverse: 5'- TCC ACG ACA TAC TCA GCA C; IFN-, forward: 5'-CTT CTT CAG CAA CAG CAA GGC GAA AA; IFN-, reverse: 5'- CCC CCA GAT ACA ACC CCG CAA TCA; TNF-, forward: 5'-CAT CTT CTC AAA ATT CGA GTG ACA A; TNF-, reverse: 5'-TGG GAG TAG ACA AGG TAC AAC CC.
We densitometrically quantified relative expressions, calculated according to the reference bands of GAPDH, by use of UVP Labworks 4.0 software.
statistical analysis
We expressed the results from the in vivo experiment as mean (SD) and performed statistical evaluation by use of the Student t-test when 2 value sets were compared. We considered P <0.05 to be significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The assignment of 1H NMR and 13C NMR data of astilbin, and its biosynthesized metabolite methylated astilbin, is shown in Tables 1
and 2
. The methylated astilbin showed chemical shifts and coupling patterns similar to those of astilbin, except for the appearance of proton (
3.88) and carbon ( 57.9) signals derived from a methyl group. The position of the methoxy groups was confirmed by the observation of a nuclear Overhauser effect between methoxy protons and H-2'. Irradiation of the resonance at 3.88 ppm (OCH3) produced a nuclear Overhauser effect at the meta-coupled doublet (d, J = 1.76) at 7.12 ppm (H-2'), which was further supported by a 13C-1H long-range COSY experiment, in which an association between C-3' and –OCH3 was observed (Table 2
).
|
|
The 13C-1H long-range COSY NMR data allowed us to identify a correlation between the proton C-3 and the carbon of the anomeric position, indicating that the linkage between the rhamnose and dihydroquercetin moieties was C-3-O-C-1.
Thus, the methylated astilbin was identified as 3'-O-methylastilbin, which had a circular dichroism spectrum with a positive Cotton effect at 329.6 nm (ellipticity = 11 m°) and negative Cotton effect at 294.6 nm (ellipticity = –30 m°), and can be assigned to 2(R):3(R) configurations, which is consistent with astilbin (18).
metabolite of astilbin in plasma, urine, and bile after oral administration to rats
We detected a new peak, with a retention time of 14 min, in the plasma and urine of rats that had been given astilbin; no peak was observed in the urine and plasma samples collected before astilbin administration (Fig. 1
). The mean recoveries for astilbin and 3'-O-methylastilbin were >80% at 1 to 5 µmol/L in plasma and >75% at 4 to 10 µmol/L in urine. Using the 3'-O-methylastilbin prepared from the culture with liver microsomal/cytosolic fractions, we confirmed that the HPLC retention time of the biosynthesized product was the same as this peak.
|
To further identify this new peak, we injected the sample on the LC/MS system and monitored it with selected ion monitoring modes. We chose m/z 473.3, the [M+Na]+ ion of AST (Mr 450), and m/z 487.3, the [M+Na]+ ion of methylated astilbin (Mr 464), to monitor the mass spectrum of plasma samples. The samples showed 2 major peaks, with retention times of 9 and 14 min, under the LC/MS elution conditions (Fig. 2
).
|
We also screened for the glucuronide metabolites of astilbin in rat plasma, urine, and bile after oral administration. As shown in Fig. 3 in the online Data Supplement, we detected the glucuronide metabolite in bile, with a retention time of 7 min. We did not detect the glucuronide metabolite of astilbin, however, when plasma or urine samples were deproteinized with methanol and analyzed by HPLC.
effect of astilbin and its metabolite on picryl chloride–induced contact hypersensitivity in mice and in vitro tnf- and ifn- expression
We injected astilbin and 3'-O-methylastilbin once intraperitoneally at the time of challenge with picryl chloride. In treated animals, 33 µmol/kg 3'-O-methylastilbin, as well as astilbin, significantly attenuated the ear swelling (Fig. 3
) compared with control animals, which had been given saline instead of drugs.
|
We collected lymph node cells from mice 5 days after the picryl chloride sensitization and cultured them with or without TNBS in the presence of astilbin and its metabolite. Twelve hours after the culture with 1 µmol/L of astilbin and its metabolite, as well as 1 µmol/L of cyclosporin A, we noted a marked decrease in TNF- and IFN- expression (Fig. 4
).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Because there is no report on the NMR spectra of the methylated derivatives of astilbin, we carried out NMR experiments to further identify the structure of methylated astilbin. The 1H NMR (Table 1
) and 13C NMR (Table 2
) data for methylated astilbin showed chemical shifts and coupling patterns similar to those of astilbin, except for the proton ( 3.88) and carbon ( 57.9) signals derived from a methyl group. The position of the methoxy group was confirmed by a nuclear Overhauser effect between methoxy protons and H-2', which was further supported by a 13C-1H long-range COSY NMR experiment, in which we observed a correlation between C-3' and –OCH3 (Table 2
).
Thus, the methylated astilbin was identified as 3'-O-methylastilbin. To our knowledge this is the first evidence for 3'-O-methylastilbin as a new flavonoid and as a metabolite of astilbin. The enzyme responsible for this metabolism may be catechol-O-methyltransferase, which is an intracellular enzyme widely distributed throughout the mammalian organs and which is able to methylate only 1 of the 2 neighbor catechol hydroxyls (19). In the structure of astilbin there are 4 phenol hydroxyls, and 2 of them are neighbors. Indeed, the in vitro O-methylation of astilbin, as described above, confirmed the selective formation of meta-O-methylated derivatives. Thus, we presume catechol-O-methyltransferase to be involved in the transformation of astilbin, and it favors 3-O-methylation over 4-O-methylation.
The characterization of this astilbin metabolite may help us to evaluate whether this unique compound can be applied for the treatment of human diseases. Indeed, the kinetic study of a candidate drug has become an increasingly important component during drug development. Metabolites that have biological activity can be used as the active form of a drug for evaluating clinical efficacy or as the lead compound for further structural optimization. Although we have previously reported the pharmacokinetic profile of astilbin in rabbits (20), there is still no in vivo knowledge about its metabolite. In this study, we found that the metabolite exists in mammals and has biologic activity.
It has been long believed that flavonoid glycosides are usually cleaved before absorption. The aglycones might then be partially absorbed or undergo further biotransformation (21). Most of these flavonoid glycosides have glucose attached to aglycones and are usually hydrolyzed by glucosidase. In the case of astilbin, however, the sugar attached to aglycon is rhamnose, which is likely difficult to hydrolyze since there have been no reports on the existence of rhamnosidase in rat gut. Indeed, we previously obtained the astilbin aglycon dihydroquercetin from RSG (15), and in this study screened for the aglycon in rat plasma and urine samples by HPLC; we did not detect the existence of the aglycon in the samples (data not shown). This finding suggests that astilbin may show a different metabolic route from other flavonoids, owing to the type and position of sugar attached. On the other hand, we did not observe any activity of the aglycon against the liver-infiltrating T cells in the previous study, suggesting the importance of the sugar at position 3 for the selective immunosuppression by astilbin (15).
We also observed a glucuronide metabolite of astilbin in rat bile after oral administration, but not in plasma or urine (see Fig. 3 in the online Data Supplement), suggesting that the glucuronide of astilbin may exist only in the bile. Furthermore, the glucuronide metabolite did not show any inhibition of picryl chloride–induced ear swelling (data not shown).
To examine whether the methylated metabolite of astilbin has any activity, we used the DTH model contact hypersensitivity and its related cytokine assay, in which proinflammatory cytokines, including TNF- and IFN-, significantly contribute to the pathogenesis of DTH (22). In addition to dermatitis, many other human diseases, such as multiple sclerosis, rheumatoid arthritis, and transplantation, have also been known to involve the DTH mechanism (23). Like the parental compound astilbin, 3'-O-methylastilbin significantly inhibited picryl chloride–induced ear swelling (Fig. 3
) and decreased both TNF- and IFN- expression in vitro in the same molar concentrations (Fig. 4
), indicating that 3'-O-methylastilbin may play an important role in in vivo immunosuppressive activity as the major active form of astilbin.
This finding also provided new evidence for the clinical efficacy of the herbal drug RSG, which mainly contains astilbin. This herbal drug has been used for the treatment of various human inflammatory diseases, as a single drug or with other herbal drugs. The use of phytomedicine is an age-old tradition in China and other countries, but there is usually a lack of scientific evidence for their efficacy. Our preclinical data may be helpful for understanding the clinical efficacy of the herbal drug RSG and its derivatives. The selective immunosuppressive feature may also provide a rationale for astilbin to be developed as a novel immunosuppressive agent, and the elucidation of its metabolic route may facilitate future clinical trials.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
, tumor necrosis factor-; SOCS, suppressor of cytokine signaling; TNBS, 2,4,6-trinitrobenzene-sulfonic acid hydrate; NMR, nuclear magnetic resonance; MS, mass spectrometry; COSY, correlation spectroscopy; LC, liquid chromatography; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN-, interferon-. 
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production and T lymphocytes adhesion. J Pharm Pharmacol 2004;56:495-502.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
