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Comparison of the Pharmacokinetics of Hawthorn Phenolics in Extract Versus Individual Pure Compound

From the School of Pharmacy (Dr Chang, Dr Zuo, Dr Chow) and the Department of Biochemistry (Dr Ho), Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, SAR, People's Republic of China, and Institute of Medicinal Plant Development, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China (Dr Chang).

Address for reprints: Dr Zhong Zuo, School of Pharmacy, Chinese University of Hong Kong, Shatin, N.T. Hong Kong.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The pharmacokinetics of an active herbal substance may be different when administered in an extract form as compared to that when administered as a pure compound. This study investigated the pharmacokinetics of 4 active compounds of hawthorn fruits—namely, (-)-epicatechin, chlorogenic acid, hyperoside, and isoquercitrin—following administration of an extract formulation (as hawthorn phenolic extract, which contained the active compounds) or equivalent doses of individual pure compound in male Sprague-Dawley rats (n = 5 per group). The hawthron phenolic extract or pure compounds were administered both orally and intravenously. Following administration, multiple plasma samples were obtained, and the plasma concentrations were determined by high-performance liquid chromatography. After the intravenous injection of hawthorn phenolic extract, higher plasma drug concentration, larger area under the plasma concentration-time curve from 0 to infinity, longer terminal elimination half-life, smaller apparent volume of distribution, lower total body clearance, and higher urinary excretion of each compound were obtained when compared to that after the pure compound. Following the oral administration of either hawthorn phenolic extract or pure compound, only epicatechin was absorbed, and their pharmacokinetics were generally not significantly different between these 2 formulations. The differences in the pharmacokinetics of the 2 formulations following intravenous but not oral administration may be attributable to the existence of other co-occurring components in the hawthorn phonolic extract (which may be present in the body after intravenous but not oral administration). The results showed that an herbal extract formulation, when administered intravenously, could potentially alter the pharmacokinetics of its active ingredients.

Key Words: Crataegus • hawthorn • pharmacokinetics • absorption • epicatechin • chlorogenic acid • hyperoside • isoquercitrin

	MATERIALS AND METHODS

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Chemicals and Reagents
The 4 active compounds from HPE are EC, ChA, HP, and IQ.¹ Chlorogenic acid, EC, and naringin (NR, used as internal standard for the high-performance liquid chromatography [HPLC] assay) were purchased from Sigma Chemical Co. (St. Louis, Mo). HP and IQ were obtained from Carl Roth GmbH (Karlsruhe, Germany). All other reagents used were of analytical grade and were purchased from Sigma or BDH Laboratory Supplies (Poole, England). Distilled and deionized water was used throughout the study.

Preparation of Hawthorn Phenolic Extract
The dried powder (1.5 kg) of hawthorn fruits (Crateagus pinnatifida Bge. var. major N.E. Br.) was extracted with 80% ethanol 3 times. The pooled filtrate was then concentrated under reduced pressure to remove the ethanol. The extract was dissolved in 4 L of water and extracted with ether (2 L x 4) to remove the lipid components and then extracted with ethyl acetate (2 L x 4). The combined ethyl acetate extract was concentrated to dryness under reduced pressure and then suspended in 4 L of water. After filtration, the resulting solution was allowed to pass through a resin Diaion HP-20 column (650 g, 6 x 40 cm, Supelco, Bellefonte, Pa) with a flow rate of 50 mL/min. The column was then washed exhaustively with 10 L water (to remove the nonphenolic compounds) and eluted with 6 L ethanol (to extract the phenolic compounds). The eluate was subsequently concentrated to a small volume and freeze-dried to yield a brown fluffy powder (9.23 g) of HPE. The prepared HPE was analyzed by HPLC. The contents corresponding to EC, ChA, HP, and IQ in HPE were 15.82%, 3.42%, 2.74%, and 2.03% (w/w), respectively. The HPLC chromatograms of HPE are shown in Figure 1.

View larger version (18K): [in this window] [in a new window]   Figure 1. High-performance liquid chromatography (HPLC) chromatograms of the hawthorn phenolic extract (HPE). The studied phenolics were detected at 278 nm for (-)-epicatechin (EC) (upper panel), 325 nm for chlorogenic acid (ChA) (middle panel), and 360 nm for both hyperoside (HP) and isoquercitrin (IQ) (lower panel).

Animals
The experiment was carried out using male Sprague-Dawley rats after approval by the Animal Ethics Committee of the Chinese University of Hong Kong. On the day before the study, a polyethylene tube (0.40 mm i.d., 0.80 mm o.d., Portex Limited, Hythe, Kent, England) was inserted into the right jugular vein of each rat under anesthesia. Afterwards, the rats were housed individually in metabolic cages. The animals were allowed to recover for 24 hours and fasted for at least 12 hours prior to drug administration.

Drug Administration
The rats were randomly divided into 6 dosing groups (2 for HPE and 1 for each of the 4 pure compounds, n = 5 rats per group). For intravenous dosing, a concentration of 44.0, 7.0, 1.6, 1.2, and 1.0 mg/mL was prepared for HPE, EC, ChA, HP, and IQ, respectively, by dissolving each in saline containing 5% DMSO. About 1 mL of the solution was given to rats by injection via the jugular vein, followed by a 0.2-mL heparinized saline flush. These correspond to a dosage of 220, 34.8, 7.5, 6.0, and 4.5 mg/kg of HPE, EC, ChA, HP, and IQ, respectively. For oral dosing, a similar dosage of each formulation was administered.

Sample Collection
Blood. For EC concentration determination, a 0.2-mL blood sample was collected at 5, 10, 20, 40, 60, 90, 120, 180, 240, and 360 minutes due to its relatively long half-life. For the rest of the 3 compounds, the samples were collected at 2, 5, 10, 15, 20, 30, 40, 50, 60, 90, and 120 minutes. At the last sampling time point, 0.4 mL blood was collected to enhance the assay sensitivity. After each blood sampling, the cannula was flushed with an equal volume of heparinized saline solution (20 IU/mL). The blood sample was immediately centrifuged at 4000 rpm for 5 minutes, and the plasma was collected and stored at -80°C until analysis.

Urine and feces. The urine and feces samples were collected over 12 and 24 hours, respectively, postdose. For the EC urine samples, 2 mL of ascorbic acid (0.2 g/mL) was added to prevent oxidation, and the urine was maintained at pH 2 to 4.² At the end of the urine collection, the metabolic cage was rinsed with 20 mL distilled water. The urine sample was combined with the rinsing solution and additional water to make up a final volume of 50 mL for concentration determination.

All the feces collected were homogenized in 100 mL of water. The mixture was sonicated for 30 minutes and centrifuged at 6000 rpm for 10 minutes. The supernatant was collected and stored at -80°C until analysis.

Stability of the Phenolics in Rat Plasma
To determine the stability of each compound, fresh rat plasma was spiked with each compound at a concentration of 20 µg/mL. After vortexing, the plasma was incubated in a 37°C water bath with continuous shaking. A 0.2-mL sample was analyzed in triplicate at multiple time intervals of up to 4 hours using HPLC.

Plasma Protein Binding of the Studied Phenolics
Plasma protein binding was determined by an ultrafiltration method³ using Whatman microcentrifuge tube filters (30K MWCO, Whatman, Inc, Clifton, NJ). Various concentrations (60.1, 30.1, and 6.0 µg/mL for EC; 13.0, 6.5, and 1.3 µg/mL for ChA; 10.4, 5.2, and 1.0 µg/mL for HP, and 7.7, 3.9, and 0.8 µg/mL for IQ) were prepared either as an extract or individual compound in plasma and phosphate buffer saline (PBS, pH 7.4). After incubation at 37°C for 30 minutes, the samples were centrifuged at 5000 rpm for 30 minutes. The filtrates (50 µL) were collected and analyzed by HPLC. Each experiment was performed in triplicate. The protein binding was calculated using the following equation: % protein binding = (1 - C_u/C) x 100%, where C_u is the concentration from the filtrate of plasma, and C is the concentration from the filtrate of PBS.

Sample Analysis
The plasma samples were prepared and analyzed as previously described.⁴ Urine (2 mL) and feces supernatant (3 mL) of EC, HP, and IQ were spiked with 50 µL of ascorbic acid (0.2 g/mL). For ChA samples, 10 µL of 50% (v/v) formic acid was added and then extracted 3 times with 3 mL ethyl acetate saturated with water. The combined ethyl acetate extract was concentrated to dryness by a centrifuge concentrator. The residue was reconstituted with 300 µL of 10% acetonitrile in sodium phosphate buffer (pH 2.4). After centrifugation at 13,200 rpm for 10 minutes, 100 µL of the supernatant was injected into HPLC for analysis.

The detection limits for EC, ChA, HP, and IQ were 200, 40, 30, and 30 ng/mL in plasma; 500, 60, 30, and 30 ng/mL in urine; and 170, 40, 15, and 15 ng/mL in feces supernatant, respectively.

Data Analysis
The plasma concentration versus time profiles were analyzed using WinNonlin software, version 2.1 (Pharsight Corporation, Mountain View, Calif). The noncompartmental method was employed to estimate the following pharmacokinetic parameters: initial plasma concentration (C₀) extrapolated to time 0, terminal elimination half-life (t_1/2,z), area under the plasma concentration-time curve from 0 to infinity (AUC_0-), total body clearance (CL), and apparent volume of distribution (V_d,z). The peak plasma concentration (C_max) and peak time (t_max) were read directly from the observed individual plasma concentration-time profiles after oral dosing. The oral bioavailability (F) was calculated based on the AUC_0- obtained from oral and IV administrations following the same dose of the same formulation.

Statistical analysis was performed by analysis of variance (ANOVA). A P value of less than .05 was considered significant. All data are expressed as mean ± SD.

	RESULTS

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Plasma Concentrations and Pharmacokinetic Parameters
The plasma drug concentration profiles and pharmacokinetic parameters of the 4 phenolics following IV administration are shown in Figure 2 and Table I. The plasma concentrations of EC, ChA, HP, and IQ were significantly higher following administration of HPE compared to that following administration of pure compounds (P < .05).

View larger version (18K): [in this window] [in a new window]   Figure 2. Plasma concentration versus time profiles of unchanged (-)-epicatechin (EC), chlorogenic acid (ChA), hyperoside (HP), and isoquercitrin (IQ) following intravenous administration of hawthorn phenolic extract (HPE) (diamonds) or individual pure compound (squares) at equivalent doses in rats. Data are expressed as mean ± SD (n = 5).

After oral administration of either HPE or 4 individual pure compounds, only EC was absorbed in the parent compound form. The plasma concentrations of EC from the extract or pure compound were similar, although there was a trend of higher C_max following administration of the pure compound (Figure 3 and Table II).

View larger version (11K): [in this window] [in a new window]   Figure 3. Plasma concentration versus time profiles of unchanged (-)-epicatechin (EC, n = 7) following oral administration of hawthorn phenolic extract (HPE, diamonds) or individual pure compound (squares) at equivalent doses in rats. Data are expressed as mean ± SD.

Stability in Plasma
HP and IQ were found to be stable in plasma, whereas EC and ChA were less stable, with 18% and 33% degradation, respectively, after a 4-hour incubation. The degradation half-lives of EC and ChA were calculated as 12.6 and 6.7 hours, respectively. Comparing the degradation half-lives of pure ChA, HP, and IQ with their in vivo elimination t_1/2,z after IV administration (9.8, 9.9, and 6.9 minutes, respectively), it could be concluded that the rapid elimination of ChA, HP, and IQ from the rat body might not be mainly caused by their chemical degradation but instead might be due to their metabolism in the liver and clearance by the kidneys.

Plasma Protein Binding
The in vitro plasma protein binding of the 4 phenolics are shown in Table III. In general, the protein binding of each compound was inversely proportional to its concentration. The binding of each compound was significantly lower in the extract formulation when compared to that in the individual pure compound, especially at high concentrations.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The results of our study clearly show that the pharmacokinetics of the 4 active hawthorn phenolics following HPE are significantly different from that following the individual pure compound administered via the intravenous route. In contrast, following oral administration, no significant differences in the pharmacokinetics between the 2 types of formulations are observed.

The mechanisms responsible for the difference in the pharmacokinetics of the hawthorn phenolics following IV but not oral administration are not clear. One potential mechanism could be the existence of the co-occurring components in HPE, which would be present in the body after intravenous but not after oral administration. It is likely that most of the co-occurring components would not have been absorbed following oral administration. This is supported by the finding that only 1 of the 4 studied components from HPE was observed following oral administration. Furthermore, even for EC, the only orally absorbable component, a much lower concentration and AUC_0- were observed following oral administration compared with that following intravenous administration. Thus, a potential mechanism could be the existence of the co-occurring components in the HPE, which would be present in the body after intravenous but not after oral administration. These co-occurring components in the extract following its IV administration could have inhibited the urinary and/or metabolic clearances or transporters. This is consistent with the observation of a higher plasma concentration and AUC and a longer t_1/2 after HPE in comparison to the pure compound following intravenous administration (Figure 2 and Table I).

Another potential mechanism could be related to tissue protein-binding displacement. In our studies, the plasma protein bindings of the studied phenolics were significantly decreased in the extract formulation in comparison to the pure individual compound. It is likely that the tissue protein binding of the phenolics is similarly displaced due to competition at the tissue binding site. Most likely, the presence of many co-occurring components in HPE following IV but not oral administration makes this tissue protein displacement interaction possible. This is consistent with the observation of smaller V_d,z and higher plasma concentration of the 4 active components following IV administration of HPE in comparison to the pure compound.

Our results on the differences of the pharmacokinetics of the 2 formulations following intravenous administration appear to be consistent with previous findings by Chen et al,⁵ who studied (-)-epigallocatechin-3-gallate (EGCG) and decaffeinated tea extract by IV administration. However, Chen et al measured the "total" concentration of the tested compounds. This may lead to imprecise calculation of the bioavailability of these compounds because the "total" concentration included not only the parent compound but also its glucuronidated and sulfated metabolites. In addition, the doses of the formulations given orally were very different from that given intravenously. These deficiencies make the interpretation of their data uncertain. In our study, the assay is specific for the parent compound, and we use the same dose intravenously and orally. Thus, our study methodology provides valid data to test the hypothesis.

In our studies, only EC was found to be absorbed in the parent form following oral administration of HPE or pure compound. The corresponding bioavailability (F) was calculated to be 34.2% following the administration of pure EC but only 10.9% following the administration of HPE. However, the AUC of EC following the oral administration of HPE or pure compound was similar. Thus, the differences in F following the 2 formulations are primarily attributed to the large difference in AUC following IV administration of HPE versus individual pure compound. These calculations indicate that the extract formulation, when administered intravenously, can significantly alter the calculation of the oral bioavailability of individual components.

In conclusion, our study results showed that different herbal formulations and different routes of administration could alter the pharmacokinetics of the herbal active substances. Such information points out the need of not only in vitro but also in vivo quality control of herbal products.

	ACKNOWLEDGEMENTS

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Financial support for this study is provided by RGC Competitive Earmarked Grant (CUHK 4122/01M) and Hong Kong Industrial Support Fund (AF/247/97).

	FOOTNOTES

 
DOI: 10.1177/0091270004270500

Submitted for publication April 26, 2004; Revised version accepted August 25, 2004.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 

1. Zhang Z, Chang Q, Zhu M, Huang Y, Ho WKK, Chen ZY. Characterization of antioxidants present in hawthorn fruits. J Nutr Biochem. 2001;12: 144-152.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

2. Zhu QY, Zhang A, Tsang D, Huang Y, Chen ZY. Stability of green tea catechins. J Agric Food Chem. 1997;45: 4624-4628.[CrossRef]

3. Kovacs SJ, Tenero DM, Martin DE, Ilson BE, Jorkasky DK. Pharmacokinetics and protein binding of eprosartan in hemodialysis-dependent patients with end-stage renal disease. Pharmacotherapy. 1999;19: 612-619.[Medline] [Order article via Infotrieve]

4. Chang Q, Zhu M, Zuo Z, Chow SSM, Ho WKK. High performance liquid chromatographic method for simultaneous determination of hawthorn active components in rat plasma. J Chromatogr B. 2001;760: 227-235.

5. Chen L, Lee MJ, Li H, Yang CS. Absorption, distribution, elimination of tea polyphenols in rats. Drug Metab Dispos. 1997;25: 1045-1050.[Abstract/Free Full Text]
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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Request Reprints Citing Articles Citing Articles via ISI Web of Science (8) Citing Articles via Google Scholar Google Scholar Articles by Chang, Q. Articles by Chow, M. S. S. Search for Related Content PubMed PubMed Citation Articles by Chang, Q. Articles by Chow, M. S. S. Social Bookmarking                   What's this?