Determination of hydroxysafflor yellow A in rat plasma and tissues by high-performance liquid chromatography after oral administration of safflower extract or safflor yellow

Copyright © 2007 John Wiley & Sons, Ltd. Biomed. Chromatogr. 21: 326–334 (2007)DOI: 10.1002/bmc

326 Y. Li et al.ORIGINAL RESEARCH ORIGINAL RESEARCH

Copyright © 2007 John Wiley & Sons, Ltd.

BIOMEDICAL CHROMATOGRAPHYBiomed. Chromatogr. 21: 326–334 (2007)Published online 15 January 2007 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/bmc.769

Determination of hydroxysafflor yellow A in rat plasmaand tissues by high-performance liquid chromatographyafter oral administration of safflower extractor safflor yellow

Yi Li,1 Zhao-yang Zhang2 and Jin-lan Zhang1*1Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Institute of Materia Medica, Chinese Academy of

Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China2Shenyang Pharmaceutical University, Shenyang 110016, Liaoning Province, People’s Republic of China

Received 6 September 2006; revised 8 October 2006; accepted 9 October 2006

ABSTRACT: A simple and reproducible HPLC method for quantification of hydroxysafflor yellow A (HSYA) in rat plasma andtissues after oral administration of safflower extract or safflor yellow (SY) was developed. Sample preparation was achieved byprotein precipitation of plasma and tissue homogenates with three volumes of methanol. p-Hydroxybenzaldehyde was used as theinternal standard (IS). HSYA and IS were separated on a Hypersil BDS-C18 column with a gradient elution system composed ofacetonitrile and aqueous acetic acid. UV detection was used at 320 nm. The calibration curves were linear in all matrices (r2 >0.999) in the concentration ranges 0.51–101.36 µg/mL for plasma, 12.27–2454.46 µg/g for intestines and 0.96–192.20 µg/g for lung.The intra-day and inter-day precision were all less than 12.5%, and the extract recovery was in the range 64.1–103.7% with RSDless than 15.6% for HSYA in all matrices. The method was used successfully to quantify HSYA in rat plasma and tissue samplesto support a pharmacokinectic study. Copyright © 2007 John Wiley & Sons, Ltd.

KEYWORDS: Carthamus tinctorius L.; hydroxysafflor Yellow A; absorption; distribution; HPLC

INTRODUCTION

The dried flower of the Safflower plant, Carthamustinctorius L. has been used extensively in traditionalChinese medicine (TCM) to treat coronary heart dis-ease, hypertension and cerebrovascular disease (Liang,2004). In traditional Chinese medicinal prescription saf-flower is mainly taken as decoction, therefore the water-soluble components are responsible for the therapeuticeffects, and results from phytochemical investigationshow that safflor yellow (SY) is the main constituent inwater-soluble extract of safflower. SY has a wide range

of pharmacological activities, including coronary dilata-tion, antioxidation, myocardial and cerebral protectionand immunosuppressive activity (Jin et al., 2004; Li andChen, 1998; Liu et al., 1992; Yang et al., 2001a).Hydroxysafflor yellow A (HSYA), the main activecomponent of SY, has been demonstrated to have theactivities of antioxidation, myocardial and cerebral pro-tective effects (Lu et al., 1991; Tian et al., 2004; Weiet al., 2005; Zhu et al., 2003, 2005a,b). Therefore,HSYA is chosen as an active marker component forcontrolling the quality of safflower in Chinese Phar-macopoeia (The State Pharmacopoeia Commission ofChina, 2005).

In recent years, some pharmacokinetic studies onHSYA have been performed after intravenous adminis-tration of pure HSYA or SY to animals (Chu et al.,2006; Liu et al., 2003, 2004; Xiong et al., 2004; Yanget al., 2001a–b). However, in traditional therapy withChinese medicine, the whole safflower not the simplebioactive component is orally administrated in the wayof decoction. In recent years the use of the safflowerextract and SY as coloring and flavoring agent hasincreased. It is important to investigate the absorption,distribution, metabolism and excretion of safflowerextract and SY after oral administration.

*Correspondence to: Jin-lan Zhang, Key Laboratory of BioactiveSubstances and Resources Utilization of Chinese Herbal Medicine,Institute of Materia Medica, Chinese Academy of Medical Sciences& Peking Union Medical College, 1# Xian Nong Tan Street, Beijing100050, People’s Republic of China.E-mail: [email protected]

Abbreviations used: HYSA, hydroxysafflor yellow A; SY, saffloryellow; TCM, traditional Chinese medicine.

Contract/grant sponsor: Ministry of Science and Technology of thePeople’s Republic of China; Contract/grant number: ke(2004-03).Contract/grant sponsor: Key Laboratory of Bioactive Substances andResources Utilization of Chinese Herbal Medicine (Peking UnionMedical College) of the Ministry of Education of the People’sRepublic of China.


Determination of hydroxysafflor yellow A in rat plasma and tissues 327ORIGINAL RESEARCH

In the present work the HPLC method to determineHSYA in biological fluids with simplicity, high sensitiv-ity and reproducibility is reported. Particular attentionis paid to directly measure the content of HSYA in ratplasma and tissues after oral administration of safflowerextract or SY.

EXPERIMENTAL

Reagents and materials

The dried flower of Carthamus tinctorius L. was purchasedfrom Hetian (Xinjiang, China). HSYA was isolated and puri-fied in our laboratory with more than 95% purity (deter-mined by HPLC). p-Hydroxybenzaldehyde (IS) was suppliedby Xudong Chemical Factory (Beijing, China, purity ≥97%).Amberlite™ XAD16 macroporous adsorption resin wasfrom Rohm and Haas Company (USA). ODS (200 µm) wasfrom Fuji Silysia Chemical (Kasugai, Japan). HPLC-gradeacetonitrile and methanol were obtained from Burdick &Jackson Company (Muskegon, MI, USA). The deionizedwater used throughout the study was purified by Milliporewater purification system (Millipore, MA, USA).

Chromatographic system

An Agilent 1100 Series HPLC system, composed of a quater-nary pump, vacuum degasser, an autosampler, thermostatedcolumn compartment and a DAD detector was used. TheHPLC separations were achieved using a Hypersil BDS-C18

column (250 × 4.6 mm, 5 µm) and an Eclipse XDB-C18 guardcolumn (12.5 × 4.6 mm, 5 µm). The mobile phase consisted ofacetonitrile (A) and 0.3% aqueous acetic acid (B) at a flowrate of 1.0 mL/min. Gradient elution was employed and thegradient program was used as follows: initial 0 min at 2%solvent A; then 0–20 min, linear increase from 2 to 10%solvent A; 20–25 min, linear increase from 10 to 17% solventA, 25–30 min, linear decrease from 17 to 2% solvent A. UVabsorption was monitored at 320 nm. The column tempera-ture was maintained at 25°C and the sample injection volumewas 10 µL.

Preparation of safflower extract, SY and HSYA

The dried flower of Carthamus tinctorius L. (1.0 kg) wasimmersed at room temperature three times with 8000, 6000and 6000 mL of deionized water for 12 h. The pooled extractwas filtered and evaporated to 2000 mL under vacuum. Then,95% ethanol was added to this 2000 mL aqueous extract tomake the final solution contain 80% ethanol, and filteredagain after 12 h. The filtrate was evaporated to dry undervacuum to get safflower extract. SY was successively separ-ated from the safflower extract by column chromatographyon macroporous adsorption resin, the aqueous elutes werecollected and concentrated, then cryo-desiccated to obtainpowder of SY. HSYA was purified from SY using ODScolumn. The column was eluted by deionized water. Theyellow fractions were collected and cryo-desiccated to obtainstandard HSYA (purity ≥95% by HPLC).

Preparation of the dosed solution of safflowerextract and SY

The orally administrated solution of safflower extract and SYwere prepared at the concentration of 607.0 and 56.1 mg/mLwith deionized water separately. The dosed solutions of twokinds of extracts were completely dissolved aqueous solu-tions. The contents of HSYA in two extracts were quantifiedby internal standard method so as to calculate the dosedamount of HSYA. The safflower extract and SY were dis-solved in deionized water to a final concentration of 3.04 and0.28 mg/mL, respectively. The volumes of 10 µL solution wereinjected into HPLC for analysis. The contents of HSYA insafflower extract and SY were 4.7 and 48.3%, respectively.The content of HSYA in dosed solution of safflower extractand SY were at 28.5 and 27.1 mg/mL, respectively.

Stock solutions

Stock solution of HSYA was prepared in 50% methanol ata concentration of approximately 5.0 mg/mL. The internalstandard solution of p-hydroxybenzaldehyde was prepared ata final concentration of 1.0 mg/mL in methanol. All solutionswere stored at −10°C prior to analysis.

Preparation of standards and quality controlsolutions

Rat plasma. Different volumes of HSYA stock solution and10 µL of IS solution were added to 1 mL plasma to preparebiological calibration curves and quality control samples.The final concentrations of biological calibration standardswere within the range 0.51–101.36 µg/mL. Quality controlsamples were prepared at concentrations of 0.51, 12.67 and101.36 µg/mL.

Rat intestines. Intestines were homogenized with threevolumes of physiological saline. The calibration curves in theconcentration range 12.27–2454.46 µg/g were prepared bydifferent volumes of HSYA stock solution and 10 µL of ISsolution to 100 µL of blank intestine homogenate. The QCsamples at three concentration levels (12.27, 245.45 and2454.46 µg/g) were prepared.

Rat lung. Lung was homogenized with two volumes of physi-ological saline. The calibration curves in the concentrationrange of 0.96–192.20 µg/g were prepared by different volumesof HSYA stock solution and 10 µL of IS solution to 1 mL ofblank lung homogenate. The QC samples at three concentra-tion levels (0.96, 24.02 and 192.20 µg/g) were also prepared.

Sample preparation

Rat plasma. To 1 mL of plasma sample, 10 µL of the IS solu-tion and 3 mL of methanol were added, then vortex-mixedfor 1 min and centrifuged at 4000 rpm for 10 min at roomtemperature. The supernatant was transferred to a clean flaskand evaporated to dryness at 32°C under vacuum. The resi-due was reconstituted in 500 µL of methanol–water (50:50,v/v), and a 10 µL aliquot was injected into the HPLC system.


328 Y. Li et al.ORIGINAL RESEARCH

Rat intestines. To 100 µL of intestine homogenate sample,10 µL of the IS solution were added. The homogenate wasdeproteinized with 300 µL of methanol, and centrifuged at4000 rpm for 10 min at room temperature, and a 10 µLaliquot of the supernatant were injected for analysis.

Rat lung. To 1 mL of lung homogenate sample, 10 µL of theIS solution were added. The homogenate was deproteinizedwith 3 mL of methanol, and centrifuged at 10,000 rpm for10 min at room temperature. The supernatant was transferredto the other clean flask and concentrated to remove solvent at32°C under vacuum. The residue was treated in the samemanner as for the plasma.

Pharmacokinetic and tissue distribution study

Male Wistar rats weighing 220 ± 10 g were obtained from theLaboratory Animal Center of Chinese Academy of MedicalSciences and Perking Union Medical College (Beijing, China).They were kept in an environmentally controlled breedingroom for 3 days before starting the experiments and fed withstandard laboratory food and water. Prior to oral administra-tion of safflower extract and SY the rats were fasted in ametabolic cage and maintained with physiological saline for24 h. Rats were randomized according to body weight, groupedeight per time point, and orally administered safflower extractor SY at the dose of 2119 mg of safflower extract/kg bodyweight or 207 mg of SY/kg body weight which was corre-spond to 100 mg HSYA/kg body weight, respectively. At eachtime point (5, 10, 15, 20, 30, 45, 60, 75, 90, 120, 150, 180 and240 min), blood samples were collected from abdominalartery under ether anesthesia into clean heparinized tubes.Plasma was separated by centrifugation at 4500 rpm for 5 min.Tissues were rapidly excised following blood collection. Allsamples were stored at −20°C until the assay was performed.

The following pharamacokimetic parameters: tmax (time tomaximum concentration), Cmax (maximum concentration),AUC (area under concentration–time curve), Vd (apparentvolume of distribution), Cl (clearance), MRT (mean resi-dence time) and AUMC (area under first moment curve)were calculated by non-compartment analysis using Win-Nonlin 5.0.1 program (Pharsight, CA, USA).

RESULTS AND DISCUSSION

Chromatography

The selection of the HPLC conditions was guided bythe requirement to obtain chromatograms with goodresolution of HSYA and internal standard within a

short time. It was found that acetic acid could improvethe separation. The gradient elution program wascarried out to successfully separate HSYA and internalstandard from interference and also to ensure that eachrun was completed in 35 min. HSYA is a compound ofchalcones; although the UV absorption spectrum ofHYSA possessed peaks at 230, 320 and 400 nm, 320 nmwas selected for the assay depending on the absorbanceand the interference from biological substances. Theinternal standard had a relatively strong UV absorptionat 320 nm like HSYA and an appropriate retentiontime without interference.

For biological samples organic precipitation in sampleprocessing could fit the quantitative analysis require-ment. However when the sample residue was dissolvedin 50% methanol, the peak shape of HSYA was greatlyimproved as well as the sensitivity. The HPLC methodsdeveloped for rat plasma and tissues suggested thatvalidation would be achieved.

Selectivity

Typical chromatograms of blank plasma, blank tissues,plasma sample and tissue samples are shown in Figs 1–3. HSYA and the internal standard in plasma andtissues were completely separated. The results pre-sented here show that a specific and selective HPLCmethod for the measurement of HSYA in rat plasmaand tissues after oral administration of safflower extractor SY has been developed.

Linearity and range

Plasma, tissue homogenates spiked with six differentconcentrations of HSYA were processed. The calibra-tion curves were constructed by calculating the peakarea ratios (y) of HSYA to IS against HSYA concen-trations (x). The calibration curves were linear withinthe concentration range 0.51–101.36 µg/mL for plasma,12.27–2454.46 µg/g for intestines, and 0.96–192.20 µg/gfor lung, with a correlation coefficient more than 0.999.The results are shown in Table 1.

Limit of detection and limit of quantification

The limit of detection (LOD) for HSYA was deter-mined when the ratio of the testing peak signal-to-noise

Table 1. Calibration curves for HSYA in rat plasma, intestines and lung

Sample matrix Standard curve Range (µg/mL or µg/g) Correlation Coefficient (r2)

Plasma y = 0.1654x − 0.0186 0.51–101.36 0.9999Intestines y = 0.0076x − 0.0263 12.27–2454.46 0.9999Lung y = 0.0999x − 0.0059 0.96–192.20 0.9996

y, peak area ratio (HYSA:internal standard); x, concentration of HSYA in samples (µg/mL or µg/g); µg/mL for plasma and µg/g for tissues.



Figure 1. Representative chromatograms of HSYA in plasma. (A) Blank plasma; (B) blankplasma spiked with HSYA (1) and internal standard (IS); (C) plasma at 10 min after oraladministration of safflower extract; (D) plasma at 20 min after oral administration of SY.

was 3. The limit of quantitation (LOQ) was the lowestconcentration of HSYA on the biological calibrationcurve with precision 20% and accuracy 80–120%.LOD for HSYA was 0.75 ng and LOQ were 0.51 µg/mL in plasma, 12.27 µg/g in intestines, and 0.96 µg/gin lung.

Precision and accuracy

The precision and accuracy of the method were studiedby analyzing the QC samples with low, medium andhigh concentrations. The measured concentrations ofQC samples were calculated from the biological



kinds of biological samples was within the range91.3–111.5%.

Recovery

The recoveries of HSYA were investigated from bio-logical samples spiked with standard solutions at low,

calibration curves. The precision was evaluated by theintra-day and inter-day variability with relative standarddeviation (RSD). The accuracy was obtained by meas-ured concentration of QC samples divided by spikedconcentration. The results are shown in Table 2. TheRSD of the overall intra- and inter-day precisionswere less than 12.5%. The accuracy for HSYA in three

Figure 2. Representative chromatograms of HSYA in intestines. (A) Blank intestines; (B)blank intestines spiked with HSYA (1) and internal standard (IS); (C) intestines at 10 minafter oral administration of safflower extract; (D) intestines at 20 min after oral administra-tion of SY.



Figure 3. Representative chromatograms of HSYA in lung. (A) Blank lung; (B) blank lungspiked with HSYA (1) and internal standard (IS); (C) lung at 10 min after oral administra-tion of safflower extract; (D) lung at 20 min after oral administration of SY.

Table 2. Intra-day and Inter-day precision of HSYA in plasma, intestines and lung

Inter-day precision and accuracy (n = 5Intra-day precision and accuracy (n = 6) for intestines and lung; n = 3 for plasma)

Spiked Measured RSD Accuracyb Measured RSD AccuracySamples (µg/mL or µg/g) (µg/mL or µg/g)a (%) (%) (µg/mL or µg/g) (%) (%)

Plasma 0.51 0.53 ± 0.03 7.9 103.9 0.48 ± 0.04 8.0 94.112.67 13.43 ± 0.25 1.9 106.0 11.89 ± 1.48 12.5 98.3

101.36 106.95 ± 4.12 4.3 105.5 93.96 ± 6.65 7.1 92.7

Intestines 12.27 13.60 ± 0.22 1.0 110.6 13.71 ± 0.24 1.7 111.5245.45 224.25 ± 1.95 0.3 91.4 224.11 ± 3.67 1.6 91.3

2454.46 2472.06 ± 6.82 0.3 100.7 2476.52 ± 6.88 0.3 100.9

Lung 0.96 1.04 ± 0.01 1.3 108.3 1.02 ± 0.10 9.4 106.324.02 24.00 ± 0.14 0.6 99.9 23.16 ± 0.75 3.3 96.4

192.20 186.41 ± 1.35 0.7 97.0 190.36 ± 3.74 2.0 99.0

a Mean ± SD; b accuracy = (mean of measured concentration/spiked concentration) × 100.



medium and high concentrations. The overall extractionrecovery of HSYA ranged from 64.1 to 103.7% withRSD less than 15.6% in the three kinds of biologicalsamples. The results are shown in Table 3.

Stability

The results of stability of HSYA in plasma and tissuehomogenates kept for 24 h at room temperature andafter three freeze (−10°C)/thaw cycles are shown inTable 4. HSYA was stable in plasma, intestines andlung within 24 h or three freeze–thaw cycles at −10°C.

Application—pharmacokinetics and tissuedistribution study

The described method was applied to analysis of plasmasamples after oral administration of safflower extract orSY. Figure 4 shows the mean plasma concentration-time profile of HSYA (n = 8). The absorption of HSYAwas rapid, with peak concentrations occurring at 10 minfor safflower extract and 20 min for SY after oraladministration.

As calculated from the mean plasma concentrationsof HSYA following oral administration of safflowerextract or SY, the non-compartmental pharmacokinetic

Table 3. Recovery of HSYA in plasma, intestines and lung (n ===== 6)

Spiked (µg/mL Measured Recoveryb RSDSamples or µg/g) (µg/mL or µg/g)a (%) (%)

Plasma 0.51 0.33 ± 0.05 64.1 15.612.67 11.31 ± 0.66 89.3 5.8

101.36 93.47 ± 0.57 92.2 0.6Intestines 12.27 8.54 ± 0.75 69.6 8.8

245.45 183.84 ± 7.90 74.9 4.32454.46 2545.27 ± 30.54 103.7 1.2

Lung 0.96 0.79 ± 0.04 82.8 4.524.02 22.49 ± 1.65 93.6 7.3

192.20 170.70 ± 8.78 88.8 5.1

a Mean ± SD; b recovery = (mean of measured concentration/spiked concentration) × 100.

Table 4. Stability of HSYA in rat plasma and tissue homogenates kept for 24 h at room temperature and during three freeze–thawcycles

Mean of percentage remaining (%)

Samples Spiked (µg/mL or µg/g) 24 h at room temperature Three freeze–thaw cycles

Plasma 0.51 81.5 84.512.67 92.9 85.7

101.36 90.1 91.6Intestines 12.27 95.7 103.5

245.45 98.1 103.42454.46 99.2 100.6

Lung 0.96 91.6 92.824.02 97.1 95.3

192.20 95.3 100.1

Figure 4. Mean plasma concentration–time profile of HSYAin rat plasma after oral administration. Each point representsthe mean ± SD.

parameters were calculated and listed in Table 5. Theseparameters indicated that HSYA was absorbed betterfrom safflower extract than SY. It was reported (Yanget al., 2001c) that the compound in safflower includedchalcone, polysaccharides, lignans, fatty acid, steroid,volatile oils and so on. SY was obtained from safflowerextract through discarding the non-chalcone components



Table 5. Mean pharmacokinetic parameters of HSYA in ratplasma (n ===== 8) after oral administration

Parameters Safflower extract SY

Tmax (min) 10 20Cmax (µg/mL) 5.84 0.82AUC0−t (min µg/mL) 246.33 114.73AUC0−∞ (min µg/mL) 337.30 269.69Vd (mL) 12800.04 34316.28Cl (mL/min) 64.72 79.72AUMC0−∞ (min min µg/mL) 58190.78 116169.13MRT0−∞ (min) 172.52 430.76

All pharmacokinetic parameters are calculated from the meanplasma concentrations.

Figure 5. HSYA in small intestine after oral administration.Each point represents the mean ± SD of eight animals. Thisfigure is available in colour online at www.interscience.wiley.com/journal/bmc

Figure 6. HSYA in cecum after oral administration. Eachpoint represents the mean ± SD of eight animals. This figureis available in colour online at www.interscience.wiley.com/journal/bmc

Figure 7. HSYA in large intestine after oral administration.Each point represents the mean ± SD of eight animals. Thisfigure is available in colour online at www.interscience.wiley.com/journal/bmc

safflower extract. It could be inferred that HSYA wasabsorbed mostly by the small intestine because the con-centration of HSYA in the small intestine was muchhigher than in the cecum and large intestine. The rea-son for the difference between the absorption after oraladministration of safflower extract and SY was prob-ably that HSYA was absorbed better from safflowerextract than SY in cecum and large intestine. Figure 8shows the HSYA in lung after oral administration ofsafflower extract. The reason why HSYA was high inlung and whether it would have efficacy or toxicityshould be investigated further.

TCMs are widely used in Asia, so the pharmacoki-netic study is a very important and useful methodto approach the pharmacological actions of TCMssince knowledge on the pharmacokinetics might helpto explain and predict a variety of events related tothe efficacy and toxicity of herbal preparations.

and concentrated chalcone compounds. These resultssuggested that the absorption of the active componentHSYA was affected by other coexisting componentsin safflower. Much attention should be paid to othercomponents which may not be active but affect thepharmacokinetic characteristics of active compoundswhen we study the TCM.

The assay described here was also successfully appliedto the investigation of tissue distribution of HSYA inrats. The pre-experiment indicated that HSYA couldbe detected in heart, liver, spleen, lung, kidney, brainand intestines after oral administration of safflowerextract or SY, and it was concluded that the distribu-tion of HSYA in the body was broad. However, onlythe HSYA in intestines after oral administration ofsafflower extract and SY and in lung after oral adminis-tration of safflower extract were over the limit of quan-tification. Figures 5–7 show the concentration of HSYAin intestines. The concentration of HSYA in small inte-stine after oral administration of safflower extract wasclose to that of SY, but in cecum and large intestine theconcentration was higher after oral administration of



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Figure 8. HSYA in lung after oral administration. Each pointrepresents the mean ± SD of eight animals. This figure isavailable in colour online at www.interscience.wiley.com/journal/bmc

Pharmacokinetic study of TCMs should join in the newdrug research as early as possible.

CONCLUSION

The method described here represents a simple, rapid andsensitive procedure for the determination of HSYA inbiological samples including plasma and tissues. Samplepreparation was simple and reliable. This HPLC methodpossessed adequate sensitivity, selectivity, accuracy andprecision. The pharmacokinetic parameters obtainedhere could provide more information for absorptionand distribution studies of HSYA. The results providescientific data for the further study of safflower andscientific clinical use of safflower extract and SY.

Acknowledgments

We thank the Ministry of Science and Technology ofthe People’s Republic of China [ke(2004-03)] and theKey Laboratory of Bioactive Substances and ResourcesUtilization of Chinese Herbal Medicine (Peking UnionMedical College) of the Ministry of Education of thePeople’s Republic of China for financial support of thiswork.

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Determination of hydroxysafflor yellow A in rat plasma and tissues by high-performance liquid chromatography after oral administration of safflower extract or safflor yellow