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Pharmacokinetic comparisons of hydroxysafflower yellow A in normal and blood stasis syndrome rats
Journal of Ethnopharmacology 129 (2010) 1–4
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Pharmacokinetic comparisons of hydroxysafflower yellow A in normal and blood stasis syndrome rats Yun Tian a,1 , Zhi-Fu Yang a,1 , Yan Li b , Yi Qiao a , Jing Yang a , Yan-Yan Jia a , Ai-Dong Wen a,∗ a b
Department of Pharmacy, Xijing Hospital, The Fourth Military Medical University, No. 127, Changle West Road, Xi’an, China Department of Comprehensive Diagnosis and Therapy, Xijing Hospital, The Fourth Military Medical University, No. 127, Changle West Road, Xi’an, China
a r t i c l e
i n f o
Article history: Received 30 November 2009 Received in revised form 10 February 2010 Accepted 18 February 2010 Available online 4 March 2010 Keywords: Hydroxysafflower yellow A Safflower extract Pharmacokinetics Blood stasis syndrome
a b s t r a c t Ethnopharmacological relevance: Safflower is a popular Traditional Chinese Medicine (TCM) to invigorate the blood and dispel ‘blood stasis’, which arises from poor blood circulation. The differences of pharmacokinetic properties between normal and blood stasis syndrome rats were seldom reported. Aim of the study: The present study was conducted to evaluate the pharmacokinetics of hydroxysafflower yellow A (HSYA) following oral administration of hydroxysafflower yellow A and safflower extract with approximately the same dose of HSYA 100 mg/kg in both normal and acute blood stasis rats. Materials and methods: The animals were orally administered with HYSA monomer and safflower extract. The blood samples were collected according to the time schedule. The concentrations of HSYA in rat plasma were determined by HPLC. Various pharmacokinetic parameters were estimated from the plasma concentration versus time data using non-compartmental methods. Results: It was found that AUC0–t , Cmax , Vd and CL of HSYA in both HSYA monomer and safflower extract in acute blood stasis rats were with significant difference (P < 0.05) comparing with that in normal rats. Conclusions: The results indicated that HSYA was with high uptake and eliminated slowly in the animals with blood stasis syndrome, suggesting that the rate and extent of drug metabolism was altered in acute blood stasis animals. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Carthamus tinctorius L., a dried flower, belongs to the Compositae family and naturally distributed over Asia and some Africa countries (Zhao et al., 2007). Carthamus tinctorius L. is the only species of this genus in China, which has been used as food additives, natural pigment and medicine, and is a centuries-old famous Traditional Chinese Medicine (TCM) (Fan et al., 2009). The florets of Carthamus tinctorius L. are popularly used for the treatment of cardiovascular, cerebrovascular and gynecological diseases (Liang, 2004). Safflower is mainly taken as decoction in traditional Chinese medicinal prescription. Hydroxysafflor yellow A (HSYA), the water soluble component, is responsible for the main curative effects of safflower. HSYA has chosen as an active marker component for controlling the quality of safflower in Chinese Pharmacopoeia (The State Pharmacopoeia Commission of China, 2005). Hydroxysafflower yellow A [Fig. 1(A)] has been demonstrated to have the activities of antioxidation, myocardial and cerebral protective effects (Zhu et al., 2003, 2005; Wei et al., 2005; Chu et al., 2006).
∗ Corresponding author. Tel.: +86 29 84773636; fax: +86 29 84773636. E-mail address: adwen [email protected] (A.-D. Wen). 1 These two authors contributed equally to this work. 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.02.023
Blood stasis was described in TCM theory as a slowing or pooling the blood due to disruption of heart Qi (Bensky and Gamble, 1993). It was explained by pathology as a state resulting from a sluggish or impeded flow of blood in the body or abnormal blood outside the vessels that remains in the body and fails to disperse (Chiu et al., 2002). What is more, it is often understood in biomedical terms of hematological disorders such as hemorrhage, congestion, thrombosis, local ischemia (microclots) and tissue changes (Bensky and Gamble, 1993). As soon as blood stasis developed, the blood circulation will further be affected and thus lead to new pathological changes. Pharmacokinetic characteristics could be affected by disease condition (Wen et al., 2001; Yang et al., 2001). Therefore, it is very important to investigate the pharmacokinetics of drugs in animals with blood stasis syndrome, which may influence absorption, metabolism and elimination of drugs in blood. In recent years, some pharmacokinetic studies on HSYA have been performed on healthy Chinese volunteers (Yang et al., 2009) and normal animals (Yu et al., 2007; Li et al., 2007; Qi et al., 2007; Chu et al., 2006). However, the pharmacokinetics of HSYA has not been investigated in detail. To our knowledge, most studies were focused on the pharmacokinetic characteristics of HSYA in healthy human beings or normal animals. And the pharmacokinetic properties of HSYA in animal with acute blood stasis and the differences between normal animals and animals with acute blood stasis syndrome were seldom reported. The primary objective of
2
Y. Tian et al. / Journal of Ethnopharmacology 129 (2010) 1–4
Fig. 1. The chemical structure of hydroxysafflor yellow A (A) and internal standard riboflavin (B).
this study was to investigate the possible pharmacokinetic differences of the compounds after oral administration of monomer and safflower extract in normal rats and rats with acute blood stasis syndrome. 2. Materials and methods 2.1. Chemicals and reagents Carthamus tinctorius L. was purchased from Xi’an Traditional Chinese Medicine Co. and authenticated by Dr. Guolian Lei (College of Pharmacy, Shaanxi University of Chinese Medicine). HSYA (98.0% purity) was provided by Shandong Lvye Natural Medicine Research and Development Center [Shandong, China]. Riboflavin [98.7% purity], the internal standard (I.S.) [Fig. 1(B)], was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile of HPLC-grade was purchased from Fisher Scientific (Pittsburgh, PA, USA). Water was distilled and purified using a Milli-Q Water Purification System (Millipore, Bedford, MA, USA). The potassium dihydrogen phosphate (KH2 PO4 ), ortho-phosphoric acid and perchloric acid were all of analytical grade. Drug-free human plasma from healthy donors was kindly provided by the Blood Center of Xijing Hospital (Xi’an, China)
2.4. Animals The investigation was conducted in accordance with the ethical principles of animal use and care (Directive 86/609/EEC on the Protection of Animals Used for Experimental and Other Scientific Purposes, 1986). A total of 32 male rats weighing 280 ± 20 g were used for this study. The rats were supplied by the animal lab of the Fourth Military Medical University (Xi’an, China). The rats were maintained in an air-conditioned animal quarter at a temperature of 22 ± 2 ◦ C and a relative humidity of 50 ± 10%. Water and food (laboratory rodent chow, Xi’an, China) were allowed ad libitum. The animals were acclimatized to the facilities for five days, and then fasted with free access to water for 12 h prior to each experiment. 2.5. In vivo study
Safflower was soaked in water for 30 min, and decocted twice by refluxing with water (1:6, w/v) for 1 h under 100 ◦ C. The solution obtained was put together and condensed under 60 ◦ C. The concentrated solution was then adjusted to pH 4.0 by HCL. Then, 60% ethanol was added. The solution was stayed overnight, centrifuged and filtered. Thereafter, the safflower extract was obtained by dried powder spraying. The dried powder was stored at −80 ◦ C before use.
Animals were randomly divided into the following four groups (n = 8): normal control group administrated with HSYA, normal control group administrated with safflower extract, acute blood stasis model group administrated with HSYA and acute blood stasis model group administrated with safflower extract, respectively. Each rat was in an individual cage. The animals were orally administered with HYSA monomer and safflower extract. The acute blood stasis group was injected with adrenaline hydrochloride injection (0.7 mg/kg). After 4 h, those rats were injected with the same injection again, waiting for 2 h, the rats were soaked in ice-water for 5 min keeping their heads outside surface. The rats were put in metabolic cages, and fed freely. An oral dose of 100 mg/kg HSYA, and 5.62 g/kg safflower extract (equal content of HSYA was contained) were administrated, all of which were suspended in 0.5% carboxymethyl cellulose sodium salt (CMC-Na) aqueous solution just before each experiment. 1 mL blood samples were collected in heparinized eppendorf tube via the caudal vein before dosing and subsequently at 0.25, 0.5, 0.75, 1, 2, 3, 4, and 6 h following oral administration. After centrifuging at 5000 rpm for 10 min, the plasma samples were obtained and frozen at −20 ◦ C until analysis.
2.3. Content of hydroxysallor yellow A in safflower extract
2.6. Instrumentation and chromatographic conditions
To calculate the administered dose, the contents of HSYA in safflower extract were quantitatively analyzed. The extract powder was ultrasonicated with 60% ethanol for 1 h, and then the suspension was diluted 100 times. After centrifuging at 10,000 rpm for 10 min, the supernatant was analyzed using a HPLC method. The contents of HSYA were determined to be 1.78% (w/w) in safflower extract.
Analyses were performed on a Shimadzu Scientific Instruments (Kyoto, Japan) liquid chromatographic system which was composed of a LC-10Avp binary pump, a SPD-10Avp variable wavelength detector and a computer system for data acquisition (LC-Solution). The analytical column employed was a Shim-pack VP-ODS C18 column (150 mm × 4.6 mm I.D., 5 m particle size) protected with a ODS guard column (10 mm × 4.6 mm I.D., 5 m
2.2. Preparations of safflower extract
Y. Tian et al. / Journal of Ethnopharmacology 129 (2010) 1–4
3
Fig. 2. Mean concentration–time profiles of (A) HSYA in normal and acute blood stasis rats and (B) safflower extract in normal and acute blood stasis rats (ABS).
Table 1 Pharmacokinetic parameters of HYSA in rats after oral administration of HYSA and safflower extract (n = 8). HYSA
Safflower extract
Normal rats AUC0–t (g h/mL) AUC0–∞ (g h/mL) MRT0–t (h) Cmax (g/mL) Tmax (h) t1/2 (h) CL (mL kg/h) Vd (L/kg)
9.52 9.79 1.76 4.82 1.00 1.15 10.21 16.95
± ± ± ± ± ± ± ±
0.89 0.78 0.23 0.22 0.00 0.28 1.27 1.53
Acute blood stasis rats 25.05 26.37 2.32 7.23 2.00 1.11 3.79 6.09
± ± ± ± ± ± ± ±
2.54* 2.25* 0.50 1.02* 0.00* 0.24 0.59* 0.66*
Normal rats 10.66 10.91 1.70 5.75 0.67 1.05 9.17 13.85
± ± ± ± ± ± ± ±
0.61 0.74 0.45 0.67 0.09 0.08 0.77 0.75
Acute blood stasis rats 27.04 29.91 2.16 9.72 1.00 1.66 3.34 7.99
± ± ± ± ± ± ± ±
1.45* 1.52* 0.70 0.94* 0.00 0.12 0.28* 0.39*
Data represents the Mean ± S.D. * P < 0.05, versus normal rats.
particle size). The mobile phase was composed of acetonitrile (A) and 0.02 M KH2 PO4 (adjusted to pH 3.5 with ortho-phosphoric acid, (B)). Gradient elution was employed and the gradient program was set as follows: initial 0 min at 10% solvent A; 0–16 min, linear increased from 10% to 22% solvent A. The system was balanced for 5 min by the initial mobile phase (A:B = 10:90) after detecting each sample. The flow rate was set at 0.8 mL/min. The injection volume was 50 L. A detection wavelength was set at 403 nm. 2.7. Sample preparation The 200 L aliquot of plasma sample was added with 40 L I.S. (20 g/mL) and 40 L 50% methanol in a 1.5 mL tube. The mixture was vortexed for 30 s. 120 L of 6% perchloric acid was added, then vortexed for 2 min. The mixture was centrifuged at 12,000 rpm for 10 min. 50 L aliquot of the supernatant was injected into the HPLC system.
2.8. Pharmacokinetic and statistical analysis Maximum plasma concentration (Cmax ) and the corresponding time (Tmax ) values were obtained directly from the observed concentrations versus time data. Area under the curve from time zero to last sampling time (AUC0–t ) was calculated by the trapezoidal method. Terminate half-life (T1/2 ) was defined as T1/2 = 0.693/Ke and the Ke was determined by unweighted linear regression of logarithmical plasma concentration versus time for the last 4–5 time points. Pharmacokinetic parameters, Cmax and Tmax , plasma clearance (CL) as well as apparent volume of distribution (Vd), were calculated, which values were given as mean ± standard deviation (S.D.). The statistical analysis was performed with SPSS Version 10.0 (SPSS Inc., Chicago, IL, USA). An unpaired Student’s t-test was used for the comparison. All statistical tests were performed at the twosided 5% level of significance.
3. Results The mean plasma concentration–time profiles of HYSA were determined after oral administration of HYSA and safflower extract. The results are illustrated in Fig. 2. The partial pharmacokinetic parameters were given in Table 1. After comparing the pharmacokinetic data of the normal group with that of blood stasis group with t-test, it could be concluded that there were significant differences (P < 0.05) between the important pharmacokinetic parameters, such as AUC0–t and Cmax , CL. From the pharmacokinetic parameters obtained using the DAS2.1 computer program with the non-compartmental method, the half-lives of HSYA in both the normal and blood stasis groups are all very short and 90% of the HSYA are eliminated within 6 h. In comparison, the pharmacokinetics of HSYA changed greatly when HSYA and safflower extracts were administered in acute blood stasis rats. The pharmacokinetic parameters of HSYA summarized in Table 1 showed that there were statistically significant differences in parameters including the Cmax , AUC0–t , Vd and CL, between the normal rats and acute blood stasis rats, both receiving HSYA (100 mg/kg) and extract of safflower containing HSYA (100 mg/kg). Particularly, in the acute blood stasis animals, the peak plasma concentration of HSYA was remarkably increased (P < 0.05), the AUC0–t was increased (P < 0.05), the CL was decreased (P < 0.05), compared to the normal animals. For safflower extract, the half-life of HSYA increased markedly. The results indicated that HSYA was with high uptake and eliminated slowly in the animals with blood stasis syndrome. 4. Discussion Many researches demonstrated that disease condition will cause the alteration of pharmacokinetic parameters (Pfeifrs, 1991a,b,c; Jinwey, 1996). It was proved by Ren et al. (2006) that the PK analysis of ferulic acid in the patients with three different syndrome, namely deficiency of spleen Qi, stagnation of liver Qi
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and spleen deficiency, and excess of stomach heat, shown obvious differences in the PK characteristics. In our study, blood stasis syndrome was chosen and the results demonstrated significant low clearance, high AUC in rats with acute blood stasis syndrome compared to the parameters obtained in the plasma of normal rats. The rats with blood stasis syndrome will appear the sluggish of blood circulation, leading to the change of hemorrheology parameters, including the increase of whole blood viscosity, plasma viscosity, hematocrit and fibrinogen. HSYA was mainly absorbed in small intestine, poor blood circulation will prolong the retention time of HSYA in small intestine and cause the increase of HSYA’s absorption. This result was supported by Tian (1989). His study has shown that the compounds absorbed from the gut following oral administration are actually affected by syndrome, therefore cause the difference of plasma concentrations. HSYA was widely distributed in many organs, such as liver and kidney. The distribution of HSYA in blood in rats with blood stasis syndrome will become longer than that in normal rats since the value of Tmax are increased significant after oral administration of HSYA (P < 0.05). And primary study in our laboratory showed that the blood stasis syndrome tended to be the inhibitor of the CYP3A4 and the inducer of CYP1A2, but the CYP2E1 was hardly affected. Owing to the above reasons, blood stasis syndrome will have effect on the pharmacokinetic properties of HSYA in vivo. 5. Conclusion In the present study, we investigated the pharmacokinetics of HSYA and safflower extract in normal and blood stasis model rats. There were statistically significant differences in pharmacokinetic parameters of HSYA including the Cmax , AUC0–t , CL, and Vd between the normal and acute blood stasis rats, both orally administered with HSYA and safflower extract. The results suggested that the rate and extent of drug metabolism was altered in animals with blood stasis syndrome. The obtained knowledge can be used to evaluate impact of the differences on the efficacy and safety of the both drugs in clinical applications. Acknowledgements We thank the grant from the National Natural Science Foundation of China for financial support (No. 30672662) and the Traditional Chinese Medicine Foundation of Shaanxi Province for financial support (No. ZY24). References Bensky, D., Gamble, A., 1993. Chinese Herbal Medicine Materia Medica, Revised Edition. Eastland Press, Washington, pp. 265–266.
Chiu, C.C., Lan, C.Y., Chang, Y.H., 2002. Objective assessment of blood stasis using computerized inspection of sublingual veins. Computer Methods and Programs in Biomedicine 69, 1–12. Chu, D.F., Liu, W.H., Huang, Z., Liu, S.S., Fu, X.Q., Liu, K., 2006. Pharmacokinetics and excretion of hydroxysafflor yellow A, a potent neuroprotective agent from safflower, in rats and dogs. Planta Medicine 72, 418–423. Directive 86/609/EEC on the Protection of Animals Used for Experimental and Other Scientific Purposes, 1986. European Commission. Fan, L., Zhao, H.Y., Xu, M., Zhou, L., Guo, H., Han, J., Wang, B.R., Guo, D.A., 2009. Qualitative evaluation and quantitative determination of 10 major active components in Carthamus tinctorius L. by high-performance liquid chromatography coupled with diode array detector. Journal of Chromatography A 1216, 2063–2070. Jinwey, 1996. The population pharmacology research on pharma and cokinetics of drugs. Pharmazie 46, 623–629. Li, Y., Zhang, Z.Y., Zhang, J.L., 2007. Determination of hydroxysafflor yellow A in rat plasma and tissues by high-performance liquid chromatography after oral administration of safflower extract or safflor yellow. Biomedical Chromatography 21, 326–334. Liang, X.T., 2004. Basic Study of Common Chinese Medicine. Part II. Science Press, Beijing, pp. 102–115. Pfeifrs, S., 1991a. The effect of disease on the pharmacokinetics of drugs. Kidney Disease Pharmazie 46, 305–312. Pfeifrs, S., 1991b. The effect of disease on the pharmacokinetics of drugs 1. Pharmazie 46, 830. Pfeifrs, S., 1991c. The effect of disease states on pharmacokinetics of drugs 2. Liver Biliary Disease Pharmazie 46, 623–628. Qi, J.P., Jin, X.F., Huang, L.S., Ping, Q.N., 2007. Simultaneous determination of hydroxysafflor yellow A and ferulic acid in rat plasma after oral administration of the co-extractum of Rhizoma chuanxiong and Flos Carthami by HPLC-diode array detector. Biomedical Chromatography 21, 816–822. Ren, P., Huang, X., Li, S.Q., 2006. Pharmacokinetic characteristics of ferulic acid in patients with different syndromes of deficiency of spleen qi, stagnation of liver qi and spleen deficiency, and excess of stomach heat. Journal of Chinese Integrative Medicine 4, 147–151. The State Pharmacopoeia Commission of China, 2005. Pharmacopoeia of the People’s Republic of China, Part I. Chemical Industry Press, Beijing, China, p. 10. Tian, Z.M., 1989. The development of blood concentration measurement on the syndrome of Chinese formula and efficacy determination. Foreign Medical Sciences 10, 58–59. Wei, X.B., Liu, H.Q., Sun, X., Fu, F.H., Zhang, X.M., Wang, J., An, J., Ding, H., 2005. Hydroxysafflor yellow A protects rat brains against ischemia-reperfusion injury by antioxidant action. Neuroscience Letters 386, 58–62. Wen, A.D., Huang, X., Jiang, Y.P., et al., 2001. Blood stasis in congestive heart failure bears relation with digoxin clinical pharmacokinetics. Journal of Fourth Mililitary Medical University 22, 631–634. Yang, Z.F., Wen, A.D., Mei, Q.B., et al., 2001. Pharmacokinetical study of safflor yellow on rat acute model of blood stasis syndrome. Journal of Chinese Medicinal Materials 24, 730–732. Yang, Z.F., Yang, J., Jia, Y.Y., Tian, Y., Wen, A.D., 2009. Pharmacokinetic properties of hydroxysafflor yellow A in healthy Chinese female volunteers. Journal of Ethnopharmacology 124, 635–638. Yu, Z.G., Gao, X.X., Zhao, Y.L., Bi, K.S., 2007. HPLC determination of safflor yellow A and three active isoflavones from TCM Naodesheng in rat plasma and tissues and its application to pharmacokinetic studies. Biomedical Chromatography 21, 577–584. Zhao, H., Wu, W., Zheng, Y.L., Wei, Y.M., Yang, Y.X., 2007. Genetic diversity of Carthamus tinctorius L. based on RAMP Markers. Journal of Plant Genetic Resources 8, 64–67. Zhu, H.B., Wang, Z.H., Ma, C.J., Tian, J.W., Fu, F.H., Li, C.L., Guo, D., Roder, E., Liu, K., 2003. Neuroprotective effects of hydroxysafflor yellow A in vivo and in vitro studies. Planta Medicine 69, 429–433. Zhu, H.B., Wang, Z.H., Tian, J.W., Fu, F.H., Liu, K., Li, C.L., 2005. Protective effect of hydroxysafflor yellow A on experimental cerebral ischemia in rats. Acta Pharmaceutica Sinica 40, 1144–1146.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Pharmacokinetic comparisons of hydroxysafflower yellow A in normal and blood stasis syndrome rats Yun Tian a,1 , Zhi-Fu Yang a,1 , Yan Li b , Yi Qiao a , Jing Yang a , Yan-Yan Jia a , Ai-Dong Wen a,∗ a b
Department of Pharmacy, Xijing Hospital, The Fourth Military Medical University, No. 127, Changle West Road, Xi’an, China Department of Comprehensive Diagnosis and Therapy, Xijing Hospital, The Fourth Military Medical University, No. 127, Changle West Road, Xi’an, China
a r t i c l e
i n f o
Article history: Received 30 November 2009 Received in revised form 10 February 2010 Accepted 18 February 2010 Available online 4 March 2010 Keywords: Hydroxysafflower yellow A Safflower extract Pharmacokinetics Blood stasis syndrome
a b s t r a c t Ethnopharmacological relevance: Safflower is a popular Traditional Chinese Medicine (TCM) to invigorate the blood and dispel ‘blood stasis’, which arises from poor blood circulation. The differences of pharmacokinetic properties between normal and blood stasis syndrome rats were seldom reported. Aim of the study: The present study was conducted to evaluate the pharmacokinetics of hydroxysafflower yellow A (HSYA) following oral administration of hydroxysafflower yellow A and safflower extract with approximately the same dose of HSYA 100 mg/kg in both normal and acute blood stasis rats. Materials and methods: The animals were orally administered with HYSA monomer and safflower extract. The blood samples were collected according to the time schedule. The concentrations of HSYA in rat plasma were determined by HPLC. Various pharmacokinetic parameters were estimated from the plasma concentration versus time data using non-compartmental methods. Results: It was found that AUC0–t , Cmax , Vd and CL of HSYA in both HSYA monomer and safflower extract in acute blood stasis rats were with significant difference (P < 0.05) comparing with that in normal rats. Conclusions: The results indicated that HSYA was with high uptake and eliminated slowly in the animals with blood stasis syndrome, suggesting that the rate and extent of drug metabolism was altered in acute blood stasis animals. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Carthamus tinctorius L., a dried flower, belongs to the Compositae family and naturally distributed over Asia and some Africa countries (Zhao et al., 2007). Carthamus tinctorius L. is the only species of this genus in China, which has been used as food additives, natural pigment and medicine, and is a centuries-old famous Traditional Chinese Medicine (TCM) (Fan et al., 2009). The florets of Carthamus tinctorius L. are popularly used for the treatment of cardiovascular, cerebrovascular and gynecological diseases (Liang, 2004). Safflower is mainly taken as decoction in traditional Chinese medicinal prescription. Hydroxysafflor yellow A (HSYA), the water soluble component, is responsible for the main curative effects of safflower. HSYA has chosen as an active marker component for controlling the quality of safflower in Chinese Pharmacopoeia (The State Pharmacopoeia Commission of China, 2005). Hydroxysafflower yellow A [Fig. 1(A)] has been demonstrated to have the activities of antioxidation, myocardial and cerebral protective effects (Zhu et al., 2003, 2005; Wei et al., 2005; Chu et al., 2006).
∗ Corresponding author. Tel.: +86 29 84773636; fax: +86 29 84773636. E-mail address: adwen [email protected] (A.-D. Wen). 1 These two authors contributed equally to this work. 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.02.023
Blood stasis was described in TCM theory as a slowing or pooling the blood due to disruption of heart Qi (Bensky and Gamble, 1993). It was explained by pathology as a state resulting from a sluggish or impeded flow of blood in the body or abnormal blood outside the vessels that remains in the body and fails to disperse (Chiu et al., 2002). What is more, it is often understood in biomedical terms of hematological disorders such as hemorrhage, congestion, thrombosis, local ischemia (microclots) and tissue changes (Bensky and Gamble, 1993). As soon as blood stasis developed, the blood circulation will further be affected and thus lead to new pathological changes. Pharmacokinetic characteristics could be affected by disease condition (Wen et al., 2001; Yang et al., 2001). Therefore, it is very important to investigate the pharmacokinetics of drugs in animals with blood stasis syndrome, which may influence absorption, metabolism and elimination of drugs in blood. In recent years, some pharmacokinetic studies on HSYA have been performed on healthy Chinese volunteers (Yang et al., 2009) and normal animals (Yu et al., 2007; Li et al., 2007; Qi et al., 2007; Chu et al., 2006). However, the pharmacokinetics of HSYA has not been investigated in detail. To our knowledge, most studies were focused on the pharmacokinetic characteristics of HSYA in healthy human beings or normal animals. And the pharmacokinetic properties of HSYA in animal with acute blood stasis and the differences between normal animals and animals with acute blood stasis syndrome were seldom reported. The primary objective of
2
Y. Tian et al. / Journal of Ethnopharmacology 129 (2010) 1–4
Fig. 1. The chemical structure of hydroxysafflor yellow A (A) and internal standard riboflavin (B).
this study was to investigate the possible pharmacokinetic differences of the compounds after oral administration of monomer and safflower extract in normal rats and rats with acute blood stasis syndrome. 2. Materials and methods 2.1. Chemicals and reagents Carthamus tinctorius L. was purchased from Xi’an Traditional Chinese Medicine Co. and authenticated by Dr. Guolian Lei (College of Pharmacy, Shaanxi University of Chinese Medicine). HSYA (98.0% purity) was provided by Shandong Lvye Natural Medicine Research and Development Center [Shandong, China]. Riboflavin [98.7% purity], the internal standard (I.S.) [Fig. 1(B)], was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile of HPLC-grade was purchased from Fisher Scientific (Pittsburgh, PA, USA). Water was distilled and purified using a Milli-Q Water Purification System (Millipore, Bedford, MA, USA). The potassium dihydrogen phosphate (KH2 PO4 ), ortho-phosphoric acid and perchloric acid were all of analytical grade. Drug-free human plasma from healthy donors was kindly provided by the Blood Center of Xijing Hospital (Xi’an, China)
2.4. Animals The investigation was conducted in accordance with the ethical principles of animal use and care (Directive 86/609/EEC on the Protection of Animals Used for Experimental and Other Scientific Purposes, 1986). A total of 32 male rats weighing 280 ± 20 g were used for this study. The rats were supplied by the animal lab of the Fourth Military Medical University (Xi’an, China). The rats were maintained in an air-conditioned animal quarter at a temperature of 22 ± 2 ◦ C and a relative humidity of 50 ± 10%. Water and food (laboratory rodent chow, Xi’an, China) were allowed ad libitum. The animals were acclimatized to the facilities for five days, and then fasted with free access to water for 12 h prior to each experiment. 2.5. In vivo study
Safflower was soaked in water for 30 min, and decocted twice by refluxing with water (1:6, w/v) for 1 h under 100 ◦ C. The solution obtained was put together and condensed under 60 ◦ C. The concentrated solution was then adjusted to pH 4.0 by HCL. Then, 60% ethanol was added. The solution was stayed overnight, centrifuged and filtered. Thereafter, the safflower extract was obtained by dried powder spraying. The dried powder was stored at −80 ◦ C before use.
Animals were randomly divided into the following four groups (n = 8): normal control group administrated with HSYA, normal control group administrated with safflower extract, acute blood stasis model group administrated with HSYA and acute blood stasis model group administrated with safflower extract, respectively. Each rat was in an individual cage. The animals were orally administered with HYSA monomer and safflower extract. The acute blood stasis group was injected with adrenaline hydrochloride injection (0.7 mg/kg). After 4 h, those rats were injected with the same injection again, waiting for 2 h, the rats were soaked in ice-water for 5 min keeping their heads outside surface. The rats were put in metabolic cages, and fed freely. An oral dose of 100 mg/kg HSYA, and 5.62 g/kg safflower extract (equal content of HSYA was contained) were administrated, all of which were suspended in 0.5% carboxymethyl cellulose sodium salt (CMC-Na) aqueous solution just before each experiment. 1 mL blood samples were collected in heparinized eppendorf tube via the caudal vein before dosing and subsequently at 0.25, 0.5, 0.75, 1, 2, 3, 4, and 6 h following oral administration. After centrifuging at 5000 rpm for 10 min, the plasma samples were obtained and frozen at −20 ◦ C until analysis.
2.3. Content of hydroxysallor yellow A in safflower extract
2.6. Instrumentation and chromatographic conditions
To calculate the administered dose, the contents of HSYA in safflower extract were quantitatively analyzed. The extract powder was ultrasonicated with 60% ethanol for 1 h, and then the suspension was diluted 100 times. After centrifuging at 10,000 rpm for 10 min, the supernatant was analyzed using a HPLC method. The contents of HSYA were determined to be 1.78% (w/w) in safflower extract.
Analyses were performed on a Shimadzu Scientific Instruments (Kyoto, Japan) liquid chromatographic system which was composed of a LC-10Avp binary pump, a SPD-10Avp variable wavelength detector and a computer system for data acquisition (LC-Solution). The analytical column employed was a Shim-pack VP-ODS C18 column (150 mm × 4.6 mm I.D., 5 m particle size) protected with a ODS guard column (10 mm × 4.6 mm I.D., 5 m
2.2. Preparations of safflower extract
Y. Tian et al. / Journal of Ethnopharmacology 129 (2010) 1–4
3
Fig. 2. Mean concentration–time profiles of (A) HSYA in normal and acute blood stasis rats and (B) safflower extract in normal and acute blood stasis rats (ABS).
Table 1 Pharmacokinetic parameters of HYSA in rats after oral administration of HYSA and safflower extract (n = 8). HYSA
Safflower extract
Normal rats AUC0–t (g h/mL) AUC0–∞ (g h/mL) MRT0–t (h) Cmax (g/mL) Tmax (h) t1/2 (h) CL (mL kg/h) Vd (L/kg)
9.52 9.79 1.76 4.82 1.00 1.15 10.21 16.95
± ± ± ± ± ± ± ±
0.89 0.78 0.23 0.22 0.00 0.28 1.27 1.53
Acute blood stasis rats 25.05 26.37 2.32 7.23 2.00 1.11 3.79 6.09
± ± ± ± ± ± ± ±
2.54* 2.25* 0.50 1.02* 0.00* 0.24 0.59* 0.66*
Normal rats 10.66 10.91 1.70 5.75 0.67 1.05 9.17 13.85
± ± ± ± ± ± ± ±
0.61 0.74 0.45 0.67 0.09 0.08 0.77 0.75
Acute blood stasis rats 27.04 29.91 2.16 9.72 1.00 1.66 3.34 7.99
± ± ± ± ± ± ± ±
1.45* 1.52* 0.70 0.94* 0.00 0.12 0.28* 0.39*
Data represents the Mean ± S.D. * P < 0.05, versus normal rats.
particle size). The mobile phase was composed of acetonitrile (A) and 0.02 M KH2 PO4 (adjusted to pH 3.5 with ortho-phosphoric acid, (B)). Gradient elution was employed and the gradient program was set as follows: initial 0 min at 10% solvent A; 0–16 min, linear increased from 10% to 22% solvent A. The system was balanced for 5 min by the initial mobile phase (A:B = 10:90) after detecting each sample. The flow rate was set at 0.8 mL/min. The injection volume was 50 L. A detection wavelength was set at 403 nm. 2.7. Sample preparation The 200 L aliquot of plasma sample was added with 40 L I.S. (20 g/mL) and 40 L 50% methanol in a 1.5 mL tube. The mixture was vortexed for 30 s. 120 L of 6% perchloric acid was added, then vortexed for 2 min. The mixture was centrifuged at 12,000 rpm for 10 min. 50 L aliquot of the supernatant was injected into the HPLC system.
2.8. Pharmacokinetic and statistical analysis Maximum plasma concentration (Cmax ) and the corresponding time (Tmax ) values were obtained directly from the observed concentrations versus time data. Area under the curve from time zero to last sampling time (AUC0–t ) was calculated by the trapezoidal method. Terminate half-life (T1/2 ) was defined as T1/2 = 0.693/Ke and the Ke was determined by unweighted linear regression of logarithmical plasma concentration versus time for the last 4–5 time points. Pharmacokinetic parameters, Cmax and Tmax , plasma clearance (CL) as well as apparent volume of distribution (Vd), were calculated, which values were given as mean ± standard deviation (S.D.). The statistical analysis was performed with SPSS Version 10.0 (SPSS Inc., Chicago, IL, USA). An unpaired Student’s t-test was used for the comparison. All statistical tests were performed at the twosided 5% level of significance.
3. Results The mean plasma concentration–time profiles of HYSA were determined after oral administration of HYSA and safflower extract. The results are illustrated in Fig. 2. The partial pharmacokinetic parameters were given in Table 1. After comparing the pharmacokinetic data of the normal group with that of blood stasis group with t-test, it could be concluded that there were significant differences (P < 0.05) between the important pharmacokinetic parameters, such as AUC0–t and Cmax , CL. From the pharmacokinetic parameters obtained using the DAS2.1 computer program with the non-compartmental method, the half-lives of HSYA in both the normal and blood stasis groups are all very short and 90% of the HSYA are eliminated within 6 h. In comparison, the pharmacokinetics of HSYA changed greatly when HSYA and safflower extracts were administered in acute blood stasis rats. The pharmacokinetic parameters of HSYA summarized in Table 1 showed that there were statistically significant differences in parameters including the Cmax , AUC0–t , Vd and CL, between the normal rats and acute blood stasis rats, both receiving HSYA (100 mg/kg) and extract of safflower containing HSYA (100 mg/kg). Particularly, in the acute blood stasis animals, the peak plasma concentration of HSYA was remarkably increased (P < 0.05), the AUC0–t was increased (P < 0.05), the CL was decreased (P < 0.05), compared to the normal animals. For safflower extract, the half-life of HSYA increased markedly. The results indicated that HSYA was with high uptake and eliminated slowly in the animals with blood stasis syndrome. 4. Discussion Many researches demonstrated that disease condition will cause the alteration of pharmacokinetic parameters (Pfeifrs, 1991a,b,c; Jinwey, 1996). It was proved by Ren et al. (2006) that the PK analysis of ferulic acid in the patients with three different syndrome, namely deficiency of spleen Qi, stagnation of liver Qi
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and spleen deficiency, and excess of stomach heat, shown obvious differences in the PK characteristics. In our study, blood stasis syndrome was chosen and the results demonstrated significant low clearance, high AUC in rats with acute blood stasis syndrome compared to the parameters obtained in the plasma of normal rats. The rats with blood stasis syndrome will appear the sluggish of blood circulation, leading to the change of hemorrheology parameters, including the increase of whole blood viscosity, plasma viscosity, hematocrit and fibrinogen. HSYA was mainly absorbed in small intestine, poor blood circulation will prolong the retention time of HSYA in small intestine and cause the increase of HSYA’s absorption. This result was supported by Tian (1989). His study has shown that the compounds absorbed from the gut following oral administration are actually affected by syndrome, therefore cause the difference of plasma concentrations. HSYA was widely distributed in many organs, such as liver and kidney. The distribution of HSYA in blood in rats with blood stasis syndrome will become longer than that in normal rats since the value of Tmax are increased significant after oral administration of HSYA (P < 0.05). And primary study in our laboratory showed that the blood stasis syndrome tended to be the inhibitor of the CYP3A4 and the inducer of CYP1A2, but the CYP2E1 was hardly affected. Owing to the above reasons, blood stasis syndrome will have effect on the pharmacokinetic properties of HSYA in vivo. 5. Conclusion In the present study, we investigated the pharmacokinetics of HSYA and safflower extract in normal and blood stasis model rats. There were statistically significant differences in pharmacokinetic parameters of HSYA including the Cmax , AUC0–t , CL, and Vd between the normal and acute blood stasis rats, both orally administered with HSYA and safflower extract. The results suggested that the rate and extent of drug metabolism was altered in animals with blood stasis syndrome. The obtained knowledge can be used to evaluate impact of the differences on the efficacy and safety of the both drugs in clinical applications. Acknowledgements We thank the grant from the National Natural Science Foundation of China for financial support (No. 30672662) and the Traditional Chinese Medicine Foundation of Shaanxi Province for financial support (No. ZY24). References Bensky, D., Gamble, A., 1993. Chinese Herbal Medicine Materia Medica, Revised Edition. Eastland Press, Washington, pp. 265–266.
Chiu, C.C., Lan, C.Y., Chang, Y.H., 2002. Objective assessment of blood stasis using computerized inspection of sublingual veins. Computer Methods and Programs in Biomedicine 69, 1–12. Chu, D.F., Liu, W.H., Huang, Z., Liu, S.S., Fu, X.Q., Liu, K., 2006. Pharmacokinetics and excretion of hydroxysafflor yellow A, a potent neuroprotective agent from safflower, in rats and dogs. Planta Medicine 72, 418–423. Directive 86/609/EEC on the Protection of Animals Used for Experimental and Other Scientific Purposes, 1986. European Commission. Fan, L., Zhao, H.Y., Xu, M., Zhou, L., Guo, H., Han, J., Wang, B.R., Guo, D.A., 2009. Qualitative evaluation and quantitative determination of 10 major active components in Carthamus tinctorius L. by high-performance liquid chromatography coupled with diode array detector. Journal of Chromatography A 1216, 2063–2070. Jinwey, 1996. The population pharmacology research on pharma and cokinetics of drugs. Pharmazie 46, 623–629. Li, Y., Zhang, Z.Y., Zhang, J.L., 2007. Determination of hydroxysafflor yellow A in rat plasma and tissues by high-performance liquid chromatography after oral administration of safflower extract or safflor yellow. Biomedical Chromatography 21, 326–334. Liang, X.T., 2004. Basic Study of Common Chinese Medicine. Part II. Science Press, Beijing, pp. 102–115. Pfeifrs, S., 1991a. The effect of disease on the pharmacokinetics of drugs. Kidney Disease Pharmazie 46, 305–312. Pfeifrs, S., 1991b. The effect of disease on the pharmacokinetics of drugs 1. Pharmazie 46, 830. Pfeifrs, S., 1991c. The effect of disease states on pharmacokinetics of drugs 2. Liver Biliary Disease Pharmazie 46, 623–628. Qi, J.P., Jin, X.F., Huang, L.S., Ping, Q.N., 2007. Simultaneous determination of hydroxysafflor yellow A and ferulic acid in rat plasma after oral administration of the co-extractum of Rhizoma chuanxiong and Flos Carthami by HPLC-diode array detector. Biomedical Chromatography 21, 816–822. Ren, P., Huang, X., Li, S.Q., 2006. Pharmacokinetic characteristics of ferulic acid in patients with different syndromes of deficiency of spleen qi, stagnation of liver qi and spleen deficiency, and excess of stomach heat. Journal of Chinese Integrative Medicine 4, 147–151. The State Pharmacopoeia Commission of China, 2005. Pharmacopoeia of the People’s Republic of China, Part I. Chemical Industry Press, Beijing, China, p. 10. Tian, Z.M., 1989. The development of blood concentration measurement on the syndrome of Chinese formula and efficacy determination. Foreign Medical Sciences 10, 58–59. Wei, X.B., Liu, H.Q., Sun, X., Fu, F.H., Zhang, X.M., Wang, J., An, J., Ding, H., 2005. Hydroxysafflor yellow A protects rat brains against ischemia-reperfusion injury by antioxidant action. Neuroscience Letters 386, 58–62. Wen, A.D., Huang, X., Jiang, Y.P., et al., 2001. Blood stasis in congestive heart failure bears relation with digoxin clinical pharmacokinetics. Journal of Fourth Mililitary Medical University 22, 631–634. Yang, Z.F., Wen, A.D., Mei, Q.B., et al., 2001. Pharmacokinetical study of safflor yellow on rat acute model of blood stasis syndrome. Journal of Chinese Medicinal Materials 24, 730–732. Yang, Z.F., Yang, J., Jia, Y.Y., Tian, Y., Wen, A.D., 2009. Pharmacokinetic properties of hydroxysafflor yellow A in healthy Chinese female volunteers. Journal of Ethnopharmacology 124, 635–638. Yu, Z.G., Gao, X.X., Zhao, Y.L., Bi, K.S., 2007. HPLC determination of safflor yellow A and three active isoflavones from TCM Naodesheng in rat plasma and tissues and its application to pharmacokinetic studies. Biomedical Chromatography 21, 577–584. Zhao, H., Wu, W., Zheng, Y.L., Wei, Y.M., Yang, Y.X., 2007. Genetic diversity of Carthamus tinctorius L. based on RAMP Markers. Journal of Plant Genetic Resources 8, 64–67. Zhu, H.B., Wang, Z.H., Ma, C.J., Tian, J.W., Fu, F.H., Li, C.L., Guo, D., Roder, E., Liu, K., 2003. Neuroprotective effects of hydroxysafflor yellow A in vivo and in vitro studies. Planta Medicine 69, 429–433. Zhu, H.B., Wang, Z.H., Tian, J.W., Fu, F.H., Liu, K., Li, C.L., 2005. Protective effect of hydroxysafflor yellow A on experimental cerebral ischemia in rats. Acta Pharmaceutica Sinica 40, 1144–1146.