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Pharmacokinetic comparisons of berberine and palmatine in normal and metabolic syndrome rats
Journal of Ethnopharmacology 151 (2014) 287–291
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Pharmacokinetic comparisons of berberine and palmatine in normal and metabolic syndrome rats Qing-Feng Liu, Xiao-Jin Shi, Zhong-Dong Li, Ming-kang Zhong n, Zheng Jiao, Bin Wang Clinical Pharmacy Laboratory, Huashan Hospital, Fudan University, 12 Wu Lu Mu Qi M Road, Shanghai 200040, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 29 August 2013 Received in revised form 1 October 2013 Accepted 22 October 2013 Available online 22 November 2013
Ethnopharmacological relevance: San-Huang formula is a popular traditional Chinese medicine (TCM) preparation to replenish Qi, resolve phlegm, dissipate blood stasis, and therapy metabolic syndrome in China. Metabolic syndrome, which is accompanied by Qi and blood stasis, mainly arises from spleen deficiency in essence. There is limited information available for differences of pharmacokinetic properties of San-Huang formula between normal and metabolic syndrome rats. The present study was conducted to compare the pharmacokinetics of berberine as well as palmatine in normal and metabolic syndrome rats following oral administration of San-Huang formula extract. Materials and methods: The animals were orally administered with San-Huang formula extract with the equivalent dose of 60.4 and 12.5 mg/kg for berberine and palmatine, respectively. The blood samples were collected according to the time schedule. The concentrations of berberine and palmatine in rat plasma were determined by LC–ESI/MS. 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 berberine and palmatine in metabolic syndrome rats were significantly different (Po 0.05) from normal rats. Conclusions: The results indicated that berberine and palmatine have higher uptake and slower elimination in the rats with metabolic syndrome, which suggests that the rate and extent of drug metabolism were altered in metabolic syndrome rats. & 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Berberine Palmatine San-Huang formula extract Pharmacokinetics Metabolic syndrome
1. Introduction San-Huang formula, which consists of Astragalus membranaceus (Fisch.) Bunge, Coptis chinensis Franch., Artemisia capillaris Thunb., Typha angustifolia L. and Alisma orientale (Sam.) Juz., is widely used in traditional Chinese medicine to prevent and treat metabolic syndrome (Wang, 2007). It is mainly administrated as decoction in traditional Chinese medicinal prescription. Berberine and palmatine, having similar molecular structure, are important ingredients that responsible for the curative effects of San-Huang formula, and have chosen as active markers for controlling the quality of Coptis chinensis Franch. in Chinese Pharmacopoeia (The State Pharmacopoeia Commission of China, 2010) and San-Huang Formula (Xu et al., 2011). It has been demonstrated that berberine [Fig. 1(A)] has the activities of anticancer (Anis et al., 2001), antiatherosclerosis (Huang et al., 2011) and treating infectious diarrhea effects (Stermitz et al., 2000), and that palmatine [Fig. 1(B)] has the activities of antitumor (Kuo et al., 1995), liver-protective
n
Corresponding author. Tel.: þ 86 21 52889381; fax: þ86 21 32120059. E-mail address: [email protected] (M.-k. Zhong).
0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.10.031
(Lee et al., 2010) and cardiovascular protective effects (Kim et al., 2009), respectively. Metabolic syndrome is a combination of medical disorders that increase the risk of cardiovascular disease and diabetes, and is prevalent in up to 25% of the US population (Ford et al., 2002) and up to 16.5% of the Chinese population (Gu et al., 2005), and has been described as a cluster of multiple, partially or fully expressed, metabolic abnormalities within the single individual. These metabolic abnormalities consist of hypertension, dyslipidemia, obesity, and impaired glucose tolerance (Skilton Michael et al., 2007). Moreover, metabolic syndrome, which mainly arised from spleen deficiency and disorders of Qi and blood transportation in traditional Chinese medicine theory, was also known as syndrome X, cardiometabolic syndrome, insulin resistance syndrome, Reaven's syndrome, CHAOS (an abbreviation for coronary artery disease, hypertension, atherosclerosis, obesity, and stroke), and the Deadly Quartet (Kaplan, 1989; Haffner et al., 1992; Schindler, 2007). With metabolic syndrome developing, the Qi and blood circulation will further be affected and thus lead to new pathological changes. Pharmacokinetic characteristics could be affected by disease condition (Ren et al., 2006; Wen et al., 2001; Hardwick et al., 2012). Therefore, it is very important to investigate the pharmacokinetics
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OCH3
O O
H3CO
N+
OCH 3
N+
H3CO OCH3
OCH3
N
Cl
N
CH3
N
N H
capillaris Thunb. was 5.9370.57 mg/g; isorhamnetin-3-O-neohesperidoside in Typha angustifolia L. was 5.8571.13 mg/g; alisol B-23-acetate in the decoction pieces of Alisma orientale (Sam.) Juz. was 5.957 0.87 mg/g, respectively. The reference standards of berberine (86.7% purity), palmatine (86.2% purity), and clozapine [IS, 99.8% purity, Fig. 1(C)] were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). HPLC-grade methanol and acetonitrile were purchased from Merck (Darmstadt, Germany); and AR-grade formic acid was purchased from Sinopharm Group Chemical Reagent (Shanghai, China). Water was distilled and purified using a Milli-Q Water Purification System (Millipore, Bedford, MA, USA). Drug-free rat plasma was gained from healthy rat provided by the Department of Animal Science of Fudan University (Shanghai, China).
Fig. 1. Chemical structures of two compounds and IS: berberine (A), palmatine (B), and internal standard and clozapine (C).
2.2. Preparations of San-Huang formula extract
of drugs in animals with metabolic syndrome, which may influence absorption, metabolism and elimination of drugs in blood. In recent years, some pharmacokinetic studies on berberine and palmatine have been performed on healthy Chinese volunteers (Hua et al., 2007; Pan et al., 2002; Zeng and Zeng, 1999) and normal animals (Deng et al., 2008; Lu et al., 2006; Wu et al., 2009). However, the pharmacokinetics of berberine and palmatine has not been investigated in detail. So far, most studies were focused on the pharmacokinetic characteristics of berberine and palmatine in healthy human beings or normal animals. And the pharmacokinetic properties of berberine and palmatine in animal with metabolic syndrome and the differences between normal animals and metabolic syndrome animals were seldom reported. The primary objective of this study was to investigate the possible pharmacokinetic differences of the compounds after oral administration of San-Huang formula extract in normal rats and metabolic syndrome rats.
Five crude herbs including Astragalus membranaceus (Fisch.) Bunge, Coptis chinensis Franch., Artemisia capillaris Thunb., Typha angustifolia L. and Alisma orientale (Sam.) Juz. (30, 9, 15, 15 and 15 g) were thoroughly soaked in water for 30 min and decocted twice with water (1:10, w/v). The first and second decoctions were respectively boiled using high heat for 60 min and 45 min, then they were put together and condensed under 60 1C. After five times of ethanol (v/v) was added, the solution was stayed overnight, filtered, condensed to 8.0 ml and stored at 4 1C before use. The quantitative analysis for San-Huang formula extract has been studied and published in our previous article (Liu et al., 2010; Xu et al., 2011), and that the contents of berberine and palmatine in the San-Huang formula extract has been determined by LC–UV to be 8.39 and 1.74 mg/ml, respectively (Xu et al., 2011). So 1.44 ml/kg dose of this decoction (according to human dosage in clinical practice and human-rat coefficient of skin surface area) was equivalent to 60.40 mg/kg dose of berberine and 12.52 mg/kg dose of palmatine.
2. Materials and methods
2.3. Animals
2.1. Chemicals and reagents
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 24 male rats weighing 180 720 g were used for this study. The rats were supplied by the Department of Animal Science of Fudan University (Shanghai, China). The rats were maintained in an air-conditioned animal quarter at a temperature of 22 72 1C and a relative humidity of 50 710%. Rats were randomly divided into the following two groups (n ¼12): normal control group and metabolic syndrome model group. Water with 20% sucrose and food with high sugar, fat and salt (laboratory rodent chow, Shanghai, China) were allowed ad libitum to metabolic syndrome model group for 3 months; however, conventional water and food (laboratory rodent chow, Shanghai, China) were allowed ad libitum to normal control group. The animals were acclimatized to the facilities for 5 days, and then fasted with free access to water for 12 h prior to each experiment.
Raw materials, including Astragalus membranaceus (Fisch.) Bunge, root, thick section; Coptis chinensis Franch. rhizome; Artemisia capillaris Thunb., aerial part; Typha angustifolia L., pollen and Alisma orientale (Sam.) Juz., tuber, thick section, all recorded in Chinese Pharmacopoeia (The State Pharmacopoeia Commission of China, 2010), were purchased from Chinese Herbal Pieces, Shanghai Hongqiao Co. Ltd., Shanghai, China, and were identified by Dr. Ying Wang (Shanghai University of Traditional Chinese Medicine, Shanghai, China). The materials, consisting of physiological traits, microscopic identification of Chinese drug powder and the contents of water, total ash, ethanolsoluble extractives, water-soluble extractives, heavy metalst, harmful elements, organochlorine pesticide residues, active compounds, were all subject to quality control according to Chinese Pharmacopoeia (The State Pharmacopoeia Commission of China, 2010). Especially, active compounds including astragaloside IV, calycosin-7-O-beta-Dglucopyranoside, berberine, epiberberine, coptisine, palmatine, chlorogenic acid, isorhamnetin-3-O-neohesperidoside and alisol B-23acetate in these materials were strictly determinded by reversedphase high-performance liquid chromatography (HPLC). The contents of astragaloside IV and calycosin-7-O-beta-D-glucopyranoside in the decoction pieces of Astragalus membranaceus (Fisch.) Bunge were 1.2270.25 and 0.4270.11 mg/g; berberine, epiberberine, coptisine and palmatine in Coptis chinensis Franch. were 83.31717.23, 12.727 3.81, 24.1774.31 and 23.0374.91 mg/g; chlorogenic acid in Artemisia
2.4. In vivo study Each of the four rats was in an individual cage, and were fasted overnight (16 h) prior to oral administration of 1.44 ml/kg SanHuang formula extract. 0.25 ml Blood samples were collected in heparinized Eppendorf tube via the tail nick before dosing and subsequently at 0.25, 0.5, 0.75, 1, 2, 3, 5, 7 and 12 h following oral administration. Following centrifugation (1441g for 10 min), the
Q.-F. Liu et al. / Journal of Ethnopharmacology 151 (2014) 287–291
The relative instrumentation and chromatographic conditions have been stated in our primary study (Liu et al., 2011) as following. The HPLC system consisted of a Surveyor MS Pump and a Surveyor Autosampler (San Jose, CA, USA). Chromatographic separation was carried out on a CAPCELL PAKC18 MG (100 mm 2.1 mm i.d., 5 μm; Shiseido, Japan) column with a Security Guard C18 column (4.0 mm 2.0 mm i.d., 5 μm; Phenomenex, USA) maintained at 25 1C. The mobile phase consisted of 0.4% formic acid solution and 0.2% formic acid solution of methanol (60:40, v/v) at a flow rate of 0.4 ml/min. The total running time was 2.4 min for each injection. In addition, an automatic washing procedure of system was incorporated during each injection to remove dirt analytical and guard column and ion source and restore the chromatgraphy system to the initial elution condition. MS detection was performed on a Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer (San Jose, CA, USA) equipped with an ESI source in the positive ionization mode. The MS operating conditions were optimized as follows: the spray voltage was 3500 V, the source CID voltage was 10 eV, the heated capillary temperature was 500 1C, the sheath gas (nitrogen) was 20 psi, the auxiliary gas (nitrogen) was 50 psi, the collision gas (argon) pressure was 1.0 mTorr (1 Torr¼ 133.3 Pa), and the collision energy was 37 eV. Data acquisition, peak integration and calibration were performed by Analyst 1.5 software (San Jose, CA, USA). Quantification was obtained using the MRM mode of the transitions at m/z 336.2-320.1 for berberine, m/z 352.3-336.2 for palmatine, and m/z 327.1-270.1 for IS with a scan time of 0.3 s/transition.
2.6. Sample preparation Acetonitrile (200 μl, containing 3% formic acid and 50 ng/ml of the IS) was added to 100 μl of plasma samples in 1.5 ml polypropylene tubes. The mixture was vigorously vortexed for 30 s and centrifuged at 20,267g for 10 min at 4 1C. The supernatant was separated and dried under a nitrogen stream in a water bath at 40 1C. The residue was reconstituted in 100 μl of 20% methanol and centrifuged as described above, and then 10 μl of the supernatant was injected onto the column.
The mean plasma concentration–time profiles of berberine and palmatine were determined after oral administration of SanHuang formula extract, as are illustrated in Fig. 2. Moreover, the partial pharmacokinetic parameters are given in Table 1. After comparing the pharmacokinetic data of the normal group with that of metabolic syndrome group with t-test, it could be concluded that there were significant differences (Po0.05) between
14 normal group
12
Concentration (ng/ml)
2.5. Instrumentation and chromatographic conditions
3. Results
MS group
10 8 6 4 2 0
0
4
8
12
Time (h)
6 normal group
Concentration (ng/ml)
plasma samples were harvested and stored at 22 1C until analysis.
289
5
MS group
4 3 2 1 0
0
4
8
12
Time (h) Fig. 2. Mean plasma concentration–time curves of berberine (A) and palmatine (B) after oral administration of San-Huang formula extract 1.44 ml/kg body weight, equivalent to 60.4 mg/kg berberine and 12.52 mg/kg palmatine, respectively. Data are shown as mean7 S.D. (n ¼12).
2.7. 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 ¼0.693/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 mean7standard deviation (S.D.). The concentration–time data were analyzed by non-compartmental methods using the Win-Nonlin 5.0.1 (provided by Mountain View, CA, USA) to obtain the pharmacokinetic parameters of berberine and palmatine, and 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 two sided 5% level of significance.
Table 1 Pharmocokinetic parameters of berberine and palmatine after an oral administration of San-Huang formula extract 1.44 ml/kg body weight (n ¼12). Berberine
AUC0-t (μg h/ml) AUC0-1 (μg h/ml) MRT0-t (h) Cmax (μg/ml) Tmax (h) t1/2 (h) CL (ml kg/h) Vd (l/kg)
Palmatine
Normal rats
MS rats
Normal rats
MS rats
27.98 7 1.40 29.447 1.74 3.59 7 0.06 7.317 0.31 1.05 7 0.12 0.62 7 0.17 2.067 0.12 7.747 0.66
54.99 7 2.06* 56.50 7 2.38* 3.647 0.06 12.81 7 0.52* 2.02 7 0.05* 0.727 0.36* 1.08 7 0.05* 3.30 7 0.48*
10.117 0.99 15.08 7 3.13 4.377 0.12 2.767 0.23 1.017 0.09 0.487 0.06 0.86 7 0.17 10.077 0.98
29.317 2.45* 32.62 7 3.35* 4.497 0.15 4.96 7 0.16* 2.067 0.05* 1.127 0.16* 0.39 7 0.05* 1.86 7 0.11*
MS represents the metabolic syndrome. Data represents the mean 7 S.D. n
P o 0.05 versus normal rats.
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the important pharmacokinetic parameters, such as AUC0–t, Cmax and CL. From the pharmacokinetic parameters obtained using the WinNonlin 5.0.1 (provided by Mountain View, CA, USA) computer program with the non-compartmental method, the half-lives of berberine and palmatine in both the normal and metabolic syndrome groups are all very short and 90% of the berberine and palmatine are eliminated within 6 h. In comparison, the pharmacokinetics of berberine and palmatine changed greatly when SanHuang formula extract was administered in metabolic syndrome rats. The pharmacokinetic parameters of berberine and palmatine 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 metabolic syndrome rats, both receiving equal extract of San-Huang formula containing berberine (60.4 mg/kg) and palmatine (12.52 mg/kg). Particularly, in the metabolic syndrome rats, the peak plasma concentration and AUC0–t of berberine and palmatine was remarkably increased (P o0.05), the t1/2 increased (P o0.05), and the CL was decreased markedly (P o0.05), compared to the normal animals. The results indicated that berberine and palmatine were with high uptake and eliminated slowly in the animals with metabolic syndrome.
4. Discussion It is believed that the occurrence, development and outcome of metabolic syndrome are closely correlatively with the spleen deficiency and correspondingly Qi and blood stasis type in the syndrome differentiation (Wang, 2007). The rats with metabolic syndrome will often appear insulin resistance, hyperglycemia, impaired glucose tolerance, hyperlipidemia, hypertension and even the sluggish of blood circulation, leading to the change of hemorrheology parameters, including the increase of whole blood viscosity, plasma viscosity, hematocrit and fibrinogen. And Qiu et al.'s (2011) 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. Berberine and palmatine were mainly absorbed in the gut and widely distributed in many organs, such as liver and kidney (Ma et al., 2012), and their distribution in the blood of rats with metabolic syndrome will become longer than that in normal rats since the value of Tmax are increased significant after oral administration of San-Huang formula extract (Po0.05). Many researches demonstrated that disease condition will cause the alteration of pharmacokinetic parameters (Ren et al., 2006; Wen et al., 2001; Rhiannon et al., 2012), such as Ren et al. proved that the PK analysis of ferulic acid in the patients with three different syndrome, namely deficiency of spleen Qi, stagnation of liver Qi and spleen deficiency, and excess of stomach heat, shown obvious differences in the PK characteristics. In our study, the results demonstrated significant low clearance, high AUC in rats with metabolic syndrome compared to the parameters obtained in the plasma of normal rats. Owing to the above reasons, metabolic syndrome will have effect on the pharmacokinetic properties of berberine and palmatine in vivo, such as higher uptake and slower elimination.
5. Conclusion In the present study, we investigated the pharmacokinetics of berberine and palmatine of San-Huang formula extract in normal and metabolic syndrome model rats. There were statistically significant differences in pharmacokinetic parameters of berberine and palmatine including the Cmax, AUC0–t, CL, and Vd between the normal and metabolic syndrome rats, both orally administered
with San-Huang formula extract. The results suggested that the rate and extent of drug metabolism was altered in rats with metabolic syndrome. The obtained knowledge can be used to evaluate impact of the differences on the efficacy and safety of San-Huang formula in clinical applications.
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Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Pharmacokinetic comparisons of berberine and palmatine in normal and metabolic syndrome rats Qing-Feng Liu, Xiao-Jin Shi, Zhong-Dong Li, Ming-kang Zhong n, Zheng Jiao, Bin Wang Clinical Pharmacy Laboratory, Huashan Hospital, Fudan University, 12 Wu Lu Mu Qi M Road, Shanghai 200040, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 29 August 2013 Received in revised form 1 October 2013 Accepted 22 October 2013 Available online 22 November 2013
Ethnopharmacological relevance: San-Huang formula is a popular traditional Chinese medicine (TCM) preparation to replenish Qi, resolve phlegm, dissipate blood stasis, and therapy metabolic syndrome in China. Metabolic syndrome, which is accompanied by Qi and blood stasis, mainly arises from spleen deficiency in essence. There is limited information available for differences of pharmacokinetic properties of San-Huang formula between normal and metabolic syndrome rats. The present study was conducted to compare the pharmacokinetics of berberine as well as palmatine in normal and metabolic syndrome rats following oral administration of San-Huang formula extract. Materials and methods: The animals were orally administered with San-Huang formula extract with the equivalent dose of 60.4 and 12.5 mg/kg for berberine and palmatine, respectively. The blood samples were collected according to the time schedule. The concentrations of berberine and palmatine in rat plasma were determined by LC–ESI/MS. 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 berberine and palmatine in metabolic syndrome rats were significantly different (Po 0.05) from normal rats. Conclusions: The results indicated that berberine and palmatine have higher uptake and slower elimination in the rats with metabolic syndrome, which suggests that the rate and extent of drug metabolism were altered in metabolic syndrome rats. & 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Berberine Palmatine San-Huang formula extract Pharmacokinetics Metabolic syndrome
1. Introduction San-Huang formula, which consists of Astragalus membranaceus (Fisch.) Bunge, Coptis chinensis Franch., Artemisia capillaris Thunb., Typha angustifolia L. and Alisma orientale (Sam.) Juz., is widely used in traditional Chinese medicine to prevent and treat metabolic syndrome (Wang, 2007). It is mainly administrated as decoction in traditional Chinese medicinal prescription. Berberine and palmatine, having similar molecular structure, are important ingredients that responsible for the curative effects of San-Huang formula, and have chosen as active markers for controlling the quality of Coptis chinensis Franch. in Chinese Pharmacopoeia (The State Pharmacopoeia Commission of China, 2010) and San-Huang Formula (Xu et al., 2011). It has been demonstrated that berberine [Fig. 1(A)] has the activities of anticancer (Anis et al., 2001), antiatherosclerosis (Huang et al., 2011) and treating infectious diarrhea effects (Stermitz et al., 2000), and that palmatine [Fig. 1(B)] has the activities of antitumor (Kuo et al., 1995), liver-protective
n
Corresponding author. Tel.: þ 86 21 52889381; fax: þ86 21 32120059. E-mail address: [email protected] (M.-k. Zhong).
0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.10.031
(Lee et al., 2010) and cardiovascular protective effects (Kim et al., 2009), respectively. Metabolic syndrome is a combination of medical disorders that increase the risk of cardiovascular disease and diabetes, and is prevalent in up to 25% of the US population (Ford et al., 2002) and up to 16.5% of the Chinese population (Gu et al., 2005), and has been described as a cluster of multiple, partially or fully expressed, metabolic abnormalities within the single individual. These metabolic abnormalities consist of hypertension, dyslipidemia, obesity, and impaired glucose tolerance (Skilton Michael et al., 2007). Moreover, metabolic syndrome, which mainly arised from spleen deficiency and disorders of Qi and blood transportation in traditional Chinese medicine theory, was also known as syndrome X, cardiometabolic syndrome, insulin resistance syndrome, Reaven's syndrome, CHAOS (an abbreviation for coronary artery disease, hypertension, atherosclerosis, obesity, and stroke), and the Deadly Quartet (Kaplan, 1989; Haffner et al., 1992; Schindler, 2007). With metabolic syndrome developing, the Qi and blood circulation will further be affected and thus lead to new pathological changes. Pharmacokinetic characteristics could be affected by disease condition (Ren et al., 2006; Wen et al., 2001; Hardwick et al., 2012). Therefore, it is very important to investigate the pharmacokinetics
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OCH3
O O
H3CO
N+
OCH 3
N+
H3CO OCH3
OCH3
N
Cl
N
CH3
N
N H
capillaris Thunb. was 5.9370.57 mg/g; isorhamnetin-3-O-neohesperidoside in Typha angustifolia L. was 5.8571.13 mg/g; alisol B-23-acetate in the decoction pieces of Alisma orientale (Sam.) Juz. was 5.957 0.87 mg/g, respectively. The reference standards of berberine (86.7% purity), palmatine (86.2% purity), and clozapine [IS, 99.8% purity, Fig. 1(C)] were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). HPLC-grade methanol and acetonitrile were purchased from Merck (Darmstadt, Germany); and AR-grade formic acid was purchased from Sinopharm Group Chemical Reagent (Shanghai, China). Water was distilled and purified using a Milli-Q Water Purification System (Millipore, Bedford, MA, USA). Drug-free rat plasma was gained from healthy rat provided by the Department of Animal Science of Fudan University (Shanghai, China).
Fig. 1. Chemical structures of two compounds and IS: berberine (A), palmatine (B), and internal standard and clozapine (C).
2.2. Preparations of San-Huang formula extract
of drugs in animals with metabolic syndrome, which may influence absorption, metabolism and elimination of drugs in blood. In recent years, some pharmacokinetic studies on berberine and palmatine have been performed on healthy Chinese volunteers (Hua et al., 2007; Pan et al., 2002; Zeng and Zeng, 1999) and normal animals (Deng et al., 2008; Lu et al., 2006; Wu et al., 2009). However, the pharmacokinetics of berberine and palmatine has not been investigated in detail. So far, most studies were focused on the pharmacokinetic characteristics of berberine and palmatine in healthy human beings or normal animals. And the pharmacokinetic properties of berberine and palmatine in animal with metabolic syndrome and the differences between normal animals and metabolic syndrome animals were seldom reported. The primary objective of this study was to investigate the possible pharmacokinetic differences of the compounds after oral administration of San-Huang formula extract in normal rats and metabolic syndrome rats.
Five crude herbs including Astragalus membranaceus (Fisch.) Bunge, Coptis chinensis Franch., Artemisia capillaris Thunb., Typha angustifolia L. and Alisma orientale (Sam.) Juz. (30, 9, 15, 15 and 15 g) were thoroughly soaked in water for 30 min and decocted twice with water (1:10, w/v). The first and second decoctions were respectively boiled using high heat for 60 min and 45 min, then they were put together and condensed under 60 1C. After five times of ethanol (v/v) was added, the solution was stayed overnight, filtered, condensed to 8.0 ml and stored at 4 1C before use. The quantitative analysis for San-Huang formula extract has been studied and published in our previous article (Liu et al., 2010; Xu et al., 2011), and that the contents of berberine and palmatine in the San-Huang formula extract has been determined by LC–UV to be 8.39 and 1.74 mg/ml, respectively (Xu et al., 2011). So 1.44 ml/kg dose of this decoction (according to human dosage in clinical practice and human-rat coefficient of skin surface area) was equivalent to 60.40 mg/kg dose of berberine and 12.52 mg/kg dose of palmatine.
2. Materials and methods
2.3. Animals
2.1. Chemicals and reagents
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 24 male rats weighing 180 720 g were used for this study. The rats were supplied by the Department of Animal Science of Fudan University (Shanghai, China). The rats were maintained in an air-conditioned animal quarter at a temperature of 22 72 1C and a relative humidity of 50 710%. Rats were randomly divided into the following two groups (n ¼12): normal control group and metabolic syndrome model group. Water with 20% sucrose and food with high sugar, fat and salt (laboratory rodent chow, Shanghai, China) were allowed ad libitum to metabolic syndrome model group for 3 months; however, conventional water and food (laboratory rodent chow, Shanghai, China) were allowed ad libitum to normal control group. The animals were acclimatized to the facilities for 5 days, and then fasted with free access to water for 12 h prior to each experiment.
Raw materials, including Astragalus membranaceus (Fisch.) Bunge, root, thick section; Coptis chinensis Franch. rhizome; Artemisia capillaris Thunb., aerial part; Typha angustifolia L., pollen and Alisma orientale (Sam.) Juz., tuber, thick section, all recorded in Chinese Pharmacopoeia (The State Pharmacopoeia Commission of China, 2010), were purchased from Chinese Herbal Pieces, Shanghai Hongqiao Co. Ltd., Shanghai, China, and were identified by Dr. Ying Wang (Shanghai University of Traditional Chinese Medicine, Shanghai, China). The materials, consisting of physiological traits, microscopic identification of Chinese drug powder and the contents of water, total ash, ethanolsoluble extractives, water-soluble extractives, heavy metalst, harmful elements, organochlorine pesticide residues, active compounds, were all subject to quality control according to Chinese Pharmacopoeia (The State Pharmacopoeia Commission of China, 2010). Especially, active compounds including astragaloside IV, calycosin-7-O-beta-Dglucopyranoside, berberine, epiberberine, coptisine, palmatine, chlorogenic acid, isorhamnetin-3-O-neohesperidoside and alisol B-23acetate in these materials were strictly determinded by reversedphase high-performance liquid chromatography (HPLC). The contents of astragaloside IV and calycosin-7-O-beta-D-glucopyranoside in the decoction pieces of Astragalus membranaceus (Fisch.) Bunge were 1.2270.25 and 0.4270.11 mg/g; berberine, epiberberine, coptisine and palmatine in Coptis chinensis Franch. were 83.31717.23, 12.727 3.81, 24.1774.31 and 23.0374.91 mg/g; chlorogenic acid in Artemisia
2.4. In vivo study Each of the four rats was in an individual cage, and were fasted overnight (16 h) prior to oral administration of 1.44 ml/kg SanHuang formula extract. 0.25 ml Blood samples were collected in heparinized Eppendorf tube via the tail nick before dosing and subsequently at 0.25, 0.5, 0.75, 1, 2, 3, 5, 7 and 12 h following oral administration. Following centrifugation (1441g for 10 min), the
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The relative instrumentation and chromatographic conditions have been stated in our primary study (Liu et al., 2011) as following. The HPLC system consisted of a Surveyor MS Pump and a Surveyor Autosampler (San Jose, CA, USA). Chromatographic separation was carried out on a CAPCELL PAKC18 MG (100 mm 2.1 mm i.d., 5 μm; Shiseido, Japan) column with a Security Guard C18 column (4.0 mm 2.0 mm i.d., 5 μm; Phenomenex, USA) maintained at 25 1C. The mobile phase consisted of 0.4% formic acid solution and 0.2% formic acid solution of methanol (60:40, v/v) at a flow rate of 0.4 ml/min. The total running time was 2.4 min for each injection. In addition, an automatic washing procedure of system was incorporated during each injection to remove dirt analytical and guard column and ion source and restore the chromatgraphy system to the initial elution condition. MS detection was performed on a Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer (San Jose, CA, USA) equipped with an ESI source in the positive ionization mode. The MS operating conditions were optimized as follows: the spray voltage was 3500 V, the source CID voltage was 10 eV, the heated capillary temperature was 500 1C, the sheath gas (nitrogen) was 20 psi, the auxiliary gas (nitrogen) was 50 psi, the collision gas (argon) pressure was 1.0 mTorr (1 Torr¼ 133.3 Pa), and the collision energy was 37 eV. Data acquisition, peak integration and calibration were performed by Analyst 1.5 software (San Jose, CA, USA). Quantification was obtained using the MRM mode of the transitions at m/z 336.2-320.1 for berberine, m/z 352.3-336.2 for palmatine, and m/z 327.1-270.1 for IS with a scan time of 0.3 s/transition.
2.6. Sample preparation Acetonitrile (200 μl, containing 3% formic acid and 50 ng/ml of the IS) was added to 100 μl of plasma samples in 1.5 ml polypropylene tubes. The mixture was vigorously vortexed for 30 s and centrifuged at 20,267g for 10 min at 4 1C. The supernatant was separated and dried under a nitrogen stream in a water bath at 40 1C. The residue was reconstituted in 100 μl of 20% methanol and centrifuged as described above, and then 10 μl of the supernatant was injected onto the column.
The mean plasma concentration–time profiles of berberine and palmatine were determined after oral administration of SanHuang formula extract, as are illustrated in Fig. 2. Moreover, the partial pharmacokinetic parameters are given in Table 1. After comparing the pharmacokinetic data of the normal group with that of metabolic syndrome group with t-test, it could be concluded that there were significant differences (Po0.05) between
14 normal group
12
Concentration (ng/ml)
2.5. Instrumentation and chromatographic conditions
3. Results
MS group
10 8 6 4 2 0
0
4
8
12
Time (h)
6 normal group
Concentration (ng/ml)
plasma samples were harvested and stored at 22 1C until analysis.
289
5
MS group
4 3 2 1 0
0
4
8
12
Time (h) Fig. 2. Mean plasma concentration–time curves of berberine (A) and palmatine (B) after oral administration of San-Huang formula extract 1.44 ml/kg body weight, equivalent to 60.4 mg/kg berberine and 12.52 mg/kg palmatine, respectively. Data are shown as mean7 S.D. (n ¼12).
2.7. 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 ¼0.693/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 mean7standard deviation (S.D.). The concentration–time data were analyzed by non-compartmental methods using the Win-Nonlin 5.0.1 (provided by Mountain View, CA, USA) to obtain the pharmacokinetic parameters of berberine and palmatine, and 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 two sided 5% level of significance.
Table 1 Pharmocokinetic parameters of berberine and palmatine after an oral administration of San-Huang formula extract 1.44 ml/kg body weight (n ¼12). Berberine
AUC0-t (μg h/ml) AUC0-1 (μg h/ml) MRT0-t (h) Cmax (μg/ml) Tmax (h) t1/2 (h) CL (ml kg/h) Vd (l/kg)
Palmatine
Normal rats
MS rats
Normal rats
MS rats
27.98 7 1.40 29.447 1.74 3.59 7 0.06 7.317 0.31 1.05 7 0.12 0.62 7 0.17 2.067 0.12 7.747 0.66
54.99 7 2.06* 56.50 7 2.38* 3.647 0.06 12.81 7 0.52* 2.02 7 0.05* 0.727 0.36* 1.08 7 0.05* 3.30 7 0.48*
10.117 0.99 15.08 7 3.13 4.377 0.12 2.767 0.23 1.017 0.09 0.487 0.06 0.86 7 0.17 10.077 0.98
29.317 2.45* 32.62 7 3.35* 4.497 0.15 4.96 7 0.16* 2.067 0.05* 1.127 0.16* 0.39 7 0.05* 1.86 7 0.11*
MS represents the metabolic syndrome. Data represents the mean 7 S.D. n
P o 0.05 versus normal rats.
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the important pharmacokinetic parameters, such as AUC0–t, Cmax and CL. From the pharmacokinetic parameters obtained using the WinNonlin 5.0.1 (provided by Mountain View, CA, USA) computer program with the non-compartmental method, the half-lives of berberine and palmatine in both the normal and metabolic syndrome groups are all very short and 90% of the berberine and palmatine are eliminated within 6 h. In comparison, the pharmacokinetics of berberine and palmatine changed greatly when SanHuang formula extract was administered in metabolic syndrome rats. The pharmacokinetic parameters of berberine and palmatine 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 metabolic syndrome rats, both receiving equal extract of San-Huang formula containing berberine (60.4 mg/kg) and palmatine (12.52 mg/kg). Particularly, in the metabolic syndrome rats, the peak plasma concentration and AUC0–t of berberine and palmatine was remarkably increased (P o0.05), the t1/2 increased (P o0.05), and the CL was decreased markedly (P o0.05), compared to the normal animals. The results indicated that berberine and palmatine were with high uptake and eliminated slowly in the animals with metabolic syndrome.
4. Discussion It is believed that the occurrence, development and outcome of metabolic syndrome are closely correlatively with the spleen deficiency and correspondingly Qi and blood stasis type in the syndrome differentiation (Wang, 2007). The rats with metabolic syndrome will often appear insulin resistance, hyperglycemia, impaired glucose tolerance, hyperlipidemia, hypertension and even the sluggish of blood circulation, leading to the change of hemorrheology parameters, including the increase of whole blood viscosity, plasma viscosity, hematocrit and fibrinogen. And Qiu et al.'s (2011) 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. Berberine and palmatine were mainly absorbed in the gut and widely distributed in many organs, such as liver and kidney (Ma et al., 2012), and their distribution in the blood of rats with metabolic syndrome will become longer than that in normal rats since the value of Tmax are increased significant after oral administration of San-Huang formula extract (Po0.05). Many researches demonstrated that disease condition will cause the alteration of pharmacokinetic parameters (Ren et al., 2006; Wen et al., 2001; Rhiannon et al., 2012), such as Ren et al. proved that the PK analysis of ferulic acid in the patients with three different syndrome, namely deficiency of spleen Qi, stagnation of liver Qi and spleen deficiency, and excess of stomach heat, shown obvious differences in the PK characteristics. In our study, the results demonstrated significant low clearance, high AUC in rats with metabolic syndrome compared to the parameters obtained in the plasma of normal rats. Owing to the above reasons, metabolic syndrome will have effect on the pharmacokinetic properties of berberine and palmatine in vivo, such as higher uptake and slower elimination.
5. Conclusion In the present study, we investigated the pharmacokinetics of berberine and palmatine of San-Huang formula extract in normal and metabolic syndrome model rats. There were statistically significant differences in pharmacokinetic parameters of berberine and palmatine including the Cmax, AUC0–t, CL, and Vd between the normal and metabolic syndrome rats, both orally administered
with San-Huang formula extract. The results suggested that the rate and extent of drug metabolism was altered in rats with metabolic syndrome. The obtained knowledge can be used to evaluate impact of the differences on the efficacy and safety of San-Huang formula in clinical applications.
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