Simultaneous Determination of Silibinin A and Silibinin B in Rat Plasma and Pharmacokinetic Study

Transcription

1 Cu Y et al. Cinese Herbal Medicines, 211, 3(4): Simultaneous Deteration of Silibinin A and Silibinin B in Rat Plasma and Parmacokinetic Study CHU Yang, L Wei, L Ziwen, L Xinxin, MA Xiaoui *, ZHU Suiping, ZHU Yongong Department of Parmacology, Tianjin Tasly nstitute, Tianjin 341, Cina Abstract: bjective To investate te bioavailability and parmacokinetics of silibinin A and silibinin B in rats, respectively. Metods Following iv and adistration of silibinin to 2 Wistar rats, te plasma samples were collected at different time points up to 12. Sample pretreatment was involved in onestep protein precipitation wit acetonitrile. Silibinin A and silibinin B were simultaneously detered by LCMS/MS. Results After dosing silibinin 28, 56, and 112 mg/kg to rats, te t 1/2β values were 5.48, 5.8, and 5.73 for silibinin A, and 4.56, 4.12, and 5.53 for silibinin B; Te C max were 674.3, , and ng/ml for silibinin A, and 671., , and ng/ml for silibinin B; Te T max were.2,.23, and.2 for silibinin A, and.2,.23, and.2 for silibinin B; Te AUC were 454.4, 845.9, and ng/ml for silibinin A, and 432., 817.1, and ng/ml for silibinin B. Te absolute bioavailabilities of silibinin A and silibinin B were 2.86% and 1.93%, respectively. Conclusion Silibinin A and silibinin B ave very low bioavailability after adistration, and tere is no snificant difference in te parmacokinetic parameters between silibinin A and silibinin B, wic indicates tat te two diastereoisomers ave similar parmacokinetic beavior in rats. Key words: bioavailability; LCMS/MS; parmacokinetics; silibinin A; silibinin B D: /j.issn ntroduction Silibinin is te main isomer of a group of flavanoids extracted from te fruit of Silybum marianum (L). Gaertn., wic as been widely used to maintain liver ealt and for te treatment of liver disorder as a common erb (Flora et al, 1998; Kidd and Head, 25; Kvasnicka et al, 23). Recent literatures ave demonstrated tat silibinin also as antiinflammatory effect (Trappoliere et al, 29; Gu et al, 27) and antitumor activity in in vitro and in vivo studies (Lin, Sukarie, and Pelletier, 29; Kim et al, 24; 29; Sing et al, 28; Raina et al, 27; Cen et al, 26; Gallo et al, 23). Silibinin, a mixture of diastereoisomers, is composed of silibinin A and silibinin B. Several papers ave been publised on te deteration of silibinin in biosamples after adistration of te drug to uman and various animals (Li et al, 26; Gatti and Perucca, 1994; Skottova et al, 21; Filburn, Kettenacker, and Griffin, 27; Wu et al, 27). However, in tese metods, silibinin was detered as a single entity due to te absence of reference substances of silibinin A and silibinin B. Altoug Rickling et al (1995) reported an HPLCECD metod for te quantification of silibinin A and silibinin B in uman plasma, tis metod made sensitivity low and sample preparation complicated. Li et al (28) detered silibinin A and silibinin B in Cinese subjects by LCMS/MS following a single oral dose of silybinpospatidylcoline complex, but tere were no parmacokinetic data after an iv adistration in te experiment, so te absolute bioavailability values of silibinin A and silibinin B were not obtained. To te best of our knowledge, te bioavailability and parma cokinetic caracteristics of silibinin A and silibinin B ave not been researced completely so far, terefore, an intensive investation on te bioavailability and parmacokinetics of te two diastereoisomers is expected. n te present study, a simple, rapid, and sensitive LCMS/MS metod was developed and validated for * Correspondence autor: Ma XH Address: Department of Parmacology, Tianjin Tasly nstitute, No. 2, Pujie East Road, Beicen District, Tianjin 341, Cina Tel: maxiaoui11@163.com Received: Marc 3, 211; Revised: May 7, 211; Accepted: June 18, 211 Fund:

2 31 Cu Y et al. Cinese Herbal Medicines, 211, 3(4): te deteration of silibinin A and silibinin B in rat plasma after iv and adistrations, and te metod was successfully applied to investate te bioavailability and parmacokinetics of silibinin A and silibinin B in rats, respectively. Materials and metods Cemical reagents Silibinin A and silibinin B (F. 1) reference substances were separated and purified by Tianjin Tasly nstitute (Tianjin, Cina) in our previous work (Li et al, 28). Teir purity was above 98.5% via HPLC analysis. Naringin, wic was used as te internal standard (S), was purcased from te National nstitute for Food and Drug Control (Beijing, Cina). Metanol (HPLC grade) was obtained from Fiser Scientific (Pittsburg, USA), wile all oter cemicals were of analytical grade and used witout furter purification. Te distilled water, prepared from deeralized water, was used trougout te study. A CH 2H Finnan, USA). Te column was Agilent Zorbax Eclipse XDBC 18 column (15 mm 2.1 mm, 5 μm). Te mobile pase was mixed wit metanolwaterformic acid (48:52:.1). Te flow rate was.25 ml/ for an isocratic elution. Te column temperature was maintained at 3. Te mass spectrometer was operated in te negative mode. Quantification was performed using selected reaction monitoring (SRM) of te transitions of m/z for silibinin A and silibinin B, and m/z for S, respectively (F. 2). Te optimized parameters were as follows: electrospay voltage 3.8 kv, capillary temperature 39, nitrogen (N 2 ) was used as seat gas and auxiliary gas at te pressures of and Pa, respectively, and te collision energy of 2 ev was used for te analytes and 34 ev for S A H CH H H 1 B H CH 2H 8 6 B H CH H H C H H H H CH 3 H H H H C H F. 1 Cemical structures of silibinin A (A), silibinin B (B), and naringin (C) LCMS/MS condition An LCMS/MS system utilized a Surveyor MS pump, a Surveyor autosampler, and a Termo Finnan TSQ Quantum triple quadrupole mass spectrometer equipped wit an ES source. Data acquisition was performed wit Xcalibur 1.3 software (Termo m/z F. 2 Mass spectra of silibinin A (A), silibinin B (B), and naringin (C) Animal experiment Male Wistar rats, weing 2426 g, were provided by Vital River Lab Animal Tecnology Co., Ltd. (Beijing, Cina) and oused wit a 12 lt/12 nt cycle at ambient temperature (about 25 ) and 45% relative umidity. All te animal experiment

3 Cu Y et al. Cinese Herbal Medicines, 211, 3(4): procedure was approved by te Animal Etics Committee of Tianjin Tasly nstitute. Silibinin was dissolved in etanolpeg2 (1:1) solution. Free access to food and water was allowed at all time except for fasting 12 before te experiment. Fifteen rats were adistered wit silibinin 28, 56, and 112 mg/kg (equivalent to silibinin A 13.3, 26.6, and 53.2 mg/kg; silibinin B 14.7, 29.4, and 58.8 mg/kg), respectively. For te absolute bioavailability study, anoter five Wistar rats were iv adistered wit silibinin 28 mg/kg (equivalent to silibinin A 13.3 mg/kg, silibinin B 14.7 mg/kg). Blood samples were collected into eparinized tubes from eac rat by te puncture of te retroorbital sinus at,.17,.33,.67, 1, 1.5, 2, 3, 4, 6, 8, and 12 after adistration, and centrifuged at 3 g for 8 to obtain plasma samples, wic were stored at 2 and analyzed witin one week. Sample preparation Tawed plasma samples were vortexed torougly at room temperature and employed as follows: n a 1.5 ml Eppendorf tube, 1 μl S solution (2 ng/ml naringin prepared by acetonitrile) and 1 μl acetonitrile were added to 1 μl plasma sample. After vortexing for 3 s, te sample was centrifuged at 12 g for 5, and 1 μl of te supernatant was injected into te LCMS/MS system for analysis. Metod validation Tis LCMS/MS assay was validated torougly in terms of specificity, matrix effect, linearity, precision, extraction recovery, and stability according to te guiding principles issued by Cina SFDA. Te quality control (QC) samples containing appropriate amount of silibinin A and silibinin B, prepared based on te procedures described above, at low, middle, and concentrations on tree sequential days, were used for validation. Data analysis Parmacokinetic parameters of silibinin A and silibinin B were calculated using te Topfit 2. program by te noncompartmental metod. Te eliation rate constant (λ z ) was obtained as te slope of te linear regression of te logtransformed concentration values versus time date in te teral pase. Te eliation alflife (t 1/2β ) was calculated as.693/λ z. Te peak plasma concentration (C max ) and te corresponding time (T max ) were directly obtained from te raw data. Te area under te curve to te last measurable concentration (AUC t ) was calculated by te linear trapezoidal rule. Te area under te curve to infinity (AUC ) was calculated as AUC = AUC t + C t /λ z, were C t is te last measurable concentration. Results Specificity and matrix effect A better resolution for silibinin A and silibinin B and a sorter run time were acieved by te combination of Agilent Zorbax Eclipse XDBC 18 column (15 mm 2.1 mm, 5 μm) and te mobile pase of metanolwater (48:52) containing.1% formic acid. Te cromatograms of blank plasma, blank plasma spiked wit silibinin A, silibinin B, and S, as well as te rat plasma sample were presented in F. 3, and no interference was observed at te retention time (t R ) of te analytes and S due to endogenous substances in rat plasma. Matrix effects for silibinin A, silibinin B, and S were evaluated by comparing te peak areas of analytes in extracted samples of blank plasma wit te corresponding areas obtained by direct injection of reference solutions. Matrix effects for silibinin A, silibinin B, and S were imal based on 9.2%17.8% of noal concentrations. Calibration curves and linearity Calibration curves were constructed by perforg weed linear regression analysis (w i = 1/C 2 ) of peak area ratios (R) of respective analyte to S versus te corresponding concentrations (C, ng/ml). Te obtained calibration curves sowed a good linearity over te range of 2. 5 ng/ml for silibinin A and silibinin B. Te regression equations and teir correlation coefficient (r) were calculated as follows: silibinin A, R =.1 25 C (r =.9981); silibinin B, R =.1 22 C (r =.9959). Te LD (S/N = 3) and LQ (S/N = 6) were detered as.1 and 2. ng/ml for silibinin A and silibinin B, respectively. Precision and accuracy Te precision and accuracy of te metod were presented in Table 1. Te intra and interbatc precisions, expressed as RSD, were obtained by analyzing QC samples containing silibinin A and silibinin B at low, middle, and concentrations in six replicates of eac concentration for eac batc and calculated by SD

4 312 Cu Y et al. Cinese Herbal Medicines, 211, 3(4): (d) m/z S m/z S S F. 3 Representative cromatograms of analytes (left) and S (rt) in rat plasma : Silibinin A (t R 6.45 ) : Silibinin B (t R 7.34 ) S: Naringin (t R 2.55 ) a: blank plasma b: blank plasma spiked wit silibinin A and S c: Blank plasma spiked wit silibinin B and S d: rat plasma sample Table 1 of measured concentrations from one batc (witin one day) and from tree batces (on tree consecutive days) divided by te mean measured concentration. Te RSD was found to be 1.9%9.1% for intrabatc precision and.8%5.7% for interbatc precision, indicating tat tis metod ad a good reproducibility. Te accuracy (relative error, RE) was calculated from te noal concentration (C nom ) and te mean value of observed concentration (C obs ) as follows: RE = [(C obs C nom )/(C nom )] 1%. Te values were estimated from 5.8% to 5.6% (intrabatc) and from 2.1% to 5.7% (interbatc), indicating accuracy of tis metod. n addition, te precision and accuracy of LQ were also investated, and te values were all less tan 12%. Recovery and stability Te mean recoveries of silibinin A, silibinin B, and S were more tan 9%. n processed samples, te analytes were stable for a period of 24 at room temperature (RE < ± 4.8%), plasma sample sowed no sn of degradation at 2 witin 6 d (RE < ± 7.4%), and no instability of analytes in spiked samples was observed over tree freeze/taw cycles (RE < ± 6.2%). Bioavailability and parmacokinetics of silibinin A and silibinin B Te mean silibinin A and silibinin B plasma Precision and accuracy of te metod Compound Concentration / (ng ml 1 ) silibinin A silibinin B nterbatc ntrabatc RSD / % RE / % RSD / % RE / % concentrations versus time curves in rat by iv and adistration were sown in F. 4, and te main parmacokinetic parameters of silibinin A and silibinin B using noncompartmental analysis wit Topfit 2. software were summarized in Tables 2 and 3. Te mean absolute bioavailability values of silibinin A and silibinin B were 2.86% and 1.93%, respectively, wic were calculated by te formula: Absolute bioavailability = (AUC Dose iv / AUC iv Dose ) 1%. Discussion n tis paper, a simple, rapid, and sensitive LCMS/MS metod was developed and validated for te deteration of silibinin A and silibinin B in rat plasma. Te LQ of te current assay was only 2. ng/ml for bot silibinin A and silibinin B, and it was demonstrated to be useful for te study of silibinin A and silibinin B disposition in rats for up to 12 after iv

5 Cu Y et al. Cinese Herbal Medicines, 211, 3(4): concentration / (ng ml 1 ) Concentration (ng/ml) A concentration Concentration / (ng ml (ng/ml) 1 ) mg kg mg/kg, B mg kg mg/kg, mg kg mg/kg, concentration / (ng ml 1 ) Concentration (ng/ml) 3 C concentration Concentration / (ng ml (ng/ml) 1 ) (d) 14.7 mg kg mg/kg, 1 D 29.4 mg kg mg/kg, mg kg mg/kg, t / F. 4 Mean plasma concentrationtime curves of silibinin A and silibinin B in rats (n = 5) A: silibinin A, iv adistration of 13.3 mg/kg B: silibinin A, adistration of 13.3, 26.6, and 53.2 mg/kg C: silibinin B, iv adistration of 14.7mg/kg D: silibinin B, adistration of 14.7, 29.4, and 58.8 mg/kg Table 2 Main parmacokinetic parameters of silibinin A in rats (n = 5) Parameters t 1/2 C max T max AUC t AUC MRT CL V d Units ng ml 1 ng ml 1 ng ml 1 1 ml 1 kg L kg 1 iv 13.3 mg kg mg kg mg kg mg kg ± ± ± ± ± ± ± ±.7.23 ±.9.2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Table 3 Main parmacokinetic parameters of silibinin B in rats (n = 5) Parameters Units iv 14.7 mg kg mg kg mg kg mg kg 1 t 1/2 C max T max AUC t AUC MRT CL V d ng ml 1 ng ml 1 ng ml 1 1 ml 1 kg L kg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 36.1 and adistration. Te plasma volumes required in our assay was only 1 μl. Tis lower volume permitted te use of serial blood sampling in studies involving a small animal species, suc as rats. Furtermore, te sample trougput was acieved by simple sample preparation and sort cromatograpic run time (witin 1 ). n conclusion, te sensitivity, small sample volume requirement, and relatively sort analytical time of te assay make it suitable for preclinical parmacokinetic studies of silibinin A and silibinin B. An exaustive investation on te bioavailability and parmacokinetics of te two diastereoisomers was first reported. t provides useful information for te furter researc of silibinin and its preparation. n te reported literature (Li et al, 28), it was

6 314 Cu Y et al. Cinese Herbal Medicines, 211, 3(4): found tat silibinin A ad better absorption and faster eliation tan silibinin B, and te velocity of absorption was similar between te two diastereoisomers in uman. Te parmacokinetic difference between silibinin A and silibinin B mt be due to stereoselective absorption, distribution, and metabolism. However, in tis study, we not only found tat silibinin A and silibinin B ad very low bioavailability after adistration, but also tere was no snificant difference in te parmacokinetic parameters between silibinin A and silibinin B, wic indicated tat te two diastereoisomers ad similar parmacokinetic beavior in rats. Compared to adistration, iv adistration of silibinin A and silibinin B sowed a sorter t 1/2β, suggesting tat silibinin A and silibinin B were eliated more rapidly from rat plasma after iv adistration. Following adistration of silibinin at 28 and 56 mg/kg, te dose and AUC/C max for silibinin A and silibinin B were positively correlated. n addition, tere was no snificant difference in systemic clearance at te two dose levels. t was demonstrated tat silibinin A and silibinin B mt ave linear parmacokinetic caracteristics in rats witin te two dose ranges tested. However, te AUC and C max of te two diastereoisomers did not increase proportionally at te dose of 112 mg/kg. We estimated tat te nonlinearity mt be due to absorption saturation at dosage. References Cen PN, Hsie YS, Ciang CL, Ciou HL, Yang SF, Cu SC, 26. Silibinin inibits invasion of oral cancer cells by suppressing te MAPK patway. J Dent Res 85(3): Filburn CR, Kettenacker R, Griffin DW, 27. Bioavailability of a silybinpospatidylcoline complex in dogs. J Vet Parmacol Ter 3(2): Flora K, Han M, Rosen H, Benner K, Milk tistle (Silybum marianum) for te terapy of liver disease. 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