Fitoterapia 101 (2015) 64–72

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Pharmacokinetic and tissue distribution profile of curculigoside after oral and intravenously injection administration in rats by liquid chromatography–mass spectrometry Ting-ting Yuan a,b,1, Hong-tao Xu a,b,1, Liang Zhao c, Lei Lv c, Yong-jing He a, Nan-dan Zhang a, Lu-ping Qin a, Ting Han a,⁎, Qiao-yan Zhang a,⁎ a b c

School of Pharmacy, Second Military Medical University, Shanghai, China Department of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, China Department of Pharmacy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China

a r t i c l e

i n f o

Article history: Received 23 September 2014 Accepted in revised form 19 December 2014 Available online 27 December 2014 Chemical compounds studied in this article: Curculigoside (PubChem CID: 158845) Naringin (PubChem CID: 442428) Keywords: Curculigoside LC–MS Pharmacokinetics Tissue distribution

a b s t r a c t Curculigoside has an extensive pharmacological activity, including estrogen-like, improving sexual behavior, antiosteoporotic, antioxidant, immunomodulatory and neuroprotective effects. However, few investigations have been conducted about the pharmacokinetics and tissue distribution of curculigoside to better understand its behavior and action mechanism in vivo. Thus, a sensitive and reliable liquid chromatography with mass spectrometry (HPLC–MS) method was established and validated for the quantification of curculigoside in rat plasma and tissue samples. Biological samples were processed with methanol precipitation, and naringin was used as the internal standard. Chromatographic separation was performed on an Agilent XDB-C18 chromatography column (3.0 mm × 50 mm, 1.8 μm) with a mobile phase consisting of acetonitrile and 0.1% formic acid. Quantification was performed by selected ion monitoring with m/z 511.1 [M + HCO2]− for curculigoside and m/z 579.1 [M − H]− for the internal standard. The validated method was successfully applied to the pharmacokinetic and tissue distribution study of curculigoside in rats. Non-compartmental pharmacokinetic parameters indicated that curculigoside had rapid distribution, extensive tissue uptake, and poor absorption into systemic circulation. The values of absolute bioavailability were 0.38%, 0.22% and 0.27% for oral doses of 100, 200 and 400 mg/kg, respectively. The results of the tissue distribution study suggested that curculigoside was distributed into the heart, lung, spleen, intestine, stomach, kidney, thymus, liver, brain, testis, and bone marrow after oral administration of 150 mg/kg. In conclusion, the present study may provide a material basis for study of the pharmacological action of curculigoside, and meaningful insights into further study on clinical application. © 2014 Elsevier B.V. All rights reserved.

1. Introduction

⁎ Corresponding authors at: Pharmacognosy of the Second Military Medical University, Shanghai 200433, China. Tel.: + 86 21 81871303; fax: + 86 21 81871306. E-mail addresses: [email protected] (T. Han), [email protected] (Q. Zhang). 1 Tingting Yuan and Hongtao Xu contributed equally to this work.

http://dx.doi.org/10.1016/j.fitote.2014.12.012 0367-326X/© 2014 Elsevier B.V. All rights reserved.

The rhizome of Curculigo orchioides Gaertn (Family: Amaryllidaceae), a commonly used traditional Chinese medicine, has been considered to have the effects of maintaining health energy and nourishing the liver and kidney and used as aphrodisiacs and tonics to treat declining strength, impotence, jaundice and asthma in China, India and Nepal. In India, C. orchioides, which is used as a substitute for “safed musli”, is usually

T. Yuan et al. / Fitoterapia 101 (2015) 64–72

combined with other herbs to treat bronchitis, chronic cough, asthma and hepatitis, and also acts as an appetite stimulant and regulates gastrointestinal function [1]. C. orchioides has showed wide spectrum pharmacological activities, including adaptive, immunostimulatory, antioxidant, antiosteoporotic, hepatoprotective and neuroprotective activities[2–4]. This effect is supposed to be attributed to phenolic glycoside from C. orchioides. Curculigoside is dominantly phenolic glycoside compound from C. orchioides Gaertn. Previous studies reported that curculigoside possessed antiosteoporotic [5], improving cognitive function in aged animals [6], antioxidant [7] and neuroprotective effects in vitro [8]. Given these remarkable functions, more attention has been paid to the pharmacological research on curculigoside and its clinical therapeutic application. Pharmacokinetics and characteristics of tissue distribution are vital to the understanding of the in vivo behavior and action mechanism. Recently, an HPLC–MS/MS method has been developed to determine curculigoside in plasma and applied to pharmacokinetic study on rats [9]. However, the absorption characteristics, oral bioavailability and tissue distribution of curculigoside in vivo have not been investigated in detail. The accumulation and target organs/tissues of this potential compound are also unknown. Here, a simple, rapid and reliable LC–MS assay for the determination of curculigoside in plasma and tissue samples was developed to explore the pharmacokinetic and tissue distribution of curculigoside in rats after oral and intravenous administrations, hoping that it could provide helpful information for the clinical application and explanation of pharmacological action mechanism of curculigoside. 2. Experimental

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administration for pharmacokinetic experiment. Thirty Sprague Dawley rats of both sexes received 150 mg/kg dose of curculigoside by oral administration for tissue distribution study. All animal experiments were carried out according to the Guidelines for the Care and Use of Laboratory Animals, and were approved by the Animal Ethics Committee of the Second Military Medical University. 2.3. Equipment and LC–MS conditions The levels of curculigoside in plasma and tissue were measured by a simple and sensitive liquid chromatography– mass spectrometry (LC–MS) method. Chromatographic analysis was performed on an Agilent 1100 HPLC system consisted of a G1322A degasser, a G1311A quaternary pump, a G1313A well-plate autos-ampler and a G1316A thermostated column compartment. A G1946D mass spectrometer (Agilent, Santa Clara, CA, USA) equipped with an electrospray source interface was used for MS detection. Data acquisition and analysis were performed using Agilent ChemStation for LC/MSD version B.02.01. Chromatographic separation was achieved on an Agilent XDB-C18 column (3.0 mm × 50 mm, i.d.:1.8 μm), and eluted with a mobile phase of acetonitrile: 0.1% formic acid aqueous solution (24:76, v/v) at a flow rate of 0.3 mL/min. The column temperature was maintained at 25 °C, the autosampler was conditioned at 4 °C and the injection volume was 5 μL. The analysis time was 4.5 min per sample. The HPLC system was connected to the mass spectrometer via an ESI interface. The mass conditions of electrospray ionization were optimized in the negative ion detection mode as follows: capillary 3500 V, nebulizer 40 p.s.i., drying gas 8 L/min, gas temperature 350 °C and fragmentor 80 V. Selected ion monitoring (SIM) was used and the fragmentation transitions were m/z 511.1 for curculigoside and m/z 579.1 for naringin.

2.1. Chemicals and reagents Curculigoside was isolated from rhizomes of C. orchioides Gaertn in our laboratory and identified by NMR, MS, UV and IR analyses. Naringin (purity N 98%) used as the internal standard was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile, formic acid and methanol were HPLC-grade reagents from Merck Company (Darmstadt, Germany). Deionized water was purified using a Milli-Q system (Millipore, Milford, MA, USA). The other reagents were of analytical grade and obtained through commercial sources. The drugfree plasma was gained from six Sprague-Dawley rats that were fasted for 12 h, with free access to water prior to blood sampling. 2.2. Animals Male Sprague-Dawley rats (220–250 g) were purchased from Shanghai Slac Laboratory Animal Co., Ltd (Shanghai, China), and were housed under standard conditions. The rats were bred in an environmentally controlled room (22 ± 2 °C, relative humidity 50 ± 20%) with free access to food and water, and in a natural light dark cycle for 7 days before the experiment was carried out. Twenty male Sprague Dawley rats received a 20 mg/kg of curculigoside by intravenous injection or 100, 200 and 400 mg/kg of curculigoside by oral

2.4. Preparation of calibration standards and quality control samples Appropriate amount of curculigoside was dissolved in methanol to prepare a stock solution 1.0 mg mL−1. Then stock solution was serially diluted with a methanol to provide working standard solutions of desired concentrations. IS stock solution was prepared at a concentration of 1.506 mg mL−1, and diluted with methanol to a final working concentration of 3012 ng mL−1 and stored at 4 °C before use. Plasma calibration standards were prepared by spiking blank plasma with appropriate amount of standard solutions and working IS solution to obtain final concentrations in the range of 1.01– 5000 ng mL−1 for curculigoside and 3012 ng mL−1 for IS. Calibration plots were prepared at concentrations of 1.01, 2.5, 5, 25, 50, 250, 500, 2500 and 5000 ng/mL. Quality control (QC) samples at three levels were also prepared in the same way at concentrations of 2.5, 250 and 2500 ng/mL. The standards and QC samples were extracted on each analysis day with the same procedure for plasma samples as described below. Calibration standards for various tissues including the heart, liver, spleen, lung, kidney, intestine and stomach were prepared by spiking 100 μL blank tissue supernatant with working standard solutions and working IS solution to obtain final concentrations in the range of 1.01–5000 ng mL−1 for curculigoside and 3012 ng mL−1 for IS.

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2.5. Sample preparation The simple organic solvent precipitation with methanol was applied for sample preparation. Plasma (100 μL) spiked with 50 μL IS solution (3012 ng/mL) and 250 μL methanol was thoroughly mixed by vortexing for 30 s. The mixture was centrifuged at 12,000 rpm for 10 min. 350 μL aliquot of supernatant was evaporated under a stream of compressed nitrogen gas for 1.5 h, the residue was dissolved in 50 μL mixture of water–methanol (40:60, v/v), vortexed for 30 s and centrifuged at 12,000 rpm for 10 min. 5 μL of the supernatant was injected into the HPLC–MS system for analysis. The tissues were ground and homogenated with 0.9% NaCl solution (1:4, w/v), then the homogenates were centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was collected, and 100 μL aliquot of supernatant was spiked with 50 μL IS solution (3012 ng/mL) and 250 μL methanol, and then treated as the same fashion as plasma sample. And also 5 μL of the supernatant was injected into the HPLC–MS system for analysis. 2.6. Method validation The method was validated for specificity, linearity, accuracy, precision, recovery and stability according to the guidelines set by the United States of Food and Drug Administration (FDA) and European Medicines Agency (EMA) [10,11]. Validation runs were conducted on three consecutive days. Each validation run consisted of one set of calibration standards and six replicates of QC samples. 2.6.1. Specificity and sensitivity The specificity of method was tested by comparing the chromatograms of blank rat plasma and tissue samples, plasma and tissue samples spiked with the analytes and IS, and plasma and tissue samples after an oral dose. Blank rat plasma and tissue samples were analyzed for endogenous interference. 2.6.2. Linearity of calibration curves and lower limit of quantification Calibration curve samples were prepared by spiking blank rat plasma or tissue samples with standard solutions (prepared in methanol) of curculigoside to the concentrations: 1.01, 2.5, 5, 25, 50, 250, 500, 2500 and 5000 ng/mL. Calibration curves were generated by plotting the peak area ratios (analyte/IS) (y) against the theoretical concentration (x) using a 1/x2 weighting. The LLOQ was defined as the lowest drug concentration that could be detected with a relative error and precision (relative standard deviation, RSD) no more than 20%.

2.6.4. Recovery, matrix effects and stability The extraction recoveries were determined by comparing the observed peak areas of curculigoside in extracted plasma or tissue samples with those of the curculigosode in nonprocessed samples at the same theoretical concentrations. The effect of rat plasma and tissue constituents on the ionization of curculigoside and IS was determined by comparing the responses of the post-extracted plasma or tissue standard QC samples (n = 5) with the response of analytes from neat standard samples at equivalent concentrations. If the peak area ratio is more than 85% or less than 115%, a matrix effect is implied. The stability tests were designed to cover the anticipated conditions that the samples might be exposed during storage and handling. The freeze/thaw stability was assessed by using threelevel QC samples, frozen at −20 °C and thawed at room temperature for a period of 12 h, the cycle was repeated three times and analysis was performed after the third time. The stability of curculigoside in processed samples in auto-sampler for 24 h was also investigated. Both the precision (RSD) of extraction recovery and matrix effect should not be more than 15%. 2.7. Pharmacokinetic study protocol and sample preparation The rats received intravenously a 20 mg kg−1 dose of curculigoside dissolved in normal saline, or orally 100, 200, and 400 mg kg−1 dose of curculigoside suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na w/v). Blood samples (0.5 mL) were serially withdrawn from the orbit venous plexus at 0.083, 0.167, 0.333, 0.5, 0.75, 1, 2, 4, 6, 8, 12, 24, 36 and 48 h after oral administration and at 0 (pre-dose), or at 0.083, 0.167, 0.333, 0.5, 1, 2, 4, 6, 8, 12 and 24 h after intravenous administration to heparinized Eppendorf tubes. The blood samples were immediately centrifuged at 12,000 rpm at 4 °C for 10 min and a 200 μL aliquot of supernatant plasma was transferred into another tube and stored at −80 °C until treatment. 2.8. Tissue distribution study Thirty rats of both sexes were orally administrated with 150 mg/kg of curculigoside. Various tissue samples of certain weight were collected from mice at 10 min, 30 min, 2 h, 4 h and 12 h post-dose. The blood samples were also collected from the carotid artery at responding time point, and plasma was acquired according to the above method. Then tissues were rinsed with saline solution to remove the blood or content, blotted with paper towel and stored at −80 °C until treatment. 2.9. Data analysis

2.6.3. Precision and accuracy The intra-day precision and accuracy were evaluated by assaying three quality control (QC) samples (n = 5) on the same day. The same procedure was performed once a day for 3 consecutive days to determine inter-day precision along with the standard calibration curve. Relative standard deviations (RSDs) were calculated and used in the estimation of the intraand inter-day precision. Accuracy was determined by comparison of the calculated mean concentrations using a calibration curve to nominal concentrations. Samples were considered stable if assay values were within the acceptable limits of accuracy (±15% bias) and precision (±15% RSD).

The concentrations of curculigoside in plasma and tissues were calculated according to the calibration curve by the ratio of the peak area to that of IS obtained from a single injection. Pharmacokinetic parameters, including the area under the blood concentration time curve (AUC0–∞, AUC0–t), body clearance (CL/F), mean residence time (MRT0–t), and elimination half-life (T1/2z) were analyzed by a non-compartmental method using the DAS 3.2.6 pharmacokinetic program (Chinese Pharmacology Society). The maximum concentration (Cmax) and time to reach the maximum concentration (Tmax) were obtained from a concentration–time curve. All results were expressed as

T. Yuan et al. / Fitoterapia 101 (2015) 64–72

mean ± standard deviation (S.D.). Absolute bioavailability was calculated according to the following equation: Bioavailability (F %) = AUC0–∞(p.o.) dose(i.v.) / AUC0–∞ (i.v.) dose(p.o.) × 100%. 3. Results and discussion 3.1. Method development Due to the high sensitivity and specificity of LC–MS, it has been widely used in biological sample analysis in recent years. The developed and validated method was successfully applied to curculigoside analysis in rat plasma and tissue samples during preclinical investigations such as pharmacokinetic and tissue distribution study. In order to develop a sensitive and specific LC–MS method for quantification of curculigoside in rat plasma and tissues, some potential factors were investigated and optimized. The response of curculigoside to ESI was evaluated by recording the full-scan mass spectra in both positive and negative ionization modes. Curculigoside exhibited a strong mass response in negative ESI mode due to the efficiency of ionization of the analyte. ESI showed that curculigoside (MW: 466.1) and IS (MW: 580.1) formed predominately molecular ions [M + HCO2]− at m/z 511.1, and mainly deprotonated molecular ions [M − H]− at m/z 579.1 in full scan mass spectra. Parameters were tuned according to the MS signal response of the target compound. The dominant product ions for quantitative detection of curculigoside and IS were at m/z 511.1 and 579.1, respectively. Chemical structures for curculigoside and naringin are shown in Fig. 1. Under the described chromatographic conditions, no any endogenous interference was observed in the plasma or tissue sample at retention time of curculigoside (3.5 min) and IS (2.5 min). Representative chromatograms obtained from the blank rat plasma, blank rat plasma spiked with the curculigoside at LLOQ level (1.01 ng/mL) and IS (3012 ng/mL), and plasma and tissue samples after oral administration are shown in Fig. 2. Chromatographic conditions, such as composition of mobile phase and type of analytical columns, might affect the ionization and the separation of curculigoside and IS. Methanol, acetonitrile, water and 0.1% formic acid were investigated to optimize the mobile phase. Compared with methanol, acetonitrile could provide sharper peak shape, lower pump pressure

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and shorter re-equilibrium time. 0.1% formic acid in water could produce appropriate ionization. Therefore, the combination of acetonitrile and 0.1% formic acid in water was chosen as mobile phase. C18 column has good separation effect for phenolic glycoside. Zorbax SB-C18 (100 mm × 2.1 mm, 3.5 μm), Zorbax SB-C18 (150 mm × 2.1 mm, 5 μm) and Agilent XBD-C18 (50 mm × 3.0 mm, 1.8 μm) were checked for chromatographic separation of curculigoside and IS. The Agilent XBD-C18 column had given the symmetrical peak shape, shorter run time and better solution. Salicin, baicalin and naringin were tested to find a suitable IS, and finally naringin was selected as internal standard for this method. Naringin and curculigoside had similar chromatographic behavior and ESI response, and both suitable for detection in negative ion electrospray ionization interface. In addition, the extraction recoveries of curculigoside were satisfactory, and it was stable during the whole analytical process. An efficient clean-up for bio-samples to remove protein and potential interferences prior to LC–MS analysis was important in the method development. The simple protein precipitation was tried in our work at initial stage, the recoveries of protein precipitation by acetonitrile, acetonitrile–methanol (1:1, v/v) and methanol were 63.7%, 85.6% and 95.7% for curculigoside, respectively. And there were some matrix effects found (around 91.5%–96.3%). Finally, methanol produced high extraction efficiency, and was used as precipitation reagent. Matrix effects were between 70% and 85% when methanol precipitation used for sample treatment. This one step protein precipitation is simpler and faster than several step protein precipitations, and produced a clean chromatogram for blank plasma samples and yielded satisfactory recovery of the analytes from rat plasma and tissue.

3.2. Method validation 3.2.1. Specificity Typical chromatograms of blank plasma and tissue homogenates, blank plasma spiked with curculigoside and IS, and plasma and heart samples after oral administration of curculigoside are presented in Fig. 2. The retention time was about 3.5 min for curculigoside and 2.5 min for IS. Due to the high selectivity of SIM mode, no significant endogenous components could interfere with the analyte and IS.

A B

Fig. 1. Chemical structure of curculigoside (A) and naringin (B).

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Fig. 2. Representative chromatograms of analytes from rat plasma and tissue: (A–L). Blank plasma and tissue sample: (A) plasma, (B) heart, (C) liver, (D) spleen, (E) lung, (F) kidney, (G) brain, (H) intestine, (I) stomach, (J) testis, (K) thymus, and (L) bone marrow; (M) plasma spiked with curculigoside (500 ng/mL) and IS (3012 ng/mL), (N) plasma sample obtained at 0.5 h after an intravenous administration of 20 mg/kg curculigoside, and (O) heart sample obtained at 0.167 h after oral administration of 150 mg/kg curculigoside. Peak 1 was naringin (IS); and peak 2 was curculigoside.

Table 1 Standard curves, linear ranges, correlation coefficients and lower limit of quantification of curculigoside in biological samples. Bio-samples

Standard curves

Plasmab Heart Liver Spleen Lung Kidney Brain Stomach Intestinum Bone marrow Testis Thymus

y y y y y y y y y y y y

a b

Unit is ng/mL. Unit is ng/mL.

= = = = = = = = = = = =

526.019x 765.667x 656.542x 627.197x 612.916x 671.939x 533.143x 524.530x 640.748x 566.615x 531.827x 543.215x

+ + + + + + + + + + + +

3840.80 6462.48 3606.50 2746.85 3282.85 3884.74 2910.96 4866.77 3731.50 2107.39 6081.15 4702.33

Linear ranges (ng/g)

Correlation coefficient (R)

LLOQ(ng/g)

1.01–5000a 1.01–5000 1.01–5000 1.01–5000 1.01–5000 1.01–5000 1.01–5000 1.01–5000 1.01–5000 1.01–5000 1.01–5000 1.01–5000

0.9983 0.9992 0.9990 0.9997 0.9994 0.9995 0.9994 0.9991 0.9995 0.9996 0.9993 0.9996

1.01b 1.01 1.01 1.01 1.01 1.01 1.01 1.01 1.01 1.01 1.01 1.01

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3.2.2. Calibration curves and LLOQ Linearity of the calibration curve was evaluated by a linear regression analysis using a 1/concentration (1/x2) weighting in the given concentration ranges of curculigoside in plasma and tissue samples. The calibration curves, determined coefficients and linear ranges of curculigoside in plasma and each tissue are listed in Table 1. The calibration curves for all matrices showed good linearity (R N 0.995) over the concentration ranges. The LLOQs were measured to be 1.01 ng/mL for plasma and 1.01 ng/g for tissue samples. 3.2.3. Precision and accuracy The plasma results for intra- and inter-day precision and accuracy are presented in Table 2. The intra- and inter-day accuracy was within −4.6% to 10.7%, respectively, while the intraand inter-day precision was less than 11.6% within the acceptable criteria of ±15%, indicating that the precision and accuracy of this assay are within the acceptable range of analysis. 3.2.4. Extraction recovery and matrix effect The extraction recoveries and matrix effect determined for curculigoside are shown in Table 3. All the variations of the matrix effect were in the range of 70.9–82.6%, which indicated that there was some matrix effect for curculigoside and IS in the plasma or tissue sample for this LC–MS determination. While this effective was relatively stable (75 ± 15%), namely relative matrix effect. The extraction recoveries ranged from 86.8% to 103.7% with RSD no more than 10% for curculigoside, which demonstrated that recoveries were consistent, precise and reproducible at different concentrations. 3.2.5. Stability The stability test was carried out under various conditions that the samples may experience. The results shown in Table 4 demonstrated that curculigoside was stable in the rat plasma after three freeze-thaw cycles, at room temperature for 12 h, at 4 °C in the auto-sampler for 24 h, and in a long term freezer set at −80 °C for 30 days. It is noteworthy that no significant degradation of curculigoside was observed under the above conditions. 3.2.6. Carry-over and dilution No detectable carry-over was observed in the current LC– MS method. The results of the dilution integrity experiments indicate that the accuracy was within ±15.0% and the precision was less than 6.9%, which could meet the requirements of dilution analysis. 3.3. Pharmacokinetic study The validated method for the quantitation of curculigoside in rat plasma was developed with a low LLOQ of 1.01 ng/mL Table 2 Precision and accuracy of curculigoside in rat plasma sample (n = 5). Concentration (ng/mL)

Precision (CV %) Intra-day

Inter-day

Accuracy (%) Intra-day

Inter-day

2.5 25 2500

9.0 8.6 8.5

5.6 11.6 7.1

−4.6 −4.5 2.7

7.9 −0.7 10.7

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Table 3 Extraction recovery and matrix effect of curculigoside and IS in rat plasma and tissue homogenates (n = 6). Bio-sample

Plasma

Nominal Extraction concentrations recovery(%) (ng/mL)

2.5 250 2500 Heart 2.5 250 2500 Liver 2.5 250 2500 Spleen 2.5 250 2500 Lung 2.5 250 2500 Kidney 2.5 250 2500 Cerebrum 2.5 250 2500 Intestinum 2.5 250 2500 Stomach 2.5 250 2500 Thymus 2.5 250 2500 Bone marrow 2.5 250 2500 Testis 2.5 250 2500 Naringin 3012

92.2 100.7 94.4 95.4 95.5 102.8 88.7 90.2 87.3 93.9 99.5 97.9 87.2 92.5 89.6 99.0 97.2 94.3 89.0 97.2 94.3 96.5 99.2 92.9 94.0 96.1 94.9 89.3 86.8 88.0 95.3 89.2 102.3 91.7 103.7 96.1 93.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

RSD (%)

3.4 3.6 5.6 5.5 8.7 9.3 2.1 2.2 3.6 3.7 4.1 3.9 7.8 8.2 8.6 8.4 7.7 8.3 5.3 5.9 8.5 8.8 5.4 5.9 4.5 4. 4 3.3 3.6 6.8 7.6 7.5 7.7 5.6 6.5 10.0 10.0 5.9 5.8 6.1 6.5 8.5 8.3 7.3 7.4 5.5 5.6 6.4 6.3 6.2 6.9 5.4 6.1 5.3 5.8 6.2 6.9 7.2 7.5 6.8 7.5 8.2 8.6 7.8 8.2 7.7 8.4 5.4 5.9 7.6 7.7 6.9 6.7 3.4 3.5

Matrix effect (%)

RSD (%)

73.2 71.2 71.3 72.6 71.3 71.3 72.1 73.2 75.5 73.7 71.6 80.1 79.4 78.2 73.5 79.7 82.6 73.5 82.2 79.4 77.7 76.3 71.9 72.5 76.4 73.8 73.4 77.6 74.9 76.4 80.8 73.7 70.9 80.8 71.1 79.3 14.5

5.5 7.8 6.4 4.3 5.7 5.9 7.7 8.8 4.9 3.9 8.0 7.2 5.7 9.1 7.5 9.2 5.5 8.9 9.5 5.2 6.5 8.7 5.9 7.1 5.0 6.0 4.5 7.2 5.7 7.5 8.9 9.6 8.8 8.9 9.5 8.8 4.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4.1 5.6 4.5 3 .1 4.0 4.2 8.2 9.1 5.3 3.8 8.1 7.1 5.7 9.4 7.9 9.7 5.5 9.1 9.4 5.7 6.7 9.1 6.1 7.4 5.2 6.3 4.8 7.1 5.9 7.9 9.2 9.2 9.3 7.9 8.2 8.0 1.0

and applied to a pharmacokinetic study in rats after oral administration of 100, 200 and 400 mg/kg curculigoside and intravenous administration of 20 mg/kg curculigoside. The major pharmacokinetic parameters were estimated using non-compartmental calculations performed with DAS (Drug and statistics) software version 3.2.6 (China). The mean plasma concentration–time curves are shown in Fig. 3. The major pharmacokinetic parameters are listed in Table 5. Upon IV administration at a dose of 20 mg/kg, the time to peak (maximum) concentration (Tmax) was at 0.17 h after intravenous administration in rats, the peak (maximum) plasma concentration (Cmax) of curculigoside was 189,328.5 ± 12,737.6 ng/mL, indicating that curculigoside could be quickly detected in plasma. Curculigoside was shown to have a moderate apparent volume of distribution (Vd = 66.1 ± 55.1 L/kg), a long half-life time (t1/2 = 121.2 ± 13.4 h) and a clearance of (0.3 ± 0.1 L/h/kg). After three dose levels of oral administration, Cmax, AUC(0–t) and AUC(0–∞) versus the curculigoside dose profile were linear with correlation coefficients of 0.9817, 0.9514, 0.9514, respectively, showing dose-dependent pharmacokinetics. The Cmax of three doses of curculigoside were (128.2 ± 46.2) ng/mL, (208.6 ± 103.6) ng/mL and (512.1 ± 136.9) ng/mL, respectively. The Tmax was observed at about

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Table 4 Stability of curculigoside in rat plasma sample (n = 5). Concentration (ng/mL)

2.5 250 2500

Autosampler (24 h, 4 °C)

Three freeze/thaw cycles

Room temperature (3 h)

Long term (30 day, −80 °C)

Precision (%)

Accuracy (%)

Precision (%)

Accuracy (%)

Precision (%)

Accuracy (%)

Precision (%)

Accuracy (%)

6.4 7.6 5.5

−10.11 −7.87 4.72

4.3 7.8 10.4

−1.25 −7.33 −3.83

5.3 5.7 4.0

−3.84 −7.76 3.52

3.4 4.5 7.1

0.61 −10.47 2.71

14–20 min after oral administration in rats, indicating that curculigoside could be quickly absorbed into the blood circulatory system. The value of Tmax and t1/2 indicated that curculigoside was rapidly distributed. The apparent volume of distribution (Vd) implied that curculigoside exhibited an obvious tissue uptake after oral administration. The values of absolute bioavailability of curculigoside were (0.38 ± 0.18) %, (0.22 ± 0.10) % and (0.27 ± 0.20) % for oral doses of 100, 200 and 400 mg/kg, respectively. These results suggested that its bioavailability was independent of the doses. A recent study [9] reported that the absolute bioavailability of curculigoside was 1.27% in rats after oral administration at dose of 32 mg/kg. Therefore, it was also concluded that curculigoside might have a low absolute bioavailability. Curculigoside is a benzylbenzoate glucoside, and similar with salicylate-type compounds in chemical structure. Comparisons of the pharmacokinetic parameters of curculigoside with those of salicylate-type compounds will help us to understand the metabolic characteristic and action mechanism. Oral administration of aspirin (100 mg/kg), the AUC of the aspirin was 2031 ± 266 min μg/mL, and t1/2 was 13.0 ± 1.27 min. However, the AUC of salicylic acid was 12,660 ± 1799 min μg/mL, and t1/2 was 269 ± 115 min, demonstrating that aspirin is quickly hydrolyzed to salicylic acid within 15 min [12]. After oral administration of guaiacol acetylsalicylate, the guaiacol salicylate concentration was found to be low in the plasma and organs, and the pharmacokinetic parameters of salicylic acid are as follows: Tmax = 3.02 h, Cmax = 331.46 μg/mL, and AUC = 2832.93 μg/mL h., indicating that guaiacol acetylsalicylate is metabolized into salicylic acid [13]. Gaultherin, 2-[(6-Oβ-D-xylopyranosyl-β-D-glucopyranosyl) oxy] benzoic acid methyl ester, a natural salicylate derivative extracted from Gaultheria yunnanensis, is administrated at a single dose of 200 mg/kg in mouse or 140 mg/kg in rat, the parent compound was not detected in the plasma of mice and rats, indicating that

gaultherin could be metabolically converted to salicylate. The maximum plasma concentration (Cmax) of salicylate in mouse and rat was 60 and 70 μg/mL, respectively and the time to reach Cmax (Tmax) was 5 at 7.5 h after dosing [14]. Curculigoside, like salicylate-type compounds, may be converted into active metabolite in gastrointestinal tract. Maybe, this is why curculigoside has a very low absolute bioavailability. Therefore, the metabolism of curculigoside should be further investigated to clarify its action mechanism. 3.4. Tissue distribution Curculigoside is widely distributed in all tissues examined after oral administration. As shown in Table 6, it extensively distributed into the extra-vascular system of animal body. Curculigoside levels are significantly reduced to be undetectable in 12 h after oral administration. The highest concentration is observed in the intestine and stomach at 10 min, followed by the lung, thymus, spleen, kidney, brain, liver, testis, heart and bone marrow, while the highest concentration is observed in the spleen, heart, kidney and testis at 30 min. Compared with the concentration in plasma, curculigoside could be detected in various tissue samples, indicating that it had a relatively high degree of tissue distribution, which is consent with the result of the pharmacokinetic study. The curculigoside is maintained at high concentration in the intestine, stomach, heart, spleen, thymus, liver, brain and testis at 4 h, and even at high level in spleen at 12 h. The lower concentration in liver suggested that curculigoside may be apt to metabolize in the liver. Meanwhile, curculigoside found in the brains implies that it could cross the blood-brain barrier. The sample collected at 12 h after administration showed that curculigoside was cleared gradually and no accumulation was observed in the tissues. The higher concentration in the lung, kidney and spleen might be attributed to the high blood flow in these organs.

Fig. 3. Mean plasma concentration–time curves after oral administration of 100, 200 and 400 mg/kg curculigoside (A) and intravenous administration of 20 mg/kg curculigoside (B) in rats.

T. Yuan et al. / Fitoterapia 101 (2015) 64–72

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Table 5 The main pharmacokinetic parameters after oral administration of 100, 200, and 400 mg/kg curculigoside or intravenous administration of 20 mg/kg curculigoside in rats (n = 5, mean ± SD). Parameter

T1/2 Tmax CL Vd Cmax AUC(0–t) AUC(0–∞) MRT(0–t) MRT(0–∞)

Oral administration (mean ± SD)

h h L/h/kg L/kg ng/mL ng/mL h ng/mL h h h

Intravenous administration (mean ± SD)

100 mg/kg

200 mg/kg

400 mg/kg

20 mg/kg

32.7 0.33 75.0 3674.4 128.2 1241.4 1374.0 26.29 39.09

27.4 0.30 118.9 4850.3 208.6 1644.4 1722.2 21.03 27.23

13.1 0.25 98.1 1879.9 512.1 4172.2 4308.8 20.89 21.79

121.2 0.17 0.3 66.1 189,328.5 74,241.9 74,323.6 0.32 0.36

± ± ± ± ± ± ± ± ±

13.5 0.12 16.0 1921.7 46.2 280.6 244.0 2.14 10.34

The tissue distribution profile of drug or compound combination in certain target organs could be used as an evidence to explain why a very low exposure of an oral drug would exert a certain efficacy. The compounds were absorbed into the blood and then distributed into target tissues, then, pharmacological effects were exerted by the unbound fraction of the drug [15]. Curculigoside distributed in the heart, liver, spleen, lung, kidney, especially in thymus and bone marrow. Therefore, we speculated that these organs may be the target points of curculigoside to exert pharmacological effects. Curculigoside showed estrogenic activity and improving sexual behavior effects [4], as we found that curculigoside distributes in testis at high concentration. Curculigoside exhibited significant antiosteoporotic effects [5], while curculigoside also distributed in the bone marrow, where osteoblast and osteoclast involved bone metabolism are formed and differentiated. Curculigoside ameliorated learning and memory capacity in aged rats [8], while curculigoside could cross blood-brain barrier and enter the brains. Curculigoside existed at high concentration in immune organs, such as the spleen and thymus and exhibited significant immunomodulatory effects [2]. 4. Conclusion A LC–MS method for the quantification of curculigoside in rat plasma and tissue samples has been established and successfully used to evaluate the pharmacokinetic and tissue distribution profile after oral and intravenous administration.

± ± ± ± ± ± ± ± ±

17.3 0.07 21.5 3298.3 103.6 298.8 274.4 4.57 4.02

± ± ± ± ± ± ± ± ±

2.8 0.17 26.5 761.9 136.9 1094.8 1088.3 1.65 1.92

± ± ± ± ± ± ± ± ±

13.4 0.22 0.1 55.1 12,737.6 26,463.6 26,484.9 0.14 0.13

The method showed high sensitivity, specificity and reliability with an LLOQ of 1.01 ng/mL. The results obtained from the pharmacokinetic and tissue distribution study suggested that curculigoside distributed in the brain, liver and gonad tissues could conduce to the therapeutic effects, but the low absolute bioavailability may limit its further application. Our ongoing study would focus on the pharmaceutical reformation and structure modification to improve bioavailability. Acknowledgment This study was supported by the National Natural Science Foundation of China (Grant No. 81274152) and Shanghai Municipal Science and Technology Commission (Grant No.12401900702, 13041900102). References [1] Chauhan NS, Sharma V, Thankar M, Dixit VK. Curculigo orchioides: the black gold with numerous health benefits [J]. J Chin Integr Med 2010;8(7): 613–23. [2] Bafna AR, Mishra SH. Immunostimulatory effect of methanol extract of Curculigo orchioides on immunosuppressed mice [J]. J Ethnopharmacol 2006;104(1–2):1–4. [3] Liu L, Guo YH, Xin HL, Nie Y, Han T, Qin LP, et al. Antiosteoporotic effects of benzylbenzoate glucosides from Curculigo orchioides in ovariectomized rats [J]. J Chin Integr Med 2012;10(12):1419–26. [4] Wang CM, Xu SY, Lai S, Geng D, Huang JM, Huo XY. Curculigo orchioides (Xian Mao) modifies the activity and protein expression of CYP3A in normal and kidney-yang deficiency model rats [J]. J Ethnopharmacol 2012;144(1):33–8.

Table 6 Curculigoside concentration (ng/g) in various tissues after oral administration of curculigoside in rats (dose = 150 mg/kg, n = 6). Tissue

10 min (ng/g)

30 min (ng/g)

2 h (ng/g)

Heart Liver Spleen Lung Kidney Brain Thymus Testis Stomach Intestine Bone marrow Plasmab

357.5 550.8 1030.3 36,294.8 885.5 569.8 3076.0 371.6 136,585.8 220,001.1 217.7 107.3

586.9 378.2 1732.7 283.6 697.1 296.2 272.8 497.5 38,937.9 10,021.0 11. 4 145. 3

127.7 100.9 65.6 945. 7 229.5 272. 7 107.1 51.5 3459.7 9024. 6 6.3 89.8

a b

Unit is ng/mL. Unit is ng/mL.

± ± ± ± ± ± ± ± ± ± ± ±

210.8 292.4 564.3 20,306.2 298.3 278.5 1792.3 283.2 58,208.2 30,594.2 250.3 40.4a

± ± ± ± ± ± ± ± ± ± ± ±

207.9 155.2 520.1 173.4 369.2 272.3 378.5 215.3 34,231.2 10,707.2 10.6 73.7a

± ± ± ± ± ± ± ± ± ± ± ±

130.6 49.6 38.2 214.2 221.6 239.9 88.3 32.1 2974.7 8853.9 4.1 52.2a

4 h (ng/g)

12 h (ng/g)

32.3 24.8 27.7 3.7 4.7 10.7 23.1 10.6 38.6 398.6 4.14 64.5

6.4 6.6 34.2 3.9 3.6 3.1 2.7 4.0 5.1 5.6 4.0 27.1

± ± ± ± ± ± ± ± ± ± ± ±

34.7 20.4 32.5 4.2 9.2 5.8 24.6 9.2 39.3 449.0 1.2 12.0a

± ± ± ± ± ± ± ± ± ± ± ±

8.5 5.3 62.7 1.2 4.3 0.9 8.3 3.0 4.8 4.2 2.3 13.7a

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[5] Jiao L, Cao DP, Qin LP, Han T, Zhang QY, Zhu Z, et al. Antiosteoporotic activity of phenolic compounds from Curculigo orchioides [J]. Phytomedicine 2009; 16(9):874–81. [6] Wu XY, Li JZ, Guo JZ, Hou BY. Ameliorative effects of curculigoside from Curculigo orchioides Gaertn on learning and memory in aged rats [J]. Molecules 2012;17(9):10108–18. [7] Wang Y, Zhao L, Wang Y, et al. Curculigoside isolated from Curculigo orchioides prevents hydrogen peroxide-induced dysfunction and oxidative damage in calvarial osteoblasts [J]. Acta Biochim Biophys Sin 2012; 44(5):431–41. [8] Tian Z, Yu W, Liu HB, et al. Neuroprotective effects of curculigoside against NMDA-induced neuronal excitoxicity in vitro[J]. Food Chem Toxicol 2012; 50(11):4010–5. [9] Zhao G, Yuan FS, Zhu JJ. An LC–MS/MS method for determination of curculigoside with antiosteoporotic activity in rat plasma and application to a pharmacokinetic study [J]. Biomed Chromatogr 2014;28(3):341–7. [10] U.S. Food, Drug Administration. Guidance for industry: bioanalytical method validation; 2001.

[11] European Medicines Agency (EMA). Guideline on bioanalytical method validation; 2011. [12] Shaw LH, Tsai TH. Simultaneous determination and pharmacokinetics of protein unbound aspirin and salicylic acid in rat blood and brain by microdialysis: an application to herbal–drug interaction [J]. J Chromatogr B 2012;895–896:31–8. [13] Qu SY, Li W, Chen YL, Sun Y, Zhang YQ, Hong T. The physiologic disposition and pharmacokinetics of guaiacol acetylsalicylate in rats [J]. Acta Pharm Sin 1990;25(9):664–9. [14] Zhang B, He XL, Ding Y, Du GH. Gaultherin, a natural salicylate derivative from Gaultheria yunnanensis: towards a better non-steroidal antiinflammatory drug [J]. Eur J Pharmacol 2006;530:166–71. [15] Liu YT, Hao HP, Xie HG, Lai L, Wang Q, Liu CX. Extensive intestinal first-pass elimination and predominant hepatic distribution of berberine explain its low plasma levels in rats [J]. Drug Metab Dispos 2010;38(10):1779–84.