Metabolites of curculigoside in rats and their antiosteoporotic activities in osteoblastic MC3T3-E1 cells

Accepted Manuscript Metabolites of curculigoside in rats and their antiosteoporotic activities in osteoblastic MC3T3-E1 cells

Liang Wang, Yong-jing He, Ting Han, Liang Zhao, Lei Lv, Yuqiong He, Qiao-yan Zhang, Hai-liang Xin PII: DOI: Reference:

S0367-326X(16)30657-8 doi: 10.1016/j.fitote.2017.01.009 FITOTE 3558

To appear in:

Fitoterapia

Received date: Revised date: Accepted date:

27 October 2016 11 January 2017 21 January 2017

Please cite this article as: Liang Wang, Yong-jing He, Ting Han, Liang Zhao, Lei Lv, Yu-qiong He, Qiao-yan Zhang, Hai-liang Xin , Metabolites of curculigoside in rats and their antiosteoporotic activities in osteoblastic MC3T3-E1 cells. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Fitote(2016), doi: 10.1016/j.fitote.2017.01.009

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ACCEPTED MANUSCRIPT Metabolites of curculigoside in rats and their antiosteoporotic activities in osteoblastic MC3T3-E1 cells

Liang Wanga#, Yong-jing Hea#, Ting Hana, Liang Zhaob, Lei Lvb, Yu-qiong Hea, Qiao-yan

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Zhang a* , Hai-liang Xin a*

School of Pharmacy, Second Military Medical University, Shanghai, China

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Department of Pharmacy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical

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University, Shanghai, China

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*Corresponding authors: Qiaoyan Zhang and Hailiang Xin; Pharmacognosy of the Second Military Medical University, Shanghai 200433, People’s Republic of China; Email:

These two authors contributed equally to this paper.

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#

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[email protected], [email protected]; Tel: 86-21-81871303; 86-21-81871300

Abstract

Curculigoside isolated from Curculiginis Rhizoma exhibits a wide spectrum of

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bioactivities. In this study, a high performance liquid chromatography/quadrupole time-of-

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flight tandem mass spectrometry (UHPLC/Q-TOF MS) method was employed to investigate the metabolism of curculigoside in rats. Plasma, bile, urine, feces and 17 tissues were collected from rats after a single PO dose of curculigoside at 100 mg/kg and prepared through methanol precipitation. Parent compound and a total of 7 metabolites were detected and identified based on their retention time and fragment ions. Metabolic pathways of curculigoside in rats include hydrolysis, demethylation and glucuronidation. Exposure of major metabolite M2 in plasma and it’s antiosteoporotic activity in osteoblastic MC3T3-E1 cells were studied to help understand that curculigoside assimilates less but works more. 1

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Key words: curculigoside; metabolic profile; UHPLC-Q-TOF MS; metabolite, osteoporosis

1. Introduction Curculiginis Rhizoma, which originated from Curculigo orchioides, has been widely

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used as traditional Chinese medicine for more than 2000 years, and officially listed in Chinese

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Pharmacopoeia [1]. Curculigoside is a representative phenolic glycosides ingredient in this

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plant[2], which are thought to be the major bioactivity constituents, possess various beneficial pharmacological effects including hepatoprotective effect [3], anti-oxidant [4],

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neuroprotective effect [5], estrogenic and antiosteoporotic effects [6], suggesting its potential application in future. Our previous investigations also found that oral administration of

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curculigoside can significantly enhance learning performance and ameliorate bone loss in APP/PS1 mutated transgenic mice and ovariectomized rat [7,8], and recover the bone

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formation activity of osteoblast damaged by H2O2 through reducing the production of ROS[4].

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However, some pharmacokinetic studies have indicated that curculigoside has poor oral bioavailability [9]. The low blood concentration and low biological availability seemed to be

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a stumbling block for the selection of curculigoside as a promising antiosteoporotic drug candidate for further evaluation. Therefore, it is necessary to clarify whether some metabolites

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of curculigoside contribute to its pharmacological activities, for example antiosteoporosis. Being a significant role in various stages of drug discovery and development, metabolic product identification not only helps to explain and predict a variety of events related to the efficacy and the toxicity of the parent drugs, but also tends to discoveries of new chemical derivatives with pharmacological activity [10]. Recently, HPLC-Q-TOF-MS has become an advantageous tool for determination of metabolites. Strengths lying in high full-spectral sensitivity of TOF analyzers make them ideal for detecting expected and unexpected

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ACCEPTED MANUSCRIPT metabolites from a single run, and their good mass resolution and mass accuracy (<3–5 ppm) enables measurement of reliable and accurate mass of any ionizable component[11]. Furthermore, the very high data acquisition speed of TOF-mass analyzers makes them most suitable for coupling with fast chromatography. Therefore, Liquid chromatography (LC)/quadrupole time-of-flight tandem mass spectrometry (QTOF-MS) has been successfully

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applied to the identification of active ingredients and metabolites of traditional Chinese

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medicine [12].

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An analytical method using HPLC/QTOF-MS was developed for detection of curculigoside and its metabolites in bile, plasma, urine and other tissues samples of dosed rats

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in this paper. Three phase I metabolites and four phase II metabolites were detected and their chemical structures were carefully elucidated based on MS/MS spectrum. Furthermore, the

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exposure of parent drug and main metabolite M2 in plasma was studied to elucidate the

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antiosteoporotic activities of curculigoside.

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2. Experimental

2.1 Materials and Reagents

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Curculigoside and metabolite M2 were isolated from rhizomes of Curculigo orchioides gaertn in our laboratory and identified by NMR, MS, UV and IR analysis. HPLC-grade of

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acetonitrile and formic acid were purchased from Fisher scientific (Tustin, CA, USA), and the water used in the test was produced by milli-Q water purification system.

2.2 Animal experiments The same batch of male and female Wistar rats bought from Shanghai SLACOM experimental animal company were housed in an air-conditioned room with 12/12 h light–dark illumination cycles for a week before starting the experiment. Rats were divided

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ACCEPTED MANUSCRIPT into two groups based on weight, including dosed group and control group with equivalent male and female rats. Curculigoside was dissolved in 0.5% CMC-Na aqueous solution and intra-gastric administrated to fasted rats (330±30mg) at the dosage of 100mg/kg body weight. All experimental procedures were reviewed and approved by the Ethics Committee of Second

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Military Medical University.

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2.3 Sample preparation

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Blood samples were collected via retro-orbital puncture at 10 and 30 min, 2, 4 and 12h after dosing and centrifuged at 12000g/min for 10 min to obtain plasma. Tissues were

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obtained at 10 and 30 min, 2, 4 and 12h from one male rat and one female rat per time. Urine was collected during 0-30 min, 0.5-4 and 4-12h and feces was collected during 0-24h. Bile

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was collected via bile duct intubation in dosed rats.

300μL methanol was added into 100μL aliquot of samples and the mixture was

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vortexed for 10 min and centrifuged at 12000g/min for 10 min. 350μL supernatant was

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collected into a new tube, dried under vacuum, redissolved in 50μL 60% methanol aqueous solution and centrifuged at 12000g/min for 10 min to obtain supernatant for analysis. 200 mg

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precisely weighed tissue or the whole tissue (less than 200 mg) samples and feces samples was homogenized with 1:3 (m: v) physiological saline, and then the homogenates were

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centrifuged at 12000g/min for 10 min to collect supernatant. Tissue and feces supernatants were further prepared in the same way as plasma samples.

2.4 Instrumental and analytical conditions The analysis was carried out on the 6210 Accurate-mass Q-TOF LC/MS (Agilent Technology, Santa Clara, CA, USA) equipped with an ESI interface. Chromatographic separations were performed on a reversed-phase column

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ACCEPTED MANUSCRIPT (Zishengtang MG-C18 column, 100×3mm, 3.0μm) with the column temperature at 25℃. A constant flow rate of 0.6 mL/min was used with a liner gradient elution from 5-90% solution A in 40 min. Solution A was acetonitrile and solution B was 0.1% formic acid. The sample injection volume was 3μL. The drying and nebulizer gas was nitrogen (N2). ESI operating parameters were

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optimized as follows: capillary voltage, 3500V; nebulizer gas pressure, 35 psig; drying gas

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flow rate, 9 L/min; gas temperature, 350℃; fragmentor and skimmer voltages, 150V and 60V

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in positive ion mode (ESI+). Q-TOF spectra parameters: mass range, 100-1000m/z; acquisition rate and time, 1.41 spectra/s and 709.6 ms/spectrum; reference mass, 121.050873

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and 922.009798. The MS/MS experiments were carried out by setting the Q-TOF premier quardrupole to allow ions of interest to pass prior to fragmentation in the collision cell with

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collision energies varying between 5 and 35 eV.

2.5 Determination of concentration of M0 and M2 in plasma

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Concentration of curculigoside and metabolite M2 in plasma were determined by a

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liquid chromatography-mass spectrometric (LC-MS) method. Chromatographic analysis was performed on an Agilent 6460 HPLC system consisted of a G1322A degasser, a

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1290-G4220A quaternary pump, a G4226A well-plate autos-ampler and a G1316C

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thermostated column compartment. G4212A mass spectrometer (Agilent, Santa Clara, CA, USA) equipped with an electrospray source interface was used. Data acquisition and analysis were carried out using Agilent ChemStation for LC/MSD version B.02.01. Chromatographic separation was performed on an Agilent Zorbax SB C18 column (3.5 μm, 2.1×100 mm) and eluted with mobile phase of acetonitrile and 0.2 % formic acid aqueous solution (25:75~30: 70, v/v) for 3 min at a flow rate of 0.3 mL/min. The column temperature was maintained at 30℃.The autosampler was conditioned at 4℃ and the injection volume was 5 μL. Working parameters of mass spectrometer were optimized as follows: capillary 4000 V for positive ion 5

ACCEPTED MANUSCRIPT model and 3000 V for negative ion model, nebulizer gas 40 psi, drying gas 10 L/min, gas temperature 325℃ and fragmentation voltage 175 V for M0 and 70 for M2. Multiple reaction monitoring (MRM) was applied and transitions 465.1→283.1 and

183.1→165.1 were

monitored for curculigoside and metabolite M2. Stock solution of curculigoside and metabolite M2 were prepared in methanol at

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1mg/mL and diluted with 10% methanol water solution to obtain working solution with a

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series of concentration. An aliquot of 100 µL working solution was added to 900 µL blank

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plasma to obtain standard samples within the final concentration range 1-1000 ng mL-1 of M0 and 10-1000 ng/mL of M2.

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2.6 The culture, assay of proliferation and alkaline phosphatase activity of osteoblastic MC3T3-E1 cells

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The osteoblastic MC3T3-E1 cells at 5.0104 cells/mL were incubated in 96 well plates at 37 °C for 24 h before being treated with M0 and M2 for 48 h. MTT was added to each well

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and incubated for additional 4 h to determine osteoblast proliferation. DMSO (150 μl) was added to each well to replace the medium. UV absorbance was measured at 550 nm on an ELx 800 universal microplate reader (Bio-Tek) and used as an indicator of osteoblast

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proliferation. osteoblastic MC3T3-E1 cells at 5.0104 cells/mL were incubated in 96 well

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plates at 37 °C for 4 days before being treated with M0 and M2 for 2 days. Cells were gently washed twice with PBS and lysed with 0.2% Triton X-100. The lysate was centrifuged at 12,000 g for 5 min. The supernatant was used for measuring the ALP activity and protein concentration using an ALP activity assay kit and a BCA-protein assay kit, respectively. The ALP activity was expressed as micromoles of p-nitrophenol liberated per-nanogram protein. The effects of M0 and M2 on the proliferation and ALP activity of osteoblast were expressed as % of control, and calculated as follows: mean value of treatment group/that of control group 100%. 6

ACCEPTED MANUSCRIPT 2.7 Data analysis The results were presented as mean values ± standard deviation. Data were analyzed by a Student's t-test or one-way ANOVA to determine statistical differences between groups using SPSS statistical software (version 18.0 for Windows, SPSS Inc, Chicago, IL, USA). The

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statistical significance of mean differences was based on a p value of 0.05.

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3 Results

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3.1 Metabolism profiles by HPLC-MS analysis

Curculigoside is an important phenolic glycosides possessing ester bond and phenolic

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glycoside bond, which are sensitive to oxidation, hydrolysis or reduction in phaseⅠchemical modification. Furthermore, phenolic hydroxyl groups and their derivative hydroxymethyl and

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benzoyloxy in parent compound and/or phaseⅠmetabolites were likely to become the potential phase II metabolic spots undergoing subsequent biotransformation such as

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glucuronidation, sulfation or methylation [13]. Curculigoside and expected metabolites were

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chosen according to selected structural classes in which the molecules give ESI+ signals as [M+H]+and [M+Na]+ ions [14]. The MS/MS data have contributed to structure elucidation

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and further backed the proposed metabolic pathways of curculigoside in rats.

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Parent compound and 7 metabolites were characterized and their calculated molecular weight, retention time, observed m/z value, molecular ion and mass accuracy were summarized in Table 1. Their EIC (extracted ion current) chromatograms and MS spectrums were shown in Fig 1 and Fig 2 respectively.

3.2 Identification of metabolites and metabolic pathways The proposed metabolic pathway of curculigoside in rats is presented in Fig.3. Compound M0 characterized in plasma was eluted at 10.268 min and gave a molecular 7

ACCEPTED MANUSCRIPT ion [M+Na]+ at m/z 489.1354 with formula C22H26O11, and it’s chromatographic and mass spectral properties are in accordance with curculigoside. Therefore, M0 could be confirmed as parent drug (Fig 4a). The measured m/z of M4 was 665.1672, which is 176 Da higher than that of M0, indicating that it may be the glucuronate conjugate of M0. The MS/MS spectrum of m/z 665.1685 (M4) gave an abundant daughter ion at m/z 489.1370 by neutral loss of 176

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Da (C6H8O6), which supported the deduction (Fig 4b). Based on the above data and

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metabolism rule of phase II in vivo, M4 should be the monoglucuronidation product of

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curculigoside at the 4-OH position. The molecular ion of M5 (m/z 651.1520) was 14 Da less than that of M4, indicating that the demethylation reaction may occur to the skeleton of M4.

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Hence, M5 was presumed to be the demethyl product of M4, and the product ion at m/z 475.1216 in its MS/MS spectrum was also generated by a loss of a C6H8O6 (176 Da), which is

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consist with that of M4. Therefore, M5 was identified as demethyl products of M4 at 2’’-O or 6’’-O position (Fig 4c).

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Compound M2, which was of high abundance, gave molecular ions [M+H]+/[M+Na]+ at

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m/z 183.0649/205.0470 with a retention time of 8.709, and M3 was observed as a molecular ion [M+Na]+ at m/z 325.0889 with a retention time of 1.982 min. M2 and M3 were deduced

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as products of the hydrolysis of ester bond between 7-O and C7’’ in curculigoside with each formula C9H10O4 and C13H18O8. The MS2 spectrum of M0 gave an abundant daughter ion

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[M+Na]+ at m/z 205.0468 (M2) via the loss of a C13H16O7 (284Da), and this fragment data assisted in elucidating the structure of M2 (Fig 5a). M2 was further identified by the daughter ion [M+H]+ 165.0546 generated by the loss of a H2O(18Da) in its MS/MS spectrum (Fig 5a). M7 gave a molecular ion [M+Na]+ at m/z 381.0788 with formula C15H18O10, and was detected at a retention time of 8.094 min. A quasi-molecular ion at m/z 381.0788 (M7) was 176 Da higher than that of the phase I abundant metabolite M2, suggesting that conjugation with one glucuronic acid occurred to M2, which possesses carboxyl groups. The MS/MS spectrum of

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ACCEPTED MANUSCRIPT M7 gave rise to a [M+Na]+ signal at m/z 205.0469 corresponding to M2 by neutral loss of 176 Da (C6H8O6), indicating that M7 should be the monoglucuronidation product of M2 (Fig 5b). M1 gave a protonated ion at m/z 305.1018 with a retention time 10.583 min and was deduced as C16H16O6 generated by hydrolysis of phenolic glycoside bond at C1 in curculigoside. In its MS2 spectrum, a fragment peak at m/z 287.0922 was formed by

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eliminating a H2O (18Da) from the mother ion at m/z 305.1042, and another fragment ion at

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m/z 183.0648 corresponding to M2 was generated by losing a C7H6O2 (122Da) (Fig 6a).

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Based on the appearances of these characteristic fragment ions in the MS2 spectra, M1was proposed to be the deglycosylated product of curculigoside. M6 was observed as a molecular

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ion [M+Na]+ at m/z 503.1156 with formula C22H24O12, which was 198Da (C6H8O6 + Na) higher than that of M1, indicating that it may be the monoglucuronidation product of M1. In

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MS2 spectrum, a [M+Na]+ fragment peak at m/z 327.0856 corresponding to M1 generated by eliminating a glucuronic acid (C6H8O6, 176Da) from mother ion backed the identification

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(Fig 6b).

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3.3 Distribution of metabolites in rat tissues and content comparison of M0 and M2 in plasma

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As shown in Table 1, M0, M1, M2, M4 and M6 were observed in plasma; M0, M1, M2, M3, M4, M5, M6 and M7 were detected in bile; M0, M1, M2, M4, M5, M6 and M7 were

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detected in urine; and no metabolites were observed in feces, indicating that these metabolites were excreted through urine and bile. Parent compound (M0), M1 and M2 were detected in most tissues, but M3, M4, M5, M6 and M7 were not observed in any tissue. Our previous pharmacokinetic study showed that the peak level of curculigoside reached 208 ng/mL at 14-20 min after oral administration in rats at dose of 200mg/kg, and it then underwent elimination within 10 h, then maintained a very low plasma concentration for 48 h Interestingly [15], curculigoside still retained antiosteoporotic effect. Why and how could this

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ACCEPTED MANUSCRIPT happen? By comparing the extract ion chromatographic peak area of curculigoside's metabolites in plasma, we found that the peak area of M2 was obviously higher than that of others in plasma. So the content of M0 and M2 were determined in plasma. As shown in Fig 7, the concentrations of M2 were much higher than that of M0 in plasma, indicating that M2 may contribute to the same biological activity as curculigoside.

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3.4 Antiosteoporotic activity of M0 and M2 in MC3T3-E1 cells

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Curculigoside possess definitely antiosteoporotic activity in animal experiment [7,8], but

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has poor oral bioavailability [15]. The content of M2 was much higher than curculigoside in plasma. Therefore, we investigated the effects of M0 and M2 on osteoblastic MC3T3-E1 cells.

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Results were showed in Fig 8 that M2, just like curculigoside, increased proliferation and alkaline phosphatase activities of osteoblastic MC3T3-E1 damaged with H2O2, indicating that

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M2, metabolite of curculigoside, also contribute to antiosteoporotic effects. Accordingly, we propose that the main active forms of curculigoside after oral administration are curculigoside

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and its metabolite M2. Both curculigoside and its metabolite M2 have antiosteoporotic

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effects.

Curculigoside is a benzylbenzoate glucoside, and shares many common chemical

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features with salicylate-type compounds, such as aspirin (acetylsalicylic acid), the most commonly used salicylate. Both curculigoside and aspirin have been reported to posses

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antiosteoporotic effects by increasing bone formation and inhibiting osteoclastic bone resorption, and have a very low bioavalability [16]. Curculigoside in rats were converted into 7 metabolites through hydrolysis, demethylation and glucuronidation, and the major metabolic product is benzoic acid derivatives 2, 6-dihydroxy benzoic acid. Aspirin is metabolized into salysilic acid, and the fall in aspirin concentration is associated with a rapid rise in salicylic acid concentration [17]. Metabolism of salicylic acid occurs through glucuronide formation to produce salicyl acyt glucuronide and salicyl phenolic glucuronide,

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ACCEPTED MANUSCRIPT conjugation with glycine to produce salicyluric acid, and oxidation to gentisic acid [17]. 2, 6-dihydroxy benzoic acid, which is the major metabolic products of curculigoside, is similar with salysilic acid in chemical structure, and has been shown to have antiosteoporotic activity in osteoblast. These findings could explaine the antiosteoporotic activity of curculigoside and salicylates to some extent at very low bioavalability. In addition, the absorption of aspirin

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follows first-order kinetics with an absorption half-life ranging from 5 to 16 minutes;

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salicylates are characterized by an extensive intestinal re-absorption to produce "second-peak"

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in PK. The difference of curculigoside with aspirin lie in the Tmax and T1/2 of curculigoside was about 14-20 min and 13-33h after oral administration in rats, and intestinal re-absorption

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is not observed [15, 17].

Curculigoside belongs to benzylbenzoate phenolic glycosides, and its metabolite, just as

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that of salicylates, is simple phenolic acid with aromatic ring structure. The compounds of benzylbenzoate phenolic glycosides widely existed in plant of genus Curculigo, including

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curculigoside and curculigoside B, piloside A and piloside B [1], and also found in the aerial

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parts of Solidago virga-aurea var. gigantea M, including 2-methoxybenzyl-2-hydroxybenzoate, benzyl-2-hydroxy-6-methoxybenzoate,

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2-methoxybenzyl-2,6-dimethoxybenzoate, 2-methoxybenzyl-2-methoxy-6-hydroxybenzoate, and benzyl-2,6-dimethoxybenzoate [18]. These benzylbenzoate phenolic glycosides have

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similar metabolic products and biological activity. The 2, 6-dihydroxy benzoic acid and salicylic acid, which respectively is the metabolites of curculigoside and salicylates, are similar with simple phenolic acid in chemical structure, such as benzoic acids, gallic acid, protocatechuic acid, ellagic acid, syringic acid, salicylic acid, ferulic acid, caffeic acid, p-coumaric acid, sinapic acid, cinnamic acid, chlorogenic acid, eugenol, and rosmarinic acid [19]. These phenolic acids are widely found in daily foods such as fruits, vegetables, cereals, legumes and wine, and provide biological, medicinal, and health properties [20]. Therefore,

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ACCEPTED MANUSCRIPT our investigation also helps us to understand the absorption, metabolism and excretion of simple phenolic acid existed in daily foods. 4. Conclusion In the present study, a HPLC-Q-TOF MS approach was employed to screen and identify the metabolites of curculigoside in rat plasma, bile, urine and selected tissues. Seven

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metabolites were elucidated and identified according to MS data. The phase I metabolites

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were mainly transformed by hydrolysis, and the phase II metabolites were mainly formed by

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demethylation and glucuronidation. The higher concentration of M2 than parent drug in plasma and protection effect of M2 on osteoblastic MC3T3 cells suggests that M2 may be an

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active metabolite. These results lead to a better understanding of the bio-transformations of

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curculigoside.

Acknowledgement

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This study was supported by the National Natural Science Foundation of China (Grant

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No. 81274152) and Shanghai Municipal Committee of Science and Technology (Grant

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ACCEPTED MANUSCRIPT Figure captions:

Fig. 1 Extracted ion current (EIC) chromatogram of curculigoside and its metabolites in rats Fig. 2 Mass spectra produced from peaks of curculigoside and its metabolites.

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Fig. 3 The proposed metabolic pathway of curculigoside in rats

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Fig. 4 Mass spectrums of metabolites M0 (a), M4 (b) and M5 (c) obtained on Q-TOF mass

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spectrometry at high collision energy and the proposed fragmentation pathways Fig. 5 Mass spectrums of metabolites M2 (a) and M7 (b) obtained on Q-TOF mass

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spectrometry at high collision energy and the proposed fragmentation pathways

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Fig. 6 Mass spectrum of metabolites M1 (a) and M6 (b) obtained on Q-TOF mass spectrometry at high collision energy and the proposed fragmentation pathways

D

Fig.7 Comparison of content between curculigoside (M0) and dominating metabolites M2 in

PT E

plasma.

Fig. 8 Effects of curculigoside (M0) and dominating metabolite M2 on osteoblastic

P<0.05, compared with control, *P<0.05, compared with H2O2 treatment group.

AC

#

CE

MC3T3-E1 cells.

22

ACCEPTED MANUSCRIPT Table 1. List of curculigoside and its metabolites detected in rats tissues. Observed Molecular No.

RT

mass(m/z

formula

Distribution in

Error（

Molecular Fragments

ion

ppm)

A

) M0

C22H26O11

10.268 （M+Na)+ 489.14

205.0468

B C D E F G H I J K L M N O P QRST

-0.29

+

+

+ -

- +

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

-0.63

+

+

+ -

- +

+ + + + - - + + - + - + ++

165.0546

-1.52

+

+

+ -

- +

/

-1.65

-

+

- -

-3.02

+

-1.78

-

287.0922 M1

C16H16O6

10.583

（M+H)+

305.1

183.0648

（M+H)+/ M2

C9H10O4

8.709

（M+Na)+

183.0649 /205.047

C13H18O8

1.982

（M+Na)+ 325.09

M4

C28H34O17

7.725

（M+Na)+ 665.17

489.1370

7.375

M6

C22H24O12

10.581 （M+Na)+ 503.12

475.1216 327.0856 205.0474

-0.8

+

+

+ -

- -

- - - - - - - - - -

- - - -

C15H18O10

8.094

（M+Na)+ 381.08

205.0469

-1.27

-

+

+ -

- -

- - - - - - - - - -

- - - -

+ -

- -

- - - -

+

+ -

- -

- - - - - - - - - -

- - - -

AC

CE

PT E

D

MA

M7

- - - -

+

NU

（M+Na)+ 651.15

C27H32O17

- - - - - - - - - - - - - - - - - - -

205.0467 M5

- -

SC

M3

+ + + + - - + + + + - + ++

RI

0

PT

165.0542

A: plasma, B: bile, C: urine, D: excrement, E: hypophysis, F: brain, G: testis, H: bone marrow, I: ovary, J: spleen, K: lungs, L: kidney, M: paranephros, N: liver, O: heart, P: thymus, Q: hypothalamus, R: uterus, S: stomach, T: small intestine

23

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Graphical abstract

24