Effects of sulfur-fumigation on the pharmacokinetics, metabolites and analgesic activity of Radix Paeoniae Alba

Journal of Ethnopharmacology 212 (2018) 95–105

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Effects of sulfur-fumigation on the pharmacokinetics, metabolites and analgesic activity of Radix Paeoniae Alba

MARK

Ming Konga,1, Huan-Huan Liua,1, Jie Wub, Ming-Qin Shenc, Zhi-Gang Wangc, Su-Min Duana, ⁎ ⁎⁎ Yan-Bo Zhangd, He Zhub, , Song-Lin Lia,b, a Department of Pharmaceutical Analysis, Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210028, PR China b Department of Metabolomics, Jiangsu Province Academy of Traditional Chinese Medicine and Jiangsu Branch of China Academy of Chinese Medical Sciences, Nanjing 210028, PR China c Department of Pharmacology, Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210028, PR China d School of Chinese Medicine, The University of Hong Kong, Hong Kong Special Administrative Region

A R T I C L E I N F O

A B S T R A C T

Compounds: Oxypaeoniflorin (PubChem CID: 21631105) Paeoniflorin (PubChem CID: 425990) Albiflorin (PubChem CID: 51346141) Gardenoside (PubChem CID: 24721095)

Ethnopharmacological relevance: Radix Paeoniae Alba (Baishao, BS), one of the most commonly used traditional Chinese medicinal herbs, has many pharmacological effects including analgesic activity. Previous studies found that sulfur-fumigation, a post-harvest handling process developed to prevent mold contamination of medicinal herbs, altered the quality of BS. However, whether sulfur-fumigation affects the pharmacokinetics, safety and efficacy of BS warrants further investigation. Aim of the study: To evaluate the feasibility of sulfur-fumigation as a post-harvest handling process of BS from the viewpoints of pharmacokinetics, safety and efficacy. Materials and methods: The pharmacokinetic behaviors of four active components of BS and one characteristic component of sulfur-fumigated BS (S-BS) were evaluated by high performance liquid chromatography triple quadrupole mass spectrometry (HPLC-TQ-MS/MS). The safety was investigated using ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS/MS) based metabolomics approach after intragastric (i.g.) administration of non-fumigated BS (N-BS) and S-BS in rats. The analgesic efficacy was compared using hot-plate test in mice, after i.g. administration of N-BS and S-BS, at both high and low dosages. Results: Systemic exposures of paeoniflorin and oxypaeoniflorin, two analgesic components of BS, were significantly decreased in the S-BS treated group compared to the N-BS treated group, while paeoniflorin sulfonate, one of the sulfur-containing derivatives of S-BS, was detected in all time-points of S-BS treated group with the area under the plasma concentration–time curve (AUC0−t) and the maximum plasma concentration (Cmax) as high as 7077.06 ± 2232.97 ng/mL*h and 1641.42 ± 634.79 ng/mL respectively, which indicated that sulfurfumigation altered the pharmacokinetic behaviors of BS. Besides, paeoniflorin sulfonate and its four metabolites with ambiguous toxicities, as well as one endogenous metabolite p-cresol glucuronide, the biomarker of disordered homeostasis of intestinal bacteria and bile acid, were identified as the characteristic metabolites in S-BS administered rats, suggesting that sulfur-fumigation reduced the safety of BS. Furthermore, the analgesic effects at both low and high dosages were decreased in different extent when compared to N-BS administered groups, indicating that sulfur-fumigation weakened the efficacy of BS.

Keywords: Radix Paeoniae Alba Sulfur-fumigation Pharmacokinetics Metabolites Analgesic P-cresol glucuronide

Abbreviations: AUC, area under the plasma concentration–time curve; BS, Radix Paeoniae Alba; CID, collision-induced dissociation; CL, clearance; Cmax, maximum plasma concentration; FDA, US Food and Drug Administration; HPLC/UV, high performance liquid chromatography/ultra violet detector; HPLC-TQ-MS/MS, high performance liquid chromatography triple quadrupole mass spectrometry; i.g., intragastric; IS, internal standard; MRM, multiple reaction monitoring; N-BS, Non-fumigated Radix Paeoniae Alba; OPLS-DA, orthogonal partial least squared discrimination analysis; PCA, Principal Component Analysis; S-BS, Sulfur-fumigated Radix Paeoniae Alba; SULT, sulphotransferase; t1/2, terminal elimination halflife; TCM, traditional Chinese medicine; Tmax, maximum concentration time; UHPLC-QTOF-MS/MS, ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry ⁎ Corresponding author. ⁎⁎ Corresponding author at: Department of Pharmaceutical Analysis, Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210028, PR China. E-mail addresses: [email protected] (H. Zhu), [email protected] (S.-L. Li). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jep.2017.10.023 Received 3 May 2017; Received in revised form 22 September 2017; Accepted 23 October 2017 0378-8741/ © 2017 Elsevier B.V. All rights reserved.

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Conclusion: Sulfur-fumigation altered the pharmacokinetics, as well as reduced the safety and efficacy of BS, suggesting that sulfur-fumigation is not recommended for post-harvest handling of BS.

1. Introduction

pharmacokinetics, safety and efficacy of BS were investigated. The findings from this research would provide important data being complementary with those from quality-based studies to comprehensively evaluate the feasibility of sulfur-fumigation as a post-harvest handling process of BS.

Sulfur-fumigation has been employed in the near decades to prevent against insects and moulds, and to shorten the drying duration during post-harvest handling process of medicinal herbs (Li et al., 2017). Accumulated studies have indicated that this processing method has impaired the quality of medicinal herbs by reducing the contents of active components (Kong et al., 2014, 2017) and generating new sulfur-containing derivatives of questionable safety (Duan et al., 2016; Pei et al., 2016). To date, the feasibility of using sulfur-fumigation for post-harvest handling of herb is still debatable (Jiang et al., 2013; Kan et al., 2011) and more evidence, beyond quality evaluation, is needed. Safety and efficacy, two essential aspects of medicinal herbs, are a reflection of the toxicities and activities of any xenobiotics, while the intensities of any toxic effects are related to their concentrations in a bodily fluid (Chaudhari et al., 2016; Shi et al., 2016). Besides, pharmacokinetics, which describes the time course of xenobiotic concentration in a body fluid, is also an important connection between quality and safety/efficacy of medicinal herbs (Ma et al., 2017; Zhu et al., 2011). Thus, feasibility evaluations of sulfur-fumigation in the processing of medicinal herbs from the viewpoints of pharmacokinetics, safety and efficacy should be performed in addition to quality-based investigation. Routine methods for safety evaluation of xenobiotics are commonly time- and money-consuming (Chaudhari et al., 2016). Moreover, characterized by multi-components against multi-targets, medicinal herbs induced toxicities are much more complex than chemical drugs. So it is a challenge to find an approach to evaluate the safety of medicinal herbs in a convenient and economical manner. Fortunately, a metabolomics approach, which could accurately and quickly provide major insights into the similarities and differences of samples by quantitatively and qualitatively measuring the dynamic change of small molecules, is a powerful tool to identify differential components (Mao et al., 2014), and provides a feasible approach to evaluate the safety of medicinal herbs through exploring characteristic markers, in particular toxic markers (Wang et al., 2017; Zhou et al., 2016). Radix Paeoniae Alba (Baishao in Chinese, BS), derived from the dried root of Paeonia lactiflora Pall., is one of the most commonly used medicinal herbs to treat blood deficiency, sallow complexion, menstrual irregularities, spontaneous sweating, night sweating, hypochondriac pain, abdominal pain, spasm and pain of limbs, headache and dizziness (Pharmacopoeia Commission of the People's Republic of China, 2015). In traditional Chinese medicine practice, BS is always used together with other herbs to constitute complex prescription, some of which showed analgesic efficacy in clinic application (Tong et al., 2010). Previous study found that sulfur-fumigation was frequently used in the post-harvest handling of BS, with up to 59% commercial BS samples being sulfur-fumigated (Kong et al., 2014). Besides, sulfur-fumigation could decrease the contents of bioactive compounds in BS and transform monoterpene glycosides (e.g. paeoniflorin), the major components of BS, into their sulfur-containing derivatives (e.g. paeoniflorin sulfonate) (Kong et al., 2014; Li et al., 2009). Recently, the influence of sulfur-fumigation on the pharmacokinetic behaviors of certain chemicals in BS was investigated after intragastric (i.g.) administration of alcohol extracts of BS (Cheng et al., 2010). However, water extraction (decoction), not alcohol extraction, is the main prescription form of traditional Chinese medicine (TCM), so it is much more meaningful to investigate the influence of sulfur-fumigation on pharmacokinetics behaviors of BS water extracts. In the present study, the influences of sulfur-fumigation on the

2. Materials and methods 2.1. Reagents and animals Reference compounds of oxypaeoniflorin and paeoniflorin were purchased from Shanghai Sunny Biotech Co., Ltd. and Chengdu Preferred Biological Technology Co., Ltd, respectively. Paeoniflorin sulfonate and gardenoside (internal standard, IS) were provided by Shanghai U-sea Biotech Co., Ltd and the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Albiflorin and benzoypaeoniflorin were isolated from BS in our lab and confirmed by MS and NMR analysis. The purities of all compounds were determined to be higher than 95% by high performance liquid chromatography/ultra violet detector (HPLC/UV) analysis. Acetonitrile and methanol (HPLC grade) were purchased from Merck (Darmstadt, Germany), and formic acid (HPLC grade) was purchased from ROE (USA). Deionized water was prepared in-house with Millipore (Millipore, MA, USA). Male Sprague-Dawley rats (220–250 g) and male Kunming mice (18–22 g) were supplied by Shanghai Slac Laboratory Animal Co., Ltd., China. All animals were housed under controlled temperature 25 ± 1 °C, relative humidity 40 − 70%. The animal care and use were in accordance with the Regulations of Experimental Animal Administration issued by the Ministry of Science and Technology of China, and the experimental protocols were approved by the Animal Affairs Committee of Jiangsu Province Academy of Traditional Chinese Medicine and Jiangsu Branch of China Academy of Chinese Medical Sciences. 2.2. Preparation of N-BS and S-BS extracts Fresh Radix Paeoniae Alba samples (Batch No. 20141010) was collected from Bozhou (Anhui Province, China), one of its indigenous cultivating regions in China. All samples were authenticated by Prof. Song-Lin Li to be the root of Paeonia lactiflora based on the morphological and histological features according to the monograph of BS documented in Chinese Pharmacopoeia (Pharmacopoeia Commission of the People's Republic of China, 2015). Non-fumigated BS sample (N-BS, JSPACM-1-K-31) was prepared according to Chinese Pharmacopoeia (Pharmacopoeia Commission of the People's Republic of China, 2015). In brief, the fresh Radix Paeoniae Alba samples were immersed in hot water (about 90 °C) for 20 min, peeled off the skins, cut into slices with the thickness of about 0.5 cm and the diameter of about 1.5–2 cm, and then dried in oven at 40 °C to get N-BS samples. The sulfur-fumigated BS sample (S-BS, JSPACM-1-K-33) was prepared from N-BS (JSPACM-1K-31) according to our previous protocol with minor modifications which imitated the sulfur-fumigation process of BS commonly utilized by the wholesalers (Kong et al., 2014). Briefly, N-BS samples were wetted with water (10:1, w/w), put into a desiccator, sulfur-fumigated for 26 h and then dried in oven at 40 °C to get S-BS samples. The voucher specimens of the N-BS and S-BS samples were deposited in Department of Metabolomics, Jiangsu Province Academy of Traditional Chinese Medicine and Jiangsu Branch of China Academy of Chinese Medical Sciences. The contents of paeoniflorin albiflorin, gallic acid, 96

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Methods were validated for selectivity, linearity, precision, accuracy, extraction recovery, matrix effect and stabilities, according to the US Food and Drug Administration (FDA) guidance for validation of bioanalytical methods (US Food and Drug Administration, 2001).

pentagalloyglucose and paeoniflorin sulfonate were quantified according to our published method (Kong et al., 2014, 2017), and the contents of paeoniflorin, albiflorin, gallic acid and pentagalloyglucose in N-BS were 3.18%, 0.74%, 0.53% and 0.23% respectively, while the contents of paeoniflorin, albiflorin, gallic acid, pentagalloyglucose and paeoniflorin sulfonate in S-BS were 1.88%, 0.71%, 0.49%, 0.20% and 1.68%, receptively. The water extracts of N-BS and S-BS were prepared as follows: N-BS or S-BS sample was immersed in water (8:1, v/w) for 30 min and extracted with boiled water for 1 h twice. The filtrates were combined and concentrated to 1.18 mL and 1.04 mL per gram of N-BS and S-BS respectively, and then stored at 4 °C before use.

2.3.4. Data analysis Pharmacokinetic parameters were determined using the Drug and Statistic (DAS) software (version 2.0, Chinese Pharmacological Society). Parameters, including terminal elimination half-life (t1/2), maximum concentration time (Tmax), maximum plasma concentration (Cmax), area under the plasma concentration–time curve (AUC0−t) and plasma clearance (CL) were determined. All the parameters were described as mean ± standard (SD) deviation, and statistical comparisons were evaluated by ANOVA test using the SPSS 16 software. Results were considered significant at p < 0.05.

2.3. Pharmacokinetics study 2.3.1. Sample collection Twelve rats were randomly divided into two groups, and were i.g. administered with N-BS and S-BS at the dosage of 13.3 g crude herbs per kilogram rat body weight (13.3 g/kg) respectively. The surgical operation of cannulation on jugular vein was operated according to our previously established model in Sprague-Dawley rats (Ma et al., 2017). Briefly, all the rats were anesthetized with an intraperitoneal injection of a mixed solution (2.0 mL/kg) containing 37.5 mg/mL ketamine and 5 mg/mL xylazine. The right jugular vein was cannulated with a polyethylene tube for blood collection. During the surgery, the body temperature was maintained at 37 °C by placing the rat on a heating pad alongside with a heating lamp. The rats were allowed to recover and fasted overnight with free access to water. After dosing, blood samples (~0.25 mL) were collected at 0, 0.08, 0.17, 0.33, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8 and 12 h, and 0.25 mL of saline containing 25 units heparin/mL was given back to rats after each collection to compensate the blood loss. The blood samples were then centrifuged at 6000 rpm for 10 min in 4 °C, and plasma was separated and stored at −80 °C for further analysis.

2.4. Metabolomics based safety evaluation 2.4.1. Sample collections Thirty-six rats were randomly divided into six groups and fasted for 12 h but allowed access to water before experiment. The collected samples were stored at −80 °C until analysis after the experiment. For bile sampling, two groups of rats were anesthetized by intraperitoneal administration of chloral hydrate (10%, w/v, 3.3 mL/kg) and inserted by cannula through cystic ducts. A heating lamp was used to maintain the rats’ body temperatures during the procedure to prevent alterations of the bile flow owing to hypothermia. Drug-containing bile samples were collected for 24 h after i.g. administration of N-BS extract and S-BS extract (13.3 g/kg) respectively. For blood sampling, two groups of rats were anesthetized by intraperitoneal administration of chloral hydrate (10%, w/v, 3.3 mL/kg) and cannulated to right jugular vein using polyethylene tubes before administration. During the experiment, the body temperature was maintained at 37 °C by placing the rat on a heating pad alongside with a heating lamp. Blood samples (~0.2 mL) were collected at 0.5, 1, 2, 3, 4, 5 and 6 h, and 0.25 mL after i.g. administration of N-BS extract and SBS extract (13.3 g/kg). Saline containing 25 units heparin/mL was given back to rats after each collection to compensate the blood loss. The samples were then centrifuged at 4000 rpm for 10 min at 4 °C, and plasma was separated. For feces and urine sampling, two groups of rats were housed in metabolic cages, and urine as well as feces samples were collected during the time period 0–24 h after i.g. administration of N-BS and S-BS extracts (13.3 g/kg). Urine samples were then centrifuged at 4000 rpm for 10 min at 4 °C and feces samples were dried in shade.

2.3.2. Sample preparation An aliquot of 50 μL plasma sample was precipitated by 200 μL methanol solution, mixed for 3 min and centrifuged for 10 min at 15,000 rpm in 4 °C after adding 10 μL of the IS (218 ng/mL). Subsequently, the supernatant was transferred into a fresh tube and evaporated to dryness at 35 °C under a nitrogen gas. The residue was redissolved by 50 μL of 50% acetonitrile and centrifuged as described above, and then an 8 μL aliquot of the supernatant was injected into the high performance liquid chromatography triple quadrupole mass spectrometer (HPLC-TQ-MS/MS) system for analysis. 2.3.3. HPLC-TQ-MS/MS determination Simultaneous determination of oxypaeoniforin, albiflorin, paeoniflorin, benzoypaeoniflorin and paeoniflorin sulfonate was performed on a Waters Alliance HPLC 2695 system combined Micromass QuattroMicro™ triple-quadrupole mass spectrometer (Waters Corp., Milford, MA, USA). Analytes were separated on a Poroshell 120 EC-C18 column (3.0 mm×100 mm, 2.7 µm, Agilent, CA, USA) maintained at 35 °C. The mobile phase was consisted of (A) 0.1% formic acid aqueous solution and (B) acetonitrile, and the gradient elution was optimized as follows: 5–18% B (0–5 min), 18% B (5–9 min), 18–90% B (9–12 min), 90% B (12–14 min), 14–15% B (14–15 min) and 5% B (15–20 min) with the flow rate at 0.3 mL/min. The auto-sampler temperature was maintained at 8 °C. The mass spectrometer was operated in negative mode using multiple reaction monitoring (MRM) for the analysis. The mass transition of the precursor/product ions for monitoring and cone voltages of each analyte were shown in Table 1. The detection parameters were optimized as follows: capillary voltage 3.50 kV; source temperature 120 °C; desolvation temperature 400 °C; desolvation gas 350 L/h; cone gas 35 L/h.

2.4.2. Sample preparations The plasma, bile and urine samples were prepared as follows: plasma samples of each rat in different time points were combined Table 1 HPLC-TQ-MS conditions used for the quantification of the analytes.

97

No.

Analyte

tR(min)

Channel Reaction

1

Paeoniflorin sulfonate

7.67

2

Oxypaeoniforin

8.16

3

Gardenoside (IS)

9.63

4

Albiflorin

10.24

5

Paeoniflorin

10.92

6

Benzoypaeoniflorin

15.15

543.0 259.2 495.1 137.0 433.2 225.1 479.0 121.0 449.1 327.1 553.1 431.1

Cone voltage (v)

Collision voltage (ev)

→

55.0

40.0

→

45.0

30.0

→

25.0

15.0

→

40.0

20.0

→

35.0

13.0

→

40.0

13.0

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respectively. An aliquot of 200 μL plasma sample was precipitated by 1 mL methanol, vortexed for 3 min and centrifuged at 15,000 rpm for 10 min. An aliquot of 1 mL urine sample or bile sample was vortex mixed for 3 min and then centrifuged at 15,000 rpm for 10 min after adding 4 mL of methanol. Subsequently, the supernatant of plasma,

urine or bile was separated and evaporated to dryness at 35 °C under a nitrogen gas. The residue of the plasma sample was re-dissolved with 200 μL of 70% methanol and that of the bile or urine sample was redissolved with 1 mL of 70% methanol. All the mixtures centrifuged as described above. Finally, a 4 μL aliquot of supernatant was injected for

Fig. 1. Mass spectra of analytes. (A) parent ions mass scan; (B) daughter ion mass scan. (1) Paeoniflorin sulfonate; (2) oxypaeoniforin; (3) gardenoside (IS); (4) albiflorin; (5) paeoniflorin; (6) benzoypaeoniflorin.

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the test on the basis of their reactivity, and finally fifty of them with response latencies of 20–28 s were included and subsequently divided into five groups for analgesic evaluation. Blank group (BLK) animals received normal saline, N-BS low dosage group (N-BS-L) and N-BS high dosage group (N-BS-H) animals received N-BS at a dosages of 11.4 g/kg and 45.4 g/kg, while S-BS low dosage group (S-BS-L) and S-BS high dosage group (S-BS-H) mice received S-BS at a dosages of 11.4 g/kg and 45.4 g/kg, receptively. After i.g. administration for three consecutive days, the response latency times of all mice were re-measured half-anhour after the last administration. A cut-off time of 60 s was used to avoid paw lesions.

ultra high performance liquid chromatography quadrupole time-offlight mass spectrometry (UHPLC-QTOF-MS/MS) analysis. The feces samples were prepared as follows: A certain amount of feces powder (1 g) was ultrasonic-extracted with 10 mL of 70% methanol for 45 min (40 kHz, 100 W), then the extracted solution was centrifuged at 15,000 rpm for 10 min. Finally, a 2 μL aliquot of supernatant was injected for UHPLC-QTOF-MS/MS analysis. 2.4.3. UHPLC-QTOF-MS/MS based metabolomics method Plasma, bile, urine and feces samples were analyzed using a Waters Acquity UHPLC system combined Synapt G2S Q‐TOF (Waters Corp., Milford, MA, USA). The separation was performed on a Waters Acquity HSS T3 column (2.1 mm × 100 mm, 1.8 µm, Waters, Milford, MA, USA). The mobile phase was consisted by (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid, and the UHPLC eluting conditions were optimized as follows: 5% B (0–1 min), 5–20% B (1–5 min), 20–30% B (5–7 min), 30–50% B (7–9 min), 50% B (9–10 min), 50–5% B (10–10.5 min), 5% B (10.5–12 min). The flow rate was 0.4 mL/min. The column and auto-sampler were maintained at 35 °C and 8 °C, respectively. The ESI source was set in negative ionization mode with m/z 50–1500, the nebulization gas was set at 1000 L/h at a temperature of 450 °C, and the cone gas was set at 40 L/h. The capillary voltage and cone voltage were set at 2500 V and 30 V, respectively. Data were collected in centroid mode, and the MSE approach was used. The QTOFMS/MS acquisition rate was 0.2 s. The energies for collision-induced dissociation (CID) were 4 V for the precursor ion and 10–50 V for fragmentation information. All MS data were acquired using the LockSprayTM to ensure mass accuracy and reproducibility. The fragmentation pathways of each component were deduced with the help of MetaboLynx software.

3. Results 3.1. Pharmacokinetics study 3.1.1. HPLC-TQ-MS/MS method optimization The mass spectrometric parameters such as collision energy, cone voltage and capillary voltage were optimized to attain the maximum sensitivity for the detection of each analyte. The precursor ions and product ions of paeoniflorin sulfonate, oxypaeoniflorin, albiflorin, paeoniflorin, benzoylpaeoniflorin and IS were clearly observed in MS and MS/MS spectra after infusing individual standard solutions into MS. The precursor ions and product ions with representative extract ions MRM chromatograms of each analyte were summarized in Table 1 and Fig. 1. No interfering endogenous peaks were observed around the retention times of detected analytes. 3.1.2. HPLC-TQ-MS/MS method validation HPLC-TQ-MS/MS method validation was conducted during the experiment. Good calibration coefficients were obtained for five analytes within the test ranges. The overall intra-day and inter-day variations (RSDs) of the analytes were less than 15%. Extraction recoveries of all analytes were in the range of 75.13–100.21%. The matrix effect of blank plasma was found to be within the acceptable range, all values were from 72.03% to 90.61%. For the stability tests, the RSDs of the peak areas for all analytes detected were lower than 15% within different conditions. All of these results demonstrated that the established method was linear, sensitive, precise, accurate, and stable enough for simultaneous quantification of paeoniflorin sulfonate, oxypaeoniflorin,

2.5. Analgesic activity study The hot-plate test method was used to evaluate the analgesic activity of BS according to published paper (Alonso-Castro et al., 2016) with minor modifications. The temperature of the metal surface was set at 55 ± 0.5 °C. The time(s) elapsed between placement until the occurrence of discomfort reactions (licking paws or jumping) was recorded as the response latency time. Ninety mice were selected prior to

Fig. 2. Plasma concentration-time profiles in rats after i.g. administration of N-BS and S-BS. (A) paeoniflorin sulfonate; (B) oxypaeoniflorin; (C) albiflorin; (D) paeoniflorin.

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mobile phase system was superior in elution resolution and efficiency than methanol/water and acetonitrile/water systems. Negative ion mode provided higher sensitivity, cleaner mass spectra and lower background. Thus, in this UHPLC-QTOF-MS/MS method, acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid mobile phase system with negative ion mode were selected.

albiflorin, paeoniflorin, benzoylpaeoniflorin in rat plasma. 3.1.3. Comparison of pharmacokinetics after i.g. administration of N-BS and S-BS decoctions The validated HPLC-TQ-MS/MS method was utilized to compare the pharmacokinetics behaviors of oxypaeoniforin, albiflorin, paeoniflorin, benzoypaeoniflorin and paeoniflorin sulfonate after i.g. administration of N-BS and S-BS decoctions at 13.3 g/kg. The mean plasma concentration-time profiles and pharmacokinetics parameters of analytes are illustrated in Fig. 2 and Table 2. After administration, typical pharmacokinetics profiles of orally administered xenobiotics were observed. AUC of paeoniflorin and oxypaeoniflorin after administration of S-BS was significantly decreased from 5575.85 ± 690.30 ng/mL*h to 3131.87 ± 642.63 ng/mL*h and 218.31 ± 37.51 ng/mL*h to 134.81 ± 43.52 ng/mL*h, respectively, compared with that of N-BS. Similarly, Cmax of paeoniflorin and oxypaeoniflorin of N-BS was higher than that of S-BS. On the contrary, t1/2 of paeoniflorin and oxypaeoniflorin after administration of S-BS water extract was obviously longer than that in N-BS, suggesting that the clearance had been altered. Besides, the pharmacokinetic behaviors of albiflorin were not altered between the two groups. Furthermore, benzoylpaeoniflorin was not detectable in the plasma after i.g. administered to the rats, so its pharmacokinetic parameters were not calculated. Paeoniflorin sulfonate, the sulfur-containing derivative of paeoniflorin, was selected as the target analyte to evaluate the systemic exposure. The pharmacokinetic behavior of paeoniflorin sulfonate was evaluated only in the S-BS administered group since it was not detected in N-BS administrated group. The plasma concentration of paeoniflorin sulfonate reached Cmax (1641.42 ± 634.79 ng/mL) at the time of 1.25 ± 0.42 h with the elimination t1/2 3.76 ± 1.11 h. In addition, the AUC0−t and AUC0-∞ of paeoniflorin sulfonate were 7077.06 ± 2232.97 ng/mL*h and 7883.69 ± 2303.39 ng/mL*h, respectively.

3.2.2. Metabolomics analysis To evaluate the influence of sulfur-fumigation on the safety of BS, the obtained UHPLC-QTOF-MS/MS data, including m/z, tR and ion intensity, were subjected to Principal Component Analysis (PCA). After Pareto scaling with mean-centering, the plots of PCA were presented in Fig. 3, which exhibited that the plasma, feces, urine and bile samples from rats administered with N-BS and S-BS were all clearly clustered into two groups. The diagram intuitively revealed that sulfur-fumigation conspicuously affected the metabolic profiles of BS. In order to rapidly identify the potential characteristic markers of SBS, the UHPLC-QTOF-MS/MS data were further processed by supervised orthogonal partial least squared discrimination analysis (OPLS-DA). In the obtained S-plot (Fig. 4), each point represented an ion tR-m/z pair. The points at the left ends of “S” that most contributed to the observed separation were selected as the potential markers for the S-BS groups, while the other side represented the N-BS group. Then the structure of the potential markers could be identified according to the retention time, precise molecular mass and fragment ions referred to our published papers (Li et al., 2009; Mao et al., 2014). For example, four ions, a2 (tR 3.55 min, m/z 543.1202), b2 (tR 3.58 min, m/z 543.1175), c2 (tR 5.51 min, m/z 283.0821) and d2 (tR 4.54 min, m/z 405.1761) were observed and regarded as the related characteristic ions in bile of rats administered with S-BS. From the ion intensity trend plots, these ions exhibited high intensities in bile samples of S-BS but were undetectable in those of N-BS, which indicated that components associated with these ions could be used as potential characteristic markers. Analogously, a1 (tR 3.55 min, m/z 543.1174), a3 (tR 3.55 min, m/z 543.1179), b3 (tR 4.98 min, m/z 381.0661), c3 (tR 3.64 min, m/z 259.0277), a4 (tR 3.39 min, m/z 705.1682), b4 (tR 3.55 min, m/z 543.1168), c4 (tR 3.58 min, m/z 543.1180), d4 (tR 4.94 min, m/z 687.1543) and e4 (tR 4.98 min, m/z 381.0661) were selected as potential characteristic markers in plasma, urine, feces samples after administered with S-BS, respectively. By comparing retention times and characteristic fragment ions with standard substances, a1, a2, a3 and b4 were identified as paeoniflorin sulfonate accurately, while b2 and c4, b3 and e4, and c3 were tentatively identified as paeoniflorin sulfonate isomer, lose one glucose metabolite of paeoniflorin sulfonate and lose one glucose and one benzoic acid metabolite of paeoniflorin sulfonate. Besides, c2 was tentatively verified as p-cresol glucuronide (Li et al., 2011). The detail information of identified components and their MS/ MS fragmentations were listed in Table 3.

3.2. Metabolomics based safety evaluation 3.2.1. UHPLC-QTOF-MS/MS method optimization for metabolomics study Metabolomics studies on plasma, bile, urine and feces samples of rats after i.g. administration of N-BS and S-BS extracts were comparatively analyzed using UHPLC-QTOF-MS/MS method. Considering the chemical complexity of the investigated biological matrixes (plasma, bile, urine and feces) which might contain original compositions, phase II metabolites and sulfur-containing derivatives of BS as well as endogenous components, the effects of chromatographic conditions including column, mobile phases and modifiers were evaluated. It was found that ACQUITY HSS T3 column, which was designed for the elution of more hydrophilic compounds, was much more suitable than other columns, such as ACQUITY BEH C18, for the separation of components in N-BS or S-BS. Therefore, ACQUITY HSS T3 column was chosen in this study. Besides, acetonitrile/water with formic acid Table 2 Pharmacokinetic parameters after gavage of S-BS and N-BS decoction (mean ± SD, n=6). Parameter

AUC(0−t) (ng/mL*h) AUC(0-∞) (ng/mL*h) MRT(0−t) (h) t1/2z (h) Tmax (h) CLz/F (L/h/kg) Cmax (ng/mL)

Paeoniflorin

Paeoniflorin sulfonate

Oxypaeoniflorin

Albiflorin

N-BS

S-BS

N- BS

S-BS

N-BS

S-BS

N-BS

S-BS

5400.29 ± 631.47 5575.85 ± 690.30 3.40 ± 0.13 2.25 ± 0.69 0.75 ± 0.16 2.42 ± 0.28 1869.57 ± 194.35

2884.00 ± 621.32** 3131.87 ± 642.63** 3.60 ± 0.10* 3.44 ± 0.88* 0.49 ± 0.15* 4.40 ± 0.90** 858.53 ± 75.29**

-

7077.06 ± 2232.97 7883.69 ± 2303.39 4.13 ± 0.36 3.76 ± 1.11 1.25 ± 0.42 1.83 ± 0.59 1641.42 ± 634.79

195.49 ± 24.23 218.31 ± 37.51 3.92 ± 0.32 3.55 ± 0.73 1.33 ± 0.26 65.61 ± 11.39 48.85 ± 6.53

105.13 ± 23.75** 134.81 ± 43.52* 4.09 ± 0.27 6.01 ± 3.50* 1.25 ± 0.27 63.19 ± 20.38 31.47 ± 13.29*

2364.35 ± 364.16 2457.34 ± 366.26 3.05 ± 0.21 2.69 ± 0.48 0.79 ± 0.19 5.53 ± 0.95 853.83 ± 141.32

2014.63 ± 372.90 2165.07 ± 291.37 3.68 ± 0.26 3.12 ± 1.04 0.63 ± 0.21 6.24 ± 0.82 700.13 ± 152.68

**: P < 0.01, *: P < 0.05; -: not detected.

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Fig. 3. PCA score plot of N-BS and S-BS administered group. (A) plasma; (B) bile; (C) urine; (D) feces.

Fig. 4. S-plot and selected ion intensity trend plot. (A) plasma; (B) bile; (C) urine; (D) feces.

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Fig. 4. (continued)

handling of BS, and many efforts have been devoted to the feasibility evaluation from quality viewpoints (Kong et al., 2014; Li et al., 2009). Nevertheless, more investigations beyond the quality evaluation are still needed. Thus, in this paper, safety and efficacy, the two most important aspects in the application of medicinal herbs, as well as pharmacokinetics were selected to evaluate the feasibility of sulfur-fumigation in the post-harvest handling process of BS. Paeoniflorin, oxypaeoniforin, albiflorin and benzoypaeoniflorin were considered to be the analgesic components of BS (Luo et al., 2014; Tong et al., 2010), while paeoniflorin sulfonate was the characteristic constituent of S-BS, so the pharmacokinetics behaviors of the five components were investigated in this paper. Compared to N-BS group,

3.3. Analgesic activity based efficacy evaluation The analgesic activities of N-BS and S-BS at two dosages assessed using the hot-plate pain test is presented in Fig. 5. N-BS demonstrated the analgesic effects in different degree, especially that at high dosage (45.4 g/kg) presented a significant (p < 0.05) analgesic activity. However, the administration of both high and low dosage of S-BS did not cause any attenuation of the pain induced by hot-plate. 4. Discussions Sulfur-fumigation is commonly practiced in the post-harvest 102

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Table 3 Characteristic absorptive and metabolic markers identified in vivo biological samples. Sample

No.

tR/min

Assigned identity

Formula

Experimental m/z [M-H]-

Error /ppm

Plasma Bile

a1 a2 b2 c2 d2 a3 b3 c3 a4 b4 c4 d4

3.55 3.55 3.58 5.51 4.54 3.55 4.98 3.64 3.39 3.55 3.58 4.98

Paeoniflorin sulfonate Paeoniflorin sulfonate Paeoniflorin sulfonate isomer P-Cresol glucuronide Unknown Paeoniflorin sulfonate Paeoniflorin sulfonate - glucose Paeoniflorin sulfonate - benzoic acid - glucose Isomaltopaeoniflorin sulfonate Paeoniflorin sulfonate Paeoniflorin sulfonate isomer Paeoniflorin sulfonate - glucose

C23H27O13S C23H27O13S C23H27O13S C13H16O7 – C23H27O13S C17H18O8S C10H12O6S C29H37O18S C23H27O13S C23H27O13S C17H17O8S

543.1174 543.1202 543.1175 283.0821 405.1761 543.1179 381.0661 259.0277 705.1682 543.1168 543.1180 381.0661

0.37 5.52 0.55 1.41 – 1.29 4.72 −0.38 −2.69 −0.74 1.47 4.72

Urine

Feces

markers, including five sulfur-containing derivatives and one endogenous metabolite p-cresol glucuronide, were identified in the S-BS group. Previous study showed that six sulfur-containing derivatives i.e. paeoniflorin sulfonate, oxypaeoniflorin sulfonate, mudanpioside E sulfonate, isomaltopaeoniflorin sulfonate, galloylpaeoniflorin sulfonate and benzoylpaeoniflorin sulfonate were generated in BS during sulfurfumigation (Li et al., 2009). In the present study, paeoniflorin sulfonate and its four metabolites with uncertain toxicities were discovered to be the characteristic markers in plasma, bile, urine and feces. As we known, sulfonate-containing metabolites, one of important phase II metabolites of xenobiotics for eliminating, could be generated with the presence of sulphotransferases (SULTs) (Zhu et al., 2015b). Given this situation, whether or not the structures of paeoniflorin sulfonate and its metabolites were similar to that of the sulfur-containing metabolites after administered N-BS is an important issue to evaluate the feasibility of sulfur-fumigation in the process of BS. So, in order to verify the origin of the sulfur-containing metabolites accurately, the metabolic profiles of paeoniflorin were further analyzed, and only one sulfur-containing metabolite was identified, which was also found in bile samples after i.g. administration of paeoniflorin. As shown in Fig. 6, the protonated ion at m/z 575.1070 gave rise to fragment ions at m/z 495.1502 and m/ z 137.0238 by the loss of SO3 and monoterpene glycosides in the MS/ MS spectrum. By comparing the data reported in the literature (Chen et al., 2014; Liang et al., 2013), this metabolite was assigned as hydroxylation and sulfation metabolite of paeoniflorin. However, the fragment ions and the fragmentation of this sulfur-containing metabolite (C23H30O15S) was entirely different from that of paeoniflorin sulfonate (C23H28O13S) produced by sulfur-fumigation of BS. The MS/MS

Fig. 5. Analgesic activities in different groups. BLK, Blank group; N-BS-L, low dosage of N-BS administered group; N-BS-H, high dosage of N-BS administered group; S-BS-L, low dosage of S-BS administered group; S-BS-H, high dosage of S-BS administered group.

the AUC values of paeoniflorin and oxypaeoniflorin were significantly decreased by 1.9- and 1.8- folds, respectively, due to the remarkable major reductions of the detected analytes in BS after sulfur-fumigation. These results were similar to those found with ginsenosides after i.g. administration of sulfur-fumigated ginseng (Ma et al., 2017). Furthermore, the AUC0−t and Cmax values of paeoniflorin sulfonate were as high as 7077.06 ± 2232.97 ng/mL*h and 1641.42 ± 634.79 ng/mL, respectively. All the results confirmed that sulfur-fumigation altered pharmacokinetic behaviors of BS. The metabolomics approach was utilized to evaluate the influence of sulfur-fumigation on the safety of BS, and seven characteristic

Fig. 6. Proposed rationalization of fragments and mass spectra. (A) sulfur-containing metabolites of paeoniflorin; (B) paeoniflorin sulfonate.

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of exposure of BS to sulfur-fumigation (Li et al., 2009; Kong et al., 2014). In the present study, we demonstrated that sulfur-fumigation which impaired the quality of BS significantly altered the pharmacokinetics fates, and consequently reduced the safety and efficacy of BS.

spectra and proposed fragmentation of this sulfur-containing compound were also listed in Fig. 6. Therefore, it is concluded that paeoniflorin sulfonate is different from the sulfur-containing metabolite of paeoniflorin generated in phase II mechanism. It is easy to understand the chemical difference between these two sulfur-containing compounds, as paeoniflorin sulfonate is generated by bisulfite addition reaction (Hayes et al., 2005), while sulfur-containing metabolite is catalyzed by SULT. P-cresol glucuronide, an endogenous biomarker that was reported to be the metabolite of gut microflora and to be correlated with bile acid metabolism (Velenosi et al., 2016; Zhang et al., 2012), was detected even after single dosage administration of S-BS, which suggested that sulfur-fumigation might change the homeostasis of intestinal bacteria and cause the turbulence of bile acid. Many diseases, including hepatotoxicity (Monte et al., 2017; Possamai et al., 2015; Zhu et al., 2015a), irritable bowel syndrome (Grayson, 2016), cancer (Thaiss et al., 2016; Yoshimoto et al., 2013) and diabetes (Cui et al., 2016; Jandhyala et al., 2017), were directly and/or indirectly relevant to the discordant homeostasis of intestinal bacteria and bile acid. All in all, sulfur-fumigation changed the metabolic profiles of BS in rats, and might decrease the safety of BS. It is hard to select a suitable animal model to evaluate the efficacy of a single herb (BS here) which is commonly used together with other herbs (complex prescription) in traditional Chinese medicine (TCM) practice. The bioactivity results obtained from any model study on one herb might be bias to the efficacy of this herb contributing to complex prescription. Paeoniflorin and albiflorin are two of the main components of BS, and paeoniflorin can be easily transformed to paeoniflorin sulfonate when BS is sulfur-fumigated. It is reported that paeoniflorin showed dose-related analgesic activity both on the early and late phases of formalin test in mice with the mechanisms related to activating kappa-opioid receptor (Tsai et al., 2001), and albiflorin exerted the analgesic activity mainly through central nervous system (Zhang et al., 2016). In the above two researches, hot-plate pain test is one of the models used to evaluate their analgesic efficacy and investigate related mechanisms. Therefore, in this study, hot-plate pain test animal model was selected to investigate the influence of sulfur-fumigation on the analgesic efficacy of BS. In China Pharmacopoeia (CP), the recommended dosage of BS is no more than 15 g per day. Referring to the ruler of equivalent dosage calculated by the surface area of the human and mouse (1: 12), the dosage of BS used in animal study should be no more than 3.0 g/kg body weight of mouse. But in our preliminary study, no significant analgesic effects were found until the dosage increased up to 45.4 g/kg. Moreover, previous study indicated that BS did not show acute toxicity even at the dosage of 80 g/kg (Huang et al., 2012). Therefore, the dosages of 11.4 g/kg (low) and 45.4 g/kg (high) were designed to investigate the influence of sulfur-fumigation on the efficacy of BS. The results showed that N-BS had significant analgesic activity at high dosage, yet S-BS had no such effects at neither high nor low dosages. It was interesting to note that high dosage of S-BS contained higher content of paeoniflorin than low dosage of N-BS, but no significant analgesic activity was found at high dosage of S-BS. In our previous study, in addition to paeoniflorin, some analogues of paeoniflorin (such as oxypaeoniforin) in BS could also be transformed to their sulfur-containing derivatives during sulfur-fumigation (Kong et al., 2014; Li et al., 2009). These sulfur-containing derivatives including paeoniflorin sulfonate might have opposite bioactivities to paeoniflorin, and thus contributes to the inactivity of S-BS. Although the animal model chosen and dosage levels investigated here could not completely represent the diverse bioactivities and human use conditions of BS in clinical practice, it is do confirmed that sulfur-fumigation could weaken the bioactivity of BS when comparatively evaluated in the same animal model with the same dosage. The pharmacokinetics, safety and efficacy of medicinal herbs are closely related to the contents of active and/or toxic components. Previous studies revealed that the contents of active components and sulfur-containing derivatives were inversely proportional to the length

5. Conclusion In this study, the feasibility of sulfur-fumigation as a post-harvest handling process of BS was investigated from the viewpoints of pharmacokinetics, safety and efficacy. The systemic exposures of two analgesic components were significantly decreased and that of the generated paeoniflorin sulfonate was detectable in all time-points after i.g. administration of S-BS, which suggested that sulfur-fumigation altered the pharmacokinetic behaviors of BS. Besides, paeoniflorin sulfonate and its four metabolites with uncertain toxicities, as well as one endogenous metabolite p-cresol glucuronide, the biomarker of disordered homeostasis of intestinal bacteria and bile acid, were identified as the characteristic markers after i.g. administration of S-BS, suggesting the potential risk to reduce the safety of BS after sulfur-fumigation. Furthermore, the analgesic effects of S-BS at both low and high dosages were decreased in different extent, indicating depressed efficacy of BS after sulfur-fumigation. Since the duration of sulfur-fumigation significantly alters the quality of BS, further studies are warranted to thoroughly evaluate the correlation between the length of sulfur-fumigation process with the compositions, pharmacokinetics, safety and efficacy of BS, so that feasibility of using sulfur-fumigation as a postharvest handling process for BS can be clarified. The limitation of the efficacy evaluation in present study by assessing single herb (BS) with only one animal model (hot-plate pain test) is the possible bias to the actual clinical application of BS-containing complex preparations. A BScontaining complex preparation with specific indications whose efficacies be evaluated with several animal models would be employed in the future studies. Acknowledgements This study was financially supported by National High Technology Research and Development Plan of China (863 Plain) (2014AA022204), National Natural Science Foundation of China (No. 81503245, No. 81573596 and No. 81503365) and National Science Foundation of Jiangsu Province, China (No. BK20151050). Declaration of interest The authors report no conflicts of interest. Author contributions Ming Kong performed the pharmacokinetics and safety studies, and drafted the manuscript. Huan-Huan Liu and Su-Min Duan prepared the BS samples, and performed the pharmacokinetics and safety studies. Jie Wu developed the HPLC-TQ-MS/MS method and calculated the pharmacokinetics parameters. Ming-Qin Shen and Zhi-Gang Wang designed and performed efficacy study. Yan-Bo Zhang designed the experiments. He Zhu designed the experiments of pharmacokinetics and safety study, and revised the manuscript. Song-Lin Li designed the experiments and revised the manuscript. References Alonso-Castro, A.J., Zapata-Morales, J.R., González-Chávez, M.M., Carranza-Álvarez, C., Hernández-Benavides, D.M., Hernández-Morales, A., 2016. Pharmacological effects and toxicity of Costus pulverulentus C. Presl (Costaceae). J. Ethnopharmacol. 180,

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