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pharmaceutics Article

Metabolic Stability and Metabolite Characterization of Capilliposide B and Capilliposide C by LC–QTRAP–MS/MS Zhongzhe Cheng 1 , Xing Zhou 2 , Zhifeng Du 2 , Wenyi Li 2 , Bingying Hu 2 , Jingkui Tian 3 , Lin Zhang 3 , Jiangeng Huang 2, * and Hongliang Jiang 2 1 2

3

*

School of Pharmacy, Weifang Medical University, 7166 Baotong West Street, Weifang 261053, Shandong, China; [email protected] School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, Hubei, China; [email protected] (X.Z.); [email protected] (Z.D.); [email protected] (W.L.); [email protected] (B.H.); [email protected] (H.J.) Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, Zhejiang, China; [email protected] (J.T.); [email protected] (L.Z.) Correspondence: [email protected]; Tel.: +86-276-666-3909

Received: 13 August 2018; Accepted: 4 October 2018; Published: 8 October 2018

Abstract: Capilliposide B (LC-B) and Capilliposide C (LC-C), two new triterpenoid saponins extracted from Lysimachia capillipes Hemsl, exhibit potential anticancer activity both in vitro and in vivo. However, their metabolic process remains unclear. In this study, the metabolic stability of LC-B, LC-C, and Capilliposide A (LC-A, a bioactive metabolite of LC-B and LC-C) was investigated in human, rat, and mouse liver microsomes, respectively. Thereafter, their metabolites were identified and characterized after oral administration in mice. As a result, species difference was found in the metabolic stability of LC-B and LC-C. All three compounds of interest were stable in human and rat liver microsomes, but LC-B and LC-C significantly degraded in mouse liver microsomes. The metabolic instability of LC-B and LC-C was mainly caused by esterolysis. Moreover, 19 metabolites were identified and characterized in mouse biological matrices. LC-B and LC-C mainly underwent deglycosylation and esterolysis, accompanied by dehydration, dehydrogenation, and hydroxylation as minor metabolic reactions. Finally, the metabolic pathway of LC-B and LC-C in mice was proposed. Our results updated the preclinical metabolism and disposition process of LC-B and LC-C, which provided additional information for better understanding efficacy and safety. Keywords: Capilliposide B; Capilliposide C; metabolic stability; metabolism; LC-MS/MS

1. Introduction Lysimachia capillipes Hemsl (Primulaceae) is traditionally used for cold and arthritis in China [1]. Capilliposides, a series of novel oleanane triterpenoid saponins, are the characteristic chemical markers for Lysimachia capillipes Hemsl (Primulaceae) [1–3]. Capilliposide B (LC-B) and Capilliposide C (LC-C), two representative compounds of them, exhibited significant cytotoxicities against ovarian carcinoma A-2780, prostate cancer PC3, and DU145 cells [2,4]. As herbal supplement, the chemosenstizing effect of the cotreatment of LC-C and oxaliplatin in esophageal squamous carcinoma cells has been reported [5]. Moreover, total saponin extracts of this herb (LCE) showed an antitumor effect in several human xenograft tumor models without obvious toxicity [6,7]. Because of its wide application and potential therapeutic effect, the standardized planting techniques of Lysimachia capillipes Hemsl have recently been developed to increase production and guarantee quality [8]. Pharmaceutics 2018, 10, 178; doi:10.3390/pharmaceutics10040178

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Pharmaceutics 2018, 10, 178

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Previously, pharmacokinetics, tissue distribution, and excretion of LC-B and LC-C were systematically evaluated by our group [9–11]. Both compounds showed poor bioavailability and low exposure in tissues after oral administration [9]. The recovery of LC-B was less than 10% in rat excreta after intravenous administration, suggesting that LC-B underwent extensive metabolism prior to excretion [11]. Similarly, extensive metabolism of LC-C in rat intestinal microflora was found and the resultant metabolite pool demonstrated a strong anticancer activity [12]. Moreover, the esterolytic metabolite of LC-C, Capilliposide A (LC-A), also exhibited a significant cytotoxicity against HepG2 cells [12]. However, the metabolic process of LC-B and LC-C remains unclear. Thus, a systemic study is required to investigate the metabolism of LC-B and LC-C. Although we have systematically evaluated the pharmacokinetics, distribution, intestinal microflora metabolism, and excretion of LC-B and LC-C [9–12], the primary aim of the present study was to study the metabolic stability of LC-B and LC-C in liver microsomes obtained from different species and to clarify their metabolites in vivo. The results will update information concerning preclinical metabolism and the disposition process of LC-B and LC-C. 2. Materials and Methods 2.1. Chemicals and Reagents Capilliposide B (LC-B) and LC-C were isolated from Lysimachia capillipes Hemsl (Primulaceae) [2]. The purity of both compounds was higher than 98%, measured by an HPLC-ELSD method [9]. LC-A was hydrolyzed from LC-C with a purity of >98%, as described in a previous report [12]. Dioscin (98% purity, Jiangxi University of Traditional Chinese Medicine, Nangchang, China) was used as internal standard (IS). Midazolam and nomifensine (IS) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Nicotinamide adenine dinucleotide phosphate (NADPH) was purchased from Dingguochangsheng Biotechnology Co., Ltd. (Beijing, China). Pooled liver microsomes originating from different species such as rats, mice, and humans were purchased from BD Gentest (Woburn, MA, USA). Acetonitrile and methanol were HPLC grade and provided by Fisher Scientific (Pittsburgh, PA, USA). Formic acid (≥96%) was purchased from Sinopharm Chemistry Reagent Co., Ltd. (Shanghai, China). The OASIS solid phase extraction (SPE) cartridges were obtained from Waters Corp. (Milford, MA, USA). 2.2. Metabolic Stability We incubated LC-A, LC-B, and LC-C separately with 0.5 mg/mL liver microsomes at a final substrate concentration of 1.0 µM. The reaction mixture contained 5.0 mM NADPH in potassium phosphate buffer (0.1 M, pH 7.4). Incubation was performed in triplicate in a final volume of 0.5 mL at 37 ◦ C. After 5 min pre-incubation, NADPH was added to start the reactions. The incubation system in the absence of NADPH was used as a negative control. Midazolam (0.5 µM) was used as a positive control. An aliquot of 0.5 mL of ice cold acetonitrile containing IS (dioscin, 200 ng/mL) was added into 20 µL of samples to terminate the reactions at 0, 5, 15, 30, 45, 60, and 90 min, respectively. For midazolam, metabolic reactions were quenched at 0, 2, 5, 10, and 20 min by adding 0.5 mL acetonitrile containing IS (nomifensine, 70 ng/mL) to 20 µL of corresponding samples. After centrifugation for 10 min at 17,000× g, the supernatant (5 µL) was subjected to analysis. 2.3. Metabolism in Mice 2.3.1. Animal Experiments Kunming mice (20 ± 2 g) were purchased from the Laboratory Animal Center of Tongji Medical College, Huazhong University of Science and Technology (HUST, Wuhan, China). The mice were housed in a standard environment with free access to food and water. The experiment protocol (Project ID: HUST-D-201410009) was approved by the Animal Ethics Committee in HUST.


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Prior to experiments, blank urinary and fecal samples were harvested from thirty mice (half male and half female). Among them, three male and three female mice were sacrificed to harvest blank plasma. Next, twenty four mice were randomly divided into four groups (half male and female) and were orally administrated LCE prepared in 0.5% carboxy methylcellulose sodium continuously for 9 days (300 mg/kg/day). After dosing, urine and feces were separately collected at 0–12 and 12–24 h. To harvest the blood samples, mice were sequentially dosed on the tenth day and then sacrificed at the predefined time intervals (30 min, 2, 6, and 12 h). After centrifugation for 10 min at 900× g at 4 ◦ C, plasma samples were obtained and stored at −80 ◦ C. 2.3.2. Sample Preparation Five hundred microliter of the pooled plasma sample was treated with 1.5 mL of acetonitrile, followed by 10 min vortex-mixing. Next, the mixtures were centrifuged for 10 min at 17,000× g at 4 ◦ C. The resultant supernatant was evaporated to dryness under a stream of nitrogen. The residue was then dissolved by 200 µL of acetonitrile:water (2:8, v/v). Urine samples were cleaned up using the previously reported solid phase extraction (SPE) method with OASIS SPE cartridges (60 mg, 30 µm, 3 mL) [11]. In brief, the cartridges were preconditioned with 1 mL of methanol and then 1 mL of water. Next, SPE columns loaded with 0.5 mL of urine sample were washed with water (1 mL) and then eluted, using methanol (1 mL). The eluate was evaporated to dryness under a stream of nitrogen at 40 ◦ C and reconstituted in 200 µL of acetonitrile:water (2:8, v/v). The pooled fecal sample (200 mg) was homogenized and extracted with methanol (2.0 mL) using ultrasonication. Then, samples were centrifuged for 10 min at 17,000× g under 4 ◦ C. The supernatant was evaporated to dryness under a stream of nitrogen at 40 ◦ C and reconstituted in 200 µL of acetonitrile:water (2:8, v/v). 2.3.3. LC-MS/MS Conditions The HPLC system consisted of a DGU-20A3 degasser, an LC-20ADXR solvent delivery system, a CTO-20AC column oven, an SIL-20ACXR autosampler, and a CBM-20A controller from Shimadzu (Kyoto, Japan). The 4500 QTRAP® mass spectrometer (SCIEX, Concord, ON, Canada) equipped with a Turboionspray® source was used. Previously, an assay for simultaneous determination of LC-A, LC-B, and LC-C was established by our group [11]. The chromatographic and MS conditions in the present study were the same as those in our previous report [11]. Briefly, the separation was achieved on an Aquasil C18 column (50 × 2.1 mm, 5 µm, Thermo Electron, Bellefont, PA, USA). Mobile Phase A was water containing 0.1% formic acid, and Mobile Phase B was methanol. The flow rate was 0.8 mL/min. The chromatographic gradient condition was as follows: 0–0.2 min 10% B, 0.2–1.5 min 10 to 90% B, 1.5–2.2 min 90% B, 2.2–2.3 min 90 to 10% B, and 2.3–3.0 min 10% B. Midazolam was monitored in positive mode. The MS parameters were used as follow: 500 ◦ C TIS temperature (TEM); 5500 V ionspray voltage; 30 psi curtain gas (CUR); 50 psi nebulizing gas (GS1) and TIS gas (GS2); 120 and 90 V declustering potential (DP) for midazolam and nomifensine (IS), respectively; 10 V entrance potential; 38 and 28 eV collision energy (CE) for midazolam and IS; 15 V collision cell exit potential. The multiple reactions monitoring (MRM) transitions were m/z 326.2→291.1 for midazolam and m/z 239.2→91.0 for IS, respectively. For in vivo metabolism study, samples analyses were performed on the same LC-MS/MS platform. Separation was achieved on the Ultimate XB-C18 column (100 × 2.1 mm, 1.7 µm, Welch, Shanghai, China), and its temperature was maintained at 40 ◦ C. Mobile Phase A was water containing 0.1% formic acid and Mobile Phase B was acetonitrile. The optimized gradient condition was as follows: 15% B from 0 to 2.0 min; 15–30% B from 2.0 to 3.0 min; 30–45% B from 3.0 to 10.0 min; 45–60% B from 10.0 to 25.0 min; 60–95% B from 25.0 to 28.0 min; 95% B from 28.0 to 32.0 min; 95–15% B from 32.0 to 32.1 min; and 15% B from 32.0 to 35.0 min. The flow rate was 0.3 mL/min. The following MS parameters were used: 600 ◦ C TEM; −4500 V ionspray voltage; 30 psi CUR; 50 psi GS1 and GS2; −200 V DP. In accordance with an information dependent acquisition (IDA) procedure, an enhanced product ion (EPI) scan was triggered by an enhanced mass spectrum (EMS) and neutral loss (NL) scan,


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respectively, to characterize the metabolic products. The EMS scan range was from m/z 100 to 1300. EPI was triggered by IDA with a threshold of 10,000 cps, −70 eV CE, and 15 eV collision energy spread (CES). The NL of 162 and 132 Da were introduced as the survey scan. The NL scan range was from m/z 500 to 1300 with a 1000 cps NL–IDA–EPI threshold. As mentioned above, the same CE and CES were used in NL–IDA–EPI mode. 2.4. Data Analysis Data analysis was carried out by Analyst software (version 1.6.1, AB SCIEX, Concord, ON, Canada). To calculate the percentage of parent compound remaining, average peak area ratio of analyte/IS of each sample was divided by that of the time zero sample. By plotting semi-logarithmic percentage of parent compound remaining versus incubation time, elimination rate constant (k) was calculated by the slope of linear regression of initial time points wherein log-linearity was observed. Half life (t1/2 ) was calculated by 0.693/k. Metabolites were identified and characterized by Peak View SoftwareTM 1.2 (AB SCIEX, Concord, ON, Canada). 3. Results and Discussion 3.1. Metabolic Stability The positive control compound midazolam was rapidly metabolized by liver microsomes with t1/2 of 2.35, 3.85, and 3.73 min in rat liver microsomes (RLM), mouse liver microsomes (MLM), and human liver microsomes (HLM), respectively (Figure 1A). This demonstrated that active microsomes and appropriate incubation conditions were used in this metabolic stability study. As shown in Figure 1B–D, LC-B, LC-C, and LC-A were stable in RLM and HLM, but MLM could remarkably metabolize LC-B and LC-C with in vitro t1/2 of 27.7 and 63.0 min, indicating that the species difference in the metabolism of LC-B and LC-C was presented. Further, LC-B and LC-C underwent obvious degradation, regardless of the presence of NADPH. Thus, the metabolism of LC-B and LC-C was not NADPH-dependent in MLM. Recently, we employed a typical esterase inhibitor (dichlorvos) to prevent esterolysis of LC-B and LC-C in rat plasma [9,13]. In the current work, dichlorvos (1 mM) was added to the same incubation system, including MLM. As expected, the degradation of LC-B and LC-C was remarkably inhibited by dichlorvos (Figure 1), suggesting that esterase was the major metabolic enzyme for the biotransformation of LC-B and LC-C in MLM. Previously, Berry et al. reported that hepatic esterase activities varied between species [14]. As such, the species differences in the metabolism of LC-B and LC-C might be associated with altered hepatic esterase activities among liver microsomes from different species. 3.2. In Vivo Metabolism in Mice 3.2.1. Metabolite Characterization by LC–QTRAP–MS/MS To gain MS sensitivity, LC–QTRAP–MS/MS was optimized by standards of LC-B and LC-C. The MS/MS fragmentation behavior of LC-B and LC-C was helpful to deduce their respective metabolic products. In positive mode, the protonated molecule ions were obtained for LC-B and LC-C, but relatively stronger MS intensity together with a larger number of fragments were detected in negative ionization mode. The optimal collision energy was applied to generate more MS/MS fragmental information. Among different survey scan triggered MS/MS approaches, the NL–IDA–EPI method demonstrated greater versatility for conjugated metabolites [15–17]. According to the MS fragment ions of LC-B, LC-C, and their metabolites, the neutral losses of sugar moieties often occurred [12]. Thus, NL–IDA–EPI and EMS–IDA–EPI assays were simultaneously employed to identify and characterize their metabolites. In this study, 19 metabolites of LC-B and LC-C were detected and characterized in mice (Table 1, Figures 2 and 3).

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A 1000

1000 HLM RLM MLM with 1 mM dichlorvos MLM

% Drug remaining

% Drug remaining

100

10

100

10

1

.1

1 0

5

10

15

20

25

0

20

40

Time (min)

60

80

100

60

80

100

Time (min)

D

C 100

% Drug remaining

% Drug remaining

100

10

10

0

20

40

60

Time (min)

80

100

0

20

40

Time (min)

Figure 1. 1. Metabolic Metabolicstability stabilityofofmidazolam midazolam(A), (A),Capilliposide CapilliposideB B(LC-B) (LC-B) (B) and Capilliposide C (LCFigure (B) and Capilliposide C (LC-C) C) in (C)human in human microsomes (HLM), ratmicrosomes liver microsomes mouse liver microsomes (C) liver liver microsomes (HLM), rat liver (RLM), (RLM), mouse liver microsomes (MLM), (MLM), MLM with diclorvos, respectively (n = 3).stability Metabolic stability (D) RLM in HLM and MLMand with diclorvos, respectively (n = 3). Metabolic of LC-A (D) of in LC-A HLM and (n =and 3). RLM (n = 3).

The LC-B, LC-C, and M1 (LC-A) were identified and confirmed according to product ion spectra − ions of LC-B, LC-C, and and retention of and theirM1 standards (Figures 2A and 3A–C). The [M-H] The LC-B,time LC-C, (LC-A) were identified and confirmed according to product ion spectra LC-A (M1) were observed m/z 1175.7, 1161.8, (Figure 3A–C). Their major of LC-B, LC-C, and LCand retention time of their at standards (Figures 2Aand and1077.6, 3A–C).respectively The [M-H]− ions fragments resulted fromatneutral loss of sugarand moieties H2 O. The (Figure [M-H]−3A–C). ion at Their m/z 1175.7 A (M1) were observed m/z 1175.7, 1161.8, 1077.6,and respectively major fragmented into m/zfrom 1043.7, 881.7, loss and 719.6, respectively 3A).The It was 132− Da its fragments resulted neutral of sugar moieties (Figure and H2O. [M-H] ionhigher at m/zthan 1175.7 product ion atinto m/zm/z 1043.7, which indicated the loss of one molecular of xylose (Xyl). product ion its at fragmented 1043.7, 881.7, and 719.6, respectively (Figure 3A). It was 132 The Da higher than m/z 881.7ion originated from neutral loss of onethe molecular xylose and glucose (Glc).(Xyl). It further lost 162 ion Da product at m/z 1043.7, which indicated loss of one molecular of xylose The product − ion of m/z 1161.8 generated the product ions at m/z (Glc) turned into m/z The [M-H] at m/zand 881.7 originated from719.6. neutral loss of one molecular xylose and glucose (Glc). It further lost 162 − ion of ions 1029.7, 1011.5, 867.5, and (FigureThe 3B).[M-H] The product at m/zgenerated 1029.7 andthe 1011.5 wereions generated Da (Glc) and turned into705.6 m/z 719.6. m/z 1161.8 product at m/z − of m/z 1161.8 fragmented into product by losing a xylose and xylose–H O, respectively. The [M-H] 1029.7, 1011.5, 867.5, and 705.6 (Figure 3B). The product ions at m/z 1029.7 and 1011.5 were generated 2 ions at m/z 867.5 and indicating Xyl-Glc and neutral loss. fragmented Interestingly,into theproduct largest by losing a xylose and705.6, xylose–H 2O, respectively. The2Glc-Xyl [M-H]− of m/z 1161.8 number of metabolites was identified in Xyl-Glc pooled feces, compared to plasma urine samples. LC-A ions at m/z 867.5 and 705.6, indicating and 2Glc-Xyl neutral loss.and Interestingly, the largest (M1) wasof detected in all was threeidentified matrices of Figurecompared 3C showed MS/MS of LC-A LC-A (M1). number metabolites in interest. pooled feces, to the plasma and spectra urine samples. The ions at m/z 945.6 were 18 Da Figure (H2 O) and 132 Da (Xyl) less thanspectra its [M-H] ion (M1)product was detected in all1059.7 three and matrices of interest. 3C showed the MS/MS of − LC-A at m/zThe 1077.6, respectively. They1059.7 further lost945.6 one molecular Xyl into m/z 927.5 (M1). product ions at m/z and were 18 Da (Hand 2O) Glc andand 132 turned Da (Xyl) less than its and [M783.5, respectively. Fragment ion at They m/z 783.5 waslost 18 one Da (H m/z 765.5.into m/z H]− ion at m/z 1077.6, respectively. further molecular Xylthan andthat Glc at and turned 2 O) higher 927.5 and 783.5, respectively. Fragment ion at m/z 783.5 was 18 Da (H2O) higher than that at m/z 765.5.


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Table 1. Characterization of the metabolites of Capilliposide B (LC-B) and Capilliposide C (LC-C) in mice by LC–QTRAP–MS/MS. Name

Metabolic Description

Formula

Retention Time (min)

[M-H]−

Fragment Ions

LC-B LC-C M1 (LC-A) M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12

Parent Parent Esterolysis LC-B dehydrogenation LC-C dehydrogenation LC-B dehydration LC-C dehydration LC-A dehydration LC-B deglycosylation of xylose LC-B deglycosylation of glucose LC-C deglycosylation of xylose LC-C deglycosylation of glucose LC-C deglycosylation of glucose and xylose LC-C hydroxylation LC-C methylation or hydroxylation with dehydrogenation LC-B hydroxylation LC-A dehydrogenation

C58 H96 O24 C57 H94 O20 C52 H86 O23 C58 H94 O24 C57 H92 O20 C58 H94 O23 C57 H92 O23 C52 H84 O22 C53 H88 O20 C52 H86 O19 C52 H86 O20 C51 H84 O19 C46 H76 O15 C58 H98 O24

11.20 9.65 5.88 9.73, 11.86, 12.13 7.68, 9.75, 11.41 10.57 10.98 6.39 11.20 12.21 11.24 11.39 13.94 8.18

1175.7 1161.8 1077.6 1173.7 1159.5 1157.7 1143.8 1059.6 1043.1 1013.6 1029.5 999.6 867.5 1177.7

C58 H96 O24

8.13

C57 H92 O26 C52 H84 O23

7.36 5.91

M13 M14 M15

Mouse Plasma

Urine

Feces

1043.7, 881.7, 719.6 1029.7, 1011.5, 867.5, 705.6 1059.7, 945.6, 927.5, 783.5, 765.5 1041.5, 1011.6, 879.5, 717.4 1027.6, 1011.7, 925.8, 879.5, 865.7, 763.3, 717.4, 703.5 1025.7, 863.6, 701.6 1011.5, 993.6, 849.5, 687.3 927.7, 765.4 881.3, 719.2 881.7, 719.4 867.5, 705.4 897.6, 867.6, 765.4, 705.5, 659.5 705.4 1045.7, 1027.8, 883.4

× × × – – – – – × – × × – –

× × × – – − – – × – × × – –

× × × × × × × × × × × × × ×

1175.7

1043.7, 881.7, 719.6

–

–

×

1191.7 1075.6

1059.7, 897.7, 735.4 943.6, 925.7, 879.7, 781.5, 763.4

– –

– –

× ×

Pharmaceutics 2018, 10, 10, 178x FOR PEER REVIEW Pharmaceutics 2018,

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A

Intensity

LC-C

LC-B 2e6

LC-A

1e6 0e0 2

4

6

8

10

12

M5 M3-2

M14

Intensity

1.5e6

M15

1.0e6

M1

16

18

20

M10

1e6

26

0

M2-3

5 10 15 20 25 30

1.5e6

M11

5.0e5

30

32

34

M12

0

5 10 15 20 25 30

1.5e6

M9

M13

M8

1.0e6

28

1e6

M7

M2-2

M3-3

M12, M13 M6 M3-1

0

Intensity

24

LC-B, M7, M9

5 10 15 20 25 30

0.0e0

C

22

M4

LC-C, M2-1

B

14

2

4

6

8

10

2

4

6

8

10

0

LC-B

5 10 15 20 25 30

12

14

16

18

20

22

24

26

28

30

32

34

12

14

16

18

20

22

24

26

28

30

32

34

8.0e5 6.0e5 4.0e5 2.0e5

0.0e0

D Intensity

LC-B, M7, M9 6e5

LC-C

M1

M10

4e5 2e5 0e0 2

4

6

8

10

2

4

6

8

10

12

14

16

18

20

22

24

12

14

16

18

20

22

24

26

28

30

32

34

2.0e4

Intensity

E

0.0e0

Intensity

LC-C

F

26

28

30

32

34

LC-B, M7, M9

M1

M10

5e5

G

Intensity

0e0 2

4

6

8

10

12

14

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

16

18

20

22

24

26

28

30

32

34

8e4

0e0

Time, min Figure 2. The representative extracted ion chromatograms (XIC) (XIC) of LC-B, their metabolites. Figure 2. The representative extracted ion chromatograms of LC-C, LC-B, and LC-C, and their (A)metabolites. The XIC of standard LC-B, and LC-B, LC-A. LC-C, (B,C) The of LC-B, and of their metabolites (A) The XIC ofLC-C, standard andXIC LC-A. (B–C)LC-C, The XIC LC-B, LC-C, in pooled feces metabolites and blank feces. (D,E) feces The XIC LC-B,feces. LC-C,(D–E) and their metabolites pooled plasma and their in pooled andofblank The XIC of LC-B,inLC-C, and their and blank plasma. (F,G) The XIC of LC-B, their metabolites in pooled urine and blank urine. metabolites in pooled plasma andLC-C, blankand plasma. (F–G) The XIC of LC-B, LC-C, and their

metabolites in pooled urine and blank urine.


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A 881

Intensity

3e6

1175.7

2e6

1043.7

1e6 0e0

881.7

719.6 200

400

B

719

600 800 Mass/Charge, Da

1000

LC-B

1200

OH O O

2e6

Intensity

1043

1161.8

1029.7

1011

O

867 OH

1029

1e6

-H2O

867.5

O

705.6 0e0

Ara

Glc

OH

Glc

200

400


1000

Xyl

1200

705

O

LC-C 765

OH

927

C

-H2O 1059

783

OH

945

Intensity

1077.6

1059.7

1.5e5

-H2O

-H2O

927.5

1.0e5

765.5

O

5.0e4

945.6

0.0e0

200

400

Ara

Glc

783.5


Glc Xyl

1000

1200

LC-A (M1) -2H

OH O

879

D

717

8e4 879.5

6e4

Intensity

O

1173.7

1011.6

4e4

O

1041

OH

717.44

O Ara

Glc

1041.5

2e4

Glc Xyl

0e0

200

400


1000

1011

1200

M2

-CO-H 2O

717 -CO-H 2O

1011

-2H 703

Intensity

E 1.0e5

717.4 763.3

5.0e4 0.0e0

865

1027.7 1011.6 1159.5 879.5 865.7

1027

925.8

703.50

200

400


1000

M3

1200

-H2O 763 -H 2O 925 -CO-H 2O 879

F 1025.7

Intensity

2e5

863

-H 2O

1157.7

1025 1e5

863.6 701.6

0e0

200

400


1000

1200 701

M4

-H2O 849

Intensity

G

993

1143.8 1011.5 5e5

849.5

993.6

687.3 0e0

200

400


1000

687

1200

M5 -H2O

H

765 3e5

Intensity

1011

-H2O

1059.6

927.7

927

2e5

765.4

1e5 0e0

200

400


1000

1200

M6

Figure 3. Typical MS/MS spectra of LC-B (A), LC-C (B), LC-A (C), M2-M6 (D–H), respectively. Figure 3. Typical MS/MS spectra of LC-B (A), LC-C (B), LC-A (C), M2-M6 (D–H), respectively.


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The M2, M3, and M15 formed [M-H]− ions at m/z 1173.7, 1159.5, and 1075.6, respectively (Figure 2B, Figure 3D,E and Figure S1). These seven metabolites were only detected in pooled feces (Figure 2B,D,F). Based on 2 mass unit difference of [M-H]− ions from LC-B, LC-C, and LC-A, M15, M2, and M3 were assigned as their corresponding dehydrogenated products. However, the position of such a metabolic process remained unknown due to limited information of fragmentation pattern. The fragment ions of M2, M3, and M15 at m/z 1041.5 (-Xyl), 1011.6 (-Glc), 879.5 (-Xyl-Glc), and 717.4 (-Xyl-2Glc), m/z 1027.6 (-Xyl), 1011.7 (-C5 H10 O2 -CO-H2 O), 925.8 (-Xyl-C5 H10 O2 -H2 O), 879.5 (-Xyl-C5 H10 O2 -CO-H2 O), 865.7 (-Xyl-Glc), 763.3 (-Xyl-Glc-C5 H10 O2 ), 717.4 (-Xyl-Glc-C5 H10 O2 -CO-H2 O), and 703.5 (-Xyl-2Glc), m/z 943.6 (-Xyl), 925.7 (-Xyl-H2 O), 879.7 (-Xyl-2H2 O-CO), 781.5 (-Xyl-Glc), and 763.4 (-Glc-Xyl-H2 O) were founded in the MS/MS spectra, which indicated that CO, H2 O, Xyl, and Glc were lost from their [M-H]− ions (Figure 3D,E and Figure S1). The M4, M5, and M6 were 18 Da less than LC-B, LC-C, and LC-A, respectively, suggesting the loss of H2 O from their parents. The three dehydrated metabolites were also only detected in pooled feces rather than in plasma and urine samples (Figure 2B,D,F). Likewise, the position of dehydration remained unclear, because their parents were multiple hydroxyl-containing compounds. Their product ions underwent a serial of neutral loss of sugar moieties, that is, the fragment ions of M4, M5, and M6 at m/z 1025.7 (-Xyl), 863.6 (-Xyl-Glc), 701.6 (-Xyl-2Glc), and m/z 1011.5 (-Xyl), 993.6 (-Xyl-H2 O), 849.5 (-Xyl-Glc), 687.3 (-Xyl-2Glc), and m/z 927.7 (-Xyl), 765.4 (-Xyl-Glc) (Figure 3F–H). The M7 and M8 demonstrated [M-H]− ions at m/z 1043.1 and 1013.6, respectively (Figure S1). These results indicated that one xylose or glucose was lost from LC-B, respectively. M7 was found in all three matrices of interest, while M8 was only detected in pooled feces (Figure 2B,D,F). The mass spectra of M7 and M8 showed the same product ions at m/z 881.3 and 719.2 after losing xylose or glucose, which resembled the mass behavior of the parent compound. The M9 gave [M-H]− ion at m/z 1029.5 (Figure S1). It was assigned as a deglycosylated product of LC-C via losing xyl. Its major product ions at m/z 867.6 and 705.4 were the same as those of LC-C (Figure S1). Similarly, M10 (m/z 999.6) was 162 Da less than LC-C. M11 (m/z 867.5) lost Glc-Xyl from its parent compound (Figure S1). Thus, they were characterized as deglycosylated products of LC-C. The M12 presented [M-H]− ion at m/z 1177.7 and main fragment ions at m/z 1045.7 and 883.4. Its [M-H]− ion and fragmental ions were 16 mass units larger than that of LC-C. Therefore, M12 was proposed to be a hydroxylated metabolite, which was only detected in pooled feces, after inserting an oxygen atom into its parent compound (Figure 2B and Figure S1). The M13 and LC-B demonstrated the same [M-H]− ion at m/z 1175.7, but it was 14 Da larger than that of the protonated LC-C. M13 was only found in feces and its retention time was found to be 8.13 min (Figure 2B). According to the mass difference from the parent compound, M13 was produced after either introduction of one oxygen atom with dehydrogenation or methylation. The main fragmental ions at m/z 1043.7 (-Xyl), 881.7 (-Xyl-Glc), and 719.6 (-Xyl-2Glc) via the neutral loss of sugar moieties (Figure S1). M14 was only found in feces and its retention time was 7.36 min (Figure 2B). The [M14-H]− ion was 16 mass units larger than that of LC-B, suggesting that hydroxylation occurred in the skeletal structure of LC-B. It fragmented into m/z 1059.8, 897.6, and 735.5, which indicated xylose, Xyl-Glc, and Xyl-2Glc were lost from its deprotonated molecular ion (Figure S1). 3.2.2. Proposed Metabolic Pathway of LC-B and LC-C in Mice To facilitate understanding of the systemic exposure of LC-B and LC-C, metabolic study was conducted in mice. A total of 19 metabolites were characterized. Figure 4 shows the proposed metabolic pathway. LC-B and LC-C mainly underwent deglycosylation, dehydrogenation, dehydration, esterolysis, and hydroxylation processes. Esterlysis and deglycosylation were found to be the major metabolic pathway in mouse plasma and urine. In this study, all of 19 metabolites were found in feces, while only four metabolites, LC-A (M1), M7, M9, and M10, were detected in all three matrices of interest (Table 1). LC-A was an esterolytic product of LC-B and LC-C. After oral administration of

deglycosylation were found to be the major metabolic pathway in mouse plasma and urine. In this study, all of 19 metabolites were found in feces, while only four metabolites, LC-A (M1), M7, M9, and M10, were detected in all three matrices of interest (Table 1). LC-A was an esterolytic product Pharmaceutics 2018, 10, 178 of LC-B and LC-C. After oral administration of LCE, LC-B and LC-C could 10 of 13 be hydrolyzed by intestinal microflora. On the other hand, the absorbed LC-B and LC-C could be metabolized by esterase in vivo. Then, LC-A was excreted from urine and feces, respectively. LCE, LC-Cincould hydrolyzed intestinal microflora. Oninthe hand, the absorbed M1LC-B was and detected all ofbe three matrices by follow oral administration theother present study, which LC-B and LC-C could be metabolized by esterase in vivo. Then, LC-A was excreted from urine was in good agreement with our previous report [9,11]. M7, M9 and M10 originated from LC-and feces, M1 was detected inofall of three matrices administration in the present B orrespectively. LC-C by losing one molecular saccharide moiety. follow M9 (m/zoral 1029.5) has been identified as study, which was in good agreement with our previous report [9,11]. M7, M9 and M10 originated from a main metabolite of LC-C, produced by intestinal microflora [12]. These results suggested that LC-B or LC-C by losing one molecular of saccharide moiety. M9 (m/z 1029.5) has been identified the terminal groups of saccharide moieties in LC-B and LC-C were readily lost both in vivo and as a main metabolite LC-C, intestinalofmicroflora [12].were These results suggested in vitro. M7, M9,ofand M10,produced as major by metabolites their parent, produced in intestinethat andthe terminal groups of saccharide moieties in LC-B and LC-C were readily lost both in vivo and in vitro. entered systemic circulation, followed by excretion from urine and feces. Possibly, it was easy M7,toM9, andsuch M10, as major metabolites of their parent, were intestine detect metabolites with relatively larger amounts in produced vivo. LC-Ain(M1), M6, and and entered M15 systemic followed excretion from urine andother feces.metabolites Possibly, itwere was easy to detect such might circulation, be derived from bothby parent compounds, while generated from metabolites with relatively larger amounts in vivo. LC-A (M1), M6, and M15 might be derived from either LC-B or LC-C. Previously, we reported that 92.7% of LC-C could be metabolized in both parent compounds, while other metabolites were generated either LC-B LC-C. Previously, intestinal microflora [12]. Subsequently, six metabolites werefrom characterized asor deglycosylation we and/or reported that 92.7% of LC-C could be metabolized in intestinal microflora [12]. esterolysis products [12]. Based on previous study, the intestine might beSubsequently, the major six metabolic metabolites were characterized as deglycosylation site of LC-B and LC-C. LC-A (M1) was and/or mainly esterolysis distributed products in mouse[12]. liverBased after on previous study, dosing the intestine might thecurrent major metabolic site of LC-B and LC-C.demonstrated LC-A (M1) was intravenous of LC-B [10].be The stability investigation mainly distributed in mouse liver after intravenous dosing of LC-B [10]. The current metabolic stability that LC-B and LC-C were subject to hydrolysis by esterase in MLM in vitro. These results investigation demonstrated that LC-B and LC-C were subject to hydrolysis bypathway esterase in vitro. indicated that esterases mediated hydrolysis could be major metabolic ofMLM LC-B in and These results indicated thattogether, esterases mediated be major metabolic pathway of LC-B LC-C in mice. Taken intestinal hydrolysis microfloracould mediated deglycosylation/hydrolysis andtogether LC-C in mice. together, intestinal microflora mediatedadeglycosylation/hydrolysis with Taken subsequent hepatic esterolysis represent significant proportion oftogether the with subsequent hepatic esterolysis a significant proportion of the biotranformation process biotranformation process for represent LC-B and LC-C in mice. Additionally, dehydration, for dehydrogenation, LC-B and LC-C inand mice. Additionally, dehydration, dehydrogenation, hydroxylation may hydroxylation may contribute, at least in part, to theand metabolic clearance contribute, at least in part, to the metabolic clearance of LC-B and LC-C in mice. of LC-B and LC-C in mice. -H2O

A

M7 (m/z 1043.1) M8 (m/z 1013.6)

-H2O

M6 (m/z 1059.6)

-Xyl

-Glc

-H2O

OH O O

-H2O O

OH

-C5H10O2

O Glc

LC-B (m/z 1175.7)

Ara Glc

M1 (m/z 1077.6)

Xyl

M4 (m/z 1157.7) -2H

-2H

OH

-2H

O

O

O

-2H

O

OH

O Glc

M2 (m/z 1173.7)

Ara Glc

Xyl

M14 (m/z 1191.7)

Figure 4. Cont.

M15 (m/z 1075.6)


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B -2H

-H2O

-2H

-H2O

M6 (m/z 1059.6)

M1 (m/z 1077.6)

-H2O

M15 (m/z 1075.6) -C5H10O2

-2H

M5 (m/z 1143.8) O

M3 (m/z 1159.5)

LC-C (m/z 1161.8) -Xyl -Glc

M9 (m/z 1029.5)

M12 (m/z 1177.7)

-Xyl-Glc

-Glc

O or

CH2 -Xyl

M11 (m/z 867.5)

M10 (m/z 999.6)

M13 (m/z 1175.7)

Figure 4. The proposed metabolic pathway of LC-B (A) and (B) in(B) mice oral dosing Figure 4. The proposed metabolic pathway of LC-B (A) LC-C and LC-C in following mice following oral of total saponins of L. capillipes Hemsl extract (LCE). dosing of total saponins of L. capillipes Hemsl extract (LCE).

In Ina aprevious demonstrated significant significantantitumor antitumoractivities activities following previousstudy, study, LCE LCE demonstrated following oraloral administration in nude mice [7]. The metabolic products of LC-C generated in intestinal microflora administration in nude mice [7]. The metabolic products of LC-C generated in intestinal also demonstrated strong anticancer effect anticancer in vitro [12]. Moreover, LC-A, metabolite of LC-Baand microflora also ademonstrated a strong effect in vitro [12].a Moreover, LC-A, LC-C, exhibited a significant cytotoxicity against HepG2 cell lines [12]. In the present study, metabolite of LC-B and LC-C, exhibited a significant cytotoxicity against HepG2 cell lines [12].two parent compounds togethertwo withparent severalcompounds previously reported products of LC-C reported produced in In the present study, togethermetabolic with several previously intestinal microflora were detected and characterized mouse plasma. These metabolites are likely to metabolic products of LC-C produced in intestinalin microflora were detected and characterized be in related with antitumor pharmacological efficacy. Future study will be required to unambiguously mouse plasma. These metabolites are likely to be related with antitumor pharmacological investigate pharmacological of such bioactive metabolites in vivo. efficacy. the Future study will beactivity required to unambiguously investigate the pharmacological activity of such bioactive metabolites in vivo. 4. Conclusions 4. In Conclusions this study, the metabolic stability of LC-A, LC-B, and LC-C in HLM, MLM, and RLM was investigated. The results showed that LC-B and LC-C were stable in HLM and RLM, but not in MLM. In this study, the metabolic stability of LC-A, LC-B, and LC-C in HLM, MLM, and RLM Their metabolic stability could be improved by inhibiting esterolysis using dichlorvos. In addition, this is was investigated. The results showed that LC-B and LC-C were stable in HLM and RLM, but the first report concerning metabolism of LC-B and LC-C in vivo. Nineteen metabolites were characterized not in MLM. Their metabolic stability could be improved by inhibiting esterolysis using in mice. A number of biotransformation reactions, including deglycosylation, dehydrogenation, dichlorvos. In addition, this is the first report concerning metabolism of LC-B and LC-C in vivo. dehydration, esterolysis, and hydroxylation were involved. Particularly, deglycosylation and esterolysis Nineteen metabolites were characterized in mice. A number of biotransformation reactions, could be the primary biotransformation reactions. Thereafter, the metabolic pathway of LC-B and including deglycosylation, dehydrogenation, dehydration, esterolysis, and hydroxylation were LC-C in mice following oral dosing of LCE was proposed. According to the current and previous involved. Particularly, deglycosylation and esterolysis could be the primary biotransformation findings, intestinal microflora mediated deglycosylation/hydrolysis together with subsequent hepatic reactions. Thereafter, the metabolic pathway of LC-B and LC-C in mice following oral dosing esterolysis played a critical role in the metabolic clearance of LC-B and LC-C in mice. of LCE was proposed. According to the current and previous findings, intestinal microflora mediated deglycosylation/hydrolysis togetheronline withatsubsequent hepatic esterolysis played a Supplementary Materials: The following are available http://www.mdpi.com/1999-4923/10/4/178/s1, Figure S1: The spectra of M7of (A), M8 (B), (C),in M10 (D), M11 (E), M12 (F), M13(G), M14(H) critical roletypical in the MS/MS metabolic clearance LC-B andM9 LC-C mice. and M15(I).

Supplementary Materials: The following areJ.H., available online at www.mdpi.com/xxx/s1, Figure S1: The Author Contributions: Conceptualization, H.J., and Z.C.; Methodology, H.J., J.H., and Z.C.; Investigation, Z.C., X.Z., MS/MS Z.D., W.L., and of B.H.; Z.C.;(F), Writing-Original Draft typical spectra M7 Resources, (A), M8 (B),J.T. M9and (C),L.Z.; M10Data (D), Curation, M11 (E), M12 M13(G), M14(H) andPreparation, M15(I). Z.C.; Writing-Review and Editing, H.J., J.H., and Z.C.; Supervision, H.J. and J.H. Author Contributions: Conceptualization, H.J., J.H., and Z.C.; Methodology, H.J., J.H., and Z.C.; Investigation, Z.C., X.Z., Z.D, W.L., and B.H.; Resources, J.T. and L.Z.; Data Curation, Z.C.; Writing-


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Funding: This work was funded by the National Science and Technology Major Project of China (2012ZX09102201-10), Program for Zhejiang Leading Team of S&T Innovation (2012R10044-10), and was also funded by National Natural Science Foundation of China (81803704), Shandong Provincial Natural Science Foundation (ZR2016HB52) and the Project of Shandong Province Higher Educational Science and Technology Program (J16LM05). Conflicts of Interest: The authors declare no conflict of interest.

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