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molecules Article

Ultra-Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS)-Based Pharmacokinetics and Tissue Distribution Study of Koumine and the Detoxification Mechanism of Glycyrrhiza uralensis Fisch on Gelsemium elegans Benth. Lin Wang, Qi Sun, Nan Zhao, Yan-Qing Wen, Yang Song and Fan-Hao Meng *

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School of Pharmacy, China Medical University, 77 Puhe Road, Shenyang 110122, Liaoning, China; [email protected] (L.W.); [email protected] (Q.S.); [email protected] (N.Z.); [email protected] (Y.-Q.W.); [email protected] (Y.S.) * Correspondence: [email protected]; Tel.: +86-24-3193-9448 Received: 11 May 2018; Accepted: 2 July 2018; Published: 11 July 2018

 

Abstract: Gelsemium elegans Benth. (G. elegans), which is a famous Chinese folk medicine, has been commonly used to treat certain types of skin ulcers and alleviate inflammation, headaches, and cancer pain. However, the extensive clinical use of G. elegans has been greatly hampered by its toxicity. As one of the most widely used herbal medicines, Glycyrrhiza uralensis Fisch, has a unique effect on detoxification of G. elegans. In the present study, a rapid and sensitive method using ultra-liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was established and validated for determination of koumine, the most abundant molecule among the alkaloids of G. elegans, in rat plasma, tissue, and liver microsome. The developed method was successfully applied to the pharmacokinetics, tissue distribution, and in vitro metabolism study in rat with or without pre-treated Glycyrrhiza uralensis Fisch extract. Meanwhile, the expression level of CYP3A1 mRNA was analyzed to explain the detoxification mechanism of Glycyrrhiza uralensis Fisch on G. elegans. As a result, our work demonstrated that Glycyrrhiza uralensis Fisch could significantly affect the pharmacokinetics and tissue distribution of koumine in rats. The detoxification mechanism of Glycyrrhiza uralensis Fisch on G. elegans may be its cytochrome enzyme up-regulation effect. Keywords: Gelsemium elegans Benth.; Glycyrrhiza uralensis Fisch; koumine; pharmacokinetics; tandem mass spectrometry; detoxification

1. Introduction Gelsemium elegans Benth. (G. elegans) is distributed in China and Southeastern Asia. It was widely used as a classic Chinese herbal medicine to treat malignant skin problems [1]. Recent years, its anti-tumor [2], anti-inflammatory [3], and anxiolytic [4] activities have also been well studied. On the other side, G. elegans was well-known for its toxicity. Typical symptoms of intoxication include vomiting, blurred vision, muscular weakness, limb paralysis, dilated pupils, breathing difficulty, and convulsion. In instances of severe poisoning, the nervous system is depressed and death is caused by respiratory depression [5,6]. Its toxicities set a limit to the application of G. elegans in clinical settings. It is critical finding effective methods to reduce the toxicity of G. elegans based on the mechanism of action. Glycyrrhiza uralensis Fisch (GU) is one of the most widely used herbal medicines. According to traditional Chinese medicine (TCM) theory, GU is primarily effective for fatigue and debilitation,

Molecules 2018, 23, 1693; doi:10.3390/molecules23071693

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asthma with2018, coughing, andREVIEW excessive phlegm. Moreover, it has a unique effect on moderating Molecules 23, x FOR PEER 2 of 13 the characteristics of toxic herbs, which could be partly interpreted as detoxification [7]. In the folk, Glycyrrhiza uralensis of Fisch one of most widely used herbal According intragastric administration GU(GU) wasisused tothe detoxify G. elegans, much medicines. of the work carried to out on traditional Chinese medicine (TCM) theory, GU is primarily effective for fatigue and debilitation, the role of GU on CYPs regulation and function, though further investigation was required for the asthma with coughing, and excessive phlegm. Moreover, it has a unique effect on moderating the mechanism of this detoxification method. characteristics of toxic herbs, which could be partly interpreted as detoxification [7]. In the folk, As the most abundant molecule alkaloids of G.much elegans, koumine (KM)out has intragastric administration of GU wasamong used tothe detoxify G. elegans, of the work carried on been demonstrated to exert numerous potent biological properties, such as anxiolytic and analgesic the role of GU on CYPs regulation and function, though further investigation was required for the effects [8]. However, also possesses inhibitory effects on splenocyte proliferation and the humoral mechanism of thisitdetoxification method. the most molecule among of G. elegans, (KM) has been immune As response [9].abundant In the previous study, the ouralkaloids group reported the koumine pharmacokinetics study of demonstrated to exert numerous biological properties, such asof anxiolytic and [10], analgesic gelsemine and koumine after oralpotent administration of the extract G. elegans but effects the tissue [8]. However, inhibitory effects on splenocytethere proliferation and the humoral distribution data itinalso thepossesses literature is limited. Furthermore, is no report about the immune influence of response [9]. In the previous study, our group reported the pharmacokinetics study of gelsemine GU on the pharmacokinetics and tissue distribution of KM and we speculate that GU may and have an koumine after oral administration of the extract of G. elegans [10], but the tissue distribution data in impact on the pharmacokinetics of KM metabolized by inducing the CYP450 system. The aim of the literature is limited. Furthermore, there is no report about the influence of GU on the this work is to evaluate the pharmacokinetics and tissue distribution properties of KM in rats and to pharmacokinetics and tissue distribution of KM and we speculate that GU may have an impact on explore these behaviors aremetabolized altered by the pre-treated GU extract. It desrved investigation thehow pharmacokinetics of KM by inducing the CYP450 system. The aim further of this work is to so asevaluate to understand the possibility the combination of KM and To achieve the pharmacokinetics andregarding tissue distribution properties use of KM in rats andGU. to explore how this, an ultra-liquid chromatography mass spectrometry method wassodeveloped these behaviors are altered bytandem the pre-treated GU extract. It(UPLC-MS/MS) desrved further investigation as to understandfor the the possibility regarding of thekoumine combination use plasma, of KM and GU. Toand achieve ultraand validated determination in rat tissues, rat this, liveranmicrosome liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed result and for (RLM). In vivo pharmacokinetic and tissue distribution study could give a straightforward validated for the determination of koumine in rat plasma, tissues, and rat liver microsome (RLM). In the influence of GU on KM. Moreover, GU is discussed for testing its potential on hepatic enzyme vivo pharmacokinetic and tissue distribution study could give a straightforward result for the inductions by both in vitro metabolism and RT-qPCR study. The results of our study would provide a influence of GU on KM. Moreover, GU is discussed for testing its potential on hepatic enzyme meaningful basis for evaluating the rationality for the detoxification effect of GU on G. elegans. inductions by both in vitro metabolism and RT-qPCR study. The results of our study would provide a meaningful basis for evaluating the rationality for the detoxification effect of GU on G. elegans.

2. Results

2. Results 2.1. Method Development 2.1. Method Development Liquid-liquid extraction (LLE) and the protein precipitation method (PPM) were compared during sample preparation. which(LLE) is a simple fast technique, obtained satisfactory recoveries Liquid-liquidPPM, extraction and theand protein precipitationhas method (PPM) were compared and reduced the endogenous-related substances in bio-matrix. The recovery data forsatisfactory both LLE and during sample preparation. PPM, which is a simple and fast technique, has obtained andinreduced the endogenous-related in bio-matrix. The recovery data for both PPM recoveries was shown supporting information Tablesubstances S1. LLE and shown in supportingexhibited information Tablesensitivity S1. Both KMPPM andwas internal standard(IS) higher in the positive mode than in the Both KM and internal standard(IS) exhibited higher in the positive mode than in themode negative mode. Quantification was performed using the sensitivity multiple reaction monitoring (MRM) negative mode. Quantification was performed using the multiple reaction monitoring (MRM) + + of the transitions m/z [M + H] 307.1→70.8 for KM and m/z [M + H] 336.0→320.4 for IS.mode The mass of the transitions m/z [M + H]+ 307.1→70.8 for KM and m/z [M + H]+ 336.0→320.4 for IS. The mass spectrometer was operated at collision energy of 25 and 15 V for KM and IS, respectively, capillary spectrometer was operated at collision energy of 25 and 15 V for KM and IS, respectively, capillary voltage of 2.0 kV, cone voltage of 44 V, source temperature of 120 ◦ C, desolvation temperature 500 ◦ C, voltage of 2.0 kV, cone voltage of 44 V, source temperature of 120 °C, desolvation temperature 500 and desolvation gas 1000 TheThe ionion pairs for the thefinal finalMRM MRM method is given °C, and desolvation gas L/h. 1000 L/h. pairswere wereselected selected for method is given in in Figure 1. Figure 1.

Figure 1. Chemical structures and full scan product ion of precursor ions of koumine (KM) (A) and IS (B).

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Molecules 2018, 23, 1693 Figure 1. Chemical structures and full scan product ion of precursor ions of koumine (KM) (A) and

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IS (B).

2.2. Method Validation

2.2. Method Validation

2.2.1. Specificity

2.2.1. Specificity

Under the the UPLC-MS/MS above,the theretention retention time of KM Under UPLC-MS/MSconditions conditionsthat that are are described described above, time of KM andand IS IS werewere 2.18 and 2.54 min, respectively. The typical chromatograms of blank plasma, blank plasma spiked 2.18 and 2.54 min, respectively. The typical chromatograms of blank plasma, blank plasma withspiked the analytes and IS, plasma collected 20 min,atand at sample 30 min at after intravenous with the analytes and IS, plasmaatcollected 20 liver min, sample and liver 30 min after administration KM are shown in Figure 2. No interference was foundwas in the intravenousof administration of KM are shown in significant Figure 2. Noendogenous significant endogenous interference elution time found in of theanalyte elution and timeIS. of analyte and IS.

Figure 2. Typical chromatograms of (A) blank rat plasma; (B) blank rat plasma spiked with KM and IS; (C) rat plasma sample at 20 min; and, (D) liver sample at 30 min after a single intravenous dose of 10 mg/kg KM. Representative MRM chromatograms of KM and IS.

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2.2.2. Linearity and Lower Limit of Quantification LLOQ Calibration curves were constructed for KM and IS by least-squares linear calibration. Moreover, the present UPLC-MS/MS method that is offered LLOQ values in rat plasma and various tissues less than 25 ng/mL. The regression equation, correlation, and linear ranges are shown in Table 1, which indicated that the method was sensitive enough for the pharmacokinetics, tissue distribution, and in vitro metabolism study. Table 1. The regression equations, linear ranges, and LLOQs for the determination of the analytes in rat biological samples. Samples Plasma Heart Liver Spleen Lung Kidney Stomach Intestine Rat Liver Microsome

Regression Equations 10−3 X

Y = 0.129 × − 0.0021 Y = 2.76 × 10−3 X − 0.3078 Y = 2.76 × 10−3 X − 0.3595 Y = 3.01 × 10−3 X − 0.0547 Y = 1.58 × 10−3 X + 0.1471 Y = 2.80 × 10−3 X − 0.1467 Y = 1.52 × 10−3 X + 0.1601 Y = 1.52 × 10−3 X + 0.05300 Y = 0.0959 × 10−3 X + 0.1471

R2

Linear Range (ng/mL)

LLOQ (ng/mL)

0.9967 0.9904 0.9978 0.9943 0.9975 0.9964 0.9968 0.9979 0.9910

10–5000 25–5000 25–5000 25–5000 25–5000 25–5000 25–5000 25–5000 50–50000

10 25 25 25 25 25 25 25 50

2.2.3. Precision and Accuracy The precision and accuracy were estimated by analyzing QC samples in six replicates. The intra-day precision was determined on the same day and the inter-day precision was determined on three consecutive days. Precisions were expressed as RSD required less than 15%, and the accuracy to be within ±15%. The intra- and inter-day precisions were less than 12.11%, and the accuracy ranged from −4.32% to 4.51%. The data in all of the biological matrices of rats were summarized in Table 2, which demonstrated that this assay was accurate and reproducible for the determination of KM in rat plasma, tissues, and RLM. Table 2. Precision and accuracy of the determination of the analytes in rat biological samples (n = 6).

Samples

Analyte Concentration

Accuracy (%)

Intra-Day Precsion (%)

Inter-Day Precsion (%)

Plasma

10 25 300 4000

−4.0 −4.3 3.2 3.9

7.8 10 9.7 9.0

9.6 11 8.5 9.7

Heart

25 100 600 4000

4.2 3.9 −3.1 2.5

8.1 12 10 11

10 9.1 7.6 10

Liver

25 100 600 4000

3.6 4.5 3.9 −3.5

8.3 11 8.5 9.5

9.2 11 9.0 8.8

Spleen

25 100 600 4000

−4.1 2.9 −2.4 3.4

8.6 12 10 9.7

10 8.7 9.2 7.6

Lung

25 100 600 4000

3.3 3.1 −2.6 3.2

9.8 12 7.2 10.0

10 9.4 10 6.6

(ng/mL)

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Table 2. Cont. Analyte Concentration

Samples

Accuracy (%)

Intra-Day Precsion (%)

Inter-Day Precsion (%)

Kidney

25 100 600 4000

−4.6 4.5 −3.8 2.2

11 9.9 10 7.4

11 8.8 7.8 9.5

Stomach

25 100 600 4000

4.3 3.0 2.5 3.3

10.2 10 5.1 8.3

8.4 9.8 7.0 7.5

Intestine

25 100 600 4000

3.7 3.8 2.2 −3.0

10 7.6 6.9 9.2

8.8 11 9.6 7.1

Rat Liver Microsome

50 250 2000 20000

2.9 3.0 2.6 2.2

10 6.2 7.7 7.4

9.2 7.2 8.1 7.2

(ng/mL)

2.2.4. Extraction Recovery and Matrix Effect The matrix effects were determined by six independent sources of matrix. It was calculated by comparing the responses of the post extracted biological samples with that of pure standard solution containing equivalent amounts of the analytes. The extraction recovery was performed by comparing the peak area rations of KM to IS of an extracted sample to the standard analytes solution of the same concentration. Extraction recovery for KM and IS in all of the biological matrices were greater than 78.58%. In addition, the matrix effect values ranged from 84.32 to 105.12% for KM. These results suggested that the evaluated method was free of matrix effects and reliable for bioanalysis. The results are shown in Table 3. Table 3. Matrix effects and extraction recovery for the analytes in rat biological samples (n = 6). Samples Plasma

Heart

Liver

Spleen

Lung

Kidney

Spiked Concentration (ng/mL) 25 300 4000 100 600 4000 100 600 4000 100 600 4000 100 600 4000 100 600 4000

Matrix Effect Mean (%) RSD (%) 92.4 96.5 102.5 88.9 93.5 87.0 94.3 94.5 87.7 101.2 105.1 94.3 88.0 90.1 84.3 104.6 92.6 103.1

9.4 6.3 8.6 10.9 7.9 8.7 9.2 8.9 10.0 9.6 7.3 9.0 8.0 8.5 9.7 9.4 7.4 9.1

Extraction Recovery Mean (%) RSD (%) 81.9 88.0 82.9 85.0 87.4 88.6 90.1 86.3 81.2 80.2 79.6 82.0 81.4 78.6 84.2 87.6 80.0 87.1

8.4 9.8 7.4 9.7 8.8 8.0 8.6 7.9 8.3 7.0 8.5 9.7 6.7 7.9 8.5 9.2 9.0 7.2

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Table 3. Cont. Spiked Concentration (ng/mL)

Samples Stomach

100 600 4000 100 600 4000 250 2000 20000

Intestine

Rat Liver Microsome

Matrix Effect Mean (%) RSD (%) 98.6 87.4 100.2 98.5 87.3 95.3 91.5 91.0 90.3

Extraction Recovery Mean (%) RSD (%)

8.2 9.1 7.6 10.0 8.9 7.5 7.8 8.3 8.6

84.3 83.1 87.1 79.0 82.0 85.3 81.3 87.1 82.3

6.4 7.4 8.2 9.7 7.3 8.5 8.0 8.2 7.7

2.2.5. Stability The results for short-term stability, three freeze-thaw cycles and long term stability are summarized in Table 4. It is illustrated that KM was stable enough in bio-matrix. Table 4. Stability data of the analytes in rat biological samples under different conditions (n = 6). Samples Plasma

Heart

Liver

Spleen

Lung

Kidney

Stomach

Intestine

Rat liver microsome

Spiked Concentration

Stability (% RE)

(ng/mL)

Three Freeze-Thaw

Short-Term

Long-Term

Post-Preparative

25 300 4000 100 600 4000 100 600 4000 100 600 4000 100 600 4000 100 600 4000 100 600 4000 100 600 4000 250 2000 20000

4.3 −3.2 4.4 −6.2 −5.3 2.4 −4.7 2.2 3.3 −3.2 4.5 3.0 −3.1 4.2 3.5 3.0 −2.8 5.0 3.2 −4.2 4.0 4.8 4.7 −3.5 2.2 3.1 3.0

−4.7 2.3 4.7 3.0 4.8 −4.4 3.6 −4.3 4.2 2.7 3.5 −4.0 2.8 4.1 4.0 −2.7 3.1 4.7 −4.1 2.6 −3.9 3.7 −3.6 3.1 3.6 2.7 2.6

3.8 4.7 −4.9 4.0 3.0 3.2 4.1 3.9 3.1 -3.1 2.7 4.3 3.6 3.8 −2.9 3.2 4.1 4.0 4.9 4.9 −3.1 3.0 3.6 3.0 3.2 2.3 3.7

4.7 −4.4 2.5 4.4 −3.9 4.7 −4.0 4.2 4.8 3.8 −4.1 −2.2 3.6 3.7 4.4 4.9 −3.0 2.8 3.1 3.8 −4.1 4.1 4.0 4.5 3.3 3.6 3.0

2.3. Pharmacokinetics Study and Tissue Distribution The pharmacokinetics parameters, including the areas under concentration-time curve (AUC0–6 and AUC0–∞ ), mean retention time (MRT0–t and MRT0–∞ ), the half-time (t1/2z ), clearance (CLZ ), apparent volume of distribution (Vz ), and maximum plasma concentration (Cmax ) were presented in Table 5. The parameters of KM in the GU + KM group, such as Cmax , t1/2z , AUC0–6 , AUC0–∞ , and CLZ statistically differed from those in control group (The p-values for Cmax , t1/2z , AUC0–6 , AUC0–∞ , CLZ , and VZ were 0.015, 0.035, 0.019, 0.039, and 0.029, p < 0.05.). Moreover, notably, the VZ showed significant differences between the two groups (The p-values for VZ was 0.002, p < 0.01).

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The results implied that GU may have effects on reducing the exposure of KM. The mean plasma concentration-time profiles were shown in Figure 3. Table 5. Pharmacokinetics parameters of KM (10 mg/kg, intravenous administration) in rat that received or did not receive GU decoctions. Parameters

Unit

KM Group

GU + KM Group

AUC0–6 AUC0–∞ MRT0–t MRT0–∞ t1/2z CLZ VZ Cmax

µg/L * h µg/L * h h h h L/h/kg L/kg µg/L

3340 ± 410 3400 ± 420 1.1 ± 0.1 1.2 ± 0.1 1.2 ± 0.1 3.0 ± 0.4 5.0 ± 0.4 5790 ± 410

2410 ± 130 * 2600 ± 200 * 1.3 ± 0.2 1.8 ± 0.4 1.7 ± 0.3 * 3.9 ± 0.3 * 9.2 ± 0.9 ** 3600 ± 1260 *

Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01 compared with the KM group.

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Figure 3. Mean ± SD plasma concentration-time concentration-time profiles of of koumine following intravenous Figure 3. Mean ± SD (n(n= =6)6)plasma profiles koumine following intravenous administration of 10 mg/kg KM that received or did not receive Glycyrrhiza uralensis Fisch (GU) (3 g/Kg, administration of 10 mg/kg KM that received or did not receive Glycyrrhiza uralensis Fisch (GU) (3 once daily for 14 days). g/Kg, once daily for 14 days).

concentrations KMininvarious various tissues tissues for areare shown in Figure 4A. At min TheThe concentrations ofofKM forboth bothgroups groups shown in Figure 4A.15At 15 min after intravenous injection, different concentrations of KM were detected in all of the rat tissues. after intravenous injection, different concentrations of KM were detected in all of the rat tissues. It It showed that KM could be distributed widely and rapidly in various tissues. With the extent of showed that KM could be distributed widely and rapidly in various tissues. With the extent of time, time, the concentrations of KM in most of the tissues decreased obviously in 2 h. This indicated the concentrations of KM in most of the tissues decreased obviously in 2 h. This indicated that there that there was no long-term accumulation of KM, which was in accordance with the change of was no long-term accumulation of KM, which was in accordance with the change of plasma plasma concentration. For the control group, the highest concentration level was observed in intestine concentration. For the control group, the (5893.27 highest ±concentration observed intestine (6109.35 ± 1795.14 ng/g), followed by lung 1383.72 ng/g),level spleenwas (2781.9 ± 348.6in ng/g), (6109.35 ± 1795.14 ng/g), followed by lung (5893.27 ± 1383.72 ng/g), spleen (2781.9 ± 348.6 kidney (2571.9 ± 82.2 ng/g), liver (2402.2 ± 1406.0 ng/g) heart (1623.2 ± 477.8 ng/g), and stomachng/g), kidney (2571.9 ± 82.2 ng/g), liver (2402.2 1406.0 ng/g) heart (1623.2 ± 477.8 stomach (655.6 ± 315.6 ng/g), which implied that±intestine and lung might be the targetng/g), organsand of KM. That is(655.6 ± 315.6 ng/g), implied that mighteffect be the target organs of KM.[11]. That is probably thewhich pharmacokinetics basisintestine of KM for and goodlung therapeutic on digestive system tumors The higher concentration of kidney and liver demonstrated that KM was mainly accumulated in liver probably the pharmacokinetics basis of KM for good therapeutic effect on digestive system tumors excretion might beof a main elimination for KM. Inthat addition, there was a accumulated statistic [11].and Therenal higher concentration kidney and liverroute demonstrated KM was mainly difference for the tissue concentration for lung and stomach between the two groups (p