Huanyu Guanab,
Xiaoming Wanga,
Shiping Wangb,
Yang Hea,
Jiajing Yuea,
Shanggao Liaob,
Yuanda Huanga and
Yue Shi*a
aInstitute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China. E-mail: shiyue1029@126.com; Fax: +86-10-57833270; Tel: +86-10-57833255
bSchool of Pharmaceutical Sciences, Guizhou Medical University, Guiyang 550004, China
First published on 11th September 2017
Shengjiang Xiexin decoction (SXD) exerts protective effects against gastrointestinal injury induced by irinotecan hydrochloride (CPT-11). The intestinal bacteria-associated in vitro pharmacokinetics of 16 components of SXD in normal rats and those with CPT-11-induced gastrointestinal toxicity were compared in this study. A sensitive and reproducible ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method was developed for the quantification of 16 components of SXD in a rat intestinal bacteria incubation system, using naringin, naringenin and tetrahydropalmatine as internal standards (ISs). The samples were prepared via salting-out assisted liquid–liquid extraction (SALLE) with NaCl to reduce matrix effects. Chromatographic separation was performed on a sub-2 μm analytical column with acetonitrile and 0.1% aqueous formic acid as mobile phase. All of the analyzed components and ISs were detected via multiple reaction monitoring (MRM) scanning with electrospray ionization. The proposed method was successfully applied for the in vitro pharmacokinetic analysis of the multiple components of a complex mixture consisting of a traditional Chinese medicine (TCM) and an intestinal bacterial incubation system. The pharmacokinetic parameters of some flavonoid glycosides and aglycones in the rats with CPT-11-induced gastrointestinal toxicity were significantly different (p < 0.05, p < 0.01) from those in the normal rats, which suggested that consumption of CPT-11 could qualitatively and/or quantitatively alter the intestinal bacteria as well as the metabolic activities of enzymes. The in vitro pharmacokinetic analysis of these components in the intestinal bacterial incubation system provided valuable information for achieving a deeper understanding of the mechanisms involved in the alteration of intestinal bacteria induced by CPT-11 and further in vivo pharmacokinetic research on SXD. The intestinal bacteria-based pharmacokinetic method could benefit the study of interactions between TCMs and chemical drugs in clinical use.
Traditional Chinese Medicine (TCM) has been used to treat or prevent cancer-related symptoms and chemotherapy-associated toxicity for thousands of years. Most of TCM are orally administered in the form of decoctions and are therefore inevitably brought into contact with bacteria and enzymes in the alimentary tract. As described in “Shang Han Lun”, Shengjiang Xiexin decoction (SXD) is a classic TCM formula to be used for the treatment of gastroenteritis, ulcerative colitis and diarrhea,14 consisting of eight herbs in the ratio of 9:
9
:
3
:
12
:
3
:
9
:
9
:
12 on a dry weight basis: Pinellia ternata (“banxia” in Chinese, the rhizome of P. ternata (Thunb.) Breit.), Glycyrrhiza uralensis (“gancao” in Chinese, the radix of G. uralensis Fisch.), Coptis chinensis (“huanglian” in Chinese, the rhizome of C. chinensis Franch.), Ziziphus jujuba (“dazao” in Chinese, the fruit of Z. jujuba Mill.), Zingiber officinale (“ganjiang” in Chinese, the rhizome of Z. officinale Rosc.), Scutellaria baicalensis (“huangqin” in Chinese, the radix of S. baicalensis Georgi.), Codonopsis pilosula (“dangshen” in Chinese, the radix of C. pilosula (Franch.) Nannf.) and Zingiberis recens (“shengjiang” in Chinese, the rhizome of Z. recens.). The combination of these herbs is based upon the rule of “Jun-Chen-Zuo-Shi”, known as “Emperor–Minister–Assistant–Courier”. Among them, C. chinensis and S. baicalensis serve as “Jun” and “Chen” to alleviate the gastrointestinal toxicity, respectively.15
Regarding modern clinical practice, when patients in several hospitals were orally administered SXD two days prior to the initiation of chemotherapy to prevent CPT-11-induced gastrointestinal toxicity, it was found that this treatment reduced the incidence of diarrhea.16 Moreover, SXD has been reported to regulate the CPT-11-induced apoptosis and necrosis of intestinal mucosal and functional cells.17 It is also noteworthy that SXD can decreased the activity of β-glucuronidase after irinotecan administration.18 Baicalin, a known flavonoid in SXD, is a β-glucuronidase inhibitor19 that inhibits the uptake of SN-38 in a concentration-dependent manner.20 Moreover, as a traditional medicines, SXD is composed of multiple components, and the flavonoids, alkaloids and triterpenoid saponins in SXD are considered the most important active components of the mixture.21–24 Some flavonoids, alkaloids and saponins can be transformed by intestinal bacteria25–28 to their metabolites, which exhibit different pharmacological activities. However, the intestinal bacteria-associated pharmacokinetics of these components in vitro are not clear. In addition, the co-existence of multiple compounds in TCMs and chemical drugs may lead to the intestinal bacteria-based metabolic and pharmacokinetic interactions. There are few available studies on such interactions involving intestinal bacteria because of the complexity of the chemical components of TCM and the intestinal bacteria system.
In a previous study, we carried out the simultaneous quantification of 14 constituents of SXD using UFLC-MS/MS.29 An analytical method has also been developed for the simultaneous quantification of flavonoids, alkaloids and triterpenoid saponins in Banxia xiexin decoction, which is analogous to SXD formula.30 Analytical conditions for the individual determination of several flavonoids,21,28,31,32 alkaloids,33,34 and triterpenoid saponins35,36 in biological matrices have been reported. However, there is little available information about the quantification and in vitro pharmacokinetics of flavonoids, alkaloids and triterpenoid saponins from TCM formulas in complex intestinal bacterial incubation systems. In addition, no data on the intestinal bacteria-associated pharmacokinetics of the major components of SXD under interaction with CPT-11 have been reported.
In the present study, a sensitive, specific and precise method was established for the simultaneous determination of oroxylin A, baicalin, baicalein, wogonoside, wogonin, chrysin, scutellarin, isoliquiritin, isoliquiritigenin, berberine, coptisine, palmatine, jatrorrhizine, glycyrrhizic acid, liquiritin and liquiritigenin (Fig. 1) in an in vitro rat intestinal bacterial incubation system, via one sample preparation combined with two chromatographic conditions. The method was validated and utilized to compare the intestinal bacteria-associated pharmacokinetics of 16 components of SXD in vitro between normal rats and those with CPT-11-induced gastrointestinal toxicity.
HPLC-grade methanol and acetonitrile were obtained from Honeywell Burdick & Jackson Company (Morristown, NJ, USA). Formic acid (MS grade) was purchased from Fisher Scientific (Madrid, Spain). Deionized water for HPLC analysis was prepared using a Milli-Q water purification system (Millipore, Milford, MA, USA). All other reagents were of analytical grade.
The UHPLC separation was achieved on an ACQUITY UPLC® BEH C18 column (2.1 mm × 100 mm, 1.7 μm) using acetonitrile (A) and 0.1% aqueous formic acid (B) as the mobile phase at a flow rate of 0.3 mL min−1. The injection volume was set to 10 μL. The auto-sampler was conditioned at 10 °C. All analyzed components were quantified in multiple reaction monitoring (MRM) mode. The optimized conditions were as follows: curtain gas (CUR): 10.0 psi; collision gas (CAD): medium; IonSpray voltage (IS): −4500 V (in negative ionization mode) and 4500 V (in positive ionization mode); source temperature: 500 °C; GS1: 40 psi; and GS2: 40 psi. The MS/MS transitions (m/z), declustering potentials (DP), collision energies (CE), entrance potentials (EP) and collision cell exit potentials (CXP) of the analyzed components and ISs are listed in Table 1. Two gradient elution programs were employed for different compounds in different ion modes. Oroxylin A, baicalin, baicalein, wogonoside, wogonin, chrysin, scutellarin, glycyrrhizic acid, liquiritin, liquiritigenin, naringin (IS1) and naringenin (IS2) were detected in negative ionization mode with elution program I: 5–5% A at 0–1 min; 5–15% A at 1–3 min; 15–15% A at 3–5 min; 15–20% A at 5–8 min; 20–20% A at 8–11 min; 20–35% A at 11–15 min; 35–45% A at 15–20 min; 45–100% A at 20–23 min; 100–100% A at 23–26 min; 100–5% A at 26–26.1 min and 5–5% A at 26.1–28 min, while isoliquiritin, isoliquiritigenin, berberine, coptisine, palmatine, jatrorrhizine, naringenin (IS2) and tetrahydropalmatine (IS3) were detected in the positive ionization mode with elution program II: 5–5% A at 0–1 min; 5–15% A at 1–3 min; 15–15% A at 3–5 min; 15–20% A at 5–9 min; 20–20% A at 9–12 min; 20–25% A at 12–16 min; 25–45% A at 16–21 min; 45–100% A at 21–23 min; 100–100% A at 23–26 min; 100–5% A at 26–26.1 min and 5–5% A at 26.1–28 min.
Compounds | Precursor | Production | DP (V) | CE (V) | EP (V) | CXP (V) |
---|---|---|---|---|---|---|
Oroxylin A | 282.9 [M − H]− | 267.8 | −90 | −26 | −8 | −19 |
Baicalin | 445.1 [M − H]− | 269.0 | −70 | −31 | −10 | −18 |
Baicalein | 268.9 [M − H]− | 222.8 | −130 | −32 | −8 | −16 |
Wogonoside | 459.0 [M − H]− | 267.9 | −90 | −43 | −11 | −19 |
Wogonin | 283.0 [M − H]− | 267.8 | −82 | −25 | −11 | −18 |
Chrysin | 252.9 [M − H]− | 142.9 | −130 | −37 | −10 | −10 |
Scutellarin | 461.0 [M − H]− | 285.0 | −95 | −30 | −8 | −20 |
Glycyrrhizic acid | 821.3 [M − H]− | 351.0 | −160 | −56 | −10 | −25 |
Liquiritin | 417.2 [M − H]− | 255.0 | −110 | −28 | −11 | −18 |
Liquiritigenin | 255.0 [M − H]− | 134.9 | −110 | −21 | −10 | −9 |
Isoliquiritin | 419.0 [M + H]+ | 257.1 | 132 | 25 | 10 | 20 |
Isoliquiritigenin | 257.1 [M + H]+ | 137.0 | 100 | 33 | 12 | 12 |
Berberine | 336.0 [M]+ | 320.1 | 105 | 41 | 9 | 24 |
Coptisine | 320.1 [M]+ | 292.0 | 95 | 40 | 8 | 20 |
Palmatine | 352.2 [M]+ | 336.0 | 100 | 40 | 11 | 26 |
Jatrorrhizine | 338.1 [M]+ | 322.1 | 100 | 40 | 5 | 26 |
Naringin (IS1) | 579.1 [M − H]− | 271.0 | −165 | −44 | −11 | −19 |
Naringenin (IS2) | 270.9 [M − H]− | 150.9 | −153 | −26 | −13 | −9 |
273.1 [M + H]+ | 153.0 | 110 | 32 | 9 | 12 | |
Tetrahydropalmatine (IS3) | 356.1 [M + H]+ | 192.1 | 120 | 35 | 9 | 16 |
Quality control (QC) samples for each compound were prepared by spiking 100 μL of the standard working solutions into 1 mL of the blank incubation solution for intestinal bacteria inactivated by acetonitrile/water-saturated n-butanol (1:
1, v/v), to obtain the following concentrations (LLOQ, low, medium and high concentrations): 10, 20, 100 and 400 ng mL−1 for oroxylin A; 20, 40, 200 and 800 ng mL−1 for baicalin, baicalein, wogonoside and wogonin; 2, 4, 40, and 160 ng mL−1 for chrysin and jatrorrhizine; 3, 6, 60 and 240 ng mL−1 for scutellarin; 12, 24, 120 and 480 ng mL−1 for glycyrrhizic acid; 8, 16, 160 and 640 ng mL−1 for liquiritin; 10, 20, 200 and 800 ng mL−1 for liquiritigenin; 3, 6, 60 and 240 ng mL−1 for isoliquiritin; 25, 50, 200 and 400 ng mL−1 for coptisine and palmatine; 50, 100, 400 and 800 ng mL−1 for berberine and 1, 2, 20 and 80 ng mL−1 for isoliquiritigenin.
The animals were randomly divided into group GT and C (n = 15). CPT-11 was administered intravenously (i.v.) at a dose of 60 mg per kg per day to the group GT rats via the tail vein for four consecutive days,3 while corresponding administration of saline was performed in group C rats. Body weight and diarrhea scores3 were monitored throughout the experimental period. Briefly, the severity of diarrhea was scored as follows: 0, normal; 1, soft feces or small black feces; 2, muddy feces; 3, watery feces or mucous feces. Animals were sacrificed by cervical dislocation under anesthesia. The colonic contents were collected aseptically in a sterile container 72 h after the final administration.
The intestinal bacterial mixture was inoculated into GAM broth in the presence of SXD extract (351.6 μg mL−1) in a ratio of 1:
4 (v/v). The cultured mixture was incubated anaerobically at 37 °C. Finally, a 1 mL aliquot of the cultured mixture was taken out, and the reaction was terminated by adding an equivalent volume of acetonitrile/water-saturated n-butanol (1
:
1, v/v) at 0, 2, 4, 6, 8, 12, 16, 20, 24, 28, 32, 36 and 48 h.
For all 16 components, each calibration curve was constructed by plotting the peak area ratio of the analyzed component to the IS versus the nominal concentration of the analyzed component using a 1/x-weighted linear least-square regression model. The LLOQ was the lowest concentration of the analyzed component on the calibration curve with an acceptable accuracy and precision. The accuracy (relative error, RE) of the LLOQ sample was within ±20% and the precision (relative standard deviation, RSD) was less than 20%.
Six batches of the blank intestinal bacterial incubation solution inactivated by acetonitrile/water-saturated n-butanol from individual rats were used to prepare QC samples at a low and a high level of concentration to evaluate the relative matrix effect. The matrix factor (MF) of each analyzed component for each batch was determined by calculating the ratio of the peak area of the analyzed component in the present of matrix to that in pure standard solutions. The IS-normalized MF was calculated by dividing the MF of the analyzed component by the MF of the IS. The RSD of the IS-normalized MF calculated from the six batches of the present matrix should not exceed 15%.
To obtain the 16 analyzed components with minimal matrix interference, PPT (acetone and acetonitrile), LLE (ethyl acetate and water-saturated n-butanol), solid-phase extraction (SPE) (Oasis HLB and Agela Cleanert PEP-SPE) and SALLE (acetonitrile with NaCl as the salting-out reagent and acetonitrile/water-saturated n-butanol (1:
1, v/v) with NaCl) were evaluated. PPT using acetone or acetonitrile and LLE using water-saturated n-butanol were not considered because the ion intensities of the analyzed components were reduced due to matrix effects, although the extraction recoveries were relatively high. LLE using ethyl acetate and SALLE using acetonitrile and NaCl provided low extraction recoveries. Although the recovery range of SPE was acceptable, it was time consuming and cost prohibitive. Moreover, matrix interference were not completely eliminated for all of the analyzed components when SPE was performed.
SALLE using acetonitrile/water-saturated n-butanol (1:
1, v/v) with NaCl could provide relatively high extraction recoveries, low matrix interferences and good repeatability for most of analyzed components satisfying the requirements of quantification for determination of pharmacokinetic parameters. Finally, the SALLE method was performed using acetonitrile/water-saturated n-butanol (1
:
1, v/v) and NaCl to prepare the samples.
However, quantification of glycyrrhetic acid, a metabolite of glycyrrhizic acid by intestinal bacteria, was compromised in this study due to its low recovery. As a saponin aglycone, the low polarity of glycyrrhetic acid makes its extraction difficult from bacterial incubation solution using acetonitrile/water-saturated n-butanol (1:
1, v/v) as the extract solvent. Fortunately, the recoveries of glycyrrhetic acid and other target components in further study can be improved by controlling the pH values and choosing appropriate solvents (acetone, methanol, ethanol and acetonitrile, etc.) for SALLE.
Compared with conventional LLE and PPT, SALLE in the present study provided a cleaner extract, effectively removing macromolecules. High extraction efficiencies were obtained for all 16 components of SXD via SALLE without the occurrence of emulsification. Compared with SPE, SALLE used much less solvent and was much faster and simpler to perform. Moreover, this was the first time that SALLE has been performed using acetonitrile/water-saturated n-butanol (1:
1, v/v) with NaCl for the extraction of target components in bacterial incubation solution.
To achieve the maximum sensitivity and response, the precursor and product ion pairs for MRM detection, as well as their corresponding DP, CE, EP and CXP values, were optimized for the quantification of each analyzed component. The results are given in Table 1.
To improve resolution and decrease runtime, methanol, acetonitrile, ammonium acetate and formic acid were tested as potential mobile phases. Acetonitrile and 0.1% aqueous formic acid were employed as the mobile phase because the best separation of all the analyzed components from each other and the minimal influence of the matrix effect were achieved. Moreover, to improve the separation efficiency for the four alkaloids and isoliquiritin, the chromatographic condition II (in which the proportion of the organic phase between 12 and 16 min was decreased compared with that in the chromatographic condition I) was performed to obtain a better separation for berberine, coptisine, palmatine, jatrorrhizine and isoliquiritin.
Components | Calibration equation | r | Linear range (ng ml−1) | LLOQ (ng ml−1) |
---|---|---|---|---|
Oroxylin A | y = 0.0518x + 0.558 | 0.9994 | 10–500 | 10 |
Baicalin | y = 0.0125x + 0.071 | 0.9987 | 20–1000 | 20 |
Baicalein | y = 0.00431x + 0.0765 | 0.9989 | 20–1000 | 20 |
Wogonoside | y = 0.0129x + 0.0155 | 0.9983 | 20–1000 | 20 |
Wogonin | y = 0.0195x + 0.167 | 0.9988 | 20–1000 | 20 |
Chrysin | y = 0.0173x + 0.0127 | 0.9993 | 2–200 | 2 |
Scutellarin | y = 0.00653x + 0.00312 | 0.9990 | 3–300 | 3 |
Glycyrrhizic acid | y = 0.00378x + 0.006 | 0.9992 | 12–600 | 12 |
Liquiritin | y = 0.00717x + 0.00829 | 0.9994 | 8–800 | 8 |
Liquiritigenin | y = 0.0234x + 0.0888 | 0.9993 | 10–1000 | 10 |
Isoliquiritin | y = 0.00153x + 0.00267 | 0.9996 | 3–300 | 3 |
Isoliquiritigenin | y = 0.0249x + 0.00376 | 0.9996 | 1–100 | 1 |
Berberine | y = 0.0115x + 0.127 | 0.9994 | 50–1000 | 50 |
Coptisine | y = 0.00253x + 0.00777 | 0.9993 | 25–500 | 25 |
Palmatine | y = 0.00623x + 0.0138 | 0.9996 | 25–500 | 25 |
Jatrorrhizine | y = 0.00585x + 0.00156 | 0.9988 | 2–200 | 2 |
Components | Conc. (ng ml−1) | Intra-day (n = 5) | Inter-day (n = 5) | ||
---|---|---|---|---|---|
Precision (RSD, %) | Accuracy (RE, %) | Precision (RSD, %) | Accuracy (RE, %) | ||
Oroxylin A | 10 | 14.70 | −3.95 | 5.02 | −2.44 |
20 | 8.60 | −5.02 | 4.77 | −8.95 | |
200 | 3.30 | 0.58 | 4.49 | −4.26 | |
400 | 3.27 | −2.38 | 6.15 | −7.46 | |
Baicalin | 20 | 19.22 | −14.70 | 18.96 | −5.90 |
40 | 2.76 | −1.98 | 7.59 | −6.34 | |
200 | 4.09 | 7.00 | 6.30 | 0.07 | |
800 | 7.45 | −5.05 | 5.33 | −9.25 | |
Baicalein | 20 | 11.74 | 10.20 | 7.68 | 16.50 |
40 | 4.59 | 4.30 | 10.17 | −6.26 | |
200 | 2.40 | 8.25 | 2.97 | 4.94 | |
800 | 3.61 | −7.58 | 5.67 | −10.21 | |
Wogonoside | 20 | 14.31 | −7.80 | 6.68 | −4.06 |
40 | 1.87 | −1.53 | 7.30 | −4.10 | |
200 | 5.57 | 5.38 | 4.87 | 0.54 | |
800 | 3.62 | −2.40 | 13.31 | −8.45 | |
Wogonin | 20 | 8.00 | −14.98 | 3.16 | −17.43 |
40 | 7.85 | −12.43 | 2.56 | −12.62 | |
200 | 2.26 | 12.00 | 5.11 | 6.70 | |
800 | 4.17 | 1.13 | 10.08 | −4.69 | |
Chrysin | 2 | 8.37 | −4.83 | 6.25 | −7.78 |
4 | 1.87 | −1.95 | 7.93 | −3.54 | |
40 | 2.12 | −2.55 | 2.89 | 0.33 | |
160 | 4.41 | −0.95 | 7.55 | −4.45 | |
Scutellarin | 3 | 19.34 | −0.70 | 9.40 | 10.90 |
6 | 2.53 | 3.75 | 7.17 | 2.88 | |
60 | 6.48 | −13.68 | 3.80 | −10.00 | |
240 | 4.69 | −9.53 | 6.26 | −4.58 | |
Glycyrrhizic acid | 12 | 4.24 | −11.95 | 3.75 | −9.55 |
24 | 2.47 | −13.90 | 1.50 | −12.98 | |
120 | 4.04 | −14.85 | 6.33 | −10.86 | |
480 | 3.65 | −8.80 | 1.12 | −8.07 | |
Liquiritin | 8 | 9.89 | 5.63 | 13.81 | −1.05 |
16 | 7.69 | −3.85 | 4.61 | −8.54 | |
160 | 7.93 | −10.23 | 14.28 | −5.81 | |
640 | 3.90 | 6.36 | 7.96 | −14.07 | |
Liquiritigenin | 10 | 5.58 | −7.65 | 1.72 | −8.76 |
20 | 4.93 | 0.88 | 4.72 | −2.45 | |
200 | 2.56 | 2.88 | 2.56 | 5.96 | |
800 | 1.38 | 0.58 | 8.86 | 0.43 | |
Isoliquiritin | 3 | 4.72 | 13.00 | 8.40 | 6.67 |
6 | 5.76 | −4.58 | 9.41 | −5.72 | |
60 | 5.32 | −9.90 | 14.23 | −2.91 | |
240 | 5.87 | −9.62 | 4.94 | −7.06 | |
Isoliquiritigenin | 1 | 14.37 | 14.00 | 8.26 | 7.71 |
2 | 9.97 | −6.90 | 6.32 | −10.88 | |
20 | 3.00 | −8.67 | 2.48 | −7.03 | |
80 | 3.89 | 0.57 | 11.26 | −7.93 | |
Berberine | 50 | 9.47 | −8.20 | 17.86 | 5.07 |
100 | 1.90 | 2.40 | 2.97 | 5.58 | |
400 | 3.24 | 3.60 | 4.70 | 1.93 | |
800 | 3.62 | −10.42 | 11.24 | −0.24 | |
Coptisine | 25 | 9.87 | −10.58 | 16.68 | 1.38 |
50 | 7.45 | 0.60 | 2.46 | 1.51 | |
200 | 3.88 | 3.00 | 1.34 | 1.76 | |
400 | 4.41 | −9.86 | 5.86 | −4.41 | |
Palmatine | 25 | 7.54 | 14.67 | 3.58 | 11.83 |
50 | 0.82 | 9.60 | 7.31 | 4.22 | |
200 | 3.98 | −1.18 | 3.41 | −3.51 | |
400 | 4.38 | −10.62 | 9.99 | −3.83 | |
Jatrorrhizine | 2 | 7.66 | 8.25 | 4.89 | 12.13 |
4 | 9.16 | 12.76 | 6.20 | 5.23 | |
40 | 4.71 | 10.61 | 13.10 | −3.91 | |
160 | 3.94 | −6.12 | 5.17 | −0.16 |
Components | Conc. (ng ml−1) | Extract recovery | At 10 °C for 48 h | At −80 °C for 2 months | Freeze–thaw cycles | ||||
---|---|---|---|---|---|---|---|---|---|
Mean (n = 5) | RSD (%) | Remain (%) | RSD (%) | Remain (%) | RSD (%) | Remain (%) | RSD (%) | ||
Oroxylin A | 20 | 93.14 | 3.19 | 107.31 | 5.01 | 93.38 | 9.37 | 112.50 | 9.08 |
100 | 86.44 | 6.33 | 95.97 | 1.89 | 84.81 | 14.12 | 110.78 | 2.41 | |
400 | 84.46 | 7.23 | 102.37 | 2.53 | 92.07 | 9.97 | 90.38 | 2.49 | |
Baicalin | 40 | 65.62 | 4.03 | 91.10 | 4.56 | 103.51 | 7.31 | 110.50 | 9.32 |
200 | 59.72 | 1.12 | 89.89 | 4.70 | 88.30 | 3.25 | 104.46 | 6.54 | |
800 | 61.22 | 2.71 | 86.68 | 3.62 | 108.15 | 5.71 | 95.20 | 1.54 | |
Baicalein | 40 | 76.96 | 9.95 | 107.26 | 1.32 | 100.77 | 8.57 | 93.07 | 1.81 |
200 | 62.94 | 8.72 | 93.02 | 4.55 | 102.76 | 8.06 | 104.55 | 9.98 | |
800 | 65.68 | 7.06 | 85.73 | 0.14 | 96.68 | 4.09 | 97.05 | 9.13 | |
Wogonoside | 40 | 94.71 | 3.62 | 86.53 | 5.92 | 94.77 | 6.87 | 97.71 | 8.63 |
200 | 83.63 | 2.01 | 87.21 | 4.19 | 102.18 | 5.78 | 103.43 | 11.55 | |
800 | 83.23 | 1.00 | 85.55 | 3.50 | 89.95 | 1.64 | 97.27 | 3.51 | |
Wogonin | 40 | 62.26 | 2.97 | 102.11 | 1.97 | 86.27 | 7.90 | 109.68 | 4.47 |
200 | 64.90 | 4.65 | 110.14 | 1.34 | 85.77 | 0.74 | 111.11 | 5.40 | |
800 | 65.95 | 7.37 | 95.93 | 4.47 | 89.68 | 4.77 | 95.28 | 1.12 | |
Chrysin | 4 | 71.84 | 2.81 | 106.90 | 3.51 | 112.85 | 2.95 | 110.45 | 4.54 |
40 | 62.01 | 4.65 | 108.11 | 3.03 | 113.00 | 1.33 | 101.64 | 6.68 | |
160 | 63.97 | 7.95 | 109.15 | 4.93 | 106.74 | 8.49 | 88.64 | 6.45 | |
Scutellarin | 6 | 56.36 | 6.15 | 106.71 | 3.83 | 98.19 | 11.28 | 89.75 | 3.82 |
60 | 59.32 | 3.54 | 103.30 | 6.92 | 85.44 | 6.07 | 86.24 | 3.27 | |
240 | 54.50 | 2.92 | 107.98 | 5.13 | 100.46 | 6.74 | 85.83 | 2.00 | |
Glycyrrhizic acid | 24 | 86.60 | 6.83 | 85.12 | 3.50 | 97.84 | 10.79 | 110.38 | 13.30 |
120 | 70.56 | 4.05 | 91.82 | 2.33 | 91.64 | 10.64 | 110.04 | 5.94 | |
480 | 68.85 | 4.12 | 90.37 | 8.97 | 102.97 | 7.01 | 94.57 | 2.70 | |
Liquiritin | 16 | 79.04 | 7.04 | 113.68 | 1.85 | 112.32 | 9.67 | 93.61 | 3.89 |
160 | 77.91 | 7.76 | 113.54 | 4.86 | 94.57 | 6.58 | 92.43 | 7.74 | |
640 | 73.79 | 6.02 | 112.25 | 3.25 | 97.63 | 4.41 | 98.45 | 3.70 | |
Liquiritigenin | 20 | 85.43 | 2.28 | 104.35 | 8.59 | 101.14 | 3.68 | 104.79 | 8.57 |
200 | 85.85 | 3.59 | 99.16 | 1.92 | 101.58 | 2.63 | 94.97 | 2.38 | |
800 | 81.55 | 4.01 | 91.31 | 6.12 | 96.58 | 7.70 | 95.87 | 1.47 | |
Isoliquiritin | 6 | 90.79 | 2.40 | 102.33 | 1.12 | 86.34 | 2.63 | 103.13 | 5.03 |
60 | 79.62 | 3.54 | 94.83 | 0.72 | 97.06 | 5.84 | 106.74 | 1.09 | |
240 | 82.39 | 4.23 | 102.77 | 2.99 | 90.85 | 3.76 | 98.77 | 4.43 | |
Isoliquiritigenin | 2 | 72.16 | 1.68 | 105.97 | 3.31 | 86.50 | 3.79 | 88.08 | 8.38 |
20 | 61.97 | 6.46 | 118.31 | 1.34 | 91.37 | 2.85 | 98.85 | 3.87 | |
80 | 58.02 | 7.28 | 109.04 | 0.74 | 94.77 | 9.77 | 98.27 | 1.53 | |
Berberine | 100 | 76.63 | 5.39 | 94.48 | 1.12 | 87.05 | 7.11 | 102.40 | 6.89 |
400 | 89.90 | 5.77 | 90.16 | 1.52 | 87.36 | 3.09 | 96.98 | 4.52 | |
800 | 71.43 | 1.95 | 99.83 | 6.59 | 85.26 | 2.58 | 104.25 | 2.88 | |
Coptisine | 50 | 64.81 | 1.65 | 104.34 | 7.64 | 86.21 | 5.33 | 97.81 | 2.69 |
200 | 72.16 | 5.47 | 87.75 | 5.92 | 100.69 | 3.49 | 91.23 | 7.61 | |
400 | 61.47 | 4.49 | 111.55 | 10.49 | 88.29 | 5.74 | 95.17 | 1.45 | |
Palmatine | 50 | 80.02 | 4.49 | 92.19 | 3.06 | 96.76 | 2.06 | 101.13 | 5.51 |
200 | 93.30 | 8.91 | 94.34 | 2.86 | 103.46 | 1.43 | 99.49 | 4.39 | |
400 | 72.75 | 5.30 | 96.86 | 6.57 | 100.34 | 4.87 | 103.47 | 2.28 | |
Jatrorrhizine | 4 | 78.53 | 2.96 | 107.31 | 0.66 | 111.02 | 2.67 | 104.77 | 5.49 |
40 | 84.20 | 3.67 | 102.46 | 4.92 | 101.85 | 2.30 | 95.85 | 1.64 | |
160 | 68.36 | 5.93 | 108.13 | 5.05 | 98.54 | 5.39 | 101.95 | 1.80 |
Components | Conc. (ng ml−1) | RSD of IS normalised MF (%) | Components | Conc. (ng ml−1) | RSD of IS normalised MF (%) |
---|---|---|---|---|---|
Oroxylin A | 20 | 7.72 | Liquiritin | 16 | 12.9 |
400 | 14.8 | 640 | 13.2 | ||
Baicalin | 40 | 13.0 | Liquiritigenin | 20 | 6.96 |
800 | 13.5 | 800 | 10.4 | ||
Baicalein | 40 | 10.4 | Isoliquiritin | 6 | 14.1 |
800 | 8.74 | 240 | 12.8 | ||
Wogonoside | 40 | 13.1 | Isoliquiritigenin | 2 | 9.31 |
800 | 14.8 | 80 | 6.42 | ||
Wogonin | 40 | 6.54 | Berberine | 100 | 8.53 |
800 | 13.8 | 800 | 10.9 | ||
Chrysin | 4 | 10.6 | Coptisine | 50 | 10.5 |
160 | 11.8 | 400 | 9.23 | ||
Scutellarin | 6 | 12.7 | Palmatine | 50 | 7.88 |
240 | 14.4 | 400 | 8.45 | ||
Glycyrrhizic acid | 24 | 12.6 | Jatrorrhizine | 4 | 11.0 |
480 | 10.6 | 160 | 12.8 |
Components | RE (%) | RSD (%) | Components | RE (%) | RSD (%) |
---|---|---|---|---|---|
Oroxylin A | 0.98 | 6.08 | Liquiritin | 8.98 | 6.28 |
Baicalin | −2.56 | 4.71 | Liquiritigenin | −0.26 | 3.46 |
Baicalein | −1.52 | 5.33 | Isoliquiritin | 14.50 | 2.61 |
Wogonoside | −6.44 | 5.49 | Isoliquiritigenin | 4.15 | 3.19 |
Wogonin | 4.92 | 4.22 | Berberine | −10.30 | 4.58 |
Chrysin | 14.60 | 4.22 | Coptisine | −5.90 | 5.93 |
Scutellarin | 3.60 | 5.45 | Palmatine | 7.50 | 4.15 |
Glycyrrhizic acid | 1.52 | 5.30 | Jatrorrhizine | 3.52 | 5.07 |
![]() | ||
Fig. 3 Body weight changes of normal rats (C) and rats with CPT-11 induced gastrointestinal toxicity (GT). |
Compounds | Group | AUC0−t (ng h ml−1) | MRT0−t (h) | T1/2 (h) | CL (ml kg−1 h−1) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
a Statistical difference between group C and GT, *p < 0.05, **p < 0.01. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Baicalin | C | 162![]() ![]() |
16.73 ± 4.49 | 15.66 ± 9.30 | 39.44 ± 20.95 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GT | 67![]() ![]() |
9.25 ± 1.85* | 8.50 ± 5.45 | 95.27 ± 24.95** | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Wogonoside | C | 66![]() ![]() |
16.00 ± 4.39 | 14.50 ± 5.06 | 57.57 ± 17.88 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GT | 23![]() ![]() |
7.55 ± 1.89** | 6.47 ± 1.77 | 262.70 ± 160.04* | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Scutellarin | C | 2000.22 ± 755.61 | 16.67 ± 3.65 | 14.92 ± 4.75 | 100.71 ± 43.50 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GT | 1014.65 ± 461.50* | 10.42 ± 2.50* | 7.96 ± 2.28* | 220.64 ± 76.74* | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Glycyrrhizic acid | C | 37![]() |
22.05 ± 0.52 | 52.69 ± 7.89 | 8.56 ± 1.23 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GT | 41![]() |
21.88 ± 1.18 | 51.35 ± 8.95 | 8.38 ± 2.27 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Liquiritin | C | 6442.02 ± 895.80 | 7.78 ± 1.45 | 5.82 ± 0.92 | 100.27 ± 13.10 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GT | 5131.50 ± 1180.01 | 5.60 ± 0.60* | 4.73 ± 0.87 | 129.50 ± 28.79 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Isoliquiritin | C | 1542.40 ± 407.50 | 16.10 ± 1.72 | 17.58 ± 5.91 | 116.41 ± 42.74 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GT | 1066.04 ± 226.65 | 9.79 ± 1.06** | 8.17 ± 2.55* | 189.66 ± 41.56* | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Berberine | C | 39![]() |
22.73 ± 0.75 | 155.45 ± 83.45 | 16.86 ± 8.32 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GT | 37![]() |
22.92 ± 0.99 | 145.29 ± 79.21 | 18.61 ± 9.87 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Coptisine | C | 19![]() |
22.98 ± 0.72 | 140.87 ± 66.10 | 18.73 ± 9.29 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GT | 17![]() |
22.85 ± 0.96 | 125.35 ± 77.86 | 23.39 ± 13.77 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Palmatine | C | 16![]() |
23.32 ± 0.49 | 178.43 ± 65.74 | 12.15 ± 3.68 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GT | 14![]() |
23.12 ± 0.61 | 118.83 ± 57.10 | 20.31 ± 9.38 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Jatrorrhizine | C | 3909.58 ± 265.03 | 23.04 ± 0.25 | 127.33 ± 46.51 | 17.27 ± 4.63 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GT | 4017.88 ± 369.03 | 22.80 ± 0.44 | 132.27 ± 44.07 | 17.67 ± 9.28 |
Compounds | Group | AUC0−t (ng h ml−1) | Cmax (ng ml−1) | Tmax (h) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
a Statistical difference between group C and GT, *p < 0.05, **p < 0.01. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Baicalein | C | 127![]() ![]() |
4082.50 ± 1123.58 | 36.00 ± 8.64 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GT | 221![]() ![]() |
6825.00 ± 1118.23* | 32.00 ± 13.47 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Wogonin | C | 72![]() ![]() |
2189.00 ± 678.59 | 43.20 ± 6.57 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GT | 104![]() ![]() |
2714.00 ± 873.32 | 21.60 ± 17.52* | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Chrysin | C | 3324.62 ± 900.04 | 98.66 ± 17.32 | 39.20 ± 13.97 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GT | 6411.08 ± 530.95** | 174.00 ± 25.93** | 28.40 ± 15.52 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Oroxylin A | C | 11![]() |
301.20 ± 149.08 | 34.40 ± 14.31 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GT | 19![]() |
591.80 ± 129.03* | 30.40 ± 6.69 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Liquiritigenin | C | 17![]() |
550.60 ± 119.37 | 42.40 ± 12.52 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GT | 21![]() |
577.60 ± 68.70 | 32.00 ± 10.20 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Isoliquiritigenin | C | 1872.57 ± 271.96 | 51.34 ± 10.80 | 26.80 ± 15.47 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GT | 1957.04 ± 463.40 | 53.16 ± 11.26 | 23.20 ± 16.59 |
A negative control experiment was carried out to demonstrate that the changes in the concentrations of the 16 analyzed components of SXD in the intestinal bacterial incubation system were caused by the bacteria. The SXD extract was anaerobically incubated in GAM broth in the absence of the intestinal bacteria for 48 h at 37 °C, then processed and analyzed via the proposed method. The peak areas obtained at 48 h were compared with those obtained at 0 h. The observed deviations in the peak area were calculated as percentages. The results revealed that the peak area percentages of the analyzed components were within the range of 92.7% to 109.3%, with RSDs of less than 3.45% (n = 5), which implied that the concentrations of the analyzed components were not affected by the anaerobic medium broth or cultured conditions.
Among the three flavonoid glycosides (scutellarin, baicalin and wogonoside) of SXD, the CL of scutellarin was greater than that of baicalin and wogonoside in the control group. The degree of metabolism was closely related to the chemical structure. Compared with the structures of baicalin and wogonoside, scutellarin exhibit one more 4′-position hydroxyl, which contributes to its excellent degree of microbial degradation.42 The lower CL of wogonoside indicated its stability to the bacteria, which resulted from the steric hindrance of methoxyl at the 8-position. In the GT group, the significantly increased CLs of baicalin, wogonoside and scutellarin implied that the bacteria from rats with CPT-11-induced gastrointestinal toxicity catalyzed the degradations of three flavonoid glycosides. CPT-11 has been reported to increase the levels of Enterococcus spp., Clostridium spp., Escherichia coli, Serratia spp., Staphylococcus spp., Peptostreptococcus spp. and Bacillus spp. in the colon.9,10 Among them, Clostridium spp., Escherichia coli and Staphylococcus spp. produced β-glucuronidase.10 The increased level of the above three species of bacteria up-regulated the expression of β-glucuronidase. The extent of the increase in the CL of wogonoside (approximately 4.6-fold) was greater than that of baicalin (approximately 2.4-fold) and scutellarin (approximately 2.2-fold), although the three flavonoid glycosides were all hydrolyzed by bacterial β-glucuronidase. The other metabolic pathway/degree of wogonoside was presumed to be altered in GT group. Moreover, the exposure levels (AUC0−t) and the MRT0−t of baicalin, wogonoside and scutellarin decreased in the GT group, which was indicative of an increase in their biotransformation rate.
In contrast to the above flavonoid glycosides, the concentration of liquiritin (a flavanone glycoside) in group C declined rapidly, reaching 10.2% of the initial level at 20 h, while the concentration in group GT declined to 4.12% at 16 h. The metabolic rate is related to the type and site of glycosidic linkage. Moreover, liquiritin undergoes deoxygenation and acetylation by bacterial enzymes besides hydrogenation, methylation and deglycosylation which are the main metabolic pathways of baicalin, wogonoside and scutellarin.43 The MRT0−t of liquiritin decreased in the group GT, while the AUC0−t of liquiritin was not significantly different from that in group C. This observation indicated that liquiritin could be completely degraded by intestinal bacteria within 48 h in both groups. However, CPT-11 could influence the bacteria-associated metabolic pathway or/and velocity of liquiritin. The increased level of Clostridium spp. by CPT-11 was speculated to accelerate the degradation rate of liquiritin.43 Similar changes were observed in the pharmacokinetic parameters of isoliquiritin, which exhibits a similar structure to that of liquiritin.
Although glycyrrhizic acid and baicalin are both glucuronide conjugates of aglycones, the CL of glycyrrhizic acid was significantly lower than that of the flavonoid glycosides. These results were supported by the report that the β-glucuronidase hydrolyzing glycyrrhizic acid might be different from the enzyme hydrolyzing baicalin, although the two compounds are both metabolized by β-glucuronidases.44 The former aimed to hydrolyze β-D-diglucuronide, while the latter might select β-D-monoglucuronide to hydrolyze. Notably, there were no significantly differences in the pharmacokinetic parameters of glycyrrhizic acid between the two groups in the present study. It can be speculated that CPT-11 alters the activity of the β-glucuronidase hydrolyzing β-D-monoglucuronide but not the enzyme hydrolyzing β-D-diglucuronide.
The concentration–time courses of the four alkaloids showed that the degradations of alkaloids by intestinal bacteria were relatively slow, and there were no significant differences in the pharmacokinetic parameters between the two groups. The slightly increased concentration of palmatine from 32 to 48 h resulted from the biotransformation of other alkaloids in Coptis chinensis.45 The increased concentration of berberine from 36 to 48 h could be attributed to the oxidization of its metabolite dihydroberberine back to berberine.27
CPT-11 may induce changes in the metabolic behavior of glycosides and aglycones due to its impact on intestinal bacteria, which accelerates the degradation rate of glycosides to improve the production of aglycones. Increased accumulation of aglycones produced by intestinal bacteria in the intestine might mean improving absorption of aglycones and increasing bioavailability of aglycones. Flavonoid aglycones resulting from the metabolism of the corresponding glycoside by intestinal bacteria, such as baicalein, chrysin, oroxylin A and wogonin, have anti-inflammatory effects,31 which may alleviate CPT-11-induced diarrhea. Moreover, chrysin has been shown to up-regulate UGT1A1 to improve the conversion of SN-38 to SN-38G in the gastrointestinal tract.47 Although CPT-11 does not alter the bacteria-associated metabolic behavior of berberine, berberine could be transformed to its intestine-absorbable form by the intestinal bacteria and enter into the blood to exert its anti-diarrheal action.27,48 Therefore, the interactions between CPT-11, intestinal bacteria and SXD are proposed, in which CPT-11 alters the intestinal bacteria qualitatively and quantitatively and thus changes metabolic behavior of SXD, resulting in protective constituents from SXD alleviating the gastrointestinal toxicity induced by CPT-11 in turn.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ra03521g |
This journal is © The Royal Society of Chemistry 2017 |