Comparative pharmacokinetic study of five flavonoids in normal rats and rats with gastric ulcer following oral administration of Mongolian medicine, Shudage-4 by UPLC – ESI – MS/MS

Purpose: To develop a simple, rapid and sensitive ultra-performance liquid chromatography electrospray ionization-mass spectrometry (UPLC–ESI–MS/MS) method was developed and fully validated for the simultaneous determination of galangin, kaempferide, galangin-3-methylether, kaempferol and quercetin in rat plasma after oral administration of Mongolian Medicine, Shudage-4 extracts. Methods: The galangin, kaempferide, galangin-3-methylether, kaempferol and quercetin were separated on a C18 column using 0.1 % formic acid at a flow rate of 0.4 mL / min and detected by a mass spectrometer in negative-ion mode with selected reaction monitoring (SRM) mode. Plasma samples were processed with a simple deproteinization technique using ethyl acetate and acetonitrile. Following the protein precipitation, the plasma samples were evaporated under gentle stream of nitrogen and analyzed by above method. Naringin was used as an internal standard (IS). Method validation was performed according to the Chinese Food and Drug Administration guidelines. Results: A good linearity (r2 ≥ 0.9990) was showed by the UPLC – ESI – MS / MS method, the low limits of quantification for galangin, kaempferide, galangin-3-methylether, kaempferol and quercetin were 229.8, 78.8, 32.0, 123.7 and 137.8 ng / mL, respectively. The results of inter-day and intra-day precisions met the experimental requirement (< 7.8 %). The matrix effect and recovery efficiency of the five analytes were more than 72.9 and 88.7 % respectively. The stability of the analytes were satisfactory. The UPLC – ESI – MS / MS method has been used for the five analytes’ pharmacokinetics study successfully after gastrointestinal route of the Mongolian Medicine Shudage-4. The pharmacokinetic parameters showed significant differences (P < 0.05) between the normal and gastric ulcer groups. The metabolism and transport of the five analytes in gastric ulcer rates were faster than in normal rats after administration of Shudage 4 extract. Double-peak phenomenon appeared in galangin, galangin – 3 methylether and quercetin. Conclusion: The results suggest that the metabolism and transport of Mongolian Medicine Shudage-4 in gastric ulcer rats is faster than in normal rats and may be enriched and acted on at the lesion site.


INTRODUCTION
On account of the cold climate, meal-rich and wine-rich diet in Mongolia area, stomach diseases occur frequently. Over the years, the Mongolian people have accumulated rich experience in treatment of stomach diseases, and some unique Mongolian medicines have shown good curative effects in the treatment of stomach diseases.
Several analytical methods based on high performance liquid chromatography (HPLC), ultraviolet spectrophotometry (UV), liquid chromatograph-mass spectrometer (LC-MS) have been reportedly used to evaluate the main flavonoids in rat plasma. In order to better separation in plasma samples, HPLC and UV methods not only required a long chromatographic process to avoid the interference, but also the unsatisfactory sensitivity for determinations was the most important problem. Furthermore, not all flavonoids have active chromophores at appropriate wavelengths or fluorescent agents [11]. Ultra-performance liquid chromatographyelectrospray ionization-mass spectrometry (UPLC-ESI-MS/MS) method is currently attracting considerable interest as a simple and sensitive method in qualitative and quantitative analysis, which can overcome most of the abovementioned limitations [12].
Pharmacokinetic studies of Mongolian medicines play an important part in evaluating clinical efficacy and guiding rational drug usage. Pharmacokinetic studies play an important role in evaluating the rationally of Mongolian medicine prescriptions.
No reported on the pharmacokinetic of Shudage -4 extract and no simultaneous determination of galangin, kaempferide, galangin-3-methylether, kaempferol and quercetin by the UPLC -ESI -MS/MS method. In consequence, the pharmacokinetics of the above analytes in gastric ulcer model and normal rat plasma were compared.
To exert a biological effect, the herbs of prescriptions must act on the target tissues after oral administration. The pharmacokinetic studies are responsible for determine concentration of the main bioactive contents in vivo, elucidate efficacy, explain drug action, and optimize dosage. . Galangin was previously isolated and identified by MS and NMR spectra. Their purity was all above 98% analyzed by HPLC. HPLCgrade methanol and formic acid were purchased from Thermo Fisher Scientific (CN, USA).

Instrumentation and chromatographic conditions
Samples were analyzed by a TSQ quantum ultramass spectrometric detector with electrospray ionization source (ESI) (Termo Scientific, USA) coupled with a Dionex Ultimate 3000 ultraperformance liquid chromatography system. The operating conditions for mass were as follows: 3000 V for spray voltage, 300 ℃ for vaporizer temperature, 35 AU for sheath gas (Arb) pressure; 10 AU for auxiliary gas (Arb) pressure; 5 AU for ion sweep gas (Arb) pressure, and 300 ℃ for capillary temperature. All the operations were under the condition of Xcalibur software (Thermo Scientific, USA).
The stock solutions and spiking blank plasma were mixed to produce a series of working solutions, which were 229.

Induction of model rats
All animal protocols were approved by the Committee of Inner Mongolia medical university on Ethics of Animal Experiments (approval no. YKD20190331). Animals were treated in accordance with Guide for the Care and Use of Laboratory Animals (8th edition, National Academies Press). Wistar rats were housed in environmentally controlled quarters (about 23 ~ 25 ℃) under a light-dark cycle (12 h: 12 h) for seven days before experiment. Twelve Wistar rats (7 ~ 8 weeks old, weighing 280 ~ 300g) were split into two, half the rats were put into model group, and another half in normal group. The model group rats were given a 56 % Chinese spirits 3 ml/day for three days by intragastric administration and the normal group rats were given physiological saline.

Plasma sample preparation
Frozen plasma was thawed at room temperature, and 400 μL rat plasma in a 10 mL of polypropylene tube was mixed with 20 μL hydrochloric acid (2 mol / mL) and vortexed for 30 s. Then the mixture was added into 100 μL of IS solution (0.986 mg / mL) and vortexed for 60 s. After being placed in a water bath at 80 ℃ for 30 min and the addition of ethyl acetate, the mixed solution was vortexed for 60 seconds and centrifuged for 10 minutes at 3000 × g. The supernatants were transferred into other polypropylene tubes, and the residues were centrifuged at 3000 × g for 10 min after adding acetonitrile and vortexing for 60 s. The two supernatants were merged firstly and then filtrated by microfiltration membrane, and finally dried under a nitrogen stream. Being dissolved in 500 μL of methanol the residues were vortexed and 20 μL solutions were analyzed in the UPLC-ESI-MS/MS system [15][16][17]. The samples were immediately centrifuged at 3000 × g for 10 min and stored at -20 ºC until analysis.

Method validation
Based on the China Food and Drug Administration guidelines, the method was validated with respect to specificity, linearity and sensitivity, precision and accuracy, stability, and recovery.

Specificity
The specificity was performed by comparing the selected reaction monitoring (SRM) chromatograms of blank plasma and spiked plasma samples (including galangin, galangin-3methylether, kaempferide, kaempferol, quercetin and naringin (IS).

Linearity and sensitivity
Seven non-zero points of standard plasma samples were assessed in duplicate and analyzed in non -stop for 3 days for preparation of calibration curves. A weighted linear-squares regression model was used to establish the peak area ratio of the concentration of analytes versus standard plasma samples. The sensitivity was assessed by the lowest concentration on the calibration curve with a signal -to -noise (S / N) ≥ 10 by analyzing six replicates.

Precision and accuracy
Intra-and inter-day precision and accuracy of the analyzed samples were selected at three levels (high, medium, and low) in five replicates on 3 non -stop days. The precision and the accuracy were determined by the relative standard deviation (RSD) and the percent relative error (RE %) respectively, and within ± 15 % for the analyzed samples.

Stability
The stability of the five analytes in rat plasma was subjected to three different circumstances (storage for 12 h at ambient temperature, store at -20 ℃ for three days, three cycles for freeze / thaw) at three different concentrations (high, medium, and low). All the samples were freshly prepared and the percentage deviation within ± 15 % were considered stable.

Extraction recovery and matrix effects
Matrix effects was assessed by comparing the peak areas of standard solutions spiked after extraction with those of the corresponding solution mixture (including galangin, galangin -3methylether, kaempferide, kaempferol, quercetin and IS) at three replicates concentrations (low, medium and high) in the same level. By comparing the peak areas of extracted samples (low, medium and high concentrations) with that of the standard solutions at the same concentrations with three replicates, the analytes' extraction recovery were assessed.

LC-MS/MS for quantitative analysis
The selected reaction monitoring (SRM) mode was selected for detection in the negative-ion mode. The optimized mass spectrometric parameters and mass spectra for all the analytes are shown in Figure 1, Figure 2 and

Specificity
As shown in Figure 4, Figure 5 and Figure 6, the specificity was assessed by comparing the chromatograms of blank plasma, spiked plasma samples and IS with good separation. The retention times were 14.46, 15.02, 14.14, 10.49 and 9.36 min respectively for the five analytes. IS was 7.38 min. There was no interference between the five compounds and IS.

Linearity and sensitivity
The linear equations of galangin, galangin-3methylether, kaempferidel, kaempferol, quercetin and their low limits of quantification (LLOQs) were listed in Table 1. The correlation coefficients (r 2 ) were all above 0.99 for the five compounds and represents good linearity for the five analytes.

Precision and accuracy
The intra and inter day precision of the five compounds were shown in Table 2, and RSD % were all less than 15 %. For the simultaneous quantitative analysis of the five analytes in rat plasma, the present method was feasible.

Stability
The stability of the five compounds were assessed under different conditions, including storage for 12 h at ambient temperature, store at -20 ℃ for 3 days, and three cycles for freeze/thaw. The UPLC -ESI -MS / MS method was satisfied in stability for the simultaneous determination for the five analytes in rat plasma.

Recovery
As shown in Table 3, all the extraction recoveries were higher than 88.7 % at three levels. No significant matrix effect was observed and the matrix ratios were in range of 72.9 -98.8 % for plasma samples.

Pharmacokinetic comparison
The pharmacokinetic process of galangin, galangin -3 -methylether, kaempferide, kaempferol and quercetin in normal and gastric ulcer rats were shown in Figure 3 and Table 4. It was found that the metabolism and transport of the five analytes in gastric ulcer rats were faster than that in normal rats after intra gastric administration of Shudage -4 extracts. For galangin, kaempferide and kaempferol, the pharmacokinetic characteristics of elimination rate constant (K) in normal rats were 9.83 ± 2.08, 29.65 ± 2.30, and 95.87 ± 17.38 (1 / min), whereas these values in gastric ulcer rats were 1.36 ± 0.43, 5.83 ± 1.80, and 40.01 ± 10.52 (1 / min), which were significantly lower (P < 0.01) than the normal group.

DISCUSSION
It has been used to treat stomach for thousands of years in Mongolia. A series of studies on the Mongolian medicine Shudage -4 has been conducted in the early stage by our groups, and the results of pharmacological experiments show that Shudage -4 has a good effect on gastritis and gastric ulcer [13][14][15]. Phytochemical studies have revealed flavonoids are one of the main ingredients in Shudage -4 [6][7][8]. The total flavonoids extracts of Shudage -4 was purify and enriched by AB -8 macroporous adsorptive resins in our research, and the content of total flavonoids were over 80% [6 -8]. The contents of galangin, galangin-3-methylether, kaempferide, kaempferol and Quercetin were all high determined by using HPLC [6][7][8]. Meanwhile, the five flavonoids ingredients had good pharmacological activities, which could be used as the representative components of Shudage-4 in the treatment of gastric diseases [13][14][15].
The pharmacokinetic parameters, including K for galangin, t1/2, K and AUC for galangin -3methylether, C max, K and AUC for kaempferide and kaempferol, C max and AUC (0-t) for quercetin showed significant difference (P < 0.05) in the normal and gastric ulcer rats. The metabolism and transport of the Mongolian Medicine Shudage -4 in gastric ulcer rats were faster than that in normal rats and one possible reason for these differences may be flavonoids ingredients of Shudage -4 concentrated and consumed in the lesion site faster than the normal group. Double peaks of galangin, galangin -3methylether, and quercetin are shown in Figure 7 A, B and E, and the results might be due to enterohepatic circulation. Flavonoids enter the blood circulation, firstly, they are carried from the liver to the small intestine, which process needs to pass through the bile duct; then be absorbed into blood by the gastrointestinal system [18]. Double-peak phenomenon of quercetin is controversial. According to one study, no doublepeak on quercetin [19], which is inconsistent with our results. Analysis of the cause may be due to the drug interactions. Drug interactions may influence the absorption and elimination of flavonoids.

CONCLUSION
The UPLC-ESI-MS/MS-based assay for the determination of galangin, kaempferide, galangin -3 -methylether, kaempferol and quercetin at the same time from effective fraction of the Mongolian Medicine Shudage-4 in plasma has been successfully developed. All results of the methodology met the criteria of the guidelines of Chinese Food and Drug Administration (CFDA). Thus, the method is specific, sensitive, precise and stable. It was applied to the pharmacokinetic studies of the five flavonoids successfully from effective fraction of Mongolian Medicine Shudage -4 in rat plasma. The in vivo pharmacokinetic data obtained in rat plasma also suggest the developed method would be suitable for use in research and clinical conditions.