# Measurement of Pharmacokinetics and Tissue Distribution of Four Compounds fromNauclea officinalis in Rat Plasma and Tissues through HPLC-MS/MS

Authors: Yuhuang Wu; Liyan Li; Guxu Ming; Xinyue Ma; Changfu Liang; Yonghui Li; Xiaoning He
Journal: Journal of Analytical Methods in Chemistry
 (2022)

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## Abstract

A rapid, sensitive, selective, and accurate HPLC–MS/MS method was developed and validated for the simultaneous determination of chlorogenic acid, naucleactonin C, khaephuoside A 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside in rat plasma and tissues after oral administration of Nauclea officinalis extracts. Chloramphenicol was used as an internal standard (IS). The plasma and tissue samples were extracted by protein precipitation with methanol-ethyl acetate (1 : 1, v/v) including 0.1% (v/v) formic acid. The chromatographic separation was achieved by using an C18 column with gradient elution using mobile phase, which consisted of 0.1% formic acid water (A) and acetonitrile (B) and the flow rate of 0.8 mL/min. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode utilizing electrospray ionization (ESI) in negative mode. The developed method exhibited good linearity (determination coefficients, R2 ≥ 0.9849), and the lower limits of quantification were 2, 5, 5, and 25 ng/mL for chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside. The intraday and interday precisions (relative standard deviation, RSD) were less than 12.65%, while the accuracy was ranged from 86.31 to 114.17%. The recovery rate were 51.85–97.06%, 75.99–106.68%, 77.46–105.35%, and 68.36–103.75% for chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside the matrix effects were 50.17–116.62%, 86.75–115.99%, 45.79–87.44%, and 51.60–92.34% for chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside in different matrix. The developed method was successfully applied to a pharmacokinetic study and tissue distribution of four compounds in rats after oral administration of Nauclea officinalis extracts.


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## Body

## 1. Introduction

Nauclea officinalis Pierre. ex Pitard, is one of the most commonly used traditional medicines in China and is mainly distributed in Hainan, Guangxi, Guangdong, and other provinces in the south of China [1]. Modern pharmacological studies reported that Nauclea officinalis exhibits various biological properties such as antibacterial, anti-inflammatory, and analgesic activity [2–6]. As a traditional Chinese medicine, the stems and twigs of Nauclea officinalis are used for the treatment of colds, fever, acute tonsillitis, sore throat, and other diseases [1, 7]. A number of different kinds of compounds have been isolated and identified from Nauclea officinalis, including alkaloids, phenolic acids, iridoids, pentacyclic triterpenoids, and flavonoids [8–12]. Previous studies showed that alkaloids were the main bioactive compound in Nauclea officinalis [13–15]. Recent literature reported that phenolic compounds such as protocatechuic acid and chlorogenic acid possess antioxidant and antibacterial effects [16–18]. Therefore, the phenolic compounds may be another kind of active ingredient in Nauclea officinalis.As a valuable work, the pharmacokinetics research could not only explain the absorption, distribution, metabolism, and excretion of bioactive compounds but also reveal the mechanism of action and the cause of toxicity [19]. Several reports have been published on the pharmacokinetics of the alkaloids of Nauclea officinalis. For example, the pharmacokinetics of alkaloids of Nauclea officinalis extracts and Danmu preparations in rat plasma [20–22] and the pharmacokinetics of Strictosamide in dog plasma [23]. However, as one of the main active compounds of Nauclea officinalis, the pharmacokinetics of phenolic acid compounds were rarely researched. To our knowledge, the quantitative detection of pharmacokinetics and tissue distribution of chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside of Nauclea officinalis extract have not been reported in rats. Hence, it is necessary to establish rapid and accurate approaches to investigate its pharmacokinetics and tissue distribution.The aim of the present investigation is to develop a reliable and sensitive method based on HPLC-MS/MS to quantify the four compounds ofNauclea officinalis extract in the plasma and tissue distribution of rats. It is expected that the results of this study can provide a useful reference for understanding the mechanism of action, safety evaluation, and clinical application of Nauclea officinalis.


## 2. Material and Methods

### 2.1. Chemicals and Reagents

The stems ofNauclea officinalis were collected from Qiongzhong County, Hainan Province, and identified by Prof. Jianping Tian of Hainan Medical University. Standards of naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside were isolated from Nauclea officinalis in our laboratory by silica gel column, semi-preparative high performance liquid chromatography, which structure was identified by NMR, and the HPLC purity was over than 98%. The standards for chlorogenic acid were purchased from Chengdu Pufeide Biological Technology Co., Ltd (HPLC >98%, Sichuan, China). Chloramphenicol was provided by the National Institute for Food and Drug Control (Beijing, China). Methanol and acetonitrile were all chromatographically pure and purchased from Fisher Scientific (Fair Lawn, NJ, USA); ethyl acetate and formic acid were all chromatographically pure and purchased from Aladdin Industrial Corporation (Shanghai, China); purified water was prepared by a LabTower EDI system (Thermo Scientific, USA).


### 2.2. Instrument and Analytical Conditions

Chromatographic analysis was performed on the LC-20ADXR System (Shimadzu, Japan). The Phenomenex Kinete EVO C18 100 Å (50 mm × 2.1 mm, 5μm) column was used as the analytical column maintained at 40°C with (A) 0.1% formic acid aqueous solution and (B) acetonitrile as the mobile phase. The gradient elution program was operated as follows: 2% B (0–0.5 min), 2–25% B (0.5–0.51 min), 25-55% B (0.51–3.0 min), 55-95% B (3.0–3.01 min), 95% B (3.01–5.0 min), 95–2% B (5.0–5.01 min), and 2% B (5.01–6.0 min). The flow rate was 0.8 mL/min, and the injection volume was 5 μL.Mass spectrometric detection was performed on an AB-SCIEX API 4000 mass spectrometer (AB SCIEX, Singapore) with ESI in negative ion multiple reaction monitoring (MRM) mode. The optimized instrument parameters were as follows: nebulizer gas: 50 psi, heated by N2 gas: 55 psi, ion spray voltage: −4500 V and temperature: 550°C; nebulizer, blowback gas, and collision gas were nitrogen; declustering potential (DP), collision energy (CE), and collision cell exit potential (CXP) of the four analytes and chloramphenicol (IS) are shown in Table 1.Table 1
Optimized multiple reaction monitoring parameters for the detection of four analytes and IS.
CompoundsRetention time (min)Precursor ion (m/z)Quantitative ion (m/z)DP (V)CE (V)CXP (V)Chlorogenic acid1.02353.0190.9−51−22.9−10.5Naucleactonin C1.65335.1182.9−110−37−9Khaephuoside A0.99477.1293.0−98−18−63,4-Dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside0.97447.1232.9−84−25−14Chloramphenicol (IS)1.16320.8175.9−85−22−9Abbreviations. DP, declustering potential; CE, collision energy; CXP, collision cell exit potential.


### 2.3. Preparation ofNauclea officinalis Extract

PowderedNauclea officinalis stems (1 kg) were accurately weighed and heated and refluxed by water (1 : 10, w/v) for 2 h. The extractions were combined and concentrated under reduced pressure to obtain the 25 g of crude water extracts.


### 2.4. Preparations of Standard Solutions, IS, and Quality Control (QC) Samples

The chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside and chloramphenicol (IS) were dissolved in methanol at a concentration of 1 mg/mL as the stock solution. A series of mixed standard working solutions were prepared by diluting the primary mixed stock solution with methanol at appropriate ratios. All the solutions were kept away from light at 4°C until analysis.The calibration standards were prepared by spiking 10μL of the mixed standard solution and 10 μL (500 ng/mL) IS solution into 90 μL rat blank matrixes at final concentrations of 2–2400 ng/mL of chlorogenic acid, 5–2400 ng/mL of naucleactonin C, 5-2000 ng/mL of khaephuoside A, and 25–2400 ng/mL of 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside.The QC samples were prepared at concentrations of 2, 5, 100, and 2400 ng/mL for chlorogenic acid; 5, 8, 140, and 2400 ng/mL for naucleactonin C; 5, 8, 120, and 2000 ng/mL for khaephuoside A; 25, 50, 350, and 2400 ng/mL for 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside, respectively. The IS concentration was 45 ng/mL in all calibration standards and QC.


### 2.5. Animals

Male Sprague–Dawley (SD) rats (250 ± 20 g weight) were purchased from Tianqin Biotechnology Co. Ltd. (License No. 4307256220100013061), Changsha, China. The animals were maintained at 22 ± 2°C and 60% ± 10% humidity with a 12 h light/dark cycle and allowed free access to food and water. All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee at the Hainan Medical University (Haikou, China).


### 2.6. Preparation of Biological Samples

An aliquot of 100μL of rat plasma and tissue homogenate were transferred to a 2 mL tube. Subsequently, 10 μL IS (500 ng/mL) were added followed by 1 mL of methanol-ethyl acetate (1 : 1, v/v) including 0.1% (v/v) formic acid. The sample was then vortexed for 1 min and centrifuged at 13,000 rpm for 5 min. Thereafter, the supernatant was dried at 37°C with nitrogen, which the residue was reconstituted with 80 μL methanol, centrifuged at 13,000 rpm for 5 min, and 5 μL of the sample were injected into the HPLC-MS/MS system for analysis.


### 2.7. Method Validation

The method validation assays were carried out according to the U.S. Food and Drug Administration (FDA) Bioanalytical Method Validation (Food and Drug Administration, 2018), including specificity, linearity, recovery, matrix effect, precision, accuracy, and stability [24].


### 2.8. Pharmacokinetic Study

Six male SD rats were tested in pharmacokinetics studies. The rats were housed at 22 ± 2°C and fasted for 12 h with free access to water prior to dosing, which were orally administeredNauclea officinalis extracts at the dose of 2 g/kg (equivalent to 32.74 mg/kg chlorogenic acid, 0.78 mg/kg naucleactonin C, 14.1 mg/kg khaephuoside A, and 10.39 mg/kg 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside). The blood samples (approximately 0.5 mL) were collected via the venous plexus of the eye socket at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 1, 12, and 24 h under anesthesia. Next, each sample was immediately centrifuged at 4000 rpm for 10 min to acquire the plasma. The plasma was transferred to new tubes and stored at −80°C until further use. The pharmacokinetics parameters of the four compounds in rat plasma were calculated by the noncompartment model using Drug and Statistics (DAS 3.3.0) software (Beijing, China).


### 2.9. Tissue Distribution

Thirty rats were divided into five groups (n = 6) at random, which were orally administered Nauclea officinalis extracts at a dose of 2 g/kg for tissue distribution conducted at 0.5, 1, 2, 4, and 6 h. During the collection, the heart, liver, spleen, lung, kidney, stomach, small intestine, and brain were rinsed with physiological saline solution to get rid of the blood or content and blotted on filter paper and then weighed. Each tissue sample was accurately weighed and homogenized by using physiological saline at four times the tissue weight (w/v). The homogenates were stored at −80°C until analysis.


## 2.1. Chemicals and Reagents

The stems ofNauclea officinalis were collected from Qiongzhong County, Hainan Province, and identified by Prof. Jianping Tian of Hainan Medical University. Standards of naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside were isolated from Nauclea officinalis in our laboratory by silica gel column, semi-preparative high performance liquid chromatography, which structure was identified by NMR, and the HPLC purity was over than 98%. The standards for chlorogenic acid were purchased from Chengdu Pufeide Biological Technology Co., Ltd (HPLC >98%, Sichuan, China). Chloramphenicol was provided by the National Institute for Food and Drug Control (Beijing, China). Methanol and acetonitrile were all chromatographically pure and purchased from Fisher Scientific (Fair Lawn, NJ, USA); ethyl acetate and formic acid were all chromatographically pure and purchased from Aladdin Industrial Corporation (Shanghai, China); purified water was prepared by a LabTower EDI system (Thermo Scientific, USA).


## 2.2. Instrument and Analytical Conditions

Chromatographic analysis was performed on the LC-20ADXR System (Shimadzu, Japan). The Phenomenex Kinete EVO C18 100 Å (50 mm × 2.1 mm, 5μm) column was used as the analytical column maintained at 40°C with (A) 0.1% formic acid aqueous solution and (B) acetonitrile as the mobile phase. The gradient elution program was operated as follows: 2% B (0–0.5 min), 2–25% B (0.5–0.51 min), 25-55% B (0.51–3.0 min), 55-95% B (3.0–3.01 min), 95% B (3.01–5.0 min), 95–2% B (5.0–5.01 min), and 2% B (5.01–6.0 min). The flow rate was 0.8 mL/min, and the injection volume was 5 μL.Mass spectrometric detection was performed on an AB-SCIEX API 4000 mass spectrometer (AB SCIEX, Singapore) with ESI in negative ion multiple reaction monitoring (MRM) mode. The optimized instrument parameters were as follows: nebulizer gas: 50 psi, heated by N2 gas: 55 psi, ion spray voltage: −4500 V and temperature: 550°C; nebulizer, blowback gas, and collision gas were nitrogen; declustering potential (DP), collision energy (CE), and collision cell exit potential (CXP) of the four analytes and chloramphenicol (IS) are shown in Table 1.Table 1
Optimized multiple reaction monitoring parameters for the detection of four analytes and IS.
CompoundsRetention time (min)Precursor ion (m/z)Quantitative ion (m/z)DP (V)CE (V)CXP (V)Chlorogenic acid1.02353.0190.9−51−22.9−10.5Naucleactonin C1.65335.1182.9−110−37−9Khaephuoside A0.99477.1293.0−98−18−63,4-Dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside0.97447.1232.9−84−25−14Chloramphenicol (IS)1.16320.8175.9−85−22−9Abbreviations. DP, declustering potential; CE, collision energy; CXP, collision cell exit potential.


## 2.3. Preparation ofNauclea officinalis Extract

PowderedNauclea officinalis stems (1 kg) were accurately weighed and heated and refluxed by water (1 : 10, w/v) for 2 h. The extractions were combined and concentrated under reduced pressure to obtain the 25 g of crude water extracts.


## 2.4. Preparations of Standard Solutions, IS, and Quality Control (QC) Samples

The chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside and chloramphenicol (IS) were dissolved in methanol at a concentration of 1 mg/mL as the stock solution. A series of mixed standard working solutions were prepared by diluting the primary mixed stock solution with methanol at appropriate ratios. All the solutions were kept away from light at 4°C until analysis.The calibration standards were prepared by spiking 10μL of the mixed standard solution and 10 μL (500 ng/mL) IS solution into 90 μL rat blank matrixes at final concentrations of 2–2400 ng/mL of chlorogenic acid, 5–2400 ng/mL of naucleactonin C, 5-2000 ng/mL of khaephuoside A, and 25–2400 ng/mL of 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside.The QC samples were prepared at concentrations of 2, 5, 100, and 2400 ng/mL for chlorogenic acid; 5, 8, 140, and 2400 ng/mL for naucleactonin C; 5, 8, 120, and 2000 ng/mL for khaephuoside A; 25, 50, 350, and 2400 ng/mL for 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside, respectively. The IS concentration was 45 ng/mL in all calibration standards and QC.


## 2.5. Animals

Male Sprague–Dawley (SD) rats (250 ± 20 g weight) were purchased from Tianqin Biotechnology Co. Ltd. (License No. 4307256220100013061), Changsha, China. The animals were maintained at 22 ± 2°C and 60% ± 10% humidity with a 12 h light/dark cycle and allowed free access to food and water. All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee at the Hainan Medical University (Haikou, China).


## 2.6. Preparation of Biological Samples

An aliquot of 100μL of rat plasma and tissue homogenate were transferred to a 2 mL tube. Subsequently, 10 μL IS (500 ng/mL) were added followed by 1 mL of methanol-ethyl acetate (1 : 1, v/v) including 0.1% (v/v) formic acid. The sample was then vortexed for 1 min and centrifuged at 13,000 rpm for 5 min. Thereafter, the supernatant was dried at 37°C with nitrogen, which the residue was reconstituted with 80 μL methanol, centrifuged at 13,000 rpm for 5 min, and 5 μL of the sample were injected into the HPLC-MS/MS system for analysis.


## 2.7. Method Validation

The method validation assays were carried out according to the U.S. Food and Drug Administration (FDA) Bioanalytical Method Validation (Food and Drug Administration, 2018), including specificity, linearity, recovery, matrix effect, precision, accuracy, and stability [24].


## 2.8. Pharmacokinetic Study

Six male SD rats were tested in pharmacokinetics studies. The rats were housed at 22 ± 2°C and fasted for 12 h with free access to water prior to dosing, which were orally administeredNauclea officinalis extracts at the dose of 2 g/kg (equivalent to 32.74 mg/kg chlorogenic acid, 0.78 mg/kg naucleactonin C, 14.1 mg/kg khaephuoside A, and 10.39 mg/kg 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside). The blood samples (approximately 0.5 mL) were collected via the venous plexus of the eye socket at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 1, 12, and 24 h under anesthesia. Next, each sample was immediately centrifuged at 4000 rpm for 10 min to acquire the plasma. The plasma was transferred to new tubes and stored at −80°C until further use. The pharmacokinetics parameters of the four compounds in rat plasma were calculated by the noncompartment model using Drug and Statistics (DAS 3.3.0) software (Beijing, China).


## 2.9. Tissue Distribution

Thirty rats were divided into five groups (n = 6) at random, which were orally administered Nauclea officinalis extracts at a dose of 2 g/kg for tissue distribution conducted at 0.5, 1, 2, 4, and 6 h. During the collection, the heart, liver, spleen, lung, kidney, stomach, small intestine, and brain were rinsed with physiological saline solution to get rid of the blood or content and blotted on filter paper and then weighed. Each tissue sample was accurately weighed and homogenized by using physiological saline at four times the tissue weight (w/v). The homogenates were stored at −80°C until analysis.


## 3. Results and Discussion

### 3.1. Optimization of Chromatographic and Mass Spectrometric Conditions

Optimization of chromatographic conditions: by analyzing the separation effect and peak shape of four analytes and the IS on different columns, we found that the Phenomenex Kinete EVO C18 column gave the best separation and peak shape. Subsequently, by exploring different mobile phase systems, such as methanol-water and acetonitrile-water, we found that the four analytes had better response values in acetonitrile. Finally, by assessing the pH of the mobile phase (0.1, 0.2, 0.5, and 0.8% formic acid solution), the response values of four analytes were highest when the concentration of formic acid was 0.1%. Therefore, 0.1% formic acid water-acetonitrile was selected for use as the mobile phase.Optimization of mass spectrometric conditions: the four analytes and the IS had strong [M-H]− peaks in the negative ion mode, which can be easily broken and used for stable fragment analysis and detection. Therefore, the ESI in negative ion mode and Q1, MS2, and MRM scan modes were adopted. The chemical structures, precursor ion, and product ion of four analytes and IS are shown in Figure 1.Figure 1
Chemical structures, precursor ion, and product ion mass spectra of four analytes and IS.


### 3.2. Optimization of the Sample Preparation

For sample processing, a simple protein precipitation method was first tried for methanol and acetonitrile, respectively, but the recoveries of chlorogenic acid and naucleactonin C were unsatisfactory. In addition, liquid-liquid extraction (LLE) was tried for various solvents tested, including n-butanol, dichloromethane, isopropanol, methyl tert-butyl ether, and ethyl acetate, but no satisfactory recovery was obtained for khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside. The extraction of chlorogenic acid should be in an acidic condition. Therefore, the extraction results of chlorogenic acid at different pH are investigated.Finally, the combination method using mixture solution of methanol-ethyl acetate (1 : 1, v/v) including 0.1% (v/v) formic acid was selected. Consequently, satisfactory and consistent recovery from plasma and tissues samples was achieved for four analytes and IS.


### 3.3. Method Validation

#### 3.3.1. Selectivity

The selectivity was assessed by analyzing chromatograms of blank rat plasma and tissue homogenates from different batches, blank plasma and tissue homogenates spiked with analytes and IS, and plasma, tissue samples obtained from rats after oral administration ofNauclea officinalis extracts (n = 6). Figure 2 shows typical chromatograms of four analytes and IS in rat plasma (Figures S1 and S2 of the Supporting Material show typical chromatograms of four analytes and IS in liver tissue and kidney tissue). Under the given condition, Chlorogenic acid, Naucleactonin C, Khaephuoside A, 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside, and IS were eluted approximately at retention time of 1.02 min, 1.65 min, 0.99 min, 0.97 min, and 1.16 min, respectively. No interfering endogenous substances were found at the respective retention times.Figure 2
Typical chromatograms of four analyte and IS in rat plasma: (a) blank plasma samples, (b) blank plasma samples spiked with four analytes (LLOQ) and IS, (c) rat plasma samples at 0.5 h after oral administration theNauclea officinalis extracts spiked with IS; (1) chlorogenic acid, (2) naucleactonin C, (3) khaephuoside A, (4) 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside, (5) chloramphenicol (IS).
(a)(b)(c)


#### 3.3.2. Linearity of Calibration Curve and Lower Limit of Quantification

The calibration curves were constructed by plotting the peak areas ratios of chlorogenic acid/IS, Naucleactonin C/IS, Khaephuoside A/IS, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside/IS versus theoretical concentrations. This method exhibited a good linear response for the range of concentrations from 2 to 2500 ng/mL in plasma and from 2 to 2500 ng/mL in tissues; all determination coefficients (R2) were greater than 0.9849. The lower limit of quantification (LLOQ) was defined as the lowest concentration of a signal-noise (S/N) ratios of 10 : 1, respectively, the precision was less than 15% and the accuracy was within ±20%. The limits were adequate for studies of pharmacokinetics and tissue distribution by oral administration of Nauclea officinalis extracts. Data from the determination are shown in Table 2.Table 2
Summary of standard curves, correlation coefficients, LLOQ, and linear ranges of four analytes in plasma and tissue samples.
CompoundsMatrixLinear regression equationR2LLOQ (ng/mL)Linear range (ng/mL)Chlorogenic acidPlasmay = 0.0288x + 0.001930.999422–2500Hearty = 0.0063x − 0.007710.984922–2500Livery = 0.0267x − 0.03230.998022–2500Spleeny = 0.0392x + 0.02770.988622–2500Lungy = 0.0442x + 0.02390.995822–2500Kidneyy = 0.0217x − 0.03020.995022–2500Small intestiney = 0.0334x − 0.04010.996422–2500Stomachy = 0.0402x − 0.01290.995222–2500Brainy = 0.0499x − 0.05620.996022–2500Naucleactonin CPlasmay = 0.00535x + 0.02070.996455–2500Hearty = 0.00313x − 0.001160.992655–2500Livery = 0.00395x − 0.01010.996455–2500Spleeny = 0.00511x − 0.005260.995055–2500Lungy = 0.00534x − 0.002630.998055–2500Kidneyy = 0.00363x − 0.00980.996055–2500Small intestiney = 0.00224x − 0.005270.996855–2500Stomachy = 0.00465x − 0.01380.997255–2500Brainy = 0.00552x − 0.01630.993255–2500Khaephuoside APlasmay = 0.00316x + 0.01530.995455–2000Hearty = 0.00673x − 0.0050.994655–2000Livery = 0.0063x − 0.005020.996255–2000Spleeny = 0.011x − 0.00540.998055–2000Lungy = 0.0099x − 0.001660.997855–2000Kidneyy = 0.00531x − 0.00470.995455–2000Small intestiney = 0.0058x − 0.01560.990655–2000Stomachy = 0.00745x − 0.008370.992055–2000Brainy = 0.0126x + 0.0003770.993855–20003,4-Dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranosidePlasmay = 0.000845x + 0.003960.99842525–2500Hearty = 0.000804x + 0.001590.98492525–2500Livery = 0.000873x − 0.005150.99682525–2500Spleeny = 0.00134x + 0.009470.98942525–2500Lungy = 0.00138x + 0.006260.99642525–2500Kidneyy = 0.000633x − 0.0005380.99302525–2500Small intestiney = 0.00109x + 0.0009660.99622525–2500Stomachy = 0.00119x + 0.005490.99642525–2500Brainy = 0.00163x − 0.01490.99522525–2500Abbreviations. LLOQ, lower limit of quantification;R2, determination coefficients.


#### 3.3.3. Accuracy and Precision

Accuracy and precision were evaluated by analyzing QC samples at high, medium, low, and LLOQ concentrations (n = 6) on the same day and three consecutive days using the standard curve, respectively. The acceptable limits of accuracy were required to be within ±15% of the actual value except when at LLOQ, and the intra- and interday precision and accuracy data of the determination were shown in Table 3. In our study, the intraday precision ranged from 0.62 to 12.65%, and intraday accuracy ranged from 86.48 to 114.17%. The interday precision ranged from 2.42 to 12.61%, and the interday accuracy ranged from 86.31 to 113.99%. The results showed the accuracy and precision were within the acceptable limits, which proved the method was reproducible, reliable, and accurate for the determination of four analytes in rat plasma and tissue samples.Table 3
Summary of intraday and interday precisions and accuracies of four analytes in plasma and tissue samples.
CompoundsMatrixConcentration (ng/mL)Intraday (n = 6)Interday (n = 18)AccuracyRSD (%)AccuracyRSD (%)Chlorogenic acidPlasma2104.02 ± 11.9111.45100.64 ± 11.6711.60497.72 ± 9.239.4596.09 ± 7.087.36100104.42 ± 6.235.97104.29 ± 10.610.162400101.22 ± 3.203.1696.59 ± 8.118.40Heart2101.90 ± 12.1411.92109.34 ± 11.7410.74499.35 ± 3.893.9193.67 ± 6.136.5410096.57 ± 4.814.9894.46 ± 7.808.262400109.00 ± 5.905.41102.88 ± 6.856.66Liver2101.65 ± 2.822.78100.88 ± 5.385.334101.45 ± 2.982.9398.59 ± 6.726.82100100.30 ± 2.892.8897.78 ± 4.895.00240095.07 ± 5.746.0497.11 ± 5.585.75Spleen296.40 ± 7.637.92102.14 ± 8.908.71495.40 ± 4.204.40101.28 ± 9.619.49100102.40 ± 2.001.95111.52 ± 7.006.27240094.93 ± 4.815.07108.26 ± 10.419.62Lung2101.53 ± 6.406.30101.22 ± 7.457.36499.75 ± 6.096.1093.33 ± 11.1711.97100108.33 ± 5.475.04111.33 ± 6.495.832400100.60 ± 3.353.33101.63 ± 5.074.99Kidney298.12 ± 5.365.4796.23 ± 5.335.544100.13 ± 2.172.1792.09 ± 7.217.8310099.37 ± 5.205.2396.10 ± 6.366.622400104.47 ± 5.795.5499.47 ± 6.066.10Small intestine293.27 ± 4.134.4396.03 ± 7.868.19486.48 ± 5.095.8993.40 ± 10.7511.5110089.75 ± 7.968.8796.61 ± 10.0610.41240092.70 ± 6.637.1693.01 ± 5.275.66Stomach2105.87 ± 7.877.43107.84 ± 7.456.90493.17 ± 7.027.5398.76 ± 6.446.52100106.00 ± 3.953.73106.35 ± 4.464.192400100.63 ± 4.184.1599.41 ± 4.814.84Brain2109.27 ± 6.746.17104.39 ± 9.479.074101.00 ± 3.813.77101.29 ± 5.425.35100106.00 ± 4.944.66106.77 ± 7.166.702400101.27 ± 2.252.22105.62 ± 7.046.67Naucleactonin CPlasma595.62 ± 12.1012.6598.24 ± 10.3010.488100.53 ± 11.7811.7196.43 ± 12.1612.6114099.95 ± 6.496.5095.76 ± 7.187.50240097.05 ± 4.804.9595.50 ± 6.236.52Heart5111.27 ± 7.326.58107.46 ± 10.589.85899.28 ± 5.485.52101.15 ± 11.4611.3314093.45 ± 4.344.6593.19 ± 5.335.722400105.92 ± 9.549.01104.46 ± 6.666.38Liver5100.90 ± 5.405.36108.30 ± 7.827.22899.47 ± 2.822.84100.52 ± 5.004.9814088.60 ± 2.602.9393.53 ± 6.336.772400102.50 ± 1.521.48101.42 ± 2.462.42Spleen5111.00 ± 6.996.29104.42 ± 9.318.91898.90 ± 3.553.59100.74 ± 6.776.7214096.07 ± 3.954.1193.72 ± 5.305.662400100.02 ± 3.393.39103.13 ± 5.064.91Lung5113.83 ± 4.073.58113.44 ± 5.374.738103.28 ± 10.4610.13102.03 ± 10.169.96140104.17 ± 3.763.61103.83 ± 4.053.902400107.83 ± 4.073.77107.06 ± 5.024.69Kidney5100.70 ± 4.284.25103.81 ± 6.616.36898.38 ± 5.315.4099.18 ± 4.364.3914095.37 ± 4.194.39100.44 ± 7.077.042400106.80 ± 9.749.12104.87 ± 6.466.16Small intestine5109.98 ± 10.979.97105.41 ± 10.199.67899.30 ± 8.949.01100.44 ± 9.189.1414088.73 ± 8.9610.1086.31 ± 7.668.882400103.30 ± 6.826.6098.26 ± 7.607.73Stomach5109.83 ± 7.056.42102.67 ± 10.099.838101.58 ± 2.962.9295.73 ± 8.078.4314094.88 ± 2.742.8892.09 ± 5.175.622400113.17 ± 4.884.31108.96 ± 7.216.62Brain5105.75 ± 10.8110.22105.82 ± 8.778.298104.78 ± 7.467.12100.57 ± 8.118.06140101.22 ± 4.594.5398.93 ± 5.735.802400113.83 ± 3.312.91111.39 ± 5.544.98Khaephuoside APlasma5101.63 ± 9.939.77105.66 ± 10.509.948101.02 ± 9.649.55101.78 ± 7.857.71120112.33 ± 4.764.24106.16 ± 6.115.761800104.08 ± 4.394.21102.52 ± 4.414.31Heart5113.50 ± 9.188.09103.33 ± 10.9210.578100.82 ± 6.196.1498.68 ± 7.067.1612096.73 ± 2.923.0289.72 ± 8.018.931800109.12 ± 9.458.66102.43 ± 8.137.94Liver599.97 ± 7.067.07101.51 ± 8.498.368104.10 ± 5.074.87101.81 ± 4.023.9512099.33 ± 0.620.62100.43 ± 4.554.531800108.00 ± 6.105.65103.94 ± 5.605.39Spleen598.73 ± 11.2111.35103.62 ± 9.479.148100.98 ± 6.256.19103.08 ± 6.756.5512099.35 ± 2.522.54109.89 ± 8.848.041800109.00 ± 4.604.22113.06 ± 5.364.74Lung5108.17 ± 10.199.42109.93 ± 7.66.928109.90 ± 11.2510.24106.91 ± 9.959.3112090.32 ± 6.126.7895.54 ± 6.536.831800100.73 ± 2.022.00103.65 ± 6.916.66Kidney5106.82 ± 7.917.40109.94 ± 6.295.728113.67 ± 2.942.59111.40 ± 6.125.49120105.30 ± 6.145.83107.78 ± 6.916.411800105.02 ± 9.819.34108.84 ± 7.606.99Small intestine5106.50 ± 8.538.01104.08 ± 10.119.71899.98 ± 9.439.13101.89 ± 11.5811.3612089.90 ± 6.847.6193.74 ± 7.808.321800104.45 ± 6.225.96105.44 ± 5.154.89Stomach596.63 ± 9.159.4795.98 ± 10.2610.69890.92 ± 7.968.7697.66 ± 8.618.82120104.00 ± 6.135.90103.24 ± 5.315.151800109.83 ± 4.834.40110.50 ± 4.544.11Brain5108.90 ± 9.748.95107.11 ± 7.947.418108.55 ± 9.909.12107.23 ± 8.017.47120113.17 ± 6.715.93110.13 ± 6.095.531800100.50 ± 3.993.97108.44 ± 7.637.033,4-Dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranosidePlasma25109.00 ± 4.053.72101.34 ± 9.108.9850105.82 ± 10.9510.35102.61 ± 7.897.69350109.83 ± 5.234.76104.49 ± 8.608.23240096.82 ± 2.762.85100.71 ± 7.507.45Heart2599.12 ± 5.065.1096.73 ± 8.088.355096.45 ± 6.757.0098.20 ± 8.288.4335089.15 ± 4.835.4290.19 ± 6.827.56240099.80 ± 10.8910.9199.33 ± 7.667.71Liver25105.07 ± 6.626.30103.78 ± 6.396.1650108.48 ± 7.396.82108.22 ± 7.386.82350112.15 ± 6.896.14113.99 ± 4.834.24240098.28 ± 3.673.73100.28 ± 3.243.23Spleen25102.03 ± 10.6810.47104.78 ± 8.638.235099.90 ± 5205.21103.82 ± 7.777.4835097.70 ± 4.124.21109.12 ± 9.078.312400100.65 ± 5.465.43110.22 ± 8.948.11Lung2596.17 ± 7.597.8999.80 ± 10.9911.0150106.78 ± 7.086.63107.49 ± 8.167.5935096.42 ± 4.494.66100.05 ± 5.505.49240099.67 ± 6.296.3199.32 ± 5.685.72Kidney2597.02 ± 9.7510.0596.23 ± 9.389.7450108.00 ± 5.665.24106.69 ± 7.006.5635099.63 ± 5.835.85100.46 ± 6.786.75240096.50 ± 7.417.6897.74 ± 6.316.46Small intestine2598.25 ± 8.929.0897.96 ± 7.737.9050101.58 ± 6.416.31101.89 ± 7.137.0035089.58 ± 4.274.7698.17 ± 8.398.552400100.18 ± 4.774.76101.32 ± 4.834.77Stomach25103.63 ± 5.215.0299.07 ± 9.649.7350101.90 ± 9.329.1497.96 ± 9.339.52350114.17 ± 3.192.79109.34 ± 6.145.622400100.62 ± 3.423.4099.42 ± 4.664.69Brain25106.95 ± 12.1311.3499.13 ± 11.2611.3650105.78 ± 7.637.22107.10 ± 7.867.34350110.00 ± 9.018.19112.56 ± 6.936.16240099.42 ± 2.902.91101.13 ± 5.445.38


#### 3.3.4. Extraction Recovery and Matrix Effect

Six batches of blank plasma from independent sources were used to obtain extracted samples, postextracted spiked samples, and unextracted samples at high, medium, low, and LLOQ concentrations. The peak areas of the three types of samples were recorded as A, B, and C, respectively. Extraction recovery was evaluated by A/B. Matrix effects were calculated by comparing the A/C of analytes. The extraction, recovery, and matrix effect of four analytes in rat plasma and tissues are shown in Table4. The recovery rate were 51.85–97.06%, 75.99–106.68%, 77.46–105.35%, and 68.36–103.75% for chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside and the matrix effects were 50.17–116.62%, 86.75–115.99%, 45.79–87.44%, and 51.60–92.34% for chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside in different matrices, indicating some ion inhibitory effects for four compounds in the biological samples, the RSD (relative standard deviation) of recovery and matrix effect were less than 15%. There were no significant differences in recoveries and matrix effects between the four compounds at different concentrations in the same matrix.Table 4
Summary of extraction recovery and matrix effect of four analytes in plasma and tissue samples (n = 6).
CompoundsMatrixConcentration (ng/mL)Matrix effect (%)RSD (%)Extraction recovery (%)RSD (%)Chlorogenic acidPlasma297.26 ± 5.355.5080.45 ± 4.365.424109.75 ± 4.404.0180.04 ± 3.083.8510082.80 ± 6.517.8669.49 ± 4.055.84240091.66 ± 1.531.6772.26 ± 1.161.61Heart2116.54 ± 3.032.6080.53 ± 9.6211.95498.55 ± 2.712.7576.82 ± 4.776.2110097.78 ± 1.761.8062.02 ± 2.243.61240093.25 ± 0.760.8163.32 ± 0.580.91Liver283.69 ± 4.245.0697.06 ± 3.753.86479.96 ± 4.795.9479.07 ± 2.903.6610063.82 ± 2.513.9466.44 ± 0.240.37240080.01 ± 0.901.1269.83 ± 1.552.22Spleen299.89 ± 2.612.6180.09 ± 1.221.524100.56 ± 1.991.9886.82 ± 2.883.3210092.05 ± 1.011.1067.29 ± 1.812.69240091.66 ± 0.810.8873.12 ± 1.612.20Lung2104.73 ± 1.781.7081.80 ± 2.523.08499.34 ± 2.072.0988.65 ± 1.551.7510098.65 ± 1.241.2580.24 ± 2.112.63240096.68 ± 1.811.8876.89 ± 1.692.20Kidney290.49 ± 5.666.2678.34 ± 4.025.13498.52 ± 4.214.2786.92 ± 3.003.4610095.03 ± 5.946.2570.73 ± 4.906.92240089.55 ± 2.383.1672.70 ± 1.502.07Small intestine256.96 ± 6.1610.8156.20 ± 3.365.98463.67 ± 2.464.1451.85 ± 1.943.7310053.67 ± 1.061.9761.26 ± 1.873.05240050.17 ± 1.723.4262.66 ± 1.061.70Stomach2114.40 ± 5.014.4685.33 ± 5.206.104116.62 ± 8.807.5591.66 ± 6.116.67100102.87 ± 3.243.1566.48 ± 3.945.93240098.38 ± 1.631.6569.41 ± 1.562.25Brain297.89 ± 6.426.5682.33 ± 4.655.654105.09 ± 2.992.8475.80 ± 3.594.7410090.02 ± 5.466.0671.37 ± 1.171.63240090.56 ± 2.162.3986.06 ± 1.681.95Naucleactonin CPlasma5104.57 ± 6.946.64100.30 ± 11.7711.748102.54 ± 2.552.48106.68 ± 6.315.91140104.07 ± 2.882.7796.41 ± 1.661.722400102.64 ± 2.092.0488.27 ± 1.051.19Heart598.86 ± 2.672.7097.71 ± 1.952.00899.69 ± 3.963.9796.33 ± 7.197.46140106.24 ± 4.874.5885.37 ± 2.903.40240099.81 ± 1.481.4886.76 ± 1.611.86Liver5101.81 ± 10.8810.6992.06 ± 9.8710.728111.16 ± 3.122.8185.03 ± 4.915.7714097.92 ± 5.095.2084.88 ± 4.525.33240097.02 ± 1.111.1480.54 ± 0.831.03Spleen590.83 ± 3.654.02100.21 ± 9.219.19893.78 ± 6.076.4799.90 ± 5.495.5014097.28 ± 2.712.7984.00 ± 1.541.832400100.67 ± 0.790.7884.60 ± 1.401.65Lung598.17 ± 7.637.77105.63 ± 3.873.668115.99 ± 7.975.0199.21 ± 12.4312.5314098.73 ± 1.391.4195.48 ± 2.883.01240099.21 ± 0.910.9284.40 ± 1.822.15Kidney599.90 ± 5.105.1094.08 ± 8.118.628100.35 ± 5.145.12105.08 ± 5.224.97140101.18 ± 0.700.6986.02 ± 4.395.102400102.49 ± 1.871.8386.64 ± 2.262.60Small intestine598.00 ± 5.515.6298.55 ± 9.619.75899.37 ± 8.488.9082.00 ± 5.036.1414095.42 ± 5.525.7991.73 ± 1.671.82240086.75 ± 2.242.5886.61 ± 3.153.63Stomach5101.53 ± 3.553.5097.93 ± 4.194.288101.21 ± 3.873.8299.25 ± 3.173.2014099.62 ± 1.191.2082.46 ± 1.882.282400101.37 ± 1.351.3375.99 ± 2.773.64Brain597.50 ± 6.506.6788.08 ± 3.123.548105.47 ± 5.144.8788.38 ± 7.328.29140102.02 ± 0.850.8397.63 ± 4.354.462400101.24 ± 2.442.4199.47 ± 1.421.43Khaephuoside APlasma564.06 ± 5.668.8488.84 ± 5.105.75865.86 ± 5.658.5792.35 ± 8.739.4512055.55 ± 4.057.2985.88 ± 2.062.40180058.01 ± 1.933.3385.24 ± 3.303.87Heart570.92 ± 8.0911.4196.07 ± 5.375.59877.13 ± 3.564.6182.29 ± 3.954.8012054.79 ± 1.142.0892.18 ± 1.641.78180059.93 ± 4.647.7586.98 ± 4.875.59Liver573.90 ± 6.849.26105.35 ± 11.0210.46853.20 ± 1.592.98103.34 ± 3.753.6312058.17 ± 1.121.9377.46 ± 2.172.80180061.29 ± 2.804.5884.49 ± 1.702.01Spleen568.33 ± 1.412.0790.99 ± 7.898.67860.36 ± 2.584.2896.38 ± 2.772.8812058.12 ± 0.420.7390.31 ± 2.803.10180061.44 ± 1.131.8486.75 ± 0.981.13Lung584.39 ± 4.204.9777.71 ± 3.734.81872.06 ± 6.899.5687.06 ± 7.308.3812059.00 ± 2.554.3386.34 ± 3.484.03180061.35 ± 4.417.1979.39 ± 6.488.17Kidney557.46 ± 1.803.1397.31 ± 4.384.50851.41 ± 2.915.6699.78 ± 3.413.4212045.79 ± 1.302.8499.65 ± 1.231.23180047.23 ± 1.382.93101.56 ± 2.172.14Small intestine556.14 ± 1.472.61102.93 ± 10.4410.15867.00 ± 5.438.1094.63 ± 6.276.6312062.69 ± 2.063.29100.39 ± 5.004.98180071.51 ± 2.082.9199.78 ± 1.061.06Stomach556.84 ± 3.215.6495.49 ± 6.606.91863.57 ± 5.598.8083.81 ± 6.898.2212054.53 ± 2.444.4877.49 ± 4.085.27180055.57 ± 1.071.9279.96 ± 0.871.09Brain576.07 ± 1.692.2293.48 ± 8.709.30876.50 ± 6.238.15102.39 ± 6.806.6412087.44 ± 3.233.7090.50 ± 0.991.09180079.81 ± 2.152.70100.48 ± 1.421.413,4-Dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranosidePlasma2564.80 ± 4.957.6488.62 ± 8.569.665065.25 ± 0.811.2489.23 ± 4.244.7635060.35 ± 3.676.0885.65 ± 1.171.37240055.98 ± 2.344.1884.82 ± 3.163.72Heart2579.81 ± 2.392.9980.56 ± 4.055.035073.97 ± 1.842.4989.91 ± 2.893.2135069.51 ± 4.045.8185.34 ± 4.204.92240064.96 ± 2.283.5185.00 ± 3.153.71Liver2555.02 ± 4.027.3188.70 ± 8.389.455053.00 ± 1.633.0894.52 ± 4.765.0435052.69 ± 2.124.0383.51 ± 2.633.15240051.60 ± 1.442.8087.94 ± 2.883.27Spleen2581.22 ± 3.274.0298.19 ± 2.872.925087.52 ± 4.515.1692.02 ± 3.153.4235081.89 ± 1.031.2689.66 ± 2.422.70240080.06 ± 3.073.8380.48 ± 2.152.68Lung2591.11 ± 4.014.4068.36 ± 4.426.475092.34 ± 3.463.7577.05 ± 2.142.7835077.28 ± 4.365.6583.53 ± 3.424.09240076.69 ± 2.733.5675.92 ± 0.730.97Kidney2563.39 ± 1.542.4386.96 ± 4.515.195064.43 ± 1.532.3899.12 ± 2.652.6735053.76 ± 3.075.72101.00 ± 1.811.79240060.75 ± 1.532.51103.75 ± 3.173.05Small intestine2564.61 ± 4.236.5598.39 ± 11.1511.335068.08 ± 3.685.4090.4 ± 3.023.3435070.33 ± 6.138.71101.86 ± 5.705.60240064.51 ± 2.353.6499.20 ± 1.011.02Stomach2568.84 ± 1.181.7292.61 ± 3.133.385062.45 ± 2.203.5288.53 ± 3.443.8935065.27 ± 1.672.5677.56 ± 1.832.36240062.11 ± 0.871.4083.08 ± 2.162.60Brain2571.85 ± 1.852.5790.54 ± 1.481.645078.40 ± 2.292.9286.20 ± 4.875.6535085.96 ± 2.913.3993.34 ± 1.882.02240075.94 ± 1.522.00101.87 ± 1.821.78


#### 3.3.5. Stability

The stability was evaluated by analyzing QC samples at high, medium, low, and LLOQ concentrations (n = 6) under different storage conditions, including room temperature for 4 h, auto-sampler (4°C) for 8 h, three freeze-thaw cycles (freeze cycle was at −20°C while thaw cycle was done at room temperature) and −80°C for 30 days. The results are shown in Table 5. It was evident from these data that four storage conditions have no significant effect on the sample. The results indicated that four analytes were stable under these four storage conditions.Table 5
Summary of stability of four analytes in plasma and tissue samples (n = 6).
CompoundsMatrixConcentration (ng/mL)Ambient temperature for 4 hAutosampler at 4°C for 8 hThree freeze-thraw cycles−80°C for 30 daysAccuracyRSD (%)AccuracyRSD (%)AccuracyRSD (%)AccuracyRSD (%)Chlorogenic acidPlasma2101.68 ± 8.398.2598.25 ± 7.837.97101.23 ± 12.4412.2993.35 ± 10.7811.55486.78 ± 6.167.0997.90 ± 11.8812.1495.02 ± 5.395.6798.92 ± 12.3212.46100112.33 ± 3.723.31111.33 ± 4.373.92100.68 ± 6.226.18113.17 ± 4.964.38240098.40 ± 5.565.6599.47 ± 7.597.6391.98 ± 4.755.17112.67 ± 7.586.73Heart2102.73 ± 12.9812.64101.70 ± 10.9110.7291.72 ± 6.336.9088.98 ± 5.736.43496.88 ± 11.0611.4299.50 ± 6.216.2499.03 ± 10.1710.27102.27 ± 10.3010.07100112.83 ± 9.118.07111.93 ± 11.7710.52111.00 ± 7.726.96112.62 ± 8.157.242400108.60 ± 8.417.75101.43 ± 10.3410.20110.55 ± 8.117.33104.85 ± 10.6910.19Liver288.47 ± 6.937.8387.82 ± 6.457.3588.47 ± 7.228.1687.43 ± 7.748.86486.27 ± 7.328.4887.33 ± 5.746.5786.00 ± 6.237.2487.30 ± 4.354.9810099.82 ± 2.892.89106.33 ± 2.882.70102.27 ± 4.974.8687.13 ± 5.776.622400104.17 ± 2.142.05100.92 ± 3.083.0599.98 ± 1.861.8687.02 ± 5.246.03Spleen297.62 ± 8.118.30109.83 ± 9.959.06105.55 ± 7.567.17109.70 ± 13.0511.904105.82 ± 9.919.37100.15 ± 9.699.67103.68 ± 9.719.3698.88 ± 4.164.20100112.00 ± 7.826.98111.12 ± 7.316.58106.82 ± 6.886.44111.17 ± 7.176.452400101.52 ± 7.737.61104.00 ± 7.457.17106.67 ± 5.435.09104.73 ± 7.046.72Lung2103.27 ± 8.017.75111.67 ± 5.434.86112.93 ± 9.578.48113.77 ± 9.208.084101.83 ± 6.416.30100.55 ± 8.588.53100.07 ± 10.3910.3898.77 ± 6.156.23100111.50 ± 5.474.90110.17 ± 10.939.92110.17 ± 7.146.48110.03 ± 9.338.462400100.67 ± 10.1710.1198.78 ± 7.017.10104.88 ± 8.237.85110.17 ± 4.544.12Kidney2110.07 ± 7.346.6788.18 ± 4.985.6497.22 ± 7.677.8989.87 ± 9.2310.27487.63 ± 7.768.8688.40 ± 6.197.0087.60 ± 9.2610.5889.60 ± 9.0510.10100112.00 ± 7.016.26111.92 ± 8.867.91111.17 ± 7.446.69103.22 ± 11.3210.972400100.55 ± 5.115.0998.10 ± 4.874.97100.45 ± 3.963.94101.23 ± 4.694.63Small intestine2105.00 ± 8.207.81102.63 ± 3.523.43103.90 ± 4.494.32101.42 ± 7.827.71491.73 ± 4.494.8999.47 ± 5.105.1399.78 ± 7.707.72106.70 ± 5.435.0910095.90 ± 12.3112.84108.67 ± 12.0411.08100.93 ± 12.3312.2299.07 ± 12.4112.522400112.00 ± 5.374.79111.67 ± 7.266.50111.67 ± 6.125.48110.83 ± 6.686.02Stomach2113.08 ± 11.069.78111.67 ± 6.255.60113.58 ± 8.227.24109.08 ± 9.979.144102.70 ± 10.179.9097.82 ± 9.059.2598.87 ± 9.829.9489.87 ± 7.047.8410093.75 ± 8.659.2294.82 ± 5.055.3393.47 ± 6.767.23107.33 ± 5.895.492400107.17 ± 8.688.1096.60 ± 3.603.72100.23 ± 4.634.6299.73 ± 10.0910.12Brain2110.33 ± 7.556.85106.15 ± 12.3611.65105.65 ± 10.9210.3490.73 ± 10.2511.30489.12 ± 9.0010.1087.57 ± 8.479.6790.68 ± 10.5111.59105.32 ± 9.729.2310091.93 ± 9.9310.80109.17 ± 7.446.82108.13 ± 9.919.17105.48 ± 6.926.562400102.93 ± 5.795.62106.72 ± 9.839.2196.57 ± 10.1210.4897.98 ± 9.569.75Naucleactonin CPlasma5101.95 ± 10.3310.14106.67 ± 10.8810.2091.87 ± 8.639.39109.57 ± 12.6611.558108.88 ± 1.871.72105.01 ± 6.326.02105.88 ± 1.871.77113.33 ± 5.324.6914087.67 ± 9.2010.5087.88 ± 4.144.71111.88 ± 1.871.67111.57 ± 9.608.60240088.93 ± 6.016.7589.25 ± 7.588.4989.17 ± 7.498.40113.83 ± 7.146.27Heart5111.97 ± 11.6310.39100.78 ± 9.639.56107.98 ± 9.458.75107.42 ± 10.249.538110.50 ± 4.424.00107.25 ± 9.258.62109.08 ± 8.257.56110.87 ± 9.048.15140102.78 ± 6.896.71104.88 ± 3.803.62100.40 ± 9.139.0998.28 ± 5.705.802400112.33 ± 10.419.26111.50 ± 5.685.10112.33 ± 5.725.09110.50 ± 4.814.35Liver5101.78 ± 11.8311.63107.70 ± 5.364.9898.32 ± 6.666.7898.38 ± 12.0712.27887.60 ± 5.906.7386.47 ± 4.525.2390.60 ± 4.915.4286.00 ± 8.6010.0014088.57 ± 2.442.7695.00 ± 4.154.3796.28 ± 4.364.5397.32 ± 7.177.36240098.93 ± 3.813.8591.37 ± 2.512.7589.35 ± 3.704.1589.97 ± 7.328.14Spleen5110.33 ± 8.717.89112.92 ± 8.727.72110.67 ± 8.577.75109.68 ± 12.5711.46899.28 ± 13.3113.4193.42 ± 8.008.5796.13 ± 11.9812.46101.90 ± 10.2510.0614093.13 ± 6.106.5595.27 ± 7.087.4389.18 ± 8.239.2392.35 ± 2.702.922400103.33 ± 8.378.10105.67 ± 8.508.05107.73 ± 8.107.51109.83 ± 4.964.51Lung5109.50 ± 11.4710.48100.40 ± 10.2610.22101.05 ± 5.335.2895.98 ± 10.4710.91894.73 ± 8.929.4191.40 ± 9.7110.63101.90 ± 10.4510.2599.03 ± 10.4910.59140102.38 ± 4.124.0394.45 ± 5.926.2793.75 ± 8.539.0992.58 ± 3.603.892400111.37 ± 9.278.33111.85 ± 9.538.52112.125 ± 9.668.61111.67 ± 3.393.03Kidney5109.33 ± 7.556.91106.08 ± 8.968.4498.92 ± 9.9110.0297.18 ± 10.4610.76899.10 ± 4.244.2797.38 ± 7.317.5191.10 ± 9.2010.1094.77 ± 7.628.0414096.08 ± 7.367.6695.43 ± 7.998.37101.10 ± 4.984.92100.25 ± 8.258.232400108.00 ± 5.625.20103.87 ± 4.684.51104.57 ± 6.095.82102.30 ± 5.905.77Small intestine5111.90 ± 12.0710.79111.67 ± 8.147.29111.17 ± 8.337.49112.67 ± 8.787.798103.33 ± 3.393.28100.03 ± 11.4611.46100.60 ± 10.2410.18104.25 ± 9.499.1014088.60 ± 9.4810.7089.80 ± 8.309.2487.88 ± 5.576.3490.17 ± 7.268.052400107.48 ± 8.237.66103.75 ± 6.686.44108.43 ± 8.657.98102.32 ± 8.658.45Stomach5111.00 ± 7.907.12112.17 ± 7.256.46113.12 ± 9.978.81113.83 ± 2.992.638107.32 ± 12.9612.0898.37 ± 10.7510.93105.90 ± 9.138.62103.55 ± 9.499.1614088.15 ± 7.918.9886.38 ± 8.389.7087.12 ± 5.246.188.28 ± 6.317.152400100.53 ± 7.297.2599.52 ± 5.855.88104.90 ± 5.535.28102.32 ± 8.228.03Brain5106.35 ± 11.8211.11109.83 ± 6.055.5198.92 ± 10.6510.77104.10 ± 10.8810.45888.87 ± 10.4311.7490.65 ± 6.377.0389.95 ± 9.2310.2688.82 ± 10.5711.9014088.33 ± 6.747.6387.92 ± 8.159.2787.20 ± 4.985.7187.93 ± 5.816.61240099.43 ± 6.386.41112.67 ± 6.625.88109.67 ± 6.445.87109.00 ± 5.224.78Khaephuoside APlasma599.10 ± 9.259.3390.97 ± 9.019.9093.93 ± 11.2611.98101.18 ± 3.533.49891.38 ± 12.7613.9694.88 ± 12.3112.9790.67 ± 9.2910.25108.17 ± 9.208.50120101.35 ± 4.334.28101.32 ± 3.403.3696.75 ± 4.554.70107.30 ± 11.4810.70180099.35 ± 6.046.08101.93 ± 6.906.7796.13 ± 4.034.19112.67 ± 7.666.80Heart5107.60 ± 11.2310.44104.15 ± 9.929.52110.67 ± 10.829.78107.45 ± 9.358.708112.67 ± 7.236.42111.83 ± 8.237.36111.33 ± 5.925.32101.00 ± 12.0511.93120111.67 ± 6.225.57112.67 ± 5.895.23108.58 ± 11.5110.60113.50 ± 7.206.351800110.83 ± 2.792.51106.58 ± 8.778.22113.67 ± 4.273.76101.90 ± 12.3012.07Liver593.87 ± 9.319.92106.17 ± 4.884.5995.62 ± 9.8210.27100.87 ± 10.2710.198108.95 ± 10.779.88101.97 ± 7.937.78105.20 ± 10.219.7091.12 ± 10.7911.84120107.67 ± 6.225.78111.50 ± 7.266.51101.62 ± 9.629.47104.23 ± 8.688.331800108.83 ± 2.322.13109.83 ± 5.384.90103.20 ± 7.527.29108.93 ± 9.528.74Spleen5110.30 ± 12.3111.16112.17 ± 7.947.07111.00 ± 10.269.24111.50 ± 8.417.548112.17 ± 5.645.02111.67 ± 6.195.54111.38 ± 7.366.58110.67 ± 7.176.48120107.85 ± 7.837.26105.45 ± 7.957.5498.83 ± 7.517.60105.42 ± 10.7910.241800109.77 ± 8.617.85111.17 ± 8.187.36113.17 ± 3.312.93111.83 ± 7.576.77Lung5113.33 ± 8.717.69106.65 ± 12.4311.6598.87 ± 9.029.12102.98 ± 9.509.238105.32 ± 7.817.41103.18 ± 10.9710.63105.03 ± 9.869.38104.00 ± 11.7311.28120101.27 ± 3.533.4999.07 ± 11.0111.11102.02 ± 6.366.23110.83 ± 5.194.691800110.83 ± 8.237.43112.80 ± 7.466.61110.00 ± 8.888.07112.50 ± 6.806.05Kidney596.42 ± 6.246.4799.65 ± 10.5310.57101.62 ± 11.3711.19112.78 ± 11.4310.138113.17 ± 6.465.71110.83 ± 7.887.11113.00 ± 6.996.18113.17 ± 7.196.36120111.67 ± 5.324.76111.83 ± 5.314.75112.00 ± 5.935.30112.00 ± 5.625.021800102.03 ± 4.444.3597.37 ± 6.847.02100.50 ± 5.535.50101.12 ± 6.566.48Small intestine5110.02 ± 8.197.44112.83 ± 3.433.04100.88 ± 9.399.3094.98 ± 9.429.918105.62 ± 9.769.24109.53 ± 10.149.25101.00 ± 10.4210.31107.70 ± 11.4710.65120103.30 ± 7.757.50106.00 ± 7.296.88102.80 ± 6.095.93101.58 ± 6.266.161800113.00 ± 6.135.43110.83 ± 8.137.34113.67 ± 8.197.20113.12 ± 12.4711.02Stomach5113.68 ± 8.427.41112.13 ± 10.239.12111.12 ± 10.439.38110.67 ± 6.095.508111.67 ± 6.595.90111.00 ± 5.224.70109.67 ± 3.333.03104.50 ± 4.714.51120113.17 ± 3.663.23111.50 ± 5.865.25112.17 ± 6.155.48110.92 ± 8.367.531800109.83 ± 7.366.70111.92 ± 10.999.82110.50 ± 6.285.69113.83 ± 5.564.89Brain5109.82 ± 10.269.34106.90 ± 12.0911.31103.05 ± 9.128.85112.50 ± 5.584.968111.00 ± 7.426.52112.50 ± 7.066.28113.33 ± 3.833.38111.33 ± 6.095.47120112.50 ± 5.134.56112.33 ± 6.475.76113.17 ± 5.314.69110.17 ± 8.287.521800105.82 ± 8.317.86112.83 ± 7.686.81110.83 ± 7.526.79113.83 ± 4.493.953,4-Dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranosidePlasma2586.25 ± 5.406.2694.95 ± 9.8010.32102.87 ± 10.3710.08105.60 ± 10.199.655087.20 ± 7.919.0798.98 ± 11.3011.3094.53 ± 9.8410.41107.88 ± 14.9613.87350112.83 ± 4.584.06112.67 ± 6.625.88108.33 ± 5.134.73109.07 ± 1.871.72240089.98 ± 4.765.2989.43 ± 7.428.3086.07 ± 3.393.93111.74 ± 3.763.37Heart2598.10 ± 9.439.61100.45 ± 10.8910.8597.55 ± 9.019.2488.38 ± 5.696.445094.60 ± 11.147.80102.70 ± 7.987.7797.05 ± 8.428.6899.12 ± 9.569.65350102.57 ± 4.924.80112.63 ± 98.937.93110.33 ± 5.825.27108.38 ± 7.346.772400102.23 ± 6.646.4992.98 ± 8.879.54101.57 ± 6.736.6399.57 ± 9.469.50Liver2591.10 ± 10.3711.39102.88 ± 10.8510.5497.32 ± 10.4410.73104.70 ± 11.9711.4350105.60 ± 9.048.56112.50 ± 5.655.02107.48 ± 10.549.8095.25 ± 11.2911.8635097.50 ± 6.636.80113.00 ± 4.003.54103.12 ± 5.265.10108.67 ± 8.988.272400104.32 ± 10.189.76100.42 ± 6.856.8396.25 ± 5.745.96107.68 ± 7.737.18Spleen2588.72 ± 9.9311.19104.47 ± 9.008.6290.93 ± 9.1510.07112.83 ± 3.603.1950113.17 ± 10.599.36108.35 ± 10.589.7795.62 ± 10.9011.40107.68 ± 10.399.65350111.33 ± 7.636.86111.68 ± 9.218.25112.17 ± 7.997.12112.17 ± 8.407.492400102.27 ± 11.5011.24112.00 ± 8.677.74105.32 ± 6.596.26111.00 ± 6.035.44Lung2590.52 ± 10.7711.9096.80 ± 11.2911.6797.07 ± 10.8711.2097.92 ± 11.3411.5950112.67 ± 7.846.96111.02 ± 7.817.04100.02 ± 12.4612.46109.25 ± 11.4610.49350111.33 ± 6.896.19109.47 ± 11.1010.14103.68 ± 11.6211.21112.33 ± 6.926.16240098.85 ± 9.839.94101.82 ± 8.678.52100.62 ± 7.717.66109.12 ± 10.699.79Kidney25100.05 ± 11.9211.92101.42 ± 8.638.5196.23 ± 9.149.50110.83 ± 6.245.635098.37 ± 7.537.65102.72 ± 10.2910.02106.75 ± 9.158.5799.88 ± 9.659.6635090.05 ± 8.479.40102.03 ± 4.614.52111.83 ± 7.036.28105.40 ± 10.289.752400104.72 ± 8.968.56106.27 ± 10.199.59107.85 ± 9.168.49104.82 ± 8.598.20Small intestine2587.47 ± 7.178.2096.42 ± 9.139.4794.10 ± 8.058.5599.25 ± 6.066.1150109.83 ± 2.562.33100.75 ± 6.116.07106.83 ± 5.345.00101.80 ± 8.788.62350111.17 ± 7.736.95113.67 ± 8.767.70113.00 ± 3.793.36106.33 ± 5.395.07240098.50 ± 5.495.57104.65 ± 7.857.5099.92 ± 5.945.9494.90 ± 8.629.08Stomach25111.83 ± 5.785.17110.50 ± 4.424.00113.00 ± 6.325.60106.32 ± 9.488.9250112.75 ± 10.979.73112.67 ± 3.673.26112.83 ± 5.604.96109.83 ± 8.667.88350113.17 ± 3.873.42108.17 ± 9.688.95111.00 ± 6.205.58111.83 ± 5.985.352400101.27 ± 7.897.8094.40 ± 6.006.3596.82 ± 8.088.34103.08 ± 9.609.32Brain2599.82 ± 9.829.84107.80 ± 12.7911.86100.68 ± 9.809.73105.37 ± 7.316.9350109.35 ± 12.1511.11112.83 ± 4.263.78113.77 ± 10.369.11113.83 ± 3.873.40350111.50 ± 8.647.75110.83 ± 7.476.74111.17 ± 7.636.27112.83 ± 7.786.90240098.95 ± 3.974.01100.10 ± 9.439.4298.23 ± 5.395.49101.50 ± 9.949.80


### 3.4. Pharmacokinetic Study

The validated analytical method was successfully applied to the quantitative estimation of chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside in the plasma samples following oral administration of Nauclea officinalis extract at a dosage of 2 g/kg. Pharmacokinetic parameters of four compounds were calculated by noncompartmental model using DAS 3.3.0 software. The mean plasma concentration–time-curve of four analytes are shown in Figure 3, and the main pharmacokinetic parameters were estimated of four analytes in Table 6.Figure 3
Concentration-time profiles of four analytes in rat plasma following oral administration ofNauclea officinalis extracts at a dose of 2 g/kg.Table 6
Summary of primary pharmacokinetic parameters of four analytes (n = 6).
ParametersUnitChlorogenic acid (mean ± SD)Naucleactonin C (mean ± SD)Khaephuoside A (mean ± SD)3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside (mean ± SD)AUC (0 −t)μg/L∗h482.76 ± 124.955281.36 ± 440.02238.70 ± 46.64212.99 ± 49.29AUC (0 − ∞)μg/L∗h554.37 ± 166.145405.02 ± 416.99262.16 ± 32.24440.17 ± 216.99MRT (0 −t)h5.33 ± 0.654.76 ± 1.573.19 ± 0.241.6 ± 0.14t1/2h10.64 ± 6.214.59 ± 1.653.12 ± 1.703.99 ± 2.87Tmaxh0.25 ± 0.001.67 ± 0.520.29 ± 0.100.33 ± 0.13CLL/h/kg62.94 ± 16.000.15 ± 0.0154.40 ± 6.0428.63 ± 13.64Cmaxμg/L242.42 ± 84.671008.58 ± 368.84106.18 ± 23.30144.13 ± 35.59Vz/FL/kg915.76 ± 451.750.966 ± 0.359253.289 ± 158.11127.43 ± 65.93Abbreviations. AUC, area under the plasma concentration–time curve; MRT, mean residence time;t1/2, elimination half-life; Tmax, time to Cmax; CL, clearance rate; Cmax, maximum plasma concentration; Vz/F, apparent distribution volume.These compounds were detected at 5 min after oral administration ofNauclea officinalis extracts, indicating four compounds were rapidly absorbed, exhibiting a therapeutic effect. Chlorogenic acid, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside were rapidly absorbed and peaked at approximately 0.5 h, while naucleactonin C peaked at 1.66 h, indicating that naucleactonin C was absorbed relatively slowly. The content of naucleactonin C was much lower than that of the other three compounds in the Nauclea officinalis extracts, but naucleactonin C was present in a much larger amount than other three compounds for AUC0−t (area under the plasma concentration–time curve) and AUC0−∞, indicating that naucleactonin C may had excellent bioavailability. However, the bioavailability of chlorogenic acid and khaephuoside A may not be satisfactory, the CL of chlorogenic acid and khaephuoside A were 62.93 ± 15.996 and 54.40 ± 6.04 L/h/kg after oral administration of Nauclea officinalis extract, respectively. Excessive clearance rate will affect the residence time of the drug in the body, thereby reducing the efficacy. In addition, literature reported that the prototype chlorogenic acid absorbed into the blood by oral chlorogenic acid accounts for only 30% [25], which greatly affected the pharmacological activity of chlorogenic acid from Nauclea officinalis. Therefore, considering the clinical application of Nauclea officinalis [26], we could design Nauclea officinalis to different dosage forms with advanced technology in order to improve the bioavailability and finally increase the efficacy.


### 3.5. Tissue Distribution Study

This method was also applied to investigate the tissue distribution of chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside in rats. The result is shown in Figure 4 that naucleactonin C and khaephuoside A could be detected in all studied tissue. However, chlorogenic acid and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside were not detected in the spleen. The concentration orders in eight different tissues were ranked as 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside > Khaephuoside A > Naucleactonin C > Chlorogenic acid in the small intestine, stomach, heart, liver, spleen, lung, and brain tissues, and naucleactonin C > 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside > Khaephuoside A > Chlorogenic acid in the kidney. The contents of the four compounds in the liver, kidney, and lung were relatively high within after oral administration of Nauclea officinalis extrac, indicating that the liver, kidney, and lung may be the main target organs for the pharmacological effects of Nauclea officinalis. This result is consistent with existing research on the inhibition of bronchitis by Nauclea officinalis [27]. The results show that all compounds except chlorogenic acid were more abundant in the small intestine than in the stomach, indicating that most of naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside possibly absorbed by the small intestine after oral administration of Nauclea officinalis extract for enter the systemic circulation system. With the exception of chlorogenic acid, the peak times and concentrations of the other three compounds in most tissues are nearly identical to those in plasma, which means that the distribution of the other three compounds depends on the blood flow or perfusion rate of the organ [28]. The peak concentration of chlorogenic acid in most tissues is much lower than in plasma and may be related to the absorption of the compound in the tissue. Drugs enter tissues that receive high blood flow first, followed by those that receive low blood flow [29]. Our study demonstrated that the four compounds are mainly distributed in organs with relatively large blood flows, such as the liver and kidneys. In addition to this, the first-pass effect of oral administration might be the main cause of high distribution in the liver.Figure 4
Mean concentration of four analytes in the heart, liver, spleen, lung, kidney, small intestine, stomach, and brain at 0.5, 1, 2, 4, and 6 h after oral administration ofNauclea officinalis extracts at a dose of 2 g/kg.


## 3.1. Optimization of Chromatographic and Mass Spectrometric Conditions

Optimization of chromatographic conditions: by analyzing the separation effect and peak shape of four analytes and the IS on different columns, we found that the Phenomenex Kinete EVO C18 column gave the best separation and peak shape. Subsequently, by exploring different mobile phase systems, such as methanol-water and acetonitrile-water, we found that the four analytes had better response values in acetonitrile. Finally, by assessing the pH of the mobile phase (0.1, 0.2, 0.5, and 0.8% formic acid solution), the response values of four analytes were highest when the concentration of formic acid was 0.1%. Therefore, 0.1% formic acid water-acetonitrile was selected for use as the mobile phase.Optimization of mass spectrometric conditions: the four analytes and the IS had strong [M-H]− peaks in the negative ion mode, which can be easily broken and used for stable fragment analysis and detection. Therefore, the ESI in negative ion mode and Q1, MS2, and MRM scan modes were adopted. The chemical structures, precursor ion, and product ion of four analytes and IS are shown in Figure 1.Figure 1
Chemical structures, precursor ion, and product ion mass spectra of four analytes and IS.


## 3.2. Optimization of the Sample Preparation

For sample processing, a simple protein precipitation method was first tried for methanol and acetonitrile, respectively, but the recoveries of chlorogenic acid and naucleactonin C were unsatisfactory. In addition, liquid-liquid extraction (LLE) was tried for various solvents tested, including n-butanol, dichloromethane, isopropanol, methyl tert-butyl ether, and ethyl acetate, but no satisfactory recovery was obtained for khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside. The extraction of chlorogenic acid should be in an acidic condition. Therefore, the extraction results of chlorogenic acid at different pH are investigated.Finally, the combination method using mixture solution of methanol-ethyl acetate (1 : 1, v/v) including 0.1% (v/v) formic acid was selected. Consequently, satisfactory and consistent recovery from plasma and tissues samples was achieved for four analytes and IS.


## 3.3. Method Validation

### 3.3.1. Selectivity

The selectivity was assessed by analyzing chromatograms of blank rat plasma and tissue homogenates from different batches, blank plasma and tissue homogenates spiked with analytes and IS, and plasma, tissue samples obtained from rats after oral administration ofNauclea officinalis extracts (n = 6). Figure 2 shows typical chromatograms of four analytes and IS in rat plasma (Figures S1 and S2 of the Supporting Material show typical chromatograms of four analytes and IS in liver tissue and kidney tissue). Under the given condition, Chlorogenic acid, Naucleactonin C, Khaephuoside A, 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside, and IS were eluted approximately at retention time of 1.02 min, 1.65 min, 0.99 min, 0.97 min, and 1.16 min, respectively. No interfering endogenous substances were found at the respective retention times.Figure 2
Typical chromatograms of four analyte and IS in rat plasma: (a) blank plasma samples, (b) blank plasma samples spiked with four analytes (LLOQ) and IS, (c) rat plasma samples at 0.5 h after oral administration theNauclea officinalis extracts spiked with IS; (1) chlorogenic acid, (2) naucleactonin C, (3) khaephuoside A, (4) 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside, (5) chloramphenicol (IS).
(a)(b)(c)


### 3.3.2. Linearity of Calibration Curve and Lower Limit of Quantification

The calibration curves were constructed by plotting the peak areas ratios of chlorogenic acid/IS, Naucleactonin C/IS, Khaephuoside A/IS, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside/IS versus theoretical concentrations. This method exhibited a good linear response for the range of concentrations from 2 to 2500 ng/mL in plasma and from 2 to 2500 ng/mL in tissues; all determination coefficients (R2) were greater than 0.9849. The lower limit of quantification (LLOQ) was defined as the lowest concentration of a signal-noise (S/N) ratios of 10 : 1, respectively, the precision was less than 15% and the accuracy was within ±20%. The limits were adequate for studies of pharmacokinetics and tissue distribution by oral administration of Nauclea officinalis extracts. Data from the determination are shown in Table 2.Table 2
Summary of standard curves, correlation coefficients, LLOQ, and linear ranges of four analytes in plasma and tissue samples.
CompoundsMatrixLinear regression equationR2LLOQ (ng/mL)Linear range (ng/mL)Chlorogenic acidPlasmay = 0.0288x + 0.001930.999422–2500Hearty = 0.0063x − 0.007710.984922–2500Livery = 0.0267x − 0.03230.998022–2500Spleeny = 0.0392x + 0.02770.988622–2500Lungy = 0.0442x + 0.02390.995822–2500Kidneyy = 0.0217x − 0.03020.995022–2500Small intestiney = 0.0334x − 0.04010.996422–2500Stomachy = 0.0402x − 0.01290.995222–2500Brainy = 0.0499x − 0.05620.996022–2500Naucleactonin CPlasmay = 0.00535x + 0.02070.996455–2500Hearty = 0.00313x − 0.001160.992655–2500Livery = 0.00395x − 0.01010.996455–2500Spleeny = 0.00511x − 0.005260.995055–2500Lungy = 0.00534x − 0.002630.998055–2500Kidneyy = 0.00363x − 0.00980.996055–2500Small intestiney = 0.00224x − 0.005270.996855–2500Stomachy = 0.00465x − 0.01380.997255–2500Brainy = 0.00552x − 0.01630.993255–2500Khaephuoside APlasmay = 0.00316x + 0.01530.995455–2000Hearty = 0.00673x − 0.0050.994655–2000Livery = 0.0063x − 0.005020.996255–2000Spleeny = 0.011x − 0.00540.998055–2000Lungy = 0.0099x − 0.001660.997855–2000Kidneyy = 0.00531x − 0.00470.995455–2000Small intestiney = 0.0058x − 0.01560.990655–2000Stomachy = 0.00745x − 0.008370.992055–2000Brainy = 0.0126x + 0.0003770.993855–20003,4-Dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranosidePlasmay = 0.000845x + 0.003960.99842525–2500Hearty = 0.000804x + 0.001590.98492525–2500Livery = 0.000873x − 0.005150.99682525–2500Spleeny = 0.00134x + 0.009470.98942525–2500Lungy = 0.00138x + 0.006260.99642525–2500Kidneyy = 0.000633x − 0.0005380.99302525–2500Small intestiney = 0.00109x + 0.0009660.99622525–2500Stomachy = 0.00119x + 0.005490.99642525–2500Brainy = 0.00163x − 0.01490.99522525–2500Abbreviations. LLOQ, lower limit of quantification;R2, determination coefficients.


### 3.3.3. Accuracy and Precision

Accuracy and precision were evaluated by analyzing QC samples at high, medium, low, and LLOQ concentrations (n = 6) on the same day and three consecutive days using the standard curve, respectively. The acceptable limits of accuracy were required to be within ±15% of the actual value except when at LLOQ, and the intra- and interday precision and accuracy data of the determination were shown in Table 3. In our study, the intraday precision ranged from 0.62 to 12.65%, and intraday accuracy ranged from 86.48 to 114.17%. The interday precision ranged from 2.42 to 12.61%, and the interday accuracy ranged from 86.31 to 113.99%. The results showed the accuracy and precision were within the acceptable limits, which proved the method was reproducible, reliable, and accurate for the determination of four analytes in rat plasma and tissue samples.Table 3
Summary of intraday and interday precisions and accuracies of four analytes in plasma and tissue samples.
CompoundsMatrixConcentration (ng/mL)Intraday (n = 6)Interday (n = 18)AccuracyRSD (%)AccuracyRSD (%)Chlorogenic acidPlasma2104.02 ± 11.9111.45100.64 ± 11.6711.60497.72 ± 9.239.4596.09 ± 7.087.36100104.42 ± 6.235.97104.29 ± 10.610.162400101.22 ± 3.203.1696.59 ± 8.118.40Heart2101.90 ± 12.1411.92109.34 ± 11.7410.74499.35 ± 3.893.9193.67 ± 6.136.5410096.57 ± 4.814.9894.46 ± 7.808.262400109.00 ± 5.905.41102.88 ± 6.856.66Liver2101.65 ± 2.822.78100.88 ± 5.385.334101.45 ± 2.982.9398.59 ± 6.726.82100100.30 ± 2.892.8897.78 ± 4.895.00240095.07 ± 5.746.0497.11 ± 5.585.75Spleen296.40 ± 7.637.92102.14 ± 8.908.71495.40 ± 4.204.40101.28 ± 9.619.49100102.40 ± 2.001.95111.52 ± 7.006.27240094.93 ± 4.815.07108.26 ± 10.419.62Lung2101.53 ± 6.406.30101.22 ± 7.457.36499.75 ± 6.096.1093.33 ± 11.1711.97100108.33 ± 5.475.04111.33 ± 6.495.832400100.60 ± 3.353.33101.63 ± 5.074.99Kidney298.12 ± 5.365.4796.23 ± 5.335.544100.13 ± 2.172.1792.09 ± 7.217.8310099.37 ± 5.205.2396.10 ± 6.366.622400104.47 ± 5.795.5499.47 ± 6.066.10Small intestine293.27 ± 4.134.4396.03 ± 7.868.19486.48 ± 5.095.8993.40 ± 10.7511.5110089.75 ± 7.968.8796.61 ± 10.0610.41240092.70 ± 6.637.1693.01 ± 5.275.66Stomach2105.87 ± 7.877.43107.84 ± 7.456.90493.17 ± 7.027.5398.76 ± 6.446.52100106.00 ± 3.953.73106.35 ± 4.464.192400100.63 ± 4.184.1599.41 ± 4.814.84Brain2109.27 ± 6.746.17104.39 ± 9.479.074101.00 ± 3.813.77101.29 ± 5.425.35100106.00 ± 4.944.66106.77 ± 7.166.702400101.27 ± 2.252.22105.62 ± 7.046.67Naucleactonin CPlasma595.62 ± 12.1012.6598.24 ± 10.3010.488100.53 ± 11.7811.7196.43 ± 12.1612.6114099.95 ± 6.496.5095.76 ± 7.187.50240097.05 ± 4.804.9595.50 ± 6.236.52Heart5111.27 ± 7.326.58107.46 ± 10.589.85899.28 ± 5.485.52101.15 ± 11.4611.3314093.45 ± 4.344.6593.19 ± 5.335.722400105.92 ± 9.549.01104.46 ± 6.666.38Liver5100.90 ± 5.405.36108.30 ± 7.827.22899.47 ± 2.822.84100.52 ± 5.004.9814088.60 ± 2.602.9393.53 ± 6.336.772400102.50 ± 1.521.48101.42 ± 2.462.42Spleen5111.00 ± 6.996.29104.42 ± 9.318.91898.90 ± 3.553.59100.74 ± 6.776.7214096.07 ± 3.954.1193.72 ± 5.305.662400100.02 ± 3.393.39103.13 ± 5.064.91Lung5113.83 ± 4.073.58113.44 ± 5.374.738103.28 ± 10.4610.13102.03 ± 10.169.96140104.17 ± 3.763.61103.83 ± 4.053.902400107.83 ± 4.073.77107.06 ± 5.024.69Kidney5100.70 ± 4.284.25103.81 ± 6.616.36898.38 ± 5.315.4099.18 ± 4.364.3914095.37 ± 4.194.39100.44 ± 7.077.042400106.80 ± 9.749.12104.87 ± 6.466.16Small intestine5109.98 ± 10.979.97105.41 ± 10.199.67899.30 ± 8.949.01100.44 ± 9.189.1414088.73 ± 8.9610.1086.31 ± 7.668.882400103.30 ± 6.826.6098.26 ± 7.607.73Stomach5109.83 ± 7.056.42102.67 ± 10.099.838101.58 ± 2.962.9295.73 ± 8.078.4314094.88 ± 2.742.8892.09 ± 5.175.622400113.17 ± 4.884.31108.96 ± 7.216.62Brain5105.75 ± 10.8110.22105.82 ± 8.778.298104.78 ± 7.467.12100.57 ± 8.118.06140101.22 ± 4.594.5398.93 ± 5.735.802400113.83 ± 3.312.91111.39 ± 5.544.98Khaephuoside APlasma5101.63 ± 9.939.77105.66 ± 10.509.948101.02 ± 9.649.55101.78 ± 7.857.71120112.33 ± 4.764.24106.16 ± 6.115.761800104.08 ± 4.394.21102.52 ± 4.414.31Heart5113.50 ± 9.188.09103.33 ± 10.9210.578100.82 ± 6.196.1498.68 ± 7.067.1612096.73 ± 2.923.0289.72 ± 8.018.931800109.12 ± 9.458.66102.43 ± 8.137.94Liver599.97 ± 7.067.07101.51 ± 8.498.368104.10 ± 5.074.87101.81 ± 4.023.9512099.33 ± 0.620.62100.43 ± 4.554.531800108.00 ± 6.105.65103.94 ± 5.605.39Spleen598.73 ± 11.2111.35103.62 ± 9.479.148100.98 ± 6.256.19103.08 ± 6.756.5512099.35 ± 2.522.54109.89 ± 8.848.041800109.00 ± 4.604.22113.06 ± 5.364.74Lung5108.17 ± 10.199.42109.93 ± 7.66.928109.90 ± 11.2510.24106.91 ± 9.959.3112090.32 ± 6.126.7895.54 ± 6.536.831800100.73 ± 2.022.00103.65 ± 6.916.66Kidney5106.82 ± 7.917.40109.94 ± 6.295.728113.67 ± 2.942.59111.40 ± 6.125.49120105.30 ± 6.145.83107.78 ± 6.916.411800105.02 ± 9.819.34108.84 ± 7.606.99Small intestine5106.50 ± 8.538.01104.08 ± 10.119.71899.98 ± 9.439.13101.89 ± 11.5811.3612089.90 ± 6.847.6193.74 ± 7.808.321800104.45 ± 6.225.96105.44 ± 5.154.89Stomach596.63 ± 9.159.4795.98 ± 10.2610.69890.92 ± 7.968.7697.66 ± 8.618.82120104.00 ± 6.135.90103.24 ± 5.315.151800109.83 ± 4.834.40110.50 ± 4.544.11Brain5108.90 ± 9.748.95107.11 ± 7.947.418108.55 ± 9.909.12107.23 ± 8.017.47120113.17 ± 6.715.93110.13 ± 6.095.531800100.50 ± 3.993.97108.44 ± 7.637.033,4-Dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranosidePlasma25109.00 ± 4.053.72101.34 ± 9.108.9850105.82 ± 10.9510.35102.61 ± 7.897.69350109.83 ± 5.234.76104.49 ± 8.608.23240096.82 ± 2.762.85100.71 ± 7.507.45Heart2599.12 ± 5.065.1096.73 ± 8.088.355096.45 ± 6.757.0098.20 ± 8.288.4335089.15 ± 4.835.4290.19 ± 6.827.56240099.80 ± 10.8910.9199.33 ± 7.667.71Liver25105.07 ± 6.626.30103.78 ± 6.396.1650108.48 ± 7.396.82108.22 ± 7.386.82350112.15 ± 6.896.14113.99 ± 4.834.24240098.28 ± 3.673.73100.28 ± 3.243.23Spleen25102.03 ± 10.6810.47104.78 ± 8.638.235099.90 ± 5205.21103.82 ± 7.777.4835097.70 ± 4.124.21109.12 ± 9.078.312400100.65 ± 5.465.43110.22 ± 8.948.11Lung2596.17 ± 7.597.8999.80 ± 10.9911.0150106.78 ± 7.086.63107.49 ± 8.167.5935096.42 ± 4.494.66100.05 ± 5.505.49240099.67 ± 6.296.3199.32 ± 5.685.72Kidney2597.02 ± 9.7510.0596.23 ± 9.389.7450108.00 ± 5.665.24106.69 ± 7.006.5635099.63 ± 5.835.85100.46 ± 6.786.75240096.50 ± 7.417.6897.74 ± 6.316.46Small intestine2598.25 ± 8.929.0897.96 ± 7.737.9050101.58 ± 6.416.31101.89 ± 7.137.0035089.58 ± 4.274.7698.17 ± 8.398.552400100.18 ± 4.774.76101.32 ± 4.834.77Stomach25103.63 ± 5.215.0299.07 ± 9.649.7350101.90 ± 9.329.1497.96 ± 9.339.52350114.17 ± 3.192.79109.34 ± 6.145.622400100.62 ± 3.423.4099.42 ± 4.664.69Brain25106.95 ± 12.1311.3499.13 ± 11.2611.3650105.78 ± 7.637.22107.10 ± 7.867.34350110.00 ± 9.018.19112.56 ± 6.936.16240099.42 ± 2.902.91101.13 ± 5.445.38


### 3.3.4. Extraction Recovery and Matrix Effect

Six batches of blank plasma from independent sources were used to obtain extracted samples, postextracted spiked samples, and unextracted samples at high, medium, low, and LLOQ concentrations. The peak areas of the three types of samples were recorded as A, B, and C, respectively. Extraction recovery was evaluated by A/B. Matrix effects were calculated by comparing the A/C of analytes. The extraction, recovery, and matrix effect of four analytes in rat plasma and tissues are shown in Table4. The recovery rate were 51.85–97.06%, 75.99–106.68%, 77.46–105.35%, and 68.36–103.75% for chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside and the matrix effects were 50.17–116.62%, 86.75–115.99%, 45.79–87.44%, and 51.60–92.34% for chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside in different matrices, indicating some ion inhibitory effects for four compounds in the biological samples, the RSD (relative standard deviation) of recovery and matrix effect were less than 15%. There were no significant differences in recoveries and matrix effects between the four compounds at different concentrations in the same matrix.Table 4
Summary of extraction recovery and matrix effect of four analytes in plasma and tissue samples (n = 6).
CompoundsMatrixConcentration (ng/mL)Matrix effect (%)RSD (%)Extraction recovery (%)RSD (%)Chlorogenic acidPlasma297.26 ± 5.355.5080.45 ± 4.365.424109.75 ± 4.404.0180.04 ± 3.083.8510082.80 ± 6.517.8669.49 ± 4.055.84240091.66 ± 1.531.6772.26 ± 1.161.61Heart2116.54 ± 3.032.6080.53 ± 9.6211.95498.55 ± 2.712.7576.82 ± 4.776.2110097.78 ± 1.761.8062.02 ± 2.243.61240093.25 ± 0.760.8163.32 ± 0.580.91Liver283.69 ± 4.245.0697.06 ± 3.753.86479.96 ± 4.795.9479.07 ± 2.903.6610063.82 ± 2.513.9466.44 ± 0.240.37240080.01 ± 0.901.1269.83 ± 1.552.22Spleen299.89 ± 2.612.6180.09 ± 1.221.524100.56 ± 1.991.9886.82 ± 2.883.3210092.05 ± 1.011.1067.29 ± 1.812.69240091.66 ± 0.810.8873.12 ± 1.612.20Lung2104.73 ± 1.781.7081.80 ± 2.523.08499.34 ± 2.072.0988.65 ± 1.551.7510098.65 ± 1.241.2580.24 ± 2.112.63240096.68 ± 1.811.8876.89 ± 1.692.20Kidney290.49 ± 5.666.2678.34 ± 4.025.13498.52 ± 4.214.2786.92 ± 3.003.4610095.03 ± 5.946.2570.73 ± 4.906.92240089.55 ± 2.383.1672.70 ± 1.502.07Small intestine256.96 ± 6.1610.8156.20 ± 3.365.98463.67 ± 2.464.1451.85 ± 1.943.7310053.67 ± 1.061.9761.26 ± 1.873.05240050.17 ± 1.723.4262.66 ± 1.061.70Stomach2114.40 ± 5.014.4685.33 ± 5.206.104116.62 ± 8.807.5591.66 ± 6.116.67100102.87 ± 3.243.1566.48 ± 3.945.93240098.38 ± 1.631.6569.41 ± 1.562.25Brain297.89 ± 6.426.5682.33 ± 4.655.654105.09 ± 2.992.8475.80 ± 3.594.7410090.02 ± 5.466.0671.37 ± 1.171.63240090.56 ± 2.162.3986.06 ± 1.681.95Naucleactonin CPlasma5104.57 ± 6.946.64100.30 ± 11.7711.748102.54 ± 2.552.48106.68 ± 6.315.91140104.07 ± 2.882.7796.41 ± 1.661.722400102.64 ± 2.092.0488.27 ± 1.051.19Heart598.86 ± 2.672.7097.71 ± 1.952.00899.69 ± 3.963.9796.33 ± 7.197.46140106.24 ± 4.874.5885.37 ± 2.903.40240099.81 ± 1.481.4886.76 ± 1.611.86Liver5101.81 ± 10.8810.6992.06 ± 9.8710.728111.16 ± 3.122.8185.03 ± 4.915.7714097.92 ± 5.095.2084.88 ± 4.525.33240097.02 ± 1.111.1480.54 ± 0.831.03Spleen590.83 ± 3.654.02100.21 ± 9.219.19893.78 ± 6.076.4799.90 ± 5.495.5014097.28 ± 2.712.7984.00 ± 1.541.832400100.67 ± 0.790.7884.60 ± 1.401.65Lung598.17 ± 7.637.77105.63 ± 3.873.668115.99 ± 7.975.0199.21 ± 12.4312.5314098.73 ± 1.391.4195.48 ± 2.883.01240099.21 ± 0.910.9284.40 ± 1.822.15Kidney599.90 ± 5.105.1094.08 ± 8.118.628100.35 ± 5.145.12105.08 ± 5.224.97140101.18 ± 0.700.6986.02 ± 4.395.102400102.49 ± 1.871.8386.64 ± 2.262.60Small intestine598.00 ± 5.515.6298.55 ± 9.619.75899.37 ± 8.488.9082.00 ± 5.036.1414095.42 ± 5.525.7991.73 ± 1.671.82240086.75 ± 2.242.5886.61 ± 3.153.63Stomach5101.53 ± 3.553.5097.93 ± 4.194.288101.21 ± 3.873.8299.25 ± 3.173.2014099.62 ± 1.191.2082.46 ± 1.882.282400101.37 ± 1.351.3375.99 ± 2.773.64Brain597.50 ± 6.506.6788.08 ± 3.123.548105.47 ± 5.144.8788.38 ± 7.328.29140102.02 ± 0.850.8397.63 ± 4.354.462400101.24 ± 2.442.4199.47 ± 1.421.43Khaephuoside APlasma564.06 ± 5.668.8488.84 ± 5.105.75865.86 ± 5.658.5792.35 ± 8.739.4512055.55 ± 4.057.2985.88 ± 2.062.40180058.01 ± 1.933.3385.24 ± 3.303.87Heart570.92 ± 8.0911.4196.07 ± 5.375.59877.13 ± 3.564.6182.29 ± 3.954.8012054.79 ± 1.142.0892.18 ± 1.641.78180059.93 ± 4.647.7586.98 ± 4.875.59Liver573.90 ± 6.849.26105.35 ± 11.0210.46853.20 ± 1.592.98103.34 ± 3.753.6312058.17 ± 1.121.9377.46 ± 2.172.80180061.29 ± 2.804.5884.49 ± 1.702.01Spleen568.33 ± 1.412.0790.99 ± 7.898.67860.36 ± 2.584.2896.38 ± 2.772.8812058.12 ± 0.420.7390.31 ± 2.803.10180061.44 ± 1.131.8486.75 ± 0.981.13Lung584.39 ± 4.204.9777.71 ± 3.734.81872.06 ± 6.899.5687.06 ± 7.308.3812059.00 ± 2.554.3386.34 ± 3.484.03180061.35 ± 4.417.1979.39 ± 6.488.17Kidney557.46 ± 1.803.1397.31 ± 4.384.50851.41 ± 2.915.6699.78 ± 3.413.4212045.79 ± 1.302.8499.65 ± 1.231.23180047.23 ± 1.382.93101.56 ± 2.172.14Small intestine556.14 ± 1.472.61102.93 ± 10.4410.15867.00 ± 5.438.1094.63 ± 6.276.6312062.69 ± 2.063.29100.39 ± 5.004.98180071.51 ± 2.082.9199.78 ± 1.061.06Stomach556.84 ± 3.215.6495.49 ± 6.606.91863.57 ± 5.598.8083.81 ± 6.898.2212054.53 ± 2.444.4877.49 ± 4.085.27180055.57 ± 1.071.9279.96 ± 0.871.09Brain576.07 ± 1.692.2293.48 ± 8.709.30876.50 ± 6.238.15102.39 ± 6.806.6412087.44 ± 3.233.7090.50 ± 0.991.09180079.81 ± 2.152.70100.48 ± 1.421.413,4-Dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranosidePlasma2564.80 ± 4.957.6488.62 ± 8.569.665065.25 ± 0.811.2489.23 ± 4.244.7635060.35 ± 3.676.0885.65 ± 1.171.37240055.98 ± 2.344.1884.82 ± 3.163.72Heart2579.81 ± 2.392.9980.56 ± 4.055.035073.97 ± 1.842.4989.91 ± 2.893.2135069.51 ± 4.045.8185.34 ± 4.204.92240064.96 ± 2.283.5185.00 ± 3.153.71Liver2555.02 ± 4.027.3188.70 ± 8.389.455053.00 ± 1.633.0894.52 ± 4.765.0435052.69 ± 2.124.0383.51 ± 2.633.15240051.60 ± 1.442.8087.94 ± 2.883.27Spleen2581.22 ± 3.274.0298.19 ± 2.872.925087.52 ± 4.515.1692.02 ± 3.153.4235081.89 ± 1.031.2689.66 ± 2.422.70240080.06 ± 3.073.8380.48 ± 2.152.68Lung2591.11 ± 4.014.4068.36 ± 4.426.475092.34 ± 3.463.7577.05 ± 2.142.7835077.28 ± 4.365.6583.53 ± 3.424.09240076.69 ± 2.733.5675.92 ± 0.730.97Kidney2563.39 ± 1.542.4386.96 ± 4.515.195064.43 ± 1.532.3899.12 ± 2.652.6735053.76 ± 3.075.72101.00 ± 1.811.79240060.75 ± 1.532.51103.75 ± 3.173.05Small intestine2564.61 ± 4.236.5598.39 ± 11.1511.335068.08 ± 3.685.4090.4 ± 3.023.3435070.33 ± 6.138.71101.86 ± 5.705.60240064.51 ± 2.353.6499.20 ± 1.011.02Stomach2568.84 ± 1.181.7292.61 ± 3.133.385062.45 ± 2.203.5288.53 ± 3.443.8935065.27 ± 1.672.5677.56 ± 1.832.36240062.11 ± 0.871.4083.08 ± 2.162.60Brain2571.85 ± 1.852.5790.54 ± 1.481.645078.40 ± 2.292.9286.20 ± 4.875.6535085.96 ± 2.913.3993.34 ± 1.882.02240075.94 ± 1.522.00101.87 ± 1.821.78


### 3.3.5. Stability

The stability was evaluated by analyzing QC samples at high, medium, low, and LLOQ concentrations (n = 6) under different storage conditions, including room temperature for 4 h, auto-sampler (4°C) for 8 h, three freeze-thaw cycles (freeze cycle was at −20°C while thaw cycle was done at room temperature) and −80°C for 30 days. The results are shown in Table 5. It was evident from these data that four storage conditions have no significant effect on the sample. The results indicated that four analytes were stable under these four storage conditions.Table 5
Summary of stability of four analytes in plasma and tissue samples (n = 6).
CompoundsMatrixConcentration (ng/mL)Ambient temperature for 4 hAutosampler at 4°C for 8 hThree freeze-thraw cycles−80°C for 30 daysAccuracyRSD (%)AccuracyRSD (%)AccuracyRSD (%)AccuracyRSD (%)Chlorogenic acidPlasma2101.68 ± 8.398.2598.25 ± 7.837.97101.23 ± 12.4412.2993.35 ± 10.7811.55486.78 ± 6.167.0997.90 ± 11.8812.1495.02 ± 5.395.6798.92 ± 12.3212.46100112.33 ± 3.723.31111.33 ± 4.373.92100.68 ± 6.226.18113.17 ± 4.964.38240098.40 ± 5.565.6599.47 ± 7.597.6391.98 ± 4.755.17112.67 ± 7.586.73Heart2102.73 ± 12.9812.64101.70 ± 10.9110.7291.72 ± 6.336.9088.98 ± 5.736.43496.88 ± 11.0611.4299.50 ± 6.216.2499.03 ± 10.1710.27102.27 ± 10.3010.07100112.83 ± 9.118.07111.93 ± 11.7710.52111.00 ± 7.726.96112.62 ± 8.157.242400108.60 ± 8.417.75101.43 ± 10.3410.20110.55 ± 8.117.33104.85 ± 10.6910.19Liver288.47 ± 6.937.8387.82 ± 6.457.3588.47 ± 7.228.1687.43 ± 7.748.86486.27 ± 7.328.4887.33 ± 5.746.5786.00 ± 6.237.2487.30 ± 4.354.9810099.82 ± 2.892.89106.33 ± 2.882.70102.27 ± 4.974.8687.13 ± 5.776.622400104.17 ± 2.142.05100.92 ± 3.083.0599.98 ± 1.861.8687.02 ± 5.246.03Spleen297.62 ± 8.118.30109.83 ± 9.959.06105.55 ± 7.567.17109.70 ± 13.0511.904105.82 ± 9.919.37100.15 ± 9.699.67103.68 ± 9.719.3698.88 ± 4.164.20100112.00 ± 7.826.98111.12 ± 7.316.58106.82 ± 6.886.44111.17 ± 7.176.452400101.52 ± 7.737.61104.00 ± 7.457.17106.67 ± 5.435.09104.73 ± 7.046.72Lung2103.27 ± 8.017.75111.67 ± 5.434.86112.93 ± 9.578.48113.77 ± 9.208.084101.83 ± 6.416.30100.55 ± 8.588.53100.07 ± 10.3910.3898.77 ± 6.156.23100111.50 ± 5.474.90110.17 ± 10.939.92110.17 ± 7.146.48110.03 ± 9.338.462400100.67 ± 10.1710.1198.78 ± 7.017.10104.88 ± 8.237.85110.17 ± 4.544.12Kidney2110.07 ± 7.346.6788.18 ± 4.985.6497.22 ± 7.677.8989.87 ± 9.2310.27487.63 ± 7.768.8688.40 ± 6.197.0087.60 ± 9.2610.5889.60 ± 9.0510.10100112.00 ± 7.016.26111.92 ± 8.867.91111.17 ± 7.446.69103.22 ± 11.3210.972400100.55 ± 5.115.0998.10 ± 4.874.97100.45 ± 3.963.94101.23 ± 4.694.63Small intestine2105.00 ± 8.207.81102.63 ± 3.523.43103.90 ± 4.494.32101.42 ± 7.827.71491.73 ± 4.494.8999.47 ± 5.105.1399.78 ± 7.707.72106.70 ± 5.435.0910095.90 ± 12.3112.84108.67 ± 12.0411.08100.93 ± 12.3312.2299.07 ± 12.4112.522400112.00 ± 5.374.79111.67 ± 7.266.50111.67 ± 6.125.48110.83 ± 6.686.02Stomach2113.08 ± 11.069.78111.67 ± 6.255.60113.58 ± 8.227.24109.08 ± 9.979.144102.70 ± 10.179.9097.82 ± 9.059.2598.87 ± 9.829.9489.87 ± 7.047.8410093.75 ± 8.659.2294.82 ± 5.055.3393.47 ± 6.767.23107.33 ± 5.895.492400107.17 ± 8.688.1096.60 ± 3.603.72100.23 ± 4.634.6299.73 ± 10.0910.12Brain2110.33 ± 7.556.85106.15 ± 12.3611.65105.65 ± 10.9210.3490.73 ± 10.2511.30489.12 ± 9.0010.1087.57 ± 8.479.6790.68 ± 10.5111.59105.32 ± 9.729.2310091.93 ± 9.9310.80109.17 ± 7.446.82108.13 ± 9.919.17105.48 ± 6.926.562400102.93 ± 5.795.62106.72 ± 9.839.2196.57 ± 10.1210.4897.98 ± 9.569.75Naucleactonin CPlasma5101.95 ± 10.3310.14106.67 ± 10.8810.2091.87 ± 8.639.39109.57 ± 12.6611.558108.88 ± 1.871.72105.01 ± 6.326.02105.88 ± 1.871.77113.33 ± 5.324.6914087.67 ± 9.2010.5087.88 ± 4.144.71111.88 ± 1.871.67111.57 ± 9.608.60240088.93 ± 6.016.7589.25 ± 7.588.4989.17 ± 7.498.40113.83 ± 7.146.27Heart5111.97 ± 11.6310.39100.78 ± 9.639.56107.98 ± 9.458.75107.42 ± 10.249.538110.50 ± 4.424.00107.25 ± 9.258.62109.08 ± 8.257.56110.87 ± 9.048.15140102.78 ± 6.896.71104.88 ± 3.803.62100.40 ± 9.139.0998.28 ± 5.705.802400112.33 ± 10.419.26111.50 ± 5.685.10112.33 ± 5.725.09110.50 ± 4.814.35Liver5101.78 ± 11.8311.63107.70 ± 5.364.9898.32 ± 6.666.7898.38 ± 12.0712.27887.60 ± 5.906.7386.47 ± 4.525.2390.60 ± 4.915.4286.00 ± 8.6010.0014088.57 ± 2.442.7695.00 ± 4.154.3796.28 ± 4.364.5397.32 ± 7.177.36240098.93 ± 3.813.8591.37 ± 2.512.7589.35 ± 3.704.1589.97 ± 7.328.14Spleen5110.33 ± 8.717.89112.92 ± 8.727.72110.67 ± 8.577.75109.68 ± 12.5711.46899.28 ± 13.3113.4193.42 ± 8.008.5796.13 ± 11.9812.46101.90 ± 10.2510.0614093.13 ± 6.106.5595.27 ± 7.087.4389.18 ± 8.239.2392.35 ± 2.702.922400103.33 ± 8.378.10105.67 ± 8.508.05107.73 ± 8.107.51109.83 ± 4.964.51Lung5109.50 ± 11.4710.48100.40 ± 10.2610.22101.05 ± 5.335.2895.98 ± 10.4710.91894.73 ± 8.929.4191.40 ± 9.7110.63101.90 ± 10.4510.2599.03 ± 10.4910.59140102.38 ± 4.124.0394.45 ± 5.926.2793.75 ± 8.539.0992.58 ± 3.603.892400111.37 ± 9.278.33111.85 ± 9.538.52112.125 ± 9.668.61111.67 ± 3.393.03Kidney5109.33 ± 7.556.91106.08 ± 8.968.4498.92 ± 9.9110.0297.18 ± 10.4610.76899.10 ± 4.244.2797.38 ± 7.317.5191.10 ± 9.2010.1094.77 ± 7.628.0414096.08 ± 7.367.6695.43 ± 7.998.37101.10 ± 4.984.92100.25 ± 8.258.232400108.00 ± 5.625.20103.87 ± 4.684.51104.57 ± 6.095.82102.30 ± 5.905.77Small intestine5111.90 ± 12.0710.79111.67 ± 8.147.29111.17 ± 8.337.49112.67 ± 8.787.798103.33 ± 3.393.28100.03 ± 11.4611.46100.60 ± 10.2410.18104.25 ± 9.499.1014088.60 ± 9.4810.7089.80 ± 8.309.2487.88 ± 5.576.3490.17 ± 7.268.052400107.48 ± 8.237.66103.75 ± 6.686.44108.43 ± 8.657.98102.32 ± 8.658.45Stomach5111.00 ± 7.907.12112.17 ± 7.256.46113.12 ± 9.978.81113.83 ± 2.992.638107.32 ± 12.9612.0898.37 ± 10.7510.93105.90 ± 9.138.62103.55 ± 9.499.1614088.15 ± 7.918.9886.38 ± 8.389.7087.12 ± 5.246.188.28 ± 6.317.152400100.53 ± 7.297.2599.52 ± 5.855.88104.90 ± 5.535.28102.32 ± 8.228.03Brain5106.35 ± 11.8211.11109.83 ± 6.055.5198.92 ± 10.6510.77104.10 ± 10.8810.45888.87 ± 10.4311.7490.65 ± 6.377.0389.95 ± 9.2310.2688.82 ± 10.5711.9014088.33 ± 6.747.6387.92 ± 8.159.2787.20 ± 4.985.7187.93 ± 5.816.61240099.43 ± 6.386.41112.67 ± 6.625.88109.67 ± 6.445.87109.00 ± 5.224.78Khaephuoside APlasma599.10 ± 9.259.3390.97 ± 9.019.9093.93 ± 11.2611.98101.18 ± 3.533.49891.38 ± 12.7613.9694.88 ± 12.3112.9790.67 ± 9.2910.25108.17 ± 9.208.50120101.35 ± 4.334.28101.32 ± 3.403.3696.75 ± 4.554.70107.30 ± 11.4810.70180099.35 ± 6.046.08101.93 ± 6.906.7796.13 ± 4.034.19112.67 ± 7.666.80Heart5107.60 ± 11.2310.44104.15 ± 9.929.52110.67 ± 10.829.78107.45 ± 9.358.708112.67 ± 7.236.42111.83 ± 8.237.36111.33 ± 5.925.32101.00 ± 12.0511.93120111.67 ± 6.225.57112.67 ± 5.895.23108.58 ± 11.5110.60113.50 ± 7.206.351800110.83 ± 2.792.51106.58 ± 8.778.22113.67 ± 4.273.76101.90 ± 12.3012.07Liver593.87 ± 9.319.92106.17 ± 4.884.5995.62 ± 9.8210.27100.87 ± 10.2710.198108.95 ± 10.779.88101.97 ± 7.937.78105.20 ± 10.219.7091.12 ± 10.7911.84120107.67 ± 6.225.78111.50 ± 7.266.51101.62 ± 9.629.47104.23 ± 8.688.331800108.83 ± 2.322.13109.83 ± 5.384.90103.20 ± 7.527.29108.93 ± 9.528.74Spleen5110.30 ± 12.3111.16112.17 ± 7.947.07111.00 ± 10.269.24111.50 ± 8.417.548112.17 ± 5.645.02111.67 ± 6.195.54111.38 ± 7.366.58110.67 ± 7.176.48120107.85 ± 7.837.26105.45 ± 7.957.5498.83 ± 7.517.60105.42 ± 10.7910.241800109.77 ± 8.617.85111.17 ± 8.187.36113.17 ± 3.312.93111.83 ± 7.576.77Lung5113.33 ± 8.717.69106.65 ± 12.4311.6598.87 ± 9.029.12102.98 ± 9.509.238105.32 ± 7.817.41103.18 ± 10.9710.63105.03 ± 9.869.38104.00 ± 11.7311.28120101.27 ± 3.533.4999.07 ± 11.0111.11102.02 ± 6.366.23110.83 ± 5.194.691800110.83 ± 8.237.43112.80 ± 7.466.61110.00 ± 8.888.07112.50 ± 6.806.05Kidney596.42 ± 6.246.4799.65 ± 10.5310.57101.62 ± 11.3711.19112.78 ± 11.4310.138113.17 ± 6.465.71110.83 ± 7.887.11113.00 ± 6.996.18113.17 ± 7.196.36120111.67 ± 5.324.76111.83 ± 5.314.75112.00 ± 5.935.30112.00 ± 5.625.021800102.03 ± 4.444.3597.37 ± 6.847.02100.50 ± 5.535.50101.12 ± 6.566.48Small intestine5110.02 ± 8.197.44112.83 ± 3.433.04100.88 ± 9.399.3094.98 ± 9.429.918105.62 ± 9.769.24109.53 ± 10.149.25101.00 ± 10.4210.31107.70 ± 11.4710.65120103.30 ± 7.757.50106.00 ± 7.296.88102.80 ± 6.095.93101.58 ± 6.266.161800113.00 ± 6.135.43110.83 ± 8.137.34113.67 ± 8.197.20113.12 ± 12.4711.02Stomach5113.68 ± 8.427.41112.13 ± 10.239.12111.12 ± 10.439.38110.67 ± 6.095.508111.67 ± 6.595.90111.00 ± 5.224.70109.67 ± 3.333.03104.50 ± 4.714.51120113.17 ± 3.663.23111.50 ± 5.865.25112.17 ± 6.155.48110.92 ± 8.367.531800109.83 ± 7.366.70111.92 ± 10.999.82110.50 ± 6.285.69113.83 ± 5.564.89Brain5109.82 ± 10.269.34106.90 ± 12.0911.31103.05 ± 9.128.85112.50 ± 5.584.968111.00 ± 7.426.52112.50 ± 7.066.28113.33 ± 3.833.38111.33 ± 6.095.47120112.50 ± 5.134.56112.33 ± 6.475.76113.17 ± 5.314.69110.17 ± 8.287.521800105.82 ± 8.317.86112.83 ± 7.686.81110.83 ± 7.526.79113.83 ± 4.493.953,4-Dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranosidePlasma2586.25 ± 5.406.2694.95 ± 9.8010.32102.87 ± 10.3710.08105.60 ± 10.199.655087.20 ± 7.919.0798.98 ± 11.3011.3094.53 ± 9.8410.41107.88 ± 14.9613.87350112.83 ± 4.584.06112.67 ± 6.625.88108.33 ± 5.134.73109.07 ± 1.871.72240089.98 ± 4.765.2989.43 ± 7.428.3086.07 ± 3.393.93111.74 ± 3.763.37Heart2598.10 ± 9.439.61100.45 ± 10.8910.8597.55 ± 9.019.2488.38 ± 5.696.445094.60 ± 11.147.80102.70 ± 7.987.7797.05 ± 8.428.6899.12 ± 9.569.65350102.57 ± 4.924.80112.63 ± 98.937.93110.33 ± 5.825.27108.38 ± 7.346.772400102.23 ± 6.646.4992.98 ± 8.879.54101.57 ± 6.736.6399.57 ± 9.469.50Liver2591.10 ± 10.3711.39102.88 ± 10.8510.5497.32 ± 10.4410.73104.70 ± 11.9711.4350105.60 ± 9.048.56112.50 ± 5.655.02107.48 ± 10.549.8095.25 ± 11.2911.8635097.50 ± 6.636.80113.00 ± 4.003.54103.12 ± 5.265.10108.67 ± 8.988.272400104.32 ± 10.189.76100.42 ± 6.856.8396.25 ± 5.745.96107.68 ± 7.737.18Spleen2588.72 ± 9.9311.19104.47 ± 9.008.6290.93 ± 9.1510.07112.83 ± 3.603.1950113.17 ± 10.599.36108.35 ± 10.589.7795.62 ± 10.9011.40107.68 ± 10.399.65350111.33 ± 7.636.86111.68 ± 9.218.25112.17 ± 7.997.12112.17 ± 8.407.492400102.27 ± 11.5011.24112.00 ± 8.677.74105.32 ± 6.596.26111.00 ± 6.035.44Lung2590.52 ± 10.7711.9096.80 ± 11.2911.6797.07 ± 10.8711.2097.92 ± 11.3411.5950112.67 ± 7.846.96111.02 ± 7.817.04100.02 ± 12.4612.46109.25 ± 11.4610.49350111.33 ± 6.896.19109.47 ± 11.1010.14103.68 ± 11.6211.21112.33 ± 6.926.16240098.85 ± 9.839.94101.82 ± 8.678.52100.62 ± 7.717.66109.12 ± 10.699.79Kidney25100.05 ± 11.9211.92101.42 ± 8.638.5196.23 ± 9.149.50110.83 ± 6.245.635098.37 ± 7.537.65102.72 ± 10.2910.02106.75 ± 9.158.5799.88 ± 9.659.6635090.05 ± 8.479.40102.03 ± 4.614.52111.83 ± 7.036.28105.40 ± 10.289.752400104.72 ± 8.968.56106.27 ± 10.199.59107.85 ± 9.168.49104.82 ± 8.598.20Small intestine2587.47 ± 7.178.2096.42 ± 9.139.4794.10 ± 8.058.5599.25 ± 6.066.1150109.83 ± 2.562.33100.75 ± 6.116.07106.83 ± 5.345.00101.80 ± 8.788.62350111.17 ± 7.736.95113.67 ± 8.767.70113.00 ± 3.793.36106.33 ± 5.395.07240098.50 ± 5.495.57104.65 ± 7.857.5099.92 ± 5.945.9494.90 ± 8.629.08Stomach25111.83 ± 5.785.17110.50 ± 4.424.00113.00 ± 6.325.60106.32 ± 9.488.9250112.75 ± 10.979.73112.67 ± 3.673.26112.83 ± 5.604.96109.83 ± 8.667.88350113.17 ± 3.873.42108.17 ± 9.688.95111.00 ± 6.205.58111.83 ± 5.985.352400101.27 ± 7.897.8094.40 ± 6.006.3596.82 ± 8.088.34103.08 ± 9.609.32Brain2599.82 ± 9.829.84107.80 ± 12.7911.86100.68 ± 9.809.73105.37 ± 7.316.9350109.35 ± 12.1511.11112.83 ± 4.263.78113.77 ± 10.369.11113.83 ± 3.873.40350111.50 ± 8.647.75110.83 ± 7.476.74111.17 ± 7.636.27112.83 ± 7.786.90240098.95 ± 3.974.01100.10 ± 9.439.4298.23 ± 5.395.49101.50 ± 9.949.80


## 3.3.1. Selectivity

The selectivity was assessed by analyzing chromatograms of blank rat plasma and tissue homogenates from different batches, blank plasma and tissue homogenates spiked with analytes and IS, and plasma, tissue samples obtained from rats after oral administration ofNauclea officinalis extracts (n = 6). Figure 2 shows typical chromatograms of four analytes and IS in rat plasma (Figures S1 and S2 of the Supporting Material show typical chromatograms of four analytes and IS in liver tissue and kidney tissue). Under the given condition, Chlorogenic acid, Naucleactonin C, Khaephuoside A, 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside, and IS were eluted approximately at retention time of 1.02 min, 1.65 min, 0.99 min, 0.97 min, and 1.16 min, respectively. No interfering endogenous substances were found at the respective retention times.Figure 2
Typical chromatograms of four analyte and IS in rat plasma: (a) blank plasma samples, (b) blank plasma samples spiked with four analytes (LLOQ) and IS, (c) rat plasma samples at 0.5 h after oral administration theNauclea officinalis extracts spiked with IS; (1) chlorogenic acid, (2) naucleactonin C, (3) khaephuoside A, (4) 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside, (5) chloramphenicol (IS).
(a)(b)(c)


## 3.3.2. Linearity of Calibration Curve and Lower Limit of Quantification

The calibration curves were constructed by plotting the peak areas ratios of chlorogenic acid/IS, Naucleactonin C/IS, Khaephuoside A/IS, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside/IS versus theoretical concentrations. This method exhibited a good linear response for the range of concentrations from 2 to 2500 ng/mL in plasma and from 2 to 2500 ng/mL in tissues; all determination coefficients (R2) were greater than 0.9849. The lower limit of quantification (LLOQ) was defined as the lowest concentration of a signal-noise (S/N) ratios of 10 : 1, respectively, the precision was less than 15% and the accuracy was within ±20%. The limits were adequate for studies of pharmacokinetics and tissue distribution by oral administration of Nauclea officinalis extracts. Data from the determination are shown in Table 2.Table 2
Summary of standard curves, correlation coefficients, LLOQ, and linear ranges of four analytes in plasma and tissue samples.
CompoundsMatrixLinear regression equationR2LLOQ (ng/mL)Linear range (ng/mL)Chlorogenic acidPlasmay = 0.0288x + 0.001930.999422–2500Hearty = 0.0063x − 0.007710.984922–2500Livery = 0.0267x − 0.03230.998022–2500Spleeny = 0.0392x + 0.02770.988622–2500Lungy = 0.0442x + 0.02390.995822–2500Kidneyy = 0.0217x − 0.03020.995022–2500Small intestiney = 0.0334x − 0.04010.996422–2500Stomachy = 0.0402x − 0.01290.995222–2500Brainy = 0.0499x − 0.05620.996022–2500Naucleactonin CPlasmay = 0.00535x + 0.02070.996455–2500Hearty = 0.00313x − 0.001160.992655–2500Livery = 0.00395x − 0.01010.996455–2500Spleeny = 0.00511x − 0.005260.995055–2500Lungy = 0.00534x − 0.002630.998055–2500Kidneyy = 0.00363x − 0.00980.996055–2500Small intestiney = 0.00224x − 0.005270.996855–2500Stomachy = 0.00465x − 0.01380.997255–2500Brainy = 0.00552x − 0.01630.993255–2500Khaephuoside APlasmay = 0.00316x + 0.01530.995455–2000Hearty = 0.00673x − 0.0050.994655–2000Livery = 0.0063x − 0.005020.996255–2000Spleeny = 0.011x − 0.00540.998055–2000Lungy = 0.0099x − 0.001660.997855–2000Kidneyy = 0.00531x − 0.00470.995455–2000Small intestiney = 0.0058x − 0.01560.990655–2000Stomachy = 0.00745x − 0.008370.992055–2000Brainy = 0.0126x + 0.0003770.993855–20003,4-Dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranosidePlasmay = 0.000845x + 0.003960.99842525–2500Hearty = 0.000804x + 0.001590.98492525–2500Livery = 0.000873x − 0.005150.99682525–2500Spleeny = 0.00134x + 0.009470.98942525–2500Lungy = 0.00138x + 0.006260.99642525–2500Kidneyy = 0.000633x − 0.0005380.99302525–2500Small intestiney = 0.00109x + 0.0009660.99622525–2500Stomachy = 0.00119x + 0.005490.99642525–2500Brainy = 0.00163x − 0.01490.99522525–2500Abbreviations. LLOQ, lower limit of quantification;R2, determination coefficients.


## 3.3.3. Accuracy and Precision

Accuracy and precision were evaluated by analyzing QC samples at high, medium, low, and LLOQ concentrations (n = 6) on the same day and three consecutive days using the standard curve, respectively. The acceptable limits of accuracy were required to be within ±15% of the actual value except when at LLOQ, and the intra- and interday precision and accuracy data of the determination were shown in Table 3. In our study, the intraday precision ranged from 0.62 to 12.65%, and intraday accuracy ranged from 86.48 to 114.17%. The interday precision ranged from 2.42 to 12.61%, and the interday accuracy ranged from 86.31 to 113.99%. The results showed the accuracy and precision were within the acceptable limits, which proved the method was reproducible, reliable, and accurate for the determination of four analytes in rat plasma and tissue samples.Table 3
Summary of intraday and interday precisions and accuracies of four analytes in plasma and tissue samples.
CompoundsMatrixConcentration (ng/mL)Intraday (n = 6)Interday (n = 18)AccuracyRSD (%)AccuracyRSD (%)Chlorogenic acidPlasma2104.02 ± 11.9111.45100.64 ± 11.6711.60497.72 ± 9.239.4596.09 ± 7.087.36100104.42 ± 6.235.97104.29 ± 10.610.162400101.22 ± 3.203.1696.59 ± 8.118.40Heart2101.90 ± 12.1411.92109.34 ± 11.7410.74499.35 ± 3.893.9193.67 ± 6.136.5410096.57 ± 4.814.9894.46 ± 7.808.262400109.00 ± 5.905.41102.88 ± 6.856.66Liver2101.65 ± 2.822.78100.88 ± 5.385.334101.45 ± 2.982.9398.59 ± 6.726.82100100.30 ± 2.892.8897.78 ± 4.895.00240095.07 ± 5.746.0497.11 ± 5.585.75Spleen296.40 ± 7.637.92102.14 ± 8.908.71495.40 ± 4.204.40101.28 ± 9.619.49100102.40 ± 2.001.95111.52 ± 7.006.27240094.93 ± 4.815.07108.26 ± 10.419.62Lung2101.53 ± 6.406.30101.22 ± 7.457.36499.75 ± 6.096.1093.33 ± 11.1711.97100108.33 ± 5.475.04111.33 ± 6.495.832400100.60 ± 3.353.33101.63 ± 5.074.99Kidney298.12 ± 5.365.4796.23 ± 5.335.544100.13 ± 2.172.1792.09 ± 7.217.8310099.37 ± 5.205.2396.10 ± 6.366.622400104.47 ± 5.795.5499.47 ± 6.066.10Small intestine293.27 ± 4.134.4396.03 ± 7.868.19486.48 ± 5.095.8993.40 ± 10.7511.5110089.75 ± 7.968.8796.61 ± 10.0610.41240092.70 ± 6.637.1693.01 ± 5.275.66Stomach2105.87 ± 7.877.43107.84 ± 7.456.90493.17 ± 7.027.5398.76 ± 6.446.52100106.00 ± 3.953.73106.35 ± 4.464.192400100.63 ± 4.184.1599.41 ± 4.814.84Brain2109.27 ± 6.746.17104.39 ± 9.479.074101.00 ± 3.813.77101.29 ± 5.425.35100106.00 ± 4.944.66106.77 ± 7.166.702400101.27 ± 2.252.22105.62 ± 7.046.67Naucleactonin CPlasma595.62 ± 12.1012.6598.24 ± 10.3010.488100.53 ± 11.7811.7196.43 ± 12.1612.6114099.95 ± 6.496.5095.76 ± 7.187.50240097.05 ± 4.804.9595.50 ± 6.236.52Heart5111.27 ± 7.326.58107.46 ± 10.589.85899.28 ± 5.485.52101.15 ± 11.4611.3314093.45 ± 4.344.6593.19 ± 5.335.722400105.92 ± 9.549.01104.46 ± 6.666.38Liver5100.90 ± 5.405.36108.30 ± 7.827.22899.47 ± 2.822.84100.52 ± 5.004.9814088.60 ± 2.602.9393.53 ± 6.336.772400102.50 ± 1.521.48101.42 ± 2.462.42Spleen5111.00 ± 6.996.29104.42 ± 9.318.91898.90 ± 3.553.59100.74 ± 6.776.7214096.07 ± 3.954.1193.72 ± 5.305.662400100.02 ± 3.393.39103.13 ± 5.064.91Lung5113.83 ± 4.073.58113.44 ± 5.374.738103.28 ± 10.4610.13102.03 ± 10.169.96140104.17 ± 3.763.61103.83 ± 4.053.902400107.83 ± 4.073.77107.06 ± 5.024.69Kidney5100.70 ± 4.284.25103.81 ± 6.616.36898.38 ± 5.315.4099.18 ± 4.364.3914095.37 ± 4.194.39100.44 ± 7.077.042400106.80 ± 9.749.12104.87 ± 6.466.16Small intestine5109.98 ± 10.979.97105.41 ± 10.199.67899.30 ± 8.949.01100.44 ± 9.189.1414088.73 ± 8.9610.1086.31 ± 7.668.882400103.30 ± 6.826.6098.26 ± 7.607.73Stomach5109.83 ± 7.056.42102.67 ± 10.099.838101.58 ± 2.962.9295.73 ± 8.078.4314094.88 ± 2.742.8892.09 ± 5.175.622400113.17 ± 4.884.31108.96 ± 7.216.62Brain5105.75 ± 10.8110.22105.82 ± 8.778.298104.78 ± 7.467.12100.57 ± 8.118.06140101.22 ± 4.594.5398.93 ± 5.735.802400113.83 ± 3.312.91111.39 ± 5.544.98Khaephuoside APlasma5101.63 ± 9.939.77105.66 ± 10.509.948101.02 ± 9.649.55101.78 ± 7.857.71120112.33 ± 4.764.24106.16 ± 6.115.761800104.08 ± 4.394.21102.52 ± 4.414.31Heart5113.50 ± 9.188.09103.33 ± 10.9210.578100.82 ± 6.196.1498.68 ± 7.067.1612096.73 ± 2.923.0289.72 ± 8.018.931800109.12 ± 9.458.66102.43 ± 8.137.94Liver599.97 ± 7.067.07101.51 ± 8.498.368104.10 ± 5.074.87101.81 ± 4.023.9512099.33 ± 0.620.62100.43 ± 4.554.531800108.00 ± 6.105.65103.94 ± 5.605.39Spleen598.73 ± 11.2111.35103.62 ± 9.479.148100.98 ± 6.256.19103.08 ± 6.756.5512099.35 ± 2.522.54109.89 ± 8.848.041800109.00 ± 4.604.22113.06 ± 5.364.74Lung5108.17 ± 10.199.42109.93 ± 7.66.928109.90 ± 11.2510.24106.91 ± 9.959.3112090.32 ± 6.126.7895.54 ± 6.536.831800100.73 ± 2.022.00103.65 ± 6.916.66Kidney5106.82 ± 7.917.40109.94 ± 6.295.728113.67 ± 2.942.59111.40 ± 6.125.49120105.30 ± 6.145.83107.78 ± 6.916.411800105.02 ± 9.819.34108.84 ± 7.606.99Small intestine5106.50 ± 8.538.01104.08 ± 10.119.71899.98 ± 9.439.13101.89 ± 11.5811.3612089.90 ± 6.847.6193.74 ± 7.808.321800104.45 ± 6.225.96105.44 ± 5.154.89Stomach596.63 ± 9.159.4795.98 ± 10.2610.69890.92 ± 7.968.7697.66 ± 8.618.82120104.00 ± 6.135.90103.24 ± 5.315.151800109.83 ± 4.834.40110.50 ± 4.544.11Brain5108.90 ± 9.748.95107.11 ± 7.947.418108.55 ± 9.909.12107.23 ± 8.017.47120113.17 ± 6.715.93110.13 ± 6.095.531800100.50 ± 3.993.97108.44 ± 7.637.033,4-Dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranosidePlasma25109.00 ± 4.053.72101.34 ± 9.108.9850105.82 ± 10.9510.35102.61 ± 7.897.69350109.83 ± 5.234.76104.49 ± 8.608.23240096.82 ± 2.762.85100.71 ± 7.507.45Heart2599.12 ± 5.065.1096.73 ± 8.088.355096.45 ± 6.757.0098.20 ± 8.288.4335089.15 ± 4.835.4290.19 ± 6.827.56240099.80 ± 10.8910.9199.33 ± 7.667.71Liver25105.07 ± 6.626.30103.78 ± 6.396.1650108.48 ± 7.396.82108.22 ± 7.386.82350112.15 ± 6.896.14113.99 ± 4.834.24240098.28 ± 3.673.73100.28 ± 3.243.23Spleen25102.03 ± 10.6810.47104.78 ± 8.638.235099.90 ± 5205.21103.82 ± 7.777.4835097.70 ± 4.124.21109.12 ± 9.078.312400100.65 ± 5.465.43110.22 ± 8.948.11Lung2596.17 ± 7.597.8999.80 ± 10.9911.0150106.78 ± 7.086.63107.49 ± 8.167.5935096.42 ± 4.494.66100.05 ± 5.505.49240099.67 ± 6.296.3199.32 ± 5.685.72Kidney2597.02 ± 9.7510.0596.23 ± 9.389.7450108.00 ± 5.665.24106.69 ± 7.006.5635099.63 ± 5.835.85100.46 ± 6.786.75240096.50 ± 7.417.6897.74 ± 6.316.46Small intestine2598.25 ± 8.929.0897.96 ± 7.737.9050101.58 ± 6.416.31101.89 ± 7.137.0035089.58 ± 4.274.7698.17 ± 8.398.552400100.18 ± 4.774.76101.32 ± 4.834.77Stomach25103.63 ± 5.215.0299.07 ± 9.649.7350101.90 ± 9.329.1497.96 ± 9.339.52350114.17 ± 3.192.79109.34 ± 6.145.622400100.62 ± 3.423.4099.42 ± 4.664.69Brain25106.95 ± 12.1311.3499.13 ± 11.2611.3650105.78 ± 7.637.22107.10 ± 7.867.34350110.00 ± 9.018.19112.56 ± 6.936.16240099.42 ± 2.902.91101.13 ± 5.445.38


## 3.3.4. Extraction Recovery and Matrix Effect

Six batches of blank plasma from independent sources were used to obtain extracted samples, postextracted spiked samples, and unextracted samples at high, medium, low, and LLOQ concentrations. The peak areas of the three types of samples were recorded as A, B, and C, respectively. Extraction recovery was evaluated by A/B. Matrix effects were calculated by comparing the A/C of analytes. The extraction, recovery, and matrix effect of four analytes in rat plasma and tissues are shown in Table4. The recovery rate were 51.85–97.06%, 75.99–106.68%, 77.46–105.35%, and 68.36–103.75% for chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside and the matrix effects were 50.17–116.62%, 86.75–115.99%, 45.79–87.44%, and 51.60–92.34% for chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside in different matrices, indicating some ion inhibitory effects for four compounds in the biological samples, the RSD (relative standard deviation) of recovery and matrix effect were less than 15%. There were no significant differences in recoveries and matrix effects between the four compounds at different concentrations in the same matrix.Table 4
Summary of extraction recovery and matrix effect of four analytes in plasma and tissue samples (n = 6).
CompoundsMatrixConcentration (ng/mL)Matrix effect (%)RSD (%)Extraction recovery (%)RSD (%)Chlorogenic acidPlasma297.26 ± 5.355.5080.45 ± 4.365.424109.75 ± 4.404.0180.04 ± 3.083.8510082.80 ± 6.517.8669.49 ± 4.055.84240091.66 ± 1.531.6772.26 ± 1.161.61Heart2116.54 ± 3.032.6080.53 ± 9.6211.95498.55 ± 2.712.7576.82 ± 4.776.2110097.78 ± 1.761.8062.02 ± 2.243.61240093.25 ± 0.760.8163.32 ± 0.580.91Liver283.69 ± 4.245.0697.06 ± 3.753.86479.96 ± 4.795.9479.07 ± 2.903.6610063.82 ± 2.513.9466.44 ± 0.240.37240080.01 ± 0.901.1269.83 ± 1.552.22Spleen299.89 ± 2.612.6180.09 ± 1.221.524100.56 ± 1.991.9886.82 ± 2.883.3210092.05 ± 1.011.1067.29 ± 1.812.69240091.66 ± 0.810.8873.12 ± 1.612.20Lung2104.73 ± 1.781.7081.80 ± 2.523.08499.34 ± 2.072.0988.65 ± 1.551.7510098.65 ± 1.241.2580.24 ± 2.112.63240096.68 ± 1.811.8876.89 ± 1.692.20Kidney290.49 ± 5.666.2678.34 ± 4.025.13498.52 ± 4.214.2786.92 ± 3.003.4610095.03 ± 5.946.2570.73 ± 4.906.92240089.55 ± 2.383.1672.70 ± 1.502.07Small intestine256.96 ± 6.1610.8156.20 ± 3.365.98463.67 ± 2.464.1451.85 ± 1.943.7310053.67 ± 1.061.9761.26 ± 1.873.05240050.17 ± 1.723.4262.66 ± 1.061.70Stomach2114.40 ± 5.014.4685.33 ± 5.206.104116.62 ± 8.807.5591.66 ± 6.116.67100102.87 ± 3.243.1566.48 ± 3.945.93240098.38 ± 1.631.6569.41 ± 1.562.25Brain297.89 ± 6.426.5682.33 ± 4.655.654105.09 ± 2.992.8475.80 ± 3.594.7410090.02 ± 5.466.0671.37 ± 1.171.63240090.56 ± 2.162.3986.06 ± 1.681.95Naucleactonin CPlasma5104.57 ± 6.946.64100.30 ± 11.7711.748102.54 ± 2.552.48106.68 ± 6.315.91140104.07 ± 2.882.7796.41 ± 1.661.722400102.64 ± 2.092.0488.27 ± 1.051.19Heart598.86 ± 2.672.7097.71 ± 1.952.00899.69 ± 3.963.9796.33 ± 7.197.46140106.24 ± 4.874.5885.37 ± 2.903.40240099.81 ± 1.481.4886.76 ± 1.611.86Liver5101.81 ± 10.8810.6992.06 ± 9.8710.728111.16 ± 3.122.8185.03 ± 4.915.7714097.92 ± 5.095.2084.88 ± 4.525.33240097.02 ± 1.111.1480.54 ± 0.831.03Spleen590.83 ± 3.654.02100.21 ± 9.219.19893.78 ± 6.076.4799.90 ± 5.495.5014097.28 ± 2.712.7984.00 ± 1.541.832400100.67 ± 0.790.7884.60 ± 1.401.65Lung598.17 ± 7.637.77105.63 ± 3.873.668115.99 ± 7.975.0199.21 ± 12.4312.5314098.73 ± 1.391.4195.48 ± 2.883.01240099.21 ± 0.910.9284.40 ± 1.822.15Kidney599.90 ± 5.105.1094.08 ± 8.118.628100.35 ± 5.145.12105.08 ± 5.224.97140101.18 ± 0.700.6986.02 ± 4.395.102400102.49 ± 1.871.8386.64 ± 2.262.60Small intestine598.00 ± 5.515.6298.55 ± 9.619.75899.37 ± 8.488.9082.00 ± 5.036.1414095.42 ± 5.525.7991.73 ± 1.671.82240086.75 ± 2.242.5886.61 ± 3.153.63Stomach5101.53 ± 3.553.5097.93 ± 4.194.288101.21 ± 3.873.8299.25 ± 3.173.2014099.62 ± 1.191.2082.46 ± 1.882.282400101.37 ± 1.351.3375.99 ± 2.773.64Brain597.50 ± 6.506.6788.08 ± 3.123.548105.47 ± 5.144.8788.38 ± 7.328.29140102.02 ± 0.850.8397.63 ± 4.354.462400101.24 ± 2.442.4199.47 ± 1.421.43Khaephuoside APlasma564.06 ± 5.668.8488.84 ± 5.105.75865.86 ± 5.658.5792.35 ± 8.739.4512055.55 ± 4.057.2985.88 ± 2.062.40180058.01 ± 1.933.3385.24 ± 3.303.87Heart570.92 ± 8.0911.4196.07 ± 5.375.59877.13 ± 3.564.6182.29 ± 3.954.8012054.79 ± 1.142.0892.18 ± 1.641.78180059.93 ± 4.647.7586.98 ± 4.875.59Liver573.90 ± 6.849.26105.35 ± 11.0210.46853.20 ± 1.592.98103.34 ± 3.753.6312058.17 ± 1.121.9377.46 ± 2.172.80180061.29 ± 2.804.5884.49 ± 1.702.01Spleen568.33 ± 1.412.0790.99 ± 7.898.67860.36 ± 2.584.2896.38 ± 2.772.8812058.12 ± 0.420.7390.31 ± 2.803.10180061.44 ± 1.131.8486.75 ± 0.981.13Lung584.39 ± 4.204.9777.71 ± 3.734.81872.06 ± 6.899.5687.06 ± 7.308.3812059.00 ± 2.554.3386.34 ± 3.484.03180061.35 ± 4.417.1979.39 ± 6.488.17Kidney557.46 ± 1.803.1397.31 ± 4.384.50851.41 ± 2.915.6699.78 ± 3.413.4212045.79 ± 1.302.8499.65 ± 1.231.23180047.23 ± 1.382.93101.56 ± 2.172.14Small intestine556.14 ± 1.472.61102.93 ± 10.4410.15867.00 ± 5.438.1094.63 ± 6.276.6312062.69 ± 2.063.29100.39 ± 5.004.98180071.51 ± 2.082.9199.78 ± 1.061.06Stomach556.84 ± 3.215.6495.49 ± 6.606.91863.57 ± 5.598.8083.81 ± 6.898.2212054.53 ± 2.444.4877.49 ± 4.085.27180055.57 ± 1.071.9279.96 ± 0.871.09Brain576.07 ± 1.692.2293.48 ± 8.709.30876.50 ± 6.238.15102.39 ± 6.806.6412087.44 ± 3.233.7090.50 ± 0.991.09180079.81 ± 2.152.70100.48 ± 1.421.413,4-Dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranosidePlasma2564.80 ± 4.957.6488.62 ± 8.569.665065.25 ± 0.811.2489.23 ± 4.244.7635060.35 ± 3.676.0885.65 ± 1.171.37240055.98 ± 2.344.1884.82 ± 3.163.72Heart2579.81 ± 2.392.9980.56 ± 4.055.035073.97 ± 1.842.4989.91 ± 2.893.2135069.51 ± 4.045.8185.34 ± 4.204.92240064.96 ± 2.283.5185.00 ± 3.153.71Liver2555.02 ± 4.027.3188.70 ± 8.389.455053.00 ± 1.633.0894.52 ± 4.765.0435052.69 ± 2.124.0383.51 ± 2.633.15240051.60 ± 1.442.8087.94 ± 2.883.27Spleen2581.22 ± 3.274.0298.19 ± 2.872.925087.52 ± 4.515.1692.02 ± 3.153.4235081.89 ± 1.031.2689.66 ± 2.422.70240080.06 ± 3.073.8380.48 ± 2.152.68Lung2591.11 ± 4.014.4068.36 ± 4.426.475092.34 ± 3.463.7577.05 ± 2.142.7835077.28 ± 4.365.6583.53 ± 3.424.09240076.69 ± 2.733.5675.92 ± 0.730.97Kidney2563.39 ± 1.542.4386.96 ± 4.515.195064.43 ± 1.532.3899.12 ± 2.652.6735053.76 ± 3.075.72101.00 ± 1.811.79240060.75 ± 1.532.51103.75 ± 3.173.05Small intestine2564.61 ± 4.236.5598.39 ± 11.1511.335068.08 ± 3.685.4090.4 ± 3.023.3435070.33 ± 6.138.71101.86 ± 5.705.60240064.51 ± 2.353.6499.20 ± 1.011.02Stomach2568.84 ± 1.181.7292.61 ± 3.133.385062.45 ± 2.203.5288.53 ± 3.443.8935065.27 ± 1.672.5677.56 ± 1.832.36240062.11 ± 0.871.4083.08 ± 2.162.60Brain2571.85 ± 1.852.5790.54 ± 1.481.645078.40 ± 2.292.9286.20 ± 4.875.6535085.96 ± 2.913.3993.34 ± 1.882.02240075.94 ± 1.522.00101.87 ± 1.821.78


## 3.3.5. Stability

The stability was evaluated by analyzing QC samples at high, medium, low, and LLOQ concentrations (n = 6) under different storage conditions, including room temperature for 4 h, auto-sampler (4°C) for 8 h, three freeze-thaw cycles (freeze cycle was at −20°C while thaw cycle was done at room temperature) and −80°C for 30 days. The results are shown in Table 5. It was evident from these data that four storage conditions have no significant effect on the sample. The results indicated that four analytes were stable under these four storage conditions.Table 5
Summary of stability of four analytes in plasma and tissue samples (n = 6).
CompoundsMatrixConcentration (ng/mL)Ambient temperature for 4 hAutosampler at 4°C for 8 hThree freeze-thraw cycles−80°C for 30 daysAccuracyRSD (%)AccuracyRSD (%)AccuracyRSD (%)AccuracyRSD (%)Chlorogenic acidPlasma2101.68 ± 8.398.2598.25 ± 7.837.97101.23 ± 12.4412.2993.35 ± 10.7811.55486.78 ± 6.167.0997.90 ± 11.8812.1495.02 ± 5.395.6798.92 ± 12.3212.46100112.33 ± 3.723.31111.33 ± 4.373.92100.68 ± 6.226.18113.17 ± 4.964.38240098.40 ± 5.565.6599.47 ± 7.597.6391.98 ± 4.755.17112.67 ± 7.586.73Heart2102.73 ± 12.9812.64101.70 ± 10.9110.7291.72 ± 6.336.9088.98 ± 5.736.43496.88 ± 11.0611.4299.50 ± 6.216.2499.03 ± 10.1710.27102.27 ± 10.3010.07100112.83 ± 9.118.07111.93 ± 11.7710.52111.00 ± 7.726.96112.62 ± 8.157.242400108.60 ± 8.417.75101.43 ± 10.3410.20110.55 ± 8.117.33104.85 ± 10.6910.19Liver288.47 ± 6.937.8387.82 ± 6.457.3588.47 ± 7.228.1687.43 ± 7.748.86486.27 ± 7.328.4887.33 ± 5.746.5786.00 ± 6.237.2487.30 ± 4.354.9810099.82 ± 2.892.89106.33 ± 2.882.70102.27 ± 4.974.8687.13 ± 5.776.622400104.17 ± 2.142.05100.92 ± 3.083.0599.98 ± 1.861.8687.02 ± 5.246.03Spleen297.62 ± 8.118.30109.83 ± 9.959.06105.55 ± 7.567.17109.70 ± 13.0511.904105.82 ± 9.919.37100.15 ± 9.699.67103.68 ± 9.719.3698.88 ± 4.164.20100112.00 ± 7.826.98111.12 ± 7.316.58106.82 ± 6.886.44111.17 ± 7.176.452400101.52 ± 7.737.61104.00 ± 7.457.17106.67 ± 5.435.09104.73 ± 7.046.72Lung2103.27 ± 8.017.75111.67 ± 5.434.86112.93 ± 9.578.48113.77 ± 9.208.084101.83 ± 6.416.30100.55 ± 8.588.53100.07 ± 10.3910.3898.77 ± 6.156.23100111.50 ± 5.474.90110.17 ± 10.939.92110.17 ± 7.146.48110.03 ± 9.338.462400100.67 ± 10.1710.1198.78 ± 7.017.10104.88 ± 8.237.85110.17 ± 4.544.12Kidney2110.07 ± 7.346.6788.18 ± 4.985.6497.22 ± 7.677.8989.87 ± 9.2310.27487.63 ± 7.768.8688.40 ± 6.197.0087.60 ± 9.2610.5889.60 ± 9.0510.10100112.00 ± 7.016.26111.92 ± 8.867.91111.17 ± 7.446.69103.22 ± 11.3210.972400100.55 ± 5.115.0998.10 ± 4.874.97100.45 ± 3.963.94101.23 ± 4.694.63Small intestine2105.00 ± 8.207.81102.63 ± 3.523.43103.90 ± 4.494.32101.42 ± 7.827.71491.73 ± 4.494.8999.47 ± 5.105.1399.78 ± 7.707.72106.70 ± 5.435.0910095.90 ± 12.3112.84108.67 ± 12.0411.08100.93 ± 12.3312.2299.07 ± 12.4112.522400112.00 ± 5.374.79111.67 ± 7.266.50111.67 ± 6.125.48110.83 ± 6.686.02Stomach2113.08 ± 11.069.78111.67 ± 6.255.60113.58 ± 8.227.24109.08 ± 9.979.144102.70 ± 10.179.9097.82 ± 9.059.2598.87 ± 9.829.9489.87 ± 7.047.8410093.75 ± 8.659.2294.82 ± 5.055.3393.47 ± 6.767.23107.33 ± 5.895.492400107.17 ± 8.688.1096.60 ± 3.603.72100.23 ± 4.634.6299.73 ± 10.0910.12Brain2110.33 ± 7.556.85106.15 ± 12.3611.65105.65 ± 10.9210.3490.73 ± 10.2511.30489.12 ± 9.0010.1087.57 ± 8.479.6790.68 ± 10.5111.59105.32 ± 9.729.2310091.93 ± 9.9310.80109.17 ± 7.446.82108.13 ± 9.919.17105.48 ± 6.926.562400102.93 ± 5.795.62106.72 ± 9.839.2196.57 ± 10.1210.4897.98 ± 9.569.75Naucleactonin CPlasma5101.95 ± 10.3310.14106.67 ± 10.8810.2091.87 ± 8.639.39109.57 ± 12.6611.558108.88 ± 1.871.72105.01 ± 6.326.02105.88 ± 1.871.77113.33 ± 5.324.6914087.67 ± 9.2010.5087.88 ± 4.144.71111.88 ± 1.871.67111.57 ± 9.608.60240088.93 ± 6.016.7589.25 ± 7.588.4989.17 ± 7.498.40113.83 ± 7.146.27Heart5111.97 ± 11.6310.39100.78 ± 9.639.56107.98 ± 9.458.75107.42 ± 10.249.538110.50 ± 4.424.00107.25 ± 9.258.62109.08 ± 8.257.56110.87 ± 9.048.15140102.78 ± 6.896.71104.88 ± 3.803.62100.40 ± 9.139.0998.28 ± 5.705.802400112.33 ± 10.419.26111.50 ± 5.685.10112.33 ± 5.725.09110.50 ± 4.814.35Liver5101.78 ± 11.8311.63107.70 ± 5.364.9898.32 ± 6.666.7898.38 ± 12.0712.27887.60 ± 5.906.7386.47 ± 4.525.2390.60 ± 4.915.4286.00 ± 8.6010.0014088.57 ± 2.442.7695.00 ± 4.154.3796.28 ± 4.364.5397.32 ± 7.177.36240098.93 ± 3.813.8591.37 ± 2.512.7589.35 ± 3.704.1589.97 ± 7.328.14Spleen5110.33 ± 8.717.89112.92 ± 8.727.72110.67 ± 8.577.75109.68 ± 12.5711.46899.28 ± 13.3113.4193.42 ± 8.008.5796.13 ± 11.9812.46101.90 ± 10.2510.0614093.13 ± 6.106.5595.27 ± 7.087.4389.18 ± 8.239.2392.35 ± 2.702.922400103.33 ± 8.378.10105.67 ± 8.508.05107.73 ± 8.107.51109.83 ± 4.964.51Lung5109.50 ± 11.4710.48100.40 ± 10.2610.22101.05 ± 5.335.2895.98 ± 10.4710.91894.73 ± 8.929.4191.40 ± 9.7110.63101.90 ± 10.4510.2599.03 ± 10.4910.59140102.38 ± 4.124.0394.45 ± 5.926.2793.75 ± 8.539.0992.58 ± 3.603.892400111.37 ± 9.278.33111.85 ± 9.538.52112.125 ± 9.668.61111.67 ± 3.393.03Kidney5109.33 ± 7.556.91106.08 ± 8.968.4498.92 ± 9.9110.0297.18 ± 10.4610.76899.10 ± 4.244.2797.38 ± 7.317.5191.10 ± 9.2010.1094.77 ± 7.628.0414096.08 ± 7.367.6695.43 ± 7.998.37101.10 ± 4.984.92100.25 ± 8.258.232400108.00 ± 5.625.20103.87 ± 4.684.51104.57 ± 6.095.82102.30 ± 5.905.77Small intestine5111.90 ± 12.0710.79111.67 ± 8.147.29111.17 ± 8.337.49112.67 ± 8.787.798103.33 ± 3.393.28100.03 ± 11.4611.46100.60 ± 10.2410.18104.25 ± 9.499.1014088.60 ± 9.4810.7089.80 ± 8.309.2487.88 ± 5.576.3490.17 ± 7.268.052400107.48 ± 8.237.66103.75 ± 6.686.44108.43 ± 8.657.98102.32 ± 8.658.45Stomach5111.00 ± 7.907.12112.17 ± 7.256.46113.12 ± 9.978.81113.83 ± 2.992.638107.32 ± 12.9612.0898.37 ± 10.7510.93105.90 ± 9.138.62103.55 ± 9.499.1614088.15 ± 7.918.9886.38 ± 8.389.7087.12 ± 5.246.188.28 ± 6.317.152400100.53 ± 7.297.2599.52 ± 5.855.88104.90 ± 5.535.28102.32 ± 8.228.03Brain5106.35 ± 11.8211.11109.83 ± 6.055.5198.92 ± 10.6510.77104.10 ± 10.8810.45888.87 ± 10.4311.7490.65 ± 6.377.0389.95 ± 9.2310.2688.82 ± 10.5711.9014088.33 ± 6.747.6387.92 ± 8.159.2787.20 ± 4.985.7187.93 ± 5.816.61240099.43 ± 6.386.41112.67 ± 6.625.88109.67 ± 6.445.87109.00 ± 5.224.78Khaephuoside APlasma599.10 ± 9.259.3390.97 ± 9.019.9093.93 ± 11.2611.98101.18 ± 3.533.49891.38 ± 12.7613.9694.88 ± 12.3112.9790.67 ± 9.2910.25108.17 ± 9.208.50120101.35 ± 4.334.28101.32 ± 3.403.3696.75 ± 4.554.70107.30 ± 11.4810.70180099.35 ± 6.046.08101.93 ± 6.906.7796.13 ± 4.034.19112.67 ± 7.666.80Heart5107.60 ± 11.2310.44104.15 ± 9.929.52110.67 ± 10.829.78107.45 ± 9.358.708112.67 ± 7.236.42111.83 ± 8.237.36111.33 ± 5.925.32101.00 ± 12.0511.93120111.67 ± 6.225.57112.67 ± 5.895.23108.58 ± 11.5110.60113.50 ± 7.206.351800110.83 ± 2.792.51106.58 ± 8.778.22113.67 ± 4.273.76101.90 ± 12.3012.07Liver593.87 ± 9.319.92106.17 ± 4.884.5995.62 ± 9.8210.27100.87 ± 10.2710.198108.95 ± 10.779.88101.97 ± 7.937.78105.20 ± 10.219.7091.12 ± 10.7911.84120107.67 ± 6.225.78111.50 ± 7.266.51101.62 ± 9.629.47104.23 ± 8.688.331800108.83 ± 2.322.13109.83 ± 5.384.90103.20 ± 7.527.29108.93 ± 9.528.74Spleen5110.30 ± 12.3111.16112.17 ± 7.947.07111.00 ± 10.269.24111.50 ± 8.417.548112.17 ± 5.645.02111.67 ± 6.195.54111.38 ± 7.366.58110.67 ± 7.176.48120107.85 ± 7.837.26105.45 ± 7.957.5498.83 ± 7.517.60105.42 ± 10.7910.241800109.77 ± 8.617.85111.17 ± 8.187.36113.17 ± 3.312.93111.83 ± 7.576.77Lung5113.33 ± 8.717.69106.65 ± 12.4311.6598.87 ± 9.029.12102.98 ± 9.509.238105.32 ± 7.817.41103.18 ± 10.9710.63105.03 ± 9.869.38104.00 ± 11.7311.28120101.27 ± 3.533.4999.07 ± 11.0111.11102.02 ± 6.366.23110.83 ± 5.194.691800110.83 ± 8.237.43112.80 ± 7.466.61110.00 ± 8.888.07112.50 ± 6.806.05Kidney596.42 ± 6.246.4799.65 ± 10.5310.57101.62 ± 11.3711.19112.78 ± 11.4310.138113.17 ± 6.465.71110.83 ± 7.887.11113.00 ± 6.996.18113.17 ± 7.196.36120111.67 ± 5.324.76111.83 ± 5.314.75112.00 ± 5.935.30112.00 ± 5.625.021800102.03 ± 4.444.3597.37 ± 6.847.02100.50 ± 5.535.50101.12 ± 6.566.48Small intestine5110.02 ± 8.197.44112.83 ± 3.433.04100.88 ± 9.399.3094.98 ± 9.429.918105.62 ± 9.769.24109.53 ± 10.149.25101.00 ± 10.4210.31107.70 ± 11.4710.65120103.30 ± 7.757.50106.00 ± 7.296.88102.80 ± 6.095.93101.58 ± 6.266.161800113.00 ± 6.135.43110.83 ± 8.137.34113.67 ± 8.197.20113.12 ± 12.4711.02Stomach5113.68 ± 8.427.41112.13 ± 10.239.12111.12 ± 10.439.38110.67 ± 6.095.508111.67 ± 6.595.90111.00 ± 5.224.70109.67 ± 3.333.03104.50 ± 4.714.51120113.17 ± 3.663.23111.50 ± 5.865.25112.17 ± 6.155.48110.92 ± 8.367.531800109.83 ± 7.366.70111.92 ± 10.999.82110.50 ± 6.285.69113.83 ± 5.564.89Brain5109.82 ± 10.269.34106.90 ± 12.0911.31103.05 ± 9.128.85112.50 ± 5.584.968111.00 ± 7.426.52112.50 ± 7.066.28113.33 ± 3.833.38111.33 ± 6.095.47120112.50 ± 5.134.56112.33 ± 6.475.76113.17 ± 5.314.69110.17 ± 8.287.521800105.82 ± 8.317.86112.83 ± 7.686.81110.83 ± 7.526.79113.83 ± 4.493.953,4-Dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranosidePlasma2586.25 ± 5.406.2694.95 ± 9.8010.32102.87 ± 10.3710.08105.60 ± 10.199.655087.20 ± 7.919.0798.98 ± 11.3011.3094.53 ± 9.8410.41107.88 ± 14.9613.87350112.83 ± 4.584.06112.67 ± 6.625.88108.33 ± 5.134.73109.07 ± 1.871.72240089.98 ± 4.765.2989.43 ± 7.428.3086.07 ± 3.393.93111.74 ± 3.763.37Heart2598.10 ± 9.439.61100.45 ± 10.8910.8597.55 ± 9.019.2488.38 ± 5.696.445094.60 ± 11.147.80102.70 ± 7.987.7797.05 ± 8.428.6899.12 ± 9.569.65350102.57 ± 4.924.80112.63 ± 98.937.93110.33 ± 5.825.27108.38 ± 7.346.772400102.23 ± 6.646.4992.98 ± 8.879.54101.57 ± 6.736.6399.57 ± 9.469.50Liver2591.10 ± 10.3711.39102.88 ± 10.8510.5497.32 ± 10.4410.73104.70 ± 11.9711.4350105.60 ± 9.048.56112.50 ± 5.655.02107.48 ± 10.549.8095.25 ± 11.2911.8635097.50 ± 6.636.80113.00 ± 4.003.54103.12 ± 5.265.10108.67 ± 8.988.272400104.32 ± 10.189.76100.42 ± 6.856.8396.25 ± 5.745.96107.68 ± 7.737.18Spleen2588.72 ± 9.9311.19104.47 ± 9.008.6290.93 ± 9.1510.07112.83 ± 3.603.1950113.17 ± 10.599.36108.35 ± 10.589.7795.62 ± 10.9011.40107.68 ± 10.399.65350111.33 ± 7.636.86111.68 ± 9.218.25112.17 ± 7.997.12112.17 ± 8.407.492400102.27 ± 11.5011.24112.00 ± 8.677.74105.32 ± 6.596.26111.00 ± 6.035.44Lung2590.52 ± 10.7711.9096.80 ± 11.2911.6797.07 ± 10.8711.2097.92 ± 11.3411.5950112.67 ± 7.846.96111.02 ± 7.817.04100.02 ± 12.4612.46109.25 ± 11.4610.49350111.33 ± 6.896.19109.47 ± 11.1010.14103.68 ± 11.6211.21112.33 ± 6.926.16240098.85 ± 9.839.94101.82 ± 8.678.52100.62 ± 7.717.66109.12 ± 10.699.79Kidney25100.05 ± 11.9211.92101.42 ± 8.638.5196.23 ± 9.149.50110.83 ± 6.245.635098.37 ± 7.537.65102.72 ± 10.2910.02106.75 ± 9.158.5799.88 ± 9.659.6635090.05 ± 8.479.40102.03 ± 4.614.52111.83 ± 7.036.28105.40 ± 10.289.752400104.72 ± 8.968.56106.27 ± 10.199.59107.85 ± 9.168.49104.82 ± 8.598.20Small intestine2587.47 ± 7.178.2096.42 ± 9.139.4794.10 ± 8.058.5599.25 ± 6.066.1150109.83 ± 2.562.33100.75 ± 6.116.07106.83 ± 5.345.00101.80 ± 8.788.62350111.17 ± 7.736.95113.67 ± 8.767.70113.00 ± 3.793.36106.33 ± 5.395.07240098.50 ± 5.495.57104.65 ± 7.857.5099.92 ± 5.945.9494.90 ± 8.629.08Stomach25111.83 ± 5.785.17110.50 ± 4.424.00113.00 ± 6.325.60106.32 ± 9.488.9250112.75 ± 10.979.73112.67 ± 3.673.26112.83 ± 5.604.96109.83 ± 8.667.88350113.17 ± 3.873.42108.17 ± 9.688.95111.00 ± 6.205.58111.83 ± 5.985.352400101.27 ± 7.897.8094.40 ± 6.006.3596.82 ± 8.088.34103.08 ± 9.609.32Brain2599.82 ± 9.829.84107.80 ± 12.7911.86100.68 ± 9.809.73105.37 ± 7.316.9350109.35 ± 12.1511.11112.83 ± 4.263.78113.77 ± 10.369.11113.83 ± 3.873.40350111.50 ± 8.647.75110.83 ± 7.476.74111.17 ± 7.636.27112.83 ± 7.786.90240098.95 ± 3.974.01100.10 ± 9.439.4298.23 ± 5.395.49101.50 ± 9.949.80


## 3.4. Pharmacokinetic Study

The validated analytical method was successfully applied to the quantitative estimation of chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside in the plasma samples following oral administration of Nauclea officinalis extract at a dosage of 2 g/kg. Pharmacokinetic parameters of four compounds were calculated by noncompartmental model using DAS 3.3.0 software. The mean plasma concentration–time-curve of four analytes are shown in Figure 3, and the main pharmacokinetic parameters were estimated of four analytes in Table 6.Figure 3
Concentration-time profiles of four analytes in rat plasma following oral administration ofNauclea officinalis extracts at a dose of 2 g/kg.Table 6
Summary of primary pharmacokinetic parameters of four analytes (n = 6).
ParametersUnitChlorogenic acid (mean ± SD)Naucleactonin C (mean ± SD)Khaephuoside A (mean ± SD)3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside (mean ± SD)AUC (0 −t)μg/L∗h482.76 ± 124.955281.36 ± 440.02238.70 ± 46.64212.99 ± 49.29AUC (0 − ∞)μg/L∗h554.37 ± 166.145405.02 ± 416.99262.16 ± 32.24440.17 ± 216.99MRT (0 −t)h5.33 ± 0.654.76 ± 1.573.19 ± 0.241.6 ± 0.14t1/2h10.64 ± 6.214.59 ± 1.653.12 ± 1.703.99 ± 2.87Tmaxh0.25 ± 0.001.67 ± 0.520.29 ± 0.100.33 ± 0.13CLL/h/kg62.94 ± 16.000.15 ± 0.0154.40 ± 6.0428.63 ± 13.64Cmaxμg/L242.42 ± 84.671008.58 ± 368.84106.18 ± 23.30144.13 ± 35.59Vz/FL/kg915.76 ± 451.750.966 ± 0.359253.289 ± 158.11127.43 ± 65.93Abbreviations. AUC, area under the plasma concentration–time curve; MRT, mean residence time;t1/2, elimination half-life; Tmax, time to Cmax; CL, clearance rate; Cmax, maximum plasma concentration; Vz/F, apparent distribution volume.These compounds were detected at 5 min after oral administration ofNauclea officinalis extracts, indicating four compounds were rapidly absorbed, exhibiting a therapeutic effect. Chlorogenic acid, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside were rapidly absorbed and peaked at approximately 0.5 h, while naucleactonin C peaked at 1.66 h, indicating that naucleactonin C was absorbed relatively slowly. The content of naucleactonin C was much lower than that of the other three compounds in the Nauclea officinalis extracts, but naucleactonin C was present in a much larger amount than other three compounds for AUC0−t (area under the plasma concentration–time curve) and AUC0−∞, indicating that naucleactonin C may had excellent bioavailability. However, the bioavailability of chlorogenic acid and khaephuoside A may not be satisfactory, the CL of chlorogenic acid and khaephuoside A were 62.93 ± 15.996 and 54.40 ± 6.04 L/h/kg after oral administration of Nauclea officinalis extract, respectively. Excessive clearance rate will affect the residence time of the drug in the body, thereby reducing the efficacy. In addition, literature reported that the prototype chlorogenic acid absorbed into the blood by oral chlorogenic acid accounts for only 30% [25], which greatly affected the pharmacological activity of chlorogenic acid from Nauclea officinalis. Therefore, considering the clinical application of Nauclea officinalis [26], we could design Nauclea officinalis to different dosage forms with advanced technology in order to improve the bioavailability and finally increase the efficacy.


## 3.5. Tissue Distribution Study

This method was also applied to investigate the tissue distribution of chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside in rats. The result is shown in Figure 4 that naucleactonin C and khaephuoside A could be detected in all studied tissue. However, chlorogenic acid and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside were not detected in the spleen. The concentration orders in eight different tissues were ranked as 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside > Khaephuoside A > Naucleactonin C > Chlorogenic acid in the small intestine, stomach, heart, liver, spleen, lung, and brain tissues, and naucleactonin C > 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside > Khaephuoside A > Chlorogenic acid in the kidney. The contents of the four compounds in the liver, kidney, and lung were relatively high within after oral administration of Nauclea officinalis extrac, indicating that the liver, kidney, and lung may be the main target organs for the pharmacological effects of Nauclea officinalis. This result is consistent with existing research on the inhibition of bronchitis by Nauclea officinalis [27]. The results show that all compounds except chlorogenic acid were more abundant in the small intestine than in the stomach, indicating that most of naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside possibly absorbed by the small intestine after oral administration of Nauclea officinalis extract for enter the systemic circulation system. With the exception of chlorogenic acid, the peak times and concentrations of the other three compounds in most tissues are nearly identical to those in plasma, which means that the distribution of the other three compounds depends on the blood flow or perfusion rate of the organ [28]. The peak concentration of chlorogenic acid in most tissues is much lower than in plasma and may be related to the absorption of the compound in the tissue. Drugs enter tissues that receive high blood flow first, followed by those that receive low blood flow [29]. Our study demonstrated that the four compounds are mainly distributed in organs with relatively large blood flows, such as the liver and kidneys. In addition to this, the first-pass effect of oral administration might be the main cause of high distribution in the liver.Figure 4
Mean concentration of four analytes in the heart, liver, spleen, lung, kidney, small intestine, stomach, and brain at 0.5, 1, 2, 4, and 6 h after oral administration ofNauclea officinalis extracts at a dose of 2 g/kg.


## 4. Conclusion

We report the development and validation of a sensitive, rapid, and reliable HPLC–MS/MS analytical method for the simultaneous determination and quantification of chlorogenic acid, naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside in the plasma and tissues of rats. This validated analytical method was assessed on the basis of the FDA guidelines for bioanalytical method validation and applied the study of pharmacokinetics and tissue distribution of oral administration of Nauclea officinalis extract in SD rats. To the best of our knowledge, this is the first study that determined the pharmacokinetic and tissue distribution of naucleactonin C, khaephuoside A, and 3,4-dimethoxyphenyl-1-O-β-apiofuroseyl(1 ⟶ 2)-β-D-glucopyranoside. In addition, the pharmacokinetics and tissue distribution of chlorogenic acid after oral administration of Nauclea officinalis extract in rats was also reported for the first time.



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*Source: 5297603-2022-12-21.xml*