Sanguisorba Officinalis

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Comparison of the pharmacokinetic profiles of thirteen phenolic acids and six triterpenes in
normal and leukopenia rats after oral administration of Sanguisorba officinalis L. extract by
LC-MS/MS.
Linzi Fan1#, Xiaotong Wang 1#, Jing Huang2, Chunli Gan2, Shuang Jiang1, Xinrong Yang1,
Chunjuan Yang1*, Meicun Yao3*.
1
Department of Pharmaceutical Analysis and Analytical Chemistry, College of Pharmacy,
Harbin Medical University
2
Department of Medicinal Chemistry and Natural Medicine Chemistry, College of Pharmacy,
Harbin Medical
University
3
Department of Pharmaceutical Analysis and Quality Assessment, School of Pharmaceutical
Sciences (Shenzhen), Sun Yat-Sen University
Received: 05 06, 2020; Revised: 08 19, 2020; Accepted: 09 07, 2020
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/jssc.202000514.
This article is protected by copyright. All rights reserved.

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*
Corresponding authors: Department of Pharmaceutical Analysis and Analytical Chemistry,
College of Pharmacy, Harbin Medical University, 157 Baojian Road, Nangang Distrct.
Harbin 150081, China. Tel: +86 45186699347; Fax: +86 45186614073; E-mail address:
chunjuanyang@hrbmu.edu.cn (C.J. Yang)
*
Department of Pharmaceutical Analysis and Quality Assessment, School of Pharmaceutical
Sciences (Shenzhen), Sun Yat-Sen University, 132 Waihuan East Road, Panyu District,
Guangzhou 510006, Guangdong, China. E-mail address: yaomeicun@gmail.com (M.C. Yao)
#
These authors contributed equally to this work.
Abbreviations: UHPLC-MS/MS, ultra-high-performance liquid
chromatography-electrospray ionization-tandem mass spectrometry; S. officinalis,
Sanguisorba officinalis L.; AUC0→t, area under the peak area–time curve from time zero to
last sampling time; AUC0→∞, area under the plasma concentration-time curve to time infinity;
Cmax, maximum plasma concentration; CTX, cyclophosphamide; Ke, elimination rate
constant; LLOQ, lower limit of quantification; MRM, multiple reaction monitoring; t1/2, the
elimination half-life; Tmax, time for maximal concentration;
Keywords: leucopenia; phenolic acid; pharmacokinetics; Sanguisorba officinalis L.;
ultra-high-performance liquid chromatography

Abstract: A selective, accurate and efficient LC-MS/MS method was developed for the
simultaneous determination of 13 phenolic acids. Additionally, for more comprehensively
determining the chemical constituents in Sanguisorba officinalis L. extract, a previously
developed method was employed for the simultaneous determination of six triterpenes. Thus,
two methods were used to ensure the comprehensiveness and reliability of this study. Based
on these methods, the pharmacokinetic profiles of the 13 phenolic acids and 6 triterpenes in
normal and leukopenia rats after oral administration of Sanguisorba officinalis L. extract
were compared for the first time in present study. Quantitative detection of the 13 phenolic
acids and 6 triterpenes was performed using the multiple reaction monitoring mode with the
electrospray ion source in negative and positive electrospray ionization, respectively.
Chromatographic separation was performed on an Agilent Eclipse Plus C18 RRHD column
(50×2.1 mm, 1.8μm), using gradient elution with a mobile phase composed of
methanol-0.1% aqueous formic acid. The pharmacokinetic results demonstrated that the
pharmacokinetic characteristics of the 19 analytes in leukopenia rats differed significantly
from those determined in normal rats, which could provide a helpful reference for the clinical
application of Sanguisorba officinalis L. in the prevention and treatment of leucopenia.

1. Introduction
Cancer is the leading cause of death that seriously threatening the lives of people
worldwide [1], and chemotherapy is considered a mainstream treatment for cancer patients.
However, chemotherapeutic drugs present several harmful side effects such as cytotoxic
effects and host immunosuppression, regardless of treatment efficacy [2-4]. Leucopenia is
one of the most common side effects, resulting in reduced resistance and repeated infection,
influencing the effects of tumor radiation and chemotherapy [5]. For centuries, traditional
Chinese medicine has been widely used in East Asia to treat diseases [6-9]. Notably, Diyv
Shengbai Tablet, whose main component is the powder of Sanguisorba officinalis L., has
been generally used to treat leukopenia in clinical practice [10-14].
Sanguisorba officinalis L. (the dried root of Sanguisorba, Rosaceae) is a traditional
medicinal herb that is widely distributed in Europe and Asia. It has been officially recorded in
the Chinese Pharmacopoeia (2015), possesses activities such as detoxification, analgesia
and hemostasis, and has been used for the prevention and treatment of various diseases in
tradition, including burns and scalds, bleeding hemorrhoids, and metrostaxis, bleeding
wounds, hematochezia, and swollen carbuncles [15-18]. Sanguiin H-4 (Ⅰ), bergenin
monohydrate (Ⅱ), 3,3'-dimethoxyellagic acid (Ⅲ), ellagic acid (Ⅳ), (+)-catechin monohydrate
(Ⅴ), methyl 3,4-dihydroxy-5-methoxybenzoate (Ⅵ), ethyl 3,4,5-trihydroxybenzoate (Ⅶ),
syringic acid (Ⅷ), ferulic acid (Ⅸ), caffeic acid (Ⅹ), gallic acid (Ⅺ), p-coumaric acid (Ⅻ),
4-hydroxybenzoic acid (XIII), ziyuglycoside I (XIV), 3β-[(α-L-arabinopyranosyl)
oxy]-urs-12,18(19)-dien-28-oic acid β-D-glucopyranosyl ester (XV), rosamultin (XVI),

alpinoside (XVII), 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-D-glucopyranosyl ester
(XVIII) and hydroxyeuscaphic acid (XIX) are all the active components isolated from S.
officinalis, and has been reported to possess an extensive range of pharmacological activities
[19, 20]. Accordingly, it has been noted that sanguiin H-4 decreases procaspase-3 associated
with human poly(ADP-ribose) polymerase (PARP) cleavage and increases the activity of
caspase-3 in HL-60 cells, without affecting normal human peripheral blood mononuclear
cells (PBMCs), suggesting that sanguiin H-4 may be a new candidate for drug development
in the prevention and treatment of cancer [21]. Ellagic acid, a special component of S.
officinalis, potentially inhibits lipopolysaccharide (LPS)-induced nitric oxide (NO),
prostaglandin E2 (PGE2) and interleukin (IL)-6 production, thus exerting its
anti-inflammatory properties [22]. Furthermore, ellagic acid prevents cognitive deficits by
normalizing lipid metabolism, increasing plasma brain-derived neurotrophic factor (BDNF)
levels, and decreasing the salivary cortisol concentration [23]. In skin carcinogenesis,
syringic acid inhibits the Nox/PTP-κ/EGFR axis, exerting a potent chemopreventive activity.
Additionally, syringic acid could be an effective chemopreventive and therapeutic agent
against UVB-mediated skin cancer [24]. Ferulic acid can reverse the inflammatory response
induced by chronic unpredictable mild stress [25] . Furthermore, it can also reduce the dose
of metformin dose required to achieve normoglycemia through the synergistic interaction
with metformin, with the dose-related side effects of metformin therapy reduced owing to the
reduced therapeutic dose [26].

Several studies have reported that various analytical methods, including colorimetry,
high-performance thin-layer chromatography (HPTLC), high-performance liquid
chromatography-ultraviolet (HPLC-UV), liquid chromatography-tandem mass spectrometry
(LC–MS/MS) and ultra-performance liquid chromatography-tandem mass spectrometry
(UPLC-MS/MS), have been used in the qualitative or quantitative analysis of the phenolic
acids and triterpenes [19, 20, 27]. Nonetheless, these methods have primarily focused on high
concentrations of ingredients in S. officinalis or other plants. Additionally, few reports are
available about the comparison of pharmacokinetic profiles of the 19 analytes in normal and
leukopenia rats.
In this study, a selective, accurate and efficient ultra-high-performance liquid
chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI MS/MS)
method was developed for the simultaneous determination of 13 phenolic acids. Moreover,
for more comprehensively determining the chemical constituents in the S. officinalis extract,
the method for simultaneous determination of 6 triterpenes in previous study was also
employed [19]. Thus, two methods were used to ensure the comprehensiveness and reliability
of this study. Accordingly, we compared the pharmacokinetic profiles of 13 phenolic acids
and 6 triterpenes in normal and leukopenia rats after oral administration of S. officinalis
extract. The results of the pharmacokinetic study provided a meaningful reference for
improving the clinical application of S. officinalis, especially for the patients with leukopenia.
The chemical structures of the 19 analytes and the internal standards are shown in Figure 1.

2. Materials and Methods
2.1. Chemicals and Reagents
The standard substances of sanguiin H-5 (Ⅰ), bergenin monohydrate (Ⅱ),
3,3'-dimethoxyellagic acid (Ⅲ), ellagic acid (Ⅳ), (+)-catechin monohydrate (Ⅴ), methyl
3,4-dihydroxy-5-methoxybenzoate (Ⅵ), ethyl 3,4,5-trihydroxybenzoate (Ⅶ), syringic acid
(Ⅷ), ferulic acid (Ⅸ), caffeic acid (Ⅹ), gallic acid (Ⅺ), p-coumaric acid (Ⅻ),
4-hydroxybenzoic acid (XIII), ziyuglycoside I (XIV), 3β,19α-dihydroxyurs-12-en-28-oic-acid
28-β-D-glucopyranosyl ester (XV), 3β-[(α-L-arabinopyranosyl)
oxy]-urs-12,18(19)-dien-28-oic acid β-D-glucopyranosyl ester (XVI), rosamultin (XVII),
1β-hydroxyeuscaphic acid (XVIII) and alpinoside (XIX), the purities of which were more
than 98%,were defined in our laboratory (identified by NMR and MS). Butylparaben
(XX)(IS-1) was used as an internal standard for the phenolic acids and was purchased from
Guangfu Fine Chemical Research Institute (Tianjin, China), and bifendate (XXI)(IS-2) (lot:
73536-69-3; purity ≥ 98%) was purchased from Chengdu Must Bio-Technology (Chengdu,
Sichuan province, China). Tinidazole (lot: 19387-91-8; purity ≥ 98%) was provided by
Shanghai Guangrui Bio-Technology (Shanghai, China). Acetic acid (purity ≥ 99%) of
HPLC-grade was purchased from CNW Technologies (Düsseldorf, Germany). Formic acid
(purity ≥ 99%) of HPLC-grade was purchased from Kermel (Tianjin, China). Methanol and
acetonitrile of HPLC grade were purchased from Dikma Tevhnologies Inc. (Beijing, China).
Ultra-pure water used throughout the experiment was prepared from a MilliQ water
purification system (Millipore, Molsheim, France). All other chemical reagents were of

analytical grade. Cyclophosphamide for injection (0.2g, 18052825) was purchased from
Jiangsu Shengdi Pharmaceutical Co. Ltd. (Jiangsu, China). S. officinalis was purchased from
the Anguo Traditional Chinese Medicine Market of Hebei and was identified by Professor
Zhenyue Wang of Heilongjiang University of Chinese Medicine in September 2018. A
voucher specimen (201809) was deposited in the Pharmaceutical Research Department of
Harbin Medical University.
2.2. Instruments and operational condition
UHPLC-ESI-MS/MS was performed on an Agilent 1290 ultra-high-performance liquid
chromatography (UHPLC) system and an Agilent 6430 QQQ-MS mass spectrometer with an
electrospray ionization (ESI) source interface (Agilent Technologies, Santa Clara, CA, USA).
Additionally, an Agilent Eclipse Plus C18 RRHD column (50×2.1 mm, 1.8μm) was selected.
Therefore, 13 phenolic acids and 6 triterpenes were separately detected under negative mode
and positive modes. As for the 13 phenolic acids, the mobile phases were 0.1% formic
acid/water (A) and methanol (B) as follows: 0–5 min, 10%–60% (B); 5–6 min, 60%–90%
(B); 6–6.5 min, 90%–10% (B). The flow rate of the mobile phase was 0.3 mL/min. The
column temperature was controlled at 30 ℃ and a 10μL sample solution was injected
accompanied with a needle wash process. For the 6 triterpenes, the mobile phases were 0.1%
formic acid/water (A) and methanol (B) as follows: 0–1 min, 10%–60%(B); 1–4 min, 60%–
65% (B); 4–4.5 min, 65%–90% (B); 4.5–5.6 min, 90%–90% (B); 5.6–6.5 min, 90%–10%
(B). The flow rate was 0.3 mL/min. The column temperature was 40 ℃. All other test
conditions adopted were the same. N2 was selected as drying gas at a flow rate of 11 L/min,

and high-purity N2 was used as the nebulizing gas. The mass spectrometer was operated in
positive or negative ion mode during each detection procedure. The capillary voltage was set
at 4500 V. The source temperature was kept at 100 ℃. And the desolvation temperature was
kept at 350 ℃.
2.3. Animals
The animal handling protocol was ratified by the Animal Ethics Committee of Harbin
Medical University and was in accordance with the principles for the Care and Use of
Laboratory Animals. Twenty-four healthy male pathogen-free Sprague-Dawley rats (200±20
g) were procured from the Laboratory Animal Center of Harbin Medical University (Harbin,
China). The animals were maintained in a SPF-grade room on a 12 h light/dark cycle with a
controlled temperature conditions (21 ± 2°C) and relative humidity (50 ± 5%) before the
experiment. During the adaptation process, all rats were provided with standard chow and
purified water each morning and evening.
2.4. Leukopenia rat model induced by cyclophosphamide
Twenty-four Sprague-Dawley rats were assigned to the normal group and model group in
random (n = 6 per group). In the model group, CTX, which is widely used to induce
leukopenia symptoms that correspond with the core symptoms of human leukopenia[28-34],
was intraperitoneally injected to rats at a dose of 30 mg/kg for 5 consecutive days. In the
normal group, rats were intraperitoneally injected with the same volume of physiological
saline. The injection volume was maintained at 0.2mL per rat. After 5 days of continuous

CTX injection, the leukopenia status was assessed by measuring the total number of white
blood cells (WBC), spleen weight coefficient and bone marrow DNA content. The total
number of white blood cells (WBC)in the peripheral blood of rats were detected using
MS9-Automatic Hematology Cell Counter (MELET CHLOESING Laboratories, France).
The bone marrow DNA content in left femur of rats were detected using Infinite F200/M200
Multifunctional Microplate Spectrophotometer (Tecan Group AG, Switzerland).
2.5. Preparation of Sanguisorba officinalis L. Extract
After crushing the dried root of S. officinalis (100 g), it was extracted by hot reflux with
1 L of a 70% ethanol-water (1:10, w/v) solution for 60 min at 80 ℃, thrice in total. All the
decoctions were combined, filtered and then evaporated into steam. The residue was added to
an appropriate amount of distilled water for complete suspension, extracted with n-butanol
solution for three times, and all the supernatant was concentrated to obtain the n-butanol
extract. The n-butanol extract was prepared in water to obtain a concentration of 0.1 g/mL of
the S. officinalis extract. The contents of S. officinalis L. extract for I–XIX were 10.60, 7.37,
35.97, 11.47, 8.74, 7.01, 3.04, 0.82, 0.02, 0.26, 47.62, 0.14, 0.16, 23.18, 3.54, 1.36, 1.97,
3.99, 0.10 mg/g, respectively.
2.6. Preparation of Calibration Standards and Quality Control Samples
The stock solutions of sanguiin H-5 (Ⅰ), bergenin monohydrate (Ⅱ),
3,3'-dimethoxyellagic acid (Ⅲ), ellagic acid (Ⅳ), (+)-catechin monohydrate (Ⅴ), methyl
3,4-dihydroxy-5-methoxybenzoate (Ⅵ), ethyl 3,4,5-trihydroxybenzoate (Ⅶ), syringic acid

(Ⅷ), ferulic acid (Ⅸ), caffeic acid (Ⅹ), gallic acid (Ⅺ), p-coumaric acid (Ⅻ),
4-hydroxybenzoic acid (XIII), ziyuglycoside I (XIV), 3β-[(α-L-arabinopyranosyl)
oxy]-urs-12,18(19)-dien-28-oic acid β-D-glucopyranosyl ester (XV), rosamultin (XVI),
alpinoside (XVII), 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-D-glucopyranosyl ester
(XVIII) and hydroxyeuscaphic acid (XIX) were prepared in methanol to yield nominal
concentration (210 ng/mL, 460 ng/mL, 200 ng/mL, 260 ng/mL, 190 ng/mL, 200 ng/mL, 200
ng/mL, 200 ng/mL, 200 ng/mL, 200 ng/mL, 200 ng/mL, 400 ng/mL, 230 ng/mL, 240 ng/mL,
110 ng/mL, 130 ng/mL, 140 ng/mL, 120 ng/mL, 120 ng/mL, respectively). The solutions
were further diluted with methanol to produce standard working solutions of the desired
concentrations. The IS-1 working solution (130 ng/mL) and the IS-2 working solution (500
ng/mL) were prepared by diluting the IS stock solution with methanol. As shown in Table 1
and Table 2, calibration standards were prepared by diluting each working stock solution at
seven concentrations. Quality control (QC) samples were prepared at: 11.4, 91.1 and 1166.4
ng/mL for I; 4.9, 39.2 and 501.4 ng/mL for II; 3.9, 31.0 and 397.0 ng/mL for III; 12.4, 98.9
and 1265.6 ng/mL for IV; 9.0, 72.2 and 924.5 ng/mL for V; 5.9, 47.3 and 606.0 ng/mL for
VI; 4.7, 37.2 and 476.4 ng/mL for Ⅶ; 12.0, 96.2 and 1231.7 ng/mL for Ⅷ; 6.7, 53.9 and 689.8
ng/mL for Ⅸ; 7.2, 57.5 and 736.3 ng/mL for Ⅹ; 6.6, 52.8 and 675.4 ng/mL for Ⅺ; 8.5, 68.3
and 873.6 ng/mL for Ⅻ; 9.6, 77.0 and 985.2 ng/mL for XIII; 8.3, 66.4 and 849.7 ng/mL for
XIV; 4.5, 35.7 and 456.4 ng/mL for XV; 4.7, 37.6 and 480.6 ng/mL for XVI; 10.6, 85.1 and
1089.3 ng/mL for XVII; 3.8, 30.7 and 392.4 ng/mL for XVIII; 7.2, 57.5 and 735.8 ng/mL for
XIX. LLOQ of I–XIX was 5.7, 2.4, 1.9, 6.2, 4.5, 3.0, 2.3, 6.0, 3.4, 3.6, 3.3, 4.3, 4.8, 4.2, 2.2,

2.4, 5.3, 1.9 and 3.6 ng/mL, respectively. All solutions were immediately stored at 4°C before
use.
2.7. Animal Experiments
The S. officinalis extract was dissolved in pure water and a single dose (0.152 g/kg) was
administrated to both the normal group and model group rats (n = 6 per group). The drug
volume was maintained at 0.2 mL per rat. All rats were fasted overnight before dosing, with
free access to water even during the experiment. Blood (0.3 mL) was collected from the
orbital venous plexus at 0, 0.08, 0.25, 0.50, 1, 1.5, 2, 3, 4, 6, 8, 12, 24 and 36 h after oral
administration. The plasma was immediately separated by centrifugation at 12000 rpm for 5
min at -4 ℃，and then stored frozen at -20 ℃ until analysis.
2.8. Plasma Sample Preparation
Frozen plasma samples were thawed at room temperature, and then an aliquot of 100 μL
rat plasma sample was mixed with 20 μL of 2 M hydrochloric acid, 10 μL of each IS solution
(130 ng/mL for IS-1, 500 ng/mL for IS-2) and 100 μL methanol by being vortexed for 30 s.
Next, 3mL ethyl acetate was immediately added and the mixture was vortex-mixed for 1 min.
After this step, the supernatant was individually pipetted into clean glass tubes and
evaporated to dryness by N2 blowing at 30 ℃ after centrifugation at 3800 rpm for 5 min. The
residue was then redissolved with 100 μL of methanol:0.1% formic acid-water (v: v, 10:90),
vortexed for 2 min and filtered through a 0.22 μm organic membrane. Next, a 10 μL aliquot

of the solution was then injected into the UHPLC-MS/MS system (Agilent Technologies,
Santa Clara, CA, USA) for analysis.
2.9. Method Validation
Method validation was performed according to the US Food and Drug Administration
(FDA) guidelines for bioanalytical method validation, available online:
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidance
s/UCM368107.pdf [35].
2.9.1. Selectivity
The selectivity of this method was evaluated by comparatively analyzing blank
biological samples from six individual sources spiked with the analytes and IS, and the
plasma samples collected after oral administration of the S. officinalis extract and spiked with
IS.
2.9.2. Linearity and Sensitivity
Calibration curves were generated by assaying standard plasma samples at seven
concentration levels and the linearity was constructed by using the plot of the peak area ratios
of each analyte to its corresponding IS versus the nominal concentration of the calibration
standard. LLOQ was defined as the lowest quantifiable concentration of calibration with
acceptable accuracy within ±20% and a precision below 20% and a corresponding
signal-to-noise ratio of approximately 10:1.

2.9.3. Accuracy and Precision
Precision and accuracy were evaluated using nine replicates of three QC samples on the
same day and three batches on three consecutive days. The accuracy was described by the
relative error percentage (RE%) within ±15%, and the intra-and inter-day precisions were
evaluated by the variability with relative standard deviation percentage (RSD) of measured
concentration, which should be less than 15%.
2.9.4. Extraction Recovery and Matrix Effect
The extraction recovery was evaluated by comparing the peak areas of samples spiked
with analytes before extraction with post-extracted samples spiked with the analytes at three
QC levels. (n=9, each concentration) The matrix effect was investigated by calculating the
mean peak areas of processed plasma samples (from nine batches of rat plasma) containing
the spiked analytes after extraction with the corresponding standard solution.
2.9.5. Stability
The stability of the analytes was evaluated by analyzing QC samples of three
concentration levels (LQC, MQC, and HQC) under various storage conditions: freeze-thaw
stability (three freeze cycles at -20 ℃ and thaw cycles), room temperature stability (4 h
storage at ambient temperature), long-term stability (storage at -20 ℃ for 2 weeks), and
post-preparation stability (storage for 12 h after sample preparation at 4 ℃). Each
concentration level utilized nine replicates.

2.10. Data Analysis
The pharmacokinetic parameters, including maximum plasma concentration (Cmax), time
to maximum concentration (Tmax), half-life (t1/2), area under the curve from time zero to last
sampling time (AUC0-t) and the total area under the curve (AUC0-∞) were calculated by a
non-compartment analysis using the Drug And Statistic 3.0 software package (Chinese
Pharmacological Association, Anhui, China). The plasma concentration-time curves were
obtained using GraphPad Prsim 6.0 (GraphPad Software, Inc., La Jolla, CA, USA).
Additionally, pharmacokinetic data were compared between the normal and model rats were
carried out using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). All data are presented
as the mean ± SD and a P-value of < 0.05 was regarded as statistically significant.
3. Results
3.1 Leukopenia rat mode induced by cyclophosphamide.
The data listed in Table 3 show that the total number of white blood cells (WBC),
spleen weight coefficient and bone marrow DNA content of the model group were
significantly lower than those observed in the normal control group (P<0.01), confirming the
successful establishment of leukopenia rats.

3.2. Operational Conditions Optimization
In the present study, a sensitive and rapid UHPLC-ESI-MS/MS method was established
to simultaneously quantify 13 phenolic acids in rat plasma. Parameters such as fragmentor
collision energy and the nitrogen flow rate were optimized to obtain the maximum sensitivity
of the MRM. The phenolic acids had good signal responses in the negative ion mode but no
responses in positive ion mode. The triterpenes could only be analyzed by positive ESI mass
spectrometry according to a previous study[19]. Thus, ESI operated in negative mode was
selected as the ionization source for the 13 phenolic acids (Ⅰ- XIII), and the positive mode
was selected as the ionization source for the 6 triterpenes (XIV - XIX). As the mass
spectrometry cleavage information for each compound changes under different operating
conditions, the mass spectrometry parameters were detected for 13 phenolic aids and 6
triterpenes to ensure the reliability of this study. In the full scan spectra, intense peaks were
observed at m/z 633.1→300.9 for sanguiin H-4 (Ⅰ), m/z 345.0→123.8 for bergenin
monohydrate (Ⅱ), m/z 328.7→314.0 for 3,3'-dimethoxyellagic acid (Ⅲ), m/z 301→145 for
ellagic acid (Ⅳ), m/z 289.2→109.2 for (+)-catechin monohydrate (Ⅴ), m/z 197.1→182.2 for
methyl 3,4-dihydroxy-5-methoxybenzoate (Ⅵ), m/z 197.0→124.1 for ethyl
3,4,5-trihydroxybenzoate (Ⅶ), m/z 197.0→122.8 for syringic acid (Ⅷ), m/z 193.1→134 for
ferulic acid (Ⅸ), m/z 179.0→135.0 for caffeic acid (Ⅹ), m/z 169.1→125.1 for gallic acid (Ⅺ),
m/z 163.0→119.2 for p-coumaric acid (Ⅻ), m/z 137.1→92.9 for 4-hydroxybenzoic acid
(XIII), m/z 784.5→437.4 for ziyuglycoside I (XIV), m/z 771.5→609.1 for
3β-[(α-L-arabinopyranosyl) oxy]-urs-12,18(19)-dien-28-oic acid β-D-glucopyranosyl ester

(XV), m/z 673.4→511.4 for rosamultin (XVI), m/z 655.4→493.0 for alpinoside (XVII), m/z
657.4→455.4 for 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-D-glucopyranosyl ester
(XVIII), m/z505.2→423.2 for hydroxyeuscaphic acid (XIX), m/z 193.1→92.2 for butyl
paraben (XX)(IS-1), and m/z 418.9→342.8 for bifendate (XXI)(IS-2). A summary of the
mass parameters for the 19 analytes and 2 ISs are shown in Table 4. The chemical structure
and product ion scan spectra of 19 analytes and 2 ISs are presented in Figure 2.
3.3. Method Validation
3.3.1. Thirteen phenolic acids
3.3.1.1. Selectivity
The selectivity of the method was assessed by comparing the MRM chromatograms of
blank plasma (A), plasma sample spiked with the analytes at LLOQ (B), IS, blank plasma
with the analytes at QCM (C) and IS, and the plasma sample obtained at 1 h after oral
administration of the S. officinalis extract (D) (Figure 3). The retention time were about
1.831, 1.982, 5.892, 4.823, 2.426, 3.780, 3.586, 3.773, 3.968, 3.058, 1.201, 3.776, 2.635 min
for Ⅰ~ XIII, respectively. The retention time for IS-1 was about 6.507 min. All results
demonstrated that the analytes and IS-1 were well separated with no interference in the
chromatogram. An Agilent Eclipse Plus C18 RRHD (2.1×50 mm, 1.8μm, Agilent
Technologies, Santa Clara, CA, USA) column was selected to avoid any crosstalk from the
interfering constituents and provided good peak efficiencies. The whole running time was 6.5
min. The column temperature was set at 30 °C, and the injection volume was 10 μL.

3.3.1.2. Linearity and Sensitivity
The typical calibration curves, correlation coefficients (r), and linear ranges of the 13
phenolic acids are summarized in Table 5. The calibration curves showed good linearity (r
≥0.9904) over the test ranges of 5.70-1458 ng/mL (Ⅰ), 2.45-626.8 ng/mL (Ⅱ), 1.94-496.3
ng/mL (Ⅲ), 6.18-1582 ng/mL (Ⅳ), 4.51-1155 ng/mL (Ⅴ), 2.96-757.5 ng/mL (Ⅵ), 2.33-595.5
ng/mL (Ⅶ), 6.01-1539 ng/mL (Ⅷ), 3.37-862.3 ng/mL (Ⅸ), 3.60-920.4 ng/mL (Ⅹ), 3.30-844.3
ng/mL (Ⅺ), 4.27-1092 ng/mL (Ⅻ), and 4.81-1231 ng/mL (XIII). The LLOQs of the method
were sufficient to be applied to in vivo studies.
3.3.1.3. Accuracy and Precision
The inter- and intra-day accuracy and precision of the method were assessed by
measuring 9 replicates of the QC samples consisting of the mixture of 13 analytes at 4
concentration levels (LLOQ, LQC, MQC and HQC) (Table 6). The relative standard
deviation (RSD) values for the intra- and inter-day were below 14.42%. The accuracies
(RE%) were between -14.23% and 7.48%. All results revealed acceptable accuracy and
precision.
3.3.1.4. Extraction Recovery and Matrix Effect
The extraction recoveries of the 13 analytes were evaluated by comparing the peak areas
of each analyte spiked before and after the extraction procedure, while the matrix effect was
assessed by comparing the peak areas of each analyte spiked after extraction with those in
solutions containing equivalent contents. As shown in Table 7, the mean recovery of the 13

phenolic acids ranged from 81.0% to 96.28%(RSD≤14.71). In the rat plasma, the IS recovery
was 94.57%. The mean matrix effect ranged from 91.02% to 104.23% (RSD≤13.77). The
matrix effect of IS was 96.42%. Stable extraction recovery and negligible endogenous
interference are indicated in the results, implying that this method is suitable for the
determination of the 13 phenolic acids in the rat biological matrix.
3.3.1.5. Stability
The stability of the 13 analytes in rat plasma was investigated by 9 complicates at 4
different conditions: freeze-thaw stability (three freeze cycles at -20 ℃ and thaw cycles),
room temperature stability (4 h storage at ambient temperature), long-term stability (storage
at -20 ℃ for 2 weeks), and post-preparation stability (storage for 12 h after sample
preparation at 4 ℃). As shown in Table 8, the 13 phenolic acids were stable under conditions
detected in this method.
3.3.2. Six Triterpenes
The method for the simultaneous determination of 6 triterpenes was well-validated in
previous study. In this study, method validation of the 6 triterpenes was also conducted. The
selectivity of the method was assessed by comparing the MRM chromatograms of blank
plasma (A), blank plasma sample spiked with LLOQ analytes and IS-2 (B), blank plasma
with QCM analytes and IS-2 (C), and the plasma sample obtained from rats at 1 h after single
oral administration of S. officinalis extract (D) (Figure 4). The results demonstrated no
significant interference from endogenous substances detected during the retention time of

those triterpenes. Other results of the method validation of 6 triterpenes in rat plasma by
UHPLC-MS/MS are presented in the Supplementary Materials. The calibration curves
revealed good linearity (r ≥0.9931) over the test ranges. The LLOQs of the method were
sufficient to be applied to in vivo studies. In the present study, the accuracy and precision
experiments were performed with three concentration levels of QCs (HQC, MQC, LQC) and
LLOQ. The RSD of intra-day and inter-day precision was within 15%, and the accuracy was
within a reasonable range. The mean recoveries of the 6 triterpenes were in the range of 77.23
- 97.01% at the concentrations of LQC, MQC, and HQC samples and the extraction recovery
of the IS-2 was 91.32%. Besides, the stability of the 6 triterpenes in rat plasma was
investigated under a variety of storage and process conditions. The measured concentrations
were all within acceptable limits (±15% of the nominal concentrations) during the entire
validation. All these results met the requirements that defined by regulatory guidelines.
3.4. Pharmacokinetic Studies
A systematic strategy was successfully applied to the pharmacokinetic study of 19
analytes in normal and leukopenia rats after oral administration of S. officinalis extract. The
concentration-time curve profiles of the 19 analytes in rat plasma are shown in Figure 5. The
main pharmacokinetic parameters, including half-time (t1/2), Cmax, Tmax, and under
concentration–time curve (AUC0-t and AUC0-∞) analysis, are illustrated in Table 9.
As shown in Figure 5, compared to the previous studies [18], the pharmacokinetic
parameters of the 6 triterpenes in S. officinalis extract in normal rats differed from those

reported in the literature. These differences could be attributed to the different extraction
methods of S. officinalis used in this study, in which a different dose of S. officinalis was
administered to rats. After single oral administration of S. officinalis extract, Ⅰ, Ⅱ, Ⅵ, Ⅶ, Ⅺ,
Ⅻ, XIII, XIV, XV and XVI were absorbed rapidly and reached Cmax in approximately 1 h, and
then eliminated at 36 h. This could be attributed to the easy dissolution of these analytes in
blood. In the present study, the Cmax of Ⅰ, Ⅳ, Ⅻ were lower than those of other compounds,
which may be attributed to their lower content in the extract. Similarly, the Cmax of Ⅱ, Ⅺ, XIII
and XIV were higher than that of other ingredients, demonstrating that Ⅱ, Ⅺ, XIII and XIV
were the major bioactive compounds in S. officinalis extract. The t1/2 of Ⅱ, Ⅲ, Ⅳ, Ⅴ, Ⅵ, Ⅻ,
XIII, and XVII were much longer than that of other ingredients, indicating they would remain
in body for a longer time to exert therapeutic action, and enhance the clinical efficacy. A
double peak phenomenon was observed for Ⅰ, Ⅴ, Ⅵ, Ⅷ, Ⅸ, Ⅹ, Ⅺ, Ⅻ, XIII and XIV, and this
phenomenon may be caused by fractionated gastric emptying, enterohepatic circulation and
separated “absorption windows”[36, 37]. Except for the reasons mentioned above, it was
suspected that the existence of multiple blood concentration peaks may be attributed to the
fact that these analytes are the structural units or aglycones of some compounds in S.
officinalis extract, and transformations between different compounds may have occurred with
similar parent structures. For example, the t1/2 of Ⅳ is longer than Ⅲ, and after 12 h, Ⅳ
attained another blood concentration, while Ⅲ was nearly eliminated. This indicated that Ⅲ
could be transformed into Ⅳin in vivo metabolites.

The pharmacokinetic profiles of 13 phenolic acids and 6 triterpenes in normal and
leukopenia rats after oral administration of the S. officinalis extract were compared for the
first time. The differences in the pharmacokinetic parameters between the normal and model
groups were significant. Statistical analysis of 19 compounds in the S. officinalis extract after
oral administration was performed using ANOVA. As demonstrated in the results, several
pharmacokinetic parameters, including AUC0-t , AUC0-∞, and Cmax for the 19 compounds in
leukopenia rats, differed from those in normal rats. The plasma concentrations of these 19
compounds in leukopenia rats were lower than those in normal rats at almost all time points.
The Cmax and AUC0-∞ values of Ⅴ, Ⅷ, Ⅸand XIV has statistical significance (p<0.05). Ⅰ, Ⅲ,
Ⅶ, Ⅹ, Ⅺ, XIII, XIV, XV, XVI, XVII, XVIII, and XIX presented a similar tendency, with no
obvious differences in Tmax and t1/2. Our findings indicated that the bioavailability of the 19
major bioactive components in was decreased in CTX-induced leucopenia rats compared
with that in normal rats. It has been reported that the function of many transporters and
enzymes related to intestinal transportation and metabolism of drugs could be affected in
pathological state [38]. Leukopenia is a prominent manifestation of bone marrow
suppression, the body's immune capacity will be weakened, severe infections and bleeding
often occur clinically [39]. It could be speculated that leukopenia is associated with multiple
physiological changes that affect the disposition of therapeutic drugs. Therefore, these
discrepancies could be attributed to the different pathological states which led to the
alterations of the absorption procedure of these compounds. For the 19 compounds, the

mechanisms resulting in decreased absorption after single oral administration of S. officinalis
extract in leukopenia rats need to be further investigated in the future.
4. Discussions
The selection of a suitable sample preparation method is essential to simultaneously and
accurately analyze the target compound. Liquid–liquid extraction (LLE), solid-phase
extraction (SPE), and protein precipitation (PPT) are the most frequently used methods for
sample preparation. PPT is utilized for its convenient operation and high recovery rate, but it
is not suitable for the analytes presenting a low content. We attempted PPT, but the recovery
of compounds was insufficient and nonrenewable. Additionally, SPE is time-consuming and
the SPE columns are expensive. Therefore, LLE was selected as the sample pretreatment
method owing to its stable extraction recoveries and negligible matrix effects. Furthermore,
selecting an appropriate organic solvent as the extract agent is key to achieve high extraction
recovery and weak matrix effect. Extraction solvent such as ethyl acetate, ether,
dichloromethane, n-butanol and acetone were evaluated, and the results indicated that ethyl
acetate could achieve higher extraction recovery and weaker matrix effect under the same
conditions. However, the target performance for phenolic acids is far from ideal. It has been
hypothesized that the phenolic acids in plasma samples were oxidized during treatment. Thus,
different volumes and concentrations of ascorbic acid and hydrochloric acid were added
during the procedures of sample treatment. Additionally, the LLE solvent volume, vortex
time, and N2 blowing temperature were considered. Eventually, the addition of 20 μL of 2 M

hydrochloric acid to rat plasma and reducing the N2 blowing temperature to 30 ℃ could to the
greatest extent improve the analytes treatment result. Furthermore, 3mL ethyl acetate could
meet the requirements of both high extraction recovery and weak matrix effect.
Obtaining a stable and sensitive analytical response for analytes is the key to optimizing
mass spectrometry parameters. The analysis performed on an Agilent series 1290 UHPLC
instrument (Agilent Technologies, Santa Clara, CA, USA) coupled with an Agilent
Technologies 6430 mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) using
an electrospray ionization (ESI) interface. The eluent was monitored using a triple
quadrupole tandem mass spectrometer (Agilent Technologies, Santa Clara, CA, USA)
equipped with ESI source and operated in negative and positive ion mode with MRM
respectively. Several different chromatographic columns were considered to reduce
interference of endogenous substances and get all analytes well-separated. At last, an Agilent
Eclipse Plus C18 RRHD (2.1×50 mm, 1.8μm, Agilent Technologies, Santa Clara, CA, USA)
column was selected. For the sensitive detection of all the 19 analytes, the positive ion mode
was chosen for the 6 triterpenes, and the negative ion mode was adopted for the 13 phenolic
acids to obtain certain signal responses.
5. Conclusions
In this study, a selective, accurate and efficient UHPLC-ESI MS/MS method was
developed for the simultaneous determination of 13 phenolic acids. On this basis, a

systematic strategy was successfully applied for the comparative analysis of pharmacokinetic
characteristics of sanguiin H-4, bergenin monohydrate, 3,3'-dimethoxyellagic acid, ellagic
acid, (+)-catechin monohydrate, methyl 3,4-dihydroxy-5-methoxybenzoate, ethyl
3,4,5-trihydroxybenzoate, syringic acid, ferulic acid, caffeic acid, gallic acid, p-coumaric
acid, 4-hydroxybenzoic acid, ziyuglycoside I, 3β-[(α-L-arabinopyranosyl)
oxy]-urs-12,18(19)-dien-28-oic acid β-D-glucopyranosyl ester, rosamultin, alpinoside,
3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-D-glucopyranosyl ester and hydroxyeuscaphic
acid in normal and CTX induced leukopenia rats after oral administration of the S. officinalis
extract. The obtained pharmacokinetic data may be benefit for a better understanding of the
pharmacological effect of S. officinalis extract, providing a substantial foundation for
investigations regarding clinical applications.
Acknowledgements
This work was supported by the Scientific Research Project of National Natural Science
Foundation of China (No. 81573551, 81973552).
Conflicts of interest
The authors declare that there are no conflicts of interest.

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Fig. 1. The chemical structures of the 19 analytes and the internal standards: sanguiin
H-4 (Ⅰ); bergenin monohydrate (Ⅱ); 3,3'-dimethoxyellagic acid (Ⅲ); ellagic acid (Ⅳ);
(+)-catechin monohydrate (Ⅴ); methyl 3,4-dihydroxy-5-methoxybenzoate (Ⅵ); ethyl
3,4,5-trihydroxybenzoate (Ⅶ); syringic acid (Ⅷ); ferulic acid (Ⅸ); caffeic acid (Ⅹ); gallic acid
(Ⅺ); p-coumaric acid (Ⅻ); 4-hydroxybenzoic acid (XIII); ziyuglycoside I (XIV);
(XV); rosamultin (XVI); alpinoside (XVII); 3β,19α-dihydroxyurs-12-en-28-oic-acid
28-β-D-glucopyranosyl ester (XVIII); hydroxyeuscaphic acid (XIX); butyl paraben
(XX)(IS-1); bifendate (XXI)(IS-2).


Fig. 2. Product ion spectra of the 19 analytes and the internal standards: sanguiin H-4
(Ⅰ); bergenin monohydrate (Ⅱ); 3,3'-dimethoxyellagic acid (Ⅲ); ellagic acid (Ⅳ);
(+)-catechin monohydrate (Ⅴ); methyl 3,4-dihydroxy-5-methoxybenzoate (Ⅵ); ethyl
3,4,5-trihydroxybenzoate (Ⅶ); syringic acid (Ⅷ); ferulic Acid (Ⅸ); caffeic acid (Ⅹ); gallic
acid (Ⅺ); p-coumaric acid (Ⅻ); 4-hydroxybenzoic acid (XIII); ziyuglycoside I (XIV);
(XV); rosamultin (XVI); alpinoside (XVII); 3β,19α-dihydroxyurs-12-en-28-oic-acid
28-β-D-glucopyranosyl ester (XVIII); hydroxyeuscaphic acid (XIX); butyl paraben
(XX)(IS-1); bifendate (XXI)(IS-2).

Fig. 3. The chromatograms of: sanguiin H-4 (Ⅰ); bergenin monohydrate (Ⅱ);
3,3'-dimethoxyellagic acid (Ⅲ); ellagic acid (Ⅳ); (+)-catechin monohydrate (Ⅴ); methyl
3,4-dihydroxy-5-methoxybenzoate (Ⅵ); ethyl 3,4,5-trihydroxybenzoate (Ⅶ); syringic acid
(Ⅷ); ferulic acid (Ⅸ); caffeic acid (Ⅹ); gallic acid (Ⅺ); p-coumaric acid (Ⅻ);
4-hydroxybenzoic acid (XIII) and IS-1(XX) in rat plasma samples. Blank plasma (A); IS-1
and plasma samples spiked with the analytes at LLOQ (B); IS-1 and blank plasma with the

analytes at QCM (C); IS-1 and the plasma samples obtained at 1h after oral administration of
Sanguisorba officinalis L. extract (D). (91.1 ng/mL for sanguiin H-4, 39.2 ng/mL for
bergenin monohydrate, 31.0 ng/mL for 3,3'-dimethoxyellagic acid , 98.9 ng/mL for ellagic
acid, 72.2 ng/mL for (+)-catechin monohydrate, 47.3ng/mL for methyl
3,4-dihydroxy-5-methoxybenzoate, 37.2 ng/mL for ethyl 3,4,5-trihydroxybenzoate, 96.2
ng/mL for syringic acid, 53.9 ng/mL for ferulic acid, 57.5 ng/mL for caffeic acid, 52.8 ng/mL
for gallic acid, 68.3 ng/mL for p-coumaric acid, 77.0 ng/mL for 4-hydroxybenzoic acid and
132.0 ng/mL for butyl paraben (IS-1)).
Fig. 4. The chromatograms of: ziyuglycoside I (XIV); 3β-[(α-L-arabinopyranosyl)
oxy]-urs-12,18(19)-dien-28-oic acid β-D-glucopyranosyl ester (XV); rosamultin (XVI);
alpinoside (XVII); 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-D-glucopyranosyl ester
(XVIII); hydroxyeuscaphic acid (XIX); and bifendate (XXI)(IS-2). in rat plasma samples.

Blank plasma (A); IS-2 and plasma samples spiked with the analytes at LLOQ (B); IS-2 and
blank plasma with the analytes at QCM (C); IS-2 and the plasma samples obtained at 1h after
oral administration of Sanguisorba officinalis L. extract (D). (66.4 ng/mL for ziyuglycoside I,
35.7 ng/mL for 3β-[(α-L-arabinopyranosyl) oxy]-urs-12,18(19)-dien-28-oic acid
β-D-glucopyranosyl ester, 37.6 ng/mL for rosamultin, 85.1 ng/mL for alpinoside, 30.7 ng/mL
for 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-D-glucopyranosyl ester, 57.5 ng/mL for
alpinoside, and 520.0 ng/mL for bifendate (IS-2)).


Fig. 5. Mean plasma concentration-time curve of nineteen analytes in normal and
CTX-induced leukopenia rats after oral administration of S. officinalis Extract. (n =12,
mean ±SD).
Table 1. Concentrations of the 13 phenolic acids for calibration curve. (n=7)
Compound/Conc 1 2 3 4 5 6 7
entration(ng/mL)
Ⅰ 5.7 11.4 22.8 91.1 182.3 729.0 1458.0
Ⅱ 2.4 4.9 9.8 39.2 78.4 313.4 626.8
Ⅲ 1.9 3.9 7.8 31.0 62.0 248.2 496.3
Ⅳ 6.2 12.4 24.7 98.9 197.8 791.0 1582.0
Ⅴ 4.5 9.0 18.1 72.2 144.5 577.8 1155.6
Ⅵ 3.0 5.9 11.8 47.3 94.7 378.8 757.5
Ⅶ 2.3 4.7 9.3 37.2 74.4 297.8 595.5
Ⅷ 6.0 12.0 24.1 96.2 192.5 769.8 1539.6
Ⅸ 3.4 6.7 13.5 53.9 107.8 431.2 862.3
Ⅹ 3.6 7.2 14.4 57.5 115.1 460.2 920.4
Ⅺ 3.3 6.6 13.2 52.8 105.5 422.2 844.3
Ⅻ 4.3 8.5 17.1 68.3 136.5 546.0 1092.0
XIII 4.8 9.6 19.2 77.0 153.9 615.8 1231.5

Table 2. Concentrations of the 6 triterpenes for calibration curve. (n=7)
Compound/Conc 1 2 3 4 5 6 7
entration(ng/mL)
XIV 4.1 8.3 16.6 66.4 132.8 531.1 1062.1
XV 2.2 4.5 8.9 35.7 71.3 285.3 570.5
XVI 2.3 4.7 9.4 37.6 75.1 300.4 600.8
XVII 5.3 10.6 21.3 85.1 170.2 680.8 1361.6
XVIII 1.9 3.8 7.7 30.7 61.3 245.3 490.5
XIX 3.6 7.2 14.4 57.5 115.0 459.9 919.8
Table 3. Effects on peripheral white blood cell count (WBC), spleen weight coefficient
and bone marrow DNA content in rats exposed to repeated CTX injection. *P ≤ 0.05,
**P ≤ 0.01 vs normal group. (n = 6)
WBC Spleen Weight Bone Marrow DNA
Dosage(mg/kg·d) Count(×109/L) Coefficient (mg/g) Content (A)
Normal Group - 10.33±0.91 4.61±0.34 0.63±0.09
Model Group 30 2.24±0.47** 3.36±0.03** 0.44±0.04**

Table 4. Mass spectrometric parameters of 19 compounds and two ISs.
Compounds Precursor Ion(m/z) Product Ion(m/z) Fragment(V) Collision Energy(V) Polarity
Ⅰ 633.1 300.9 90 13 Negative
Ⅱ 345 123.8 175 29 Negative
Ⅲ 328.7 314 127 21 Negative
Ⅳ 301 145 187 40 Negative
Ⅴ 289.2 109.2 141 16 Negative
Ⅵ 197.1 182.2 100 20 Negative
Ⅶ 197 124.1 119 20 Negative
Ⅷ 197 122.8 92 13 Negative
Ⅸ 193.1 134 95 15 Negative
Ⅹ 179 135 95 12 Negative
Ⅺ 169.1 125.1 98 18 Negative
Ⅻ 163 119.2 90 13 Negative
XIII 137.1 92.9 83 16 Negative
XIV 784.5 437.4 150 10 Positive
XV 771.5 609.1 300 48 Positive
XVI 673.4 511.4 330 40 Positive
XVII 655.4 493 290 34 Positive
XVIII 657.4 455.4 130 20 Positive
XIX 505.2 423.2 110 15 Positive
XX(IS-1) 193.1 92.2 96 18 Negative
XXI(IS-2) 418.9 342.8 78 18 Positive

Table 5. The calibration curve, linear ranges and LLOQs for the determination of 13
phenolic acids in rat plasma. (n=7)
Compounds Calibration Curve r Linear Range(ng/mL) LLOQ(ng/mL)
Ⅰ Y=6.496×10-4Х-1.67×10-3 0.9962 5.70-1458 5.70
Ⅱ Y=1.314×10-3Х-4.092×10-4 0.9990 2.45-626.8 2.45
Ⅲ Y=1.073×10-2Х-3.793×10-3 0.9959 1.94-496.3 1.94
Ⅳ Y=1.624×10-4Х+3.581×10-4 0.9932 6.18-1582 6.18
Ⅴ Y=5.520×10-4Х+1.502×10-3 0.9927 4.51-1155 4.51
Ⅵ Y=6.742×10-3Х+2.020×10-3 0.9992 2.96-757.5 2.96
Ⅶ Y=9.501×10-3Х-7.021×10-4 0.9987 2.33-595.5 2.33
Ⅷ Y=3.740×10-4Х+8.097×10-4 0.9904 6.01-1539 6.01
Ⅸ Y=2.018×10-3Х-1.266×10-3 0.9983 3.37-862.3 3.37
Ⅹ Y=9.521×10-3Х-1.499×10-2 0.9995 3.60-920.4 3.60
Ⅺ Y=2.272×10-3Х+1.101×10-3 0.9983 3.30-844.3 3.30
Ⅻ Y=7.857×10-3Х-6.187×10-5 0.9973 4.27-1092 4.27
XIII Y=4.311×10-3Х-4.320×10-6 0.9984 4.81-1231 4.81

Table 6. Intra-day and inter-day precisions and accuracies for the determination of 13
phenolic acids in rat plasma. (n=9)
Accuracy
Nominal Measured Intra-Day Inter-Day
Compound
Concentration(ng/ml) Concentration(ng/ml) Precision (RSD%) Precision (RSD%)
(RE%)
5.7 5.27±0.48 -8.55 6.30 6.98
11.4 11.24±1.25 -4.28 7.77 9.36

Ⅰ
91.1 86.73±6.07 -3.48 4.91 5.28
1166.4 1086.50±158.86 -6.25 10.14 11.67
2.4 2.72±0.46 4.29 10.83 12.75
4.9 4.92±0.79 3.46 11.40 10.80

Ⅱ
39.2 39.73±7.22 -5.15 11.83 14.30
501.4 497.52±32.06 -2.07 7.88 5.35
1.9 2.05±0.24 6.43 13.25 11.15
3.9 3.93±0.84 4.17 11.91 13.26

Ⅲ
31.0 29.76±2.37 -2.31 10.92 7.07
397.0 370.43±26.89 -5.07 5.43 4.75
6.2 6.67±0.33 6.61 8.18 3.59
12.4 12.81±1.70 -1.56 7.55 9.98

Ⅳ
98.9 94.09±5.26 -4.05 4.34 4.85
1265.6 1250.73±115.02 1.01 8.94 7.60
4.5 4.40±0.36 -2.13 7.07 6.06
Ⅴ 9.0 9.34±0.69 3.44 10.79 4.68
72.2 75.80±10.93 3.20 11.01 11.31

924.5 863.57±56.34 -8.31 7.56 4.69
3.0 3.04±0.31 4.28 11.48 8.70
5.9 6.11±0.98 4.27 10.57 12.31

Ⅵ
47.3 42.63±6.11 -3.96 9.38 9.79
606.0 558.10±33.98 -7.11 5.07 4.65
2.3 2.06±0.38 -14.23 12.02 13.55
4.7 4.61±0.37 -2.25 8.90 7.36

Ⅶ
37.2 36.66±4.28 -1.26 9.23 10.29
476.4 438.56±17.42 -9.71 6.22 3.17
6.0 5.76±0.69 -4.39 9.54 11.03
12.0 10.98±0.58 -8.68 3.59 3.97

Ⅷ
96.2 86.00±6.85 -9.32 5.14 5.60
1231.7 1131.95±57.23 -10.01 5.48 3.92
3.4 3.43±0.54 1.66 10.45 11.90
6.7 6.91±0.85 2.03 6.39 8.44

Ⅸ
53.9 51.31±3.66 -3.29 9.50 5.43
689.8 628.51±35.11 -9.63 5.91 4.69
3.6 3.28±0.47 -11.48 10.09 10.52
7.2 6.44±0.31 -10.26 5.78 3.62

Ⅹ
57.5 53.89±2.79 -6.02 7.01 4.60
736.3 718.11±43.60 -4.31 5.60 4.65
3.3 3.16±0.57 -4.8 12.16 14.42

Ⅺ
6.6 6.21±0.77 -6.51 9.08 9.46

52.8 54.16±6.33 4.35 9.01 10.22
675.4 625.96±53.29 -6.93 6.16 6.81
4.3 4.27±0.54 5.53 10.91 9.12
8.5 8.00±0.91 -3.16 9.26 7.95

Ⅻ
68.3 59.50±4.03 -11.68 7.08 4.66
873.6 787.71±53.02 -9.10 5.02 4.82
4.8 4.19±0.68 -10.47 8.82 10.62
9.6 9.09±0.58 -6.36 6.04 4.89

XIII
77.1 80.25±9.73 6.10 11.99 9.74
985.2 1046.78±102.35 7.48 8.85 8.63
Table 7. Matrix effect and extraction recoveries of 13 phenolic acids and IS-1 in rat
plasma. (n=9)
Matrix Effect (%) Extraction Recovery (%)

Spiked
Compound
Concentration(ng/mL)
Mean (%) RSD (%) Mean (%) RSD (%)
11.4 91.86 11.87 85.48 8.14
Ⅰ 91.1 97.81 4.91 92.86 8.31
1166.4 95.44 9.42 90.90 9.81
4.9 93.46 13.33 81.01 3.69
Ⅱ 39.2 101.43 9.19 93.15 10.69
501.4 97.71 8.28 95.60 9.02
3.9 91.27 13.77 82.68 14.71

Ⅲ
31.0 97.89 11.08 93.93 9.49

397.0 91.67 5.95 93.67 5.54
12.4 94.99 9.43 89.01 9.06
Ⅳ 98.9 96.48 4.21 92.58 8.45
1265.6 93.67 9.26 90.48 14.51
9.0 99.52 10.28 90.52 11.18
Ⅴ 72.2 91.81 6.85 96.28 12.43
924.5 103.62 3.90 91.69 4.69
5.9 99.37 8.75 87.38 12.06
Ⅵ 47.3 101.24 9.05 92.52 12.61
606.0 91.18 5.76 91.51 4.32
4.7 93.95 10.62 87.01 10.96
Ⅶ 37.2 101.97 8.74 93.37 16.14
476.4 95.72 7.22 90.29 3.17
12.0 95.30 1.52 88.55 5.88
Ⅷ 96.2 95.16 3.71 90.33 5.38
1231.7 91.38 6.99 89.99 3.92
6.7 98.55 3.02 87.18 9.30
Ⅸ 53.9 104.23 11.30 93.61 11.65
689.8 94.49 6.48 90.37 4.69
7.2 95.48 6.01 89.74 3.62
Ⅹ 57.5 91.02 9.10 93.98 4.60
736.3 97.90 6.61 93.43 3.11
Ⅺ 6.6 99.63 8.28 90.97 7.10

52.8 101.17 8.16 94.87 5.89
675.4 93.45 6.09 93.07 6.81
8.5 95.24 11.17 90.98 8.21
Ⅻ 68.3 97.55 5.35 93.20 9.45
873.6 94.90 4.56 91.28 5.37
9.6 97.44 6.77 86.71 7.61
XIII 77.0 97.36 11.88 90.94 4.60
985.2 101.63 9.35 88.87 6.79
XX(IS-1) 130.2 96.42 3.22 94.57 7.98
Table 8. Stability of 13 phenolic acids in rat plasma under various conditions.
(n=9)
Stability(%RE)
Spiked
Compound
Concentration(ng/mL) Three Post-Preparati
Short-Term Long-Term
Freeze-Thaw on
11.4 4.12 3.83 -3.08 5.03
Ⅰ 91.1 -6.44 -3.34 -2.34 -2.19
1166.4 -1.01 -5.24 -5.56 -4.56
4.9 8.84 5.32 1.81 3.67
Ⅱ 39.2 -0.11 2.16 -5.88 1.43
501.4 2.51 5.54 0.07 7.68
Ⅲ 3.9 9.01 6.68 1.65 4.17

31.0 5.44 2.86 8.84 14.01
397 -3.43 -7.56 -5.84 -8.33
12.4 2.46 3.23 -0.36 4.43
Ⅳ 98.9 -4.41 -3.34 -4.23 -3.52
1265.6 -4.72 -0.33 -5.01 -6.33
9.0 11.04 3.55 8.64 8.75
Ⅴ 72.2 0.91 3.43 -8.42 -8.19
924.5 -2.52 0.84 -5.55 3.60
5.9 8.67 5.79 -2.14 -0.63
Ⅵ 47.3 0.99 0.49 -3.21 1.24
606.0 -10.52 -6.99 -8.94 -8.82
4.7 -3.80 -3.53 -4.77 -6.05
Ⅶ 37.2 -3.53 0.65 0.07 1.97
476.4 -5.48 -10.12 -3.87 -4.28
12.0 -6.56 -7.34 -6.05 -4.70
Ⅷ 96.2 -5.28 -6.75 -7.41 -4.84
1231.7 -11.35 -9.48 -9.15 -8.62
6.7 1.22 -0.98 1.55 -1.45
Ⅸ 53.9 -0.79 1.22 -0.28 4.23
689.8 -8.18 -10.52 -4.62 -5.51
7.2 -7.42 -5.52 -9.26 -4.52
Ⅹ 57.5 -5.50 -8.21 -6.78 -8.98
736.3 -5.03 -4.73 -1.68 -2.10

6.6 -1.07 -8.54 1.66 -0.37
Ⅺ 52.8 0.67 2.06 3.46 1.17
675.4 -7.16 -9.94 -3.54 -6.55
8.5 -1.41 -4.31 -3.61 -4.76
Ⅻ 68.3 -6.38 -9.12 -5.02 -2.45
873.6 -5.55 -5.89 -8.30 -5.10
9.6 -6.91 -2.89 -6.03 -2.56
XIII 77.0 4.01 4.23 5.72 3.85
985.2 9.33 5.99 9.89 6.71
Table 9. Pharmacokinetic parameters of 19 analytes in normal and CTX-induced
leukopenia rats after oral administration of S. officinalis extract. (n = 12, mean ±SD)
Compounds Group Cmax(ng/mL) Tmax(h) T1/2(h) /AUC0→t(ng·h/mL) AUC0→∞(ng·h/mL)
Normal 94.83±12.79 1.13±0.92 5.03±3.56 443.45±39.761 470.48±91.11

Ⅰ
Model 69.69±8.77** 1.08±0.94 25.73±33.85 387.56±76.65 644.86±438.67
Normal 527.78±77.33 0.25±0 16.93±9.82 1550.77±569.39 1897.90±869.81

Ⅱ
Model 391.22±68.31** 0.79±0.10** 3.20±1.60** 1191.71±200.38 1197.72±200.27
Normal 310.78±44.34 3.01±2.45 10.94±7.57 3775.38±1582.53 4231.49±1877.78

Ⅲ
Model 277.92±37.18 3.21±0.23 13.84±4.72 1590.72±135.46** 1782.88±149.55**
Normal 113.96±20.17 3.83±0.41 14.23±3.05 1832.47±179.61 2239.36±261.79

Ⅳ
Model 111.45±12.41 2.83±0.41** 13.74±8.30 1265.90±190.60** 1546.06±554.62*
Ⅴ Normal 374.40±92.16 1.38±2.27 11.64±2.95 5363.24±1340.01 6166.10±1814.77

Model 286.48±54.37 3.38±1.53 7.49±1.43* 2969.58±342.78** 3074.93±313.28**
Normal 417.68±28.14 0.50±0.21 12.39±2.62 2325.84±574.14 2659.07±720.05

Ⅵ
Model 296.71±83.63** 0.50±0.11 5.24±0.23** 1697.78±70.01* 1735.13±53.23*
Normal 369.77±70.94 0.75±0.32 10.64±8.03 1923.89±902.47 2244.96±1181.74

Ⅶ
Model 312.48±52.34 1.01±0.32 10.28±4.90 1499.18±247.49 1628.24±296.95
Normal 437.03±74.04 0.96±0.10 9.40±3.26 2521.73±224.21 2719.07±310.11

Ⅷ
Model 354.17±19.88 2.32±0.11** 9.02±1.33 2100.70±290.91* 2221.19±317.82*
Normal 417.33±73.01 6.79±2.96 10.25±5.57 3889.66±1007.48 4417.36±1623.91

Ⅸ
Model 285.99±55.22** 0.71±0.10** 6.84±1.93 2233.53±313.54** 2335.57±318.23*
Normal 320.95±46.32 8.12±0.31 9.84±6.65 3166.99±576.89 3565.10±995.14

Ⅹ
Model 253.59±16.20** 8.22±0.12 8.83±1.97 2463.82±201.67* 2654.61±316.33
Normal 544.61±33.19 0.75±0.01 7.46±3.87 3728.68±911.79 3939.81±958.15

Ⅺ
Model 334.24±38.04** 1.42±2.25 8.33±4.43 2331.15±472.38** 2467.97±527.18**
Normal 155.77±19.09 0.54±0.10 20.65±24.43 1335.17±299.39 1784.97±614.16

Ⅻ
Model 118.86±16.23** 0.50±0.11 14.53±4.66 1454.65±94.01 1718.83±197.15
Normal 775.66±119.17 0.25±0.12 18.49±6.51 7613.11±1403.10 10024.16±2865.07

XIII
Model 658.42±126.02 0.25±0.32 13.89±2.61 8262.14±896.98 9767.07±1146.56
Normal 710.50±95.32 0.75±0.44 5.23±3.19 2435.30±180.79 2563.38±292.99

XIV
Model 521.69±84.74** 0.75±0.32 3.31±1.65 1910.97±333.70** 1950.15±382.95*
Normal 379.8±60.15 0.92±0.13 9.87±4.69 1876.46±513.17 2439.43±880.12

XV
Model 360.518±37.79 1.01±0.22 6.34±2.58 1552.00±284.99 1708.22±394.81
Normal 386.78±51.14 1.12±0.44 8.25±7.97 1262.61±250.41 1408.88±462.89

XVI
Model 401.87±36.74 1.11±0.32 6.42±8.55 1455.39±116.11 1577.60±351.80

Normal 463.06±57.99 0.79±0.10 20.33±24.54 2405.33±340.63 4330.11±3132.10

XVII
Model 375.839±52.06* 0.79±0.10 6.34±2.68 1608.41±640.58* 1743.23±683.64
Normal 290.01±50.24 0.75±0.44 4.42±3.06 1216.52±229.12 1280.86±240.03

XVIII
Model 265.945±63.04 0.75±0.32 4.63±2.80 1145.16±115.59 1205.87±165.16
Normal 509.72±57.14 0.75±0.12 7.44±9.32 1801.83±305.31 2073.94±763.38

XIX
Model 442.475±96.36 0.75±0.26 3.79±1.07 1367.07±401.40 1387.83±420.93
*p≤0.05 and **p≤0.01, according to a t-test, versus normal rats.

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com Page 1 Journal of Separation Science

Chunjuan Yang1*, Meicun Yao3*.

Harbin Medical University

Sciences (Shenzhen), Sun Yat-Sen University

Received: 05 06, 2020; Revised: 08 19, 2020; Accepted: 09 07, 2020

This article is protected by copyright. All rights reserved.

chunjuanyang@hrbmu.edu.cn (C.J. Yang)

Guangzhou 510006, Guangdong, China. E-mail address: yaomeicun@gmail.com (M.C. Yao)

Abbreviations: UHPLC-MS/MS, ultra-high-performance liquid

chromatography-electrospray ionization-tandem mass spectrometry; S. officinalis,

Cmax, maximum plasma concentration; CTX, cyclophosphamide; Ke, elimination rate

elimination half-life; Tmax, time for maximal concentration;

Keywords: leucopenia; phenolic acid; pharmacokinetics; Sanguisorba officinalis L.;

ultra-high-performance liquid chromatography

This article is protected by copyright. All rights reserved.

simultaneous determination of 13 phenolic acids. Additionally, for more comprehensively

determining the chemical constituents in Sanguisorba officinalis L. extract, a previously

electrospray ion source in negative and positive electrospray ionization, respectively.

pharmacokinetic characteristics of the 19 analytes in leukopenia rats differed significantly

application of Sanguisorba officinalis L. in the prevention and treatment of leucopenia.

This article is protected by copyright. All rights reserved.

effects and host immunosuppression, regardless of treatment efficacy [2-4]. Leucopenia is

been generally used to treat leukopenia in clinical practice [10-14].