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Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Metabolites of Dietary Acteoside: Profiles, Isolation, Identification, and Hepatoprotective Capacities Qingling Cui,† Yingni Pan,*,†,‡ Wei Zhang,† Yanan Zhang,† Shumeng Ren,† Dongmei Wang,§ Zhenzhong Wang,‡,∥ Xiaoqiu Liu,*,† and Wei Xiao*,‡,∥ †

School of Traditional Chinese Medicine, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China Jiangsu Kanion Pharmaceutical Company Ltd., Lianyungang 222001, China § School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China ∥ State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, Lianyungang 222001, China ‡

S Supporting Information *

ABSTRACT: In recent years, cistanche tea has been increasingly used as a major herbal supplement in functional drinks, and it has attracted a growing number of consumers because of its excellent tonic effects and medicinal properties. Acteoside (ACT), which is the principal bioactive component of Chinese cistanche tea, possesses various pharmacological effects. This study profiled, isolated, identified, and investigated the hepatoprotective capacities of metabolites in rat urine after the administration of ACT. Eleven metabolites, including one new compound (M8), were obtained and identified by nuclear magnetic resonance (NMR) spectroscopy for the first time. Compared with native ACT, ACT metabolites such as hydroxytyrosol (HT), 3hydroxyphenylpropionic acid (3-HPP), and caffeic acid (CA) exhibited higher hepatoprotective activities by regulating oxidative stress, lipid peroxidation, and inflammatory responses in a GalN/LPS-induced-acute-hepatic-injury mouse model. The HT treatment markedly reduced the levels of TNF-α to 280 ± 14.3 ng/L compared with the model group (429 ± 9.20 ng/L, p < 0.01). The results obtained indicated that cistanche tea could be developed as a functional drink for the prevention of hepatic injuries and that ACT metabolites could be responsible for the potent hepatoprotective activity as well as the other therapeutic effects. KEYWORDS: cistanche tea, acteoside, urine metabolites, isolation, hepatoprotective



INTRODUCTION Herba cistanche is found in North Africa, Arabia, and certain Asian countries. In China, it is used as a traditional medicine for treating kidney deficiencies, amnesia, and constipation, and it has been already applied in energetic drinks, such as cistanche tea, cistanche coffee, and cistanche wine, to improve immunity and for its antiaging and antifatigue effects.1−3 Moreover, a dietary supplement composed of herba cistanche and gingko, developed by Nutrilite to improve memory and protect the brain, has been attracting more and more consumers all over the world.4 The main active components of dietary supplements and functional drinks containing herba cistanche are phenylethanoid glycosides, especially acteoside (ACT, Figure 1), all of which contain three chemical groups: caffeic acid (CA), glycosyl, and hydroxytyrosol (HT). There have also been numerous studies on the biological activity of ACT, which have

shown that in functional drinks, ACT has can potentially protect against diabetes,5 liver injuries,6 and obesity.7 There have also been a number of publications describing the pharmacokinetics of ACT,8,9 and it has been shown that therapeutic effects are still present even when the oral bioavailability of the prototype is only 0.12%. Therefore, it is considered a prodrug with metabolites that may be active in vivo.10,11 In previous studies involving the metabolism of ACT, it was shown that ACT was metabolized in the gastrointestinal tract and that hydrolysis was one of the most important metabolic pathways. ACT was biotransformed to HT, 3hydroxyphenylpropionic acid (3-HPP), and CA by intestinal microflora prior to being absorbed into the blood; then, it was converted into conjugated derivatives of HT and 3-HPP.12,13 Therefore, it is speculated that the metabolites of ACT might have been responsible for the pharmacological activities when the oral bioavailability of the prototype was extremely low. Although a number of phenomena related to ACT absorption, bioavailability, and metabolism have already been described, the activities of ACT’s metabolites after its ingestion have not yet been fully characterized and understood. Received: Revised: Accepted: Published:

Figure 1. Structure of acteoside (ACT). © XXXX American Chemical Society

A

October 8, 2017 February 22, 2018 February 24, 2018 February 24, 2018 DOI: 10.1021/acs.jafc.7b04650 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

were kept in an environmentally controlled room (25−28 °C, 50−60% humidity, 12 h dark−light cycles) for 4 days. A total of 48 rats were fed standard laboratory food and water ad libitum and fasted overnight before the experiments were started. Urine and Plasma Collection. Urine was collected after administration of ACT at 200 mg per kilogram of body weight (BW). ACT was administrated at 200 mg/kg BW (about 2 times the bioequivalent dose in humans) in order to enrich enough metabolites as soon as possible. During the process of urine collection, the 48 rats were kept in metabolic cages to collect their urine 0−48 h after the intragastric administration of ACT. The rats fasted but had free access to water. In order to promote the rats’ consumption of water and thus allow the collection of as many metabolites as possible, 0.5 g of glucose was added per 100 mL of water. Conical bottles were used to pick up the urine. In total, 9 L of urine was collected before and after dosing, and the pH of the urine was adjusted to 3−5 with 0.5% phosphoric acid. Some of the urine was used for UPLC-Q-TOF-MS analysis, and the remaining urine was stored at −40 °C until the separation of the metabolites. Blood samples were collected at 0−36 h from the infraorbital vein after the administration of ACT; then, the plasma was obtained by centrifugation for 10 min at 2746g. The urine samples (200 μL) were purified by solid-phase extraction (Bond Elut C18, 500 mg, 3 mL; Agilent, Santa Clara, CA), in which they were eluted with 2 mL of distilled water followed by 3 mL of methanol. The plasma samples (350 μL) underwent protein precipitation (3 volumes of acetonitrile), followed by centrifugation at 9000g for 10 min. Then, samples of 2 μL of urine and 5 μL of plasma were subjected to UPLCQ-TOF-MS for analysis. Chromatographic separation was achieved on an Agilent ZORBAX Extent-C18 column (50 × 2.1 mm, 1.8 μm). UPLC/ESI-Q-TOF-MS analyses were recorded on a 6538 quadrupole time-of-flight (Q-TOF) mass spectrometer (Agilent). The chromatographic and mass spectrometric conditions were as follows:10 A mobile phase composed of A (a mixture of 0.1% formic acid in water) and B (acetonitrile) was pumped at a flow rate of 0.4 mL/min. After a linear gradient of 2−5% B for 0−5 min, 5−25% B for 5−20 min, 90% B for 20−25 min, 90−2% B for 25−27 min, the column was reconditioned with 2% B for 27−30 min. The MS analyses were in negative-ion mode. The capillary voltage was 3.5 kV, and fragmentor voltage was 135 V. The flow of N2 was 10.0 L/min, and the temperature of the drying gas was 350 °C. The raw data were acquired from 50 to 1500 Da and processed using the Agilent Mass Hunter Workstation Version B.03.01 software. Isolation and Purification of Urine Metabolites. The frozen urine was thawed and concentrated on a rotary evaporator (Rotavapor RE-52AA, Yarong, Shanghai, China) at 55 °C from 9 L to around 500 mL, and then extracted three times with ethyl acetate. The resulting ethyl acetate mixture was evaporated under a vacuum at 55 °C to obtain a residue. The remaining solution was then passed through a macroporous-resin D101 column. The column was eluted consecutively with water and 10, 30, 50, and 70% (v/v) ethanol, using a total volume 5000 mL. The 70% ethanol elution was concentrated in a vacuum to obtain a 70% ethanol residue. Then, the 70% ethanol and ethyl acetate residues were dissolved in 60% (v/v) methanol, loaded on a Sephadex LH-20 column, and eluted with 40% (v/v) methanol to give four fractions (fractions 4.1−4.4) and six fractions (fractions 1− 6), respectively. Purification of the ACT metabolites was carried out on a Waters 1525 HPLC-UV semipreparative column (C18, 250 × 10 mm, 5 μm, YMC Prep, Kyoto, Japan). The HPLC-UV system was composed of two pumps and a 2489 UV detector set at 280 nm. All of the chromatographic determinations were carried out at 35 °C with a flow rate of 1.0 mL/min and an injection volume of 200 μL in a system composed of water/formic acid (99:1, v/v) and methanol. Fraction 3 was eluted with 35% methanol to give compounds 1 and 2 (10 and 7 mg). Fraction 4 was eluted with 55% methanol to give compounds 3 and 4 (8 and 30 mg). Fractions 5 and 6 were eluted with 51% methanol to give compounds 5, 6, and 7 (1.5, 1.2, and 2 mg). Fraction 4.2 underwent chromatography on a semipreparative column with 55% methanol to obtain compounds 8, 9, 10, and 11 (3, 4.0, 3, and 2

In recent years, studies have shown that a liver injury is a complex process involving multiple factors, and oxygen-freeradical responses, lipid peroxidation, and inflammatory responses14,15 play important roles in liver injuries and liver disease. At present, the prevention and treatment of liver injuries remains a serious problem. It would be of great practical significance to establish a liver-injury animal model similar to that of human liver disease to screen new drugs with potential hepatoprotective activities. Experimental liver-injury models involving D-galactosamine combined with lipopolysaccharide (GalN/LPS) have been widely used because of their close relationship to human viral hepatitis in terms of their morphologies and functional characteristics. Although there have also been some studies of the hepatoprotective activity of ACT, the mechanisms and hepatoprotective activities of the metabolites of ACT have not been completely investigated. In our study reported here, GalN and LPS were employed to establish an acute-liver-injury model in mice in order to evaluate the hepatoprotective effects of ACT and its metabolites. In general, for the sake of understanding the mechanisms of the biological activities of dietary ACT and the physiological functions of ACT’s metabolites, the determination of ACT’s metabolites and their subsequent isolation are essential. Therefore, in the present research, the in vivo metabolic profiles of ACT in rat urine and plasma were first evaluated using UPLC-Q-TOF-MS/MS; then, urine was collected from 48 rats after they were given ACT, and the ACT metabolites were isolated, purified, and identified by NMR. Finally, the hepatoprotective activities of ACT and its metabolites were studied. The main metabolites of ACT in rat urine were isolated and identified in an attempt to reveal the hepatoprotective activities that correlate with the identified metabolites.



MATERIALS AND METHODS

Chemicals. Dried plant materials of Cistanche tubulosa (Schenk) Wight were purchased from Hetian Dichen Pharmaceutical Biotechnology Company, Ltd. (Hetian, China) and were authenticated by Associate Professor Yingni Pan of Shenyang Pharmaceutical University. The plant materials were extracted by 70% ethanol on the basis of previous studies that indicated that such extracts were rich in ACT. ACT was isolated from the extraction of Cistanche tubulosa by a silica-gel column, polyamide-column chromatography, and preHPLC in our laboratory. Its purity was above 98% as shown by UPLCUV. The structure of ACT was confirmed by 13C NMR. Caffeic acid (CA) was obtained from Chengdu Pufeide Biological Technology Company, Ltd. (Chengdu, China). LC-MS-grade acetonitrile and formic acid were obtained from Fisher Chemicals (Fair Lawn, NJ). Macroporous resin D101 was obtained from Baoen Adsorption Material Technology Company, Ltd. (Cangzhou, China). A Sephadex LH-20 was obtained from GE Healthcare (Chalfont, U.K.). GalN and LPS were obtained from Sigma Chemical Company (St. Louis, MO), and silybin was obtained from Tasly Pharmaceutical Company, Ltd. (Tianjin, China). The diagnostic kits used to measure alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), high mobility group box 1 (HMGB1), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) were obtained from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Animals and Experimental Design. Male Sprague-Dawley rats (220−250 g) were obtained from the Animal Experiment Center in Shenyang, China [license number of the experimental animals: SCXK (Liao 2015-001)]. All of the animal experimental procedures were performed according to the protocols issued by the Institutional Guidelines for Animal Use in Research, Beijing, China. The animals B

DOI: 10.1021/acs.jafc.7b04650 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 2. Structures of metabolites M1−M11 isolated from the rat urine. mg). The UPLC chromatogram of fraction 4.2 is shown in Figure S2 in the Supporting Information. Identification of Urine Metabolites. We used nuclear magnetic resonance (NMR) spectroscopy, including 1H-NMR, 13C-NMR, HMQC, and HMBC spectroscopy, to identify the active metabolites in the rat urine. NMR spectroscopy was carried out on a Bruker 600 AVIII HD instrument with tetramethylsilane (TMS) as the internal standard. The pure metabolites were dissolved in CD3OD, and the data were processed using the Bruker Topspin 2.1 software. Hepatoprotective Capacities. Male KM mice (22−25 g) were obtained from the Laboratory Animal Center of Shenyang Pharmaceutical University (Shenyang, China). They were randomly divided into seven groups: (1) the control group (n = 8), (2) the model group (n = 8), (3) the ACT group (n = 8), (4) the HT group (n = 8), (5) the CA group (n = 8), (6) the 3-HPP group (n = 8), and (7) the positive-control silybin group (n = 8). Rats in the first group were given normal saline (NS) only. Groups 2−7 received intraperitoneal injections of GalN (700 mg/kg) and LPS (10 μg/kg) to induce acute liver injuries. One and a half hours before the GalN/ LPS treatment, the mice were orally given normal saline, ACT, HT, CA, 3-HPP, or silybin at a dose of 0.15 mmol/kg BW. The prototype drug was administrated in a dose bioequivalent to what a human would receive, and the metabolites had concentrations equimolar to that of the prototype. Blood and liver samples were collected 7 h after the intraperitoneal injection of GalN/LPS; then, plasma was obtained by centrifugation (2746g, 10 min) and stored at −80 °C for further study. The liver tissue was stored at −80 °C for the histological assays. Determination of Serum ALT and AST. Serum ALT and AST levels were measured according to standard spectrophotometric procedures using AST and ALT commercial kits (Nanjing Jiancheng Bioengineering Institute). In duplicate, each serum sample was added to the matrix liquid, dinitrophenylhydrazine and sodium hydroxide

were added, and the samples were then measured on a microplate reader at 510 nm. Determination of Serum HMGB1, TNF-α, and IL-6. Serum HMGB1, TNF-α, and IL-6 levels were measured at 450 nm with a microplate reader according to standard spectrophotometric procedures using HMGB1, TNF-α, and IL-6 commercial kits (Nanjing Jiancheng Bioengineering Institute). Each sample was measured twice. Determination of Hepatic SOD, GSH, MDA, and MPO. Liver tissues were homogenized in 9-fold volumes of frozen normal saline using an automatic homogenizing device (DY89-II, Ningbo Scientz Biotechnology Company, Ltd., Zhejiang, China). Then, each homogenate was centrifuged for 10 min at 2746g, and in duplicate, the SOD, GSH, MDA, and MPO levels in the supernatant were determined by standard spectrophotometric procedures and commercial kits (Nanjing Jiancheng Bioengineering Institute). Histological Analysis. Samples were embedded in paraffin, sliced into 5 μm sections, and stained. The hematoxylin and eosin (H&E)stained sections were examined under an optical microscope (Olympus Optical, Tokyo, Japan). All of the liver samples were evaluated by a single pathologist in nonconsecutive, randomly chosen fields at 400× magnification. Statistical Analysis. The data were exhibited as the means ± the standard deviations (SD) and were analyzed by Student’s t-tests. The overall significance of the data was analyzed by one-way analysis of the variance, and results were considered statistically significant when p < 0.05.



RESULTS AND DISCUSSION Analysis of Metabolites of ACT in Urine and Plasma Based on UPLC-Q-TOF-MS. Compared with the blank samples, a total of 28 potentially active metabolites of ACT, including HT derivatives, CA derivatives, the parent derivatives, and structural isomers, were detected in urine and plasma. The C

DOI: 10.1021/acs.jafc.7b04650 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. NMR Assignments of Metabolites M8, M9, M10, and M11 (CD3OD)a M8 H-NMR δH (J in Hz)

1

position

13

C-NMR δC

M9 1

HMBC (H to C)

M10

H-NMR δH (J in Hz)

13

C-NMR δC

H-NMR δH (J in Hz)

1

M11 13

C-NMR δC

H-NMR δH (J in Hz)

1

13

C-NMR δC

Aglycone 1 2 3 4 5 6 7 8 −OCH3

6.69 (d, 2.0)

6.70 (d, 8.4) 6.62 (dd, 8.4, 2.0) 2.80 (t) 3.73, 3.94 (m) 3.75 (s)

132.7 117.0 147.3 147.5 112.8 121.1

6.69 (d, 8.0) 6.64 (dd, 8.0, 2.0) 2.84 (t) 3.74, 3.94 (m) 3.78 (s) Glc 4.34 (d, 8.4) 3.33 (m) 3.52 (m) 4.01 (m) 3.57 (m) 4.51 (dd), 4.35 (dd) Rha 5.18 (d, 1.2)

104.4 75.7 84.0 70.0 75.4 64.7

4.34 (d, 7.8) 3.32 (m) 3.52 (m) 4.01 (m) 3.57 (m) 4.51 (dd), 4.37 (dd)

104.5 75.7 84.0 70.0 75.4 64.7

4.34 (d, 7.8) 3.32 (m) 3.52 (m) 4.01 (m) 3.57 (m) 4.50 (dd), 4.37 (dd)

104.4 75.7 84.0 70.0 75.4 64.7

102.7

5.17 (d, 1.2)

102.7

5.17 (d, 1.8)

102.7

3.97 (m) 3.69 (m)

72.4 72.4

3.97 (m) 3.69 (m)

72.4 72.5

3.98 (m) 3.69 (m)

72.4 72.5

74.0 70.5 17.9

3.41 (m) 3.41 (m) 1.25 (d, 6.0)

74.0 70.5 17.9

3.41 (m) 3.41 (m) 1.24 (d, 6.0)

74.0 70.5 17.9

7.05 (d, 1.8)

128.8 114.6

7.15 (d, 2.0)

127.6 111.5

7.16 (d, 1.8)

127.6 111.5

6.91 (d, 8.4)

148.0 151.6 112.5

6.79 (d, 8.0)

150.7 149.4 116.5

6.79 (d, 7.8)

150.7 149.4 116.5

C-2, -6, 117.0, 121.1 C-1, 132.7 C-4, 147.5

4.33 (d, 7.8) 3.32 (m) 3.52 (m) 4.01 (m) 3.57 (m) 4.51 (dd), 4.37 (dd)

104.4 75.7 84.0 70.0 75.4 64.7

C-8, 72.3 C-4′, 70.5 C-1′, -5′, 104.4, 75.4 C-2′, -6′, 75.7, 64.7 C-3′, 84.0 C-4′, 70.5

1′′

5.17 (d, 1.2)

102.7

2″ 3′′

3.96 (m) 3.69 (m)

72.3 72.4

4′′ 5′′ 6′′

3.41 (m) 3.41 (m) 1.27 (d, 6.0)

74.0 70.5 17.9

C-3′′, -3′′, 72.4, 84.0 C-4′′, 74.0 C-1′′, -5′′, 102.7, 70.0 C-2′′, 72.3 C-3′′, 72.4 C-4′′, 74.0

1′′′ 2′′′

7.06 (d, 1.8)

128.8 114.7

6.92 (d, 7.8)

148.0 151.6 112.5

6′′′ 7′′′ 8′′′ 9′′′ −OCH3 a1

6.98 (dd, 7.8, 1.8) 7.58 (d, 15.9) 6.34 (d, 15.9) 3.89 (s)

123.0 146.8 115.9 168.9 56.4

131.4 117.0 147.4 147.2 112.5 121.1

C-1, -3, 132.7, 147.3 C-2, -4, 117.0, 147.5

36.7 72.3 56.4

C-4′′′, -6′′′, 151.6, 123.0

36.9 72.3 56.3

3.41 (m) 3.41 (m) 1.25 (d, 6.0) Caffeoyl

C-1′′′, -3′′′, 148.0, 128.8 C-2′′′, -4′′′, 151.6, 114.7 C-5′′′, -9′′′, 123.0, 168.9 C-1′′′, 128.8

6.94 (dd, 8.4, 1.8) 7.57 (d, 15.9)

123.0

6.32 (d, 15.9)

C-4′′′, 151.6

3.89 (s)

115.8 168.9 56.3

146.8

6.79 (d, 2.0)

131.4 113.6 148.8 145.9 116.1 122.4

6.82 (d, 2.0)

1′ 2′ 3′ 4′ 5′ 6′

3′′′ 4′′′ 5′′′

131.4 113.6 148.8 145.9 116.1 122.4

C-4, -6, 147.5, 121.1

6.67 (d, 8.0) 6.64 (dd, 8.0, 2.0) 2.84 (t) 3.74, 3.94 (m) 3.78 (s)

36.9 72.3 56.3

7.00 (dd, 8.0, 2.0) 7.62 (d, 15.9)

124.3

6.38 (d, 15.9)

115.2 169.0 56.4

3.86 (s)

147.1

6.69 (d, 2.0)

6.70 (d, 8.0) 6.65 (dd, 8.0, 2.0) 2.80 (t) 3.74, 3.94 (m) 3.75 (s)

36.9 72.2 56.3

7.01 (dd, 7.8, 1.8) 7.62 (d, 15.9)

124.3

6.39 (d, 15.9)

115.2 168.9 56.4

147.0

3.86 (s)

13

H NMR: 600 MHz, C NMR: 150 MHz.

Table 2. Effects of ACT and Its Metabolites on the Serum Levels of ALT and AST in the GalN/LPS-Induced-AcuteHepatic-Injury Mouse Model (n = 8) group control model ACT HT CA 3-HPP silybin

Figure 3. Key correlations observed in the HMBC spectrum of the new compound, M8.

dosage

0.15 0.15 0.15 0.15 0.15

  mmol/kg mmol/kg mmol/kg mmol/kg mmol/kg

ALT (IU/L) 9.55 153 126 111 123 133 120

± ± ± ± ± ± ±

0.492 0.920*** 0.851## 0.780## 0.573## 2.05## 0.851##

AST (IU/L) 16.4 141 120 113 118 125 117

± ± ± ± ± ± ±

0.420 0.713*** 1.85## 1.84## 0.701## 1.20## 0.724##

***p < 0.001 versus the control group, ##p < 0.01 versus the model group.

metabolites were tentatively identified on the basis of their accurate deprotonated-molecular-ion ([M − H]−)-massspectral-fragmentation patterns and retention times. Information on the masses of the ACT metabolites, their relative

a

D

DOI: 10.1021/acs.jafc.7b04650 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 4. Protective effects of ACT and its metabolites on serum levels of HMGB1 (A), TNF-α (B), and IL-6 (C) in the GalN/LPS-induced-acutehepatic-injury mouse model. The results are expressed as the mean values ± SD for 8 mice in each group. ***p < 0.001 versus the control group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus the model group.

than homovanillic acid (m/z 181.0508). Thus, it was authenticated as a sulfated derivative of homovanillic acid. M6a exhibited [M − H]− at m/z 181.0506. The typical fragment ion at m/z 137.0614 was observed and resulted in m/ z 181.0506 by the loss of CO2. It was suggested that it was homovanillic acid. M8a had [M − H]− at m/z 165.0557 and showed typical fragment ions at m/z 121.0662 from 165.0557 from the loss of CO2. Thus, it was considered 3-hydroxyphenylpropanoic acid (3-HPP). M7a showed [M − H]− at m/z 245.0125 and was assigned as the sulfated derivative of M8a because of its increase of 80 Da (SO3) compared with M8a. Both M7a and M8a had the same typical fragment ions, m/z 165.0565 and 121.0666. Therefore, M7a was the sulfated conjugate of M8a. M9a had [M − H]− at m/z 163.0401 and was assigned as dehydroxy caffeic acid because of the decrease of 16 Da (−OH) compared with CA. M10a and b had [M − H]− at m/z 813.2458 and were 176 and 14 Da (−CH2) larger than ACT, indicating that they were methyl glucuronide products of ACT. M11a had the same [M − H]−, m/z 623.1990, and product ions as ACT. M11a was considered isoacteoside, the isomer of ACT. M12a−h had [M − H]− at m/z 827.2615. They were identified as the dimethyl and glucuronide conjugations of acteoside because of their increases of 176 and 28 Da (−2CH2) compared with ACT.

contents, and their in vivo extracted ion chromatograms were given in the Supporting Information (Table S1 and Figure S1). ACT had characteristic ions at m/z 623.1981, 461.1675, and 161.0254 ([M − H]−, [M − CA]−, and [CA − H2O]−, respectively) in the MS and MS2 spectra. However, the prototype drug, ACT, was not found in the urine or plasma because of intestinal metabolism. M1a and b had [M − H]− at m/z 329.0878 in the mass spectra, which were 176 Da more than HT (m/z 233.0125), and were identified as glucuronide-conjugate products of HT. The typical ions m/z 153.0568 and 123.0466 resulted from HT, and m/z 175.0256 and 113.0258 resulted from glucuronide. M2a and b showed [M − H]− at m/z 233.0124, which were increases of 80 Da (SO3) compared with HT, and were considered the sulfated derivatives of HT. The main fragmentions, m/z 153.0557 and 123.0472, were also observed, which further provided further evidence that M2a and b were the sulfated derivatives of HT. M3a and b had [M − H]− at m/z 343.1036, which were increases of 176 Da compared with homovanillic alcohol (m/z 167.0714), and exhibited the characteristic ions m/z 167.0713 and 113.0252, which were identified as methyl and glucuronide hydroxytyrosol conjugate products. M4a and b had deprotonated molecular ions at m/z 247.0282 and were identified as methyl and sulfated derivatives owing to their increases of 80 Da (SO3) and 14 Da (−CH2) compared with HT. M5a was detected in rat plasma with a deprotonated molecular ion at m/z 261.0074, which was 80 Da (SO3) more E

DOI: 10.1021/acs.jafc.7b04650 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 5. Protective effects of ACT and its metabolites on the hepatic levels of SOD (A), GSH (B), MPO (C), and MDA (D) in the GalN/LPSinduced-acute-hepatic-injury mouse model. The results are expressed as the mean values ± SD for 8 mice in each group. **p < 0.01, ***p < 0.001 versus the control group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus the model group.

of ACT at a dose of 200 mg/kg BW. Most of the ACT was metabolized by intestinal bacteria into phase I metabolites such as homovanillic acid, CA, and HT. Then, these metabolites were absorbed into the blood by the intestinal epithelial cells through the hepatic portal vein, and then phase II metabolism occurred as reported.11 In our study, we found that the relative contents of the sulfate hydroxytyrosol conjugates (M2a and b) and sulfate 3-hydroxyphenylpropionic conjugates in rat plasma were 56.44 and 25.54%, respectively. The relative contents of M2a and b were 23.36% in rat urine. It is probable that HT and CA were the active substances in vivo, as previously reported.12,13 However, the hepatoprotective capacities of HT, CA, and 3-hydroxyphenylpropionic acid remain unknown. Structural Determination of Metabolites Isolated from Acteoside Urine. In order to obtain as many and as much of the metabolites of ACT as possible, a large volume of urine was collected. The urine was first concentrated in vacuo and then extracted with ethyl acetate, and the remaining solution was then subjected to chromatography on a macroporous-resin D101 column. Our preliminary experiments had shown that the metabolites were stable for 4 h at 55 °C, so the metabolites in the urine were concentrated (from 9 L to 0.5 L) for 3 h by vacuum reflux in order to further aid the separation and purification of the metabolites. On the basis of the above methods, one new compound and seven metabolites were obtained, and the spectral (1D and 2D NMR and MS) analyses showed that the isolated metabolites were the hydroxytyrosol derivatives hydroxytyrosol (M1), homovanillic acid (M2); the

Figure 6. Protective effects of ACT and its metabolites on histological changes in mice following GalN/LPS injection (original magnification: ×400, scale bar: 50 μm).

M13a−d had [M − H]− at m/z 651.2294 and were identified as the dimethyl and glucuronide conjugations of ACT because they were 28 Da (−2CH2) larger than ACT. The typical fragment ions at m/z 475.1689 and 193.0525 suggested that the two methyls were connected to the hydroxy groups of HT (m/ z 475.1689) and CA (m/z 193.0525), respectively. The relative contents of the metabolites in the rat plasma and urine were calculated by the ratios of the peak areas of each metabolite and the total peak areas based on the literature.10 The prototype drug ACT was undetected in the plasma and urine. It was completely metabolized in the rat plasma and urine within 36 h. The main metabolic pathways of ACT in vivo included isomerization, hydrolysis, hydrogenation, dehydroxylation, methylation, sulfation, and glucuronidation. The hydroxytyrosol and caffeic acid derivatives were the major metabolites in the rat plasma and urine after the administration F

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agents. ALT and AST are two vital parameters for monitoring liver necrosis. As shown in Table 2, the levels of serum ALT and AST in the control group were 9.55 ± 0.490 and 16.4 ± 0.420 IU/L, respectively, and these had significantly increased 7 h after the injection of GalN/LPS (153 and 141 IU/L, respectively). Doses of 0.15 mmol/kg HT, 3-HPP, and silybin reduced these increases. Serum HMGB1, TNF-α, and IL-6 Levels. High mobility group box 1 (HMGB1), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) play important roles in liver injuries.24,25 Once the liver is injured, liver Kupffer cells will release HMGB1 to further increase the macrophage-related immune response, thereby promoting the synthesis and release of ROS and inflammatory cytokines such as TNF-α, IL-1β, and IL-6, which can lead to a series of inflammatory cascades. This increases the damage caused by cell death.26 As shown in Figure 4, the serum HMGB1, TNF-α, and IL-6 levels were significantly increased to 1913 ± 50.6, 429 ± 6.12, and 43.0 ± 3.05 in the model group compared with those of the control group (p < 0.001). However, administration of the metabolites CA, HT, and 3HPP at doses of 0.15 mmol/kg prior to GalN/LPS-treatment markedly ameliorated the induced rise in HMGB1, TNF-α, and IL-6 levels (varying significances of p < 0.05, p < 0.01, and p < 0.001). In addition, the positive-control group (silybin) and the parent-drug group (ACT) also exhibited reduced levels of HMGB1, TNF-α, and IL-6 in the liver-injury mouse model. Hepatic SOD, GSH, MDA, and MPO Levels. A defense system of antioxidant enzymes, such as SOD and GSH, has a significant effect in preventing damage due to the GalN/LPSinduced production of reactive oxygen species in the liver.27,28 These enzymes form a mutually supportive defense system to protect against the harmful effects of reactive oxygen species. However, lipid peroxides and reactive oxygen species can easily inactivate these antioxidant enzymes under toxic conditions. Therefore, changes in the hepatic levels of these antioxidant enzymes are closely related to the ability of the liver to cope with oxidative stress following the toxic effects of GalN/LPS administration. As shown in Figure 5, the activities of hepatic SOD and GSH in the model group were significantly lower than those of the normal group (p < 0.001). In the model group, the levels of the hepatic antioxidant enzymes SOD and GSH were reduced to 139.6 ± 13.1 U/mg of protein and 2.93 ± 0.200 μmol/g of protein, respectively. The corresponding values in the normal group were 217 ± 23.0 U/mg of protein and 6.22 ± 0.240 μmol/g of protein, respectively. In contrast, the groups treated with 0.15 mmol/kg ACT, CA, HT, 3-HPP, and silybin exhibited significantly increased (p < 0.05 and p < 0.01) levels of SOD and GSH, and the protective effects of the CA, HT, and 3-HPP treatments were similar to that of silybin. Liver lipid peroxidation was significantly increased in the GalN/LPS-treated group compared with that in the control group (p < 0.01). The administration of CA, HT, and 3-HPP at doses of 0.15 mmol/kg prior to the GalN/LPS treatment markedly reduced the increases in MDA levels (p < 0.05 and p < 0.01). Similarly, treatment with silybin, the positive-control compound, also produced a good protective effect. Neutrophil infiltration is also an important pathological process in the emergence and transmission of inflammatory injuries.29 The level of MPO was used as a biochemical index to investigate the extent of neutrophil infiltration. We found that GalN/LPS administration markedly increased MPO levels in liver tissues, indicating significant macrophage infiltration,

caffeic acid derivatives dihydrocaffeic acid (M3), 3-hydroxyphenylpropionic acid (M4), feralic acid (M5), isoferalic acid (M6), and caffeic acid (M7); and the parent compounds 4,4′′′dimethoxy-isoacteoside (M8), 3,4′′′-dimethoxy-isoacteoside (M9), epimeridinoside A (M10), and isomartynoside (M11). Their chemical structures are shown in Figure 2. The raw data from the characterizations of these metabolites are given in the Supporting Information. Metabolite M8 was obtained as a light-yellow powder (MeOH). Its molecular formula was found to be C31H40O15 by HR-ESI-MS with [M − H]− at m/z 651.2286 (calcd for C31H40O15, m/z 651.2294). In the 1H-NMR spectrum, M8 (Table 1) showed the typical proton signals of two ABX systems: δ 6.98 (1H, dd, J = 1.8, 7.8 Hz, H-6′′′), 6.92 (1H, d, J = 7.8 Hz, H-5′′′), and 7.06 (1H, d, J = l.8 Hz, H-2′′′) and δ 6.62 (1H, d, J = 2.4, 8.4 Hz, H-6), 6.70 (1H, d, J = 8.4 Hz, H5), and 6.69 (1H, d, J = 2.4 Hz, H-2). It also showed two transolefinic protons at δ 6.34 (1H, d, J = 15.9 Hz, H-8′′′) and 7.58 (1H, d, J = 15.9 Hz, H-7′′′). In addition, the characteristic signals for the two methoxy groups were also observed at δ 3.89 (3H, s, −OCH3) and 3.75 (3H, s, −OCH3). The protons resonated at δ 5.17 (1H, d, J = 7.8 Hz) and 4.33 (1H, d, J = 7.8 Hz), which together with the signals of the oxygenated carbons in the 13C-NMR spectrum suggested a glucosyl group and a rhamnosyl group, respectively. The 13C-NMR spectrum of M8 (Table 1) showed 31 carbon resonances, including 14 olefinic, 1 carbonyl, and 17 aliphatic carbons. A detailed spectroscopic analysis showed that M8 was an isoacteoside derivative with two methoxy groups. To determine the structure, HSQC and HMBC experiments (Figure 3) were performed. Long-range correlations were observed between δ 3.89 (3H, s, −OCH3) and δ 151.6 (C-4′′′) and between δ 3.75 (3H, s, −OCH3) and δ 147.5 (C-4), which identified the sites of the two methoxy groups. The structure of M8 was confirmed as 4′′′-dimethoxy-isoacteoside, and it was a new compound that had not been reported in the literature. The full spectral data and original charts for this compound are supplied in the Supporting Information (Figures S3 and S4). M9, M10, and M11 were found to have the same deprotonated ions as M8 at m/z 651.2294 as well as the molecular formula, C31H40O15. The 1H-NMR spectra of M9, M10, and M11 (Table 1) showed the same characteristic proton signals of the two methoxy groups, two ABX systems, two trans-olefinic protons, and sugar. The 13C-NMR spectra of M9, M10, and M11 also showed 31 carbon resonances, including 14 olefinic, 1 carbonyl, and 17 aliphatic carbons. On the basis of these detailed spectroscopic features combined with reports in the literature, M9, M10, and M11 were deduced to be 3,4′′′-dimethoxy-isoacteoside, epimeridinoside A, and isomartynoside,16,17 respectively. For metabolites M1−7, by comparing their physical and spectroscopic data with the values reported in the literature, their structures were identified as hydroxytyrosol (M1),18 homovanillic acid (M2),19 dihydrocaffeic acid (M3),20 3hydroxyphenylpropionic acid (M4),20 feralic acid (M5),21 isoferalic acid (M6)22 and caffeic acid (M7).23 The 1H-NMR and 13C-NMR spectral data for M1−11 are supplied in the Supporting Information. Serum ALT and AST Levels. GalN and LPS are often administered in combination to prepare an animal model of a liver injury that is very similar to that of human viral hepatitis in terms of its morphology and functional characteristics. It is usually used for demonstrating the efficacy of hepatoprotective G

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whereas the CA, HT, and 3-HPP pretreatments reduced the levels of MPO (Figure 5C), and this may be another mechanism of CA-, HT-, and 3-HPP-mediated protection against liver injuries. Histological Analysis. Histopathological analysis demonstrated that the hepatic lobular architecture remained intact without any abnormalities in the control group. Seven hours after the GalN/LPS treatment, we found great changes in the liver cells, including degeneration and edema, in the model mice (Figure 6). Strong positive signals of apoptosis, such as chromatin condensation and marginalization, nuclear-membrane disruption, the fragmentation of chromatin, cholestasis, and hemorrhages, were also observed. These pathological changes were reduced in the treatment groups, including those with levels of ACT, HT, CA, and 3-HPP in the liver. The wide range of pharmacological activities of ACT have been confirmed by a number of reports on its pharmacodynamics.6,30,31 Also, the pharmacokinetics of ACT have suggested that it could be considered a prodrug, and its metabolites could be responsible for the pharmacologic effects.10,11 It has been reported that 10 metabolites were found in plasma, and 43 metabolites were found in rat feces, indicating that most of ACT was excreted via the intestinal tract. This was confirmed by previous reports on the poor oral bioavailability but high biological activities of ACT.32 In addition, another study has shown that 6 metabolites were detected in rat plasma within 36 h that were hydrolysis metabolites, such as 3-HPP + SO3 and HT + SO3.10 However, previous studies only tentatively identified metabolites of ACT by MS and speculated about the active metabolites, and studies on the metabolites that reversed pathologies were missing. In our study reported here, metabolites of ACT were not only isolated but also evaluated for their hepatoprotective activities and compared with previous works.10,32 Eleven metabolites, including one new metabolite, were separated and identified for the first time in rat urine after ACT administration. Then, a pharmacological experiment involving acute liver injuries induced by GalN/LPS was carried out in order to evaluate the hepatoprotective efficacies of the metabolites. It was found that the hydrolysis products of ACT, including HT, CA, and 3HPP, combatted the pathological changes, as shown by observably reductions in plasma ALT and AST levels. Also, the HT, CA, and 3-HPP treatments prevented the oxidative stress and inflammation associated with GalN/LPS-induced liver injuries. In the liver tissues, the metabolite treatments increased the contents of GSH and SOD, reduced the levels of MPO and MDA, and improved histopathological changes, and in sera, the treatments regulated the levels of the serum inflammatory cytokines HMGB1, TNF-α, and IL-6. These results showed why ACT exhibited therapeutic effects when its oral bioavailability was extremely low. Our results also provided strong support for the idea that the metabolites of ACT were responsible for the liver-injury-related and other therapeutic effects. In previous studies, we found that ACT metabolites such as HT, CA, and 3-HPP were produced by intestinal bacteria.12 This showed that the therapeutic effects of dietary acteoside from cistanche tea were directly related to a steady state of intestinal bacteria. It should not be forgotten that the therapeutic effects of cistanche tea disappear when the intestinal bacteria are destroyed, in particular when taken in combination with antibiotics.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b04650. Detailed mass data of the ACT metabolites and their relative contents in rat urine and plasma; extracted-ion chromatograms of the ACT metabolites in rat urine and plasma; UPLC chromatogram of fraction 4.2; 1H-NMR, 13 C-NMR, HSQC, and HMBC spectra of M8; and 1HNMR and 13C-NMR data for M1−M8 (PDF)



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +86-24-23986469. Fax: +86-24-23986469 (Y.N.P.). *E-mail: [email protected] (X.Q.L). *E-mail: [email protected] (W.X.). ORCID

Qingling Cui: 0000-0002-3336-5968 Funding

The authors are grateful for financial support from the National Natural Science Foundation of China (81202892 and 81470176) and the Project of Science and Technology of the Education Department of Liaoning Province (2017LQN11). Notes

The authors declare no competing financial interest.



REFERENCES

(1) Xiong, W. T.; Gu, L.; Wang, C.; Sun, H. X.; Liu, X. Antihyperglycemic and hypolipidemic effects of Cistanche tubulosa in type 2 diabetic db/db mice. J. Ethnopharmacol. 2013, 150, 935−945. (2) Dai, S. H.; Li, L.; Zou, S. D.; Cheng, W. D. Development of Cistanche coffee health drink. Food Res. Dev. (Tianjin, China) 2012, 33, 113−116. (3) Wu, X. J. Development a health wine from Cistanche black rice. Food Res. Dev. (Tianjin, China) 2014, 35, 73−75. (4) Li, L.; Low, M. Y.; Aliwarga, F.; Teo, J.; Ge, X. W.; Zeng, Y.; Bloodworth, B. C.; Koh, H. L. Isolation and identification of hydroxythiohomosildenafil in herbal dietary supplements sold as sexual performance enhancement products. Food Addit. Contam., Part A 2009, 26, 145−151. (5) Morikawa, T.; Ninomiya, K.; Imamura, M.; Akaki, J.; Fujikura, S.; Pan, Y.; Yuan, D.; Yoshikawa, M.; Jia, X.; Li, Z.; Muraoka, O. Acylated phenylethanoid glycosides, echinacoside and acteoside from Cistanche tubulosa, improve glucose tolerance in mice. Journal of Natural Medicines. 2014, 68, 561−566. (6) Lee, K. J.; Woo, E. R.; Choi, C. Y.; Shin, D. W.; Lee, D. G.; You, H. J.; Jeong, H. G. Protective effect of acteoside on carbon tetrachloride-induced hepatotoxicity. Life Sci. 2004, 74, 1051−1064. (7) Wu, X.; He, W.; Zhang, H.; Li, Y.; Liu, Z.; He, Z. Acteoside: A lipase inhibitor from the Chinese tea Ligustrum purpurascens kudingcha. Food Chem. 2014, 142, 306−310. (8) Li, Y.; Gan, L.; Li, G. Q.; Deng, L.; Zhang, X.; Deng, Y. Pharmacokinetics of plantamajoside and acteoside from Plantago asiatica in rats by liquid chromatography-mass spectrometry. J. Pharm. Biomed. Anal. 2014, 89, 251−256. (9) Wu, Y. T.; Lin, L. C.; Sung, J. S.; Tsai, T. H. Determination of acteoside in Cistanche deserticola and Boschniakia rossica and its pharmacokinetics in freely-moving rats using LC−MS/MS. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2006, 844, 89−95. (10) Cui, Q. L.; Pan, Y. N.; Bai, X. W.; Zhang, W.; Chen, L. X.; Liu, X. Q. Systematic characterization of the metabolites of echinacoside and acteoside from Cistanche tubulosa in rat plasma, bile, urine and H

DOI: 10.1021/acs.jafc.7b04650 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry feces based on UPLC-ESI-Q-TOF-MS. Biomed. Chromatogr. 2016, 30, 1406−1415. (11) Tao, J. H.; Zhao, M.; Wang, D. G.; Yang, C.; Chen, G. T.; Zhao, X.; Pu, X. L.; Jiang, S. UPLC-Q-TOF/MS-based screening and identification of two major bioactive components and their metabolites in normal and CKD rat plasma, urine and feces after oral administration of Rehmannia glutinosa Libosch extract. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2015, 1001, 98−106. (12) Cui, Q. L.; Pan, Y. N.; Xu, X. T.; Zhang, W. J.; Wu, X.; Qu, S. H.; Liu, X. Q. The metabolic profile of acteoside produced by human or rat intestinal bacteria or intestinal enzyme in vitro employed UPLCQ-TOF-MS. Fitoterapia 2016, 109, 67−74. (13) Li, Y.; Zhou, G.; Peng, Y.; Tu, P.; Li, X. Screening and identification of three typical phenylethanoid glycosides metabolites from Cistanches Herba by human intestinal bacteria using UPLC/QTOF-MS. J. Pharm. Biomed. Anal. 2016, 118, 167−176. (14) Wei, L. L.; Ren, F.; Zhang, X. Y.; Wen, T.; Shi, H. B.; Zheng, S. J.; Zhang, J.; Chen, Y.; Han, Y. P.; Duan, Z. P. Oxidative stress promotes D-GalN/LPS-induced acute hepatotoxicity by increasing glycogen synthase kinase increasing glycogen synthase kinase 3β activity. Inflammation Res. 2014, 63, 485−494. (15) Li, X.; Gou, C.; Yang, H.; Qiu, J.; Gu, T.; Wen, T. Echinacoside ameliorates D-galactosamine plus lipopolysaccharide-induced acute liver injury in mice via inhibition of apoptosis and inflammation. Scand. J. Gastroenterol. 2014, 49, 993−1000. (16) Wang, Y. L.; Luan, X. Phenethyl alcohol glycosides in Epimeredi indica. Chin. Tradit. Herb. Drugs 2004, 12, 8−10. (17) Tao, L.; Huang, J.; Zhao, Y.; Li, C. Chemical constituents in Buddleja albiflora. China J. Chin. Mater. Med. 2009, 34, 3043−3046. (18) Vo, Q. H.; Nguyen, P. H.; Zhao, B. T.; Ali, M. Y.; Choi, J. S.; Min, B. S.; Nguyen, T. H.; Woo, M. H. Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw. Fitoterapia 2015, 103, 113−121. (19) Samsonowicz, M.; Kowczyk-Sadowy, M.; Regulska, E.; Lewandowski, W. Molecular structure and spectroscopic analysis of homovanillic acid and its sodium salt-NMR, FT-IR and DFT studies. Spectrochim. Acta, Part A 2014, 118, 1068−1074. (20) Takagaki, A.; Nanjo, F. Catabolism of (+)-catechin and (−)-epicatechin by rat intestinal microbiota. J. Agric. Food Chem. 2013, 61, 4927−4935. (21) Song, K.; Zheng, X. K.; Zhang, J. K.; Zhang, Y. L.; Li, M.; Wang, J. C.; Zou, Z. M.; Feng, W. S. Chemical constituents of Erigeron annuus (L.) Pers. China J. Chin. Mater. Med. 2016, 51, 1462−1466. (22) Yu, M. F.; Shao, J.; Tao, Y. D. Chemical constituents of Tibetan herbal medicine Sibiraea laevigata. Chin. Tradit. Herb. Drugs 2014, 45, 3528−3531. (23) Chen, Y. H.; Chang, F. R.; Lin, Y. J.; Wang, L.; Chen, J. F.; Wu, Y. C.; Wu, M. J. Identification of phenolic antioxidants from Sword Brake fern (Pteris ensiformis Burm.). Food Chem. 2007, 105, 48−56. (24) Oshima, G.; Shinoda, M.; Tanabe, M.; Ebinuma, H.; Nishiyama, R.; Takano, K.; Yamada, S.; Miyasho, T.; Masugi, Y.; et al. Increased plasma levels of high mobility group box 1 in patients with acute liver failure. Eur. Surg. Res. 2012, 48, 154−162. (25) Rolando, N.; Wade, J.; Davalos, M.; Wendon, J.; PhilpottHoward, J.; Williams, R. The systemic inflammatory response syndrome in acute liver failure. Hepatology 2000, 32, 734−739. (26) Yang, Q.; Liu, Y.; Shi, Y.; Zheng, M.; He, J.; Chen, Z. The role of intracellular high-mobility group box 1 in the early activation of Kupffer cells and the development of Con A-induced acute liver failure. Immunobiology 2013, 218, 1284−1292. (27) Gong, X.; Yang, Y.; Huang, L.; Zhang, Q.; Wan, R. Z.; Zhang, P.; Zhang, B. Antioxidation, anti-inflammation and anti-apoptosis by paeonol in LPS/d-GalN-induced acute liver failure in mice. Int. Immunopharmacol. 2017, 46, 124−132. (28) Yang, Y.; Gong, X. B.; Huang, L. G.; Wang, Z. X.; Wan, R. Z.; Zhang, P.; Zhang, Q. Y.; Chen, Z.; Zhang, B. S. Diosmetin exerts antioxidative, anti-inflammatory and anti-apoptotic effects to protect against endotoxin-induced acute hepatic failure in mice. Oncotarget 2017, 8, 30723−30733.

(29) Ramaiah, S. K.; Jaeschke, H. Role of neutrophils in the pathogenesis of acute inflammatory liver injury. Toxicol. Pathol. 2007, 35, 757−766. (30) Wang, J.; Ma, C. H.; Wang, S. M. Effects of acteoside on lipopolysaccharide-induced inflammation in acute lung injury via regulation of NF-κB pathway in vivo and in vitro. Toxicol. Appl. Pharmacol. 2015, 285, 128−135. (31) Shiao, Y. J.; Su, M. H.; Lin, H. C.; Wu, C. R. Acteoside and isoacteoside potect amyloid β peptide induced cytotoxicity, cognitive deficit and neurochemical disturbances in vitro and in vivo. Int. J. Mol. Sci. 2017, 18, 895. (32) Su, D.; Li, W.; Xu, Q. M.; Liu, Y. L.; Song, Y. G.; Feng, Y. L. New metabolites of acteoside identified by ultra-performance liquid chromatography/quadrupole-time-of-flight MSE in rat plasma, urine, and feces. Fitoterapia 2016, 112, 45−55.

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