Asparagus officinalis L. - ACS Publications - American Chemical Society

Article pubs.acs.org/JAFC

Antihepatic Fibrosis Effect of Active Components Isolated from Green Asparagus (Asparagus officinalis L.) Involves the Inactivation of Hepatic Stellate Cells Chunge Zhong,†,∥ Chunyu Jiang,§,∥ Xichun Xia,† Teng Mu,† Lige Wei,§ Yuntian Lou,§ Xiaoshu Zhang,§ Yuqing Zhao,*,§,# and Xiuli Bi*,† †

College of Life Science, Liaoning University, Shenyang 110036, China Department of Traditional Chinese Medicine and #Key Laboratory of Structure-based Drug Design and Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China

§

ABSTRACT: Green asparagus (Asparagus officinalis L.) is a vegetable with numerous nutritional properties. In the current study, a total of 23 compounds were isolated from green asparagus, and 9 of these compounds were obtained from this genus for the first time. Preliminary data showed that the ethyl acetate (EtOAc)-extracted fraction of green asparagus exerted a stronger inhibitory effect on the growth of t-HSC/Cl-6 cells, giving an IC50 value of 45.52 μg/mL. The biological activities of the different compounds isolated from the EtOAc-extracted fraction with respect to antihepatic fibrosis were investigated further. Four compounds, C3, C4, C10, and C12, exhibited profound inhibitory effect on the activation of t-HSC/Cl-6 cells induced by TNFα. The activation t-HSC/Cl-6 cells, which led to the production of fibrotic matrix (TGF-β1, activin C) and accumulation of TNF-α, was dramatically decreased by these compounds. The mechanisms by which these compounds inhibited the activation of hepatic stellate cells appeared to be associated with the inactivation of TGF-β1/Smad signaling and c-Jun N-terminal kinases, as well as the ERK phosphorylation cascade. KEYWORDS: Asparagus officinalis L, active components, antihepatic fibrosis, TGF-β1/Smad signaling, c-Jun N-terminal kinases, ERK

■

INTRODUCTION

intake, or successful viral hepatitis treatment can control fibrosis. Because the prevalence of chronic liver diseases is predicted to increase, partially owing to the rising prevalence of obesity and metabolic syndrome, especially in developed countries,8 there is a need to find more efficient ways to prevent or retard the eventual progression from fibrosis to cirrhosis in the vast majority of patients. On the basis of the liver protective effects of green asparagus reported previously, the current study was designed to identify and clarify the chemical constituents isolated from green asparagus through chromatographic fractionation, chemical analysis, and bioactivity assays and to investigate the potential mechanism associated with the antihepatic fibrosis activity of these constituents.

Asparagus officinalis L., the so-called “king of vegetables”, is a well-known healthy vegetable that is mainly consumed for its edible shoots. It is known to contain steroids, saponins, flavonoids, and many other valuable constituents, such as protein, fat, sugar, amino acids, vitamins, and minerals.1 Green asparagus has recently received increasing research interest because of its important nutritional and medicinal effects. Hafizur et al. reported that asparagus extract exerts antidiabetic effects by improving insulin secretion and β-cell function in streptozotocin-induced type 2 diabetic rats.2 It has also been reported that A. of f icinalis can potentially be used in therapy designed to protect the liver from various harmful insults.3 Liver fibrosis is a public health problem that results in significant morbidity and mortality. It is caused by inappropriate tissue repair via connective tissue deposition, and results from chronic liver injuries, including those caused by alcohol, chronic viral hepatitis, autoimmune diseases, parasites, metabolic diseases, or toxins or other drugs.4,5 Although important progress has been made during the past years regarding our understanding of the pathogenesis of hepatic fibrosis, the most common concept is that after acute liver damage, parenchymal cells are regenerated to replace the necrotic and apoptotic cells. This regenerative process is associated with an inflammatory response and a limited deposition of extracellular matrix (ECM). Hepatic stellate cells (HSC) are the main source of ECM.6 So far, there is no standard treatment for liver fibrosis,7 but a reduction of liver injury events, change of lifestyle such as cessation of alcohol © XXXX American Chemical Society

■

MATERIALS AND METHODS

Reagents and Plant Material. 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT), TNF-α, and DMSO were purchased from Sigma Chemical Co. (St. Louis, MO, USA). TGF-β1, p-Smad2, activin C, p-ERK, p-JNK, c-Jun, c-Fos, and β-actin monoclonal antibodies were purchased from Bio Basic Inc., Canada. All other chemical agents were of analytical grade, and all cell culture reagents were obtained from Gibco/Invitrogen (Grand Island, NY, USA). The stems of green asparagus were collected in Caoxian, Shandong province, China, and then kept in a desiccator after air-drying. A Received: March 24, 2015 Revised: June 10, 2015 Accepted: June 11, 2015

A

DOI: 10.1021/acs.jafc.5b01490 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Schematic representation of the extraction and isolation procedure. voucher specimen was deposited in Shenyang Pharmaceutical University. General Experimental Procedures. Column chromatography (CC) was performed with silica gel (SiO2 300−400 mesh, Qingdao Marine Chemical Group, Co. Qingdao, Shandong, China), reversed phase ODS (300−400 mesh, Agela Technologies Co. Tianjin, China), and Sephadex LH-20 (Pharmacia, Co. Tokyo, Japan). NMR spectra were recorded with a Bruker ARX-600 (1H, 600 MHz; 13C, 150 MHz) spectrometer in DMSO-d6 or CDCl3 with tetramethylsilane as internal standard. All other chemical agents, including CH3OH, ethanol, petroleum ether, ethyl acetate, and n-butanol, were of analytical grade and were obtained from Tianjin DaMao Chemical Reagents Co. (Tianjin, China) Extraction and Isolation. To obtain the active constituents, the stems of green asparagus (30 kg) were extracted with 75% EtOH (bp 78.1 °C) under reflux at 80 °C, on the basis of the results of several published references.9,10 After evaporation of the alcohol, the residual aqueous solution was subsequently extracted with water, petroleum ether (PE, 388 g), ethyl acetate (EtOAc, 260 g), and n-butanol (nBuOH, 200 g). The extraction and isolation scheme is shown in Figure 1. Phytochemical study of green asparagus led to the isolation of 23 compounds, including 7 steroids (1−7), 2 esters (8, 11), 3 arynes (9, 10, 12), 1 lignin (13), 3 flavonoids (14−16), 4 alkaloids (17−20), and 3 aliphatic series (21−23): 24(R)-24-ethyl-5α-cholestane-3β,5,6β-triol (1),11 stigmasterol (2),12 (22E,24S)-5α,8α-epidioxy-24-methylcholesta-6,22-dien-3β-ol (3),13 5α,8α-epidioxy-(20S,22E,24R)-ergosta-6,22dien-3β-ol (4),14 24(S)-cycloart-25-ene-3β,24-diol (5),15 α-spinasterol (6),16 yamogenin (7),17 1,3-O-diferuloylglycerol (8),18 methyl phydroxycinnamate (11),19 4-[5-(4-methoxyphenoxy)-3-penten-1-ynyl] phenol (9),20 4-[5-(4-hydroxyphenoxy)-3-penten-1-ynyl]phenol (10),20 4,4-dihydroxy-2-methyl-3-[(4-hydroxyphenyl)ethynyl]biphene (12),21 syringaressinol (13),22 quercetin (14),23 kaempferol (15),24 quercitrin (16),25 aurantiamide acetate (17),26 neoechinulin A (18),27 3-pyridinecarboxylic acid, 1,6-dihydro-6-oxo-methyl ester (19), 2-(2hydroxytetracosanoylmino)-1,3,4-octadecanetriol (20),28 hexadecanoic acid, 2,3-dihydroxypropyl ester (21),29 12,13,15-trihydroxy-9-octadecenoic acid (22),30 and 2-octadecendioic acid (23).31 The known compounds 1−23 (Figure 2) were identified by comparison of their NMR data with those reported in the literature. The purity of each compound ranged from 95 to 98%, as determined by HPLC analysis.

Furthermore, compounds 1, 3, 5, 11, 12, and 17−20 were extracted from the Asparagus genus for the first time. Cell Culture. Murine hepatic stellate cells (t-HSC/Cl-6) were routinely maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum and antibiotics (100 U/mL penicillin and streptomycin) in a humidified atmosphere containing 5% CO2 at 37 °C. Cell Viability Assay. t-HSC/Cl-6 cells were cultured in 96-well plates (1 × 104 cells/well) overnight and then treated with the individual compounds at various concentrations with or without TNFα for another 48 h. Cell viability was then assessed by the MTT assay. After removal of all of the remaining liquid in the wells, 100 μL of fresh medium containing MTT reagent (5 μg/mL) was added to each well and incubated for 3 h at 37 °C. After that, the formazan crystals were dissolved with dimethyl sulfoxide, and the reduction of cell viability was determined by measuring the absorbance at 490 nm using a microplate reader. For this experiment, each of the 23 compounds was dissolved in dimethyl sulfoxide to give a 0.1 M stock solution. RNA Extraction and Quantitative RT-PCR. Total RNA was extracted from the treated cells. The quality and quantity of RNA were determined. Quantitative real-time PCR was carried out using the ABI Prism 7900-HT sequence detection system (96 wells) as described.32 The following primers were designed for mouse cytokine analysis: tumor necrosis factor-α (TNF-α), forward 5′-CCCTCACACTCAGATCATCTTCT-3′ and reverse 5′-GCTACGACGTGGGCTACAG-3′; IL-6, forward 5′-TAGTCCTTCCTACCCCAATTTCC-3′ and reverse 5′-TTGGTCCTTAGCCACTCCTTC-3′; TGF-β1, forward 5′-ACCTGCAAGACCATCGACAT-3′ and reverse 5′-GGTTTTCTCATAGATGGCGT-3′; activin C, forward 5′-CCGTGAGATTGGCTGGAAT-3′ and reverse 5′-GCGTTGGCTTTGAGCAGAT-3′. β-Actin was used as internal control. Western Blotting Analyses. In addition to RNA, total protein was also extracted from the treated cells. In brief, the cells were lysed in RIPA buffer (Cell Signaling Technology, USA) for 15 min on water ice. After brief sonication, the cell lysate was centrifuged at 14000g for 15 min at 4 °C. The supernatant was resolved by SDS-PAGE using 12% gel. The proteins in the gel were then transferred to a nitrocellulose membrane. The following primary antibodies were used for immunoblotting: anti-TGF-β1 (1:1000), anti-p-Smad2 (1:1000), anti-activin C (1:1000), anti-p-ERK (1:1000), anti-p-JNK (1:1000), anti-c-Jun (1:1000), anti-c-Fos (1:1000), and anti-β-actin B


Article


Figure 2. Chemical structures of isolated compounds 1−23 and silymarin. (1:3000). Signals were detected by an enhanced chemiluminescence technique (Amersham Life Science, USA). Quantification of the Active Compounds from Green Asparagus. For HPLC analysis, the top four active compounds (3, 4, 10, 12) were analyzed using a HPLC system (LC3000, Beijing ChuangXinTongHeng Science & Technology Co., Ltd., Beijing, China) equipped with a UV lamp (Beijing ChuangXinTongHeng Science & Technology Co., Ltd.) and a C18 reversed-phase analytical column (Agilent HC-C18, 4.6 × 250 mm, 5 μm; Agilent Technologies Inc., Santa Clara, CA, USA). The HPLC mobile phase was composed

of methanol (solvent A) and water (solvent B). The linear gradient elution system was as follows: 0−20 min, 53−65% (v/v) A; 20−30 min, 65−80% (v/v) A; 30−45 min, 80−90% (v/v) A; 45−60 min, 90−100% (v/v) A, and 60−90 min, 100% (v/v) A, at a flow rate of 1 mL/min. The quantification of the top four active compounds by UV was performed at 210 nm. The column temperature was maintained at 30 °C, and the injection volume was 20 μL. In addition, to quantitate the top four active compounds, namely, (22E,24S)-5α,8α-epidioxy-24methylcholesta-6,22-dien-3β-ol, 5α,8α-epidioxy-(20S,22E,24R)-ergosta-6,22-dien-3β-ol, 4-[5-(4-hydroxyphenoxy)-3-penten-1-ynyl]phenol, C


Article

Journal of Agricultural and Food Chemistry and 4,4-dihydroxy-2-methyl-3-[(4-hydroxyphenyl)ethynyl]biphene, were measured at concentrations of 0.025, 0.05, 0.1, 0.2, and 0.5 g/ L to prepare a calibration curve for each compound. The percentage of the top four active compounds was calculated using eq 1:

Table 1. Compounds Isolated from the Stems of Green Asparagus kind steroids

15 16 17

esters

8 11

1,3-O-diferuloylglycerol methyl p-hydroxycinnamate

18 19

arynes

9

4-[5-(4-methoxyphenoxy)-3-penten-1-ynyl] phenol 4-[5-(4-hydroxyphenoxy)-3-penten-1-ynyl] phenol 4,4-dihydroxy-2-methyl-3-[(4-hydroxyphenyl) ethynyl]biphene

20

1 2 3 4

■

RESULTS Phytochemical Characterization. Phytochemical investigation of the stem of green asparagus resulted in the isolation of 23 compounds, including 7 steroids, 2 esters, 3 arynes, 1 lignan, 3 flavonoids, 4 alkaloids, and 3 aliphatic series. Among these, compounds 1, 3, 5, 11, 12, and 17−20 were obtained from this genus for the first time. The structures of compounds 1−23 were determined using a combination of NMR spectra and then identified by comparison of the spectral data with those reported in the literature (Table 1). Among the tested compounds, compounds 3, 4, 7, 8, 10−12, 16, and 20 showed potent antihepatic fibrosis activity, and the relative order of potency was 10 > 12 > 3 > 8 > 4 > 20 > 16 > 7 > 11 as depicted in Table 2. We concluded that the high activity of compounds 10 and 12 could be due to their acetylenic bonds, whereas a significant difference was observed between the activity of compounds 9 and 10, both of which had the same skeleton, except for the 16-hydroxyl (10) or methoxyl (9), suggesting that the hydroxyls may be more important for the antihepatic fibrosis activity. In addition, comparison of compounds 1−7 showed that the antihepatic fibrosis capacities of steroidal compounds may be influenced by the ring containing oxygen. Quantification of the Top Four Active Compounds. The contents of 3, 4, 10, and 12 in A. of f icinalis L. were determined by HPLC-UV analysis and are shown in Table 3. Preliminary Screening of Inhibitory Activity against Self-Activated t-HSC/Cl-6 Cells. All of the extracts and compounds isolated were assayed for their inhibitory activities against self-activated t-HSC/Cl-6 cells, and the results are shown in Table 2. The PE- and EtOAc-extracted fractions greatly inhibited the activation of t-HSC/Cl-6 cells, with IC50 values of 87.77 and 45.52 μg/mL, respectively. At the same time, compounds 3, 4, 7, 8, 10−12, 16, and 20 isolated from the EtOAc-extracted fraction were more potent than the positive control drug silymarin (IC50 = 352.11 μM) or Fufang BiejiaRuangan Pian (IC50 = 212.34 μg/mL) at inhibiting the activation of t-HSC/Cl-6 cells, with IC50 values of 76.70, 111.08, 172.70, 95.22, 36.65, 196.30, 56.04, 166.34, and 125.11 μM, respectively. The inhibitory activity of these compounds against the activation of t-HSC/Cl-6 cells suggested that they might contribute to the inhibitory activity of the EtOAcextracted fraction against hepatic fibrosis. Furthermore, the strong inhibitory activity of compounds 10 and 12 toward the activated t-HSC/Cl-6 cells could possibly be related to their different phytochemical properties.

ref

5 6 7

(1)

Statistical Analysis. All of the results were expressed as means ± SDs of three determinations at each concentration for each sample. Analysis of variance (ANOVA) was performed, and the mean separation was done by LSD (p ≤ 0.05) using the SPSS 16.0 program for Windows (SPSS Inc., Chicago, IL, USA). The HPLC chromatograms were recorded and analyzed using a CXTH-3000 Data Station (CXTH Co., Ltd., Beijing, China), and the results were calculated using Windows Excel 2010.

name 24(R)-24-ethyl-5α-cholestane-3β,5,6β-triol stigmasterol (22E,24S)-5α,8α-epidioxy-24-methylcholesta6,22-dien-3β-ol 5α,8α-epidioxy-(20S,22E,24R)-ergosta-6,22dien-3β-ol 24(S)-cycloart-25-ene-3β,24-diol α-spinasterol yamogenin

% of compd = A(peak area of compd) /A(total peak areas of compd) × 100

compd

10 12

11 12 13 14

20 21

lignan

13

syringaressinol

22

flavonoids

14 15 16

quercetin kaempferol quercitrin

23 24 25

alkaloids

17 18 19

aurantiamide acetate neoechinulin A 3-pyridinecarboxylic acid, 1,6-dihydro-6-oxomethyl ester 2-(2-hydroxytetracosanoylmino)-1,3,4octadecanetriol

26 27

21

hexadecanoic acid, 2,3-dihydroxypropyl ester

29

22 23

12,13,15-trihydroxy-9-octadecenoic acid 2-octadecenedioic acid

30 31

20

aliphatic series

28

Growth Inhibitory Effects of Compounds 3, 4, 10, and 12 toward TNF-α-Activated t-HSC/Cl-6 Cells. HSC activation, which includes initiation and perpetuation, is an early event in liver fibrogenesis. The activation of HSCs converts normal, quiescent vitamin A-rich cells into myofibroblast-like cells characterized by proliferation, chemotaxis, fibrogenesis, contractility, matrix degradation, retinoid loss, and the release of white blood cell chemoattractant/ cytokine.33−37 On the basis of the preliminary growth inhibitory screening data and considering the yield of phytochemical isolation, compounds 3, 4, 10, and 12 were subjected to further investigation (Figure 3); 8 was not evaluated due to its low yield, although it also has better inhibitory activity on self-activated t-HSC/Cl-6 cells. t-HSC/ Cl-6 cells were treated with these compounds (one at a time) plus TNF-α for 48 h, and the growth of the t-HSC/Cl-6 cells was determined. According to the data shown in Figure 3, compounds 3, 4, and 10, but not 12, could significantly decrease the growth of activated t-HSC/Cl-6 in a dosedependent manner, and the extents of growth inhibition exerted by these compounds were even greater than that exerted by the positive control silymarin. D


Article


involved in the fibrogenic response. The levels of transforming growth factor beta 1 (TGF-β1) and activin C (Figure 4), both of which are HSC activation-associated factors, were increased in TNF-α activated t-HSC/Cl-6 cells. In addition, the levels of TNF-α and IL-6 were also increased in these cells. However, treatment of these cells with compounds 3, 4, 10, and 12 decreased the transcript levels of both TGF-β1 and activin C, as well as inhibiting the release of TNF-α, while having no effect on the level of IL-6. Compounds 3, 4, 10, and 12 Change the Expression Levels of HSC Activation-Associated Genes. TGF-β1 produced by HSCs acts as a potent fibrogenic signal as it increases the production of collagen I and other matrix constituents such as fibronectin and proteoglycans.35,38 These effects are induced by the interaction between TGF-β1 and the membrane receptor complex formed by TβRI and TβRII, leading to the phosphorylation of intracellular mediators, namely, Smad proteins (Figure 5). The antifibrotic mechanism is mainly the result of the down-regulation of the TGF-β1/ Smad signaling pathway. To further investigate the antifibrotic mechanism of the tested compounds, changes in the expression levels of genes associated with TGF-β1/Smad signaling pathway and its downstream target genes were determined. Upon stimulation with TNF-α, the expression levels of TGFβ1, activin C, and p-Smad2 were increased. At the same time, increased levels of phosphorylated extracellular signal-regulated kinsase (ERK) and c-Jun N-terminal kinases (JNKs) were also detected. Down-regulation of the expression of TGF-β1 and activin C and modulation of its intracellular mediators Smad2 by 3, 4, 10, and 12 were observed in the present study. The above-mentioned compounds attenuated TNF-α-induced ERK and JNK phosphorylations and also the expression level of cFos and c-Jun, the downstream targets of JNK.

Table 2. Inhibitory Activities of Crude Extract and Isolated Compounds from Green Asparagus extract/compd

IC50 (μM),a hepatic fibrosis

1 2 3 4 5 6 7 8 9 10 11

>200 >200 76.70 ± 1.68b 111.08 ± 3.90b >200 >200 172.70 ± 1.33b 95.22 ± 1.61c >200 36.65 ± 1.49b 196.30 ± 15.94b

12 13

56.04 ± 1.49b >200

14 15

>200 >200

extract/compd

IC50 (μM/μg/mL),a hepatic fibrosis

16 17 18 19 20 21 22 23 PE extractc EtOAc extractc n-BuOH extractc water extractc positive control silymarin Fufang Biejia Ruangan Pianc

166.34 ± 1.13b >200 >200 >200 125.11 ± 1.55b >200 >200 >200 87.77 ± 0.62d 45.52 ± 0.71d 160.56 ± 8.96d >200

352.11 ± 0.19 261.29 ± 2.46

IC50 values, expressed as means ± SDs, were calculated from the least-squares regression equations in the plot of the logarithm of three graded concentrations vs % inhibition using SPSS 16.0 program for Windows (SPSS Inc., Chicago, IL, USA). bSignificant differences with respect to positive control (silymarin) at the p < 0.01 level. cThe concentration unit of these extracts is μg/mL. dSignificant differences with respect to positive control (Fufang Biejia Ruangan Pian) at the p < 0.01 level. a

Table 3. Retention Times and Contents of 3, 4, 10, and 12 As Resolved by HPLC Analysis compd 3 4 10 12

regression eq y y y y

= = = =

36.95x 14.94x 436.4x 161.2x

− 0.062 + 0.132 − 1.865 − 0.112

R2

retention time (min)

%

0.999 0.999 0.999 0.999

57.048 62.043 33.848 14.385

0.012 0.016 0.069 0.016

■

DISCUSSION In present study, 23 compounds (purity > 95%, % is referred to HPLC analysis) were isolated from green asparagus, and 9 of them were obtained from this genus for the first time. The structures of compounds 1−23 were determined using a combination of 1H and 13C NMR spectra and then identified by comparison of the spectral data with those reported previously in the literature. During chronic liver injury, HSCs are activated, and their subsequent proliferation can cause ECM deposition, leading to scar formation and fibrosis. During the early stages of liver disease and hepatic fibrosis, the infiltrated immune cells produce and release high levels of TNF-α and IL-1β. Therefore, TNF-α has been implicated as a key effector molecule in the activation of HSCs through regulating the levels of cytokines, chemokines, and other adhesion molecules. Medicinal plants are often safe, cost-effective, and versatile and are therefore popular potential antifibrotic agents. There are two ways by which medicinal plants and their bioactive compounds and extracts could reduce liver fibrosis: inhibition of HSC activation and reduction of ECM deposition. In the present study, we investigated the effect of the crude extract of green asparagus as well as of the pure compounds isolated from the extract on TNF-α-activated quiescent HSCs. Perpetuation of HSC activation results in the maintenance of the activated phenotype and therefore the generation of fibrosis.39 Scars are formed through changes in HSC behavior, such as proliferation, fibrogenesis, contractility, chemotaxis, retinoid loss, white blood cell chemoattractant/cytokine release,

Figure 3. Effects of compounds 3, 4, 10, and 12 on the viability of murine hepatic stellate cells (t-HSC/Cl-6). t-HSC/Cl-6 cells were seeded in 96-well plates (1 × 104 cells/well) for 24 h and then treated with TNF-α (10 ng/mL) and the four compounds at various concentrations for 48 h. Silymarin with TNF-α (10 ng/mL) was used as positive control. Viable cell numbers were estimated via MTT assay. Data represent the means ± SDs of three independent experiments, each performed in triplicate. (∗) p ≤ 0.05; (∗∗) p ≤ 0.01.

Compounds 3, 4, 10, and 12 Decrease Cytokine Released by TNF-α-Activated t-HSC/Cl-6 Cells. Besides HSC activation, ECM deposition and other events like oxidative stress, inflammation, and immune responses are also E


Article


Figure 4. Changes in the expression levels of marker genes involved in inflammation (TNF-α, IL-6) and fibrosis (TGF-β1, activin C) in t-HSC/Cl-6 cells induced by the four compounds (3, 4, 10, and 12). RNA was extracted from t-HSC/Cl-6 cells with or without TNF-α (10 ng/mL) and the four compounds or silymarin (positive control) at different concentrations for 48 h and subjected to real-time PCR analysis. Asterisks indicate significant differences in mRNA levels of TNF-α, IL-6, TGF-β1, or activin C with respect to TNF-α treatment only at the (∗) p ≤ 0.05 and (∗∗) p ≤ 0.01 levels, respectively.

hepatic fibrosis. The compounds from green asparagus could reduce TNF-α level in a time- and dose-dependent manner. This suggested that the inhibition of HSC activation by the compounds purified from green asparagus were probably due to their anti-inflammation capacity, stemming from the suppression of TNF-α expression. Activation of TGF-β1/Smad signaling is one of the most important pro-fibrogenic pathways.40 The disruption of this pathway is a common mode of action exerted by antifibrotic plants. Our findings were consistent with findings reported by other investigators. The compounds from green asparagus could decrease the level TGF-β1 and the phosphorylation of Smad2. Meanwhile, the phosphorylated forms of ERK and JNK, as well as the expression levels of c-Fos and c-Jun, the downstream targets of JNK, were also decreased. Impressively, activin C, a closely related member of the TGF-β superfamily of growth and differentiation factors, has been shown to inhibit regenerative DNA synthesis in hepatic cells and liver regeneration and to induce apoptosis.41−45 In direct contrast, Wada et al. reported that overexpression or recombinant activin C stimulates DNA synthesis and growth of AML12 and AML12tAR cells and accelerates liver regeneration.46,47 The present data showed that in TNF-α-activated t-HSC/Cl-6 cells, the level of activin C was increased, and after treatment with the compounds, it was reduced, but only at certain concentrations of the compounds. In summary, our results demonstrated that compounds from green asparagus could inhibit HSC activation by inhibiting the proliferation of activated HSCs and attenuating TNF-α level. Moreover, TGF-β1/Smad signaling and MAPKs (ERK and JNK) signaling pathways were also involved in the antifibrosis mechanism of the tested compounds. Therefore, the current tested compounds from green asparagus may prove to be good

Figure 5. Inhibition of TGF-β/Smad-signaling-associated proteins mediated by the four compounds (3, 4, 10, and 12) in t-HSC/Cl-6 cells. The levels of TGF-β/Smad signaling-associated proteins in tHSC/Cl-6 cells activated with TNF-α (10 ng/mL) with or without the compounds for 48 h were determined and compared. β-Actin was used as a loading control. All experiments were performed three times. Representative data are shown.

and matrix degradation.35 Cell proliferation further promotes liver fibrosis by increasing the number of collagen-producing cells. However, the growth of TNF-α-activated t-HSC/Cl-6 cells was significantly inhibited by compounds 3, 4, and 10, and in dose-dependent manner, which suggested that bioactive compounds from green asparagus could improve liver fibrosis via inhibition of the proliferation of activated HSCs. Inflammation and immune responses are involved in the initiation and perpetuation of fibrosis.33,34,36,37 These pathways are potential targets for therapy aiming to reduce hepatic fibrosis. High levels of TNF-α released by immune cells are usually detected during the early stages of liver disease and F


Article

Journal of Agricultural and Food Chemistry potential agents for the therapy of hepatic fibrosis. However, it should be noted that liver fibrosis treatment should take into account the versatility of its pathogenesis and should act upon all pathways involved, beginning with HSC activation and ECM deposition. As a widely consumed vegetable worldwide, the current findings will no doubt have a positive effect on the search for protection against hepatic fibrosis, However, considering the contents analysis results in the current study, it may be difficult to acquire the necessary amount of the active compounds to protect against hepatic fibrosis unless a sufficient intake of asparagus can be ensured. It may therefore be necessary to produce green asparagus extracts or compounds and use them as a food supplement. Further study of the bioactive components from green asparagus on suppressing the development of hepatic fibrosis in vivo and at the clinical level will be necessary.

■

(6) Reeves, H. L.; Friedman, S. L. Activation of hepatic stellate cells − a key issue in liver fibrosis. Front. Biosci., Landmark Ed. 2002, 7, d808−d826. (7) Bataller, R.; Brenner, D. A. Liver fibrosis. J. Clin. Invest. 2005, 115, 209−218. (8) Lim, Y. S.; Kim, W. R. The global impact of hepatic fibrosis and end-stage liver disease. Clin. Liver Dis. 2008, 12, 733−746 vii. . (9) Sun, Z.; Huang, X.; Kong, L. A new steroidal saponin from the dried stems of Asparagus of f icinalis L. Fitoterapia 2010, 81, 210−213. (10) Terada, K.; Honda, C.; Suwa, K.; Takeyama, S.; Oku, H.; Kamisako, W. Acetylenic compounds isolated from cultured cells of Asparagus of f icinalis. Chem. Pharm. Bull. 1995, 43, 564−566. (11) Notaro, G.; Piccialli, V.; Sica, D. 3β,5α,6β-6P-Trihydroxylated sterols with a saturated nucleus from two populations of the marine sponge clzona copiosa. J. Nat. Prod. 1991, 54, 1570−1575. (12) Ge, F.; Wang, Y.; Wang, J.; Yu, H.; Li, Z. Research on main bioactive components in red fermented rice. J. Kunming Univ. Sci. Technol. 2012, 37, 61−64. (13) Ioannou, E.; Abdel-Razik, A. F.; Zervou, M.; Christofidis, D.; Alexi, X.; Vagias, C.; Alexis, M. N.; Roussis, V. 5α,8α-Epidioxysterols from the gorgonian Eunicella cavolini and the ascidian Trididemnum inarmatum: isolation and evaluation of their antiproliferative activity. Steroids 2009, 74, 73−80. (14) Xu, Y.; Zhang, H. W.; Yu, C. Y.; Lu, Y.; Chang, Y.; Zou, Z. M. Norcyperone, a novel skeleton norsesquiterpene from Cyperus rotundus L. Molecules 2008, 13, 2474−2481. (15) Pei, Y.; Wu, Q.; Shi, Y. Triterpenoids and other constituents from Euphorbia humif usa. J. Chin. Chem. Soc. 2007, 54, 1565−1572. (16) Wang, B.; Li, J.; Zhao, Y.; Hu, C. Study of triterpenes from Buddleia of f icinalis. J. Beijing Med. Univ. 1996, 28, 472−477. (17) Yang, Q.; Yang, C. Steroidal constituents of Aspidistra elatior from Yongshan, Yunnan. Acta. Bot. Yunnanica 2000, 22, 109−115. (18) Luo, J.; Li, L.; Kong, L. Preparative separation of phenylpropenoid glycerides from the bulbs of Lilium lancifolium by highspeed counter-current chromatography and evaluation of their antioxidant activities. Food Chem. 2012, 131, 1056−1062. (19) Pitchai, P.; Vlagi, R.; Mohan, P. S.; Gengan, R. M. A novel alkaloid,four alkaloid precursors and a coumarin form Feronia Iimonia. Indian J. Chem., Sect. B: Org. Chem. Incl. Med. Chem. 2012, 51B, 1771− 1775. (20) Terada, K.; Honda, C.; Suwa, K.; Takeyama, S.; Oku, H.; Kamisako, W. Acetylenic compounds isolated from cultured cells of Asparagus of f icinalis. Chem. Pharm. Bull. 1995, 43, 564−566. (21) Xu, K. P.; Zou, H.; Li, F. S.; Xiang, H. L.; Zou, Z. X.; Long, H. P.; Li, J.; Luo, Y. J.; Li, Y. J.; Tan, G. S. Two new selaginellin derivatives from Selaginella tamariscina (Beauv.) Spring. J. Asian Nat. Prod. Res. 2011, 13, 356−360. (22) Cheng, Y.; Zhou, J.; Ding, Z. Phenolic Constituents from Manglietia crassipes. Acta Bot. Yunnanica 2000, 22, 365−367. (23) Ding, Y.; Guo, Y.; Ren, Y.; Dou, D.; Li, Q. Isolation of flavonoids from male flowers of Eucommia ulmoides and their antioxidantive activities. Chin. Tradit. Herb. Drugs 2014, 45, 323−327. (24) Ni, F.; Chen, Z.; Xu, Q.; Yang, S.; Chen, D. Chemical constituents from Rhodiola sachalinensis. Chin. Tradit. Herb. Drugs 2013, 44, 798−802. (25) Wang, P.; Luo, J.; Yang, M.; Kong, L. Chemical constituents of Lobelia chinensis. Chin. Tradit. Herb. Drugs 2013, 44, 794−797. (26) Xu, J.; Zeng, S.; Qu, H. Chemical constituents from Violayedoensis. Chin. Tradit. Herb. Drugs 2010, 41, 1423−1425. (27) Li, Y.; Li, X.; Kim, M. K.; Jung, S.; Choi, H. D.; Rho, J. R.; Son, B. W. Golmaenone, a new diketopiperazine alkaloid from the marinederived fungus Aspergillus sp. Chem. Pharm. Bull. 2004, 52, 375−376. (28) Qin, L.; Li, B.; Guan, J.; Zhang, G. Chemical study on Aspergillus sp. Chin. J. Appl. Environ. Biol. 2007, 13, 66−68. (29) Li, F.; Deng, S.; Rao, Z.; Wu, H.; Xu, J.; Liu, X.; Zhong, H.; Zhao, D. A study on the chemical constituents of the South China sea hard coral Montipora ramosa bernard. Chin. J. Mar. Drugs 1997, 61, 4− 6.

AUTHOR INFORMATION

Corresponding Authors

*(Y.Z.) Mail: Department of Traditional Chinese Medicine, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China. Phone: +86-24-23986521. E-mail: [email protected]. *(X.B.) Mail: College of Life Science, Liaoning University, 66 Chongshan Road, Shenyang 110036, China. Phone: +86-2462202232. Fax: +86-24-62202232. E-mail: [email protected], [email protected]. Author Contributions ∥

C.Z. and C.J. contributed equally.

Funding

The work was financially supported by a program of Liaoning Excellent Talents in University to X.B. (LETU LR 2014001); the National Science Foundation of China to X.B. (Grants 81272333 and 81472821); and the Construction of R&D institute of state original new drug at Benxi of Liaoning Province to Y.Z. (2009ZX09301-012-105B). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We express our gratitude to Senior Engineer Wen Li and Yi Sha from Shenyang Pharmaceutical University for the measurements of NMR spectra. We thank Dr. Alan K. Chang (Liaoning University) for revising the language of the manuscript.

■

REFERENCES

(1) Li, C.; Mao, R.; Li, Z.; Wang, Y.; Liu, X. Composition analysis and nutritional evaluation of the asparagus. Mod. Food Sci. Technol. 2011, 27, 1260−1263. (2) Hafizur, R. M.; Kabir, N.; Chishti, S. Asparagus officinalis extract controls blood glucose by improving insulin secretion and β-cell function in streptozotocin-induced type 2 diabetic rats. Br. J. Nutr. 2012, 108, 1586−1595. (3) Kim, B. Y.; Cui, Z. G.; Lee, S. R.; Kim, S. J.; Kang, H. K.; Lee, Y. K.; Park, D. B. Effects of Asparagus of f icinalis extracts on liver cell toxicity and ethanol metabolism. J. Food Sci. 2009, 74, H204−H208. (4) Mormone, E.; George, J.; Nieto, N. Molecular pathogenesis of hepatic fibrosis and current therapeutic approaches. Chem.-Biol. Interact. 2011, 193, 225−231. (5) Sanchez-Valle, V.; Chavez-Tapia, N. C.; Uribe, M.; MendezSanchez, N. Role of oxidative stress and molecular changes in liver fibrosis: a review. Curr. Med. Chem. 2012, 19, 4850−4860. G


Article

Journal of Agricultural and Food Chemistry (30) Zhang, W.; Xiao, K.; Yang, G.; Chen, H. New compounds named sanleng acid from Sparganiun stoloniferum. Chin. Tradit. Herb. Drugs 1995, 26, 125−126. (31) Sharma, A.; Chattopadhyay, S.; Mamdapur, V. R. PPL catalyzed monoesterification of α,ω-dicarboxylic acids. Biotechnol. Lett. 1995, 17, 939−942. (32) Bi, X.; Xia, X.; Mou, T.; Jiang, B.; Fan, D.; Wang, P.; Liu, Y.; Hou, Y.; Zhao, Y. Anti-tumor activity of three ginsenoside derivatives in lung cancer is associated with Wnt/β-catenin signaling inhibition. Eur. J. Pharmacol. 2014, 742, 145−152. (33) Friedman, S. L. Mechanisms of hepatic fibrogenesis. Gastroenterology 2008, 134, 1655−1669. (34) Rockey, D. C. Translating an understanding of the pathogenesis of hepatic fibrosis to novel therapies. Clin. Gastroenterol. Hepatol. 2013, 11, 224−231 e1−5. . (35) Friedman, S. L. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol. Rev. 2008, 88, 125−172. (36) Lee, U. E.; Friedman, S. L. Mechanisms of hepatic fibrogenesis. Best. Pract. Res. Clin. Gastroenterol. 2011, 25, 195−206. (37) Jiao, J.; Friedman, S. L.; Aloman, C. Hepatic fibrosis. Curr. Opin. Gastroenterol. 2009, 25, 223−229. (38) George, J.; Roulot, D.; Koteliansky, V. E.; Bissell, D. M. In vivo inhibition of rat stellate cell activation by soluble transforming growth factor β type II receptor: a potential new therapy for hepatic fibrosis. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 12719−12724. (39) Safadi, R.; Friedman, S. L. Hepatic fibrosis − role of hepatic stellate cell activation. MedGenMed 2002, 4, 27. (40) Mauviel, A. Transforming growth factor-β: a key mediator of fibrosis. Methods Mol. Med. 2005, 117, 69−80. (41) Gold, E. J.; Zhang, X.; Wheatley, A. M.; Mellor, S. L.; Cranfield, M.; Risbridger, G. P.; Groome, N. P.; Fleming, J. S. βA- and βCactivin, follistatin, activin receptor mRNA and βC-activin peptide expression during rat liver regeneration. J. Mol. Endocrinol. 2005, 34, 505−515. (42) Takamura, K.; Tsuchida, K.; Miyake, H.; Tashiro, S.; Sugino, H. Activin and activin receptor expression changes in liver regeneration in rat. J. Surg. Res. 2005, 126, 3−11. (43) Chabicovsky, M.; Herkner, K.; Rossmanith, W. Overexpression of activin β(C) or activin β(E) in the mouse liver inhibits regenerative deoxyribonucleic acid synthesis of hepatic cells. Endocrinology 2003, 144, 3497−3504. (44) Lau, A. L.; Kumar, T. R.; Nishimori, K.; Bonadio, J.; Matzuk, M. M. Activin βC and βE genes are not essential for mouse liver growth, differentiation, and regeneration. Mol. Cell. Biol. 2000, 20, 6127−6137. (45) Vejda, S.; Erlach, N.; Peter, B.; Drucker, C.; Rossmanith, W.; Pohl, J.; Schulte-Hermann, R.; Grusch, M. Expression of activins C and E induces apoptosis in human and rat hepatoma cells. Carcinogenesis 2003, 24, 1801−1809. (46) Wada, W.; Maeshima, A.; Zhang, Y. Q.; Hasegawa, Y.; Kuwano, H.; Kojima, I. Assessment of the function of the βC-subunit of activin in cultured hepatocytes. Am. J. Physiol.−Endocrinol. Metab. 2004, 287, E247−E254. (47) Wada, W.; Medina, J. J.; Kuwano, H.; Kojima, I. Comparison of the function of the β(C) and β(E) subunits of activin in AML12 hepatocytes. Endocr. J. 2005, 52, 169−175.

H


Asparagus officinalis L. - ACS Publications - American Chemical Society

Recommend Documents