Euryachincoside, a Novel Phenolic Glycoside with Anti-Hepatic Fibrosis Activity from Eurya chinensis

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• Plant Materials
• Extraction and isolation
• Acid hydrolysis of ECS and absolute configuration determination of sugar
• Cell line culture and treatments
• Intracellular ROS measurement
• Animals and treatments
• Histological analysis
• mRNA expression and Western-blot analysis
• Elisa assays
• Statistical Analysis
• Contributorsʼ Statement
• References

Abstract

Eurya chinensis has been recorded as a folk medicine traditionally used for treatment of a variety of symptoms. However, the phytochemical and pharmacological investigations of this plant are still scarce. A novel phenolic glycoside named Euryachincoside (ECS) was isolated by chromatographic separation from E. chinensis, and its chemical structure was identified by analysis of HRMS and NMR data. Its anti-hepatic fibrosis effects were evaluated in both HSC-T6 (rat hepatic stellate cells) and carbon tetrachloride (CCl₄)-induced mice with Silybin (SLB) as the positive control. In an in vitro study, ECS showed little cytotoxicity and inhibited transforming growth factor-beta (TGF-β)-induced Collagen I (Col1) along with alpha-smooth muscle actin (α-SMA) expressions in HSC-T6. An in vivo study suggested ECS significantly ameliorated hepatic injury, secretions of inflammatory cytokines, and collagen depositions. Moreover, ECS markedly mediated Smad2/3, nuclear factor kappa B (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways both in vitro and vivo. These present findings confirmed that ECS is a novel phenolic glycoside from E. chinensis with promising curative effects on hepatic fibrosis, and its mechanisms may include decreasing extracellular matrix accumulation, reducing inflammation and attenuating free radicals via Smad2/3, NF-κB and Nrf2 signaling pathways, which may shed light on the exploration of more effective phenolic glycoside-based anti-fibrotic agents.

Introduction

Chronic liver diseases, one of the leading causes for death with an estimated 2 million deaths annually, ranging from mild steatosis, nonalcoholic steatohepatitis, fibrosis, liver cirrhosis to end-stage carcinoma, have attracted substantial attention [1], [2]. Among these maladies, hepatic fibrosis is an indispensable phase in the progression of chronic hepatic diseases, representing a major public health problem [3]. Fibrosis is a pathophysiological progression of abnormal hyperplasia of syndesm during healing, specifically manifested as extravagant accumulation of ECM responding to chronic liver injury [4], which can interfere with the structure and function of hepatic cells [5]. At present, the cure of hepatic fibrosis is still difficult and the current treatment is just to guard against its further deterioration. Meanwhile, drug therapies are often accompanied by side effects in clinical practice [6], [7], [8]. Therefore, it is urgent to develop novel therapeutic agents for hepatic fibrosis.

Eurya chinensis R.Br belongs to the genus Eurya, the family Thaceae. It grows all over southern China and its stems and leaves are often added to tea or pastry in daily life [9], [10]. It has been also recorded as an ethnodrug to cure a diversity of ailments especially hepatic diseases in Guangdong, Fujian, and Jiangxi provinces by Chinese Regional Pharmacopoeia [11], [12]. It was reported that inflammation-related signaling pathways might be affected in the liver tissues of mice treated with E. chinensis extract [13]. Recently, phytochemistry studies on the E. chinensis started to afford a series of novel diterpenes which have been proved to possess promising anti-inflammatory activities [14], [15], [16]. However, as a great potential phytomedicine, the investigations of its phytochemical and pharmacological properties are still scarce, and the scientific basis for liver protection of this plant has never been reported.

With the intent of exploring for structurally various and biologically potent secondary metabolites from E. chinensis, one novel phenolic glycoside named ECS was isolated from its branches. To the best of our knowledge, there are no reports of phenolic acid glycosides in the treatment of liver fibrosis. However, phenolic acids have been demonstrated to possess the effects of anti-liver fibrosis by anti-inflammation and anti-oxidation [17], and wetherefore, speculate that ECS may also have the similar abilities. Thus, this present study was aimed to isolate and structural elucidate of ECS from E. chinensis and investigate its anti-hepatic fibrosis effects as well as its possible underlying mechanisms.

Results and Discussion

The compound ([Fig. 1]) was obtained as a yellow amorphous powder, and its molecular formula was deduced as C₂₉H₂₂O₁₄ from its ¹³C NMR data and HRESIMS data wherein a deprotonated molecular ion [M – H]⁻ was observed at m/z 593.0934, indicating an unsaturation equivalence of 19. The existence of a trans-cinnamoyl moiety in the compound was evidenced by trans-olefinic proton signals at δ _H 6.71 and 7.80 (each 1H, d, J = 16.0 Hz), aromatic proton signals at δ _H 7.78 (2H, overlapped) and 7.47 (3H, overlapped) and by carbon resonances at δ _C 164.9, 146.4, 133.9, 130.9, 129.1 (2C), 128.6 (2C), 117.2 ([Table 1]). A sugar group was readily indicated by the observed signals at δ _H 5.74 (1H, d, J = 8.2 Hz), 4.88 (1H, t, J = 8.8 Hz), 4.63 (1H, m), 4.05 (1H, m), 3.96 (1H, m), 3.78 (1H, t, J = 8.8 Hz), 3.47 (1H, t, J = 8.4 Hz), and carbon signals at δ _C 94.0, 75.3, 72.6, 72.4, 69.5, 66.0. The sugar moiety was established as D-configuration by acid hydrolysis of the compound and sequential derivatization with L-cysteine methyl ester and arylisothiocyanate followed by HPLC analysis (Fig. 9S, Supporting Information). Besides, the relatively large coupling constant, 8.2 Hz, of the anomeric proton revealed that the glucose was in β-configuration. Apart from the signals of the groups of trans-cinnamoyl and glucose, the remaining ones could be ascribed to 2 carbonyl and 12 aromatic signals. Among these signals, one characteristic pair of signals with close chemical shifts occurred in the downfield where NMR spectra could be detected. Additionally, the correlations from C-7″ (δ _C 169.2) to H-3″ (δ _H 7.20, s), as well as from C-7‴ (δ _C 167.4) to H-3‴ (δ _H 7.07, s) could be observed in the HMBC experiment. These aforementioned data suggested the presence of a 1,1′-(3,3′,4,4′-tetrahydroxy) dibenzofurandicarboxyl group in the molecule. It was firstly discovered in the plants belonging to the Mallotus (Euphorbiaceae) [18], [19], [20]. The anomeric proton at δ _H 5.74 (H-1) showed correlation to C-1′ (δ _C 164.9) in the HMBC spectrum, suggested the trans-cinnamoyl moiety located at C-1. The trans-cinnamoyl, glucose, and 1,1′-(3,3′,4,4′-tetrahydroxy) dibenzofurandicarboxyl groups accounted for 18 indices of hydrogen deficiency, the remaining one thus requiring the compound to be monocyclic. According to the downfield chemical shift of H-4 and H-6, the position of the 1,1′-(3,3′,4,4′-tetrahydroxy) dibenzofurandicarboxyl moiety was determined to be situated in C-4 and C-6 hydroxyls of glucose unit, and thus, a ring moiety was constructed in the molecule. Consequently, the structure of the compound was identified as 1-O-(E)-cinnamoyl-4,6-[1,1′-(3,3′,4,4′-tetrahydroxy) dibenzofurandicarboxyl]-β-D-glucopyranose. Hence the compound is a phenolic glycoside isolated from E. chinensis, it was named as Euryachincoside (ECS).

In the development of hepatic fibrosis, HSCs are considered the strategic targets for the treatment [21], [22]. Hence, MTT assay was firstly launched to evaluate the cytotoxicity of ECS. As shown in [Fig. 2 a], the cell viabilities under the treatments ranging from 1.25 to 50 µM of ECS had no significant difference from that of the blank control, suggesting its cytosafety and excluding that its anti-fibrosis effects were attributed to the inhibition of cell viabilities. Since the in vitro study of high drug concentration is of little significance, 5, 10, 20 µM of ECS were therefore used as low, medium, and high concentrations, respectively, in the following in vitro experiments.

Activated by stimulations, the resting HSCs will express extracellular matrix proteins causing liver fibrosis, such as α-SMA and Col1 [23], and SLB treatment could alleviate these symptoms [24]. Hence, SLB was selected as the positive control to evaluate the anti-hepatic fibrosis activities of ECS. As illustrated in [Fig. 2 b, c], TGF-β induced significant elevations of α-SMA and Col1 at both gene and protein levels, while treatment with ECS or SLB remarkably reduced them. Meanwhile, the inhibition effects of ECS on ROS were also investigated. The results indicated that ECS or SLB treatment remarkably lowered the ROS production even though TGF-β significantly raised the intracellular ROS level in HSC-T6 cells ([Fig. 2 d]).

To further reveal the underlying mechanism of ECS, key proteins of Smad2/3, Nrf2 and NF-κB signaling pathways were then detected in vitro. Smad signaling pathway is the most prestigious one, giving rise to the ECM proliferation and crucial to the progression of hepatic fibrosis. The results of [Fig. 3 a] suggested that the phosphorylation of Smad2/3 was significantly increased by TGF-β in vitro. However, treatments with different concentrations of ECS lowered the phosphorylation levels. Generally, oxidative stress has been considered as an important factor in the pathophysiology of hepatic fibrosis [25], and Nrf2 has been recognized as the crucial transcription factor governing several antioxidant genes such as HO-1 and NQO-1 [26]. Given the positive results of ROS assay, the expressions of Nrf2, NQO-1, and HO-1 were then detected. As a result, the expression levels of these proteins were significantly upregulated by TGF-β and ECS further enhanced the levels in a concentration-dependent manner. These evidences suggested ECS may exert the ability of anti-oxidative stress by activating this pathway ([Fig. 3 b]). Moreover, since inflammation is often accompanied with liver fibrosis, the P65 expression in NF-κB signaling was detected and shown in [Fig. 3 c], expectedly, ECS suppressed the rasied nuclear P65 level caused by TGF-β.

Next, the anti-fibrotic effects of ECS were verified in animal model. The hepatic tissue sections of CCl₄ group displayed serious damages of hepatic architecture and obvious ECM depositions ([Fig. 4 a]). However, treatment with ECS or SLB showed significant alleviations in the histopathological changes. It is widely known that the functional enzymes such as ALT and AST abnormally arise due to liver structural lesions [27], and thus the levels of them are of great significance to diagnose hepatic functions [28]. In our case, CCl₄ gave rise to significant rises in serum ALT and AST compared to those of control group, while ECS or SLB treatment remarkably lowered these values ([Fig. 4 b]). Besides, ECS or SLB also significantly reduced the raised liver/body weight ratio induced by CCl₄ ([Fig. 4 c]). Moreover, the levels of IL-6 and TNF-α in hepatic tissue were rose significantly in CCl₄-treated mice, while these two cytokines were significantly declined by ECS or SLB treatment ([Fig. 4 d]). All these results manifested that ECS therapy could effectively reverse hepatic fibrosis processes induced by CCl₄.

To further explore whether the anti-fibrotic effects of ECS in vivo were also involved with the Smad2/3, Nrf2 or NF-κB signaling pathways, related protenins were determined by western blot assays. Similarly, CCl₄ treatment induced significant increases in the expressions of α-SMA and Col1, while treatment by ECS or SLB significantly lowered these expressions ([Fig. 5]). Consistent with the in vitro study, the phosphorylation levels of Smad2/3 and nuclear P65 were significantly suppressed by ECS or SLB treatment and ECS also upregulated the protein levels in the Nrf2 signaling pathway. These findings suggested ECS as a promising agent for hepatic fibrosis treatments and shed light on the exploration of more effective phenolic glycoside-based anti-fibrotic agents from traditional folk medicine. Meanwhile, this study could explain the ethnopharmacological effects of Eurya chinensis for its liver protection, at least in part.

Materials and Methods

Plant Materials

Stems of E. chinensis were collected from Enping District of Guangdong Province, China. The voucher specimen (No. 2 020 032 701) was identified by Dr. Yi Tong of Guangzhou University of Chinese Medicine (GZUCM) and has been deposited in GZUCM (Guangzhou, China).

Extraction and isolation

The stems of E. chinensis were dried and chopped (20.0 kg) followed by extracting with 60 L of 95% ethanol for 3 times at room temperate. The extraction was concentrated to dryness under a lowered pressure condition to yield a brown crude residue (800 g) followed by being suspended over 5 L of water and then separated with 5 L of EtOAc. Subsequently, the EtOAc-soluble portion (512 g) was subjected to CC over silica gel eluted with a gradient system comprised of petroleum ether/EtOAc to afford 12 fractions (A1 to A12). The obtained Fraction A4 (54 g) was applied to CC using a step gradient elution of CH₂Cl₂ and CH₃OH to obtain 6 fractions (A4A-A4F). Fraction A4B was further separated into 4 subfractions (A4B1-A4B4) by CC over MCI gel (EtOH/H₂O, 10% to 50%). Subsequently, 3 subfractions (A4B2A-A4B2C) were obtained from fraction A4B2 (12.6 g) over Sephadex LH-20 (MeOH) by CC. Subfraction A4B2B (1.8 g) was selected for purification by CC over Sephadex LH-20 (MeOH), followed by preparative HPLC (254 nm, CH₃CN/H₂O from 5% to 50%), yielding ECS (155 mg).

ECS: Yellow amorphous powder; [α]_D ²⁵ + 12.5 (c 0.1, MeOH); IR (KBr) ν _max 3400, 1723, 1635, 1531, 1335, 1160, 1098, 770 cm⁻¹; ¹H NMR and ¹³C NMR data, see [Table 1]; HRESIMS m/z 593.0934 [M – H]⁻ (calcd for C₂₉H₂₁O₁₄, 593.0925).

Acid hydrolysis of ECS and absolute configuration determination of sugar

ECS was subjected to hydrolysis with HCl for 6 h at 60 °C. After dying under a vacuum, the hydrolyzed product was dissolved in pyridine containing L-cysteine methyl ester hydrochloride, and heated for 60 min at 60 °C [29]. Arylisothiocyanate was then added to the mixture and incubated for another 60 min at 60 °C. After that, the reaction mixture was analyzed by Reversed – Phase HPLC and detected at 250 nm. The absolute configuration of ECS was confirmed by the comparative HPLC analysis with the derivatives of standard D-glucose and L-glucose, which were treated at the same way.

Cell line culture and treatments

HSC-T6 cells were purchased from the CAS Cell Bank and cultured in an incubator at 37 °C within the DMEM medium system with 10% FBS and 1% penicillin/streptomycin (Gibco). Before treatments, cells were seeded at the density of 4 × 10⁴ · mL⁻¹. MTT assay was carried out to evaluate the cell viabilities under a series of ECS concentrations (1.25, 2.5, 5, 10, 20, 50 µM) for 24 h. In mRNA expression and western-blot assays, 5 µM, 10 µM, 20 µM of ECS were added into medium of seeded cells 2 h prior to TGF-β stimulation (10 ng/mL) (PeproTech). After 24 h-treatment, cells were collected for further analysis.

Intracellular ROS measurement

Briefly, after 24 h-treatment, cells were then loaded with 0.1 µM of DCFH-DA (Biotine) at 37 °C for 20 min. After that, cells were carefully rinsed by PBS 3 times. The fluorescence values were detected by a microplate reader with 488 nm as excitation wavelength and 525 nm as emission wavelength at room temperature.

Animals and treatments

28 female Kunming mice (aged 6 – 8 weeks) were purchased from Laboratory Animal Center of GZUCM. All animals were raised under the specific pathogen-free condition with free access to standard chow as well as sterile water. All animal housing process and experiments were strictly reviewed, approved, and performed under the guidelines of the GZUCM Animal Care and Use Committee the approval number is 00 252 421, dated 2020 – 12 – 11. After one week of acclimatization, the animals were divided into 4 groups randomly indicated as below: the control group, CCl₄ group, CCl₄ + ECS group, and CCl₄ + SLB group. Animals in the CCl₄ group, CCl₄ + ECS group and CCl₄ + SLB group were treated with intraperitoneal injection of 20% CCl₄ soluted in olive oil (0.05 mL/g) twice a week. Animals in the CCl₄ + ECS group and CCl₄ + SLB group received ECS or SLB at the dose of 20 mg/kg daily. Animals in the control group were treated with an isovolumetric olive oil like those of other groups. After four weeks, all animals were sacrificed and their liver tissues, as well as serum, were harvested for further assays.

Histological analysis

The hepatic tissues were harvested and filled in the 4% paraformaldehyde buffer and then stained with H&E, Masson trichrome, along with Sirius red staining by standard procedures, respectively.

mRNA expression and Western-blot analysis

The mRNA expression assays were performed as previously described [30]. RNA was extracted using a RNA extraction kit (Tiangen) according to the manufacturerʼs protocol, and then reversely transcribed using PrimeScript^™ RT reagent Kit with gDNA Eraser (TaKaRa) and qPCR was further carried out with designed primers (Table 1S, Supporting Information) on the Applied Biosystems 7500 Real Time PCR System. The GAPDH as an internal reference was utilized to normalize the expressions of targeted genes. Western blot analysis was launched as described before [31] with GAPDH and PCNA as the internal references. The membrane was probed with antibodies for α-SMA (Cell Signaling Technology, mAb #19245), Col1 (Affinity Biosciences, AF0134), Smad2/3 (Affinity Biosciences, AF6367), p-Smad2/3 (Abcam Biosciences, ab272332), Nrf2 (Abcam Biosciences, ab137550), HO-1 (Cell Signaling Technology, mAb#82206), NQO1 (Cell Signaling Technology, mAb#62262), P65 (Cell Signaling Technology, mAb#3033), GAPDH (Affinity Biosciences, AF7021) and PCNA (Affinity Biosciences, AF0239) followed by exposure to horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies as appropriate (Cell Signaling Technology, #7074 and #7076). Protein expression was determined using an Enhanced Chemiluminescence kit (Annoron Biosciences, 1824c03). Electrophoresis and blotting were carried on a Bio-rad western blot system (1 645 050 and 1 703 930), and exposure was performed on a Tanon 4200SF gel imager.

Elisa assays

The concentrations of IL-6 and TNF-α in hepatic tissues were determined by Elisa kits (Dakewe). The methods of the assays were accroding to manufacturersʼ instructions.

Statistical Analysis

The data were expressed as mean ± standard deviation (SD) in this work. One-way analysis of variance [1] together with Tukey multiple comparison tests were performed with GraphPad Prism 5 software.

Contributorsʼ Statement

B. L. Li provided experimental design and manuscript drafting. H. J. Liang carried out chemical extraction, and purification. Q. R. Li critically discussed and confirmed data. Q. Wang performed in vitro experiments. Z. Y. Ao., Y. W. Fan, W. J. Zhang, X. Lian and J. Y. Chen performed in vivo experiments together. J. Yuan and J. W. Wu provided the final approval of the article.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

This present work was financially supported by the National Natural Science Foundation (No. 81 903 509).

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Correspondence

Publication History

Received: 04 January 2022

Accepted after revision: 14 April 2022

Accepted Manuscript online:
19 April 2022

Article published online:
23 January 2023

© 2022. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 Younossi ZM, Stepanova M, Younossi Y, Golabi P, Mishra A, Rafiq N, Henry L. Epidemiology of chronic liver diseases in the USA in the past three decades. Gut 2020; 69: 564-568
  CrossrefPubMedSearch in Google Scholar
• 2 Pimpin L, Cortez-Pinto H, Negro F, Corbould E, Lazarus JV, Webber L, Sheron N, Comm EHS. Burden of liver disease in Europe: Epidemiology and analysis of risk factors to identify prevention policies. J Hepatol 2018; 69: 718-735
  CrossrefPubMedSearch in Google Scholar
• 3 Balsano C, Alisi A, Nobili V. Liver fibrosis and therapeutic strategies: The goal for improving metabolism. Curr Drug Targets 2009; 10: 505-512
  CrossrefPubMedSearch in Google Scholar
• 4 Lee UE, Friedman SL. Mechanisms of hepatic fibrogenesis. Best Pract Res Clin Gastroenterol 2011; 25: 195-206
  CrossrefPubMedSearch in Google Scholar
• 5 Arteel GE, Naba A. The liver matrisome – looking beyond collagens. JHEP Rep 2020; 2: 100115
  CrossrefPubMedSearch in Google Scholar
• 6 Tan Z, Sun HB, Xue TX, Gan CL, Liu HY, Xie YT, Yao YQ, Ye TH. Liver fibrosis: Therapeutic targets and advances in drug therapy. Front Cell Dev Biol 2021; 9: 730176-730193
  CrossrefPubMedSearch in Google Scholar
• 7 Fang YY, Hegazy L, Finck BN, Elgendy B. Recent advances in the medicinal chemistry of farnesoid x receptor. J Med Chem 2021; 64: 17545-17571
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 9 Chang ZF, Chen JL, Fan DM. Study on the effect of medical plants of Theaceae. J Beijing Univ TCM 1996; 19: 28-30
  PubMedSearch in Google Scholar
• 10 Yu B, Zeng GF. Flora reipublicae popularis sinicae. Beijing: Science Press; 1998
  Search in Google Scholar
• 11 Committee HR. Chinese Herbal Medicine in Huiyang Area of Guangdong Province. Huiyang City: Guangdong Huiyang area garrison, Guangdong Huiyang District Revolutionary Committee; 1969
  Search in Google Scholar
• 12 Editorial Board of Chinese Materia Medica of State Administration of Traditional Chinese Medicine. Chinese Materia Medica. Shanghai: Shanghai Science and Techology Press; 1999
  Search in Google Scholar
• 13 Fang J, Hu YX, Ying HZ, Yu WY, Jin XY, Yu CH. Identification of MicroRNAs from Eurya chinensis and function analysis on their target genes. Chin Tradi and Herb Drugs 2015; 46: 80-85
  PubMedSearch in Google Scholar
• 14 Song JL, Yuan Y, Nie LH, Li BL, Qin XB, Li Y, Wu JW, Qiu SX. A new ent-kaurane diterpene derivative from the stems of Eurya chinensis R.Br. Nat Prod Res 2018; 32: 182-188
  CrossrefPubMedSearch in Google Scholar
• 15 Song JL, Yuan Y, Nie LH, Li BL, Qin XB, Li Y, Wu JW, Qiu SX. Two new ent-kaurane diterpenes from the stems of Eurya chinensis . J Asian Nat Prod Res 2018; 20: 962-968
  CrossrefPubMedSearch in Google Scholar
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