The antioxidant and anti-hepatic fibrosis activities of acorns (Quercus liaotungensis) and their natural galloyl triterpenes

Journal of Functional Foods 46 (2018) 567–578

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The antioxidant and anti-hepatic fibrosis activities of acorns (Quercus liaotungensis) and their natural galloyl triterpenes

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Jing Xua,b, Xude Wanga,b, Guangyue Sua,b, Jiayin Yuea, Yuanyuan Suna,b, Jiaqing Caoa,b, ⁎ ⁎ Xiaoshu Zhanga,b, , Yuqing Zhaoa,b, a b

School of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China Key Laboratory of Structure-based Drug Design and Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Acorn Galloyl triterpene esters Antioxidant Anti-hepatic fibrosis

Acorn is an edible plant and it has also been used as traditional food. In our investigation of the antioxidant and anti-hepatic fibrosis constituents of acorns (Quercus liaotungensis), the novel galloyl triterpenes were discovered, their potential mechanisms were investigated by microscope observation, and western blot. Evaluation of their antioxidant and antiproliferation against t-HSC/Cl-6 cells indicated that acorn can be used as antioxidative and anti-hepatic fibrosis food, and the galloyl triterpenes may represent attractive ingredients. Furthermore, the novel galloyl triterpenes were rationally designed by incorporation of gallic acid into phyto-triterpenes through esterification, and obtained 9 compounds. With radical scavenging and ferrous ion chelating methods revealed their excellent antioxidant activities. Evaluation of anti-hepatic fibrosis activities indicated that galloyl triterpenes exhibited excellent inhibition activities than substrate. The galloyl triterpenes offers an intriguing solution for naturally derived antioxidants and liver protector, and maybe is invaluable for further development of anti-fibrosis drugs.

1. Introduction Antioxidants are a class of molecules able to effectively eliminate free radicals derived from oxygen, and are widely used as ingredients in dietary supplement for preventing diseases (Birosová, Mikulásová, & Vaverková, 2005). Under environmental stress, free radicals levels increased dramatically. This might result in a significant insult to cellular structures, causing tissue damage (Rufino, Fernandes, Alves, & Brito, 2009). Many scientists are currently dedicated to discovering naturally derived and safe antioxidants. Liver fibrosis is a public health problem that results in significant morbidity and mortality. It indicates progress of early phase cirrhosis. Oxidative stress is a key regulator of liver fibrosis, which damages hepatocytes and initiates the synthesis of proinflammatory mediators (Yang et al., 2016). Most studies have confirmed that the activation of hepatic stellate cells (HSC) is the reason for liver fibrosis. Study showed oxidative stress, as a key trigger, can stimulate HSC activation, cause cell proliferation and overproduction of extracellular matrix (ECM), and finally exacerbate hepatic damage and lead to liver fibrosis (Hazra et al., 2004). As well known, liver fibrosis is reversible. However, once further deterioration leads to severe cirrhosis and liver cancer, it will be irreversible (Sanchez-Valle, Chavez-Tapia, Uribe, & Sanchez, 2012). It

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is important to prevent the progress of liver fibrosis or reversing its pathological process. Therefore, the discovery of novel HSC inhibitors may potentially suppress the progression of pathological process. Acorn, which is the nut of the oak, is a ubiquitous natural resource. Many of its species play important roles in food production and livestock breeding (Cantos, Espin, Lopez, & Barberan, 2003). In the ongoing research, acorn as functional food not only rich contain nutrients, but also have various biological activities, such as antioxidant, antibacterial, antitumor, neuroprotection, prevention of degenerative diseases (Cantos et al., 2003; Luísa et al., 2013; Luísa et al., 2015). In the previous study, phytochemical investigations of the acorn afforded three new galloyl triterpenes and other triterpenes (Jing, Jiaqing, Jiayin, Xiaoshu, & Yuqing, 2018). In this paper, we are based on previous chemical studies, it is the first to analysis the anti-hepatic fibrosis activities of acorns (Quercus liaotungensis), these chemical and biological results indicated that galloyl triterpenes in functional foods may represent attractive candidates for remedy anti-hepatic fibrosis levels. As one of the most abundant natural compounds, gallic acid are ubiquitously present in acorn and other foods. It is well known that ester derivatives of gallic acid usually exhibit improved biological activities. Methyl gallate is a potent inhibitor for oral bacteria and the widely used lauryl gallate is an effective inhibitor for xanthine oxidase

Corresponding authors at: School of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China. E-mail addresses: [email protected] (X. Zhang), [email protected] (Y. Zhao).

https://doi.org/10.1016/j.jff.2018.05.031 Received 7 February 2018; Received in revised form 14 May 2018; Accepted 19 May 2018 Available online 26 May 2018 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.

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DMEM medium and FBS were bought from Thermo Fisher Scientific Co., Ltd. Cl-caspase-3 (Cell Singaling Technology, Inc., Danvers, MA, USA), Cl-caspase-9 (Cell Singaling Technology Inc), Bcl-2 (Santa Cruz Biotechnology, Inc), BAX (Proteintech Group, Inc), Nrf2, HO-1 (Abcam, UK), β-actin (Nanjing, China).

(Ortega et al., 2003). Triterpenes with their basic common structure, exist in nature and show interesting biological activities (Fontanay, Grare, & Mayer, 2008). Meanwhile, the study and design of new triterpenes derivatives with different structural modifications for better therapeutic activity is a growing ongoing research field (Mishra & Tiwari, 2011). Hence, we assumed that combining gallic acid and natural triterpene via esterification may yield valuable results, and these galloyl triterpenes may naturally exist with better safety (Ogata, Saito, Matsuo, Maeda, & Tanaka, 2016). Several kinds of galloyl triterpenes derivatives have not been synthesized together, making the study of their structural/functional relationship particularly challenging. This study is the first to analysis the anti-hepatic fibrosis activities of acorns, and gallic acid derivatives of oleanolic acid (1), ursolic acid (2), hederagenin (3), betulinol (4), and betulinic acid (5) have been design and semi-synthesised to test for their biological activities. The synthetic method used was relatively mild and easily controllable. A total of 9 galloyl triterpene derivatives were obtained. These compounds were evaluated for antioxidant activity by DPPH, ABTS and FRAP, and for antifibrosis activity. Meanwhile, the potential mechanisms of acorn extract and the most active compound were also investigated by microscope observation, and western blot for the first time.

2.2. Plant materials The acorns of Quercus liaotungensis were collected from Jilin in China, in 2011, and identified as the fruits of Quercus liaotungensis by Prof. Jincai Lu (Shenyang Pharmaceutical University, Shenyang, China). After peeling, the kernels of acorn dried at 50 °C for 3 days, placed in our lab, where a specimen (No. XZ2011) was stored. 2.3. Preparation of triterpenoid-enriched Acorn extract (TAE) using macroporous resins The dried acorns (2.0 kg) were powered and extracted with 75% EtOH at 90 °C three times (each for 3 h). The extracts were filtered and evaporated under reduced pressure to obtain. The obtained acorn extracts (AE, 56.2 g) was then suspended in 2 L of 30% ethanol using ultrasound for 30 min in a sonication water bath (KQ 2200B, Kunshan Ultrasonic Equipment Co., Ltd., Kunshan, China). Subsequently, 1 kg (dry weigh) HPD100 macroporous resins was placed into 5 L conical flask. The flask was shaken (110 rpm) at 25 °C for 12 h. Then the macroporous resins were loaded into the glass column (80 × 1200) for triterpenoids. The column was eluted using 30% and 80% (V/V) ethanol concentrations for 10BV respectively. The 80% fraction (8.5 g) was evaporated under reduced pressure, to obtain triterpenoid-enriched acorn extract (TAE).

2. Materials and methods 2.1. Reagents and chemicals Oleanolic acid (1), ursolic acid (2), 3β, 23-dihydroxyolean-12-en28-oic acid (3), 3-O-galloyloleanolic acid (6), 3-O-galloylursolic acid (7), 23-acetoxy-3-O-galloyloleanolic acid (8), 3-acetoxy-23-O-galloyloleanolic acid (9), arjunolic acid (10), 2α,3β,19α,23-tetrahydroxyolean12-en-28-oic acid (11), 3-O-acetyloleanolic acid (12), 3β,23-O-acetylolean-12-en-28-oic acid (13), arjunglucoside II (14), arjunglucoside I (15), rotundic acid (16), 3-O-acetylursolic acid (17), 2α,3β,19α,23tetrahydroxyurs-12-en-28-oic acid (18), niga-ichigoside F1 (19), bayogenin (20), methyl barbinervate (21), germanicol acetate (22) were isolated from the extracts of the acorns (Quercus liaotungensis) in our previous studies (Jing et al., 2018). The dried acorns of Quercus liaotungensis (20 kg) were extracted with 75% EtOH (80 L × 3, 2 h each) under reflux, filtered and concentrated to give 15 L aqueous residue. The aqueous residue was partitioned with petroleum ether (PE), ethyl acetate (EtOAc), and n-butyl alcohol (n-BuOH) (15 L × 3 in each case), respectively. The EtOAc extract (200 g) was fractionated by silica gel column (300 mm × 1200 mm, 10 kg) using a gradient of CH2Cl2/MeOH (1:0 to 0:1) to obtain 10 fractions, A-J. Fraction A (17 g) was subjected to chromatography on silica gel (70 mm × 800 mm, 400 g) and then eluted with PE/EtOAc in increasing polarity to yield betulinol (4, 75 mg) and betulinic acid (5, 90 mg). Fractions F (20 g) were subjected to chromatography on silica gel (70 mm × 800 mm, 500 g) and then eluted with CH2Cl2/MeOH in increasing polarity to yield gallic acid (GA, 300 mg). Their structures were illustrated in Fig. 1. The identity of these compounds were confirmed by 1H NMR and 13C NMR, 2D NMR, and MS. Silica gel (mesh: 200-300, Qingdao Marine Chemistry Co., China). Purified water was purchased from Wahaha (Hangzhou, China). Methanol and other solvents (Analytical grade) were purchased from Kangkede (Tianjin, China). DPPH (2,2-diphenyl-1-picrylhydrazyl, purity > 98.0%), 2,2′azinobis (3-ethylbenzothiazo line-6-sulphonic acid) (ABTS), 2,4,6-Tris (2-pyridyl)-S-triazine (TPTZ) were obtained from Sigma Company (St. Louis, MO, USA). NMR (1D and 2D) spectra were measured on a Bruker ARX-300 spectrometer using TMS as the internal standard. HR-ESI-MS was measured on a Bruker micro-TOF-Q mass spectrometer. Optical rotations were measured on a Perkin-Elmer 241MC polarimeter (PerkinElmer Co., Waltham, USA) using methanol as the solvent. MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and DMSO were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

2.4. Triterpenoid content The total triterpenoid content was determined by using ultraviolet spectrophotometry with oleanolic acid as the standard (Li et al., 2017). An accurately weighed sample of the extract was transferred to a 25 ml volumetric flask to make a stock solution in methanol. One milliliter of the stock solution was removed and placed into a 5 ml volumetric flask and the solvent was evaporated at 40 °C. After this, 0.4 ml 5% vanillinglacial acetic acid solution and 0.8 ml perchloric acid were added to volumetric flask and further incubated at 60 °C for 30 min. After incubation, the volumetric flask was immediately cooled in an ice bath to room temperature and made up to mark with glacial acetic acid. The sample and standard (oleanolic acid) were processed identically, and the experiments were done in triplicate. Absorbance of the sample was recorded at 550 nm on a WFZ UV-2800AH spectrophotometer (UNIC, Shanghai, China) and the final concentration was calculated from the standard curve. 2.5. Analytical HPLC-UV HPLC-UV analyses were conducted using a YMC-Pack ODS-A column (150 × 4.6 mm, 5 μm) and HPLC (CXTH LC3000, China) with a flow rate of 0.8 ml/min and injection volume of 20 μl. The column temperature was maintained at 25 °C and the wavelength of detection was set at 203 nm. A gradient solvent system consisting of solvent A (0.1% acetonitrile formic acid) and solvent B (0.1% aqueous formic acid) was used as follows: 0–10 min, 30–50% A; 10–25 min, 50–80% A; 25–28 min, 80–100% A; 28–45 min, 100% A. Fig. 2 shows the HPLC-UV chromatograms of AE extract (5 mg/ml in MeOH; A), seventeen of the isolated compounds (combined into one single injection; B), and TAE (5 mg/ml in MeOH; C). 2.6. Quantification of major components in TAE by HPLC-UV A solution of TAE was freshly prepared in MeOH (5 mg/ml). 568

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Fig. 1. The chemical structures of the triterpenoids isolated from acorns (Quercus liaotungensis).

Appropriate amounts of compounds were dissolved in MeOH and filtered (0.22 μm) before HPLC analysis. HPLC-UV conditions were the same as described above.

yield obtained was around 91.5%. 0.0125 mol of 3,4,5-triacetyl benzoic acid in 30 ml dichloromethane, then 0.0025 mol triterpenoid substrates (1–5) were added, respectively. Further, DCC (0.015 mol) in 30 ml tetrahydrofuran solution was added. The reaction lasted at room temperature for 24 hours. The white and insoluble N,N-dicyclohexylurea (DCU) was filtered off and the intermediate product was obtained. Hydrazine hydrate (0.0375 mol) was added into the intermediate solution and allowed to react at room temperature for 0.5 h. Then, the mixture was poured into acetic acid (0.0375 mol) and H2O (100 ml), and extracted with EtOAc (100 ml × 3 in each case). The organic phase was washed to neutral, dried over anhydrous MgSO4, and concentrated in vacuo. The crude product was purified on silica gel column chromatography eluting with petroleum ether/EtOAc system to obtain 1a–5a.

2.7. General synthetic methods The synthetic procedures of target compounds were illustrated in Scheme 1. Firstly, 3,4,5-triacetyl benzoic acid was prepared by reacting acetic anhydride and gallic acid with concentrated sulfuric acid as the catalyst (i). The intermediate was then prepared with 3,4,5-triacetyl benzoic acid and triterpenoids (1–5) under DCC/DMAP conditions (ii). All the reactions were monitored by TLC analysis. Analytical TLC was carried out on silica gel plates GF254 (Qindao Haiyang Chemical, China), and spots were visualized with 10% alcoholic solution of sulfuric acid as the coloration. Commercial silica gel (200-300 mesh) was used for column chromatography. The final products 1a–5a were obtained with intermediates on hydrazine hydrate deacetylation. Their structures were illustrated in Fig. 3.

2.8.1. (3β)-3,4,5-trihydroxy-phenyl-olean-12-en-28-oic acid (1a) Yellow powder, 26 mg, yield: 23 %, mp: 179-181 °C, [α ]20 D +16.2° (c 0.1, MeOH); HRMS (ESI) m/z C37H52O7Na 631.3597 [M+Na]+, calculated for C37H52O7Na 631.3605. 1H NMR (C5D5N, 400 MHz) δ (ppm): 7.90 (s, 2H, H-2′,6′), 5.48 (t-like, 1H, H-12), 4.89 (dd, 1H, J = 11.8, 4.5 Hz, H-3), 3.31 (dd, 1H, J = 3.7, 13.7 Hz, H-18), 1.28 (s, 3H), 1.02 (s, 3H), 1.00 (s, 3H), 0.96 (s, 3H), 0.93 (s, 3H), 0.89 (s, 3H), 0.83 (s, 3H). 13 C NMR (C5D5N, 100 MHz) δ (ppm): 38.6 (C-1), 28.6 (C-2), 81.1 (C-3), 40.0 (C-4), 55.9 (C-5), 18.8 (C-6), 30.3 (C-7), 40.0 (C-8), 48.2 (C-9), 37.5 (C-10), 24.3 (C-11), 122.7 (C-12), 145.2 (C-13), 42.0 (C-14), 28.6 (C-15), 24.0 (C-16), 47.0 (C-17), 42.3 (C-18), 46.8 (C-19), 31.3 (C-20), 34.6 (C-21), 33.6 (C-22), 28.6 (C-23), 17.5 (C-24), 15.7 (C-25), 17.7 (C26), 26.5 (C-27), 180.6 (C-28), 33.6 (C-29), 24.1 (C-30), 122.2 (C-1′), 110.5 (C-2′, 6′), 148.1 (C-3′, 5′), 141.2 (C-4′), 167.2 (C-7′).

2.8. Preparation of compounds 1a–5a A 1000 ml three-necked flask was installed with a motor stirrer, water bath cooler, return flow cooling and thermometer. Acetic anhydride (2.6 mol) was added with few concentrated sulfuric acid as the catalyst. GA (0.5 mol) was then added in batches while stirring. The feeding speed was then controlled to maintain the temperature of the reaction system at around 60 °C. After feeding, it was stirred for 5-10 min. The reaction system was then placed in a 90 °C heating jacket to incubate for 2 h. The trace reaction progress was monitored using TLC. The solution was cool down, then filtered and dried in vacuum to obtain a white solid substance, which was 3,4,5-triacetyl benzoic acid. The 569

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Fig. 2. HPLC-UV chromatogram (detection wavelength 203 nm) of (A) EtOH extract of acorn (AE), (B) 17 triterpenoids isolated and identified in AE, and (C) triterpenoid-enriched acorn extract (TAE). The names of labelled peaks correspond to the compounds as shown in Fig. 1.

2.8.2. (3β)-3,4,5-trihydroxy-phenyl-ursen-12-en-28-oic acid (2a) Yellow powder, 21 mg; yield: 20%, mp: 195-197 °C, [α ]20 D +21.2° (c 0.1, MeOH); HRMS (ESI) m/z C37H52O7Na 631.3608 [M+Na]+, calculated for C37H52O7Na 631.3605. 1H NMR (C5D5N, 400 MHz) δ (ppm): 7.91 (s, 2H, H-2′,6′), 5.49 (t-like, 1H, H-12), 4.90 (dd, 1H, J = 11.6, 4.4 Hz, H-3), 2.65 (d, 1H, J = 11.5, H-18), 1.24 (s, 3H), 1.04 (s, 3H), 1.02 (s, 3H), 0.98 (s, 3H), 0.95 (s, 3H), 0.90 (br. s, 3H), 0.84 (br. s, 3H). 13C NMR (C5D5N, 100 MHz) δ (ppm): 38.6 (C-1), 28.7 (C-2), 81.2 (C-3), 39.9 (C-4), 55.9 (C-5), 18.8 (C-6), 30.3 (C-7), 40.3 (C-8), 48.2 (C-9), 37.4 (C-10), 24.3 (C-11), 125.9 (C-12), 139.7 (C-13), 42.9 (C-14), 28.7 (C-15), 23.9 (C-16), 48.4 (C-17), 53.9 (C-18), 39.8 (C-19), 38.7 (C-20), 33.7 (C-21), 30.4 (C-22), 29.1 (C-23), 15.9 (C-24), 17.9 (C-25), 17.6 (C26), 25.3 (C-27), 180.3 (C-28), 17.8 (C-29), 21.8 (C-30), 122.3 (C-1′), 110.5 (C-2′, 6′), 148.1 (C-3′, 5′), 141.3 (C-4′), 167.3 (C-7′).

Hz, H-3), 4.38 (d, 1H, J = 11.5 Hz, H-23), 4.08 (d, J = 11.5 Hz, H-23), 3.26 (dd, J = 3.7, 13.7 Hz, H-18), 2.00 (s, 3H, H-OAc), 1.23 (s, 3H), 0.99 (s, 3H), 0.95 (s, 3H), 0.93 (s, 3H), 0.86 (s, 3H), 0.83 (s, 3H). 13C NMR (C5D5N, 100 MHz) δ (ppm): 38.2 (C-1), 28.4 (C-2), 75.3 (C-3), 41.6 (C-4), 49.0 (C-5), 18.7 (C-6), 33.2 (C-7), 40.0 (C-8), 48.6 (C-9), 37.1 (C-10), 23.6 (C-11), 122.5 (C-12), 145.3 (C-13), 42.4 (C-14), 28.4 (C-15), 24.1 (C-16), 47.0 (C-17), 42.3 (C-18), 46.7 (C-19), 31.3 (C-20), 34.5 (C-21), 33.5 (C-22), 65.9 (C-23), 13.5 (C-24), 16.0 (C-25), 17.7 (C26), 26.3 (C-27), 180.5 (C-28), 33.6 (C-29), 23.9 (C-30), 121.5 (C-1′), 110.5 (C-2′, 6′), 148.1 (C-3′, 5′), 141.5 (C-4′), 167.2 (C-7′), 170.7 (C-8′), 21.4 (C-9′). Linkage position of galloyl of 3a-1 was determined through examination of HMBC cross peak correlations between H-23 (δH 4.08 and 4.38) and C-7′ (δc 167.2). Acetyl was attached to C-3 because of HMBC correlation between H-3 (δH 5.12) and C-8′ (δc 170.7).

2.8.3. (3β, 4α)-3-acetyl-23-3,4,5-trihydroxy-phenyl-olean-12-en-28-oic acid (3a-1) Yellow powder, 41 mg; yield: 39%, mp: 201-203 °C, [α ]20 D +28.6° (c 0.1, MeOH); HRMS (ESI) m/z C39H54O9Na 689.3673 [M+Na]+, calculated for C39H54O9Na 689.3660. 1H NMR (C5D5N, 400 MHz) δ (ppm): 7.90 (s, 2H, H-2′,6′), 5.45 (t-like, 1H, H-12), 5.12 (dd, 1H, J = 11.6, 4.6

2.8.4. (3β, 4α)-3-3,4,5-trihydroxy-phenyl-23-acetyl-olean-12-en-28-oic acid (3a-2) Yellow powder, 27 mg; yield: 25%, mp: 211-213 °C, [α ]20 D +73.1° (c 0.1, MeOH); HRMS (ESI) m/z C39H54O9Na 689.3663 [M+Na]+, calculated for C39H54O9Na 689.3660. 1H NMR (C5D5N, 400 MHz) δ (ppm): 7.90 (s, 2H, H-2′,6′), 5.49 (t-like, 1H, H-12), 5.34, (dd, 1H, J = 11.8, 570

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Scheme 1. i: Ac2O, H2SO4, 2h; ii: DCC, DMAP, THF, DCM, 24h; iii: for 1a–5a: hydrazine hydrate, 30min

110.6 (C-2′, 6′), 148.1 (C-3′, 5′), 141.4 (C-4′), 167.0 (C-7′), 170.9 (C-8′), 21.0 (C-9′). Linkage position of galloyl of 3a-2 was determined through examination of HMBC cross peak correlations between H-3 (δH 5.34) and C-7′ (δc 167.0). Acetyl was attached to C-23 because of HMBC correlation between H-23 (δH 3.96 and 4.16) and C-8′ (δc 170.9).

4.8 Hz, H-3), 3.96 (d, 1H, J = 11.4 Hz, H-23), 4.16 (d, 1H, J = 11.4 Hz, H-23), 3.31 (dd, J = 3.3, 13.6 Hz, H-18), 1.97 (s, 3H, H-OAc), 1.31 (s, 3H), 1.02 (s, 3H), 1.02 (s, 3H), 0.95 (s, 3H), 0.87 (s, 3H), 0.84 (s, 3H). 13 C NMR (C5D5N, 100 MHz) δ (ppm): 38.2 (C-1), 28.6 (C-2), 75.2 (C-3), 41.6 (C-4), 48.9 (C-5), 18.7 (C-6), 33.2 (C-7), 40.1 (C-8), 48.5 (C-9), 37.4 (C-10), 23.9 (C-11), 122.8 (C-12), 145.2 (C-13), 42.5 (C-14), 28.6 (C-15), 24.1 (C-16), 47.1 (C-17), 42.4 (C-18), 46.8 (C-19), 31.3 (C-20), 34.6 (C-21), 33.6 (C-22), 66.6 (C-23), 13.8 (C-24), 16.1 (C-25), 17.8 (C26), 26.4 (C-27), 180.6 (C-28), 33.6 (C-29), 24.0 (C-30), 122.0 (C-1′),

2.8.5. (3β, 4α)-3-hydroxy-23-3,4,5-trihydroxy-phenyl-olean-12-en-28-oic acid (3a-3) Yellow powder, 19 mg; yield: 17%, mp: 210-212 °C, [α ]20 D +26.5° (c

Fig. 3. The chemical structures of synthetic galloyl triterpenes and their starting substrates. 571

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0.1, MeOH); HRMS (ESI) m/z C37H51O8 623.3601 [M-H]−, calculated for C37H51O8 623.3609. 1H NMR (C5D5N, 400 MHz) δ (ppm): 7.93 (s, 2H, H-2′,6′), 5.50 (t-like, 1H, H-12), 5.15 (br. s, 1H, H-3), 4.68 (d, 1H, J = 11.5 Hz, H-23), 4.53 (d, 1H, J = 11.5 Hz, H-23), 3.30 (1H, H-18), 1.26 (s, 3H), 1.25 (s, 3H), 1.00 (s, 3H), 0.99 (s, 3H), 0.95 (s, 3H), 0.91 (s, 3H). 13C NMR (C5D5N, 100 MHz) δ (ppm): 39.1 (C-1), 28.4 (C-2), 71.9 (C-3), 42.4 (C-4), 49.0 (C-5), 19.0 (C-6), 33.3 (C-7), 40.0 (C-8), 48.8 (C-9), 37.4 (C-10), 24.0 (C-11), 122.6 (C-12), 145.3 (C-13), 42.4 (C-14), 28.4 (C-15), 24.1 (C-16), 47.0 (C-17), 42.4 (C-18), 46.7 (C-19), 31.3 (C-20), 34.5 (C-21), 33.6 (C-22), 67.0 (C-23), 13.2 (C-24), 16.2 (C25), 17.8 (C-26), 26.3 (C-27), 180.6 (C-28), 33.5 (C-29), 24.1 (C-30), 121.9 (C-1′), 110.4 (C-2′, 6′), 148.1 (C-3′, 5′), 141.3 (C-4′), 167.5 (C-7′). The observed long range correlations from H-23 (δH 4.53 and 4.68) to C-7′ (δc 167.5), indicated that galloyl of 3a-3 attached at C-23.

correlations between H-28 (δH δH 4.22 and 4.71) and C-7′ (δc 167.9). Acetyl was attached to C-3 because of HMBC correlation between H-3 (δH 4.68) and C-8′ (δc 171.0). 2.8.9. (3β)-3-3,4,5-trihydroxy-phenyl-20(29)-lupaene-28-oic acid (5a) Yellow powder, 18 mg; yield: 16%, mp: 200-202 °C, [α ]20 D +16.2° (c 0.1, MeOH); HRMS (ESI) m/z C37H51O7 607.3671 [M-H]−, calculated for C37H51O7 607.3679. 1H NMR (C5D5N, 400 MHz) δ (ppm): 7.91 (s, 2H, H-2′,6′), 5.52 (t-like, 1H, H-3), 4.91 (d, 1H, J = 4.3, H-29), 4.89 (d, 1H, J = 4.3, H-29), 1.80 (s, 3H), 1.08 (s, 3H), 1.04 (s, 3H), 0.92 (s, 3H), 0.88 (s, 3H), 0.71 (s, 3H). 13C NMR (C5D5N, 100 MHz) δ (ppm): 38.3 (C1), 27.9 (C-2), 80.5 (C-3), 38.3 (C-4), 56.4 (C-5), 19.2 (C-6), 34.3 (C-7), 40.8 (C-8), 50.4 (C-9), 37.4 (C-10), 22.7 (C-11), 25.8 (C-12), 38.1 (C13), 42.6 (C-14), 27.3 (C-15), 34.3 (C-16), 47.5 (C-17), 49.5 (C-18), 47.5 (C-19), 151.5 (C-20), 29.9 (C-21), 30.9 (C-22), 16.7 (C-23), 30.0 (C-24), 16.1 (C-25), 14.0 (C-26), 14.6 (C-27), 178.7 (C-28), 109.8 (C29), 18.2 (C-30), 121.6 (C-1′), 109.9 (C-2′, 6′), 147.5 (C-3′, 5′), 140.7 (C-4′), 166.7 (C-7′).

2.8.6. (3β, 4α)-23-hydroxy-3-3,4,5-trihydroxy-phenyl-olean-12-en-28-oic acid (3a-4) Yellow powder, 11 mg; yield: 8%, mp: 208-210 °C, [α ]20 D +31.3° (c 0.1, MeOH); HRMS (ESI) m/z C37H51O8 623.3632 [M-H]−, calculated for C37H51O8 623.3609. 1H NMR (C5D5N, 400 MHz) δ (ppm): 7.92 (s, 2H, H-2′,6′), 5.50 (t-like, 1H, H-12), 5.19 (br. s, 1H, H-3), 4.68 (d, 1H, J = 11.5 Hz, H-23), 4.53 (d, 1H, J = 11.5 Hz, H-23), 3.30 (1H, H-18), 1.25 (s, 6H), 1.00 (s, 3H), 0.99 (s, 3H), 0.94 (s, 3H), 0.90 (s, 3H). 13C NMR (C5D5N, 100 MHz) δ (ppm): 39.1 (C-1), 28.5 (C-2), 71.9 (C-3), 42.5 (C-4), 49.0 (C-5), 19.0 (C-6), 33.4 (C-7), 40.1 (C-8), 48.8 (C-9), 37.5 (C-10), 24.0 (C-11), 123.0 (C-12), 145.3 (C-13), 42.5 (C-14), 28.5 (C-15), 24.1 (C-16), 47.1 (C-17), 42.5 (C-18), 47.1 (C-19), 31.3 (C-20), 34.6 (C-21), 33.7 (C-22), 67.0 (C-23), 13.3 (C-24), 16.2 (C-25), 17.8 (C26), 26.4 (C-27), 180.3 (C-28), 33.5 (C-29), 24.1 (C-30), 121.9 (C-1′), 110.5 (C-2′, 6′), 148.1 (C-3′, 5′), 141.1 (C-4′), 167.3 (C-7′). The observed long range correlations from H-3 (δH 5.19) to C-7′ (δc 167.3), indicated that galloyl of 3a-4 attached at C-3.

2.9. Assay for DPPH radical scavenging activity The DPPH radical scavenging activity was carried by method reported with some modifications (Sá et al., 2014). 201.8 μmol/L DPPH stock solution was prepared with methanol and stored at 4 °C. The absorbance of DPPH stock solution was adjusted to 0.88 ± 0.02 to obtain DPPH working solution. 200 μl of DPPH working solution and 5 μl of test solution or Trolox solution at varied concentrations were successively added into each well of a 96-well plate. With 200 μl of DPPH working solution added into 5 μl of methanol as the blank, 3 duplicate wells were set for each concentration. The plate was placed in a dark room at room temperature for 30 min. The absorbance of the sample at 517 nm was measured by microplate reader (imark, BIORAD, USA).Three parallel operations were done. Trolox was used as positive control. Percentage DPPH radical scavenging activity = (A0 − Ai)/ A0 × 100, where A0: absorbance of the blank solution; Ai: absorbance of the reference or test solution.

2.8.7. (3β)-3-hydroxy-28-3,4,5-trihydroxy-phenyl-20(29)-lupaene (4a-1) Yellow powder, 37 mg; yield: 34%, mp: 226-228 °C, [α ]20 D +9.2° (c 0.1, MeOH); HRMS (ESI) m/z C37H53O6 593.3877 [M-H]−, calculated for C37H53O6 593.3899. 1H NMR (C5D5N, 400 MHz) δ (ppm): 7.91 (s, 2H, H-2′,6′), 4.88 (br. s, 1H, H-29), 4.74 (br. s, 1H, H-29), 4.71 (br. s, 1H, H-28), 4.24 (d, 1H, J = 11.1, H-28), 3.45 (t, 1H, J = 7.6 Hz, H-3), 1.74 (s, 3H), 1.23 (s, 3H), 1.03 (s, 3H), 1.00 (s, 3H), 0.99 (s, 3H), 0.85 (s, 3H). 13C NMR (C5D5N, 100 MHz) δ (ppm): 38.1 (C-1), 27.8 (C-2), 78.4 (C-3), 39.6 (C-4), 56.2 (C-5), 19.1 (C-6), 34.9 (C-7), 39.8 (C-8), 51.0 (C-9), 37.8 (C-10), 21.2 (C-11), 25.9 (C-12), 37.8 (C-13), 43.3 (C14), 27.8 (C-15), 35.3 (C-16), 43.3 (C-17), 49.4 (C-18), 48.3 (C-19), 150.9 (C-20), 30.6 (C-21), 41.5 (C-22), 28.6 (C-23), 15.2 (C-24), 16.5 (C-25), 16.7 (C-26), 16.7 (C-27), 63.0 (C-28), 110.6 (C-29), 19.7 (C-30), 121.7 (C-1′), 110.5 (C-2′, 6′), 148.1 (C-3′, 5′), 141.2 (C-4′), 167.9 (C-7′). The observed long range correlations from H-28 (δH 4.24 and 4.71) to C-7′ (δc 167.9), indicated that galloyl of 4a-1 attached at C-28.

2.10. Assay for ABTS radical scavenging activity The ABTS radical scavenging activity was employed to measure activity of compounds according to the method reported with a few modifications (Re et al., 1999). In this method, an antioxidant was added to a pre-formed ABTS radical solution and after a fixed time period the remaining ABTS+ was measured at 734 nm. ABTS+ was produced by reacting ABTS with oxidant solution (K2S2O8) at the volume ratio of 1:1, stored in the dark at room temperature for 12–16 h. The ABTS+ solution was diluted to give an absorbance at 0.80 ± 0.025 in 80% EtOH. 10 μl of test solution or Trolox solution at varied concentrations were added successively to 200 ul ABTS+ solution in each well of a 96-well plate, with 10 μl of methanol as the blank. 3 duplicate wells were set for each concentration. The plate was placed at room temperature for 30 min. The absorbance of the sample at 734 nm was measured by microplate reader (imark, BIO-RAD, USA).Three parallel operations were done. Trolox was used as positive control. Percentage ABTS radical scavenging activity = (A0 − Ai)/ A0 × 100, where A0: absorbance of the blank solution. Ai: absorbance of the reference or test solution.

2.8.8. (3β)-3-acetyl-28-3,4,5-trihydroxy-phenyl-20(29)-lupaene (4a-2) Yellow powder, 43 mg; yield: 40%, mp: 240-242 °C, [α ]20 D +19.7° (c 0.1, MeOH); HRMS (ESI) m/z C37H51O8 635.3986 [M-H]−, calculated for C37H51O8 635.4058. 1H NMR (C5D5N, 400 MHz) δ (ppm): 7.92 (s, 2H, H-2′,6′), 4.81(br. s, 1H, H-29), 4.74 (br. s, 1H, H-29), 4.71 (br. s, 1H, H-28), 4.68 (dd, 1H, J = 11.6, 4.5 Hz, H-3), 4.22 (d, 1H, J = 11.1, H-28), 2.07 (s, 3H), 1.73 (s, 3H), 0.98 (s, 3H), 0.96 (s, 3H), 0.87 (s, 3H), 0.86 (s, 3H), 0.77 (s, 3H). 13C NMR (C5D5N, 100 MHz) δ (ppm): 38.1 (C1), 27.8 (C-2), 81.0 (C-3), 38.8 (C-4), 55.9 (C-5), 18.7 (C-6), 34.6 (C-7), 39.8 (C-8), 50.7 (C-9), 37.5 (C-10), 21.2 (C-11), 25.8 (C-12), 37.5 (C13), 43.2 (C-14), 27.8 (C-15), 35.3 (C-16), 43.2 (C-17), 49.4 (C-18), 48.3 (C-19), 150.8 (C-20), 30.5 (C-21), 41.4 (C-22), 28.3 (C-23), 15.2 (C-24), 16.4 (C-25), 16.6 (C-26), 16.6 (C-27), 63.0 (C-28), 110.6 (C-29), 21.5 (C-30), 121.7 (C-1′), 110.5 (C-2′, 6′), 148.1 (C-3′, 5′), 141.4 (C-4′), 167.9 (C-7′), 171.0 (C-8′), 21.5 (C-9′). Linkage position of galloyl of 4a2 was determined through examination of HMBC cross peak

2.11. Assay for ferric reducing antioxidant power (FRAP) Ferric reducing antioxidant power (FRAP) assay was employed to measure activity of compounds according to the method reported with slight modifications (Sun et al., 2001). Firstly, 300 mmol/L acetic acid sodium acetate buffer solution (pH 3.6), 10 mmol/L TPTZ (40 mmol/L HCl) solution and 20 mmol/L FeCl3 solution were prepared. They were 572

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3. Results and discussion

mixed in a ratio of 5:1:1 to obtain the FRAP working solution. FeSO4 solution was prepared at varied concentrations to obtain a standard curve. 180 μl of FRAP working solution, 5 μl of standard solutions series, test solution or Trolox at varied concentrations were successively added into each well of a 96-well plate, with methanol as the blank, reacted at 37 °C for 30 min. The absorbance of the sample at 593 nm was measured by microplate reader (imark, BIO-RAD, USA). A standard curve was drawn with concentrations of FeSO4 solution.

3.1. Phytochemical studies of acorn Twenty two pentacyclic triterpenoids (1–22) were isolated from acorn. On the basis of their structural skeleton, these triterpenoids could be classified into three subclasses including oleanane, ursane and lupane derivatives. Fig. 2A shows the HPLC-UV profile (detection wavelength at 203 nm) of Acorn-EtOH extract (AE). Fig. 2B shows the HPLC-UV profile of the seventeen purified isolates (all combined into a single injection), other five compounds (4, 12, 13, 17, and 22) because of their small polarity are very late in peak time, which is difficult to detect under this HPLC condition. On the basis of the chromatograms shown in Fig. 2A and B, it was apparent that pentacyclic triterpenoids of oleanane type were the major compounds present in the AE. Based on our phytochemistry studies, triterpenoid-enriched acorn extract (TAE) was prepared from acorn powder as described above with a yield of 4.3%. UV-quantitative analysis indicated that the total triterpenoid content of TAE was 26.5% on the basis of dry weight, however, the total triterpenoid content of AE was just 5.6%. From the HPLC-UV (at 203 nm) profile of TAE (Fig. 2C), seventeen peaks were assigned based on comparison of their retention times with isolated standards. It is notable that triterpenoids were present as the major constituents in TAE (Fig. 2C). HPLC-UV was used to quantification of the major compounds in TAE, calibration curves were constructed for the major compounds (Supplementary information, Table S1). Because there are a lot of isomers that are difficult to separate under the same conditions, such as 1 and 2, 6 and 7, 8 and 9, 10 and 20, 11 and 18, 15 and 19, we measure the content of the isomer mixture. The quantification analyses based on % dried weight (DW) revealed that oleanolic acid and ursolic acid (5.2%, DW); 2α,3β,19α,23-tetrahydroxyolean-12en-28-oic acid and 2α,3β,19α,23-tetrahydroxyurs-12-en-28-oic acid (10.7%, DW); arjunglucoside I and niga-ichigoside F1 (7.9%, DW) were the major triterpenoids present in TAE (Table 1). The content and degree of separation of the galloyl triterpenes in TAE were low/poor. But the activities of them were predominant. Therefore, we used mild synthesis methods to abtain a series of galloyl triterpene derivatives. Oleanolic acid (1), ursolic acid (2), hederagenin (3), betulinol (4), and betulinic acid (5) represent three different types of pentacyclic triterpenes were obtained from acorn as a model set for esterification, gallic acid as the ester group (synthetic method in the above). Moreover, the activities in vitro were comparison between the galloyl triterpenes naturally occurring in acorn (6–9) and the synthetic ones (1a, 2a, 3a-1, and 3a-2). Other five new galloyl triterpenes (3a-3, 3a-4, 4a-1, 4a-2, and 5a) were synthesized to test that esterify with gallic acid whether can improve the activity of antioxidation and anti liver fibrosis.

2.12. Assay for anti-proliferative activities The t-HSC/Cl-6 cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). t-HSC/Cl-6 cells were cultured in DMEM media, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% 100 μg/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. Cell viability was measured using the MTT assay. Cells were seeded in each well containing 100 μl medium in 96 well micro titer plates at identical conditions. All compounds tested were dissolved and eventually further diluted in DMSO. After overnight incubation, the cells were treated with different test concentrations or carrier solvent alone in a final volume of 100 μl with five replicates each. The concentration of DMSO did not exceed 0.1%. After 48 h, 10 μl of MTT (5 mg/ml) was added to each well and the plate was incubated at 37 °C in the dark for 4 h. Supernatants were removed and the formazan crystals were dissolved in DMSO (100 μl/well). The solution was agitated with a vortex mixer for 10 min, and the absorbance was measured at 490 nm by microplate reader (imark, BIO-RAD, USA) to calculate 50% inhibition concentration (IC50).

2.13. DAPI staining t-HSC/Cl-6 cells were exposed to 40 µg/ml TAE, 4 µM compound 6, and 0.1% DMSO for 12 h and fixed with 4% (v/v) paraformaldehyde for 30 min at 4 °C, stained with DAPI at 37 °C in the dark for 30 min and then observed under a fluorescent microscope.

2.14. Western blotting analysis RIPA lysis buffer was employed to extract total cellular proteins, and the protein concentration was determined using the bicinchoninic acid. SDS-PAGE was performed in 10% gel with equal loading amount of protein per lane. After electrophoresis, the resolved protein bands were transferred to a polyvinylidene difluoride (PVDF) membrane, and the membrane was blocked with 5% bovine serum albumin (BSA) in TBST buffer for 1 h. After blocking, the membrane was incubated with a 1:1000 dilution of primary antibody against Cl-caspase-3, Cl-caspase-9, Bcl-2, BAX, Nrf2, HO-1 in 5% BSA and at 4 °C for overnight. Next, the PVDF membrane was washed with TBST containing 0.1% Tween-20 and was then incubated with a 1:5000 dilution of horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h. Positive bands were visualized on an X-ray film using an enhanced chemiluminescence system (Kodak).

3.2. Antioxidant activity The antioxidant activity of AE, TAE and all compounds were measured as free radical scavenging activity (DPPH, ABTS methods) and ferric reducing capacity by the FRAP method, the results are listed in Table 2. EC50 indicates the antioxidant concentration required to reduce the original free radical concentration to 50%. Usually, the antioxidant activity is higher if EC50 is lower. In the DPPH assay, the EC50 for AE and TAE were 20.15 and 35.12 µg/ml, the positive control Trolox with EC50 was 7.42 µg/ml, this demonstrates that the ability of scavenging free radicals have diminished after the enrichment

2.15. Statistical analysis All data represent the mean ± S.D. of three independent experiments. Analysis of variance was performed by two-way ANOVA procedures, using Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). Western blot band intensity was analyzed using ImageJ and statistical analyses were conducted using SPSS software package (Version 16.0, IBM Corp., Armonk, NY, USA).

Table 1 The content of compounds in TAE.

573

Peak No.

1+2

3

5

6+7

8+9

10+20

11+18

14

15+19

Content (%)

5.2

1.7

2.6

3.4

0.8

0.7

10.7

2.6

7.9

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Table 2 Antioxidant activity of compounds by DPPH, ABTS, FRAP assays and the effects of the compounds on cell viability against t-HSC/Cl-6 cell line Compounds

DPPHa

ABTSa

AE TAE 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 1a 2a 3a-1 3a-2 3a-3 3a-4 4a-1 4a-2 5a GA Trolox

20.15 ± 3.26d 35.12 ± 4.19d > 100e > 100e > 100e > 100e > 100e 26.13 ± 1.79e 36.27 ± 3.86e 59.16 ± 4.61e 57.32 ± 4.96e > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e 28.43 ± 2.09e 34.86 ± 3.19e 56.46 ± 4.11e 58.92 ± 4.56e 56.04 ± 3.98e 52.14 ± 2.38e 38.10 ± 2.16e 36.68 ± 1.96e 32.74 ± 3.92e 30.64 ± 2.16e 29.66 ± 5.22e 7.42 ± 1.31d – –

39.67 ± 47.16 ± > 100e > 100e > 100e > 100e > 100e 47.26 ± 55.98 ± 46.28 ± 39.66 ± > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e 44.86 ± 53.08 ± 37.59 ± 44.36 ± 38.16 ± 39.19 ± 78.08 ± 83.79 ± 61.66 ± 22.11 ± 54.01 ± 13.51 ± – –

Silymarin

a b c d e

FRAPb 4.92d 3.22d

2.83e 2.82e 2.49e 2.79e

2.02e 1.98e 1.56e 1.89e 1.36e 3.26e 3.67e 4.72e 3.16e 1.59e 9.85e 2.46d

42.28 ± 59.27 ± > 100e > 100e > 100e > 100e > 100e 65.11 ± 70.52 ± 68.12 ± 76.02 ± > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e > 100e 67.41 ± 69.62 ± 74.32 ± 65.09 ± 62.95 ± 60.05 ± 78.95 ± 84.94 ± 91.75 ± 22.25 ± 93.47 ± 23.37 ± – –

t-HSC/Cl-6c 5.15d 8.98d

5.73e 5.81e 5.16e 4.59e

5.12e 5.26e 4.96e 4.55e 5.16e 3.02e 4.92e 4.87e 6.62e 1.43e 13.66e 3.41d

60.03 ± 7.65d 26.79 ± 4.15d 11.82 ± 2.64e 23.54 ± 1.74e 31.42 ± 4.11e 35.53 ± 3.12e 29.18 ± 3.36e 3.12 ± 0.55e 6.98 ± 1.13e 9.62 ± 1.98e 16.13 ± 2.61e 40.56 ± 4.16e 126.55 ± 9.25e 39.15 ± 3.96e 45.69 ± 4.26e 112.47 ± 8.83e 160.52 ± 11.92e 130.17 ± 9.13e 50.26 ± 6.53e 129.55 ± 10.15e 150.52 ± 10.22e 45.56 ± 5.63e 167.12 ± 12.29e 182.52 ± 15.02e 2.26 ± 0.67e 6.18 ± 1.43e 11.82 ± 2.68e 16.93 ± 3.01e 21.75 ± 3.22e 24.16 ± 2.12e 29.50 ± 2.82e 21.19 ± 3.21e 19.23 ± 2.70e 122.30 ± 10.06e – – 197.37 ± 19.60e 95.22 ± 9.46d

EC50: the antioxidant concentration required to reduce the original free radical concentration to 50%. FRAP: The amount of sample reduce 1 mM Fe3+ to Fe2+. IC50: Half inhibitory concentrations measured by the MTT assay. μg/mL. μM. Data represent the mean ± S.D. of three independent experiments.

Fig. 4. Cells were incubated with TAE (40 μg/ml) and Com.6 (4 μM) for 24 h, and the cellular morphological changes were observed by fluorescence microscopy with DAPI staining.

Moreover, the triterpenoids from acorn does not show strong antioxidant activity with the three methods, except for the galloyl triterpenes. This further illustrates acorn exhibit strong antioxidant activity maybe because of their high amounts of phenolic acids (Cantos et al., 2003), rather than triterpenoids. GA is endowed with a strong antioxidant activity, amazingly, the galloyl triterpene derivatives have a similar capacity. The results are

triterpenoid constituents. Similar as in the ABTS assay, the AE with EC50 of 39.67 µg/ml showed the higher free radical scavenging activities than TAE, with EC50 was 47.16 µg/ml. Ferric reducing antioxidant power (FRAP) test was extensively used for antioxidant analysis in food and health care products. The reducing capacity of samples could be reflected by detecting the production of the blue colored Fe2+-TPTZ complex. It can see that the reducing capacity of AE higher than TAE.

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Fig. 5. (A–F) The apoptosis mechanism of induction by TAE, Com.6 and Com.1a in t-HSC/Cl-6 cells. (A–C) The apoptosis by examining the expression of the hallmark proteins, CL-caspase-9 and CL-caspase-3. (D–F) The expression levels of apoptosis-related proteins, including Bcl-2 and BAX. (G–I) Effects of TAE, Com.6 and Com.1a on the expression of Nrf2 and HO-1 in t-HSC/Cl-6 cells. (G–I) The expression levels of Nrf2-mediated antioxidant proteins. They were all detected by Western blot analysis. (J–L) Quantification of CL-caspase-9, CL-caspase-3, Bcl-2, BAX, Nrf2 and HO-1 protein expression. (M–P) Effects of TAE, Com.6 and Com.1a on the ratio of Bcl-2/BAX in t-HSC/Cl-6 cells. Data are presented as the means ± SDs of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with actin.

stronger t-HSC/Cl-6 inhibition than AE. Among triterpenes (1–22), compounds 1–9 showed significant antiproliferative effects against t-HSC/Cl-6 cells at IC50 values from 3.12 to 35.53 µM, compared with IC50 of positive control, silymarin, at 197.37 µM. Moreover, galloyl-substituted compounds 6, 7, 8, and 9, showed much stronger activity than compounds 1, 2, and 3, which do not have galloyl group. C-3-acetylated compounds 12, 13, and 17 showed moderate anti-proliferative effects compared with unacetylated compounds 1, 2, and 3. On the other hand, glycosylated and α-hydroxylated triterpenes at C-28 and C-19 positions, respectively, did not exhibit excellent activity. Oleanane and ursane types did not show significant difference in anti-proliferative effects against t-HSC/Cl-6 cells under the same substituent group. These suggest the importance of galloyl substituent in triterpene derivatives to inhibit t-HSC/Cl-6 cells. Hence, the synthetic galloyl triterpenes were also assessed via their anti-proliferative effects against t-HSC/Cl-6 cells. After the synthesis, the anti-fibrosis activity of the esterification products (1a–5a) of triterpenes and GA resulted better, compared to the substrates (1–5). The IC50 values of 1a–5a were all less than those of the positive reference drug silymarin, particularly the esterification product (1a) of oleanolic acid and gallic acid, which showed a strong activity (IC50 = 2.26 ± 0.67 μM), while the IC50 of silymarin was 197.37 ± 19.60 μM. Analyzing the structure-activity relationship, we observed that anti-fibrosis activity was not affected when the same site was substituted with hydroxyl or acetyl group in the same triterpene. The activity of compounds was slightly higher with acetyl group, compared with compounds with hydroxyl group at the same position. For example, compounds 3a-1, 3a-2 and 4a-2 had better anti-fibrosis activity than those of 3a-3, 3a-4 and 4a-1. Moreover, in the anti-proliferative effects against t-HSC/Cl-6 cells there was no significant

listed in Table 2, and indicated that the antioxidant activity were improved in the esterification products (1a–5a) of triterpenes and GA, as compared to their starting triterpenes. Analyze the antioxidant activity, there was no significant difference between the galloyl triterpenes naturally occurring in acorn and the synthetic ones. The DPPH radical scavenging activity experiment indicated that the free radical scavenging activity was slight reduced in the esterification products, with respect to GA and Trolox, but the EC50 values for 1a–5a compounds were lower than 60 μM. All products were also tested for ABTS radical scavenging activity, and esterification products (1a–5a) of triterpenes and GA overall exhibited a higher free radical scavenging activity, but slightly lower than that of GA. However, the EC50 values of compounds 1a, 2a and 3a-1-4 were lower than those of the used reference Trolox. In the FRAP assay, the reducing capacity of all the esterification products (1a–5a) of triterpenes and GA resulted higher than the reference Trolox. On the basis of these analyses, the esterification products (1a–5a) of triterpenes and GA had free radical elimination capacity and reducing activity, thus they have excellent antioxidant properties. 3.3. Anti-fibrosis activity The activation of hepatic stellate cells (HSC) is strictly related to liver fibrosis, hence the acorn extract and all compounds were assessed via their anti-proliferative effects against t-HSC/Cl-6 cells. Results are reported in Table 2. Potent anti-proliferative effects against t-HSC/Cl-6 were observed among AE and TAE with IC50 values of 60.03 and 26.79 µg/ml, compared with silymarin at IC50 of 95.22 µg/ml. Meanwhile, our results showed that the most of triterpenoids from acorn exhibited stronger anti-proliferative effects against t-HSC/Cl-6 cells than the reference silymarin, these information may explain why TAE yielded 575

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Fig. 5. (continued)

staining indicated that nuclear condensation had occurred.

difference between the galloyl triterpenes naturally occurring in acorn and the synthetic ones. Interestingly, we observed that the synthesized products had similar trend between anti-fibrosis and antioxidant activities, which increased after esterification with GA. It was preliminarily assumed that antioxidant activity might be related with antifibrosis, and good antioxidant activity might promote anti-fibrosis effects. These naturally derived galloyl triterpene derivatives might have better safety profile and could be used in opposing liver fibrosis.

3.5. Western blot analysis 3.5.1. TAE, Com.6, and Com.1a induce t-HSC/Cl-6 cells apoptosis Based on the results acquired from the morphological changes, western blots were used to determine the possible effects of the TAE, Com.6, and Com.1a on the regulatory proteins associated with the apoptosis pathway. We determined the expression level of two caspase family proteins, caspase 9 and caspase 3, because activated caspases are well known as biochemical markers of apoptosis and play a major role in chromatin condensation (Chu, Rothfuss, & Ad’ Avignon, Zeng C.B., Zhou D., Hotchkiss R.S., , 2007). As Fig. 5A–C exhibited that the expression levels of cleaved caspase-9 and cleaved caspase-3 were activated in a dose-dependent manner after compounds treatment. The Bcl2 family proteins, including pro-apoptosis (such as BAX) and antiapoptosis (such as Bcl-2) proteins (Gross, McDonnell, & Korsmeyer, 1999), are the key regulators of the mitochondrial apoptosis pathway (Martinou & Youle, 2011). The ratio of Bcl-2/BAX is important in the induction of apoptosis. Hence, we also evaluated the expression levels

3.4. Analysis of DAPI staining To clarify how TAE and Com.6 affect t-HSC/Cl-6 cells growth, we observed the morphological changes of the treated t-HSC/Cl-6 cells. Compared with the control group, some of the TAE or Com.6-treated cells exhibited chromatin condensation, a hallmark of apoptosis (Fig. 4). The results suggest that TAE and Com.6 cause t-HSC/Cl-6 cells to undergo apoptosis. Apoptosis is an orderly and synchronized death process, it is characterized by morphological changes such as cell shrinkage, chromatin condensation, DNA fragmentation, as well as membrane blebbing (Plenchette et al., 2004). In this study, DAPI 576

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Acknowledgments

of the apoptosis related proteins, BAX and Bcl-2. Both compounds increased the expression of BAX protein and decreased the expression of Bcl-2 protein significantly (Fig. 5D–F). In addition, the Bcl-2/BAX ratio was dramatically down regulated in a dose-dependent manner (Fig. 5M5P). These data indicated that TAE, Com.6, and Com.1a induced t-HSC/ Cl-6 cells apoptosis through caspase-dependent pathway. It can see that the galloyl triterpene naturally occurring in acorn (Com.6) and the synthetic one (Com.1a) have consistent role.

The research was supported by National Nature Science Foundation of China (Grant Nos. 81703389 and 81703386), Education Fund Item of Liaoning Province (201610163L35), Youth Development Support Plan of Shenyang Pharmaceutical University (ZQN2016023), E&T Modern Center for Natural Products of Liaoning Province of China (No. 2008402021), Construction of R&D Institute of State Original New Drugs at Benxi of Liaoning Province (2009ZX09301-012-105B).

3.5.2. TAE, Com.6, and Com.1a up-regulated Nrf2-mediated antioxidant expression Nuclear factor erythroid-2-related factor 2 (Nrf2) is a transcription activator binding to antioxidant response elements in the promoter regions of target genes (Dinkova-Kostova et al., 2002). Under stress conditions, Nrf2 can be translocated into nucleus to activate the expression of antioxidant-responsive genes and induce phase II detoxifying enzymes, which is considered as a major regulator of oxidant resistance (Gan & Johnson, 2014). Heme oxygenase-1 (HO-1), a rate limiting enzyme in heme catabolism, has been identified as an important endogenous protective factor induced in many cell types by various stimulants (Kovac et al., 2015). The Nrf2/HO-1 pathway plays an important role against oxidative stress. Thus, we examined the effects of TAE, Com.6, and Com.1a on the expression levels of Nrf2 and HO-1, and result showed that treatment with the increased concentration, they could significantly increase the expression of Nrf2 and HO-1 in a dose-dependent manner by Western blot analysis (Fig. 5G-5I). Maybe the antioxidant can inhibit the proliferation of t-HSC/Cl-6 cells. Moreover, there was no significant difference between the galloyl triterpene naturally occurring in acorn (Com.6) and the synthetic one (Com.1a), they have the same function.

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jff.2018.05.031. References Birosová, L., Mikulásová, M., & Vaverková, S. (2005). Antimutagenic effect of phenolic acids. Biomedical Papers of the Medical Faculty of the University Palacky Olomouc Czech Repubic, 149(2), 489–491. Cantos, E., Espin, J., Lopez, C., & Barberan, F. A. (2003). Phenolic compounds and fatty acids from acorns (Quercus spp.): The main dietary constituent of free ranged Iberian pigs. Journal of Agricultural and Food Chemistry, 51(21), 6248–6255. Chu, W. H., Rothfuss, J., Ad’ Avignon, Zeng, C. B., Zhou, D., Hotchkiss, R. S., et al. (2007). Isatin sulfonamide analogs containing a Michael addition acceptor: A new class of caspase-3/7 inhibitors. Journal of Medicinal Chemistry, 50, 3751–3755. Dinkova-Kostova, A. T., Holtzclaw, W. D., Cole, R. N., Itoh, K., Wakabayashi, N., Katoh, Y., et al. (2002). Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proceedings of the National Academy of Sciences of the United States of America, 99(18), 11908–11913. Fontanay, S., Grare, M., & Mayer, J. (2008). Ursolic, oleanolic and betulinic acids: Antibacterial spectra and selectivity indexes. Journal of Ethnopharmacology, 120(2), 272–276. Gan, L., & Johnson, J. A. (2014). Oxidative damage and the Nrf2-ARE pathway in neurodegenerative disease. Biochimic et Biophysica Acta, 1842(8), 1208–1218. Gross, A., McDonnell, J. M., & Korsmeyer, S. J. (1999). BCL-2 family members and the mitochondria in apoptosis. Genes & Development, 13, 1899–1911. Hazra, S., Xiong, S., Wang, J., Rippe, R. A., Krishna, V., Chatterjee, K., et al. (2004). Peroxisome proliferator-activated receptor gamma induces a phenotypic switch from activated to quiescent hepatic stellate cells. Journal of Biological Chemistry, 279, 11392–11401. Jing, X., Jiaqing, C., Jiayin, Y., Xiaoshu, Z., & Yuqing, Z. (2018). New triterpenoids from acorns of Quercus liaotungensis and their inhibitory activity against α-glucosidase, αamylase and protein-tyrosine phosphatase 1B. Journal of Functional Foods, 41, 232–239. Kovac, S., Angelova, P. R., Holmström, K. M., Zhang, Y., Dinkova-Kostova, A. T., & Abramov, A. Y. (2015). Nrf2 regulates ROS production by mitochondria and NADPH oxidase. Biochimic et Biophysica Acta, 1850(4), 794–801. Li, Y. Y., Xu, J. L., Yuan, C., Ma, H., Liu, T., Liu, F., et al. (2017). Chemical composition and anti-hyperglycaemic effects of triterpenoid enriched Eugenia jambolana Lam. berry extract. Journal of Functional Foods, 28, 1–10. Luísa, C., João, P., Fernando, A., Nuno, R. N., José, M. F. N., & Anabela, R. (2013). Extracts from Quercus sp. acorns exhibit in vitro neuroprotective features through inhibition of cholinesterase and protection of the human dopaminergic cell line SHSY5Y from hydrogen peroxide-induced cytotoxicity. Industrial Crops and Products, 45, 114–120. Luísa, C., João, P., Fernando, A., Nuno, R. N., José, M. F. N., & Anabela, R. (2015). Phenolic composition, antioxidant potential and in vitro inhibitoryactivity of leaves and acorns of Quercus suber on key enzymes relevantfor hyperglycemia and Alzheimer’s disease. Industrial Crops and Products, 64, 45–51. Martinou, J. C., & Youle, R. J. (2011). Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics. Developmental Cell, 21, 92–101. Mishra, B. B., & Tiwari, V. K. (2011). Natural products: An evolving role in future drug discovery. European Journal of Medicinal Chemistry, 46, 4769–4807. Ogata, M., Saito, Y., Matsuo, Y., Maeda, H., & Tanaka, T. (2016). Triterpene galloyl esters from edible acorn of Castanopsis cuspidate. Natural Product Communications, 11(2), 179–181. Ortega, E., Sadaba, M. C., Ortiz, A. I., Cespon, C., Rocamora, A., & Escolano, J. M. (2003). Tumoricidal activity of lauryl gallate towards chemically induced skin tumours in mice. British Journal of Cancer, 88, 940–943. Plenchette, S., Filomenko, R., Logette, E., Solier, S., Buron, N., Cathelin, S., et al. (2004). Analyzing markers of apoptosis in vitro. Methods in Molecular Biology, 281, 313–331. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26, 1231–1237. Rufino, M. S. M., Fernandes, F. A. N., Alves, R. E., & Brito, E. S. D. (2009). Free radical scavenging behavior of some north-east Brazilian fruits in a DPPH system. Food Chemistry, 114, 693–695. Sá, M. D., Justino, V., Spranger, M. I., Zhao, Y. Q., Han, L., & Sun, B. S. (2014). Extraction

4. Conclusions The present experiments demonstrate for the first time the antihepatic fibrosis activities of acorns. The apoptosis mechanism of the effects on t-HSC/Cl-6 cells is researched firstly. In addition, acorns and their galloyl triterpene (Com. 6) up-regulated the expression levels of Nrf2 and HO-1 in t-HSC/Cl-6 cells. In summary, they showed antioxidative effects on anti-hepatic fibrosis, indicating its potential for being developed into anti-hepatic fibrosis food or medicine. Meanwhile, this research involves the study of a series of naturally derived galloyl triterpene derivatives that have been synthesized to assess their biological activities. Antioxidant properties resulted improved when triterpenoid compounds esterified with gallic acid, and some products showed levels higher than the reference Trolox. While assessing the antifibrosis activity of target products, we observed a similar trend between antifibrosis and antioxidant activities, both increasing after esterification with GA. The synthetic method used to obtaind these galloyl triterpene derivatives is mild and controllable, and can be used to prepare large quantities. Particularly, they as naturally derived may had a better safety profile and are invaluable for further development of antifibrosis drug. This research can also shed light on the structural modifications of other substrates (e.g., sterol, flavone, etc.). In summary, the synthetic design of galloyl triterpene derivatives provides a good strategy to discover new lead compounds with high performance and low toxicity profiles. 5. Conflict of interest The authors declare that there are no conflicts of interest. 6. Ethics statement This study was approved by Shenyang Pharmaceutical University. All procedures performed in studies were in accordance with the ethical standards. 577

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(2001). Evolution of phenolic composition of red wine during vinification and storage and its contribution to wine sensory properties and antioxidant acticity. Journal of Agricultural and Food Chemistry, 59, 6550–6557. Yang, J. H., Kim, S. C., Kim, K. M., Jang, C. H., Cho, S. S., Kim, S. J., et al. (2016). Isorhamnetin attenuates liver fibrosis by inhibiting TGF-beta/Smad signaling and relieving oxidative stress. European Journal of Pharmacology, 783, 92–102.

yields and anti-oxidant activity of proanthocyanidins from different parts of grape pomace: Effect of mechanical treatments. Phytochemical Analysis, 25, 134–140. Sanchez-Valle, V., Chavez-Tapia, N. C., Uribe, M., & Sanchez, N. M. (2012). Role of oxidative stress and molecular changes in liver fibrosis: A review. Current Medicinal Chemistry, 194, 850–4860. Sun, B. S., Neves, A. C., Fernandes, T. A., Fernandes, A. L., Mateus, N., Freitast, V. D., et al.

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