New triterpenoids from acorns of Quercus liaotungensis and their inhibitory activity against α-glucosidase, α-amylase and protein-tyrosine phosphatase 1B

Journal of Functional Foods 41 (2018) 232–239

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New triterpenoids from acorns of Quercus liaotungensis and their inhibitory activity against α-glucosidase, α-amylase and protein-tyrosine phosphatase 1B ⁎

Jing Xua, Jiaqing Caoa,b, Jiayin Yuea, Xiaoshu Zhanga,b, , Yuqing Zhaoa,b, a b

T

⁎

School of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang 110016, 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 L E I N F O

A B S T R A C T

Keywords: Acorn Quercus liaotungensis Triterpenoids Hypoglycaemic α-Glucosidase and PTP1B inhibitory activities

Acorn has been used as a functional food with numerous nutritional and active ingredients. In this study, 3 new triterpenoids, together with 22 known compounds were isolated from acorns (Quercus liaotungensis) and further confirmed by NMR. In order to disclose the hypoglycaemic ingredients, the extracts and all the discovered compounds were researched for enzyme inhibition using in-vitro assays on α-amylase, α-glucosidase, and proteintyrosine phosphatase 1B (PTP1B). The results suggested that 75% EtOH, PE, EtOAc, and n-BuOH extracts of acorns all exhibited potent inhibitory activities against PTP1B and α-glucosidase. Most of the compounds showed strong inhibitory effects on PTP1B and α-glucosidase, especially new compounds and compound 12, their IC50 values were about 6-fold to 20-fold lower than positive control, all showed competitive inhibitory pattern. The result suggested that triterpenoids from acorns are potential functional food ingredients that can be used as new anti-diabetic agent.

1. Introduction Sustained high blood glucose patients without proper and timely controls, they have higher risks for nephropathy, cardiovascular disease, neuropathy, retinopathy, cognitive decline, and premature death (Colhoun et al., 2004). Type 2 diabetes is characterized by high blood glucose level and is complicated metabolic disorder arising from insulin deficiency, insulin resistance, or both (Imam, 2012). The world health organization (WHO) predicts around 439 million people worldwide will suffer from diabetes by 2030 (Shaw, Sicree, & Zimmet, 2010). The intensive researches on development of effective hypoglycaemic methods have provided discoveries of molecular therapy targets, such as αamylase, α-glucosidase and PTP1B. α-Amylase and α-glucosidase are important enzymes in breakdown of starch and intestinal absorption. Human α-amylase is secreted by pancreas and salivary glands to hydrolyze starch molecules by cleaving internal α-D-(1-4) glycosidic bonds to yield different types of products, including dextrins, oligosaccharides, and monosaccharides (Lili, 2003). α-Glucosidase is found on surface of brush-border membrane of intestinal cells and catalyzes last step of carbohydrate hydrolysis to release monosaccharides (Baron, 1998). Modern pharmacological research demonstrated that anti-hyperglycemic drugs, such as acarbose, miglitol, and voglibose, decreased

⁎

post-prandial hyperglycemia by inhibiting α-glucosidase and α-amylase. However, anti-hyperglycemic inhibitor drugs may cause various side effects, such as abdominal cramping, flatulence, diarrhea, and liver disorders (Bischoff, 1994). Hence, studies attempted to discover foods which could function as α-amylase and α-glucosidase inhibitors. PTP1B is intracellular non-transmembrane enzyme, it primarily catalyzes dephosphorylation of activated insulin receptors and their corresponding substrate proteins, functions as a negative regulator of insulin action in insulin receptor signaling pathway. PTP1B inhibitors can reduce blood glucose level by increasing insulin sensitivity. Since its discovery, PTP1B has received much attention in discovery of novel PTP1B inhibitors from foods because of the safety and structure diversity (Combs, 2010). Foods with active ingredients provide valuable sources for the development of dietary supplements for preventing disease. Acorn is fruit of oak (Quercus) tree, which belongs to Fagaceae family. Quercus liaotungensis is native in China, where the tree mainly grows in northern region (Yin, Zhou, Sui, He, & Li, 2013). Acorn is nutritionally rich in vitamins, minerals, trace element, lipids, amino acids, proteins, and carbohydrates (Cantos, Espin, Lopez, & Barberan, 2003), many countries produce flour, bread, jelly made from acorn (Jaroslaw, Mariusz, Rafal, & Leslaw, 2015). Recently acorns have attracted intensive

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

https://doi.org/10.1016/j.jff.2017.12.054 Received 7 April 2017; Received in revised form 13 December 2017; Accepted 21 December 2017 1756-4646/ © 2017 Published by Elsevier Ltd.

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2.3. Extraction and isolation

attention as functional food because of their various biological activities, such as antioxidant, antibacterial, antitumor, neuroprotection, prevention of degenerative diseases (Cantos et al., 2003; Luísa et al., 2013, 2015). Acorns have also been researched for their phytochemical contents. It is well known that acorns contain a good deal of phenolic compounds, such as tannins, phenolic acids, and flavonoids, which are considered to be the main bioactive ingredients. There have been many reports focused on the correlational analysis between the biological activities and chemical composition of phenolic compounds from acorns (Ana, Joao, Anabela, & Oliveira, 2016; Cantos et al., 2003; Kim et al., 2004; Luísa et al., 2015). In fact, the biological activity of plants or foods is usually generated by different types of phytochemical composition. Triterpenoids and sterols are also important bioactive substances in natural products, but few reports refer to chemical constituents of triterpenoids and sterols from acorns (Ana et al., 2016). Furthermore, the reports on anti-diabetic activity of acorns were few, especially on the target of PTP1B. In a recent study, acorns obtained from Q. brantii Lindl tree were identified as potential hypoglycaemic source (Abdulahad, Ismail, & Mehmet, 2015). However, effective chemical components responsible for hypoglycaemic activity of acorn have not been fully study. This study is the first to identify active hypoglycaemic constituents of acorns, 20 triterpenoids and 5 sterols were isolated and identified, and 3 of them were new compounds. Also the inhibition activity of extracts and all compounds from acorns against α-amylase, α-glycosidase, and PTP1B were tested. Additionally, this is the first time that acorn was researched for their enzyme inhibition on PTP1B.

To obtain the active constituents, the dried acorns of Quercus liaotungensis (15 kg) were extracted with 75% EtOH (70 L × 3, 2 h each) under reflux, filtered and concentrated to give 10 L aqueous residue. The aqueous residue (10 L) was partitioned with petroleum ether (PE), ethyl acetate (EtOAc), and n-butyl alcohol (n-BuOH) (10 L × 3 in each case), respectively. The yields of PE, EtOAc, and n-BuOH extracts were 40 g, 100 g, and 220 g, respectively. The schematic representation of the extraction and isolation procedure is shown in supplementary data. The EtOAc fraction (100 g) was subjected to a silica gel column chromatography (SGCC) with a gradient of PE: EtOAc (100:0 to 0:100) and 100% MeOH to afford seven fractions (Fr. A-G) based on TLC analysis. Fraction B (5.0 g) was chromatographed over SGCC eluted with PE: Acetone at 10:1, 8:1, 5:1, 2.5:1, 1:1, and 100% MeOH to give compounds 20 (7.3 mg), 21 (33.2 mg) and four fraction (fr. B-1 to B-4). Fr. B-2 was subjected to chromatography via semi-preparative HPLC (MeOH: H2O, 95:5) to obtain compounds 22 (14.6 mg) and 24 (15.2 mg). Fr. B-3 was repeatedly separated by SGCC, fr. B-3-3 further purified by semi-preparative HPLC (MeOH: H2O, 88:12, added with 0.3% formic acid and 1.998 g/L β-cyclodextrin) to afford compounds 8 (9.5 mg) and 15 (8.5 mg), and fr. B-3-4 further purified by semi-preparative HPLC (MeOH: H2O, 85:15, added with 0.3% formic acid and 1.998 g/L β-cyclodextrin) to get compounds 4 (26.1 mg) and 13 (19.4 mg). Fraction C (7.4 g) was subjected to a SGCC and eluted with a gradient of CH2Cl2: MeOH (100:0 to 0:100) to give four fractions (fr. C1-C4). Fr. C-1 was subjected to a SGCC and eluted with a gradient of cyclohexane: EtOAc (20:1 to 1:1, v/v) to obtain compounds 5 (27.1 mg), 9 (7.8 mg), and 19 (6.5 mg). Fr. C-3 was chromatographed by Sephadex LH-20 (CH2Cl2: MeOH, 50:50) and then purified by semipreparative HPLC (MeOH: H2O, 70:30, added with 0.3% formic acid) to obtain compounds 6 (9.2 mg), 14 (6.4 mg), and 18 (9.8 mg). Fraction D (9.4 g) was subjected to an ODS column chromatography with MeOH: H2O (10–100%) to obtain eight sub-fractions. Fr. D-4 was subjected to a Sephadex LH-20 column chromatography with MeOH, and then separated by semi-preparative HPLC (MeOH: H2O, 57:43, added with 0.3% formic acid and 1.998 g/L β-cyclodextrin) to get compounds 7 (28.7 mg) and 16 (18.4 mg). With the same way, fr. D-7 was finally purified by semi-preparative HPLC (MeOH: H2O, 90:10) to give compounds 23 (11.4 mg) and 25 (9.3 mg). Fraction E (6.4 g) was subjected to an ODS with MeOH: H2O (10–100%), fr. E-4 was separated by SGCC repeatedly and eluted with a gradient of CH2Cl2: MeOH (15:1 to 1:1) to obtain compound 10 (22.8 mg), then fr. E-4-6 was separated by Sephadex LH-20 with MeOH, subsequently, it was purified by semipreparative HPLC (MeOH: H2O, 46:54, added with 0.3% formic acid and 1.998 g/L β-cyclodextrin) to yield compounds 11 (20.2 mg) and 17 (13.2 mg). Fr. F was applied to Sephadex LH-20, subsequently, it was subjected to an ODS with MeOH: H2O (10–100%) to obtain six subfractions. Fr. f-4 was separated by semi-preparative HPLC (MeOH: H2O, 75:25, added with 0.3% formic acid and 1.998 g/L β-cyclodextrin) to yield compounds 2 (8.2 mg) and 3 (6.1 mg). With the same way, compounds 1 (9.2 mg) and 12 (6.5 mg) were obtained from semi-preparative HPLC (MeOH: H2O, 85:15, added with 0.3% formic acid and 1.998 g/L β-cyclodextrin).

2. Materials and methods 2.1. General experimental procedure Silica gel (mesh: 300–400, Qingdao Marine Chemistry Co., China). RP C18 silica gel (mesh: 300–400, Agela Technology, China). Sephadex LH-20 (amersham, Sweden, USA). Semi-preparative HPLC (CXTH LC3000, China). Octadecyl silica chromatographic column [YMC-Pack ODS A (5 mm, 250 mm × 10 mm)]. The flow rate of semi-preparative HPLC was 3.0 mL/min, HPLC column temperature was at 25 °C. The ultraviolet spectrophotometric detection of semi-preparative HPLC was at 203 nm. Purified water was purchased from Wahaha (Hangzhou, China). Methanol, formic acid (HPLC-grade) and other solvents (Analytical grade) were purchased from Kangkede (Tianjin, China). PTP1B (human, recombinant), porcine pancreatic α-amylase, yeast αglucosidase, dithiothreitol (DTT), p-nitrophenyl phosphate (pNPP), pnitrophenyl-α-D-glucopyranoside (pNPG), β-mercaptoethanol were purchased from Sigma-Aldrich. Other chemicals (analytical grade) were purchased from Sinopharm. Nuclear magnetic resonance (NMR) spectra were recorded with a Bruker AVANCE (Bruker Co., Karlsruhe, Germany) spectrometer in C5D5N/CDCl3 with tetramethylsilane as internal standard. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) spectra were recorded on a Bruker Solarix 7.0 T system. It equipped electrospray ionization (ESI) interface at a nebulizing gas pressure of 4.0 bar, a dry gas flow rate of 8.0 L/min, with a capillary voltage of −3.0 kV, an end plate offset of −500 V and a transfer capillary temperature of 200 °C.

2.4. Assay for PTP1B inhibitory activity The method of PTP1B inhibition assay was performed according to the reported method (Fang, Cao, Duan, Tang, & Zhao, 2014). A total of 83 μL enzyme in buffer (pH 7.5) consist of 2 mM β-mercaptoethanol, 25 mM Tris-HCl, 1 mM DTT, 1 mM EDTA, 10 uL of test compounds or extracts solution at various concentrations (in 1% DMSO) were incubated in 96-well plate at 37 °C for 10 min, subsequently, 4 μL pNPP (10 mM) as a substrate in buffer was added. Following incubation at 37.0 °C for 30 min, 5 μL NaOH (2 mol/L) was added to stop the reaction. To blank control group was added 10 μL of 1% DMSO instead of the

2.2. Plant material 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.

233

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Fig. 1. Chemical structures of isolated compounds 1–25.

sample solution. Dephosphorylation of pNPP generated product pNP, which can be monitored at 405 nm, therefore, we measured the absorbance of the sample at 405 nm by microplate reader (imark, BIORAD, USA). Na3VO4 was used as positive control.

and determined by microplate Reader (imark, BIORAD, USA). Acarbose was used as positive control.

2.5. Assay for α-glycosidase inhibitory activity

The method of α-amylase inhibition assay with slight modification was used (Yu, Yin, Zhao, Liu, & Chen, 2012). 10 μL of α-amylase solution (in water, 1 unit/ml) was mixed with 10 μL of the purified compound or extracts solution at various concentrations (in 1% DMSO) and incubated at 37.0 °C for 15 min, subsequently, the reaction was started when 500 μL of 1% starch solution [in 20 mmol/L sodium phosphate buffer (pH 6.9)] was added, then the sample was incubated at 37 °C for 10 min. 300 μL of DNS reagent (1% 3,5-dinitrosalicylic acid, 12% Na-K tartrate in 0.4 mol/L NaOH) was added to terminate the reaction. The test tubes were then incubated in a boiling water bath for 10 min and cooled to room temperature, subsequently, the reaction mixture was then diluted by addition of 5 ml of distilled water and 150 μL was removed from each tube and transferred to the wells of a 96-well microplate. To blank control group was added 10 μL of 1% DMSO instead

2.6. Assay for α-amylase inhibitory activity

A slightly modified and developed method of the α-glucosidase assay was used (Xu, Wu, Zhang, Chen, & Wang, 2014). 30 μL of the αglycosidase solution (in 0.1 mol/L potassium phosphate buffer, 2 units/ mL, pH 6.8) was mixed with 20 μL of the purified compound or extracts solution at various concentrations (in 1% DMSO) and incubated at 37.0 °C for 5 min, subsequently, the reaction was started when 150 μL of p-nitrophenyl glucopyranoside (pNPG, 10 mM) and 800 μL of 0.1 M potassium phosphate buffer were added. Following incubation at 37.0 °C for 30 min, 2 mL of Na2CO3 (1 M) was added to stop the reaction. To blank control group was added 20 μL of 1% DMSO instead of the sample solution. The absorbance at 405 nm was measured to quantify the amount of p-nitro phenol, which was the released product 234

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of the sample solution. The absorbance of the reaction sample was measured at 540 nm by microplate Reader (imark, BIORAD, USA). Acarbose was used as positive control. 2.7. Kinetic of PTP1B inhibition Compounds (1, 2, 3, and 12) were further analyzed for type of PTP1B inhibition. The type of inhibition of the active constituents against PTP1B was determined using increasing concentrations of pNPP as a substrate in the absence or presence of compounds as inhibitors at different concentrations. Concentration of inhibitors was fixed at 0, 1.0, 3.0, and 10.0 μM, while concentration of substrate was varied from 0.2 to 2.0 mM (0.2, 0.25, 0.33, 0.5, 1.0, and 2.0 mM). The type of inhibition was determined using a Lineweaver–Burk plot analysis (Graphpad Prism version 4.02). 2.8. Kinetic of α-glucosidase inhibition

Fig. 2. The key HMBC correlations of compounds 1–3.

Compounds (1, 2, 3, and 12) were further analyzed for type of αglucosidase inhibition. The reaction mixture consisted of different concentrations of pNPG as a α-glucosidase substrate in the absence or presence of compounds at different concentrations. Concentration of inhibitors was fixed at 0, 0.25, 1.0 and 3.0 μM, while concentration of substrate was varied from 0.125 to 1.0 mM (0.125, 0.2, 0.25, 0.33, 0.5, and 1.0 mM). The type of inhibition was determined using a Lineweaver–Burk plot analysis (Graphpad Prism version 4.02).

25), 0.95 (H-29), 1.02 (H-26), 1.02 (H-30), and 1.31 (H-27), one trisubstituted olefinic proton at δH 5.49 (H-12), and methine proton at δH 3.31 (H-18). 3β, 23-Dihydroxyolean-12-ene type triterpenoid was detected through presence of carbonyl carbon at δc 180.6 (C-28), two olefinic carbons at δc 122.8 (C-12) and 145.2 (C-13), one hydroxylated methine carbons at δc 75.2 (C-3) and one hydroxylated methylene carbon at δc 66.6 (C-23). 1H and 13C NMR spectra of compound 2 were closely related to those of compound 1, with exception of acetyl group signal at δH 1.97, δc 21.0, and δc 170.9. Acetylation was confirmed by long-range correlation observed in HMBC spectrum between proton H23 (δH 3.96 and 4.16) and carbonyl carbon C-8′ (δc 170.9) of acetyl group. Moreover, downfield signal of C-23 (δc 66.6) proves presence of acetyl group at this position. Thus, structure of 2 was elucidated as 23acetoxy-3-O-galloyloleanolic acid. Total assignments of protons and carbons of compound 2 were performed based on HSQC and HMBC, as shown in Table 1. Compound 3 was difficult to separate from compound 2, and purification of former was only achieved by reversed phase HPLC using C18 silica column to obtain yellowish amorphous powder. Compounds 2 and 3 have identical molecular formula, which is C39H54O9, according to FT-ICR-MS. Based on comparison of NMR data of compound 3 with that of compound 2, similar spectra was observed in 1H and 13C NMR, therefore suggesting possible difference in location of acetyl and galloyl substituents in two compounds. Linkage positions of galloyl and acetyl units were determined through examination of HMBC cross peak correlations between H-23 (δH 4.08 and 4.38) and C-7′ (δc 167.2); H-3 (δH 5.12) and C-8′ (δc 170.7), respectively. Therefore, compound 3 was determined to be 3-acetoxy-23-O-galloyloleanolic acid. Total assignments of protons and carbons of compound 3 were performed based on HSQC and HMBC, as shown in Table 1. Twenty-two known compounds (see Fig. 1) were characterized by their corresponding NMR spectra. The obtained data was compared with the data reported in the literature. The known compounds were identified as oleanolic acid (4) (Yang, Tian, Xiao, Han, & Jiang, 2012), 3β, 23-dihydroxyolean-12-en-28-oic acid (5) (Liu, Zheng, Yu, Zhang, & Lin, 2005), arjunolic acid (6) (Li, Zhou, & Kong, 2009), 2α,3β,19α,23tetrahydroxyolean-12-en-28-oic acid (7) (Xia et al., 2010), 3-O-acetyloleanolic acid (8) (Akira & Hideji, 1989), 3β,23-O-acetylolean-12-en28-oic acid (9) (Ilye et al., 1989), arjunglucoside II (10) (Eder, Walmir, Lidilhone, Caroline, & Fernanda, 2008), arjunglucoside I (11) (Zhou et al., 1992), 3-O-galloylursolic acid(12), ursolic acid (13) (Zhou, Wu, Han, & Wang, 2016), rotundic acid (14) (Hiroshi et al., 2007), 3-Oacetylursolic acid (15) (Feng, Feng, Wang, & Cui, 2011), 2α,3β,19α,23tetrahydroxyurs-12-en-28-oic acid (16) (Li, Shen, Li, & Yu, 2003), nigaichigoside F1 (17) (David, Liu, & Michael, 1994), bayogenin (18) (Masaki et al., 2009), methy barbinervate (19) (Luo et al., 2016), germanicol acetate (20) (Michael, Michelle, & Seiichi, 2000), stigmast-4-

2.9. Statistical analysis All data of the inhibitory activities were expressed as the mean of triplicate determinations and standard deviation (SD). Analysis of IC50 values were performed by using SPSS 16.0 program. 3. Results and discussion 3.1. Phytochemical investigation The structure of 3 new (1–3) pentacyclic triterpenes and 22 known (4–25) compounds see in Fig. 1. Compound 1 was obtained as a yellowish amorphous powder. Its molecular formula was determined as C37H52O7 by HR-ESI–MS at m/z 631.3599 [M + Na]+ (calcd for C37H52O7Na, 631.3605). 1H NMR spectrum of compound 1 showed signals corresponding to seven tertiary methyl groups at δH 0.83 (H-25), 0.89 (H-24), 0.93 (H-23), 0.96 (H-29), 1.00 (H-26), 1.02 (H-30), and 1.28 (H-27), one oxygen-bearing methine protons at δH 4.89 (dd, J = 11.8, 4.5 Hz, H-3) and olefinic proton at δH 5.48 (H-12). The 13C NMR showed six olefinic signals at δc 110.5, 122.2, 122.7, 141.2, 145.2, and 148.1, in which δc 122.7 and 145.2 were typical of double bond at C-12 and C-13 of an ole-12-enetype triterpene. Additionally, two carbonyl carbons at δc 180.6 (C-28) and δc 167.2 (C-7′), respectively, and one oxygenated carbon at δc 81.1 (C-3). Spectral data were compared with those from oleanolic acid, and strong agreement was observed, with exception of chemical shift corresponding to C-3 and presence of galloyl group. Considering molecular formula, compound 1 was presumed as triterpenoid gallate. Galloyl group was attached to C-3 because of HMBC correlation between H-3 (δH 4.89) and C-7′ (δc 167.2), as shown in Fig. 2. Large coupling constant of H-3 (J = 11.8 and 4.5 Hz) indicate that hydroxyl moiety at C-3 were oriented in β-position (Bisoli, Garcez, Hamerski, Tieppo, & Garcez, 2008). Thus, compound 1 was identified as 3-O-galloyloleanolic acid. Total assignments of protons and carbons of compound 1 were performed based on HSQC and HMBC, as shown in Table 1. Compound 2 was obtained as a yellowish amorphous powder. Its molecular formula was determined as C39H54O9 by FT-ICR-MS at m/z 689.36684 [M + Na]+ (calcd for C39H54O9Na, 689.3660). 1H NMR spectra showed six tertiary methyl signals at δH 0.84 (H-24), 0.87 (H235

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Table 1 NMR data of compound 1–3 in C5D5N (1H: 600 MHz,

13

Table 2 Inhibitory activities of extracts and isolated compounds.

C: 100 MHz).

Position

1 δc δH, mult. (J)

2 δc δH, mult. (J)

3 δc δH, mult. (J)

1

38.6

38.2

38.2

2 3

28.6 81.1

Compounds

PTP1B

α-Glucosidase

α-Amylase

0.34 ± 0.01a 0.39 ± 0.03a 0.70 ± 0.03a 5.54 ± 0.18a 7.18 ± 0.21a 82.79 ± 3.21a > 100a 11.72 ± 0.79a 12.71 ± 0.87a > 100a > 100a 0.35 ± 0.03a 6.37 ± 0.26a > 100a 9.69 ± 0.34a > 100a > 100a 25.92 ± 1.59a > 100a > 100a > 100a > 100a 53.39 ± 2.56a > 100a 75.90 ± 1.22a 1.24 ± 0.09b 0.97 ± 0.04b 0.75 ± 0.07b 4.73 ± 0.47b

> 100a > 100a > 100a 77.24 ± > 100a > 100a > 100a > 100a > 100a > 100a > 100a > 100a 84.43 ± > 100a > 100a > 100a > 100a > 100a > 100a > 100a > 100a > 100a > 100a > 100a > 100a 88.16 ± > 100b > 100b 80.97 ±

5.10 ± 0.18a 3.29 ± 0.12b

25.67 ± 0.63a 16.58 ± 0.41b

IC50

4 5 6

40.0 55.9 18.8

7 8 9 10 11 12 13 14 15 16

30.3 40.0 48.2 37.5 24.3 122.7 145.2 42.0 28.6 24.0

17 18

47.0 42.3

19

46.8

20 21

31.3 34.6

22

33.6

23

28.6

0.94, m 1.44, m 2.15, m 4.89, dd J = 11.8, 4.5 Hz – 0.86, m 1.29, m 1.44, m 1.26, m – 1.63, m – 1.65, m 5.48, t-like – – 1.19, m 1.81, m 2.15, m – 3.31, dd J = 3.7, 13.7 Hz 1.31, m 1.82, m – 1.20, m 1.45, m 1.85, m 2.06, m 0.93, s

24 25 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6′ 7′ OAc –CH3

17.5 15.7 17.7 26.5 180.6 33.6 24.1 122.2 110.5 148.1 141.2 148.1 110.5 167.2

0.89, 0.83, 1.00, 1.28, – 0.96, 1.02, – 7.90, – – – 7.90, –

s s s s s s s.

s.

28.6 75.2

41.6 48.9 18.7 33.2 40.1 48.5 37.4 23.9 122.8 145.2 42.5 28.6 24.1 47.1 42.4

46.8 31.3 34.6 33.6 66.6

13.8 16.1 17.8 26.4 180.6 33.6 24.0 122.0 110.6 148.1 141.4 148.1 110.6 167.0 170.9 21.0

0.96, m 1.46, m 2.16, m 5.34, dd J = 11.8, 4.8 Hz – 1.35, m 1.34, m 1.48, m 1.29, m – 1.71, m – 1.72, m 5.49, t-like – – 1.20, m 1.89, m 2.15, m – 3.31, dd J = 3.3, 13.6 Hz 1.32, m 1.82, m – 1.23, m 1.45, m 1.85, m 2.05, m 3.96, d J = 11.4 Hz 4.16, d J = 11.4 Hz 0.84, s 0.87, s 1.02, s 1.31, s – 0.95, s 1.02, s – 7.90, s – – – 7.90, s – – 1.97, s

28.4 75.3

41.6 49.0 18.7 33.2 40.0 48.6 37.1 23.6 122.5 145.3 42.4 28.4 24.1 47.0 42.3

46.7 31.3 34.5 33.5 65.9

13.5 16.0 17.7 26.3 180.5 33.6 23.9 121.5 110.5 148.1 141.5 148.1 110.5 167.2 170.7 21.4

0.50, m 1.29, m 2.07, m 5.12, dd J = 11.6, 4.6 Hz – 1.48, m 1.31, m 1.55, m 1.54, m – 1.58, m – 1.59, m 5.45, t-like – – 1.04, m 1.81, m 1.98, m – 3.26, dd J = 3.7, 13.7 Hz 1.26, m 1.75, m – 1.18, m 1.42, m 1.78, m 2.01, m 4.08, d J = 11.5 Hz 4.38, d J = 11.5 Hz 0.86, s 0.83, s 0.95, s 1.23, s – 0.93, s 0.99, s – 7.90, s – – – 7.90, s – – 2.00, s

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 75% EtOH extract PE extract EtOAc extract n-BuOH extract Na3VO4 Acarbose

a b

2.10 ± 0.17a 4.17 ± 0.38a 4.52 ± 0.20a 17.25 ± 0.84a 47.60 ± 2.76a > 100a > 100a 53.01 ± 1.46a 61.59 ± 2.36a > 100a > 100a 1.86 ± 0.21a 17.37 ± 0.95a > 100a 48.47 ± 2.53a > 100a > 100a 99.11 ± 5.09a > 100a > 100a 47.58 ± 0.88a > 100a > 100a > 100a > 100a 4.04 ± 0.16b 4.49 ± 0.18b 3.27 ± 0.20b 4.18 ± 0.15b 23.90 ± 1.78a 4.40 ± 0.33b

2.13a

1.62a

1.31b

2.46b

μM. μg/mL.

significant PTP1B inhibitory activities at IC50 values of 2.10, 4.17, 4.52, 17.25, 1.86, and 17.37 µM, respectively, compared with IC50 of positive control, Na3VO4, at 23.90 µM. Moreover, galloyl-substituted compounds 1, 2, 3, and 12, showed much stronger activity than compounds 4, 5, 9, and 13, which do not have galloyl group. As shown in Fig. 3A, the abilities of compounds 1, 2, 3, 12, and Na3VO4 to inhibit PTP1B were evaluated at five different doses (0.03, 0.30, 3.00, 10.00 and 30.00 µM). The test samples exhibited a dose dependent inhibition. The compounds 1, 2, 3, and 12 showed significant high PTP1B inhibitory activities (1: 80.12%; 2: 75.77%; 3: 74.82%; 12: 81.36%, at 30 µM), even at lower doses (1: 57.23%; 2: 42.6%; 3: 49.75%; 12: 64.89%, at 3 µM). C-3-acetylated compounds 8, 9, and 15 showed moderate inhibition compared with unacetylated compounds 4, 5, and 13. On the other hand, glycosylated and α-hydroxylated triterpenes at C-28 and C19 positions, respectively, did not exhibit activity with IC50 > 100 µM, suggesting importance of galloyl substituent in triterpene derivatives to inhibit PTP1B. Oleanane and ursane types did not show significant difference in PTP1B inhibitory activities under the same substituent group. Among sterols (21–25), only compound 21 with di-keto groups at C-3 and C-6 displayed moderate inhibition with IC50 of 47.58 µM, indicating that ketone moieties in sterols may enhance PTP1B inhibitory activity. Potent inhibitory effects on PTP1B were observed among extracts of 75% EtOH, PE, EtOAc, and n-BuOH with IC50 values of 4.04, 4.49, 3.27, and 4.18 µg/mL, respectively, compared with Na3VO4 at IC50 of 4.40 µg/mL. Meanwhile, our results showed that compounds 1, 2, 3, and 12 inhibited PTP1B better than Na3VO4, these information may also explain why EtOAc extract yielded strong PTP1B inhibition.

ene-3,6-dione (21) (Elvira & Higuinaldo, 1993), 22,23-dihydrospinasterol (22) (Hisashi, Noriko, Akiko, & Haruo, 1990), 22,23dihydrospinasterol-3-O-β-D-glucoside (23) (Wu, Xie, Li, Bai, & Cheng, 2013), α-spinaterol (24) (Zhang, Yang, Xu, Zou, & Yang, 2005), 3-O-βD-glucopyranosylspinasterol (25) (Tae et al., 2011). 3.2. Bioactivity assay All the extracts and compounds from acorns of Quercus liaotungensis were researched for their enzyme inhibition using in-vitro assays on PTP1B, α-glucosidase, and α-amylase. The results are presented in Table 2. 3.2.1. PTP1B inhibition assay All isolates (1–25) were analyzed for in vitro inhibition of PTP1B. Among triterpenes (1–20), compounds 1, 2, 3, 4, 12, and 13 showed 236

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Fig. 3. Enzyme inhibitory effects of compounds 1, 2, 3, 12, and the reference. (A) Inhibitory activities against PTP1B. (B) Inhibitory activities against a-glucosidase. Values are expressed as the mean ± SD (n = 3). Bars with different letters are significantly different (p < 0.05).

Fig. 4. Lineweaver-Burk plot analysis of the inhibition kinetics of PTP1B inhibitory effects by isolated compounds 1, 2, 3, and 12. The data are expressed as the mean reciprocal of initial velocity for n = 3 replicates at each substrate concentration.

3.2.2. α-Glucosidase inhibition assay The analysis of α-glucosidase inhibition, compounds 1, 2, 3, and 12 significantly inhibited α-glucosidase enzyme at IC50 values of 0.34, 0.39, 0.70, and 0.35 µM, respectively. The inhibitory activity was measured for compounds (1, 2, 3, and 12) and acarbose at 0.03, 0.30, 3.00, 10.00 and 30.00 µM concentration. All the test samples exhibited a dose dependent inhibition as shown in Fig. 3B. Consistent with the results obtained for PTP1B inhibition determined in this study, the same trend of matter-dependent α-glucosidase inhibition values were observed. The compounds 1, 2, 3, and 12 showed significant high αglucosidase inhibitory activities (1: 83.01%; 2: 83.06%; 3: 82.59%; 12:

82.29%, at 10 µM), even at lower doses (1: 51.05%; 2: 50.41%; 3: 39.06%; 12: 55.88%, at 0.3 µM). Compounds 4, 5, 8, 9, 13, 15, and 18 also showed moderate inhibition at IC50 values of 5.54, 7.18, 11.72, 12.71, 6.37, 9.69, and 25.92 µM, respectively. Compounds 6, 23, and 25 displayed weak inhibitions at IC50 values of 82.79, 53.39, and 75.90 µM, respectively, whereas positive control, acarbose, has IC50 value of 5.10 µM. Among isolates, galloyl unit linked at C-3 or C-23 may be responsible for increased inhibitory activity of pentacyclic triterpene skeleton. By contrast, α-hydroxyl moiety at C-19 presented no activity at IC50 > 100 µM. Inhibitory activity was not observed in pentacyclic triterpene substituted with sugar moiety at C-28. Compound 6 is

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Fig. 5. Lineweaver-Burk plot analysis of the inhibition kinetics of a-glucosidase inhibitory effects by isolated compounds 1, 2, 3, and 12. The data are expressed as the mean reciprocal of initial velocity for n = 3 replicates at each substrate concentration.

different from compound 18 in terms of orientation of hydroxyl moiety at C-2. With hydroxyl moiety at C-2 at β-position, compound 18 shows stronger activity at IC50 value of 25.92 µM compared with IC50 of 82.79 µM for compound 6. Notably, same trend was also reflected in PTP1B inhibition activity of these compounds. Based on above results, galloyl substituent on triterpene derivative is most important factor in inhibition of PTP1B and α-glucosidase activities. With same substituent, oleanane and ursane types did not show significant difference in αglucosidase inhibitory activity. Among sterols (21–25), with sugar moiety attached at C-3, compounds 23 and 25 showed stronger activity than aglycone (compounds 22 and 24). However, this trend is opposite to that of triterpenoids results, where triterpenoid aglycone showed better inhibitory activities than glucoside. As shown in Table 2, 75% EtOH, PE, EtOAc, and n-BuOH extracts significantly inhibited α-glucosidase in dose-dependent manner at IC50 values of 1.24, 0.97, 0.75, and 4.73 µg/mL, respectively, compared with acarbose at IC50 of 3.29 µg/mL. Among compounds tested, compounds 1, 2, 3, and 12 resulted in better α-glucosidase inhibition compared with acarbose, which might contribute to the inhibitory effect of EtOAc.

3.2.4. Enzyme kinetic assay In our continued efforts to identify the type of PTP1B and αGlucosidase inhibition, we employed a Lineweaver-Burk plot. The reciprocal of the rate of the reaction was plotted against the reciprocal of the substrate concentration to monitor the effect of the inhibitor on both Km and Vmax. The kinetics studies of compounds 1, 2, 3, and 12 revealed competitive inhibition toward to the substrate with pNPP (PTP1B) and pNPG (α-Glucosidase) (Figs. 4 and 5). Figs. 4 and 5 all revealed that, with increasing concentration of compounds 1, 2, 3, and 12, Km significantly increased, whereas the Vmax remained unaltered, which indicated their competitive inhibition for PTP1B and α-Glucosidase.

4. Conclusion The extract of acorn was seperated by various chromatographic methods, 3 new triterpenoids and 22 known compounds were purified and identified. To evaluate their hypoglycemic activity, the inhibitory activity of the extracts and isolated compounds were tested against three different targets including α-glucosidase, α-amylase and PTP1B, which are directly related to the absorption or metabolism of glucose. Furthermore, based on structure-activity relationship observation, we found the effects of minor structural differences on inhibitory activity which may provide a new basis for development of new hypoglycemic agent. In conclusion, our studies provide foundation for future research on potential hypoglycaemic function of acorn and development of dietary supplements for prevention of diabetes mellitus.

3.2.3. α-Amylase inhibition assay Table 2 shows weak inhibition of α-amylase by extracts of 75% EtOH and n-BuOH at IC50 values of 88.16 and 80.97 µg/mL, respectively, compared with acarbose (IC50 = 16.58 µg/mL). Among all compounds tested, compounds 4 and 13 weakly inhibited α-amylase. For various compounds, different inhibitory activities are possibly related to their different phytochemical identities. However, only limited information is available for α-amylase inhibitory activity of compounds from acorn. Therefore, further study is necessary to identify active compounds from extracts.

Notes The authors declare that there are no conflicts of interest. 238

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Acknowledgments

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