Structural characterization, hepatoprotective and antihyperlipidemic activities of alkaloid derivatives from Murraya koenigii

Phytochemistry Letters 35 (2020) 135–140

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Structural characterization, hepatoprotective and antihyperlipidemic activities of alkaloid derivatives from Murraya koenigii

T

Rongrui Weia, Qinge Maa,*, Guoyue Zhonga, Yulun Sub, Jianbo Yangb, Aiguo Wangb, Tengfei Jib, Hongxia Guoa, Meiling Wanga, Ping Jianga, Haichao Wua a

State Key Laboratory of Innovative Drugs and High Efficiency Energy Saving and Consumption Reduction Pharmaceutical Equipment, Research Center of Natural Resources of Chinese Medicinal Materials and Ethnic Medicine, Key Laboratory of Modern Preparation of TCM of Ministry of Education, Jiangxi University of Traditional Chinese Medicine, Nanchang, 330004, China b State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, China

ARTICLE INFO

ABSTRACT

Keywords: Murraya koenigii Alkaloid derivatives Hepatoprotective Antihyperlipidemic

Three new alkaloids (1-3) and a new natural alkaloid (4), named (1′R,3′R,4′R,6′S)-endocycliomurrayamine-A (1), 3-formyle-7-hydroxy-9H-carbazole-1-O-β-D-glucopyranoside (2), 4′-hydroxyphenyl-6-ethyl-1H-pyrrole-2carboxaldehyde (3), and 4-hydroxyphenoxy-N-methyl-propanamide (4), together with thirteen known alkaloid derivatives (5–17) were isolated from Murraya koenigii. Among them, compounds (8–17) were isolated from this plant for the first time. The structures of compounds (1–17) were elucidated on the basis of their spectroscopic analysis and references. Compounds 1, 15, 16, and 17 (10 μM) showed moderate hepatoprotective activities against D-galactosamine-induced HL-7702 cells damage. Compounds 2, 3, 9, and 13 showed moderate activation of PPARα and PPARγ receptors.

1. Introduction Murraya koenigii (L.) Spreng, belonging to the Rutaceae family, is popular as an ingredient in the Southeast Asia (Ma et al., 2013). It is mainly distributed Yunnan, Guangxi, and Hainan provinces in China. The leaf of M. koenigii is well known as curry leaf, and its dried powder is traditionally added to Indian gravy and other vegetables for the delicious taste (Mani and Milind, 2009). Previous phytochemical studies on M. koenigii afforded structurally-diverse compounds, such as phenylpropanoids, alkaloids, sesquiterpenes, alkenes, and volatile oils (Ma et al., 2019). M. koenigii has been known for its medical properties for the treatment of nephroprotective (Mahipal and Pawar, 2017), antiinflammatory (Rautela et al., 2018), hepatoprotective (Ma et al., 2014), antioxidative (Ma et al., 2016), antilisterial (Kumar et al., 2017) PTP1B inhibitory (Rahman and Gray, 2005), immunomodulatory (Priyanka et al., 2011) inhibitory activity by measuring IL-6-induced STAT3 promoter activity in HepG2 cells, and inhibition against lipopolysaccharide (LPS)-induced NO production in RAW264.7 macrophages functions (Ma et al., 2019). Inspired by the functions of M. koenigii in the treatment of many diseases, we carried out a bioactivity-guided investigation of M. koenigii in order to evaluate their further pharmacological potentials. As a result, three new alkaloids (1–3) and a new ⁎

natural alkaloid (4), together with thirteen known alkaloids derivatives (5–17) were isolated from the active fractions of M. koenigii. Meanwhile, compounds (1–17) were evaluated for their hepatoprotective and antihyperlipidemic activities in this work. Described herein are the isolation, structural elucidation, and bioactivity evaluation of these alkaloid derivatives (1–17) from M. koenigii in this paper (Supporting information: Fig. 1). 2. Results and discussion 2.1. Structure elucidation Compound 1 was obtained as colorless oil. Its molecular formula was determined to be C23H25NO3, established by HR-ESI-MS at m/z 364.1905 [M+H]+ (calcd. for C23H26NO3, 364.1907) corresponding to twelve degrees of unsaturation. The UV spectrum showed at λmax 202, 237, 267, and 319 nm, indicating the presence of the carbazole alkaloid skeleton (Sim and Teh, 2011). The IR spectrum implied the presence of amino (3343 cm−1), hydroxyl (2923 cm−1), benzene ring (1620 cm−1) functionalities. In the 1H NMR spectrum of compound 1, there was an ABX system that showed at δH 7.23 (1H, d, J =8.4 Hz, H-5), 6.68 (1H, dd, J = 8.4, 2.4 Hz, H-6), 6.96 (1H, d, J =2.4 Hz, H-8), and a single

Corresponding author. E-mail address: [email protected] (Q. Ma).

https://doi.org/10.1016/j.phytol.2019.11.001 Received 30 September 2019; Received in revised form 1 November 2019; Accepted 4 November 2019 1874-3900/ © 2019 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

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Fig. 1. Structures of compounds 1-17.

peak at δH 7.62 (1H, s, H-4) in the aromatic field. Moreover, there were four methyl signals at δH 2.25 (3H, s, 10−CH3), 1.70 (3H, s, 8′−CH3), 1.34 (3H, s, 9′−CH3), and 1.32 (3H, s, 10′−CH3) in the high field of 1H NMR spectrum. The above information coupled with biogenetic considerations and literature indicated that the H-7 was substituted by hydroxyl in the alkaloid skeleton (Tian et al., 1995). In the 13C NMR spectrum of compound 1, there were twenty-three carbon signals consisting of twelve aromatic carbons, four methyl carbons, three linked oxygen carbons, and four other carbons (Table 1). Meanwhile, two methylenes (C-2′, C-7′) and two methines (C-4′, C-6′) were confirmed by the HSQC and DEPT spectra. The fragments of compound 1 were connected by the HMBC correlations of H-4/C-2, C-9a; H-5/C-4a, C-7, C-8a; H-6/C-4b, C-8; H-8/C-4b, C-6; H-1′/C-1, C-2, C-3′, C-6′,C-9a; H2′/C-1, C-4′; H-4′/C-2′, C-6′; H-6′/C-1′, C-4′; H-7′/C-3′, C-5′ (Fig. 2) and the 1H-1H COSY spectrum correlations of H-5/H-6; H-1′/H-2′; H-6′/H7′; H-4′/H-7′ (Fig. 2). The absolute configuration of compound 1 was determined by circular dichroism (CD) method. In the CD spectrum of compound 1, there were a positive cotton effect at 218 nm and a negative cotton effect at 247 nm. According to the reference (Tian et al., 1995), the absolute configuration of C-3′ was determined as 3′R. In addition, according to the 2D-NOESY spectrum correlations of H-1′/H4′ and H-4′/H-6′ and the rule of Cahn-Ingold-Prelog, the absolute configurations of C-1′, C-4′, and C-6′ were determined as 1′R, 4′R, and 6′S. Therefore, compound 1 was identified as (1′R,3′R,4′R,6′S)-

endocycliomurrayamine-A. Compound 2 was obtained as colorless powder, the molecular formula of C19H19NO8 based on HR-ESI-MS at m/z 390.1190 [M+H]+ (calcd. for C19H20NO8, 390.1183), indicating eleven degrees of unsaturation. The UVspectrum showed absorptions at λmax: 203, 237, 285 nm, indicating the presence of the carbazole alkaloid skeleton (Chihiro et al., 1988), and the IR spectrum showed the absorption bands of amino (3288 cm−1), hydroxyl (2928 cm−1), benzene ring (1660, 1615 cm−1) functionalities. In the 1H NMR spectrum of compound 2, there were an ABX system showed at δH 6.81 (1H, dd, J = 8.7, 2.1 Hz, H-5), 7.95 (1H, d, J =8.7 Hz, H-6), 6.95 (1H, d, J =2.1 Hz, H-8) and two single peaks at δH 7.70 (1H, s, H-2), 8.27 (1H, s, H-4). The above information coupled with biogenetic considerations and literature indicated the carbazole alkaloid skeleton was in compound 2 (Chihiro et al., 1988). Furthermore, there were some characteristic signals of the fragment of glucose: δH 5.08 (1H, d, J =8.4 Hz, H-1′), 3.45–3.61 (4H, m, H-2′,3′,4′,5′), 4.00 (1H, dd, J = 12.0, 4.8 Hz, H-6a), 3.82 (1H, dd, J = 16.0, 4.8 Hz, H-6b) (Table 1) in the high field of 1H NMR spectrum. The 13C NMR spectrum of compound 2 showed the presence of a carbonyl carbon, twelve aromatic carbons attributed to alkaloid skeleton, and six carbons of the glucose. In the HMBC spectrum of compound 2, the correlation of H-1′/C-1 indicated that H-1 was substituted by the glucose unit. The fragments of compound 2 were connected by some key correlations of H-2/C-4, C-9a, C-10; H-4/C-2, C-9a, C-10; H-5/C-4a, 136

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Table 1 1 H NMR,

13

C NMR, and HMBC correlations of compounds 1 and 2. 1a

No.

1 2 3 4 4a 4b 5 6 7 8 8a

9 9a 10-CH3 1′ 2′ 3′ 4′ 5′ 6′ – 7′ 8′-CH3 9′-CH3 10′-CH3

2b

δH

δC

HMBC(1H -13C)

δH

δC

HMBC(1H-13C)

– – – 7.62(s) – – 7.23(d,8.4) 6.68(d,8.4,2.4) – 6.96(d,2.4) – 9.89(brs) – 2.25(s) 5.28(d,3.6) 1.50(m) – 1.96(t,9.0,4.8) – 1.83(m) – 1.58(m) 1.70(s) 1.34(s) 1.32(s)

107.2 148.5 118.2 120.9 117.7 117.0 120.2 108.9 156.2 97.8 142.5–

– – – C-2,C-9a – – C-4a,C-7,C-8a C-4b,C-8 – C-4b,C-6 ––

– 7.70(s) – 8.27(s) – – 6.81(dd,8.7,2.1) 7.95(d,8.7) – 6.95(d,2.1) ––

114.9 110.0 136.8 120.4 126.6 143.6 111.5 122.2 158.7 98.2 130.6–

– C-4,C-9a,C-10 – C-2,C-9a,C-10 – – C-4a,C-7,C-8a C-4b,C-8 – C-4b,C-6 ––

139.4 16.6 70.7 28.6 78.2 51.6 43.3 82.3 – 27.7 30.9 28.2 23.2

– – C-1,C-2,C-3′,C-6′,C-9a C-1,C-4′ – C-2′,C-6′ – C-1′,C-4′ – C-3′,C-5′– – – –

– 9.96(s) 5.08(d,8.4) 3.61(m) 3.55(m) 3.45(m) 3.51(m) 4.00(12.0,4.8) 3.82(16.0,4. 8) – – – –

117.7 193.9 104.1 75.1 78.4 71.4 77.8 62.5 – – – – –

– – C-1,C-3′,C-5′ C-4′ C-1′,C-5′ C-6′ C-1′,C-3′ C-4′ C-4′ – – – –

a 1

H NMR (600 MHz, CD3COCD3), 13C NMR (150 MHz, CD3COCD3). H NMR (300 MHz, MeOD), 13C NMR (125 MHz, MeOD).

b 1

C-7, C-8a; H-6/C-4b, C-8; H-8/C-4b, C-6; H-1′/C-1, C-3′, C-5′; H-2′/C-4′; H-3′/C-1′, C-5′; H-4′/C-6′; H-5′/C-1′, C-3′; H-6′/C-4′ in the HMBC spectrum (Fig. 2) and some key correlations of H-5/H-6 in the 1H-1H COSY spectrum (Fig. 2). Therefore, compound 2 was elucidated as 3formyle-7-hydroxy-9H-carbazole-1-O-β-D-glucopyranoside. Compound 3 was isolated as pale yellow powder, the molecular formula of C13H13NO2 based on HR-ESI-MS at m/z 216.1016 [M+H]+ (calcd for C13H14NO2, 216.1069), indicating eight degrees of unsaturation. Its UV spectrum showed absorbances at 203, 223, 286 nm (Yang et al., 2002) it was concluded that the pyrrolidine alkaloid skeleton was in compound 3. Its IR absorptions indicated the existence of hydroxyl (3220 cm−1), carbonyl (1723 cm−1), benzene ring (1637, 1615, 1517 cm−1) functionalities. There was an AA´BB´ system at δH 7.00 (2H, d, J =8.4 Hz, H-2´/6´), 6.75 (2H, d, J =8.4 Hz, H-3´/5´), an ABC system at δH 6.98 (1H, d, J =2.7 Hz, H-3), 6.16 (1H, dd, J = 3.9, 2.7 Hz, H-4), 6.99 (1H, d, J =2.7 Hz, H-5), and a broad single peak at δH 9.57 (1H, brs, CHO) in the 1H NMR spectrum of compound 3 (Table 2). Furthermore, two typical triple peaks at δH 4.49 (2H, t, J =7.2 Hz, H-6), 2.92 (2H, t, J =7.2 Hz, H-7) in the 1H NMR spectrum indicated that the fragment of −CH2−CH2- was in compound 3. The fragments of compound 3 were connected by some key correlations of H-3/C-5; H-4/C-2; H-5/C-2, C-3; H-6/C-1´, C-5; H-7/C-2´, C-6´; H-2´/C4´, C-6´, C-7; H-3´/C-1´, C-5´; H-5´/C-1´, C-3´; H-6´/C-2´, C-4´ in the HMBC spectrum of compound 3, and correlations of H-3/H-4; H-4/H-5; H-2´/H-3´; H-5´/H-6´ in the 1H-1H COSY spectrum (Fig. 2). Consequently, compound 3 was determined as 4′-hydroxyphenyl-6-ethyl-1Hpyrrole-2-carboxaldehyde (3). Additionally, other fourteen known alkaloid derivatives (4-17) were isolated from M. koenigii, and their structures were identified as 4-hydroxyphenoxy-N-methyl-propanamide (4) (Kato and Kitajima, 1988), cyclomahanimbine (5) (Adebajo et al., 2005), murrayazolinine (6) (Chakraborty et al., 2009), 3-formylcarbazole (7) (Chihiro et al., 1988), pyrrolezanthine-6-methyl ether (8) (Xu et al., 2009), pyrolezanthine (9) (Yang et al., 2002), 5-hydroxymethyl-1-methylpyrrol-2-carbaldehyde (10) (Jurch and Tatum, 1970), 2-formyl-5-hydroxymethyl-pyrrole (11) (Hiermann et al., 2002), N-trans-feruloyl-3′-O-methyldopamine (12)

(Zhao et al., 2018), portulacatone (13) (Yue et al., 2015), claulansium B (14) (Peng et al., 2018), claulansiums A (Peng et al., 2018), 1′-Omethylclaulamine B (16) (Peng et al., 2018), Dunnine E (17) (Cao et al., 2018) by comparison of their physical and spectroscopic data with those reported in the references. 2.2. Conclusion In this work, compounds (1-17) were bioassayed for hepatoprotective activities against D-galactosamine-induced toxicity in HL-7702 cells, and the hepatoprotective activity drug bicyclol was used as the positive control. As shown in Table 3, compounds 1, 15, 16, and 17 (10 μM) showed moderate hepatoprotective activities. Moreover, compounds (1-17) were assayed for their antihyperlipidemic activities in terms of PPARα and PPARγ activation through a reporter gene assay. Among them, compounds 2 and 13 showed moderate activation of PPARα receptors, and compounds 3 and 9 showed moderate activation of PPARγ receptors (Table 4). However, the residual compounds showed no activation of PPARα and PPARγ. These findings shed much light on a better understanding of the hepatoprotective and antihyperlipidemic activities of these alkaloid derivatives and provided new insights into developing better hepatoprotective and antihyperlipidemic drugs in the future. 3. Experimental 3.1. General experimental procedures The IR spectra were recorded on a Nicolet 5700 FT-IR microscope spectrometer (Shanghai Xiangrun Industry Co. Ltd., Shanghai, China). The UV spectra were taken with a Hitachi UV-240 spectrophotometer (GBC Scientific Equipment Pty. Ltd., Braeside, Australia). The optical rotations were recorded on a Perkin-Elmer 241 digital polarimeter at 20℃ (PerkinElmer, America). The NMR spectra were performed on Varian Mercury-300, Inova-501, DD2-500,VNS-600 spectrometers with tetramethylsilane as an internal standard. The ESI-MS data were 137

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Table 3 Hepatoprotective effects of compounds 1, 15, 16, and 17 (10 μM) against Dgalactosamine-induced toxicity in HL-7702 cellsa. compound

cell survival rate(% of normal)

inhibition(% of control)

normal control bicyclol 1 15 16 17

100 .0 ± 2.3 56.2 ± 1.9 70.3 ± 5.6* 87.5 ± 3.4*** 68.8 ± 2.7* 71.3 ± 3.8* 79.5 ± 2.7**

– – 32.2 71.5 28.8 34.5 53.2

a Results were expressed as means ± SD (n = 3; for normal and control, n = 6); bicyclol was used as positive control (10 μM). * p < 0.05. ** p < 0.01. *** p < 0.001.

Table 4 Activation of PPARα and PPARγ by compounds 2, 3, 9, and 13a. compound

2 3 9 13

fold induction in PPARα activitya

fold induction in PPARγ activityb

50 μg/ mL

25 μg/ mL

12.5 μg/ mL

50 μg/ mL

25 μg/ mL

12.5 μg/ mL

1.68 NA NA 1.42

1.36 NA NA 1.22

1.18 NA NA 1.06

NAc 2.03 1.66 NA

NA 1.75 1.34 NA

NA 1.36 1.12 NA

a

Ciprofibrate (1.47 μg/mL) caused a 2.2-fold induction in PPARα activity. Rosiglitazone (1.81 μg/mL) caused a 3.5-fold induction inactivity PPARγ activity. c NA = No Activation. b

California, America). The HPLC separation was performed by an Agilent 1200 series with a DIKMA (4.6 × 250 mm) analytical column packed with C18 (5 μm) (Agilent Technologies, California, America) and the preparative HPLC was conducted by a Shimadzu LC-6AD instrument with a SPD-20A detector and an YMC-Pack ODS-A column (250 × 20 mm, 5 μm) (Shimadzu Corporation, Japan). The column chromatography was performed on Sephadex LH-20 and silica gel (100–200, 200–300 mesh) (Qingdao Marine Chemical Inc., Qingdao, China). The TLC experiment was carried out on precoated silica gel GF254 plates and the spots were visualized under UV light (254 or 365 nm) or by spraying with 10 % sulfuric acid in EtOH followed by heating (Ma et al., 2013). 3.2. Plant material M. koenigii was collected from Xi Shuang Ban Na Tropical Botanical Garden, the Chinese Academy of Sciences, Kunming, China, in August 2010. This plant was authenticated by Prof. Lin Ma (a specialist in herbal medicine, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College). The voucher specimen (No.ID-S-2436) was deposited at the Herbarium of Institute of

Fig. 2. Key HMBC and 1H-1H COSY correlations of 1-3.

measured by a Q-Trap LC/MS/MS spectrometer (Turbo Ionspray Source) and HR-ESI-MS data were were recorded on an Agilent 1100 series LC/MSD Trap SL mass spectrometer (Agilent Technologies, Table 2 1 H NMR (300 MHz, CD3COCD3), No.

2 3 4 5 6 7

13

C NMR (125 MHz, CD3COCD3), and HMBC correlations of compounds 3.

3

3 1

13

δH

δC

HMBC( H - C)

δH

– 6.98(d,2.7) 6.16(dd,3.9,2.7) 6.99(d,2.7) 4.49(t,7.2) 2.92(t,7.2)

130.7 125.2 109.8 132.5 51.3 37.7

– C-5 C-2 C-2,C-3 C-1′,C-5 C-2′,C-6′

1′ 2′ 3′ 4′ 5′ 6′

138

– 7.00(d,8.4) 6.75(d,8.4) – 6.75(d,8.4) 7.00(d,8.4)

δC

HMBC(1H-13C)

129.9 130.7 116.0 156.9 116.0 130.7

– C-4′,C-6′,C-7 C-1′,C-5′ – C-1′,C-3′ C-2′,C-4′

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Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.

on cytotoxicity induced by D-Galactosamine in HL-7702 cells by a 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay, using the hepatoprotective activity drug bicyclol as the positive control (Wang et al., 2013). Each cell suspension of 2 × 104 cells in 200 μL of RPMI 1640 containing fetal calf serum (10 %), penicillin (100 U/mL), and streptomycin (100 μg/mL) was placed in a 96-well microplate and precultured at 37 °C under a 5 % CO2 atmosphere for 24 h. Fresh medium (100 μL) containing bicyclol and test samples was added, and the cells were cultured for 1 h. The cultured cells were exposed to D-galactosamine (25 mM) for 24 h. Then, the MTT (100 μL, 0.5 mg/mL) was added to each well after the withdrawal of the culture medium and incubated for an additional 4 h. The resulting formazan was dissolved in DMSO (150 μL) after aspiration of the culture medium. The optical density (OD) of the formazan solution was measured on a microplate reader at 492 nm. Inhibition (%) was obtained by the following formula: inhibition (%) = [(OD(sample) OD(control))/(OD(normal) - OD(control))] × 100 (Ma et al., 2018).

3.3. Extraction and isolation The air-dried whole plant of M. koenigii (22.50 kg) was extracted with 95 % EtOH (60 L) heating under reflux for 3 h each time. The extracts were concentrated by rotary evaporator under reduced pressure resulting in a dark brown residue (2.10 kg). The residue was suspended in 1.5 % HCl (7 L) and filtered. The filtrate was tuning with ammonia into pH = 8–9 and pH = 9–10, then partitioned with CHCl3 (18 L), separately. After this, the remaining filtrate was tuned with 1.5 % HCl into pH = 7, and partitioned with EtOAc (18 L) and n-BuOH (18 L) to obtain the CHCl3 soluble fraction (33.50 g), the EtOAc soluble fraction (24.90 g), the n-Butanol soluble fraction (221.20 g) (Ma et al., 2013). According to the screening results of bioactivity-guided investigation, the CHCl3 soluble fraction exhibited potential hepatoprotective and antihyperlipidemic activities. The CHCl3 soluble fraction was fractionated over silica gel (100–200 mesh) eluting with petroleum ether-acetone (10:1, 5:1, 1:1, 1:5, 1:10) to afford twenty fractions, A1A20. The fraction A5 (3.15 g) was chromatographed over silica gel (200–300 mesh) eluting with petroleum ether-EtOAc gradients (9:1, 7:1, 5:1) to give five sub-fractions, A5a-A5e. The separation of A5b (1.86 g) was chromatographed over Sephadex LH-20 eluting with 95 % MeOH to give three sub-fractions, A5b1-A5b3. Separation of fraction A5b2 (1.02 g) by MPLC (45–100 % MeOH) and preparative HPLC (λmax = 203 nm, 6 mL/min), successively, yielded 1 (1.35 mg), 2 (2.00 mg), 4 (5.36 mg), 5 (5.27 mg), 6 (1.44 mg), 7 (2.37 mg), 12 (7.58 mg), 13 (5.85 mg), and 16 (4.05 mg). In the same way, the fraction A7 (2.20 g) was repeatedly chromatographed over silica gel (100–200, 200–300 mesh), Sephadex LH-20, and preparative HPLC, successively, yielded 3 (1.17 mg), 8 (2.28 mg), 9 (13.00 mg), 10 (2.88 mg), 11 (1.00 mg), 14 (6.53 mg), 15 (7.60 mg), and 17 (4.88 mg). The structures of compounds (1-17) were shown in Supporting information: Fig. 1. (1′R,3′R,4′R,6′S)-endocycliomurrayamine-A (1): colorless soil; [α]20 D +4.02 (c 0.02, MeOH); UV (MeOH) λmax: 202, 237, 267, 319 nm; CD (MeOH): 218 (Δε +0.63), 247(Δε -0.04) nm; IRνmax: 3343, 2923, 1620 cm−1; 1H NMR (CD3COCD3, 600 MHz) and 13C NMR (CD3COCD3, 150 MHz) data, see Table 1; HR-ESI-MS m/z 364.1905[M+H]+(calcd. for C23H26NO3, 364.1907). (2): 3-formyle-7-hydroxy-9H-carbazole-1-O-β-D-glucopyranoside colorless powder; [α]20 D -2.50 (c 0.10, MeOH); mp: 235.2-236.7 °C; UV (MeOH) λmax: 203, 237, 285 nm; IRνmax: 3288, 2928, 1660, 1615 cm−1; 1 H NMR (CD3OD, 300 MHz) and 13C NMR (CD3OD, 125 MHz) data, see Table 1; HR-ESI-MS m/z 390.1190 [M+H]+(calcd. for C19H20NO8, 390.1183). 4′-hydroxyphenyl-6-ethyl-1H-pyrrole-2-carboxaldehyde (3): pale yellow powder, mp: 102.6-104.2 °C; UV (MeOH) λmax: 203, 223, 286 nm; IR νmax: 3220, 1723, 1637, 1615, 1517 cm−1; 1H NMR (CD3COCD3, 300 MHz) and 13C NMR (CD3COCD3, 125 MHz) data, see Table 2; HRESI-MS m/z 216.1016 [M+H]+(calcd. for C13H14NO3, 216.1019).

3.3.3. Antihyperlipidemic effects assay The activations of PPARα and PPARγ of compounds (1-17) were determined in human hepatoma (HepG2) cells with Ciprofibrate and Rosiglitazone used as positive controls which described previously (Yang et al., 2013). The HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum(10 %), penicillin G sodium (100 units/mL), and streptomycin (100 μg/mL). The HepG2 cells were transfected with pSG5-PPARα and PPRE X3-tk-luc or pCMV-rPPARγ and pPPREaP2-tk-luc plasmid DNA (25 μg/1.5 mL cell suspension), respectively, by electroporation at 160 V for a single 70 ms pulse using a BTX Electro Square Porator T 820 to assay for the PPARα and PPARγ activation. The transfected cells were plated in 96-well tissue culture plates with a density of 5 × 104 cells/ well and grown for 24 h. The cells were treated with compounds (1-17) after incubation for 24 h, they were lysed and the luciferase activity was measured. The fold induction of luciferase activity in treated cells was calculated in comparison to the vehicle control (Safa et al., 2018). Declaration of Competing Interest There are no conflicts of interest to declare. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (81803843), the Standard Revision Research Project of National Pharmacopoeia Committee (2018Z090), the Key Scientific Research Project of Colleges and Universities in Henan Province (19A350006), the Science and Technology Project of Jiangxi Health Commission (20195648, 20195650), the Science and Technology Project of Jiangxi Provincial Department of Education (GJJ180662, GJJ180688), the Scientific Research Project of First-class Discipline of Chinese Materia Medica of Jiangxi University of TCM (JXSYLXK-ZHYAO027, JXSYLXK-ZHYAO032), and the Doctoral Research Initiation Fund Project of Jiangxi University of TCM (2018BSZR007, 2018BSZR010).

3.3.1. Acid hydrolysis and sugar analysis of compound 2 Compound 2 (1.0 mg) was treated in 5 % HCl (0.5 mL) and heated at 90 °C for 2 h (Chang and Case, 2005). The reaction mixture was extracted with EtOAc after cooling, and the aqueous layer was neutralized with 0.1 M NaOH. The type of sugar of compound 2 was identified by the TLC method with authentic sugar (β-D-glucose). Under the condition of CHCl3:MeOH:H2O = 14:6:1 (Kieselgel 60 F254 plate), the Rf value of β-D-glucose was 0.13. Meanwhile, the Rf value (n-BuOH/ pyridine/H2O = 6:4:3, Cellulose 60 F plate) of β-D-glucose was 0.37. Consequently, the type of sugar of compound 2 was confirmed as β-Dglucose (Ma et al., 2014).

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3.3.2. Hepatoprotective effects assay Compounds (1-17) were assayed for their hepatoprotective effects 139

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