Novel nortriterpenoids with new skeletons and limonoids from the fruits of Evodia rutaecarpa and their bioactivities

Journal Pre-proof Novel nortriterpenoids with new skeletons and limonoids from the fruits of Evodia rutaecarpa and their bioactivities

Yu-Sheng Shi, Hong-Min Xia, Chuan-Hai Wu, Chun-Bin Li, Chen-Chen Duan, Chang Che, Xue-Jing Zhang, Hao-Tian Li, Yan Zhang, Xu-Fu Zhang PII:

S0367-326X(20)30085-X

DOI:

https://doi.org/10.1016/j.fitote.2020.104503

Reference:

FITOTE 104503

To appear in:

Fitoterapia

Received date:

28 December 2019

Accepted date:

8 February 2020

Please cite this article as: Y.-S. Shi, H.-M. Xia, C.-H. Wu, et al., Novel nortriterpenoids with new skeletons and limonoids from the fruits of Evodia rutaecarpa and their bioactivities, Fitoterapia (2020), https://doi.org/10.1016/j.fitote.2020.104503

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© 2020 Published by Elsevier.

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Novel nortriterpenoids with new skeletons and limonoids from the fruits of Evodia rutaecarpa and their bioactivities Yu-Sheng Shia, Hong-Min Xiab , Chuan-Hai Wuc, Chun-Bin Lia, Chen-Chen Duana, Chang Che a, a

a

d,*

Xue-Jing Zhang , Hao-Tian Li , Yan Zhang , and Xu-Fu Zhang a

d,*

Key Laboratory of Biotechnology and Bioresources Utilization, Educational of Minister, College

of Life Science, Dalian Nationalities University, Dalian 116600, People’s Republic of China b

Taishan Scholar-Distinguished Experts Position, Key Disciplines on Analysis of Traditional

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f

Chinese Medicine, Shandong Key Laboratory of Chinese Medicine Quality Standard Research, Shandong Academy of Chinese Medicine, Jinan 250014, People’s Republic of China Department of Ocean Science, Division of Life Science and Hong Kong Branch of the Southern

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c

e-

Marine Science and Engineering Guangdong Laboratory, The Hong Kong University of Science

d

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and Technology, Hong Kong, People’s Republic of China

School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515,

rn

al

People’s Republic of China

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ABSTRACT: Two novel nortriterpenoids together with 7 known compounds were isolated from the fruits of Evodia rutaecarpa. The structures of the new compounds were elucidated by spectroscopic analysis, X-ray, and electronic circular dichroism (ECD) calculations. Compound 1 is the first example of triterpenoid with a 27(17→12)-abeo-five-ring skeleton. In turn, compound 2 possesses a unique C/D/E linear fused ring system and a methyl on C-21. Plausible biogenetic pathway for the new compounds 1 and 2 are also proposed. Compound 1 exhibited significantly antitumor activity against A549 and LoVo cells with IC50 values of 2.0 μM and 1.9 μM, respectively. Colony formation inhibition, cell cycle arrest and cell apoptosis of compound 1 were also evaluated. Compound 2, 6, 7 and 9 showed potent neuroprotective activities against serum-deprivation induced P12 cell damage. Key words: Evodia rutaecarpa; Nortriterpenoid; Limonoid; Antitumor activity;

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Neuroprotective activity

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Fig. 1. Structures of 1−9 from the Fruits of Evodia rutaecarpa.

1. Introduction

Evodia rutaecarpa (Juss.) Benth. (Rutaceae) is a shrub or tree, which is widely distributed in the south of China [1]. Its fruits have been used as a traditional Chinese medicine (named “Wu Zhu Yu”) for thousands of years for the treatment of dysentery, headache, abdominal pain, and postpartum hemorrhage [2]. Previous phytochemical studies of E. rutaecarpa mainly revealed a large number of alkaloids [3, 4], limonoids [5], flavonoids [6] and some triterpenoids [7] from the methanol or aqueous ethanol extracts. Among them, triterpenoid together with limonoid which is a highly oxygenated and polycyclic triterpenoid, have variable chemical structures and showed a broad spectrum of biological activities, such as antimicrobial [8], anti- inflammatory

Journal Pre-proof [9], anti-cancer [10], neuroprotective effect [11]. Through an ongoing search for bioactive constituents from E. rutaecarpa, two novel aromatic C29 nortriterpenoids with new skeletons, evoditrilone A (1), which is the first example of an unprecedented 27(17→12)-abeo-five-ring triterpenoid skeleton, and evoditrilone B (2), possessing a unique C/D/E linear fused ring system and a methyl on C-21, together with 7 known compounds,

including

oleanic

acid

(3)

[12],

ursolic

acid

(4)

[13] ，

3β-hydroxyoleana-11,13(18)-diene (5) [14], limonin (6) [15], 12α-hydroxylimonin (7) [16], 7-deacetylproceranone (8) [17], and nomilin (9) [18] were obtained from the

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plant. All compounds were evaluated for antitumor and neuroprotective activities. Reported herein are the isolation, structural elucidation, and bioactivities of these

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compounds.

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2.1 General Experimental Procedures

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2. Materials and methods

Optical rotations were measured on a JASCO P-2000 polarimeter (JASCO, Tokyo,

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Japan). CD spectra were obtained on a JASCO J-815 spectrometer (JASCO, Tokyo,

rn

Japan). NMR spectra were obtained on a Bruker 600 spectrometer (Bruker Corp.

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Karlsruhe, Germany). HRESIMS or HRAPCIMS data were acquired by a Q Exactive Focus LC-MS/MS spectrometer (Thermo Fisher, MA, USA). The MPLC system (Biotage, Uppsala, Sweden) was equipped with a YMC-Pack ODS-A column (500 mm × 50 mm, 50 µm, YMC, Tokyo, Japan). Preparative HPLC (Shimadzu, Tokyo, Japan) was performed using a Shimadzu LC-6AD instrument equipped with SPD-10A detector, using a YMC-Pack ODS-A column (250 × 20 mm, 5µm). Column chromatography was conducted with MCI GEL CHP20P resin (75–150 µm, Mitsubishi, Tokyo, Japan). Silicagel (200–300 mesh, Qingdao Marine Chemical Inc.,

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Qingdao,China). Spots were visualized under UV light or by spraying with 5% H2 SO4 in 95% EtOH followed by heating. 2.2 Plant Material The fruits of E. rutaecarpa (50 kg) were collected in September of 2016 in Tongren of Guizhou Province, P. R. China, and identified by vice-professor X.- Z Chen from

f

Heilongjiang university of Chinese medicine. Voucher specimen (No. 2016090806)

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has been deposited in College of Life Science of Dalian Nationalities University. 2.3 Extraction and Isolation

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The dried and powdered ripe fruits of E. rutaecarpa (50 kg), were extracted with

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95% ethanol (150 L × 2 h × 3), and the solvent was evaporated in vacuo. The residue (3.5kg) was dissolved in water (30 L), and partitioned successively with EtOAc and

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n-BuOH, respectively. The EtOAc extract (380 g) was subjected to silica gel CC (20 × 60 cm) and eluted with CHCl3 –MeOH gradient system (9/1 to 1/9, v/v) as the eluent

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to yield 8 subfractions E1-E8. Subfraction E2 (35 g) was separated by

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RP-semipreparative MPLC with a MeOH-H2 O gradient system (1/9 to 95/5, v/v) to

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get subfraction E2(1-65). Compounds 1 (12.0 mg), 2 (8.0 mg) and 5 (6.0 mg) were purified by RP-semipreparative HPLC (85% MeOH/H2 O) from subfraction E2(50). Compound 3 (9.3 mg) and 4 (12.1 mg) were obtained by RP-semipreparative HPLC (80% MeOH/H2 O) from subfraction E2(30). Fraction E3(50 g) was subjected to column chromatography over MCI resin, eluted with water, 30%, 60% and 95% ethanol to obtain subfractions E3(1-4). Subfraction E3(3) (15 g) was further chromatographed over RP-semipreparative MPLC to get fractions E3(3) (1-37). Compound 7 (6.5 mg) was obtained by RP-semipreparative HPLC (60% MeOH/H2 O) from subfraction E3(3)(16). Compounds 6 (12.0 mg), 8 (8.0 mg) and 9 (6.0 mg) were purified by RP-semipreparative HPLC (70% MeOH/H2 O) from subfraction E3(3)(25). 2.4. Spectral data of compounds

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2.4.1 evoditrilone A (1) C29 H44O, white, amorphous powder; [α]D25 −15.0 (c 0.10, CHCl3 ); UV (MeOH) λmax (log ε) 205 (1.43) nm; 1 H NMR and

13

C NMR see Table 1; HRAPCIMS m/z

391.33597 ([M–H2 O+H]+, calcd for 391.33593, C29 H43 ); CDMeOH, Δεnm : Δε232 −0.06. 2.4.2 evoditrilone B (2) C29 H40O, colorless crystals; [α]D25 −20.0 (c 0.10, CHCl3 ); 1 H NMR and

13

C NMR

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see Table 1; HRESIMS m/z 405.31396 [M + H]+ (calcd for 405.31519, C29 H41O);

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CDMeOH, Δεnm: Δε241 +2.90，Δε308 −0.36.

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Crystal data for 2: C29 H40 O (M =404.61 g/mol): tetragonal (MeOH–CHCl3 ), space group P43 (no. 78), a = 19.3166(6) Å, c = 7.6873(4) Å, V = 2868.4(2) Å3 , Z = 4, T =

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100.00(10) K, μ(CuKα) = 0.410 mm-1 , Dcalc = 0.937 g/cm3 , 20623 reflections

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measured (4.574° ≤ 2Θ ≤ 146.938°), 5290 uniq ue (Rint = 0.0659, Rsigma = 0.0519)

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which were used in all calculations. The final R1 was 0.0736 (I > 2σ(I)) and wR2 was

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0.2163 (all data). Crystallographic data have been deposited at The Cambridge Crystallographic Data Centre and allocated the deposition number CCDC 1884357. The

data

can

be

obtained

free

of

charge

via

www.ccdc.cam.ac.uk/products/csd/request. 2.5 Antitumor activity assay 2.5.1 Antibodies and other materials Antibodies specific to cleaved caspase-3 and PARP, were purchased from Cell Signaling Technology (Cell Signaling Technology, Inc, USA). Antibodies specific to Bcl-2, BAX, N-cadherin, E-cadherin, β-actin and all the secondary antibodies were purchased from Proteintech Group (Proteintech, Inc, USA). Antibodies specific to

Journal Pre-proof MMP2 were purchased from Abcam. RPMI 1640 media, fetal bovine serum (FBS), and Trypsin were purchased from Gibico. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified. 2.5.2 Cell culture Human non-small cell lung cancer cell A549, human lung fibroblasts HLF and human colorectal cancer cell LoVo were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI 1640 media, supplemented with 10% fetal bovine serum (FBS), and grown at 37°C in a humidified

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atmosphere with 5% CO 2 . The cell lines have been confirmed to be free of mycoplasma by Mycoplasma Detection Kit-Quick Test (Biotool).

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2.5.3 CCK-8 assay

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Cell viability was measured using CCK-8 assay. Briefly, 6.0×103 cells were counted and seeded into 96-well culture plates followed by adhesion for overnight,

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and then treated with appropriate concentrations of compound 1. The quintuplicate was set for each concentration group. Followed by incubation for 48h, 10 μL of

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CCK-8 was added to each well. For 2-4 h incubation at 37 °C, the absorbance was

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measured at 450 nm by EnSpire® Multimode Plate Reade (Perkin Elmer, USA). Each experiment was repeated at least three times.

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2.5.4 Colony formation assay

A549 cells were treated with compound 1 for 48 h, and then trypsinized into single cells and seeded into a 6-well plate at 1000 cells/well. After being incubated at 37 °C with 5% CO2 for 10 days until colonies were large enough to be visualized, the cells were washed with PBS and fixed with the mixture (methanol: glacial acetic: ddH 2 O = 1:1:8) for 10 mins, and stained with 0.1% crystal violet for 30 mins. 2.5.5 Wound healing assay Wound healing assay (scratch assay) was performed to detect cell migration. Briefly, A549 and LoVo cells were grown to full confluence in 6-well culture plates. After 6 h of serum starvation, the confluent cell monolayer was scraped with a sterile 100 uL pipette tip and treated with appropriate does of compound 1 after washed with PBS.

Journal Pre-proof 2.5.6 Flow cytometry analysis To determine the distribution of the cells in the cell cycle and the proportion of apoptotic cells, flow cytometry analysis was performed using a flow cytometer (BD FACS Accuri C6, CA, USA). Briefly, the treated cells were collected and fixed with ice-cold 70% ethanol at 4°C for 4 h, and then stained according to Cell Cycle kit protocol (Beyotime). The cell cycle distribution of A549 cells was determined using a FACS analysis system. 2.5.7 Western blot analysis

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Proteins from cell lysates were subjected to SDS-PAGE and then transferred to a PVDF membrane. Protein bands were visualized by enhanced chemiluminescence

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(ECL) and integrated optical density of bands was quantitated by the ImageQuant

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software (GE Healthcare). The concentration of proteins was determined in the cell

2.6 P12 Cell protection assay

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lysates using BCA method. Similar experiments were performed at least three times.

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The MTT method were used for testing the neuroprotective effect of compounds

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1–9 against serum deprivation-induced P12 cell injure [19]. PC12 cells at a density of

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5 × 103 cells per well in 96-well plates were cultured in DMEM media (Hyclone) supplemented with 5% fetal bovine serum (FBS, Hyclone), serum (Hyclone), and L-glutamine (2 μM). The system was maintained at 37 ℃ in 5% CO 2 for 24 h. The P12 cells were cultured with or without compounds (10 μM) without serum. After incubation for another 48 h, 10 μL of the 5 mg/mL MTT (Sigma) was added and maintained for 4 h. NFG (Nerve Growth Factor, 0.05 μg/mL) was a positive control for the model of serum deprivation- induced P12 cells damage. Cultures were maintained at 37 ℃ in 5% CO 2 in a humidified incubator for 48 h. Compounds at concentrations of 10 and/or 4 μM rotenone were added to the cells. After incubation

Journal Pre-proof for another 48 h, 10 μL of the 5 mg/mL MTT was added and maintained for 4 h. Absorbance was measured at 570 nm using an Ultramark microplate reader. Cell viability was evaluated. 3. Results and discussion 3.1. Isolation and structural characterization Compound 1 was obtained as a white amorphous powder. Its molecular formula

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was determined as C 29 H44O from the positive HRAPCIMS at m/z 391.33597

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([M–H2 O+H]+, calcd for 391.33593, C29 H43 ) analysis，and was consistent with its 1 H

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and 13 C NMR (Table 1) evidence, accounting for eight indices of hydrogen deficiency. The 1 H NMR data of 1 (Table 1) showed seven methyls at δH 1.18 (3H, d, J = 6.8 Hz),

e-

1.13 (3H, s), 1.01 (3H, s), 1.00 (3H, s), 0.95 (3H, s), 0.91 (3H, s), and 0.82 (3H, s), a

The

13

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pair of aromatic protons at δH 7.05 (1H, d, J = 8.2 Hz) and 6.91 (1H, d, J = 8.2 Hz). C NMR data of 1 (Table 1) totally exhibited 29 carbon resonances, including

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six aromatic carbons (δC 146.6, 138.6, 134.1, 133.1, 126.9 and 122.1), and one oxygenated carbon (δC 79.0). The aforementioned information suggested that the

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structure of 1 was related to oleanolic acid [12]. The main differences between 1 and

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oleanolic acid were that the double bond 12 and C-28 carboxyl groups were missing, the C-27 methyl is now on C-12, rather than on C-14, and the ring D was aromatized. The Me-27 at δH 1.18 (3H, d, J = 6.8 Hz) exhibited the HMBC correlations (Fig. 2) with C-11, C-12 and C-13 confirming that the methyl group was linked at C-12. The HMBC correlations from Me-23 and Me-24 to C-3, from Me-26 to C-7, C-9 and C-14, from H-15 to C-8, C-13 and C-17, from H-16 to C-14, C-18 and C-22, from H-21 to C-17, and from H-22 to C-16, C-17 and C-18 in combination with the 1 H- 1 H COSY cross-peaks constructed the structure of 1. All the 1 H and

13

C NMR data of 1 were

unambiguously assigned by means of the HSQC, HMBC and

1

H- 1 H COSY

experiments. The relative configuration of 1 was determined through an NOESY experiment (Fig. 3) which showed the following correlations: H-3α ↔ H-5 and Me-23, H-9 ↔ H-5, H-11α and Me-27 requiring that H-3α, Me-23, H-5, H-9 and Me-27 were

Journal Pre-proof α-oriented. One the other hand, the NOESY correlations of H-11β/H-12, H-11β/Me-25, H-11β/Me-26, Me-24/Me-25

indicated these were β-oriented.

According to the relative configuration determined for 1. According to the relative configuration determined for 1, alternative absolute configurations could be proposed as (3S,5R, 8R,9R,10R,12S) (1a) or (3R,5S, 8S,9S,10S,12R) (1b). The calculated ECD spectrum of 1a displayed CD curves similar to the experimental spectrum of 1 (Fig. 4). Thus, the structure of 1 was established as shown in Fig. 1. As far as we know, compound 1 represents a new class of triterpenoid for which the name evoditrilone A

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is proposed. It is the first example of triterpenoid with a 27(17→12)-abeo-five-ring

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skeleton, in which Me-27 migrated from C-17 to C-12 to form a rare skeleton. Compound 2 was obtained as colorless crystals, and had a molecular formula of

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C29 H40O, as inferred by a pseudo molecular ion peak showed at m/z 405.31396 [M +

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H]+ (calcd for 405.31519, C29 H41 O) in the positive HRESIMS and by the number of CH3 , CH2 , CH, and C groups determined by the Analysis of the

13

13

C NMR and DEPT experiments.

C NMR data and HSQC spectrum revealed the presence of seven

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methyls, five sp3 methylenes, three sp3 methines, four sp3 quaternary carbons, five sp2

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methines, and five sp2 quaternary carbons. In the

13

C NMR spectrum, ten olefinic

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carbon signals at δ 141.8, 134.9, 134.0, 132.1, 131.3, 129.9, 129.4, 128.1, 127.4 and 123.4, and one oxygenated methine carbon signal at δ 78.9 were observed. The above spectral evidence suggested 2 to be an aromatic nortriterpenoid containing seven methyls, two double bonds and one hydroxy group. A close comparison of the

13

C

chemical shifts of 2 with 3β-hydroxyoleana-11,13(18)-diene [14] suggested the same A/B/C/D rings as oleanane system with an unprecede nted E ring. The E ring moiety was deduced as an o-xylene and fused at C-16 and C-17 on the basis of the following HMBC correlations (Fig. 2): from H-18 to C-16 and C-19, H-19 to Me-29, C-16, C-18 and C-21, and from H-22 to Me-30, C-15, C-17 and C-20. In addition, the HMBC correlations from Me-29 to C-19, C-20 and C-21, coupled with the correlations from Me-30 to C-20, C-21 and C-22 revealed the two methyls were attached at C-20 and C-21, respectively. The relative configuration of 2 was mainly

Journal Pre-proof deduced by analysis of the NOESY spectrum (Fig. 3). Accordingly, the key NOESY correlations of H-3α/H-5/H-9/H-15α/Me-23/Me-27 suggested that they were on the same face. In contrast, the correlations of H-15β/Me-24/Me-25/Me-26 indicated that they were on the β- face. The X-ray crystallographic analysis of 2 (Fig. 5; CCDC 1884357) suggested the absolute configuration of 2 as 3S,5R, 8R,9R,10S. However, the Flack parameter [−0.2 (5)] is not good enough to unambiguously establish the absolute configuration. For this deduction to be confirmed, the ECD experiment of 2 was carried out. According to the relative configuration apparent for 2, possible

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absolute configurations of 2 could be proposed as (3S,5R, 8R,9R,10S) (2a) or (3R,5S, 8S,9S,10R) (2b). The calculated ECD spectrum of 2a displayed a CD curve similar to

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the experimental spectrum of 2 (Fig. 4). Thus, the absolute configuration of 2 was

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determined to be the same as 2a, which was established as depicted in Fig. 1 and named as evoditrilone B. Compound 2 has a unique C/D/E linear fused ring system

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and aromatic nortriterpenoid skeleton with 29 carbons and a methyl on C-21. As far as we know, it has no precedent among known natural products or synthetic compounds.

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Since the structure of 2 is partially similar to 3β-hydroxyoleana-11,13(18)-diene, we

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deduced that 2 was likely derived from 3β-hydroxyoleana-11,13(18)-diene by a series of biochemical reactions including acidation, decarboxylation, ring cleavage and ring

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formation, methyl migration, and aromatization. A hypothetical biosynthetic pathway of 2 was postulated as shown in Scheme 2.

Fig.2. Key 1 H−1 H COSY and HMBC correlations of 1 and 2.

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Fig. 3. Optimized structures and key NOESY correlations of 1 and 2.

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Fig. 4. Calculated and experimental ECD spectra of 1 and 2.

Fig. 5. X-ray crystal structure of 2. 3.2. Proposed biosynthetic pathway of compounds 1 and 2 As far as we know, it has no precedent of 1 and 2 among known natural products or synthetic compounds. A plausible biogenetic route toward the novel carbon skeleton of 1 is proposed and shown in Scheme 1. It might be generated from oleanolic acid as a precursor. These steps involve decarboxylation, methyl migration, and aromatization to produce compound 1.

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Scheme 1. Plausible biogenetic pathway for 1.

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Since the structure of 2 is partially similar to 3β-hydroxyoleana-11,13(18)-diene,

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we deduced that 2 was likely derived from 3β-hydroxyoleana-11,13(18)-diene by a series of biochemical reactions such as acidation, decarboxylation, ring cleavage and

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ring formation, methyl migration, and aromatization. A hypothetical biosynthetic

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pathway of 2 was postulated as shown in scheme 2.

Scheme 2. Plausible biogenetic pathway for 2.

Journal Pre-proof Table 1 The 1 H and

13

C-NMR spectroscopic data for compounds 1 and 2 in CDCl3 .

1

2

No.

1

13

1

1.02 (1H, m)

H-NMR

1

C-NMR

38.1 (t)

H-NMR

1.05 (1H, m)

1.83 (1H, dt, 12.9, 3.5)

13

C-NMR

38.3 (t)

1.90 (1H, dt, 13.1, 3.5)

2

1.60–1.74 (2H, m)

27.3 (t)

1.62–1.74 (2H, m)

27.1 (t)

3 4

3.25 (1H, dd, 11.5, 4.9) ―

79.0 (d) 39.0 (s)

3.25 (1H, dd, 11.4, 4.9) ―

78.9 (d) 38.9 (s)

5 6

0.88 (1H, m) 1.59 (1H, m)

55.3 (d) 19.0 (t)

0.84 (1H, m) 1.50 (1H, m)

54.8 (d) 18.1 (t)

1.74 (1H, m)

1.67 (1H, m)

8 9

― 1.50 (1H, m)

38.9 (s) 49.2 (d)

10 11

― 1.53 (1Hα, m)

36.8 (s) 26.0 (t)

13

―

14

―

15

7.05 (1H, d, 8.2)

40.5 (s) 53.7 (d) 36.9 (s) 128.1 (d)

6.15 (1H, dd, 10.0, 2.9)

129.4 (d)

138.6 (s)

―

141.8 (s)

146.6 (s)

―

43.0 (s)

122.1 (d)

2.28 (1H, d, 15.7)

33.7 (t)

126.9 (d)

3.09 (1H, d, 15.7) ―

132.1 (s)

133.1 (s) 134.1 (s)

― 6.10 (1H, s)

131.3 (s) 123.4 (d)

30.2 (d)

Pr

3.12 (1H, m)

32.7 (t)

― 5.68 (1H, dd, 10.0, 1.8)

al

12

― 2.15 (1H, dd, 2.9, 1.8)

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1.86 (1Hβ , td, 12.6, 5.5)

1.48–1.57 (2H, m)

f

40.6 (t)

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1.48 (1H, m) 2.40 (1H, m)

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7

6.91 (1H, d, 8.2)

17 18

― ―

19

2.41 (1H, d, 16.3) 2.52 (1H, d, 16.3)

40.0 (t)

6.80 (1H, s)

127.4 (d)

20

―

29.7 (s)

―

134.0 (s)

21

1.52 (2H, t, 6.8)

35.5 (t)

―

134.9 (s)

22

2.76 (2H, t, 6.8)

27.1 (t)

6.84 (1H, s)

129.9 (d)

23

1.00 (3H, s)

27.9 (q)

1.02 (3H, s)

27.8 (q)

24

0.82 (3H, s)

15.3 (q)

0.81 (3H, s)

15.1 (q)

25

0.91 (3H, s)

16.7 (q)

0.96 (3H, s)

17.8 (q)

26

1.13 (3H, s)

26.8 (q)

1.02 (3H, s)

17.8 (q)

27

1.18 (3H, d, 6.8)

0.94 (3H, s)

17.7 (q)

2.20 (3H, s) 2.21 (3H, s)

19.3 (q) 19.5 (q)

29 30

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rn

16

ex

1.01 (3H, s) 0.95 (3H, s) ex

ex = exchange 3.3. Cytotoxic activity

22.2 (q) ex

28.6 (q) 28.5 (q) ex

Journal Pre-proof All isolated compounds 1−9 were evaluated for their antitumor activity against two cancer cell lines, Human non-small cell lung cancer cell A549 and human colorectal cancer cell LoVo. As shown in Fig 6A, treatment with compound 1 resulted in the dose-dependent growth inhibition of above tumor cells. The IC 50 value of compound 1 was 2.00 ± 0.29 μM (A549) and 1.95 ± 0.15 μM (LoVo) (Fig. 6B). Other compounds were inactive in the assay. Furthermore, we also detected the cytotoxicity of compound 1 in human normal lung cell line (HLF cells), and IC50 value was 12.97 ± 1.42 μM (HLF) (Fig. 6B). Interestingly, compound 1 could only cause about 10%

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cytotoxicity in HLF cells at IC50 value of cancer cells (2.0 μM) (Fig. 6A), that implied that compound 1 had selective antitumor activity. Taxol was as a positive control, the

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IC50 values were 243.7 ± 36.2 nM (A549), 386.3 ± 18.1 nM (LoVo), and 653.2 ± 37.6

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nM (HLF) (Fig. 6C).

3.4 Inhibition mechanism of compound 1 against cancer cell lines

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3.4.1 Compound 1 induced colony formation inhibition, cell cycle arrest and cell apoptosis

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We next investigated the effect of compound 1 on colony formation in A549 cells

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by a clonogenic cell survival assay. Consistent with cell viability inhibition, compound 1 also significantly inhibited colony formation (Fig. 7A). Cell proliferation

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inhibition often accompanies changes in cell cycle progressio n [20]. We next evaluated the degree of compound 1 on cell cycle arrest. As shown in Fig. 7B, the percentages of cells in the S phase increased from 19.97 to 31.72% after compound 1 treatment in A549 cells compared with the control group, whereas the percentages of cells in the G0/G1 phase decreased from 70.29 to 31.12% (Fig.7B and 7C). Furthermore, to ascertain detailed mechanisms, the expression of ke y proteins involved in cell cycle (CDK4 and cyclin A) were evaluated by western blotting after treatment with compound 1. The results indicated that compound 1 obviously reduced the expression of CDK4 and cyclin A (Fig. 7D). These data provide evidence that compound 1 inhibits the proliferation of tumor cells by inducing cell-cycle arrest at the S phase.

Journal Pre-proof In addition, treatment with compound 1 dose-dependently increased the sub-G1 population, which represented the apoptotic cells (Fig. 7E). To confirm the underlying mechanism of compound 1 on apoptosis in A549 cells, we detected the levels of apoptosis-relative proteins (PARP, caspase-3, BAX and Bcl-2) in 48 h-treated cells by Western blot. As shown in Fig. 7F, treatment with compound 1 resulted in an increased protein levels of cleaved PARP (C-PARP) and cleaved caspase 3 (C-caspase3), and Bax; and a decreased protein level of Bcl-2. As the ratio of Bax/Bcl-2 was a "molecular switch" that initiates apoptosis, we also calculated and

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found that compound 1 could effectively increase the ratio. These results showed that compound 1 could efficiently induced tumor cell apoptosis.

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3.4.2 Compound 1 induced the inhibition of cell migration

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Wound healing assays were employed to detect the effect of compound 1 on tumor cell mobility. As shown in Fig. 8A, the part of gap or wounding space between cell

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layers after making a scratch was occupied almost (in A549 cells) or completely (in LoVo cells) by the migrating cells without compound 1 treatment. By contrast,

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compound 1 treated cells failed to occupy the scraped space through migration due to

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their impaired migration capability. Quantitative analysis revealed that the inhibition of migration was in a dose-dependent manner (Fig. 8B). Further, to ascertain detailed

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underlying mechanisms, the key protein markers including matrix metalloproteinases (MMP2), N-Cadherin (N-Ca) and E-Cadherin (E-Ca) were evaluated. As shown in Fig. 8C and 8D, compound 1 treatment could effectively result in an increased protein level of E-Ca, and a decreased protein levels of MMP2 and N-Ca. These results suggest that compound 1 has the perfect properties in suppressing tumor cell migration.

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Fig.6. Effects of compound 1 on cell proliferation. (A). A549, LoVo, and HLF cells were treated with compound 1

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at the indicated doses. At 48 h after treatment, the cell viability was determined by a CCK-8 assay. The IC50 values

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of compound 1 (B) and positive control taxol (C) for cell viability inhibition was calculated.

Fig.7. Effects of compound 1 on colony formation, cell cycle arrest and cell apoptosis. (A). A549 cells were treated with compound 1 at the indicated doses. (B and C). The A549 cells cycle analysis was performed after 48 h of treatment with compound 1 by BD Accuri C6 Flow Cytometer. (D). The expression of the cyclin A and CDK4

Journal Pre-proof proteins were analyzed by Western blot. (E). The sub G1 accumulation (apoptotic cells) were also calculated. (F). The expression of the PARP, caspase-3, Bcl-2 and Bax proteins in were analyzed by Western blot. The ratio of

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Bax/Bcl-2 was also calculated.

Fig.8. Effect of compound 1 on cell migration. (A). Cell migration was analyzed by a scratch assay in A549 and

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LoVo cells. After 48h of treatment with compound 1 at the indicated doses, the wound gap was observed and photographed. (B). The percentage of migration cells were calculated relative to the original gap. (C). The expression of N-cadherin, E-cadherin and MM P-2 proteins were analyzed by Western blot in different treatment group. (D). The quantitative analysis of the proteins in C was performed.

3.5 Neuroprotective effect assay Compounds 1–9 were tested for the neuroprotective effect of against serum deprivation- induced (Table 2). Compounds 2, 6, 7 and 9 exhibited neuroprotective activity against P12 injured by serum deprivation, increasing the cell viability in the model from 55.5 ± 5.0% to 80.3 ± 6.1%, from 55.5 ± 5.0% to 83.5 ± 5.3%, from 55.5

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± 5.0% to 81.0 ± 3.7%, and from 55.5 ± 5.0% to 88.6 ± 5.6%, at the concentration of 10 μM. Table 2 Neuroprotective activities of compounds 1−9 on the survival rate against serum-deprivation induced P12 cell damage (10 μM, mean ± SD, n=6) Group

Serum-deprivation induced P12 cells

100±6.6

Model

55.5 ± 5.0###

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Control

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damage model

78.9± 7.3 ***

2

80.3 ± 6.1 ***

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NFG

6

83.5 ± 5.3 *** 81.0 ± 3.7 ***

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7

88.6 ± 5.6 ***

NFG as a positive control;

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9

p < 0.001 vs control; ***p < 0.001 vs model; **p <

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0.001 vs model. 4. Conclusion

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Phytochemical investigation of 95% enthanol extract of the fruits of Evodia

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rutaecarpa lead to the characterization of two novel nortriterpenoids 1 and 2, together with 7 known compounds (3–9). Among them, Compound 1 demonstrated potent antitumor activity against A549 and LoVo cells with IC 50 values of 2.0 μM and 1.9 μM, respectively. More importantly, compound 1 could efficiently induce tumor cell apoptosis and inhibit tumor cell migration in vitro. Additionally, compound 2, 6, 7 and 9 showed potent neuroprotective activities against serum-deprivation induced P12 cell damage. This work may provide the basis for further phytochemical study on the fruits of Evodia rutaecarpa. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgments

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The authors thank the support from the National Natural Science Foundation of China (grants no. 81773975). Supporting information Supplementary data associated with this article can be found in the online version via the Internet.

(X.-F. Zhang)

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*E-mail: [email protected]

(Y. Zhang)

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*E-mail: [email protected]

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Corresponding Author

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