Coumarins from the roots of Angelica dahurica with antioxidant and antiproliferative activities

Journal of Functional Foods 20 (2016) 453–462

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Coumarins from the roots of Angelica dahurica with antioxidant and antiproliferative activities Yan Bai a,b,1, Dahong Li a,c,1, Tingting Zhou a, Ningbo Qin a, Zhanlin Li a, Zhiguo Yu b,*, Huiming Hua a,** a

Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China b School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China c State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Processes, Jiangsu Kanion Pharmaceutical Co. Ltd, Lianyungang 222001, China

A R T I C L E

I N F O

A B S T R A C T

Article history:

Angelica dahurica has been frequently used as a food additive and a folk medicinal herb in

Received 16 September 2015

Asian countries. The aim of this study was to isolate key active compounds from A. dahurica

Received in revised form 27 October

with antioxidant and anticancer activities. Three new furocoumarin dimers and twenty known

2015

coumarins were obtained from A. dahurica. Their structures were identified by extensive 1D

Accepted 2 November 2015

and 2D NMR, CD, and HR-ESIMS spectroscopic analyses and screened by UPLC-MS/MS.

Available online

Imperatorin oxypeucedanin hydrate, xanthotoxol, bergaptol, and 5-methoxy-8-hydroxypsoralen exhibited moderate DPPH• scavenging activity and strong ABTS•+ scavenging activity.

Keywords:

Isoimperatorin, phelloptorin, and pabularinone showed significant inhibition on HepG2 cells

Angelica dahurica

with IC50 values of 8.19, 7.49, and 7.46 µM, respectively. Furthermore, pabularinone also showed

Furanocoumarins

moderate inhibition on HeLa cells with an IC50 value of 13.48 µM. These results suggested

Antioxidant activity

that A. dahurica could be explored as new and potential natural antioxidants and cancer

Antiproliferative activity

prevention agents for use in functional foods.

UPLC-MS/MS

1.

Introduction

Currently, people not only care about the life span, but also concern more about life quality. So people pay growing attention to healthy problems. Cancer is one of the largest single causes of human death as more than one million people are diagnosed with cancer each year (Howlader et al., 2012). Surgery,

© 2015 Elsevier Ltd. All rights reserved.

chemotherapy and radiotherapy are conventional therapies, while chemotherapy agents can cause injury to normal cells. Oxidative damage is considered to play a pivotal role in the occurrence of various human diseases including inflammationrelated cancers (Seril, Liao, Yang, & Yang, 2003; Sosa et al., 2013). In recent years, many researchers focus on searching for novel effective anticancer agents with less toxic effects (Adamson, 2015). And more attention has been paid to natural products

* Corresponding author. School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China. Tel.: +86 024 23986295; fax: +86 024 23986295. E-mail address: [email protected] (Z. Yu). ** Corresponding author. Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China. Tel.: +86 024 23986465; fax: +86 024 23986465. E-mail address: [email protected] (H. Hua). 1 These authors contributed equally to this work. Abbreviations: DPPH, 2, 2-Diphenyl-1-picrylhydrazyl; ABTS+, 2, 2′-Azinobis-(3-ethylbenzothiazoline-6-sulfonate) radical cation; HeLa, Human cervical cancer; HepG2, Human hepatoblastoma; MCF-7, Human breast cancer http://dx.doi.org/10.1016/j.jff.2015.11.018 1756-4646/© 2015 Elsevier Ltd. All rights reserved.

454

Journal of Functional Foods 20 (2016) 453–462

O 5

10

6

3 2

9

O

7

8

8a

O

O

1

O 11

O

O

4

4a

O

O

O

O

OH

O

O O

O

12 13

14

O 14’

O

15

13’ 12’

O

OH

HO

OH

HO

11’

HO 15’ 10’ 6’

O

O

O

5’ 4'a 4’

3’

9’

2’

O

7’

8'a 8’

O

O

O

1’

O

O

O

2

1

R1

O

O

3

O H3CO HO

O

O

O

R2

O

O

O

O

O

O

O

23

22

21 4 R1 = a, R2 = H; 5 R1 = H, R2 = a; 6 R1 = OCH3, R2 = a; 7 R1 = b, R2 = OH; 8 R1 = c, R2 = H; 9 R1 = H, R2 = c; 10 R1 = OCH3, R2 = c; 11 R1 = d R2 = H; 12 R1 = e, R2 = OCH3;

HO

O

O

13 R1 = OCH3 , R2 = e; a 14 R1 = f, R2 = H; 15 R1 = OCH3 , R2 = f; b 16 R1 = g, R2 = H; 17 R1 = H, R2 = OH; 18 R1 = OH, R2 = H; c 19 R1 = OCH3, R2 = OH; 20 R1 = OH, R2 = OCH3; d

e

O

O OH

f

OH O OH

O O g O OH

OH O OH

Fig. 1 – Chemical structures of the isolated compounds 1–23 from the roots of Angelica dahurica.

for preventing the development and progression of cancer (Saha & Khuda-Bukhsh, 2013). Reactive oxygen species (ROS) include superoxide (O2−), hydrogen peroxide (H2O2) and hydroxyl radical (OH), which cause oxidative injury to living organisms accompanying many lifestyle-related diseases, such as arthritis, cancer, diabetes, arthrosclerosis, and neurodegenerative diseases (Rajendran et al., 2014). Nowadays, several synthetic antioxidants have been developed and used widely. However, several undesirable effects have been found on human health (Williams, Iatropoulos, & Whysner, 1999). Therefore, identifying and characterising natural antioxidants with anticancer activities are beneficial to the study of effective and less toxic anticancer drugs (Roleira et al., 2015). The roots of Angelica dahurica (Fisch. ex Hoffm) Benth. et Hook. were used as spices and condiments in many foods, such as in hot pot or in soup. It could also be used in cosmetics, for example, face masks, which has skin whitening function. As a food additive, the roots of A. dahurica contain abundant nutrients with biological activities, including coumarins, steroids, and essential oil. Among them, coumarins were considered as the dominant chemical constituents. As is known to all, the roots of A. dahurica were served as a traditional medicine to

treat cough, headache, rhinobyon, nasosinusitis, toothache, leukorrhea, and asthma in Asian countries (Korea, China, and Japan) for many years (Deng et al., 2015; Li et al., 2014; Sarker & Nahar, 2004). Coumarins in the roots of A. dahurica are considered particularly relevant for application as antiinflammatory, anti-mutagenic, antitumor and antioxidant agents (Deng et al., 2015; Liu et al., 2011; Piao et al., 2004; Thanh, Jin, Song, Bae, & Kang, 2004). However, to the best of our knowledge, there were few studies about the relationship between the antitumor and antioxidant activities and isolated compounds extracted from the roots of A. dahurica. Therefore, the researches of its bioactive compounds would have a highly practical value. In this context, as part of an effort to identify more structurally and biologically diverse coumarins from the roots of A. dahurica, three new furocoumarin dimers, named angdahuricaols A–C (1–3), along with twenty known coumarins (4–23), (Fig. 1) were isolated and identified by NMR spectroscopy, and the contents of part of the individual coumarins were determined by UPLC-MS/MS. Moreover, their respective antioxidant as well as the growth inhibitory effects against three selected human cancer cell lines (HeLa, HepG2, and MCF-7) was also evaluated.

Journal of Functional Foods 20 (2016) 453–462

2.

Materials and methods

2.1.

Plant material and chemicals

Plant material was purchased from Beijing Tongrentang drugstore in Shenyang, Liaoning Province, People’s Republic of China, in September, 2012, which was identified by Prof. Jincai Lu (Division of TCM Resource, Shenyang Pharmaceutical University), and a voucher sample (AD20120915) was deposited at room temperature in the Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China. All the solvents for the extraction were purchased from Yuwang Chemicals Industries, Ltd. Methanol and glacial acetic acid (chromatography grade) was purchased from Concord Chemical Reagents Co. (Tianjin, China). The water used during HPLC analysis and for sample preparation was obtained from Wahaha Group Co. Ltd. (Hangzhou, China). 2,2-Diphenyl-1picrylhydrazyl (DPPH), 2,2′-azinobis(3-ethylbenzothiazoline-6sulphonic acid) diammonium salt (ABTS), 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT), and foetal bovine serum (FBS) were obtained from Sigma Aldrich (Sigma Chemical Co., Shanghai, China). Column chromatography was performed with silica gel (100–200 and 200–300 mesh; Qingdao Ocean Chemical Co. Ltd., Qingdao, China), C18 reversed-phase silica gel (50 µm; YMC Co. Ltd., Kyoto, Japan), and Sephadex LH20 (GE Healthcare). TLC plates were precoated with silica gel GF 254 (Qingdao Ocean Chemical Co. Ltd., Qingdao, China).

2.2.

Instruments

Optical rotations were measured with a Perkin-Elmer 241MC polarimeter. 1H NMR (300 MHz and 600 MHz), 13C NMR (75 MHz and 150 MHz) and 2D NMR were acquired on Bruker-ARX-300 and Bruker-AV-600 NMR spectrometers using TMS as an internal standard. HR-ESI-MS spectra were obtained using Bruker micrOTOF-Q mass spectrometer. CD spectra were obtained using MOS-450 detector from BioLogic. RP-HPLC separations were carried out on a LC-6AD instrument with an SPD-20A VP UV/ vis detector using YMC-Pack-ODS-A column (250 × 20 mm, 5 µm). The UPLC-MS/MS analysis was carried out on an Acquity UPLC I-Class System (Waters Corp., Milford, MA, USA) combined with a Xevo TQ-S mass spectrometer (Waters Corp., Milford, MA, USA). The compositions of samples were analysed by a Thermo Hypersil GOLD C18 column (2.1 × 50 mm, 1.9 µm).

2.3.

455

of increasing polarity with petroleum ether-ethyl acetate (from 100:0 to 1:1, v/v) to afford seven fractions (A–G). 4 (1.4 g) was recrystallised from fraction A, while 5 (1.4 g) and 6 (1.5 g) were obtained from fraction B, and 7 (26.5 mg) and 8 (106.5 mg) from fraction C by recrystallisation, respectively. Fraction D (7 g) was subjected to silica gel column chromatography (70 g, 3.5 × 50 cm) eluting with petroleum etheracetone (from 100:0 to 10:3, v/v) to afford three fractions (D1– D3). Fraction D1 (1 g) was separated by preparative HPLC (MeOH/ H2O, 61:39) to yield 10 (137.3 mg, 32 min) and 13 (28.8 mg, 28 min). Silica gel column chromatography (25 g, 2 × 30 cm) of fraction D2 (1.2 g) with step gradient elution of petroleum etheracetone (from 10:1 to 10:3, v/v) led to the isolation of 9 (7.2 mg), 17 (194.8 mg), 19 (80.9 mg), and 21 (19.3 mg). Fraction D3 was applied to a C18 silica gel column (50 µm, 3.5 × 40 cm) eluting with a MeOH-H2O gradient (from 40:60 to 80:20, v/v), followed by preparative HPLC (MeOH/H2O, 67:33) to yield 11 (232.0 mg, 29 min) and 18 (278.8 mg, 24 min). Fraction E (9 g) was applied to a C18 column (50 µm, 3.5 × 40 cm) eluting with a MeOH-H2O gradient (from 3:7 to 4:1, v/v) to afford three fractions (E1–E3), of which each was subjected to Sephadex LH-20 column chromatography (1.5 × 50 cm, eluted with MeOH), then purified by preparative TLC (20 × 20 cm), eluted with petroleum ether-acetone (3:1, v/v) to provide 12 (5.5 mg), 20 (6.8 mg), and 22 (4.4 mg), respectively. Fraction G (12 g) was further subjected to silica gel column chromatography (162 g, 5 × 130 cm) eluting with dichloromethane-acetone (from 100:0 to 2:1, v/v) to afford three fractions (G1–G3) and to yield 15 (452.7 mg) and 16 (643.6 mg). Fraction G1 was further purified by preparative TLC (20 × 20 cm), eluted with petroleum ether-acetone (3:1, v/v) to provide 23 (3.1 mg). Fraction G3 was separated by preparative HPLC (MeOH/ H2O, 68:32) to afford 14 (8.3 mg, 25 min), 1 (4.6 mg, 32 min), 2 (9.8 mg, 38 min) and 3 (12.8 mg, 41.3 min), respectively.

2.4.

UPLC-MS/MS analysis

The mass spectrometry was operated in positive scan mode. The mobile phase was composed of 0.1% formic acid in water (solvent A) and methanol (solvent B). The gradient elution program was as follows: 0–8 min, 45%–75% B, and then 75% B for 2 min. The total running time was 10 min with a flow rate of 0.3 mL/min. The main working parameters were set as follows: corn voltage, 30 V; the desolvation temperature, 350 °C; ion source temperature, 150 °C; gas flow (N2), 700 L/h.

2.5.

Antioxidant assay

2.5.1.

Assay of DPPH radical scavenging activity

Extraction and isolation

The air-dried roots of A. dahurica (20 kg) were powdered and extracted three times with 95% EtOH (3 × 7.5 L) under reflux (3 h each). The combined EtOH extracts were concentrated under reduced pressure to give a residue (800 g), which was suspended in water (1.5 L). The water layer was successively partitioned with petroleum ether (3 × 1.5 L), ethyl acetate (3 × 1.5 L), and n-butanol (3 × 1.5 L) to afford petroleum ether (350 g), ethyl acetate (81 g), and n-butanol (310 g) fractions, respectively. The ethyl acetate fraction (81 g) was chromatographed over a silica gel column (480 g, 5 × 100 cm) using a gradient system

The DPPH free radical scavenging activity of isolated compounds was determined using the previously reported method with minor modification (Pinela et al., 2012). Briefly, different concentrations (1, 2, 5, 10, and 20 mg/mL) of obtained compounds were further dissolved in ethanol to obtain the following concentrations: 5, 10, 20, 50, 100, and 200 µg/mL. Sample solution was added to DPPH. The content was vortexed for 1 min and incubated in the dark for 30 min at room temperature. After incubation, the absorbance value of all samples was read at 517 nm. The antioxidant activity was measured according to the formula:

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Journal of Functional Foods 20 (2016) 453–462

scavenging effect (% ) = {1 − ( A sample − A blank A control )} × 100%

expressed as mean ± SD (standard deviations). An ANOVA test using SPSS 16.0 (Statistical Program for Social Sciences, SPSS Inc., Chicago, IL, USA) was used to analyse the experimental data.

where Asample was absorbance of sample, Ablank was absorbance of sample blank, and Acontrol was absorbance of the control. EC50 (µg/mL, concentration to scavenge 50% of free radicals) was calculated from the regression equation. All samples were analysed in triplicate using a calibration curve of trolox.

3.

Result and discussion

2.5.2.

3.1.

Phytochemical investigation

Assay of ABTS radical scavenging activity

The ABTS decolourisation assay was carried out using the previously reported method with minor modification (Liu et al., 2009). The ABTS radical cation solution was prepared by mixing 7 mM ABTS stock solution and 2.45 mM potassium persulphate and incubated in the dark for 12 h at room temperature. The ABTS radical cation solution was then diluted to obtain an absorbance of 0.70 ± 0.02 at 734 nm. ABTS solution was added to the test samples with various concentrations (1, 5, 10, 20, 50, and 100 µg/mL) and mixed vigorously. The absorbance was measured at 734 nm after 10 min against the corresponding blank. The antioxidant activity was measured according to the formula:

scavenging effect (% ) = {1 − ( A sample − A blank A control )} × 100% where Asample was absorbance of sample, Ablank was absorbance of sample blank, and Acontrol was absorbance of the control. EC50 (µg/mL, concentration to scavenge 50% of free radicals) was calculated from the regression equation. All samples were analysed in triplicate using a calibration curve of trolox.

2.6.

Determination of antiproliferative activity

2.6.1.

Cell culture

HeLa (human cervical cancer), HepG2 (human hepatoblastoma), and MCF-7 (human breast cancer) cell lines were obtained from the American Type Culture Collection. The cell lines were cultured in RPMI 1640 or DMEM with high glucose or low glucose supplemented with 10% FBS at 37 °C in 5% CO2.

2.6.2.

Antiproliferative activity assay

The antiproliferative activity assay was performed by the MTT method (Zhu et al., 2011). In brief, HeLa (5 × 104), HepG2 (5 × 104) and MCF-7 (3.4 × 104) cells were placed in 96-well plates, respectively, and incubated for 24 h. Concentrations (5–80 µM) of the compounds (purities > 98%) were added to the well, and then incubated for 96 h. 50 µL of MTT solution was added, and the absorbance at 570 nm was measured with a microplate reader spectrophotometer. 5-Fluorouracil (80 mM/L) was used as the positive control. The results were obtained from three independent experiments carried out in duplicate. The inhibition rate was calculated using the following formula:

Inhibition rate (% ) = [( A control − A sample ) ( A control − A blank )] × 10 00% where Acontrol was absorbance of the control, Asample was absorbance of sample, and Ablank was absorbance of blank.

2.7.

Statistical analysis

All measurements of antioxidant, antiproliferative activities and coumarin contents were repeated in triplicate and data were

Compound 1 was obtained as yellowish oil. The positive HRESI-MS spectrum revealed an [M + Na]+ peak at m/z 643.1782 (calcd. 643.1791 for C33H32O12Na), suggesting a molecular formula of C33H32O12 with eighteen degrees of unsaturation. Its IR spectrum showed the presence of hydroxy (3453 cm−1) and lactone carbonyl (1742 cm−1) groups. The 1H NMR spectrum (Table 1) indicated the presence of two sets of proton signals for furocoumarin skeletons. The proton signals at δ 8.06 (1H, d, J = 9.7 Hz), 6.21 (1H, d, J = 9.7 Hz), 7.27 (1H, d, J = 1.7 Hz), and 7.93 (1H, d, J = 1.7 Hz) were assigned to a 5,8-disubstituted lineartype furocoumarin unit, while the signals at δ 8.05 (1H, d, J = 9.7 Hz), 6.07 (1H, d, J = 9.7 Hz), 7.22 (1H, s), 7.95 (1H, d, J = 1.7 Hz), and 7.24 (1H, d, J = 1.7 Hz) were attributed to 5-monosubstituted linear-type furocoumarin unit. The 1H NMR spectrum also exhibited the presence of two trioxygenated isopentyl groups [δ 4.64 (1H, dd, J = 9.7, 2.9 Hz), 4.55 (2H, m), 4.20 (1H, dd, J = 9.7, 7.9 Hz), 3.75 (2H, m), 1.26 (3H, s), 1.22 (3H, s), 1.07 (3H, s), and 1.15 (3H, s)] and a methoxy group at δ 4.15 (3H, s). The 13C NMR spectrum (Table 1) exhibited 33 carbon signals including 22 aromatic and 11 aliphatic carbons, which are consistent with the above deduced moieties. The above data indicated that 1 is a furocoumarin dimer. Compared with the known coumarins, two sets of the 1H and 13C NMR data of 1 were nearly identical to those of byakangelicin and oxypeucedanin hydrate or aviprin, respectively (Boyd et al., 2002; Yin, Cheng, & Lou, 2004), except for a few different chemical shifts of the side chain carbons. The connections of two furocoumarin moieties and the side chain were determined on the basis of the HMBC experiment. HMBC (Fig. 2) correlations from the H-11 proton signals at δH 4.20 and 4.55 to C-8 at δC 126.5, as well as from a methoxy group signal at δH 4.15 to C-5 at δC 143.9, support the existence of byakangelicin moiety. Significant correlations were observed between the H-11′ proton signals at δH 4.55 and 4.64, and the carbon signals at δC 148.7 (C-5′) prove the presence of hydrated oxypeucedanin moiety. On the basis of the downfield shifts of C-13 at δC 77.8 and C-12′ at δC 76.9 compared with the corresponding carbon signals of byakangelicin and oxypeucedanin hydrate (Boyd et al., 2002; Yin et al., 2004), and the HMBC correlations from the H-12′ proton signals at δH 3.75 to C-13 at δC 77.8, the byakangelicin and oxypeucedanin hydrate moieties were connected by ether linkage at C-13 and C-12′. The determination of the absolute configuration of C-12 in 1 was carried out by the [Rh2(OCOCF3)4]induced CD spectrum (Frelek & Szczepek, 1999), which showed the positive Cotton at ca. 347 nm, indicating the S configuration of C-12. The structure of compound 1 was therefore characterised as a new furocoumarin dimer and named angdahuricaol A (Fig. 1). Compound 2 was obtained as yellowish oil, with the molecular formula C33H32O12 based on the positive HR-ESI-MS (m/z

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Journal of Functional Foods 20 (2016) 453–462

Table 1 – 1H and 13C NMR data for 1–3 (DMSO-d6). Position

1 δC

2 3 4 4a 5 6 7 8 8a 9 10 11

159.4 112.2 139.5 106.5 143.9 114.1 149.4 126.5 143.0 145.9 105.5 73.8

12 13 14 15 2′ 3′ 4′ 4′a 5′ 6′ 7′ 8′ 8′a 9′ 10′ 11′

76.3 77.8 21.4 23.5 159.9 111.9 139.1 105.4 148.7 112.4 157.6 92.9 152.0 145.7 105.7 75.4

12′ 13′ 14′ 15′ 5-OCH3

76.9 70.8 25.2 27.9 60.6

2 δH 6.21 d (9.7) 8.06 d (9.7)

7.93 d (1.7) 7.27 d (1.7) 4.55 m 4.20 dd (9.7, 7.9) 3.75 m 1.22 s 1.26 s 6.07 d (9.7) 8.05 d (9.7)

7.22 s 7.95 d (1.7) 7.24 d (1.7) 4.64 dd (9.7, 2.9) 4.55 m 3.75 m 1.07 s 1.15 s 4.15 s

δC 159.4 112.3 139.4 106.6 143.9 114.1 149.1 126.1 142.8 145.9 105.5 75.2 77.6 71.0 25.9 26.7 160.1 111.5 139.6 105.9 149.1 112.9 157.5 92.8 151.8 145.6 105.5 74.8 75.0 77.7 21.8 24.0 60.6

3 δH 6.22 d (9.8) 8.04 d (9.8)

7.88 d (2.0) 7.23 d (2.0) 4.27 d (3.8) 3.79 t-like (4.0) 1.12 s 1.16 s 6.08 d (9.8) 8.13 d (9.8)

7.11 s 7.91 d (2.0) 7.23 d (2.0) 4.87 d (9.5) 4.38 t-like (8.9) 3.92 d (8.0) 1.25 s 1.35 s 4.12 s

δC 159.4 112.4 139.5 106.7 144.2 114.2 149.4 126.2 143.1 146.1 105.6 75.6 77.3 70.9 26.0 26.6 160.2 112.0 140.0 106.5 149.2 113.4 157.5 93.2 152.0 145.6 105.4 74.8 76.0 77.8 21.6 23.1 60.7

δH 6.26 d (9.8) 8.09 d (9.8)

7.99 d (2.3) 7.23 d (2.3) 4.43 dd (9.9, 3.4) 4.20 dd (9.9, 4.0) 3.77 t-like (3.9) 1.14 s 1.16 s 6.26 d (9.8) 8.31 d (9.8)

7.24 s 7.84 d (2.2) 7.20 d (2.2) 4.86 dd (9.8, 1.7) 4.35 dd (9.8, 7.4) 3.79 d (7.4) 1.29 s 1.33 s 4.14 s

NMR spectroscopic data were recorded at 600 MHz (1H NMR) and 150 MHz (13C NMR).

643.1784 [M + Na]+, calcd. 643.1791 for C33H32O12Na). Its IR spectrum showed the presence of hydroxy (3407 cm−1) and lactone carbonyl (1736 cm−1) groups. Extensive comparison of 1H and 13 C NMR spectra (Table 1) of 2 with 1, and analysis of HMBC spectrum of 2 suggested that 2 was also a furocoumarin dimer derived from byakangelicin and oxypeucedanin hydrate or aviprin with the difference at the connection position of the side chain. Significant long-range correlation between the proton signal at δH 3.79 (H-12) and the carbon signal at δC 77.7 (C-13′) assigned the ether linkage of the byakangelicin and oxypeucedanin hydrate moieties at C-12 and C-13′. The absolute configuration of C-12′ in 2 was determined as R by the [Rh2(OCOCF3)4]-induced CD spectrum of in situ formed Rhcomplexes of 2 with the negative Cotton effect at ca. 344 nm (E band). Thus, the structure of compound 2 was characterised and given the trivial name angdahuricaol B. Compound 3 was obtained as yellowish oil. Its molecular formula was determined as C33H32O12 on the basis of its quasimolecular ion peak [M + Na]+ in the HR-ESI-MS (m/z 643.1786 [M + Na]+, calcd. 643.1791 for C33H32O12Na). Its IR, 1H, 13C NMR (Table 1), and HMBC spectra were similar to 2, suggesting that

3 is the epimer or enantiomer possessing the same skeleton as 2. The only difference between 3 and 2 was the absolute configuration of C-12 and C-12′. The CD data of in situ formed Rh-complexes of 3 showed the positive Cotton effect at ca. 350 nm, assigning the absolute configuration of C-12′ as S. Thus, the structure of compound 3 was characterised and named angdahuricaol C. The known compounds were identified by spectroscopic analyses and their data were consistent with those reported in the literature. Compounds 4–23 were determined to be isoimperatorin (4), imperatorin (5) (Tian, Zhang, & Xu, 2013), phelloptorin (6) (Ola et al., 1997), alloimperatorin (7) (Ru, Jin, Jin, & Chen, 2007), isooxypeucedanin (8) (Mi, Shi, Li, Qiao, & Wang, 1997), pabularinone (9) (Xiao et al., 1995), anhydrobyakangelicin (10) (Adebajo & Reisch, 2000), pabulenol (11) (Tian et al., 2013), apaensin (12) (Sun, Ding, Lin, Yi, & Fu, 1982; Sun, Lin, Niu, & Ding, 1981), neobyakangelicol (13) (Tian et al., 2013), aviprin (14) (Boyd et al., 2002), byakangelicin (15) (Ahua et al., 2004; Yu & Zhang, 2006), oxypeucedanin hydrate (16) (Boyd et al., 2002), xanthotoxol (17) (Yu & Zhang, 2006), bergaptol (18) (Li, Luo, Peng, Liang, & Ding, 2006),

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Journal of Functional Foods 20 (2016) 453–462

O

O

O

O

O

O

O

O

H H

OH

H

O

H

O 1

O

O

O H

O

O

H

O

O

O

H H OH

HO H

H

O

O O

OH

HO

H H

O

H H

O H

O HO

O

O

O

O

O

O

3

2 HMBC, H to C

Fig. 2 – Key HMBC correlation of compounds 1–3.

5-methoxy-8-hydroxypsoralen (19) (Liang, Xu, Zhou, & Yang, 2005), 5-hydroxy-8-methoxy-psoralen (20) (Jiang, Liu, Shi, & Tu, 2007; Wang, Chen, & Li, 2012a), oxyalloimperatorin (21) (Liu et al., 2011), marmesin (22) (Okuyama, Takata, & Shibata, 1989), and scopoletin (23) (Duan, Shi, Wu, Gui, & Wang, 2007), respectively.

3.2.

ESI-MS analysis

Usually, coumarins in the roots of A. dahurica are linear furanocoumarins, and the differences of structures are the substituents at C-5 and C-8. Some examples were chosen to discuss the ESI-MS behaviours of linear furanocoumarins in order to identify the coumarins in the roots of A. dahurica or other herbal medicines rapidly and conveniently. If coumarins possess isopentyloxy or its oxygenated substituent at C-5 or C-8, such as 4, 5, 8, 11, and 16, the fragment m/z 203 due to a neutral loss of the substituent could be observed in Fig. 3. If coumarins possess hydroxyl substituent at C-5 or C-8, such as 17 and 18, sequential cleavage to lose molecules of CO produced the typical fragments m/z 147 [203 − 2 × CO]+ (see Fig. 3). 7 had a isopentyl substituent at C-5, and produced quite different ESI-MS fragmentation pattern with a fragment m/z 215 [M + H-C4H8]+. Moreover, for coumarins with the substituents both at C-5 and C-8, such as 10 and 19, there was a tendency to lose the larger fragment preferentially (Fig. 3).

3.3.

Antioxidant activity

The antioxidant activities of ethyl acetate fraction and isolated compounds from the roots of A. dahurica were measured by two different methods, DPPH• and ABTS•+ scavenging assays (see Table 2).

3.3.1.

DPPH scavenging activity

DPPH• is stable in ethanolic solutions and has a maximum absorbance at 517 nm (Modena) because of its odd electron ion.

DPPH• can be scavenged when it encounters a proton-donating substance, which leads to a decreased absorbance at 517 nm. Ethyl acetate fraction showed DPPH• scavenging activity with EC50 value of 0.23 mg/mL. The scavenging activities of 5 and 16–19 were superior to other isolated compounds with EC50 values of 19.48, 15.00, 86.86, 26.66, and 45.24 µM, respectively. These results illustrated that some compounds from the roots of A. dahurica showed strong DPPH scavenging activity. Thus, it might have the ability to turn DPPH• into less harmful compounds and interrupt the radical chain reaction.

3.3.2.

ABTS•+ scavenging activity

The ABTS radical scavenging assay has been extensively applied to evaluate the total antioxidant activity of various materials. Specific absorbance at 734 nm is used as an index reflecting the antioxidant activity of tested materials. The antioxidants can donate its labile hydrogen atom to peroxyl radical to form stable radicals, which can terminate the radical reaction. Ethyl acetate fraction showed ABTS•+ scavenging activity with an EC50 value of 80.45 µg/mL. 5 and 16–19 also exhibited the strong ABTS•+ scavenging activity which were in accordance with DPPH• scavenging assay. 16 may be the most promising compound with an EC50 value of 6.44 µM. Primary structure–activity relationship study showed that the antioxidative activity of furanocoumarins on DPPH• and ABTS•+ scavenging assays was probably related to the presence of hydroxyl group at C-5 or C-8. The result was consistent with previous study (Jovanovic, Steenken, Tosic, Marjanovic, & Simic, 1994; Piao et al., 2004).

3.4.

Determination of antiproliferative activity

All the compounds were evaluated for their growth inhibitory effects against HeLa, HepG2, and MCF-7 cell lines by the MTT method. The antiproliferative activities were summarised in Table 2. 4–7, 10, 17–19, 21 and 23 showed potential antiproliferative activities against HeLa and HepG2 than MCF-7

Journal of Functional Foods 20 (2016) 453–462

459

Fig. 3 – The fragmentation pathway of ten linear furanocoumarins.

cells. In addition, 4, 6 and 19 showed higher inhibition on HepG2 cells than other compounds, with IC50 values 8.19, 7.49, and 7.46 µM, respectively. Moreover, 19 also showed the moderate inhibitory activity against HeLa cell line with an IC50 value of 13.48 µM. The result revealed that a prenyl or prenyloxy group (4–7) at C-5 or C-8 and a hydroxy group at C-8 (17 and 19) play an important role in the growth inhibition on cancer cells, which were consistent with previous study (Wang, Chen, & Li, 2012b; Yang, Wang, Chen, & Wang, 2003). Previous studies also showed that high antioxidant activities may effectively prevent cancer formation or progression (Valko et al., 2007). These results can be as a support that 19 could be explored as novel and potential natural antioxidant and cancer prevention agent.

the coumarins, the content of 5 with 6.24 mg/g was the highest. The second one is 4 with 4.53 mg/g. In the remaining coumarins, 16 showed content with 2.54 mg/g, followed by 17 with 1.51 mg/ g, 19 with 0.84 mg/g, 10 with 0.68 mg/g, 7 with 0.30 mg/g, 11 with 0.20 mg/g, while 8 and 10 showed the same lowest content with 0.19 mg/g (see Table 3). Above all, 4, 5, 16, 17, and 19 were the major components in the roots of A. dahurica. It is noteworthy that the five furanocoumarins showed obvious antioxidant (except for 4) and antiproliferative activities. It is suggested that the five furanocoumarins were key antioxidant and antiproliferative principles of the roots of A. dahurica.

3.5. Determination of coumarin contents in the roots of A. dahurica

4.

Quantitation is the key issue for quality control of functional foods. In this study, the contents of ten coumarins in the 95% ethanol extract of the roots of A. dahurica were measured by UPLC-MS/MS analysis. The retention times of ten coumarins were as follows: 4 tR = 6.83 min; 5 tR = 4.74 min; 7 tR = 5.11 min; 8 tR = 2.89 min; 10 tR = 2.94 min; 11 tR = 3.19 min; 16 tR = 1.56 min; 17 tR = 0.95 min; 18 tR = 1.57 min; 19 tR = 1.22 min. The contents of ten coumarins exhibited remarkable differences. Among

In this study, 23 coumarins were isolated from the ethyl acetate fraction of the roots of A. dahurica, including three new furocoumarin dimers, angdahuricaols A (1), B (2), and C (3), and their in vitro antioxidant and antiproliferative activities were tested for the first time. 5 and 16–19 showed moderate radical scavenging activity against DPPH• and strong radical scavenging activity against ABTS•+, of which 16 exhibited significant ABTS•+ scavenging activity with an EC50 value of 6.44 µM. In

Conclusion

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Table 2 – The antioxidant activity and in vitro activity against HeLa, HepG2, and MCF-7 cells of ethyl acetate fraction and isolated compounds from the roots of Angelica dahurica. EC50 (µM)a

Sample

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 EtOAc fractiond VE 5-Fu

IC50 (µM)b •+

DPPH assay

ABTS assay

HeLa

HepG2

MCF-7

>100 >100 >100 >100 19.48 ± 1.11 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 15.00 ± 0.14 86.86 ± 10.01 26.66 ± 1.58 45.24 ± 2.22 >100 >100 >100 >100 232.12 ± 15.40 6.79 ± 0.83 NDb

78.91 ± 2.66 40.33 ± 1.24 56.77 ± 0.87 >100 10.93 ± 0.04 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 6.44 ± 0.49 22.82 ± 3.62 13.55 ± 1.51 17.86 ± 2.18 >100 >100 >100 >100 80.45 ± 1.63 4.22 ± 0.62 NDb

>80 >80 >80 17.77 ± 1.17 22.79 ± 1.00 25.60 ± 0.25 22.86 ± 1.00 >80 >80 70.04 ± 0.73 >80 >80 >80 >80 >80 >80 23.59 ± 0.25 58.57 ± 0.53 13.48 ± 0.85 >80 68.83 ± 0.90 >80 >80 54.01 ± 0.77 NDc 3.73 ± 0.58

>80 >80 >80 8.19 ± 0.64 14.66 ± 0.10 7.49 ± 0.27 19.58 ± 0.61 >80 >80 17.97 ± 0.87 >80 >80 >80 >80 >80 >80 15.57 ± 1.34 68.42 ± 0.43 7.46 ± 0.03 >80 48.74 ± 0.37 >80 41.81 ± 0.34 39.23 ± 1.87 NDc 3.13 ± 0.52

56.14 ± 2.03 35.24 ± 0.26 44.00 ± 0.66 >80 37.94 ± 0.16 33.35 ± 0.42 >80 >80 >80 >80 >80 >80 >80 >80 >80 >80 >80 >80 26.59 ± 1.23 >80 >80 >80 >80 80.96 ± 2.24 NDc 4.74 ± 0.02

EC50 and bIC50 values represent the means ± SD of three parallel measurements. cND means not determined. dµg/mL.

a

Table 3 – Calibration and quantitation of 10 coumarins. No.

Analytes

Regression equation

Correlation coefficient (r)

X ranges (µg/mL)

Contents (mg/g)

4 5 7 8 10 11 16 17 18 19

Isoimperatorin Imperatorin Alloimperatorin Isooxypeucedanin Anhydrobyakangelicin Pabulenol Oxypeucedanin hydrate Xanthotoxol Bergaptol 5-Methoxy-8-Hydroxypsoralen

Y = 1E + 08x + 3E + 07 Y = 1E + 08x + 151,426 Y = 1E + 06x + 20,820 Y = 7E + 06x + 540,380 Y = 7E + 06x + 1E + 06 Y = 6E + 06x + 666,769 Y = 7E + 07x + 1E + 07 Y = 5E + 06x − 4E + 06 Y = 1E + 07x + 257,984 Y = 1E + 07x − 5E + 06

0.9958 0.9940 0.9952 0.9948 0.9972 0.9957 0.9953 0.9922 0.9957 0.9955

0.0061–0.0303 0.0027–0.0136 0.0250–0.1248 0.0181–0.0906 0.0008–0.0041 0.0101–0.0508 0.0007–0.0037 0.0033–0.0167 0.0012–0.0060 0.0008–0.0038

4.53 ± 1.22 6.24 ± 2.20 0.30 ± 0.05 0.19 ± 0.07 0.19 ± 0.09 0.20 ± 0.04 2.54 ± 0.50 1.51 ± 0.25 0.68 ± 0.19 0.84 ± 0.13

addition, 4–7, 10, 17–19, 21 and 23 showed higher potential antiproliferative activities against HeLa and HepG2 than MCF-7 cells. Moreover, 4, 6, and 19 possessed significant inhibitory effects on the growth of HepG2 cells, with IC50 values 8.19, 7.49, and 7.46 µM, respectively. 19 also displayed moderate inhibitory effect on the growth of HeLa cells with an IC50 value of 13.48 µM. By UPLC-MS/MS analysis, it was found that 4, 5, 16, 17 and 19 were the major components in the roots of A. dahurica. These findings suggested that coumarins from the roots of A. dahurica were new sources of natural active compounds, and they could not only be used as natural antioxidants, but also administered to prevent cancer. It is worth mentioning that the in vitro bioactivities of these isolated

compounds are not necessarily equal to their actual beneficial effects in vivo. Therefore, further studies on the bioactivities of the isolates in vivo will be necessary to increase understanding of their function.

Acknowledgment This work was financially supported by the National Natural Science Foundation of China (31570350, 21502121), General Scientific Research Projects of Department of Education in Liaoning Province (L2014382), Young Teachers’ Scientific Research Fund

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Project of Shenyang Pharmaceutical University (QNJJ 2013501) and Career Development Support Plan for Young and Middleaged Teachers in Shenyang Pharmaceutical University.

Appendix: Supplementary material Supplementary data to this article can be found online at doi: 10.1016/j.jff.2015.11.018. REFERENCES

Adamson, P. C. (2015). Improving the outcome for children with cancer: Development of targeted new agents. CA – A Cancer Journal for Clinicians, 65, 212–220. Adebajo, A. C., & Reisch, J. (2000). Minor furocoumarins of Murraya koenigii. Fitoterapia, 71, 334–347. Ahua, K. M., Ioset, J. R., Ransijn, A., Mauel, J., Mavi, S., & Hostettmann, K. (2004). Antileishmanial and antifungal acridone derivatives from the roots of Thamnosma rhodesica. Phytochemistry, 65, 963–968. Boyd, D. R., Sharma, N. D., Loke, P. L., Malone, J. F., Colin McRoberts, W., & Hamilton, J. T. G. (2002). Absolute configuration assignment and enantiopurity determination of chiral alkaloids and coumarins derived from O- and C-prenyl epoxides. Chemical Communications, 24, 3070–3071. Deng, G. G., Wei, W., Yang, X. W., Zhang, Y. B., Xu, W., Gong, N. B., Lü, Y., & Wang, F. F. (2015). New coumarins from the roots of Angelica dahurica var. formosana cv. Chuanbaizhi and their inhibition on NO production in LPS-activated RAW264.7 cells. Fitoterapia, 101, 194–200. Duan, C. H., Shi, B. J., Wu, L. H., Gui, X., & Wang, Z. T. (2007). Chemical constituents of Gentiana waltonii. Chinese Journal of Natural Medicines, 5, 417–420. Frelek, J., & Szczepek, W. J. (1999). [Rh2(OCOCF3)4] as an auxiliary chromophore in chiroptical studies on steroidal alcohols. Tetrahedron, Asymmetry, 10, 1507–1520. Howlader, N., Noone, A. M., Krapcho, M., Neyman, N., Aminou, R., Altekruse, S. F., Kosary, C. L., Ruhl, J., Tatalovich, Z., Cho, H., Mariotto, A., Eisner, M. P., Lewis, D. R., Chen, H. S., Feuer, E. J., & Cronin, K. A. (2012). Coumarins from the roots of Angelica dahurica with antioxidant and antiproliferative activities. SEER Cancer Statistics Review. Jiang, Y. Y., Liu, B., Shi, R. B., & Tu, G. Z. (2007). Isolation and structure identification of chemical constituents from Saposhnikovia divaricata (Turcz.) Schischk. Acta Pharmaceutica Sinica, 42, 505–510. Jovanovic, S. V., Steenken, S., Tosic, M., Marjanovic, B., & Simic, M. G. (1994). Flavonoids as antioxidants. Journal of the American Chemical Society, 116, 4846–4851. Li, B., Zhang, X., Wang, J., Zhang, L., Gao, B., Shi, S., Wang, X., Li, J., & Tu, P. (2014). Simultaneous characterisation of fifty coumarins from the roots of Angelica dahurica by off-line two-dimensional high-performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry. Phytochemistry Analysis, 25, 229–240. Li, Y. H., Luo, F., Peng, S. L., Liang, J., & Ding, L. S. (2006). A new dihydroisocoumarin from the rhizomes of Notopterygium forbesii. Natural Products Research, 20, 860–865. Liang, B., Xu, L. Z., Zhou, Z. M., & Yang, S. L. (2005). Chemical constituents isolated from Angelica dahurica var. formosana. Chinese Traditional and Herbal Drugs, 36, 1132–1135. Liu, D. P., Luo, Q., Wang, G. H., Xu, Y., Zhang, X. K., Chen, Q. C., & Chen, H. F. (2011). Furocoumarin derivatives from radix

461

Angelicae dahuricae and their effects on RXRα transcriptional regulation. Molecules, 16, 6339–6348. Liu, L., Sun, Y., Laura, T., Liang, X., Ye, H., & Zeng, X. (2009). Determination of polyphenolic content and antioxidant activity of kudingcha made from Ilex kudingcha C.J. Tseng. Food Chemistry, 112, 35–41. Mi, C. F., Shi, H. L., Li, F. X., Qiao, B. L., & Wang, C. D. (1997). Chemical study of root of Angelica polymorpha Maxim. (II). Natural Products Research & Development, 9, 43–45. Okuyama, T., Takata, M., & Shibata, S. (1989). Structures of linear furano- and simple-coumarin glycosides of Bai-Hua Qian-Hu. Planta Medica, 55, 64–67. Ola, B., Kim, D., Mogens, N., Rudong, S., Robin, W., Jinglu, A., & Olov, S. (1997). Furanocoumarins with affinity to brain benzodiazepine receptors in vitro. Phytochemistry, 44, 1121– 1124. Piao, X. L., Park, I. H., Baek, S. H., Kim, H. Y., Park, M. K., & Park, J. H. (2004). Antioxidative activity of furanocoumarins isolated from Angelica dahurica. Journal of Ethnopharmacology, 93, 243– 246. Pinela, J., Barros, L., Dueñas, M., Carvalho, A. M., Santos-Buelga, C., & Ferreira, I. C. F. R. (2012). Antioxidant activity, ascorbic acid, phenolic compounds and sugars of wild and commercial Tuberaria lignosa samples: Effects of drying and oral preparation methods. Food Chemistry, 135, 1028–1035. Rajendran, P., Nandakumar, N., Rengarajan, T., Palaniswami, R., Gnanadhas, E. N., Lakshminarasaiah, U., Gopas, J., & Nishigaki, I. (2014). Antioxidants and human diseases. Clinica Chimica Acta, 436, 332–347. Roleira, F. M. F., Tavares-da-Silva, E. J., Varela, C. L., Costa, S. C., Silva, T., Garrido, J., & Borges, F. (2015). Plant derived and dietary phenolic antioxidants: Anticancer properties. Food Chemistry, 183, 235–258. Ru, J., Jin, L., Jin, Y. S., & Chen, H. S. (2007). Chemical constituents in roots of Angelica dahurica var. formosana. Academic Journal of Second Military Medical University, 28, 294–298. Saha, S. K., & Khuda-Bukhsh, A. R. (2013). Molecular approaches towards development of purified natural products and their structurally known derivatives as efficient anti-cancer drugs: Current trends. European Journal of Pharmacology, 714, 239– 248. Sarker, S. D., & Nahar, L. (2004). Natural medicine: The genus Angelica. Current Medicinal Chemistry, 11, 1479–1500. Seril, D. N., Liao, J., Yang, G. Y., & Yang, C. S. (2003). Oxidative stress and ulcerative colitis associated carcinogenesis: Studies in humans and animal models. Carcinogenesis, 24, 353– 362. Sosa, V., Moliné, T., Somoza, R., Paciucci, R., Kondoh, H., & Leonart, M. E. (2013). Oxidative stress and cancer: An overview. Ageing Research Reviews, 12, 376–390. Sun, H. D., Ding, J. K., Lin, Z. W., Yi, Y. F., & Fu, J. (1982). The 13CNMR spectrum of some natural occurring coumarins. Acta Pharmaceutica Sinica, 17, 835–840. Sun, H. D., Lin, Z. W., Niu, F. D., & Ding, J. K. (1981). The study of the Chinese drugs of Umbelliferae IV. The structure of apaensin. Acta Botanica Yunnancia, 3, 279–281. Thanh, P. N., Jin, W. Y., Song, G. Y., Bae, K. H., & Kang, S. S. (2004). Cytotoxic coumarins from the root of Angelica dahurica. Archives of Pharmacal Research, 27, 1211–1215. Tian, Y. Q., Zhang, Z. X., & Xu, H. H. (2013). Laboratory and field evaluations on insecticidal activity of Cicuta virosa L. var. latisecta Celak. Industrial Crops and Products, 41, 90–93. Valko, M., Leibfritz, D., Moncol, J., Croninm, M. T. D., Mazur, M., & Telser, J. (2007). Free radicals and antioxidants in normal physiological functions and human disease. The International Journal of Biochemistry & Cell Biology, 39, 44–84. Wang, M., Chen, J. W., & Li, X. (2012a). Chemical constituents of supercritical carbon dioxide extraction from the root bark of

462

Journal of Functional Foods 20 (2016) 453–462

Changium smyrnioides Wolff. Natural Products Research & Development, 24, 764–767. Wang, M., Chen, J. W., & Li, X. (2012b). Study on antitumor activity of five furanocoumarins from the root bark of Changium smyrnioides in vitro. Chinese Journal of Experimental Traditional Medical Formulae, 18, 203–205. Williams, G., Iatropoulos, M., & Whysner, J. (1999). Safety assessment of butylated hydroxyanisole and butylated hydroxytoluene as antioxidant food additives. Food and Chemical Toxicology, 37, 1027–1038. Xiao, Y. Q., Cui, S. L., Liu, X. H., Yang, L. X., Liu, D., Kimiye, B., & Masahiko, T. (1995). Studies on chemical components of Pleurospermum rivulorum (Diels). China Journal of Chinese Materia Medica, 20, 423–424.

Yang, L. L., Wang, M. C., Chen, L. G., & Wang, C. C. (2003). Cytotoxic activity of coumarins from the fruits of Cnidium monnieri on leukemia cell lines. Planta Medica, 69, 1091– 1095. Yin, F., Cheng, L., & Lou, F. C. (2004). Studies on the constituents of Citrus medica L. var. sarcodactylis (Noot.) Swingle. Chinese Journal of Natural Medicines, 2, 149–151. Yu, L., & Zhang, D. M. (2006). Studies on chemical constituents from fruits of Paliurus ramosissimus. China Journal of Chinese Materia Medica, 31, 2049–2052. Zhu, F., Di, Y. T., Li, X. Y., Liu, L. L., Zhang, Q., Li, Y., Hao, X. J., & He, H. P. (2011). Neoclerodane diterpenoids from Scutellaria barbata. Planta Medica, 77, 1536–1541.