Polyhydroxytriterpenoids and Phenolic Constituents from Forsythia

Dec 22, 2015 - In traditional Chinese medicines, Fructus Forsythiae (the fruit of F. suspensa) has mainly been used for the treatment of gonorrhea, er...
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Polyhydroxytriterpenoids and Phenolic Constituents from Forsythia suspensa (Thunb.) Vahl Leaves Ying Ge,† Yazhen Wang,§ Pingping Chen,⊗ Yufang Wang,† Congcong Hou,† Yibing Wu,† Manli Zhang,† Ligeng Li,† Changhong Huo,*,†,‡ Qingwen Shi,†,# and Haixia Gao*,⊥ †

School of Pharmaceutical Sciences, ‡Institute of Chinese Integrative Medicine, #Collaborative Innovation Center of Forensic Medical Molecular Identification, and ⊥Department of Pharmacology, Hebei Medical University, Shijiazhuang 050017, China § Medical Examination Center, Hebei General Hospital, Shijiazhuang 050051, China ⊗ Hebei University of Chinese Medicine, Shijiazhuang 050200, China S Supporting Information *

ABSTRACT: Forsythia suspensa (Thunb.) Vahl leaves have been consumed in China as a health-promoting functional tea for centuries. Three new polyhydroxytriterpenoid glycosides named suspensanosides A−C (1−3), seven known polyhydroxytriterpenoids (4−10), and 12 known phenolics (11−22) were identified from F. suspensa leaves. Compounds 1−10, 15−17, and 22 have not been found in the Forsythia genus previously, whereas compound 14 was first reported to be isolated from the leaves of F. suspensa. All isolates were tested for their antiproliferative activities on BGC-823 and MCF-7 human tumor cell lines, whereas all phenolics were further investigated for their antioxidant activities by a DPPH assay. The results clearly demonstrate that triterpenoids, especially ursane-type triterpenoids, and the diverse phenolic components are crucial for the beneficial effects of F. suspensa leaves. KEYWORDS: Forsythia suspensa (Thunb.) Vahl, leaves, triterpenoids, phenolics, antioxidant activity, antiproliferative activity

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INTRODUCTION Forsythia suspensa (Thunb.) Vahl, a well-known medicinal and ornamental plant, is widely grown in Asia and Europe. In traditional Chinese medicines, Fructus Forsythiae (the fruit of F. suspensa) has mainly been used for the treatment of gonorrhea, erysipelas, inflammation, and ulcers. The leaves of F. suspensa have been used as a health-promoting functional tea by Chinese people for hundreds of years, mainly in Hebei, Shanxi, and Shaanxi Provinces. In the past few years, an increasing number of researchers have become interested in F. suspensa leaves and tea infusions prepared from F. suspensa leaves. A broad spectrum of pharmacological activities, such as hypoglycemic, hypolipidemic, hepatoprotective, antiobesity, antioxidant, antiaging, antifatigue, antiallergy, antiproliferative, and heart protection effects have been reported.1−4 Although the extract of F. suspensa leaves has been used as a succedaneum of the extract of F. suspensa fruits in medicinal preparations in recent years, such replacement is still highly controversial.5 Therefore, extensive research on the bioactive components of F. suspensa leaves is highly necessary to justify their use as a functional tea that may prevent chronic diseases and cancers and as the succedaneum of Fructus Forsythiae. Previous chemical studies on F. suspensa leaves were mainly focused on the characterization of phenolics. The following compounds have been isolated and identified including quercetin, rutin, pinoresinol, phillygenin, (+)-pinoresinol 4-Oβ-D-glucoside, forsythoside A, forsythoside I, forsythenside F, calceolarioside C, calceolarioside D, and suspensaside A.1,6 To the best of our knowledge, apart from phenolic derivatives, Forsythia species and Fructus Forsythiae also contain other classes of constituents.1 Except for ursolic acid reported by © XXXX American Chemical Society

Fang et al., the only study that investigated the nonphenolic contituents of F. suspensa leaves including eight triterpenoids and two steroids were previously published by our team.1,7 To identify more bioactive substances responsible for the beneficial effects of F. suspensa leaves, further chemical investigation was conducted. As a result of that, 3 new polyhydroxytriterpenoid glycosides along with 7 known polyhydroxytriterpenoids and 12 known phenolic derivatives were obtained. Herein we describe the structural determination of three new compounds and assess the antiproliferative and antioxidant activities of the isolated compounds. These results will enrich our knowledge about the bioactive components of F. suspensa leaves and facilitate their use as dietary supplements.

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MATERIALS AND METHODS

General. ESIMS and HRESIMS were acquired on an AB SCIEX 3200Q TRAP spectrometer and an AB SCIEX TripleTOF 5600+ system, respectively. UV spectra were operated on a Shimadzu UV2201 machine. Optical rotations were performed on an Autopol IV polarimeter. NMR data were obtained with a Bruker Avance DRX-500 instrument. GC analysis was carried out in an Agilent 7890A system coupled with a FID. The GC analytical conditions were as follows: HP-5 column; carrier gas, N2 (1.4 mL/min); column temperature, from 120 to 280 °C (8 °C/min); injection temperature, 250 °C. Plant Material. F. suspensa leaves were obtained from Taihang Mountain on May 19, 2009. A voucher specimen with the access number of NMC-2009-FSL-2 has been deposited in our Herbarium. Received: September 14, 2015 Revised: December 11, 2015 Accepted: December 22, 2015

A

DOI: 10.1021/acs.jafc.5b04509 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. NMR Data of Suspensanosides A−Ca (CD3OD)a 1

a

position

δC

1

47.6

2 3 4 5 6

69.4 78.1 44.0 48.1 19.1

7

33.5

8 9 10 11

40.9 48.4 38.8 24.5

12 13 14 15

129.3 139.3 42.8 29.2

16

26.9

17 18 19

49.3 55.4 73.5

20 21

156.1 28.7

22

38.6

23

66.2

24 25 26 27 28 29

13.5 17.4 17.4 23.8 177.9 106.0

30 1′ 2′ 3′ 4′ 5′ 6′

27.4 95.6 73.7 77.7 70.8 78.1 61.8

2 δH

1.95 0.92 3.70 3.36

dd (12.8, 4.4) t (12.3) m d (9.6)

1.30 1.43 1.66 1.66 1.31

m m m m m

1.78 m 2.01 m 5.34 br s

1.04 1.80 2.74 1.75

m m m m

2.65 br s

2.10 2.78 1.96 1.68 3.27 3.51 0.70 1.04 0.79 1.35

dt (13.5, 4.6) m m m d (11.0) d (11.0) s s s s

4.70 4.90 1.37 5.33 3.31 3.40 3.33 3.33 3.80 3.67

s s s d (8.2) m t (8.8) m m dd (12.0, 2.1) dd (12.0, 4.6)

3

δC

δH

47.3 69.4 77.6 44.2 48.4 19.2 33.3 40.7 49.2 39.6 25.2

1.88 0.97 3.71 3.51

dd (12.4, 4.6) t (11.7) m d (9.7)

1.45 1.44 1.67 1.45 1.33

m m m m m

1.95 m 2.21 m 1.99 m 5.53 br s

122.7 141.4 42.0 24.4

1.13 1.37 2.85 1.64

24.2 41.0 50.1 36.1

m m m m

1.77 d (10.0) 2.12 m

85.3 30.4 29.0 73.3 13.7 17.0 16.6 21.9 181.1 64.6 17.7 104.7 75.2 78.3 71.8 78.0 62.9

1.86 1.76 2.15 1.52 3.40 3.71 0.73 1.02 0.77 1.10

m m m br d (12.6) d (9.9) d (9.9) s s s s

3.62 3.67 1.00 4.27 3.22 3.35 3.30 3.30 3.88 3.68

d (11.6) d (11.6) d (7.1) d (7.8) dd (9.1, 7.9) t (8.9) m m dd (11.8, 2.1) dd (11.8, 5.4)

δC 47.4 69.7 78.2 44.2 48.1 19.1 32.9 41.9 55.9 38.7 127.1 126.8 137.6 43.7 26.5 34.6 47.3 133.9 35.8 38.0 83.5 42.4 66.1 13.3 20.0 17.3 20.4 182.3 25.6 27.3 105.8 75.4 77.9 71.4 77.5 62.7

δH 2.21 0.96 3.76 3.39

dd (12.5, 4.7) t (12.3) td (11.1, 4.3) d (9.6)

1.33 1.44 1.53 1.44 1.30

d (10.1) m m m m

2.07 br s 5.67 d (10.6) 6.53 dd (10.6, 2.8)

1.06 1.53 2.08 1.62

m m m m

2.33 d (14.6) 2.46 d (14.6) 3.38 m 2.65 1.69 3.28 3.52 0.69 1.01 0.78 0.99

dd (14.0, 3.4) dd (14.0, 1.4) d (11.2) d (11.2) s s s s

0.81 s 1.04 4.27 3.18 3.33 3.32 3.18 3.84 3.70

s d (7.7) m m m m dd (11.9, 2.1) dd (11.9, 5.0)

From HSQC, HMBC, and COSY results.

Extraction and Isolation. F. suspensa leaves were macerated with 10 times the amount of EtOH at room temperature. EtOH extraction of 2.3 kg was diluted with water, and the lipids were removed with petroleum ether. Subsequently, the aqueous phase was fractionated in CH2Cl2, EtOAc, and n-BuOH. The CH2Cl2-soluble fraction (288 g) was applied for repeated silica gel CC to afford compound 14 (11 mg). The EtOAc extraction (115 g) was chromatographed over silica gel (CH2Cl2/MeOH, 98:2−1:1) to furnish fractions E1−E6. Fraction E2 gave compound 7 (36 mg). Fraction E3 afforded compounds 6 (20 mg) and 10 (7 mg) by an ODS column (MeOH/H2O, 25:75−100:0).

Compounds 5 (8 mg), 9 (5 mg), and 16 (20 mg) were purified from fraction E6 according to the same method. Compounds 12 (3.0 g) and 13 (4.7 g) were obtained by recrystallization from fractions E4 and E5, respectively. The separation of n-BuOH extraction (668 g) was carried out on an HP-20 column to get A (30% EtOH eluate) and B (50% EtOH eluate) parts. Part A was chromatographed over a silica gel column (CHCl3/MeOH, 95:5−15:1) to give fractions BA1−BA4. Fraction BA2 yielded compound 22 (100 mg). Fraction BA4 furnished compounds 15 (9 mg) and 21 (57 mg). Part B was carried out on a silica gel CC to afford fractions BB1−BB9. Fraction BB4 was B

DOI: 10.1021/acs.jafc.5b04509 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Structures of compounds 1−22. Suspensanoside B (2): white amorphous powder; [α]25 D −28.6 (c 0.10, MeOH); UV (MeOH) λmax 200 nm; ESIMS m/z 687 [M − H2O + Na]+; HRESIMS m/z 663.3748 [M − H2O − H]− (calcd for C36H55O11, 663.3744); 1H and 13C NMR data are given in Table 1. Suspensanoside C (3): white amorphous powder; [α]25 D −66.0 (c 0.09, MeOH); UV (MeOH) λmax 242, 250, and 259 nm; ESIMS m/z 687 [M + Na]+; HRESIMS m/z 663.3749 [M − H]− (calcd for C36H55O11, 663.3744); 1H and 13C NMR data are given in Table 1. Acid Hydrolysis of Suspensanosides A−C. After heating at 80 °C for 2 h, a 2 N HCl solution containing each suspensanoside (0.75 mg/mL) was extracted with EtOAc. Next, the concentrated aqueous phase was mixed with 1.0 mL of pyridine and 2.0 mg of L-cysteine

fractionated over ODS and Sephadex LH-20, yielding compounds 1 (8 mg), 4 (9 mg), and 8 (6 mg). Fractions BB1, BB5, and BB9 gave compounds 11 (199 mg), 17 (3 mg), and 20 (956 mg), respectively. From fraction BB6, compounds 2 (13 mg) and 3 (7 mg) were isolated by ODS CC (MeOH/H2O, 25:75−100:0). Compounds 18 (480 mg) and 19 (83 mg) were obtained from fraction BB7 over silica gel and Sephadex LH-20. Suspensanoside A (1): white amorphous powder; [α]25 D +166.4 (c 0.12, MeOH); UV (MeOH) λmax 211 nm; ESIMS m/z 687 [M + Na]+; HRESIMS m/z 663.3746 [M − H]− (calcd for C36H55O11, 663.3744); 1H and 13C NMR data are given in Table 1. C

DOI: 10.1021/acs.jafc.5b04509 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry methyl ester hydrochloride at 60 °C for 2 h. 1-(Trimethylsilyl)imidazole (0.5 mL) was then added into the mixture and treated for the same time at 60 °C. The reaction mixture was analyzed by GC. The retention times of these trimethylsilylated products were all consistent with that of D-glucose (20.1 min). DPPH Radical Scavenging Assay. Samples with different concentrations in EtOH (100 μL) and 0.2 mM DPPH in EtOH (100 μL) were incubated at 37 °C for 30 min, and the absorbance (517 nm) was determined using a Sunrise microplate reader (Tecan, Grödig, Austria). The efficient concentration of samples that inhibits 50% of the DPPH radicals (IC50) was calculated and expressed in micromolar.8 L-Ascorbic acid was applied as the positive reference. Tests were performed in triplicate. Antiproliferative Activity Assay. Antiproliferation assay was performed against BGC-823 and MCF-7 cells utilizing MTT method.9 5-FU, an anticancer agent, was applied as positive control. Tests were repeated three times.

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RESULTS AND DISCUSSION Structural Elucidation. Suspensanoside A (1) has an elemental composition of C36H56O11, on the basis of its HRESIMS at m/z 663.3746 [M − H]−. The NMR data (Table 1) suggested that 1 was a triterpene monosaccharide possessing 5 tertiary methyl, 10 methylene, 6 methine, 8 quaternary, and 1 carbonyl carbon, along with a hexose moiety. One broad singlet at δH 5.34, two carbons at δC 129.3 and 139.3, and one carbonyl at δC 177.9 are characteristic of an urs-12-en-28-oic acid skeleton. Furthermore, a pair of isolated AB protons at δH 4.70 and 4.90 and two carbons at δC 106.0 and 156.1 indicated the presence of an exocyclic double bond. Additionally, four oxygenated carbons at δC 66.2, 69.4, 73.5, and 78.1 were typical for carbons of alcoholic functions. Exact assignment of these hydroxy groups and the exocyclic double bond was elucidated using extensive 2D NMR results. Two vicinal protons at δH 3.36 and 3.70 and a pair of oxygenated geminal methylene protons at δH 3.27 and 3.51 exhibited HMBC correlations to C4 at δC 44.0, and a methyl singlet at δH 0.70 (3H, s) due to Me24 exhibiting strong NOESY correlation to β-oriented Me-25 at δH 1.04 (3H, s), which undoubtedly implied that the structure of 1 had a 2,3,23-trihydroxy-substituted pattern. Due to good consistency of 13C NMR data between C-2, C-3, C-23, and those reported for related compounds, 2-OH and 3-OH were assigned as equatorial α-orientation and equatorial βorientation, respectively,10 which was further supported by the coupling constant J2,3 (9.6 Hz) and the NOESY correlation of H-2 with Me-24. In the upfield 1H NMR spectrum, the absence of two characteristic doublet signals for Me-29 and Me30 of usual ursane type triterpenes was indicative of the fact that C-19 and -20 emerged as quaternary carbons and these two carbons were the possible positions of the remaining hydroxy group and the exocyclic sp2 methylene group. The HMBC correlations between H-18, H2-29, Me-30, and related carbons located the position of 19-OH and an exocyclic methylene group at C-20. Furthermore, the NOESY cross peaks of Me-24/Me-25, Me-25/Me-26, and Me-26/H-18 indicated H-18 with β-orientation. The NOESY correlations of H-18/Me-30 and H-12, as well as Me-30/H-12, suggested that the orientation of 19-OH should be identical with that of coussaric acid. The absolute configuration of the latter was established in 2003 (Figure 2).11 A β-D-glucopyranosyl unit in compound 1 was identified by NMR and GC analyses. The HMBC cross peak of Glc H-1/C-28 revealed the sugar moiety linked to C-28. Consequently, 1 was characterized as 2α,3β,19α,23-tetrahydroxyurs-12,20(29)-dien-28-oic acid β-D-

Figure 2. Key NOESY correlations of suspensanosides A and B.

glucopyranosyl ester (Figure 1), the aglycon of which was reported here for the first time. Suspensanoside B (2) had the molecular formula C36H58O12, as determined from NMR and HRESIMS data. The 13C NMR spectrum of 2 (Table 1) indicated 36 carbons including 5 methyl, 10 methylene, 7 methine, 7 quaternary, and 1 carbonyl carbon and a glucose moiety. The 1H NMR data showed the presence of 49 protons, implying that 9 protons occurred in the form of hydroxy groups. Two oxymethine carbons at δC 69.4 and 77.6, two oxymethylene carbons at δC 64.6 and 73.3, one oxygenated quaternary carbon at δC 85.3, a carboxylic acid carbon at δC 181.1, and two olefinic carbons at δC 122.7 and 141.4, along with other spectral features, supported that the aglycon of 2 had a pentahydroxyurs-12-en-28-oic acid skeleton with the same 2α,3β,23-trihydroxy-substituted pattern as 1. A detailed analysis of HMBC data showed that the two remaining hydroxy groups substituted at C-20 and C-29. The large coupling constant J18,19 (10.0 Hz) indicated that H-18 and H-19 were trans-diaxial. The NOESY cross peaks of H-18/Me-30 and H-19/H2-29 suggested an equatorial α-orientation of 20CH2OH (Figure 2). The HMBC cross peak of Glc H-1/C-23 suggested that a glucose linked with C-23. Accordingly, 2 was assigned as 23-O-β-D-glucopyranosyl 2α,3β,20β,23,29-pentahydroxyurs-12-en-28-oic acid (Figure 1). To our knowledge, this is the first report of such an aglycon. Suspensanoside C (3) had the same molecular formula as that of suspensanoside A. Extensive NMR analysis indicated that 3 was a triterpenoid β-D-glucopyranoside with a Δ11,13(18)oleanolic acid aglycon (Table 1)10,12 and also possessed the same 2α,3β,23-hydroxylation pattern in the triterpene core as that of 1. The presence of three oxymethine carbons at δC 69.7, 78.2, and 83.5 and one oxymethylene carbon at δC 66.1 revealed that the aglycon of 3 had four hydroxy groups. The D

DOI: 10.1021/acs.jafc.5b04509 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Antioxidant Activities. The antioxidant activity of the isolated phenolic derivatives, including phenylethanoid glycosides (18, 19), flavonoids (20−22), lignans (11−14), phenylpropionic acid esters (15, 16), and phenolic acid (17), were evaluated by DPPH radical scavenging assay (Table 2). Except

fourth hydroxy substituent was attached at C-21 by HMBC experiment. The small coupling of 3.4 and 1.4 Hz observed between two protons at C-22 and H-21 allowed us to assign the equatorial orientation of H-21. Furthermore, the NOESY correlations of H-12/H-19eq, H-19eq/Me-29, Me-29/H-21, and Me-30/H-19ax,H-21 supported the axial β-orientation of 21OH. The HMBC cross peak of Glc H-1/C-21 implied that the sugar unit connected to C-21. Hence, 3 was characterized as 21-O-β- D-glucopyranosyl 2α,3β,21β,23-tetrahydroxyolean11,13(18)-dien-28-oic acid (Figure 1). Its aglycon has never been reported before in the literature. The other isolates were nigaichigoside F1 (4), quadranoside IV (5), esculentic acid (6), corosolic acid (7), arjunglucoside I (8), arjunglucoside II (9), hovenic acid (10), (+)-phillygenin (11), forsythin (12), (+) epipinoresinol 4′-β-D-glucoside (13), forsythenin (14), (E)-caffeic acid methyl ester (15), 1-Ocoumaroyl-β-D-glucopyranose (16), protocatechuic acid (17), forsythoside A (18), forsythoside I (19), rutin (20), quercetin (21), and kaempferol (22) (Figure 1), the spectral data of which agreed with reported values. In the present work, 3 new polyhydroxytriterpenoid glycosides (suspensanosides A−C), along with 7 known polyhydroxytriterpenoids and 12 known phenolics, were isolated and identified from the leaves of F. suspensa. In particular, the aglycons of compounds 1−3 have never been reported, whereas compounds 4−10, 15−17, and 22 have not been found in the Forsythia genus previously. Compound 14 was also first reported to be isolated from F. suspensa leaves. An interesting fact is that nine of the isolated triterpenoids (1−5, 7−10) bear a 2α,3β,23-trihydroxyl functionality, and five of them (1−4 and 8) also bear one or two hydroxy groups in ring E, even though their skeleton types involve ursane, oleanane, and lupane. Up to now, more than 140 compounds have been isolated from the F. suspensa genus. The aglycons of triterpenoids isolated so far from this genus include ursane, oleanane, lupine, and dammarane types,1 but the triterpenoid skeleton bearing a 2α,3β,23-trihydroxyl functionality with the addition of substituted hydroxy groups in ring E is reported for the first time. Therefore, this unique structure could be recognized as a chemotaxonomic feature for F. suspensa leaves. Furthermore, dammarane-type triterpenoids previously reported occurring in the fruits of F. suspensa were not confirmed in the leaves. This difference of contents of triterpenoids in two plant organs may not only imply a specially differentiated biosynthetic pathway but also provide phytochemical evidence for the argument regarding whether F. suspensa leaves could be used as the succedaneum of Fructus Forsythiae in the medicinal preparations. Among the isolated known triterpenoids, nigaichigoside F1 (4) could relieve pain and inflammation and had hypoglycemic and hypolipidemic effects.13,14 Quadranoside IV (5) exhibited hepatoprotective activity.15 Esculentic acid (6) displayed antiangiogenic and anti-inflammatory effects and can treat bronchitis, hepatitis, and rheumatoid arthritis.16,17 Corosolic acid (7) exerted hypotensive, hypoglycemic, hypolipidemic, anti-inflammatory, antioxidative, and hepatoprotective activities. Moreover, it can ameliorate obesity and hepatic steatosis.18−21 Arjunglucoside I (8) had antimicrobial activity,22 whereas hovenic acid (10) showed cytotoxic activity.23 This information demonstrates that triterpenoids, especially ursane-type triterpenoids, have a significant role in the health-promoting function of F. suspensa leaves as well as tea infusions prepared from F. suspensa leaves.

Table 2. Antioxidant Activities of 11−22 compound 11 12 13 14 15 16 17 18 19 20 21 22 L-ascorbic acid a

IC50a (μM) 149.67 627.49 185.22 657.85 12.17 55.60 30.21 10.11 16.29 18.05 13.27 65.58 35.35

± ± ± ± ± ± ± ± ± ± ± ± ±

4.15 15.24 4.36 10.26 0.25 1.67 1.11 0.59 0.72 0.83 0.98 2.03 1.52

Values represent means ± SD.

for compounds 12 and 14, all phenolics exhibited potent antioxidative activity. In particular, (E)-caffeic acid methyl ester (15), protocatechuic acid (17), forsythiaside (18), forsythoside I (19), rutin (20), and quercetin (21) were more potent than Lascorbic acid (IC50 = 35.35 ± 1.52 μM). The result revealed that the antioxidant property of F. suspensa leaves should be attributed to the combination effect of diverse phenolic phytochemicals. Antiproliferative Activities. The antiproliferative activities of compounds 1−22 were assessed in human BGC-823 (gastric cancer) and MCF-7 (breast cancer) cell lines in vitro (Table 3). Compound 1 exhibited potent inhibitory activity against MCF7 cells (IC50 = 12.44 ± 0.69 μM) and BGC-823 cells (IC50 = 15.44 ± 0.99 μM), which were similar to the positive control 5FU with IC50 values of 17.88 ± 0.96 and 20.88 ± 0.97 μM. Compounds 4, 7, 11−14, and 22 displayed potent antiproliferative activities against MCF-7 cells (IC50 = 11.22−39.23 μM), whereas the rest of the compounds had no promising cytotoxicity. It is worth noting that the antiproliferative activities of all tested lignans (11−14) against MCF-7 cells were similar to that of 5-FU. The data obtained in this biological test suggested that bisepoxylignan and its analogues could be a promising class of antitumor agents toward human breast tumor cell lines. This result was in agreement with a previous report that lignans could markedly reduce breast cancer risk.24 Structure−activity relationship analysis of three lignans (11−13) suggested that methylation or glycosylation of the hydroxy groups on two benzene rings might be favorable for antiproliferative activity. Besides, three ursane-type triterpenoids (1, 4, and 7) displayed significant activity against BGC-823 and/or MCF-7 cells, whereas the oleanane- and lupane-type triterpenoids had no promising antiproliferative potency. These data further supported the important role of ursane-type triterpenoids in the beneficial effects of F. suspensa leaves. In conclusion, we report here the structures of 22 compounds isolated from F. suspensa leaves, as well as their antioxidant and antiproliferative activities. The results indicated that the major difference of secondary metabolite classes E

DOI: 10.1021/acs.jafc.5b04509 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Table 3. Antiproliferation Effects of 1−22

a

BGC-823

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 5-FU

15.44 ± 0.99 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 20.88 ± 0.97

MCF-7 12.44 >100 >100 19.02 >100 >100 39.23 >100 >100 >100 20.57 11.22 20.64 24.23 >100 >100 >100 >100 >100 >100 >100 24.30 17.88

± 0.69

± 0.76

± 0.12

± ± ± ±

0.92 0.59 0.91 0.83

± 1.01 ± 0.96

Data represent means ± SD.

between F. suspensa leaves and fruits is triterpenoid components, whereas the health benefits of F. suspensa leaves are attributable to the complex mixture of bioactive compounds present in leaves. Our research provides a valid justification for the use of F. suspensa leaves as a health-promoting functional tea.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b04509. NMR and HRESIMS spectra of suspensanosides A−C (PDF)

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REFERENCES

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IC50a (μM) compound

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AUTHOR INFORMATION

Corresponding Authors

*(C.H.H.) Phone: +86311 86265634. Fax: +86311 86265634. E-mail: [email protected]. *(H.X.G.) E-mail: [email protected]. Funding

This work was supported by the National Program on Key Basic Research Project of China (973 Program, No. 2012CB518601), the Natural Science Foundation of China (81072551, 81201642), and the Natural Science Foundation of Hebei Province (H2015206113). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Françoise Sauriold (Queen’s University, Canada) for recording the NMR spectra and Dr. Lei Wang and Dr. Yucheng Gu for proofreading. F

DOI: 10.1021/acs.jafc.5b04509 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.5b04509 J. Agric. Food Chem. XXXX, XXX, XXX−XXX