Bioactive terpenoid constituents from Eclipta prostrata

Phytochemistry 170 (2020) 112192

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Bioactive terpenoid constituents from Eclipta prostrata a,b

b

Shu-Juan Yu , Jin-Hai Yu , Zhi-Pu Yu Hua Zhangb,∗ a b c

a,b

a,b

, Xue Yan

b

c

, Jun-Sheng Zhang , Jin-yue Sun ,

T

School of Chemistry and Chemical Engineering, University of Jinan, 336 West Road of Nan Xinzhuang, Jinan 250022, China School of Biological Science and Technology, University of Jinan, 336 West Road of Nan Xinzhuang, Jinan 250022, China Institute of Agro-Food Science and Technology, Shandong Academy of Agricultural Sciences, 202 Gongye North Road, Jinan 250100, China

ARTICLE INFO

ABSTRACT

Keywords: Eclipta prostrata Compositae Terpenoid α-glucosidase Cytotoxicity Antibacterial

Chemical fractionation of the ethanolic extract of Eclipta prostrata yielded a series of unreported terpenoid constituents, including a rare 6/6/6/6-fused tetracyclic triterpenoid, a pentacyclic triterpenoid, two pentacyclic triterpenoid saponins, a diterpenoid and a sesquiterpenoid. Structures were assigned to these compounds on the basis of comprehensive spectroscopic analyses, with the absolute configurations of the tetracyclic triterpenoid, the diterpenoid and the sesquiterpenoid being determined via explanation of electronic circular dichroism data. Screening of these isolates in an array of bioassays revealed antibacterial, cytotoxic and α-glucosidase inhibitory activities for selective compounds. Of particular interest, the tetracyclic triterpenoid showed very strong inhibition against α-glucosidase with an IC50 of 0.82 ± 0.18 μM, being 103-fold as active as the positive control acarbose.

1. Introduction Eclipta prostrata (L.) L. (family Compositae), the only Eclipta species of China, is an annual herb widely distributed in the tropical and subtropical areas all over the world (Flora of China Editorial Committee, 2011). The whole plants have been used as folk medicine in Japan, China, India and other tropical regions for the treatment of several diseases, such as infectious hepatitis, hemorrhagic diseases, cardiovascular ailments and respiratory disorders including cough and asthma. The extracts of this medicinal plant have been reported to exhibit a broad spectrum of biological properties, including antitumor (Chung et al., 2017), anti-inflammatory (Kim et al., 2017), antihyperglycemic (Rahman et al., 2011), antihyperlipidemicand (Zhao et al., 2015) antioxidant (Chan et al., 2014) activities, as well as ameliorating the cognitive impairment (Jung et al., 2016). Previous phytochemical investigations into E. prostrata have revealed the presence of flavonoids, thiophenes, steroids, triterpenoids and saponins (Fang et al., 2015; Jeong et al., 2013; Liu et al., 2012; Ryu et al., 2013; Tabata et al., 2015; Tewtrakul et al., 2011; Xi et al., 2014) with the bioactivities of antitumor (Cho et al., 2016; Kim et al., 2015), anti-inflammation (Ryu et al., 2013; Tewtrakul et al., 2011), antibacterial (Khanna et al., 2009; Ray et al., 2013) and against dipeptidyl peptidase IV (Xi et al., 2014). Mount Kunyu is located in the east of Shandong Peninsula and is blessed with a unique miroclimate from the interaction of external

∗

marine climate and internal complex topography, thus breeding an extremely diverse source of plant species (Xu et al., 1988). Supported by the Natural Science Foundation of Shandong Province, we started a project since 2017 aiming to explore the bioactive constituents in the herbal plants from Mount Kunyu area. We have subsequently completed the phytochemical investigations on Dianthus superbus var. superbus (Sun et al., 2019a, 2019b), Ipomoea nil (Song et al., 2019) and Inula japonica (Yu et al., 2019), which led to a fruitful reward of a number of cytotoxic and α-glucosidase inhibitory natural molecules. Then our attention turned to E. prostrata, a widely distributed medicinal species in most Chinese provinces, to examine the bioactive chemicals from this herb growing in the special environment of Mount Kunyu. Consequently, the current work resulted in the separation and identification of a panel of terpenoid compounds (see Fig. 1) including an unusual lemmaphyllanetype triterpenoid (1), an oleanane-type triterpenoid lactone (2), two oleanane-type triterpenoid saponins (3 & 4), a beyerane-type diterpenoid (5) and a guaiane-type sesquiterpenoid acid (6), as well as two previously reported triterpenoid saponins (7 & 8). Details of the isolation, structural characterization and biological assessments are presented below . 2. Results Compound 1, a white amorphous powder, had a molecular formula of C30H50O2 as determined by (+)-HRESIMS analysis at m/z 425.3794

Corresponding author. E-mail address: [email protected] (H. Zhang).

https://doi.org/10.1016/j.phytochem.2019.112192 Received 1 June 2019; Received in revised form 27 August 2019; Accepted 29 October 2019 0031-9422/ © 2019 Elsevier Ltd. All rights reserved.

Phytochemistry 170 (2020) 112192

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Table 1 1 H and 13C NMR data of 1 and 2 in CDCl3. No.

1

2

δC

δH (J in Hz)

δC

δH (J in Hz)

1

35.5

39.6

2

28.2

3 4 5 6

79.2 39.1 50.7 19.3

α 1.18, td (13.1, 3.8) β 1.79, dt (13.1, 3.5) α 1.68, m β 1.58, m 3.23, dd (11.8, 4.5)

α 1.38, ddd (13.1, β 1.87, ddd (13.1, α 2.37, ddd (15.9, β 2.56, ddd (15.9,

7

27.3

8 9 10 11

134.2 134.5 37.8 20.1

12

33.4

13 14 15

35.0 39.9 26.4

16

34.6

17 18 19

45.4 21.5 20.2

20 21 22

33.2 32.4 43.9

23 24 25 26 27 28 29 30

124.4 140.8 71.1 30.2 30.2 28.3 15.8 22.8

1.06, dd (12.6, 2.0) α 1.68, m β 1.39, m α 1.89, m β 2.11, m

α 1.88, m β 2.09, m α 1.10, dd (13.0, 8.6) β 1.52, m α 1.61, m β 1.37, m α 1.37, m β 1.49, td (14.1, 4.6) 1.19, m (2H) 0.94, s 0.95, s 0.84, 1.95, 2.35, 5.65, 5.57,

s dd (14.4, 6.7) dd (14.4, 7.3) dt (15.5, 7.3) d (15.5)

1.32, 1.32, 1.00, 0.80, 0.97,

s s s s s

34.5 218.0 47.9 55.9 19.9 32.0 40.6 46.7 37.1 23.9 125.3 139.7 43.7 36.6 67.9 50.1 40.9 44.1 34.3 84.2 26.9 26.8 21.9 15.7 16.1 29.4 181.5 29.4 24.2

11.8, 6.7) 7.3, 3.7) 6.7, 3.7) 11.1, 7.3)

1.31, dd (8.0, 5.9) α 1.55, m β 1.55, m α 1.58, m β 1.48, m 1.53, m 1.97, m (2H) 5.40, t (3.6)

α 1.12, dd (14.0, 12.5) β 2.08, dd (14.0, 4.9) 4.00, dd (12.5,4.9) 2.61, dd (12.9, 6.8) α 1.73, dd (14.5, 12.9) β 1.43, dd (14.5, 6.8) 4.25, d (5.5) α 2.10, d (11.9) β 2.42, dd (11.9, 5.5) 1.09, s 1.06, s 1.05, s 0.94, s 1.24, s

Fig. 1. Chemical structures of 1–8.

respective chemical shifts at δC 79.2 and 71.0. Therefore, the planar structure of 1 was delineated to bear a rare 6/6/6/6-fused triterpenoid framework. A search in the literature revealed that 6/6/6/6 tetracyclic triterpenoids represent a very unusual and small group of natural products, with five types, namely, baccharane, D:B-friedobaccharane, lemmaphyllane, shionane and montecrinane, being reported to date, whereas few examples have been recorded for any type of skeleton (Purino et al., 2016). To the best of our knowledge, compound 1 is only the third natural member of lemmaphyllane-type triterpenoids, and the other two (lemmaphylla-7,21-diene and 3β-hydroxy-lemmaphylla-7,21diene) were first reported in 1983 (Masuda et al., 1983) and 1997 (Akihisa et al., 1997), respectively. The relative configuration of 1 was elucidated by analyses of 1H–1H couplings and ROESY data as described below. The coupling pattern of H-3 (dd, J = 11.8, 4.5 Hz) suggested that H-3 were axially bonded in the chair-like conformation of ring-A and was assigned to be α-oriented (Nakamura et al., 2009), thus leaving 3-OH β-equatorially directed. Then the ROESY correlations (see Fig. 2B) of H-3/H-5 and H-5/H-7α (δH 1.89) indicated that H-5 and H-7α were also axially bonded and α-positioned, while those of H-2β (δH 1.58) with H3-19 and H3-29 supported the β-orientation for Me-19 and Me-29. Subsequently, the cross-peaks of H-7β (δH 2.11) with H3-30, H3-30 with H-12β (δH 1.52) and H-16β (δH 1.49), and H3-19 with H-11β (δH 2.09), uncovered that these protons and proton-bearing groups were coplanar and thus β-directed. Furthermore, the correlations of H-11α (δH 1.88) with H3-18, H3-18 with H-15α (δH 1.61) and H-22 at δH 1.95 allowed the α-direction assignment for H-15α, Me-18 and the C-20 sidechain, thus leaving Me-21 β-positioned. Finally, the magnitude of J23,24 (15.5 Hz) suggested an E-configuration for Δ23, which was also supported by the ROESY correlations of H-24/H2-22. The absolute configuration of 1 was further established

1.00, s 1.03, s

([M + H − H2O]+, calcd 425.3778) and 13C NMR data, corresponding to six indices of hydrogen deficiency. Analysis of the NMR data (Table 1) for 1 revealed 30 carbon resonances consistent with eight methyls, ten methylenes, four methines (one oxygenated and two olefinic) and eight quaternary carbons (one oxygenated and two olefinic). Four carbon signals at δC 124.4, 134.2, 134.5 and 140.8 indicated the presence of two double bonds, accounting for two out of six indices of hydrogen deficiency, and the remaining four suggested a tetracyclic framework for 1. Comprehensive analysis of 1H–1H COSY and HMBC data (see Fig. 2A) enabled the establishment of the planar structure of 1 as shown. In detail, the 1H–1H COSY data helped to build up five spinspin coupling systems (a−e) as drawn in bold bonds (see Fig. 2A), which were further connected via eight quaternary carbons to generate the whole gross structure by the HMBC correlations from H3-19 to C-1 (δC 35.5), C-5 (δC 50.7), C-9 (δC 134.5) and C-10 (δC 37.8); H3-28 to C-3 (δC 79.2), C-4 (δC 39.1), C-5 and C-29 (δC 15.8); H2-7/H2-11 to C-8 (δC 134.2) and C-9; H3-18 to C-12 (δC 33.4), C-13 (δC 35.0), C-14 (δC 39.9) and C-17 (δC 45.4); H3-21 to C-16 (δC 34.6), C-17, C-20 (δC 33.2) and C22 (δC 43.9); H3-30 to C-8, C-13, C-14 and C-15 (δC 26.4); and H3-26 to C-24 (δC 140.8), C-25 (δC 71.1) and C-27 (δC 30.2). Particularly, the HMBC correlations from H2-7/H2-11 to C-8 and C-9 located one double bond at Δ8, while Δ23 was assigned via the COSY correlations of H2-22 with H-23 and the HMBC correlation of H3-26 with C-24. Lastly, the molecular composition of 1 required the existence of two hydroxyl groups which were attached at C-3 and C-25 as supported by their 2

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Fig. 2. (A) 1H–1H COSY and key HMBC correlations for 1; (B) Selected ROESY correlations for 1.

(Kinjo et al., 1992) indicative of a didehydro analogue. Comparison of the NMR data (Table 1) for 2 with those for acacic acid lactone revealed that their structures were closely related, and the only difference occurred to ring-A, where 3-OH in acacic aid lactone was oxidized to 3keto in 2. Careful examination of the 2D NMR data [see Fig. S1A, Supplementary data (SM)] confirmed the aforementioned deduction, with five proton-bearing fragments (a−e, acquired from 1H–1H COSY data) being connected by diagnostic HMBC correlations to afford an oleanane backbone for 2. The relative configuration of 2 was identified to be the same as that of acacic aid lactone at all corresponding chiral centers based on excellent NMR comparisons, which was further corroborated by analysis of ROESY data (see Fig. S1B, SM). Compound 2 was thereby characterized to be 16α-hydroxy-olean-12-en-3-on-28,21βolide. Compound 3 was assigned a molecular formula of C35H56O7 by analysis of 13C NMR and (+)-HRESIMS (m/z 589.4102 [M + Na]+, calcd 589.4099) data, indicative of an isomer of its cometabolite 3βhydroxy-28-norolean-12-en-16-one 3-O-β-D-glucopyranoside (7) (Li et al., 2016). Detailed examination of the 1H and 13C NMR data (Table 2) for 3 supported this hypothesis and also revealed a 28-noroleanane type triterpenoid saponin. The major NMR differences between the two compounds were observed to occur to signals of D and E rings, indicating different configuration(s) of the chiral center(s) in the two rings. Further inspection of 2D NMR data including 1H–1H COSY, HSQC and HMBC (see Fig. S2A, SM) confirmed that 3 possessed an identical planar structure with 7. Subsequent analysis of ROESY data (see Fig. S2B, SM) indicated that the two isolates incorporated the same configurations at all chiral centers but C-17, as supported by diagnostic correlations of H3-26/H-15β (δH 2.63), H-15β/H-18 and H3-27/H-17 for

Fig. 3. Experimental ECD spectrum of 1 (black) compared with the calculated ECD spectra of 1 (red) and its enantiomer (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

by comparing its experimental and calculated ECD spectra, the latter being acquired by using the time-dependent density functional theorybased computational method at B3LYP/6-311G(d,p) level in vacuo, where the measured ECD curve of 1 presented an excellent match with the theoretically predicted one (see Fig. 3). Compound 2 was obtained as a white amorphous powder. Its molecular formula of C30H44O4 was deduced from the (+)-HRESIMS ion peak at m/z 451.3209 ([M + H − H2O]+, calcd 451.3207) and 13C NMR data, which was two mass units less that of acacic acid lactone 3

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cometabolite eclalbasaponin A (8) (Yahara et al., 1994), suggestive of a crotonyl derivative. Further examination of the NMR data (Table 2) for 4 corroborated this hypothesis, with characteristic signals for a crotonyloxy group at δC 167.9, 146.6, 123.5 and 18.2, as well as at δH 7.01 (dq, J = 15.5, 6.9 Hz), 5.89 (dq, J = 15.5, 1.6 Hz) and 1.90 (dd, J = 6.9, 1.6 Hz). The crotonyloxy group was connected to C-6′ as indicated by the HMBC correlations from H2-6′ (δH 4.38, 4.32) to the carbonyl carbon at δC 167.9 (C-7′). Excellent comparisons of the remaining NMR data between 4 and 8, including key proton coupling constants, suggested common structural features between the two cometabolites, including configurations at all chiral centers. The assignment of β-D-glucose for the sugar unit was accomplished via the same procedure as 3 (see Fig. S57, SM). Compound 4 was hence characterized to be the 6′-O-crotonyl derivative of 8, which was further confirmed by detailed check of full 2D NMR data including 1H–1H COSY, HSQC, HMBC and ROESY spectra (see Figs. S29–S32, SM). The molecular formula of C20H32O2 for compound 5 was deduced from the (+)-HRESIMS ion peak at m/z 287.2375 ([M + H − H2O]+, calcd 287.2369), corresponding to five degrees of unsaturation. The 1H NMR data (Table 3) showed signals for a disubstituted olefin (δH 5.84, 5.61), an oxygenated methine (δH 3.81) and an oxygenated methylene (δH 3.57, 3.50) functionalities. The 13C and DEPT135 NMR data (Table 3) revealed 20 carbon resonances in agreement with three methyls, eight methylenes (one oxygenated), five methines (one oxygenated and two olefinic) and four quaternary carbons. In addition to the degree of unsaturation occupied by the double bond, the remaining four ones indicated a tetracyclic skeleton which was further confirmed to be a beyerane diterpenoid by analysis of 2D 1H–1H COSY and HMBC data (see Fig. 4A). Two hydroxyl groups were located at C-11 and C-17 as supported by the chemical shifts at δC 69.4 and δC 68.3 for the two carbons, respectively, and this assignment was further corroborated by the COSY correlations of H-11 with H-9 and H2-12, along with the HMBC correlations from H2-17 to C-12, C-13, C-14 and C-16. The

Table 2 1 H and13C NMR data of 3 (in DMSO‑d6) and 4 (in methanol-d4). No.

3

4

δC

δH (J in Hz)

δC

δH (J in Hz)

1

38.1

40.2

2

25.7

3 4 5 6

88.3 38.9 55.5 18.1

α 0.96, m β 1.49, m α 1.84, m β 1.52, m 3.04, m

α 0.95, m β 1.57, m α 1.84, m β 1.64, m 3.10, dd (11.7, 4.4)

7

32.6

8 9 10 11

38.3 46.9 36.8 23.0

12 13 14 15

117.1 142.4 42.5 43.8

16 17 18 19

214.4 48.9 36.2 42.3

20 21

30.5 38.1

22

23.3

23 24 25 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6′

27.7 16.6 15.4 16.7 25.2

7′ 8′ 9′ 10′ 1″ 2″ 3″ 4″ 5″ 6″

33.2 24.9 105.5 74.0 76.7 70.2 76.8 61.3

α β α β

1.51, 1.34, 1.31, 1.27,

m m m m

1.61, dd (10.6, 7.2) α 2.01, m β 1.83, m 5.39, dd (6.8, 3.4) α 1.86, d (15.5) β 2.63, d (15.5) 1.79, m 2.31, m α 0.90, m β 1.82, m α 1.12, β 1.37, α 1.92, β 1.22, 0.96, s 0.75, s 0.85, s 0.77, s 0.91, s 0.91, 0.84, 4.14, 2.94, 3.11, 3.02, 3.05, 3.40, 3.62,

td (13.2, 1.7) m m m

s s d (7.8) dd (9.0, 7.8) dd (9.0, 8.6) m m dd (11.7, 5.8) dd (11.7, 2.1)

27.1 91.0 40.1 57.2 19.3 34.2 40.9 48.3 37.9 24.6 123.5 144.8 42.7 36.3 74.9 50.0 42.1 47.8 31.3 36.5 31.7 28.5 17.0 16.1 17.8 27.3 177.2 33.3 25.0 106.7 75.5 78.3 72.2 75.1 64.7 167.9 123.5 146.6 18.2 95.7 74.0 78.2 71.1 78.7 62.4

0.76, m α 1.52, m β 1.35, m α 1.52, m β 1.33, m 1.62, m 1.90, m (2H) 5.32, t (3.7) α 1.36, m β 1.87, m 4.53, dd (3.3, 3.3) 2.99, dd (14.5, 4.5) α 2.29, dd (14.5, 12.8) β 1.06, m α 1.16, m β 1.90, m α 1.93, m β 1.77, m 1.04, s 0.84, s 0.95, s 0.79, s 1.37, s 0.84, 0.95, 4.29, 3.20, 3.32, 3.25, 3.47, 4.38, 4.32,

s s (7.8) dd (9.2, 7.8) m dd (9.8, 8.9) ddd (9.7, 7.2, 2.4) dd (11.7, 2.4) dd (11.7, 7.1)

5.89, 7.01, 1.90, 5.35, 3.29, 3.32, 3.33, 3.33, 3.81, 3.67,

dq (15.5, 1.6) dq (15.5, 6.9) dd (6.9, 1.6) d (8.2) m m m m m m

Table 3 1 H and13C NMR data of 5 and 6 in CDCl3. No.

3, which assigned an α-orientation for H-17 and a trans-conjunction relationship for D and E rings. The sugar moiety of 3 was determined to be β-D-glucose based on the coupling pattern of H-1′ (J = 7.8 Hz) and the comparison of HPLC analysis of the (S)-(−)-1-phenylethylamine derivatives for the hydrolyzed sugar and authentic D- and L-glucoses (see Fig. S57, SM). Compound 3 was thus identified to be the C-17 epimer of 7 and the NMR data of 7 in DMSO‑d6 were also reported for the first time (Table S4, SM). Analysis of the 13C NMR and (+)-HRESIMS (m/z 887.4765 [M + Na]+, calcd 887.4763) data for compound 4 returned a molecular formula of C46H72O15, which was 68 mass units more than that of its 4

5

6

δC

δH (J in Hz)

δC

δH (J in Hz)

1

41.8

47.2

3.17, m

2

18.9

26.8

3

42.0

α 1.91, m β 1.69, m 1.74, m (2H)

4 5 6

33.6 56.0 20.3

α β α β α β

7

38.0

8

49.7

9

61.5

10 11 12

39.2 69.4 40.1

13

50.0

14

55.2

15 16 17

138.1 132.8 68.3

18 19 20

34.0 22.1 16.5

2.02, 1.16, 1.57, 1.41, 1.36, 1.16,

m m m m m m

0.93, d (11.3) α 1.40, m β 1.61, m α 1.64, m β 1.42, m 1.11, d (8.7) 3.81, m α 2.02, m β 1.28, dd (12.1,9.0) α 1.54, d (9.9) β 1.20, d (9.9) 5.84, d (5.7) 5.61, d (5.7) a 3.57, d (10.7) b 3.50, d (10.7) 0.88, s 0.84, s 0.89, s

40.3 82.1 54.1 33.0 41.8 37.2 37.3 152.6 146.4 171.8 124.9 110.5 24.5

2.10, ddd (11.8, 9.3, 2.2) α 1.61, m β 1.19, m 2.60, td (10.8, 3.4) α 1.93, β 1.30, α 2.01, β 2.52,

m m m m

6.27, s 5.61, s a 4.88, s b 4.78, s 1.20, s

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Fig. 4. (A) 1H–1H COSY and key HMBC correlations for 5; (B) Selected ROESY correlations for 5.

Fig. 6. (A) 1H–1H COSY and key HMBC correlations for 6; (B) Selected ROESY correlations for 6.

(+)-HRESIMS analysis at m/z 273.1469 ([M + Na]+, calcd 273.1461), requiring five indices of hydrogen deficiency. The NMR data of 6 exhibited diagnostic resonances for a conjugated carboxyl functionality (δC 171.8), two 1,1-disubstituted vinyl groups (δC 152.6, 110.5 and δH 4.88, 4.78; δC 146.4, 124.9 and δH 6.27, 5.61), an oxygenated quaternary carbon (δC 82.1) and a tertiary methyl (δC 24.5 and δH 1.20). These data accounted for three out of five indices of hydrogen deficiency, thus suggesting the presence of two rings in the structure of 6. The planar structure of 6 was readily established by analysis of 1H–1H COSY and HMBC data (see Fig. 6A). The COSY cross-peaks enabled the establishment of a long coupling system from H2-3 to H-1 which extended via H-5 sequentially to H2-9 as shown in bold bonds. Then the HMBC correlations from H2-13 to C-7 (δC 41.8), C-11 (δC 146.4) and C12 (δC 171.8); H2-14 to C-1 (δC 47.2), C-9 (δC 37.3) and C-10 (δC 152.6); and H3-15 to C-3 (δC 40.3), C-4 (δC 82.1) and C-5 (δC 54.1), constructed a guaiane-type sesquiterpene skeleton for 6. Further check of ROESY data (see Fig. 6B) revealed strong interaction of H-1 with H-5, indicating that they were co-facial and were randomly assigned to be αoriented. It was followed by the assignment of α-orientation for H-3α, H-7, H-9α and Me-15 as determined by the ROSEY correlations of H-1 with H-9α (δH 2.01), H-5 with H-6α (δH 1.61), H-7 and H3-15 (δH 1.20), and H-7 with H-9α. Finally, the absolute configuration of 6 was established as shown by comparing its measured ECD spectrum with the calculated one (Fig. 7). Hyperglycemia is the most common characteristic of type 2 diabetes mellitus (Ríos et al., 2015), while targeting hyperglycemia with α-glucosidase inhibitors has proven to be beneficial to glycemic control and thus to the treatment of type 2 diabetes (Joshi et al., 2015). In our previous work, we have acquired natural products as potent α-glucosidase inhibitors from both botanical (Song et al., 2019) and microbial (He et al., 2019) origins. As a continuation of the earlier efforts for antidiabetes solutions from natural resources, the antihyperglycemic

Fig. 5. Experimental ECD spectrum of 5 (black) compared with the calculated ECD spectra of 5 (red) and its enantiomer (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

relative configuration of 5 was established by examination of ROESY data (see Fig. 4B). The ROESY correlations of H-5 with H-3 at δH 1.16, H-7 at δH 1.42 and H-9, as well as H-9 with H-1 at δH 1.16 and H-14 at δH 1.20, indicated that these protons were axially bonded in their respective rings and they were assigned to be co-facial and β-oriented. Subsequently, the interactions of H3-19 with H-2 at δH 1.57 and H-6 at δH 1.40, as well as H3-20 with the same H-2 and H-6 protons, H-11 and H-15, revealed that these protons or proton-bearing groups were positioned at the opposite side of the molecule and thus α-oriented. Accordingly, the above-mentioned observations allowed the assignment of β-orientation for 11-OH and the CH2-14 bridge groups. In order to establish the absolute configuration of 5, the theoretical ECD spectra of its two enantiomers were computed and compared with the experimentally measured one (see Fig. 5), thus leading to the assignment of (5R,8R,9S,10R,11S,13S)-configuration for 5. Compound 6 was assigned a molecular formula of C15H22O3 by 5

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et al., 2019; Yang et al., 2015; Tundis et al., 2010) reveals that compound 1 represents an undescribed type of triterpenoid inhibitor and is one of the most potent natural ones, and it thus could serve as a template compound for future anti-diabetes drug development. Giving the limited isolation yield of 1 and recent progress in the biomimetic synthesis of 6/6/6/6 tetracyclic triterpenoids (Terasawa et al., 2017; Hoshino et al., 2014), an effort to acquire more 6/6/6/6-fused triterpenoid analogues for further pharmacological investigations remains an ongoing work in our lab. 4. Experimental 4.1. General experimental procedures Optical rotations were measured on a Rudolph VI polarimeter (Rudolph Research Analytical, Hackettstown, USA) with a 10 cm length cell. ECD spectra were obtained on a Chirascan Spectrometer (Applied Photophysics Ltd., Leatherhead, UK) with a 0.1 cm pathway cell. NMR experiments were performed on a Bruker Avance DRX600 spectrometer (Bruker BioSpin AG, Fällanden, Switzerland) and referenced to solvent peaks (δC 49.00 and δH 3.31 ppm for CD3OD; δC 77.23 and δH 7.26 ppm for CDCl3; δC 39.51 and δH 2.50 ppm for DMSO‑d6). ESIMS analyses were carried out on an Agilent 1260–6460 Triple Quad LC-MS instrument (Agilent Technologies Inc., Waldbronn, Germany). HR-ESIMS data were acquired on an Agilent 6545 Q-TOF mass spectrometer (Agilent Technologies Inc., Waldbronn, Germany). All HPLC analyses were performed on an Agilent 1260 series LC instrument (Agilent Technologies Inc., Waldbronn, Germany), while all HPLC separations were performed on a Shimadzu 20 A series LC instrument (Shimadzu, Tokyo, Japan). Agilent SB-C18 column (5 μm, 9.4 × 250 mm, Agilent Technologies Inc., Santa Clara, USA) and YMC-Pack ODS-A column (5 μm, 10 × 250 mm, YMC Co. Ltd., Tokyo, Japan) were used for HPLC separations. Column chromatography (CC) was performed on D101macroporous absorption resin (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and silica gel (300–400 mesh, Qingdao Marine Chemical Co. Ltd., Qingdao, China). Pre-coated silica gel GF254 plates (Qingdao Marine Chemical Co. Ltd., Qingdao, China) were used for thin-layer chromatography (TLC) analyses. All solvents used for CC were of analytical grade (Tianjin Fuyu Fine Chemical Co. Ltd., Tianjin, China) and solvents used for HPLC were of HPLC grade (Oceanpak Alexative Chemical Ltd., Goteborg, Sweden). All solvent mixtures used for analyses and separations (HPLC & CC) were presented in the ratio of volume to volume, unless otherwise specified.

Fig. 7. Experimental ECD spectrum of 6 (black) compared with the calculated ECD spectra of 6 (red) and its enantiomer (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Table 4 α-Glucosidase inhibitory and cytotoxic activities of 1–7 (IC50 in μM). Compds No.

1 2 3 4 5 6 7 acarbose adriamycin

α-glucosidase

0.82 ± 0.18 NT 15.5 ± 2.7 NT NT NT NT 824 ± 129 –

tumor cell lines MDA-MB-231

Hela

NT NT 14.1 ± 2.2 NT NT NT 19.6 ± 1.8 – 0.58 ± 0.08

NT NT 7.5 ± 1.3 NT NT NT 18.7 ± 2.5 – 1.26 ± 0.17

NT: IC50 not tested.

activity of compounds 1–7 was evaluated in vitro by testing their αglucosidase inhibitory effects with acarbose as positive control. While the others were less active, compounds 1 and 3 showed significant inhibitory activity at the initial 100 μM screening concentration (Table S5, SM). The two most active ones were further subjected to IC50 measurements and proved to be strong α-glucosidase inhibitors (Table 4). The remarkably different biological effects between compounds 3 and 7 indicated the importance of the conjunction mode of D/ E rings to the inhibitory activity. Of particular note, the rare tetracyclic triterpenoid 1 showed very potent inhibition against α-glucosidase with an IC50 of 0.82 ± 0.18 μM, which was a thousand times more active than the positive control. Extending our biological investigations, the cytotoxicity of 1–7 toward two human female cancer cell lines MDA-MB-231 (breast) and Hela (cervical), along with the antibacterial activity against two Grampositive strains Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633, were also tested. While only compound 4 showed antibacterial activity against S. aureus with an IC50 of 37.36 μM, both 3 and 7 displayed moderate cytotoxicity against the two cell lines with IC50 values ranging from 7.5 to 19.6 μM (Table 4).

4.2. Plant material The aerial parts of Eclipta prostrata (L.) L. (Compositae) were collected in July 2016 in Mount Kunyu area, Shandong Province, China, and were authenticated by Prof. Jie Zhou from University of Jinan. A voucher specimen has been deposited at School of Biological Science and Technology, University of Jinan (Accession number: npmc-008). 4.3. Extraction and isolation The air-dried aerial parts of Eclipta prostrata (30.2 kg) were smashed into powder and then extracted with 95% EtOH at room temperature for four times (one week per time). After evaporation of solvent under reduced pressure, the residue (3.1 kg) was suspended in 5.0 L water and then partitioned with EtOAc (3 × 5.0 L). The combined EtOAc layer was evaporated under reduced pressure to give a dark material (634 g) which was then subjected to CC over D-101 macroporous resin (EtOH–H2O, 30%, 50%, 80% and 95%) to return three fractions. The 80% EtOH elution was separated on silica gel eluted with petroleum ether (PE)EtOAc (15:1 to 1:3) to give eight fractions (A–H). Fraction B was separated by RP-18 CC, eluted with MeOH–H2O (70%–100%) to obtain two

3. Discussion In summary, our chemical fractionation of the ethanol extract of E. prostrata has afforded an array of terpenoid constituents comprising six triterpenoids (1–4, 7 & 8), one diterpenoid (5) and one sesquiterpenoid (6), among which triterpenoid 1 with a rare lemmaphyllane skeleton displays particularly strong α-glucosidase inhibitory activity. A literature search of recent reviews on natural α-glucosidase inhibitors (Alam 6

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fractions (B1 and B2) which were further purified by silica gel CC eluted with CH2Cl2–MeOH (200:1 to 80:1) and semi-preparative HPLC (3.00 mL/min, 85% MeCN–H2O) to yield 1 (2.8 mg, tR = 20.5 min) and 2 (2.5 mg, tR = 11.0 min). Fraction C was separated by Sephadex LH-20 CC, eluted with CH2Cl2–MeOH (1:1), to obtain three fractions (C1–C3). Fraction C1 was then subjected to a RP-18 CC, eluted with MeOH–H2O (70%–100%) to afford four fractions (C1a−C1d). Fraction C1b was first separated by silica gel CC eluted with CH2Cl2–MeOH (200:1 to 60:1) and then purified by semi-preparative HPLC (3.00 mL/min, 60% MeCN–H2O) to obtain 6 (4.7 mg, tR = 10.0 min). Fraction D was submitted to Sephadex LH-20 CC, eluted with CH2Cl2–MeOH (1:1) to afford three fractions (D1−D3). Fraction D1 was then separated by a RP-18 CC using MeOH–H2O (60%–100%) to furnish five major fractions D1a−D1e. The fourth fraction D1d was subjected to silica gel CC eluted with PE-EtOAc (8:1 to 1:1) and then purified by semi-preparative HPLC using 55% MeCN–H2O (3.00 mL/min) as the mobile phase, to yield compound 5 (2.5 mg, tR = 16.0 min). Fraction E was chromatographed on a RP-18 CC, eluted with MeOH–H2O (60%–100%), to afford three fractions (E1−E3). E2 was subjected to a silica gel CC eluted with CH2Cl2–MeOH (150:1 to 20:1) and then purified by semi-preparative HPLC (3.00 mL/ min, 80% MeCN–H2O) to obtain 3 (6.7 mg, tR = 17.0 min) and 7 (18.3 mg, tR = 19.5 min). Fraction F was separated by silica gel CC, eluted with EtOAc-MeOH-H2O (30:1:1 to 5:1:1), to obtain six fractions (F1–F6), and the second fraction F2 was then submitted to RP-18 CC eluted by MeOH–H2O (40%–100%) to afford four fractions (F2a−F2d). F2c was fractionated on a silica gel CC eluted with CH2Cl2–MeOH–H2O (10:1:1 to 1:1:1) to obtain two fractions (F2c1 and F2c2) which were then purified with semi-preparative HPLC (3.00 mL/min, 55% MeCN–H2O) to afford compounds 4 (4.0 mg, tR = 17.5 min) and 8 (12.5 mg, tR = 9.0 min), respectively.

4.4. Determination of D-glucose for 3 and 4

4.3.1. 3β,25-dihydroxy-23E-lemmaphyll-8,23-diene (1) White solid; [α]D24 6.8 (c 0.28, CHCl3); 1H and 13C NMR data (CDCl3) see Table 1; ECD (c 0.04, MeOH) λ (Δε) 202 (−4.6) nm; (+)-ESIMS: m/z 425.1 [M + H − H2O]+; (+)-HRESIMS: m/z 425.3794 [M + H − H2O]+ (calcd for C30H49O, 425.3778).

4.5.2. Cytotoxic assay The cytotoxicity toward two human female cancer cell lines, MDAMB-231 (breast) and Hela (cervical), was screened by the MTT method as reported formerly (He et al., 2017). A single concentration of 30 μM was chosen for the primary screening, and only compounds showing > 50% growth inhibitory activity were further tested for their IC50 values. Adriamycin was used as the positive control.

Compound 3 (1.5 mg) was dissolved in 3.0 mL 1.0 mol/L HCl and stirred at 90 °C for 4 h. Following removal of excessive HCl under reduced pressure, the residual aqueous mixture was filtered to remove the aglycone part and the monosaccharide was obtained by evaporating the water in vacuo. The acquired glucose, (S)-(−)-1-phenylethylamine and NaBH3CN were dissolved in ethanol and then stirred at 40 °C for 3 h with a few drops of acetic acid. After evaporation of the solvent, the residue and acetic anhydride were stirred in pyridine at room temperature for 5 h to afford the amino derivative. Following the same procedure, the amino derivatives of authentic D- and L-glucoses were obtained. These derivatives were subjected to HPLC analysis on an Agilent ZORBAX SB-C18 column (5 μm, 4.6 × 250 mm, detection at 210 nm) with 50% MeOH–H2O (v/v) as mobile phase at 1.00 mL/min. As shown in Fig. S57 (SM), the retention times of authentic D- and Lglucose derivatives were observed at tR 22.927 and 20.762 min, respectively, while those of 3 and 4 were observed at tR 23.103 and 23.029 min, respectively, thus assigning the D-configuration for both sugar moieties. 4.5. Bioassays 4.5.1. α-Glucosidase inhibitory assay The α-glucosidase inhibitory activity was examined by a previously described method (Omar et al., 2012) with slight modification as reported in our recent article (Sun et al., 2019a, 2019b). All the isolates were first tested at a primary concentration of 100 μM and only those with > 50% inhibition ratio were further sent for IC50 measurements. Acarbose was as used as the control drug.

4.3.2. 16α-hydroxy-olean-12-en-3-on-28,21β-olide (2) White solid; [α]D24 14.1 (c 0.28, CHCl3); 1H and 13C NMR data (CDCl3) see Table 1; (+)-ESIMS: m/z 491.0 [M + Na]+; (+)-HRESIMS: m/z 451.3209 [M + H − H2O]+ (calcd for C30H43O3, 451.3207).

4.5.3. Antibacterial assay The antibacterial activity against two Gram-positive trains, Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633, was tested using liquid growth inhibition method as we described previously (Bao et al., 2018). The initial testing concentration was set at 50 μM, and only compounds with > 50% growth inhibition rate were further subjected for IC50 measurements. Cephalosporin was used as the positive control.

4.3.3. 3β-hydroxy-17-epi-28-norolean-12-en-16-one 3-O-β-D-glucopyranoside (3) White solid; [α]D24 20.9 (c 0.10, CHCl3–CH3OH 1:1); 1H and 13C NMR data (DMSO‑d6) see Table 2; (−)-ESIMS: m/z 623.6 [M + Cl]−; (+)-HRESIMS: m/z 589.4102 [M + H]+ (calcd for C35H57O7, 589.4099). 4.3.4. 3β-O-(6-O-crotonyl-β-D-glucopyranosyl)-16α-hydroxy-olean-12-en28-oic acid 28-O-β-D-glucopyranosyl ester (4) White solid; [α]D24 −4.4 (c 0.50, CH3OH); 1H and 13C NMR data (CD3OD) see Table 2; (+)-ESIMS: m/z 887.6 [M + Na]+; (+)-HRESIMS: m/z 887.4765 [M + Na]+ (calcd for C46H72O15Na, 887.4763).

Declaration of competing interest

4.3.5. 11β,17-dihydroxy-beyer-15-ene (5) White solid; [α]D24 17.2 (c 0.22, CHCl3); 1H and 13C NMR data (CDCl3) see Table 3; ECD (c 0.03, MeOH) λ (Δε) 192 (−10.8) nm; (+)-ESIMS: m/z 287.2 [M + H − H2O]+; (+)-HRESIMS: m/z 287.2375 [M + H − H2O]+ (calcd for C20H31O, 287.2369).

This project was financially supported by Natural Science Foundation of Shandong Province [No. JQ201721], the Young Taishan Scholars Program [No. tsqn20161037], Innovation Team Project of Jinan Science & Technology Bureau (No. 2018GXRC003) and Shandong Talents Team Cultivation Plan of University Preponderant Discipline [No. 10027].

The authors declare no conflict of interest. Acknowledgements

4.3.6. 4β-hydroxy-guai-10(14),11(13)-dien-12-oic acid (6) White solid; [a]D24 −23.3 (c 0.43, CHCl3); 1H and 13C NMR data (CDCl3) see Table 3; ECD (c 0.03, MeOH) λ (Δε) 198 (−10.3), 214 (3.0) nm; (+)-ESIMS: m/z 273.1 [M + Na]+; (+)-HRESIMS: m/z 273.1469 [M + Na]+ (calcd for C15H22O3Na, 273.1461).

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.112192. 7

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