Cytotoxic Cardiac Glycoside Constituents of Vallaris glabra Leaves

J. Nat. Prod. , 2017, 80 (11), pp 2987–2996. DOI: 10.1021/acs.jnatprod.7b00554. Publication Date (Web): October 26, 2017. Copyright © 2017 The Amer...
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Cytotoxic Cardiac Glycoside Constituents of Vallaris glabra Leaves Sudarat Kruakaew,† Chonticha Seeka,† Thitima Lhinhatrakool,‡ Sanit Thongnest,§ Jantana Yahuafai,⊥ Suratsawadee Piyaviriyakul,⊥ Pongpun Siripong,⊥ and Somyote Sutthivaiyakit*,† †

Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Ramkhamhaeng University, Hua Mark, Bangkapi, Bangkok 10240, Thailand ‡ College of Oriental Medicine, Rangsit University, Muang Ake, Pathumthani 12000, Thailand § Chulabhorn Research Institute, Kamphang Phet 6 Road, Bangkok 10210, Thailand ⊥ Natural Products and Integrative Medicine Research Section, Research Division, National Cancer Institute, Rama VI Road, Bangkok 10400, Thailand S Supporting Information *

ABSTRACT: Thirteen cardenolide glycosides (1−13) were isolated from the CH2Cl2 and MeOH extracts of Vallaris glabra leaves. The structures of the new compounds (2−13) were identified by spectroscopic methods, with the absolute configurations of the sugar moieties determined by acid hydrolysis. All compounds were evaluated for their cytotoxic activity against human cervix adenocarcinoma, lung carcinoma, and colorectal adenocarcinoma cell lines. The two most potent compounds [2′-Oacetylacoschimperoside P (1) and oleandrigenin-3-O-α-L-2′-O-acetylvallaropyranoside (2)] exhibited IC50 values in the range of 0.03−0.07 μM.

schimperoside P (1),3 together with three cardenolide glycosides (2−4), while nine cardenolide glycosides (5−13) were isolated from the MeOH extract. We report herein the spectroscopic identification of these cardenolides (2−13) together with the 1H and 13C NMR spectroscopic data of 1 in MeOH-d4 and their cytotoxic activities against three cancer cell lines.

Vallaris glabra (L.) Kuntze (Apocynaceae), commonly known as bread flower and named “com-ma-nat” in Thailand, is a shrub growing up to 8 m in height.1 Its latex was reported to possess wound-healing and strong laxative properties and can cause vomiting, stimulate striated muscle, increase blood pressure, and induce uterine contractions.2 A previous study on this species demonstrated the presence of two cardenolide glycosides, 2′-O-acetylacoschimperoside P (1) and 1-acetoxydigitoxigenin-3-O-α-D-2′-O-acetylacofrioside, with hedgehog signaling inhibitory and cytotoxic activities.3 Subsequently, the isolation of ursolic acid, stearic acid, and caffeoyl esters of quinic acid from the leaves was reported.4 Several monoterpenoids and 2-acetyl-1-pyrroline were also found to occur in the flowers.5 In the present work, a TLC investigation, using Kedde’s reagent as a staining reagent, revealed the presence of several cardenolides in the CH2Cl2 and MeOH extracts of the fresh leaves of V. glabra. The CH2Cl2 extract showed cytotoxic activity against human cervix adenocarcinoma (HeLaS3), human lung carcinoma (A549), and human colorectal adenocarcinoma (HT-29) cells with IC50 values of 1.3 ± 0.1, 1.6 ± 0.1, and 1.9 ± 0.1 μg/mL, respectively. The MeOH extract was inactive against these three cell lines (IC50 > 20 μg/ mL). Thorough chromatographic purification of the CH2Cl2 extract led to the isolation of 10 known compounds, 3β,28dihydroxyurs-12-ene (uvaol),6 3β-hydroxyolean-12-en-28-oic acid,7 3β-hydroxyurs-12-en-28-oic acid,7 3β,27-dihydroxyurs12-en-28-oic acid,8 cycloeucalenol,9 lupeol,7 stigmast-4-en-3one, β-sitosterol, β-sitosteryl glucoside, and 2′-O-acetylaco© 2017 American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION Compound 2 was obtained as a colorless solid, mp 172−173 °C, with a molecular formula of C34H50O11 based on its HRESIMS [M + Na]+ ion at m/z 657.3263 (calcd for C34H50O11Na, 657.3353). The IR absorption bands at νmax 1732 (CO stretch) and 1650 cm−1(CC stretch), the 1H NMR resonances at δH 4.99 and 4.96 (both as dd, J = 18.5 and 1.7 Hz, H2-21) and δH 5.97 (t, J = 1.7 Hz, H-22), and 13C NMR resonances at δC 175.4, 170.9, 120.3, and 76.7 indicated the presence of an α,β-unsaturated-γ-lactone moiety in 2 (Tables 3 and 4). The cardenolide aglycone was characterized as oleandrigenin based on the 1H and 13C NMR spectroscopic data, particularly the 1H NMR resonances at δH 3.90 (brs, W1/2 = 8.0 Hz, H-3α),10 2.07 (s, OCOCH3-2′), 1.93 (s, OCOCH316), 5.46 (ddd, J = 9.5, 8.7, and 2.0 Hz, H-16), and 3.25 (d, J = 8.7 Hz, H-17) as found in 2′-O-acetylacoschimperoside P Received: June 29, 2017 Published: October 26, 2017 2987

DOI: 10.1021/acs.jnatprod.7b00554 J. Nat. Prod. 2017, 80, 2987−2996

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3′′), 71.7 (C-4′′), 77.8 (C-5′′), and 62.9 (C-6′′)]. The key HMBC cross-peak between H-1′′/C-4′ revealed the connectivity of C-1′′ to C-4′-O. Upon acid hydrolysis of 3 with 5% HCl at 70 °C for 6 h and subsequent chromatographic purification, the aglycone 14,16-dianhydrogitoxigenin,11 together with L-acofriose (3-O-methyl-L-rhamnose)12 and D-glucose,13 was obtained. On the basis of its NMR spectroscopic data and the acid hydrolysis procedure, compound 3 was thus elucidated as oleandrigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-2′-O-acetylacofriopyranoside. Compound 4 was isolated as a colorless solid with mp 183− 184 °C. The high-resolution ESIMS suggested a molecular formula of C40H60O16, as indicated from the [M + Na]+ ion at m/z 819.3793 (calcd for C40H60O16Na, 819.3773). Most of the 1 H and 13C resonances were similar to those of compound 3 (Tables 1−4), except for the data of H-3′ (δH 3.73, dd, J = 5.6 and 3.3 Hz) and H-4′ (δH 3.90, dd, J = 6.7 and 3.3 Hz) and a deshielded quintet at δH 4.28 (J = 6.7 Hz) of H-5′. This indicated a β-oriented OCH3-3′, thus establishing the presence of a vallaropyranosyl moiety. A glucopyranosyl unit was detected from 1H and 13C NMR resonances particularly at δH 4.39 (d, J = 7.7 Hz, H-1″) and δC 103.0 (C-1″) and of an oxymethylene group at δH 3.86 and 3.65 and δC 62.8. The key HMBC cross-peak between H-1″/C-4′ indicated a connectivity of C-1′′ to C-4′-O. Based on the acid hydrolysis of 2 and 3, which furnished L-vallarose, and L-acofriose and D-glucose, respectively, compound 4 was therefore assigned as oleandrigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-2′-O-acetylvallaropyranoside. Compound 5 was isolated as a colorless, amorphous solid with a molecular formula of C38H56O14, as indicated from the HRESIMS {m/z 759.3580 [M + Na]+ (calcd for C38H56O14Na 759.3562)}. Patterns of the 1H and 13C NMR resonances of 5 resembled those of 3 (Tables 1, 2, and 5) except that the 1H NMR resonances of H-16 at ca. δH 5.46 and a singlet at ca. δH 1.93 (CH3CO-16) as encountered in 3 were replaced by resonances at δH 6.25 (brs, H-16), together with a doublet at δH 2.77 and a doublet of doublets at δH 2.31 of H2-15, indicating the cardenolide aglycone as 16-anhydrogitoxigenin.14 Based on the 1H−1H COSY, HSQC, and HMBC data and the acid hydrolysis of 3, which provided L-acofriose and D-glucose, the structure of compound 5 was thus proposed as 16anhydrogitoxigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-2′-Oacetylacofriopyranoside. Compound 6, a colorless, amorphous solid with a molecular formula of C36H54O13, exhibited a molecular mass 42 amu lower than that of 5 {HRESIMS [M + Na]+ ion at m/z 717.3462 (calcd for C36H54NaO13, 717.3447)}. The 1H NMR and 1H−1H COSY spectra revealed the absence a singlet of an O-acetyl group at ca. δH 2.09, and a resonance of H-2′ at ca. δH 5.19 (dd, J = 3.3 and 1.6 Hz), as found in 5, was replaced by a shielded resonance of H-2′ at δH 4.00 (dd, J = 3.1 and 1.8 Hz), thus indicating the deoxy sugar as an acofriose instead of 2′-Oacetylacofriose. The remainder of the 1H and 13C NMR resonances, particularly of the aglycone, resembled those of 5 (Table 5). On the basis of acid hydrolysis of 3, which gave Dglucose and L-acofriose, the structure of 6 was proposed as 16anhydrogitoxigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-acofriopyranoside. Compound 7 was obtained as an amophous solid showing the same molecular mass as 6. The 1H and 13C NMR spectra exhibited similar resonances to those of 6 (Table 5), except that the resonances of an acofriopyranosyl moiety were replaced by

(oleandrigenin-3-O-α-L-2′-O-acetylacofriopyranoside, 1),3 previously isolated from this plant and also in the present study. The 1H NMR resonances of the deoxy sugar unit and its coupling constants [δH 4.70 (brs, H-1′), 5.08 (dd, J1′,2′ = 1.8 Hz, J2′,3′ = 3.5 Hz, H-2′), 3.49 (t, J2′,3′ = J3′,4′ = 3.5 Hz, H-3′), 3.45 (dd, J3′,4′ = 3.5 Hz, J4′,5′ = 8.7 Hz, H-4′), 4.08 (dq, J4′,5′ = 8.7 Hz, J5′,6′ = 6.4 Hz, H-5′), and 1.20 (d, J5′,6′ = 6.4 Hz, H3-6′)] were slightly different from those of the 2′-O-acetylacofriopyranosyl group [δH 3.45 (dd, J2′,3′ = 3.4 Hz, J3′,4′ = 9.5 Hz, H-3′), 3.35 (t, J3′,4′ = J4′,5′ = 9.5 Hz, H-4′)] in 1 (Tables 1 and 3), revealed the orientation of the OCH3-3′ as β, and thus established the sugar moiety as a 2′-O-acetylvallaropyranosyl group. In addition, the ROESY spectrum exhibited cross-peaks between OCH3-3′/H-5′, H-3/H-1′, H-16/H-17, and H3-18/H22. The absolute configuration of the sugar was assigned after acid hydrolysis of 2 with 5% HCl at 70 °C for 6 h to give the aglycone 14,16-dianhydrogitoxigenin [3-hydroxycarda14,16,20(22)-trienolide]11 and L-vallarose (3-O-methyl-6-des12 oxy-L-altrose) showing [α]26 [α]23 D −38.9 (c 0.04, H2O), lit. D −17.2 (c 0.90, H2O). The structure of compound 2 was thus elucidated as oleandrigenin-3-O-α-L-2′-O-acetylvallaropyranoside. This compound was obtained from vallarosolanoside, previously reported in V. solanacea,12 after acetylation using acetic anhydride in pyridine; however, there were no NMR spectroscopic data reported for this derivative. Compound 3, a colorless solid with mp 179−181 °C, was assigned a molecular formula of C40H60O16 as deduced from the HRESIMS, which gave a [M + Na]+ ion at m/z 819.3796 (calcd for C40H60O16Na, 819.3773). The 1H and 13C NMR spectra showed similar resonance patterns to those found for 1 (Tables 1 and 2), with additional resonances of a glucopyranosyl ring [(δH 4.57, d, J = 7.8 Hz, H-1″), 3.15 (dd, J = 9.1 and 7.8 Hz, H-2″), 3.35 (H-3′′), 3.29 (H-4′′), 3.24 (H-5′′), and 3.86 (dd, J = 11.9 and 2.1 Hz, H-6a″), and 3.67 (dd, J = 11.9 and 5.3 Hz, H-6b″), δC 104.9 (C-1″), 75.6 (C-2′′), 77.8 (C2988

DOI: 10.1021/acs.jnatprod.7b00554 J. Nat. Prod. 2017, 80, 2987−2996

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Table 1. 1H NMR Spectroscopic Data (400 MHz) of 1, 3, 8, 12, and 13 (in MeOH-d4) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 1′ 2′ 3′ 4′ 5′ 6′ OCOCH3-16 OCOCH3-2′ OCH3-3′ 1″ 2″ 3″ 4″ 5″ 6″

1

3

8

12

13

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

1.91, 1.43 1.73, 1.59 3.96 (w1/2 = 9.0) 1.64, 1.48 1.69 1.88, 1.29 1.82, 1.25 1.62 1.68

1.88, 1.32 1.62a 3.95 brs (w1/2 = 9.0) 1.66, 1.46 1.67 1.29 1.23 1.62a 1.69

1.44, 1.56 191, 1.31 3.97 (w1/2 = 8.0) 1.89, 1.50 1.70 1.65, 1.60 1.86, 1.24 1.68 1.75

1.79, 1.48 1.91, 1.27 3.97d 1.87, 1.28 1.67 1.57 1.81 1.62 1.73

1.59, 1.25 1.59, 1.25 3.95 (w1/2 = 9.0) 1.88 1.65 1.56 1.87, 1.23 1.62 1.69

1.44 1.55, 1.41

1.44 1.54, 1.43

1.48, 1.29 1.57, 1.46

1.41, 1.23 1.44

1.79, 1.44 1.54, 1.41

2.78 dd (15.6, 9.6), 1.79 dd (15.6, 2.4) 5.46 ddd (9.6, 8.7, 2.4) 3.25 d (8.7) 0.93 s 0.97 s

2.78 brd (15.6, 9.6), 1.79 dd (15.6, 2.4) 5.46 ddd (11.2, 9.4, 2.3) 3.26b 0.93 s 0.96 s

2.81 dd (15.5, 9.7), 1.83 dd (15.5, 2.4) 5.49 dt (9.7, 2.4) 3.30 d (9.7)i 0.96 s 0.98 s

1.82 dd (15.4, 2.3), 2.81 dd (15.4, 9.6) 5.50 ddd (9.6, 7.3, 2.3) 3.30i 0.96 s 0.98 s

1.79 dd (15.6, 2.2), 2.75 dd (15.6, 9.5) 5.47 dt (9.5, 2.2) 3.26 d (9.5) 0.93 s 0.96 s

4.99 dd (18.2, 1.8), 4.96 dd (18.2, 1.8) 5.97

4.97 dd (18.2, 1.8), 4.9j

5.04 dd (18.5, 1.7), 5.00 dd (18.5, 1.7) 6.00 t (1.7)

5.04 dd (18.6, 1.8), 4.9 dd (18.6, 1.8) 6.00 s

5.01 dd (18.6, 1.7), 4.94 dd (18.6, 1.7) 5.97 s

4.80 5.17 3.45 3.35 3.70 1.24 1.93 2.07 3.39

4.81 d (1.7) 5.19 dd (3.3, 1.7) 3.73 dd (9.4, 3.3) 3.61 t (9.4) 3.76 dq (9.4, 6.2) 1.30 d (6.2) 1.93 s 2.08 s 3.38 s 4.57 d (7.8) 3.15 dd (9.1, 7.8) 3.35 3.29 3.24b 3.86 dd (11.9, 2.1), 3.67 dd (11.9, 5.3)

4.83 3.99 3.62 3.72 3.74 1.31 1.96

4.83 d (1.8) 3.99 dd (3.0, 1.8)d 3.65 dd (8.9, 3.0) 3.75e 3.76 quint (5.6)e 1.28 d (5.6) 1.96 s

d (1.7) dd (3.4, 1.7) dd (9.5, 3.4) t (9.5) dq (9.5, 6.2) d (6.2) s s s

5.97 dd (1.8, 1.48)

d (1.7) dd (3.1, 1.7) dd (8.8, 3.1) t (9.6, 8.8)c dq (9.6, 5.5)c d (5.5) s

3.46 s 4.61 d (7.7) 3.19 dd (8.7, 7.7) 3.39 t (8.7) 3.31 t (8.7) 3.25 ddd (8.7, 5.5, 2.2) 3.87 dd (12.1, 2.2), 3.69 dd (12.1, 5.5)

1‴ 2‴ 3‴ 4‴ 5‴ 6‴ a−i

4.80 d (1.7) 5.18 dd (3.8, 1.7) 3.73 dd (9.3, 3.8)g 3.62 t (9.3)h 3.76 dq (6.1, 9.2)g 1.30 d (6.1) 1.93 s 2.11 s 3.46 s 3.39 s 4.62 d (7.7) 4.58 d (7.8) 3.16 dd (8.7, 7.7) 3.16 dd (8.4, 7.8) 3.36 t (8.7) 3.36i 3.30 t (8.7) 3.32 dd (9.3, 7.1)i 3.42 ddd (8.7, 5.9, 2.1) 3.42 ddd (9.3, 7.1, 2.0) 4.16 dd (11.7, 2.1), 3.78 dd 4.14 dd (11.7, 2.0), 3.78f (11.7, 5.9) 4.40 d (7.8) 4.37 d (7.8) 3.19 dd (8.6, 7.8) 3.28 dd (9.4, 7.8) 3.36 t (8.6) 3.36 t (9.4) 3.27 t (8.6)f 3.28 t (9.4) 3.26 ddd (10, 5.4, 2.2)f 3.27 dt (9.4, 4.0) 3.88 brd (11.9), 3.69 dd 3.86 dd (12.0, 1.9), 3.66 dd (11.9, 5.4) (12.0, 4.0)h

Overlapped signals. jObscured by solvent signal.

those of a vallaropyranosyl moiety [δH 4.65 (d, J = 3.1 Hz, H1′), 3.92 (dd, J = 6.9 and 3.1 Hz, H-2′), 3.63 (dd, J = 6.9 and 3.1 Hz, H-3′), 3.95 (dd, J = 6.9 and 3.1 Hz, H-4′), 4.21 (dq, J = 6.9 and 6.6 Hz, H-5′), and 1.23 (d, J = 6.6 Hz, H-6′)]. A glucopyranosyl moiety was deduced from the 1H and 13C NMR resonances at δH 4.40 (d, J = 7.7 Hz, H-1″) and δC 102.6 (C1″) and an oxymethylene group at δH 3.85 and 3.66 and δC 62.8. The key HMBC cross-peak between H-1″/C-4′ indicated the connectivity of C-1′′ to C-4′-O. Based on the acid hydrolyses of 2 and 3, which provided L-vallarose, and L-

acofriose and D-glucose, respectively, the structure of 7 was thus assigned as 16-anhydrogitoxigenin-3-O-β-D-glucopyranosyl(1→4)-α-L-vallaropyranoside. Compound 8 was proposed with a molecular formula of C38H58O15, based on its HRESIMS. Comparison of its NMR spectroscopic data with those of 6 (Tables 1, 2, and 5) revealed the 1H and 13C resonances of sugar moieties to have almost identical patterns, while the resonances of the aglycone showed the corresponding resonances of OCOCH3-16 (δH 1.96), H-16 (δH 5.49, dt, J = 9.7 and 2.4 Hz), and H-17 (δH 3.30, d, J = 9.7 2989

DOI: 10.1021/acs.jnatprod.7b00554 J. Nat. Prod. 2017, 80, 2987−2996

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Table 2. 13C NMR Spectroscopic Data (100 MHz) of 1, 3, 8, 12, and 13 (in MeOH-d4) 1

3

8

12

13

position

δC, type

δC, type

δC, type

δC, type

δC, type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 1′ 2′ 3′ 4′ 5′ 6′ OCOCH3-16 OCOCH3-2′ OCH3-3′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴

29.4, CH2 25.9, CH2 73.1, CH 30.2, CH2 36.8, CH 26.3, CH2 20.8, CH2 41.4, CH 35.3, CH 34.9, C 20.6, CH2 38.6, CH2 50.0, C 83.6, C 39.9 CH2 74.6 CH 56.1, CH 15.0, CH3 22.9, CH3 170.8, C 76.2, CH2 120.4, CH 175.4, C 96.0, CH 69.0, CH 79.3, CH 71.8, CH 68.7, CH 16.6, CH3 170.6, C; 19.5, CH3 170.3, C; 19.4, CH3 56.7, CH3

31.6, CH2 27.3, CH2 74.6, CH 30.9, CH2 38.6, CH 27.7, CH2 22.2, CH2 42.7, CH 36.6, CH 36.3, C 22.0, CH2 39.9, CH2 51.4, C 85.0, C 41.3, CH2 76.0, CH 57.4, CH 16.4, CH3 24.4, CH3 171.6, C 77.6, CH2 121.7, CH 176.8, C 97.3, CH 70.1, CH 81.0, CH 79.2 CH 68.8, CH 18.2, CH3 172.1,a C; 20.9, CH3 172.2,a C; 20.8, CH3 57.7, CH3 104.9, CH 75.6, CH 77.8, CHb 71.7, CH 77.8, CHb 62.9, CH2

31.6, CH2 27.7, CH2 74.0, CH 30.9, CH2 38.1, CH 27.4, CH2 22.2, CH2 42.7, CH 36.6, CH 36.3, C 22.0, CH2 39.9, CH2 51.4, C 84.9, C 41.3, CH2 76.0, CH 57.4, CH 16.4, CH3 24.3, CH3 171.6, C 77.5, CH2 121.7, CH 176.7, C 99.8, CH 68.5, CH 82.6, CH 79.3, CH 68.8, CH 18.2, CH3 21.0, CH3, 172.2, C

31.6, CH2 27.7, CH2 74.0, CH 30.9,CH2 38. 2, CH 27.4, CH2 22.2, CH2 42.7, CH 39.7, CH 36.3, C 22.0, CH2 39.9 CH2 51.4, C 85.0, C 41.4, CH2 76.0, CH 57.4, CH 16.4, CH3 24.3, CH3 171.6, C 77.6, CH2 121.7, CH 176.8, C 99.8, CH 68.6, CH 82.7, CH 79.0, CH 68.8, CH 18.4, CH3 172.2, C, 20.9, CH3

56.9, CH3 105.0, CH 75.8, CH 77.8, CH 71.7, CH 77.9, CH 62.9, CH2

56.9, CH3 104.9, CH 75.7, CH 77.9, CH 71.6, CH 77.0, CH 70.5, CH2 105.0, CH 75.2, CH 78.0, CH 71.9, CH 77.9, CH 62.8, CH2

30.9, CH2 27.3, CH2 74.7, CH 31.6, CH2 38.2, CH 27.6, CH2 22.2, CH2 42.7, CH 36.7, CH 36.3, C 22.0, CH2 40.0, CH2 51.4, C 84.9, C 41.4, CH2 76.0, CH 57.5, CH 16.4, CH3 24.3, CH3 171.6, C 77.6, CH2 121.8, CH 176.8, C 97.3, CH 70.2, CH 81.0, CH 79.3, CH 68.8, CH 18.4, CH3 172.3, C; 20.9, CH3 172.2, C; 20.9, CH3 57.7, CH3 104.9, CH 75.6, CH 78.0, CH 71.8, CH 77.0, CH 70.5, CH2 105.1, CH 75.2, CH 78.0, CH 71.6, CH 77.8, CH 62.2, CH2

a,b

Overlapped signals.

field than H-5′ of 8. Considering the 1H NMR resonances and J values of H-1′, H-2′, H-3′, H-4′, and H-6′, which were detected at δH 4.68 (d, J = 3.1 Hz), 3.95 (dd, J = 5.4 and 3.1 Hz), 3.65 (dd, J = 5.4 and 3.3 Hz), 3.98 (dd, J = 6.7 and 3.3 Hz), and 1.29 (d, J = 6.7 Hz), respectively, it could be deduced that one of the sugar moieties is a α-vallaropyranosyl unit. Characteristic resonances of a β-glucopyranosyl group were also present. The HMBC cross-peaks between H-3/C-1′ and H-1″/C-4′ indicated connectivities between the oxygen atom at C-3 to C1′, and C-4′-O to C-1″. On the basis of acid hydrolyses of 2 and 3, which provided L-vallarose, and L-acofriose and D-glucose, respectively, compound 9 could thus be proposed as

Hz), as in the oleandrigenin unit of 1−3. On the basis of acid hydrolysis of 3, which provided L-acofriose and D-glucose, and further perusal of the 1H,1H−COSY, HSQC, HMBC, and 1DTOCSY data, the structure of compound 8 was elucidated as oleandrigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-O-acofriopyranoside. Compound 9 was isolated as a colorless, amorphous solid, having the same molecular formula, C38H58O15, as that of 8 based on the HRESIMS. The 1H and 13C NMR spectra (Tables 3 and 4) showed characteristic resonances of an oleandrigenin aglycone, with two sugar moieties quite similar to those of 8. The 1H NMR resonance of H-5′ was notably detected as a wellseparated quintet at δH 4.24 (J = 6.7 Hz), at somewhat lower 2990

DOI: 10.1021/acs.jnatprod.7b00554 J. Nat. Prod. 2017, 80, 2987−2996

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Table 3. 1H NMR Spectroscopic Data (400 MHz) of 2, 4, 9, 10, and 11 (in MeOH-d4) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 1′ 2′ 3′ 4′ 5′ 6′ OCOCH3-16 OCOCH3-2′ OCH3-3′ 1″ 2″ 3″ 4″ 5″ 6″

2

4

9

10

11

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

1.73, 1.27 1.58 3.90 (w1/2 = 8.0) 1.89. 1.49 1.63 1.87, 1.25 1.82, 1.18 1.51 1.59

1.81 1.57 3.91a 1.49 1.65 1.87, 1.24 1.44 1.61 1.67

1.89 1.93, 1.29 4.00b 1.80, 1.53 1.75 1.64 1.85, 1.26 1.67 1.72

1.31 1.94, 1.29 4.00c 1.86, 1.53 1.79 1.63 1.82 1.56 1.66

1.86, 1.57 1.90, 1.27 3.92e 1.51 1.75 1.60 1.80 1.63 1.69

1.51 1.45, 1.33

1.77, 1.18 1.52, 1.42

1.49, 1.27 1.60, 1.46

1.46, 1.59, 1.39

1.48, 1.24 1.54, 1.44

2.77 dd (15.4, 9.5), 1.79 dd (15.4, 2.0) 5.46 ddd (9.5, 8.7, 2.0) 3.25 d (8.7) 0.93 s 0.95 s

2.77 dd (15.7, 9.6), 1.78 dd (15.7, 2.1) 5.46 ddd (9.6, 8.2, 2.1) 3.26 d (8.2) 0.93 s 0.94 s

2.88 dd (15.6, 9.6), 1.82 dd (15.6, 2.2) 5.49 dt (9.6, 2.2) 3.30 d (9.6)e 0.96 s 0.98 s

2.64 dd (14.9, 8.4), 1.72 dd (14.9, 2.2), 4.65 dt (8.2, 2.1) 3.15 d (8.2)e 0.94 s 0.97 s

1.79 dd (15.6, 2.4), 2.78 dd (15.6, 9.4) 5.46 dt (9.4, 2.4) 3.26 d (9.4) 0.91 s 0.93 s

4.99 dd (18.5, 1.7), 4.96 dd 5.01 dd (18.5, 1.7), 4.94 dd 5.09 dd (18.5, 1.7), 4.97 dd 5.19 dd (1.76, 18.6), 5.13 (18.5, 1.7) (18.5, 1.7) (18.5, 1.7) dd (1.6, 18.5) 5.97 t (1.7) 5.97 brs 6.00 s 5.96 brs

5.01 dd (18.5, 1.7), 4.94 dd (18.5, 1.7) 5.97 s

4.70 5.08 3.49 3.45 4.08 1.20 1.93 2.07 3.48

4.66 3.93 3.62 3.81 4.20 1.24 1.93

brs dd (3.5, 1.8) t (3.5) dd (8.7, 3.5) dq (8.7, 6.4) d (6.4) s s s

4.73 d (3.3) 5.11 dd (5.6, 3.3) 3.73 dd (5.6, 3.3) 3.90 dd (6.7, 3.3)a 4.28 quint (6.7) 1.25 d (6.7) 1.93 s 2.07 s 3.47 s 4.39 d (7.7) 3.21 dd (9.0, 7.7) 3.36 3.27b 3.27b 3.86 d (11.8), 3.65 dd (11.8, 5.4)

4.68 3.95 3.65 3.98 4.24 1.29 1.96

d (3.1) dd (5.4, 3.1) dd (5.4, 3.3) dd (6.7, 3.3)b quint (6.7) d (6.7) s

3.51 s 4.43 d (7.7) 3.24 dd (9.4, 7.7) 3.39 t (9.4) 3.32 t (9.4) 3.29 ddd (9.4, 5.4, 1.9) 3.88 dd (12.1, 1.9), 3.69 dd (12.1, 5.4)

1‴ 2‴ 3‴ 4‴ 5‴ 6‴ a−f

4.68 3.95 3.65 3.97 4.24 1.26

d (3.1) dd (5.4, 3.1) dd (5.4, 3.2) dd (6.7, 3.2)c quint (6.7) d (6.7)

3.51 s 4.43 d (7.7) 3.21 dd (9.2, 7.7) 3.37 t (9.2) 3.32 dd (9.6, 9.2)d,g 3.28 ddd (9.6, 4.2, 1.2)d 3.88 dd (12.0, 1.2), 3.69 dd (12.0, 4.2)

d (2.9) dd (5.2, dd (5.2, dd (7.0, dq (7.0, d (6.6) s

2.9)e 3.3) 3.3) 6.6)

3.47 s 4.39 d (7.4) 3.22 dd (8.8, 7.4) 3.36 t (8.8) 3.31 t (8.8) 3.46.ddd (8.8, 6.1, 2.1) 4.14 dd (11.7, 2.1), 3.77 dd (11.7, 6.1) 4.39 d (7.6) 3.19 dd (8.7, 7.6) 3.37 t (8.7) 3.28 obs dd (8.7, 7.3)f 3.24f 3.86 dd (11.6, 1.9) 3.66 dd (11.6, 5.2)

Overlapped signals. gObscured by solvent signal.

(gitoxigenin). The structure of compound 10 was thus elucidated to be gitoxigenin-3-O-β-D-glucopyranosyl-(1→4)-αL-vallaropyranoside. Compound 11 was isolated as an amorphous solid with a molecular formula of C44H68O20 based on the HRESIMS. The 1 H and 13C NMR spectra showed some complex sets of resonances, however, with recognizable resonances of the aglycone oleandrigenin and three dioxygenated methine groups being evident at δH 4.66 (d, J = 2.9 Hz), 4.39 (d, J = 7.4 Hz), and 4.39 (d, J = 7.6 Hz) and δC 100.3, 102.4, and 105.0 (Tables

oleandrigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-vallaropyranoside. Compound 10 was isolated as an amorphous solid with a molecular formula of C36H56O14 as detected by HRESIMS, a molecular mass of 42 amu lower than that of 9. The 1H and 13C NMR spectra of 10 were similar to those of 9 (Tables 3 and 4), but a singlet of OCOCH3-16 at ca. δH 1.96 was absent, and double triplets at δH 5.49 (assigned for H-16 in 9) were replaced by shielded double triplets of H-16 at δH 4.65, thus indicating the aglycone of 10 to be 16-desacetyloleandrigenin 2991

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Table 4. 13C NMR Spectroscopic Data (100 MHz) of 2, 4, 9, 10, and 11 (in MeOH-d4) 2

4

9

10

11

position

δC, type

δC, type

δC, type

δC, type

δC, type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 1′ 2′ 3′ 4′ 5′ 6′ OCOCH3-16 OCOCH3-2′ OCH3-3′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴

29.9, CH2 26.2, CH2 73.2, CH 30.1, CH2 36.7, CH 26.4, CH2 20.8, CH2 41.3, CH 35.3, CH 34.9, C 20.6, CH2 38.6, CH2 50.0, C 83.6, C 40.0 CH2 74.6 CH 56.0, CH 15.0, CH3 23.0, CH3 170.9, C 76.7, CH2 120.3, CH 175.4, C 96.0, CH 69.1, CH 77.4, CH 69.7, CH 65.2, CH 16.4, CH3 170.2,a C; 19.6, CH3 170.2,a C; 19.5, CH3 56.4, CH3

31.3, CH2 27.5, CH2 75.0, CH 31.5, CH2 38.1, CH 27.8, CH2 22.2, CH2 42.7, CH 36.7, CH 36.4, C 22.0, CH2 40.0, CH2 51.4, C 85.0, C 41.4, CH2 76.0, CH 57.5, CH 16.4, CH3 24.4, CH3 171.6, C 77.6, CH2 121.7, CH 176.8, C 97.2, CH 71.0, CH 76.8, CH 77.2 CH 67.7, CH 17.4, CH3 172.2, C; 24.4,b CH3 171.7, C; 24.4,b CH3 57.8, CH3 103.0, CH 75.3, CH 77.8, CH 71.6, CH 78.0, CH 62.8, CH2

31.5, CH2 27.8, CH2 74.9, CH 31.4, CH2 37.6, CH 27.5, CH2 22.2, CH2 42.7, CH 36.7, CH 36.3, C 22.0, CH2 40.0, CH2 51.4, C 85.0, C 41.3, CH2 76.0, CH 57.4, CH 16.4, CH3 24.3, CH3 171.6, C 77.5, CH2 121.7, CH 176.7, C 100.2, CH 69.3, CH 79.0, CH 76.7, CH 67.4, CH 17.6, CH3 172.1, C; 20.9, CH3

31.5, CH2 27.9, CH2 75.4, CH 31.5, CH2 38. 0, CH 27.6, CH2 22.5, CH2 42.9, CH 36.8, CH 36.3, C 22.1, CH2 41.0, CH2 51.3, C 85.7, C 43.8, CH2 73.2, CH 59.7, CH 17.1, CH3 24.4, CH3 173.7, C 77.8, CH2 120.6, CH 178.0 C 100.3,CH 69.3, CH 79.1, CH 76.9, CH 67.5, CH 17.7, CH3

31.5, CH2 27.8, CH2 74.8, CH 31.5, CH2 38.0, CH 27.6, CH2 22.2, CH2 42.7, CH 36.7, CH 36.3, C 22.0, CH2 40.0, CH2 51.4, C 85.0, C 41.4, CH2 76.0, CH 57.4, CH 16.4, CH3 24.3, CH3 171.6, C 77.5, CH2 121.7, CH 176.8, C 100.3, CH 69.2, CH 78.9, CH 76.7, CH 67.2, CH 17.8, CH3 172.2, C; 20.9, CH3

56.9, CH3 105.0, CH 75.8, CH 77.8, CH 71.7, CH 77.9, CH 62.9, CH2

57.9, CH3 102.6, CH 74.9, CH 71.8, CH 71.7, CH 78.1, CH 62.9, CH2

57.8, CH3 102.4, CH 75.2, CH 77.9, CH 71.7, CH 77.3, CH 62.7, CH2 105.0 CH 75.3, CH 77.7, CH 71.6, CH 78.0, CH 70.2, CH2

a,b

Overlapped signals.

3 and 4). The 1H−1H COSY cross-peaks helped identify the 1H NMR resonances of a vallaropyranosyl unit at δH 4.66 (d, J = 2.9 Hz, H-1′), 3.93 (dd, J = 5.2 and 2.9 Hz, H-2′), 3.62 (dd, J = 5.2 and 3.3 Hz, H-3′), 3.81 (dd, J = 7.0 and 3.3 Hz, H-4′), 4.20 (dq, J = 7.0 and 6.6 Hz, H-5′), and 1.24 (d, J = 6.6 Hz, H-6′). The presence of two glucopyranosyl moieties was shown from the resonances of two anomeric protons and two oxymethylene groups. The HMBC cross-peaks between H-1′/C-3, H-1″/C4′, and H-1‴/C-6′′ indicated connectivities of C-1′-O to C-3, C-1″-O to C-4′, and C-1‴-O to C-6″. On the basis of the 1 H−1H COSY, HSQC, HMBC, and 1D-TOCSY spectra and the acid hydrolyses of 2 and 3, which gave L-vallarose, and Lacrofriose and D-glucose, respectively, the structure of

compound 11 was therefore elucidated as oleandrigenin-3-Oβ-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-α-L-vallaropyranoside. Compound 12 exhibited the same molecular formula as 11 based on the HRESIMS. The 1H and 13C NMR spectra resembled those of 11 (Tables 1−4). However, the 1H,1HCOSY spectrum showed H-5′ resonating at δH 3.76 (as quint, J = 5.6 Hz), at higher field than that in 11, and occurred together with resonances at δH 4.83 (d, J = 1.8 Hz, H-1′), 3.99 (dd, J = 3.0 and 1.8 Hz, H-2′), 3.65 (dd, J = 8.9 and 3.0 Hz, H-3′), 3.75 (H-4′), and 1.28 (d, J = 5.6 Hz, H-6′), similar to those of an acofriopyranosyl moiety as displayed in 8. The 1H and 13C NMR resonances of two glucosyl units were also encountered 2992

DOI: 10.1021/acs.jnatprod.7b00554 J. Nat. Prod. 2017, 80, 2987−2996

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Table 5. 1H (400 MHz) and 13C (100 MHz) NMR Spectroscopic Data of 5−7 (in MeOH-d4) 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 1′ 2′ 3′ 4′ 5′ 6′ OCOCH3-2′ OCH3-3′ 1″ 2″ 3″ 4″ 5″ 6″ a−f

6

δH (J in Hz)

position

1.59, 1.45 1.95, 1.29 3.95 brs (w1/2 = 9) 1.90, 1.49 1.68 1.60 1.82, 1.29 1.75 1.67 1.45, 1.18 2.05, 1.18

2.77 brd (17.8), 2.31 dd (17.8, 3.2) 6.25 brs 1.27 s 0.99 s 5.12 dd (16.8, 1.5), 5.02 dd (16.8, 1.5) 5.99 brs 4.80 5.19 3.73 3.61 3.76 1.29 2.09

d (1.6) dd (3.3, 1.6) dd (9.4, 3.3) t (9.4) dq (9.4, 6.3) d (6.3) s

3.39 s 4.57 d (8.0) 3.15 dd (8.8, 8.0) 3.39 dd (9.4, 8.8) 3.31 dd (9.4, 8.2)a 3.27a 3.86 dd (11.8, 2.1), 3.73 dd (11.8, 5.2)

δC, type 31.7, 27.6, 74.6, 30.9, 37.6, 27.4, 22.2, 42.1, 38.2, 36.3, 21.1, 39.4, 53.4, 86.7, 41.1,

CH2 CH2 CH CH2 CH CH2 CH2 CH CH C CH2 CH2 C C CH2

134.9, CH 145.1, C 17.0, CH3 24.6, CH3 162.0, C 73.6, CH2 112.0 CH 177.4, C 97.3, CH 68.8, CH 81.0, CH 79.3, CH 70.1, CH 18.2, CH3 172.1, C; 20.9, CH3 57.7, CH3 105.0, CH 75.6, CH 77.9, CHb 71.8, CH 77.9, CHb 62.9, CH2

δH (J in Hz) 1.57 1.94 3.97c 1.89, 1.50 1.65 1.59 1.80, 1.29 1.75 1.66 1.84, 1.12 2.06 dt (9.4, 2.1), 1.17 t (9.4)

2.80 d (17.5), 2.34 dd (17.5, 2.9) 6.28 t (2.9) 1.29 s 1.01 s 5.17 dd (16.7, 1.6), 5.03 dd (16.7, 1.6) 6.01 s 4.83 4.00 3.62 3.73 3.74 1.31

d (1.8) dd (3.1, 1.8)c dd (9.2, 3.1) t (9.2)d dq (9.2, 5.7)d d (5.7)

3.46 s 4.62 d (7.8) 3.19 dd (9.1, 7.8) 3.39 dd (9.1, 8.8) 3.31 t (8.8) 3.25 ddd (8.8, 5.4, 2.4) 3.87 dd (11.8, 2.4), 3.69 dd (11.8, 5.4)

7 δC, type 31.7, 27.7, 73.9, 30.9, 37.6, 27.5, 22.2, 42.1, 38.1, 36.3, 21.0, 39.4, 53.4, 86.7, 41.1,

CH2 CH2 CH CH2 CH CH2 CH2 CH CH C CH2 CH2 C C CH2

134.9, CH 145.0, C 17.0, CH3 24.6, CH3 162.0, C 73.5, CH2 111.9, CH 177.3, C 99.8, CH 68.5, CH 82.6, CH 79.3, CH 68.8, CH 18.2, CH3

56.9, CH3 105.0, CH 75.8, CH 77.9, CH 71.7, CH 77.9, CH 62.9, CH2

δH (J in Hz) 1.50, 1.93, 3.94e 1.87, 1.77 1.60 1.83, 1.74 1.67

1.55 1.27 1.80

1.28

1.46, 1.14 2.05, 1.17

2.77 brd (18.2), 2.31 dd (18.2, 3.4) 6.25 brs 1.27 s 0.98 s 5.14 dd (16.7, 1.4), 5.00 dd (16.7, 1.4) 5.99 brs 4.65 3.92 3.63 3.95 4.21 1.23

d (3.1) dd (6.9, dd (6.9, dd (6.9, dq (6.9, d (6.6)

3.1)e 3.1) 3.1) 6.6)

3.48 s 4.40 d (7.7) 3.21 dd (9.1, 7.7) 3.36 t (9.1) 3.29 t (9.1)f 3.27f 3.85 dd (11.9, 1.8), 3.66 dd (11.9, 5.3)

δC, type 31.6, 27.7, 74.9, 31.5, 38.0, 27.6, 22.2, 42.2, 37.6, 36.3, 21.1, 39.5, 53.4, 86.8, 41.1,

CH2 CH2 CH CH2 CH CH2 CH2 CH CH C CH2 CH2 C C CH2

134.9, CH 145.1, C 16.9, CH3 24.6, CH3 162.0, C 73.6, CH2 111.9, CH 177.4, C 100.2, CH 69.3, CH 79.0, CH 76.8, CH 67.4, CH 17.6, CH3

57.8, CH3 102.6, CH 75.3, CH 77.8, CH 71.6, CH 78.1, CH 62.8, CH2

Overlapped signals.

3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-α-

(Tables 1 and 2). The HMBC cross-peaks between H-1′/C-3, H-1″/C-4′, and H-1‴/C-6′′ indicated connectivities between C-1′-O and C-3, C-1″-O and C-4′, and C-1‴-O and C-6″. On the basis of acid hydrolysis of 3, which provided L-acofriose, and D-glucose, the structure of compound 12 could be deduced as oleandrigenin-3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-α-L-acofriopyranoside. Compound 13 was obtained as a colorless solid with a molecular formula of C46H70O21, based on HRESIMS, of molecular mass 42 amu higher than that of 12. The 1H and 13C NMR spectra exhibited 1H and 13C NMR resonances similar to those of 12, but the resonances due to an acofriopyranosyl moiety were replaced by those of a 2′-O-acetylacofriopyranosyl unit (Tables 1 and 2). After further investigation of the 1H,1HCOSY, HSQC, HMBC, and 1D-TOCSY data, and on the basis of acid hydrolysis of 3 providing L-acofriose and D-glucose, the structure of compound 13 could be assigned as oleandrigenin-

L-2′-O-acetylacofriopyranoside.

Compounds 1−13 were evaluated for their cytotoxicity against HeLaS3 [human cervix adenocarcinoma (ATCC; CCL2.2)], A549 [human lung carcinoma (ATCC: CCL-185)], HT29 [human colorectal adenocarcinoma (ATCC; HTB-38)], and Vero (normal African green monkey kidney, ATCC; CCL-81) cells (Table 6). Among the test compounds, 1 (the monoglycoside of oleandrigenin with a 2′-O-acetylacofriosyl moiety) was the most potent against HeLaS3 and HT-29 cells, showing IC50 values of 0.04 ± 0.00 and 0.07 ± 0.01 μM, respectively, and 2 (the monoglycoside of oleandrigenin with a 2′-O-acetylvallarosyl moiety) was the most potent against A549 cells, exhibiting an IC50 value of 0.03 ± 0.02 μM. When comparing the diglycosides of oleandrigenin (3 and 4), 4 (with a 2′-O-acetylvallarosyl moiety) was more active than 3 (with a 2′-O-acetylacofriosyl moiety) against all cell lines. Among the 2993

DOI: 10.1021/acs.jnatprod.7b00554 J. Nat. Prod. 2017, 80, 2987−2996

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0:100)) to give four subfractions (5.1−5.4). Also obtained was subfraction 5.2 (360 mg) after CC (silica gel, hexanes−EtOAc, 97:3) and passage over Sephadex LH-20 (CH2Cl2−MeOH, 5:95) to afford lupeol (70 mg). Subfraction 5.3 (1.93 g) was further purified by CC (silica gel, hexanes−EtOAc, 96:4, then Sephadex LH-20, MeOH) to give 3β,27-dihydroxyurs-12-en-28-oic acid (13 mg). Fraction 6 (3.98 g) was fractionated by CC (silica gel, hexanes−EtOAc, 92:8 to 80:20) to give seven subfractions (6.1−6.7), and subfraction 6.2 (380 mg) after CC (silica gel, hexanes−EtOAc, 95:5) furnished stigmast-4-en-3one (4 mg) and an additional quantity of lupeol (69 mg). Subfraction 6.3 (421 mg) was purified (CC, RP-18, MeOH) to give five subfractions (6.3.1−6.3.5), and subfraction 6.3.4 after CC (RP-18, MeOH, then silica gel, hexanes−CH2Cl2, 35:65 to 25:75) gave cycloeucalenol (161 mg). Subfraction 6.4 (349 mg) was purified by CC (silica gel, hexanes−EtOAc, 97:3 to 96:4) to give three subfractions (6.4.1−6.4.3), and subfraction 6.4.2 (168 mg) after further CC (silica gel, hexanes−CH2Cl2, 45:55) gave β-sitosterol (367 mg). Fraction 7 (3.94 g) after CC (silica gel, CH2Cl2−MeOH, 99.6:0.4 to 97:3) gave seven subfractions (7.1−7.7), and subfraction 7.2 (1.67 g) was further purified (CC, silica gel, hexanes−EtOAc, 92:8 to 80:20, then hexanes−CH2Cl2, 20:80) to give uvaol (urs-12-ene-3β,28-diol) (19 mg). Subfraction 7.5 (80 mg) after CC (silica gel, CH2Cl2− MeOH, 99.2:0.8) gave oleanolic acid (31 mg) and ursolic acid (20 mg). Fraction 8 (4.2 g) was fractionated (CC, CH2Cl2−MeOH, 99.6:0.4 to 97:3) to give 10 subfractions (8.1−8.10). Subfraction 8.5 (143 mg) was purified by reversed-phase CC (RP-18, MeOH−H2O, 65:35−100:0) to give five subfractions (8.5.1−8.5.5), and subfraction 8.5.2 gave 2 (37 mg), while subfraction 8.5.4 (37 mg) after CC (Sephadex LH-20, MeOH) gave an additional quantity of 3β,27dihydroxyurs-12-en-28-oic acid (7 mg). Subfraction 8.6 (131 mg) after CC (RP-18, MeOH−H2O, 70:30) gave three subfractions (8.6.1− 8.6.3), and the polar fraction of those (41 mg) after CC (Sephadex LH-20, MeOH) furnished 2 (9 mg). Subfraction 8.7 (210 mg) after CC (RP-18, MeOH−H2O, 60:40 to 100:0) gave four subfractions (8.7.1−8.7.4), and subfraction 8.7.2 (86 mg) was further purified by CC (Sephadex LH-20, MeOH) to give additional quantities of 2 (3 mg) and 1 (16 mg). Fraction 11 (10.3 g) was fractionated by CC (silica gel, CH2Cl2−MeOH, 98.5:1.5−86:14) to give six subfractions (11.1−11.6), and subfraction 11.2 gave an additional quantity of ursolic acid (700 mg). Subfraction 11.6 (922 mg) after CC (RP-18, MeOH−H2O, 55:45 to 100:0) gave β-sitosteryl glucoside (64 mg). Fraction 12 (1.70 g) was fractionated by CC (silica gel, CH2Cl2− MeOH, 99:1−93:7) to give three subfractions (12.1−12.3), and subfraction 12.3 (700 mg) was further purified by CC (Sephadex LH20, MeOH) to give four subfractions, (12.3.1−12.3.4). Subfraction 12.3.3 (176 mg), after reversed-phase CC (RP-18, MeOH−H2O, 60:40 to 100:0), gave four additional subfractions (12.3.3.1−12.3.3.4). Compounds 4 (9 mg) and 3 (19 mg) were obtained after subfraction 12.3.3.2 (70 mg) was purified by reversed-phase CC (RP-18, MeOH− H2O, 50:50 to 55:45). Fraction 13 (13.4 g) was fractionated by CC (silica gel, CH2Cl2−MeOH, 96:4 to 50:50) to give three subfractions (13.1−13.3), and subfraction 13.2 (1.34 g) after further purification by reversed-phase CC (RP-18, MeOH−H2O, 80:20−100:0) gave four subfractions (13.2.1−13.2.4). Subfraction 13.2.3 (424 mg) after CC (CH2Cl2−MeOH, 96:4 to 92:8) gave four subfractions (13.2.3.1− 13.2.3.4), and subfraction 13.2.3.2 (34 mg) after further CC (Sephadex LH-20, MeOH) furnished an additional quantity of 3 (13 mg). Subfraction 13.2.3.3 (156 mg) after purification by CC (silica gel, CH2Cl2−MeOH, 90:10, then reversed-phase RP-18 CC (MeOH− H2O, 70:30−100:0), gave 5 (7 mg). The crude MeOH extract (100 g) was fractionated by CC (Diaion HP20, H2O−MeOH, 100:0 to 0:100) to obtain six fractions (1−6). An aliquot of the less polar fraction 5 (1.4 g) was further fractionated (CC, Sephadex LH-20, MeOH) to obtain six subfractions (5.1−5.6). Subfraction 5.2 (135 mg) was purified by reversed-phase CC (H2O− MeOH, 55:45 to 0:100) to give 12 (7 mg) and 13 (7 mg). Subfraction 5.3 (220 mg) after CC (RP-18, H2O−MeOH, 65:35 to 0:100) gave seven subfractions (5.3.1−5.3.7), and further purification of subfraction 5.3.3 (13 mg) by reversed-phase CC (RP-18, H2O−MeOH, 45:65 to 0:100) gave 11 (7 mg). Subfraction 5.3.6 (59 mg) was

Table 6. Cell Growth Inhibitory Activities of Crude Extracts and Cardenolides 1−13 against Cancer and Noncancerous (Vero) Cellsa compound

HeLaS3 (IC50)

Crude Extract (μg/mL) VG-L-CH2Cl2 1.2 ± VG-L-MeOH >20 Compound (μM) 1 0.04 ± 2 0.1 ± 3 1.4 ± 4 0.4 ± 5 >10 6 0.7 ± 7 0.7 ± 8 0.2 ± 9 0.1 ± 10 6.8 ± 11 0.5 ± 12 0.4 ± 13 1.6 ± doxorubicin 20

1.9 ± 0.1 >20

5.1 ± 1.3 >20

0.00 0.0 0.1 0.0

0.5 0.03 1.5 0.6 >10 0.3 1.6 0.1 0.1 3.8 1.2 0.6 2.1 5.0 >10

0.1 0.1 0.0 0.0 0.9 0.1 0.0 0.1 0.1

± ± ± ±

0.1 0.01 0.2 0.1

± ± ± ± ± ± ± ± ±

0.1 0.1 0.0 0.0 0.3 0.1 0.1 0.2 1.7

0.1 0.1 1.9 0.6 >10 0.8 1.9 0.2 0.2 6.4 1.6 1.4 5.0 0.4 7.6

± ± ± ±

0.0 0.0 0.2 0.0

± ± ± ± ± ± ± ± ± ±

0.1 0.1 0.0 0.0 0.9 0.1 0.0 1.0 0.0 0.7

0.2 0.3 5.8 2.2 >10 2.5 6.2 0.7 0.6 >10 6.9 4.8 >10 6.6 >10

± ± ± ±

0.0 0.0 0.4 0.2

± ± ± ±

0.2 0.4 0.1 0.1

± 1.0 ± 1.4 ± 0.2

a Data are represented as mean ± SD (n = 6); IC50 = 50% inhibition concentration.

diglycosides possessing similar glycosyl units (with an Lvallarosyl moiety) of oleandrigenin (9), 16-anhydrogitoxigenin (7), and gitoxigenin (10), the order of cytotoxic activity was found to be 9 > 7 > 10, and furthermore among the diglycosides possessing similar glycosyl units (with an Lacofriosyl moiety) of oleandrigenin (8) and 16-anhydrogitoxigenin (6), 8 was more potent. Based on these results, it could be concluded that the aglycone oleandrigenin moiety was the most crucial for higher cytotoxic potency and the gitoxigenin moiety less effective.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured using an Electrothermal melting point apparatus and are uncorrected. Optical rotations were recorded on a JASCO DIP 1020 polarimeter. The IR spectra were obtained on a PerkinElmer 1760x FT-IR spectrophotometer. The 1H and 13C NMR spectra were recorded with Bruker AVANCE 400 MHz and Bruker AVANCE III HD 400 MHz NMR spectrometers. Chemical shifts are referenced to the residual solvent signals (MeOH-d4: δH 3.30 and δC 49.0 ppm). HRESIMS were recorded on a Bruker Daltonics microTOF mass spectrometer. Plant Material. The plant Vallaris glabra was collected from a garden in Lat Phrao District, Bangkok, in March 2012. The plant was identified by Assoc. Prof. Dr. Nijsiri Ruangrungsi, Department of Pharmacognosy, Faculty of Pharmaceutical Science, Chulalongkorn University, Bangkok, Thailand. A voucher specimen (SSVG-1/2012) is maintained at the Department of Chemistry, Ramkhamhaeng University. Extraction and Isolation. Fresh V. glabra leaves (6.2 kg) were ground and soaked in MeOH (36 L) at room temperature for 3 weeks. The filtrate was concentrated and partitioned successively with hexanes and CH2Cl2 (8 L) to obtain hexanes (dark green solid, 82.6 g), CH2Cl2 (dark green solid, 124.8 g), and MeOH (reddish-brown, 212.4 g) extracts. The CH2Cl2 extract was fractionated by column chromatography (CC, silica gel, hexanes−CH2Cl2, 80:20 to CH2Cl2− MeOH, 40:60) to obtain 14 fractions. The moderately polar fraction 5 (2.7 g) was purified by CC (silica gel, hexanes−CH2Cl2 (40:60 to 2994

DOI: 10.1021/acs.jnatprod.7b00554 J. Nat. Prod. 2017, 80, 2987−2996

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Article

NMR (MeOH-d4, 100 MHz) data see Tables 3 and 4; HRESIMS m/z 735.3564 [M + Na]+ (calcd for C36H56NaO14, 735.3552). Oleandrigenin-3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-α-L-vallaropyranoside (11): [α]32 D −45.8 (c 0.33, MeOH); FT-IR (ATR) νmax 3350, 2924, 2878, 2855, 1733, 1628, 1449, 1378, 1257, 1165, 1102, 1068, 1033, 1014, 905, 801 cm−1; 1H NMR (MeOH-d4, 400 MHz), 13C NMR (MeOH-d4, 100 MHz) data see Tables 3 and 4; HRESIMS m/z 939.4209 [M + Na]+ (calcd for C44H68NaO20, 939.4182). Oleandrigenin-3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-α-L-acofriopyranoside (12): [α]32 D −40.6 (c 0.35, MeOH); FT-IR (ATR) νmax 3369, 2932, 2876, 1733, 1618, 1447, 1378, 1248, 1162, 1101, 1066, 1033, 1018, 986, 907, 889 cm−1; 1H NMR (MeOHd4, 400 MHz), 13C NMR (MeOH-d4, 100 MHz) data see Tables 1 and 2; HRESIMS m/z 939.4208 [M + Na]+ (calcd for C44H68NaO20, 939.4182). Oleandrigenin-3-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-α-L-2′-O-acetylacofriopyranoside (13): colorless solid, mp 182−184 °C (MeOH/EtOAc); [α]32 D −37.9 (c 0.33, MeOH); FT-IR (ATR) νmax 3381, 2931, 2894, 2868, 1735, 1728, 1609, 1445, 1370, 1235, 1168, 1130, 1073, 1051, 1022, 985, 912 cm−1; 1H NMR (MeOH-d4, 400 MHz), 13C NMR (MeOH-d4, 100 MHz) data see Tables 1 and 2; HRESIMS m/z 981.4306 [M + Na]+ (calcd for C46H70NaO21, 981.4287). Acid Hydrolysis of 2. A mixture of compound 2 (4.2 mg) and 5% HCl (5 mL) was heated at 70 °C for 5 h. The cold reaction mixture was extracted with EtOAc (3 × 10 mL). The combined EtOAc extract, after washing with water, was concentrated to give the agylcone, 14,16dianhydrogitoxigenin, Rf 0.5 (CH2Cl2−MeOH, 99:1). The aqueous extract, after removal of water, gave L-vallarose (0.7 mg), Rf 0.7 (silica gel TLC, thickness 0.2 mm, CH2Cl2−MeOH, 92:8). The optical rotation value was measured after 24 h of dissolution in H2O: L12 23 vallarose [α]26 D −38.9 (c 0.04, H2O) [lit. [α]D −17.2 (c 0.90, H2O)]. Acid Hydrolysis of 3. A mixture of compound 3 (4.2 mg) and 5% HCl (5 mL) was heated at 70 °C for 5 h, then left to cool to room temperature. The reaction mixture was then partitioned with EtOAc to give an EtOAc extract, from which the aglycone, 14,16-dianhydrogitoxigenin (1.0 mg), Rf 0.72 (CH2Cl2−MeOH, 98:2), was obtained after concentration. The aqueous phase was neutralized with saturated NaHCO3 and concentrated to give an aqueous extract (3.1 mg). Reversed-phase column chromatography (RP-18, MeOH−H2O, 15:85) gave D-glucose (1.0 mg, Rf 0.1 (CH2Cl2−MeOH, 90:10) and L-acofriose, 0.5 mg) Rf 0.8 [silica gel TLC, thickness 0.2 mm, CH2Cl2− MeOH (86:14)]. The optical rotation values were measured after 24 h 13 of dissolution in H2O: D-glucose [α]29 D +40.2 (c 0.1, H2O) [lit. +53.2 12 29 21 (c 0.1, H2O)] and L-acofriose, [α]D +30.0 (c 0.05) [lit. [α]D +37.3 (c 0.95, H2O)]. Bioassay. The antiproliferative effects of the isolated compounds on HeLaS3 [human cervix adenocarcinoma (ATCC; CCL-2.2)], A549 [human lung carcinoma (ATCC; CCL 185)], HT-29 [human colorectal adenocarcinoma (ATCC HTB-38)], and nontumorigenic (Vero, normal African green monkey kidney, ATCC; CCL-81) cells using a microculture tetrazolium (MTT) assay were determined according to a previously reported protocol.15

fractionated by CC (silica gel, EtOAc−MeOH, 93:7) to give four subfractions (5.3.6.1−5.3.6.4), and subfraction 5.3.6.3 gave an additional quantity of 13 (17 mg). Subfraction 5.4 (336 mg) was purified by reversed-phase CC (RP-18, H2O−MeOH, 75:5 to 0:100) to give seven subfractions (5.4.1−5.4.7). Subfraction 5.4.4 gave 10 (5 mg), and subfraction 5.4.5 after further purification by CC (Sephadex LH-20, MeOH) gave five subfractions (5.4.5.1−5.4.5.5). Subfraction 5.4.5.2 gave 9 (8 mg), and subfraction 5.4.5.3 (82 mg) after further purification by CC (RP-18, H2O−MeOH, 75:5 to 0:100, then silica gel, CH2Cl2−MeOH, 92:8) gave 7 (5 mg). Subfraction 5.4.6 (17 mg) after CC (silica gel, CH2Cl2−MeOH, 92:8) gave 6 (9 mg) and 8 (5 mg). Oleandrigenin-3-O-α-L-2′-O-acetylvallaropyranoside (2′-O-acetylvallarosolanoside, 2): colorless solid, mp 172−173 °C (MeOH/ EtOAc); [α]25 D −55.6 (c 0.30, MeOH); FT-IR (ATR) νmax 3451, 2932, 1732, 1650, 1371, 1234, 1096, 1034, 1012 cm−1; 1H NMR (MeOH-d4, 400 MHz) and 13C NMR ((MeOH-d4, 100 MHz) data see Tables 3 and 4; HRESIMS m/z 657.3263 [M + Na]+ (calcd for C34H50NaO11, 657.3353). Oleandrigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-2′-O-acetylacofriopyranoside (3): colorless solid, mp 179−181 °C (MeOH/ EtOAc); [α]25 D −24.1 (c 0.35, MeOH); FT-IR (ATR) νmax 3369, 2929, 1731, 1620, 1371, 1234, 1071, 1053, 1033, 1025 cm−1; 1H NMR (MeOH-d4, 400 MHz), 13C NMR (MeOH-d4, 100 MHz) data see Tables 1 and 2; HRESIMS m/z 819.3796 [M + Na]+ (calcd for C40H60NaO16, 819.3773). Oleandrigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-2′-O-acetylvallaropyranoside (4): colorless solid, mp 183−184 °C (MeOH/ EtOAc); [α]25 D −15.2 (c 0.37, MeOH); FT-IR (ATR) νmax 3396, 2929, 1732, 1630, 1238, 1033, 1026, 1015 cm−1; 1H NMR (MeOH-d4, 400 MHz), 13C NMR (MeOH-d4, 100 MHz) data see Tables 3 and 4; HRESIMS m/z 819.3793 [M + Na]+ (calcd for C40H60NaO16, 819.3773). 16-Anhydrogitoxigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-2′-Oacetylacofriopyranoside (5): [α]25 D −9.6 (c 0.15, MeOH); FT-IR (ATR) νmax 3389, 2927, 2881, 2852, 1728, 1619, 1449, 1374, 1235, 1161, 1105, 1051, 1029, 981, 902, 888 cm−1; 1H NMR (MeOH-d4, 400 MHz), 13C NMR (MeOH-d4, 100 MHz) data see Table 5; HRESIMS m/z 759.3580 [M + Na]+ (calcd for C38H56O14Na 759.3562). 16-Anhydrogitoxigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-acofriopyranoside (6): colorless solid, mp 198−200 °C (MeOH/EtOAc); [α]31 D −18.6 (c 0.45, MeOH); FT-IR (ATR) νmax 3528, 3371, 2930, 2880,1724, 1695, 1623, 1449, 1379, 1260, 1108, 1072, 1047, 1030, 982, 887 cm−1; 1H NMR (MeOH-d4, 400 MHz), 13C NMR (MeOHd4, 100 MHz) data see Table 5; HRESIMS m/z 717.3462 [M + Na]+ (calcd for C36H54NaO13, 717.3447). 16-Anhydrogitoxigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-vallaropyranoside (7): colorless solid, mp 204−206 °C (MeOH/ EtOAc); [α]31 D −18.3 (c 0.25, MeOH); FT-IR (ATR) νmax 3476, 3383, 2933, 2885, 2851, 1727, 1621, 1447, 1378, 1255, 1163, 1076, 1030, 1015, 984, 891, 829 cm−1; 1H NMR (MeOH-d4, 400 MHz), 13C NMR (MeOH-d4, 100 MHz) data see Table 5; HRESIMS m/z 717.3455 [M + Na]+ (calcd for C36H54NaO13, 717.3447). Oleandrigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-O-acofriopyranoside (8): [α]31 D −49.1 (c 0.26, MeOH); FT-IR (ATR) νmax 3376, 2928, 2874, 1732, 1631, 1447, 1377, 1246, 1163, 1107, 1070, 1031, 983, 892 cm−1; 1H NMR (MeOH-d4, 400 MHz), 13C NMR (MeOHd4, 100 MHz) data see Tables 1 and 2; HRESIMS m/z 777. 3667 [M + Na]+ (calcd for C38H58NaO15, 777.3657. Oleandrigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-vallaropyranoside (9): [α]31 D −39.5 (c 0.40, MeOH); FT-IR (ATR) νmax 3377, 2926, 2873, 2856, 1732, 1630, 1447, 1375, 1246, 1162, 1071, 1032, 1013, 896 cm−1; 1H NMR (MeOH-d4, 400 MHz), 13C NMR (MeOHd4, 100 MHz) data see Tables 3 and 4; HRESIMS m/z 777. 3661 [M + Na]+ (calcd for C38H58NaO15, 777.3657). Gitoxigenin-3-O-β-D-glucopyranosyl-(1→4)-α-L-vallaropyranoside (10): [α]31 D −21.4 (c 0.38, MeOH); FT-IR (ATR) νmax 3351, 2925, 2854, 1759, 1733, 1627, 1597, 1451, 1376, 1261, 1160, 1072, 1028, 1008, 961, 889 cm−1; 1H NMR (MeOH-d4, 400 MHz), 13C

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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.jnatprod.7b00554. 1

H and 13C NMR spectra of compounds 1−13, 2D NMR spectra of 2, and 1D TOCSY spectra of 5, 7, 11, and 12 (PDF) HMBC data of compounds 1−13 (PDF)

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Article

AUTHOR INFORMATION

Corresponding Author

*Tel: (662) 319-0931. Fax: (662) 319-1900. E-mail: somyote_ [email protected]; [email protected]. ORCID

Somyote Sutthivaiyakit: 0000-0002-3249-1380 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Ramkhamhaeng University, the Thailand Research Fund, and the Center of Excellence for Innovation in Chemistry (PERCH−CIC), Commission on Higher Education, Ministry of Education, for financial support. We acknowledge Miss W. Hadtugvong, Miss P. Pongpinit, Miss S. Karoonborirak, Miss N. Polteera, Miss N. Tepnuan, Miss J. Khambudda, Miss T. Srithong, and the late Mr. W. Komkhunthot for technical assistance.

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REFERENCES

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