Isolation and Structural Characterization of Echinocystic Acid

Article Cite This: J. Nat. Prod. 2017, 80, 2692-2698

pubs.acs.org/jnp

Isolation and Structural Characterization of Echinocystic Acid Triterpenoid Saponins from the Australian Medicinal and Food Plant Acacia ligulata Diana Jæger,†,‡ Chi P. Ndi,† Christoph Crocoll,§ Bradley S. Simpson,⊥ Bekzod Khakimov,∥ Ruth Marian Guzman-Genuino,†,∇,□ John D. Hayball,†,∇,□ Xiaohui Xing,◆,# Vincent Bulone,◆,# Philip Weinstein,¶ Birger L. Møller,‡ and Susan J. Semple*,† †

Sansom Institute for Health Research, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia 5000, Australia ‡ Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark § DynaMo Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark ⊥ Flinders Centre for Innovation in Cancer, Flinders University, Bedford Park, South Australia 5042, Australia ∥ Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 26, DK-1958 Frederiksberg C, Denmark ∇ Experimental Therapeutics Laboratory, Hanson Institute and Sansom Institute, Adelaide, South Australia 5000, Australia □ Robinson Research Institute and Adelaide Medical School, University of Adelaide, Adelaide, South Australia 5005, Australia ◆ ARC Centre of Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, Urrbrae, 5064, Australia # Division of Glycoscience, Royal Institute of Technology (KTH), School of Biotechnology, AlbaNova University Centre, Stockholm, SE-10691, Sweden ¶ Department of Ecology and Environmental Sciences, School of Biological Sciences, The University of Adelaide, Adelaide, South Australia 5005, Australia S Supporting Information *

ABSTRACT: The Australian plant Acacia ligulata has a number of traditional food and medicinal uses by Australian Aboriginal people, although no bioactive compounds have previously been isolated from this species. Bioassay-guided fractionation of an ethanolic extract of the mature pods of A. ligulata led to the isolation of the two new echinocystic acid triterpenoid saponins, ligulatasides A (1) and B (2), which differ in the fine structure of their glycan substituents. Their structures were elucidated on the basis of 1D and 2D NMR, GC-MS, LC-MS/MS, and saccharide linkage analysis. These are the first isolated compounds from A. ligulata and the first fully elucidated structures of triterpenoid saponins from Acacia sensu stricto having echinocystic acid reported as the aglycone. Compounds 1 and 2 were evaluated for cytotoxic activity against a human melanoma cancer cell line (SK-MEL28) and a diploid fibroblast cell line (HFF), but showed only weak activity.

T

chemical constituents of the pods of Acacia ligulata A. Cunn. ex Benth. were examined. This species is extensively distributed across mainland Australia.6 Its leaves and bark have been used in traditional medicine by Aboriginal Australians to treat coughs, colds, and chest infections and to wash the skin,7−9 whereas the seeds and possibly the pods have been used as a food source.10−12 However, A. ligulata has only been used during dry periods in some areas of Australia, and eating the

he genus Acacia sensu stricto (Fabaceae: Mimosoideae) is a large genus consisting of 1073 species with 1063 naturally occurring in Australia and others native to Southeast Asia, the islands of the Pacific, and the Mascerene Islands.1 Previously, pods of the Australian species Acacia victoriae Benth. were found to contain triterpenoid saponins called avicins, which were found to exhibit potent cytotoxic activity against cancerous cells.2−4 Therefore, it has been suggested that the distribution and chemistry of saponins in the genus Acacia should be more fully investigated in order to understand their structure−activity relationships.5 In the present study the © 2017 American Chemical Society and American Society of Pharmacognosy

Received: May 19, 2017 Published: October 4, 2017 2692

DOI: 10.1021/acs.jnatprod.7b00437 J. Nat. Prod. 2017, 80, 2692−2698

Journal of Natural Products

Article

Chart 1

Table 1. 1H NMR Spectroscopic Data (CD3OD, 600 MHz) for Ligulatasides A (1) and B (2)a position

1 δH (J in Hz)

2 δH (J in Hz)

position

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 GlcNAc 1 2 3 4 5 6

1.67, 1.02, m 1.96, 1.71, m 3.14, dd (11.5, 4.0)

1.66, 1.01, m 1.96, 1.71 m 3.13, dd (11.6, 4.1)

0.81, m 1.63, 1.43, m 1.61, 1.44, m

0.81, m 1.63, 1.43, m 1.61, 1.44, m

1.67, m

1.68, m

1.94, m 5.39, m

1.94, m 5.39, m

1.76, 1.48, m 4.53, br t (3.0)

1.75, 1.48, m 4.53, br t (3.0)

3.07, m 2.34, 1.09, m

3.06, m 2.33, 1.08, m

1.98, 1.19, m 1.96, 1.80, m 1.01, br s 0.82, br s 1.00, br s 0.83, br s 1.42, s

1.98, 1.18, m 1.95, 1.80, m 1.01, br s 0.81, br s 0.99, br s 0.82, br s 1.41, s

0.92, s 1.00, br s

0.91, s 0.99, br s

4.49, d (8.8) 3.76, m 3.65, m 3.63, m 3.42, m 3.90, 3.68, m

4.49, d (8.5) 3.76, m 3.64, m 3.63, m 3.42, m 3.90, 3.67, m

t-Glc 1 2 3 4 5 6 Ara 1 2 3 4 5 Rha 1 2 3 4 5 6 t-Xyl 1 2 3 4 5 t-Ara 1 2 3 4 5 CH3CONH −OCOCH3

1 δH (J in Hz)

2 δH (J in Hz)

4.46, d (8.0) 3.37, m 3.39, m 3.32, m 3.25, m 3.90, m

4.47 d (7.9) 3.37, m 3.37, m 3.32, m 3.25, m 3.90, m

5.60, d (4.7) 3.86, m 3.90, m 3.85, m 3.93, 3.56, m

5.60, d (4.4) 3.86, m 3.90, m 3.85, m 3.92, 3.55, m

5.16, br s 4.03, m 3.86, m 3.67, m 3.82, m 1.33, d (6.2)

5.17, br s 4.03, m 3.86, m 3.70, m 3.83, m 1.35, d (5.9)

4.51, d (7.6) 3.30, m 3.36, m 3.53, m 3.90, 3.26, m

4.51, d (7.6) 3.30, m 3.36, m 3.53, m 3.90, 3.26, m

5.36, br s 4.12, m 3.83, m 4.14, m 4.32, 4.18, m 1.98, br s 2.09, s

5.37, br s 4.11, m 4.02, m 3.88, m 3.76, 3.67, m 1.96, br s

a

d = doublet, dd = doublet of doublets, br t = broad triplet, br s = broad singlet, s = singlet, m = multiplet. t = terminal. GlcNAc = Nacetylglucosamine. Glc = glucose. Ara = arabinose. Rha = rhamnose. Xyl = xylose.

seeds of this plant has been observed to “make your hair fall out” by people of the Yankunytjatjara language group in Central Australia.13 This observation of possible toxicity

following consumption of the plant made A. ligulata interesting for cytotoxic screening and chemical investigations. Preliminary screening studies of crude extracts from different devel2693



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Table 2. 13C NMR Spectroscopic Data (CD3OD, 600 MHz) for Ligulatasides A (1) and B (2)a

opmental stages of seeds and pods from A. ligulata showed a cytotoxic effect of pod extracts, particularly against melanoma cancer cells (SK-MEL28).14 The extract of the mature pods showed the best activity, with an IC50 value at 40.4 μg/mL.14 Further, metabolite screening indicated a similar metabolite profile between the different pod extracts, suggesting the active component(s) was the same in all pod extracts. This paper describes the bioassay-guided isolation, structural elucidation, and cytotoxic analysis of the new triterpenoid saponin compounds 1 and 2 from the mature pods of A. ligulata.

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RESULTS AND DISCUSSION The pods of A. ligulata were extracted using 80% (v/v) aqueous ethanol. The crude extract was subjected to bioassay-guided fractionation using cytotoxicity in SK-MEL28 cells. The extract was initially fractionated by normal-phase solid-phase extraction followed by radial centrifugal TLC and column chromatography to yield an active fraction. The two major compounds in this fraction were purified by RP-HPLC. They were identified as triterpenoid saponins 1 and 2 and given the trivial names ligulataside A and ligulataside B, respectively, with their structures elucidated on the basis of 1D and 2D NMR, GCMS, LC-MS/MS, and saccharide linkage analysis studies (Figures S1−S27, Supporting Information). The 1H and 13C NMR data are shown in Tables 1 and 2, respectively, and the spectra are given in the Supporting Information (Figures S1−S16). Compound 1 was obtained as a white, amorphous solid. The molecular formula, C67H107NO31, was deduced from the HRESIMS data (m/z 1422.6950 [M + H]+, calcd 1422.6900) (Figure S17, Supporting Information). Compound 1 showed one olefinic proton in the 1H NMR spectrum (δH 5.39 ppm) and seven methyl singlets (δH 0.82, 0.83, 0.92, 1.00, 1.00, 1.01, and 1.42 ppm) with one of them (δH 1.42 ppm) showing an HMBC correlation to the quaternary olefinic carbon at δC 145.2. The 13C NMR spectrum also had two characteristic signals at δC 23.6 and 173.8 ppm, which indicated the presence of a CH3CONH group in the compound.15 Further, the 1H NMR shifts of protons at δH 5.60 (d, J = 4.7 Hz), 5.36 (br s), 5.16 (br s), 4.51, (d, J = 7.6 Hz); 4.49, (d, J = 8.8 Hz); and 4.46 ppm (d, J = 8.0 Hz) together with their correlations in the HSQC spectrum to oxygenated methines at δC 94.8, 111.4, 101.6, 106.8, 105.4, and 105.1 ppm, respectively, suggested them to be anomeric protons. LC-MS/MS analysis of 1 in the positive-ion mode indicated a sequenced loss of masses corresponding to saccharide units, which could be observed in the fragmentation spectra (Figure S18 A and B, Supporting Information). The presence of three pentoses (neutral loss of −132), one methyl pentose (−146), one hexose (−162), an N-acetylglucosamine (−204), and an acetyl group (−42) was indicated by the fragments observed at m/z 1422 [M + H]+, 1290 [M − 132 + H]+, 1248 [M − 132− 42 + H]+, 1116 [M − 132 − 132 − 42 + H]+, 970 [M − 132 − 132 − 42 − 146 + H]+, 838 [M − 132 − 132 − 42 − 146 − 132 + H]+, 366 [M − 132 − 132 − 42 − 146 − 132 − 472 + H]+, and 204 [M − 132 − 132 − 42 − 146 − 132 − 472 − 162 + H]+. The presence of a hexose linked to an N-acetylglucosamine residue was strongly suggested by the fragments of m/z 366, 221, 204, and 186.16,17 Acid hydrolysis of 1 with HCl followed by GC-MS suggested the aglycone was echinocystic acid [(3β,16α)-3,16-dihydroxyolean-12-en-28-oic acid] (3) (Figures S21 and S22, Supporting Information) (Figure 1)

position

1 δC, type

2 δC, type

1 δC, type

2 δC, type

1 2

40.3, CH2 27.4, CH2

40.3, CH2 27.4, CH2

t-Glc 1

105.1, CH

3 4 5 6 7

91.7, CH 40.5, C 57.6, CH 20.1, CH2 35.0, CH2

91.8, CH 40.5, C 57.6, CH 20.1, CH2 35.0, CH2

2 3 4 5 6

78.7, CH 78.4, CH 71.9, CH 75.5, CH 62.6, CH2

105.1, CH 78.7, CH 78.4, CH 71.9, CH 75.5, CH 62.5, CH2

8 9 10 11 12 13

41.3, C 48.7, CH 38.4, C 25.0, CH2 124.2, CH 145.2, C

41.3, C 48.7, CH 38.4, C 25.0, CH2 124.1, CH 145.2, C

Ara 1 2 3 4 5

94.8, CH 75.4, CH 72.5, CH 68.2,CH 65.0, CH2

94.8, CH 75.7, CH 72.6, CH 68.2, CH 65.1, CH2

14 15

43.2, C 36.94, CH2 75.1, CH 50.8, C 42.7, CH 48.3, CH2 31.8, C

Rha 1

101.6, CH

16 17 18 19 20

43.2, C 36.94, CH2 75.1, CH 50.8, C 42.7, CH 48.3, CH2 31.8, C

2 3 4 5 6

72.8, CH 81.9, CH 79.6, CH 69.5, CH 18.8, CH3

101.6, CH 72.9, CH 82.1, CH 79.4, CH 69.6, CH 18.9, CH3

21 22

37.0, CH2 32.5, CH2

36.9, CH2 32.5, CH2

t-Xyl 1

106.8, CH

23 24 25 26

29.1, 17.6, 16.7, 18.5,

29.1, 17.6, 16.7, 18.5,

2 3 4 5

75.7, CH 78.4, CH 71. 6, CH 67.5, CH2

27 28

27.7, CH3 177.4, C

27.7, CH3 177.4, C

t-Ara 1

111.4, CH

29 30 GlcNAc 1

33.9, CH3 25.5, CH3

33.9, CH3 25.5, CH3

105.4, CH

105.4, CH

2 3 4 5

84.7, CH 79.6, CH 82.4, CH 65.8, CH2

2 3 4 5 6

57.8, CH 74.6, CH 81.6, CH 76.8, CH 63.0, CH2

57.8, CH 74.6, CH 81.6, CH 76.8, CH 63.0, CH2

CH3 CH3 CH3 CH3

CH3 CH3 CH3 CH3

position

CH3CONH CH3CONH −OCOCH3 −OCOCH3

23.6 173.8 21.2 173.0

106.8, CH 75.7, CH 78.3, CH 71.6, CH 67.5, CH2 111.4, CH 84.7, CH 85.7, CH 79.0, CH 63.5, CH2 23.6 173.8

a

t = terminal. GlcNAc = N-acetylglucosamine. Glc = glucose. Ara = arabinose. Rha = rhamnose. Xyl = xylose.

and indicated the presence of D-glucose, L-rhamnose, D-xylose, and L-arabinose (two units) (Figures S23 and S24, Supporting Information). An N-acetylglucosamine residue could not be detected by initial GC-MS possibly due to degradation caused by the strong HCl treatment. In summary, the presence of six anomeric protons in the 1H NMR spectrum and the mass spectrometric fragmentation pattern in the LC-MS/MS suggested a sequential loss of six sugar moieties. This was also supported by the GC-MS analysis of an acidic hydrolyzed sample that confirmed the saccharide moieties. Together, this evidence suggested that compound 1 possesses six saccharide units. 2694



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Figure 1. Hydrolysis of ligulataside A (1). Acid hydrolysis resulted in the aglycone echinocystic acid and free saccharide units analyzed by GC-MS. Alkaline hydrolysis gave the prosaponin (4), analyzed by LC-MS, indicating the tetrasaccharide chain to be attached at C-28.

Figure 2. Key HMBC and ROESY correlations for 1.

which was analyzed by LC-MS (Figure S25, Supporting Information) with fragments found at m/z 838 corresponding to echinocystic acid (m/z 472), the N-D-acetylglucosamineglucose (m/z 366), suggesting the two saccharides to be linked at C-3. A tetrasaccharide chain was therefore proposed at C-28 through an ester bond, which was supported by the HMBC data showing a linkage between the anomeric proton of the arabinose unit of the chain and C-28 of the echinocystic acid unit (Figure 2). The connectivities of each of the saccharides and the aglycone were established further using information from the 1H, 13C, DEPT, COSY, HSQC, HMBC, ROESY, and

The identity of the aglycone as echinocystic acid was further confirmed by comparison of the NMR data with previously reported echinocystic acid-based triterpenoid saponins.18,19 The NMR data were consistent with 1 being a bisdesmosidic triterpenoid glycoside with two linkage sites (C-3 and C-28), as indicated by the chemical shifts of C-3 (δC 91.7) and C-28 (δC 177.4) of the aglycone. This was also shown by the HMBC cross-correlation peaks between the doublet at δH 4.49 (H-1 of GlcNAc) and δC 91.7 (C-3 of the aglycone) as well as between the doublet at δH 5.60 (H-1 of Ara) and δC 177.4 (C-28 of the aglycone) (Figure S11, Supporting Information). Alkaline hydrolysis of 1 (Figure 1) produced the prosaponin (4), 2695



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1

H−1H TOCSY experiments. The 13C NMR spectroscopic data suggested one of the arabinose residues to be in the furanose form, as indicated by the chemical shift of the anomeric carbon (δC 111.37 ppm),3 while the remaining five monosaccharides are in the pyranose form.20 The coupling constants of each of the anomeric protons, the glucose, N-acetylglucosamine, and the xylose were consistent with a beta-configuration (δH 4.46 (d, J = 8.0 Hz), 4.49 (d, J = 8.8 Hz), and 4.51 (d, J = 7.6 Hz)), while the signals arising from the two arabinose units and the rhamnose residue (δH 5.60 (d, J = 4.7 Hz), 5.36 (br s), and 5.16 (br s), respectively) were consistent with an alpha configuration.21,22 The saccharide sequences and linkage sites were obtained from TOCSY, HMBC, and ROESY experiments (Figures S11, S13, S15, Supporting Information). The occurrence of the esterified saccharide substituent at C-28 was inferred from the following significant C−H correlations obtained in the HMBC spectrum: H-1 (δH 5.60) of Ara with C-28 (δC 177.4) of the aglycone; H-1 (δH 5.16) of Rha with C-2 (δC 75.4) of Ara; H-1 (δH 4.51) of Xyl with C-3 (δC 81.9) of Rha; and H-1 (δH 5.36) of the terminal Ara with C-4 (δC 79.6) of Rha. Further supporting evidence came from the ROESY analysis, as illustrated in Figure 2. The sequence of the disaccharide chain at the C-3 position was indicated by the long-range coupling in the HMBC spectrum of H-1 (δH 4.46) of Glc with C-4 (δC 81.6) of GlcNAc as well as the key correlation between H-1 (δH 4.49) of GlcNAc with C-3 (δC 91.7) of the aglycone, which further supported the attachment of the disaccharide moiety at C-3 of the aglycone. The 1H NMR spectrum of 1 also showed a signal for an acetyl methyl group (δH 2.09). HMBC correlations between H-5 of the furanosyl Ara and the carbonyl carbon (δC 173.0) of this acetyl group indicated that it was attached to the C-5 oxygen of the Ara saccharide unit. The types of glycosidic linkages were confirmed by permethylation analysis and GC-MS. Based on the MS fragmentation pattern of partially methylated alditol acetates (Figure S26, Supporting Information), the glycosidic linkages in the samples were identified to be terminal arabinofuranose (tAraf), terminal xylopyranose (t-Xylp), terminal glucopyranose (t-Glcp), 2-linked arabinopyranose (2-Arap), 3,4-linked rhamnopyranose (3,4-Rhmp), and 4-linked N-acetylglucosaminopyranose (4-GlcNAcp). Due to the experimental conditions during the methylation (strong alkaline environment caused by the presence of sodium hydroxide), the acetyl group attached to the t-Araf residue of compound 1 was cleaved off during the analysis. The deacetylated compounds 1 and 2 had the same glycosidic linkage types (Figure S27, Supporting Information). The complete structure of 1 was assigned as 3-O-β-Dglucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosylechinocystic acid 28-O-(5-O-acetyl)-α-L-arabinofuranosyl(1→4)-[β-D-xylopyranosyl-(1→3)]-α-L-rhamnopyranosyl-(1→ 2)-α-L-arabinopyranosyl ester (ligulataside A). Compound 2 was obtained as a white, amorphous solid. The molecular formula, C65H105NO30, was deduced from the HRESIMS data (m/z 1380.6851 [M + H]+, calcd 1380.6794) (Figure S19, Supporting Information), which was 42 mass units less than 1. The 1D NMR data for 2 indicated this compound displays identical 1H NMR and 13C NMR signals as 1, except for the chemical shifts assigned to C-3, C-4, and C-5 of the terminal Ara in 1 (δC 79.6, 82.4, and 65.8 ppm in 1 and δC 85.7, 79.0, and 63.5 ppm in 2) as well as the attached protons (δH 3.83, 4.14, and 4.32, 4.18 ppm in 1 compared to δH 4.02, 3.88,

and 3.76, 3.67 ppm in 2). Further, the chemical shifts of the acetyl group of 1 at δC 21.2, 173.0 and δH 2.09 ppm could not be observed for 2. LC-MS/MS analysis of 2 in the positive-ion mode gave similar sequential loss of saccharide masses to those observed for 1. The mass loss of m/z 42 corresponding to the acetyl unit seen for 1 (Figure S18, Supporting Information) was also absent (Figure S20, Supporting Information), which suggested that 2 is a deacetylated derivative of 1. GC-MS and linkage analysis of the hydrolyzed compound indicated echinocystic acid as the aglycone and the type of saccharides and their linkages to be identical to 1 (Figures S22−S27, Supporting Information). Accordingly, the complete structure of 2 was determined as 3-O-β-D-glucopyranosyl-(1→4)-2acetamido-2-deoxy-β-D-glucopyranosylechinocystic acid 28-Oα-L-arabinofuranosyl-(1→4)-[β-D-xylopyranosyl-(1→3)]-α-Lrhamnopyranosyl-(1→2)-α-L-arabinopyranosyl ester (ligulataside B). The cytotoxicity of the two purified ligulatasides was tested against the SK-MEL28 cells using the sulforhodamine B (SRB) assay to determine the IC50 values of the pure compounds. Compounds 1 and 2 gave IC50 values of 25.0 and 15.0 μM, respectively. These values indicated a lack of significant cytotoxicity (IC50 > 10 μM). The compounds were further tested against noncancerous diploid human foreskin fibroblast (HFF) cells. It was shown that the activity toward the HFF cells was similar to the activity against SK-MEL28 cells, with IC50 values of 13.4 and 14.2 μM for 1 and 2, respectively. This suggested no selectivity between the cancerous and noncancerous cell lines tested. The most common aglycone for saponins found in Acacia sensu stricto has been acacic acid, which is structurally related to echinocystic acid, except for the presence of an additional glycosylation site. Acacic acid-based saponins are not restricted to the genus Acacia, but have been found in the closely related genera Albizia, Entada, and Archidendron from the Mimosoideae subfamily of the Fabales.23 To the best of our knowledge echinocystic acid-based saponins have only been described in Acacia sensu stricto from Acacia pulchella,24 but the complete structures of any saponins from this species have not been published. Saponins also consisting of echinocystic acid and with N-acetylglucosamine attached at C-3 similar to ligulatasides A (1) and B (2) have been found in Albizia.25−28 According to recent molecular studies, the genera Albizia and Acacia sensu stricto are closely related.29 In contrast to acacic acid-based saponins, echinocystic acid-based triterpenoid saponins have been found to be more widely distributed throughout the kingdom Plantae in families such as Campanulaceae, Asteraceae, and Sapindaceae30−32 and are not restricted to the Mimosoideae. In conclusion, we here report the first compounds isolated and identified from A. ligulata and the first fully elucidated echinocystic-type triterpenoid saponins from Acacia sensu stricto.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on an Anton Paar MCP 100 polarimeter at 25 °C. UV spectroscopy was conducted using an Agilent Technologies Cary 6 UV−vis spectrophotometer. The 1D and 2D NMR data were acquired on a Varian INOVA 600 MHz spectrometer in CD3OD (SigmaAldrich, St Louis, MO, USA). Chemical shifts in the NMR spectra were assigned by reference to the signals from the residual solvents. LC-MS/MS was performed on a Dionex UltiMate 3000 Quaternary Rapid Separation UHPLC+ focused system (Thermo Fisher Scientific, 2696



Article

Germering, Germany). Separation was achieved on a Kinetex 1.7 μm XB-C18 column (100 × 2.1 mm, 1.7 μm, 100 Å, Phenomenex). For eluting 0.05% (v/v) formic acid in H2O and MeCN [supplied with 0.05% (v/v) formic acid] were employed as mobile phases A and B, respectively. Gradient conditions were as follows: 0.0−1.0 min 10% B; 1.0−7.0 min 10−40% B; 7.0−20.0 min 40−80% B, 20.0−26.0 min 80% B, 26.0−27.0 min 80−100%, 27.0−30.0 min 100% B, 30.0−31.0 min 100−10% B, and 31.0−34.0 min 10% B. The flow rate of the mobile phase was 300 μL/min. The column temperature was maintained at 30 °C. UV spectra for each sample were acquired at 205, 210, 250, and 310 nm. The UHPLC was coupled to a Compact micrOTOF-Q mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ion source (ESI) operated in the positive or negative ionization modes. The ion spray voltage was maintained at 4500 or 3900 V in the positive and negative ionization modes, respectively. The dry temperature was set to 200 °C, and the dry gas flow was set to 8 L/min. Nitrogen was used as the dry gas, nebulizing gas, and collision gas. The nebulizing gas was set to 2.5 bar and collision energy to 15 eV. Na-formate clusters were used for calibration. HRESIMS and MS/MS spectra were acquired in a m/z range from 50 to 3000 amu at a sampling rate of 3 Hz. All files were automatically calibrated by postprocessing. The free echinocystic acid fraction and acidic and alkaline hydrolyzed fractions were analyzed using GC-MS. The GC-MS consisted of an Agilent 7890A GC and an Agilent 5975C series MSD (Agilent Technologies, Glostrup, Denmark). GC separation was performed on an Agilent HP-5MS column (30 m × 250 μm × 0.25 μm) using hydrogen as carrier gas at a constant flow rate of 1.2 mL/min. Details of sample preparation and experimental conditions for the GC-MS are provided in the Supporting Information. All solvents used for extractions from plant tissues and separations were analytical grade (Merck, Darmstadt, Germany, and Univar, Ajax Finechem, Auckland, New Zealand) or HPLC grade (Merck) for HPLC separations. TLC plates (normal-phase silica gel 60 F254) were from Merck. Silica gel 60 (70−230 mesh/0.063−0.200 mm, Merck), silica gel 60 (2−25 μm, Sigma-Aldrich), and silica gel 60 (230−400 mesh/0.040−0.063 mm, Merck) were used for solid-phase extraction (SPE), radial centrifugal thin-layer chromatography (TLC), and column chromatography, respectively. All HPLC experiments were carried out on a Shimadzu SIL 20-AHT UFLC system with an autoinjector using a Phenomenex Gemini 3 μm NX-C18 column (250 mm × 4.40 mm, 3 μm, 110 Å, Phenomenex, Torrance, CA, USA). Plant Material. Mature pods of Acacia ligulata were collected in the Southern Flinders Ranges, South Australia, in December 2013. The pods were collected from more than 20 individual trees. A voucher specimen was recorded and lodged at the State Herbarium of South Australia (AD271669), and species identity authenticated by botanist Mr. Martin O’Leary. Fresh plant material was stored at −20 °C until used. Extraction and Isolation. A. ligulata mature pods (120.9 g, separated from seeds) were ground and extracted at room temperature in 80% (v/v) aqueous EtOH (Univar, Ajax Finechem, Auckland, New Zealand) using a solvent to plant material ratio of 5:1. After 24 h, the extract was decanted and filtered through a Whatman No. 1 filter paper (GE Health Life Sciences, UK). A second equivalent portion of 80% (v/v) EtOH was added to the plant material and allowed to extract for an additional 24 h before being decanted and filtered. The filtered extract was concentrated using a rotary evaporator (Buchi, Switzerland) with a water bath temperature of 40 °C, then freeze-dried (Christ Alpha 2-4LD). The dried extract was stored at −20 °C until further fractionation. An initial separation of 10 g of the crude extract was performed by normal-phase solid-phase extraction (NP-SPE) using a 150 mL cartridge (⦶ 3.8 cm, length 15 cm, Grace Davison Discovery Sciences, Deerfield, IL, USA) and silica gel 60 (0.063−0.200 mm, Merck). The column was attached to a vacuum manifold for each separation step. The compounds were eluted with increasing amounts of MeOH in an EtOAc/MeOH system (90 mL 0%, 90 mL 20%, 240 mL 40%, 90 mL 60%, 90 mL 80%, and 150 mL 100%) with collection of 10 mL fractions. Based on TLC analysis the eluted fractions were pooled into

larger fractions (ALP1−7), of which each was tested in the SRB assay. Fractions ALP4 (1130.2 mg), ALP5 (1155.7 mg), and ALP6 (4017.7 mg) all showed activity against the SK-MEL28 cells. Fraction ALP5 was further separated by radial centrifugal thin-layer chromatography using a Chromatotron centrifugal thin-layer chromatograph (T-squared Technology, San Bruno, CA, USA) with a 1 mm silica plate (silica gel 60, 2−25 μm, Sigma-Aldrich). Fractions were eluted from the plate using increasing amounts of MeOH in a CH2Cl2/MeOH, 0.1% (v/v) acetic acid system (100 mL per concentration increasing from 0 to 25% MeOH in 5% increments, then 200 mL 30%), with the collection of approximately 5 mL fractions. The remaining compounds were washed off with 100% acetone. Based on the TLC analysis of every second fraction, fractions were pooled into 10 larger fractions (ALP5.1−10). Fraction ALP5.10 (266 mg) showed the most potent activity against SK-MEL28 cells. The TLC analysis indicated that fraction ALP5.10 consisted of more than one compound, and a further purification using a short bed column was performed. A short glass column (⦶ 10 mm, packing height 200 mm) was wet loaded (in CH2Cl2) with Merck silica (0.040−0.063 mm, Merck, Darmstadt, Germany). The compounds were eluted with increasing amounts of MeOH in a CH2Cl2/MeOH, 0.1% (v/v) acetic acid system (100 mL per concentration increasing from 0 to 25% MeOH in 5% increments and 200 mL 70%) with approximately 5 mL fractions being collected. The remaining compounds were washed off with 100% MeOH. A TLC analysis of every second fraction from the column showed that at least three major compounds were eluted from ALP5.10. A final purification by reversed-phase HPLC using a mobile phase of CH3CN/H2O (3:7) with 0.1% (v/v) formic acid was performed with manual collection of eluting peaks. This yielded 13.8 mg of compound 1 as a white, amorphous solid, 15.6 mg of compound 2 as a white, amorphous solid, and minor fractions of other impure compounds. Ligulataside A (1): white, amorphous solid; [α]25 D −17.7 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 261 (2.21) nm (sh); 1H and 13C NMR data (CD3OD, 600 MHz), see Table 1 and Table 2; HRESIMS m/z 1422.6950 [M + H]+ (calcd for C67H107NO31, 1422.6900). Ligulataside B (2): white, amorphous solid; [α]25 D −32.7 (c 0.16, MeOH); UV (MeOH) λmax (log ε) 279 (2.57) nm; 1H and 13C NMR data (CD3OD, 600 MHz), see Table 1 and Table 2; HRESIMS m/z 1380.6851 [M + H]+ (calcd for C65H105NO30, 1380.6794). Acid and Alkaline Hydrolysis. In order to identify the aglycone and the attached saccharides of the saponins in A. ligulata pod extracts, HPLC-purified saponin fractions were subjected to acid and alkaline hydrolysis. Details of these procedures are given in the Supporting Information. Methylation-GC-MS Analysis. Pure compounds 1 and 2 (∼0.5 mg) were subjected to glycosidic linkage analysis by permethylation and GC-MS. The compounds were consecutively methylated in the presence of iodomethane and sodium hydroxide in dimethyl sulfoxide, hydrolyzed with trifluoroacetic acid, reduced with sodium borodeuteride, and peracetylated using acetic anhydride to produce partially methylated alditol acetates (PMAAs), as described previously.33 PMAAs from neutral saccharides and amino saccharides were analyzed on an Agilent 6890/5973 GC-MS system (Agilent Technologies, Santa Clara, CA, USA) fitted with an SP-2380 capillary column (30 m × 0.25 mm i.d., Sigma-Aldrich) and an Agilent CP-Sil 5 CB capillary column (30 m × 0.25 mm i.d.; Agilent Technologies), respectively.34 Experiments were conducted in duplicate. Cytotoxicity Assays. The SK-MEL28 human melanoma cancer cell line (ATCC HTB-72) (ATCC, Manassas, VA, USA) was cultured in RPMI-1640 GlutaMAX medium (Gibco, ThermoFisher, Australia) supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo Scientific, MA, USA). The diploid human fibroblast cell line HFF (ATCC SCRC-1041) was cultured in DMEM (SA Pathology, Adelaide, Australia) supplemented with 2 mM glutamine and 10% (v/v) FBS (Thermo Scientific). All cells were cultured in humidity at 37 °C with 5% CO2. The cytotoxic activity of fractions and compounds was measured using a sulforhodamine B-based cell viability assay kit (TOX6, SigmaAldrich). Cells were distributed in 96-well plates (Sarstedt, Australia) 2697



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at a density of 2 × 105 cells/mL and preincubated for 24 h. The following day, cells were treated with fractions or compounds in various concentrations and incubated for 48 h. Controls for cell growth (without treatment) in the maximum DMSO concentration (0.4% for all assays), fraction absorbance controls of all concentrations, and medium controls were also included. A positive control of staurosporine as a known growth inhibitor was also included. After 48 h, the cells were fixed and stained following the manufacturer’s protocol. Absorbance was measured using an Ascent Multiskan plate reader (Labsystems, Helsinki, Finland) at 540 nm and at 690 nm (background). The concentration of each fraction or pure compound that inhibited cell viability by 50% compared to the control (IC50) was determined by dose−response curve fitting using the GraphPad Prism software (version 6 for Mac OS X, GraphPad, San Diego, CA, USA).

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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.7b00437. Experimental details, GC-MS analysis, the acid and alkaline hydrolysis of saccharides, NMR spectra, mass spectra, GC-MS traces, and MS fragmentation pattern of PMAAs (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +61 8 830 22395. Fax: +61 8 830 21087. E-mail: Susan. [email protected]. ORCID

Susan J. Semple: 0000-0001-5988-3993 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially funded through a Sansom Institute for Health Research, University of South Australia, Collaborative Grant and University of South Australia Scholarship to D.J. B. Dueholm is thanked for his assistance with the collection of plant material. P. Clements (University of Adelaide) is thanked for running the NMR experiments. H. C. Lam and A. Day (University of Adelaide) are thanked for assistance in measuring the specific rotation.

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REFERENCES

(1) Maslin, B. Gard. Bull. Singapore 2015, 67, 231−250. (2) Haridas, V.; Higuchi, M.; Jayatilake, G. S.; Bailey, D.; Mujoo, K.; Blake, M. E.; Arntzen, C. J.; Gutterman, J. U. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 5821−5826. (3) Jayatilake, G. S.; Freeberg, D. R.; Liu, Z.; Richheimer, S. L.; Blake, M. E.; Bailey, D. T.; Haridas, V.; Gutterman, J. U. J. Nat. Prod. 2003, 66, 779−783. (4) Mujoo, K.; Haridas, V.; Hoffmann, J. J.; Wächter, G. A.; Hutter, L. K.; Lu, Y.; Blake, M. E.; Jayatilake, G. S.; Bailey, D.; Mills, G. B. Cancer Res. 2001, 61, 5486−5490. (5) Seigler, D. S. Conservation Sci. Western Australia 2002, 4, 109− 116. (6) Chapman, A.; Maslin, B. Nuytsia 1992, 8, 249−283. (7) Webb, L. J. Mankind 1969, 7, 137−146. (8) Barr, A.; Chapman, J.; Smith, N.; Wightman, G.; Knight, T.; Mills, L.; Andrews, M.; Alexander, V. Traditional Aboriginal Medicines in the Northern Territory of Australia by Aboriginal Communities of the Northern Territory; Conservation Commission of the Northern Territory of Australia: Darwin, Australia, 1993. 2698


Isolation and Structural Characterization of Echinocystic Acid

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