Effects of Oleanane-Type Triterpene Saponins from the Leaves of

Jul 11, 2016 - Subsequent bioassay-guided fractionation afforded seven new oleanane-type triterpene saponins, ezoukoginosides A–G (1–7), along wit...
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Effects of Oleanane-Type Triterpene Saponins from the Leaves of Eleutherococcus senticosus in an Axonal Outgrowth Assay Yue-Wei Ge,† Chihiro Tohda,‡ Shu Zhu,† Yu-Min He,† Kayo Yoshimatsu,§ and Katsuko Komatsu*,† †

Division of Pharmacognosy and ‡Division of Neuromedical Science, Institute of Natural Medicine, University of Toyama, Toyama 930-0194, Japan § Research Center for Medicinal Plant Resources, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki 567-0085, Japan S Supporting Information *

ABSTRACT: An aqueous extract of Eleutherococcus senticosus leaves exerted a beneficial effect in restoring the neurite outgrowth from Aβ25−35-induced degeneration using an axonal density assay. Subsequent bioassay-guided fractionation afforded seven new oleanane-type triterpene saponins, ezoukoginosides A−G (1−7), along with nine known analogues. The structures of 1−7 were elucidated through chemical and spectroscopic approaches, and their effects on restoring the neurite outgrowth from Aβ25−35-induced degeneration were investigated. The results revealed that hydrophilic oleanane-type saponins substituted with a free carboxylic acid, hydroxy, or formyl group in the aglycone, especially when the oxidation occurred at C-29, not only restrained Aβ25−35-induced degeneration but also restored axonal outgrowth significantly. Compounds 2 (−COOH at C-29) and 3 (−CH2OH at C-29) showed the most potent bioactivity among the isolates.

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Araliaceae.13 The rhizomes and roots of E. senticosus are recorded in the Chinese and Japanese pharmacopoeias as a treatment for neurasthenia, hypertension, chronic coughing, and ischemic heart disease. Our previous studies have shown that a water extract of the rhizomes of E. senticosus had protective activity against Aβ25−35-induced axonal atrophy, and the active constituents were identified as eleutheroside B, eleutheroside E, and isofraxidin.9,14 Moreover, multiple bioactivities such as antibacterial and glucosidase inhibitory effects of E. senticosus leaves have been reported.15−18 Phytochemical investigation revealed that the major constituents in the leaves of this plant are triterpene saponins, caffeoylquinic acids, flavonoids, and polysaccharides, which are different from the rhizome constituents.19 The neuron-related bioactivity of E. senticosus leaves has not been investigated so far. The aim of the present study was to investigate the effect of an extract from E. senticosus leaves on Aβ25−35-induced neurite degeneration. A bioassay-guided isolation procedure was performed on the active constituents, and several bioactive triterpene saponins were elucidated.

lzheimer’s disease (AD) is the most common neurodegenerative disease and is characterized by a decline in the memory and other cognitive functions of patients. Epidemiological surveys have indicated that 50−60% of individuals older than 85 years old may develop AD.1 The number of patients suffering from AD worldwide is expected to increase from 30.8 million in 2010 to more than 106.2 million in 2050.2 The current therapies for AD mainly rely on cholinesterase inhibitors (rivastigmine, galantamine, and donepezil) and memantine, which have weak beneficial effects on cognitive function.3 The discovery of new drugs or lead compounds from natural source for AD is urgently required.4 The generation of AD is known to be associated with some abnormal protein deposits including senile plaques that are formed by the β-amyloid (Aβ) peptides. These Aβ peptides are the sequential endoproteolytic cleavage products of amyloid precursor proteins, and Aβ1−40 and Aβ1−42 are the major components of pathogenic plaques.5 Aβ25−35, corresponding to a fragment of Aβ1−40 and Aβ1−42, is a synthetic peptide of 11 amino acids retaining both the physical and biological properties of full-length Aβ,6,7 and it has been adopted to represent the pathogenic behavior of Aβ in the drug screening for new AD lead compounds.8−12 Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. (synonymous with Acanthopanax senticosus), also known as “Siberian Ginseng” (English), “Ciwujia” (Chinese), or “Ezoukogi” (Japanese), is a species of woody shrub in the family © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 13, 2016

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DOI: 10.1021/acs.jnatprod.6b00329 J. Nat. Prod. XXXX, XXX, XXX−XXX

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and olefinic (1635 cm−1) groups. Analysis by HPLC-MS2 showed a loss of 512 Da, corresponding to a characteristic oligosaccharide chain composed of two gluconosyl units, a rhamnosyl unit, and a C-6 acetyl-substituted glucosyl unit.19,23 Another neutral loss of 146 and 132 Da occurred in an HPLCMSn (n = 3−4) experiment, suggesting the presence of a deoxy sugar moiety (146 Da) and a pentose moiety (132 Da). Acid hydrolysis of 1 yielded a mixture of sugars that were identified as D-glucose, L-rhamnose, and L-arabinose, as well as an aglycone that was identified as settatagenic acid by comparing its NMR data with values from the literature.22 The 1H and 13C NMR spectroscopic data of 1 (Tables 1−3) exhibited signals for seven methyl groups (δH 0.89, 1.08, 1.10, 1.16, 1.24, 1.48, and 1.93; δC 15.5, 17.3, 16.8, 27.9, 25.8, 19.8, and 20.4), an olefinic group (δH 5.46, 1H, br s; δC 122.9), an oxymethine group (δH 3.24, dd, J = 12.0, 4.0 Hz; δC 88.1), a free carboxylic acid carbon (δC 181.0), and two acyl carbons (δC 176.0 and 170.4). Five anomeric proton signals at δH 4.91 (d, J = 4.0 Hz), 6.16 (br s), 6.29 (d, J = 8.0 Hz), 5.00 (d, J = 7.5 Hz), and 5.55 (br s) were observed, which were correlated in the HMQC spectrum with anomeric carbons at δC 104.7, 101.5, 95.4, 104.7, and 102.7, respectively. The assignment of the proton and carbon signals on the basis of the DEPT, HMQC, and HMBC spectra supported the presence of an α-arabinopyranosyl, two α-rhamnopyranosyl, and two β-glucopyranosyl moieties, in agreement with the acid hydrolysis results. Two sugar chains were characterized as α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl (β-chain) and α-L-rhamnopyranosyl-(1→4)-6-Oacetyl-β- D -glucopyranosyl-(1→6)-β- D -glucopyranosyl (αchain), respectively, based on the correlations between δH 6.16 (Rha-H-1) and δC 75.6 (Ara-C-2), between δH 5.55 (Rha′-H-1) and δC 78.9 (Glc″-C-4), and between δH 5.00 (Glc″-H-1) and δC 69.3 (Glc′-C-6) in the HMBC spectrum. An acetyl group (δH 1.93, δC 20.4, 170.4) was confirmed as a substituent at C-6 of the outer glucopyranosyl unit of the βchain due to the cross-peaks from δH 4.54 and 4.64 (Glc′-H-6) to the acyl carbon (δC 170.4) in the HMBC spectrum. The linkages between the sugar chains and the aglycone were confirmed by the correlations between δH 4.91 (Ara-H-1) and δC 88.1 (C-3) and between δH 6.29 (Glc′-H-1) and 176.0 (C28) in the HMBC spectrum, which suggested the β-chain is attached at C-3 and the α-chain is attached at C-28. The free carboxylic acid group (C-29) was determined as being in an equatorial conformation (α) from the chemical shift of the axial (β, C-30) methyl group signal that was deshielded and appeared at δC 19.8.24 In contrast, an equatorial (α, C-29) methyl group signal would appear at approximately δC 26 with an axial (β, C-30) carboxylic acid group.25 Accordingly, 1 was assigned as 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-3β-hydroxyolean-12-ene-28,29-dioic acid 28-O-α-L-rhamnopyranosyl-(1→4)-6-O-acetyl-β-D-glucopyranosyl-(1→6)-β-Dglucopyranosyl ester. Ezoukoginoside B (2) was obtained as an amorphous powder, and its elemental formula of C55H86O24 was evident on the basis of a negative HRESIMS [M − H]− ion peak at m/z 1129.5437 (calcd 1129.5436). Acid hydrolysis of 2 suggested the presence of a sugar mixture containing D-glucose, Lrhamnose, and L-arabinose. The HPLC-MSn (n = 1−3) experiment showed fragment ions at m/z 617 [M − H − 512]− and 411 [M − H − 512 − 44 − 132]−, corresponding to the loss of a sugar chain [-Glc-Glc(Ac)-Rha], a carbon dioxide molecule, and an arabinosyl unit. The 1H and 13C NMR spectroscopic data of 2 (Tables 1−3) showed similar

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RESULTS AND DISCUSSION A preliminary experiment revealed that a hot water extract of E. senticosus leaves restrained the Aβ25−35-induced degeneration of neurites in cultured cortical neurons from the embryos of a pregnant ddY mouse and promoted axonal outgrowth. To determine the axonal regenerative constituents, a bioassayguided extraction and fractionation procedure on the dried E. senticosus leaves was conducted (Figure S1, Supporting Information). The hot water (85 °C) extract showed greater bioactivity than the 50% aqueous MeOH extract, from which polysaccharides, caffeoylquinic acids, flavonoids, and triterpene saponins as the major constituents were enriched in four fractions. Among these, the triterpene saponin fraction (TSF) showed promising neurite regeneration activity. Subsequent screening led to the identification of the most active chromatographic subfraction, TSF-1. Purification of this subfraction yielded seven new triterpene saponins, ezoukoginosides A−G (1−7), and tauroside H2 (8).20 Moreover, ciwujianosides B (14),21 C2 (15),21 C3 (9),21 C4 (11),21 and D2 (13),21 acanthopanaoxides B (12)22 and C (16),22 and eleutheroside M (10)21 were also isolated. The structures of compounds 1−7 were determined as follows. Ezoukoginoside A (1) was obtained as an amorphous powder with [α]25 D −20.7 (c 0.1, MeOH). The negative HRESIMS showed a deprotonated molecular ion peak at m/z 1275.6022 [M − H]−, indicating a molecular formula of C61H96O28 (calcd 1275.6015). The IR spectrum showed absorptions for hydroxy (1390 cm−1), carbonyl (1730 cm−1), B

DOI: 10.1021/acs.jnatprod.6b00329 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 13C NMR Data of the Aglycone Moieties of Compounds 1−7a 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 a

1 38.7, 26.4, 88.1, 39.3, 55.7, 18.3, 32.9, 39.7, 47.8, 36.8, 23.6, 122.9, 143.4, 41.9, 28.0, 23.2, 46.8, 40.6, 40.6, 42.2, 28.9, 31.5, 27.9, 16.8, 15.5, 17.3, 25.8, 176.0, 181.0, 19.8,

2 CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH C C CH2 CH2 C CH CH2 C CH2 CH2 CH3 CH3 CH3 CH3 CH3 C C CH3

38.6, 26.5, 88.4, 39.4, 55.6, 18.3, 32.9, 39.7, 47.8, 36.8, 23.6, 122.9, 143.4, 41.9, 28.0, 23.3, 47.0, 40.8, 40.8, 42.2, 28.9, 31.6, 28.0, 16.8, 15.4, 17.3, 25.8, 176.1, 181.2, 19.8,

3 CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH C C CH2 CH2 C CH CH2 C CH2 CH2 CH3 CH3 CH3 CH3 CH3 C C CH3

38.7, 26.3, 88.6, 39.8, 55.7, 18.3, 32.9, 40.2, 48.4, 36.8, 23.6, 123.7, 144.6, 41.9, 28.1, 23.2, 46.8, 40.7, 40.9, 36.2, 28.6, 31.9, 27.9, 16.8, 15.5, 17.3, 25.9, 175.9, 73.7, 19.6,

4 CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH C C CH2 CH2 C CH CH2 C CH2 CH2 CH3 CH3 CH3 CH3 CH3 C CH2 CH3

38.7, 26.6, 88.5, 39.5, 55.8, 18.4, 33.3, 39.9, 47.0, 36.9, 23.7, 122.5, 144.3, 41.9, 35.7, 73.8, 49.0, 41.1, 47.0, 30.6, 35.9, 32.1, 27.9, 16.8, 15.6, 17.4, 27.1, 175.8, 33.0, 24.4,

5 CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH C C CH2 CH C CH CH2 C CH2 CH2 CH3 CH3 CH3 CH3 CH3 C CH3 CH3

38.8, 26.3, 88.7, 39.3, 55.7, 18.3, 32.9, 39.7, 47.8, 36.8, 23.6, 123.1, 142.9, 41.8, 27.9, 23.1, 46.7, 39.9, 36.7, 46.1, 25.6, 30.6, 27.9, 16.8, 15.4, 17.3, 25.8, 175.7, 205.5, 16.3,

6 CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH C C CH2 CH2 C CH CH2 C CH2 CH2 CH3 CH3 CH3 CH3 CH3 C CH CH3

38.9, 26.5, 89.2, 39.9, 55.9, 19.1, 33.0, 39.5, 48.0, 37.5, 23.8, 123.1, 143.6, 42.0, 28.1, 23.3, 46.9, 40.4, 37.0, 46.3, 25.7, 30.7, 28.1, 17.0, 15.6, 17.4, 26.0, 176.5, 205.7, 16.5,

7 CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH C C CH2 CH2 C CH CH2 C CH2 CH2 CH3 CH3 CH3 CH3 CH3 C CH CH3

38.8, 26.7, 88.6, 39.6, 55.8, 19.0, 33.1, 39.9, 48.0, 37.0, 23.8, 123.1, 143.2, 42.0, 28.2, 23.3, 46.9, 40.0, 37.0, 46.3, 25.8, 30.7, 28.2, 17.0, 15.6, 17.5, 26.0, 176.1, 205.7, 16.5,

CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH C C CH2 CH2 C CH CH2 C CH2 CH2 CH3 CH3 CH3 CH3 CH3 C CH CH3

Recorded in pyridine-d5 at 125 MHz. The assignments were based on DEPT, HMQC, and HMBC NMR experiments.

of 1 on the basis of the DEPT, HMQC, and HMBC spectra. Finally, 3 was identified as 3-O-α-L-rhamnopyranosyl-(1→2)-αL-arabinopyranosyl-3β-29-hydroxyolean-12-en-28-oic acid 28O-α-L-rhamnopyranosyl-(1→4)-6-O-acetyl-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester. Ezoukoginoside D (4) was obtained as an amorphous powder, and its molecular formula of C59H96O27 was suggested by the negative HRESIMS [M − H]− ion peak at m/z 1235.6039 (calcd 1235.6066). Acid hydrolysis of 4 yielded a sugar mixture of D-glucose, L-rhamnose, and L-arabinose. The HPLC-MSn (n = 1−4) experiment showed fragments at m/z 765 [M − H − 470]−, 603 [M − H − 470 − 162]−, and 471 [M − H − 470 − 162 − 132]−, supporting the presence of a sugar chain [-Glc-Glc-Rha], an L-arabinosyl unit, and an Lrhamnosyl unit. Comparing the 1H and 13C NMR spectroscopic data of 4 (Tables 1−3) with those of 3, the H-27 signal (δH 1.82) of 4 was obviously deshielded, while downfield shifts were observed for H-16 and C-16 (δH 5.28, δC 73.8), C-15 (δC 35.7), and C-17 (δC 49.0), which suggested the substitution of a hydroxy group at C-16 of the aglycone as in echinocystic acid, which was identified by comparing the 1H and 13C NMR data of 4 with values from the literature.20,27,28 The anomeric carbon of an arabinosyl unit was downfield shifted to δC 107.2, and the Ara-C-3 was deshielded and appeared at δC 83.9, which supported a sugar moiety being linked at Ara-C-3.27 This linkage was further determined by the correlation between δH 4.19 (Ara-H-3) and δC 106.2 (Glc-C-1) in the HMBC spectrum. Thus, 4 was identified as 3-O-β-D-glucopyranosyl-

resonances to those of 1, apart from the lack of a rhamnosyl unit. When compared with 1, a downfield shift of Ara-C-1 (δC 107.3, +2.6 ppm) and an upfield shift of Ara-C-2 (δC 72.6, −3.0 ppm) were observed, which supported the lack of a sugar moiety linked at Ara-C-2. Further assignments of the signals of an arabinosyl moiety suggested there was no extended sugar unit linked with it.21 Accordingly, 2 was determined to be 3-Oa-L-arabinopyranosyl-3β-hydroxyolean-12-ene-28,29-dioic acid 28-O-α-L-rhamnopyranosyl-(1→4)-6-O-acetyl-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester. Ezoukoginoside C (3) was obtained as an amorphous powder. The negative HRESIMS showed a deprotonated molecular ion peak at m/z 1261.6216 [M − H]−, indicating the molecular formula to be C61H98O27 (calcd 1261.6223). The presence of an oligosaccharide chain [-Glc-Glc(Ac)-Rha], a deoxy sugar unit, and a pentose unit was suggested by fragment ions at m/z 749 [M − H − 512]−, 603 [M − H − 512 − 146]−, and 471 [M − H − 512 − 146 − 132]− in an HPLC-MSn (n = 1−4) experiment. Acid hydrolysis of 3 yielded a sugar mixture containing D-glucose, L-rhamnose, and L-arabinose. The 1H and 13 C NMR spectra of 3 (Tables 1−3) revealed signals for seven methyl groups, an olefinic group, an oxymethine group, and two acyl carbons. The HMBC spectrum of 3 showed a crosspeak from the δH 1.11 (s, H-30) to a methylene at δC 73.7, which indicated a hydroxy group substituted at C-29. The aglycone of 3 was identified as mesembryanthemoidigenic acid by comparing its NMR data with values from the literature.26 The sugar chains of 3 were determined to be the same as those C

DOI: 10.1021/acs.jnatprod.6b00329 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 13C NMR Data of the Sugar Moieties of Compounds 1−7a position 3-OAra 1 2 3 4 5 Rha 1 2 3 4 5 6 Glc 1 2 3 4 5 6 28-OGlc′ inner 1 2 3 4 5 6 Glc″ outer 1 2 3 4 5 6 Rha′ 1 2 3 4 5 6 CH3CO CH3CO a

1 104.7, 75.6, 73.7, 68.5, 64.5, 101.5, 72.2, 72.4, 73.9, 69.7, 18.4,

95.4, 73.6, 78.6, 70.7, 77.8, 69.3, 104.7, 74.9, 76.1, 78.9, 73.7, 63.4, 102.7, 72.2, 72.5, 73.6, 70.5, 18.4, 20.4, 170.4,

2 CH CH CH CH CH2 CH CH CH CH CH CH3

CH CH CH CH CH CH2 CH CH CH CH CH CH2 CH CH CH CH CH CH3 CH3 C

107.3, 72.6, 74.5, 69.3, 66.6,

95.5, 73.7, 78.6, 70.8, 77.8, 69.4, 104.7, 74.5, 76.2, 78.9, 73.6, 63.5, 102.8, 72.2, 72.6, 73.7, 70.5, 18.5, 20.6, 170.6,

3 CH CH CH CH CH2

CH CH CH CH CH CH2 CH CH CH CH CH CH2 CH CH CH CH CH CH3 CH3 C

104.6, 75.7, 73.7, 68.5, 64.5, 101.5, 72.2, 72.4, 73.8, 69.7, 18.4,

95.4, 73.6, 78.5, 70.7, 77.8, 69.2, 104.6, 74.9, 76.1, 78.9, 73.6, 63.4, 102.7, 72.2, 72.5, 73.7, 70.4, 18.4, 20.4, 170.4,

4 CH CH CH CH CH2 CH CH CH CH CH CH3

CH CH CH CH CH CH2 CH CH CH CH CH CH2 CH CH CH CH CH CH3 CH3 C

5

107.2, 71.8, 83.9, 69.0, 66.8,

CH CH CH CH CH2

106.2, 75.6, 78.0, 71.4, 78.5, 62.5,

CH CH CH CH CH CH2

95.6, 73.7, 78.5, 70.6, 77.9, 69.0, 104.7, 75.1, 76.3, 78.2, 77.0, 61.1, 102.6, 72.4, 72.6, 73.8, 70.1, 18.4,

CH CH CH CH CH CH2 CH CH CH CH CH CH2 CH CH CH CH CH CH3

6

7

104.7, 75.7, 73.7, 68.6, 64.6, 101.6, 72.2, 72.4, 73.9, 69.7, 18.4,

CH CH CH CH CH2 CH CH CH CH CH CH3

104.9, 75.9, 74.1, 68.7, 64.7, 101.8, 72.4, 72.6, 74.1, 69.9, 18.6,

CH CH CH CH CH2 CH CH CH CH CH CH3

107.6, 72.8, 74.7, 70.0, 66.8,

CH CH CH CH CH2

95.5, 73.6, 78.6, 70.7, 77.9, 69.3, 104.7, 74.9, 76.2, 78.9, 73.7, 63.4, 102.8, 72.2, 72.5, 73.6, 70.5, 18.4, 20.4, 170.4,

CH CH CH CH CH CH2 CH CH CH CH CH CH2 CH CH CH CH CH CH3 CH3 C

95.8, 73.9, 78.7, 70.9, 78.1, 69.3, 104.9, 75.3, 76.5, 78.3, 77.2, 61.3, 102.7, 72.4, 72.6, 73.9, 70.3, 18.6,

CH CH CH CH CH CH2 CH CH CH CH CH CH2 CH CH CH CH CH CH3

95.8, 73.9, 78.8, 70.9, 78.1, 69.6, 105.0, 75.4, 76.6, 78.3, 77.2, 61.3, 102.8, 72.6, 72.8, 73.9, 70.4, 18.6,

CH CH CH CH CH CH2 CH CH CH CH CH CH2 CH CH CH CH CH CH3

Recorded in pyridine-d5 at 125 MHz. The assignments were based on DEPT, HMQC, and HMBC NMR experiments.

and δC 205.5. The cross-peaks between signals of δH 1.12 (H30) and δC 205.5 (C-29), and between δH 9.54 (H-29) and δC 16.3 (C-30), and at 46.1 (C-20) in the HMBC spectrum suggested that the formyl group is located at C-20. The conformation of C-29 was deduced as equatorial by the chemical shift of axial methyl (δC 16.3, C-30).24 The two oligosaccharide chains at C-3 and C-28 were both confirmed to be the same as those of 1 from the DEPT, HMQC, and HMBC spectra. Thus, 5 was identified as 3-O-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl-3β-29-alolean-12-en-28-oic acid 28-O-α-L-rhamnopyranosyl-(1→4)-6-O-acetyl-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester. Ezoukoginoside F (6) was obtained as an amorphous powder. The negative HRESIMS showed a deprotonated molecular ion peak at m/z 1217.5967 [M − H]−, indicating the molecular formula to be C59H94O26 (calcd 1217.5961). Acid hydrolysis of 6 yielded a sugar mixture containing D-glucose, Lrhamnose, and L-arabinose. The HPLC-MSn (n = 1−4) analysis of 6 showed fragments at m/z 747 [M − H − 470]−, 601 [M −

(1→3)-α-L-arabinopyranosyl-3β-16-hydroxyolean-12-en-28-oic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester. Ezoukoginoside E (5) was obtained as an amorphous powder. The negative HRESIMS showed a deprotonated molecular ion peak at m/z 1259.6060 [M − H]−, indicating the molecular formula to be C61H96O27 (calcd 1259.6066). The IR spectrum showed absorptions for hydroxy (3381 cm−1), formyl (1740, 2820 cm−1), and olefinic (1664 cm−1) groups. Acid hydrolysis of 5 yielded a sugar mixture containing D-glucose, Lrhamnose, and L-arabinose. The aglycone was identified as 3βhydroxy-29-alolean-12-en-28-oic acid by comparing its 1H and 13 C NMR data with values from the literature.24 An HPLC-MSn (n = 1−4) experiment showed fragments at m/z 747 [M − H − 512]−, 601 [M − H − 512 − 146]−, and 469 [M − H − 512 − 146 − 132]−, suggesting the presence of an oligosaccharide chain [-Glc-Glc(Ac)-Rha], an L-arabinosyl unit, and an Lrhamnosyl unit. The 1H and 13C NMR spectroscopic data of 5 (Tables 1−3) showed signals of a formyl group at δH 9.54 (s) D

DOI: 10.1021/acs.jnatprod.6b00329 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 3. Selected 1H NMR Data of Compounds 1−7a position 3 5 12 16 18 23 24 25 26 27 29 30 3-OAra 1 Rha 1 Glc 1 28-OGlc′ inner 1 Glc″ outer 1 Rha′ 1 CH3CO

1 3.24 0.76 5.46 2.02 2.23 3.31 1.16 1.10 0.89 1.08 1.24

dd (12.0, 4.0) d (11.5) br s br d (11.0) br d (13.0) dd (12.0, 4.0) s s s s s

2 3.33 0.77 5.46 2.01 2.23 3.31 1.17 1.10 0.89 0.97 1.25

dd (12.0, 5.0) d (11.0) br s br d (10.5) br d (13.0) dd (12.0, 5.0) s s s s s

1.48 s

1.48 s

4.91 d (4.0) 6.16 br s

4.77 d (8.0)

3 3.22 0.76 5.44 1.99 2.20 3.29 1.16 1.08 0.89 1.11 1.26 3.56 1.11

dd (11.5, 4.0) d (11.5) br s br d (11.0) m dd (12.0, 4.0) s s s s s s s

4.90 d (4.5) 6.13 br s

4

5

3.35 0.83 5.57 5.28

dd (11.5, 3.5) (11.5) br s br s

3.47 1.26 0.96 0.89 1.11 1.82 0.96 1.02

dd (13.5, 4.0) s s s s s s s

4.74 (6.5)

3.25 0.77 5.40 2.03 2.30 3.19 1.18 1.11 0.89 1.06 1.21 9.54 1.12

dd (13.5, 3.5) d (11.5) t (3.0) br d (11.0) td (11.0, 2.5) dd (14.0, 4.0) s s s s s s s

6 3.24 0.76 5.44 2.05 2.30 3.16 1.16 1.08 0.87 0.91 1.23 9.55 1.11

7

dd (12.0, 4.0) d (11.5) br s br d (11.0) br d (11.5) dd (14.0, 3.5) s s s s s s

3.33 0.78 5.39 2.18 2.29 3.17 1.18 1.06 0.86 0.95 1.25 9.51 1.07

dd (11.5, 4.0) d (11.5) br s (10.5) br d (11.0) dd (14.0, 3.5) s s s s s s s

4.92 d (5.0) 6.17 br s

4.90 (5.0) 6.18 br s

4.76 d (6.0)

6.26 d (8.0) 5.02b 5.54 br s 1.93 s

6.25 (8.0) 5.01b 5.86 br s

6.24 d (8.0) 5.01b 5.85 br s

5.38 (8.0) 6.29 5.00 5.55 1.93

d (8.0) d (7.5) br s s

6.30 d (8.0) 5.01b 5.55 br s

6.24 5.04 5.53 1.92

d (8.0) d (7.5) br s s

6.24 (8.0) 4.96 (7.5) 5.84 br s

a

Recorded in pyridine-d5 at 500 MHz, J in Hz. The assignments were based on DEPT, HMQC, and HMBC NMR experiments. bSignal partially obscured.

H − 470 − 146]−, and 469 [M − H − 470 − 146 − 132]−, suggesting the presence of an oligosaccharide chain [-Glc-GlcRha], an L-arabinosyl unit, and an L-rhamnosyl unit. The 1H and 13C NMR spectroscopic data of 6 (Tables 1−3) showed similar signals to those of 5, apart from the lack of signals of an acetyl moiety and the upfield shift of Glc″-C-6 (δC 61.3). Finally, 6 was identified as 3-O-α-L-rhamnopyranosyl-(1→2)-αL-arabinopyranosyl-3β-29-alolean-12-en-28-oic acid 28-O-α-Lrhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester. Ezoukoginoside G (7) was obtained as an amorphous powder, and its negative HRESIMS showed a deprotonated molecular ion peak at m/z 1071.5375 [M − H]−, indicating the molecular formula to be C53H84O22 (calcd 1071.5381). Acid hydrolysis of 7 gave a sugar mixture of D-glucose, L-rhamnose, and L-arabinose. An HPLC-MSn (n = 1−4) analysis of 7 showed fragments at m/z 601 [M − H − 470]− and 469 [M − H − 470 − 132] − , suggesting the presence of an oligosaccharide chain [-Glc-Glc-Rha] and an L-arabinosyl unit. The 1H and 13C NMR spectra of 7 were similar to those of 6, apart from the lack of signals of a rhamnopyranosyl unit. Similar to 2, the chemical shift of arabinosyl carbons (δC 107.6, 72.8, 74.7, 70.0, and 66.8) suggested that there were no more extended sugar units linked to it. Accordingly, 7 was identified as 3-O-α-L-arabinopyranosyl-3β-29-alolean-12-en-28-oic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)β-D-glucopyranosyl ester. To investigate the neuron-related bioactivity of the extracts, fractions and isolates from E. senticosus leaves, an axonal density assay was performed to evaluate the effects on restoring axonal outgrowth from Aβ25−35-induced degeneration. As shown in Figure 1a, the Aβ/WE group (water extract, 10 μg/mL) showed positive effects in restraining Aβ25−35-induced toxicity, compared with the Aβ/Veh group. However, two major fractions of the aqueous-soluble extract, the caffeoylquinic

acid fraction and the flavonoid fraction, did not show positive effects. In contrast, the triterpene saponin fraction (Aβ/TSF group) showed a significant effect on attenuating the Aβ25−35induced degeneration, and it was fractionated to afford a hydrophilic fraction, TSF-1, from which compounds 1−8 were isolated and which significantly restored the outgrowth of a neurite from withering. As shown in Figure 1b, compared with the other compounds (8−16), compounds 1−7 showed greater activity, and they not only attenuated Aβ25−35-induced degeneration but also restored axonal outgrowth. On the basis of structural comparison, a common feature of compounds 1−7 is that the aglycones are based on oleanolic acid and have hydrophilic groups, such as a carboxylic acid group (1, 2), a hydroxy group (3, 4), or a formyl group (5−7), especially when oxidation occurred at C-29. Compounds 2 and 3 were found to have the highest activity among all saponins isolated. Thus, these results suggested that the triterpene saponins formed by oleanane-type aglycones substituted with hydrophilic groups are promising lead compounds for the development of anti-AD drugs.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a JASCO DIP-140 digital polarimeter, whereas IR spectra were measured on a JASCO FT/IR-460Plus infrared spectrometer. NMR spectra were obtained using a JEOL JNM-LA500 spectrometer with TMS as reference, and the chemical shifts are indicated as δ values (ppm). HPLC-HRMS measurements were performed on a Thermo LTQ Orbitrap XL mass analyzer. Column chromatography was performed with C18 silica gel (ODS-A and ODS-AQ, YMC, Japan). Preparative HPLC was conducted on an ODS-AQ column (25 × 250 mm, S-5 μm, 12 nm) equipped with a Waters Delta 600 pump and a Waters 2489 UV/visible detector. All solvents used for isolation were of an analytical grade (Wako Pure Chemical Industries, Japan). Plant Material. Fresh leaves of E. senticosus were collected from Fujiyoshida, Yamanashi, Japan, in June 2014. A voucher specimen (S. E

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Figure 1. Effects of E. senticosus extract, fractions (a), and isolates (b) on axonal extension after Aβ25−35-induced atrophy. The cortical neurons were cultured for 3 days, and then the cells were treated with (Veh) or without (Cont) 10 μM Aβ25−35. Three days later, each of the extract, fractions or isolates, NGF (10 ng/mL), or Veh (0.1% DMSO) was added to the neurons. Four days after the treatment, the neurons were fixed and immunostained. The lengths of the pNF-H-positive neurites were measured. The values are the means ± SEMs of the data [*p < 0.05 vs Veh (oneway ANOVA post hoc Dunnett’s test, n = 15−20 photographs)]. Scale bar = 100 μm. WE, water extract; CQAF, caffeoylquinic acid fraction; FLF, flavonoid fraction; TSF, triterpene saponin fraction; 1−16, isolated compounds; IHC, immunohistochemistry. Isoda 201401) is kept at the Museum of Materia Medica, Institute of Natural Medicine, University of Toyama (TMPW). Extraction and Isolation. The dried powder of E. senticosus leaves (268 g) was extracted in hot water (85 °C, 3000 mL) for 30 min, with this stage repeated once. The liquid portion was then combined, filtered, and lyophilized to yield a water-soluble extract (WE). The WE (82.5 g) was fractionated by MPLC on an ODS-AQ-packed column by gradient elution (20 mL/min, aqueous MeCN 2−95% over 60 min) to yield a caffeoylquinic acid fraction (CQAF), a flavonoid fraction (FLF), and a triterpene saponin fraction (TSF). The TSF (11.2 g) was further separated into eight subfractions (TSF-1 to -8) by gradient

elution (10 mL/min, aqueous MeCN 2−95% over 50 min) using preparative HPLC equipped with an ODS-A-packed C18 column. Of these subfractions, TSF-1 (2.1 g) was further separated into nine subfractions (TSF-1-1 to -1-9) by preparative HPLC on a C18 column by gradient elution (10 mL/min, aqueous MeCN 10−55% over 50 min). Compound 1 (24.8 mg) was afforded by TSF-1-1 by preparative HPLC on an ODS-AQ-packed column (4 mL/min, aqueous MeCN 15−28% containing 0.2% acetic acid, over 60 min). Compounds 2 (17.3 mg) and 3 (157.9 mg) were obtained from subfraction TSF-1-4 by preparative HPLC (4 mL/min, aqueous MeCN 15−28% containing 0.2% acetic acid, over 60 min). In turn, compounds 4 F

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pyridine (200 μL), to which 2 mg of L-cysteine methyl ester hydrochloride was added. After stirring the mixture for 1 h at 60 °C, 50 μL of o-tolylisothiocyanate was added, and the mixture was stirred for another 1 h at 60 °C. The reaction mixture was directly analyzed by RP-HPLC that was coupled with a Kinetex PFP column (4.6 × 250 mm, S-5 μm, 12 nm) with the elution of MeCN/H2O (15:85, v/v, 1.0 mL/min) and detected under UV (254 nm). The derivatives of monosaccharides (D-glucose, L-arabinose, and L-rhamnose) in compounds 1−7 were identified by the comparison of their retention times with those of authentic samples (tR: D-glucose 23.9 min, Lglucose 21.4 min, L-arabinose 27.3 min, D-arabinose 30.5 min, Lrhamnose 14.5 min, D-xylose 27.8 min, and L-xylose 25.7 min). Primary Culture. The embryos of a pregnant ddY mouse (Japan SLC, Shizuoka, Japan) were removed on the 14th day of gestation.30 Then, the dura mater was removed from the dissected cerebral cortices. The tissues were minced, dissociated, and cultured with neurobasal medium (Gibco BRL, Rockville, MD, USA) containing 12% horse serum, 0.6% D-glucose, and 2 mM L-glutamine on eight-well chamber slides (Falcon, Franklin Lakes, NJ, USA) coated with 5 μg/ mL poly-D-lysine (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a humidified incubator with 10% CO2. The seeding cell density was 2.94 × 104 cells/cm2. The medium was replaced with the neurobasal medium containing 2% B-27 (Gibco BRL) 3−12 h after neuronal culture. All the above experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the University of Toyama. The approval number for the animal experiments was A2014INM-1. Axonal Density Assay. The neurons were stimulated for 3 days with 10 μM Aβ25−35 (Sigma-Aldrich), which was preincubated previously for 4 days at 37 °C for aggregation. After the stimulation, the Aβ25−35-containing medium was removed, and then the neurons were treated with fresh medium containing E. senticosus extracts, fractions, or isolates and vehicle (0.1% DMSO) for 4 days. The final concentrations of E. senticosus extracts and fractions were set at 10 μg/ mL, and those of the isolates were set at 10 μM. NGF (10 ng/mL; Alomone, Jerusalem, Israel) served as a positive control. After the coculturing, the neurons were fixed with 4% paraformaldehyde for 90 min and were immunostained with a monoclonal antibody against phosphorylated neurofilament-H (1:250, SMI-35, Covance, Dedham, MA, USA) as an axonal marker. A polyclonal antibody against microtubule-associated protein 2 (MAP2, 1:4000, Abcam, Cambridge, UK) was used as a neuronal marker. Alexa Fluor 594-conjugated goat anti-mouse IgG (1:200) and Alexa Fluor 488-conjugated goat antirabbit IgG (1:200) were used as the secondary antibodies (Molecular Probes, Eugene, OR, USA). The nuclear counterstaining was performed using DAPI (1 μg/mL, Sigma-Aldrich). The fluorescent images were captured at 644 μm × 855 μm using a fluorescent microscope system (Cell Observer, Carl Zeiss, Tokyo, Japan), as previously described.30 Ten images were captured per treatment. Ten to 79 photos per treatment were measured using a MetaMorph analyzer (Molecular Devices, Sunnyvale, CA, USA). Statistical Analysis. Statistical comparisons were performed by one-way analysis of variance (ANOVA) with Dunnett’s post hoc test using GraphPad Prism software, version 5 (GraphPad Software, La Jolla, CA, USA). Values of p < 0.05 were considered significant. The data are expressed as the means ± SEM.

(5.1 mg), 6 (4.9 mg), and 7 (4.6 mg) were purified by preparative HPLC from TSF-1-6 with isocratic elution (4 mL/min, 26% aqueous MeCN). Fraction TSF-1-9 was subjected to preparative HPLC with isocratic elution (4 mL/min, 28% aqueous MeCN) to yield compounds 5 (23.8 mg) and 8 (3.9 mg). Fraction TSF-3 was loaded on the C18 preparative HPLC column with a linear gradient (4 mL/ min, 2−100% aqueous MeCN, over 60 min), yielding compounds 14 (7.1 mg) and 16 (14.9 mg). Fraction TSF-5 was subject to preparative HPLC on a C18 column by gradient elution (4 mL/min, aqueous MeCN 20−55%, over 40 min) to yield compounds 9 (8.6 mg), 12 (12.5 mg), and 15 (9.5 mg). Compounds 10 (9.6 mg), 11 (15.6 mg), and 13 (6.5 mg) were purified by preparative HPLC from fraction TSF-6 with gradient elution (4 mL/min, aqueous MeCN 10−50%, over 40 min). Ezoukoginoside A (1): amorphous powder; [α]25 D −20.7 (c 0.1, MeOH); IR (KBr) νmax 3425, 2930, 1730, 1635, 1560, 1459, 1390, 1247, 1135, 1060 cm−1; 1H and 13C NMR data, see Tables 1−3; negative HRESIMS m/z 1275.6022 (calcd for C61H96O28 [M − H]−, 1275.6015); negative ESI-LTQ-MSn fragments m/z (MS1) 1275.6 (100) [M − H]−, (MS2) 763.5 (100), (MS3) 719.5 (100), 617.4 (97), (MS4) 573.4 (100), 441.4 (10). Ezoukoginoside B (2): amorphous powder; [α]25 D −22.9 (c 0.1, MeOH); IR (KBr) νmax 3404, 2934, 1734, 1648, 1563, 1453, 1382, 1257, 1247, 1061 cm−1; 1H and 13C NMR data, see Tables 1−3; negative HRESIMS m/z 1129.5437 (calcd for C55H86O24 [M − H]−, 1129.5436); negative ESI-LTQ-MSn fragments m/z (MS1) 1129.5 (100) [M − H]−, (MS2) 573.4 (100), 617.4 (47), (MS3) 441.4 (100). Ezoukoginoside C (3): amorphous powder; [α]25 D −29.4 (c 0.1, MeOH); IR (KBr) νmax 3419, 2937, 1729, 1637, 1453, 1388, 1239, 1137, 1058 cm−1; 1H and 13C NMR data, see Tables 1−3; negative HRESIMS m/z 1261.6216 (calcd for C61H98O27 [M − H]−, 1261.6223); negative ESI-LTQ-MSn fragments m/z (MS1) 1307.6 (100) [M + HCOO]−, (MS2) 1261.6 (100) [M − H]−, (MS3) 749.5 (100), (MS4) 471.4 (100), 603.4 (41), 585.5 (36). Ezoukoginoside D (4): amorphous powder; [α]25 D −19.6 (c 0.1, MeOH); IR (KBr) νmax 3392, 2934, 1733, 1654, 1459, 1387, 1129, 1059 cm−1; 1H and 13C NMR data, see Tables 1−3; negative HRESIMS m/z 1235.6039 (calcd for C59H96O27 [M − H]−, 1235.6066); negative ESI-LTQ-MSn fragments m/z (MS1) 1281.6 (100) [M + HCOO]−, (MS2) 1235.4 (100) [M − H]−, (MS3) 765.4 (100), (MS4) 603.5 (100), 585.5 (32), 471.6 (4). Ezoukoginoside E (5): amorphous powder; [α]25 D −25.5 (c 0.1, MeOH); IR (KBr) νmax 3425, 2937, 1724, 1648, 1453, 1385, 1247, 1135, 1057 cm−1; 1H and 13C NMR data, see Tables 1−3; negative HRESIMS m/z 1259.6060 (calcd for C61H96O27 [M − H]−, 1259.6066); negative ESI-LTQ-MSn fragments m/z (MS1) 1305.6 (100) [M + HCOO]−, (MS2) 1259.4 (100) [M − H]−, (MS3) 747.4 (100), (MS4) 469.4 (100), 601.4 (45). Ezoukoginoside F (6): amorphous powder; [α]25 D −28.3 (c 0.1, MeOH); IR (KBr) νmax 3368, 2934, 1717, 1654, 1456, 1376, 1319, 1261, 1075 cm−1; 1H and 13C NMR data, see Tables 1−3; negative HRESIMS m/z 1217.5967 (calcd for C59H94O26 [M − H]−, 1217.5961); negative ESI-LTQ-MSn fragments m/z (MS1) 1263.0 (100) [M + HCOO]−, (MS2) 1217.5 (100) [M − H]−, (MS3) 747.4 (100), (MS4) 469.4 (100), 601.5 (46). Ezoukoginoside G (7): amorphous powder; [α]25 D −19.9 (c 0.1, MeOH); IR (KBr) νmax 3431, 2929, 1731, 1632, 1382, 1072, 1030 cm−1; 1H and 13C NMR data, see Tables 1−3; negative HRESIMS m/ z 1071.5375 (calcd for C53H84O22 [M − H]−, 1071.5381); negative ESI-LTQ-MSn fragments m/z (MS1) 1117.5 (100) [M + HCOO]−, (MS2) 1071.4 (100) [M − H]−, (MS3) 601.4 (100), 469.2 (13). Acid Hydrolysis and Absolute Configuration Determination of Monosaccharides. An HPLC-UV-based method was performed for monosaccharide determination.29 Compounds 1−7 (each 1.0 mg) were dissolved in 2 mL of 2 M HCl (dioxane/H2O, 1:1, v/v), and each solution was heated at 90 °C for 3 h. After removing the dioxane under vacuum, the solution was diluted with H2O and extracted with EtOAc (3 × 1 mL). The aqueous layer was dried down under vacuum and then redissolved in water and neutralized with Amberlite IRA-400 (OH− form). After drying under vacuum, the residue was dissolved in

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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.6b00329. Bioassay-guided isolation procedure and 1D and 2D NMR spectra of compounds 1−7 (PDF) G

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(22) Jiang, W.; Li, W.; Han, L.; Liu, L.; Zhang, Q.; Zhang, S.; Nikaido, T.; Koike, K. J. Nat. Prod. 2006, 69, 1577−1581. (23) Guo, M.; Song, F.; Liu, Z.; Liu, S. Anal. Chim. Acta 2006, 557, 198−203. (24) Spengel, S. M. Phytochemistry 1996, 43, 179−182. (25) Miyakoshi, M.; Isoda, S.; Sato, H.; Hirai, Y.; Shoji, J.; Ida, Y. Phytochemistry 1997, 46, 1255−1259. (26) Shao, C. J.; Kasai, R.; Xu, J. D.; Tanaka, O. Chem. Pharm. Bull. 1989, 37, 42−45. (27) Song, S. J.; Nakamura, N.; Ma, C. M.; Hattori, M.; Xu, S. X. Chem. Pharm. Bull. 2000, 48, 838−842. (28) Melek, F. R.; Miyase, T.; Abdel-Khalik, S. M.; Hetta, M. H.; Mahmoud, I. I. Phytochemistry 2002, 60, 185−195. (29) Tanaka, T.; Nakashima, T.; Ueda, T.; Tomii, K.; Kouno, I. Chem. Pharm. Bull. 2007, 55, 899−901. (30) Tohda, C.; Lee, Y. A.; Goto, Y.; Nemere, I. Sci. Rep. 2013, 3, 3395−3395.

AUTHOR INFORMATION

Corresponding Author

*Tel (K. Komatsu): +81-76-434-7601. Fax: +81-76-434-5064. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Associate Professor K. Toume of the University of Toyama for help with the NMR operation. We also thank Dr. S. Isoda, at the Medicinal Plant Garden, School of Pharmaceutical Sciences, Showa University, for the collection of the plant material. This work was supported by JSPS KAKENHI Grant Numbers 24406005 and 15H05268, Japan Agency for Medical Research and Development, AMED (Research on Development of New Drug, No. 15ak0101034h0001), and a grant from the President’s Discretion Expenses of the University of Toyama.

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