Amphipaniculosides A-D, triterpenoid glycosides, and amphipaniculoside E, an aliphatic alcohol glycoside from the leaves of Amphilophium paniculatum.

Phytochemistry xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Amphipaniculosides A–D, triterpenoid glycosides, and amphipaniculoside E, an aliphatic alcohol glycoside from the leaves of Amphilophium paniculatum Mamdouh Nabil Samy a,b, Hany Ezzat Khalil b, Sachiko Sugimoto a, Katsuyoshi Matsunami a,⇑, Hideaki Otsuka a,c,⇑, Mohamed Salah Kamel b a

Department of Pharmacognosy, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia 61519, Egypt c Department of Natural Products Chemistry, Faculty of Pharmacy, Yasuda Women’s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153, Japan b

a r t i c l e

i n f o

Article history: Received 10 September 2014 Received in revised form 4 January 2015 Accepted 23 February 2015 Available online xxxx Keywords: Amphilophium paniculatum Bignoniaceae Triterpenoids Amphipaniculosides Aliphatic alcohol glycoside

a b s t r a c t Four new triterpenoids; One oleanane-, one ursane- and two cycloartane-type triterpenoids, named amphipaniculosides AD, in addition to one new aliphatic alcohol glycoside, named amphipaniculoside E, were isolated from the 1-BuOH fraction of the leaves of Amphilophium paniculatum (L.) Kunth., together with five known compounds, (+)-lyoniresinol 3a-O-b-D-glucopyranoside, ()-lyoniresinol 3a-O-b-Dglucopyranoside, acteoside (verbascoside), isoacteoside (isoverbascoside), and luteolin 7-O-b-D-glucopyranoside. Their structures were elucidated by spectroscopic methods including 1D and 2D NMR experiments (1H, 13C, DEPT, COSY, ROESY, HSQC, HMBC) in combination with HR-ESI-MS and by comparisons of their physical and spectroscopic data with literature values. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Bignoniaceae is a family of flowering plants comprising about 800 species in 120 genera. Some species are cultivated as ornamentals, being distributed in tropical and subtropical areas of South America, Africa, and India (Gentry, 1980, 1992). Nearly all members of this family have woody stems. The genus Amphilophium has woody liana, 2–3 foliolate leaves, and fruits are smooth, flat, and somewhat woody. Amphilophium paniculatum (L.) Kunth. has 2 foliolate leaves, sometimes with a tendril; leaflets are ovate to sub-round, acuminate to obtuse, membranous, and palmately veined at the base. The inflorescence is caducous bracteate with terminal panicle racemose on a short lateral branch. Fruits are rounded oblong, flat, and somewhat warty-lenticellate (Gentry, 1973). Preliminary studies on this plant have led to the isolation of flavonoids (Nasser et al., 2013a) and a eudesmane phenolic acid ⇑ Corresponding authors at: Department of Pharmacognosy, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Tel./fax: +81 82 257 5335 (K. Matsunami). Department of Natural Products Chemistry, Faculty of Pharmacy, Yasuda Women’s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153, Japan. Tel./fx: +81-82-878-9496 (H. Otsuka). E-mail addresses: [email protected] (K. Matsunami), otsuka@ yasuda-u.ac.jp (H. Otsuka).

ester (Nasser et al., 2013b). However, there have been no reports on triterpenoidal glycosides of Amphilophium species, although a number of triterpenoidal glycosides were isolated from other genera in the family Bignoniaceae. The present study deals with the isolation and structural elucidation of five new compounds (1–5), including four new triterpenoidal glycosides, amphipaniculosides A–D (1–4) and one aliphatic alcohol glycoside, amphipaniculoside E (5), together with five known compounds from the leaves of this plant (Fig. 1). 2. Results and discussion The 1-BuOH fraction obtained from the 95% EtOH extract of leaves of Amphilophium paniculatum was fractionated by repeated column chromatography on porous polystyrene resin (Diaion HP-20), silica gel, and octadecylsilanized (ODS) silica gel and then further purified by HPLC, yielding five new compounds (1–5) (Fig. 1). Their structures were established mainly by spectroscopic methods including NMR experiments and mass spectrometry. Furthermore, five known isolated compounds were identified by comparison of their spectroscopic data with literature values as (+)-lyoniresinol 3a-O-b-D-glucopyranoside (6) (Ohashi et al., 1994), ()-lyoniresinol 3a-O-b-D-glucopyranoside (7) (Ohashi et al., 1994), acteoside (verbascoside) (8) (Kanchanapoom et al., 2002), isoacteoside (isoverbascoside) (9) (Wu et al., 2004), and

http://dx.doi.org/10.1016/j.phytochem.2015.02.020 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Samy, M.N., et al. Amphipaniculosides A–D, triterpenoid glycosides, and amphipaniculoside E, an aliphatic alcohol glycoside from the leaves of Amphilophium paniculatum. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.02.020

2

M.N. Samy et al. / Phytochemistry xxx (2015) xxx–xxx

Fig. 1. Structures of the isolated compounds (1–10).

luteolin 7-O-b-D-glucopyranoside (10) (Orhan et al., 2012), respectively. Amphipaniculoside A (1) [a]24 D of 17.5 was obtained as an amorphous powder, and its molecular formula was determined to be C42H68O16 by HR-ESI-MS. The IR spectrum showed absorptions indicating the presence of hydroxy group (3361 cm1), and carbonyl group (1714 cm1). The 1H NMR spectrum of 1 (Tables 1 and 2) displayed signals corresponding to an olefinic proton (dH 5.48, br s), five tertiary methyls (dH 1.02, 1.05, 1.092, 1.097, 1.18) and two anomeric proton resonances at dH 5.75 (1H, d, J = 7.7 Hz) and 6.24 (1H, d, J = 8.2 Hz). These spectroscopic data suggested that 1 was a triterpene diglycoside. This was supported by the 13 C NMR spectrum (Tables 1 and 2), which showed 42 carbon signals. The chemical shifts of the B, C, and D rings and the olefinic carbons (dC 123.2 and 144.7) suggested that 1 should have an oleanane skeleton with a double bond at C-12 (Bisoli et al., 2008). The carbon resonances corresponding to ring A, including the two oxymethine groups (dC 68.9 and 78.1) and an oxymethylene group (dC 66.4), suggested the presence of three hydroxy groups at C-2, C-3, and C-23, which was supported by the HMBC spectrum that showed long-range correlations between the methine protons H-2 and C-3, H-3 and C-2, H-3 and C-4, and the correlations of oxymethylene protons H2-23 with C-3, C-4, C-5 and the methyl carbon C-24 (Fig. 2). In addition, the 1H and 13C NMR spectra of 1 indicated the presence of one more oxymethylene groups (dH 3.52, 2H, br s; dC 73.8, t). The oxymethylene protons had HMBC correlations with C-19, C-20, and C-21 and the methyl carbon at dC 19.8 (C-30), suggesting the position of the second oxymethylene group was at C-20 (Adnyana et al., 2000). Acid hydrolysis of 1 yielded D-glucose as the sugar component. On the other hand, the C-20 signal of the inner

glucopyranosyl unit of 1 was shifted downfield at dC 79.2 (dC 74.1 for the mono glucoside, quadranoside III (Adnyana et al., 2000)). Thus, it was apparent that the terminal sugar was linked to the 2-hydroxy group of the inner glucose. This was confirmed in the HMBC spectrum (Fig. 2), showing the long-range correlation between the anomeric proton of the terminal glucopyranosyl unit (dH 5.75, d, J = 7.7 Hz) and C-2 (dC 79.2) of the inner moiety. Moreover, the HMBC data displayed the correlation between the anomeric proton of the inner glucopyranosyl unit (dH 6.24, d, J = 8.2 Hz) and C-28 (dC 176.7) of the aglycone, giving evidence that the glycosylation of this aglycone was on the hydroxy group at C28. The stereochemistry of 1 was determined by the coupling constant (9.3 Hz) between H-2 and H-3, indicating that the hydroxy groups have a 2a,3b -orientation, which was further supported by the ROESY correlations (Fig. 3). The intense cross-peaks between H3-24 and H-2, between H-2 and H3-25, and between H-3 and H-5 led the relative configuration of ring A to be determined as 2a-OH, 3b-OH, and 4a-CH2OH. Furthermore, the cross-peak between H-18 and H3-30 indicated that CH2OH-20 should be a (C-29). The aglycone of 1 was not obtained successfully by enzymatic or acid treatment in our experiment. However, a closely related mono-glucoside, quadranoside III, has essentially the same chemical shift values in aglycone and inner glucose (Adnyana et al., 2000). From these data, compound 1 was concluded to be 2a,3b,23,29-tetrahydroxyolean-12-en-28-oic acid 28-O-[b-D-glucopyranosyl-(100 ?20 )]-b-D-glucopyranosyl ester, named amphipaniculoside A as a glucoside of quadranoside III. Amphipaniculoside B (2), [a]24 D of 2.43 was obtained as an amorphous powder, having the molecular formula C42H68O16 determined by HR-ESI-MS. The IR spectrum of 2 showed bands at


3

M.N. Samy et al. / Phytochemistry xxx (2015) xxx–xxx Table 1 H (600 MHz) and

1

No.

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

13

C (150 MHz) NMR spectroscopic data for aglycone moieties of amphipaniculosides AD (1–4) (pyridine-d5).

1

2

3

4

dH (m, J in Hz)

dC

dH (m, J in Hz)

dC

dH (m, J in Hz)

dC

dH (m, J in Hz)

dC

2.20 (dd, 12.0, 4.8) 1.37 (m) 4.23 (m)

47.8

2.25 (dd, 12.4, 5.0) 1.32 (m) 4.22 (m)

48

1.39 1.16 2.14 1.72 4.35 – 2.13 1.20 1.11 2.28 2.10 1.47 – – 1.88 1.09 1.58 – – 1.88 1.64 4.46

31.7

1.33 1.13 2.09 1.70 4.25 – 2.13 1.26 1.15 2.27 2.11 1.51 – – 1.87 1.10 1.59 – – 1.88 1.64 4.45

(m) (m) (m) (m) (dd, 12.5, 4.8)

31.5

1.97 1.04 0.53 0.29 1.73 0.96

(dd, 10, 6.8) (s) (d, 3.9) (d, 3.9) (m) (d, 6.6)

2.28 1.58 1.33 0.91 4.66 – 1.64 1.65 – 1.33 1.13

(m) (m) (m) (m) (br s)

4.20 – 1.78 1.72 1.41 1.87 1.58 – 1.80 – 2.03

(d, 9.3) (br d,12.0) (m) (m) (m) (m) (m) (2H, m)

5.48 (br s) – – 2.30 (m) 1.23 (m) 2.34 (m) 2.20 (m) – 3.26 (dd, 13.7, 4.5) 2.07 (m) 1.42 (m) – 1.71 (m) 1.31 (m) 1.61 (m) 1.25 (m) 4.14 (d, 10.5) 3.64 (d, 10.5) 1.02 (s) 1.05 (s) 1.097 (s) 1.18 (s) – 3.52 (2H, br s) 1.092 (s)

68.9 78.1 42.2 48.2 18.6 31.8 40.1 47.9 38.5 24 123.2 144.7 43.7 28.9 23.1 47.5 41.3 41 36.4 29.2 32.9 66.4 14.4 17.7 17.5 26.1 176.7 73.8 19.8

4.16 – 1.81 1.71 1.41 1.76 1.64 – 2.03

(d, 9.3) (br d, 12.0) (m) (m) (m) (m) (m)

2.11 (2H, m)

68.9 78.3 43.7 48 18.7 33.2 40.8 47.9 38.4 24.2

5.51 (br s) – – 2.40(ddd, 17.6, 13.6, 3.7) 1.32 (m) 3.08 (ddd, 17.1, 12.6, 4.3) 2.26 (m) – 2.90 (br s) –

128.2 139.6 42.2 29.9

1.42 1.99 1.19 2.01 1.86 4.11 3.62 0.99 1.06 1.12 1.59 – 1.37 1.05

42.2 26.8

(m) (m) (m) (m) (m) (d, 10.3) (d, 10.3) (s) (s) (s) (s) (s) (d, 7.9)

25.9 48.8 54.6 72.7

37.5 66.4 14.4 17.5 17.5 24.6 177 27 16.7

3362 and 1735 cm1 corresponding to hydroxy and carbonyl group absorptions, respectively. The 1H NMR spectrum of 2 (Tables 1 and 2) displayed signals corresponding to five tertiary methyls (dH 0.99, 1.06, 1.12, 1.37, 1.59), one secondary methyl (dH 1.05), an olefinic proton (dH 5.51, br s), and two anomeric protons at dH 5.68 (1H, d, J = 7.7 Hz) and dH 6.19 (1H, d, J = 8.2 Hz). The 13C NMR spectrum (Tables 1 and 2) showed 42 carbon resonances, which led to the conclusion that 2 is also a triterpene diglycoside. The presence of one secondary methyl group (dC 16.7) and the chemical shifts of the olefinic carbons (dC 128.2, 139.6) suggested that 2 is an ursane-type triterpene with a double bond at C-12 (Li et al., 1998). The signals at dC 66.4, 68.9, 72.7 and 78.3 indicated the presence of four hydroxy groups. The carbon resonances corresponding to ring A, including the two oxymethine groups (dC 68.9, 78.3) and an oxymethylene group (dC 66.4), were identical with those of 1, suggesting the presence of three hydroxy groups at C-2, C-3, and C-23. This was supported by the HMBC spectrum (Fig. 4). In addition, the 1H and 13C NMR spectra of 2 indicated the presence of one oxygenated quaternary carbon at dC 72.7. The methyl protons H329 and H3-30 had HMBC correlations with C-19, suggesting the position of this carbon was at C-19 (Cai et al., 2011). Furthermore, the sugar moiety attached to the carboxyl group was concluded to be a b-glucopyranosyl-(100 ?20 )-b-glucopyranose by comparing its 1H and 13C NMR spectroscopic data with those of 1. Thus, structure of 2 was deduced, and the relative

(m) (m) (m) (m) (dd, 12.5, 4.8) (m) (m) (m) (m) (m) (m)

(m) (m) (2H, m)

(dd, 7.2, 5.5) (m) (dd-like, 7.2, 6.7)

1.98 1.04 0.52 0.30 1.75 0.97

(dd, 9.4, 6.7) (s) (d, 3.9) (d, 3.9) (m) (d, 6.6)

2.28 1.59 1.32 0.88 4.66 – 1.64 1.65 – 1.32 1.14

(m) (m) (m) (m) (br s) (s) (s) (s) (s)

28.8 84.5 55.6 45.6 26 26 47.6 19 25.7 27 33.1 47.1 48 43 82.8 59.4 19.6 30 35 19.6 35.6 23.1 126.8 130.8 18.1 26.2 175.7 11 20.2

(m) (m) (m) (m) (m) (m)

(m) (m) (2H, m)

(m) (m) (dd-like, 6.9, 6.8)

(s) (s) (s) (s)

28.5 84.6 55.5 45.2 25.9 25.7 47.3 18.8 25.6 26.9 33 47 47.9 42.8 82.7 59.3 19.6 29.7 34.9 19.3 35.5 22.9 126.6 130.7 18.1 26.2 175.3 10.9 20

stereochemistry of ring A was found to be identical with that of 1, i.e., 2a-OH, 3b-OH, 4a-CH2OH from the ROESY spectrum. The configurations of the OH at C-19 and the methyl groups at C-20 were also determined to be 19a and 20a, respectively, from the ROESY correlations between H-18 and H3-29 (Fig. 5). Thus, the structure of compound 2 was elucidated to be 2a,3b,19a,23-tetrahydroxyurs-12-en-28-oic acid 28-O-[b-D-glucopyranosyl(100 ?20 )]-b-D-glucopyranosyl ester, named amphipaniculoside B as a glucoside of niga-ichigoside F1 (Seto et al., 1984). Amphipaniculoside C (3), [a]24 D of 29.9 was isolated as an amorphous powder and had a molecular formula C47H76O16 as determined by HR-ESI-MS. Its IR spectrum showed strong absorption bands at kmax 3361 (hydroxy) and 1747 cm1 (carboxyl). The 1 H NMR spectrum (Tables 1 and 2) showed five singlet methyls at dH 1.04, 1.14, 1.32, 1.64 and 1.65, one doublet methyl at dH 0.97, and an olefinic proton at dH 4.66. Two doublet signals at dH 0.30 and 0.52 (each J = 3.9 Hz) were assignable to a methylene group in the cyclopropane ring and three anomeric proton signals at dH 4.64 (1H, d, J = 7.1), 5.47 (1H, br s) and 6.81 (1H, br s) suggested the presence of three sugars in 3. The 13C NMR spectrum of 3 (Tables 1 and 2) indicated the presence of 47 carbon signals, which led to the conclusion that 3 is a triterpene triglycoside. It showed two downfield resonances at dC 126.8 and 130.8, corresponding to C-24 and C-25, respectively, two signals at dC 82.8 and 84.5 for two oxygenated methines at C-16 and


4


Table 2 H (600 MHz) and

1

No.

13

C (150 MHz) NMR spectroscopic data for sugar moieties of amphipaniculosides AD (1–4) (pyridine-d5; Glc: Glucose; Rha: Rhamnose; Ara: Arabinose).

1

2

dH (m, J in Hz) 28-O-Glc 6.24 (d, 8.3) 4.50 (dd, 8.5, 8.3) 4.21 (m) 4.28 (m) 4.32 (m) 4.42 (dd, 11.7,1.6) 4.37 (m) Glc 5.75 (d, 7.7) 4.12 (m) 4.28 (m) 4.11 (m) 3.94 (m) 4.64 (dd, 10.8,1.9) 4.38 (m)

10 20 30 40 50 60

100 200 300 400 500 600

3

dH (m, J in Hz)

dC

28-O-Glc 6.19 (d, 8.2) 4.46 (m) 4.30 (m) 4.22 (m) 4.24 (m) 4.59 (dd, 10.8, 1.9) 4.35 (m) Glc 5.68 (d, 7.7) 4.09 (m) 3.94 (m) 4.06 (m) 3.99 (m) 4.43 (m) 4.34 (m)

93.7 79.2 78.3 70.8 78.9 62

104.7 76 78.4 72.9 79 63.9

dC

dH (m, J in Hz)

93.8 79.2 78.2 70.9 79.1 62.2

3-O-Rha 5.47 (br s) 4.56 (m) 4.25 (m) 4.50 (m) 4.25 (m) 1.66 (d, 6.2)

104.8 75.9 78.2 73 79.2 64

16-O-Ara 4.64 (d, 7.1) 4.33 (m) 4.13 (m) 4.67 (m) 4.30 (m) 3.75 (br d, 11.8) 28-O-Rha 6.81 (br s) 4.55 (m) 4.17 (m) 4.42 (m) 4.26 (m) 1.66 (d, 6.2)

10 0 0 20 0 0 30 0 0 40 0 0 50 0 0 60 0 0

4 dC

dH (m, J in Hz)

dC

104 73.1 74.4 73 70.4 18.9

3-O-Rha 5.54 (br s) 4.49 (m) 4.40 (m) 4.52 (m) 4.20 (m) 1.66 (d, 6.2)

103.3 82.5 73.4 73 70 18.8

103.4 73.2 75 72 67.6

95.9 73.1 73.7 73 70 18.8

Glc 5.29 (d, 7.8) 4.00 (t, 8.2) 3.96 (m) 4.13 (m) 4.14 (m) 4.59 (br d, 11.8) 4.35 (d, 11.8, 3.6) 16-O-Ara 4.64 (d, 7.1) 4.31 (m) 4.12 (m) 4.69 (m) 4.29 (m) 3.74 (br d, 10.7) 28-O-Rha 6.93 (br s) 4.52 (m) 4.17 (m) 4.52 (m) 4.27 (m) 1.60 (d, 6.2)

10 0 0 0 20 0 0 0 30 0 0 0 40 0 0 0 50 0 0 0 60 0 0 0

HOH2C

O

HO

CH2OH

O

O HO HOH2C

OH OH CH2OH

O

O H

C HMBC 1H-1H COSY

OH OH OH

Fig. 2. Selected COSY and HMBC Correlations of 1.

C-3, respectively, and a characteristic resonance at dC 30.0 belonging to C-19 (Kasai et al., 1999; Radwan et al., 2004; Facundo et al., 2008; Zhao et al., 2008, 2011, 2013). All these above-mentioned data indicated that the aglycone belonged to a cycloartane-type of triterpenoid. The location of the carboxyl group at C-4 was established by the HMBC experiment (Fig. 6), which showed a clear

107.3 76.1 78.8 71.5 78.7 62.6

102.4 72.8 74.9 71.7 67.4

95.7 73.1 74.8 72.9 69.8 18.6

correlation between H3-29 (dH 1.32) and the C-28 signal at dC 175.7. The presence of a hydroxy group at C-16 (dC 82.8) was confirmed by the correlations observed in the HMBC of 3 between H16 (dH 4.46) and the carbon atoms at C-14 (dC 48.0), C-15 (dC 43.0), C-17 (dC 59.4) and C-20 (dC 35.0). Acid hydrolysis of 3 afforded Lrhamnose and L-arabinose. The 1H and 13C NMR spectra of 3 demonstrated the presence of two a-rhamnopyranosyl and one a-arabinopyranosyl units. One of the two a-rhamnopyranosyl units was identified as an ester-linked rhamnose from its characteristic chemical shifts of the anomeric proton (dH 6.81) and carbon (dC 95.9) signals, which was confirmed by the correlation between H-1000 (dH 6.81) and C-28 (dC 175.7) in the HMBC. Another rhamnose is attached to the hydroxy group at C-3, which was shifted significantly downfield at dC 84.5. Furthermore, the HMBC experiment showed the correlation of the anomeric proton H-10 at dH 5.47 to C-3 (dC 84.5) of the aglycone, confirming the 3-O-glycosylation. The arabinose was allocated to C-16 from the downfield shift of this carbon at dC 82.8 (Verotta et al., 1998), which was indicated by the cross-peak between H-100 (dH 4.64) and C-16 (dC 82.8) in the HMBC. The orientation of the hydroxy group at C-3 was concluded to be b on the basis of the coupling constant of H-3, since the H-3 (dH 4.35) signal was observed as a doublet of doublets due to axialaxial (J = 12.5 Hz) and axial–equatorial coupling (J = 4.8 Hz) with H-2ax and H-2 eq, respectively. In the case of C-16-OH, the doublet of doublets-like resonance for H-16 (dH 4.46 (dd-like, J = 7.2, 6.7 Hz)) clearly indicated the a-configuration for the hydroxy group (for b-OH: doublet of triplets should be appeared (Yoshikawa et al., 2000)). This estimation was further confirmed by ROESY analysis, i.e. the correlation between H3-18 and H-16, instead of the absence of the correlation between H3-30 and H-16. Thus, compound 3 was identified as 3b,16adihydroxycycloart-24-en-28 oic acid 3-O-a-L-rhamnopyranoside,


5


H

Me Me

HO HO

COOGlc2_1 Glc

H

Me H

HO HO

Me

HO H2C H

H

Me

H

H

CH2OH

Me

COOGlc2-1Glc

H

Me

HO H2C H

H

Me

H

H

Me

Me HO

H

Me

Fig. 5. Significant correlations observed in the ROESY spectrum of 2. Fig. 3. Significant correlations observed in the ROESY spectrum of 1.

HO O

HO

CH2OH

O

O HO

OH

HOH2C

OH CH2OH

O

O H

C HMBC 1H-1H COSY

OH OH OH

Fig. 4. Selected COSY and HMBC Correlations of 2.

16-O-a-L-arabinopyranoside, 28-O-a-L-rhamnopyranosyl ester, named amphipaniculoside C. Amphipaniculoside D (4), [a]24 D of 22.9 was isolated as an amorphous powder and had the molecular formula C53H86O21, deduced from positive-ion mode HR-ESI-MS. The 1H and 13C NMR spectra of 4 (Tables 1 and 2) were similar to those of 3 except for the presence of one anomeric proton signal at dH 5.29 (1H, d, J = 7.8 Hz), and six carbon signals at dC 107.3, 78.8, 78.7, 76.1, 71.5 and 62.6, in addition to the downfield carbon resonances at dC 82.5 instead of 73.1 in 3, suggesting the presence of a terminal glucose attached to C-20 of the rhamnose. The position of the new glucose was confirmed to be at C-20 on the basis of the long range correlation between the anomeric proton of the glucose (dH 5.29) and C-20 (dC 82.5) of the rhamnose in the HMBC spectrum (Fig. 7). Consequently, the structure of compound 4 was established as 3b,16a-dihydroxycycloart-24-ene-28 oic acid 3-O-b-D-glucopyranosyl-(100 ?20 )-a-L-rhamnopyranoside, 16-O-aL-arabinopyranoside, 28-O-a-L-rhamnopyranosyl ester, named amphipaniculoside D. Amphipaniculoside E (5), [a]24 D of 36.8 was isolated as an amorphous powder and possessed the molecular formula C19H34O10 as determined by HR-ESI-MS. The 13C NMR and DEPT spectra (Table 3) showed the presence of eight carbon signals for the aglycone, including one methyl (dC 14.4) and five methylenes (dC 23.7, 25.7, 33.1, 35.8 and 116.2) as well as two methines (dC 82.8 and 141.0). The chemical shifts of the aglycone were

almost the same as those of 3-O-substituted (3R)-1-octen-3-ol (Yamamura et al., 1998; Kanchanapoom et al., 2001). The 1H and 13 C NMR spectra (Table 3) displayed two anomeric proton signals at dH 4.33 (1H, d, J = 7.4 Hz) on dC 105.4 and 4.67 (1H, d, J = 7.9 Hz) on dC 100.8, indicating the presence of b-xylopyranose and b-galactopyranose moieties, respectively (Table 3). The linkage of these sugars was determined to be xylopyranosyl-(1?6)-galactopyranoside, because a 13C NMR signal, which was assignable to the 6-position of galactose, was shifted significantly downfield (dC 69.8) compared with that of an unsubstituted b-galactopyranoside (Agrawal, 1992), which was concluded from the HMBC correlation between the anomeric proton (H-100 ) of the xylopyranosyl moiety at dH 4.33 and C-60 of the galactopyranosyl moiety at dC 69.8. The HMBC spectroscopic data also displayed the correlation between the anomeric proton of the galactopyranosyl moiety (dH 4.67) and C-3 (dC 82.8) of the aglycone, giving evidence that the glycosylation was on the hydroxy group at C-3 as shown in Fig. 8. Acid hydrolysis gave D-xylose and D-galactose,

which were identified by comparison of the optical rotation with authentic samples. Therefore, the structure of compound 5 was assigned as (3R)-1-octen-3-ol 3-O-b-D-xylopyranosyl-(100 ?60 )b-D-galactopyranoside, named amphipaniculoside E. These four new triterpenes (1–4) are the first examples for the isolation of polyglycosyl derivatives of the known triterpenes such as quadranoside III and niga-ichigoside F1. In addition, compounds 3 and 4 are trisdesmoside having glycoside at three different positions, which can serve as chemotaxonomic marker for this plant. Compounds 1–10 were examined for their 1,1-diphenyl-2picrylhydrazyl (DPPH) radical-scavenging activity and tumor cell growth inhibitory activities toward A549 by means of the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. None of the tested compounds (1–10) showed any significant activity toward A549 (IC50 > 100 lM). The results were compared with those of doxorubicin as a positive control (IC50: 0.90 ± 0.02 lM). Compounds 6–10 showed potent DPPH radical scavenging activity (IC50: 14.2 ± 0.30, 13.6 ± 1.30, 7.42 ± 1.56, 5.51 ± 0.69 and 4.70 ± 0.90 lM, respectively) comparable with that of the standard trolox (16.6 ± 2.2 lM).

3. Experimental 3.1. General Experimental Procedures Optical rotation data were measured on a JASCO P-1030 polarimeter. IR spectra were obtained on a Horiba FT-710 Fourier transform infrared spectrophotometer. 1H and 13C NMR spectra were recorded on a BRUKER AVANCE 600 MHz spectrometer. HRESI mass spectra were taken on a LTQ Orbitrap XL mass spectrometer. The highly porous synthetic resin, Diaion HP-20, was purchased from Mitsubishi Chemical Co., Ltd. (Tokyo, Japan). Silica gel column chromatography (CC) was performed on silica gel 60 [(E. Merck, Darmstadt, Germany), 70–230 mesh]. Reversed-phase [Octadecylsilanized silica gel (ODS)] open CC (RPCC) was


6


O

O OH

OH

O OH

O

O

O

OH

O

OH

O

OH

O

OH

O

O OH OH OH

OH

O H

HOH2C OH O O

OH

C HMBC 1H- 1H COSY

OH OH

OH

OH OH

O H

OH

C HMBC 1H- 1H COSY

O

OH

Fig. 6. Selected COSY and HMBC correlations of 3. Fig. 7. Selected COSY and HMBC Correlations of 4.

performed on Cosmosil 75C18-OPN (Nacalai Tesque, Kyoto, Japan) (U = 20 mm, L = 40 cm). High-performance liquid chromatography (HPLC) was performed on an ODS column [Inertsil ODS-3; GL Science, Tokyo, Japan; (U = 10 mm, L = 25 cm, flow rate: 1.0 ml/min)], using a refractive index detector. Precoated silica gel 60 F254 plates (E. Merck; 0.25 mm in thickness) were used for TLC analyses, and visualized by spraying with a 3% H2SO4 solution in EtOH and heating to around 150 °C on a hotplate. 3.2. Plant material The leaves of A. paniculutum were collected in May 2012 from Orman Garden, Giza, Egypt. A voucher specimen of the plant is deposited in the Herbarium of the Faculty of Pharmacy, Minia University, Egypt (Minia-12-May-AP). 3.3. Extraction and isolation The air-dried powdered leaves (2.30 kg) of A. paniculutum were exhaustively extracted with EtOH:H2O (5 l 5, 95:5 V/V) and then concentrated under reduced pressure to yield a viscous gummy material (292.9 g). This residue was dissolved in H2O (500 ml) and defatted with n-hexane (1 l 5). The aqueous layer was evaporated to remove a trace amount of organic solvent, and then successively extracted with EtOAc and 1-BuOH (1 l 5 each). The EtOAc and 1-BuOH fractions were concentrated under reduced pressure to give 50.6 g and 47.9 g of residues, respectively. The remaining aqueous layer was concentrated to furnish a watersoluble fraction (132.5 g). The 1-BuOH fraction (47.9 g) was fractionated by CC on a highly porous synthetic resin, Diaion HP-20 (U = 60 mm, L = 36 cm). The column was eluted initially with H2O (3 l), then with a MeOH-H2O stepwise gradient with increasing MeOH content using 40% (2 l), 80% (2 l) and 100% MeOH (3 l). The effluents were collected in fractions (500 ml each). Similar fractions were combined to provide five fractions in total. The fourth fraction B-4 (11.3 g) was subjected to silica gel CC (360 g), (U = 60 mm, L = 33 cm) using a CHCl3:MeOH gradient system; 300 ml fractions were collected and similar fractions were combined to yield a total of eleven fractions. Fraction B-4–7 (6.16 g) was purified on RPCC, affording 16 fractions. The second fraction B-4–7-2 (115.2 mg) was purified by HPLC (MeOH:H2O, 30:70, V/V) to produce compounds 6 (14.1 mg) and 7 (9.5 mg). The sixth fraction B-4–7-6 (253.4 mg) was purified by HPLC

Table 3 H (600 MHz) and 13C (150 MHz) NMR spectroscopic data of amphipaniculoside E (5) (CD3OD).

1

No.

dH (m, J in Hz)

dC

1

5.21 5.10 5.86 4.10 1.66 1.49 1.35 1.31 1.30 0.89 4.67 3.32 3.54 4.04 3.20 3.99 3.71 4.33 3.19 3.31 3.76 3.85 3.16

116.2

2 3 4 5 6 7 8 1’ 20 30 40 50 60 100 200 300 400 500

(br d,17.3) (br d, 10.3) (ddd, 17.3, 10.3, 7.0) (quartet, 13.0, 7.0) (m) (m) (2H, m) (2H, m) (2H, m) (t, 7.1) (d, 7.9) (m) (dd, 9.7, 3.0) (t, 3.0, 3.0) (m) (dd, 11.5, 2.2) (dd, 11.5, 5.4) (d, 7.4) (m) (m) (m) (dd, 11.4, 5.2) (m)

141.0 82.8 35.8 25.7 33.1 23.7 14.4 100.8 72.4 73.0 68.9 74.5 69.8 105.4 74.9 77.6 71.2 66.9

(MeOH:H2O, 45:55, V/V) to give compounds 8 (94.8 mg), 9 (29.4 mg), and 10 (5.3 mg). The eighth fraction B-4–7-8 (80.2 mg) was purified by HPLC (MeOH:H2O, 50:50, V/V) to afford compound 5 (5.3 mg). Fraction B-4-9 (2.19 g) was purified on RPCC, affording 16 fractions. The eighth fraction B-4–9-8 (165 mg) was purified by HPLC (MeOH:H2O, 50:50, V/V) to produce compounds 1 (12.2 mg) and 2 (10.5 mg). The fifth fraction B-5 (5.56 g) was applied to a silica gel coloumn(150 g), (U = 35 mm, L = 45 cm) using a CHCl3-MeOH gradient system 300 ml fractions were collected as above, with similar fractions combined to finally yield 15 fractions. Fraction B-5–9 afforded 3 (25.8 mg), and fraction B-5-12 produced 4 (88.3 mg). 3.4. Amphipaniculoside A (1) White amorphous powder; [a]24 D 17.5 (c 0.48, MeOH); IR (film) mmax 3361, 2927, 1714, 1649, 1070, 1024 cm–1; 1H NMR (600 MHz, pyridine-d5) and

13

C NMR (150 MHz, pyridine-d5): Table 1 and 2;



OH

and 6.10 min, respectively. Compound 4 gave peaks for L-()rhamnose, L-(+)-arabinose and D-(+)-glucose with retention times of 5.38, 6.10 and 8.32 min, respectively. Compound 5 gave peaks for D-(+)-xylose and D-(+)-galactose with retention times of 6.34 and 8.28 min, respectively.

H

O O

OH

O

O

3.10. DPPH radical scavenging activity

OH

OH OH

OH

7

H

C HMBC

Fig. 8. Selected HMBC Correlations of 5.

HR-ESI-MS (positive-ion mode) m/z: 851.4390 [M + Na]+ (Calcd for C42H68O16Na: 851.4400). 3.5. Amphipaniculoside B (2) White amorphous powder; [a]24 D 2.43 (c 0.37, MeOH); IR (film) mmax 3362, 2927, 1735, 1649, 1072, 1035 cm–1; for 1H NMR (600 MHz, pyridine-d5) and 13C NMR (150 MHz, pyridine-d5) spectroscopic data, see Tables 1 and 2; HR-ESI-MS (positive-ion mode) m/z: 851.4401 [M + Na]+ (Calcd for C42H68O16Na: 851.4400). 3.6. Amphipaniculoside C (3) White amorphous powder; [a]24 D 29.9 (c 1.64, MeOH); IR (film) mmax 3361, 2931, 1747, 1649, 1088, 1051 cm–1; for 1H NMR (600 MHz, pyridine-d5) and 13C NMR (150 MHz, pyridine-d5) spectroscopic data, see Tables 1 and 2; HR-ESI-MS (positive-ion mode) m/z: 919.5030 [M + Na]+ (Calcd for C47H76O16Na: 919.5026). 3.7. Amphipaniculoside D (4) White amorphous powder; [a]24 D 22.9 (c 1.47, MeOH); IR (film) mmax 3361, 2932, 1747, 1649, 1072, 1051 cm–1; for 1H NMR (600 MHz, pyridine-d5) and 13C NMR (150 MHz, pyridine-d5) spectroscopic data, see Tables 1 and 2; HR-ESI-MS (positive-ion mode) m/z: 1081.5560 [M + Na]+ (Calcd for C53H86O21Na: 1081.5554). 3.8. Amphipaniculoside E (5) White amorphous powder; [a]24 D 36.8 (c 0.33, MeOH); IR (film) mmax 3361, 2927, 1508, 1456, 1038 cm–1; for 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz, CD3OD) spectroscopic data, see Table 3; HR-ESI-MS (positive-ion mode) m/z: 445.2040 [M + Na]+ (Calcd for C19H34O10Na: 445.2044). 3.9. Analysis of the sugar moiety Compounds 1–5 (circa/1 mg) were hydrolyzed individually with 1 M HCl (1.0 ml) at 80 °C for 2 h. The reaction mixtures were each neutralized with Amberlite IRA96SB (OH), then partitioned with an equal amount of EtOAc (1.0 ml), with the aqueous layers analyzed for their sugar components. The sugars were determined by HPLC on an amino column [Shodex Asahipak NH2P-50 4E (4.6 mm 250 mm) with CH3CN-H2O (3:1), 1 ml/min], using a chiral detector (JASCO OR-2090plus), with comparison to authentic sugars (D-glucose, D-galactose, D-xylose, L-arabinose and L-rhamnose). Compounds 1 and 2 each gave a peak for D-(+)-glucose with a retention time of 8.32 min, whereas compound 3 gave peaks for L-()-rhamnose and L-(+)-arabinose with retention times of 5.38

The absorbance with various concentrations of the test compounds dissolved in MeOH (100 lL) in 96-well microtiter plates was measured at 515 nm as Ablank. Then, a 200 lM DPPH solution (100 lL) was added to each well, followed by incubation at room temperature for 30 min. The absorbance was then measured again as Asample. The % inhibition was calculated using the following equation:

% Inhibition ¼ ½1 ðAsample Ablank Þ=ðAcontrol Ablank Þ 100 where Acontrol is the absorbance of the control reaction mixture containing DMSO and all reagents except for the test compound. IC50 was determined as the concentration of sample required to inhibit the formation of the DPPH radical by 50%. (Samy et al., 2014). 3.11. Human cancer cell growth inhibition assay This assay was performed using a human lung cancer cell line (A549) and viability was estimated by means of the colorimetric MTT assay. Dulbecco’s modified Eagle medium supplemented with 10% heat-inactivated FBS and 100 lg/mL kanamycin was used as the cell culture medium. The test compounds were dissolved in DMSO and then added to the wells of 96-well microtiter plates to a final concentration of 1%. A549 cells (5 103 cells/well) were cultured in a 5% CO2 incubator at 37 °C for 72 h and then the MTT solution was added to each well and the plates were incubated for a further 1.5 h. Then the formazan precipitates were dissolved in DMSO and the optical density of each well was measured at 540 nm with a microplate reader. Doxorubicin was used as a positive control. Cell growth inhibition was calculated using the following equation:

% Inhibition ¼ ½1 ðAsample Ablank Þ=ðAcontrol Ablank Þ 100 where Acontrol is the absorbance of the control reaction mixture containing DMSO and all reagents except for the test compound. IC50 was determined as the concentration of sample required to inhibit the formation of MTT formazan by 50% (Samy et al., 2014). Acknowledgements This work was supported by JSPS KAKENHI Grant Number 26460122. It was also supported by the Ministry of Higher Education, Egypt through the Scientific Mission System. The authors are grateful for access to the superconducting NMR instrument, UV and ESI-MS at the Analytical Center of Molecular Medicine, the Analysis Center of Life Science and the Natural Science Center for Basic Research and Development of the Graduate School of Biomedical and Health Sciences, Hiroshima University. References Adnyana, I.K., Tezuka, Y., Banskota, A.H., Xiong, Q., Tran, K.Q., Kadota, S., 2000. Quadranosides I–V, new triterpene glucosides from the seeds of Combretum quadrangulare. J. Nat. Prod. 63, 496–500. Agrawal, P.K., 1992. NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides. Phytochemistry 31, 3307–3330.


8


Bisoli, E., Garcez, W.S., Hamerski, L., Tieppo, C., Garcez, F.R., 2008. Bioactive pentacyclic triterpenes from the stems of Combretum laxum. Molecules 13, 2717–2728. Cai, W.-H., Matsunami, K., Otsuka, H., Takeda, Y., 2011. Symplocosionosides A–C, three megastigmane glycosides, a neolignan glucoside, and symplocosins A and B, two triterpene glycosyl esters from the leaves of Symplocos cochinchinensis var Philippinensis. Am. J. Plant Sci. 2, 609–618. Facundo, V.A., Rios, K.A., Moreira, L.S., Militao, J.S.L.T., Stabelli, R.G., Braz-Filho, R., Silveir, E.R., 2008. Two new cycloartanes from Combretum leprosum Mart. (Combretaceae). Rev. Latinoamer. Quim. 36, 76–82. Gentry, A.H., 1973. Flora of Panama. Part IX. Family 172. Bignoniaceae. Ann. (M)issouri Bot. Garden 60, 781–977. Gentry, A.H., 1980. Flora Neotropica: Bignoniaceae - part I (Tribes Crescentieae and Tourrettieae). Flora Neotropica Monograph 25(1). The New York Botanical Garden, New York, pp. 1–150. Gentry, A.H., 1992. Flora Neotropica: Bignoniaceae - part II (Tribes Tecomeae). Flora Neotropica Monograph 25(2). The New York Botanical Garden, New York, pp. 1– 130. Kanchanapoom, T., Kasai, R., Picheansoonthon, C., Yamasaki, K., 2001. Megastigmane, aliphatic alcohol and benzoxazinoid glycosides from Acanthus ebracteatus. Phytochemistry 58, 811–817. Kanchanapoom, T., Kasai, R., Yamasaki, K., 2002. Phenolic glycosides from Markhamia stipulata. Phytochemistry 59, 557–563. Kasai, R., Sasaki, A., Hashimoto, T., Kaneko, T., Ohtani, K., Yamasaki, K., 1999. Cycloartane glycosides from Trichosanthes tricuspidata. Phytochemistry 51, 803–808. Li, B.-Z., Wang, B.-G., Jia, Z.-J., 1998. Pentacyclic triterpenoids from Rubus xanthocarpus. Phytochemistry 49, 2477–2481. Nasser, I.N., Aboutabl, M.A., Eskander, D.M., Grace, M.H., El-Khrisy, E.-D.A., Sleem, A.A., 2013a. Flavonoid glycosides and pharmacological activity of Amphilophium paniculatum. Pharm. Res. 5, 17–21. Nasser, I.N., Eskander, D.M., Abdel-Razik, A.F., El-Khrisy, E.-D.A., Aboutabl, M.A., ElBeih, A.A., 2013b. A new eudesmane phenolic acid ester from Amphilophium Paniculatum (L.) Kunth. Nat. Prod. Chem. Res. 1, 1–4.

Ohashi, K., Watanabe, H., Okumura, Y., Uji, T., Kitagawa, I., 1994. Indonesian medicinal plants. XII. Four isomeric lignan-glucosides from the bark of Aegle marmelos (Rutaceae). Chem. Pharm. Bull. 42, 1924–1926. Orhan, F., Barıs, O., Yanmıs, D., Bal, T., Guvenalp, Z., Gulluce, M., 2012. Isolation of some luteolin derivatives from Mentha longifolia (L.) Hudson subsp. longifolia and determination of their genotoxic potencies. Food Chem. 135, 764–769. Radwan, M.M., El-Sebakhy, N.A., Asaad, A.M., Toaima, S.M., Kingston, D.G.I., 2004. Kahiricosides II–V, cycloartane glycosides from an Egyptian collection of Astragalus kahiricus. Phytochemistry 65, 2909–2913. Seto, T., Tanaka, T., Tanaka, O., Naruhashi, O., 1984. b-Glucosyl esters of 19ahydroxyursolic acid deribatives in leaves of Rubus species. Phytochemistry 23, 2829–2834. Samy, M.N., Sugimoto, S., Matsunami, K., Otsuka, H., Kamel, M.S., 2014. Bioactive compounds from the leaves of Eugenia uniflora. J. Nat. Prod. (India) 7, 37–47. Verotta, L., Orsini, F., Tatò, M., El-Sebakhy, N.A., Toaima, S.M., 1998. A cycloartane triterpene 3b, 16b diglucoside from Astragalus trigonus and its non natural 6hydroxy epimer. Phytochemistry 49, 845–852. Wu, J., Huang, J., Xiao, Q., Zhang, S., Xiao, Z., Li, Q., Long, L., Huang, L., 2004. Complete assignments of 1H and 13C NMR data for 10 phenylethanoid glycosides. Magn. Reson. Chem. 42, 659–662. Yamamura, S., Ozawa, K., Ohtani, K., Kasai, R., Yamasaki, K., 1998. Antihistaminic flavones and aliphatic glycosides from Mentha spicata. Phytochemistry 48, 131– 136. Yoshikawa, K., Katsuta, S., Mizumori, J., Arihara, S., 2000. New cycloartane triterpenoids from Passiflora edulis. J. Nat. Prod. 63 (10), 1377–1380. Zhao, Z., Matsunami, K., Otsuka, H., Shinzato, T., Aramoto, M., Takeda, Y., 2008. Tareciliosides A–G: cycloartane saponins from the leaves of Tarenna gracilipes (Hayata) Ohwi. Chem. Pharm. Bull. 56, 1153–1158. Zhao, Z., Matsunami, K., Otsuka, H., Shinzato, T., Takeda, Y., 2011. Tareciliosides H– M: further cycloartane glycosides from the leaves of Tarenna gracilipes. Chem. Pharm. Bull. 59, 902–905. Zhao, Z., Matsunami, K., Otsuka, H., Shinzato, T., Takeda, Y., 2013. Tareciliosides N– S: further cycloartane saponins from the leaves of Tarenna gracilipes, and cytotoxicity of saponins and triterpenes. J. Nat. Med. 67, 503–511.


A new triterpenoid glycoside from Vitex negundo.

Aliphatic Glucoside, Zanthoionic Acid and Megastigmane Glucosides: Zanthoionosides A-E from the Leaves of Zanthoxylum ailanthoides.

Triterpenoid glycosides from the bark of Mimosa tenuiflora.

Cytotoxic triterpenoid glycosides from the roots of Camellia oleifera.

Leptocarposide: a new triterpenoid glycoside from Ludwigia leptocarpa (Onagraceae).

Minor diterpene glycosides from the leaves of Stevia rebaudiana.

Complex flavonol glycosides from the leaves of Ginkgo biloba.

Two triterpenoid glycosides from the roots of Camellia oleifera and their cytotoxic activity.

Nine phenethyl alcohol glycosides from Stachys sieboldii.

New neolignan glycoside and an unusual benzoyl malic acid derivative from Maytenus senegalensis leaves.

Isolation and study of the aurone glycoside leptosin from the leaves of Flemengia strobilifera.

Two new flavonol glycosides from Dimocarpus longan leaves.

[Flavonol glycosides from Euphorbia helioscopia, E. stricta, E. verrucosa and E. dulcis (author's transl)].

New cytotoxic spirostane saponin and biflavonoid glycoside from the leaves of Acacia saligna (Labill.) H.L. Wendl.

Glycosides of marine invertebrates--V. A comparative study of glycoside fractions from starfish.

Biological and taxonomic perspective of triterpenoid glycosides of sea cucumbers of the family Holothuriidae (Echinodermata, Holothuroidea).

Triterpenoid glycosides from Medicago sativa as antifungal agents against Pyricularia oryzae.

A new triterpenoid compound from stems and leaves of American ginseng.

Galloyl-oxypaeoniflorin, suffruticosides A, B, C, and D, five new antioxidative glycosides, and suffruticoside E, A paeonol glycoside, from Chinese moutan cortex.

Ypsiyunnosides A-E, Five New Cholestanol Glycosides from Ypsilandra yunnanensis.

Three new trixane glycosides obtained from the leaves of Jungia sellowii Less. using centrifugal partition chromatography.

Antioxidant and antibacterial activities of flavonoid glycosides from Ficus exasperata Vahl-Holl (moraceae) leaves.

An acylated isorhamnetin glycoside from Aerva javanica.

One lignanoid compound and four triterpenoid compounds with anti-inflammatory activity from the leaves of Elaeagnus oldhamii maxim.