Flavonoid glycosides from the aerial parts of Acacia pennata in Myanmar

Phytochemistry 118 (2015) 17–22

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Flavonoid glycosides from the aerial parts of Acacia pennata in Myanmar Anna Kim a,1, Janggyoo Choi b,1, Khin Myo Htwe c, Young-Won Chin d, Jinwoong Kim b, Kee Dong Yoon a,⇑ a

College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, Bucheon 420-743, Republic of Korea College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul 151-742, Republic of Korea c Popa Mountain Park, Forest Department, Kyaukpadaung Township, Mandalay Division, Myanmar d College of Pharmacy and RFIND-BKplus Team, Dongguk University-Seoul, Goyang 410-820, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 16 April 2015 Received in revised form 21 July 2015 Accepted 2 August 2015

Keywords: Acacia pennata Mimosaceae Myanmar Flavonoid glycosides

a b s t r a c t Phytochemical investigations of the aerial parts of Acacia pennata (Mimosaceae) from Myanmar led to the isolation of five flavonoid glycosides and six known compounds. The new compounds were identified as (2R, 3S)-3,5,7-trihdyroxyflavan-3-O-a-L-rhamnopyranoside, (2S)-5,7-dihydroxyflavan-7-Ob-D-glucopyranoside-(4a ? 8)-epiafzelechin-3-O-gallate, (2R)-40 ,7-dihydroxyflavan-(4a ? 8)-(2R, 3S)-3, 5,7-trihdyroxyflavan-300 -O-a-L-rhamnopyranoside, 5,7-dihydroxyflavone 6-C-b-boivinopyranosyl-7-O-bD-glucopyranoside, and 5,7-dihydroxyflavone 7-O-b-D-glucopyranosyl-8-C-b-boivinopyranoside based on interpretation of spectroscopic data. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Acacia pennata (L.) Willd., belonging to the Mimosaceae family, is a perennial woody climber with bipennate leaves, and is distributed in regions of south and southeast Asia including Bangladesh, Bhutan, India, Myanmar, Sri Lanka, southwest China and Thailand (Lalchhandama, 2013; Terangpi et al., 2013). Traditionally, A. pennata has been used as a medicinal plant to treat cough, headaches, rheumatism and fever in certain regions of Myanmar. Several studies have determined multiple biological activities of A. pennata including anti-nociceptive and antiinflammatory, (Dongmo et al., 2005, 2007), antioxidant, (Sowndhararajan et al., 2013), anti-parasitic (Lalchhandama, 2013), and anti-transcription activities (Rifai et al., 2010). With regard to the constituents of A. pennata, only two reports have described terpenoids (teapeenin D, (+)-drim-8-ene, labdanolic acid, 8,15-labdanediol) and quercetin-, apigenin- and kaempferoldigycosides, isorhamnetin mono-glycoside and isovitexin (Dongmo et al., 2007; Rifai et al., 2010). As part of an ongoing search for chemical constituents of medicinal plants from Myanmar, the phytochemicals contained in the aerial parts (leaves and twigs) of A. pennata were investigated, and five new flavonoid

⇑ Corresponding author at: College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea. E-mail address: [email protected] (K.D. Yoon). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.phytochem.2015.08.001 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.

glycosides as well as six known compounds were isolated. The new flavonoids have the characteristic feature of containing a nonsubstituted B-ring in the aglycone. 2. Results and discussion Eleven compounds, including five new (1–5) and six known (6–11) compounds, were obtained from the ethyl acetate-soluble fraction of A. pennata. The six known compounds were elucidated as quercetin-3-O-b-D-glucopyranoside 6 (Liu et al., 2010), quercetin-3-O-a-L-rhamnopyranoside 7 (Mok and Lee, 2013), chrysin-7-O-b-D-glucopyraniside 8 (Liu et al., 2010), kaempferol 3-O-a-L-rhamnopyranoside 9, koaburanin 10 (Ogawa et al., 1970) and pinocembrin-7-O-b-D-glucopyranoside 11 (Hammami et al., 2004) (Fig. 1) by comparisons with spectroscopic evidence in the literature. Compound 1 was obtained as a brown amorphous powder with a molecular formula of C21H24O8 from a pseudomolecular ion peak at m/z 427.1378 [M + Na]+ (calcd. for C21H24O8Na, 427.1369). The 1 H NMR spectrum established a flavan-3-ol skeleton with monosubstituted benzene protons at dH 7.32–7.39 (5H, m), two metacoupled aromatic protons [dH 5.96 (1H, d, J = 2.3 Hz, H-6) and 5.89 (1H, d, J = 2.3 Hz, H-8)], two methylene protons at dH 2.86 (1H, dd, J = 16.1, 5.6 Hz, H-4a) and 2.69 (1H, dd, J = 16.1, 8.0 Hz, H-4b) and two methine protons [dH 4.80 (1H, d, J = 7.7 Hz, H-2), and 3.99 (1H, ddd, J = 8.0, 7.7, 5.6 Hz, H-3)]. The 2,3-trans configuration was deduced from the coupling constant of H-2 (J = 7.7 Hz) and the chemical shift of C-2 (dC 81.0) (Ishimaru et al., 1987)

18

A. Kim et al. / Phytochemistry 118 (2015) 17–22

Fig. 1. Structures of compounds 1–11.

(Tables 1 and 2). The absolute configuration was assigned to be 2R, 3S from a negative Cotton effect at 277 nm in the CD spectrum (Korver and Wilkins, 1971; Slade et al., 2005) (Fig. 2). In addition, an anomeric proton signal at dH 4.24 (1H, d, J = 1.4 Hz), as well as six aliphatic carbon resonances at dC 102.2, 73.9, 72.2, 72.0, 70.4 and 17.9 indicated the presence of a a-rhamnopyranosyl moiety. The glycosidic linkage was determined to be at the C-3 position, deduced from the HMBC correlation of H-100 (dH 4.24) to C-3 (dC 76.2). Therefore, the chemical structure of 1 was established as (2R, 3S)-3,5,7-trihdyroxyflavan-3-O-a-L-rhamnopyranoside. Compound 2 was isolated as a brownish amorphous powder, and a positive ion QTOF-MS showed its molecular formula as C43H40O17 from the pseudomolecular ion peak at m/z 851.2179 [M + Na]+ (calcd. for C43H40O17Na, 851.2163). Exhaustive analyses of 1D and 2D NMR spectroscopic data including 1H, 13C, 1H–1H COSY, HSQC and HMBC demonstrated that 2 consisted of a flavan glycoside as an upper (U) unit and a flavan-3-ol gallate as a lower (L) unit. The 1H NMR spectra of the U unit displayed a monosubstituted benzene ring at dH 7.21–7.25 (5H, m, H-20 -H-60 ), two meta-coupled aromatic protons at dH 6.30 (1H, J = 2.3 Hz, H-8) and 6.00 (1H, J = 2.3 Hz, H-6), a methylene group at dH 2.26 (1H, m, H-3a) and 2.07 (1H, ddd, J = 13.2, 7.2, 1.7 Hz, H-3b) and two methine protons [dH 4.86 (1H, dd, J = 11.1, 1.3 Hz, H-2) and 4.79 (1H, dd, J = 11.6, 7.1 Hz, H-4)]. A proton resonance at dH 5.26 (1H, d, J = 7.6 Hz, H-10000 ) along with six carbon signals at dC 100.1, 77.3, 76.7, 75.1, 71.1, 62.4 and the sugar analysis result by GC demonstrated the presence of a b-D-glucopyranosyl moiety, and a HMBC correlation of dH 5.26 (H-10000 ) to dC 157.3 established that the glucosyl group was linked to the C-7 position. Comparing these data to those in the literature, the skeleton of the U unit was determined to be 5,7-dihydroxyflavan-7-O-b-D-glucopyranoside (Ogawa et al., 1970). With regard to the L unit, the 1H NMR spectrum showed a 1,4-disubstituted aromatic ring at dH 6.73 (2H,

J = 8.6 Hz, H-2000 , H-6000 ) and 6.62 (2H, J = 8.6 Hz, H-300 0 , H-500 0 ), a penta-substituted aromatic ring at dH 6.11 (1H, s, H-600 ), two methylene protons [dH 2.96 (1H, dd, J = 17.1, 4.6 Hz, H-400 a), 2.85 (1H, brd, J = 17.1 Hz, H-400 b)] and two methine protons at dH 5.36 (1H, m, H-300 ) and 4.99 (1H, s, H-200 ). In addition, one singlet at dH 6.89 (2H, s, H-20000 0 , H-600 000 ) indicated the presence of a 1,3,4,5tetra-substituted aromatic ring characteristic of gallic acid (Liu et al., 2008). A broad singlet dH 4.99 (H-2) of the lower flavan-3ol skeleton indicated a 2,3-cis configuration (De Mello et al., 1996). Therefore, the lower unit was elucidated to be ()epiafzelechin-3-O-gallate (Zhang et al., 2012). The HMBC correlation of dH 4.79 (H-4) to dC 155.1 (C-700 ), 112.8 (C-800 ) and 154.9 (C-900 ) established a (4 ? 8) connectivity between the U and L units, and the a-orientation of the interflavan connectivity at C-4 (U) was assigned from the positive and negative Cotton effect at 201 and 218 nm in the circular dichroism (CD) spectrum. Furthermore, the CD spectrum showed a negative Cotton effect at 235, 267 and 289 nm, which was consistent with those of procyanidin B-4 (catechin-(4a ? 8)-epicatechin) (Barrett et al., 1979). Based on the above spectroscopic evidence, compound 2 was determined to be (2S)-5,7-dihydroxyflavan-7-O-b-Dglucopyranoside-(4a ? 8)-epiafzelechin-3-O-gallate. The molecular formula of compound 3 was established to be C42H46O16 from the positive ion QTOF-MS peak at m/z 829.2711 (calcd. for C42H46O16Na [M + Na]+, 829.2684). A comparison of the 1H and the 13C NMR spectroscopic data of 3 with those of 2 suggested that 3 possessed a flavan-dimer skeleton similar to 2 except for the L unit (Table 1). The 1H NMR spectra of the L unit showed flavan-3-ol resonances including five mono-substituted benzene protons at dH 6.93–7.28 (5H, m, H-200 0 -H-6000 ), a penta-substituted aromatic ring at dH 6.11 (1H, s, H-600 ), a methylene group at dH 2.89 (1H, dd, J = 16.1, 5.9 Hz, H-400 a) and dH 2.61 (1H, dd, J = 16.1, 9.2 Hz, H-400 b) and two methine protons at dH 4.59 (1H, d,

Table 1 H NMR spectroscopic data of compounds 1–5 (d in CD3OD, 500 MHz).

1

1

2

Position

1

2 3 4a 4b 6 8 20 60

4.80 3.99 2.86 2.69 5.96 5.89 7.39

30 50 40

H

3

Position (1H, d, J = 7.7 Hz) (1H, ddd, J = 8.0, 7.7, 5.6 Hz) (1H, dd, J = 16.1, 5.6 Hz) (1H, dd, J = 16.1, 8.0 Hz) (1H,d, J = 2.3 Hz) (1H,d, J = 2.3 Hz) (2H, o)a

4.86 2.26 2.07 4.79 6.00 6.30

7.37 (2H, o)a

20 ,60

7.32 (1H, tt, J = 7.1, 1.5 Hz)

30 ,50 0

100

4.24 (1H, d, J = 1.4 Hz)

L-unit

200

3.47 (1H, dd, J = 3.2, 1.7 Hz)

200

3.57 (1H, dd, 9.5, 3.3 Hz)

3

400 500

3.30 (1H, t, J = 9.5 Hz) 3.68 (1H, dq, J = 9.5, 6.3 Hz)

600

1.25 (3H, d, J = 6.3 Hz)

00

Position

4 1

H

5

Position

1

H

U-unit 2 3a 3b 4 6 8

4.77 2.20 2.04 4.72 5.89 6.16

7.23 (2H, o)a

20 ,60

7.12 (2H, dd, J = 7.8 1.7 Hz)

200 ax

3.07 (1H, ddd, J = 14.0, 12.4, 3.1 Hz)

7.25 (2H, o)a

30 ,50

7.25 (2H, o)a

200 eq

1.45 (1H, brd, J = 14.0 Hz, 2.4 Hz)

(1H)* (1H, m) (1H, ddd, J = 13.2, 7.2, 1.7 Hz) (1H, dd, J = 11.6, 7.2 Hz) (1H, d, J = 2.3 Hz) (1H, d, J = 2.3 Hz)

7.21 (1H, o)

a

0

4

(1H, (1H, (1H, (1H, (1H, (1H,

dd, J = 11.7, 1.4 Hz) m) ddd, J = 13.2, 7.6, 1.4 Hz) dd, J = 11.2, 7.6 Hz) d, J = 2.3 Hz) d, J = 2.4 Hz)

7.20 (1H, o)

b

L-unit 4.99 (1H, s)

200 00

4.59 (1H, d, J = 8.4 Hz) c

3 6.85 8 7.08 20 ,60 8.04 30 ,50 7.57 0 4 7.59 6-C-Boivinosyl 100 5.53

Position (1H, (1H, (2H, (2H, (1H,

s) s) brd, J = 7.0 Hz) o)a o)a

(1H, dd, J = 12.4, 2.4 Hz)

00

4.04 (1H, brs)

400

3.40 (1H, brs)

500

4.10 (1H, brq, J = 6.6 Hz)

3

6

00

5.36 (1H, m)

3

3.66 (1H, o)

1.23 (3H, d, J = 6.6 Hz)

400 a 400 b

2.96 (1H, dd, J = 17.1, 4.6 Hz) 2.85 (1H, brd, J = 17.1 Hz)

400 a 400 b

2.89 (1H, dd, J = 16.1, 5.9 Hz) 2.61 (1H, dd, J = 16.1, 9.2 Hz)

7-O-Glucosyl 100 0 4.94 (1H, d, J = 7.7 Hz)

600

6.11 (1H, s)

600

6.11 (1H, s)

200 0

3.60 (1H, o)b

200 0 ,600 0 300 0 ,500 0

6.73 (2H, d, J = 8.6 Hz) 6.62 (2H, d, J = 8.6 Hz)

200 00 ,600 00 300 00 ,500 0

6.93 (2H, dd, J = 7.7, 2.0 Hz) 7.28 (2H, o)a

300 0 400 0

3.53 (1H, t, J = 9.1 Hz) 3.37 (1H, o)*

7-O-Glucosyl 100 00 5.26 (1H, d, J = 7.6 Hz)

400 00 7.21 (1H, o)b 7-O-Glucosyl

500 0 600 0 a

3.60 (1H, o)b 4.02 (1H, dd, J = 12.1, 2.0 Hz)

200 00

100 00

600 0 b

3.52 (1H, dd, J = 12.1, 6.4 Hz)

00 00

3.16 (1H, dd, J = 7.6, 7.2 Hz)

3 3.53 400 00 3.36 500 00 3.36 600 00 a 3.88 600 00 b 3.72 300 -O-Gallaoyl 00 00 0 00 00 0 2 ,6 6.89

(1H, (1H, (1H, (1H, (1H,

m) o)a o)a dd, J = 12.0, 2.2 Hz) dd, J = 12.0, 4.9 Hz)

(1H, s)

00 00

4.80 (1H, d, J = 7.7 Hz)

2 3.26 (1H, 300 00 3.40 (1H, 400 00 3.40 (1H, 500 00 3.40 (1H, 600 00 a 3.88 (1H, 600 00 b 3.73 (1H, 300 -O-Rhamnosyl 00 00 0 1 3.94 (1H, 200 00 0 3.46 (1H, 300 00 0 3.55 (1H, 400 00 0 3.26 (1H, 500 00 0 3.66 (1H, 600 00 0 1.22 (1H,

d

1

H

3 6.86 (1H, s) 6 6.74 (1H, s) 20 ,60 8.20 (2H, brs) 30 ,50 7.56 (2H, o)a 0 4 7.59 (1H, o)a 8-C-Boivinosyl 100 5.71 (1H, dd, J = 12.5, 1.6 Hz) 200 ax 2.99 (1H, brt, J = 12.5 Hz) 200 eq 1.54 (1H, brd, J = 14.0 Hz) 00 3 4.07 (1H, d-like, J = 3.0 Hz) 400 3.43 (1H, d-like, J = 3.5 Hz) 500 4.15 (1H, brq, J = 6.5 Hz) 00 6 1.27 (3H, d, J = 6.5 Hz) 7-O-Glucosyl 100 0 4.95 (1H, d, J = 7.6 Hz) 200 0 3.59 (1H, t, J = 8.4 Hz) 00 0 3 3.50 (1H, o)b 00 0 4 3.45 (1H, t, J = 9.2 Hz) 500 0 3.53 (1H, o)b 600 0 a 3.95 (1H, dd, J = 12.1, 1.9 Hz) 600 0 b 3.50 (1H, dd, J = 12.1, 5.4 Hz)

A. Kim et al. / Phytochemistry 118 (2015) 17–22

4

3

H

U-unit 2 3a 3b 4 6 8

3-O-Rhamnosyl

00

1

o) o)e o)e o)e brd, J = 11.5 Hz) dd, J = 11.5, 3.2 Hz) d, J = 1.4 Hz) dd, J = 3.3, 1.4 Hz) dd, J = 9.5, 3.3 Hz) o)d o)c d, J = 6.2 Hz)

a,b,c,d,e *

These signals were overlapped to each other; m, o: unresolved (m) or overlapped (o) signal. Signal was overlapped by water or solvent peak.

19

20

A. Kim et al. / Phytochemistry 118 (2015) 17–22

Table 2 C NMR spectroscopic data of compounds 1–5 (d in CD3OD, 125 MHz).

13

1 Position

2 13

C

2 81.0 3 76.2 4 27.8 5 157.6 6 96.5 7 158.0 8 95.5 9 156.7 10 100.6 0 1 140.5 20 60 128 129.3 30 50 40 129.2 3-O-Rhamnosyl 100 102.2 200 72.0 00 3 72.2 73.9 400 500 70.4 600 17.9

Position U-unit 2 3 4 5 6 7 8 9 10 10 20 ,60 30 ,50 40 L-unit 200 300 400 500 600 700 800 900 1000 10 00 20 00 ,60 00 300 0 ,50 00 40 00

3 13

C

79.6 38.6 30.1 158.8 99.5 157.3 96.2 156.8 109.5 143.5 127.0 129.2 128.5

Position

13

C

7-O-Glucosyl 100 00 100.1 200 00 75.1 300 00 77.3 400 00 71.1 500 00 76.7 600 00 62.4 300 -O-Gallaoyl 00 00 0 1 121.7 200 00 0 ,600 00 0 110.7 300 00 0 ,500 00 0 146.1 400 00 0 139.5 C@O 167.9

78.4 69.9 27.1 154.6 96.2 155.1 112.8 154.9 99.7 130.6 129.0 115.6 157.5

Position U-unit 2 3 4 5 6 7 8 9 10 10 20 ,60 30 ,50 40 L-unit 200 300 400 500 600 700 800 900 1000 10 00 20 00 ,60 00 300 0 ,50 00 40 00

Fig. 2. CD spectra of compounds 1–3.

J = 8.4 Hz, H-200 ) and dH 3.66 (1H, m, H-300 ). The coupling constant of H-200 (J = 8.4 Hz) indicated the 2,3-trans configuration (Ishimaru et al., 1987). In addition, anomeric proton resonance at dH 3.94 (1H, d, J = 1.4 Hz, H-100 000 ) and six carbon signals at dC 102.2, 73.9 (C-40000 0 ), 72.3 (C-300 000 ), 71.8 (C-200 000 ), 70.4 (C-500000 ) and 17.9 (C-600 000 ) indicated the presence of an a-L-rhamnopyranosyl group which was further confirmed by acid hydrolysis of 3 followed by GC analysis of its hydrolysate. HMBC correlation of H-200 000 (dH 3.94) to C-3 (dC 76.3) indicated that the rhamnosyl moiety was linked to C-300 of the L unit. Therefore, the L unit of compound 3 was elucidated to be 3,5,7-trihdyroxyflavan-3-O-a-L-rhamnopyranoside. The (4 ? 8) interflavan linkage between the U and L units was established by the HMBC correlations of H-4 (dH 2.85) to C-700 (154.9) C-800 (112.8) and C-900 (154.7). Hatano et al. (1997) synthesized several flavan dimers. Flavan dimers with a (2R)-configuration of the U unit showed a positive Cotton effect at 286–287 nm, while compounds with a (2S)-configuration of the upper unit showed a negative Cotton effect at 286–293 nm regardless of the configuration of the (4 ? 8) interflavan linkage. The CD spectrum of 3 showed

4 13

C

Position

79.6 38.5 29.5 159.1 157.4 157.4 99.5 157.4 96.0 157.0 109.7 143.4 127.0 129.2 128.5 81.0 76.3 29.1 154.7 96.1 154.9 112.8 154.7 101.9 139.7 128.2 128.9 128.8

13

C

7-O-Glucosyl 100 00 101.4 200 00 74.9 300 00 77.9 400 00 70.9 500 00 77.1 600 00 62.3 300 -O-Rhamnosyl 00 00 0 1 102.1 200 00 0 71.8 300 00 0 72.3 400 00 0 73.9 500 00 0 70.3 600 00 0 17.9

Position

5 13

C

2 166.2 3 106.5 4 184.4 5 160.1 6 114.4 7 165.0 8 96.4 9 158.7 10 107.2 0 1 132.3 20 ,60 127.6 30 ,50 130.3 40 133.3 6-C-Boivinosyl 100 67.1 200 31.3 00 3 69.4 400 71.6 500 72.3 600 17.5 7-O-Glucosyl 100 0 103.7 200 0 75.0 00 0 3 77.0 400 0 71.1 500 0 78.7 600 0 62.7

Position

13

C

2 166.2 3 105.9 4 184.6 5 160.5 6 100.7 7 163.0 8 110.5 9 156.8 10 107.2 0 1 132.3 20 ,60 128.1 30 ,50 130.3 40 133.3 8-C-Boivinosyl 100 67.9 200 32.2 00 3 69.6 400 71.3 500 73.3 600 17.5 7-O-Glucosyl 100 0 103.2 200 0 74.9 00 0 3 77.2 400 0 71.0 500 0 78.5 600 0 62.4

positive and negative Cotton effects at 201 and 218 nm, indicating a (4a ? 8) configuration, as well as a negative Cotton effect at 237 nm and a positive Cotton effects at 290 nm, which was very close to those of the (2R)-40 ,7-dihydroxyflavan-(4a-8)-(2R,3S)catechin (Hatano et al., 1997). Therefore, compound 3 was (2R)-4 0 ,7-dihydroxyflavan-(4a ? 8)-(2R,3S)-3,5,7-trihdyroxyflavan-3-Oa-L-rhamnopyranoside. Compound 4 was obtained as a pale yellow amorphous powder, and the molecular formula was deduced to be C27H30O12 from its QTOF-MS spectrum at m/z 569.1645 (calcd. for C27H30O12Na [M + Na]+, 569.1635). The 1H NMR spectrum of 4 displayed resonances consistent with a 5,7-dihydroxy-flavone skeleton including a mono-substituted benzene ring at dH 8.04 (2H, brd, J = 7.0 Hz, H20 , H-60 ), 7.59 (1H, m, H-40 ) and 7.57 (2H, m, H-30 , H-50 ), as well as two singlets at dH 7.08 (1H, s, H-8) and 6.85 (1H, s, H-3). Furthermore, two anomeric proton resonances were observed at dH 5.53 (1H, dd, J = 12.5, 2.4 Hz, H-100 ) and 4.94 (1H, d, J = 7.7 Hz, H-100 0 ) and were correlated with dC 67.1 and 103.7 in the HSQC spectra, respectively. These HSQC data indicated that one sugar moiety was linked through a C-linkage and one was connected via an O-linkage. The anomeric proton of the sugar moiety was observed at dH 4.94 (J = 7.7 Hz), and the 13C NMR showed six aliphatic carbon signals at dC 103.7 (C-1000 ), 78.7 w(C-500 0 ), 77.0 (C3000 ), 75.0 (C-200 0 ), 71.1 (C-4000 ) and 62.7 (C-6000 ), suggesting that 2 possessed a b-glucopyranosyl moiety. The HMBC correlation of dH 4.94 to dC 165.4 established that the C-7 was linked to a bglucopyranosyl group via an O-linkage. The C-linked sugar moiety was analyzed using 1D and 2D NMR spectra. In 1H–1H COSY spectrum, the correlations between H-100 /H-200 /H-300 /H-400 /H-500 /H-600 were unambiguously assigned. The anomeric proton (dH 5.53, H100 ) was coupled with two proton resonances at dH 3.07 (1H, dt, J = 14.0, 12.5, 2.6 Hz, H-200 ax) and 1.45 (1H, dt, J = 14.0 Hz, 2.6 Hz, H-200 eq). The coupling constant (J = 12.5 Hz) between H-100 and H200 ax demonstrated that H-100 was located at the axial position. The H-300 and H-400 were detected as a broad doublet indicating an equatorial position. In the ROESY spectrum, the H-500 (dH 4.10)

A. Kim et al. / Phytochemistry 118 (2015) 17–22

was correlated to the H-100 (dH 5.53), suggesting the axial position of H-500 . The 13C NMR spectrum had six carbon signals assigned at dC 67.1 (C-100 ), 31.1 (C-200 ), 69.4 (C-300 ), 71.1 (C-400 ), 72.3 (C-500 ) and 17.5 (C-600 ). Therefore, the C-linked sugar part was determined to be b-boivinopyranoside (Suzuki et al., 2003; Takeda et al., 2004; Xu et al., 2013), and the HMBC spectrum established that the b-boivinopyranose was linked to C-6 from the correlations of H-100 (dH 5.53) to C-5 (dC 160.1), C-6 (dC 114.4) and C-7 (dC 165.0). Based on the above spectroscopic evidence, compound 4 was determined to be 5,7-dihydroxyflavone 6-C-bboivinopyranosyl-7-O-b-D-glucopyranoside. The QTOF-MS spectrum of 5 gave the molecular formula of C27H30O12 from the pseudomolecular ion peak at m/z 569.1638 [M + Na]+. The 1D and 2D NMR spectroscopic patterns of 5 were similar to those of 4, displaying a 5,7-dihydroxy-flavone, a C-linked b-boivinopyranoside and an O-linked b-D-glucopyranoside. The difference between 4 and 5 was the C-glycosidic linkage of the b-boivinopyranosyl group to the aglycone, which was confirmed by the HMBC correlations of H-100 (dH 5.71) to C-700 (dC 160.1), C-800 (dC 160.1) and C-900 (dC 160.1), indicating that the boivinosyl group was linked to the C-8 position. Thus, compound 5 was identified as 5,7-dihydroxyflavone 7-O-b-D-glucopyranosyl8-C-b-boivinopyranoside. 3. Conclusions Phytochemical studies of the aerial parts of A. pennata from Myanmar led to isolation of five new flavonoid glycosides (1–5) and six known compounds (6–11). Their chemical structures were determined by interpreting 1D and 2D NMR, MS, UV, and CD data. Only two previous reports have described the constituents of A. pennata and identified terpenes and flavonoid glycosides. The new flavonoid glycosides described herein feature a nonsubstituted B-ring in the flavonoid aglycone. 4. Experimental 4.1. General experimental procedures 1 H NMR and 13C NMR spectra were measured on a Bruker AscendTM 500 spectrometer. Mass spectra (Q-TOF MS) were recorded on an Agilent 6530 ESI-Q-TOF mass spectrometer. UV spectra were measured using a Shimadzu UV-1800 spectrometer, and optical rotation data were obtained on a Jasco P-2000 polarimeter. A Gilson preparative HPLC system was used for the high-performance column chromatography (HPLC), and was equipped with binary pumps, an UV/VIS-155 detector, and a GX-271 liquid handler. HPLC separation was performed using an YMC-Pack ODS-A column (250  20 mm; ID, 5 lm; Japan). GC analysis was performed on a GC353B-FSL gas chromatography coupled with a flame ionization detector and BPX50 column (0.25 mm  30 m). The semi-preparative and preparative highperformance countercurrent chromatography (HPCCC) instruments were Dynamic Extractions Spectrum and Midi, respectively, and were combined with an ECOM IOTA S 300 pump, a Sapphire 600 UV–VIS variable wavelength detector, a Teledyne Foxy R2 fraction collector, and an Eyela CCA-1111 circulatory temperature regulator. Organic solvents for column chromatography (CC) were analytical grade and purchased from Daejung Chemical and Metals (Gyunggido, Korea). Deionized H2O was produced using a Millipore Milli-Q water purification system (Millipore, Billerica, MA, USA). CH3CN, MeOH, and H2O for HPLC were obtained from Fisher Scientific Korea (Seoul, Korea). Silica gel and reversedphase silica gel were purchased from Merck (Germany), and Sephadex LH-20 was obtained from Pharmacia Co. (Sweden).

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4.2. Plant material Aerial parts of A. pennata were collected from Popa Mountain National Park (Mandalay, Myanmar) in August 2011, and identified by Khin Myo Htwe (Popa Mountain Park, Myanmar). A voucher specimen (#FBRSVP0000270438) was deposited at the herbarium of the National Institute of Biological Resources (NIBR) in Korea. 4.3. Extraction and isolation The dried and ground aerial parts of A. pennata (951 g) were extracted with MeOH (12 L) in an ultrasonic bath (4 L  3 h  3 times) and evaporated under reduced pressure to obtain the corresponding extract (64 g). The MeOH extract was suspended in H2O and continuously partitioned with organic solvents to give n-hexane (8.2 g), EtOAc (17.1 g), and n-BuOH (28.5 g)-soluble extracts. The EtOAc fraction (12 g) was subjected to a preparative HPCCC using n-hexane:EtOAc:MeOH:H2O system (1:10:1:10, v/v, reversed-phase mode; rotational speed: 1,400 rpm; flow rate: 28 mL/min; UV detection at 280 nm) to give 10 subfractions (EA 1–EA 10). EA 4 (2.1 g) was successively separated on a semi-preparative HPCCC (EtOAc:n-BuOH:H2O, 8:2:10, v/v, reversed-phase mode; rotational speed: 1600 rpm; flow rate: 3 mL/min; UV detection at 280 nm) to give seven subfractions (EA 4-1 to EA 4-7). EA 4-3 was subjected to RP-HPLC (15 ? 50% CH3CN in 60 min) to obtain compound 4 (2.8 mg) and 5 (3.3 mg). EA 7 was loaded on Sephadex LH-20 CC using MeOH as an eluent and gave four subfractions (EA 7-1 to EA 7–4). EA 7-2 was subjected to RP-HPLC (15 ? 40% CH3CN in 60 min) to yield compounds 1 (5.3 mg), 10 (11.7 mg) and 11 (1.5 mg). EA 7-4 (251 mg) was subjected to silica gel CC using CHCl3:MeOH mixtures (6:1, 3:1, 2:1, 1:1, v/v) as eluents to give four subfractions (EA 7-4-1 to EA 7-4-4). Compounds 6 (39.6 mg) and 7 (4.2 mg), 8 (2.7 mg) 9 (2.3 mg) were obtained from EA 7-4-1 by RP-HPLC using CH3CN:H2O (27:73, v/v) as eluent, and EA 7-4-3 (49 mg) was subjected to RP-HPLC to give compounds 2 (2.1 mg) and 3 (8.4 mg). 4.3.1. (2R,3S)-3,5,7-trihdyroxyflavan-3-O-a-L-rhamnopyranoside (1) Brown amorphous powder: [a24 D ] 3.9° (c 0.02, MeOH); UV (MeOH) kmax (log e) 208 (3.83), 232 (sh), 270 (2.42); CD (MeOH) [h] (nm): 42,283 (200), 57,823 (214), +4042 (239), +23,390 (276); for 1H and 13C NMR (CD3OD) spectroscopic data, see Tables 1 and 2; ESI-Q-TOF MS: m/z 427.1378 (calcd. for C21H24O8Na [M + Na]+, 427.1369, error D 2.11 ppm). 4.3.2. (2S)-5,7-dihydroxyflavan-7-O-b-D-glucopyranoside-(4a ? 8)epiafzelechin-3-O-gallate (2) Brown amorphous powder: [a24 D ] 129.9° (c 0.03, MeOH); UV (MeOH) kmax (log e) 211 (3.75), 281 (2.86); CD (MeOH) [h] (nm): +45,960 (201), 37,430 (219), 15,539 (235), 3050 (267), 6006 (289); for 1H and 13C NMR (CD3OD) spectroscopic data, see Tables 1 and 2; ESI-Q-TOF MS: m/z 851.2179 (calcd. for C43H40O17Na [M + Na]+, 851.2163, error D 1.88 ppm). 4.3.3. (2R)-40 ,7-dihydroxyflavan-(4a ? 8)-(2R,3S)-3,5,7trihdyroxyflavan-3-O-a-L-rhamnopyranoside (3) Brown amorphous powder: [a24 D ] 178.8° (c 0.05, MeOH); UV (MeOH) kmax (log e) 211 (3.88), 272 (2.77); CD (MeOH) [h] (nm): +43,505 (201), 39,965 (218), 7713 (237), +5627 (266), +11,613 (290); for 1H and 13C NMR (CD3OD) spectroscopic data, see Tables 1 and 2; ESI-Q-TOF MS: m/z 829.2711 (calcd. for C42H46O16Na [M + Na]+, 829.2684, error D 3.26 ppm).

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4.3.4. 5,7-dihydroxyflavone 6-C-b-boivinopyranosyl-7-O-b-Dglucopyranoside (4) Yellow amorphous powder: [a24 D ] +65.8° (c 0.01, MeOH); UV (MeOH) kmax (log e) 213 (3.42), 272 (3.46); for 1H and 13C NMR (CD3OD) spectroscopic data, see Tables 1 and 2; ESI-Q-TOF MS: m/z 569.1645 (calcd. for C27H30O12Na [M + Na]+, 569.1635, error D 1.05 ppm). 4.3.5. 5,7-dihydroxyflavone 7-O-b-D-glucopyranosyl-8-C-bboivinopyranoside (5) Yellow amorphous powder: [a]. 36.6° (c 0.01, MeOH); UV (MeOH) kmax (log e) 213 (3.54), 271 (3.54); for 1H and 13C NMR (CD3OD) spectroscopic data, see Tables 1 and 2; ESI-Q-TOF MS: m/z 569.1638 (calcd. for C27H30O12Na [M + Na]+, 569.1635, error D 0.53 ppm). 4.4. Acid hydrolysis Compounds 1–5 (1–2 mg) were dissolved in 1 N HCl (1 mL) and heated at 80 °C under condition of reflux for 3 h. The solution was neutralized with Ag2CO3 and evaporated under N2 gas. The residue was dissolved in H2O and partitioned with CH2Cl2. The aqueous layer was concentrated and the H2O was thoroughly removed using N2 gas. The residue was dissolved in pyridine (200 lL), and then L-cysteine methyl ester hydrochloride in pyridine (0.06 M, 200 lL) was added to the solution. The reaction mixture was incubated at 60 °C for 2 h, and trimethylsilylimidazole (200 lL) was added followed by heating at 60 °C for 1.5 h. After drying the mixture, the residue was partitioned with n-hexane and H2O (0.1 mL, each) and the n-hexane layer was subjected to GC under the following conditions; column BPX50 (0.25 mm  30 m); carrier gas He; column temperature 210 °C; injector temperature 270 °C; detector temperature 300 °C. Under the above conditions, authentic D-glucose, L-rhamnose gave GC peaks at tR 11.87 and 9.21 min, respectively, and the tR of the sugars of the isolates obtained by acid hydrolysis gave similar results as those of standard sugars. Acknowledgements This work was supported by a National Institute of Biological Resources (NIBR) Grant, the National Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011–0011899), and a research fund of the Catholic University of Korea (2011). The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/adBO13. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2015. 08.001.

References Barrett, M.W., Klyne, W., Scopes, P.M., Fletcher, A.C., Porter, L.J., Haslam, E., 1979. Circular dichroism of procyanidins and related compounds. J. Chem. Soc. Perkin Trans. 1, 2375–2377. De Mello, J.P., Petereit, F., Nahrstedt, A., 1996. Flavan-3-ols and prodelphinidins from Stryphnodendron adstringens. Phytochemistry 41, 807–813. Dongmo, A.B., Miyamoto, T., Yoshikawa, K., Arihara, S., Lacaille-Dubois, M.A., 2007. Flavonoids from Acacia pennata and their cyclooxygenase (COX-1 and COX-2) inhibitory activities. Planta Med. 73, 1202–1207. Dongmo, A.B., Nguelefack, T., Lacaille-Dubois, M.A., 2005. Antinociceptive and antiinflammatory activities of Acacia pennata wild (Mimosaceae). J. Ethnophamacol. 98, 201–206. Hammami, S., Jannet, H.B., Bergaoui, A., Ciavatta, L., Cimino, G., Mighri, Z., 2004. Isolation and structure elucidation of a flavanone, a flavanone glycoside and vomifoliol from Echiochilon fruticosum growing in Tunisa. Molecules 9, 602–608. Hatano, T., Yamashita, A., Hashimoto, T., Ito, H., Kubo, N., Yoshiyama, M., Shimura, S., Itoh, Y., Okuda, T., Yoshida, T., 1997. Flavan dimers with lipase inhibitory activity from Cassia nomame. Phytochemistry 46, 893–900. Ishimaru, K., Nonaka, G.-I., Nishioka, I., 1987. Flavan-3-ol and procyanidin glycoside from Quercus miyagii. Phytochemistry 26, 1167–1170. Korver, O., Wilkins, C.K., 1971. Circular dichroism spectra of flavonols. Tetrahedron 27, 5459–5465. Lalchhandama, K., 2013. Efficacy and structural effects of Acacia pennata root bark upon the avian parasitic helminth, Raillietina echinoboththrida. Pharmacogn. J. 5, 17–21. Liu, J.-X., Di, D.-L., Shi, Y.-P., 2008. Diversity of chemical constituents from Saxifraga Montana H. J. Chin. Chem. Soc. 55, 863–870. Liu, H., Mou, Y., Zhao, J., Wang, J., Zhou, L., Wang, M., Wang, D., Han, J., Yu, Z., Yang, F., 2010. Flavonoids from Halostachys caspica and their antimicrobial and antioxidant activities. Molecules 15, 7933–7945. Mok, S.-Y., Lee, S., 2013. Identification of flavonoids and flavonoid rhamnosides from Rhododendron mucronulatum var. albiflorum and their inhibitory activities against aldose reductase. Food Chem. 136, 969–974. Ogawa, M., Hisada, S., Inagaki, I., 1970. Studies of the constituents of Enkianthus nupides. I. New flavan glucosides of the leaves and its absolute configuration. Yakugaku Zasshi 90, 1081–1087. Rifai, Y., Arai, M.A., Koyano, T., Kowithayakon, T., Ishibashi, M., 2010. Terpenoid and a flavonoid glycoside from Acacia pennata leaves as hedgehog/GLI-mediated transcriptional inhibitors. J. Nat. Prod. 73, 995–997. Slade, D., Ferreira, D., Marais, J.P., 2005. Circular dichroism, a powerful tool for the assessment of absolute configuration of flavonoid. Phytochemistry 66, 2177– 2215. Sowndhararajan, K., Joseph, J.M., Manian, S., 2013. Antioxidant and free radical scavenging activities of indan Acacias: Acacia leucophloea (Roxb.) Willd., Acacia ferruginea DC., Acacia dealbata Link., and Acacia pennata (L.) Willd. Int. J. Food. Prop. 16, 1717–1729. Suzuki, R., Okada, Y., Okuyama, T., 2003. Two flavones C-glycosides from the style of Zea mays with glycation inhibitory activity. J. Nat. Prod. 66, 564–565. Takeda, Y., Okada, Y., Masuda, T., Hirata, E., Shinkazu, T., Otsuka, H., 2004. C, Obisglycosylapigenins from the leaves of Rhamnella inaequlatera. Phytochemistry 65, 463–468. Terangpi, R., Basumatary, R., Tamuli, A.K., Teron, R., 2013. Pharmacognostic and physiochemical evaluation of stem bark of Acacia pennata (L.) Willd., a folk plant of the Dimasa tribe of Assam. J. Pharmacogn. Phytochem. 2, 134–140. Xu, F., Wang, C., Yang, L., Luo, H., Fan, W., Zi, C., Dong, F., Hu, J., Zhou, J., 2013. Cdideoxy flavons from the stems and leaves of Passiflora edulis Sims. Food Chem. 136, 94–99. Zhang, H.M., Wang, C.F., Shen, S.M., Wang, G.L., Liu, P., Liu, Z.M., Wang, Y.Y., Du, S.S., Liu, Z.L., Deng, Z.W., 2012. Antioxidant phenolic compounds from Pu-erh tea. Molecules 17, 14037–14045.