Acylphloroglucinolated Catechin and Phenylethyl Isocoumarin

Sep 2, 2016 - ... synthesized using Agrimonia pilosa. Maheshkumar Prakash Patil , Yong Bae Seo , Gun-Do Kim. Microbial Pathogenesis 2018 116, 84-90 ...
3 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/jnp

Acylphloroglucinolated Catechin and Phenylethyl Isocoumarin Derivatives from Agrimonia pilosa Hyun Woo Kim, Junha Park, Kyo Bin Kang, Tae Bum Kim, Won Keun Oh, Jinwoong Kim, and Sang Hyun Sung* College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Gwanak-gu, Seoul 08826, Republic of Korea S Supporting Information *

ABSTRACT: Eight new compounds (1−8), including five acylphloroglucinolated catechins (1−5) and three phenylethyl isocoumarin glycosides (6−8), were isolated from Agrimonia pilosa along with six other known compounds (9−14). The new compounds were characterized structurally by NMR, MS, and ECD analyses. Compounds 4 and 5 were assigned as acylphloroglucinolated procyanidin derivatives, which are described for the first time from Nature. The absolute configuration of compound 8 was elucidated by computational analysis of its ECD spectrum. The isolated compounds were evaluated for their inhibitory activity against lipopolysaccharide-induced NO production in BV2 microglial cells.

■

RESULTS AND DISCUSSION A methanol extract of the whole plant of A. pilosa was suspended in H2O and partitioned successively with n-hexane, CHCl3, EtOAc, and n-butanol. Further separation of the EtOAc-soluble fraction by chromatographic methods yielded eight acylphloroglucinolated catechins (1−5 and 12−14) and six phenylethyl isocoumarins (6−11). In addition, six compounds (9−14) were identified as known compounds, namely, agrimonolide (9), agrimonolide-6-O-glucoside (10), desmethylagrimonolide-6-O-glucoside (11), and pilosanols A− C (12−14), by comparison of their spectroscopic data with literature values.4,5 Compound 1 was obtained as a brownish, amorphous solid. The HRESIMS of this substance showed a deprotonated ion peak at m/z 539.1909 [M − H]− (calcd for C29H31O10, 539.1917), indicating a molecular formula of C29H32O10. The 1 H NMR spectrum showed an ABX aromatic system [δH 7.17 (1H, dd, J = 8.1, 1.8 Hz, H-2′), 7.21 (1H, d, J = 8.1 Hz, H-3′), and 7.59 (1H, d, J = 1.8 Hz, H-6′)], two oxygenated methine protons [δH 5.19 (1H, d, J = 7.5 Hz, H-2) and 4.58 (1H, ddd, J = 8.2, 7.5, 5.4 Hz, H-3)], a methylene group [δH 3.55 (1H, dd, J

Agrimonia pilosa Ledeb. (Rosaceae) is a perennial herb that is distributed widely in East Asia. The aerial parts of this species have been used traditionally for the treatment of hemostasis, inflammation, and diarrhea in Korea, mainland China, and Japan. In previous studies, extracts and pure compounds isolated from A. pilosa exhibited various bioactivities including antioxidative, antiobesity, α-glucosidase inhibitory, and antitumor effects.1−3 Flavonoids, tannins, and triterpenoids are the main reported constituents of A. pilosa.1,4,5 Two classes of compounds, acylphloroglucinolated catechins and phenylethyl isocoumarins, have also been isolated before and are observed characteristically in Agrimonia species.4,5 These compounds were reported to show antimicrobial, hepatoprotective, and anti-inflammatory activities.4,6,7 In a search for these types of compounds from A. pilosa, five new acylphloroglucinolated catechins (1−5) and three new phenylethyl isocoumarins (6− 8) were isolated along with six known compounds (9−14). The structures of 1−8 were elucidated by interpreting their NMR, HRESIMS, and ECD spectra. All isolated compounds were evaluated for their inhibitory activities against LPSinduced NO production in BV2 microglia cells. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: June 20, 2016

A

DOI: 10.1021/acs.jnatprod.6b00566 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

quaternary carbons [δC 112.0 (C-1″), 112.6 (C-3″), and 109.5 (C-5″)], a methylene carbon (δC 18.3, C-7″), an aryl methyl carbon (δC 10.1, C-8″), a methoxy carbon (δC 62.6, OCH3-4″), a carbonyl carbon (δC 211.3, C-9″), and four aliphatic carbons [δC 46.2 (C-10″), 27.9 (C-11″), 12.6 (C-12″), and 17.8 (C13″)], suggesting the presence of an acylphloroglucinol moiety (D ring). The structure of the acylphloroglucinol moiety was characterized further by 2D NMR data analysis (Figure 1). The

Figure 1. Key 1H−1H COSY, HMBC, and ROESY correlations of compound 1.

= 16.1, 5.4 Hz, H-4a) and 3.24 (1H, dd, J = 16.1, 8.2 Hz, H4b)], and an aromatic singlet proton [δH 6.58 (1H, s, H-8)], indicating that 1 is a catechin derivative, in which C-6 or C-8 was also substituted (Table 1). The 13C NMR spectrum showed resonances for three oxygenated aromatic carbons [δC 162.6 (C-2″), 160.1 (C-4″), and 159.9 (C-6″)], three aromatic

HMBC correlation of the methylene proton signals at δH 4.27 (2H, s, H-7″) with resonances at δC 112.0, 162.6, and 159.9 suggested their assignments as C-1″, C-2″, and C-6″, respectively. The HMBC correlations from the aryl methyl

Table 1. 1H and 13C NMR Data of Compounds 1−3 in Pyridine-d5 (1H 600 MHz, 13C 150 MHz) isopilosanol A (1) position

δC, type

2 3 4a 4b 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 11″ 12″ 13″ OCH3-4″

83.6, CH 68.3, CH 30.1, CH2 155.6, 107.2, 154.3, 96.2, 155.8, 103.1, 132.3, 120.0, 116.8, 147.6, 147.6, 116.4, 112.0, 162.6, 112.6, 160.1, 109.5, 159.9, 18.3, 10.1, 211.3, 46.2, 27.9, 12.6, 17.8, 62.6,

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

5.19, 4.58, 3.55, 3.24,

δC, type

d (7.5) ddd (8.2, 7.5, 5.4) dd (16.1, 5.4) dd (16.1, 8.2)

83.6, CH 68.3, CH 30.1, CH2

6.58, s

7.17, dd (8.1, 1.8) 7.21, d (8.1)

7.59, d (1.8)

4.27, s 2.26, s 3.76, 1.90, 0.90, 1.22, 3.69,

isopilosanol B (2)

δH (J in Hz)

dd (13.4, 6.7) m 1.44, m t (7.4) d (6.8) s

155.6, 107.3, 154.4, 96.3, 155.8, 103.1, 132.3, 120.0, 116.8, 147.6, 147.6, 116.4, 112.1, 162.5, 112.6, 160.0, 109.2, 159.7, 18.3, 10.2, 211.4, 39.6, 20.2, 20.2,

5.20, 4.59, 3.55, 3.25,

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

δC, type

d (7.5) ddd (8.2, 7.5, 5.4) dd (16.1, 5.4) dd (16.1, 8.2)

83.6, CH 68.3, CH 30.0, CH2

6.59, s

7.17, dd (8.1, 1.9) 7.21, d (8.1)

7.59, d (2.0)

4.27, s 2.26, s 3.86, dt (13.5, 6.8) 1.23, d (1.4) 1.22, d (1.4)

62.6, CH3

3.68, s B

isopilosanol C (3)

δH (J in Hz)

155.6, 107.1, 154.3, 96.2, 155.8, 103.1, 132.4, 120.0, 116.8, 147.6, 147.6, 116.4, 111.7, 163.2, 112.5, 160.7, 109.0, 160.4, 18.1, 10.1, 207.0, 45.0, 18.8, 14.5,

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

62.3, CH3

δH (J in Hz) 5.21, 4.59, 3.57, 3.27,

d (7.5) ddd (8.2, 7.5, 5.4) dd (16.2, 5.4) dd (16.2, 8.2)

6.59, s

7.17, dd (8.1, 1.9) 7.2, d (8.1)

7.59, d (1.9)

4.25, s 2.25, s 3.13, t (7.2) 1.76, h (7.2) 0.94, t (7.2) 3.67, s DOI: 10.1021/acs.jnatprod.6b00566 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

protons at δH 2.26 (H-8″) to the aromatic carbons at δC 162.6 (C-2″), 160.1 (C-4″), and 112.6 (C-3″) and from the methoxy protons at δH 3.69 (OCH3-4″) to 160.1 (C-4″) supported the location of the aryl methyl and the methoxy group at C-3″ and C-4″, respectively. The acyl group was identified as a 2methylbutanoyl group from the 1H−1H COSY spectrum (Figure 1), which did not show any interactions with the aromatic carbons in the HMBC spectrum. In the ROESY spectrum, however, the α-methine proton of the acyl group (H10″) showed a NOE correlation with the methoxy proton (δH 3.69, OCH3-4″), suggesting that the acyl group is connected to C-5″ (δC 109.5) (Figure 1). The HMBC correlation between the methylene protons at δH 4.27 (2H, s, H-7″) and the catechin carbons at δC 107.2 (C-6) indicated a linkage between the catechin and acylphloroglucinol moieties. Both H-2 and H8 of the catechin moiety showed HMBC correlations with δC 155.8 (C-9), demonstrating that the phloroglucinol moiety is substituted at C-6 instead of C-8. The absolute configuration of 1 was determined from the ECD spectrum. The ECD spectrum of (+)-catechin was reported to show a negative Cotton effect at 270−280 nm.8 The ECD spectrum of 1 also showed a negative Cotton effect in this region, so the configurations at C2 and C-3 were determined to be (2R,3S). From these results, the structure of 1 was elucidated as shown. While the functional groups were identical to those of pilosanol A (12), the acylphloroglucinol moiety was substituted at C-6 instead of C8. Accordingly, compound 1 has been named isopilosanol A. From its HRESIMS and the deprotonated ion at m/z 525.1746 [M − H]−, the molecular formula of 2 was suggested as C28H30O10. On comparing the 1H and 13C NMR spectra of 2 with those of 1, the presence of two methyl group proton signals at δH 1.23 (3H, d, J = 1.4 Hz, H-11″) and 1.22 (3H, d, J = 1.4 Hz, H-12″), and a methine proton signal at δH 3.86 (1H, dt, J = 13.5, 6.8 Hz, H-10″) indicated that compound 2 contains a 2-methylpropanoyl group instead of the 2methylbutanoyl group of 1 (Table 1). The HMBC spectrum also showed the acylphloroglucinol moiety to be substituted at C-8. The ECD spectrum of 2 showed a negative Cotton effect at 270−280 nm, indicating the absolute configuration of this compound to be (2R,3S). Owing to the similarity between the structure of 2 and pilosanol B (13), compound 2 was named isopilosanol B. The molecular formula of 3, C28H30O10, was concluded from the HRESIMS, which showed a deprotonated molecular ion at m/z 525.1765 [M − H]−. The 1H and 13C NMR spectra of 3 were similar to those of 1, but one methyl group was absent (Table 1). The 1H−1H COSY spin system of three proton peaks [(δH 3.13 (H-10″), 1.76 (H-11″), and 0.94 (H-12″)] supported the presence of a butanoyl group in the D ring. The absolute configuration of 3 was also identified by the electronic circular dichroism (ECD) negative Cotton effect at 270−280 nm. By comparison with the structure of pilosanol C (14), compound 3 was named isopilosanol C. Compound 4 was isolated as a pale pink, amorphous powder. Its molecular formula was determined to be C43H42O16 from the HRESIMS (m/z 813.2407 [M − H]−, calcd for C43H41O16, 813.2395). Interpretation of the 1H and 13C NMR spectra confirmed this compound to be a procyanidin derivative. From the two ABX spin systems in the aromatic region (δH 5.6−7.0), the methine protons with large coupling constants in the region of δH 4.4−4.7 (H-2/H-3/H-4), and the 13C NMR resonances of two oxygenated carbons at δC 85.9 (C-2u) and 81.3 (C-2t), two catechin units could be identified (Table 2). The coupling

Table 2. 1H and 13C NMR Data of Compounds 4 and 5 in CD3OD (1H 600 MHz, 13C 150 MHz) pilosanidin A (4)

pilosanidin B (5)

position

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

2u 3u

85.9, CH 73.7, CH

4.41, d (10.1) 4.68, dd (10.1, 7.1) 4.47, d (7.1)

84.6, CH 72.2, CH

4.45, d (10.1) 4.78, dd (10.0, 7.0) 4.50, d (6.9)

4u 5u 6u 7u 8u 9u 10u 1′u 2′u 3′u 4′u 5′u 6′u 1″u 2″u 3″u 4″u 5″u 6″u 7″u a 7″u b 8″u 9″u 10″u 11″u 12″u OCH34″u 2t 3t 4t a

38.8, 155.9, 98.7, 155.2, 106.3, 155.1, 108.1, 131.3, 121.3, 116.4, 146.2, 146.9, 116.7, 111.2, 161.2, 112.2, 159.7, 110.2, 158.3, 17.4,

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

9.4, 212.9, 40.4, 20.2, 19.6, 62.8,

CH3 C CH CH3 CH3 CH3

81.3, CH 67.6, CH 26.7, CH2

4t b 5t 6t 7t 8t 9t 10t 1′t 2′t 3′t 4′t 5′t 6′t

6.03, s

6.80, br s 6.79, br s

6.97, br s

3.64, d (15.8), 3.51, d (15.8) 1.91, s 3.61, 1.09, 1.13, 3.60,

m d (7.0) d (7.0) s

4.91, d (5.1) 4.10, dd (10.3, 5.1) 2.51, m

37.2, 153.5, 97.4, 154.4, 104.7, 154.0, 107.3, 129.8, 120.0, 114.8, 145.4, 144.8, 115.1, 109.2, 160.6, 110.8, 159.4, 106.5, 158.2, 15.6,

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

7.9, 206.7, 44.1, 17.6, 12.9, 60.6,

CH3 C CH2 CH2 CH3 CH3

C CH C C C C C CH CH C C CH

6.04, s

5.67, br d (8.1) 6.32, d (8.1)

6.37, br s

6.86, d (7.8) 6.82, d (7.8)

7.03, br d

3.58, br d 3.58, br d 1.93, s 3.05, 1.70, 0.99, 3.58,

t (7.3) m t (7.5) s

79.4, CH 65.8, CH

5.03, d (3.8) 4.18, m

24.2, CH2

2.56, dd (16.6, 3.9) 2.38, dd (16.6, 4.2)

2.51, m 156.0, 95.9, 155.1, 108.8, 154.0, 101.6, 131.8, 119.1, 115.9, 145.4, 145.5, 113.8,

6.04, s

154.5, 111.6, 153.6, 108.0, 152.2, 99.8, 130.1, 117.1, 114.2, 143.9, 143.7, 94.3,

C CH C C C C C CH CH C C CH

6.29, s

5.46, d (7.8) 6.20, d (8.2)

6.04, br s

constants of H-2u (δH 4.41, d, J = 10.1 Hz) and H-2t (δH 4.91, d, J = 5.1 Hz) suggested these two catechin units are 2,3-transcatechin and 2,3-cis-epicatechin, respectively. An aromatic proton at δH 6.04 (1H, s, H-6t) and a methine proton at δH 4.47 (1H, d, J = 7.1 Hz, H-4u) indicated the presence of an interflavan bond between the two catechin units. The position of the interflavan bond was determined from the HMBC spectrum. The cross-peak between the proton at δH 4.47 (1H, C

DOI: 10.1021/acs.jnatprod.6b00566 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

d, J = 7.1 Hz, H-4u) and the aromatic quaternary carbon at δC 108.8 (C-8t) signified a C4→C8 interflavan linkage of a B-type procyanidin (Figure 2). Three aliphatic protons at δH 3.61 (1H,

with a 2-methylpropanoyl group, similar to 2. The regiostructure of the D ring was confirmed by the HMBC spectrum (Figure 2). The HMBC correlations of the methylene protons [δH 3.64 (1H, d, J = 15.8 Hz, H-7″u a) and 3.51 (1H, d, J = 15.8 Hz, H-7″u b)] with δC 111.2 (C-1″u), 161.2 (C2″u), and 158.3 (C-6″u) showed their relative positions. The arylmethyl group at δH 1.91 and the methoxy group at δH 3.60 exhibited cross-peaks with resonances at δC 161.2 (C-2″u), 112.2 (C-3″u), and 159.7 (C-4″u), which suggested an arylmethyl group and methoxy group to be located at C-3″u and C-4″u, respectively. The position of the 2-methylpropanoyl unit was assigned as C-5″u by the NOE interactions of H-11″u and H-12″u with this methoxy group (OCH3-4″u) in the ROESY spectrum, in a similar manner to compound 1 (Figure 2). The HMBC cross-peak of H-7″ and δC 106.3 (C-8u) showed the D ring to be substituted at C-8u. The absolute configuration of the interflavan linkage was determined by the ECD spectrum. From the literature, the Cotton effect at 220− 240 nm was used to investigate the orientation of the 4-flavanyl linkage.9−12 In the ECD spectrum of 4, a strong negative Cotton effect was seen at 220−240 nm, which indicated the configuration at C-4 to be R. To the best of our knowledge, this is the first time that this type of acylphloroglucinolated

Figure 2. Key 1H−1H COSY, HMBC, and ROESY correlations of compound 4.

m, H-10″u), 1.09 (3H, d, J = 7.0 Hz, H-11″u), and 1.13 (3H, d, J = 7.0 Hz, H-12″u), an arylmethyl proton at δH 1.91 (3H, s, H8″u), and a methoxy proton at δH 3.60 (3H, s, OCH3-4″u) revealed the presence of an acylphloroglucinol moiety (D ring)

Table 3. 1H and 13C NMR Data of Compounds 6−8 in CD3OD (1H 600 MHz, 13C 150 MHz) (3S)-desmethylagrimonolide-4′-O-β-Dglucopyranoside C (6) position

172.4, C 80.6, CH 34.7, CH2

5 6 7 8 9 10 1′ 2′,6′ 3′,5′ 4′ OCH3-4′ 1″a 1″b 2″a 2″b Glc-1 Glc-2 Glc-3 Glc-4 Glc-5 Glc-6

108.9, 167.5, 103.1, 166.4, 102.2, 144.2, 137.2, 131.2, 118.7, 158.3,

Ara-1 Ara-2 Ara-3 Ara-4 Ara-5 a

δC, type

1 3 4

CH C CH C C C C CH CH C

38.5, CH2 32.0, CH2 103.3, 78.9, 78.8, 75.7, 72.2, 63.3,

CH CH CH CH CH CH2

δH (J in Hz) 4.44, m 2.86, d (7.3) 6.19, d (2.2) 6.18, d (2.2)

7.15, d (8.6) 7.02, d (8.6)

2.07, m 1.97, m 2.85, m 2.66, m 4.86, d (7.7)a 3.48−3.36, m 3.48−3.36, m 3.48−3.36, m 3.48−3.36, m 3.87, dd (12.0, 1.9) 3.68, dd, (12.0, 5.2)

(3S)-agrimonolide-6-O-α-L-arabinofuranosyl-(1→6)β-D-glucopyranoside (7) δC, type

δH (J in Hz)

171.4, C 80.2, CH 34.0, CH2 107.7, 165.2, 103.1, 165.0, 104.1, 143.5, 134.4, 130.4, 115.0, 159.6, 55.6, 37.8,

4.53, 3.01, 2.94, 6.50, 6.54,

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

7.15, d (8.6) 6.83, d (8.6) 3.75, s 2.08, m 1.99, m 2.83, m 2.72, m 4.96, d (7.4) 3.43−3.46, m 3.43−3.46, m 3.36−3.31, m 3.67, m 4.06, dd (11.1, 2.0) 3.59, m 4.91, d (1.3) 4.00, dd (3.4, 1.5) 3.81, dd (6.0, 3.4) 3.97, td (5.6, 3.3) 3.71, dd, (11.9, 3.2), 3.61, m

31.1, CH2 101.3, 74.7, 77.9, 71.7, 77.2, 68.2,

CH CH CH CH CH CH2

110.1, 83.4, 78.9, 85.8, 63.1,

CH CH CH CH CH2

m m m d (2.2) d (2.2)

(3S,4R)-4-hydroxyagrimonolide-6-O-β-Dglucopyranoside (8) δC, type

δH (J in Hz)

170.1, C 84.0, CH 68.3, CH

4.31, ddd (9.0, 8.0, 3.3) 4.58, d (8.0)

107.1, 165.8, 104.5, 165.2, 102.6, 146.4, 130.7, 130.5, 115.0, 159.7, 55.8, 35.0,

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

31.3, CH2 101.6, 78.5, 78.0, 74.8, 71.4, 62.6,

CH CH CH CH CH CH2

6.77, d (2.2) 6.60, d (2.2)

7.15, d (8.6) 6.84, d (8.6) 3.76, 2.16, 1.96, 2.87, 2.73, 5.02, 3.49, 3.48, 3.47, 3.40, 3.90, 3.70,

s m m m, m d (7.3) m m m m dd (12.1, 2.3) dd, (12.2, 5.5)

The signal was partially masked by the water signal in CD3OD; thus, the J value was obtained in DMSO-d6 (300 MHz). D

DOI: 10.1021/acs.jnatprod.6b00566 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 3. Key 1H−1H COSY and HMBC correlations of compounds 6 and 7.

268 nm, so the absolute configuration at C-3 was found to be 3S. Consequently, the structure of 6 was determined as (3S)desmethylagrimonolide-4′-O-β-D-glucopyranoside. Compound 7 was purified as a pale white, amorphous powder, for which the molecular formula was determined to be C29H36O14 by the HRESIMS, which exhibited a deprotonated molecular ion peak at m/z 607.2027 [M − H]− and an additional fragment ion at m/z 313.1081 [M − Glc − Ara − H]−. This suggested that 7 is a diglycosidic compound containing both a hexose and a pentose unit. The 1H and 13 C NMR spectra showed that compound 7 has a similar structure to 6 (Table 3). Aromatic AA′XX′ proton resonances at δH 7.15 (2H, d, J = 8.6 Hz, H-2′, H-6′) and 6.83 (2H, d, J = 8.6 Hz, H-3′, H-5′) and two meta-coupled aromatic proton resonances at δH 6.50 (1H, d, J = 2.2 Hz, H-5) and 6.54 (1H, d, J = 2.2 Hz, H-7) were consistent with the two aromatic groups of an agrimonolide derivative. A methoxy group at δH 3.75 (3H, s, OCH3-4′) was also observed as being different from compound 6. Two anomeric proton resonances at δH 4.96 (1H, d, J = 7.4 Hz, H-Glc-1) and 4.91 (1H, d, J = 1.3 Hz, HAra-1) supported the presence of two sugars on the molecule. The HMBC correlation of H-Ara-1 and C-Glc-6 (δC 68.2) supported a glucopyranosyl-(1→6)-arabinoside moiety, and the cross-peak between H-Glc-1 and C-6 (δC 165.2) indicated the location of the diglycosidic moiety to be C-6 (Figure 3). The sugar units were confirmed as D-glucose and L-arabinose by the hydrolytic method used for compound 6.13 The cyclic form of arabinose was determined to be arabinofuranose by comparison of NMR data with literature values. 14 The absolute configuration at C-3 was confirmed by the ECD positive Cotton effect at 268 nm to be 3S. Therefore, compound 7 was elucidated as (3S)-agrimonolide-6-O-α-L-arabinofuranosyl-(1→ 6)-β-D-glucopyranoside. Compound 8 was isolated as a pale yellow powder. From the analysis of the HRESIMS spectrum, the molecular formula of 8 was determined to be C24H28O11, or one more oxygen atom than 6. This suggested 8 has an additional hydroxy group when compared to 6. In comparison with the 1H NMR spectrum of 6, 8 showed two shifted peaks, with one at δH 4.31 (1H, ddd, J = 9.0, 8.0, 3.3 Hz, H-3) and the other at δH 4.58 (1H, d, J = 8.0, H-4). A hydroxylated carbon at δC 68.3 was observed, and it was assigned as C-4 by the HMBC correlation with H-3 and H5. These results showed that C-4 is substituted with a hydroxy group. The relative configurations at C-3 and C-4 were assigned by analysis of the J value of H-4. In the literature on 3substituted 4-hydroxy isocoumarin derivatives, the J value of H4 is above 6 Hz for 3,4-trans isomers and under 2.5 Hz for 3,4cis isomers.15 Thus, the relative configuration of compound 8 was confirmed as 3,4-trans. In the HMBC spectrum, the anomeric proton at δH 5.02 (1H, d, J = 7.3 Hz, H-Glc-1)

procyanidin has been isolated from Nature. Compound 4 was named pilosanidin A. Compound 5 was isolated as a pale pink, amorphous powder, for which the molecular formula was determined as C43H42O16 from the HRESIMS data. This elemental formula was the same as that of 4, and the NMR spectra of these two compounds were also similar. One carbonyl carbon at δC 206.7 and three aliphatic proton signals at δH 3.05 (2H, t, J = 7.3 Hz, H-10″u), 1.70 (2H, dd, J = 14.1, 6.9 Hz, H-11″u), and 0.99 (3H, t, J = 7.8 Hz, H-12″u) revealed the presence of a butanoyl group. The absolute configuration of the interflavan linkage in 5 was also determined by the ECD spectrum. The ECD spectrum of 5 showed a negative Cotton effect at 220−240 nm, again suggesting the configuration at C-4 to be R. From these results, the structure of 5 (pilosanidin B) was determined as shown. Compound 6 was isolated as a pale white powder. Its molecular formula was determined to be C23H26O10 from its HRESIMS. In the IR spectrum, an absorption peak at 1657 cm−1 showed the presence of a lactone and was supported by the 13C NMR signal at δC 171.35 (C-1). The 1H NMR spectrum of 6 exhibited an AA′XX′ aromatic system [δH 7.15 and 7.02] and two meta-coupled aromatic protons at δH 6.19 (1H, d, J = 2.2 Hz, H-5) and δH 6.18 (1H, d, J = 2.2 Hz, H-7), suggesting the presence of 1,4-disubstituted and 1,3,4,5tetrasubstituted aromatic substructures, respectively (Table 3). The 1H−1H COSY spin system of H-2″s (δH 2.85, 2.66), H-1″s (δH 2.07 and 1.97), H-3 (δH 4.44), and H-4s (δH 2.86) supported the presence of an aliphatic unit of 6 (Figure 3). The HMBC correlations of H-2″, H-3′, and H-5′ (δH 7.02) with C1′ (δC 137.2) showed that the 1,4-disubstituted aromatic ring is connected to the aliphatic group at C-2″. The methylene protons at δH 2.86 (H-4) showed a cross-peak with δC 108.9 (C-5), which supported the aromatic ring as having metacoupled protons and being connected to C-4. In addition, the correlations of H-3 and H-7 with a carbonyl carbon (δC 172.4, C-1) suggested that the lactone oxygen could be located to C-3 (Figure 3). From these results, compound 6 was assigned as a demethylated agrimonolide derivative. The presence of an anomeric proton at δH 4.86 (1H, d, J = 7.7 Hz, H-Glc-1) and other protons at 3.36−3.87 suggested the presence of an O-βglucosyl moiety. In the HMBC spectrum, the cross-peak of HGlc-1 with C-4 supported the placement of this βglucopyranosyl group at C-4. The glucose moiety was confirmed as D-glucose by acid hydrolysis of 6 and subsequent conversion to an arylthiocarbamoyl-thiazolidine derivative by reaction with L-cysteine and o-tolyl isothiocyanate.13 The absolute configuration at C-3 of 6 was confirmed from the ECD spectrum. Previously, a Cotton effect at 268 nm was correlated with the C-3 configuration of agrimonolide (11) and its derivatives.5 Compound 6 showed a positive Cotton effect at E

DOI: 10.1021/acs.jnatprod.6b00566 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

NO production with IC50 values of 43.6 and 38.1 μM, respectively.

showed a cross-peak with C-6 (δC 165.8), indicating a 6-O-βglucopyranosyl substitution (Figure 4). The glucosyl moiety

Table 4. Inhibition of LPS-Induced NO Production in BV2Microglial Cellsa compound 4 5 12 13 14 NAMEb

Figure 4. Key 1H−1H COSY and HMBC correlations of compound 8.

IC50 (μM) 38.1 52.4 60.4 54.6 43.6 24.7

± ± ± ± ± ±

1.6 1.4 1.5 1.4 1.6 1.1

a

Nitrite (NO) concentrations of the control and LPS-treated groups (500 ng/mL) were 4.9 ± 0.9 and 41.6 ± 5.5 μM, respectively. Values are the mean ± SD of three experiments. bNAME (ω-nitro-L-arginine methyl ester) was used as a positive control.

was identified as a D-glucosyl unit by the method described above for compound 6.13 The absolute configurations at C-3 and C-4 were determined by comparing the ECD spectrum of aglycone 8a with the computationally calculated ECD spectrum. Enzymatic hydrolysis of 8 by β-glucosidase was carried out to afford the aglycone 8a. The ECD spectrum of 8a was recorded and compared with density functional theory (DFT)-calculated spectra for the 3S,4R stereoisomer of 8a.16 First, a conformational search of 3S,4R-8a using the molecular mechanics force field (MMFF) was performed. Since 8a has many rotatable points such as C-1″ and C-2″, up to 300 conformers were created. These were sorted by their expected population, and the 10 major conformers were selected. The sum of their expected population was above 50%. The conformers were followed by DFT geometry optimizations at the B3LYP/def-SV(P) level in the gas phase. After optimization, the ECD spectra of the optimized conformers were calculated using time-dependent DFT (TDDFT) at the B3LYP/def-TZVP level. The Boltzmann-averaged computed ECD spectrum of the major 8a conformers (population above 1%) showed a similar fit to the experimental spectrum of 8a, which exhibited one positive and two negative Cotton effects at 215, 230, and 256 nm, respectively (Figure 5). These results suggested the absolute configuration of 8a to be 3S,4R. Therefore, the structure of 8 was elucidated as (3S,4R)-4hydroxyagrimonolide-6-O-β-D-glucopyranoside. Compounds 1−14 were evaluated for their inhibitory activity against LPS-stimulated NO production in BV2 microglial cells with the Griess assay. Among them, compounds 4, 5, 12, 13, and 14 showed an inhibitory effect without cytotoxicity. As shown in Table 4, compounds 5 and 14 exhibited inhibition of

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a JASCO P-2000 polarimeter (JASCO, Easton, MD, USA). All UV and ECD spectra were obtained with a Chirascan ECD spectrometer (Applied Photophysics, Leatherhead, UK). IR spectra were taken on a JASCO FT/IR-4200 spectrometer. The NMR spectra were recorded on an AVANCE-600 NMR spectrometer (Bruker, Billerica, MA, USA), which was equipped with a cryogenic probe. All HRESIMS data were measured on a Waters Xevo G2 QTOF mass spectrometer (Waters Co., Milford, MA, USA). Column chromatography was performed with Kieselgel 60 silica gel (40−60 μm, 230−400 mesh, Merck, Darmstadt, Germany) and Sephadex LH-20 (25−100 μm, Pharmacia, Piscataway, NJ, USA). TLC was carried out using Kieselgel 50 F254 coated normal-phase silica gel TLC plates (Merck). The preparative HPLC system was equipped with a G-321 pump (Gilson, Middleton, WI, USA), a G-151 UV detector (Gilson), and a Kintex C18 column (250 mm × 10 mm i.d.; 5 μm, Phenomedex, Torrance, CA, USA). All solvents were purchased from Daejung Chemicals & Metals Co. Ltd. (Siheung, Korea). The reagents for aldose discrimination (L-cysteine methyl ester hydrochloride, o-tolyl isothiocyanate) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The conformation search was performed with Conflex 7 (Conflex Corp., Tokyo, Japan). The geometrical optimization of the computed conformers was carried out and visualized with TmoleX 3.4 and Turbomole (COSMOLogic GmbH, Leverkusen, Germany). Plant Material. Agrimonia pilosa was collected in July 2014 at the Medicinal Plant Garden, Seoul National University, Goyang, Korea, and authenticated by Prof. Tae-Jin Yang (College of Agricultural and Life Sciences, Seoul National University). A voucher specimen (SNU0724) of the plant has been deposited at the Herbarium of the Medicinal Plant Garden of the College of Pharmacy, Seoul National University. Extraction and Isolation. Dried whole parts of A. pilosa (3.9 kg) were ground and extracted three times with MeOH (4 L × 3) with ultrasonication at 40 °C. The methanol extract was concentrated using a rotary evaporator to give a crude extract (120 g). This extract was suspended in H2O and partitioned successively with n-hexane, CHCl3, EtOAc, and n-BuOH to yield n-hexane (10 g), CHCl3 (28 g), EtOAc (39 g), and n-BuOH (20 g) fractions, respectively. The EtOAc fraction was subjected to silica gel CC (40 × 10 cm), eluted with mixtures of CHCl3−MeOH (50:1 to 10:1), to yield seven fractions (A−G). Fraction A was separated over Sephadex LH-20 by elution with MeOH (100 × 2 cm), to yield three subfractions (A1−A3). Compounds 1−3 (4, 5, and 4 mg, respectively), 12 (30 mg), 13 (45 mg), and 14 (25 mg) were isolated from subfraction A3 using semipreparative HPLC with 50% aqueous acetonitrile. Fraction B was subjected to Sephadex LH-20 CC eluted with MeOH to yield compound 10 (412 mg). Fraction D was purified by Sephadex LH-20

Figure 5. Comparison of the experimental ECD spectrum of compound 8a and the calculated ECD spectrum. F

DOI: 10.1021/acs.jnatprod.6b00566 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(10.4), 230 (−2.6), 240 (0.5), 260 (−1.5), 280 (−0.3); 1H NMR (600 MHz, CD3OD) δ 7.12 (2H, d, J = 8.6 Hz, H-2′ and 6′), 6.82 (2H, d, J = 8.6 Hz, H-3′ and 6′), 6.50 (1H, d, J = 2.7 Hz, H-5′), 6.24 (1H, d, J = 2.3 Hz, H-7′), 4.50 (1H, d, J = 8.2 Hz, H-4′), 4.24 (1H, ddd, J = 9.2, 8.2, 3.1 Hz, H-3′), 3.73 (3H, s, OCH3-4′), 2.85 (1H, ddd, J = 14.2, 9.5, 4.7 Hz, H-2″a), 2.76−2.64 (1H, m, H-2″b), 2.19−2.09 (1H, m, H1″a), 1.99−1.89 (1H, m, H-1″b); HRESIMS m/z 329.1028 [M − H]− (calcd for C18H17O6, 329.1025) Cell Culture and Viability Assay. BV2 microglial cells, originally developed by Prof. V. Bocchini at the University of Perugia (Perugia, Italy), were generously provided by Prof. Sun-yeou Kim at Kyunghee University (Suwon, Korea). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) with penicillin (100 IU/mL) and streptomycin (10 mg/ mL) at 37.8 °C in a humidified atmosphere of 95% air with 5% CO2. Cell viability was evaluated using the MTT assay. The cells were incubated in a 96-well plate for 24 h, and the medium was replaced with fresh medium containing MTT (0.2 mg/mL) followed by incubation at 37.8 °C for 3 h. The supernatant was then removed, and 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan produced. The absorbance at 540 nm was measured using a microplate reader. Data are expressed as the percent cell viability relative to the control group. Evaluation of Inhibitory Activity on NO Production in LPSStimulated BV2 Microglial Cells. Isolated compounds were dissolved in DMSO for the assay, and the final concentration of DMSO in the cultures was under 0.1%. BV2 cells were seeded in a 96well plate at a concentration of 4 × 104 cells/well. Cells were treated with the test samples for 1 h before exposure to 500 ng/mL of lipopolysaccharide. After a 24 h incubation period, the nitrite in the culture medium was measured to evaluate the NO production in the BV2 cells with the Griess assay. The assay was done as previously described.17 L-NAME (ω-nitro-L-arginine methyl ester; Sigma) was used as a positive control.

CC to yield three subfractions (D1−D3). Subfraction D2 was purified using semipreparative HPLC under isocratic conditions (45% aqueous acetonitrile, 4 mL/min) to yield compounds 6−8 (15, 8, and 2, respectively) and 11 (10 mg). Fraction E was separated by Sephadex LH-20 CC into three subfractions (E1−E3). Subfraction E2 was subjected to semipreparative HPLC to yield compounds 4 (3 mg) and 5 (3 mg). Compound 9 (105 mg), which was the major compound of the CHCl3 extract, was isolated by normal-phase MPLC (CHCl3− MeOH, 50:1 → 30:1), followed by recrystallization with MeOH. Isopilosanol A (1): brown, amorphous powder; [α]25 D +27 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 236 (sh), 282 (3.2), 330 (3.2) nm; ECD (MeOH) λmax (Δε) 207 (−1.7), 247 (0.3), 284 (−0.3) nm; IR νmax 3704, 2980, 1608 cm−1; 1H (600 MHz) and 13C (150 MHz) NMR data, see Table 1; HRESIMS m/z 539.1909 [M − H]− (calcd for C29H41O10, 539.1917). Isopilosanol B (2): brown, amorphous powder; [α]25 D +28 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 235 (sh), 286 (3.2), 332 (3.2) nm; ECD (MeOH) λmax (Δε) 208 (−1.8), 243 (0.5), 285 (−0.3) nm; IR νmax 3701, 2972, 1605 cm−1; 1H (600 MHz) and 13C (150 MHz) NMR data, see Table 1; HRESIMS m/z 525.1746 [M − H]− (calcd for C28H39O10, 525.1761). Isopilosanol C (3): brown, amorphous powder; [α]25 D +19 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 236 (sh), 283 (3.2), 332 (3.2) nm; ECD (MeOH) λmax (Δε) 207 (−2.3), 241 (0.4), 284 (−0.3) nm; IR νmax 3705, 2938, 1608 cm−1; 1H (600 MHz) and 13C (150 MHz) NMR data, see Table 1; HRESIMS m/z 525.1765 [M − H]− (calcd for C28H39O10, 525.1761). Pilosanidin A (4): pale pink, amorphous powder; [α]25 D −19 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 236 (sh), 283 (3.2), 332 (3.2) nm; ECD (MeOH) λmax (Δε) 215 (−14.8), 233 (−5.4), 240 (−5.6), 274 (0.0), 287 (−0.3) nm; IR νmax 3704, 2971, 1689 cm−1; 1H (600 MHz) and 13C (150 MHz) NMR data, see Table 2; HRESIMS m/z 813.2407 [M − H]− (calcd for C43H41O16, 813.2395). Pilosanidin B (5): pale pink, amorphous powder; [α]25 D −32 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 236 (sh), 280 (3.2), 331 (3.2) nm; ECD (MeOH) λmax (Δε) 214 (−14.8), 232 (−4.0), 242 (−4.8), 276 (0.0), 287 (−0.3) nm; IR νmax 3702, 2972, 1606 cm−1; 1H (600 MHz) and 13C (150 MHz) NMR data, see Table 2; HRESIMS m/z 813.2409 [M − H]− (calcd for C43H41O16, 813.2395). (3S)-Desmethylagrimonolide-4′-O-β- D -glucopyranoside (6): white, amorphous powder; [α]25 D −71 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 217 (4.3), 269 (3.9), 302 (3.6) nm; ECD (MeOH) λmax (Δε) 211 (2.4), 227 (−1.5), 242 (0.5), 252 (0.0), 272 (1.0) nm; IR νmax 3369, 2921, 1657 cm−1; 1H (600 MHz) and 13C (150 MHz) NMR data, see Table 3; HRESIMS m/z 461.1451 [M − H]− (calcd for C23H25O10, 461.1448). (3S)-Agrimonolide-6-O-α-L-arabinofuranosyl-(1→6)-β-D-glucopyranoside (7): white, amorphous powder; [α]25 D −63 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (3.9), 265 (3.5), 302 (3.0) nm; ECD (MeOH) λmax (Δε) 211 (1.4), 226 (−0.1), 235 (0.1), 245 (−0.2), 269 (0.3), 285 (−0.3); IR νmax 3398, 1669 cm−1; 1H (600 MHz) and 13C (150 MHz) NMR data, see Table 3; HRESIMS m/z 607.2027 [M − H]− (calcd for C29H35O14, 607.2027). (3S,4R)-4-Hydroxyagrimonolide-6-O-β-D-glucopyranoside (8): white, amorphous powder; [α]D25 −102 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 215 (4.5), 265 (4.1), 303 (3.8); ECD (MeOH) λmax (Δε) 210 (10.2), 227 (−2.7), 237 (−0.3), 256 (−2.4), 278 (−0.5); IR νmax 3705, 2971, 1670 cm−1; 1H (600 MHz) and 13C (150 MHz) NMR data, see Table 3; HRESIMS m/z 491.1551 [M − H]− (calcd for C24H27O11, 491.1553). Enzymatic Hydrolysis of 8. Compound 8 (0.5 mg) was dissolved in 0.1 M NaOAc−acetate buffer (pH 4.0, 1 mL), and then βglucosidase from almonds (1.0 U, Sigma-Aldrich Co., St. Louis, MO, USA) was added. After the reaction solution was incubated at 36 °C for 1 h, it was separated on a Sep-Pak C18 cartridge (H2O → acetonitrile). Aglycone 8a was afforded from the acetonitrile fraction and identified as 4-hydroxyagrimonolide by comparison with the UV spectrum, 1H NMR spectrum, and LC-MS data of 8. (3S,4R)-4-Hydroxyagrimonolide (8a): UV (MeOH) λmax (log ε) 215 (4.3), 265 (4.1), 303 (3.7) nm; ECD (MeOH) λmax (Δε) 210

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00566. NMR data of compounds 1−8, data of the computationally calculated conformers of 8a, and bioactivity data of all the isolated compounds (1−14) (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Tel: 82-2-880-7859. Fax: 82-2-877-7859. E-mail: shsung@snu. ac.kr. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), which is funded by the Ministry of Science, ICT, and Future Planning (NRF-2013R1A2A2A01016296). We would like to thank Mr. S. I. Han (Medicinal Plant Garden of the College of Pharmacy, Seoul National University) for kindly providing the plant material.

■

REFERENCES

(1) Liu, X.; Zhu, L. C.; Tan, J.; Zhou, X. M.; Xiao, L.; Yang, X.; Wang, B. C. BMC Complementary Altern. Med. 2014, 14, 12. (2) Lee, J. A.; Ahn, E. K.; Hong, S. S.; Oh, J. S. Han'guk Sikp'um Yongyang Kwahak Hoechi 2012, 42, 161−167.

G

DOI: 10.1021/acs.jnatprod.6b00566 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(3) Miyamoto, K.; Kishi, N.; Koshiura, R. Jpn. J. Pharmacol. 1987, 43, 187−195. (4) Kasai, S.; Watanabe, S.; Kawabata, J.; Tahara, S.; Mizutani, J. Phytochemistry 1992, 31, 787−789. (5) Kato, H.; Li, W.; Koike, M.; Wang, Y. H.; Koike, K. Phytochemistry 2010, 71, 1925−1929. (6) Park, E. J.; Oh, H.; Kang, T. H.; Sohn, D. H.; Kim, Y. C. Arch. Pharmacal Res. 2004, 27, 944−946. (7) Taira, J.; Ohmine, W.; Ogi, T.; Nanbu, H.; Ueda, K. Bioorg. Med. Chem. Lett. 2012, 22, 1766−1769. (8) Korver, O.; Wilkins, C. K. Tetrahedron 1971, 27, 5459−5465. (9) Ding, Y. Q.; Li, X. C.; Ferreira, D. J. Nat. Prod. 2010, 73, 435− 440. (10) Wang, C. M.; Hsu, Y. M.; Jhan, Y. L.; Tsai, S. J.; Lin, S. X.; Su, C. H.; Chou, C. H. Molecules 2015, 20, 12787−12803. (11) Botha, J. J.; Ferreira, D.; Roux, D. G. J. Chem. Soc., Chem. Commun. 1978, 698−700. (12) Lokvam, J.; Coley, P. D.; Kursar, T. A. Phytochemistry 2004, 65, 351−358. (13) Tanaka, T.; Nakashima, T.; Ueda, T.; Tomii, K.; Kouno, I. Chem. Pharm. Bull. 2007, 55, 899−901. (14) Yasukawa, K.; Ogawa, H.; Takido, M. Phytochemistry 1990, 29, 1707−1708. (15) Hobson, S. J.; Parkin, A.; Marquez, R. Org. Lett. 2008, 10, 2813−2816. (16) Rivera-Chavez, J.; Figueroa, M.; Gonzalez, M. C.; Glenn, A. E.; Mata, R. J. Nat. Prod. 2015, 78, 730−735. (17) Lee, K. Y.; Jeong, E. J.; Sung, S. H.; Kim, Y. C. Rec. Nat. Prod. 2016, 10, 109−112.

H

DOI: 10.1021/acs.jnatprod.6b00566 J. Nat. Prod. XXXX, XXX, XXX−XXX