Iridoids from the Roots of Patrinia scabra and Their Inhibitory Potential

(1−5) However, the anti-inflammatory activity of the roots of P. scabra has not been ... and (8S) models were calculated to determine the absolute c...
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Article Cite This: J. Nat. Prod. 2018, 81, 1468−1473

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Iridoids from the Roots of Patrinia scabra and Their Inhibitory Potential on LPS-Induced Nitric Oxide Production Da Hye Lee,† Ji-Sun Shin,† Shin-Young Kang,† Seung-Bin Lee,† Jin Su Lee,† Seung Mok Ryu,‡ Kyung Tae Lee,† Dongho Lee,*,‡ and Dae Sik Jang*,† †

Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University, Seoul 136-713, Korea Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 130-701, Republic of Korea

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ABSTRACT: An activity-guided fractionation procedure of the 70% aqueous EtOH extract from the roots of Patrinia scabra led to the isolation and characterization of five new iridoids, patriscabrins A−E (1−5), along with 13 known compounds. The structures of 1−5 were determined by interpretation of spectroscopic data, particularly by 1D and 2D NMR, ECD, and VCD studies. Thereafter, isolates were evaluated for their inhibitory effects on lipopolysaccharideinduced nitric oxide production in RAW 264.7 cells. Of these, the new iridoids 2 and 5 and the known lignan patrineolignan B (6) exhibited IC50 values of 14.7 to 17.8 μM.

Patrinia scabra Bunge is a perennial herb that belongs to the family Valerianaceae. The roots of P. scabra have been used in traditional Chinese medicine to treat malaria, dysentery, leukemia, gastric cancer, typhoid fever, injuries from falls, and leukorrhea.1−5 However, the anti-inflammatory activity of the roots of P. scabra has not been reported previously. According to prior investigations, there are various types of secondary metabolites in P. scabra such as iridoids, lignans, sesquiterpenes, triterpenoids, steroids, coumarins, flavonoids, and other compounds.5−18 As a part of an our ongoing project to search for novel, plant-derived anti-inflammatory agents, a 70% aqueous EtOH extract of the roots of P. scabra was found to exhibit inhibitory activity in a preliminary in vitro screening procedure using lipopolysaccharide (LPS)-induced nitric oxide (NO), a pro-inflammatory mediator, in RAW 264.7 cells. The extract was fractionated using column chromatography to afford eight pooled fractions (E1−E8). These then were evaluated for their inhibitory activity against NO production in RAW 264.7 cells. In the present study, chemical investigation of the most active fraction (E2) from the 70% aqueous EtOH extract led to the isolation and characterization of five new iridoids, patriscabrins A−E (1−5), and 14 known compounds. The structures of the known compounds were identified from measurement of their spectroscopic data (1H NMR, 13C NMR, 2D NMR, and MS) and by comparison with published values. The isolation and structural elucidation of 1−5 and inhibitory activity of all of the isolates on LPS-induced NO production in RAW 264.7 cells are described herein. © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Compound 1 was isolated as a yellowish oil, and its molecular formula was established as C15H17O5Cl by HRESIMS (m/z 313. 0827 [M + H]+; calcd for C15H18O5Cl, 313.0843). The isotopic ion peak was observed at m/z 315.0833 [M + H]+ in Received: March 20, 2018 Published: May 25, 2018 1468

DOI: 10.1021/acs.jnatprod.8b00229 J. Nat. Prod. 2018, 81, 1468−1473

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(TDDFT) method, and the (8R) and (8S) models were calculated to determine the absolute configuration of C-8. Conformational searches were performed using the MMFF force field with Spartan’14 software, and geometry optimization for selected conformers was carried out at the DFT[B3LYP/631+G(d,p)] basis set level in the Gaussian 09 software. Following optimization, the theoretical ECD spectra were calculated at the TDDFT(CAM-B3LYP/SVP) basis set level with a CPCM solvent model in CH3CN. The experimental ECD spectrum of 1 exhibited a negative Cotton effect (CE) at 216 nm (Δε −6.3), a positive CE at 327 nm (Δε +6.9), and a negative CE at 366 nm (Δε −8.1). The calculated ECD spectrum of the (8S) model was in agreement with the experimental version (Figure 2), suggesting the absolute

the ratio of ca. 3 to 1, suggesting the presence of a chlorine atom, which was also supported by NMR data. The 1H and 13C NMR spectroscopic data of 1 (Table 1) exhibited signals for Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Spectroscopic Data for 1 and 2 in CDCl3 1 position

δC

1 3 4 5 6 7 8 9 10

140.2 150.7 114.8 159.0 105.6 201.1 75.06 127.4 48.3

11 1′ 2′ 3′ 4′/5′ 1″ 2″ 3″ 4″/5′′

66.6 172.7 43.1 25.7 22.4

δH mult. (J in Hz) 7.43 d (1.0) 7.39 s

5.46 d

3.55 d (11.5) 3.74 d (11.5) 4.84 s 2.20 d (7.0) 2.09 sept (6.0) 0.94 d (7.0)

2 δC 140.0 150.5 114.7 158.4 106.1 201.9 74.7 127.3 66.2 59.9 172.8 43.2 25.7 22.4 172.8 43.2 25.7 22.4

δH mult. (J in Hz) 7.23 d (2.0) 7.33 s

5.44 d (1.5)

3.96 d (11.5) 4.44 d (11.5) 4.82 d (2.5) 2.20 d (6.5) 2.07 sept (7.0) 0.93 overlap 2.20 d (6.5) 2.07 sept (7.0) 0.93 overlap

two CH3 protons [δH 0.94 (6H, d, J = 7.0 Hz)] and resonances for a CH group (δC 25.7), a CH2 group (δC 43.1), and an ester carbon atom (δC 172.7), indicating the presence of an isovalerate group.20,21 This was confirmed by the observed HMBC correlations for 1 (Figure 1). Besides the isovalerate Figure 2. Comparison of experimental and calculated ECD spectra of 1.

configuration of 1 to be (8S). Thus, from the analysis of the above data, the structure of the new compound 1 was elucidated as (S)-(7-(chloromethyl)-7-hydroxy-6-oxo-6,7dihydrocyclopenta[c]pyran-4-yl)methyl 3-methylbutanoate and was named patriscabrin A. Compound 2 was indicated as C20H26O7 by HRESIMS (m/z 379.1760 [M + H]+, calcd for C20H27O7, 379.1757). The proton and carbon signals in the 1H and 13C NMR spectra of 2 were similar to those of 1 (Table 1). However, preliminary inspection of the 1H and 13C NMR spectroscopic data of 2 revealed the presence of an additional isovalerate group [δH 2.20 (4H, d, J = 6.0 Hz), 2.07 (2H, sept, J = 7.0 Hz), and 0.93 (12H, overlap)] at C-10 of 2, instead of a chlorine atom in 1. The absence of a chlorine atom in 2 was also supported by the observed 13C NMR data, which showed a large downfield shift of C-10 in 2 (from δC 48.3 to δC 66.2). The locations of the two isovalerate groups were confirmed by the HMBC correlations from H-10 (δH 4.44 and 3.96) and H-11 (δH 4.82) to C-1′ and C-1″ (δC 172.8). The absolute configuration of 2 was determined as (8R) from the ECD spectrum, which exhibited the same pattern as that of 1. Therefore, the structure of 2 (patriscabrin B) was elucidated as (R)-(7-hydroxy-6-oxo6,7-dihydrocyclopenta[c]pyran-4,7-diyl)bis(methylene) bis(3methylbutanoate).

Figure 1. Selected correlations observed in the COSY (bold lines) and HMBC (→) NMR spectra of 1 and 2 (in CDCl3).

group, the remaining 10 carbon signals, including those for two CH2 units, one linked to oxygen (δC 66.6) and another to chlorine (δC 48.3), a quaternary carbon (δC 75.1), six olefinic carbons (δC 159.0, 150.7, 140.2, 127.4, 114.8, and 105.6), and a conjugated ketone carbon (δC 201.1), were consistent with an iridoid structure of 1.19 An HMBC experiment on 1 showed a long-range correlation between H-11 (δH 4.84) and the C-1′ ester (δC 172.7) of the isovalerate group, revealing the isovalerate group to be located at C-11 (Figure 1). The locations of the chlorine atom, conjugated ketone, and hydroxy and olefinic groups were determined unambiguously by COSY and HMBC experiments (Figure 1). The absolute configuration of 1 was established by comparison of its experimental electronic circular dichroism (ECD) spectrum with those calculated using the time-dependent density functional theory 1469

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located at C-11 and C-1, respectively (Figure 3). Comparison of the coupling constant of H-1 (δH 6.17, J1,9 = 10.5 Hz) with

The molecular formula of 3 was assigned as C20H30O8 by HRESIMS (m/z 399.2031 [M + H]+, calcd for C20H31O8, 399.2019). The 13C NMR spectroscopic data of 3 (Table 2) Table 2. 1H (500 MHz) and 13C NMR (125 MHz) Spectroscopic Data for 3−5 in CDCl3

position

δC

1

93.1

3 4 5 6

147.9 138.4 108.8 119.9

7

84.0

8 9

81.6 47.6

10

65.4

11

60.8

1′ 2′

173.3 43.4

3′ 4′/5′

25.8 22.5

1″ 2″

171.2 43.3

3″ 4″/5′′

25.7 22.5

3

4

δH mult. (J in Hz)

δH mult. (J in Hz)

6.17 d (10.5) 6.66 s

5.72 dd (3.0, 3.0)

4.59 d (3.0) 2.87 dd (10.5, 2.5) 3.99 d (11.5) 3.73 d (11.5) 4.66 d (12.5) 4.72 d (12.5) 2.19 d (7.0)

2.09 m 0.94 d (7.0) 2.30 d (7.5)

2.17 m 1.00 d (6.5)

δC 86.7 142.0 115.1 69.5 42.4

72.3 79.3 57.1

66.0

61.1

173.1 43.2

25.8 22.5 171.0 43.5

25.8 22.5

1‴ 2‴

173.8 43.2

3‴ 4‴/5″′

25.8 22.5

5 δC

δH mult. (J in Hz)

90.6

6.32 d (4.5) 6.45 s

Figure 3. Key correlations observed in the COSY (bold lines), HMBC (→), and NOESY (↔) NMR spectra of 3−5 (in CDCl3).

2.14 m

142.3 116.2 75.5 44.7

2.35 dd (14.0, 5.0) 3.76 brt

77.8

2.13, overlap 2.37 dd (14.0, 5.0) 3.88 brt

83.3 54.9

2.57 d (4.0)

those of the structurally similar compounds indicated that H-1 and H-9 are trans-diaxial configured.22−24 The NOESY experiment (Figure 3) exhibited a clear correlation between H-9 and H-10, but there was no NOE effect between H-7 and H-10. The absolute configuration of 3 was suggested as 1R, 7S, 8R, and 9S from the above results and by analogy with cyclopentanopyran iridoids of known stereochemistry at C-1 and C-9.25,26 This suggestion was supported by ECD experiments of 3. The experimental ECD spectrum of 3 exhibited positive CEs at 216 nm (Δε +4.2) and 255 nm (Δε +8.5). The calculated ECD spectrum of the (1R, 7S, 8R, and 9S) model matched well with the experimental results (Figure 4). Thus, the structure of 3 (patriscabrin C) was proposed as

6.61 d (2.0) 6.42 s

2.77 d (1.5) 4.12 d (12.0) 4.16 d (12.0) 4.65 d (12.5) 4.69 d (12.5) 2.19 d (7.5) 2.25 d (7.0) 2.08 m 0.95 m

2.07 dd (6.5, 4.0) 2.20 dd (7.0, 4.0) 2.07 m 0.95 m

2.19 d (7.5) 2.25 d (7.0) 2.08 m 0.95 m

66.6

61.6

4.26 d (11.5) 4.65 d (12.0) 4.67 d (13.5) 4.74 d (12.0)

173.1 44.7

2.20 d (7.0)

25.8 22.5

2.11 m 0.97 m

174.3 43.3

2.24 dd (7.5, 3.0)

25.8 22.5

2.11 m 0.97 m

171.2 43.3

2.30 d (7.5)

25.8 22.5

2.11 m 0.97 m

exhibited two isovalerate groups (δC 173.3, 171.2, 43.4, 43.3, 25.8, and 25.7), which were further confirmed by the HMBC correlations of 3. The remaining 10 carbon signals showed two CH2 units linked to oxygen (δC 65.4 and 60.8), a quaternary carbon (δC 81.6), two olefinic CH carbons (δC 147.9 and 119.9), two olefinic quaternary carbons (δC 138.4 and 108.8), and three CH carbons (δC 93.1, 84.0, and 47.6), which revealed that 3 is an iridoid with two isovalerate groups.19 The HMBC experiment of 3 showed long-range correlations from H-11 (δH 4.66 and 4.72) to C-1′ (δC 173.3) and from H-1 (δH 6.17) to C-1″ (δC 171.2), indicating that the isovalerate groups are

Figure 4. Comparison of experimental and calculated ECD spectra of 3.

((1S,6S,7R,7aS)-6,7-dihydroxy-7-(hydroxymethyl)-1-((3methylbutanoyl)oxy)-1,6,7,7a-tetrahydrocyclopenta[c]pyran-4yl)methyl 3-methylbutanoate. The molecular formula of 4 was established as C25H40O10 by HRESIMS (m/z 523.2534 [M + Na]+, calcd for C25H40O10Na, 523.2519). The mass spectrometric and NMR spectroscopic data of 4 indicated that this compound is an iridoid with three 1470

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above results and knowledge of the normal β,β-cis-fused cyclopentanopyran ring system of iridoids,25,26 the absolute configuration of 4 was suggested to be 1R, 7R, 8R, and 9S. To determine the remaining configuration of the hydroxy group at C-5, the ECD method was found to be unsuitable since no significant absorption was detected due to the lack of a UV chromophore in the structure. Therefore, the vibrational circular dichroism (VCD) spectrum of 4 was measured with the aim of establishing the absolute configuration at C-5. Conformational searches and geometry optimizations were carried out by the same procedure as conducted for ECD calculations, and theoretical VCD spectra were calculated at the DFT[B3LYP/6-31+G(d,p)] basis set level in Gaussian 09 software. The experimental IR and VCD spectra of 4 matched well with the theoretical results of the 5R isomer (Figure 6). Thus, the structure of 4 (patriscabrin D) was suggested as ((1R,6R,7R,4aR,7aS)-6,7,4a-trihydroxy-1-((3-methylbutanoyl)oxy)-1,5,6,7,4a,7a-hexahydrocyclopenta[c]pyran-4,7-diyl)bis(methylene) bis(3-methylbutanoate). The 1H and 13C NMR spectra of 5 (Table 2) were very similar to those of 4, and their molecular formulas [5, HRESIMS (m/z 523.2502 [M + Na] + , calcd for C25H40O10Na, 523.2519)] were found to be the same. The HMBC correlations from H-1 to C-1″′, H-10 to C-1″, and H11 to C-1′ indicated three isovalerate groups as being substituted at C-1, C-10, and C-11 and with the planar structure of 5 being the same as that of 4. A NOESY experiment showed correlations of H-10 with H-7 and H-9, and the absolute configuration at C-7 was determined as R by the modified Mosher’s method (Figures 3 and 5). Thus, it was proposed that 5 has an epimeric structure at the C-5 position with 4. To confirm this suggestion, a similar VCD technique was attempted, but an appropriate VCD spectrum could not be obtained due to the paucity of 5. Accordingly, the structure of 5 (patriscabrin E) was proposed as ((1R,6R,7R,7aS)-6,7,4atrihydroxy-1-((3-methylbutanoyl)oxy)-1,5,6,7,4a,7a-hexahydrocyclopenta[c]pyran-4,7-diyl)bis(methylene) bis(3-methylbutanoate). The known compounds were identified via a comparison of their NMR data with literature values as patrineolignan B (6),27 patrineolignan A, 27 homobaldrinal,28 (+)-matairesinol,29 (+)-nortrachelogenin,30 (+)-lariciresinol,31 caryolane-1,9βdiol,32 clovanediol,32 caryophyllene oxide,32 vanillin,33 epoxyconiferyl alcohol,34 3-carbomethoxy-5-(1′-hydroxyethyl)pyridine,35 and scopoletin.36

isovalerate groups (Table 3). The 13C NMR and DEPT spectra of 4 showed the presence of an olefinic CH group (δC 142.0), a Table 3. Inhibitory Effects of 1−6 Isolated from the Roots of P. scabra on Nitric Oxide Production in LPS-Induced RAW 264.7 Cellsa compound

IC50 (μM)

compound

IC50 (μM)

1 2 3

27.6 17.1 >50

4 5 6

26.0 14.7 17.8

a IC50 values are defined as the concentration that results in a 50% decrease production of nitric oxide. The values represent the means of the results from three independent experiments with similar patterns. The other compounds isolated were not active (IC50 > 50 μM) in the nitric oxide production assay. L-N6-(1-Iminoethyl)lysine (L-NIL) was used as a positive control substance for NO production (IC50 value = 15.8 μM).

quaternary carbon atom (δC 115.1), and two CH2 groups connected to oxygen (δC 61.1 and 66.0). In addition to these signals, there were two oxygenated CH groups (δC 86.7 and 72.3), two oxygenated quaternary carbon atoms (δC 69.5 and 79.3), a CH2 (δC 42.4), and a CH (δC 57.1) in an iridoid skeleton. The HMBC cross-peaks of H-1 to C-1″′, H-10 to C1″, and H-11 to C-1′ indicated that the three isovalerate groups are located at C-1, C-10, and C-11, respectively (Figure 3). A NOESY experiment showed clear correlations of H-10 with H7 and H-9, suggesting they are located on the same face of the ring system (Figure 3). The absolute configuration at C-7 of 4 was determined by the modified Mosher’s method with 1H NMR, COSY, and HSQC data as 7R (Figure 5). Based on

Figure 5. Δδ Values (δS − δR) in ppm obtained for the two MTPA esters derived from 4 and 5 in pyridine-d5.

Figure 6. Comparison of experimental and calculated VCD spectra of 4. 1471

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was fractionated by Sephadex CC with a CH2Cl2−MeOH mixture (1:1 v/v) to yield 12 further fractions (E2-5-1−E2-5-12). Fraction E2-5-3 was separated further using a flash chromatography system with a Redi Sep-C18 column (26 g, MeOH−H2O, 2:8 to 9:1 v/v), to obtain compounds 4 (29.2 mg) and 5 (11.8 mg). Compound 1 (119.4 mg), caryolane-1,9β-diol (31.0 mg), and clovanediol (16.4 mg) were obtained from fraction E2-5-7 by using a reversed-phase HPLC system with a YMC Pack ODS-A column. Fraction E2-7 was chromatographed by silica gel CC with a CH2Cl2−EtOAc gradient (from 9:1 to 3:7 v/v; final stage, MeOH 100%), to afford 23 fractions (E2-7-1−E27-23). Fraction E2-7-3 was further subdivided with a flash chromatography system (Redi Sep-C18 cartridge, 26 g, MeOH− H2O, 2:8 to 9:1 v/v), yielding patrineolignan B (6, 35.1 mg) and epoxyconiferyl alcohol (74.8 mg). Compounds 2 (14.2 mg) and 3 (10.4 mg) were isolated from fraction E2-7-17 by flash chromatography (Redi Sep-C18 cartridge, 13 g, MeOH−H2O, 2.5:7.5 to 8:2 v/v). Patrineolignan A (14.7 mg), (+)-matairesinol (30.0 mg), and scopoletin (5.5 mg) were obtained from fraction E2-7-2 through repeated chromatographic separation. 3-Carbomethoxy-5-(1′hydroxyethyl)pyridine (4.3 mg) was purified using reversed-phase HPLC with a YMC Pack ODS-A column from fraction E2-7-21. (+)-Nortrachelogenin (189.9 mg) and (+)-lariciresinol (28.4 mg) were obtained by Sephadex CC eluted with CH2Cl2−MeOH (1:1 v/v) from fraction E2-9. Fraction E2-3-1 (537.0 mg) was further fractionated using silica gel CC with a CH2Cl2−MeOH−H2O mixture (9:0.9:0.1 v/ v/v) to afford homobaldrinal (17.4 mg). Fraction E2-4 was chromatographed by silica gel CC as stationary phase with a CH2Cl2−EtOAc gradient (from 9:1 to 3:7 v/v; final stage, MeOH 100%) as mobile phase to afford 16 fractions (E2-4-1−E2-4-16). Homobaldrinal (5.6 mg) and vanillin (39.2 mg) were isolated from fraction E2-4-3 with a Redi Sep-C18 cartridge (26 g, MeOH-H2O, 3.5:6.5 to 9:1 v/v). Caryophyllene oxide (119.2 mg) was isolated by silica gel CC (n-hexane−EtOAc, 9:1 to 5:5 v/v) from fraction E2-2. Patriscabrin A (1): yellowish oil; [α]23D −109.7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (3.50), 290 (3.36), 346 (3.55) nm; ECD (c 0.2 mM, CH3CN) λmax 216 (−6.3), 327 (6.9), 366 (−8.1); IR (ATR) νmax 3346, 1659, 1543 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS (positive mode) m/z 313.0827 [M + H]+ (calcd for C15H18ClO5, 313.0843). Patriscabrin B (2): yellowish oil; [α]23D −24.9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 227 (3.39), 290 (3.23), 346 (3.49) nm; ECD (c 0.2 mM, CH3CN) λmax 215 (−17.6), 328 (20.5), 367 (−24.1); IR (ATR) νmax 3380, 1733, 1663, 1544 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS (positive mode) m/z 379.1760 [M + H]+ (calcd for C20H27O7, 379.1757). Patriscabrin C (3): colorless solid; [α]23D 7.1 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (3.83), 245 (3.70) nm; ECD (c 0.2 mM, CH3CN) λmax 216 (4.2), 225 (3.7), and 255 (8.5); IR (ATR) νmax 3402, 2958, 1717 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS (positive mode) m/z 399.2031 [M + H]+ (calcd for C20H31O8, 399.2019). Patriscabrin D (4): colorless crystal; [α]23D −98.5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (3.87) nm; VCD (c 0.2 M, CDCl3) (Figure 6); IR (ATR) νmax 3458, 2958, 1730, 1089 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS (positive mode) m/z 523.2534 [M + Na]+ (calcd for C25H40O10Na, 523.2519). Patriscabrin E (5): colorless crystal; [α]23D −140.1 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (3.86) nm; IR (ATR) νmax 3396, 2958, 1719, 1024 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS (positive mode) m/z 523.2502 [M + Na]+ (calcd for C25H40O10Na, 523.2519). Computational Methods. Conformer distributions, optimizations, and ECD and VCD calculations of compounds 1, 3, and 4 were performed as described previously.37,38 Preparation of (S)- and (R)-MTPA Ester Derivatives of Compounds 4 and 5. Preparation of (S)- and (R)-MTPA ester derivatives of patriscabrins D and E (4 and 5) was performed as described previously.37 S-MTPA ester of 4 (4a): 1H NMR (pyridine-d5, 500 MHz) δH 5.92 (1H, dd, J = 5.5, 4.0 Hz, H-7), 4.84 (2H, m, H-10), 3.15 (1H, dd, J =

All of the isolates from the roots of P. scabra were evaluated for their inhibitory effects on LPS-induced NO production in RAW 264.7 cells at nontoxic concentrations (Table 3). Of these, the new iridoids patriscabrins B (2) and E (5) and the known lignan patrineolignan B (6) markedly decreased LPSinduced NO production in a concentration-dependent manner, with observed IC50 values of 17.1, 14.7, and 17.8 μM, respectively (Table 3). Patrineolignan B (6) has been isolated from P. scabra in a previous study.27 Although 6 has been found to have strong cytotoxicity against the human cervical carcinoma HeLa cell line and gastric carcinoma MNK-45 cell line using the microculture tetrazolium assay,27 no biological activity data related to its anti-inflammatory effects have been reported to date.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-2000 polarimeter, using a 10 cm microcell. UV spectra were obtained on an Optizen Pop instrument (Mecasys, Daejeon, Korea). ECD and VCD spectra were calculated by Spartan’14 (Wavefunction, Inc., Irvine, CA; 2014) and Gaussian 09 (Revision E.01; Gaussian, Inc., Wallingford, CT; 2009) software. ECD and VCD spectra were measured with a J-1100 circular dichroism spectrophotometer (JASCO, Tokyo, Japan) and a ChiralIR-2X TM FT-VCD spectrometer (BioTools, Jupiter, FL, USA), respectively. IR spectra were obtained using an Agilent Cary 630 FTIR spectrometer (Agilent Technologies, Santa Clara, CA, USA). NMR spectra were obtained using a JEOL 500 MHz NMR spectrometer using tetramethylsilane as an internal standard, and chemical shifts are expressed as δ values. HRESIMS were obtained using a Q-TOF micro mass spectrometer (Waters, Milford, MA, USA). TLC analysis was performed on silica gel 60 F254 (Merck) and RP-18 F254S (Merck) plates. Compounds were visualized by dipping plates into 20% (v/v) H2SO4 reagent (Aldrich) and then heated at 110 °C for 5−10 min. Silica gel (Merck 60A, 70−230 or 230−400 mesh ASTM), Sephadex LH-20 (Amersham Pharmacia Biotech), and reversed-phase silica gel (YMC Co., ODS-A 12 nm S-150 μm) were used for column chromatography. Flash chromatography was performed using the flash purification system (Combi Flash Rf, Teledyne Isco, Lincoln, NE, USA). Prepacked cartridges Redi Sep-Silica and Redi Sep-C18 were used for flash chromatography. HPLC was performed using the Gilson purification system (Gilson Inc., Middleton, WI, USA) with a YMC Pack ODS-A column (250 × 20 mm i.d., 5.0 μm, YMC), a J’sphere ODS-M80 column (250 × 20 mm i.d., 4.0 μm, YMC), and a Luna 10 μm C18(2) 100A column (250 × 20 mm i.d., 10.0 μm, Phenomenex). All solvents used for the chromatographic separations were distilled before use. Plant Material. The roots of P. scabra were obtained from Hyunjin Pharmaceutical Co. (Dongdaemun-gu, Seoul, Republic of Korea), in March 2014. The identity of the plant material was checked by D.S.J., and a representative specimen (PASC1-2014) has been deposited in the Laboratory of Natural Product Medicine, College of Pharmacy, Kyung Hee University, Republic of Korea. Extraction and Isolation. The dried plant material (6.0 kg) was extracted twice with 120 L of 70% EtOH at 80 °C in a water bath for 3 h, and the solvent was evaporated in vacuo at 45 °C. The 70% EtOH extract (822.0 g) was absorbed onto silica gel (70−230 mesh) and finely ground. The powdered extract was fractionated using column chromatography (CC) over silica gel and eluted with a stepwise gradient of CH2Cl2−MeOH−H2O (from 9:0.9:0.1 to 6:3.6:0.4 v/v/v; final stage, MeOH 100%) to afford eight pooled fractions (E1−E8). The fractions were evaluated for their inhibitory activity against nitric oxide production in RAW 264.7 cells. Only fractions E1−E3 exhibited promising inhibitory activity, with observed IC50 values of 41.4, 18.9, and 45.2 μg/mL, respectively. Based on these results, the most active fraction, E2 (56.4 g), was further separated into 10 major fractions (E2-1−E2-10), using silica gel CC (n-hexane−EtOAc−MeOH, 8:1.8:0.2 to 5:4:1 v/v/v; final stage, MeOH 100%). Fraction E2-5 1472

DOI: 10.1021/acs.jnatprod.8b00229 J. Nat. Prod. 2018, 81, 1468−1473

Journal of Natural Products

Article

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15.0, 5.5 Hz, H-6b), 2.86 (1H, m, overlap, H-6a); ESIMS (positive) m/z 739 [M + Na]+. R-MTPA ester of 4 (4b): 1H NMR (pyridine-d5, 500 MHz) δH 5.88 (1H, t, J = 5.0 Hz, H-7), 4.78 (2H, m, H-10), 3.19 (1H, dd, J = 15.0, 5.0 Hz, H-6b), 3.05 (1H, dd, J = 15.0, 4.5 Hz, H-6a); ESIMS (positive) m/z 739 [M + Na]+. S-MTPA ester of 5 (5a): 1H NMR (pyridine-d5, 500 MHz) δH 5.98 (1H, dd, J = 8.0, 6.0 Hz, H-7), 4.89 (2H, br s, H-10), 3.34 (1H, m, overlap, H-6b), 2.71 (1H, dd, J = 13.0, 8.0 Hz, H-6a); ESIMS (positive) m/z 739 [M + Na]+. R-MTPA ester of 5 (5b): 1H NMR (pyridine-d5, 500 MHz) δH 6.02 (1H, t, J = 6.0 Hz, H-7), 4.85 (1H, d, J = 11.0 Hz, H-10b), 4.73 (1H, d, J = 11.0 Hz, H-10a), 3.34 (1H, m, overlap, H-6b), 2.90 (1H, m, overlap, H-6a); ESIMS (positive) m/z 739 [M + Na]+. Measurement of Nitric Oxide Production. Cell viability studies were performed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide] assay.39 Nitrite levels were measured using a Griess reaction assay as described previously.39 L-N6-(1Iminoethyl)lysine was used as a positive control for NO production.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00229. 1D and 2D NMR spectra for 1−5 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel (D. Lee): +82-2-3290-3017. Fax: +82-2-953-0737. E-mail: [email protected]. *Tel (D. S. Jang): +82-2-961-0719. Fax: +82-2-961-9580. Email: [email protected]. ORCID

Dongho Lee: 0000-0003-4379-814X Dae Sik Jang: 0000-0001-5472-5232 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2016R1D1A1B03930222).

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REFERENCES

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DOI: 10.1021/acs.jnatprod.8b00229 J. Nat. Prod. 2018, 81, 1468−1473