Clerodane Diterpenoids from - ACS Publications - American Chemical

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neo-Clerodane Diterpenoids from Scutellaria barbata and Their Inhibitory Effects on LPS-Induced Nitric Oxide Production Eung Tae Yeon,† Jin Woo Lee,† Chul Lee,† Qinghao Jin,† Hari Jang,† Dongho Lee,‡ Jong Seog Ahn,§ Jin Tae Hong,† Youngsoo Kim,† Mi Kyeong Lee,† and Bang Yeon Hwang*,† †

College of Pharmacy, Chungbuk National University, Cheongju 28644, Korea Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea § Korea Research Institute of Bioscience and Biotechnology, Cheongju 28116, Korea ‡

Downloaded by UNIV OF SUSSEX on September 2, 2015 | http://pubs.acs.org Publication Date (Web): September 2, 2015 | doi: 10.1021/acs.jnatprod.5b00126

S Supporting Information *

ABSTRACT: Three new neo-clerodane diterpenoids (1−3) along with 12 known compounds (4−15) were isolated from a methanol extract of the aerial parts of Scutellaria barbata. The structures of 1−3 were determined by interpretation of their 1D and 2D NMR spectroscopic data as well as HRESIMS values. All isolated compounds were tested for their inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 macrophages. Compounds 1−4, 7, and 10−12 were found to inhibit nitric oxide production with IC50 values ranging from 20.2 to 35.6 μM. isolates were examined for their inhibitory effects on LPSinduced NO production in RAW264.7 cells. Compound 1, obtained as a yellow amorphous powder, exhibited a molecular formula of C39H40N2O9, as determined from the HRESIMS (m/z 703.2630 [M + Na]+; calcd for C39H40N2O9Na, 703.2632) in combination with its NMR data (Table 1). The 1H and 13C NMR spectra showed the presence of four tertiary methyl groups [δH 1.14 (s, H3-20), 1.15 (s, H317), 1.51 (s, H3-19), 1.62 (s, H3-18); δC 21.2 (q, C-20), 19.9 (q, C-17), 16.8 (q, C-19), 20.2 (q, C-18)], an olefinic group [δH 5.30 (br s, H-3); δC 120.2 (d, C-3), 143.3 (s, C-4)], an oxygenbearing methylene group [δH 4.06 (d, J = 9.0 Hz, Hα-16), 4.13 (d, J = 9.0 Hz, Hβ-16); δC 76.5 (t, C-16)], an isolated methylene group [δH 2.61 (d, J = 17.3 Hz, Hα-14), 3.07 (d, J = 17.3 Hz, Hβ-14); 44.4 (t, C-14)], and an 8,13-ether linkage [δC 76.6 (C-13) and 81.1 (C-8)], which are characteristic signals for a neo-clerodane diterpenoid with a 3-ene-13-spiro-15,16-γlactone moiety.3,4 The 1H and 13C NMR spectra of 1 also exhibited signals for two nicotinoyloxy groups [δH 7.11 (dd, J = 8.0, 4.8 Hz, H-6″), 7.38 (dd, J = 8.0, 4.8 Hz, H-6′), 7.89 (dt, J = 8.0, 1.8 Hz, H-7″), 8.18 (dt, J = 8.0, 1.8 Hz, H-7′), 8.54 (dd, J =

Scutellaria barbata D. Don, a member of the family Lamiaceae, is distributed widely in Korea, mainland China, India, and other Asian countries. The dried herb of S. barbata has been used in traditional medicine as an anti-inflammatory, antihepatitis, and antitumor agent.1 This plant is known as a source of neoclerodane diterpenoids, and more than 150 neo-clerodane diterpenoids have been isolated from this genus.2−6 Previous biological studies of these neo-clerodane diterpenoids have demonstrated their promising cytotoxic activity against several cancer cell lines.7−11 In the course of a research program for the isolation of anti-inflammatory constituents from medicinal plants, a MeOH extract of the aerial parts of S. barbata showed significant inhibitory effects against LPS-induced nitric oxide (NO) production (IC50 value of 32.5 μg/mL). Subsequently, the dried aerial parts of S. barbata were extracted with MeOH, and the resultant methanolic extract was suspended in water and then partitioned successively with n-hexane, dichloromethane, and ethyl acetate. Bioassay-guided fractionation of the CH2Cl2-soluble fraction led to the isolation of three new neoclerodane diterpenoids (1−3) along with 12 known compounds (4−15). The structures of 1−3 were elucidated on the basis of spectroscopic data interpretation, especially using 2D NMR COSY, HSQC, HMBC, and NOESY methods. All © XXXX American Chemical Society and American Society of Pharmacognosy

Received: February 6, 2015

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DOI: 10.1021/acs.jnatprod.5b00126 J. Nat. Prod. XXXX, XXX, XXX−XXX


Journal of Natural Products

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15,16-olide skeleton. The only difference between these compounds was shown to be the location of the substituents. The benzoyloxy group was located at C-1, and two nicotinoyloxy groups were located at C-6 and C-7 from the observed HMBC correlations from H-1 (δH 5.85) to C-1′ (δC 165.7), H-6 (δH 5.89) to C-1″ (δC 164.3), and H-7 (δH 5.89) to C-1‴ (δC 165.0). The configuration of 2 was deduced to be the same as that of 1 based on comparison of their NMR chemical shifts, coupling constants, and NOESY data. Therefore, compound 2 (scutebatin B) was determined to be 13(S)-1βbenzoyloxy-6α,7β-dinicotinoyloxy-8β,13-epoxy-3-neocleroden15,16-olide. Compound 3 was obtained as a yellow amorphous powder. Its molecular formula of C32H36N2O7 was determined from the HRESIMS and NMR data. The 1H and 13C NMR data of 3 were similar to those of 1 and 2, except for the absence of both an oxygenated functionality at the C-7 position and a benzoyloxy moiety. Detailed analysis of COSY, HSQC, and HMBC demonstrated that compound 3 has the same neoclerodane diterpenoid skeleton with a 3-ene-13-spiro-15,16-γlactone moiety and bearing two nicotinoyloxy groups. The HMBC correlations from H-1 (δH 5.75) to C-1′ (δC 164.5) and H-6 (δH 5.37) to C-1″ (δC 164.1) suggested that the two nicotinoyloxy groups are attached at the C-1 and C-6 positions (Figure 1). The NOESY correlations of H-1/H3-19, H-1/H320, H-7α/H3-17, and H-6/H-10 in compound 3 indicated that these all have the same configuration and suggested that H-1, H3-17, H3-19, and H3-20 are α-oriented and H-6 and H-10 are β-oriented (Figure 2). However, the configuration of C-13 was inverted in comparison with those of compounds 1 and 2, which was determined to be R by the NOESY correlation between H2-14 and H3-17 (Figure 2).3 This was established further by the comparison of the signals arising from H2-14 (δH 2.51, 2.68, Δ = 0.17 ppm) and C-14 (δC 42.5) and C-16 (δC 80.0), which were markedly different than those observed in compounds 1 and 2. In addition, the absolute configuration of 3 was confirmed by comparing its CD data with those of scutolide K, for which absolute configuration was determined by single-crystal X-ray diffraction analysis.12 Therefore, compound 3 (scutebatin C) was proposed structurally as 13(R)-1β,6α-dinicotinoyloxy-8β,13-epoxy-3-neocleroden15,16-olide. The 12 known neo-clerodane diterpenoids isolated in this study were identified as scutebarbatine W (4),3 scutebata G (5),3,5 scutebata D (6),5 scutebata P (7),13 6-O-nicotinoylscutebarbatine G (8),3,14 scutebarbatine F (9),3,15 scutebarbatine B (10),16 scutebarbatine A (11),6 scutebarbatine Y (12),3 scutebata C (13),5 scutebarbatine X (14),3 and scutebata B (15),5 by comparison of their observed and reported spectroscopic data. All compounds were evaluated for their inhibitory effects on the NO production in LPS-induced RAW 264.7 macrophage cells. Compounds 1−4, 7, and 10−12 exhibited inhibitory effects against NO production with IC50 values ranging from 20.2 to 35.6 μM, as compared with the positive control, aminoguanidine (IC50 value of 20.6 μM) (Table 2). Cell viability assay results indicated that none of the test compounds showed evident cytotoxicity at their effective concentrations (data not shown). Compounds 10−12 (IC50 values of 22.8−27.7 μM), all possessing a conjugated double bond with a α,β-unsaturated γlactone moiety, exhibited activity more potent than that of compounds 13−15 (IC50 values of >50 μM), implying that the

4.8, 1.8 Hz, H-5″), 8.76 (dd, J = 4.8, 1.8 Hz, H-5′), 8.85 (d, J = 1.8 Hz, H-3″), 9.11 (d, J = 1.8 Hz, H-3′); δC 123.1 (d, C-6″), 123.6 (d, C-6′), 125.8 (s, C-2″), 125.9 (s, C-2′), 136.7 (d, C7″), 136.9 (d, C-7′), 150.7 (d, C-3″), 150.8 (d, C-3′), 153.3 (d, C-5″), 153.9 (d, C-5′), 164.3 (s, C-1″), 164.5 (s, C-1′)] and a benzoyloxy group [δH 7.25 (br t, J = 8.0 Hz, H-4‴, 6‴), 7.40 (br t, J = 8.0 Hz, H-5‴), 7.75 (br d, J = 8.0 Hz, H-3‴, 7‴); δC 128.3 (d, C-4‴, 6‴), 128,8 (s, C-2‴), 129.8 (d, C-3‴, 7‴), 133.4 (d, C-5‴), 166.3 (s, C-1‴)]. Analysis of the 1H−1H COSY spectrum suggested the proton spin systems from C-1 to C-3, C-1 to C-10, C-6 to C-7, and C-11 to C-12. The positions of two nicotinoyloxy groups and a benzoyloxy group, located at C-1, C-6, and C-7, were determined on the basis of key HMBC correlations from H-1 (δH 5.80) to C-1′ (δC 164.5), H-6 (δH 5.82) to C-1″ (δC 164.3), and H-7 (δH 5.60) to C-1‴ (δC 166.3). Furthermore, HMBC correlations of H-11 with C-13, H-12 and H-14 with C-16, and H-16 with C-15 supported the proposed 13-spiro-15,16-γ-lactone skeleton of 1 (Figure 1). The configuration of 1 was deduced from the interpretation of the NOESY spectrum and comparison of NMR data with reported values.3,5,6 The chemical shifts at δH 2.61, 3.07 (H2-14, Δ = 0.46 ppm) and δC 44.4 (C-14) and 76.5 (C-16) were indicative of a 13S form, whereas the chemical shift for the 13R form in compound 3 would be at δH 2.51 and 2.68 (H2-14, Δ = 0.17 ppm) and at δC 42.5 (C-14) and 80.0 (C-16).3 The 13S*spiro chiral center was further determined by a NOESY correlation between H2-16 and H3-17 (Figure 2). The coupling constant (10.2 Hz) between H-6 and H-7 indicated that both of the protons have a trans diaxial relationship.4 The NOESY correlations observed for H-7/H3-17, H3-19, H3-20, H-1/H319, H3-20, and H-6/H-10 suggested that H-1, H-7, H3-17, H319, and H3-20 are α-oriented and H-6 and H-10 β-oriented (Figure 2). Therefore, compound 1 was determined as 13(S)1β,6α-dinicotinoyloxy-7β-benzoyloxy-8β,13-epoxy-3-neocleroden-15,16-olide and was given the trivial name scutebatin A. Compound 2 was obtained as a yellow amorphous powder. The molecular formula, C39H40N2O9, was deduced from its HRESIMS and NMR data. Comparison of the 1D and 2D NMR spectra of 2 and 1 indicated that both compounds have the same 1β,6α,7β-trisubstituted 8β,13-epoxy-3-neoclerodenB



Note

Table 1. 1H NMR (700 MHz) and 13C NMR (175 MHz) Data of Compounds 1−3 in CDCl3a


1

a

2

3

no.

δC

δH

δC

δH

δC

δH

1 2 3 4 5 6 7 8 9 10 11 12 13 14α 14β 15 16α 16β 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ 3″ 5″ 6″ 7″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴

71.7 33.1 120.2 143.3 44.6 74.4 74.4 81.1 38.9 43.5 28.7 29.1 76.6 44.4

5.80 (m) 2.72 (m), 2.17 (m) 5.30 (br s)

70.8 33.1 120.7 142.9 44.6 74.5 75.3 81.1 38.9 43.4 28.5 29.1 76.7 44.4

5.85 (m) 2.77 (m), 2.24 (m) 5.36 (br s)

72.3 32.8 120.1 144.1 43.4 74.6 38.1 79.8 37.8 43.9 28.3 29.7 76.0 42.5

5.75 (m) 2.69 (m), 2.14 (m) 5.29 (br s)

173.6 76.5 19.9 20.2 16.8 21.2 164.5 125.9 150.8 153.9 123.6 136.9 164.3 125.8 150.7 153.3 123.1 136.7 166.3 128.8 129.8 128.3 133.4 128.3 129.8

5.82 (d, 10.2) 5.60 (d, 10.2)

2.81 (d, 9.5) 1.59 (m), 2.03 (m) 1.70 (m), 2.09 (m) 2.61 (d, 17.3) 3.07 (d, 17.3) 4.06 4.13 1.15 1.62 1.51 1.14

(d, 9.0) (d, 9.0) (s) (s) (s) (s)

9.11 (d, 1.8) 8.76 (dd, 4.8, 1.8) 7.38 (dd, 8.0, 4.8) 8.18 (dt, 8.0, 1.8)

8.85 8.54 7.11 7.89

(d, 1.8) (dd, 4.8, 1.8) (dd, 8.0, 4.8) (dt, 8.0, 1.8)

7.75 7.25 7.40 7.25 7.75

(br (br (br (br (br

d, 8.0) t, 8.0) t, 8.0) t, 8.0) d, 8.0)

5.89 (d, 10.3) 5.89 (d, 10.3)

2.87 (d, 9.4) 1.63 (m), 2.17 (m) 1.76 (m), 2.20 (m) 2.69 (d, 17.3) 3.14 (d, 17.3)

173.6 76.5 20.0 20.2 16.8 21.2 165.7 130.0 129.5 128.8 133.5 128.8 129.5 164.3 125.6 150.7 153.6 123.3 136.7 165.0 124.8 151.1

4.12 4.20 1.22 1.68 1.57 1.21

(d, 9.0) (d, 9.0) (s) (s) (s) (s)

7.98 7.49 7.62 7.49 7.98

(br (br (br (br (br

8.96 8.65 7.22 7.99

(d, 1.8) (d, 4.8, 1.8) (dd, 8.0, 4.8) (dt, 8.0, 1.8)

d, 7.5) t, 7.5) t, 7.5) t, 7.5) d, 7.5)

174.9 80.0 24.1 20.5 15.3 21.6 164.5 126.0 150.8 153.6 123.5 136.9 164.1 126.5 150.8 153.8 123.6 137.1

5.37 (m) 1.97 (m), 1.82 (m)

2.61 (d, 9.0) 1.50 (m), 1.84 (m) 1.48 (m), 2.03 (m) 2.51 (d, 16.8) 2.68 (d, 16.8) 4.14 4.27 1.18 1.61 1.35 1.01

(d, 8.9) (d, 8.9) (s) (s) (s) (s)

9.10 (br s) 8.73 (br d, 5.0) 7.36 (dd, 8.0, 5.0) 8.17 (br d, 8.0)

9.16 8.74 7.36 8.23

(br s) (br d, 5.0) (dd, 8.0, 5.0) (br d, 8.0)

9.00 (d, 1.8)

153.8 123.3 137.1

8.69 (d, 4.9, 1.8) 7.29 (dd, 7.9, 4.9) 8.10 (dt, 7.9, 1.8)

Assignments are supported with COSY, HSQC, and HMBC experiments.

presence of these functionalities might be required for activity. However, the inhibitory effects of NO production in the 13spiro neo-clerodane compounds 1−9 were found to vary considerably with the position and type of the acyl functionalities present. Recently, the 70% ethanol extract of S. barbata was found to have inhibitory effects on NO production in macrophage RAW 264.7 cells.17 However, this is the first report that purified neoclerodane diterpenoid constituents of this plant possess inhibitory activity on NO production. In conclusion, the results suggested that neo-clerodane diterpenoids could partly account for the use of S. barbata in the treatment of inflammatory diseases, indicating that this plant might have the potential for further investigation as an anti-inflammatory agent.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO DIP-1000 polarimeter (Tokyo, Japan). UV and IR spectra were obtained using a JASCO UV-550 and JASCO FTIR 4100 spectrometer, respectively. CD spectra were obtained on a JASCO J-710 spectropolarimeter. NMR spectra were recorded on Bruker AVANCE III 700 MHz NMR spectrometer using CDCl3 as a solvent. HRESIMS data were recorded on a Bruker maXis 4G spectrometer. Column chromatography was performed on silica gel (70−230 mesh, Merck), Sephadex LH-20 (25−100 μm, Pharmacia), and Lichroprep RP-18 (40−63 μm, Merck). MPLC was performed on a Biotage Isolera Prime chromatography system. Semipreparative HPLC was performed using a Waters HPLC system equipped with two Waters 515 pumps with a 2996 photodiode array detector using a YMC J’sphere ODS-H80 column (4 μm, 150 × 20 mm, i.d., flow rate 6 mL/min). TLC was performed using precoated silica gel 60 F254 (0.25 C



Note


Figure 1. 1H−1H COSY () and HMBC (→) correlations of compounds 1−3. HPLC (H2O−MeCN, 40:60 to 0:100) to yield compounds 8 (12.0 mg, tR 17.5 min), 13 (7.2 mg, tR 21.0 min), and 14 (6.9 mg, tR 27.0 min). Fraction SBC6-8 (550 mg) was chromatographed further on a Sephadex LH-20 column (CH2Cl2−MeOH, 1:1) to give compound 11 (200 mg). Fraction SBC3 (2.1 g) was chromatographed on a silica gel column eluted with a step gradient of CH2Cl2−MeOH (50:1 to 0:100) to give eight fractions (SBC3-1 to SBC3-8). Fraction SBC3-4 (200 mg) was further purified by HPLC (H2O−MeOH, 45:55 to 0:100) to afford compounds 6 (8.6 mg, tR 15.6 min) and 7 (6.2 mg, tR 19.0 min). Fraction SBC5 (3.7 g) was subjected to passage over a RP-18 MPLC column eluted with a step gradient of H2O−MeOH (80:20 to 0:100) to give four fractions (SBC5-1 to SBC5-4). Fraction SBC5-1 (250 mg) was purified by HPLC (H2O−MeCN, 30:70 to 10:90) to afford compounds 5 (4.5 mg, tR 17.0 min) and 10 (60.1 mg, tR 21.5 min). Fraction SBC5-3 (400 mg) was further purified by HPLC (H2O− MeCN, 40:60 to 0:100) to afford compounds 9 (15.8 mg, tR 18.2 min), 12 (7.0 mg, tR 23.5 min), and 15 (12.0 mg, tR 26.0 min). Scutebatin A (1): yellow amorphous powder; [α]25D −57.7 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 220 (4.08), 264 (3.52) nm; CD (MeOH) λmax (Δε) 220 (+0.9), 235 (−5.2) nm; IR (KBr) νmax 2966, 1784, 1723, 1590, 1373, 1278, 1113, 957 cm−1; 1H and 13C NMR (CDCl3, 700 and 175 MHz), see Table 1; HRESIMS m/z 703.2630 [M + Na]+ (calcd for C39H40N2O9Na, 703.2632). Scutebatin B (2): yellow amorphous powder; [α]25D −82.4 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 221 (4.07), 262 (3.50) nm; CD (MeOH) λmax (Δε) 217 (+0.1), 231 (−2.1) nm; IR (KBr) νmax 2980, 1786, 1725, 1592, 1426, 1279, 1107, 950, 843 cm−1; 1H and 13C NMR (CDCl3, 700 and 175 MHz), see Table 1; HRESIMS m/z 703.2630 [M + Na]+ (calcd for C39H40N2O9Na, 703.2632). Scutebatin C (3): yellow amorphous powder; [α]25D −14.0 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 218 (4.10), 264 (3.54) nm; CD (MeOH) λmax (Δε) 207 (−4.5), 222 (−2.2), 246 (2.0) nm; IR (KBr) νmax 2929, 1785, 1718, 1594, 1442, 1375, 1279, 1108, 1029, 852, 706 cm−1; 1H and 13C NMR (CDCl3, 700 and 175 MHz), see Table 1; HRESIMS m/z 583.2417 [M + Na]+ (calcd for C32H36N2O7Na, 583.2420). Measurement of LPS-Induced NO Production and Cell Viability. The nitrite concentration in the medium was measured as an indicator of NO production according to the Griess reaction. Briefly, RAW 264.7 cells were seeded into 96-well tissue culture plates at 2 × 106 cells/mL and stimulated with 1 μg/mL of LPS in the presence or absence of compounds. After incubation at 37 °C for 24 h, 100 μL of cell-free supernatant was mixed with 100 μL of Griess reagent containing equal volumes of 2% (w/v) sulfanilamide in 5% (w/v) phosphoric acid and 0.2% (w/v) of N-(1-naphthyl)ethylenediamine solution to determine nitrite production. Absorbance was measured at 550 nm against a calibration curve with sodium nitrite standards. Cell viability of the remaining cells was determined by a cell counting kit (Dojindo, Kumamoto, Japan) based colorimetric assay.

Figure 2. Key NOESY correlations of compounds 1 and 3.

Table 2. Inhibition of LPS-Induced NO Production in Macrophage RAW 264.7 Cells of Compounds 1−15 compound

IC50 (μM)a

compound

IC50 (μM)a

1 2 3 4 5 6 7 8

21.5 20.2 35.6 34.2 >50 >50 20.6 >50

9 10 11 12 13 14 15 AGb

>50 22.8 26.3 27.7 >50 >50 >50 20.6

Results are expressed as the mean IC50 values in μM from triplicate experiments. bAG = aminoguanidine was used as the positive control. a

mm, Merck) plates, and spots were detected by a 10% vanillin−H2SO4 in water spray reagent. Plant Material. The dried aerial parts of Scutellaria barbata were purchased from Kyungdong Oriental Herbal Market in Seoul, Korea, in March 2011. The plant material was identified by Emeritus Professor Kyong Soon Lee, and a voucher specimen of this plant was deposited at the Herbarium of College of Pharmacy, Chungbuk National University, Korea (CBNU-2011-03-ST). Extraction and Isolation. The dried and powered aerial parts of S. barbata (5.4 kg) were extracted with MeOH (3 × 10 L) at room temperature. After evaporation of the solvent, the residue was suspended in H2O and partitioned successively with n-hexane, CH2Cl2, and EtOAc. The CH2Cl2-soluble extract (33.5 g) was chromatographed over a silica gel column eluted with a step gradient of n-hexane−EtOAc (50:1 to 0:1) to give seven fractions (SBC1− SBC7). Fraction SBC6 (3.5 g) was further subjected to passage over a RP-18 MPLC column eluted with a step gradient of H2O−MeCN (80:20 to 10:90) to produce nine fractions (SBC6-1 to SBC6-9). Fraction SBC6-9 (300 mg) was purified by semipreparative HPLC (H2O−MeCN, 30:70 to 0:100) to afford compounds 1 (3.9 mg, tR 18.5 min), 2 (3.1 mg, tR 20.2 min), 3 (2.0 mg, tR 22.5 min), and 4 (5.0 mg, tR 26.0 min). Fraction SBC6-6 (400 mg) was purified further by D



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Note

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00126. 1D NMR, 2D NMR, and HRESI mass spectra of compounds 1−3 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-43-261-2814. Fax: +82-43-268-2732. E-mail: [email protected]. Notes

The authors declare no competing financial interest.


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ACKNOWLEDGMENTS This research was supported by a grant from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program and by the Medical Research Center Program (MRC, 2008-0062275) through the National Research Foundation of Korea.

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REFERENCES

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