New Iridoids from Scrophularia ningpoensis

Abstract

Five new compounds including five iridoids (1–5) and six known compounds were isolated from the rhizomes of Scrophularia ningpoensis. Their structures were determined by extensive NMR and IR, MS spectroscopic data analyses. The anti-inflammatory, antibacterial, antifungal, and cytotoxic activities of the isolated compounds were evaluated. Compound 11 exhibited significant inhibitory effects on lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophage cells.

Scrophularia ningpoensis HEMSL., belonging to the family Scrophulariaceae, is a kind of perennial herbal medicine widely distributed in China. Its dried roots have been used as an important traditional Chinese medicine named “Xuan Shen” for the treatment of bacteria, atopy, pimple, tonsillitis, angina, encephalitis, and various inflammation diseases.^1,2) Previous phytochemical studies on S. ningpoensis showed that iridoids^3–8) and phenylpropanoid glycosides^7–9) are the main chemical constituents of S. ningpoensis. Moreover, saponins¹⁰⁾ and alkaloids¹¹⁾ were also isolated from S. ningpoensis. Iridoids as the main chemical constituents are assumed to be the predominant active pharmaceutical ingredients of S. ningpoensis.^1,12,13)

As our search for unique and bioactive compounds from traditional Chinese medicine, the chemical constituents of the dried roots of S. ningpoensis were investigated. In present study, we reported the isolation and structural elucidation of compounds 1–11. The anti-inflammatory, cytotoxic and antimicrobial activities of isolated compounds were evaluated.

Results and Discussion

Seventy percent EtOH extract from the dried roots of S. ningpoensis was subjected to silica gel, Sephadex LH-20, and octadecyl silica (ODS) column chromatography to afford 11 pure compounds. They were three new iridoids, 1β,6β-dimethoxy-dihydrocatalpolgenin (1), ningpogeniridoid (2), 1-ethyoxyl-3-hydroxy-2,3-seconingpogenin (4), two pairs of new inseparable isomers, 1β-hydroxy-6β-methoxy-dihydrocatalpolgenin (3a)/1α-hydroxy-6β-methoxy-dihydrocatalpolgenin (3b) and ningpopyrrosides A/B (5a/b) together with six known compounds, 5-(methoxymethyl)-1H-pyrrole-2-carbaldehyde (6),¹⁴⁾ 5-methoxypyrrolidin-2-one (7),^15,16) ningpogenin (8),^3,17) pedicularis lactone (9),¹⁸⁾ iridolactone (10),¹⁹⁾ and scrophularianine A (11)¹¹⁾ (Fig. 1). All compounds were identified based on detailed spectroscopic data analysis. Among them, compounds 5 and 7 were 5-hydroxypyrrolidin-2-ones, which had a rare five-membered heterocyclic lactam with a hemiaminal group in natural source including plants and microorganisms. To our best knowledge, this is the first report of the isolation of 5-hydroxypyrrolidin-2-ones from S. ningpoensis.

Fig. 1. Structures of Compounds 1–11

Compound 1 was acquired as a colorless oil and its molecular formula was deduced as C₁₁H₁₈O₅ on the basis of high resolution-electrospray ionization (HR-ESI)-MS data (m/z 253.1045 [M+Na]⁺, Calcd C₁₁H₁₈NaO₅, 253.1052). The ¹H-NMR data of 1 (Table 1) presented five methine protons including three oxygenated methines at δ 4.10 (1H, d, J=8.6 Hz), 3.78 (1H, br d, J=9.2 Hz), 3.62 (1H, br s) and two aliphatic methines at δ 1.97 (1H, t, J=8.3 Hz), 1.92 (1H, br q, J=7.5 Hz); three methylenes at δ 3.76 (1H, dd, J=12.1, 5.5 Hz), 3.41 (1H, td, J=12.1, 2,2 Hz), δ 3.70 (1H, dd, J=12.8, 5.3 Hz), 3.49 (1H, dd, J=12.8, 6.8 Hz), and δ 1.60 (1H, m), 1.40 (1H, br d, J=14.1 Hz); two methoxyls at δ 3.37 (3H, s), 3.35 (3H, s). In addition, an active proton was observed at δ 4.46 (1H, dd, J=6.8, 5.3 Hz). ¹³C-NMR data gave eleven carbons, except for the carbons corresponding to the proton signals, one more carbon without directly bonded proton was presented at δ 65.4 (C-8) with the help of heteronuclear single quantum coherence (HSQC) experiment. The ¹H- and ¹³C-NMR data of 1 was very similar to those of dihydrocatalpolgenin²⁰⁾ except for two more methoxyls appeared in 1. Heteronuclear multiple bond connectivity (HMBC) correlations from 1-OMe (δ 3.35) to C-1 (δ 102.0), from 6-OMe (δ 3.37) to C-6 (δ 80.9), from H-1 (δ 4.10) to 1-OMe (δ 55.8), and from H-6 (δ 3.78) to 6-OMe (δ 57.3) indicated the two methoxyls were located at C-1 and C-6, respectively. The relative configuration of 1 was determined by analysis of the coupling constants between H-1 and H-9 (J_H1–H9=8.6 Hz), H-5 and H-9 (J_H5–H9=8.3 Hz), H-5 and H-6 (J_H5–H6=9.2 Hz), and H-6 and H-7 (J_H6–H7 about 0 Hz) suggesting 1 possessed the same relative configuration as dihydrocatalpolgenin.^21,22) The nuclear Overhauser effect (NOE) correlations between 10-OH (δ 4.46) and H-1 (δ 4.10), H-6 (δ 3.78), H-10a (δ 3.70), H-10b (δ 3.49), between H-1 (δ 4.10) and H-7 (δ 3.62), H-10b (δ 3.49), H-3b (δ 3.41), H-9 (δ 1.97), between 1-OMe (δ 3.35) and H-5 (δ 1.92), between 6-OMe (δ 3.37) and H-9 (δ 1.97) also confirmed H-1, H-6, H-7, and hydroxymethyl at C-8 were α-oriented, H-5, H-9, 1-OMe, and 6-OMe were β-oriented (Fig. 2). Thus, the structure of 1 was determined and named 1β,6β-dimethoxy-dihydrocatalpolgenin.

Table 1. ¹H-NMR (600 MHz) and ¹³C-NMR (150 MHz) Data for 1–3 in DMSO-d₆

Position	1		2		3a		3b
	δC	δH mult. (J in Hz)	δC	δH mult. (J in Hz)	δC	δH mult. (J in Hz)	δC	δH mult. (J in Hz)
1	102.0	4.10, d (8.6)	94.9	5.30, s	94.0	4.32, dd (8.2, 6.2)	98.4	5.18, t (4.0)
3	61.4	3.76, dd (12.1, 5.5)	57.9	3.66, t (11.6)	60.3	3.68, dd (11.9, 5.3)	52.3	3.83, dd (12.5, 2.8)
		3.41, td (12.1, 2.2)		3.53, dd (11.6, 3.3)		3.40, td (11.9, 2.0)		3.30, td (12.5, 5.5)
4	23.4	1.60, m	32.6	1.97, dd (12.8, 5.9)	22.5	1.57, m	21.9	1.66, m
		1.40, br d (14.1)		1.34, qd (12.8, 4.6)		1.37, br d (13.9)		1.47, br d (13.6)
5	35.2	1.92, br q (7.5)	42.8	2.70, m	34.3	1.90, br q (7.5)	31.3	1.76, br q (7.5)
6	80.9	3.78, br d (9.2)	87.6	3.21, t (6.1)	79.8	3.78, br d (8.4)	81.7	3.82, br d (9.0)
7	56.6	3.62, br s	70.8	4.38, br t (6.4)	55.4	3.61, br s	56.7	3.50, br s
8	65.4		138.5		64.5		65.0
9	42.4	1.97, t (8.3)	133.5		43.0	1.93, br t (8.0)	38.4	2.29, br t (5.5)
10	59.3	3.70, dd (12.8, 5.3)	55.5	4.11, dd (13.5, 4.4)	58.4	3.77, dd (13.0, 5.3)	59.2	3.95, dd (12.5, 6.4)
		3.49, dd (12.8, 6.8)		3.49, ddd (13.5, 6.2, 2.9)		3.60, dd (13.0, 7.2)		3.30, dd (12.5, 5.5)
1-OMe	55.8	3.35, s	54.2	3.25, s
6-OMe	57.3	3.37, s	57.5	3.31, s	56.2	3.36, s	56.3	3.35, s
1-OH						6.71, d (6.1)		6.71, br d (4.0)
7-OH				4.43, d (7.0)
10-OH		4.46, dd (6.8, 5.3)		4.72, dd (6.2, 4.0)		4.37, dd (7.2, 5.3)		4.37, t (6.2)

Fig. 2. Key NOE Correlations of Compounds 1 and 2

Compound 2 was isolated as a colorless oil and its molecular formula was deduced as C₁₁H₁₈O₅ from HR-ESI-MS and NMR spectroscopic data. The ¹H- and ¹³C-NMR data of 2 were characteristic of an iridoid. Comparing NMR data of 2 (Table 1) with those of a related iridoid, 1β,6β,7α,8α,10-pentahydroxy-cis-2-oxabicyclo[4.3.0]nonane,²¹⁾ one more olefinic double bond and two additional methoxyls were presented and one methine and one oxygenated quaternary were disappeared in 2. HMBC correlations between H-1 (δ 5.30), 10-OH (δ 4.72), 7-OH (δ 4.43), H-7 (δ 4.38), H-10 (δ 4.11, 3.49) and C-8 (δ 138.5), between H-7 (δ 4.38), H-10 (δ 4.11, 3.93) and C-9 (δ 133.5), between 1-OMe (δ 3.25) and C-1 (δ 94.9), between 6-OMe (δ 3.31) and C-6 (δ 87.6) showed the olefinic double bond was located at C-8, C-9 and two methoxyls were linked with C-1 and C-6, respectively (Fig. 3). The coupling constants of H-5 and H-6 (J_H5–H6=6.1 Hz), H-6 and H-7 (J_H6–H7=6.4 Hz) indicated both of H-5/H-6 and H-6/H-7 were anti configurations.²²⁾ NOE correlations observed between 1-OMe (δ 3.25) and H-7 (δ 4.38), H-3β (δ 3.66), between H-3β (δ 3.66) and H-5 (δ 2.70) suggested methoxy at C-1 was β configuration (Fig. 2). Thus, the structure of 2 was determined and named ningpogeniridoid.

Fig. 3. Key HMBC Correlations of Compounds 2, 4 and 5

Compounds 3a and b were obtained as an inseparable mixture of two epimers in a 5 : 3 ratio according to ¹H-NMR integral signals. The molecular formulas of 3a and b were established as C₁₀H₁₆O₅ by HR-ESI-MS. Two sets of signals could be obviously assigned in the ¹H- and ¹³C-NMR spectra according to their intensity as well as correlation spectroscopy (COSY) and HSQC experiments. The ¹H- and ¹³C-NMR data of 3a and b (Table 1) were characteristic of iridoids and close resemblance to dihydrocatalpolgenin.²³⁾ The difference between them was an additional methoxy signal presented both in 3a (δ 56.2, δ 3.36) and 3b (δ 56.3, δ 3.35). The methoxyls located at C-6 both in 3a and b were confirmed by HMBC correlations between 6-OMe (δ 3.36 in 3a)/C-6 (δ 79.8 in 3a) and 6-OMe (δ 3.35 in 3b)/C-6 (δ 81.7 in 3b). Except for the configurations of C-1, the chiral carbons configuration of 3a and b were identical to those of compound 1 and dihydrocatalpolgenin according to the coupling constants and NOE correlations. The larger coupling constant between H-1 and H-9 (J_H1–H9=8.2 Hz) of the major compound 3a was consistent with a β position of the hemiacetal hydroxy at C-1. While, a small value of J_H1–H9=4.0 Hz showed the hemiacetal hydroxyl was α configuration in 3b.^20,23) Therefore, the structures were elucidated as 1β-hydroxy-6β-methoxy-dihydrocatalpolgenin (3a) and 1α-hydroxy-6β-methoxy-dihydrocatalpolgenin (3b), respectively.

Compound 4 was assigned the molecular formula C₁₁H₂₀O₄ by HR-ESI-MS (m/z 239.1259 [M+Na]⁺). The ¹H-NMR data of 4 (Table 2) showed signals for one olefinic proton at δ 5.93 (1H, s), four couples of oxygenated methylenes at δ 4.14 (1H, dd, J=15.4, 4.4 Hz), 3.99 (1H, dd, 15.4, 5.5 Hz), δ 3.50 (1H, m), 3.34 (1H, m), δ 3.48 (1H, m), 3.35 (1H, m), and δ 3.47 (1H, m), 3.43 (1H, m), an oxygenated methine at δ 4.03 (1H, br d, J=5.7 Hz), a pair of methylene at δ 1.61 (1H, m), 1.55 (1H, m), two methines at δ 2.45 (1H, q, J=6.4 Hz) and 2.20 (1H, quint, J=7.2 Hz), and one methyl at δ 1.06 (3H, t, J=6.0 Hz), in addition to three active protons at δ 4.83 (1H, t, J=5.3 Hz), 4.37 (1H, t, J=5.3 Hz), and δ 4.09 (1H, dd, J=6.4, 3.5 Hz). The ¹³C-NMR spectrum (Table 2) manifested eleven carbons including two olefinic carbons (δ 155.1, 124.9), five oxygenated aliphatic carbons (δ 81.6, 63.6, 61.7, 60.2, 60.1), two methines (δ 48.9, 42.7), one methylene (δ 29.1), and one methyl (δ 16.0) with the aid of HSQC experiment. The NMR data of 4 showed a close similarity to those of a related iridoid, ningpogenin,^3,17) which also isolated from the same herbal medicine. The difference between 4 and ningpogenin (8) were one more ethoxyl and one more hydroxyl arising in 4 on the basis of NMR data and molecular formula. COSY and HMBC correlations deduced 4 was a 2,3-seco-ring ningpogenin derivative with an ethyoxyl group. HMBC correlations from H-1′ (δ 3.48, 3.35) to C-1 (δ 81.6), from H-1 (δ 4.03) to C-1′ (δ 63.6) revealed ethyoxy located a C-1 (Fig. 3). The relative configuration of 4 was identical with ningpogenin deduced from the coupling constant of H-1 and H-5 (J_H1–H5=7.2 Hz), H-5 and H-6 (J_H5–H6=6.4 Hz). Consequently, the structure of 4 was determined to be 1-ethyoxyl-3-hydroxy-2,3-seconingpogenin.

Table 2. ¹H-NMR (600 MHz) and ¹³C-NMR (150 MHz) Data for 4 and 5 in DMSO-d₆

Position	4		5a/5b
	δC	δH mult. (J in Hz)	δC	δH mult. (J in Hz)
1	81.6	4.03, br d (5.7)	86.6	4.90, br d (7.0)
3	60.1	3.47, m 3.43, m	66.7	3.61, m, 3.45, m
4	29.1	1.61, m 1.55, m	28.3	1.74, m, 1.66, m
5	42.7	2.20, quint (7.2)	42.8	2.90, m
6	48.9	2.45, q (6.4)	46.0	2.85, m
7	155.1		149.8/149.5
8	124.9	5.93, s	124.8/125.0	5.48, d (1.5)
9	61.7	3.50, m	66.1	3.62, m, 3.44, m
		3.34, m		3.45, m, 3.28, dd (9.0, 8.0)
10	60.2	4.14, dd (15.4, 4.4)	59.4	3.98, dd (15.4, 6.8)
		3.99, dd (15.4, 5.5)		3.89, d (15.4)
1′	63.6	3.48, m, 3.35, m, 3.34, m		8.68, s
2′	16.0	1.06, t (6.0)	177.9
3′			28.5	2.23, m, 2.02, m
4′			28.1	2.17, m, 1.85, m
5′			85.8	4.85, d (7.0)/4.87, d (7.0)
3-OH		4.37, t (5.3)
9-OH		4.09, dd (6.4, 3.5)
10-OH		4.83, t (5.3)		4.76, br s

Compound 5 being as a pair of inseparable stereoisomers (5a/5b) were isolated as a mixture. The molecular formulas were established to be C₁₃H₁₉NO₄, based on their HR-ESI-MS data as well as ¹³C-NMR data. The ¹H-NMR and ¹³C-NMR spectrum (Table 2) of 5a showed characteristic signals for ningpogenin moiety and a set of more signals containing one carbonyl (δ 177.9), one oxygenated methine (δ 86.6, δ 4.90), two methylenes (δ 28.5, 28.1, δ 2.23/2.02, 2.17/1.85), and an active proton (δ 8.68). The fragment was confirmed as 5-hydroxypyrrolidin-2-one residue based on the COSY, HSQC, and HMBC experiments.^15,24) HMBC correlations from H-5′ (δ 4.85) to C-9 (δ 66.1) and from H-9 (δ 3.62/3.44) to C-5′ (δ 85.8) revealed the connection of the two moieties (Fig. 3), ningpogenin and 5-hydroxypyrrolidin-2-one, as in Fig. 1.

The ¹H- and ¹³C-NMR spectra of 5 presented another set of signals for 5b, with a ratio of nearly 1 : 1 with 5a. The ¹H- and ¹³C-NMR data of 5b were almost identical with 5a, except for chemical shift changes at H-9 (δ 3.62/3.44), H-5′ (δ 4.85), C-7 (δ 149.8) and C-8 (δ 124.8) for 5a, H-9 (δ 3.45/3.28), H-5′ (δ 4.87), C-7 (δ 149.5) and C-8 (δ 125.0) for 5b, indicating 5b is a C-1′ isomer of 5a. The HMBC correlations from H-5′ (δ 4.87) to C-9 (δ 66.1) and from H-9 (δ 3.45/3.28) to C-5′ (δ 85.8) confirmed the structure. Alike 4, the relative configurations of 5a/b were established by analysis on the coupling constant values of J_H1–H5=7.0 Hz. Even though the ¹H-NMR spectrum didn’t give a clear coupling constant of H-5 and H-6, a weak NOE correlation of H-1 (δ 4.90) and H-6 (δ 2.85) could be assigned both syn relationships of H-1 and H-5, H-5 and H-6. Therefore, the structures of 5a/b were determined, and named as ninpopyrrosides A and B.

All of the isolated compounds (1–11) were assayed for their anti-inflammatory, antibacterial, antifungal, and cytotoxic activities. Anti-inflammatory activities of isolated compounds were tested by examining their ability to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells. Among the tested compounds, only 11 showed a dose-dependent inhibition of NO production at the tested concentrations (11, 33, 100 µM) (Fig. 4). The other compounds show very weak activity with an inhibitory ratio lower than 20% at 100 µM. The cytotoxic activities and antimicrobial activities were also assessed for compounds 1–11, and no compounds exhibited obvious inhibitory activity.

Fig. 4. Effects of Compound 11 on the Inhibition of LPS-Induced NO in RAW264.7 Cell

Cells were treated by LPS with or without different concentrations of compounds, respectively (11, 33, 100 µM) for 24 h. Minocycline (50 µM) was used as a positive control. Nitrite–nitrate levels were measured in the medium and expressed as % activity over LPS stimulation. All conditions were run in triplicate, and data show mean±S.D. values. * p<0.01 compared to LPS.

Experimental

General Experimental Procedures

Optical rotations were determined using an AntonPaar MCP200 automatic polarimeter. Ultraviolet spectra were measured using a Beckman Coulter DU 730 nucleic acid/protein analyzer. IR spectra were recorded using a Bruker Tensor 27 FT-IR spectrometer (film). One and two dimensional (1D- and 2D)-NMR spectra were recorded using a Bruker AV-600 spectrometer, δ in ppm rel. to tetramethylsilane (TMS), J in Hz. ESI-MS were measured using an Agilent 1290-6420 triple quadrupole LC-MS spectrometer. HR-ESI-MS were measured using a Bruker micro TOF-Q mass instrument (Bruker Daltonics, Billerica, MA, U.S.A.). Silica gel (100–200 mesh and 200–300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), Sephadex LH-20 (GE Healthcare Biosciences AB, Uppsala, Sweden), and YMC-GEL ODS-A (S-50 µm, 12 nm) (YMC Co., Ltd., Kyoto, Japan) were used for column chromatography. Biological assays were analyzed using a microplate reader (BioTek Synergy H1, BioTek Instruments, Inc., Vermont, U.S.A.).

Plant Material

Dried rhizomes of S. ningpoensis were purchased from the Anguo Huirui Pharmaceutical Co., Ltd., Hebei Province, China, in August 2014 (batch No. 141101) and were identified by Professor Jinhui Wang from Shenyang Pharmaceutical University, Shenyang, China. A voucher specimen (No. 141101) was deposited at the Institute of Microbial Pharmaceuticals, College of Life and Health Sciences, Northeastern University.

Extraction and Isolation

Dried rhizomes of S. ningpoensis (19 kg) were extracted with 70% EtOH (50 L×3) under reflux, and the solvent was evaporated in vacuo. This concentrate was dissolved in water and partitioned with ethyl acetate to obtain an ethyl acetate extract (200 g). The dried EtOAc extract was subjected to silica gel column chromatography, eluting with a gradient of CH₂Cl₂–MeOH to afford 8 fractions (fractions 1–8). Fraction 4 (7 g) was subjected to Sephadex LH-20 chromatography (MeOH) to produce 12 subfractions (fractions 4.1–4.12). Fraction 4.5 (970 mg) was separated by silica gel column chromatography, eluting with CH₂Cl₂–MeOH=30 : 1 to afford 8 (20.0 mg) and the residues was subjected to ODS column chromatography, eluting with methanol–water (40 : 60) to yield 7 (18.0 mg). Fraction 4.6 (1.5 g) was separated by silica gel column chromatography, eluting with CH₂Cl₂–MeOH=30 : 1 to produce 16 subfractions (fractions 4.6.1–4.6.16). Subfraction 4.6.13 (190 mg) was subjected to silica gel column chromatography, eluting with CH₂Cl₂–acetone=3 : 1 to afford a mixture of 3a and b (7.0 mg). Subfraction 4.6.16 (65 mg) was subjected to silica gel column chromatography, eluting with CH₂Cl₂–MeOH=30 : 1 to afford a mixture of 5a and b (16.0 mg). Fraction 4.7 (198 mg) was separated by silica gel column chromatography, eluting with CH₂Cl₂–acetone=7 : 1 to yield 8 subfractions (fractions 4.7.1–4.7.8). Subfraction 4.7.2 (30 mg) was subjected to ODS column chromatography, eluting with methanol–water (45 : 55) to yield 6 (2.0 mg). Fraction 5 (8 g) was separated using Sephadex LH-20 chromatography (MeOH) to produce 7 subfractions (fractions 5.1–5.7). Fraction 5.4 (2 g) was subjected to silica gel column chromatography (CH₂Cl₂–MeOH=20 : 1) to produce 9 subfractions (fractions 5.4.1–5.4.9). Subfraction 5.4.1 (200 mg) was subjected to silica gel column chromatography (CH₂Cl₂–acetone=10 : 1) to yield 1 (2.7 mg). Subfraction 5.4.8 (60 mg) was subjected to silica gel column chromatography (CH₂Cl₂–acetone=2 : 1) to yield 9 (3.3 mg). Fraction 6 (7 g) was separated using Sephadex LH-20 chromatography (MeOH) to produce 7 subfractions (fractions 6.1–6.7). Fraction 6.2 (970 mg) was subjected to silica gel column chromatography (CH₂Cl₂–MeOH=25 : 1) to produce 9 subfractions (fractions 6.2.1–6.2.9). Subfraction 6.2.3 (18 mg) was subjected to silica gel column chromatography (CH₂Cl₂–acetone=3 : 1) to yield 2 (1.5 mg). Subfraction 6.2.8 (30 mg) was subjected to silica gel column chromatography (CH₂Cl₂–acetone=3 : 1) to yield 4 (3.3 mg). Fraction 6.3 (930 mg) was subjected to silica gel column chromatography (CH₂Cl₂–MeOH=25 : 1) to produce 10 subfractions (fractions 6.3.1–6.3.10). Subfraction 6.3.7 (64 mg) was subjected to silica gel column chromatography (CH₂Cl₂–MeOH=12 : 1) to yield 10 (5.0 mg). Fraction 6.6 (100 mg) was subjected to silica gel column chromatography (CH₂Cl₂–MeOH=30 : 1) to yield 11 (2.0 mg)

1β,6β-Dimethoxy-dihydrocatalpolgenin (1)

Colorless oil; [α]_D²⁰ −80.0 (c=0.4, MeOH); IR (film) cm⁻¹: 3457, 2934, 2852, 1393, 1202, 1120, 1086, 1057, 991, 968. ¹H-NMR (600 MHz, dimethyl sulfoxide (DMSO)-d₆) and ¹³C-NMR (150 MHz, DMSO-d₆) data, see Table 1; ESI-MS m/z 253 [M+Na]⁺; HR-ESI-MS m/z 253.1045 [M+Na]⁺ (Calcd for C₁₁H₁₈O₅Na, 253.1052).

Ningpogeniridoid (2)

Colorless oil; [α_D²⁰ −80.0 (c=0.3, MeOH); IR (film) cm⁻¹: 3385, 2927, 2855, 1643, 1453, 1238, 1179, 1111, 1090, 1038, 974, 955. ¹H-NMR (600 MHz, DMSO-d₆) and ¹³C-NMR (150 MHz, DMSO-d₆) data, see Table 1; ESI-MS m/z 253 [M+Na]⁺; HR-ESI-MS m/z 253.1054 [M+Na]⁺ (Calcd for C₁₁H₁₈O₅Na, 253.1052).

1β-Hydroxy-6β-methoxy-dihydrocatalpolgenin (3a) and 1α-Hydroxy-6β-methoxy-dihydrocatalpolgenin (3b)

Colorless oil; [α]_D²⁰ −45.5 (c=1.0, MeOH); IR (film) cm⁻¹: 3385, 2936, 1648, 1107, 1083, 1057, 1017, 994. ¹H-NMR (600 MHz, DMSO-d₆) and ¹³C-NMR (150 MHz, DMSO-d₆) data, see Table 1; ESI-MS m/z 239 [M+Na]⁺; HR-ESI-MS m/z 239.0891 [M+Na]⁺ (Calcd for C₁₀H₁₆O₅Na, 239.0895).

1-Ethyoxyl-3-hydroxy-2,3-seconingpogenin (4)

Colorless oil; [α]_D²⁰ +62.9 (c=0.7, MeOH); IR (film) cm⁻¹: 3373, 3333, 2959, 2926, 1650, 1443, 1331, 1160, 1123, 1061, 992. ¹H-NMR (600 MHz, DMSO-d₆) and ¹³C-NMR (150 MHz, DMSO-d₆) data, see Table 2; ESI-MS m/z 239 [M+Na]⁺; HR-ESI-MS m/z 239.1259 [M+Na]⁺ (Calcd for C₁₁H₂₀O₄Na, 239.1259).

Ninpopyrrosides A and B (5a/b)

Colorless oil; [α]_D²⁰ +7.7 (c=1.04, MeOH); IR (film) cm⁻¹: 3393, 3275, 2949, 2927, 2872, 1692, 1453, 1422, 1333, 1280, 1058, 958. ¹H-NMR (600 MHz, DMSO-d₆) and ¹³C-NMR (150 MHz, DMSO-d₆) data, see Table 2; ESI-MS m/z 254 [M+H]⁺, 276 [M+Na]⁺; HR-ESI-MS m/z 254.1390 [M+H]⁺(Calcd for C₁₃H₂₀NO₄, 254.1392).

Inhibition of NO Production Assay

The anti-inflammatory activity of the isolated compounds 1–11 were evaluated through inhibition of NO production in lipopolysaccharide-induced RAW264.7 macrophage cells using the Griess method²⁵⁾ and experiments were carried out as our previous descriptions.²⁶⁾ Minocycline was used as a positive control.

Cytotoxicity Assay

The cytotoxicities of 1–11 were assayed against three human carcinoma cell lines including a human gastric carcinoma cell line (SGC-7901), a human lung carcinoma cell line (H1975), and a human hepatocellular carcinoma cell line (SMMC-7721) in a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.²⁶⁾ Etoposide (VP-16) was assayed as a positive control.

Antimicrobial Assay

A micro broth dilution assay²⁷⁾ was used to evaluate antimicrobial activity of 1–11 against Bacillus subtilis ATC C 6633, Staphylococcus aureus ATC C 25923, Escherichia coli ATC C 25922, Candida albicans ATC C MYA-2876, and Candida parapsilosis ATC C 22019. The minimum inhibitory concentrations (MICs) were defined as the lowest concentration of the antimicrobial agent that completely inhibited the visual growth of an organism. Ciprofloxacin and amphotericin B were used as positive controls against bacteria and fungi, respectively.

Acknowledgments

This work was funded by National Natural Science Foundation of China (No. U1608282) and Basic Scientific Research Fund of Northeastern University, China (No. N142002001 and N152006002).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

References

• 1) Yang S., Li J., Zhao Y., Chen B., Fu C., J. Agric. Food Chem., 59, 1612–1621 (2011).
• 2) Lee M. K., Choi O. G., Park J. H., Cho H. J., Ahn M. J., Kim S. H., Kim Y. C., Sung S. H., J. Sep. Sci., 30, 2345–2350 (2007).
• 3) Qian J., Hunkler D., Rimpler H., Phytochemistry, 31, 905–911 (1992).
• 4) Chen X., Liu Y. H., Chen P., Nat. Prod. Res., 21, 1187–1190 (2007).
• 5) Nguyen A. T., Fontaine J., Malonne H., Claeys M., Luhmer M., Duez P., Phytochemistry, 66, 1186–1191 (2005).
• 6) Niu Z. R., Wang R. F., Shang M. Y., Cai S. Q., Nat. Prod. Res., 23, 1181–1188 (2009).
• 7) Chen B., Liu Y., Liu H. W., Wang N. L., Yang B. F., Yao X. S., Chem. Biodivers., 5, 1723–1735 (2008).
• 8) Hua J., Qi J., Yu B. Y., Fitoterapia, 93, 67–73 (2014).
• 9) Li Y. M., Jiang S. H., Gao W. Y., Zhu D. Y., Phytochemistry, 54, 923–925 (2000).
• 10) Li J., Huang X., Du X., Sun W., Zhang Y., Nat. Prod. Res., 23, 775–780 (2009).
• 11) Zhu L. J., Hou Y. L., Shen X. Y., Pan X. D., Zhang X., Yao X. S., Fitoterapia, 88, 44–49 (2013).
• 12) Georgiev M. I., Ivanovska N., Alipieva K., Dimitrova P., Verpoorte R., Phytochemistry, 92, 8–15 (2013).
• 13) Huang T. H., Tran V. H., Duke R. K., Tan S., Chrubasik S., Roufogalis B. D., Duke C. C., J. Ethnopharmacol., 104, 149–155 (2006).
• 14) Don M. J., Shen C. C., Lin Y. L., Syu W. J., Ding Y. H., Sun C. M., J. Nat. Prod., 68, 1066–1070 (2005).
• 15) Ma G., Liu H., Zhang W., J. Photochem. Photobiol. Chem., 187, 377–383 (2007).
• 16) Cheng Y. X., Zhou J., Tan N. H., Teng R. W., Lu Y., Wang C., Zheng Q. T., J. Nat. Prod., 65, 750–752 (2002).
• 17) Kaneko T., Ohtani K., Kasai R., Yamasaki K., Duc N. M.; Nguyen Minh Duc, Phytochemistry, 46, 907–910 (1997).
• 18) Yang L., Wang C., Jia Z., Phytochemistry, 40, 491–494 (1995).
• 19) Lin S. J., Tan C. H., Jiang S. H., Li Y. M., Zhu D. Y., Helv. Chim. Acta, 89, 2789–2793 (2006).
• 20) Gao J., Yang L., Jia Z. J., Planta Med., 63, 248–250 (1997).
• 21) Valladares M. G., Rios M. Y., J. Nat. Prod., 70, 100–102 (2007).
• 22) Chaudhuri R. K., Sticher O., Helv. Chim. Acta, 64, 3–15 (1981).
• 23) Esposito P., Iavarone C., Sen A., Trogolo C., Phytochemistry, 23, 803–807 (1984).
• 24) Hong S. S., Lee S. A., Kim N., Hwang J. S., Han X. H., Lee M. K., Jung J. K., Hong J. T., Kim Y., Lee D., Hwang B. Y., Bioorg. Med. Chem. Lett., 21, 1279–1281 (2011).
• 25) Hunter R. A., Storm W. L., Coneski P. N., Schoenfisch M. H., Anal. Chem., 85, 1957–1963 (2013).
• 26) Ma Q. J., Han L., Bi X. X., Wang X. B., Mu Y., Guan P. P., Li L. Y., Huang X. S., Phytochemistry, 131, 150–157 (2016).
• 27) Ding N., Jiang Y., Han L., Chen X., Ma J., Qu X., Mu Y., Liu J., Li L., Jiang C., Huang X., J. Nat. Prod., 79, 799–805 (2016).