Anti-inflammatory Prenylated Flavonoids from Helminthostachys zeylanica

Abstract

Two new [neougonins A (1) and B (2)] and nine known prenylated flavonoids were isolated from the whole plants of Helminthostachys zeylanica. The structures of the new isolates were elucidated by extensive NMR techniques, including one and two dimensional (1D)- and (2D)-NMR experiments, as well as comparison with spectroscopic data of known analogous compounds. Moreover, compound 1 inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells with an IC₅₀ value of 3.32 µM.

Helminthostachys zeylanica (L.) HOOK. (Ophioglossaceae) is a rare monotypic plant found in southeastern Asia.¹⁾ The rhizomes of H. zeylanica are used in a popular antipyretic and anti-inflammatory Chinese herbal medicine known as “Daodi-Ugon.”^1,2) This plant has also been shown to exhibit hepatoprotective activity.^3,4) Numerous bioactive flavonoids containing prenyl or geranyl groups have been isolated from the rhizomes of H. zeylanica.^2–6) Our current systematic phytochemical investigation of this plant led to the isolation of two new prenylflavonoids, neougonins A (1) and B (2), along with nine known flavonoids (Fig. 1). In addition, compound 1 showed significant inhibition of nitric oxide (NO) production with an IC₅₀ value of 3.32 µM. We report herein the isolation, structural elucidation and biological evaluation of the isolated compounds.

Fig. 1. Structures of Compounds 1–11

Results and Discussion

The dried whole plants of H. zeylanica were extracted with 95% EtOH, and the concentrated extract was suspended in water and partitioned with EtOAc. The EtOAc fraction was subjected to repeated column chromatography (MCI CHP 20P, 75–150 µm), Sephadex LH-20, and octadecyl silica (ODS)) and semi-preparative HPLC to yield two new prenylated flavonoids, neougonins A (1) and B (2), together with nine known flavonoids.

Compound 1 was isolated and purified as a yellow amorphous powder. A molecular ion peak at m/z 424.1887 in the high resolution-electron ionization (HR-EI)-MS indicated a molecular formula of C₂₅H₂₈O₆, corresponding to 12 degrees of unsaturation. The IR spectrum of 1 showed the presence of hydroxy (ν_max 3407 cm⁻¹) and carbonyl (ν_max 1637 cm⁻¹) groups. The UV spectrum showed a disparate course of spectra with maxima at 230 (sh) nm, 298 (sh) nm. Generally, the spectra corresponded to the π→π* and n→π* electronic transitions of the flavanone skeleton.⁷⁾ A pair of double doublets at δ_H 2.98 (1H, J=17.1, 13.2 Hz, H-3α) and 2.62 (1H, J=17.1, 2.7 Hz, H-3β), as well as a double doublet at δ_H 5.14 (1H, J=13.2, 2.7 Hz, H-2β) in the ¹H-NMR spectrum (Table 1) further confirmed the presence of a flavanone skeleton, and it was also verified by the ¹H–¹H correlation spectroscopy (COSY) correlations between H-2 and H-3. Moreover, the key heteronuclear multiple bond connectivity (HMBC) correlations from H-2 and H-8 to C-8a as well testified it. An aromatic proton at δ_H 5.91 (1H, s) showed HMBC correlations (Fig. 2) with carbons at C-6 (δ_C 112.9), C-7 (δ_C 167.8), C-4a (δ_C 104.0) and C-8a (δ_C 163.3), and was assigned as H-8 of ring A. There remaining aromatic protons at δ_H 6.92 (1H, br s, H-2′) and 6.78 (2H, overlapped, H-5′, 6′), which showed HMBC corrections with C-1′ (δ_C 131.3), were ascribed to ring B. In addition, three tertiary methyls at δ_H 1.46 (3H, s, Me-18), 1.47 (3H, s, Me-15) and 1.58 (3H, s, Me-14) were also observed in the ¹H-NMR spectrum. Furthermore, a set of coupled signals at 6.24 (1H, dd, J=17.5, 10.7 Hz, H-16), 4.72 (1H, dd, J=17.5, 1.1 Hz, H-17a) and 4.69 (1H, dd, J=10.7, 1.1 Hz, H-17b) were attributed to a mono-substituted double bond. The ¹³C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra of 1 (Table 1) showed resonances for 25 carbons: three tertiary methyls [δ_C 25.9, q, C-14; 17.7, q, C-15; 28.2, q, C-18], four methylenes including a terminal double bond (δ_C 107.8, t, C-17), seven methines including six unsaturated and one oxygenated (δ_C 80.9, d, C-2), together with eleven quaternary carbons [one carbonyl (δ_C 198.9, s, C-4), one sp³ (δ_C 45.5, s, C-9) and nine sp²]. Fifteen carbons were assigned to the flavanone skeleton, and the remaining ten carbons were ascribed to a monoterpene unit. On careful investigation, the ¹H- and ¹³C-NMR spectroscopic data of 1 were quite similar to those of 6-(1,1-dimethylallyl)eriodictyol,⁸⁾ except for the added presence of a prenyl group (δ_C 25.2, t, C-11; 126.5, d, C-12; 131.9, s, C-13; 25.9, q, C-14; 17.7, q, C-15). The HMBC correlations from H-10 (δ_H 1.94, m; 1.72, m) to C-11 and C-12, from H-11 (δ_H 1.85, m; 1.80, m) to C-10 (δ_C 41.8), and from H-12 (δ_H 4.99, overlapped) to C-14 and C-15, combined with one H-atom spin system (H-10/H-11/H-12) deduced from the ¹H–¹H COSY correlations, suggested that the prenyl group was attached to a methyl of the 1,1-dimethylallyl group. Moreover, the signals of H-10, H-16 and Me-18 all exhibited HMBC correlations with C-6 (δ_C 114.2), which confirmed the assigned linkage shown in Fig. 1.

Table 1. The ¹H- and ¹³C-NMR Data for Compounds 1 and 2 in CD₃OD [δ (ppm), J (Hz)]

No.	1		2
	δH (400 MHz)	δC (100 MHz)	δH (600 MHz)	δC (150 MHz)
2	5.14, dd (13.2, 2.7)	80.9, CH		147.9, C
3	2.98, dd (17.1, 13.2)	44.6, CH2		137.4, C
	2.62, dd (17.1, 2.7)
4		198.9, C		177.7, C
4a		104.0, C		105.5, C
5		163.5, C		153.4, C
6		112.9, C		114.2, C
7		167.8, C		166.4, C
8	5.91, s	97.9, CH	6.17, s	94.7, CH
8a		163.3, C		163.3, C
9		45.5, C		45.0, C
10	1.94, m	41.8, CH2	4.55, q (6.6)	92.1, CH
	1.72, m
11	1.85, m	25.2, CH2	1.13, d (6.6)	19.7, CH3
	1.80, m
12	4.99, overlapped	126.5, CH	1.30, s	22.0, CH3
13		131.9, C	1.61, s	26.6, CH3
14	1.58, s	25.9, CH3
15	1.47, s	17.7, CH3
16	6.24, dd (17.5, 10.7)	151.2, CH
17	4.72, dd (17.5, 1.1)	107.8, CH2
	4.69, dd (10.7, 1.1)
18	1.46, s	28.2, CH3
1′		131.3, C		124.3, C
2′	6.92, br s	114.9, CH	7.75, d (1.8)	116.1, CH
3′		146.4, C		146.5, C
4′		146.7, C		149.0, C
5′	6.78, overlapped	116.1, CH	6.90, d (8.4)	116.4, CH
6′	6.78, overlapped	119.4, CH	7.62, dd (8.4, 1.8)	121.6, CH

Fig. 2. Key HMBC Correlations for Compounds 1 and 2

The relative configuration of H-2 was determined by comparing the chemical shifts and the coupling constants of H-2 and H-3 of 1 with those of 6-(1,1-dimethylallyl)eriodictyol.^8–11) And the absolute configuration at stereogenic center C-2 could be determined using circular dichroism (CD) spectroscopic analysis. Compound 1 depicted a negative Cotton effect originating from a π→π* transition at 290 nm, and this was adequately indicative of a 2S configuration.^12,13) The stereochemistry of C-9 still remains to be determined since the inadequate rotating frame nuclear Overhauser enhancement spectroscopy (ROESY) correlations and the failure to obtain suitable crystal for single X-ray diffraction analysis. Based on these results, the structure of 1 (neougonin A) was assigned as 5,7,3′,4′-tetrahydroxy-6-(2,6-dimethyl-6-vinyl-2-hexenyl)flavanone.

Compound 2 was obtained as a yellow amorphous powder. The negative HR-electrospray ionization (ESI)-MS exhibited a quasi-molecular ion peak at m/z 369.0979 [M−H]⁻, consistent with the molecular formula C₂₀H₁₈O₇, indicating 12° of unsaturation. The ¹³C-NMR and DEPT spectra (Table 1) indicated the presence of twelve quaternary carbons [one carbonyl (δ_C 177.7, s, C-4), one sp³, ten sp²], five methines [four aromatic, one aliphatic], and three methyls, which suggested the presence of a flavonoid skeleton with a prenyl unit. In the ¹H-NMR spectrum (Table 1), three aromatic protons were present as an ABX system at δ_H 7.75 (1H, d, J=1.8 Hz, H-2′), 6.90 (1H, d, J=8.4 Hz, H-5′) and 7.62 (1H, dd, J=8.4, 1.8 Hz, H-6′), and were assigned to a 1,3,4-trisubstituted ring B. A singlet signal at δ_H 6.17 was attributed to H-8 of ring A, based on the HMBC correlations (Fig. 2) of δ_H 6.17 with δ_C 114.2 (C-6), 166.4 (C-7), 105.5 (C-4a) and 163.3 (C-8a). Proton resonances at δ_H 4.55 (1H, q, J=6.6 Hz, H-10), 1.13 (3H, d, J=6.6 Hz, Me-11), 1.30 (3H, s, Me-12) and 1.61 (3H, s, Me-13), indicated the presence of a 2,3-dihydro-2,3,3-trimethylfuran ring,³⁾ which was located at C-6 (δ_C 114.2) based on the HMBC correlations from Me-12 and Me-13 to C-6. A side-by-side comparison of the ¹H- and ¹³C-NMR data of 2 with those of ugonin T³⁾ showed that the two compounds were extremely similar, except for the ring C signals. Moreover, the chemical shifts of C-2 (δ_C 147.9), C-3 (δ_C 137.4) and C-4 in 2 were comparable with those of the flavonol ugonin F,²⁾ while the molecular weight of 2 was 16 mass units less than that of ugonin T. Based on the above evidence, the structure of compound 2 (neougonin B) was unambiguously elucidated as 4″,5″-dihydro-3,5,3′,4′-tetrahydroxy-4″,4″,5″-trimethylfurano[2″,3″:6,7]flavonol.

The remaining isolated isoprenylated flavonoids were identified based on comparison of their spectroscopic data with the literature values for ugonin D (3),⁶⁾ 2-(3,4-dihydroxyphenyl)-6-((2,2-dimethyl-6-methylenecyclohexyl)methyl)-5,7-dihydroxy-chroman-4-one (4),⁴⁾ ugonins E (5),²⁾ J (6),²⁾ L (7),²⁾ and S (8),³⁾ 4″a,5″,6″,7″,8″,8″a-hexahydro-5,3′,4′-trihydroxy-5″,5″,8″a-trimethyl-4H-chromeno[2″,3″:7,6]flavone (9),⁴⁾ 4″a,5″,6″,7″,8″,8″a-hexahydro-5,3′,4′-trihydroxy-5″,5″,8″a-trimethyl-4H-chromeno[2″,3″:7,8]flavone (10)⁴⁾ and ugonin N (11).³⁾

All compounds were examined for inhibition of NO production in lipopolysaccharide (LPS)-treated RAW264.7 cells to evaluate their anti-inflammatory potential. NO production was increased by LPS (1 µg/mL) treatment for 24 h as compared with control group. This increase was markedly suppressed in a dose dependent manner by treatment with compound 1 (IC₅₀=3.32 µM), while the IC₅₀ value of the positive control (NG-monomethyl-L-arginine, monoacetate salt (L-NMMA)) was 17.42 µM. Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, only compound 1 exhibited moderate cytotoxicity against RAW264.7 cells with IC₅₀ values of 16.13 µM, which suggested that the NO inhibiting effects may be not due to the cytotoxicity. The remaining isolates were not active.

Experimental

General Experimental Procedures

Optical rotations were measured with a Jasco DIP-370 digital polarimeter (JASCO Corporation, Tokyo, Japan). CD spectra were obtained using a Jasco J715 spectropolarimeter. IR spectra were acquired on a Bio-Rad FTS-135 spectrophotometer as KBr pellets (Bio-Rad Corporation, CA, U.S.A.). UV spectra were recorded using a Shimadzu UV-2401 A spectrophotometer (Shimadzu Corporation, Kyoto, Japan). One and two dimensional (1D)- and (2D)-NMR spectra were recorded by using Bruker AM-400, DRX-500 and AVANCE III-600 instruments, with tetramethylsilane (TMS) as an internal standard (Bruker BioSpin Group, Karlsruhe, Germany). ESI-MS and HR-ESI-MS were obtained with an API-Qstar-TOF instrument (Allen-Bradley, Milwaukee, MI, U.S.A.), and HR-EI-MS were measured on a VG Auto Spec-3000 spectrometer (Micromass, Manchester, U.K.). Column chromatography (CC) was carried out on silica gel (200–300 mesh, 100–200 mesh, 80–100 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), MCI gel (CHP 20P, 75–150 µm, Mitsubishi Chemical Corporation, Tokyo, Japan), ODS (75 µm, YMC Co., Ltd., Japan) and Sephadex LH-20 (Amersham Biosciences AB, Uppsala, Sweden). Semi-preparative HPLC was performed on an Agilent 1200 liquid chromatograph with a ZORBAX SB-C18 (5 µm, 9.4×250 mm, Agilent, U.S.A.) column. TLC was carried out on silica gel GF₂₅₄ coated glass plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) using various solvent systems, and spots were detected by spraying with 5% H₂SO₄–EtOH, followed by heating on a hot plate.

Plant Material

The whole plants of Helminthostachys zeylanica were collected from Xishuangbanna, Yunnan Province, China, in August 2011, and identified by Prof. Hai-Zhou Li, Kunming University of Science and Technology. A voucher specimen (KMUST 20110801) was deposited at the Laboratory of Phytochemistry, Faculty of Life Science and Technology, Kunming University of Science and Technology.

Extraction and Isolation

The dried and pulverized whole plants of H. zeylanica (500 g) were extracted with 95% EtOH (3×3 L, 48 h, each) at room temperature and filtered. The filtrate was concentrated in vacuo and the resulting residue was partitioned between EtOAc and H₂O. The EtOAc extract (8.0 g) was subjected to MCI CC and eluted with MeOH/H₂O in a stepwise gradient of 30, 60, 90, 100% MeOH to produce fractions A–D. Fraction C (4.0 g) was subjected to Sephadex LH-20, eluting with the same MeOH–H₂O gradient to provide subfractions C-1–C-4. Fraction C-1 (605 mg) was applied to a Sephadex LH-20 column and eluted with MeOH to yield three fractions, C-1-1–C-1-3. Fraction C-1-2 (290 mg) was again applied to Sephadex LH-20 (MeOH) and then purified using preparative TLC (CHCl₃–MeOH–H₂O, 8 : 2 : 0.2) to afford compounds 11 (2.3 mg) and 8 (3.0 mg). Fraction C-2 (1.6 g) was subjected to ODS CC eluting with MeOH–H₂O (10 : 90, 30 : 70, 60 : 40, 90 : 10, 100 : 0) and then purified on semi-preparative HPLC (43% MeOH in H₂O, flow rate 3 mL/min) to obtain compounds 2 (1.8 mg, t_R=10.3 min), 7 (1.5 mg, t_R=14.0 min) and 4 (1.2 mg, t_R=17.5 min). Fraction C-3 (1.9 g) was chromatographed over ODS, eluting with MeOH–H₂O (10% stepwise gradient from 20–70% MeOH) to provide three subfractions, C-3-1–C-3-3. Fraction C-3-1 (156 mg) was separated by silica gel CC eluted with a CHCl₃–MeOH gradient (10 : 1–1 : 1) to yield compound 6 (12.0 mg). Fraction C-3-2 (960 mg) was further repeatedly subjected to Sephadex LH-20 CC (MeOH eluant) to afford compound 5 (2.3 mg) and a mixture, which was further purified over Sephadex LH-20 (MeOH) followed by semi-preparative HPLC (33% CH₃CN in H₂O, flow rate 3 mL/min) to give compounds 1 (4.5 mg, t_R=12.5 min) and 3 (2.3 mg, t_R=15.0 min). Fraction C-3-3 (415 mg) was subjected to Sephadex LH-20 CC eluting with MeOH and then purified by preparative TLC (CHCl₃–MeOH, 9 : 1) to produce compounds 9 (3.2 mg) and 10 (3.5 mg).

Neougonin A (1)

Yellow amorphous powder; −84.1 (c=0.13 MeOH); UV (MeOH) λ_max (log ε) 298 (4.33), 204 (4.73) nm; CD (0.000199 M) λ_max (Δε) 326 (+1.35), 290 (−17.69), 227 (+9.86) nm; IR (KBr) ν_max: 3407, 2924, 1637, 1522; ¹H- and ¹³C-NMR data, see Table 1; HR-EI-MS m/z 424.1887 [M]⁺ (Calcd for C₂₅H₂₈O₆, 424.1886).

Neougonin B (2)

Yellow amorphous powder; −7.2 (c=0.12 MeOH); ¹H- and ¹³C-NMR data, see Table 1; ESI-MS m/z 369 [M−H]⁻; HR-ESI-MS (negative mode) m/z 369.0979 [M−H]⁻ (Calcd for C₂₀H₁₇O₇, 369.0974).

Inhibition of Nitric Oxide Production Assay

Murine macrophage RAW264.7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (high glucose) supplemented with 10% (v/v) fetal bovine serum, 100 µg/mL penicillin and streptomycin and 10 mmol/L N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) at 37°C in a 5% CO₂ atmosphere. RAW264.7 cells were purchased from Kunming Institute of Zoology. Inhibition of nitric oxide production assay was performed as described previously.¹⁴⁾ Cells were pre-treated with compounds 1–11 at the concentrations of 0, 0.39, 1.56, 6.25, 25 and 100 µM, and co-incubated with 1 µg/mL of LPS for 24 h. Wells with dimethyl sulfoxide (DMSO) were used as a negative control, and L-NMMA was used as a positive control. Cytotoxicity was determined by the MTT assay as described.¹⁵⁾

Acknowledgments

This research work was supported financially by Grants from the National Natural Science Foundation of China (Nos. 21262021, 21572082), the State Key Laboratory of Phytochemistry and Plant Resources in West China (No. P2015-KF01), and the Talent Cultivation Project Foundation of Yunnan Province (Nos. 14118650, 14118631).

Conflict of Interest

The authors declare no conflict of interest.

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