Lignans and Terpenoids From the Fruits of Vitex kwangsiensia and Their Inhibitory Activity on Nitric Oxide Production in Macrophages

Abstract

Phytochemical investigation of the fruits from Vitex kwangsiensia led to the isolation of 18 constituents, including 6 lignans (1-6), 3 diterpenoids (7-9), 2 sesquiterpenoids (10, 11), 2 triterpenoids (16, 17), and 5 other compounds. Their structures were identified by spectroscopic methods (mass spectroscopy, infrared, UV, 1-dimensional and 2-dimensional nuclear magnetic resonance) and comparison with reported data in the literatures. Among these constituents, compound 1 was a new lignan and named as vitekwangin A. The spectroscopic data of 2 was reported herein for the first time, and given a trivial name vitekwangin B. The isolated lignans, diterpenoids, and sesquiterpenoids were evaluated for their inhibitory activity to prevent nitric oxide production in lipopolysaccharide-stimulated RAW264.7 macrophages.

Inflammation is the response of an organ triggered by the upregulated production of an array of proinflammatory cytokines and chemokines. ¹ Macrophages play an important role in inflammation reactions by producing a variety of inflammatory mediators including nitric oxide (NO). ^2,3 Inducible isoform of NO synthase (iNOS or NOS-2) isolated from macrophages synthesizes NO on l-arginine. ^4,5 In general, the free radical NO as an essential component of the host innate immune not only emerged for nonspecific host defense but also acted as a potent mediator of cellular damage. ¹ Excess NO will be unnecessarily expressed in macrophages when the iNOS is induced strongly by proinflammatory stimuli, which may lead to inflammation symptoms and other diseases such as cancer, atherosclerosis, and Alzheimer’s disease. ⁶ Therefore, reducing NO production or NOS gene expression in macrophages is usually taken as therapeutic mark in the course of anti-inflammatory drug screening.

The genus Vitex (Verbenaceae family) consists of about 250 species with 14 species, 7 varieties, and 3 subtypes occurring in China. ⁷ The mature fruits of several species are used as traditional Chinese medicine (TCM) for the treatment of algomenorrhea, migraine, eye pain, colds, headaches, arthritis, and so on. ⁸ Previous phytochemical investigations on this genus revealed that diterpenoids are the characteristic secondary metabolites of their fruits. ^9-11 In addition, other kinds of constituents, including flavonoids, ^12,13 sesquiterpenoids, ¹⁴ lignans, ^15-18 triterpenoids, ¹⁹ and iridoids, ²⁰ have been isolated from the fruits of some species. And many isolated diterpenoids and lignans showed potent inhibitory activity against NO production. ^10,21 Vitex kwangsiensia C. Pei is distributed only in very few regions of Guangxi Zhuang Autonomous Region of China. Locally, its fruits are usually used instead of that of Vitex negundo to be TCM “Bujingzi” or “Huangjingzi” mainly for the remedy of inflammation. Up to now, there is no chemical investigation on this plant in the literatures to our knowledge. This work was conducted for the phytochemical investigation of its fruits and inhibitory activity assay of isolated lignans, diterpenoids, and sesquiterpenoids against NO production in RAW264.7 cells.

The CHCl₃-soluble fraction of the MeOH extract of the fruits of V. kwangsiensia was repeatedly isolated by column chromatography (CC; SiO₂ and ODS) to afford 6 lignans (1-6), 3 diterpenoids (7-9), 2 sesquiterpenoids (10, 11), 2 phenylpropanoids (12, 13), 2 polymethoxyflavonoids (14, 15), 2 triterpenoids (16, 17), and a sterol (18), of which 1 was a new lignan (Scheme 1).

Scheme 1

Structures of the compounds isolated from Vitex kwangsiensia.

Compound 1 was isolated as pale yellow gum. Its high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) spectrum displayed quasimolecular ion peak at m/z 371.1491 ([M+H]⁺), consistent with the molecular formula C₂₁H₂₂O₆ corresponding to 11 indices of hydrogen deficiency. UV absorptions at 225.7, 254.5, and 355.5 nm were similar to that of 3, ²² suggesting the presence of aromatic moieties and an α,β-unsaturated ketone system. The infrared (IR) spectrum displayed OH group absorption at 3424 cm⁻¹. The presence of formyl groups was indicated by IR bands at 1662 cm⁻¹ and confirmed by ¹³C-NMR signals at δ(C) 194.5 (Table 1). The ¹H-NMR spectrum (Table 1) showed resonances for 1 conjugated olefinic proton δ(H) 7.39 (s, H-C(1)); 1 formyl proton δ(H) 9.35 (s, H-C(2α)); a 1,3,4-trisubstituted aromatic ring δ(H) 6.62 (d, J = 8.4, H-C(5′)), 6.56 (d, J = 2.0, H-C(2′)), 6.29 (dd, J = 8.4, 2.0, H-C(6′)); a 1,2,4,5-tetrasubstituted aromatic ring (δ(H) 6.59 (s, H-C(5)), 7.00 (s, H-C(8)); 3 methoxyl groups 3.81 (s, 7-OMe), 3.63 (s, 4′-OMe), 3.61 (s, 3′-OMe); and several signals in high field. The ¹³C-NMR spectrum (Table 1) exhibited 21 carbon signals, including 1 formyl carbon δ(C) 194.5 (C(2α)), 14 unsaturated carbons, 3 methoxyl carbons, 1 oxygenated methylene carbon δ(C) 62.7 (C(3α)), and 2 saturated methine carbons δ(C) 44.3 (C(4)), 43.9 (C(3)). These data were very similar with that of 3 except for the presence of one more methoxyl group. The structure of 1 was finally confirmed by heteronuclear multiple bond coherence (HMBC) and nuclear Overhauser effect spectroscopy (NOESY) experiments (Figure 1). The HMBC correlations δ(H) 9.35 (H-C(2α))/δ(C) 149.4 (C(1)), 136.1 (C(2)), and 43.9 (C(3)) indicated that formyl group was located at C(2), which was also supported by the NOESY correlations δ(H) 9.35 (H-C(2α))/δ(H) 7.39 (H-C(1)). The HMBC correlations δ(H) 3.38 and 3.05 (H-C(3α))/δ(C) 136.1 (C(2)) and 44.3 (C(4)) indicated the hydroxymethyl group attached at C(3). The NOESY correlations δ(H) 7.00 (H-C(8))/δ(H) 3.81 (7-MeO), δ(H) 6.56 (H-C(2′))/δ(H) 3.61 (3′-MeO), and δ(H) 6.62 H-C(5′)/δ(H) 3.63 (4′-MeO) suggested that three methoxyl groups were attached to C(7), C(3′), and C(4′) respectively. The NOESY correlations δ(H) 4.26 (H-C(4))/δ(H) 3.38 and 3.05 (H-C(3α)) suggested the trans-configuration of hydroxymethyl group and 3,4-dimethoxyphenyl group. From these data, 1 was established as 6-hydroxy-4-(3,4-dimethoxyphenyl)-3-hydroxymethyl-7-methoxy-3,4-dihydro-2-naphthaldehyde and named as vitekwangin A.

Table 1

¹H- and ¹³C-NMR Data of 1 and 2. (In CD₃OD; δ in ppm, J in Hz; 500 MHz for ¹H-NMR and 125 MHz for ¹³C-NMR.)

	1		2
	δ H	δ C	δ H	δ C
1	7.39 s	149.4	7.37 s	149.1
2	-	136.1	-	135.9
3	3.09 dd (10.0, 3.5)	43.9	3.11 dd (10.0, 4.5)	38.6
4	4.26 brs	44.3	4.78 brs	43.6
5	6.59 s	118.6	-	149.1
6	-	151.6	-	155.6
7	-	148.6	6.77 d (8.0)	116.7
8	7.00 s	113.9	7.07 d (8.0)	127.9
9	-	125.0	-	125.9
10	-	134.4	-	134.3
1′	-	138.9	-	138.2
2′	6.56 d (2.0)	112.8	6.63 d (1.5)	113.0
3′	-	150.4	-	150.4
4′	-	149.1	-	147.9
5′	6.62 d (8.4)	113.0	6.60 d (8.5)	113.1
6′	6.29 dd (8.4, 2.0)	120.9	6.32 dd (8.5, 1.5)	121.0
2α	9.35 s	194.5	9.34 s	194.5
3α	3.38 dd (10.0, 3.5) 3.05 dd (10.0, 10.0)	62.7	3.38 dd (10.0, 4.5) 2.98 dd (10.0, 10.0)	62.9
3′-MeO	3.61 s	56.5	3.61 s	56.5
4′-MeO	3.63 s	56.6	3.61 s	56.6
5-MeO	-	-	3.43 s	61.0
7-MeO	3.81 s	56.8	-	-

Figure 1

Key HMBC and NOESY correlations for 1. HMBC, heteronuclear multiple bond coherence; NOESY, nuclear Overhauser effect spectroscopy.

Compound 2 was identified as 6-hydroxy-4-(3,4-dimethoxyphenyl)-3-hydroxymethyl-5-methoxy-3,4-dihydro-2-napthaldehyde. It was previously isolated from the seeds of V. negundo, though the authors did not provide any spectral data in their approved patent. ²³ Herein, we report its MS, ¹H-, and ¹³C-NMR data and give it a trivial name as vitekwangin B. On the basis of spectroscopic data and comparison with that in literatures, other compounds were identified as 6-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-7-methoxy-3,4-dihydro-2-naphthaldehyde (3) ^15,22 vitedoin A (4) ¹⁶ balanophonin (5) ¹⁷ syringaresinol (6) ¹⁸ nakamurol C (7) ⁹ negundoin E (8) ¹⁰ vitexilactone (9) ¹¹ negunfurol (10) ¹⁴ schensianol A (11) ¹⁴ 4-hydroxy-3-methoxy-cinnamic aldehyde (12) ²⁴ sinapaldehyde (13) ²⁵ chrysosplenol-D (14) ¹² penduletin (15) ¹³ uvaol (16) ²⁶ ursolic acid (17) ²⁷ and erost-6,22-diene-3β,5α,8α-triol (18). ²⁸

Vitex spp belongs to Verbenaceae family in which chemotaxonomic markers are thought to be iridoids and ecdysteroids. ²⁹ Recently, this genus was considered to be segregated from Verbenaceae to Lamiaceae due to their morphological differences and identifications of a large number of diterpenoids and polymethoxyflavonoids. ³⁰ In previous investigations of Vitex plants, iridoids and ecdysteroids were almost identified from their roots bark, stems bark, or leaves. ^20,31 The fruits of Vitex are always used as folk medicine and the predominant constituents of their fruits or seeds were reported to be diterpenoids and polymethoxyflavanoids which have been regarded as specific taxonomic markers of this genus. ³² In this investigation, the diterpenoid (7-9) and polymethoxyflavonoid (14, 15) constituents of V. kwangsiensia are in agreement with those of other members of the genus Vitex and this study reinforced the viewpoint that the secondary metabolites of Vitex fruits mainly consisted of diterpenoids and polymethoxyflavonoids. ^33,34

Lignans are not common secondary metabolites of Vitex species and have ever been isolated from V. trifolia, ³⁵ V. cannabifolia, ³⁶ V. altissima, ³⁷ and V. negundo. ^16,38 Vitex cannabifolia, synonymous V. negundo Linn. var. cannabifolia, was regarded as a variety of V. negundo. There are only minor morphological differences between V. cannabifolia, V. negundo, and V. kwangsiensia. The isolation of lignans 1 to 6 from V. kwangsiensia suggested that these three plants are close species in chemotaxonomy. In fact, the fruits of all these three plants are always used as TCM “Bujingzi” for their anti-inflammatory effects. Before this research, sesquiterpenoids 10 and 11 were isolated only from the fruits of V. negundo. ¹⁴ The occurrence of these two compounds in both V. negundo and V. kwangsiensia further supports the close genetic relationship between these two plants.

The lignans (1-6), diterpenoids (7-9), and sesquiterpenoids (10, 11) were evaluated for their inhibitory activity on the lipopolysaccharide (LPS)-induced NO production using RAW 264.7 cells. The cell viability was determined in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and all test compounds showed no or minor cytotoxicity on RAW 264.7 cells with IC₅₀ greater than 80 µM. At lower concentrations, all the compounds exhibited minor inhibitory effects on NO production (Figures 2 and 3). At higher concentrations, diterpenoids, especially compound 7, exhibited higher activity than lignans (1-6). Under the condition of 40 µM, compound 7 inhibited 51.1% NO production. Compound 7 is the main anti-inflammatory constituent of V. kwangsiensia. In general, lignans from this plant showed lesser inhibitory activity (Figure 2). At the concentration of 40 µM, compounds 5 and 6 exhibited stronger inhibitory activities than compounds 1 to 4, which suggests that the presence of furan ring in lignans may enhance the NO production inhibitory activity. The activity of sesquiterpenoids (10, 11) is weakest in tested compounds.

Figure 2

NO production in RAW246.7 cells treated with lignans 1 to 6. LPS-induced NO production was used for control and represented at 100%. Data are expressed as mean±SD from triplicate experiments. *P < 0.05 and **P < 0.01 vs LPS control group. NO, nitric oxide; LPS, lipopolysaccharide.

Figure 3

NO production in RAW246.7 cells treated with diterpenoids 7 to 9 and sesquiterpenoids 10 and 11. LPS-induced NO production was used for control and represented at 100%. Data are expressed as mean±SD from triplicate experiments. *P < 0.05 and **P < 0.01 vs LPS control group. NO, nitric oxide; LPS, lipopolysaccharide.

Experimental

General Experimental Procedures

Thin-layer chromatography: silica gel GF₂₅₄ precoated-plates (Qindao Haiyang Chemical Group Co., Qingdao, China). Column chromatography: silica gel (SiO₂, 200-300 or 300-400 mesh; Qindao Haiyang Chemical Group Co.) and silica gel C-18 (Merck, Darmstadt, Germany). UV: Agilent 8453E UV-visible spectroscopy system (USA); optical rotation: Gyromat HP polarimeter (Germany); IR spectrum: Thermo Nicolet Avatar-360-ESP spectrophotometer (USA); nuclear magnetic resonance: Bruker AVANCE 500 spectrometer (USA); atmospheric pressure chemical ionization/electrospray ionization-mass spectroscopy: Thermo MSQ Plus LC/MS instrument (USA); in m/z. HR-ESI-MS: LTQ-Orbitrap XL.

Plant Material

The fruits of V. kwangsiensia were collected in September 2011, in the suburbs of Liuzhou, Guangxi Zhuang Autonomous Region of China. The plant was identified by Dr Hong Zhao from the College of Marine Science, Shandong University at Weihai. Voucher specimen (No. VK201109) was deposited at the herbarium in the Laboratory of Botany, College of Marine Science, Shandong University.

Extraction and Isolation

The air-dried and powdered fruits (10 kg) of V. kwangsiensia were extracted with methanol 3 times (7 day each time) at room temperature. The solvent was evaporated under vacuum, the residue (970 g) was suspended in water and partitioned with hexane to remove chlorophylls and essential oil, and then partitioned with CHCl₃. The CHCl₃ extract (210 g) was submitted to silica gel CC, eluted with hexane/acetone (10:1, 5:1, 2:1, 0:1 v/v): fractions (Frs.) 1 to 4. Fr. 1 (with hexane/acetone 10:1; 15 g) was separated by CC eluting with hexane/AcOEt in linear gradient (20:1, 15:1, 8:1, 5:1) to obtain subfractions Fr. 1a-Fr. 1c. Fr. 1a (with hexane/acetone 20:1, 90 mg) was purified by silica gel CC eluting with hexane/AcOEt 5:1 to give 16 (13 mg). Fr. 1b (with hexane/acetone 15:1, 2 g) was purified by CC with petroleum ether/AcOEt gradient elution (20:1, 10:1 v/v) to give compound 17 (245 mg). Fr. 1c (with hexane/acetone 8:1, 500 mg) was subjected to silica gel CC eluted with hexane/acetone 10:1 to afford Fr. 1c-1 and Fr. 1c-2. Fr. 1c-1 (110 mg) was purified by silica gel CC eluted with CHCl₃/AcOEt 15:1 to give 10 (13 mg) and 11 (40 mg). A fraction (15 mg) from Fr. 1c-1 was further purified by ODS eluting with MeOH/H₂O 1.5:1 to give 7 (3 mg). Fr. 1c-2 (110 mg) was isolated by silica gel CC with hexane/acetone elution (40:1, 30:1, 20:1, 10:1 v/v), and further purified by ODS CC, eluted with MeOH/H₂O (1:1, 1.5:1, 2:1, 1:0 v/v) to give 8 (8.5 mg) and 18 (4 mg). Fr. 2 (with hexane/acetone 5:1, 27 g) was separated with petroleum ether/AcOEt (8:1, 5:1, 2:1, 0:1) to give subfractions Fr. 2a-Fr. 2d. Fr. 2a (with petroleum ether/AcOEt 8:1, 250 mg) was purified by silica gel CC with hexane/acetone 18:1 elution to give 9 (38 mg). Fr. 2b (with petroleum ether/AcOEt 5:1, 376 mg) was purified by ODS CC eluting with MeOH/H₂O 2:1 to give 12 (10 mg). Fr. 2c (with petroleum ether/AcOEt 2:1, 60 mg) was submitted to silica gel CC eluting with CHCl₃/MeOH 100:1 to afford 13 (23 mg). Fr. 2d (with petroleum ether/AcOEt 2:1, 140 mg) was isolated by silica gel CC with CH₂Cl₂/AcOEt 15:1 elution, and further purified by ODS CC eluting with MeOH/H₂O 1.5:1 to give 15 (11 mg). Fr. 3 (with hexane/acetone 2:1, 3.8 g) was subjected to a silica gel CC and eluted with petroleum ether/AcOEt (3:1, 2:1, 1:1 v/v) to yield Fr. 3a and Fr. 3b. Fr. 3a (with petroleum ether/AcOEt 3:1, 1.08 g) was fractionated by silica gel CC eluting with hexane/AcOEt 2:3 to yield subfractions Fr. 3a1-Fr. 3a3. Fr. 3a1 (580 mg) was purified by silica gel CC eluting with CHCl₃/MeOH 10:1 to give 2 (27 mg) and 4 (120 mg). Fr. 3a2 (170 mg) was purified by silica gel CC eluting with CHCl₃/MeOH 10:1 to give 5 (70 mg). Fr. 3b (with petroleum ether/AcOEt 2:1, 510 mg) was isolated by silica gel CC with CHCl₃/MeOH (50:1, 20:1 v/v) elution to give 3 (22 mg) and a mixture (150 mg) of 1 and 6. The mixture was purified repeatedly by silica gel CC with CHCl₃/MeOH 50:1 and petroleum ether/AcOEt 3:2 elution to give 1 (25 mg) and 6 (18 mg).

Vitekwangin A: 4-(3,4-Dimethoxyphenyl)-6-Hydroxy-3-(Hydroxy-Methyl)-7-Methoxy-3,4-Dihydronaphthalene-2-Carbaldehyde (1)

Pale yellow gum.

[α]²⁰ D: −71.2 (c 0.025, MeOH).

IR (KBr): 3424 (OH), 1662 (C=O), 1622, 1567, and 1514 (aromatic ring C=C).

UV/Vis λ _max (MeOH) nm (log ε): 225.7 (3.55), 254.5 (3.55), 355.5 (3.42).

¹H- and ¹³C-NMR: Table 1.

APCI-MS: 371.12 ([M+H]⁺).

HR-ESI-MS: 371.1491 ([M+H]⁺ , C₂₁H₂₃O₆ ⁺; calc. 371.1495).

Vitekwangin B: 4-(3,4-Dimethoxyphenyl)-6-Hydroxy-3-(Hydroxy-Methyl)-5-Methoxy-3,4-Dihydronaphthalene-2-Carbaldehyde (2)

Pale yellow gum.

[α] ${\displaystyle {}_D^{20}}$ : −68.2 (c 0.025, MeOH).

¹H- and ¹³C-NMR: Table 1.

APCI-MS: 371.12 ([M+H]⁺).

Cytotoxicity Assays

Cytotoxicity of compounds to RAW264.7 cells was tested by MTT assays similar to our previous research. ³⁹ Briefly, the cells were placed at 5 × 10³ per well in 96-well plates, overnight, and then exposed to tested compounds at given concentrations. DMSO was used as a negative control. After another 24 hours, MTT solutions (5 mg/mL) were added to each well and incubated for 4 hours. Absorbance was recorded at a wavelength of 570 nm. Cytotoxicity was calculated from the plotted results using untreated cells as 100%.

Assay for Inhibition Ability Against LPS-Induced NO Production

The production of NO was assessed by determining the nitrite concentration in supernatants of cultured RAW 264.7 macrophages with an NO assay kit. ³⁹ Cells were seeded in 96-well culture plates, co-incubated with tested compounds, and stimulated with LPS (1 mg/mL) for 24 hours. Aliquots of supernatants were reacted with Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2.5% phosphoric acid) for 5 minutes. The absorption was read at 570 nm. Sodium nitrite (NaNO₂) was used to calibrate the absorption coefficient.