Anti-inflammatory phenolics isolated from Juniperus rigida leaves and twigs in lipopolysaccharide-stimulated RAW264.7 macrophage cells

Abstract

Inflammation is an essential host defense system particularly in response to infection and injury; however, excessive or undesirable inflammatory responses contribute to acute and chronic human diseases. A high-throughput screening effort searching for anti-inflammatory compounds from medicinal plants deduced that the methanolic extract of Juniperus rigida S. et L. (Cupressaceae) inhibited significantly nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Activity-guided fractionation and isolation yielded 13 phenolic compounds, including one new phenylpropanoid glycosides, 3,4-dimethoxycinnamyl 9-O-β-D-glucopyranoside (1). Among the isolated compounds, phenylpropanoid glycosides with p-hydroxy group (2, 4) and massoniaside A (7), (+)-catechin (10), amentoflavone (11) effectively inhibited LPS-induced NO production in RAW264.7 cells.

Keywords::

• Juniperus rigida
• Cupressaceae
• nitric oxide
• inflammation
• RAW264.7

Introduction

While nitric oxide (NO) acts as pivotal messenger and effector in a variety of tissues, it also participates in a number of pathologies, especially in inflammation and sepsis, depending on the relative concentration of NO and the surrounding milieu in which NO is produced¹. Chronic inflammation, an undesirable phenomenon, can ultimately lead to development of inflammatory diseases, such as rheumatoid arthritis, bronchitis, gastritis, multiple sclerosis and inflammatory bowel disease². Macrophages, which are widely distributed in the body, play an important role in inflammation and immune responses by providing an immediate defense against foreign agents prior to leukocyte migration³. Hence, the pharmacological reduction of lipopolysaccharide (LPS)-inducible inflammatory mediators is regarded as an effective therapeutic strategy for alleviating inflammatory conditions caused by macrophage activation.

High throughput screening assay searching for inhibitory compounds on NO production from natural sources using LPS-stimulated RAW264.7 macrophage cells as an in vitro system found that the 80% methanolic extract of the leaves and twigs of Juniperus rigida Sieb. et Zucc. significantly inhibited LPS-induced NO production in RAW264.7 cells. J. rigida is juniper species in Cupressaceae family, which is native to northern China and Korea. The dried berries of this tree, “Dudongsil” have been traditionally used for the treatment of neuralgia and arthritis in Korean folk medicine. Monoterpenes, sesquiterpenes, phenylpropanoid glycosides and flavonoids have been reported as chemical constituents of J. rigida⁴; however, there has been no report on anti-inflammatory constituents of this plant. Thus, we have attempted to investigate active compounds mediate anti-inflammatory effects of J. rigida using bioactivity-guided isolation techniques.

Methods and materials

Plant material

The leaves and twigs of J. rigida were collected in Herbarium of the Medicinal Plant Garden, College of Pharmacy, Seoul National University, Koyang, Korea. The plant was authenticated by Dr. Jong Hee Park, professor of Pusan National University

Extraction and isolation

The air-dried plant material (10.1 kg) was extracted three times with 80% MeOH in an ultrasonic apparatus. Removal of the solvent in vacuo yielded a methanolic extract (971.3 g). The methanolic extract was then suspended in distilled water and partitioned successively with n-Hexane, CHCl₃, EtOAc and n-BuOH. The EtOAc soluble fraction (53.5 g) and n-BuOH soluble fraction (120.0 g), which showed the potent inhibitory activity on NO production in RAW264.7 cells, were used to elucidate bio-active compounds. The EtOAc fraction was subjected to silica gel column using mixtures of CHCl₃−MeOH of increasing polarity as eluents to give 7 fractions (EI–VII). The EII was subjected to repeated ODS gel column chromatography with a gradient elution of MeOH−Water yielding compound 8 (32 mg). The EIII was also applied to ODS gel column chromatography with a gradient elution of MeOH−Water to give 16 sub-fractions (EIII-1–16). Compound 3 (15 mg) was obtained from EIII-9 by additional C₁₈ RP HPLC (MeOH−H₂O 40:60, 2.0 mL/min, 254 nm). Compound 11 (21.5 mg) was isolated from EIII-16 by recrystallization with MeOH. The EIV was subjected to ODS gel column chromatography with a gradient elution of MeOH−Water to give 11 sub-fractions (EIV-1–11). Compounds 10 (842 mg), 2 (574 mg), 5 (698 mg) and 9 (12.6 mg) were obtained from EIV-2, EIV-6, EIV-8 and EIV-11, respectively, by additional C₁₈ RP preparative LC (MeOH−H₂O 40:60, 4.0 mL/min, 220 nm). The EVII was applied on ODS gel column eluting with a gradient of MeOH−Water to yield 5 sub-fractions (EVII-1–5). Compounds 4 (22.3 mg) and 13 (12.7 mg) were obtained from EVII-2 through RP C₁₈ preparative LC (MeOH−H₂O 75:25, 4.0 mL/min, 220 nm). Compounds 6 (19.5 mg) and 7 (39.2 mg) were obtained from EVII-5 by additional RP C₁₈ HPLC (MeOH−H₂O 40:60, 2.0 mL/min, 254 nm).

The n-BuOH (120 g) fraction was subjected to column chromatography on Sephadex LH-20 (MeOH) to give 8 fractions (BI−VIII). Silica gel column chromatography of BV with a gradient elution of CHCl₃−MeOH afforded 13 sub-fractions (BV-1−13). Compound 1 (8 mg) was isolated from BV-3 by additional RP C₁₈ preparative LC (MeOH−H₂O 50:50, 4.0 mL/min, 220 nm). BV-13 was further purified by recrystallization with MeOH to yield compound 13 (1104 mg).

3,4-dimethoxycinnamyl 9-O-β-D-glucopyranoside (1): Colourless amorphous powder, HRESIMS (positive) m/z 356.1468 [M+Na]⁺; [α]²⁵_D −30.2° (c 1.0, MeOH); IR (KBr) v_max cm⁻¹: 3397, 1514, 1264, 1159, 1076, 1024, 621; 1H-NMR (500 MHz) δ 7.04 (1H, d, J = 1.8 Hz, H-2), 6.95 (1H, d, J = 1.8, 8.2 Hz, H-6), 6.88 (1H, d, J = 8.4 Hz, H-5), 6.60 (1H, d, J = 15.9 Hz, H-7), 6.24 (1H, dt, J = 6.6, 15.9 Hz, H-8), 4.49 (1H, dd, J = 5.4, 12.4 Hz, H-9a), 4.35 (1H, d, J = 7.7 Hz, H-19), 4.30 (1H, dd, J = 5.6, 13.7 Hz, H-9b), 3.89–3.81 (1H, m, H-69), 3.83 (3H, s, 3-OMe), 3.81 (3H, s, 4-OMe), 3.74–3.63 (1H, m, H-69), 3.33-3.18 (4H, overlapped, H-29, 39, 49, 59); ¹³C-NMR (500 MHz) δ 150.6 (C-3), 150.5 (C-4), 137.1 (C-1), 133.9 (C-7), 124.8 (C-8), 121.2 (C-6), 112.9 (C-5), 110.8 (C-2), 103.3 (C-19), 78.2 (C-39), 78.1 (C-59), 75.2 (C-29), 71.8 (C-49), 71.0 (C-9), 62.9 (C-69), 56.5 (3-OMe, 4-OMe).

Cell cultures

The RAW264.7 macrophage cells were obtained from Korea Cell Line Bank (Seoul, Korea). The cell line was maintained in DMEM containing 20mM HEPES, 2mM L-Glutamine, 10% FBS with penicillin (100 IU/mL) and streptomycin (10 mg/mL) at 37°C in a humidified atmosphere of 95% air-5% CO₂.

Determination of NO content

The RAW264.7 cells were seeded in 48 well plates (1 × 10⁵ cells/well) and incubated at 37°C for 24 h. Then, the cell culture was washed and the medium was replaced with phenol red free medium to remove any trace of phenol red, and treated with sample to be tested for 1 h before exposure to 0.1 μg/mL of LPS. After 24 h incubation, nitrite concentration in culture medium was measured to assess NO production in RAW264.7 cells using Griess reagent. The absorbance at 550 nm was measured on a microplate reader and the concentration was determined using nitrite standard curve.

Estimation of cell viability

After 100 μL of sample aliquot was collected for Griess assay, MTT (0.2 mg/mL) was directly added to cultures followed by incubation at 37°C for 2 h. The supernatant was then aspirated and 100 μL of DMSO was added to dissolve the formazan. After insoluble crystals were completely dissolved, absorbance at 540 nm was measured using a microplate reader. Data were expressed as percentage cell viability relative to control cultures.

Results and discussion

The 80% methanolic extract of J. rigida leaves and twigs was suspended in water and successively partitioned with n-Hexane, CHCl₃, EtOAc and n-BuOH. Each fraction was evaluated for its inhibitory activity on NO production in LPS-stimulated RAW264.7 cells. The EtOAc and n-BuOH soluble fractions effectively attenuated LPS-induced NO production in RAW264.7 cells without cytotoxicity at the concentration ranging from 10μg/mL to 100μg/mL (Figure 1A and B). These two fractions were further subjected to repeated column chromatography to elucidate inhibitory compounds on NO production. Thirteen compounds, including six phenylpropanoid glycosides (1−6), three lignan glycosides (7−9), three flavonoids (10−12) and coumarin (13) were obtained.

Figure 1.  Inhibitory effects on NO production and cytotoxicity of total extract and each fraction (A and B), and compounds 1−13 (C and D) isolated from J. rigida leaves and twigs in LPS-stimulated RAW264.7 cells. The RAW264.7 cells were pretreated with each sample to be tested for 1 h before exposure to LPS for 24 h. The concentration of nitrite in culture medium was measured by Griess reaction and sodium nitrite was used as a standard. The values shown are mean ± SD of three independent experiments. The results differ significantly from LPS-only treated; *p < 0.01, **p < 0.001.

Compound 1 was obtained as colourless amorphous powder with [α]²⁵_D −30.2 (c 0.1, MeOH). The molecular formula of 1 was determined as C₁₇H₂₄O₈ from [M+Na]⁺ ion peak at m/z 356.1468 (calc. 356.1471) in HRESIMS. The ¹H-NMR spectrum of 1 displayed signals due to aromatic protons of ABX system [δ_H 7.00 (1H, d, J = 1.7 Hz, H-2), 6.84 (1H, dd, J = 1.7, 8.2 Hz, H-4), 6.72 (1H, d, J = 8.0 Hz, H-5)], (E)-3-hydroxy-1-propenyl group [δ_H 6.56 (1H, d, J = 15.8 Hz, H-7), 6.18 (1H, dt, J = 6.5, 15.8 Hz, H-8), 4.48 (1H, dd, J = 5.8, 12.4 Hz, H-9), 4.28 (1H, dd, J = 6.8, 12.3 Hz, H-9) and two methoxy groups. The signals characteristic for glucose unit were also found in ¹H and ¹³C-NMR spectrum. Comparison of NMR spectra of 1 with the literature values previously reported indicated that 1 was similar to citrusin D except for the presence of an additional methoxy group. From the correlations in NOSEY spectrum between H-5/4-OMe and H-2/3-OMe, it was deduced that two methoxyl groups are located at C-3 and C-4. On the basis of above data, compound 1 was determined as 3,4-dimethoxycinnamyl 9-O-β-D-glucopyranoside.

Twelve known compounds were also identified as citrusin D (2)^4,5, juniperoside (3)^5,6, sachaliside I (4)^5,6, rosin (5)⁵, rosarin (6)⁶, massoniaside A (7)^7,8, isothujastadin (8)^9,10, (+)-pinoresinol 4-O-β-D-glucopyranoside (9)^11,12, (+)-catechin (10)^4,13, amentoflavone (11)^14,15, quercetin 3-O-α-L-rhamnopyranosyl (1-–6-)-β-D-glycopyranoside (12)^4,8, skimmin (13)^6,16, respectively, by comparison of spectroscopic data with the literature values. Compounds 3−9 and 13 were reported for the first time from this plant.

Inhibitory effects of compounds 1−13 on LPS-induced NO production in RAW264.7 cells were evaluated using the Griess reaction described in Methods (Figure 1C and D). The increased concentration of nitrite in the medium induced by LPS was effectively reduced by pretreatment of compounds 2, 4, 7, 10 and 11. Among the phenylpropanoid glycosides 1–6, compounds 2 and 4 only showed the inhibitory effects on NO production in LPS-stimulated RAW264.7 cells, while the others were inactive. Naturally occurring phenylpropanoid glycosides have been received much attention for its antioxidant, anti-tumour, antivirus, anti-inflammatory, hepato-protective, immune-suppressive and neuro-protective activities. The current studies demonstrated that pharmacological activities and action mechanisms of phenylpropanoid are closely related to their structure as well as the location and number of substituents attached. Ethyl cinnamate and structurally related esters were found to be more potent than the corresponding carboxylic acids as antifeedants¹⁷ and even small changes in the structure of benzoate derivatives induced dramatic effects in the antifeedant activity for H. abietis¹⁸. Phenylpropanoids from Scrophularia buergeriana showed entirely different activity in primary cultures of rat cortical cells speculating that α, β-unsaturated ester moiety and the p-methoxy group might be important for their neuro-protective activity¹⁹. In inhibitory activity of phenylpropanoids on NO production herein, compounds 2 and 4 which possess p-hydroxy group only showed the activity, while the others were almost inactive. At the concentration of 100 μM, the NO production was decreased to 40.1 ± 2.3% and 44.5 ± 3.1% of control level by the pretreatment of compounds 2 and 4, respectively. Structure–activity relationship study applying the related diverse derivatives might be needed to elucidate the key moiety in phenylpropanoid for inhibitory activity on NO production.

Massoniaside A (7), (+)-catechin (10) and amentoflavone (11) also showed the inhibitory activities on LPS-induced NO production, which was reduced to about 50% of control level at the concentration of 100 μM. The L-Nitro-Arginine Methyl Ester (L-NAME), the NOS inhibitor, was used as positive control. To verify whether the reduced cell numbers which were caused by the cytotoxicity of these compounds resulted in decrease of NO production, cell viability was measured employing MTT assay. Cell viability was not significantly altered by the treatment of compounds 1–13 at the concentrations used.

Declaration of interest

The authors report no declarations of interest. This research was supported by a grant (2011K000290) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Education, Science and Technology, the Republic of Korea.