In vitro neuroprotective activity of sesquiterpenoids from the flower buds of Tussilago farfara

Abstract

We have isolated four sesquiterpenoids from Tussilago farfara, a traditional herbal medicine in Korea and China, and investigated the protective effects on LPS-induced neuronal cell death. Four sesquiterpenoids inhibited the production of nitric oxide, prostaglandin E₂ and tumor necrosis factor-α in LPS-treated BV-2 cells through the inhibition of NF-κB pathway. These sesquiterpenoids also inhibited reactive oxygen species (ROS) generation in LPS-treated BV-2 cells. Furthermore, they inhibited LPS-induced neuronal cell death in co-culture system through the inhibition of NF-κB pathway and scavenging of ROS. These results indicated that sesquiterpenoids from Tussilago farfara may have beneficial therapeutic potential for the treatment of neurodegenerative diseases through inhibition of microglial activation.

• Neuroprotection
• sesquiterpenoids
• Tussilago farfara

Introduction

Microglia is the principal immune defense system in the brain1–3. As an immunocompetent cells in the central nervous system (CNS), microglia is activated by immunogenic reactions such as infection, inflammation, trauma, ischemia and neurodegeneration in the CNS4^,5. Under these pathological conditions, activated microglia releases cytotoxic factors including nitric oxide (NO), pro-inflammatory cytokines and reactive oxygen species (ROS), and induce neuronal cell injuries and death6^,7. These reactions, as the processes of microgliosis, implicate in neurological disorders such as Alzheimer’s disease and Parkinson’s disease4–6. Activation of microglia is mediated by various transcription factors and protein kinases. For example, lipopolysaccharide (LPS) activates toll-like receptors and increases the transcriptional activity of nuclear factor-κB (NF-κB) and activating protein-1 (AP-1), and also increases the release of pro-inflammatory molecules such as NO, prostaglandin E₂ (PGE₂) and cytokines, through upregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-25^,8^,9. Thus, inhibition of microgliosis induced by activated microglia may be a good strategy to protect neuronal cell from harmful stresses.

Tussilago farfara is an herbaceous perennial plant and wildly grows in Asia, America and Europe. The flower bud of Tussilago farfara has been used as an important traditional medicine in Korea and China to treat cough and wheezing10. The extracts of Tussilago farfara have several biological activities including anti-inflammatory, antioxidant, antimicrobial and anti-colon cancer activities11–14. Previously, we have reported that tussilagone, one of the major components of Tussilago farfara, suppresses iNOS and COX-2 expression in LPS-stimulated BV-2 microglial cells15. In this study, we have isolated four anti-inflammatory principles (compounds 1–4) including tussilagone (compound 4) from this plant, and investigated the protective effects on LPS-induced microgliosis.

Materials and methods

Reagents

Tussilago farfara was purchased from Kyungdong traditional herbal market in Seoul, Korea in March 2012. COX-2 antibody was purchased from Cayman Chemical Co. (Ann Arbor, MI); iNOS antibody from Transduction Laboratories (Lexington, KY); Iκ-B antibody from Santa Cruz Biotechnology (Santa Cruz, CA) and β-actin antibody from Sigma-Aldrich (St Louis, MO). Horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) antibody and horseradish peroxidase-conjugated anti-mouse IgG antibody were purchased from Assay Designs (Ann Arbor, MI). Enhanced chemiluminescence (ECL) detection system was purchased from GE Healthcare Life Science (Uppsala, Sweden). LPS, 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA-AM), nerve growth factor (NGF), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and other chemical reagents were purchased from Sigma-Aldrich.

Extraction and isolation

The flower buds of Tussilago farfara (1.2 kg) was extracted with methanol (3 × 2 L) to yield crude methanol extract (250 g). The extracts were suspended in water and successively partitioned with EtOAc and BuOH. The EtOAc soluble fraction was followed by activity-guided chromatography for the purification of active principles. The EtOAc fraction (34 g) was subjected to CC (SiO₂, 70–230 mesh, 1500 g) eluting with a gradient mixture of n-hexane: EtOAc (70:1–1:1, 4 L each) to give 15 fractions. Fraction 6 (1.9 g) was chromatographed on reverse phase (RP)-C18 column (40–63 μm, 3 × 25 cm) with a gradient elution (60–90% MeOH, 500 mL each) to give Fraction 6–4 (1.2 g) that was further separated by CC (SiO₂, 230–400 mesh, 120 g) eluting with n-hexane:EtOAc (80:1–5:1, 1000 mL each) as eluent to give compound 1 (135.3 mg). Another Fraction 9 (1.5 g) was chromatographed on RP-C18 column (40–63 μm, 3 × 25 cm) with a gradient elution (60–90% MeOH, 500 mL each) to give Fraction 9–8 (611 mg). Fraction 9–8 was further purified by semi-preparative high-performance liquid chromatography (HPLC) (μ-Bondapak C18 column, 10 × 300 cm, 75% MeOH, 2 mL/min, 254 nm) to yield compound 2 (15 mg) and compound 3 (9 mg). Fraction 11 (3.7 g) was further separated by RP-C18 column (40–63 μm, 3 × 25 cm) with a gradient elution (50–90% MeOH, 500 mL each) to give Fraction 11–6 (143.2 mg) that was further purified by semi-preparative HPLC (μ-Bondapak C18 column, 10 × 300 cm, 90% MeOH, 2 mL/min, 254 nm) to yield compound 4 (36.2 mg).

Cell culture

Murine microglial cell line (BV-2) was kindly provided by Prof. Hee-Sun Kim at Ewha Women’s University, Korea. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 2 mM glutamine, 1 mM pyruvate, penicillin (100 U/mL) and streptomycin (10 μg/mL) at 37 °C in a humidified incubator with 5% CO₂. Rat neuron-like pheochromocytoma cell line, PC-12 cells were obtained from American Type Culture Collection (ATCC), and cultured in DMEM containing 5% fetal bovine serum, 10% horse serum, 2 mM glutamine, 1 mM pyruvate, penicillin (100 U/mL) and streptomycin (10 μg/mL) at 37 °C in a humidified incubator with 5% CO₂.

Nitrite assay

NO released from BV-2 cells was measured as described in previous study11 by the determination of nitrite () concentration in culture supernatant. Samples (100 μL) of culture media were incubated with a 150 μL of Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine in 2.5% phosphoric acid solution) at room temperature for 10 min in 96-well microplate16. Absorbance at 540 nm was read using an enzyme-linked immunosorbent assay (ELISA) plate reader (Molecular Devices, Sunnyvale, CA). Standard calibration curve was prepared using sodium nitrite as standard.

Measurement of TNF-α and prostaglandin E₂

TNF-α levels were measured with a sandwich ELISA. Hundred microliters of supernatant of culture medium were put into the 96-well plate which was coated with Hamster anti-murine TNF-α monoclonal antibody as the capture antibody. The plate were incubated for 90 min at room temperature, and washed with phosphate-buffered saline (pH 7.4) containing 0.05% Tween-20. And then, 100 μL of polyclonal rabbit anti-murine TNF-α antibody was added to each well as the second antibody. For detection, a peroxidase-conjugated goat anti rabbit antibody was added, followed by the addition of substrate for peroxidase (1 mg/mL of p-nitrophenylphosphonate). The absorbance at 405 nm was read using a microplate reader. The concentration of TNF-α were calculated from a standard curve generated with recombinant murine TNF-α. The accumulated PGE₂ in culture media were determined using ELISA kit from Cayman Chemical Co. according to the manufacturer’s instruction. A standard curve was prepared simultaneously with PGE₂ standard ranging from 0.06 to 6 ng/mL.

Measurement of ROS generation

BV-2 cells were incubated with LPS (0.1 μg/mL) and test compounds for 18 h. The generation of ROS was measured by fluorescence-activated cell sorting (FACS) analysis after incubation with DCF-DA-AM.

Western blot analysis

The treated cells were rinsed with phosphate-buffered saline and lysed by boiling with RIPA lysis buffer [1% sodium dodecyl sulfate (SDS), 1.0 mM sodium vanadate, 10 mM Tris, pH 7.4]. The protein of cell lysates was run on SDS–polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membrane by the standard method. The membrane was probed with antibodies against mouse iNOS, COX-2, Iκ-Bα and actin. The Western blot was visualized using an ECL detection kit according to manufacturer’s instruction.

Reverse-transcription polymerase chain reaction

BV-2 cells were treated with and without test samples for 4 h together with LPS (0.1 μg/mL). The whole RNA was isolated with TRIzol® reagent (Invitrogen, Carlsbad, CA). Each RNA extract (2 μg) was reverse transcribed into cDNA using superscript III reverse transcriptase (Invitrogen). Reverse-transcription polymerase chain reaction (RT-PCR) was performed in 25 μL of a solution containing Go Taq™ DNA polymerase (Promega, Madison, WI), primers and cDNA. The sense and antisense oligonucleotides for iNOS were 5′-CCCTTCCGAAGTTTCTGGCAGCAGC-3′ and 5′-GGCTGTCAGAGCCTCGTGGCTTTGG-3′, respectively. The sense and antisense primers for COX-2 were 5′-CCAGATGCTATCTTTGGGGAGAC-3′ and 5′-TTGCATTGATGGTGGCTG-3′, respectively. The sense and antisense primers for β-actin were 5′-TGTGATGGTGGGAATGGGTCAG-3′ and 5′-TTTGATGTCACGCACGATTTCC-3′, respectively. After pre-incubation for 3 min at 94 °C, 26 cycles of amplification cycles were performed for iNOS and β-actin, and 30 cycles for COX-2. Each PCR product was run on 2% agarose gel and stained with ethidium bromide.

Neuronal differentiation and co-culture

To investigate the protective effects of active compounds, we used co-culture model of microglia and neuronal cells17. PC-12 cells were plated with 1000 cells/well into 48-well plate and media was changed into the differentiation media (0.5% horse serum, 100 ng/mL NGF) after 18 h incubation. The differentiation media were changed every 2 days and incubated for 1 week.

BV-2 cells were seeded at 3 × 10⁴ cells/well in 48-well plates and treated with indicated concentrations of samples in the presence of LPS for 18 h. The supernatant was collected as conditioned media and immediately transferred to the differentiated PC-12 cells and incubated for 18 h in the presence of NGF (100 ng/mL). To normalize the effect of LPS on neuronal cell viability, 0.1 μg/mL LPS was directly added to differentiated PC-12 cells as a control. Cell viability was evaluated by MTT assay.

Statistics

The results were expressed as mean ± standard deviation (SD) of three experiments, and statistical analysis was performed by Student’s t-test, and a p value of <0.01 was considered to be significantly different.

Results and discussion

We isolated four sesquiterpenoids (1–4) from ethyl acetate soluble fraction of Tussilago farfara by activity-guided purification procedures. Their purities were determined by HPLC analysis as higher than 95%. Their structures were identified as 7β-(3′-ethyl-cis-crotonoyloxy)-1α-(2′-methylbutyryloxy)-3,14-dehydronotonipetranone (compound 1), 14-acetoxy-7β-(3′-ethyl-cis-crotonoyloxy)-1α-(2′-methylbutyryloxy)-notonipetranone (compound 2), 1α,8-bisangeloyloxy-3β,4β-epoxy-bisabola-7(14),10-diene (compound 3) and 14-acetoxy-7β-(3′-ethyl-cis-crotonoyloxy)-notonipetranone (compound 4, tussilagone) by MS and NMR spectroscopic data analyses and by comparing with reported data (Figure 1A)15^,18–20. These compounds purified from Tussilago farfara inhibited the NO production in LPS-activated BV-2 cells (Figure 1B). The concentration required for inhibiting the NO production by 50% (IC₅₀ value) was calculated on the basis of concentrations of nitrite released into culture media. The IC₅₀ values of compounds 1–4 were 3.4, 3.8, 7.9 and 8.7 μM, respectively.

Figure 1. Isolation of sesquiterpenoids (compounds 1–4) from Tussilago farfara. (A) The structures of sesquiterpenoids isolated from Tussilago farfara. (B) NO was measured in LPS-treated BV-2 cells after treatment of test compounds from Tussilago farfara. **p < 0.01 compared with LPS-treated group (med: media).

To further investigate the neuroprotective effects of sesquiterpenoids, we evaluated their effects on the production of several neurotoxic factors in LPS-treated BV-2 cells. LPS treatment significantly increased the level of TNF-α and PGE₂ in BV-2 cells. Treatment of test compounds significantly inhibited the production of TNF-α and PGE₂ (Figure 2A and B). LPS treatment increased TNF-α level from 2.40 (control) to 25.09 ng/mL, and treatment of compounds 1–4 reduced LPS-induced TNF-α to 9.88, 9.95, 11.58 and 15.12 ng/mL, respectively. And PGE₂ was increased from basal level 0.12 to 8.20 ng/mL by treatment of LPS, while treatment of compounds 1–4 reduced LPS-induced PGE₂ to 0.41, 0.87, 2.33 and 1.73 ng/mL, respectively. Compounds 1–4 also significantly inhibited the LPS-induced ROS generation in BV-2 cells Figure 2C). LPS treatment induced ROS in 46.89% of cells, while co-treatment of compounds 1–4 induce ROS in 3.69, 6.18, 19.04 and 28.89% of total cells, respectively. Compounds 1–4 (10 μM) were not cytotoxic against BV-2 cells as shown in Figure 2(B). Taken together, these four compounds inhibit generation of neurotoxic factors in BV-2 cells but not affect cell numbers.

Figure 2. The inhibitory effects of sesquiterpenoids (compounds 1–4) on LPS-induced production of cytotoxic factors in BV-2 cell. (A) TNF-α was measured by the ELISA after treatment of test compounds in BV-2 cells. (B) PGE₂ was measured by ELISA with the treatment of test compounds in LPS-activated BV-2 cells. (C) ROS generation was measured by FACS after treatment of test compounds in BV-2 cells. (D) BV-2 cell viability was measured by MTT after treatment of test compounds (10 μM) for 24 h. Values are mean ± SD of three experiments. **p < 0.01 compared with LPS-treated group (med: media).

To investigate the mechanism of sesquiterpenoids for the inhibitory production of LPS-induced neuronal cytotoxic factors, we have determined the level of iNOS, COX-2 and Iκ-Bα in LPS-activated BV-2 cells. Treatment of 10 μM of each compounds decreased both protein and mRNA expression of iNOS and COX-2 (Figure 3A and B). The gene expression of iNOS and COX-2 can be modulated by NF-κB which can be activated by the degradation of Iκ-Bα through phosphorylation. Iκ-Bα was fully degraded by 30-min exposure of LPS (0.1 μg/mL) and followed by the recovery in microglia. Treatment of four compounds suppressed the degradation of Iκ-Bα in LPS-stimulated BV-2 cells (Figure 3C) and this might be the mechanism for their suppression of iNOS and COX-2 expression and the production of NO and PGE₂.

Figure 3. The inhibitory effects of sesquiterpenoids (compounds 1–4) on the expression of iNOS and COX-2 in LPS-activated BV-2 cells. (A) The protein level of iNOS and COX-2 were measured by Western blotting. (B) The mRNA level of iNOS and COX-2 were measured by RT-PCR. (C) The protein level of Iκ-B was measured by Western blotting.

To investigate whether sesquiterpenoids protect neuronal cell death, we used conditioned media in neural and microglial co-culture system. The cell viabilities of PC-12 cells were suppressed by the treatment of LPS-conditioned media from BV-2 cells, but not by direct treatment of LPS (Figure 4). The treatment of compounds 1–4 (10 μM) significantly protected the PC-12 cells against LPS-conditioned media induced toxicity. The respective NF-κB inhibitor and ROS scavenger, NDGA and Trolox also protected cell death as shown in Figure 4. These results indicate that compounds 1–4 suppress the neuronal cell death in co-culture systems without changing microglia cell viability. Compounds 1, 2 and 4 share same backbone structure, but compounds 1 and 2 inhibit more efficiently the production of cytotoxic factors including ROS, NO, TNF-α and PGE₂ (Figures 1 and 2) and the expression of iNOS and COX-2 (Figure 3). This difference of efficacy might come from the additional substituent of 2′-methylbutyryloxy at 1α-position of ring B. The substitution of 14-acetoxy group of compounds 2 and 4 might have no effect on the activity (Figure 1A). Even though compound 3 has two angeloyloxy substituents at 1α and 8, it showed weaker activity than compounds 1 and 2. This may suggest that ring opening is not favorable for the biological activity. These findings would be very informative in the process of structural optimization for the drug development, even though the information was derived from very limited structures.

Figure 4. The inhibitory effect of sesquiterpenoids (compounds 1–4) on microglia-induced neuronal cell death. Cell viability of PC12 cells was measured by MTT assay after treatment of conditioned media from BV-2 cells prepared as described in material and methods. The viability was represented as the fold of media control of PC12 cells. **p < 0.01 compared with LPS-treated group.

Tussilago farfara, an important traditional medicine in Korea and China has been reported to have antioxidant, antimicrobial and anti-colon cancer activities11–14. In previous study, we have reported that Tussilago farfara inhibits NO synthesis in LPS-stimulated macrophages13. We also reported that the extracts of Tussilago farfara protect neurons from the damages induced by arachidonic acid, spermine NONOate, A_β, glutamate, N-methyl-d-aspartic acid, H₂O₂, xanthine/xanthine oxidase and Fe²⁺/ascorbic acid through inhibition of NO generation and scavenging free radical11^,13. Tussilagone (compound 4), one of major components of Tussilago farfara, suppresses iNOS and COX-2 expression in LPS-stimulated BV-2 microglial cells through inhibition of NF-κB activation15. In the present study, we found four sesquiterpenoids from Tussilago farfara that exhibit protective effects against LPS-induced neuronal cell death (Figure 4) through the inhibition of NF-κB activation. However, the exact activity mechanism and their structural optimization should be further investigated for the successful drug development for neurodegenerative diseases.

In conclusion, four sesquiterpenoids from the flower buds of Tussilago farfara have inhibitory effects on the production of ROS, NO, TNF-α and PGE₂ in activated microglia. They exhibit cytoprotective activity through the inhibition of I-κBα degradation and subsequent inhibition of iNOS and COX-2 expression. Taken together, Tussilago farfara may have beneficial therapeutic potential for neurodegenerative diseases through the inhibition of microgliosis.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2011-0030074) and the SRC Research Center for Women’s Diseases of Sookmyung Women’s University (2011).