Evaluation of an Aqueous Extract from Horseradish Root (Armoracia rusticana Radix) against Lipopolysaccharide-Induced Cellular Inflammation Reaction (2024)

2.1. Materials

Lymphoprep™ gradient was purchased from Progen (Heidelberg, Germany). RPMI 1640 medium, fetal calf serum, trypsin 10x (25 mg/mL), trypsin-EDTA 10x (5 mg/mL trypsin and 2.2 mg/mL EDTA), and phosphate buffered saline (PBS, without Ca and Mg) were from PAA Laboratories Gmbh (Coelbe, Germany). L-Glutamine, penicillin, and streptomycin were from Invitrogen (Karlsruhe, Germany). Camptothecin was from Tocris (Eching, Germany); Triton-X 100, milk powder, and N,N,N′,N′-tetramethyl-1-,2-diaminomethane (Temed) were from Carl Roth (Karlsruhe, Germany); DMSO, lipopolysaccharide (LPS), phorbol, 12-myristate 13-acetate, CFSE, ammonium persulfate, bovine serum albumin, ethanol absolute, hydrochloric acid (37%), leupeptin hemisulfate, p-coumaric acid, pepstatin A, Ponceau S, trypan blue, Tween 20, and ionomycin were from Sigma-Aldrich (Taufkirchen, Germany).

Antibodies against p-38 (Thr180/Tyr182), p-ERK1/2 (Thr202/Tyr204), p-JNK, and p-c-Jun (Ser73) and the horseradish peroxidase- (HRP-) labelled secondary antibodies, anti-mouse and anti-rabbit, were from Cell Signaling Technology (Boston, USA); anti-human COX-2 was from R&D Systems (Wiesbaden, Germany); anti-human COX-1 and anti-human 5-LOX (mouse monoclonal, clone 33) were from Santa Cruz Biotechnology (Heidelberg, Germany); and mAb against β-actin was from Sigma-Aldrich (Taufkirchen, Germany).

2.2. Isolation of Human PBMC and Cell Culture

The study was approved by the Ethical Committee of the University of Freiburg, Germany (EK-Freiburg: 597/14). Human PBMC were obtained with written consent from volunteers according to the guidelines of the local ethics committee. PBMC were isolated by centrifugation on a Lymphoprep gradient (density: 1.077 g/cm³, 20 min, 500 ×g). Cells were washed twice with prewarmed PBS and cell concentration and viability were determined using trypan blue. Fresh RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin was added to PBMC (2 × 10⁶ cells/mL). Cells were treated either with solvent (10% water or 0.1% DMSO) or with aqueous plant extracts, washed twice with prewarmed PBS, and subsequently stimulated with 1 μg/mL LPS and/or 1 μM fMLP for different time points at 37°C in a humidified incubator with 5% CO₂/95% air atmosphere. Subsequently, cells were washed with PBS and used for the bioassays as described below.

2.3. Plant Powder Extraction

Standardized lyophilized plant powder of the root of horseradish (Armoracia rusticana Radix, batch number PN19869) that is used in pharmacological remedy was provided by Repha GmbH (Langenhagen, Germany). Plant powder extraction was done as described before [10]. Briefly, one gram of plant powder was mixed with 10 mL double-distilled water or DMSO and directly incubated at 37°C for 30 min at 100 rpm. For the water extract, only glassware like vials, fennels, and flasks was used. The extract was strained through gauze and sterile filtered using a Millex syringe driven filter unit, 0.2 μm (Merck Millipore, Darmstadt, Germany), and 6 serial dilutions were prepared in a 1 : 3 ratio. The initial concentration of the extracts was 33.33 mg/mL. 1 mL PBMC (1 × 10⁶ cells) suspension, prepared in a 24-well plate, was treated with 10 μL of water extract. For the samples exposed to DMSO extract, 1 μL was used in 1 mL cell suspension. The final concentration of DMSO in the cell suspension did not exceed 0.1%.

2.4. Separation of Plant Extracts Using Preparative HPLC

The separation of aqueous plant extracts using preparative HPLC was described before [10].

2.5. Nontargeted Analysis of Nonvolatile Metabolites by UHPLC-QToF-MS

The metabolites from 25 mg of finely powdered tissue were extracted with 250 μL water at 37°C for 30 min. After this period, a defined volume of 70% methanol (70°C) (Carl Roth GmbH and Co. KG, Karlsruhe, Germany) was added. An aliquot of 100 μL was used for the nontarget analysis of the metabolites [13]. In addition, fractions H1–H4 were analyzed concerning their metabolite profile. The aqueous plant extracts were filtered through a 0.2 μm PTFE membrane (Amchro GmbH, Hattersheim, Germany) and analyzed with a 1290 Infinity UHPLC coupled with an Agilent 6250 QToF LC-MS (Agilent Technologies GmbH, Germany). Samples (5 μL) were injected into a C18 column (2.1 × 50 mm, 1.8 μm; Agilent Zorbax Entend-C18-Rapid Resolution HT). Column and sample temperatures were kept at 30°C and 10°C, respectively. The chromatographic gradient (solvent A: 0 min 98%, hold 3 min, 15 min 15%, 18 min 0%) was composed of 2 solutions (solvent A, 0.01% aqueous formic acid; solvent B 0.01% formic acid in acetonitrile) that were used to elute the compounds at a flow rate of 0.4 mL/min. An electron spray (ESI) source was used and spectra were collected in positive and negative ionization mode (acquisition rate: 1 spectrum/s) over a 100 to 1700 m/z range (capillary voltage: 3.5 kV; source temperature: 300°C; nebulizer gas flow: 8 L/min at 35 psi, skimmer 65 V; fragmentor voltage: 175 V). For MS/MS experiments, spectra were collected over a 100 to 1700 m/z range for MS and 50 to 900 m/z for MS/MS selecting a maximum of 3 precursor ions per cycle. The collision energy was ramped with a slope of 4 and offset of 6. The raw data from the UHPLC-QToF-MS analysis were converted and processed by Mass Profiler Professional (MPP; Version 13.1, Agilent Technologies) using molecular feature extraction (MassHunter B.07.00) with the following settings: small molecules, minimum 500 counts for feature extraction and 5 000 counts for MPP analysis, ion species [M + H]⁺, [M − H]⁻, [M + Na]⁺, [M + NH₄]⁺, [M + HCOO]⁻, and H₂O as neutral loss, 15 ppm extraction width, and a quality score based on mass accuracy, isotope abundance, and isotope spacing of 80%. Raw data files were imported to MPP for recursive workflow. The formulas were then generated using the abovementioned ions and neutral loss with match tolerance of 20 ppm and 0.2 min.

The compounds were identified tentatively by comparing the mass spectra with MassHunter METLIN PCDL (Agilent, 79609 compounds) and public available databases for nonvolatile compounds.

2.6. Analysis of the Glucosinolate Profile of the Extract

The compounds were analyzed as previously reported [14].

2.7. Protein Analysis by Immunoblotting

Analysis of proteins by immunoblotting was performed as described before [16]. In brief, 15 μg of protein was mixed with SDS-containing sample buffer and loaded onto acrylamide gel. Proteins were then transferred to a nitrocellulose membrane (Hybond ECL, GE Healthcare Life Sciences, Freiburg, Germany) using wet blotting (0.7 mA/cm²,  90 min). Membrane was blocked with 5% low fat milk in TBS/Tween 0.1% and incubated with primary antibody diluted with 5% low fat milk in TBS/Tween 0.1% or 5% BSA in TBS/Tween 0.1% for 1 h at RT or overnight at 4°C. Afterwards, horseradish peroxidase labelled secondary antibodies were incubated for 1 h at RT. Then, the signals were detected using ECL Advance Western Blotting Detection Kit (Hybond ECL, GE Healthcare Life Sciences, Freiburg, Germany) and the gel documentation system Molecular Imager® ChemiDoc™ XRS system (Bio-Rad, Munich, Germany). As loading control, the structural protein β-actin was detected on each membrane.

2.8. Quantification of TNF-α Release by ELISA Assay

To analyze TNF-α release, isolated PBMC (2 × 10⁶ cells/mL) were pretreated with aqueous plant extracts in the concentration of 1–333 μg/mL for 3 h or solvent control (10% water) for 3 h and subsequently stimulated with 1 μg/mL LPS for 3 h at 37°C in a humidified incubator with 5% CO₂/95% air atmosphere. Supernatants were then used for photometric quantification of TNF-α using the TNF-α ELISA kit supplied from eBioscience (Frankfurt, Germany) according to the manufacturer's instructions.

2.9. Quantification of PGE₂ Release by ELISA Assay

To quantify PGE₂ release, isolated PBMC (2 × 10⁶ cells/mL) were analyzed with or without LPS stimulation for 24 h after pretreatment with the plant extracts (1–333 μg/mL) or solvent control (10% water) for 6 h at 37°C in a humidified incubator with 5% CO₂/95% air atmosphere. Supernatants were then used for photometric quantification of PGE₂ using the PGE₂ ELISA kit from R&D Systems (Wiesbaden, Germany) and Cayman (Hamburg, Germany) and were used for PGE₂ quantification according to the manufacturer's instructions.

2.10. Quantification of LTB4 by ELISA Assay

To quantify LTB4 release, isolated PBMC (2 × 10⁶ cells/mL) were pretreated for 3 h with the plant extracts (333 μg/mL) or subfraction H4, followed by 15 min incubation with 1 μg/mL LPS and subsequently with 1 μM fMLP for 15 or 30 min at 37°C in a humidified incubator with 5% CO₂/95% air atmosphere. LTB4 release was photometrically quantified in the supernatants using the LTB ELISA kit from Cayman (Hamburg, Germany) according to the manufacturer's instructions.

2.11. COX-2 Enzyme Activity Assay

To analyze the direct inhibition of the COX-2 enzyme activity mediated by horseradish, the COX Inhibitor Screening Assay kit from Cayman (Hamburg, Germany) according to the manufacturer's instructions was used. Briefly, COX-2 enzyme was incubated with aqueous extract or fraction H4 for 10 min at 37°C and subsequently the reaction was initiated by adding arachidonic acid and incubating the mixture for 2 min at 37°C. Enzyme catalysis was stopped by adding saturated stannous chloride solution. Afterwards, PGF_2-α release was quantified using ELISA. As positive control, 25 or 50 μM indomethacin was used.

2.12. Data Analysis

Data were analyzed using GraphPad Prism 5.0 software (La Jolla, CA). Data are presented as mean ± standard error of the mean (SEM) of at least three independent experiments and statistical significance was determined by the ordinary one-way ANOVA. p values < 0.05 (∗) were considered statistically significant and values <0.01 (∗∗) were considered highly statistically significant, compared to the respective controls.

3.1. The Aqueous Extract from Horseradish Root Selectively Inhibits COX-2 Protein Expression and PGE₂ Release but without Impacting COX-2 Enzyme Activity

First, we analyzed whether the aqueous extract from horseradish root has toxic effects on human immune cells in the presence of LPS. For this, the trypan blue exclusion assay was used. None of the extract concentrations impaired the cell viability (Figure 1(a)) which ranged between 96.4 and 97.6% at all concentrations tested.

In an attempt to elucidate the anti-inflammatory potential of the plant, we studied its effect on the cyclooxygenase isoforms COX-1 and COX-2. Therefore, primary human PBMC were incubated with the aqueous extract at different concentrations prior to stimulation with LPS and subjected to immunoblot analysis of COX-1 and COX-2. Pretreatment of cells for 3 h strongly inhibited LPS-induced COX-2 expression at a broad concentration range from 1 μg/mL to 333 μg/mL (Figure 1(b)). To ascertain that the fraction of primarily water soluble compounds did account for the observed COX-2 regulation, a DMSO extract was additionally tested using the same experimental settings as described above but this had no impact on LPS stimulated COX expression (Figure 1(b)). This result indicated that the bioactive compounds were only present in the polar aqueous plant extract. The isoform COX-1, which is thought to be responsible for normal physiological functions in the human body, was not altered by any of the extracts tested (Figure 1(b)). In cells pretreated for 6 h with the aqueous extract and subsequent LPS treatment for 3 h, we found that higher concentrations (≥37 μg/mL) stimulated COX-2 protein expression beyond the LPS effect (Figure 1(c)). To examine the contribution of the aqueous plant extract to this observation, we repeated the experiment in the absence of LPS. As shown in Figure 1(c), the extract induced COX-2 expression independent of LPS.

The enzyme COX-2 converts endogenous arachidonic acid to PGE₂ in PBMC. The impact of horseradish on PGE₂ release from LPS stimulated PBMC was next measured by ELISA. Despite the observed COX-2 induction by the aqueous plant extract after 6 h preincubation, our analysis demonstrated significant PGE₂ inhibition in LPS-induced cells by the extract at >1 μg/mL with a maximum of 87% at 37 μg/mL (Figure 1(d)). To limit the number of potential relevant bioactive compounds, we then examined different subfractions prepared by HPLC fractionation. Only compounds from subfraction H4 accounted for the strong inhibitory effect on PGE₂ release as observed by the whole plant extract (Figure 1(e)). The potency of H4 was then equal to the combination of all four fractions (Figure 1(e)). Next, we analyzed whether the aqueous extract directly inhibits COX-2 enzyme activity. However, neither the complete extract nor its bioactive subfraction H4 could block the activity of COX-2 enzyme (Figure 1(f)). In contrast, indomethacin, which was used as reference, inhibited COX-2 enzyme activity (Figure 1(f)).

3.2. The Aqueous Extract from Horseradish Inhibits LTB4 Release

To determine the effect on the 5-LOX pathway, 5-LOX protein expression was next studied. As shown in Figure 2(a), 5-LOX protein expression was not regulated at any extract concentrations tested. We then analyzed the impact of the aqueous extract and subfraction H4 on LTB4 release from PBMC, stimulated with LPS and fMLP. The cotreatment of cells with LPS and fMLP induced LTB4 release of 78% (15 min fMLP) and 86% (30 min fMLP) as compared to the solvent control. Pretreatment of cells with the whole aqueous extract or subfraction H4 significantly attenuated the release of LTB4 as stimulated by LPS/fMLP (63% for whole extract and 65% for H4 at 15 min fMLP and 77% for whole extract at 30 min fMLP) (Figure 2(b)).

3.3. The Aqueous Extract from Horseradish Interferes with LPS-Activated ERK Signalling

The MAPK signalling pathway is one of the important early upstream cascades involved in inflammatory responses. Cells treated with the aqueous plant extract were therefore next subjected to analysis of MAPK activation. Concentration-dependent inhibition of LPS-triggered ERK1/2 activation was evident after exposure to the aqueous extract; the highest concentration (333 μg/mL) almost completely inhibited ERK1/2 phosphorylation (Figure 3(a)). In contrast, the phosphorylation status of p38 was not altered by water extract treatment at any concentration tested. The activated form of ERK1/2 triggers transcriptional factors that alter the expression level of the target gene COX-2. In this, the transcription factor AP-1 plays a critical role in response to the inflammatory stimuli LPS. We therefore next investigated whether this transcription factor is blocked by pretreatment of LPS-activated PBMC with the aqueous extract. As depicted in Figure 3(b), pretreatment with the extract at 333 μg/mL reduced the activation of c-Jun, the major component of AP-1, in a time-dependent manner.

Moreover, to assess whether the anti-inflammatory potential of horseradish also contributed to the inhibition of inflammatory cytokines, quantification of TNF-α was finally conducted. In Figure 3(c), it is shown that, similar to PGE₂, the release of TNF-α from PBMC was also inhibited by whole extract treatment in a concentration-dependent manner. A concentration of ≥37 μg/mL led to significant inhibition. At 333 μg/mL, TNF-α release was inhibited by 87%.

3.4. Characterization of the Aqueous Extract of Horseradish Root Using UHPLC-QToF and GC-MS

To characterize the whole aqueous plant extract and the subfractions, we performed metabolomic screening and a targeted analysis of glucosinolates (GLS) [14] of the aqueous plant extract. Prominent compounds in the extract were the amino acids arginine and proline, citric acid, phenolic compounds including caffeic acid and kaempferol derivatives, the main GLS, 2-propenyl-GLS and 3-methylsulfinyl-propyl-GLS, and fatty acid derivatives (Figure 4). Targeted analysis revealed 2-propenyl-GLS and 2-phenylethyl-GLS as major GLS derivatives (Figure 5). Analysis of subfractions H1–H4 revealed in fraction H1, for instance, GLS; in H2, kaempferol derivatives; in H3, caffeic acid and its derivatives as well as sugars; in H4, fatty acid and derivatives (Table 1).

Additionally, we determined the volatile compounds present in the aqueous plant extract (Figure 6). We identified various glucosinolate derived volatiles including ITC, isocyanates, and nitriles; further compounds derived from the phenylpropanoid pathway (e.g., benzaldehyde and phenylethanol) or from fatty acids (e.g., hexanal and nonanal).

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