Sesquiterpene Lactone Composition and Cellular Nrf2 Induction of Taraxacum officinale Leaves and Roots and Taraxinic Acid β

Abstract

Taraxacum officinale, the common dandelion, is a plant of the Asteraceae family, which is used as a food and medical herb. Various secondary plant metabolites such as sesquiterpene lactones, triterpenoids, flavonoids, phenolic acids, coumarins, and steroids have been described to be present in T. officinale. Dandelion may exhibit various health benefits, including antioxidant, anti-inflammatory, and anticarcinogenic properties. We analyzed the leaves and roots of the common dandelion (T. officinale) using high-performance liquid chromatography/mass spectrometry to determine its sesquiterpene lactone composition. The main compound of the leaf extract taraxinic acid β-d-glucopyranosyl ester (1), a sesquiterpene lactone, was isolated and the structure elucidation was conducted by nuclear magnetic resonance spectrometry. The leaf extract and its main compound 1 activated the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in human hepatocytes more significantly than the root extract. Furthermore, the leaf extract induced the Nrf2 target gene heme oxygenase 1. Overall, present data suggest that compound 1 may be one of the active principles of T. officinale.

Introduction

The genus Taraxacum belongs to the family Asteraceae and is native to temperate regions of the Northern Hemisphere.¹ The species Taraxacum officinale, the common dandelion, is well known as a wild flower worldwide.^2,3 The name dandelion comes from the French word “dent de lion” (“lion's tooth”).¹ The perennial weed dandelion has an average length of 15–30 cm.¹ The taproots reach a length of 60–100 cm.¹ The serrate leaves (5–40 cm) are ordered in a rosette at the base of the stem, which carries the yellow inflorescence.¹ The fruits are achenes in a crown with white hairy pappus, which are distributed by wind.¹ In Figure 1, dandelion with inflorescence (Fig. 1A) and achene crowned by a white hairy pappus (Fig. 1B) are shown. Taraxacum stems and leaves exude milky latex after cell rupture.⁴

FIG. 1.

Taraxacum officinale with inflorescence (A) and achene crowned by a white hairy pappus (B).

Moreover, in its entirety, that is, inflorescences, leaves, and roots, dandelion is an edible weed.¹ It is consumed raw (salad), cooked, or roasted (used as coffee substitute). Furthermore, as flavoring it is used in, for example, alcoholic drinks, candy, and cheese.^1,2,5

Different dandelion tissues contain sesquiterpene lactones, triterpenoids, steroids, flavonoids, phenolic acids, and coumarins in various amounts.⁶ Sesquiterpenes are probably responsible for the bitter taste of the dandelion.⁷ T. officinale roots contain various sesquiterpene lactones such as eudesmanolides, that is, tetrahydroridentin B and taraxacolide-O-β-glucopyranoside, guaianolides, that is, 11β,13-dihydrolactucin and ixerin D, and esterified germacranolide acids, that is, taraxinic acid β-glucopyranosyl ester, its 11,13-dihydroderivative and ainslioside.^8,9 Also, the presence of taraxacoside (acylated γ-butyrolactone glycoside),¹⁰ triterpenes, phytosterols,³ and various phenolic acids, for example, chicoric acid, monocaffeoyltartaric, chlorogenic, and caffeic acids,¹¹ is known. Moreover, beside the secondary plant metabolites, roots contain considerable amounts of inulin.¹² The leaves contain the bitter sesquiterpene lactones taraxinic acid β-d-glucopyranoside and 11β,13-dihydrotaraxinic acid.⁹ The polyphenol content in the leaves is higher than in the roots. Chicoric acid is one of the main polyphenols in T. officinale. ² Flavonoids, phenolic acids (hydroxycinnamic acid derivatives) such as chlorogenic, chicoric, and monocaffeoyltartaric acids, and coumarins were detected in the leaves.⁶ Moreover, dandelion leaves are an important source of potassium, which is probably responsible for its diuretic activity.¹³

In the past decades, the perennial weed was used as a medicinal plant for the treatment of various diseases, for example, anorexia and hepatitis. Various health benefits have been described so far, such as antioxidative, anti-inflammatory, anticarcinogenic, antihyperglycemic, anticoagulatory, analgesic, diuretic, choleretic, and prebiotic properties.¹ However, a search with the term “Taraxacum” or “dandelion” in the medical database PubMed under the category clinical trials (www.ncbi.nlm.nih.gov/pubmed/?term=Taraxacum or www.ncbi.nlm.nih.gov/pubmed/?term=DANDELION, last accessed on June 13, 2016) resulted only in about five articles.

The Keap1 (Kelch-like ECH-associated protein 1)–Nrf2 (nuclear factor erythroid 2-related factor 2) pathway plays a central role in cellular stress response and antioxidant defense mechanisms.^14,15 Nrf2 orchestrates the expression of genes encoding antioxidant and phase II enzymes such as heme oxygenase 1 (HO-1 or Hmox1), glutathione S-transferase (Gst), NAD(P)H quinone dehydrogenase 1 (Nqo1).^15,16 Under basal conditions, Nrf2 is bound to its repressor Keap1 in the cytoplasm.¹⁷ Oxidants and electrophiles are able to inactivate Keap1 and stop the ubiquitination of Nrf2, leading to the release of Nrf2 from Keap1 and activation of Nrf2.^14,17 Thus, Nrf2 translocates into the cell nucleus, where it binds to the so-called antioxidant response element (ARE), thereby inducing the expression of Nrf2 target genes.^14,17 One important Nrf2 target gene is Hmox1, which catalyzes the degradation of heme to iron, biliverdin, and carbon monoxide.¹⁸

In the current study, the sesquiterpene composition of T. officinale leaves and roots was analyzed by high-performance liquid chromatography/mass spectrometry (HPLC/MS). It is known that T. officinale may counteract oxidative stress.¹⁹ Therefore, the objective of our cell culture experiments was to determine Nrf2 transcription factor activity in response to a leaf and root extract treatment. The leaf sesquiterpenes induced Nrf2 and its target gene Hmox1. The most active Nrf2 inducer of the leaves, taraxinic acid β-d-glucopyranosyl ester (compound 1), was isolated by semipreparative HPLC, and the structure was elucidated by nuclear magnetic resonance (NMR) spectroscopy and MS data. Nrf2 activity was monitored by a dual luciferase reporter gene assay.

Experimental

Plant material

T. officinale leaves and roots were collected in spring 2012 in the Fulda valley (Germany).

Chemicals

If not indicated otherwise, chemicals were obtained from Carl Roth (Karlsruhe, Germany), Sigma-Aldrich (Seelze, Germany), or Merck Millipore (Darmstadt, Germany). All chemicals were used as received from the supplier. Chemicals for cell culture such as l-glutamine, penicillin, and streptomycin were purchased from PAA (Coelbe, Germany), and dimethyl sulfoxide (DMSO) was purchased from Carl Roth.

Extraction and isolation of 1 from T. officinale

Leaves and roots were extracted according to the same procedure. About 350 g of T. officinale leaves and about 100 g roots were freeze-dried to yield 41.6 or 21.8 g of dry powder, respectively. The freeze-dried leaves or roots were extracted twice with 350 mL of dry methanol for 60 min. Sodium sulfate was added and the mixture was stirred for 40 min, filtered, and evaporated. One milliliter of the residue was applied onto a silica gel flash chromatography (Carl Roth). Acetone was used as an elution solvent. The fractions were collected in test tubes by a fraction collector. After analysis of the eluate by thin-layer chromatography (TLC; see Thin-layer chromatography), test tubes were combined. Two leaf extracts (combined fractions 7–13 and 15–22) were collected. About 100 mg of the fraction 15–22 was obtained, which was enriched with compound 1 (R_f value of 0.85). The latter fraction was further separated by semipreparative HPLC (see High-performance liquid chromatography/mass spectrometry). Fractions were collected every 30 sec and analyzed by HPLC/MS. Fractions containing pure compound 1 were combined and yielded 3 mg of 1. Structure elucidation was done by NMR spectroscopy (see Nuclear magnetic resonance spectroscopy).

Thin-layer chromatography

Silica gel 60G F254 applied on aluminum (Merck Millipore) was used as stationary phase. A mixture of acetone:methanol (80:20, v/v) was applied as mobile phase. After development, the detection was conducted by UV detection and derivatization with the sodium permanganate solution.

High-performance liquid chromatography/mass spectrometry (HPLC/ESI-MSⁿ)

An HPLC-MS instrument equipped with a PE 200 pump, PE 200 autosampler (PerkinElmer, Rodgau, Germany), a G1315B PDA detector (Agilent Technologies, Waldbronn, Germany), and a light scattering detector (Sedex 75; Sedere, Alfortville Cedex, France) controlled by Analyst 1.3 Software (AB Sciex, Darmstadt, Germany) was used. An API150ex mass spectrometer (AB Sciex) with electrospray ionization in negative and positive fast switching mode was used for analysis.

LC analyses were performed on a LiChrospher^® 60 RP-Select B column (250 × 4 mm, 5 μm; Merck Millipore) equipped with a precolumn of the same material at 23°C. Mobile phases consisted of 5 mM ammonium formiate and 0.1% formic acid (A) and acetonitrile:methanol (50:50, v/v) with 5 mM ammonium formiate and 0.1% formic acid (B). The following gradient program was used: 0 min 15% B, 30 min 100% B, 40 min 100% B, and 45 min 15% B. The flow rate was set at 1.0 mL/min. All compounds were dissolved in methanol, and 20 μL was injected into the system. Analyst 1.3 software was used for data analyses.

Isolation of taraxinic acid β-d-glucopyranosyl ester by semipreparative HPLC

The semipreparative isolation was performed using an HPLC system from Hitachi (Düsseldorf, Germany) equipped with HPLC Pump l7150, autosampler L7250, UV detector L-4000A, and a semipreparative HPLC column (LiChrospher 60 RP-select B, 250 × 16 mm, 10 μm; Merck Millipore). HSM software (Dießen am Ammersee, Germany) was used for data analyses. Water (A) and methanol:acetonitrile (50:50, v/v) (B) were used as mobile phases. The linear gradient starting with 22% B up to 48% B in 57 min was used. The flow rate was set at 15 mL/min. The sample was dissolved in methanol, and 1 mL of the sample was injected. The chromatograms were recorded at λ 250 nm. Fractions were collected every 30 sec from 7.7 to 57.7 min. Fractions 29 to 31 were combined, evaporated, and analyzed by HPLC/MS and NMR.

Nuclear magnetic resonance spectroscopy

A DRX500 (500 MHz; Bruker Biospin, Rheinstetten, Germany) was used to measure 1D- ¹H and ¹³C and 2D-NMR (HSQC, HMBC, HH-COSY) spectra of the sesquiterpene lactone glycoside 1 in methanol-d₄ at 298 K. Spectra were referenced to the residual solvent signal (δ _H 3.30 ppm and δ _C 49.2 ppm, respectively). All data were in accordance with the data from the literature.⁴

Cell culture

Huh7 human hepatoma cells were obtained from the Institute of Applied Cell Culture, Munich, Germany. Huh7 cells were maintained in high glucose (4 g/L) Dulbecco's Modified Eagle's Medium supplemented with 10% (v/v) fetal bovine serum, 4 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (all PAA, Coelbe). Cells were incubated in a humidified incubator at 37°C and 5% CO₂. Stock solutions for cell culture assays were dissolved at a concentration of 100 mg/mL of B7-13, B15-22, and W33-47 and 100 mM of compound 1 in DMSO and stored at −80°C until further use. For all cell culture experiments, DMSO controls (0.1%) have been performed.

Neutral red assay for cell viability determination

Huh7 cells were seeded at a density of 0.15 × 10⁶ cells/well in a 24-well plate for 24 h and treated with 1–100 μg/mL B7-13, B15-22, or W33-47 extracts or 1–100 μM compound 1 for an additional 24 h. For further procedure see Wagner et al. (2011).²⁰

Transient transfection and luciferase reporter gene analysis for Nrf2 transactivation

Transient transfection of Huh7 cells (24-well plate at a density of 0.15 × 10⁶ cells for 24 h) and luciferase reporter gene analysis for Nrf2 were conducted according to Wagner et al. (2011).²⁰ Huh7 cells were treated with 1 and 50 μg/mL B7-13, B15-22, or W33-47 and in case of compound 1 with 1, 5, 10, 25, 50, and 100 μM. Resveratrol (Resv; 25 μM) and allyl isothiocyanate (AITC; 25 μM) were used as positive controls. Three or two independent experiments were performed in triplicate.

RNA isolation and quantitative reverse transcription-polymerase chain reaction

Huh7 cells were seeded at a density of 0.9 × 10⁶ cells/well in a six-well plate for 24 h and treated with 1 and 50 μg/mL B7-13 for 6 h. Resveratrol (25 μM) was used as positive control. Subsequently, cells were harvested with TriFAST (VWR International GmbH, Erlangen, Germany), and RNA was isolated according to the manufacturer's instructions. mRNA expression levels of human Hmox1 were determined. Primer3 Input software version 0.4.0 was used for primer design, and primers were ordered from Eurofins MWG (Ebersberg, Germany). Primer sequence was as follows: forward 5′-CCAGGCAGAGAATGCTGAGT-3′ and reverse 5′-GTAGACAGGGGCGAAGACTG-3′. Real-time PCR was performed with a one-step procedure using the SensiFAST™ SYBR No-ROX One-Step Kit (Bioline, Luckenwalde, Germany) with SYBR Green detection on a Rotorgene 6000 cycler (Corbett Life Science, Sydney, Australia). Quantitation was done by an external standard curve. Results were normalized to glyceraldehyde 3-phosphate dehydrogenase (Gapdh) (forward 5′-CAATGACCCCTTCATTGACC-3′ and reverse 5′-GATCTCGCTCCTGGAAGATG-3′), which served as a housekeeping gene.

Statistical analysis

Statistical analysis was performed using SPSS software version 23.0 (SPSS, Inc., Munich, Germany). Normal distribution of data was tested by Kolmogorov–Smirnov and Shapiro–Wilk tests. Student's t-test or one-way analysis of variance with a Dunnett-T post hoc test was applied. Results are presented as mean + SEM. Significance was accepted at P-values <.05.

Results and Discussion

HPLC/MS analysis of the leaf and root extracts of T. officinale

The freeze-dried leaves and roots of T. officinale were extracted with dry methanol. These evaporated extracts were applied on a silica gel flash chromatography and eluted with acetone. All fractions were analyzed by TLC. Two leaf extracts B7-13 and B15-22 and a root extract W33-47 were obtained and analyzed by LC/MS. HPLC chromatograms of the leaf and root extracts of T. officinale are displayed in Figure 2.

FIG. 2.

HPLC chromatograms of (A) collected leaf extract fractions B7-13, (B) collected leaf extract fractions B15-22, and (C) root extracts W33-47 of T. officinale.

About 3 mg of pure compound 1 was obtained from the crude extract (B15-22) after semipreparative HPLC in high purity of >98%. Main compounds of the collected two leaf extracts B7-13 and B15-22 and the root extract W33-47 of T. officinale identified by comparison of t_R and MS spectra with those of authentic samples are given in Table 1 and the chemical structures are shown in Figure 3.

FIG. 3.

Chemical structures of sesquiterpenes from T. officinale.

Table 1.

Sesquiterpene Composition of the Two Leaf Extracts B7-13 and B15-22 and the Root Extract W33-47 of Taraxacum officinale Collected by Silica Gel Column Chromatography

Fraction	t_R (min)	Compound	Molecular formula	Molecular weight (g/mol)
B7-13	8.85	1,5-bis(4-hydroxyphenylacetyl)-l-chiro-inositol	C₂₂H₂₄O₁₀	448.1291
	17.27	taraxinic acid (2)	C₁₅H₁₈O₄	262.3010
	17.56	11β,13-dihydro-taraxinic acid (3)	C₁₅H₂₀O₄	264.3010
B15-22	12.76	taraxinic acid β-d-glucopyranosyl ester (1)	C₂₁H₂₈O₉	424.4416
W33-47	12.88	taraxacolide 1-O-β-d-glucopyranoside (4)	C₂₁H₃₂O₉	428.2046

In fraction B7-13, the inositol derivative 1,5-bis(4-hydroxyphenylacetyl)-l-chiro-inositol, taraxinic acid (2), a hydrolysate of sesquiterpene lactone glycoside, and a related sesquiterpene, 11β,13-dihydro-taraxinic acid (3), were enriched, whereas in the second leaf extract (B15-22) the sesquiterpene lactone taraxinic acid β-d-glucopyranosyl ester (1) was the main compound. The main compound in the root extract was the terpene glycoside taraxacolide 1-O-β-d-glucopyranoside (4).

In a recent study, differences between Taraxacum species in terms of their chemical composition and antioxidant activity have been described.² In the present study, the inositol 1,5-bis(4-hydroxyphenylacetyl)-l-chiro-inositol was identified in T. officinale leaves. Kenny et al. isolated further 4-hydroxyphenylacetic acid derivatives of inositol from T. officinale roots. We found the sesquiterpene lactone taraxinic acid β-d-glucopyranosyl ester (1) in our T. officinale leaf samples. In comparison to our results, Kisiel et al. identified taraxinic acid β-d-glucopyranosyl ester (1) as the major compound in the roots.⁹ Taraxinic acid (2) and 11β,13-dihydrotaraxinic acid (3) were detected in the leaves. Previously, these acids were found in roots⁸ and their derivatives are known for T. officinale. ^4,9 We detected eudesmanolide taraxacolide 1-O-β-d-glucopyranoside (4) in the roots of T. officinale. This result is in line with literature data.⁸

Cytotoxicity of the leaf and root extracts of T. officinale

The neutral red assay was applied to determine the cytotoxicity after incubation with B7-13, B15-22, or W33-47 extracts and the pure compound 1. Extracts and compound 1 did not exhibit any cytotoxicity up to 100 μg/mL or 100 μM, respectively (Fig. 4).

FIG. 4.

Cytotoxicity of the leaf and root extracts and compound 1 determined by the neutral red assay. Human Huh7 cells were seeded at a density of 0.15 × 10⁶ cells/well for 24 h and treated with the leaf and root extracts (1–100 μg/mL) or compound 1 (1–100 μM) for an additional 24 h.

Induction of the transcription factor Nrf2 and its target gene heme oxygenase 1

The Nrf2 transactivation activity of the B7-13, B15-22, and W33-47 extracts was determined as shown in Figure 5. The leaf extracts exhibited a more potent Nrf2 transactivation activity compared to the root extract. Supplementation of Huh7 cells with 50 μg/mL B7-13 extract resulted in a significant increase in Hmox1 mRNA levels (Fig. 6).

FIG. 5.

Effects of the leaf and root extracts of T. officinale on Nrf2 transactivation in human Huh7 cells. Human liver Huh7 cells (0.15 × 10⁶ cells/well) were supplemented after transient transfection with 1 and 50 μg/mL B7-13, B15-22, or W33-47 extracts for further 24 h. Resveratrol (Resv; 25 μM) and allyl isothiocyanate (AITC; 25 μM) served as positive controls. Data are mean + SEM of at least three experiments performed in triplicate (n = 3). * and *** indicate statistically significant differences between DMSO control cells and supplemented cells with extracts P < .05 and P < .001, respectively; Student's t-test. DMSO, dimethyl sulfoxide.

FIG. 6.

mRNA levels of heme oxygenase 1 (Hmox1) in human hepatocytes (Huh7). Huh7 cells (0.9 × 10⁶ cells/well) were supplemented with 1 and 50 μg/mL B7-13 extract after 24 h incubation for 6 h. Resveratrol (Resv; 25 μM) was used as positive control. The data are expressed as the mean + SEM (n = 3). *** significant differences in B7-13-treated cells compared with DMSO control cells P < .001, Student's t-test. SEM, standard error of the mean.

The main compound of the B15-22 extract, compound 1, also affected Nrf2 transactivation in a dose-dependent manner (Fig. 7).

FIG. 7.

Effects of compound 1 on Nrf2 transactivation. Huh7 cells (0.15 × 10⁶ cells/well) were supplemented after transient transfection with 1, 5, 10, 25, 50, and 100 μM of compound 1 for further 24 h. Resveratrol (Resv; 25 μM) and allyl isothiocyanate (AITC; 25 μM) served as positive controls. Data are mean + SEM of at least two experiments performed in triplicate (n = 2). *** statistically significant differences between compound 1-supplemented cells and DMSO control cell extracts P < .001, Dunnett-T-test.

Some Taraxacum species may decrease cellular levels of reactive oxygen species and may also exhibit free radical scavenging activities.² Furthermore, T. officinale leaf extracts have been reported to decrease lipid peroxidation in mice.²¹ Our results indicate that the leaf extracts and the compound 1 isolated from leaves are relatively potent inducers of Nrf2 transactivation. Some sesquiterpenes belong to the chemical class of activated Michael acceptors, electrophiles capable to S-alkylate redox-sensitive cysteine thiols.²² It is suggested by Fischedick et al. ²³ that the sesquiterpene lactones guaianolides (such as ixerin D) are more potent Nrf2/ARE inducers than the germacranolides [such as taraxinic acid β-d-glucopyranosyl ester (1)] and eudesmanolides [such as taraxacolide-O-β-glucopyranoside (4)].²³ In our cell culture studies, also the Nrf2 target gene Hmox1 was induced following the supplementation of human liver Huh7 cells with the leaf extract. Beside the protection against oxidative stress, the preventive effect of the T. officinale leaf extract against hepatotoxicity and genotoxicity is known.²⁴ Moreover, it is suggested that T. officinale leaves may mediate anti-inflammatory activities through the suppression of inducible NO synthase gene expression via the inhibition of the transcription factor NF-κB.¹⁹ Since Nrf2 partly antagonizes NF-κB,²⁵ it seems plausible that the anti-inflammatory properties of T. officinale may be partly mediated by the induction of Nrf2.

Structure elucidation

Compound 1, isolated from the leaf fraction B15-22, shows typical signals for a glucose moiety and exomethylene protons of a sesquiterpene lactone with a γ-butyrolactone moiety. Searching these features together with the molecular weight of m/z 424 determined by ESI-LC/MS and the genus Taraxacum in the Dictionary of Natural products on DVD (CRC Press Taylor & Francis Group 2014) hinted to taraxinic acid β-d-glucopyranosyl ester. The spectral data were identical with those reported in the literature.⁸

Conclusions

The sesquiterpene lactone composition in the root and leaf extracts of T. officinale was determined by HPLC/MS. T. officinale leaf and root extracts were potent inducers of Nrf2. Moreover, the leaves of T. officinale induced the Nrf2 target gene Hmox1. The pure compound 1 taraxinic acid β-d-glucopyranosyl ester isolated from T. officinale leaves was found to increase Nrf2 transactivation in a dose-dependent manner. Thus, we suggest that compound 1 may be one of the active principles of T. officinale. Overall, the present study indicates that T. officinale exhibits antioxidant properties in human liver cells partly due to the induction of Nrf2 and Hmox1. Our data regarding the biological activity of T. officinale extracts and the various sesquiterpene lactones should be verified in appropriate in vivo models in the future.

Footnotes

Acknowledgments

We thank Vivien Schmuck, Gaby Steinkamp, and Inga Richter for excellent technical help. We are thankful for the financial support of the “Interne Forschungsförderung” from the University of Applied Sciences Fulda.

Author Disclosure Statement

No competing financial interests exist.

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Sesquiterpene Lactone Composition and Cellular Nrf2 Induction of Taraxacum officinale Leaves and Roots and Taraxinic Acid β - d -Glucopyranosyl Ester

Abstract

Introduction

Experimental

Plant material

Chemicals

Extraction and isolation of 1 from T. officinale

Thin-layer chromatography

High-performance liquid chromatography/mass spectrometry (HPLC/ESI-MSn)

Isolation of taraxinic acid β-d-glucopyranosyl ester by semipreparative HPLC

Nuclear magnetic resonance spectroscopy

Cell culture

Neutral red assay for cell viability determination

Transient transfection and luciferase reporter gene analysis for Nrf2 transactivation

RNA isolation and quantitative reverse transcription-polymerase chain reaction

Statistical analysis

Results and Discussion

HPLC/MS analysis of the leaf and root extracts of T. officinale

Cytotoxicity of the leaf and root extracts of T. officinale

Induction of the transcription factor Nrf2 and its target gene heme oxygenase 1

Structure elucidation

Conclusions

Footnotes

Acknowledgments

Author Disclosure Statement

References

High-performance liquid chromatography/mass spectrometry (HPLC/ESI-MSⁿ)