Three Novel Triterpenoids from Taraxacum officinale Roots

Three novel lupane-, bauerane-, and euphane-type triterpenoids (1–3), in addition to seven known triterpenoids (4–10)—18β,19β-epoxy-21β-hydroxylupan-3β-yl acetate (4), 21-oxolup-18-en-3β-yl acetate (5), betulin (6), officinatrione (7), 11α-methoxyolean-12-en-3-one (8), eupha-7,24-dien-3-one (9), and 24-oxoeupha-7,24-dien-3β-yl acetate (10)—were isolated from the roots of Taraxacum officinale. Their structures were elucidated on the basis of spectroscopic analyses using 1D and 2D-NMR spectra and electron ionization mass spectrometry (EIMS). The effects of compounds 1–10 on the production of nitric oxide (NO) in lipopolysaccharide (LPS)-activated mouse peritoneal macrophages were evaluated. Compounds 4, 6, and 10 exhibited similar NO inhibitory activities to NG-monomethyl-l-arginine acetate (l-NMMA). These compounds did not exhibit cytotoxicity at an effective concentration. The results of present study suggest that compounds 4, 6, and 10 have potential as anti-inflammatory disease agents.


Introduction
Taraxacum officinale (commonly known as dandelion) has a number of culinary and medicinal uses, despite being generally regarded as a weed. Dandelion is the general name of approximately two thousand species of plants that belong to the genus Taraxacum (Compositae). Young leaves and inflorescences are used as ingredients in salads and stir-fries. Dandelion roots have long been largely used on the continent, and the plant is cultivated largely in India as a remedy for liver complaints. Dandelion roots contain bitter principles that have a tonic effect on the liver and digestive system [1]. It is also a gentle laxative and natural diuretic that is rich in natural potassium, which enriches the body s supply. It has been used as a tonic to treat rheumatic issues and also acts as a blood purifier [2]. In recent studies, it was revealed that dandelion extract may play a significant role during adipogenesis and lipid metabolism, and may have the potential for the treatment of obesity [3]. Dandelion is available as capsules, liquid extracts, and tea forms [4]. We previously reported five novel lupan-type triterpenoids isolated from the roots of T. officinale. Of these, officinatrione, a (17S)-17,18-seco-lupane was identified as a new carbon skeletal triterpenoid [5]. We herein reported three new triterpenoids: 18β,19β-epoxy-21β-methoxylupan-3β-yl acetate (1), 3β-acetoxybauer-7-en-6-one (2), 3β-acetoxyeupha-7,24-dien-6-one (3), in addition to seven known compounds (4-10) (Figure 1). The isolated compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10) were evaluated for their inhibitory activities on macrophage activation using an inhibitory assay of nitric oxide (NO) production in RAW 264.7 mouse macrophages stimulated by lipopolysaccharide (LPS).

Results and Discussion
The methanol extract of the roots of T. officinale (5.81 kg) was partitioned between EtOAc and water. The organic layer was condensed in vacuo to give a yellowish residue (890 g) and separated by silica gel chromatography followed by HPLC [octadecyl silica (ODS), MeOH-H2O (95: 5)] to afford the novel lupane, bauerane, euphane-type triterpenes, as well as seven known triterpenes.

Results and Discussion
The methanol extract of the roots of T. officinale (5.81 kg) was partitioned between EtOAc and water. The organic layer was condensed in vacuo to give a yellowish residue (890 g) and separated by silica gel chromatography followed by HPLC [octadecyl silica (ODS), MeOH-H 2 O (95:5)] to afford the novel lupane, bauerane, euphane-type triterpenes, as well as seven known triterpenes.
Compound (2) Figure 3). The planar structure of 2 was established by a comprehensive analysis of 1D and 2D NMR spectra, particularly the HMBC spectrum. Selected HMBC correlations are shown in Figure 3. Strong correlations were observed from Me-23 (δ H 1.20 (s)) and Me-24 (δ H 1.19 (s)) to C-3 (δ C 80.6 (d)), C-4 and C-5; from Me-25 (δ H 0.88 (s)) to C-1, C-5, C-9 and C-10; from Me-26 (δ H 1.06 (s)) to C-8 (δ C 170.3 (s)), C-13, C-14 and C-15; from Me-27 (δ H 0.96 (s)) to C-12, C-13, C-14 and C-17; from Me-28 (δ H 1.06 (s)) to C-16, C-17, C-18 and C-22; from Me-29 (δ H 1.07 (d)) to C-18, C-19 and C-20; from Me-30 (δ H 0.92 (d)) to C-19, C-20 and C-21; from H-7 (δ H 5.84 (d)) to C-5, C-6 (δ C 199.8 (s)), C-8, C-9 and C-14; and from H-18 (δ H 1.33) to C-12, C-13, C-14, C-18, C-19, C-20, C-27 and C-28. Based on the above spectral data, in addition to the 1 H-NMR chemical shift in 2 compared with previous data on baurerenyl acetate [7,8], the plain structure was assumed to be D:C-friedours-7-ene-6-one (baueran-7-en-6-one). The relative configuration of 2 was established in the NOESY experiment. Strong NOESY correlations between Me-23 and H-3α; between Me-25 and Me-24 and Me-26; between Me-27 and H-9α and Me-30; between H-5α and H-9α: between H-7 and H-15α and H-15β; between H-18 and Me-26, Me-28 and Me-29; between H-19 and Me-27; and between H-20 and Me-28 revealed an acetyl group at C-3 in the β orientation, H-18 in the β orientation, and Me-19 and Me-20 in the β and α orientations, respectively. Therefore, the structure of 2 was established as 3β-acetoxybauer-7-en-6-one. Natori et al. synthesized 3β-acetoxy-9α-bauer-7-en-6-one (XIII) from the oxidation of bauerenyl acetate, and confirmed the structure of XIII from a comparison between 3β-acetoxy-9β-bauer-7-en-6-one (XII) and XIII [9]. However, a marked difference was found in the 1 H-NMR chemical shifts in 2 and XIII; therefore, the structure of XIII does not appear to be reasonable due to the lack of sufficient evidence. On the other hand, Yen isolated 3β-acetoxy-9β-bauer-7-en-6-one from the stem bark of Hiptage benghalensis, and the chemical shift value of 1 H-NMR was similar to that described above for compound XII [10].    [11]. The 13 C-NMR chemical shift values of the side chain of 3 had similar literature data of butyrospermol [12] and kansenone [13]. Based on these results and the biogenetical consideration [14], 3 was established as 3β-acetoxyeupha-7,24-dien-6-one, isolated for the first time. Although euphane-type triterpenoids have been known for a long time, they are not present in abundant amounts in nature.   [11]. The 13 C-NMR chemical shift values of the side chain of 3 had similar literature data of butyrospermol [12] and kansenone [13]. Based on these results and the biogenetical consideration [14], 3 was established as 3β-acetoxyeupha-7,24-dien-6-one, isolated for the first time. Although euphane-type triterpenoids have been known for a long time, they are not present in abundant amounts in nature.  from Melia dubia, and the NOESY spectrum of Meliastatins resembles that of 3 [11]. The 13 C-NMR chemical shift values of the side chain of 3 had similar literature data of butyrospermol [12] and kansenone [13]. Based on these results and the biogenetical consideration [14], 3 was established as 3β-acetoxyeupha-7,24-dien-6-one, isolated for the first time. Although euphane-type triterpenoids have been known for a long time, they are not present in abundant amounts in nature. Macrophages may be a potential therapeutic target for inflammatory diseases [15]. Activated macrophages release pro-inflammatory mediators, such as NO, reactive oxygen species, interleukin-1 beta, tumor necrosis factor-alpha, and other inflammatory mediators, which play important roles in biological defenses. However, the overexpression of these mediators has been implicated in diseases such as osteoarthritis, rheumatoid arthritis, and diabetes, because the increased production of pro-inflammatory mediators has been shown to induce severe or chronic inflammation [15]. Ten triterpenoids and L-NMMA-an inducible nitric oxide synthase (iNOS) inhibitor-were evaluated for their inhibitory effects on NO production ( Table 2). Among the compounds tested, 4, 6, 7, 8, and 10 exhibited NO inhibitory activities. Of these, 7 and 8 exhibited no cytotoxicity at 1-30 μM. Although 4, 6, and 10 exhibited some cytotoxicity at higher concentrations, they had similar inhibitory effects on NO production by triterpenoids from roots of Taraxacum officinale superior inhibitory activities to L-NMMA at non-toxic concentrations (4 at 3-10 μM; 6 at 1 and 3 μM; 10 at 3 and 10 μM).

Plant Material
The roots of T. officinale (Compositae) were collected in Takatsuki city, Osaka, Japan in April, 2014. A voucher specimen (TR-01) was deposited in the Herbarium of the Laboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences. Macrophages may be a potential therapeutic target for inflammatory diseases [15]. Activated macrophages release pro-inflammatory mediators, such as NO, reactive oxygen species, interleukin-1 beta, tumor necrosis factor-alpha, and other inflammatory mediators, which play important roles in biological defenses. However, the overexpression of these mediators has been implicated in diseases such as osteoarthritis, rheumatoid arthritis, and diabetes, because the increased production of pro-inflammatory mediators has been shown to induce severe or chronic inflammation [15]. Ten triterpenoids and L-NMMA-an inducible nitric oxide synthase (iNOS) inhibitor-were evaluated for their inhibitory effects on NO production ( Table 2). Among the compounds tested, 4, 6, 7, 8, and 10 exhibited NO inhibitory activities. Of these, 7 and 8 exhibited no cytotoxicity at 1-30 µM. Although 4, 6, and 10 exhibited some cytotoxicity at higher concentrations, they had similar inhibitory effects on NO production by triterpenoids from roots of Taraxacum officinale superior inhibitory activities to L-NMMA at non-toxic concentrations (4 at 3-10 µM; 6 at 1 and 3 µM; 10 at 3 and 10 µM).

Plant Material
The roots of T. officinale (Compositae) were collected in Takatsuki city, Osaka, Japan in April 2014. A voucher specimen (TR-01) was deposited in the Herbarium of the Laboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences.