Characterization of a New Sesquiterpene and Antifungal Activities of Chemical Constituents from Dryopteris fragrans (L.) Schott

One new sesquiterpene and six known compounds were isolated from Dryopteris fragrans (L.) Schot. They were identified as 3-O-β-D-glucopyranosylalbicanol-11-O-β-D-glucopyranoside (1), dihydroconiferylalcohol (2), (E)-3-(4-hydroxyphenyl)acrylic acid (3), esculetin (4), 5,7-dihydroxy-2-hydroxymethylchromone (5), eriodictyol (6) and isoorientin (7) by UV, MS, 1D-NMR and 2D-NMR spectroscopy. The antifungal activities of the seven isolated compounds were screened. Compounds 2, 3, 4 and 5 showed obvious activities against Microsporum canis and Epidermophyton floccosum.


Introduction
Dryopteris fragrans (L.) Schott, a deciduous perennial herb from the genus Dryopteris (Dryopteridaceae), is mainly distributed in Northeast China, Russia, Japan, Korea and North America. The herb is used for treatment of skin diseases such as psoriasis, rashes, dermatitis, other skin diseases, barbiers and arthritis [1][2][3][4]. Previous research had discovered phloroglucins, terpenes, flavonoids, saponins, essential oils and sterols in this plant, and activity screenings of the related constituents have become popular [2,5,6]. Our research group has reported one new phenolic acid from the herb [7,8].
In this paper, we report the isolation and structural identification of one new sesquiterpene together with six known compounds which were obtained from genus Dryopteris for the first time and the assay of their antifungal activity in order to identify the active compounds.

Chemical Structure Identification and Spectroscopic Data
Compound 1, obtained as a light yellow oil, had a molecular formula of C 27 H 46 O 12 based on the HRESIMS ([M+Na] + 585.2890), which indicated five degrees of unsaturation. The UV max absorption at 205.242 nm indicated an isolated chromophore in the structure. Four methyl groups (δ H 0.80, 1.17, 1.26, 2.05), one olefinic proton (δ H 5.47), one oxygenated methine proton (δ H 4.06) and two oxygenated methylenes (δ H 4.01) were observed. Furthermore, we deduced the presence of two sugar residues from the signal of two anomeric protons at δ H 4.84 (1H, d, J = 7.8 Hz) and δ H 4.90 (1H, d, J = 7.8 Hz, Table 1). The acid hydrolysis of 1 with aqueous 2 M HCl yielded D-glucose, which was identified by GC comparison with a sugar standard. In the 13 C-NMR spectrum (Table 1) 27 carbon signals were resolved. Besides the carbon signals of the two D-glucoses there were also 15 carbon signals comprising four methyls (δ C 22.5, 14.8, 16.6, 28.2), four methylenes (δ C 37.8, 27.9, 24.6, 69.9), four methines (δ C 89.0, 50.1, 122.7, 54.9), and three quaternary carbons (δ C 39.4, 134.4, 35.6) as classified by their chemical shifts and from the HSQC spectrum. All of the signals above suggested the aglycone of compound 1 was a sesquiterpene.

Plant Material
The

Acid Hydrolysis
Compound 1 (5 mg) was hydrolyzed with 2 mol/L HCl (5 mL) for 5 h at 90 °C. After cooling to room temperature, the reaction mixture was extracted with EtOAc (5 mL) three times. Each remaining aqueous layer was neutralized with 0.5 N NaOH and then freeze-dried to give a residue. The residue was dissolved in pyridine (2 mL) and L-cysteine methyl ester hydrochloride (3 mg) was added to the solution. The solution was kept at 60 °C for 1 h. Then trimethylchlorosilane (0.5 mL) was added to the reaction mixture and heated at 60 °C for another 30 min. After centrifugation, the supernatant was analyzed by GC. The sugar derivatives obtained from compounds 1 showed a single peak at 32.3 min. The retention time was similar to that of a D-glucose derivative, so the sugar was identified as D-glucose. [8][9][10] M. canis and E. floccosum were obtained from the fungus preservation center in the China Academy of Sciences and the Institute of Medicine of Dermatology, respectively. Antifungal tests were performed using the method of Dilution Antifungal Susceptibility Testing of Filamentous Fungi as described by the National Committee for Clinical Laboratory Standards [9].

Conclusions
A new sesquiterpene 1 and known compounds 2-7 were isolated from the genus Dryopteris for the first time. The antifungal activity screening results with Microsporum canis and Epidermophyton floccosum showed that compounds 2, 3, 4 and 5 have remarkable activities against both species.