Polar Bioactive Constituents from Aerial Parts of Thymus longicaulis

Thymus longicaulis C. Presl., belonging to the Lamiaceae family, is a small aromatic perennial herb typical of the Mediterranean vegetation. This species, known in traditional phytoteraphy of Italy, has been extensively investigated in terms of chemical analysis and biological activity of its essential oils. Nevertheless, few data are available in the literature, regarding the chemical characterization of polar components of T. longicaulis. In this study, the phytochemical investigation of methanol extract of T. longicaulis through different chromatographic techniques, led to the isolation of thirteen compounds. The structures of rosmarinic acid and two derivatives, as well as that of flavones, triterpenes and a lignan have been elucidated on the basis of extensive NMR spectroscopic analyses. The evaluation of DPPH radical scavenging activity of pure compounds has been performed. Corresponding authors: Brigida, D’Abrosca. Department of Environmental Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Via Vivaldi 43, Caserta, Italy; Tel: +39-0823-274564; E-mail: brigida.dabrosca@unina2.it Received Date: 22 February, 2016 Accepted Date: 29 February, 2016 Published Date: 3 March, 2016 Citation: D’Abrosca, B., et al. Polar Bioactive Constituents from Aerial Parts of Thymus Longicaulis C. Presl. (2016) Lett Health Biol Sci 1(1): 14. Lett Health Biol Sci | volume 1: issue 1 www.ommegaonline.com

vent signal, J (coupling constant) are given in Hz. Standard pulse sequences and phase cycling from Varian library were used for 1 H, 13 C, DEPT, DQF-COSY, COSY, TOCSY, HSQC, H2BC, HMBC and CIGAR-HMBC experiments. 1 H NMR spectra were acquired over a spectral window from 14 to 2 ppm, with 1.0 s relaxation delay, 1.70 s acquisition time (AQ), 90° pulse width = 13.8 μs. The initial matrix was zero-filled to 64 K. 13 C-NMR spectra were recorded in 1H broadband decoupling mode, over a spectral window from 235 to 15 ppm, 1.5 s relaxation delay, 90° pulse width = 9.50 μs, AQ = 0.9 s. The number of scans for both 1 H and 13 C-NMR experiments was chosen depending on the concentration of the samples. Also for homonuclear and heteronuclear 2D-NMR experiments, data points, number of scan and of increments were adjusted according to the sample concentrations. Correlation spectroscopy (COSY) and double quantum filtered COSY (DQF-COSY) spectra were recorded with gradient enhanced sequence at spectral widths of 3000 Hz in both f2 and f1 domains; the relaxation delays were of 1.0s. The total correlation spectroscopy (TOCSY) experiments were performed in the phase-sensitive mode with a mixing time of 90 ms. The spectral width was 3000 Hz. Nuclear Overhauser effect spectroscopy (NOESY) experiments were performed in the phase-sensitive mode. The mixing time was 500 ms and the spectral width was 3000 Hz. For all the homonuclear experiments, the initial matrix of 512 x 512 data points was zero-filled to give a final matrix of 1 k x 1 k points. Proton-detected heteronuclear correlations were measured.
Heteronuclear single-quantum coherence (HSQC) experiments (optimized for 1 J (H,C) = 140 Hz) were performed in the phase sensitive mode with field gradient; the spectral width was 12,000 Hz in f1 ( 13 C) and 3000 Hz in f2 ( 1 H) and 1.0 s of relaxation delay; the matrix of 1 k x 1 k data points was zero-filled to give a final matrix of 2 k x 2 k points. Heteronuclear 2 bond correlation (H2BC) spectra were obtained with T = 30.0 ms, and a relaxation delay of 1.0 s; the third-order low-pass filter was set for 130 < 1 J(C,H) < 165 Hz. Heteronuclear multiple bond coherence (HMBC) experiment (optimized for nJ (H,C) = 8 Hz) was performed in the absolute value mode with field gradient; typically, 1 H-13 C gHMBC were acquired with spectral width of 18,000 Hz in f1 ( 13 C) and 3000 Hz in f2 (1H) and 1.0 s of relaxation delay; the matrix of 1 k x 1 k data points was zero-filled to give a final matrix of 4 k x 4 k points. Constant time inverse-detection gradient accordion rescaled heteronuclear multiple bond correlation spectroscopy (CIGAR-HMBC) spectra (8 > nJ (H,C) > 5) were acquired with the same spectral width used for HMBC. Heteronuclear single-quantum coherence-total correlation spectroscopy (HSQC-TOCSY) experiments were optimize for nJ (H,C) = 8 Hz, with a mixing time of 90 ms.

Plant material
Thymus longicaulis C. Presl was collected in a garrigue on the calcareous hills of Durazzano, (41°3′N, 14°27′E; southern Italy) in the vegetative state and identified by Dr. Assunta Esposito of the Dept. of Environmental, Biological and Pharmaceutical Sciences and Technologies of Second University of Naples (SUN). A voucher specimen (CE235) has been deposited at the Herbarium of the Department. Leaves of Thymus longicaulis were harvested and immediately frozen in liquid N 2 in order to avoid unwanted enzymatic reactions and stored at −80°C up to the freeze drying process. Once freeze dried they were powdered in liquid nitrogen and stored at −20°C until the extraction process was carried out.

Extraction and isolation of compounds
Dried leaf material was powdered and extracted by ultrasound assisted extraction (Elma ® Transonic Digitals) one hour with methanol. The extract was filtered on Whatman paper and concentrated under vacuum. After removal of the solvent, a dried crude extract was obtained (4.1 g) which was stored at -20°C until its purification. The methanol extract, dissolved in distilled water and shaken with EtOAc, give an aqueous and an organic fraction.
The first was chromatographed on Amberlite XAD-4 and eluted first with water, to eliminate sugars, peptides, free amino acids and other primary metabolites, and then with methanol. The alcoholic eluate furnished 1. Fraction B, was chromatographed by RP-18 CC furnishing two fractions: fraction B1 contained pure 6 (20.1 mg), while B2 gave pure compound 7 (4.5 mg). Fraction C contained pure compound 4 (89 mg), fraction D, instead, re-chromatographed by RP-18 CC furnished a fraction identified as compound 9 (10.9 mg). Fraction E was purified by SiO 2 TLC (0.5 mm), eluting with the lower phase of the biphasic solution CHCl 3 /MeOH/0,1% TFA (13:7:2), and gave two spots. The first spot was identified as pure 11 (2.8 mg), while the second spot as the metabolite 10 (1.5 mg). Finally, fraction F after re-chromatography on RP-18 CC (MeOH: H 2 O polarity decreasing solutions) furnished a fraction identified as compound 12 (4.5 mg).

Results and Discussion
The phytochemical investigation of the methanol extracts of thyme led to isolation and characterization of thirty compounds (figures 1 and 2) belonging to different classes of secondary metabolites: isoprenoids, cinnamic acid derivatives, and flavones. In particular compounds 1 and 2 were identified as tuberonic acid 13-O-β-D-glucopyranoside and 4, 7-megastigmadien-3-one 9-O-β-D-glucopyranoside, respectively, both isolated from seeds of Astragalus complanatus [10] .  Metabolites 3 and 4 are two ursane triterpenes. In particular, 3 was identified as 3β-hydroxy-urs-12-en-28-oic acid, known as ursolic acid, while compound 4 has been character-ized as pomolic acid, by comparison of its spectral data with those reported in literature data. Both triterpenes were as reported as constituent of Annurca apple fruits [11] . Metabolites 5-7 were identified as caffeic acid derivatives (figure 2); in particular, compound 5 was recently reported as constituent of several aromatic Mediterranean plant species [8] . Compound 6 was elucidated as salvianolic acid K, previously isolated from Salvia deserta [12] .
NMR data of 7, known as 4-O-β-D-glucopyranosyl rosmarinic acid, was in good accordance with [13] , that reported this compound as constituent of Sanicula lamelligera while metabolite 8 was identified as methyl caffeoate.
In order to evaluate the antioxidant efficacy of the isolated pure metabolites from T. longicaulis leaves, the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) spectrophotometric method was performed as previously reported using five decreasing concentrations of pure metabolites. The results are reported in ( figure  3). The polyophenols showed the highest radical scavenging activity. In particular rosmarinic acid (5) and salvialonic acid (6) reducing DPPH concentration of 94 % and 67 %, respectively at highest concentration, with considerable radical scavenging activity also at 100μM. Rosmarinic acid is distributed in 26 plant families, and its biological activity has been extensively examined. Earlier studies ascribed to rosmarinic acid antiviral, antibacterial, anti-inflammaroty and antioxidant properties [19] . Recently, others interesting biological effects of rosmarinic acid have been reported: antifibrotic activity, protection of neurons against insults, suppression of UVB-induced alterations to human keratinocytes, inhibition of bone metastasis from breast carcinoma [20] .
Salvianolic acid K is reported in the literature for its antioxidant [21] and aldose reductase inhibitory activities [22] .
Isocsutellarein derivatives have also been described for its important beneficial activities: antioxidant [27] antinociceptive and anti-inflammatory [28] and significant inhibitory activity against osteoclast differentiation [29] .
Spices are widely investigated for their essential oils content in term of chemical composition and biological activity. Nevertheless, spices are also abundant sources of polyphenols which have interesting biological properties as recently reported also by Kindl, et al (2015) [30] that report antioxidant and acetylcholinesterase inhibitory activities of the ethanolic extracts of six selected Thymus species growing in Croatia. Consumption of spices has been implicated in the prevention of cardiovascular diseases, carcinogenesis, and inflammation mainly due to the presence of polyphenols.