New bioactive labdane diterpenoids from Marrubium aschersonii

Abstract A phytochemical investigation of the ethanol extract of Marrubium aschersonii Magnus (Lamiaceae) collected from Tunisia led to the isolation and identification of two new labdane diterpenoids, marrubaschs A (1) and B (2), along with two known compounds (3 and 4). Their structures were elucidated by spectroscopic methods including HRESIMS and NMR techniques. All compounds were evaluated for their inhibitory effects on the nitric oxide (NO) production induced by lipopolysaccharide in RAW 264.7 macrophage cells. Compound 2 exhibited weak inhibition of NO production with an IC50 value of 35 ± 1.0 μM.


Introduction
The genus Marrubium (Lamiaceae) comprising about 40 species is distributed in Europe, North Africa and Asia (Mabberley 1997;Meyre-Silva and Cechinel-Filho V 2010). Plants of Marrubium have been found to be rich in diterpenoids, sterols, flavonoids and phenylpropanoids, which possessed a wide range of pharmaceutical uses such as antinociceptive, antispasmodic, antioedematogenic, antimicrobial, antiviral and anti-inflammatory activities (De Jesus et al. 2000;Sahpaz et al. 2002;Stulzer et al. 2006;Zaabat et al. 2011;Sanna et al. 2015). Marrubium aschersonii Magnus is a perennial plant endemic to the north-west and south of Tunisia in sunny sites with low to medium altitudes or on low steep slopes. Covered by thick tomentum on its stems and branches, the leaves of this species are flabellate and whitish on both sides. Its flowers are pink with tubular calyx topped with 6-8 irregular, divergent, piquant teeth at the base. To the best of our knowledge, only the chemical constituents of the essential oil of M. aschersonii have been studied (Hamdaoui et al. 2013). In our continuing search for structurally diverse and biologically significant metabolites from medicinal plants (Dong et al. 2014;Zhu et al. 2015), two new diterpenoids (1 and 2) together with two known compounds (3 and 4) were isolated from the ethanolic extract of this plant collected from the Kroumiria region in north-west of Tunisia. All compounds were evaluated for their inhibitory effects on the nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW 264.7 macrophage cells. Compound 2 exhibited weak inhibition of NO production with an IC 50 value of 35 ± 1.0 μM. Herein, this paper focuses on the isolation and structure elucidation of labdane diterpenoids from M. aschersonii, as well as their anti-inflammatory activities.  (Yin et al. 2007). In addition, ten other carbons including six methylenes (one oxygenated), one methine and three quaternary carbons (one oxygenated and two high-field ones) were observed in the 13 C NMR and DEPT spectra of 1. The collective data implied that 1 possessed a labdane diterpenoid skeleton and showed high similarities to those of leoleorin A (Wu et al. 2013), a labdane diterpenoid isolated from Leonurus species. Comparison of the NMR data of 1 with those of leoleorin A revealed the presence of an additional acetyl group and a hydroxymethyl (δ C 71.2) in 1 instead of the methyl group in leoleorin A. The acetyl group was linked to the hydroxymethyl (C-19) by the correlations of H 2 -19 (δ H 4.11, q, J = 11.0 Hz) to the acetyl carbonyl (δ C 172.5). Detailed 2-D NMR analyses of 1 (see S9 in Supplementary Material) supported its planar structure as depicted. The relative configuration of 1 was determined to be the same as that of leoleorin A by a NOESY experiment and by comparison of their 13 C NMR data. In particular, the NOESY correlations of H 2 -19/H 3 -20, H 3 -20/H-8 and H-8/H 2 -11 indicated that these protons were co-facial and were arbitrarily assigned as β-oriented, while H 3 -18 and H 3 -17 were α-oriented. The orientation at C-8, C-9 and C-10 was further confirmed by comparison of the 13 C NMR data of 1 with those of leoleorin A [C-8 (δ C 48.1 in 1; δ C 47.8 in leoleorin A), C-9 (δ C 80.9 in 1; δ C 79.2 in leoleorin A), and C-10 (δ C 47.2 in 1; δ C 46.5 in leoleorin A)]. Thus, the structure of 1 was established and named marrubasch A ( Figure 1).

Results and discussion
Compound 2 exhibited a molecular formula of C 21 H 34 O 6 as determined by HRESIMS at m/z 365.2320 [M − H 2 O + H] + (Calcd 365.2323). The NMR data of 2 bore a resemblance to those of sibiricinone C (Boalino et al. 2004), except for the presence of a methylene and hydroxymethyl (δ C 69.1) in 2 instead of the keto and methyl group in sibiricinone C. 1 H-1 H COSY between H-6 (δ H 4.21) and H-7 (δ H 1.53, 1.67), HSQC correlation from H-7 (δ H 1.53, 1.67) to the methylene (δ C 38.9) and HMBC correlation from CH 3 -17 to the methylene (δ C 38.9) located the methylene at C-7. This was further supported by the significant upfield-shifted carbon signals for C-6 and C-8 in 2 with respect to those in sibiricinone C (δ C 65.6 and 31.3 in 2; δ C 75.9 and 46.1 in sibiricinone C). HMBC correlations from H-19a [δ H 3.17 (1H, d, J = 11.5 Hz)] to C-3 (δ C 40.6) and C-5 (δ C 49.4) located the hydroxymethyl at C-19. The relative configuration of 2 was assigned to be the same as that of sibiricinone C by comparison of their 1-D NMR and NOESY data. The NOESY correlations of H-8/H 3 -20 and H 3 -20/H 2 -19 indicated H-8, H 2 -19 and H 3 -20 were co-facial and assigned in β-oriented, while H 3 -18 and H-6 were α-oriented. Therefore, compound 2 was given the trivial name marrubasch B.
The known compounds marrubiin (3) (Hussein et al. 2003) and marrubenol (4) (EL Bardai et al. 2003) were identified by comparison of their NMR data with those in the literature.
Compounds 1-4 were evaluated for their inhibitory effects on NO production in LPSactivated RAW 264.7 macrophages. Quercetin, a natural NO inhibitor, was used as the positive control (IC 50 = 16.1 ± 1.3 μM). The cell viability was determined initially by the MTT method to determine whether the inhibition of NO production was caused by the cytotoxicity of the tested compounds. As a result, all compounds showed no obvious cytotoxic effects (over 90% cell survival) at the concentration of 50 μM on RAW 264.7 cells. Subsequent NO inhibition assay showed that compound 2 could inhibit NO release, with an IC 50 values of 35.0 ± 1.0 μM, while the other compounds were considered to be inactive (IC 50 > 100 μM).

Plant material
The aerial parts of M. aschersonii were collected during its flowering period from May to June in the wet sandy and rubble localities of the Kroumiria region (north-west of Tunisia), and was identified by Dr. Ridha El Mokni, botanist in the Laboratory of Botany and Plant Ecology, Faculty of Sciences, university of Bizerta, Jarzouna, Bizerta, Tunisia. A voucher specimen (accession number: OXZC201409) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen university.

Cell culture
The RAW 264.7 mouse macrophage cell line was purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China), and was cultured in Dulbecco's modified Eagle medium (DMEM, Gibco Invitrogen Corp., Carlsbad, CA, uSA) which was supplemented with 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin. The cells were placed at 37 °C in a humidified incubator containing 5% CO 2 . The inhibitory activity of the isolated compounds towards NO production was determined using the Griess reagent system (Beyotime, Beijing, China). Cells were cultured in each well of 96-well plates in the density of 5 × 10 4 cells/well with 100 μL DMEM for 24 h. Then, test samples were diluted with DMEM，and the diluted samples stocked in DMSO were added to wells as well as LPS 2 μg/mL as stimuli. The final drug concentration was 50 μM with 1% DMSO. Wells treated with only LPS were served as model control and wells treated with neither LPS nor test samples were served as blank control (all contained 1% DMSO). After 24 h incubation, half of the medium (50 μL) in each well was harvested

MTT assay
The cytotoxicity of the isolated compounds towards RAW 264.7 cells was determined by MTT assay. RAW264.7 cells were planted in 96-well plates (5 × 10 3 /well) for 24 h. Then, they were treated with test samples which dissolved in DMSO and diluted in 100 μL DMEM making the final drug concentration 50 μM and 1% DMSO. 1% DMSO served as solvent control. Wells without cells containing only 100 μL DMEM were served as blank control. Twenty-four hours later, 20 μL solution MTT was added to each well. After incubation for 4 h, the medium was removed and 100 μL DMSO was added to each well, then the absorbance (A) was detected at 490 nm using a microplate reader. The inhibition of cell growth was calculated according to the following formula: % Inhibition = [1 − (A sample − A blank )/(A solvent − A blank )] × 100.

Inhibitory activity towards NO
NO release was assessed by a colorimetric assay based on a diazotisation reaction using the Griess reagent system. After 50 μL Griess reagent I and 50 μL Griess reagent II were added in each well, the absorbance (A) was measured at 540 nm using a microplate reader. The inhibition of NO release was calculated according to the following formula: % Inhibition = [1 − (A sample − A blank )/(A model − A blank )] × 100.

Conclusions
Two new labdane diterpenoids, marrubaschs A (1) and B (2), along with two known analogues, marrubiin (3) and marrubenol (4), were isolated from the plants of M. aschersonii. The structures of the new compounds were determined by extensive NMR spectroscopic analysis. As the isolated diterpenoids shared the same scaffold with that of andrographolide, a well-known anti-inflammatory natural product (Low et al. 2015), a subsequent anti-inflammatory bioassay regarding the NO inhibitory ability of these compounds was conducted. Only compound 2 exhibited weak inhibition with an IC 50 value of 35 ± 1.0 μM. It is possible that the lack of lactone features in the current structures caused the significant reduce of the anti-inflammatory activity. This is the first report of the chemistry of the aerial part of M. aschersonii.

Disclosure statement
No potential conflict of interest was reported by the authors.