Anti-Inflammatory and Cytotoxic Potential of New Phenanthrenoids from Luzula sylvatica

Phenanthrenoids have been widely described, in the Juncaceae family, for their biological properties such as antitumor, anxiolytic, anti-microbial, spasmolytic, and anti-inflammatory activities. The Juncaceae family is known to contain a large variety of phenanthrenoids possessing especially anti-inflammatory and cytotoxic properties. Luzula sylvatica, a Juncaceae species, is widely present in the Auvergne region of France, but has never been studied neither for its phytochemical profile nor for its biological properties. We investigated the phytochemical profile and evaluated the potential anti-inflammatory activities of L. sylvatica aerial parts extracts. A bioassay-guided fractionation was carried out to identify the most active fractions. Nine compounds were isolated, one coumarin 1 and eight phenanthrene derivatives (2–9), including four new compounds (4, 5, 8 and 9), from n-hexane and CH2Cl2, fractions. Their structures were established by HRESIMS, 1D and 2D NMR experiments. The biological properties, especially the anti-inflammatory/antioxidant activities (ROS production) and antiproliferative activity on THP-1, a monocytic leukemia cell line, of each compound, were evaluated. Three phenanthrene derivatives 4, 6, and 7 showed very promising antiproliferative activities.


Introduction
Juncaceae represents a large family of plants with nearly 500 different species distributed in seven genera, the most representative ones are the Juncus and the Luzula genera (around 350 and 110 species respectively).
Molecules 2020, 25 Plants belonging to this family are widespread all around the world, the Juncus and Luzula genera grow in both hemispheres, generally in badly drained soils. Several studies described the use of Juncaceae species for the traditional treatment of dysuria, fidgetiness, irritability, insomnia, and inflammation [1]. In China, J. inflexus is commonly used for its sedative effect, as J. effusus and J. conglomeratus are used for skin diseases [2,3] in the Basque region. Considering Luzula species, no ethnopharmacological uses have been reported yet. The biological activities of Juncaceae seem mostly due to the presence of flavonoids [4], coumarins [5], terpenoids [6], and phenolic acid derivatives [7]. In addition, phenanthrene derivatives exerting potent anti-inflammatory [8,9] and cytotoxic activities [10,11] have also been identified in several members of the family. Such constituents are scarcely distributed in the plant kingdom and have mainly been reported in the Orchidaceae [11] and the Combretacae families [12,13]. Regarding Juncaceae, most of the chemical studies focused on the Juncus genus leading to the identification of more than 100 phenanthrenoids [1]. Even if a large variety of phenanthrenoids have been described, most of them possess a phenanthrene or a 9,10-dihydrophenanthrene backbone, often substituted at positions 2, 5 and 7 with hydroxyl, methyl, or vinyl groups. A vinyl group at position 5 on the phenanthrene skeleton could be considered as a chemotaxonomic marker for Juncus species [11].
To our knowledge, very limited investigations have been reported on the Luzula species especially on Luzula sylvatica (L. sylvatica) [10]. This species is widely present in the Auvergne area, a region of France which possesses diversified flora, particularly due to the variety of landscapes and climatic conditions. L. sylvatica, commonly named Great Wood-Rush, is a perennial plant, measuring between 30 and 110 cm, and growing mostly in wet soils (humid forest, in the shade). The numerous dark green basal leaves, possessing small white cils, are between 6 and 12 mm wide and 10-25 cm long. The inflorescence is loose, subdivided in open panicles, and flowers are brownish, sessile and group by 2-5 in glomerules [14].
Like most of the Juncaceae plants, L. sylvatica seems to contain phenanthrenoids [1,15]. This class of compounds is known to different have biological properties such as antitumor, anxiolytic, antimicrobial, spasmolytic and anti-inflammatory activities [11]. Considering the strong chemotaxonomical and biological interests of phenanthrenoids, the present study aimed to investigate the chemical composition of a methanolic extract from L. sylvatica aerial parts focusing particularly on this chemical class. In addition, anti-inflammatory and antiproliferative properties of the studied extract and its isolated compounds were evaluated.

Results and Discussion
A methanolic extract, prepared from the aerial parts of L. sylvatica, was partitioned using increasing polarity liquid-liquid extraction. The five fractions ((n-hexane), dichloromethane (CH 2 Cl 2 ), ethyl acetate (EtOAc), n-butanol (n-BuOH) and water) were tested for their anti-inflammatory/antioxidant activities by evaluating the production of ROS (reactive oxygen species) from blood leucocytes. All the fractions were able to decrease significantly the ROS production ( Figure 1). The EtOAc, CH 2 Cl 2 and n-Hex fractions were the most active ones (with a decrease of 60 to 75% compared to control). Analysis of the EtOAc fraction by LC-MS, LC-MS 2 , and UV spectrometry, in comparison with standards, led to the characterisation of luteolin, as its major constituent, as well as 1,3-O-dicaffeoylglycerol (ananasate) [16][17][18] and 3',5,5',7-tetrahydroxyflavanone [19]. Thus, the activity of EtOAc fraction could be due to the presence of luteolin, a flavone known for its anti-inflammatory properties [20,21]. . Data are shown as means ± SEM (n = 3-6); * p < 0.05 compared with Control. All results are expressed as a percentage, with control (i.e. cells with PMA but without extract) normalized as 100%.
The HRESIMS of compound 4, isolated as a pale-yellow oil, furnished deprotonated ion peak [M − H] − at m/z 247.1127 (calcd 247.1128) corresponding to a molecular formula of C 18 H 16 O. The 1 H NMR data showed the presence of three vinylic protons, six aromatic methine protons, two methyl groups and a hydroxyl group. A comparison of the NMR data of compound 4 with those obtained for juncunol and the HRESIMS data, suggested the presence of a phenanthrene backbone. The 13 C NMR data confirm the presence of the vinylic function (δ C 142.0 and 114.2 ppm) and the hydroxyl group (δ C 151.1 ppm). HMBC correlations from H-13 (δ H 5.78 and 5.44) to C-5 (δ C 137.1) and from H-12 (δ H 7.47) to C-6 (δ C 130.6) suggested that the vinyl group was located at C-5. The HMBC correlations from H-6 (δ H 7.45) and H-8 (δ H 7.60) to C-14 (δ C 21.3) suggested that the first methyl group was located at position 7. Moreover, a HMBC correlation from H-14 (δ H 2.54) to C-7 (δ C 134.9) confirmed this hypothesis. The HMBC correlation from H-8 (δ H 7.60) to C-9 (δ C 127.8) and the downshield chemical shifts of H-9 and H-10 protons (δ H 7.69 and 7.89 ppm respectively) allowed us to confirm the skeleton of compound 4 as a phenanthrene backbone. The chemical shift of C-5a (δ C 127.3) was determined based on the HMBC correlations from H-4 (δ H 8.65), H-6 (δ H 7.45), H-8 (δ H 7.60) and H-9 (δ H 7.69) to C-5a. The chemical shift of C-1a (δ C 133.4) was confirmed by HMBC correlations from H-4 (δ H 8.65) and H-9 (δ H 7.69) to C-1a. The positions of the second methyl and the hydroxyl groups were determined by HMBC data. Correlations from H-11 (δ H 2.61) to C-1 (δ C 117.3), C-1a (δ C 133.4) and C-2 (δ C 151.1), from H-3 (δ H 7.08) to C-1 (δ C 117.3) and from H-4 (δ H 8.65) to C-2 (δ C 151.1) suggested that the second methyl group was located at C-1 and the hydroxyl group at C-2. The chemical shift of C-4a (δ C 125.8) was confirmed by HMBC correlation from H-3 (δ H 7.08) and H-10 (δ H 7.89) to C-4a. The chemical shift of C-8a (δ C 132.0) was confirmed by HMBC correlation from H-10 (δ H 7.89) to C-8a. All these data confirmed the structure of compound 4 as 1,7-dimethyl-5-vinylphenanthrene-2-ol, named dehydrojuncunol.
Concerning compound 5, the HRESIMS furnished deprotonated ion peak [M − H] − at m/z 265.1242 corresponding to a molecular formula of C 18 H 18 O 2 (calculated 265.1234). Comparison with the 1 H and the 13 C NMR spectra of compound 3 suggested that compound 5 possesses a 9,10-dihydrophenanthrene backbone with a vinyl group located at C-5, a methyl group at C-7 and a hydroxyl group at C-2. The 1 H and 13 C data established the presence of a hydroxylmethyl group (δ H 5.01), singlet for two protons (δ C 60.2). HMBC correlations from H-11 (δ H 5.01) and C-2 (δ C 155.3), C-1a (δ C 138.4) and C-3 (δ C 113.4) and the correlation from H-3 (δ H 6.78) and C-2 (δ C 155.3), C-1a (δ C 138.4) suggested that the hydroxyl group is located at C-2 and the hydroxyl methylene group at C-1. The structure of the new isolated compound 5 was confirmed as 1-hydroxymethyl-7-methyl-5-vinyl-9,10-hydrophenanthrene-2-ol, named sylvaticin A.
For compound 8, isolated as a pale-yellow oil, the molecular formula of C 19 H 20 O 2 was established by HRESIMS (m/z 263.1426, [M + H − H 2 O] + ; calculated m/z 263.1430) as well as the presence of a hydroxyl group. The chemical shift of a methine at δ C 64.0 suggested the presence of the hydroxyl group on a non-aromatic carbon. 1 H NMR data showed the presence of three vinylic protons, two methyl groups and a methoxy group. Comparison with the 1 H and the 13 C NMR spectra of compound 3 suggested that compound 8 is a 9,10-dihydrophenanthrene with a vinyl group located at C-5 and two methyl groups at C-1 and at C-7. The position of the methoxy group was determined by HMBC data. Correlations from H-4 (δ H 7.56) and H-12 (δ H 3.88) to C-2 (δ C 157.1) suggested that the methoxy group is located at C-2. The structure of compound 8 was confirmed by NOESY. This experiment was performed in CD 3 OD in order to obtain two different chemical shifts for the protons of the two methyl groups (δ H 2. 33 5.28). The structure of the new compound 8 was determined as 2-methoxy-1,7-dimethyl-5-vinyl-9,10-dihydrophenanthrene-10-ol, named sylvaticin B. The CD spectrum of 8 showed a negative Cotton effect at 239 nm and a positive Cotton effect at 280 nm, in agreement with the (S) configuration of carbon 10 according to literature [27].
The HRESIMS of compound 9 furnished a deprotonated ion peak [M − H] − at m/z 251.1077 (calcd 251.1078) corresponding to a molecular formula of C 17 H 16 O 2 . As the 1 H and 13 C NMR data were strongly similar to compound 3 NMR data, we could identify compound 9 as a 9,10-dihydrophenanthrene backbone possessing three substituents, two methyl groups at C-1 and C-7, and a hydroxyl group at C-2. The 1 H and 13 C NMR data showed the presence an aldehyde function with the corresponding carbon at δ C 193.5 ppm (CH) and a singlet proton at δ H 10.07 ppm. As previously reported in literature [28][29][30], the aldehyde function was located on C-5. This was confirmed by the HMBC correlations from H-12 (δ H 10.07) to C-6 (δ C 127.2). The position of all the substituents and the chemical shifts of all the protons and the carbons were attributed using HMBC and COSY correlations as described on Figure 3. The structure of the new compound 9 was determined as 2-hydroxy-1,7-dimethyl-9,10-dihydrophenanthrene-5-carbaldehyde, named sylvaticin C.
Molecules 2020, 25, x 5 of 10 previously reported in literature [28][29][30], the aldehyde function was located on C-5. This was confirmed by the HMBC correlations from H-12 (δH 10.07) to C-6 (δC 127.2). The position of all the substituents and the chemical shifts of all the protons and the carbons were attributed using HMBC and COSY correlations as described on Figure 3. The structure of the new compound 9 was determined as 2-hydroxy-1,7-dimethyl-9,10-dihydrophenanthrene-5-carbaldehyde, named sylvaticin C. The nine isolated compounds were tested on leucocytes ROS production as described above for the five fractions. Four of them inhibited ROS production significantly in a dose-dependent manner: compounds 2, 4, 8, and 1 (Figure 4), although the last one had to be tested at higher concentration to be effective. In addition to their anti-inflammatory activity, phenanthrenes were known to exhibit promising in vitro antiproliferative activities on various cancer cell lines [9,31,32]. Thus, the cytotoxicity of the isolated compounds was evaluated with a resazurin assay on THP-1, a monocytic leukemia cell line. The nine isolated compounds were tested on leucocytes ROS production as described above for the five fractions. Four of them inhibited ROS production significantly in a dose-dependent manner: compounds 2, 4, 8, and 1 (Figure 4), although the last one had to be tested at higher concentration to be effective.
Molecules 2020, 25, x 5 of 10 previously reported in literature [28][29][30], the aldehyde function was located on C-5. This was confirmed by the HMBC correlations from H-12 (δH 10.07) to C-6 (δC 127.2). The position of all the substituents and the chemical shifts of all the protons and the carbons were attributed using HMBC and COSY correlations as described on Figure 3. The structure of the new compound 9 was determined as 2-hydroxy-1,7-dimethyl-9,10-dihydrophenanthrene-5-carbaldehyde, named sylvaticin C. The nine isolated compounds were tested on leucocytes ROS production as described above for the five fractions. Four of them inhibited ROS production significantly in a dose-dependent manner: compounds 2, 4, 8, and 1 (Figure 4), although the last one had to be tested at higher concentration to be effective. In addition to their anti-inflammatory activity, phenanthrenes were known to exhibit promising in vitro antiproliferative activities on various cancer cell lines [9,31,32]. Thus, the cytotoxicity of the isolated compounds was evaluated with a resazurin assay on THP-1, a monocytic leukemia cell line. Data are shown as means ± SEM (n = 3); * p < 0.05 compared with Control. All results are expressed as a percentage, with control (i.e., cells with PMA but without extract) normalized as 100%.
In addition to their anti-inflammatory activity, phenanthrenes were known to exhibit promising in vitro antiproliferative activities on various cancer cell lines [9,31,32]. Thus, the cytotoxicity of the isolated compounds was evaluated with a resazurin assay on THP-1, a monocytic leukemia cell line.  1 and 9, all components revealed a strong cytotoxic activity, with IC 50 below 15 µM. The most effective compounds were 4, 6, and 7 (IC 50 of 3, 6, and 5 µM, respectively). Table 1. Cytotoxic effect of compounds 1-9 in THP-1 cells reported as IC 50 in µM. Cells metabolic activity was determined after 24 h of incubation with or without compounds at different concentrations (2, 5, 10 and 20 µM for compounds 2-9 and 10, 25, 50 and 100 µM for compound 1) by resazurin assay (n = 3). Doxorubicine was used as a positive control at 1 µM for our experiments. At 1 µM, the viability of THP-1 decreased at around 80% (21.4 ± 3% of the fluorescence after 24 h of incubation, with negative control normalized as 100%).
Flash chromatography was performed on a SPOT II FLASH (Interchim) with a monochromator detector. All chemical standards and references were purchased from Extrasynthese (Genay, France).

Plant Material
The aerial parts of L. sylvatica were collected in June 2017 (Cantal). A voucher specimen (CLF 110940) was deposited at the UniVegE herbarium of the Clermont Auvergne University.

Extraction and Isolation
Aerial parts of L. sylvatica (1.6 kg) were air-dried at room temperature in a dark room, powdered, then extracted with methanol (3 × 20 L each 24 h) and dried under vacuo to yield a greenish solid crude extract (195 g). This crude extract was then dissolved in water and extracted three times with increase solvent polarity to give five fractions: n-hexane (16.3%), CH 2 Cl 2 (0.5%), EtOAc (2.0%) n-butanol (20.6%) and water (60.6%).

Cytotoxicity Evaluation of the Compounds
The human monocytic leukemia cell line, THP-1 (American Type Culture Collection) was cultured and propagated at 37 • C in a humidified atmosphere of 5% CO 2 in a RPMI 1640 medium supplemented with 10% FBS, gentamicin 50 µg/mL and Gln 2 mM. Cells were placed in 96-well plates and incubated with the compounds for 24 h. Then resazurin (25 µg/mL) was added to the medium to track their viability. Fluorescence (excitation/emission: 544/590 nm) was recorded for 2 h using a microplate fluorometric reader (Tecan Spark®) [35].

Conclusions
Phenanthrenoids are common secondary metabolites in plants of the Juncaceae family, possessing antiproliferative activities. From aerial parts of L. sylvatica, a Juncaceae species widely present in Auvergne region in France, we isolated nine compounds from the apolar n-hexane and CH 2 Cl 2 fractions: coumarin 1 and eight phenanthrenoids (2-9). Among them, four new compounds were characterized: dehydrojuncunol 4, sylvaticin A 5, sylvaticin B 8, uncommonly substituted at C-10, and sylvaticin C 9. We demonstrated that phenanthrenoids 2, 4, and 8 inhibited ROS production significantly in a dose-dependent manner.
Compounds 4, 6, and 7 showed moderate cytotoxic effects on THP-1 cells with IC 50 lower than 6 µM. Further investigations will be carried out on the most active compounds, especially on new phenanthrenoid 4 in order to study its antiproliferative activity on other cancer cells and to explore its mechanism of action.