A new biphenanthrene with cytotoxic activity from Liparis nervosa (Thunb.) Lindl

Abstract 2,7,2′-Trihydroxy-3,4,4′7′-tetramethoxy-1,1′-biphenanthrene (1), a previously undescribed biphenanthrene, and five known phenanthrenes, i.e. 2,5-dihydroxy-4-methoxy-9,10-dihydroxyphenanthrene (2), 2,4-dihydroxy −7-methoxy-9,10-dihydroxyphenanthrene (3), 7-hydroxy-2-methoxy-phenanthrene-1,4-dione (4), 7-hydroxy-2-methoxy-9,10-dihydrophenanthrene-1,4-dione (5), and 4,4′,7,7′-tetrahydroxy-2,2′-dimethoxy-9,9′,10,10′-tetrahydro−1,1′-biphenanthrene (6) were isolated from the whole plant (stems, leaves, roots and fruits) of Liparis nervosa (Thunb.) Lindl., which is a medicinal plant of the genus Liparis in the Orchidaceae family. The structures of isolates were identified using spectroscopic methods, including NMR and mass spectrometry. Additionally, the cytotoxic potency of all the isolates against human lung cancer A549 cell line was evaluated by an MTT assay. All the isolated compounds showed cytotoxic activities with IC50 values in the range of 10.20 ± 0.81 to 42.41 ± 2.34 μM. The obtained data highlight the importance of L. nervosa as a source of natural lead compounds for cancer therapy. GRAPHICAL ABSTRACT


Introduction
Liparis nervosa (Thunb.)Lindl.belongs to the Orchidaceae family.It has a cylindrical stem, two to five entire leaves and capsules.The raceme usually has not more than 10 flowers (Flora of China 1999).The whole plant of L. nervosa is a kind of traditional Chinese medicine called 'Jian-Xue-Qing' in China (Chinese Materia Medica 1999).It has a long history of clinical use in China with the function of clearing heat and removing toxins, and they are usually decocted with water to get the fluid for later use.(Chinese Materia Medica 1999).L. nervosa has been reported to have phenanthrenes, which are a type of well-known secondary metabolites for their multiple biological activities, including cytotoxic (Liu et al. 2016;Liu et al. 2019;Fu et al. 2022), anti-inflammatory (Wang et al. 2014), and antioxidant activities (Wang et al. 2014;Boudjada et al. 2019).The family Orchidaceae, one of the largest flowering plant families, is an abundant source of phenanthrenes, which are one of the characteristic and effective constituents of this family as well.Previous phytochemical investigations on L. nervosa have led to the isolation of several cytotoxic phenanthrenes (Liu et al. 2016).In a continuation of our work aiming at searching for more biologically active phenanthrenes from the Liparis genus, one of the most represented genera, the whole plant of L. nervosa were extracted and isolated, which resulted in the isolation of a new biphenanthrene (1), together with five known phenanthrenes (2-6) (Figure 1).In the present study, we reported the isolation, structure elucidation and cytotoxic activity of the isolates so as to provide more useful data for the further investigation and utilization of L. nervosa.
since phenanthrenes have been reported to have cytotoxic activity (Liu et al. 2016;Liu et al. 2019;Fu et al. 2022), the cytotoxic potency of 1-6 against human lung cancer A549 cell line was evaluated by an MTT assay.The results were shown in Table s2, all the isolated compounds showed cytotoxic activities with IC 50 values ranging from 10.20 ± 0.81 to 42.41 ± 2.34 μM.The IC 50 values of the two identified biphenanthrenes (1 and 6) and the two phenanthraquinones (4 and 5) were in the range of 10.20 ± 0.81 to 17.56 ± 0.69 μM, much lower than those of the two monophenanthrenes (2 and 3, IC 50 = 42.41 ± 2.34/35.73± 3.55, respectively).Previous study revealed that dimerization of phenanthrenes is a very important factor for the inhibition of cancer cell growth (Zhang et al. 2005) and the quinone substructure is crucial for enhancement of cytotoxic activity of phenanthrenes (sanchez-Duffhues et al. 2009) our investigation is consistent with these reports.

General
All spectra were recorded on a Bruker Avance 600 NMR spectrometer (14.1 T) and equipped with a broad band observe (BBO) 5 mm probe head. 1 H spectra were acquired with 32 transients, a spectral width of 9013.7 Hz (corresponding to 15 ppm) and 64K data points for an acquisition time of 7,3 s.Heteronuclear bidimensional 1 H-13 C HsQC and 1 H-13 C HMBC experiments were acquired with a spectral width of 15 and 200 ppm for proton and carbon dimension, respectively.HResIMs spectral data were acquired using a Bruker Maxis mass spectrometer (Germany) with a Waters esI source (Micromass, Manchester u.K.).The infusion experiments were carried out at the syringe flow rate of 5 μL/min, with source parameters optimized as follows: capillary voltage 2.7 kV in negative ion mode, cone voltage 27 V, source temperature 100 °C, desolvation temperature 150 °C, cone gas flow 40 L/h, desolvation gas flow 400 L/h.spectral data were acquired in scan mode for 2 min in the mass range 100-1000 Da for compounds 1-6.silica gel (200-300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), Merck,Germany) and sephadex LH-20 (Ge Healthcare, sweden) were used for column chromatography.A CXTH LC3000 instrument (Beijing, China) equipped with an uV Detector and an ODs column (5 C 18 -Ms-ІІ column, 250 mm × 10 mm, 5 μM, Cosmosil, Japan) was used for semi-preparative HPLC separations.

Cell line and cell culture
A549 cancer cell line was purchased from the Cell Bank of Chinese Academy of sciences (shanghai, China).Cells were cultured in RPMI-1640 medium containing 10% FBs, 100 u/mL penicillin, and 100 mg/mL streptomycin in a 5% CO 2 -humidified incubator at 37 °C.

Plant material
The plant materials (stems, leaves, roots, and fruits) were collected from Wuling Mountain (1025 metres above sea level) of China, located on GPs 29°33′6.54″N and 107°34′10.3″e, in Feb 2014.They were identified by Prof. Hu-Yin Huai, school of Biological science and Technology of Yangzhou university of China.A voucher specimen (No.JXQ20140205) was deposited at the Herbarium of Pharmacy Department, College of Medicine of Yangzhou university.