Lignans and indole alkaloids from the seeds of Centaurea vlachorum Hartvig (Asteraceae), growing wild in Albania and their biological activity

Abstract The present phytochemical investigation of the seeds of Centaurea vlachorum led to the isolation and characterisation of four compounds including two indole alkaloids N-(p-coumaroyl) serotonin (1) and moschamine (2) and two dibenzylbutyrolactone lignans matairesinol (3) and arctiin (4). This is the first report on the isolation of non-volatile secondary metabolites from C. vlachorum. The chemataxonomic significance of these compounds was summarised. Moreover, the isolated compounds were tested for their free radical scavenging activity using the following in vitro assays: (i) interaction with the free stable radical of DPPH (1,1-diphenyl-2-picrylhydrazyl), (ii) inhibition of linoleic acid peroxidation with the dihydrochloric acid of 2,2-Azobis-2-amidinopropane (AAPH). Finally, their inhibitory activity towards soybean lipoxygenase was evaluated, using linoleic acid as substrate.


Introduction
The large polymorphous genus Centaurea L. (Asteraceae, Cardueae) known also as Knapweeds comprises about 600 species; it includes grassy plants, from annual to perennial, rarely suffruticose. Centaurea vlachorum Hartvig was considered as Greece's endemic species known from only two mountains, Milea and Aftia of Northern Pindos (Hartvig 1981) until 2009 when Shuka and Kit Tan reported its occurrence in NE Albania, exceeding its distribution range more in north (Shuka & Tan 2009). In Albania, it occurs in two localities, Kunora of Lura Mt, Dibra district, at altitudes 1600-2000 m and Qafa e Kumbulles, Kukesi district, at altitudes 1100-1300 m (Shuka et al. 2011). The existing information on the phytochemicals produced by C. vlachorum is scarce. Only the volatiles constituents from the leaves, flowers and stems has been analysed to date (Hodaj et al. 2015). To the best of our knowledge, there are no reports in the literature concerning the chemical profile of non-volatile compounds of C. vlachorum. To fill the existing gap and continuing our research on the chemical constituents of Centaurea spp. (Lazari et al. , 2000Skaltsa et al. 1999Skaltsa et al. , 2000aSkaltsa et al. , 2000bKarioti et al. 2002;Panagouleas et al. 2003), and our search for new compounds of pharmacological interest, we now report the investigation of the seeds of C. vlachorum Hartvig.

Chemotaxonomic significance
The present phytochemical investigation of the seeds of C. vlachorum led to the isolation and characterisation of four compounds including two indole alkaloids: N-(p-coumaroyl) serotonin (1) Shoeb et al. 2006), and moschamine (2) (Sarker et al. 1997), and two dibenzylbutyrolactone lignans: matairesinol (3) (Rahman et al. 1990) and arctiin (4) (Nishibe et al. 1993). The structures of the isolates are given in Figure 1 and were elucidated on the basis of 1-D and 2-D NMR spectral analyses (Tables S1-S4 and Figures S1-S19). This is the first report on the isolation of chemical constituents from the seeds of C. vlachorum. The distribution of these four compounds within the genus Centaurea is summarised in Table S5.
The two isolated lignans, matairesinol (3) and arctiin (4), belong to the dibenzylbutyrolactone group of lignans, and have previously been isolated from several other Centaurea species (Table S5). Also, several lignans of this group occur in various species of the Asteraceae family (Ayres & Loike 1990;Zhang et al. 2014). The co-occurrence of these lignans in the species of Centaurea might has some chemotaxonomic significance.
The chemical profile of C. vlachorum seeds is in a good agreement with the previous reports of Centaurea genus.

Biological activity
All the isolated compounds were evaluated for their antioxidant and antiiflammatory activities (Table S6). Matairesinol (3) is presenting the highest interaction with the stable radical DPPH, followed by moschamine (2) and N-(p-coumaroyl) serotonin (1). For the samples (2) and (3), the reducing activity is increased by the time e.g. it enhances after 20 min of interaction and it is higher after 60 min. It is time dependent with the exception of N-(p-coumaroyl) serotonin which presents higher interaction at 20 min and lower after 60 min. Arctiin (4) did not show any reducing ability. Concerning the free radical scavenging activity of arctiin (4) and matairesinol (3), our results are in agreement with the bibliography (Shoeb et al. 2005). The most prominent antioxidant activity of matairesinol could be attributed to the presence of the highest number of phenolic hydroxyl groups (four -OH) in the molecule. Arctiin (4) and matairesinol (3) were also reported to be active compounds against colon cancer cells in vitro. The presence of a sugar moiety in the molecule tends to reduce significantly the anticancer activity of these compounds. It can be assumed that the presence of a sugar group could prevent the effective transport of these compounds through the cell membrane, hence their reduced biological activities (Shoeb et al. 2005). Matairesinol has been found to have strong inhibitory effect on the proliferation of cultured human cell lines such as skin epidermoid carcinoma (A431) cell line, cervix epithelial adenocarcinoma (HeLa) and breast epithelial adenocarcinoma (MCF7) cell lines. Arctiin was also found to be active against the HeLa and A431 tumour cells, while moschamine exerted high or moderate potency selectively against the A431 cell line (Csapi et al. 2010).
Also for N-(p-coumaroyl) serotonin (1) and moschamine (2), our results are in good agreement with the previous reported data, as they have shown significant free radical scavenging (antioxidant) activity and also were found to be toxic in brine shrimp lethality . N-(p-coumaroyl) serotonin (1) and moschamine (2) have been proven to be potent melanin synthesis inhibitors and thus may be considered as promising candidates for novel whitening agents (Roh et al. 2004). N-(p-coumaroyl) serotonin has been reported to possess antifungal activity against Aciculosporium take (pathogenic fungi to bamboo) (Tanaka et al. 2003).
While the interaction with the free stable radical of DPPH of all the compounds were in good agreement to that of recently published data, they have never been tested for the inhibition of lipid peroxidation of sodium linoleate induced by the dihydrochloric acid of 2,2-Azobis-2-amidinopropane (AAPH), and for their inhibitory activity towards soybean lipoxygenase, using sodium linoleate as a substrate. N-(p-coumaroyl) serotonin (1) and arctiin (4) showed high antilipid peroxidation activity ranged from 82.3 to 81.6% higher than the reference compound trolox (73%) whereas, moschamine (2) did not present any effect. Matairesinol (3) presented lower activity of 72.2%.
The tested compounds (3) and (2) significantly inhibit soybean lipoxygenase (~80%), whereas compounds (4) and (1) did not exhibit any inhibition. This action could be attributed to the presence of methoxy groups in both active compounds (2) and (3) and to the lack of the sugar moiety in compound (3), as lipophilicity is one of the important factors to biological response.