Phenolic compounds from Glycyrrhiza pallidiflora Maxim. and their cytotoxic activity

Abstract Twenty-one phenolic compounds (1–21) including dihydrocinnamic acid, isoflavonoids, flavonoids, coumestans, pterocarpans, chalcones, isoflavan and isoflaven, were isolated from the roots of Glycyrrhiza pallidiflora Maxim. Phloretinic acid (1), chrysin (6), 9-methoxycoumestan (8), isoglycyrol (9), 6″-O-acetylanonin (19) and 6″-O-acetylwistin (21) were isolated from G. pallidiflora for the first time. Isoflavonoid acetylglycosides 19, 21 might be artefacts that could be produced during the EtOAc fractionation process of whole extract. Compounds 2–4, 10, 11, 19 and 21 were evaluated for their cytotoxic activity with respect to model cancer cell lines (CEM-13, MT-4, U-937) using the conventional MTT assays. Isoflavonoid calycosin (4) showed the best potency against human T-cell leukaemia cells MT-4 (CTD50, 2.9 μM). Pterocarpans medicarpin (10) and homopterocarpin (11) exhibit anticancer activity in micromolar range with selectivity on the human monocyte cells U-937. The isoflavan (3R)-vestitol (16) was highly selective on the lymphoblastoid leukaemia cells CEM-13 and was more active than the drug doxorubicin


Introduction
Licorice (liquorice), obtained from the Glycyrrhiza species, is one of the most frequently used natural drugs in Europe, China, Japan and United states . The genus Glycyrrhiza comprises around 15 known species, half of which are pharmacopoeial ingredients for herbal preparations. Among all the Glycyrrhiza species, the most widely used in commerce are Glycyrrhiza glabra ('European licorice') and Glycyrrhiza uralensis ('Chinese licorice' , 'Gan Cao') (Chevallier 1996;Fiore et al. 2005). In China, only three species, G. uralensis, G. glabra and Glycyrrhiza inflata, are officially used as Gan-Cao; among them, G. uralensis is the mainstream species, and constitutes more than 90% of total licorice production. Some other species, including Glycyrrhiza glandulufera and Glycyrrhiza aspera, are also used as Gan-Cao in local areas (Li et al. 2011).
Our attention was attracted to Glycyrrhiza pallidiflora Maxim. which belongs to the section of false licorices Pseudoglycyrriza Krug. (Kruganova 1955). All species belong to this section do not contain glycyrrhizin and the roots of the species of Pseudoglycyrrhiza section are un-sweet (hayashi et al. 2000;Farag et al. 2012).
Chemical investigation in Glycyrrhiza has mostly focused on G. glabra, G. uralensis and G. inflata with little information on other species' chemical composition (Kinoshita et al. 1997;Dey et al. 2009;Zhang et al. 2009;Farag et al. 2012;Fu et al. 2013). G. pallidiflora from Russian Far East has not been systematically studied. According to literature (Liu et al. 1992;hayashi et al. 2000;Ammosov & Litvinenko 2007;Li et al. 2011), the composition of chemical constituents (triterpene saponins, chalcones and flavonoids) and morphological characteristics of the fruits in G. pallidiflora and G. glabra or G. uralensis were very different.
The aim of this study was to examine the constituents of extractive compounds, especially flavonoids, of the root of G. pallidiflora Maxim. collected in the Russian Far East (Primorsky Territory, near the south shore of Khanka Lake). One of the objectives of the present paper was to investigate the cytotoxic activity of the accessible flavonoids from G. pallidiflora. Recently, the flavonoids of licorice have attained a considerable interest because of their structural diversity and potent pharmacological actions, including anticancer, anti-inflammatory, immunomodulatory, hepatoprotective and cardioprotective activities. For example, chalcone isoliquiritigenin was reported to exhibit a variety of biological properties, such as anti-inflammatory, antioxidative and antitumour activities (Park et al. 2009). Licochalcone A, a major chalcone in licorice, was reported to possess antitumor activity (Fu et al. 2004). Chalcone echinatin (Fu et al. 2013) and isoflavonoid formononetin (Wang et al. 2013) are shown strong antioxidant activity by an (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) (ABTS)-based total antioxidant capicity assay. Some isoflavan-derived flavonoids in licorice, such as glabridin, licoricidin and licorisoflavan A, showed anti-inflammatory, antiulcer, antioxidative, antimicrobial and antitumour activities (Toshio et al. 2002;Fukai et al. 2003;La et al. 2011;Asha et al. 2013;Eerdunbayaer et al. 2014). Glycyrol, a coumestan-derived compounds, was also reported to possess anti-inflammatory activities (Shin et al. 2008). Quite recently, Glycyrrhiza flavonoids may become a valid alternative of potential basis for new hypoglycaemic and antidiabetic agents (Gou et al. 2015)

Isolation and identification of the compounds
The dried roots of G. pallidiflora were successively extracted with hexane, diethyl ether, tert-butyl methyl ether, ethyl acetate and 95% ethanol. Each extract was combined and concentrated under reduced pressure to give the whole extract. The components extracted with 95% ethanol were then partitioned between ethyl acetate (EA) and water, which separated the reserve sugars and dissolve flavonoids.
Structures of all compounds were elucidated on the basis of detailed spectroscopic analyses and by comparison with those of related model compounds or authentic materials.
Compounds 2-5, 7, 10-18 and 20 were isolated previously from G. pallidiflora. Phloretinic acid 1, flavones 6, coumestans 8, 9, 6″-O-acetylononin (19) and 6″-O-acetylwistin (21) are isolated from this plant for the first time. To our knowledge, 21 had never been found previously neither from Glycyrrhiza sp., nor from any other natural resources. Acetylglycosides 19, 21 might be artefacts which could be produced during the EtOAc extraction and fractionation process of whole extract rather than be biogenetic precursors of 18, 20, respectively. 6″-O-Acetylwistin (21) was isolated as a yellowish powder. The mass spectra showed the molecular ion peak m/z 502 consistent with the molecular formula C 25 h 26 O 11 . The signals of 1 h and 13 C NMR supported 21 to be an isoflavone. The 1 h NMR spectrum revealed the characteristic h-atom signals at δ 8.31 (1h, s) of an isoflavone framework, 7.65 (d, J = 2.2 hz, 1h) and 7.34 ppm (s, 1h) of h-5, and h-8 of A-ring and 7.75 (brd, J = 7.6 hz, 2h) and 6.89 ppm (brd, J = 7.6 hz, 2h) belonging to a typical A 2 X 2 system in the B-ring. The sugar in 21 was identified as a β-d-glucose from the 1 h and 13 C NMR spectra. In comparison of the 1 h and 13 C NMR spectra of 21 with those of wistin 20 (Tostes et al. 1999;Ding et al. 2004), similar data were obtained for the aglycone moiety. however, in the 13 C NMR spectrum of the β-glucose unit in 21, a difference was observed with respect to wistin. The new two C-atoms in 21 appeared at δ (C) 172.2, and 19.8 ppm (acetoxy group). A downfield shift of the C(6″) signal (2.7 ppm) and an upfield shift of the C(5″) signal (2.5 ppm) were observed in the β-glucose moiety of 20, with similar signals for C(6″) and C(5″) in 6″-O-acetylwistin (21). The h(6″) in COLOC spectra showed correlations with carbonyl carbon of acetoxy group, which supported unambiguously that an acetyl group was linked to the hydroxyl group of C(6″) of 21. This data indicating that the acetoxy group is located at C(6″).

Bioactivity of the compounds
The easy of isolation of some flavonoids from G. pallidiflora cause our interest in the cytotoxic activity investigation of the accessible compounds.
The cytotoxic activity of some isolated compounds was determined by measuring the concentration inhibiting tumour cell viability by 50% (cell toxicity dose CTD 50 ). The CTD 50 was determined using the conventional MTT assay, which allows estimating the number of survived cells (Lipeeva et al. 2014). The results are listed in Table 1. Isoflavonoid calycosin 4 possessed the best activity on the lymphoblastoid leukaemia cells CEM-13 and human T-cell leukaemia cells MT-4 compared with formononetin 2, afromosin 3, and their 7-O-glycosides 19, 21. The structure of the phenolic substituent in 3rd position of isoflavonoids seems to have an important role in this class of compounds; indeed 3-hydroxy-4-methoxyphenyl substituent in compound 4 demonstrated increase in potency against MT-4 and CEM-13 human cancer cells. The potency of calycosin 4 on the lymphoblastoid cell lines was comparable with the activity of the drug doxorubicin. The glycosidation will led to the activity decreasing obviously, such as glycosides 19, 21 lost the activities.
In the series of pterocarpans, medicarpin 10 was active in all three tested cell lines, whereas homopterocarpin 11 shown selectivity on the human monocyte lines U-937.
high cytotoxic activity was also observed for the isoflavan (3R)-vestitol 16. This compound is highly selective against CEM-13 cell line and was twice more active in this assay as the comparative drug doxorubicin.

Conclusion
As a conclusion, both phytochemical and biological evaluations of the G. pallidiflora belong to the section of false licorices, may be rewarding in the discovery of new and potent metabolites. Such compounds as (-)-medicarpin 10, (-)-vestitol 16, bolusanthin III 17, ononin 18 and wistin 20 occur in the roots in significant quantities (isolated in yields of 0.34, 0.31, 0.25, 0.32 and 0.28%, respectively). Of interest was the high contents of pterocarpans in this species and also the isolation of acetylglycosilated isoflavones. The cytotoxicity findings in this study urge for further mechanistic and toxicological evaluations.

Supplementary material
Supplementary material relating to this paper is available online and included some experimental details (General experimental procedures; Extraction and isolation; and Cell Culture and determination of cytotoxicity).

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