molecules Article Phenolic Compounds and Bioactive Properties of Ruscus aculeatus L. (Asparagaceae): The Pharmacological Potential of an Underexploited Subshrub Joana P. B. Rodrigues , Ângela Fernandes * , Maria Inês Dias , Carla Pereira ,Tânia C. S. P. Pires , Ricardo C. Calhelha, Ana Maria Carvalho , Isabel C. F. R. Ferreira and Lillian Barros * Centro de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal; [email protected] (J.P.B.R.); [email protected] (M.I.D.); [email protected] (C.P.); [email protected] (T.C.S.P.P.); [email protected] (R.C.C.); [email protected] (A.M.C.); [email protected] (I.C.F.R.F.) * Correspondence: [email protected] (Â.F.); [email protected] (L.B.); Tel.: +351-273-330-904 (Â.F.); +351-273-330-901 (L.B.) Abstract: Ruscus aculeatus L. is a subshrub used in traditional medicine in different parts of the world, namely in Europe and the Iberian Peninsula. According to reported folk knowledge, the aerial parts are mainly used as diuretics and the underground organs are used for the treatment of disorders of the urinary system and as a laxative. In this work, the aerial part and the roots and rhizomes of R. aculeatus were chemically characterized with regard to the content of phenolic compounds and Citation: Rodrigues, J.P.B.; bioactive properties. Aqueous (infusions and decoctions) preparations and hydroethanolic extracts Fernandes, Â.; Dias, M.I.; Pereira, C.; from the two mentioned parts of the plant were prepared. Nine phenolic compounds were detected Pires, T.C.S.P.; Calhelha, R.C.; in all the extracts. Apigenin-C-hexoside-C-pentoside isomer II was the major compound in aqueous Carvalho, A.M.; Ferreira, I.C.F.R.; extracts and, in the hydroethanolic extract was quercetin-O-deoxyhexoside-hexoside followed by Barros, L. Phenolic Compounds and apigenin-C-hexoside-C-pentoside isomer II. All extracts revealed antioxidant activity and potential Bioactive Properties of Ruscus to inhibit some of the assayed bacteria; aqueous extracts of the aerial part and infusions of roots and aculeatus L. (Asparagaceae): The rhizomes did not show cytotoxic effects on a non-tumor primary cell culture. This preliminary study Pharmacological Potential of an provides suggestions of the biological potential associated with the empirical uses and knowledge of Underexploited Subshrub. Molecules 2021, 26, 1882. https://doi.org/ this species, in particular its bioactivities. 10.3390/molecules26071882 Keywords: Ruscus aculeatus L.; aerial part; roots and rhizomes; phenolic compounds; bioactivities Academic Editors: Raffaele Capasso and Lorenzo Di Cesare Mannelli Received: 4 March 2021 1. Introduction Accepted: 23 March 2021 In addition to providing oxygen, plants are a source of natural compounds that are Published: 26 March 2021 also used by humans since they have aromatic, medicinal, and food capabilities [1]. The use of plants for medicinal purposes is based on ancient knowledge, passed down from Publisher’s Note: MDPI stays neutral generation to generation, and for centuries they have been the only resource in terms of with regard to jurisdictional claims in medical, curative, or preventive care for many populations [2]. Medicinal plants have been published maps and institutional affil- and are still used in the development of new drugs for the treatment of several diseases. iations. Their popular use is mostly based on empirical knowledge and beliefs; therefore, some of their therapeutic applications lack scientific foundation [3]. However, such species are called medicinal plants because they have therapeutic benefits, which must be linked to particular substances, namely active ingredients that Copyright: © 2021 by the authors. might have a recognized pharmacological action. Thus, it is important to know which parts Licensee MDPI, Basel, Switzerland. of the plants are traditionally used for medicinal purposes, how are they processed and This article is an open access article applied, and which chemical compounds are responsible for their therapeutic properties. distributed under the terms and The increased interest in new herbal medicines has led to the discovery of new compounds conditions of the Creative Commons of therapeutic interest, such as steroids, alkaloids, saponins, terpenoids, and glycosides [4]. Attribution (CC BY) license (https:// The nature of a plant-based medicine is determined by the content of different active creativecommons.org/licenses/by/ components, and the potential that each plant has in these components can contribute to 4.0/). Molecules 2021, 26, 1882. https://doi.org/10.3390/molecules26071882 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 1882 2 of 13 an excellent therapeutic complement to conventional medicine [2]. Thus, according to the World Health Organization (WHO), wild or cultivated species are used as medicinal, both in traditional/folk medicine and in complementary medicine [5]. Ruscus aculeatus L. is a Eurasian species of the Monocotyledon group, currently part of the Asparagaceae botanical family reported as a medicinal species in European folk medicine [6–9]. The plant is a small subshrub, always green, with thick underground rhi- zomes [10]. The empirical medicinal uses are related with aerial parts are used empirically as diuretics and, the underground parts (roots and rhizomes) to alleviate the symptoms of several disorders of venous insufficiency, edema, urinary system, premenstrual syndrome, and hemorrhoids [8,11]. Few research groups have already studied this species [11–13]; the same authors report that the main active ingredients found in R. aculeatus are steroidal saponins (rucogenin and neoruscogenin), which are responsible for its pharmacological effects; other constituents have been isolated, including sterols, triterpenes, flavonoids, coumarins, sparteine, tyra- mine, and glycolic acid. Moreover, some of the traditional applications of the species seem related to particular compounds, since the highest concentration of ruscogenins is located in the rhizome [11]. This work represents the detailed characterization of R. aculeatus regarding the phe- nolic compounds of the aerial (laminar stems and lateral branches) and the underground (rhizomes with roots) parts. In addition, the bioactive potential of the hydroethanolic and aqueous extracts was also assessed in terms of their antioxidant, anti-tumor, anti- inflammatory, hepatotoxicity, and antimicrobial properties. Therefore, this work intends to contribute to the knowledge of the chemical composition of this species and to relate it to its documented empirical uses. 2. Results and Discussion The identification of individual phenolic compounds was carried out considering their retention times, whenever possible in comparison with commercially available standards, and both UV and MS spectra. Data obtained by HPLC-DADESI/MS analysis (retention − 2 time, λmax, pseudomolecular ion ([M − H] ), and main fragment ions in MS ), phenolic compounds’ tentative identifications, and respective quantification are present in Table1. The study revealed the presence of nine phenolic compounds, one caffeic acid deriva- tive, and eight flavonoids, namely six C-glycosylated derivatives of apigenin and two O-glycosylated derivatives of quercetin and kaempferol. Peak 1 showed a pseudomolecular ion [M − H]− at m/z 341 and MS2 fragments at m/z 179 and 135 consistent with the loss of a caffeic acid, therefore, being tentatively identified as caffeic acid hexoside. Peaks 2/5/7 ([M − H]− at m/z 563) were all tentatively identified as apigenin-C-hexoside-C-pentoside isomers I, II, and III, respectively. On the other hand, peaks 3/4/6, also showing a pseudomolecular ion at [M − H]− at m/z 563 were tentatively identified as apigenin-C-pentoside-C-hexoside isomers I, II, and III, respectively. The differentiation between isomers and sugar position took into account what was previously described by some authors Tahir et al. [14] and Ferreres et al. [15]. Finally, peaks 8 ([M − H]− at m/z 609) and 9 ([M − H]− at m/z 593) presented a single MS2 fragment at m/z 301 and 285, aglycones of quercetin and kaempferol, respectively, corresponding to the loss of a 308 u (146 u + 162 u, deoxyhexoside + hexoside moieties, respectively), and therefore, being tentatively identified as quercetin-O-deoxyhexoside-hexoside and kaempferol-O- deoxyhexoside-hexoside, respectively. The hydroethanolic extracts of the aerial part presented the highest levels of phenolic compounds (107 ± 3 mg/g extract, Figure1), followed by the aqueous extracts, the decoction (18 ± 1 mg/g extract), and the infusion (14.6 ± 0.3 mg/g extract). All extracts performed with aerial parts showed the same phenolic profile; however, the same was not verified for the roots, where no phenolic compounds were identified. Molecules 2021, 26, 1882 3 of 13 Table 1. Retention time (Rt), wavelengths of maximum absorption in the visible region (λmax), mass spectral data, tentative identification and quantification of the phenolic compounds (mg/g of extract) found in hydroethanolic extracts, and infusion and decoction preparations of R. aculeatus (mean ± SD, n = 9). Rt λmax [M − H]− Quantification Peaks MS2 (m/z) Tentative Identification (min) (nm) (m/z) Hydroethanolic Infusion Decoction 1 5.96 320 341 179 (100), 135 (20) Caffeic acid hexoside 1.42 ± 0.03 a 0.013 ± 0.001 c 0.091 ± 0.005 b 545 (21), 473 (100), 443 (91), 413 2 10.49 334 563 Apigenin-C-hexoside-C-pentoside isomer I 2.8 ± 0.1 a 0.446 ± 0.007 c 0.87 ± 0.01 b (*), 383 (36), 353 (41), 297 (*) 545