RSC Advances PAPER View Article Online View Journal | View Issue Flavonoids from duckweeds: potential applications in the human diet† Cite this: RSC Adv., 2020, 10, 44981 a ab a Debora´ Pagliuso, Carmen Eusebia Palacios Jara, Adriana Grandis, c b a Eric Lam, Marcelo Jose´ Pena Ferreira and Marcos Silveira Buckeridge * Duckweeds are the smallest free-floating flowering aquatic plants. Their biotechnological applications include their use as food, bioenergy, and environmental sustainability, as they can help clean polluted water. The high growth capacity and their chemical properties make them suitable for human health applications. Here we evaluated the ethanolic extracts from five species of duckweeds by HPLC-DAD/ MS-MS for chemical characterization. Sixteen compounds were identified and quantified, in which three were chlorogenic acid derivatives and eleven apigenin and luteolin derivatives. We describe for the first time the presence in duckweeds of 5-O-(E)-caffeoylquinic acid (1), 3-O-(E)-coumaroylquinic acid (2), luteolin-7-O-glucoside-C-glucoside (3), 4-O-(E)-coumaroylquinic acid (4), luteolin-6-C-glucoside-8-C- rhamnoside (5), and luteolin-8-C-glucoside-6-C-rhamnoside (6). The flavonoids diversity showed Creative Commons Attribution-NonCommercial 3.0 Unported Licence. a significant content of luteolin and its derivatives, except for Landoltia punctata that had significant Received 4th August 2020 apigenin content. Flavones identified in duckweeds were mostly C-glycosides, which can benefit human Accepted 21st November 2020 diets, and its abundance seems to be related to the higher antioxidant and anticancer capacities of DOI: 10.1039/d0ra06741e Wolffiella caudata, Wolffia borealis, and Landoltia punctata. Our findings reinforce the idea that rsc.li/rsc-advances duckweeds could be valuable additives to the human diet, and their potential should be further explored. Introduction Among the secondary metabolites, the avonoids are a group of compounds synthesized from cinnamic acid derivatives This article is licensed under a Duckweed is the given name for the smallest owering aquatic coupled with three acetate units that display several biological 15 plants that belong to Lemnaceae, which comprises 36 species roles. These polyphenol compounds are characterized by the grouped in ve genera: Landoltia, Lemna, Spirodela, Wolffia, and C15 skeleton arranged in two phenyl rings and a heterocyclic 1,2 – – Open Access Article. Published on 21 December 2020. Downloaded 10/2/2021 7:59:21 AM. Wolffiella. Furthermore, these plants are subdivided into the ring (C6 C3 C6). The substitution pattern of the central ring subfamilies Lemnoideae and Wolffioideae, which contain along with the degree of unsaturation and the oxidation of the respectively the species of Landoltia, Lemna and Spirodela, and avonoid structure resolve this group of compounds into six the rootless species of Wolffia and Wolffiella.3 These plants are main classes: avonol, avone, avanonol, avanone, antho- 14,16,17 capable of duplicating their biomass in 96 hours, conferring cyanin, and isoavonoid. Despite their grossly similar enormous applicability in biotechnology as water remediators, chemical properties, the avonoids in plants are involved in for environmental monitoring, food, biofuels, and cosmetic diverse processes such as growth, photosynthesis, and protective production as well as pharmaceutical intakes.4–10 The evolution biological systems such as seed development, pollination, light trend in Lemnaceae has a relationship with secondary metab- screening, defence against pathogens, protection against UV- olites and cell wall composition despite its diversity.11,12 damage, temperature acclimation, and drought resistance. Also, In plants, the secondary metabolites consist of a broad group some avonoids can act as signaling molecules for allelopathic 13,14 of compounds produced to benet the organism for different interactions, such as phytoalexins and detoxifying agents. purposes: those related to the internal functioning of the plant Besides the natural bioactive properties in plants, avonoids and others selected by their applications for human health.13,14 are phytochemicals with several medically-relevant biological activities such as antiviral, antifungal, antibacterial anti- hepatotoxic, anti-osteoporotic, antiulcer, anti-prolifera- a Laboratory of Plant Physiological Ecology, Department of Botany, Institute of 16,18–21 ˜ tive. Flavonoids display several other properties such as Biosciences, University of Sao Paulo, Brazil ff bLaboratory of Phytochemistry, Department of Botany, Institute of Biosciences, immunomodulation, apoptotic e ects, and consolidation of University of Sao˜ Paulo, Brazil applications on carcinogenesis, in ammation, atherosclerosis, 16,18,19 cDepartment of Plant Biology and Pathology, Rutgers, The State University of New and thrombosis. Flavonoids are also considered func- Jersey, New Brunswick, New Jersey, USA. E-mail: [email protected] tional foods that benet human health.16,18,19 Considering the † Electronic supplementary information (ESI) available. See DOI: applications for human food, the antioxidant capacity of 10.1039/d0ra06741e This journal is © The Royal Society of Chemistry 2020 RSC Adv.,2020,10,44981–44988 | 44981 View Article Online RSC Advances Paper avonoids has an essential action in preventing the formation (ThermoScientic® Savant SC 250 EXP), and resuspended in À of reactive oxygen and nitrogen species, causing damage in 2mgmL 1 in a hydroethanolic solution of 80% ethanol. DNA, proteins, lipids, and other biomolecules.22,23 Remarkable knowledge is found in the literature about the wine and tea phenolic compounds, which are responsible for their activity in the HPLC-DAD-ESI-MS/MS analysis of phenolic compounds in prevention of heart diseases and cancer.24–27 Thus, the intake of duckweeds polyphenols present in plants is desirable for human health. Samples diluted in ethanol 80% were ltered through a 0.45 mm Recently, avonoidssuchasquercetinandrutinhavebeenshown Nylon syringe, and reversed-phase HPLC-DAD analyzed the to interfere with the entrance of the SARS-Cov-2 in cells, high- extracts of duckweeds on an Agilent 1260. The phenolic 28 lighting their potential to treat the COVID-19 disease. compounds were separated using a Zorbax Eclipse Plus C18 Duckweeds can have high levels of avonoids and together column (4.6 Â 150 mm, 3.5 mm) maintained at 45 C. The with its nutritional value of amino acids and proteins make it injection volume of the sample was 5 mL. The solvents used in 29–34 suitable for human consumption with health bene ts. the mobile phase were 0.1% acetic acid in Milli-Q water (A) and Duckweeds have been sources of human food in several Asian acetonitrile (B), with the following concentration gradient of B: 35 countries. Moreover, one duckweed species (Spirodela polyrhiza) 0–3 min, 0%, 3–8 min, 0–10%, 8–8.1 min, 10–11%, 8.1–15 min, is being used to treat urticaria, acute nephritis, in uenza, and 11%, 15–15.1 min, 11–12%, 15.1–20 min, 12%, 20–20.1 min, 12– 36,37 in ammation in Japan, Korea, and China. Several studies 13%, 20.1–25 min, 13%, 25–30 min, 13–23%, 30–38 min, 23– identi ed avonoids mainly quercetin, apigenin, and luteolin 30%, 38–43 min, 30–50%, 43–65 min, 50–100 min, 65–70 min. 18,31,38 derivatives as compounds in Spirodela polyrhiza extracts and The phenolic acids and avonoids were detected at 325 nm and these components are probably related to the pharmacological 352 nm, respectively. Identication of compounds from duck- applications of this duckweed in traditional medicine. weeds was carried out through HPLC-DAD-MS/MS (Agilent 1260 Based on the importance of the duckweed family for phar- coupled to an Esquire 3000 Plus mass spectrometer) using the Creative Commons Attribution-NonCommercial 3.0 Unported Licence. macology and nutrition, this paper aimed at investigating the same chromatographic conditions and a negative ionization phenolic composition of ethanolic extracts and antioxidant mode. Quantication of each phenolic compound was carried out activity from ve species of duckweeds in order to characterize by comparing the peak areas obtained from HPLC analysis with À further the range of natural variations in the varieties of avo- calibration curves of standards at the range of 1.5–150 mgmL 1, noids produced by this plant family. according to the phenolic class. Chlorogenic acid, vitexin, and orientin were used to quantify phenolic acids, apigenin, and Experimental luteolin derivatives. Duckweed strains, cultivation method, and sample preparation This article is licensed under a Antioxidant activity Five-species of duckweeds were investigated in this study. Three The antioxidant activity was evaluated by the 1,1-diphenyl-2- Lemnoideae species (Landoltia punctata, Lemna gibba, and Spi- picryl-hydrazyl (DPPH) (Sigma-Aldrich®) method that is based rodela polyrhiza) and two Wolffioideae species (Wolffia borealis 39 Open Access Article. Published on 21 December 2020. Downloaded 10/2/2021 7:59:21 AM. upon the elimination of the free radical by the samples. For and Wolffiella caudata) were obtained from the Rutgers Duck- the assay, a 0.2 mM DPPHc solution in methanol was freshly weed Stock Cooperative (RDSC) collection. Landoltia punctata prepared, and 200 mL of DPPHc solution was reacted with 20 mL (7624), Lemna gibba (DWC128), Spirodela polyrhiza (9509), À of the plants' extracts (2 mg mL 1) diluted in 10, 20, 50, 100, and Wolffia borealis (9144) and Wolffiella caudata (9139) were culti- À1 À 200 mgmL . The reaction was incubated in the dark for 20 min, vated on Schenk–Hildebrandt medium (1.6 g L 1) supple- and the absorbance was measured at 515 nm with EPOCH mented with 0.5% of sucrose, pH 6.5 at 25 C with À equipment (Sellex Inc.). All samples were analyzed with three a photoperiod of 16 h of light in the intensity of 20 mmol m 2 À biological replicates and three technical replicates. The cali- s 1.Aer 21 days of cultivation, the plants were frozen in liquid bration curve was performed with a methanol Trolox (6-hydroxy- nitrogen, freeze-dried and pulverized by Geno/Grinder®2010 2,5,7,8-tetramethylchroman-2-carboxylic acid) (Sigma-Aldrich®) SPEX SamplePrep.