Dalea Genus, Chemistry, and Bioactivity Studies

Chapter 8

Dalea Genus, Chemistry, and Bioactivity Studies M.A. Peralta*,†,2, M.D. Santi*,†,2, J.L. Cabrera*,† and M.G. Ortega*,†,1 *

Departamento de Ciencias Farmac euticas, Facultad de Ciencias Quımicas, Universidad Nacional de Co´rdoba, Co´rdoba, Argentina † Instituto Multidisciplinario de Biologıa Vegetal (IMBIV-CONICET), Co´rdoba, Argentina 1 Corresponding author: e-mail: [email protected]

Chapter Outline Introduction Dalea Genus Chemical and Biological Studies on North and Central American Dalea Species Dalea emoryi A. Gray [Syn.: Psorothamnus emoryi (A. Gray) Rydb.]; Dalea polyadenia F. Heller, and Dalea tinctoria Brandegee [Syn.: Parosela tinctoria (Brandegee) Standl] Dalea scandens var. paucifolia (J.M. Coult.) Barneby [Syn.: D. thyrsiflora A. Gray] Dalea purpurea Vent. [Syn.: Petalostemon purpureum (Vent.) Rydb] Dalea filiciformis B.L. Rob. & Greenm [Syn.: Parosela filiciformis (B.L. Rob. & Greenm) Rose] Dalea carthagenensis var. barbata (Oerst.) Barneby

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[Syn.: Psoralea carthagenensis Jacq] 313 Dalea aurea Nutt. ex Fraser [Syn.: Parosela aurea (Nutt. ex Pursh) Britton] 314 Dalea formosa Torr. [Syn.: Parosela formosa (Torr.) Vail] 315 Dalea spinosa A. Gray [Syn.: Psorothamnus spinosus (A. Gray) Barneby] 316 Dalea searlsiae (A. Gray) Barneby [Syn.: Petalostemon searlsiae A. Gray] 317 Dalea frutescens A. Gray [Syn.: Parosela frutescens (A. Gray) Vail Ex Rose] 317 Dalea ornata (Hook.) Eaton & J. Wright [Syn.: Petalostemon lagopus Rydb] 318 Dalea versicolor Zucc. var. sessilis (A. Gray) Barneby [Syn.: Dalea sessilis (A. Gray) Tidestr] 318

2. M.A.P. and M.D.S. contributed equally to this publication. Studies in Natural Products Chemistry, Vol. 62. https://doi.org/10.1016/B978-0-444-64185-4.00008-3 Copyright © 2019 Elsevier B.V. All rights reserved.

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308 Studies in Natural Products Chemistry Dalea greggii A. Gray [Syn.: Dalea fulvosericea (Rydb.) Gentry], Dalea lumholtzii Robins and Fern. [Syn.: Dalea arizonica (Vail) K. Schum] 319 Dalea foliolosa (Aiton) Barrneby [Syn.: Dalea citriodora (Cav.) Willd] 320 Chemical and Biological Studies on South American Dalea Species 320 Dalea caerullea L. f. [Accepted Name: Dalea coerulea (L. f.) 320 Schinz & Thell]

Dalea strobilacea Barneby Dalea boliviana Britton [Syn.: Dalea calliantha Ulbr] Dalea elegans Gillies Ex Hook. & Arn. [Syn.: Parosela eosina J.F. Macbr] Dalea pazensis Rusby [Syn.: Parosela pazensis (Rusby) J.F. Macbr] Discussion Concluding Remarks Acknowledgments References

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INTRODUCTION Dalea Genus Dalea L. (Fabaceae) is an exclusively American genus, with more than 172 species [1]. Its habitat extends from Canada to the central region of Argentina and Chile [2]. The Dalea L. genus includes plants of fine foliage and showy flowers of purplish color. They are perennial herbs or shrubs (and, rarely, annuals) [3] and are found in deserts, grasslands, thorny scrubs, tropical dry forests, and tropical mountain forest areas. Figs. 8.1 and 8.2 illustrate examples of Daleas species that grow in North and South America. Some species that belong to this genus were informed in relation to the traditional use. Ethnomedicinal studies reported that the North American Dalea spp. were used for different illnesses by native tribes, such as the Apache, Wanalicha, and Dakota [4,5]. The Dakota tribes used Dalea purpurea Vent., to which they gave the native name “Wanalicha,” for the treatment of heart diseases [4,5], while Dalea aurea was used for gastrointestinal disorders, among other uses [5]. Additionally, Dalea polyadenia was used against smallpox by native communities of the Colorado Desert in California. Another species used was Dalea carthagenensis var. barbata, which has been reported to be used for the treatment of dermatological conditions by the Yucatecan Maya in Mexico [6,7]. Furthermore, D. carthagenensis is used by people of the Tehuaca´n-Cuicatla´n Valley in Mexico as an antiinflammatory agent and to treat gastrointestinal infections [8]. Another of these species was Dalea foliolosa, employed in some regions of Mexico for its antiinflammatory properties [9], and Dalea formosa, used by Native American tribes for its analgesic properties; they used its leaves for this purpose, among other traditional medicinal uses [10]. In Ecuador, the stems, leaves, and flowers of Dalea caerullea (L. f.) Schinz & Thell are used for the treatment of stomach ailments [11].

Dalea Genus, Chemistry, and Bioactivity Studies Chapter

Dalea emoryi A. Gray

Dalea purpurea Vent.

D. searlsiae (A. Gray) Barneby

D. frutescens A. Gray

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Dalea aurea Nutt. ex Fraser

Dalea formosa Torr.

FIG. 8.1 North american Dalea species [Girish Mahajan (2018), Matt Lavin (2011), Mason Brock (2017), Blaine Hansel (2005), Miwasatoshi, (2008), Jerry Friedman (2010)].

Other uses reported include insecticidal use (D. caerullea), and as a dyestuff for fibers and animal skin (D. emoryi, D. polyadenia, and D. tinctorea). Ethnobotanical and ethnomedical research has provided information on the uses of this genus in traditional medicine. These data have led to the chemical and biological study of Dalea species. This chapter presents an exhaustive review on the chemical and bioactivity investigations that have been carried out about this genus. These studies could corroborate some of the traditional uses of the Dalea species and allow us to postulate that this genus is a potential source of natural compounds in the search of new drugs.

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Dalea caerullea (L. f.) Schinz & Thell.

Dalea boliviana Britton

Dalea elegans Gillies ex. Hook. & Arn.

Dalea pazensis Rusby FIG. 8.2 South American Dalea species [Dick Culbert (2013), Cabrera Jose Luis (2005, 2011, 2013)].

CHEMICAL AND BIOLOGICAL STUDIES ON NORTH AND CENTRAL AMERICAN DALEA SPECIES Dalea emoryi A. Gray [Syn.: Psorothamnus emoryi (A. Gray) Rydb.]; Dalea polyadenia F. Heller, and Dalea tinctoria Brandegee [Syn.: Parosela tinctoria (Brandegee) Standl] The first chemical study in the Dalea genus was performed in 1975. Dreyer et al. [12] investigated two species from the United States and Mexico: D. emoryi A. Gray and D. polyadenia F. Heller, both of them known by the common name

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indigo bush. These two species grow in drier regions of the southwestern United States and Mexico. They were employed by the native communities of the region for coloring animal skins and dying fibers employed in basket-making. Coumarin (1), 5-methoxycoumarin (2) and two red pigments called dalrubone (3) and methoxydalrubone (4) were isolated from benzene extract of D. emoryi [12]. Coumarins 1 and 2 are biogenetically derived from the C6–C3–C6 flavonoid system; the cooccurrence of 1 and 2 with the dalrubones 3 and 4 allowed the authors to suggest that the coumarins are degradation products of the dalrubones [12]. D. polyadenia is used by native communities of the Colorado Desert not only for its pigments, but also in the treatment of numerous ailments, such as smallpox. Hot tea prepared with the fresh or dried stems is used as a cure for colds and coughs [13]. Coumarins 1 and 2, as well as the red pigment 3, have also been reported to be present in D. polyadena; however, methoxydalrubone (4) could not be detected. The presence of demethoxymatteucinol (5) in D. polyadena was the first evidence of a flavonoid-type structure in Dalea species. A possible biosynthetic relationship between dalrubones and demethoxymatteucinol has been suggested [12]. The species D. tinctoria grows in Mexico. The chemical study carried out by Dreyer reported the isolation of compounds 1–4 [14]. Only chemical studies have been performed for these species so far.

Dalea scandens var. paucifolia (J.M. Coult.) Barneby [Syn.: D. thyrsiflora A. Gray] The habitat of D. scandens var. paucifolia (Syn. D. thyrsiflora), a small shrub native to Mexico, extends from southern Texas to Chiapas, Mexico. No reports about medicinal traditional uses have been found for this species. Two isoprenylflavanones designated as louisfieserone (6) and its 2R-stereoisomer isolouisfieserone (7), together with the chalcone aurentiacin A (8) and the flavanone alpinetin (9), were isolated from the petroleum ether extract of the whole plant [15]; 6 and 7 were the first prenylfavonoids reported from the Dalea genus. The bioassay-guided fractionation of the ethyl acetate extract from the roots of D. scandens var. paucifolia resulted in the isolation of two prenyl flavanones, 20 ,40 -dihydroxy-50 -(1000 ,1000 -dimethylallyl)-8-prenylpinocembrin (10) and 40 -hydroxy-20 -methoxy-50 -(1000 ,1000 -dimethylallyl)-8-prenylpinocembrin (11), as well as a prenyl flavone 20 ,40 -dihydroxy-50 -(1000 ,1000 -dimethylallyl)-8prenilpinocembrin (12) [16]. Metabolites 10, 11, and 12 demonstrated significant antibacterial activity when tested against methicillin-susceptible and methicillin-resistant strains of Staphylococcus aureus. Prenylflavanone 10 was the most potent, with a minimum inhibitory concentration (MIC) of 1.53 mg/mL, while flavanone 11 and flavone 12 showed MICs of 3.13 mg/mL against both strains. According to Kuete [17], antimicrobial activity of natural products can be set as follows:

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for extract, significant (MIC < 100 mg/mL), moderate (100 < MIC  625 mg/ mL), or weak (MIC > 625 mg/mL). For compounds, the end point criteria are significant (MIC < 10 mg/mL), moderate (10 < MIC  100 mg/mL), and low or negligible (MIC > 100 mg/mL) [17]. The MIC values obtained for compounds from D. scandens var. paucifolia indicated significant antimicrobial activity. With respect to structure–activity relationships, the authors suggested that dihydroxylation in B-ring, such as in 20 , 40 or 20 , 60 -positions, is important for significant antimicrobial activity of prenyl flavonoids. Furthermore, 20 hydroxyl could be methylated and the flavanone nucleus could be a flavone without losing its activity [16]. This first biological approach about the activity of prenyl flavonoids from the Dalea genus demonstrated the potential therapeutic effect of these compounds on resistant bacterial strains.

Dalea purpurea Vent. [Syn.: Petalostemon purpureum (Vent.) Rydb] This species, known as purple prairie clover, was used by Native Americans for the treatment of heart trouble, diarrhea, measles, and pneumonia [18]. In addition, antecedents have reported that the Dakotas used it to treat heart disease [4,5]. Ethanolic root extracts of P. purpureum demonstrated antimicrobial activity against bacteria and fungi. Bioassay-directed fractionation led to the isolation of petalostemumol (13) as the active constituent. That compound showed potent activity against the Gram-positive bacteria Bacillus subtilus (MIC ¼ 0.78 mg/mL) and S. aureus (MIC ¼ 3.12 mg/mL), as well as moderate activity against Gram-negative bacteria, Escherichia coli (MIC ¼ 6.25 mg/mL), and Mycobacterium intracellulare (MIC¼ 6.25mg/mL). Petalostemumol exhibited moderate activity against Candida albicans (MIC ¼ 12.5 mg/mL) and marginal activity against Cryptococcus neoformans (MIC ¼ 25mg/mL) [18]. Belofsky et al. [19] reported that the methanol extract of D. purpurea showed moderate activity in the opioid assay. Its fractionation by silica gel vacuum liquid chromatography (VLC) led to the isolation of three new geranyl stilbenes, pawhuskins A–C (14–16), and one known compound, petalostemumol (13), which was previously isolated from this plant. The activities of these compounds were evaluated in an opioid receptor assay in vitro, which was used as a nonselective radioligand ([3H]-naloxone) to perform the receptor binding assays, because naloxone is an antagonist with similar affinities for the mu, kappa, and delta opioid receptors. This nonselective antagonist allowed the authors to perform an initial screen against each of the three subtypes without considering their different affinity states, and to investigate the compounds binding to multiple opioid receptor subtypes simultaneously. Pawhuskin A (14) showed the strongest activity of the four compounds, with its KI value of 0.29  0.11 mM being 14-fold more potent than the followed compound 15, KI ¼ 4.2  3.6 mM. Compound 16 presented a KI value of 11.4  7.9 mM,

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and the least active compound, 13, exhibited a KI value >100 mM [19]. The authors suggested that compound 14 could be used as an analgesic agent. Previous to this, it had been demonstrated that D. purpurea contained high levels of condensed tannins with potent activity against E. coli O157:H7, so feeding farm animals with that species has considerable potential as a dietary strategy to reduce the prevalence of that bacterium in the ruminant digestive tract without adversely affecting digestion [20,21]. Liu et al. [22] informed that condensed tannins (CTs) from that species can react with proteins and liposome, cause cell aggregation, and alter the outer membrane morphology and permeability of E. coli. In addition, bacteriostatic activity against E. coli 0157:H7 of these CT was demonstrated at levels of up to 200 mg/mL, and the mechanism may involve alteration of the fatty acid composition and disruption of the outer membrane of the cell. Jin et al. informed that the incorporation of D. purpurea in forage reduced the fecal shedding of E. coli in grazing cattle, probably due to the anti-E. coli properties of CT [20].Other studies carried out by Peng et al. concluded that D. purpurea had a higher nutritive value than alfalfa hay owing to its greater dry matter, organic matter and protein digestibility, but it did not improve lamb growth [23].

Dalea filiciformis B.L. Rob. & Greenm [Syn.: Parosela filiciformis (B.L. Rob. & Greenm) Rose] This species is distributed throughout various areas in Mexico and is part of the screening investigation of natural products with potential use as antihypertensives, but it does not have any data on its folk medicinal use. First, the methanol/dichloromethane extract obtained from its roots was evaluated on the endothelin-converting enzyme (ECE), showing an important inhibition. Patil et al., following the bioguided fractionation of this extract, isolated a new phytoalexin named daleformis (17) using various chromatographic techniques, with a significant IC50 of 9 mM on ECEs. It could be a candidate to prove for further in vivo antihypertensive studies (Fig. 8.1). Until now, this has been the only study on this species [24].

Dalea carthagenensis var. barbata (Oerst.) Barneby [Syn.: Psoralea carthagenensis Jacq] D. carthagenensis var. barbata is found in Central America, the West Indies, and Florida [1]. Its leaves were traditionally used for a number of dermatological conditions, including injuries caused by venomous animals [25]. In 2002, nonpolar extracts of D. carthagenensis leaves have been evaluated for their cytotoxicity activity on the KB cell line (ATCC CCL 17; human nasopharyngeal carcinoma) and for its nuclear factor-кB (NF-кB) inhibitory activity, a proinflammatory transcriptional factor. That extract showed cytotoxic activity against KB cells (IC50 31mg/mL) and elicited inhibition of NF-кB at 150 mg/mL [25].

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Montes-de-Oca Ma´rquez et al. [26] obtained the hexane, acetone, and methanol extracts of flowers and stems of this species by maceration, and evaluated them for their antibacterial and antifungal activity. The results showed that the hexane extract presented the highest antibacterial activity on S. aureus ATCC 29213 (flowers’ hexane extract, MIC ¼ 125 mg/mL and stem, MIC ¼ 500 mg/ mL). The same extract was the most active as an antifungal on C. albicans ATCC 14065 (MIC ¼ 125 mg/mL). The acetone and hexane flowers and stem extracts inhibited more than 90% of the radial growth of the Trichophyton mentagrophytes fungus at the concentration of 125 mg/mL. The chemical composition of the species was determined by qualitative tests and revealed the presence of terpenes (hexanic flower and stem extracts), phenols (all extracts), alkaloids (hexanic stem extract), and saponins and tannins (acetonic and methanolic stem extracts and methanolic flower extract). The highest antimicrobial and antifungal activities were observed in the hexanic flower extract that possess terpenes and flavonoids, so the authors have suggested that these metabolites would be responsible for the biological activity observed. With these results, Montes de Oca Ma´rquez et al. validate the medicinal use of D. carthagenensis in the treatment of diseases of infectious origin [26].

Dalea aurea Nutt. ex Fraser [Syn.: Parosela aurea (Nutt. ex Pursh) Britton] This species is a perennial subshrub grown in North America and is known as “golden prairie clover”. Native American tribes such as the Dakotas have used leaves of this species to prepare an infusion or decoction against dysentery, stomachaches, colic and gastrointestinal disorders [5]. Belofsky et al. isolated two isoflavones from the methanolic extract of D. aurea: a new one called 7-methoxymanuifolin (18) and manuifolin K (19) [27]. The antiamebic activity against Naegleria fowleri, the ameba responsible for primary amebic meningoencephalitis (PAM), was performed on the methanolic extract (10 mg/mL) and compounds 18 and 19 (at 30 mM for each one). Even though the methanolic extract did not show activity, compounds 18 and 19 were active, inhibiting the growth of N. fowleri, and showing important activity compared to N. fowleri treated with amphotericin B (AMB), the control (0.1 mM). The activity of both compounds was superior, compared to AMB after day 4 from a period of 7 days of assays. Then, in vivo assay using a mouse model of PAM was performed, which tested the isoflavan 19 at 25 mg/kg/day over 5 days. Compound 19 produced no protective effect against N. fowleri infection. The authors postulated that the results could be different if the compound was given rather than by intraperitoneal injection. Considering the traditional use of this species, it was suggested that more experimental assays, focused on animal amebiasis models produced by pathogenic intestinal ameba, such as Entamoeba hystolitica, should be evaluated [27].

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Dalea formosa Torr. [Syn.: Parosela formosa (Torr.) Vail] D. formosa Torr (Fabaceae) is known by the tribal name of “feather plume.” It is a shrub o subshrub growing in the southwestern United States and northern Mexico. This plant was used in traditional medicine by several Native American tribes; for example, its leaves were used as a cathartic, emetic, and strengthener [10]. The twigs and leaves were used as an analgesic, and the whole plant was used to treat influenza and other respiratory infections [28]. The first chemical studies of D. formosa reported on the composition of the essential oil, indicating the presence of 58 known components, with a-pinene (31.7%), camphene (8.4%), and limonene (8.1%) being the most abundant constituents [29]. In relation to biological activity, in an antifungal screening performed with extracts obtained from plants used in folk medicine, the ethyl acetate extract obtained from aerial parts of D. formosa exhibited antifungal activity against Candida sp. strains, with significant MICs of 20 mg/mL on Candida glabrata and >80 mg/mL on C. albicans [30]. Belofsky et al. obtained a methanolic extract from D. formosa roots. It showed an important antifungal activity on yeast strains, expressing different conditions of efflux-mediated resistance mechanisms [31]. This extract was fractionated and eight new compounds were isolated, six of them being isoflavonoids: Sedonans A–F (20–25), along with but-2-enolide, 40 -O-methylpuerol A (26), and pterocarpan ent-sandwicensin (27). All of these were identified by NMR and HRMS experiments. In relation to the antifungal activity evaluated on the methanolic extract and its compounds, C. albicans, C glabrata, and several strains of Saccharomyces cerevisiae were employed. The methanolic extract showed growth inhibition against both C. glabrata and S. cerevisiae strains, with significant MICs of 30 mg/mL, while an MIC of 60 mg/mL was reported for C. albicans inhibition [31]. Multidrug resistance pumps (MDR-pumps) were shown to mediate resistance to many structurally varied compounds, including most therapeutically used antifungal drugs. PDR5 and its homologous SNQ2 and YOR1 are the three major MDR transporter-encoding genes involved in the yeast pleiotropic drug resistance (PDR), as well as their major transcriptional activators, Pdr1p and Pdr3p. Inactivation of PDR1 and PDR3, as well as PDR5, SNQ2, and YOR1, resulted in a marked increase in activity of the D. formosa root extract against S. cerevisiae (MIC ¼ 3.8 and 7.6 mg/mL respectively), which allowed the authors to suggest the presence of growth inhibitor substrates of Pdr5p, Snq2p, and Yor1p in the crude extract. On the other hand, a large increase in the MIC was observed upon overproduction of Cdr1p, suggesting that the presence of this ABC multidrug transporter of C. albicans induced a reduction in growth-inhibition effects of the crude extract (MIC ¼ 244 mg/mL) [31].

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Dalea spinosa A. Gray [Syn.: Psorothamnus spinosus (A. Gray) Barneby] D. spinosa A. Gray, known as “smoke tree”, is a perennial shrub that grows in North America and Mexico [32]. For this species, no reports about its traditional use in folk medicine were found. Successive fractionation by chromatographic techniques of the methanolic extract obtained from the lower bark of D. spinosa produced several compounds: two new compounds, Spinosan A (28) and B (29), in addition to the known pterocarpans, (+)-Melilotocarpan A (30) and (+)-Medicarpin (31), and the known isoflavone 6,40 -dimethoxy-7,20 -dihydroxyisoflavone (32). The identification was performed by NMR spectroscopic and mass spectrometric methods [33]. All the compounds were tested against several microorganisms, S. aureus, Enterococcus faecalis, E. coli, Pseudomonas aeruginosa, S. cerevisiae, and C. albicans, in order to evaluate the antibacterial activity per se and the presence of synergy with antibiotics used in therapy. Although none of them showed any direct antimicrobial activity (MIC > 140 mM), some compounds, such as 28, 31, 32, and 33 (acetylated derivative of 28), weak natural antimicrobials, showed potentiation activity against only S. aureus when combined with berberine. Other assays related to MDR-pump inhibition were performed using strains of S. aureus overexpressed and knockout isogenic efflux mutants for the Nor A pump. Berberine was tested against wild-type S. aureus, showing an MIC of 372 mM. When tested against the isogenic NorA mutant of S. aureus, an MIC of 89 mM was observed, while an MIC > 1488 mM was observed against the NorA-overexpressing S. aureus mutant. Then, berberine was evaluated in the presence of each compound tested at concentrations between 42 and 56 mM. The MICs of berberine were 45 and 6 mM against wild-type S. aureus in the presence of compound 28 and its acetate (33), respectively, showing decreases of 8- and 62-fold with respect to those obtained with berberine alone. A fourfold decrease of MICs of berberine was observed (MIC ¼ 89 mM) by combining it with compounds 31 and 32, indicating various degrees of berberine potentiation under these conditions. The combinations were evaluated against the S. aureus knockout efflux mutant, showing the potential of berberine in the presence of compounds 28 and 31 with MICs of 6 and 21 mM (15-fold and fourfold decreases in MIC), respectively. Given that the MIC of berberine was 45mM in the presence of compounds 32 and 33, its effects were lower. The berberine effect against the NorA overexpression S. aureus mutant was augmented in the presence of compound 33, which also produced a decrease in the MIC of berberine in the wild-type strain compared to the knockout mutant, suggesting NorA-associated activity. For compounds 28, 31, and 32, the results obtained could not be sufficient to conclude NorA-associated activity. For these reasons, other efflux systems present in S. aureus may be related to their activities [33].

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Dalea searlsiae (A. Gray) Barneby [Syn.: Petalostemon searlsiae A. Gray] This species is an herbaceous perennial plant, native to the western United States [1], known by the common name Searle’s prairie clover. No traditional medicinal uses have been reported for this species. From the methanolic extract of roots, Belofsky et al. obtained new prenylated and genarylated flavanones, malheurans A–D (34–37), together with two known flavanones, (2S)-50 -(2-methylbut-3-en-2-yl)-8-(3-methylbut-2-en-1-yl)5,7,20 ,40 -tetrahydroxyflavanone (38) and prostratol F (39). On the other hand, the known pterocarpans tephrosin and milletosin (40 and 41) and two isoflavones, griffonianone E (42) and calopogonium A (43), were isolated from methanolic extracts of aerial parts. These compounds (34–43) were evaluated for their antimicrobial and antiinsect activity [34]. All compounds obtained from roots (34–39) showed antimicrobial activity and had significant MIC values of 2.0–5.3 mg/mL against the cariogenic bacterium Streptococcus mutans, suggesting a potential application of these compounds for oral health. In addition, these compounds showed MICs of 2.3–8.0 mg/mL against the endemic soil bacterium Bacillus cereus and MIC values of 3.1–6.8 mg/mL, against oxacillin sensitive, and MICs of 3.4–6.5 mg/mL resistant to S. aureus. The evaluation of the structure–activity relationship for this activity established that activity increased due to the presence of lipophilic groups (e.g., geranyl or prenyl). Compounds 40–43, isolated from the aerial parts, were inactive against these bacterial strains. The minimum bactericidal concentration (MBC) values were slightly higher than the MICs, so the authors suggested that these compounds show a bactericidal mechanism of action. Larvicidal activities toward Spodoptera frugiperda were demonstrated for compounds 37, 40, and 41, with compound 40 being the most active, with an associated mortality of 66%. A differential allocation of antimicrobial and antiinsectant plant resources to root and aerial parts of the plant, respectively, was suggested after these studies [34].

Dalea frutescens A. Gray [Syn.: Parosela frutescens (A. Gray) Vail Ex Rose] This species is a low-mounding shrub with feathery foliage that grows in dry limestone, and is known as black Dalea. No uses have been reported in folk medicine. The supercritical CO2 extract of leaves and stems from a native Texas species (D. frutescens) [1] was tested against PC-3 and DU145 prostate cancer cell lines, which are androgen independent and provide a model of castration-resistant prostate cancer (CRPC). The study demonstrated activity at concentrations <10 mg/mL, and the bioassay-guided fractionation of that extract led to the isolation of two new isoprenylated chalcones, sanjuanolide

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(44) and sanjoseolide (45), which were evaluated in the same model using the sulforhodamine B (SRB) assay. The results obtained for the antiproliferative effect against PC-3 and DU145 cells show that compound 44 was 3.2- and 3.6-fold more potent than 45, respectively. These compounds differ only in the 300 -substituent; in this way, it can be observed how minor structural changes highly affect the biological activity. Additionally, cytotoxicity was assessed by evaluating the cell density. Compound 44 was approximately two fold more cytotoxic than compound 45, exhibiting 87% of cytotoxic activity at 61.7 mM, as opposed to 46% generated by compound 45 at 61.3mM. In addition, compound 44 diminished DU145 prostate cell line colonies at 44% at its IC90. Furthermore, compound 44 interferes in cellular mitosis, causing the development of abnormal mitotic spindles and, thus, preventing cell division [35]. Thus, compound 44 would be a very interesting candidate for prostate cancer treatment.

Dalea ornata (Hook.) Eaton & J. Wright [Syn.: Petalostemon lagopus Rydb] It is a perennial subshrub or herb found in the Great Basin region of the western United States, in the states of California, Idaho, Oregon, Nevada, and Washington [1], and it is known by the common name handsome prairieclover and blue mountain prairie-clover. This species does not have documented medicinal use. The crude methanol extract of aerial parts of D. ornata was informed by Deardorff et al. as antihelmintic toward Ancylostoma ceylanicum. From this extract, phenolic metabolites were isolated (46–55) after being fractionated by silica gel VLC. These compounds were tested ex in vivo against the adult hookworm, A. ceylanicum. The new compound, (2S)-8-(3-methylbut-2-en-1-yl)-6,7,40 -trihydroxyflavanone (46), showed weak antihelmintic activity, with survival percentages between 97% and 83%. The rotenoids deguelin (54) and tephrosin (55) were the most active, with complete mortality (0% of survival) at 6.3 and 6.0 mM, respectively, so they were presented as the best candidates in that work [36].

Dalea versicolor Zucc. var. sessilis (A. Gray) Barneby [Syn.: Dalea sessilis (A. Gray) Tidestr] Another studied species of this genus was D. versicolor var. sessilis, with habitat in Mexico, Guatemala, and North America. No data reporting traditional use was found for this species. The organic extracts obtained from its whole plant showed important antimicrobial activity on S. aureus, with activity also observed in the presence of berberine. The MICs were 7.8 mg/mL in both conditions. For this reason, the fractionation of these extracts was initiated and seven phenolics compounds were obtained: four flavonoids, a new isoflavone,

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5,7,20 ,40 -tetrahidroxi-50 -(1000 ,1000 -dimetilalil)-8-prenilisoflavona (Dalversinol A, 56), and three known ones, 2(S)-50 -(10000 ,1000 -Dimethylallyl)-8-(300 ,300 -dimethylallyl)-20 ,40 ,5,7-tetrahydroxyflavanone (57), 2(S)-50 -(1000 ,1000 -Dimethylallyl)-8(300 ,300 -dimethylallyl)-20 -methoxy-40 ,5,7-tetrahydroxyflavanone (58), and 40 ,60 Dihydroxy-30 ,50 -dimethyl-20 -methoxychalcone (59); one pterocarpin, (+)Medicarpin (60); and two stilbenes, 3,5-dimethoxy-40 -hydroxy-trans-stilbene (61) and 3,5,40 -trimethoxy-trans-stilbene (62), using several spectroscopic methods, including NMR 1H and 13C and HMRS techniques. They were evaluated for their antimicrobial direct activity and for their capacity to inhibit the MDR-pump by using a combination of berberine at its subinhibitory concentration [37]. Of all of them, compounds 56 and 57 showed antimicrobial activity, with MICs of 31.3 and 7.8 mg/mL, respectively. While compounds 59 and 62 demonstrated low MICs at 250 and 500 mg/mL, respectively, they showed a relevant inhibition of S. aureus, in combination with berberine, at very low concentrations (approximately 3.3 mg/mL), showing differential activity with relation to the MDR-pump inhibition. Also, compounds 59 and 62 were evaluated against B. cereus at the same conditions described previously and with other antibiotics, potentiating the activity of the antibiotics used. As the study continued, compound 59, alone and in combination with antibiotics, was evaluated against a S. aureus NorA knockout mutant, without the NorA efflux pump, in comparison with wild-type S. aureus. This study demonstrated the potential for antibiotic activity in the wild-type S. aureus strain and showed MDR inhibition as a possible action mechanism of this activity. So, it is probable that the important antibacterial activity observed in the extract of this species is due to the presence of several compounds: ones with direct antibacterial activity and others with inhibitory activity on the MDR-pump [38]. We want to highlight this particular condition because it could be an important strategy for the treatment of bacterial resistance in several associated pathologies.

Dalea greggii A. Gray [Syn.: Dalea fulvosericea (Rydb.) Gentry], Dalea lumholtzii Robins and Fern. [Syn.: Dalea arizonica (Vail) K. Schum] D. greggii A. Gray (common name of “peabush”) and Dalea lumholtzii Robins. and Fern. (common name of “lemon peabush”) are native species of the desert and mountain areas of southern Arizona. A survey was made in order to determine the amount, physical constants, and chemical properties of the essential oil obtained by steam distillation of the fresh leaf and stem material of both mentioned Dalea species. D. greggii yielded 0.15% of oil extract with a light yellow color and faint lemon odor, while D. lumholtzii yielded 0.40% of essential oil with a yellow and lemon verbena odor. Dalea extracts presented oxygenated terpenes (78%). D. lumholtzii essential oil contains a relatively high amount of oxygenated terpenes [39].

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Dalea foliolosa (Aiton) Barrneby [Syn.: Dalea citriodora (Cav.) Willd] This Mexican species is traditionally boiled to prepare aqueous infusions against type 2 diabetes mellitus or is locally administered to treat contusions and abrasions. It is frequently found in patches of rainfed crops along roadsides, from Mexico to Honduras [9]. Dried leaves collected in three consecutive years (2014–2016) were extracted by hydrodistillation in order to obtain the essential oil, which was evaluated by gas chromatography-mass spectrometry (GC-MS) and gas chromatography with flame ionization detector (GC-FID). The chemical composition showed the presence of 29 known compounds, with a dominance of monoterpenes over sesquiterpenes and aliphatic hydrocarbons. Of the constituents of this essential oil, the principal component was cryptone (22%–30%), an oxygenated monoterpene, followed by linalool (10%–17%), caryophyllene oxide (4%–15%), ascaridole (4%–8%) and b-citronellol (3%–7%). The authors observed changes in the endogenous levels of some constituents of essential oil obtained in different years, so they hypothesized that this fact could be associated with the interaction of wild D. foliolosa plants with various biotic and abiotic factors. With regard to biological activity, the antioxidant, the anti-a-glucosidase enzyme, and the antibacterial activities were evaluated. The IC50 value (mg/mL) for each annual essential oil was estimated. For the antioxidant, using DPPH radical, the IC50 values for the years 2014–2016 were 156.3  12.9, 127.8  15.6, and 45.7  2.6, respectively and for the anti-a-glucosidase enzyme, they were 133.6  2.3, 47.2  5.2, and 14.4 1.4, respectively. Both activities were maintained during the studied years, with the hydrodistillated extract from 2016 being more effective than the others (P < 0.01) in both in vitro systems. In addition, the annual essential oils exhibited a significant antibacterial activity against Pseudomonas syringae pv. tabaci TBR2004 and P. syringae pv. tomato DC3000, and that activity was maintained in the 3 years of the study. The authors proposed that further studies in successive years could reveal changes in the chemical profile and biological activities [9].

CHEMICAL AND BIOLOGICAL STUDIES ON SOUTH AMERICAN DALEA SPECIES Dalea caerullea L. f. [Accepted Name: Dalea coerulea (L. f.) Schinz & Thell] This Dalea species grows in Colombia and Ecuador. It is commonly known as “pispura,” and the decoction of its leaves and flowers has been used by Colombians to ward off lice and fleas [40]. Its use as an insecticide attracted the attention of researchers, who studied the chemical composition of the aerial part extracts and the essential oil of this plant.

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For instance, Gonzalez et al. reported the presence of the C-methyl chalcone 40 ,60 -dihydroxy-30 ,50 -dimethyl-20 -methoxychalcone (63) in the petroleum ether extract of D. caerullea leaves. To establish this structure unequivocally by spectroscopic methods, the diacetate-derived compound was obtained from this chalcone [41]. Two years later, in their search for compounds with insecticidal activity from the petroleum ether extract of aerial parts from D. caerullea, Arango and Gonza´lez reported the isolation of four prenylated flavanones: 5, 7-dihydroxy-8-prenylflavanone (64), 5,7-dihydroxy-6-methyl-8-prenylflavanone (65), 5,7-dihydroxy-6-prenylflavanone (66), and 5-hydroxy-7methoxy-8-prenylflavanone (67). These compounds did not show significant activity in response to the Artemia salina toxicity test [42]. The petroleum ether extract was obtained from the aerial parts of D. caerullea. This extract was subjected to chromatographic separations guided by a general bioassay of toxicity with A. salina. The active fractions were purified by repetitive chromatographic techniques, and the products were evaluated by bioassays of A. salina and of specific toxicity (pupicide against Spodoptera sunia and larvicidal on Galleria mellonella and Achroia grisella). The most active fraction was comprised of 6 monoterpenes and 12 sesquiterpenes, being the most important components, b-ocimene and caryophyllene, and showed repellent and insecticidal action on Xenopsylla sp. and Macrosiphum rosae. The four flavanones previously mentioned (64–67) and a complex mixture of terpenoid compounds were obtained from another fraction. The pure flavanones did not show significant toxic activity; however, the mixture presented high toxic action on the larvae of S. sunia and larvicidal action on G. mellonella and A. grisella. This study suggests that the insecticide uses of D. caerullea could be due to its terpenoid components, while the flavanones did not have insecticidal effects [43].

Dalea strobilacea Barneby D. strobilacea Barneby, with a habitat in the northern Peruvian Andes, is known as “hierba de chil”. People have used this species for digestive disorders (e.g., stomach distress and indigestion). To determine the essential oil’s composition and evaluate its biological activity, Benites et al. have obtained it by hydrodistillation of fresh aerial parts (0.90% w/w). The results of the CG-MS analysis revealed the presence of 51 known compounds, with monoterpene hydrocarbons being the principal constituents (82%), while the oxygenated monoterpenes were present in concentrations of 5%. In lower concentrations were sesquiterpene hydrocarbons (7%), oxygenated sesquiterpenes (3%), and phenylpropanoids (0.1%). It is important to highlight that b-phellandrene (44%), a-pinene (18%), limonene (3%), and d-cadinene (3%) were the main compounds present in this essential oil.

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In relation to the biological activity, antibacterial evaluation was performed. Several strains were tested to cover a wider spectrum of antimicrobial activity: Gram-negative ones [E. coli (ATCC 25922), Klebsiella pneumoniae (ATCC 23357), and P. aeruginosa (ATCC 27853)]; and Gram-positive ones [S. aureus (ATCC 6538), Staphylococcus epidermidis (ATCC 12228), Enterococcus faecalis (ATCC 106996), Enterococcus hirae (ATCC 10541), and Bacillus subtilis (ATCC 6633)]. The essential oil showed significant activity only for three strains: E. faecalis (7.81mg/mL), K. pneumoniae (59.5 mg/mL), and S. aureus (62.5mg/mL). In accordance with these results, the presence of several degrees of inhibition against the microorganisms was evaluated. The authors indicated that the antimicrobial activity that they observed could be related to the chemical proportion of each compound (main and minor) present in the essential oil, but it needed more experiments in order to establish the relevance of each one in the antimicrobial activity observed in the essential oil without disregarding the presence of a synergistic effect in the activity observed [44].

Dalea boliviana Britton [Syn.: Dalea calliantha Ulbr] In our continuous search for new bioactive compounds from Dalea species, we approached the chemical and biological study of D. boliviana. This species is a small shrub that inhabits northwestern Argentina, Bolivia, and Peru at elevations from 2600 to 4000 m above sea level. A study on the Apillapampa community (in the Bolivian Andes) reported that the Quechua use D. boliviana for medicinal and animal food purposes [45]. From the n-hexane extract of D. boliviana roots, we isolated and elucidated three new prenylated flavonoids (68–70), together with the known obovatin (71) that previously had been isolated from the Tephrosia genus. Their structures were established by one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy, as well as HRMS analysis. These structures were established as (2S)-5,7,20 -trihydroxy-50 -(1000 ,1000 -dimethylallyl)-8-prenylflavanone (68), (2S)-5,7,20 -trihydroxy-8,30 -diprenylflavanone (69), and (2S)-5, 20 -dihydroxy-600 ,600 -dimethylchromeno-(7,8:200 ,300 )-30 -prenylflavanone (70), as well as the known chromeno (dimethylpyrano) flavanone, obovatin (71) [46]. Tyrosinase is an enzyme widely distributed from microorganisms to plants and animals. It is a cupro-glycoprotein and catalyzes the two first stages of the melanogenic pathway: hydroxylation of L-tyrosine to L-DOPA (the Monophenolase reaction) and its oxidation to dopaquinone (the Diphenolase reaction) [47]. Its overproduction can develop hyperpigmented disorders such as melasma, age spots, sites of actinic damage, and other skin disorders. To date, the most effective hypopigmenting agents are tyrosinase inhibitors, but most of them have serious side effects. Kojic acid, a compound used as a depigmenting agent, is genotoxic and hepatocarcinogenic and produces allergic dermatitis [48]. Therefore, considering that the drugs currently used present several adverse effects, the search for new tyrosinase inhibitors is a viable alternative.

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For this reason, all these compounds were evaluated as tyrosinase inhibitors by using the spectrophotometric methods according to Rahman et al. [49] with slight modifications. Only the flavonoids that showed a relevant inhibition on tyrosinase enzyme (more than 50% at 100 mM) had estimated their IC50. The IC50 values of 68 and 69 were 27.1 and 68.5 mM, respectively. Compounds 70 and 71 demonstrated very low tyrosinase inhibition even at 100 mM (6.4 and 16.2% inhibition, respectively). Kojic acid was used as a positive control (IC50 ¼ 10.2 mM). The chemical importance of this research relies on the fact that it provided new structure–activity relationships for prenyl flavonoids as tyrosinase inhibitors: the presence of a phloroglucinol A-ring with two free hydroxy groups in their structures (Fig. 8.2). The flavanones 68 and 69 obtained from D. boliviana presented this moiety, and its importance is emphasized by the weak activity exerted by compounds 70 and 71, which lack this chemical characteristic as a consequence of the formation of the chromene ring [46].

Dalea elegans Gillies Ex Hook. & Arn. [Syn.: Parosela eosina J.F. Macbr] D. elegans Gillies ex Hook & Arn. is the only specimen of this genre that grows in the province of Cordoba (central region of Argentina) [50]. No medicinal traditional use has been reported on this species. Our research group performed the first chemical study in this species in the 1990s. The aerial parts and roots of this species were processed and two prenylated flavanones derived from pinocembrin were isolated: 20 ,40 dihydroxy-50 -(1000 ,1000 -dimethylallyl)-6-prenylpinocembrin (72) from root extracts and 6-prenylpinocembrin (73) from aerial part extracts. They were identified by ultraviolet-visible (UV–vis), 1D 1H, and 13C and EI-MS [51]. Another investigation deepened the phytochemical study of D. elegans and evaluated the isolated compounds as potential tyrosinase inhibitors [52]. Consequently, three C-methyl flavanones, one methyl chalcone and the two prenylated flavanones mentioned previously (72 and 73), were obtained from roots and aerial part extracts of D. elegans. The structures were established by 1D and 2D NMR spectroscopy, as well as HRMS analysis. The analysis of 2D NMR spectroscopic data for 72 and 73 provided new chemical evidence, especially on the substitution pattern of an A ring, in relation to the position of the prenyl group. This investigation proposed the reassignment of both structures. In fact, the new data obtained from 2D NMR experiments provided evidence that the substitution of the prenyl group on the A ring is at C-8, instead of C-6 as it was reported for compound 72 [51]. This new information supported the reassignment of compound 72 as (2S)-20 ,40 dihydroxy-50 -(1000 , 1000 -dimethylallyl)-8-prenylpinocembrin (10). This structure was previously reported in other species of Dalea genus as well [14,31]. In a similar way, the spectral data of HMBC and HSQC supported the reassignment of compound 73 as (2S)-8-prenylpinocembrin (64) [52].

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In addition, the fractionation of benzene extract from D. elegans aerial parts led to the isolation of four other known compounds that were identified by means of spectroscopic methods and by comparing experimental data with those previously described in the literature [52]. Compounds 20 ,60 -dihydroxy40 -methoxy-30 -methylchalcone (triangularin, 74) [53]; 6,8-dimethylpinocembrin (demethoxymatteucinol, 5) [54]; 7-hydroxy-5-methoxy-6-methylflavanone (comptonin, 75) [55]; and 7-hydroxy-5-methoxy-6,8-dimethylflavanone (76) [56] were informed. On the basis of 2D NMR data analysis of the last compound, several differences on the assignments of the oxygenated carbons C-5, C-7, and C-9, as well as the aromatic carbons of a B ring, were found when compared to those previously reported [56]. The first report on the biological activity of the prenylflavanones isolated from D. elegans (compounds 72 and 73), further reassigned as compounds 10 and 64, respectively [52], evaluated the antibacterial and antifungal activity of these compounds. Antimicrobial activity was performed on the bacterial strains Salmonella anatum, B. subtilis (ATCC 6633), K. pneumoniae, E. coli, and S. aureus (ATCC 6538P); and the fungal strains S. cerevisiae (ATCC 9763), Aspergillus parasiticus (NRRL 2999), Penicillum citrinum, and C. albicans (ATCC 14231). Streptomycin was used as a reference. Only compound 10 showed relevant antibacterial activity against S. aureus and B. subtilis at 2.5 mg/mL, as well as fungal growth inhibition of S. cerevisiae at 20 mg/mL. The reference antimicrobial showed inhibitory activity at 10mg/mL [57]. Compound 10 was tested on growth inhibition of bacterial and fungal strains with clinical relevance from various human biological sources (hemoculture, urine culture) obtained from acquired immunodeficiency syndrome (AIDS) patients. This prenylflavanone was active, with significant MICs against Micrococcus luteus ATCC 9341 (MIC 15 mg/mL), S. aureus oxacillin-sensitive and oxacillin-resistant strains (both with MIC  15 mg/mL). In relation to the antifungal activity, compound 10 was active against C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, T. mentagrophytes, and Cryptococcus neoformans strains [58]. Further biological studies were performed in order to deepen the antifungal properties of compound 10 [59]. The effect of fluconazole (FLZ) and compound 10 on rhodamine 6G (Rh 6G) efflux was evaluated in both azole-sensitive and azole-resistant C. albicans strains. A flow cytometry assay was utilized in order to measure the efflux of Rh 6G, with a fluorescent dye used as a substrate of the membrane transporters, in the azoleresistant C. albicans 12–99 (RCa) strain, which expresses the multidrug transporters Cdr1p, Cdr2p, and Mdr1p [60,61]. Verapamil was applied as a reference inhibitor of the ABC transporters. The residual fluorescence was quantified. Compound 10 inhibited Rh 6G efflux only in RCa in a concentration-dependent manner, with IC50 ¼ 119 mM, while FLZ inhibited the efflux with an IC50 756 mM. Lineweaver–Burk analysis performed at various concentrations of the substrate Rh 6G revealed that compound 10

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competitively inhibited the efflux and produced a fourfold increase in the apparent Km [(11.75  2.45) mM toward (49.29  10.76) mM], but it did not affect Vmax. This prenyl flavanone also showed selectivity of efflux inhibition in comparison with other flavanones, such as compound 64, isolated from the aerial parts of D. elegans, pinocembrin, naringenin, and hesperetin. In antifungal assays, MIC values were 150 mM for 10 and higher than 400 mM for FLZ. The combination of both compounds at either inhibitory or subinhibitory concentrations was significantly more effective than each compound by itself. In fact, in the presence of compound 10 at 100 mM, the MIC for FLZ decreased by more than 400 times, showing MIC values similar to those of azolesensitive C. albicans and suggesting that the azole-resistance in RCa was reversed by the combination of compound 10 with FLZ [59]. In a subsequent investigation, the effects of the combination of both compounds on cell growth were evaluated. The checkerboard method was used, and MIC, FIC, and FICI values were calculated. MIC was defined as the lowest concentration capable of producing growth inhibition higher than 90% when the viable counts were compared with those of the control without the tested samples. FIC was calculated as the MIC of FLZ or compound 10 when combined divided by the MIC of each compound alone. FICI was the sum of the FIC of each substance. Compound 10 showed additive interactions with FLZ regarding cell growth, with FIC values of 0.11 and 0.5 for compound 10 and FLZ, respectively (FICI ¼ 0.61). These interactions were revealed in the fungicidal effect on C. albicans. Compound 10 presented a dual action: fungicidal per se, and by means of an increase in FLZ potency and efficacy. This last effect would involve the inhibition of CDR transporters, with the consequent increase in intracellular azole concentrations and responses, in a way similar to the one reported previously [59,62]. In preliminary in vivo studies on acute toxicity on mice at a single dose schedule, administered by the intraperitoneal injection (i.p.) route, LD50 values suggested an acceptable level of safety for compound 10 in comparison with other compounds, and it would not induce hepatotoxicity [62]. Due to the antifungal properties of compound 10 against C. albicans, it was evaluated as a biofilm inhibitor. A complete evaluation of the effects of compound 10 on formed biofilms and its impact on the imbalance in oxidative stress of the biofilms was performed [63]. Biofilm formation is an important virulence factor that allows C. albicans to cause many types of candidiasis at both mucosal and systemic sites [64]. Candida biofilms are 30–2000 times more resistant to several antifungal agents than their planktonic (free-living) counterparts, and the antifungal drugs available to treat these infections are becoming increasingly limited. For this reason, developing novel strategies to inactivate Candida biofilms has great clinical relevance in treating candidiasis.

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Compound 10 had similar antibiofilm effects against sensitive and resistant C. albicans strains. The biofilm formation was strongly inhibited (>85%) by compound 10 at 100 mM. The biofilm inhibition performed by this compound was associated with an increase of oxidative and nitrosative stress and an increase in total antioxidant defenses as a response to oxidative stress provoked by compound 10 in the treated biofilms. This is the first study that has attempted to correlate biofilm inhibition with changes in the oxidative balance by flavonoid 10 [63]. Compound 10 was also evaluated for its antioxidant and mitochondrial toxicity properties showing both antioxidant and antiradical activities. Furthermore, it inhibited the enzymatic lipid peroxidation, exhibited significant scavenging activity, and demonstrated significant antioxidant activity by decreasing the reduction of Mo(VI)–Mo(V) in rat liver microsomes. Furthermore, compound 10 was cytotoxic in a concentration-dependent manner in cytotoxic assays performed on the cell line derived from a laryngeal epidermoid carcinoma, HEp-2 cells. This flavanone also collapsed the rat liver mitochondrial membrane potential in a concentration-dependent manner and inhibited the ATPase activity in coupled mitochondria and submitochondrial particles. In the latter, this compound also inhibited the enzymatic activities of nicotinamide adenine dinucleotide dehydrogenase (NADH) oxidase and succinate dehydrogenase. It was suggested that compound 10 impairs the hepatic energy metabolism by acting as a mitochondrial uncoupler and by inhibiting enzymatic activities linked to the respiratory chain. Due to its cytotoxicity, this compound could be considered as a potentially promising therapeutic agent in such applications as cancer chemotherapy [65]. In a recent study, the relationship between antibiofilm and antioxidant properties of compound 10 was evaluated. At high concentrations (1000 mM or higher), an antioxidant effect of compound 10 was detected, and levels of oxidant metabolites remained low in C. albicans biofilms. Thus, a concentration-dependent antioxidant action of compound 10, which can alter its antifungal activity on biofilms, was reported [66]. Another biological activity evaluated for compounds isolated from D. elegans (10, 64, 5, 74–76) was the inhibitory activity on tyrosinase. Of all compounds tested at 100 mM, compounds 10 and 74 displayed significant inhibition [(95.0  0.6) % and (98.6  0.6) %, respectively] on the tyrosinase enzyme. As for compounds 64 and 5, the activity was moderate [(73.7  0.6)% and (50.0  0.6)%, respectively]. Comptonin (75) and 7-hydroxy-5-methoxy-6,8-dimethylflavanone (76) showed very low tyrosinase inhibitory activity (22.7  0.6% and 6.97 0.6%, respectively). Consequently, the IC50 values were estimated only for the most active compounds. Compound 10 showed important tyrosinase inhibition activity [IC50 (2.32  0.01) mM], almost twice as active than Kojic acid, the positive control [IC50 (4.93  0.01) mM], followed by compound 74 [(33.3  0.1) mM], while compounds 66 and 5 showed weak inhibition [(80.6 0.3) and (97.6  0.3) mM, respectively].

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Then, the study of the inhibitory mechanism of compounds 10 and 74 was performed. The Lineweaver–Burk plottings suggested a reversible inhibition on the enzyme for both compounds, with compound 10 being a competitive inhibitor, with a KI of (2.94  0.01) mM, and compound 74 being an uncompetitive inhibitor, with a KIS of (7.8  2.3) mM. The structural pattern observed for compound 10 is in accord with the structural requirements reported for flavanones as tyrosinase inhibitors, such as the 4-substituted resorcinol moiety in the B ring, and 4-substituted phloroglucinol moiety with two free hydroxyl groups when it is part of the A ring [49]. These results allowed us to present this compound as a potential agent for the treatment of skin pigmentation disorders [52]. Chiari et al. also studied aerial parts of D. elegans. They demonstrated antityrosinase activity in an ethanol extract, and through bioguided fractionation of it, they isolated a new compound 5, 20 ,40 -trihydroxy-200 ,200 -dimethylchromene-(6,7: 500 ,600 )-flavanone (dalenin, 77). They performed the in vitro tyrosinase inhibition using both L-tyrosine and L-DOPA, showing relevant activity, with IC50 values of 0.26 and 18.61 mM, respectively. Also, they determined the kinetic mechanism on both substrates. A reversible inhibition of the enzyme was observed with L-tyrosine and with L-DOPA, and it was a mixed-I type or noncompetitive. Also, molecular modeling evaluation was realized to support the inhibitory potency of compound 77 demonstrated in the in vitro assays. The interaction of the 20 ,40 -dihydroxy substituents with the enzyme was relevant to show this activity. The authors considered that this compound could be a potential candidate for a pharmaceutical and cosmetic agent for use in dermatological disorders associated with melanin [67].

Dalea pazensis Rusby [Syn.: Parosela pazensis (Rusby) J.F. Macbr] This Bolivian endemic species is a shrub with yellow tap roots and violet flowers. Like D. boliviana, D. pazensis has used in traditional medicine and as animal food by the Quechua community in Apillapampa, Bolivia [45]. Our research group performed chemical and pharmacological studies of that species, employing the column chromatography (CC) technique, with silica gel as the stationary phase and n-hexane/ethyl acetate as the mobile phase. From roots’ benzene extract, we isolated two new prenylated flavanones, (2S)-30 ,40 dihydroxy-6,200 -diprenylpinocembrin (pazentin A, 78) and (2S)-40 -hydroxy20 -methoxy-50 -(1000 , 1000 -dimethylallyl)-6-prenylpinocembrin (pazentin B, 79), as well as two known ones, 20 ,40 -dihydroxy-50 -(1000 ,1000 -dimethylallyl)-8prenylpinocembrin (10) and 40 -hydroxy-20 -methoxy-50 -(1000 ,1000 -dimethylallyl)8-prenylpinocembrin (11), previously isolated from D. elegans [34,52] and from D. scandens var. paucifolia [16,34], respectively. These compounds were evaluated for their mushroom tyrosinase inhibitory activity for compounds 11, 78, and 79. Compounds 78 and 79 were weakly

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active, and compound 11 presented an IC50 of (49.80  0.09) mM [68]. As was observed, compound 11 showed lower activity than the one previously reported for compound 10. These results reinforced the characteristics necessary to show antityrosinase activity: the presence of the 4-substituted phloroglucinol moiety when it is part of the A ring, and the presence of resorcinol moiety when it is in the B ring (10). Instead, compound 11 only presents the first of these requirements. With regard to compounds 78 and 79, we hypothesized that the replacement of 4-substituted phloroglucinol by a 6-substituted phloroglucinol, added to the absence of the resorcinol moiety in B ring, would be responsible for the weak activity observed. Subsequently, the inhibition of melanin production in B16 cells was evaluated for the tyrosinase inhibitory activity of intracellular murine B16F0 melanoma cells [68]. Those evaluations were performed in order to approach the behavior that would occur in humans. Due to the high homology between B16 murine melanoma tyrosinase and human melanoma tyrosinase, the first of these represents a valid model. First, the cellular viability was determined, and the maximum noncytotoxic concentration, which is the concentration that the cells are maintained at 90% of viability (MNCC), was estimated. The values of their MNCC were (50.0  8.0) mM for 79, (10.0  1.0) mM for 10, (5.0  0.5) mM for 11, and (1.0  0.3) mM for 78. Kojic acid showed an MNCC of (5000.0  10.0) mM. All compounds were cytotoxic in a concentration-dependent manner. Then, the inhibition of melanin in B16 cells was evaluated. The extracellular melanin content, after 24 h of incubation with the compounds, was measured at 510 nm. The culture medium of untreated cells was considered as 100% of melanin production. The concentration of each prenylated flavanone that induced a 50% inhibition of melanin production was compound 78 (0.75  0.2) mM > compound 10 (1.0  0.4) mM > compound 79 (5.0  1.0) mM ¼ compound 11 (5.0  1.8) mM > Kojic acid (2000.0  5.0) mM [68]. All the compounds were more active than the reference inhibitor. These results showed the importance of the prenyl and hydroxyl groups on both A and B rings for the inhibition of melanin production in B16 cells, in concordance with the findings reported by Arung et al. [69]. In order to investigate the mechanism involved in the diminished melanin content, we evaluated the murine B16 melanoma intracellular tyrosinase inhibitory effect of these compounds. B16F0 cells were treated with various concentrations of each compound for 24 h; after that, the cells were solubilized and the supernatant was mixed with L-DOPA and MBTH. The formation of a stable, dark pink adduct between 3-methyl-2-benzothiazolinone (MBTH) and dopaquinone was measured at 490 nm, as previously described by Winder et al. [70]. Compound 10 was more active than Kojic acid and the tyrosinase inhibition could be related to the decrease observed in melanin production in B16 melanoma cells. Compounds 78 and 79 were inactive on murine intracellular tyrosinase; therefore, we conclude that the diminished melanin content

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achieved by compounds 78, 79, and 11 was independent of the tyrosinase inhibitory activity. We hypothesized that other stages related to melanin biosynthesis, such as protein and/or transcriptional expression of the enzymes that regulates its biosynthesis (Tyrp1 and Tyrp2) or signaling pathways, could be implicated in the action of those compounds. However, other studies should be conducted in order to determine the mechanism of action of the prenyl flavonoids from D. pazensis. These are the first chemical and biological studies on D. pazensis Rusby [68].

DISCUSSION The Dalea genus has been widely used in traditional medicine by Native American tribes. It is noteworthy that, despite its medicinal attributes, only a few species of this genus have been investigated in chemical and biological studies to correlate their medicinal uses with bioactivity of their chemical components. Table 8.1 summarizes the data informed in this chapter. The chemical studies in this genus have revealed the presence of prenylated flavonoids (Fig. 8.3). Despite the wide variety of natural flavonoids, the distribution in the plant kingdom of flavonoid-type metabolites, which present prenyl moieties in their structures, is limited to certain genera, such as Sophora (Fabaceae), Morus, and Artocarpus (Moraceae). Given that the distribution of this type of structure in nature is so limited, prenyl flavonoids could be proposed as chemotaxonomic markers for the Dalea genus. Several studies have established a close relationship between the presence of this kind of flavonoid and the taxonomic classification in some other genera, such as Sophora. In order to establish the subclassification in this genus, it has been proposed that prenyl flavonoids could be a chemotaxonomic feature.A study revealed that prenylated flavonoid compounds in the roots of Sophora could be classified according to their carbon skeletons (flavanone, isoflavanone, etc.), oxygenation patterns on the B-ring, and variation of prenyl groups as C5, C10 substituent, or both. The chemotaxonomy of the genus Sophora, based on the presence of prenyl flavonoid compounds in the species, showed a close relationship with the morphological classification of Sophora, with only a few exceptions [71]. Another study that reveals the chemotaxonomic importance of the presence of prenylated flavonoids was performed in the Artocarpus genus. This study revealed that the restricted occurrence of 3-isoprenyl 20 ,40 -dioxygenated or 20 ,40 ,50 -trioxygenated flavones only in the Artocarpus species of the Moraceae family is of chemotaxonomic significance, and it allowed for proposing these chemical features in the flavonoids as a taxonomic marker for the classification of the Artocarpus species [72]. Although chemical studies in species of the Dalea genus have demonstrated the presence of prenylated flavonoids, more studies are needed in other

TABLE 8.1 Summary of Chemical and Biological Studies on the Dalea Genus Origin

Species

Chemical Constituents

Biological Activity

References

North American Species

Dalea emoryi

1–4

n.d.

[12]

Dalea polyadenia

1–5

n.d.

[12]

Dalea tinctoria

1–4

n.d.

[14]

Dalea scandens var. paucifolia

6–12

Antibacterial activity against methicillin-susceptible and methicillin-resistant Staphylococcus aureus strains

[15–17]

Dalea purpurea

13–16

Antimicrobian activity against Bacillus subtilis, S. aureus, Mycobacterium intracellulare, Candida albicans, and Cryptococcus neoformans

[18–22]

Analgesic activity on opioid receptor Bacteriostatic activity against E. coli

North American Species

Dalea filiciformis

17

Inhibitory activity on endothelin converting enzyme (ECE)

[24]

Dalea carthagenensis

n.d.

Anticancer activity on the KB cell line Antiinflammatory activity Antifungal activity on Candida albicans and Trichophyton mentagrophytes

[25,26]

Dalea aurea

18, 19

Antiamebic activity against Naegleria fowleri both in vitro and in vivo

[27]

Dalea formosa

Essential oil 20–27

Antifungal activity against Candida spp. Antifungal activity on yeast strains expressing different conditions of efflux mediated resistance mechanisms

[29–31]

Dalea spinosa

28–32

Antibacterial activity against S. aureus, Enterococcus faecalis, E. coli, Pseudomonas aeruginosa, S. cerevisiae, and C. albicans

[33]

Antimicrobial potentiation when combined with berberine against an MDR S. aureus strain overexpressing a NorA efflux pump Dalea searlsiae

34–43

Antimicrobial against Streptococcus mutans

[34]

South American Species

Dalea frutescens

44,45

Anticancer on prostate cancer cell lines

[35]

Dalea ornata

46–55

Antihelmintic against Ancylostoma ceylanicum

[36]

Dalea versicolor

56–62

Antimicrobial direct activity and MDR-pump inhibition by combination with berberine

[37]

Dalea greggii

Essential oil

n.d.

[39]

Dalea lumholtzii

Essential oil

n.d.

Dalea foliolosa

Essential oil

Antioxidant activity Antibacterial activity against Pseudomonas syringae

[9]

Dalea caerullea

Essential oil 63–67

Repellent and insecticidal action on Xenopsylla sp. and Macrosiphum rosae. Toxic action on larvae of Spodoptera sunia and larvicidal action on Galleria mellonella and Achroia grisella

[41–43]

Dalea strobilacea

Essential oil

Antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus hirae, and Bacillus subtilis

[44]

Dalea boliviana

68–71

Antityrosinase activity

[46]

Dalea elegans

5, 10, 64, 74–76, 77

Antityrosinase activity on mushroom tyrosinase

[51,52, 57–59, 62,63, 65–67]

Antibacterial and antifungal activity Inhibition of MDR-eflux pumps in azole-resistant C. albicans strain Fluconazole antifungal potentiation Antibiofilm effects against sensitive and resistant C. albicans strains Antioxidant-prooxidant concentration-dependent dual action on C. albicans biofilms Antioxidant and antiradical activities. Inhibition of the enzymatic lipid peroxidation.

Dalea pazensis n.d., not determined.

10, 11, 78, 79

Antityrosinase activity Antimelanogenic activity

[68]

332 Studies in Natural Products Chemistry

FIG. 8.3 Chemical structures of natural compounds isolated from species in the Dalea genus.

species from various regions of the Americas to support a chemosystematic classification based on the occurrence of prenylated flavonoid compounds in this genus. Due to the relatively narrow distribution of prenyl flavonoids in the plant kingdom the promising biological properties that they have will be important all the efforts in order to obtain these compounds by either total synthesis or semisynthesis. In this sense, Rao et al. reported for the first time the total synthesis of the prenylated flavonoids (68–70) isolated from D. boliviana. They did so by using a seven-step synthesis that began with 2,4,6-trihydroxyacetophenone as the starting reagent, and posterior prenylation with isoprenylbromide [73]. Other approaches to obtain prenyl flavonoids include techniques as semisyntheic

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8 333

FIG. 8.3—Cont’d

pathways by using isoprenylbromide as reagent [74] and also biosynthesis by prenyl transferases [75]. These could be strategic methodologies to use to obtain several prenyl flavonoids present in this genus. Other chemical constituents isolated from some Dalea species are the essential oils that have oxygenated terpenes as principal components. Because the pharmacological properties of essential oil components have been widely investigated, the essential oils from Dalea have been demonstrated antimicrobial, antioxidant, anti-a-glucosidase enzyme, and insecticidal properties. With respect to prenylflavonoids, the wide spectrum of their biological activities supports their pharmacological potential. The biological studies carried out in Dalea have been mainly oriented toward the antimicrobial activity against various

334 Studies in Natural Products Chemistry

FIG. 8.3—Cont’d

pathogenic microorganisms, including resistant bacterial and yeast strains. These investigations have shown the great potential of the Dalea genus as a source of new antimicrobial drugs. Microbial resistance mechanisms are emerging and spreading globally, threatening our ability to treat infectious diseases. There is, then, an urgent need for new drugs to treat infections caused by resistant microorganisms. The investigation of new antimicrobial drugs of natural origin, with the promising results obtained with Dalea compounds, is one important action to be done to cope with MDR problems. In this sense, most advanced research has come to elucidate some of the molecular mechanisms involved in the antimicrobial activity of the most potent compounds. It is important to point out that the Dalea compounds could be evaluated on other nonpathogenic strains, especially those related to the gastrointestinal

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FIG. 8.3—Cont’d

microbiota, taking into account studies demonstrating that CTs obtained from purple prairie clover (D. purpurea) decreased the bacterial (Lactobacillus genus) diversity during both ensiling and aerobic exposure [76]. Therefore, further studies could focus on areas related to nutritional and pharmaceutical uses. Another interesting biological activity of the prenylated flavonoids obtained from Dalea is inhibition of the tyrosinase enzyme, which allows for proposing them as new candidates for research with the aim of discovering new drugs that could be used in pathologies that involve skin pigmentation disorders. It is important to consider that the clinical projection of these compounds should be developed, taking into account the promising results obtained in the biological activities that were evaluated in vitro. In this sense, and considering the relevant activity observed by compound 10, we performed preliminary

336 Studies in Natural Products Chemistry

FIG. 8.3—Cont’d

in vivo studies on acute toxicity on a single dose schedule. The results obtained led us to indicate a relatively low toxicity in albino mice, as suggested by LD50 values, without toxicity symptoms on various organs or cavities [62]. These relevant results suggest an acceptable level of safety for this prenylated flavonoid and encourage the beginning to the experimental studies with a clinical projection.

CONCLUDING REMARKS This chapter summarizes all the chemical and biological studies on Dalea species. This genus is an important source of species with medicinal traditional uses. From chemical studies, it is interesting to note the presence of

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8 337

FIG. 8.3—Cont’d

prenylated flavanoids, compounds that have an exclusive distribution in a few families and genus in the plant kingdom. Some Dalea species have also been studied on the chemical composition of their essential oils. Biological research has been directed principally toward the search for compounds with antimicrobial properties. In addition, prenyl flavonoids have several relevant biological activities, such as antimicrobial activity, inhibition of mechanisms of fungal and bacterial resistance, and inhibition of the tyrosinase enzyme. These properties allow us to postulate this genus as an important source for the search and development of new drugs of natural origin.

ACKNOWLEDGMENTS We wish to acknowledge the assistance of Universidad Nacional de Co´rdoba and Consejo Nacional de Investigaciones Cientıficas y Tecnicas (CONICET), both of which support facilities used in this investigation. M.A.P., M.G.O., and J.L.C. are members of the Research Career of CONICET. M.D.S. is a posdoctoral fellow of CONICET. We are indebted to Claudia Vulcano, a professional English reviewer. This work was supported by ANPCyT BID–PICT 1576, CONICET D32/10, SeCyTUniversidad Nacional de Co´rdoba (05/C375), and MINCyT Cba PID.

338 Studies in Natural Products Chemistry

ABBREVIATIONS AMB CC CRPC CT DPPH ECE EI-MS FIC FICI FLZ GC-FID GC-MS HRMS i.p. IC50 KI KIS Km LD50 MBC MBTH MDR MIC mL MNCC NADH NF-кB NMR NorA P PAM PDR RCa Rh 6G spp. SRB VLC Vmax mg mM

amphotericin B column chromatography castration-resistant prostate cancer condensed tannin 2,2-diphenyl-1-picrylhydrazyl endothelin-converting enzyme electron ionization-mass spectrometry fractional inhibitory concentration index sum of fractional inhibitory concentration individually indexes fluconazole gas chromatography with flame ionization detector gas chromatography-mass spectrometry high-resolution mass spectrometry intraperitoneal injection media inhibitory concentration kinetic inhibition constant apparent constant of inhibition Michaelis–Menten constant lethal doses media minimum bactericidal concentration 3-methyl-2-benzothiazolinone multidrug resistance minimum inhibitory concentration milliliter maximum noncytotoxic concentration nicotinamide adenine dinucleotide dehydrogenase nuclear factor-кB nuclear magnetic resonance staphylococcus aureus multidrug transporter probability value primary amebic meningoencephalitis pleiotropic drug resistance azole-resistant Candida albicans rhodamine 6G species sulforhodamine B vacuum liquid chromatography maximum reaction rate microgram micromolar

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