Bioassay-Guided Isolation of Fungistatic ... - ACS Publications

Article Cite This: J. Nat. Prod. 2019, 82, 1496−1502

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Bioassay-Guided Isolation of Fungistatic Compounds from Mimosa caesalpiniifolia Leaves Marcelo J. Dias Silva,‡ Ana M. Simonet,† Naiara C. Silva,§ Amanda L. T. Dias,§ Wagner Vilegas,‡ and Francisco A. Macías*,†

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†

Grupo de Alelopatía, Departamento de Química Orgánica, Instituto de Biomoléculas (INBIO), Facultad de Ciencias, Universidad de Cádiz, C/República Saharaui, 7, 11510-Puerto Real (Cádiz), Spain ‡ Biosciences Institute, UNESP−Univ Estadual Paulista, Coastal Campus of São Vicente, Praça Infante Dom Henrique, s/n, 11330-900, São Vicente, São Paulo, Brazil § Faculty of Pharmaceutical Sciences, UNIFAL−Univ Federal de Alfenas, Rua Gabriel Monteiro da Silva, 714, Centro, 37130-000, Alfenas, Minas Gerias, Brazil S Supporting Information *

ABSTRACT: A bioassay-guided phytochemical study of a Mimosa caesalpiniifolia leaf extract with antifungal activity permitted the identification of 28 compounds, including the new 6-(β-boivinopyranosyl)apigenin (1), 8-(β-oliopyranosyl)apigenin (2), (E)-6-(2-carboxyethenyl)apigenin (3), (E)-8-(2carboxyethenyl)apigenin (4), and 7,5″-anhydro-6-(α-2,6dideoxy-5-hydroxyarabinohexopyranosyl)apigenin (5). The structures of the new compounds were determined by comprehensive spectroscopic analysis, including 1D and 2D NMR techniques, and by mass spectrometry. Compound 3 showed promising activity and selectivity against Candida krusei (IC50 44 nM), which exhibits resistance to azoles. The association of the major components 3-β-D-glucopyranosyloxysitosterol (8) and ethyl gallate (10) was synergistic against C. krusei, especially the IC values of compound 10, which were reduced by more than 100-fold.

T

M. caesalpiniifolia Benth. is a medicinal plant that is commonly known as “sabiá” or “sansão-do-campo”. It is a rugged and fast-growing native perennial tree from the Brazilian northeast, and it contributes greatly to the production of pollen and honey; indeed, it is considered to be an important honey plant in this region.11 Owing to their therapeutic effects, the leaves, stem bark, and flowers have been used in traditional medicine for the treatment of bronchitis, skin infections, and injuries and for inflammation and hypertension.12 The plant extract exhibits cytotoxic activity against the human breast cancer cell line MCF-713 and reduces oxidative DNA damage produced by cadmium.14 Furthermore, the plant attenuated lesions of the colon, reduced inflammation, and modulated the expression of COX-2 and TNF-α during chronic colitis caused by TNBS acid (2,4,6trinitrobenzenesulfonic acid).15 Preliminary studies on the antifungal effects of an EtOAc extract from the liquid−liquid extraction of the 70% aqueous ethanolic extract of M. caesalpiniifolia Benth. leaves showed promising growthinhibition results in the case of yeasts such as Candida glabrata (ATCC 90030) and C. krusei (ATCC 6258) at concentrations of 20 and 40 μg/mL, respectively.16 As a continuation of these

he Mimosa genus (Fabaceae: Mimosoideae) comprises about 530 species native to South America. The major center of diversification for Mimosa is Central Brazil, where numerous species are found in Caatinga and in the Cerrado vegetation.1 Mimosaceae species have been investigated in relation to their therapeutic properties based on traditional knowledge. Crude extracts of these plants showed diverse biological activities, including anticonvulsant,2 anti-inflammatory, and wound-healing3 effects in M. pudica, antibacterial and antifungal4,5 in M. tenuiflora and M. hamata, and attenuation of the development of pulmonary arterial hypertension6 in M. pigra. The Mimosa genus has a wide variety of chemical compounds, including some flavonoid glycosides7 such as 2″(α-L-rhamnopyranosyloxy)isovitexin, 2″-(α-Lrhamnopyranosyloxy)vitexin, 3-(α-L-arabinofuranosyloxy)quercetin, and 3-(α-L-xylopyranosyloxy)quercetin isolated from M. xanthocentra extracts.8 The aerial parts of M. pudica allowed the isolation of isoquercitrin, avicularin, 7-(β-Dglucopyranosyloxy)apigenin, 4″-hydroxymaysin, cassiaoccidentalin B, orientin, and isoorientin.9 3-(2″-Galloyloxy-α-Lrhamnopyranosyloxy)myrcetin, 3-(2″-galloyloxy-α- L rhamnopyranosyloxy)quercetin, 3-(α-L-rhamnopyranosyloxy)myricetin, 3-(α-L-rhamnopyranosyloxy)quercetin, and 3-(α-Larabinopyranosyloxy)quercetin have been isolated from M. pigra.10 © 2019 American Chemical Society and American Society of Pharmacognosy

Received: December 5, 2018 Published: June 18, 2019 1496

DOI: 10.1021/acs.jnatprod.8b01025 J. Nat. Prod. 2019, 82, 1496−1502

Journal of Natural Products

Article

to be identified as another 2,6-dideoxyhexopyranosyl moiety, and the coupling constants for H-1″ (δ 5.48, dd, J = 12.2; 2.7 Hz) are consistent with this proton being in an axial position. The coupling constants between H-2″ and H-3″ (2.9 and 3.0 Hz) and H-3″ and H-4″ (3.7 Hz) indicated equatorial positions for H-3″ and H-4″. The 2D-ROESY spectrum shows a correlation between H-1″ and H-5″ (δ 4.14, qd, 6.6, 1.2 Hz), thus indicating that both protons are in axial positions. All of these values indicate the relative configuration of the sugar, which corresponds to a β-boivinopyranosyl moiety. The 13C NMR assignments were made by analysis of the HSQC and HMBC correlations and confirmed the presence of boivinopyranosyl35 and apigenin units. The connection of these units was established by means of the HMBC correlations between H-1″ (δ 5.48) and C-5 (δ 158.7), C-6 (δ 111.6), and C-7 (δ 164.6). The structure of mimocaesalpin A (1) was thus defined as 6-(βboivinopyranosyl)apigenin. Compound 2 had a deprotonated molecule at m/z 399.1060 [M − H]− (calcd 399.1080) in the HRESIMS data, which, in conjunction with the 13C NMR data, are consistent with the molecular formula, C21H20O8. This compound showed the same signals for the sugar moiety protons as torosoflavone A (6),17 and an oliopyranosyl unit was determined to be part of the molecule. The 1H NMR singlet corresponding to the Aring of apigenin was shielded, in agreement with a connection of the oliopyranosyl unit with C-8 of apigenin. Analysis of the HSQC and HMBC data was done to assign the 1H and 13C NMR signals and the HMBC correlations, which were observed between H-1″ (δ 5.24) and C-7 (δ 164.4), C-8 (δ 107.1), and C-9 (δ 156.1) and H-2ax″ (δ 2.34) and C-8 (δ 107.1), thus confirming the C-8 connection. The structure of mimocaesalpin B (2) was thus defined as 8-(β-oliopyranosyl)apigenin. Compounds 3 and 4 are two isomers that exhibited a deprotonated molecular ion at m/z 339.0500 ([M − H]−, calcd 339.0505) in the HRESI-TOFMS negative mode, and this corresponds to the molecular formula C18H12O7. Both compounds showed signals for apigenin in the 1H NMR spectrum and two additional doublets with a coupling constant of 16.2 Hz and chemical shifts reminiscent of a trans olefinic bond. The 13C NMR spectrum in pyridine-d5 of the major isomer 3 contained signals for apigenin and three signals at δ 122.1, 134.8, and 170.8. The HSQC correlations of the first two carbon signals with the 1H NMR signals at δ 7.91 (d, J = 16.2 Hz) and 9.05 (d, J = 16.2 Hz), along with the HMBC correlation of the 1H NMR signals with 13C NMR signals of the A-ring of apigenin and the signal at 170.8 ppm, are consistent with the presence of a 2-carboxyethenyl moiety. Its location was defined at C-6 of the apigenin moiety via the HMBC correlations between the signal of the 5-hydroxy proton (15.1 ppm) and the H-2″ signal (7.91 ppm) with C-6 (δ 107.7). Thus, the structure of mimocaesalpin C (3) was established as (E)-6-(2-carboxyethenyl)apigenin. The methyl ester of this compound was isolated from Anadenanthera colubrine,36 and the published spectroscopic data are consistent with those of compound 3. Compound 4 comprises a minor component of fraction B. The 1H NMR data (Table 2) of compounds 3 and 4 showed as the main difference for compound 4 the absence of the signal at δ 6.29 (H-8) of compound 3 and the presence of a singlet at δ 6.03, which can be assigned to H-6. Thus, the structure of mimocaesalpin D (4) was proposed to be (E)-8-(2-

studies it is important to ascertain the chemical composition of the EtOAc extract. With this aim in mind, a bioassay-guided isolation of fungistatic compounds from M. caesalpiniifolia Benth. leaves was carried out.

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RESULTS AND DISCUSSION Dried and ground leaves of M. caesalpiniifolia Benth. were extracted with EtOH/H2O (7:3). The extract was partitioned in EtOAc/H2O, and the organic phase was subjected to chromatographic fractionation to give five fractions. Evaluation of the antifungal activity of these fractions showed that fractions B, C, and D had the best activity in terms of minimum inhibitory concentrations for 50% growth (Table 1). Table 1. Inhibitory Concentrations for 50% (IC50) and 90% (IC90) of the Microbial Growth of Candida spp. for the Main Fractionsa sample fluconazole mimocaesalpin A mimocaesalpin B mimocaesalpin C mimocaesalpin D mimocaesalpin E EtOAc

IC (μg/ mL)

C. krusei ATCC 6258

C. glabrata ATCC 90030

IC50 IC50 IC90 IC50 IC90 IC50 IC90 IC50 IC90 IC50 IC90 IC50 IC90

16 250 1000 7.8 250 62.5 − 125 250 62.5 250 31.3 125

4 1000 − 3.9 500 3.9 − 3.9 250 7.8 500 15.6 500

a

(−) absence of activity at the analyzed concentrations.

The active fractions were refractionated by chromatography to obtain five new flavonoids, named mimocaesalpin A−E (1−5), and 23 known compounds, torosoflavone A (6),17 7demethylaciculatin (7),18 3-(β-D-glucopyranosyloxy)sitosterol (8),19 gallic acid (9),14 ethyl gallate (10),11 trans-coumaric acid (11),20 epicatechin (12),21 luteolin (13),22 quercetin (14),23 cassiaoccidentalin A (15),24 cassiaoccidentalin B (16),25 tetrastigma B (17),24 8-[β-6-deoxy-2-(α-L-rhamnopyranosyloxy)-xylohexopyranos-3-ulosyl]luteolin (18),26 D-pinitol (19),27 cis-coumaric acid,20 isoquercitrin,28 hyperoside,29 astragalin,28 trifolin,30 7-(β-glucopyranosyloxy)kaempferol,31 drymariatin A,32 6-(β-boivinopyranosyl)luteolin,33 and malaferin B.34 Seventeen compounds were identified from fraction B including four isomers. The 1H NMR spectroscopic data for these compounds showed characteristic signals35 for Cglycosylated apigenins previously described from this genus.9 The sugar proton signals were characteristic of a 2,6dideoxyhexopyranoside, and, for two of the compounds, their 1 H NMR spectra are in good agreement with the reported data for torosoflavone A (6),17 which has an oliopyranosyl unit at C-6, and 7-de-O-methylaciculatin (7),18 with a digitopyranosyl moiety at C-8. Compound 1 was isolated as the major isomer, and it gave a deprotonated molecule at m/z 399.1083 [M − H]− (calcd 399.1080) in the HRESIMS, which, in conjunction with the 13C NMR data, are consistent with the molecular formula C21H20O8. The NMR features of compound 1 (Table 2) for the sugar moiety allowed the spin system for this sugar 1497



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Table 2. 1H NMR (500 MHz) and 13C (125 MHz) Spectroscopic Data for Mimocaesalpins A−E (1−5) (δ ppm, Methanol-d4) mimocaesalpin A (1) position

δC, type

2 3 4 5 6 7 8 9 10 1′ 2′/6′ 3′/5′ 4′

166.4, C 103.8, CH 184.0, C 158.7, C 111.6, C 164.6, C 96.0, CH 158.3, C 104.9, C 123.2, C 129.5, CH 117.0, CH 162.8, C

mimocaesalpin B (2)a

δH (J in Hz) 6.60, s

6.49, s

7.85, d (8.8) 6.92, d (8.8)

δC 166.4 103.5 184.1 162.3 100.6, CH 164.2 107.1, C 156.1 105.2 123.2 130.1 117.1 162.9

β-boivinopyranoside 1″ 2″

3″ 4″ 5″ 6″ a1

70.0, CH 32.8, CH2

68.8, CH 70.6, CH 72.7, CH 17.4, CH3

mimocaesalpin C (3)

δH (J in Hz) 6.63, s

6.23, s

8.03, d (8.8) 6.94, d (8.8)

β-oliopyranoside

δC b 164.5 103.8 182.8 163.4 107.8, C 165.4 94.3, CH 158.3 104.5 122.0 129.0 116.8 162.8

mimocaesalpin D (4)

δH (J in Hz)b 6.90, s

7.86, d (8.8) 7.15, d (8.8)

72.8

5.24, dd (11.9, 2.4)

134.9

2.26, ddd (14.7, 12.2, 2.9) 1.72, ddd (14.7, 3.0, 2.7) 4.00, ddd (3.7, 3.0, 2.9) 3.39 brd (3.7)

34.1

122.1 CH

71.4

2.34, ddd (13.2, 11,9, 11.7) 1.78, ddd (13.2, 4.9, 2.4) 3.91, ddd (11.7, 4.9, 3.0) 3.67 brd (3.0)

4.14, qd (6.6, 1.2)

77.0

3.78, brq (6.4)

104.3, C

1.28, d (6.6)

17.8

1.35, d (6.4)

24.9

170.9, C

δC

δH (J in Hz)

7.93, d (16.2) 7.10, d (16.2)

6.48, s

5.48, dd (12.2, 2.7)

71.1

9.05, d (16.2) 7.91, d (16.2)

mimocaesalpin E (5)

δH (J in Hz)

166.7 103.8 6.64, s 184.2 157.1 6.03, s 107.6 159.5 6.29, s 95.1 6.57, s 157.9 105.2 123.1 7.77, d (8.8) 7.92, d (8.8) 129.6 7.87, d (8.8) 6.84, d (8.8) 6.90, d (8.8) 117.1 6.93, d (8.8) 163.0 2α-2,6-dideoxy-5carboxyethenyl hydroxyarabinohexopyranoside 6.44, s

6.78, s

2-carboxyethenyl

δH (J in Hz)

8.04, d (16.0)

67.1

5.22, dd (4.6, 2.0)

7.15, d (16.0)

38.8 CH2

2.26, ddd (13.1, 5.3, 2.0) 2.02, ddd (13.1, 11.3, 4.6) 3.57, ddd (11.3, 9.3, 5.2) 3.44 d (9.3)

68.3 CH 80.4

1.63, s

H NMR (600 MHz) and 13C (150 MHz). bPyridine-d5.

The coupling constants of the anomeric proton signal at δ 5.22 (dd, 4.6, 2.0 Hz) indicate that there is no diaxial arrangement with either of the H-2″ protons, and it was therefore considered that H-1″ had an equatorial position. The H-2″ signal at δ 2.02, ddd (13.1, 11.3, 4.6 Hz) is consistent with an axial position for this proton and H-3″, and the coupling constant between signals H-3″ and H-4″ (9.3 Hz) indicated that both are in axial positions. The correlations between the signal at H-4″ and H-6″ and H-2″ax in the 2DROESY spectrum placed them on the same side of the monosaccharide moiety, so an equatorial position was fixed for C-6″. The relative configuration deduced for the sugar moiety was α-2,6-dideoxy-5-hydroxyarabinohexopyranoside. The C-6 position of the apigenin moiety is proposed for the glycosylation site on the basis of the correlation in the HMBC spectrum between H-1″ (δ 5.22, dd, 4.6, 2.0 Hz) and C-5 (δ 157.1). The HMBC spectrum showed a weak correlation between H-6″ (δ 1.63, s) and C-7 (δ 159.5). The chemical shifts of C-7 and C-5″ show that both connected to a heteroatom, which is consistent with oxygen when the MS data are considered. The latter confirms the cyclic ether linkage. Another O,C-fused glycosidic apigenin has been described from Serjania marginata together with C-glycopyranosylapigenins.24 Mimocaesalpin E (5) was identified as 7,5″-anhydro-6-α(2,6-dideoxy-5-hydroxyarabinohexopyranosyl)apigenin (Figure 1). Evaluation of the Antifungal Activity of the Isolated Compounds. Having identified fractions B, C, and D to be the most promising, the available compounds 1, 3, and 8−19

carboxyethenyl)apigenin. The difference between the chemical shifts of the A-ring signals for both isomers is consistent with the reported data.26,37 Compound 5 has the molecular formula C21H18O8, as determined from its deprotonated HRESIMS (ion at m/z 397.0919 [M − H]− (calcd 397.0923)). The 1H and 13C NMR spectra showed, as for compounds 1 and 2, characteristic signals for a C-glycosylated apigenin. An anomeric proton signal, at δ 5.22, dd (4.6, 2.0 Hz), that correlates with the 13C NMR signal at δ 67.1 in the HSQC spectrum is consistent with the presence of a C-glycosidic bond. Analysis of the coupling constants of the sugar signals in the spectra allowed the spin system for this sugar to be identified as [−O−CHR−CH2− CHOH−CHOH−], and the following assignments were made: H-1″ (δ 5.22, dd, J = 4.6; 2.0 Hz); H-2″ (δ 2.26, ddd, 13.1, 5.3, 2.0 Hz and δ 2.02, ddd, 13.1, 11.3, 4.6 Hz); H3″ (δ 3.57, ddd, 11.3, 9.3, 5.3); H-4″ (δ 3.44, d, 9.3 Hz). Furthermore, correlations with the 13C NMR signals at C-1″ (δ 67.1); C-2″ (δ 38.8); C-3″ (δ 68.3); and C-4″ (δ 80.4) are consistent with a 2-deoxysugar. A three-proton singlet at δ 1.63 showed HMBC correlations with C-4″ and the deoxygenated secondary carbon at δ 104.3, which suggests the presence of a 6″-deoxy unit and a dioxygenated C-5″. The pyranoside form of the sugar was determined by the HMBC correlation between the anomeric proton signal (δ 5.22) and C-5″ (δ 104.3). One natural hexopyranoside monosaccharide with a dioxygenated C-5″ has been described, namely, α-2,6-dideoxy5-hydroxyribohexopyranoside, but the sugar moiety of compound 5 was epimeric to the sugar moiety reported for serjanone A.24 1498



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Figure 1. Structures of compounds 1−10 and 13−18.

was obtained for compound 13 (52 nM), whereas compound 3 exhibited an IC50 equal to 44 nM against C. krusei. Among the tested substances from the EtOAc extract, flavonoids with a catechol B-ring, i.e., compounds 13, 14, 16, and 18, were more active than phenol B-ring derivatives 1, 15, and 17. Compound 16 and its isomer 18 gave similar inhibitory concentrations against C. glabrata (±2log2 IC), although they are different in terms of absolute values. The esterification of compound 9 to compound 10 led to an increase in antifungal activity for Candida spp. (IC50 equal to 76 nM), which has a high incidence in infections and has been linked to the use of broad spectrum antibiotics.38 The antifungal activities of gallate derivatives were previously evaluated,39 and in these tests the dermatophytes were the most sensitive microorganisms. According to the authors, the hydroxy group appears to be necessary but not sufficient to confer activity. From an analysis of these studies, it is believed that the lipophilicity of each gallate could play an important role in its antifungal activity. Compound 3 showed promising activity and selectivity for C. krusei (IC50 equal to 44 nM), which has developed resistance to azoles. It is interesting to note that compound 3 was the only active apigenin derivative, and the carboxyethenyl moiety therefore seems to play an important role. The carboxyethenyl moiety was present in compound 11, but this was inactive, so the combination of both substructures appears to be crucial for activity. Compounds 8 and 10 were the major components of fraction B, and they were evaluated in combination against C. krusei and C. glabrata. The association between these compounds elicited a positive interaction against both species

were submitted to evaluation of their fungal activity against the same strains of Candida glabrata and C. krusei (Table 3). Table 3. Inhibitory Concentrations for 50% (IC50) of the Microbial Growth of Candida spp. by Compounds Present in Fractions B, C, and Da C. krusei ATCC 6258

C. glabrata ATCC 90030

sample

IC50 (μg/mL)

IC50 (nM)

IC50 (μg/mL)

IC50 (nM)

fluconazole 3 8 9 10 13 14 16 18 EtOAc

16 15 150 − 15 − 100 − 100 31.3

52 44 260 − 76 − 331 − 173

4 − 150 60 15 15 60 100 60 15.6

13 − 260 353 76 52 199 173 104

a

Compounds 1, 11, 12, 15, 17, and 19 did not show activity at the concentrations analyzed. (−) absence of activity at the concentrations analyzed. IC90 was not observed for any pure compound.

It can be seen from the results in Table 3 that compounds 3, 10, and 13 showed the most interesting results for the Candida strains evaluated, and these are closer to those shown by the EtOAc extract. Compounds 3, 10, and 13 showed relatively low IC50 values that are comparable to those of fluconazole, the standard antifungal used in the test. Compounds 9, 13, and 16 were only active against C. glabrata, and the lowest IC50 value 1499



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Table 4. Association between Compounds 8 and 10, Their IC50 Values Alone and Combined, the ∑FIC Value for the Association, and the Type of Interaction compound 8 IC50 (nM)

compound 10 IC50 (nM)

combination

strain

alone

combined

alone

combined

∑FIC

interaction

compound 8 with compound 10

Candida krusei ATCC 6258 Candida glabrata ATCC 90030

260 260

33 130

76 76

1 2,5

0.1 0.5

synergistic additive

(Table 4). For the checkerboard assay,40 the results were considered as being due to a synergistic effect when the ∑FIC was ≤0.5 and an additive effect if the ∑FIC was between 0.5 and 4. In this way the association was synergistic against C. krusei, and additive effects were observed against C. glabrata. IC50 values for the compounds were markedly reduced, especially the IC50 values for compound 10, which were reduced in excess of 100 times. Compound 8 has a steroidal structure; that is, it is different from phenolic acids such as ethyl gallate (10), and this may have an influence on synergistic effects in combination with compound 10 (Table 4). Tests involving antifungal combinations were recognized as an important clinical strategy nearly 25 years ago, with an increased therapeutic profile observed for the combination of amphotericin B (AmB) with 5-fluorocytosine (5FC) in the treatment of Cryptococcal meningitis.41 Since then, associative studies between AmB and fluconazole (FLZ) against Candida spp. have been reported.42 In the course of time, different types of compound associations have been considered in an effort to identify new therapeutic possibilities and to make such combinations more promising and effective in the treatment of Candida infections,43,44 especially when these associations result in a reduction in side effects and clinical improvement. In our study, positive interactions were observed in the association selected to test and antagonistic effects were not observed. It should be noted that, even though the association of the compounds against C. glabrata cannot be considered synergistic, the association is satisfactory given that there is reduction in the required concentration of the compounds, which indicates the viability and effectiveness of the association. Reported here is the biological activity of the extracts, fractions, and compounds isolated from extracts of M. caesalpiniifolia against emerging species of non-albicans Candida, namely, C. krusei and C. glabrata. The findings of the present study are encouraging and highlight the promising role of natural bioactive compounds isolated from extracts, and their combinations, to treat resistant infections caused by resistant Candida spp., especially species of non-albicans Candida. The most promising result was obtained for C. krusei, but the result obtained for the association against C. glabrata was also interesting given that there was a reduction in the IC50 values. Such results highlight the importance of studies such as that reported here regarding the discovery and development of new therapeutic entities as an appropriate combination that may allow a considerable reduction in the required concentration of drugs, and this may, in turn, minimize the side effects and toxicity observed for antimicrobial agents in conventional use as well as the final cost of treatment and the possibilities of microbial resistance manifestation. In this context, prospective

studies in appropriate animal models are still required to develop therapeutic strategies for these combinations.

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EXPERIMENTAL SECTION

General Experimental Procedures. UV spectra were recorded on a Jasco V-630 spectrophotometer. 1D and 2D NMR spectra were recorded on an Agilent 600 spectrometer equipped with a 5 mm 1 H−13C−15N cryoprobe and an Agilent 500 spectrometer equipped with a 5 mm 1H−19F/15N−31P OneNMR PFG probe. 1H (599.772 and 499.720 MHz) and 13C (150.826 and 125.666 MHz) NMR spectra were recorded in pyridine-d5 and methanol-d4 at 25 °C. Chemical shifts are given on the δ scale and are referenced to residual pyridine (δH 8.70, 7.55, 7.18 and δC 149.84, 135.50, 123.48) or methanol (δH 3.30 and δC 49.00). The Varian pulse sequence with a gradient was applied, and all 2D spectra, except for HMBC spectra, were recorded in the phase-sensitive mode. Exact masses were measured on a UPLC-QTOF ESI (Waters Synapt G2, Manchester, UK) high-resolution mass spectrometer (HRESI-TOFMS). Mass spectra were recorded in negative or positive ion mode in the range m/z 100−2000 with a mass resolution of 20 000 and an acceleration voltage of 0.7 kV. The solvents used for the preparation of extracts and chromatographic fractionation were purchased from Prolabo VWR. TLC plates (silica 60 F254, Merck) were used to monitor the isolation process. Preparative silica gel TLC60 F254 (Merck, 0.25 mm) and Si gel RP-18 TLC F254S (Merck, 0.25 mm) were used to purify some of the flavonoid fractions. Compounds were visualized under UV254/366 light and by spraying with sulfuric H2SO4/H2O/HOAc (4:16:80 v/v/v). Sephadex LH-20 (Sigma-Aldrich) and Kieselgel 60 silica gel (200−60 μm, Merck) were used for column chromatography. Plant Material. Leaves of M. caesalpiniifolia Benth. were collected in Alfenas (Minas Gerais, Brazil) in February 2012 in an area of Cerrado located at a latitude of 21°24′44.1″ S, longitude of 45°55′24.19,9″ W. The plant was identified by Dr. Marcelo Polo and Dr. Geraldo Alves da Silva, and a voucher specimen (no. 695) has been deposited at the Herbarium of the Universidade Federal de Alfenas, Brazil. Extraction and Isolation. The dried leaves (450 g) were extracted successively by percolation at room temperature with EtOH/H2O (7:3, v/v). The hydroalcoholic extract was filtered, concentrated under vacuum at approximately 40 °C, and lyophilized to yield 105 g (23%) of the powdered extract. The crude extract (77g) was suspended in H2O/EtOAc (1:1, v/v) and extracted with EtOAc. The solvent was removed to give 20 g (26%) of EtOAc extract. This extract (20 g) was purified on a silica gel column (23 × 8.5 cm) with CHCl3/MeOH as eluent to give five principal fractions: A (4.2 g, 21%, 90:5), B (1.5 g, 7.5%, 90:10), C (1.6 g, 8.0%, 90:10), D (1.8 g, 9.0%, 80:20), and E (5.6 g, 28.0%, 100:0). Fraction B (300 mg) was chromatographed on a Sephadex LH-20 column (50 × 2.5 cm); with MeOH to give 12 fractions (BA−BL), BA: 3-(β-D-glucopyranosyloxy)sitosterol19 (8) (20 mg), BB: ethyl gallate13 (10) (19 mg), BC: trans-coumaric acid20 (11) (4.5 mg) and cis-coumaric acid,20 BF: mimocaesalpin A (1) (4 mg), BH: epicatechin21 (12) (15 mg), BJ: luteolin22 (13) (5.5 mg). Subfractions BE, BG, and BK were purified by preparative Si gel TLC RP-18 F 254 , acetone/H 2 O (5:4), to afford from BE mimocaesalpin B (2) (2 mg) and 7-demethylaciculatin18 (7) (1 mg), from BG mimocaesalpin E (5) (1 mg) and torosoflavone A17 (6) (1 mg), and from BK malaferin B34 (2.0 mg). Subfractions BD 1500



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and BI were purified by preparative Si gel TLC CHCl3/MeOH (8.5:1.5) to afford mimocaesalpin C (3) (4 mg) and mimocaesalpin D (4) (1 mg). Subfraction BL were purified by preparative Si gel TLC RP-18 F254, ACN/H2O (4:6), to afford drymariatin A32 (1 mg) and 6(β-boivinopyranosyl)luteolin33 (1 mg). Fraction C (300 mg) was chromatographed on a Sephadex LH-20 column (50 × 2.5 cm) with MeOH to give seven fractions (CA−CG). Subfractions CA and CE were purified by preparative Si gel TLC RP-18 F254, H2O/MeOH (3:7), to afford from CA ethyl gallate (10) (10 mg) and from CE cassiaoccidentalin A24 (15) (5 mg) and tetrastigma B24 (17) (4 mg). The subfraction CG was a mixture of astragalin, trifolin,30 and 7-(βglucopyranosyloxy)kaempferol.31 Fraction D (300 mg) was chromatographed on a Sephadex LH-20 column (50 × 2.5 cm) with MeOH to give 10 fractions (DA−DJ). DD: D-pinitol27 (19) (15 mg), DF: a mixture of isoquercitrin28 and hyperoside,29 and DJ: quercetin23 (14) (5 mg). Subfractions DC and DE were purified by preparative Si gel TLC RP-18 F254, H2O/MeOH (45:55), to afford from DC gallic acid7 (9) (5 mg) and from DE cassiaoccidentalin B25 (16) (15 mg) and its isomer 8-[β-6-deoxy-2-(α-L-rhamnopyranosyloxy)xylohexopyranos-3ulosyl]luteolin26 (18) (4 mg). Mimocaesalpin A (1): yellow oil, [α]25D +51 (c 0.2, MeOH); UV λmax (log ε) 213.9 (1.40), 270.9 (1.19), 332.9 (1.25) nm; 1H and 13C NMR, see Table 2; HRESIMS m/z 399.1083 ([M − H]−) (calcd for C21H19O8, 399.1080). Mimocaesalpin B (2): yellow oil, [α]25D +32 (c 0.1, MeOH); UV λmax (log ε) 202.0 (1.16), 270.9 (0.54), 331.1 (0.66) nm; 1H and 13C NMR, see Table 2; HRESIMS m/z 399.1060 ([M − H]−) (calcd for C21H19O8, 399.1080). Mimocaesalpin C (3): yellow oil, UV λmax (log ε) 201.1 (0.82), 312.9 (0.74) with shoulders at 278.6 and 342.9 nm; 1H and 13C NMR, see Table 2; HRESIMS m/z 339.0500 ([M − H]−) (calcd for C18H11O7, 339.1505). Mimocaesalpin D (4): yellow oil, 1H NMR, see Table 2; HRESIMS m/z 339.0501 ([M − H]−) (calcd for C18H11O7, 339.1505). Mimocaesalpin E (5): yellow oil, [α]25D +85 (c 0.1, MeOH); UV λmax (log ε) 213.9 (1.00), 270.0 (0.77), 335 (0.84) nm; 1H and 13C NMR, see Table 2; HRESIMS m/z 397.0919 ([M − H]−) (calcd for C21H17O8, 397.0923). Antifungal Activity Evaluation. The antifungal activities of the EtOAc extract, fractions A−E, and the 14 compounds mimocaesalpin A (1), mimocaesalpin C (3), 3-(β-D-glucopyranosyloxy)sitosterol (8), gallic acid (9), ethyl gallate (10), p-coumaric acid (11), epicatechin (12), luteolin (13), quercetin (14), cassiaoccidentalin A (15), cassiaoccidentalin B (16), tetrastigma B (17), 8-[β-6-deoxy-2-(α-Lrhamnopyranosyloxy)xylohexopyranos-3-ulosyl]luteolin (18), and Dpinitol (19) were evaluated in vitro for antifungal activity against Candida spp. through the broth microdilution method according to the methodology and interpretative criteria proposed by the document M27 A345 and supplementary document M27 S4.46 First, concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.81, 3.9, and 1.95 μg mL−1 were tested for the EtOAc extract and fractions A− E. Concentrations of 100, 60, 30, 15, 7.5, 3.75, 1.875, 0.468, 0.23, and 0.06 μg mL−1 were tested for pure compounds. DMSO was used as the diluent for all tested compounds with no more than 1% DMSO concentration at the final concentration. The standard drug fluconazole was applied as a control for fungistatic action. The microplates were incubated at 37 °C for 24 h, and the results were visualized and analyzed by spectrophotometry at 530 nm in an Anthos Zenyth 200rt microplate reader. The inhibitory concentration for microbial growth was determined at 50% (IC50) and 90% (IC90) for each compound against the Candida species evaluated. Checkerboard Assay. 3-(β-D-Glucopyranosyloxy)sitosterol (8) and ethyl gallate (10) were evaluated in combination against C. krusei and C. glabrata. Briefly, each compound was serially diluted in Mueller−Hinton broth, and the ranges of drug concentrations to be used were determined so that after addition of 100 μL of inocula their concentrations were the average values of the ranges. The microtiter plates were incubated for 24 h at 37 °C, and the growth was quantified by using the spectrophotometric readings as in the

susceptibility test. The results for the effects of compounds 8 and 10 in association were calculated and expressed in terms of a fractional inhibitory concentration (FIC), which is the ratio between the IC of the compound evaluated in combination and its IC evaluated alone. The FIC index is usually calculated as a sum of the FIC values, and it indicates the type of association that exists between the evaluated compounds. From the results obtained, a synergistic effect was assumed when the ∑FIC was ≤0.5, no interaction when the ∑FIC was between 0.5 and 4, or an antagonistic effect when the value was >4.47

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b01025. HRESIMS and 1D and 2D NMR spectra for compounds 1 to 5 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +34 956 012770. Fax: +34 956 016193. ORCID

Francisco A. Macías: 0000-0001-8862-2864 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Sao Paulo State Research Foundation (FAPESP) for funding and CNPq for fellowships (grant 2009/ 52237-9 to W.V. and grants 2012/18760-9 and 2015/21479-8 to M.J.D.S.). This research was supported by the Ministerio de Economiá Industria y Competitividad (MINEICO) (Project AGL2017-88083-R).

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REFERENCES

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