Minutifloroside, a New Bis-Iridoid Glucoside with Antifungal and Antioxidant Activities and Other Constituents from Palicourea minutiflora

A new bis-iridoid glucoside minutifloroside (1) was isolated from Palicourea minutiflora, together with asperuloside, (−)-epicatechin, catechin, quercetin, rutin, ursolic acid, oleanolic acid, daucosterol and two monoterpenic indole alkaloids strictosidinic acid and vincosamine. Structural characterization of the compounds was established on their spectral data basis, mainly mass spectrometry (MS) and 1D and 2D nuclear magnetic resonance (NMR). The bis-iridoid showed high activity against Candida albicans strain and antioxidant activity.


Introduction
Iridoids are monoterpenoids found in plenty of plants families and are present in a number of folk medicinal species. 1,2 These bioactive metabolites were considered chemotaxonomic markers in the Rubiaceae family and exhibit remarkable biological and pharmacological properties such as neuroprotective, antitumor, antiinflammatory, antiviral, antibacterial, antifungal, antioxidant, antiprotozoal and antiallergic. 3,4 The presence of dimer iridoids was found in species of Rubiaceae, mainly those belonging to the genus Saprosma, Paederia, Mussaenda, Lasianthus, Randia and Asperula. 5 The genus Palicourea (Rubiaceae) is taxonomically complex and previous phytochemical studies demonstrate the remarkable presence of quinolinic and monoterpenoid indole alkaloids, 6-8 flavonoids, 9 coumarins 10 and terpenoids. 11 Therefore, as part of the investigative efforts to find compounds from Rubiaceae of the Northeastern Brazil flora, this work reports the first chemical study of aerial parts from Palicourea minutiflora, endemic species of Atlantic Forest from Brazil. The MeOH extract was investigated resulting in the isolation and structural elucidation of a new bisiridoid glucoside, minutifloroside (1), and the known iridoid asperuloside (2), the two monoterpenic indole alkaloids strictosidinic acid (3) and vincosamine (4), the flavonoids (−)-epicatechin, catechin, quercetin, and rutin, along with ursolic acid, oleanolic acid and daucosterol. The isolation of the vincosamine (4) and (−)-epicatechin has been reported for the first time in genus Palicourea. This paper deals with the isolation and structure elucidation of the new compound and the assessment of antifungal and antioxidant activities. Optical rotations were measured in H 2 O in a PerkinElmer 343 digital polarimeter at 20 °C and 589 nm, with an optical cell path of 10 mm. High-resolution electrospray ionization Fourier transform mass spectrometry (ESI-FT-MS) data were performed using a Q-Exactive system (Thermo Fischer Scientific) constituted of a heated electrospray ionization (HESI-II) probe source and a hybrid mass analyzer, quadrupole-Orbitrap. The samples mass spectra were acquired in negative mode. The extract solution was injected by direct infusion with a flow rate of 10 µL min -1 . The experimental parameters to ESI(−)-FT-MS analysis were as follows: spray voltage of 4.5 kV, capillary temperature of 300 °C and s-lens of 70 V. For collision, it was applied high collision dissociation (HCD) of 10 and 20 eV. The Xcalibur 3.0.63 software (Thermo Fisher Scientific) was used to acquire and process the data. The exact mass from the m/z of each ion was compared with theoretical m/z to assign the molecular formula with an error less than 5 ppm. ESI(+)-MS data was acquired using a Premier XE triple quadrupole mass spectrometer (Waters Co.) running in the positive and negative ion mode. Major ESI(+)-and ESI(−)-S source parameters were as follow: capillary voltage of 2.0-3.5 kV, cone voltage of 10-45 V and the quadrupole was set to unitary resolution.

Experimental
The nuclear magnetic resonance (NMR) spectra were recorded on a Varian-Mercury plus spectrometer operating at 300.06 ( 1 H) and at 75.45 MHz ( 13 C), respectively, and D 2 O as solvent.

Plant material
The aerial parts (leaves and branches) of P. minutiflora were collected in January 2014, at the Reserva Particular do Patrimônio Natural Serra Bonita (Camacan, Bahia State, Northeast of Brazil, geographical coordinates 15°23'30"S; 39°33'55"W). Voucher specimens (No. 141.214) were deposited at the Centro de Pesquisa do Cacau (CEPEC) and identified by Dr A. M. Amorim (Universidade Estadual de Santa Cruz) and authenticated by C. M. Taylor (Missouri Botanical Garden, MO).

Extraction and isolation
Dried powdered aerial parts (600.0 g) were extracted by maceration with MeOH at room temperature and concentrated under vacuum to yield 85.3 g of MeOH crude extract. Part of MeOH crude extract (10.0 g) was dissolved in a mixture of MeOH:H 2 O (1:1 v/v) and then successively partitioned with different solvents to give n-hexane (HF, 0.96 g), CHCl 3 (CF, 0.12 g), EtOAc (AcF, 3.95 g) and the remaining hydromethanolic (HMF, 4.97 g) fractions.
The HF fraction (0.96 g) was fractionated by CC over silica gel eluted with n-hexane and mixtures of n-hexane/ EtOAc in order of increasing polarity (Hex, Hex:EtOAc 10-90%) providing the steroid daucosterol (15.2 mg).

Antifungal assay
For susceptibility testing, we used the broth microdilution method according to the standards of the Clinical and Laboratory Standards Institute (M27-A3), 12 with some modifications for natural products. 13 The experiment was performed with standard strains: Candida albicans (ATCC 90028) and C. glabrata (ATCC 90030). The final cellular density of the yeast was adjusted to 0.5-5 × 10 3 colony-forming units (CFU) mL -1 in RPMI (Roswell Park Memorial Institute, Gibco) with L-glutamine (without sodium bicarbonate) and 0.165 M 3-(N-morpholino) propanesulfonic acid (pH 7.2). The test was performed in flat-bottom 96-well microtiter plates (Techno Plastic Products, Switzerland). For the assay with compound 1, we tested concentrations of 1250 to 9.765 µg mL -1 and fluconazole (Sigma) was used as reference antifungal drug. The plates were incubated at 35 °C for 48 h. The minimum inhibitory concentration (MIC) of compound 1 was considered the lowest concentration at which no fungal growth was evident, by visual reading.

Antioxidant assay
The radical scavenging capacity of the bis-iridoid (1) was investigated from their ability to reduce the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH • ) by TLC bioautography analysis. 14 The experiment was performed with Macherey-Nagel precoated silica gel 60 F254 plates (Düren, Germany) as the stationary phase and CHCl 3 :MeOH:H 2 O (6:3:1) as the mobile phase. After application of the compound 1 (1.0 mg mL -1 ) and development, the plate was immersed for 2 s in 0.25% (m/v) DPPH • methanolic solution. The antiradical activity results appeared as yellow spots against the purple-blue background. The flavonoid rutin was used as positive control. 15 Minutifloroside (

Results and Discussion
Iridoids glucosides minutifloroside (1) and asperuloside (2), 16 indole alkaloids strictosidinic acid (3) 17 and vincosamine (4) 18 (Figure 1), along with (−)-epicatechin, 19 catechin, 20 quercetin, 21 rutin, 22 ursolic acid, 23 oleanolic acid, 24 and daucosterol, 25 were isolated from the aerial parts extract of Palicourea minutiflora. The structures of known compounds were identified and elucidated using a combination of spectroscopic techniques ( 1 H, 13 C NMR and 2D NMR) and by comparisons with literature data. [16][17][18][19][20][21][22][23][24][25] The structure of the new compound 1 was elucidated by spectrometric methods, including 1D and 2D NMR experiments and HRESIMS.  26,28 The fragment of m/z 227.0556 may be formed from the precursor ion and the product ion of m/z 389.1084. 28,29 This ion presents several fragments, providing more information of the structure resulting in two possible ions pathways, one by its decarboxylation resulting in the ions of m/z 183.0657 ([C 9 H 11 O 4 ] -) and the other fragment of m/z 209.0450 ([C 10 H 9 O 5 ] -) was formed by elimination of a water. 28 The 1 H and 13 C NMR spectra indicated the presence of the signals of two distinct moieties of an iridoid glucoside structure and suggested this compound is a bis-iridoid glucoside which is hereafter referred to as units A and B ( Table 1)  comparing their spectroscopic data with those reported for deacetylasperulosidic acid. 30 The remaining spectral data revealed a second iridoid unit (part B) of the new bis-iridoid. Signals at  H-10a,b). The terminal group of the sugar was revealed by signals at d 4.79 in the 1 H NMR and at d 101.6 in the 13 C NMR spectra. In the NOESY spectrum, a signal at d 3.14 (H-9) correlated with 5.29 (H-1) and 3.04 (H-5) indicating the cis-junction between the two rings and the O-glycosyl residue C-1" with a β configuration. The 1 H and 13 C NMR data of the moiety indicated signals similar to those of scandoside, 30 and further confirmed by the detailed analyses of 1 H-1 H COSY and HMBC spectra. Further proof of the linkage was obtained from the HMBC correlations between H-6' at d 3.77 (m) of the unit A and C-6" d 66.8 of precoated unit B, and by extensive analysis in mass (MS/MS) experiment.
The compound 1 was assayed for their antifungal activities against the yeasts C. albicans and C. glabrata by broth microdilution method. This compound exhibited the highest antifungal activity in C. albicans than C. glabrata species. The MIC was 9.765 µg mL -1 for C. albicans and killed at C. glabrata at 1250 µg mL -1 . Vol. 31, No. 3, 2020 The capacity to scavenge DPPH • radical using TLC bioautography method was also carried out with compound 1. 14 The antioxidant activity was positive for compound 1 at 1 mg mL -1 by visualization of a yellow spot against the purple background.

Conclusions
In summary, in this study two monoterpenic indole alkaloids, four flavonoids and three terpenoids were isolated from the methanolic extract from P. minutiflora Müll. Arg., including asperuloside and a novel bis-iridoid, minutifloroside. This bis-iridoid exhibited high antifungal activity against C. albicans and showed antioxidant capacity.

Supplementary Information
1D and 2D NMR spectra for compound 1 are available online free of charge at http://jbcs.sbq.org.br as a PDF file.