New Polyhydroxylated Steroidal Saponins from Solanum paniculatum

Article Cite This: J. Agric. Food Chem. 2018, 66, 8703−8713

pubs.acs.org/JAFC

New Polyhydroxylated Steroidal Saponins from Solanum paniculatum L. Leaf Alcohol Tincture with Antibacterial Activity against Oral Pathogens Alexander B. Valerino-Díaz,† Daylin Gamiotea-Turro,† Ana C. Zanatta,† Wagner Vilegas,‡ Carlos Henrique Gomes Martins,§ Thayná de Souza Silva,§ Luca Rastrelli,∥ and Lourdes Campaner dos Santos*,† Downloaded via UNIV OF SOUTH DAKOTA on August 29, 2018 at 07:48:23 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Institute of Chemistry, UNESP − São Paulo State University, Rua Prof. Francisco Degni, 55, 14800-060 Araraquara, São Paulo, Brazil ‡ Institute of Biosciences, UNESP − São Paulo State University, Praça Infante Dom Henrique, s/n, 11330-900 São Vicente, São Paulo, Brazil § Laboratory of Research in Applied Microbiology, UNIFRAN − University of Franca, Av. Dr. Armando Salles Oliveira, 201, 14404-600 Franca, São Paulo, Brazil ∥ Dipartimento di Farmacia − University of Salerno, Via Giovanni Paolo II, 84084 Fisciano, Salerno, Italy S Supporting Information *

ABSTRACT: Solanum paniculatum L. is widely used in Brazilian folk medicine for the treatment of liver and gastrointestinal disorders as well as for culinary purposes and beverage production. Fractionation of hydroalcoholic [ethanol (EtOH) 70%] tincture from S. paniculatum leaves led to the isolation of six new spirostanic saponins which included 6-O-α-Lrhamnopyranosyl-(1′′→3′)-β-D-quinovopyranosyl-(22S,23R,25S)-3β,6α,23-trihydroxy-5α-spirostane (1), 6-O-β-D-xylopyranosyl-(1′′→3′)-β-D-quinovopyranosyl-(22S,23R,25R)-3β,6α,23-trihydroxy-5α-spirostane (4), 3-O-α-L-rhamnopyranosyl-(1′′→ 3′)-β-D-quinovopyranosyl-(22S,23S,25R)-3β,6α,23-trihydroxy-5α-spirostane (5), 3-O-β-D-xylopyranosyl-(1′′→3′)-β-D-quinovopyranosyl-(22S,23S,25R)-3β,6α,23-trihydroxy-5α-spirostane (6), 6-O-α-L-rhamnopyranosyl-(1′′→3′)-β-D-quinovopyranosyl(22S,25S)-1β,3β,6α-trihydroxy-5α-spirostane (7), and 6-O-β-D-xylopyranosyl-(1′′→3′)-β-D-quinovopyranosyl-(22S,25S)3β,4β,6α-trihydroxy-5α-spirostane (8) together with two known spirostanic saponins (2, 3). The structures of these compounds were determined by one-dimensional (1D) and two-dimensional (2D) NMR experiments in addition to highresolution electrospray ionization mass spectrometry (HRESIMS) analyses. The 70% alcohol tincture, used as phytomedicine, exhibited promising activities against oral pathogens, including, Steptococcus sanguinis, St. oralis, St. mutans, St. mitis, and Lactobacillus casei with minimal inhibitory concentration (MIC) values ranging from 6.25 to 50 μg/mL. The saponin fraction, nonetheless, showed lower activity against all the strains tested (from 100 to >400 μg/mL). KEYWORDS: Solanum paniculatum, spirostanic saponins, steroidal saponins, antibacterial activity, oral pathogens

■

INTRODUCTION The Solanaceae family is known to have about 3000 species. The Solanum genus, which belongs to this family, is considered one of the most complex and numerous genera, with about 1500 species. These species are part of the ecological systems typically prevalent in the tropical and subtropical regions, with their center of diversity and distribution in South America estimated to be between 1000 and 1100 species of the genus.1 The genus is found to be essentially unique by virtue of the economic importance of many of its species which include potato (S. tuberosum),2 tomato (S. lycopersum),3 and eggplant ( S. melongena).4 Many other species belonging to the genus Solanum, including S. americanum, are used in Brazilian popular medicine for the treatment of gastric dysfunctions, such as gastric ulcer.5 Solanum plants have the ability to biosynthesize steroids,6 saponins,7 alkaloids,3 glycosylated flavonoids,8,9 as well as other structurally diversified and complex secondary metabolites. 10,11 S. paniculatum L., popularly referred to as jurubeba,12 is commonly consumed © 2018 American Chemical Society

both as food and medicinal plant (with a bitter taste). Remarkably, it is the only species of the genus Solanum that has been included in the Brazilian Ministry of Health list of medicinal plants of interest for phytotherapic formulation.13 S. paniculatum is used in the Brazilian folk medicine for the treatment of respiratory tract diseases, for the mitigation of fever, and as tonic.14 It is also used for the treatment of liver and gastric dysfunctions.15 In traditional culinary use, there are recipes that involve the consumption of S. paniculatum fruits after cooking accompanied by rice, pickles, and other cereals. Moreover, fruits from the S. paniculatum plant are used in manufacturing a commercial wine beverage with antioxidant properties.16 The plant is used as a component of various phytotherapic formulations including infusion, decoctions, and Received: Revised: Accepted: Published: 8703

March 13, 2018 July 10, 2018 July 26, 2018 July 26, 2018 DOI: 10.1021/acs.jafc.8b01262 J. Agric. Food Chem. 2018, 66, 8703−8713

Article

Journal of Agricultural and Food Chemistry 70% alcohol tincture.16 The ethanolic extract exhibits antiulcerogenic effect.16 Indeed, numerous works have been published in the literature related to the chemical studies conducted on S. paniculatum, and many steroidal compounds are reported to have been isolated from this species.17 As part of our contribution toward understanding the uses and benefits derived from this species, the present work aims to better investigate the hydroalcoholic leaf tincture (70%) of S. paniculatum, used in Brazil as a nutritional supplement. The phytochemical study carried out in this work allowed the isolation and identification of eight saponins (1−8) with polyhydroxylated spirostane skeleton. Among the isolated substances, six new natural products were discovered (1, 4−8). Considering the traditional use of S. paniculatum, we also evaluated the antibacterial activity of the alcoholic tincture (70%) and saponin fraction (F16−26) against oral pathogens.

■

The tincture obtained 1:10 was a homogeneous brown liquid. The concentration of solid material was 21.1 mg mL−1. Extraction and Isolation. Beginning with the tincture, the solvent was evaporated to dryness under a low pressure. This yielded 52.7 g of the crude ethanol extract (EE) (21.1%). The EE (45 g) was dissolved in 2.25 L of water, and a liquid−liquid partition with 0.75 L of n-butanol (three times) was carried out. After removing the solvent, n-butanol extract (21.9 g, 48.7%) and aqueous extract (15 g, 33.3%) were derived from the extraction. The n-butanol extract (3 g) was fractionated using Sephadex LH-20 gel, column (85 cm × 2.5 cm; H × i.d.) and eluted with methanol. The fractionation of this extract yielded 120 fractions (5 mL each) which were combined into six major fractions (F1−15, F16−26, F27−43, F44−52, F53−59, and F60−76) according to their TLC profiles. The fraction F16−26 (250 mg) was analyzed by HPLC with a refractive index detector (HPLCRI) using an analytical C18 column with MeOH/H2O (7:3 v/v, 1 mL/ min) as mobile phase. This enabled us to establish a better chromatographic condition for the isolation of the compounds in the fraction. After that, the fraction F16−26 was subjected to HPLCRI with the aid of a semipreparative C18 column (Figure S1, Supporting Information) using MeOH/H2O (7:3 v/v, 2 mL/min) as mobile phase; this gave rise to compounds 1 (13.6 mg), 2 (4.7 mg), 3 (17.4 mg), 4 (3.4 mg), 5 (4 mg), 6 (3.1 mg), 7 (5.4 mg), and 8 (3.9 mg). 6-O-α- L -Rhamnopyranosyl-(1′′→3′)-β- D -quinovopyranosyl(22S,23R,25S)-3β,6α,23-trihydroxy-5α-spirostane (1). Yellow amorphous solid: [α]D25 −34° (c 0.1, MeOH); 1H and 13C NMR data (CD3OD), see Tables 1 and 2. HRESIMS m/z :741.4410 [M + H]+ (calcd for C39H65O13, 741.4420). ESIMS/MS m/z: 723.4315 [M + H − H2O]+, 595.3829 [M + H − Rha]+, 431.3150 [M + H − Rha − H2O]+. 6-O-α- L -Rhamnopyranosyl-(1′′→3′)-β- D -quinovopyranosyl(23R,25R)-3β,6α,23-trihydroxy-5α-spirostane (2). Yellow amorphous solid: [α]D25 −26° (c 0.1, MeOH). 1H NMR (CD3OD): aglycone, δH 1.00−1.69 (2H, m, H-1), 1.15−2.37 (2H, m, H-2), 3.45 (1H, m, H3), 1.40−1.74 (2H, m, H-4), 1.15 (1H, m, H-5), 3.37 (1H, m, H-6), 0.94−2.16 (2H, m, H-7), 1.64 (1H, m, H-8), 0.67 (1H, m, H-9), 1.30−1.54 (2H, m, H-11), 1.13−1.73 (2H, m, H-12), 1.15 (1H, m, H-14), 1.25−1.96 (2H, m, H-15), 4.45 (1H, m, H-16), 1.67 (1H, m, H-17), 0.81 (3H, s, H-18), 0.87 (3H, s, H-19), 2.24 (1H, q, J = 7.0 Hz, H-20), 1.09 (3H, d, J = 7.0 Hz, H-21), 3.51 (1H, m, H-23), 1.60 (2H, m, H-24), 2.03 (1H, m, H-25), 3.35−3.45 (2H, m, H-26), 0.76 (3H, d, J = 6.6 Hz, H-27); β-D-quinovose, 4.26 (1H, d, J = 7.9 Hz, H1′), 3.25 (1H, m, H-2′), 3.40 (1H, m, H-3′), 3.00 (1H, d, J = 9.1 Hz, H-4′), 3.29 (1H, m, H-5′), 1.27 (3H, d, J = 6.1 Hz, H-6′); α-Lrhamnose, 5.13 (1H, d, J = 1.3 Hz, H-1′′), 3.92 (1H, m, H-2′′), 3.68 (1H, dd, J = 3.2 Hz, H-3′′), 3.37 (1H, m, H-4′′), 3.98 (1H, m, H-5′′), 1.23 (3H, d, J = 6.2 Hz, H-6′′). 13C NMR (CD3OD): aglycone, δc 37.1 (CH2, C-1), 31.3 (CH2, C-2), 70.4 (CH, C-3), 30.5 (CH2, C-4), 50.4 (CH, C-5), 78.9 (CH, C-6), 40.2 (CH2, C-7), 33.9 (CH, C-8), 53.7 (CH, C-9), 36.1 (C, C-10), 20.6 (CH2, C-11), δC 39.3 (CH2, C12), 40.7 (C, C-13), 56.1 (CH, C-14), 31.6 (CH2, C-15), 81.0 (CH, C-16), 64.3 (CH, C-17), 15.3 (CH3, C-18), 12.4 (CH3, C-19), 40.3 (CH, C-20), 15.6 (CH3, C-21), 108.6 (C, C-22), 69.7 (CH, C-23), 36.2 (CH2, C-24), 23.6 (CH, C-25), 66.1 (CH2, C-26), 16.0 (CH3, C-27); β-D-quinovose, 103.7 (CH, C-1′), 75.1 (CH, C-2′), 82.7 (CH, C-3′), 74.3 (CH, C-4′), 71.6 (CH, C-5′), 17 (CH3, C-6′); α-Lrhamnose, 101.4 (CH, C-1′′), 71.0 (CH, C-2′′), 70.8 (CH, C-3′′), 72.6 (CH, C-4′′), 68.6 (CH, C-5′′), 16.5 (CH3, C-6′′). HRESIMS m/z: 741.4421 [M + H]+ (calcd for C39H65O13, 741.4420), 763.4240 [M + Na]+, 723.4316 [M + H − H2O]+. 6-O-β- D -Xylopyranosyl-(1′′→3′)-β- D -quinovopyranosyl(23R,25S)-3β,6α,23-trihydroxy-5α-spirostane (3). Yellow amorphous solid: [α]D25 −34° (c 0.1, MeOH). 1H NMR (CD3OD): aglycone, δH 1.02−1.73 (2H, m, H-1), 1.16−2.39 (2H, m, H-2), 3.46 (1H, m, H3), 1.40−1.75 (2H, m, H-4), 1.16 (1H, m, H-5), δH 3.40 (1H, m, H6), 0.95−2.16 (2H, m, H-7), 1.66 (1H, m, H-8), 0.68 (1H, td, J = 2.1 Hz, 3.7 Hz, 3.9 Hz, H-9), 1.31−1.53 (2H, m, H-11), δH 1.15−1.75 (2H, m, H-12), 1.16 (1H, m, H-14), 1.28−1.97 (2H, m, H-15), 4.47 (2H, m, H-16), 1.70 (1H, m, H-17), 0.81 (3H, s, H-18), 0.86 (3H, s,

MATERIALS AND METHODS

Chemicals. Methanol, HPLC grade, was purchased from Tedia Company (Fairfield, OH). The water used for all high-performance liquid chromatography (HPLC) mobile phases in the experiments was purified using a Milli-Q system (Millipore, Billerica, MA). Other organic solvents (n-butanol and chloroform) used in the experiments were of analytical grade (acquired from Synthlab, São Paulo, Brazil). All solutions prepared for HPLC were filtered with a 0.22 μm GHP filter (Waters, Milford, MA) before use. Plant Material. S. paniculatum L. leaves were collected from the orchard of medicinal plants of the UNESP−São Paulo State University, situated at the Faculty of Pharmaceutical Sciences of Araraquara, São Paulo State, Brazil, located on GPS 21°48′52.44′′ S and 48°12′07.13 W. The leaves were identified by Dr. Luis Vitor Sacramento. A voucher specimen (HRCB 60754) was deposited at the herbarium of UNESP−São Paulo State University, Institute of Biosciences, Rio Claro, São Paulo State, Brazil. General Apparatus. The optical rotations were carried out using a PerkinElmer Instruments model 341LC polarimeter (l = 10 cm, 589 nm). One-dimensional (1D) and two-dimensional (2D) NMR spectra were collected from a Bruker Advance III HD 600 spectrometer (14.1 T) using an inverse detection 5 mm (1H, 13C, 15 N) cryoprobe. The samples were dissolved in CD3OD (≥99.8) acquired from Sigma-Aldrich. Tetramethylsilane (TMS) was used as reference. High-resolution electrospray ionization mass spectrometry (HRESIMS) data analysis was conducted in the positive ion mode using a Bruker Maxis impact mass spectrometer with the configuration ESI-QqTOF-MS. HPLC analysis in isocratic mode was performed using a Knauer Azura apparatus equipped with smart line 2300 refractive index detector. A Synergy Hydro-RP C18 column (4 μm, 80 Å, 250 mm × 4.6 mm i.d) was employed for analytical purposes, while a Synergy Hydro-RP C18 column (4 μm, 80 Å, 250 mm × 10 mm i.d) was used to conduct the semipreparative analyses. Size exclusion column chromatography was carried out using Sephadex LH-20 (Pharmacia) in order to purify the n-butanol extract. Thin-layer chromatography (TLC) analysis was performed using Merck silica gel 60 plates (>230 mesh). The spots on the TLC plates were revealed by sprinkling the plates with anisaldehyde− H2SO4 reagent, followed by heating at 120 °C. The determination of absolute configuration of monosaccharides was performed using gas chromatography/mass spectrometry (GC/MS) analysis under the following conditions: Agilent 7890B gas chromatograph equipped with a 5977A mass detector (detection temperature 220 °C); column, HP-5MS capillary column (50 m, 0.25 mm i.d., 0.25 μm); column temperature, 150−260 °C at the rate of 8 °C/min; carrier gas, He (0.8 mL/min); split ratio, 1/10; injection temperature, 250 °C; injection volume, 0.5 μL. Preparation of Tincture. On the basis of the traditional Brazilian tincture preparation, dried leaves (250 g) of S. paniculatum were extracted by percolation at room conditions with 70% EtOH (2.5 L). 8704

DOI: 10.1021/acs.jafc.8b01262 J. Agric. Food Chem. 2018, 66, 8703−8713

8705

4.26, 3.25, 3.41, 3.00,

1′ 2′ 3′ 4′

27

m m m s s m d (7.0)

m

m m m d (3.7) d (7.2) Quic d (7.9) m m t (9.2)

3.56, t (3.6)

1.15, 1.27 1.96, 4.46, 1.69, 0.81, 0.87, 2.34, 1.11,

1.52 2.06, 1.66, 3.31, 3.95, 1.20,

25 26

m m td (2.5, 3.9, 4.0)

m m m

m m

m

1.30 1.53, m 1.01 1.74, m

1.13 1.74, 1.15 2.35, 3.45, 1.38 1.74, 1.15, 3.37, 0.94 2.16, 1.64, 0.68, m m td (2.3, 3.8, 3.8)

m m m

m m

m

m m m s s m d (7.0)

m

2.03, m 3.35 3.46, m 0.76, d (6.7) Qui 4.31, d (7.8) 3.34, m 3.42, m 3.04, t (9.1)

1.60, m

3.52, m

1.16, 1.25 1.97, 4.45, 1.68, 0.81, 0.87, 2.25, 1.09,

1.31 1.54, m 1.13 1.74, m

1.01 1.71, 1.16 2.39, 3.45, 1.40 1.74, 1.16, 3.39, 0.95 2.16, 1.65, 0.68,

4 δH (J in Hz)

1

δH (J in Hz)b

24

16 17 18 19 20 21 22 23

13 14 15

12

8 9 10 11

5 6 7

3 4

2

1

position

m m td (2.0, 3.8, 4.1)

m m m

m m

m

m m dd (6.4, 9.1) s s m d (7.3)

m

4.26, 3.26, 3.41, 3.00,

1.47, 1.70 m 1.70, 3.29 3.43, 0.80,

m d (6.54) Qui d (7.9) m m t (9.2)

m

d (11.9)

3.62, dd (2.8, 4.7)

1.10, 1.95 2.30, 4.70, 1.83, 0.87, 0.86, 2.19, 1.15,

1.33 1.54, m 1.15 1.73, m

1.15 1.79, 1.15 2.37, 3.38, 0.93 2.15, 1.15, 3.44, 1.40 1.76, 1.65, 0.68,

δH (J in Hz)

5

Table 1. 1H NMR Spectroscopy Data (14.1 T, CD3OD) of Compounds 1, 4−8a 6

m m m m

m m

m

m m dd (6.3) s s m d (7.3)

m

1.46, dd (11.9) 1.70 m 1.72, m 3.30 3,43, m 0.81, d (6,5) Qui 4.30, d (7.8) 3.34, m 3.42, m 3.05, t (9.1)

3.62, m

1.10, 1.95 2.30, 4.71, 1.83, 0.86, 0.87, 2.18, 1.15,

1.33 1.54, m 1.15 1.74, m

1.65, m 0.68, td (2.1, 3.8, 4.0)

1.02 1.78, 1.16 2.38, 3.39, 0.94 2.16, 1.16, 3.45, 1.39,

δH (J in Hz)

m m d (10.8) 3.84, m

m

m m m s s q (7.0) d (7.0)

m

1.09, d (7.1) Qui 4.26, d (7.8) 3.25, dd (8.0, 8.94) 3.41, m 3.00, t (9.0)

1.15 1.39, 1.64 1.89, 1.89, 3.18,

1.16, 1.40 1.75, 4.42, 1.74, 0.85, 0.87, 2.53, 0.95,

1.31 1.54, m 1.02 1.67, m

m m td (2.4, 3.8, 3.9)

m m m

m m

3.65, m 1.16 1.74, 3.45, 1.95 2.37, 1.16, 3.37, 0.94 2.17, 1.66, 0.68,

7 δH (J in Hz)

m m td (2.2, 3.8, 3.9)

m m

m m m

m

4.30, 3.34, 3.42, 3.05,

1.15 1.39, 1.66 1.89, 1.89, 3.20, 3.84, 1.10,

1.17, 1.40 1.75, 4.43, 1.75, 0.84, 0.87, 2.52, 0.95,

m m m dd (1.9) d (7.0) Qui d (8.0) m m t (9.1)

m

m m m s s q (7,0) d (7.0)

m

1.32 1.54, m 1.01 1.70, m

1.15, 3.40, 0.96 2.17, 1.69, 0.69,

1.15 1.75, 1.92 2.39, 3.67, 3.45,

8 δH (J in Hz)

Journal of Agricultural and Food Chemistry Article


Article

1.23, d (6.0) 1.23, d (6.2) 1.23, d (6.2) 6′′

Assignments were made by 1H ̵ 1H gCOSY, gHSQC, gHMBC, TOCSY-1D, and NOESY-1D data. b1H−1H coupling constants were measured from 1H NMR spectra in hertz. cβ-D-Quinovopyranoside. d α-L-Rhamnopiranoside. eβ-D-Xylopyranoside.

1′′ 2′′ 3′′ 4′′ 5′′

H-19), 2.34 (1H, q, J = 7.0 Hz, H-20), 1.11 (3H, d, J = 7.0 Hz, H-21), 3.57 (1H, t, J = 3.7 Hz H-23), 1.52−2.06 (2H, m, H-24), 1.67 (1H, m, H-25), 3.32−3.95 (2H, m, dd, J = 3.5; 11.0 Hz, H-26), 1.20 (3H, d, J = 7.3 Hz, H-27); β-D-quinovose, 4.30 (1H, d, J = 7.8 Hz, H-1′), 3.35 (1H, dd, J = 8.0, H-2′), 3.42 (1H, m, H-3′), 3.05 (1H, d, J = 9.1 Hz, H-4′), 3.32 (1H, m, H-5′), 1.26 (3H, d, J = 6.1 Hz, H-6′); β-Dxylose, 4.47 (1H, d, J = 7.6 Hz, H-1′′), 3.26 (1H, m, H-2′′), 3.33 (1H, m, H-3′′), 3.50 (1H, m, H-4′′), 3.23−3.89 (2H, m, dd, J = 5.4 Hz, H5′′). 13C NMR (CD3OD): aglycone: δC 37.1 (CH2, C-1), 31.3 (CH2, C-2), 70.4 (CH, C-3), 30.5 (CH2, C-4), 50.4 (CH, C-5), 78.9 (CH, C-6), 40.2 (CH2, C-7), 33.9 (CH, C-8), 53.7 (CH, C-9), 36.1 (C, C10), 20.6 (CH2, C-11), 39.4 (CH2, C-12), 40.7 (C, C-13), 56.1 (CH, C-14), 31.5 (CH2, C-15), 81.2 (CH, C-16), 64.0 (CH, C-17), 15.4 (CH3, C-18), 12.4 (CH3, C-19), 39.6 (CH, C-20), 15.5 (CH3, C-21), 109.9 (C, C-22), 69.6 (CH, C-23), 33.2 (CH2, C-24), 26.6 (CH, C25), 64.8 (CH2, C-26), 18.8 (CH3, C-27); β-D-quinovose, 103.4 (CH, C-1′), 73.7 (CH, C-2′), 86.3 (CH, C-3′), 73.8 (CH, C-4′), 71.3 (CH, C-5′), 16.9 (CH3, C-6′); β-D-xylose, 104.6 (CH, C-1′′), 73.8 (CH, C2′′), 76.3 (CH, C-3′′), 69.7 (CH, C-4′′), 65.7 (CH2, C-5′′). HRESIMS m/z: 727.4263 [M + H]+ (calcd for C38H63O13, 727.4263), 749.4080 [M + Na]+, 709.4160 [M + H − H2O]+. 6-O-β- D -Xylopyranosyl-(1′′→3′)-β- D -quinovopyranosyl(22S,23R,25R)-3β,6α,23-trihydroxy-5α-spirostane (4). Yellow amorphous solid: [α]D25 −21° (c 0.1, MeOH). 1H and 13C NMR data (CD3OD), see Tables 1 and 2. HRESIMS m/z: 727.4262 [M + H]+ (calcd for C38H63O13, 727.4263). ESIMS/MS m/z: 749.4083 [M + Na]+, 709.4156 [M + H − H2O]+, 595.3843 [M + H − Xyl]+, 431.3150 [M + H − Xyl − Qui − H2O]+, 413.3037 [M + H − Xyl − Qui − 2H2O]+. 3-O-α- L -Rhamnopyranosyl-(1′′→3′)-β- D -quinovopyranosyl(22S,23S,25R)-3β,6α,23-trihydroxy-5α-spirostane (5). Yellow amorphous solid: [α]D25 −14° (c 0.1, MeOH). 1H and 13C NMR data (in CD3OD), see Tables 1 and 2. HRESIMS m/z: 741.4407 [M + H]+ (calcd for C39H65O13, 741.4420). ESIMS/MS m/z: 723.4300 [M + H − H2O]+, 595.3827 [M + H − Rha]+, 431.3148 [M + H − Rha − Qui − H2O]+. 3-O-β- D -Xylopyranosyl-(1′′→3′)-β- D -quinovopyranosyl(22S,23S,25R)-3β,6α,23-trihydroxy-5α-spirostane (6). Yellow amorphous solid: [α]D25 +21° (c 0.1, MeOH). 1H and 13C NMR data (CD3OD), see Tables 1 and 2. HRESIMS m/z: 727.4262 [M + H]+ (calcd for C38H63O13, 727.4263). ESIMS/MS m/z: 749.4083 [M + Na]+, 709.4160 [M + H − H2O]+, 431.3150 [M + H − Xyl − Qui − H2O]+. 6-O-α- L -Rhamnopyranosyl-(1′′→3′)-β- D -quinovopyranosyl(22S,25S)-1β,3β,6α-trihydroxy-5α-spirostane (7). Yellow amorphous solid: [α]D25 −35° (c 0.1, MeOH). 1H and 13C NMR data (CD3OD), see Tables 1 and 2. HRESIMS m/z: 741.4414 [M + H]+ (calcd for C39H65O13, 741.4420). ESIMS/MS m/z: 763.4231 [M + Na]+, 723.4307 [M + H − H2O]+, 595.3835 [M + H − Rha]+, 431.3155 [M + H − Rha − Qui − H2O]+, 413.3042 [M + H − Rha − Qui − 2H2O]+. 6-O-β- D -Xylopiranosyl-(1′′→3′)-β- D -quinovopyranosyl(22S,25S)-3β,4β,6α-trihydroxy-5α-spirostane (8). Yellow amorphous solid: [α]D25 −22° (c 0.1, MeOH). 1H and 13C NMR data (CD3OD), see Tables 1 and 2. HRESIMS m/z: 727.4254 [M + H]+ (calcd for C38H63O13, 727.4263). ESIMS/MS m/z: 749.4071 [M + Na]+, 709.4150 [M + H − H2O]+, 595.3838 [M + H − Xyl]+, 431.3150 [M + H − Xyl − Qui − H2O]+. Acid Hydrolysis of Saponins 1−8 and Determination of Absolute Configuration of Monosaccharides. To determine the absolute configuration of saponins 1−8, acid hydrolysis was performed as described by Hara et al.18 For the determination of the Rf value of the hydrolyzed sugars, TLC analysis was performed using the solvent system EtOAc/MeOH/H2O/AcOH (11:2:2:2). The Rf values obtained for the hydrolyzed sugars were compared to those of sugar standards. The following Rf values were obtained for Dquinovose, L-rhamnose, and D-xylose: 0.65, 0.50, and 0.75, respectively. The aqueous layer was concentrated and analyzed by GC/MS as described by Hara et al.18 The absolute configurations of D-quinovose,

a

Qui 3.31, m 1.26, d (6.0) Xyl 4.47, d (7.6) 3.26, m 3.33, m 3.49, m 3.23, m 3.89, dd (5.3) Qui 3.30, m 1.26, d (6.0) Rha 5.13, d (1.2) 3.92, dd (1.7, 3.2) 3.67, m 3.37, m 3.98, m Qui 3.32, m 1.27, d (6.2) Xyl 4.47, d (7.0) 3.26, m 3.33, m 3.49, m 3.23, d (2.6) 3.88, dd (5.4) Qui 3.30, m 1.27, d (6.3) Rha 5.12, d (1.5) 3.92, dd (1.8) 3.68, dd (3.4, 9.5) 3.37, m 3.99, m Qui 3.31, m 1.27, d (6.1) Xyle 4.47, d (7.6) 3.25, m 3.33, m 3.50, m 3.23, m 3.89, dd (5.4) 3.30, m 1.27, d (6.2) Rhad 5.13, d (1.6) 3.92, dd (1.8, 3.3) 3.68, dd (3.4) 3.38, m 3.99, m 5′ 6′

Quic

8 7

δH (J in Hz) δH (J in Hz)

6 5

δH (J in Hz)

4

δH (J in Hz)

1

δH (J in Hz)b position

Table 1. continued

δH (J in Hz)

Journal of Agricultural and Food Chemistry

8706


Article

Journal of Agricultural and Food Chemistry Table 2.

13

C NMR Spectroscopy Data (14.1 T, CD3OD) of Compounds 1, 4−8a 1

4

5

6

7

8

position

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

37.1, CH2 31.3, CH2 70.4, CH 30.5, CH2 50.4, CH 78.9, CH 40.2, CH2 33.9, CH 53.7, CH 36.2, C 20.6, CH2 39.4, CH2 40.7, C 56.1, CH 31.5, CH2 81.2, CH 64.0, CH 15.4, CH3 12.4, CH3 39.6, CH 15.5, CH3 109.9, C 69.7, CH 33.2, CH2 26.6, CH 64.9, CH2 18.8, CH3 Quib 103.7, CH 75.0, CH 82.8, CH 74.3, CH 71.6, CH 16.9, CH3 Rhac 101.4, CH 71.0, CH 70.8, CH 72.6, CH 68.6, CH 16.5, CH3

37.1, CH2 31.3, CH2 70.4, CH 30.5, CH2 50.4, CH 78.9, CH 40.2, CH2 33.9, CH 53.7, CH 36.2, C 20.6, CH2 39.3, CH2 40.7, C 56.1, CH 31.5, CH2 81.0, CH 64.3, CH 15.3, CH3 12.4, CH3 40.3, CH 15.6, CH3 108.6, C 69.6, CH 36.2, CH2 23.6, CH 66.1, CH2 15.,9, CH3 Qui 103.4, CH 73.7, CH 86.3, CH 73.8, CH 71.3, CH 16.9, CH3 Xyld 104.6, CH 73.8, CH 76.3, CH 69.7, CH 65.7, CH2

37.0, CH2 31.3, CH2 79.0, CH 40.3, CH2 50.4, CH 70.5, CH 30.5, CH2 33.6, CH 53.7, CH 36.2, C 20.7, CH2 39.8, CH2 40.9, C 55.1, CH 33.3, CH2 83.9, CH 62.9, CH 15.4, CH3 12.4, CH3 42.4, CH 15.0, CH3 112.1, C 69.4, CH 37.1, CH2 30.5, CH 68.1, CH2 15.8, CH3 Qui 103.7, CH 75.0, CH 82.8, CH 74.3, CH 71.6, CH 16.9, CH3 Rha 101.4, CH 71, CH 70.8, CH 72.6, CH 68.6, CH 16.5, CH3

37.0, CH2 31.3, CH2 78.9, CH 40.2, CH2 50.4, CH 70.4, CH 30.5, CH2 33.6, CH 53.7, CH 36.2, C 20.7, CH2 39.8, CH2 40.9, C 55.3, CH 33.3, CH2 83.9, CH 62.9, CH 15.4, CH3 12.4, CH3 42.4, CH 15.0, CH3 111.1, C 69.4, CH 37.1, CH2 30.5, CH 68.1, CH2 15.7, CH3 Qui 103.4, CH 73.7, CH 86.3, CH 73.8, CH 71.3, CH 16.8, CH3 Xyl 104.0, CH 73.8, CH 76.3, CH 69.6, CH 65.8, CH2

62.5, CH 39.8, CH2 70.4, CH 31.3, CH2 50.4, CH 79.0, CH 40.3, CH2 33.7, CH 53.7, CH 36.2, C 20.6, CH2 37.0, CH2 40.8, C 56.0, CH 30.5, CH2 81.2, CH 61.7, CH 15.6, CH3 12.4, CH3 35.6, CH 12.9, CH3 111.2, C 31.1, CH2 34.5, CH2 29.9, CH 63.6, CH2 16.2, CH3 Qui 103.7, CH 75.0, CH 82.8, CH 74.3, CH 71.6, CH 16.9, CH3 Rha 101.4, CH 71.0, CH 70.8, CH 72.6, CH 68.6, CH 16.5, CH3

39.9, CH2 31.1, CH2 62.5, CH 70.4, CH 50.4, CH 78.9, CH 40.2, CH2 33.7, CH 53.7, CH 36.2, C 20.7, CH2 37.1, CH2 40.8, C 55.9, CH 30.5, CH2 81.2, CH 61.7, CH 15.6, CH3 12.4, CH3 35.6, CH 12.9, CH3 111.2, C 31.3, CH2 34.5, CH2 29.9, CH 63.6, CH2 16.2, CH3 Qui 103.4, CH 73.7, CH 86.3, CH 73.8, CH 71.3, CH 16.8, CH3 Xyl 104.6, CH 73.8, CH 76.3, CH 69.6, CH 65.7, CH2

1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″

Assignments were made by DEPT 135°, TOCSY-1D, HSQC, and HMBC data. bβ-D-Quinovopyranoside. cα-L-Rhamnopiranoside. dβ-DXylopyranoside.

a

33478), St. mutans (ATCC 25175), St. mitis (ATCC 49456), and Lactobacillus casei (ATCC 11578 and CI).

L-rhamnose, and D-xylose were confirmed through the analyses of their retention times and by comparison with standard samples. The retention times of monosaccharides derivatives are as follows: Dquinovose (12.51 min), L-rhamnose (12.67 min), and D-xylose (11.89 min), respectively. Antibacterial Activity Evaluation. The evaluation of the antimicrobial activity and the minimal inhibitory concentration (MIC) was performed in triplicate by the microdilution method, as described in the Clinical and Laboratory Standards Institute (CLSI)19 and by Sarker et al.20 The bacterial strains employed were maintained in the culture collection of the Research Laboratory of Applied Microbiology (LaPeMA), University of Franca, São Paulo Brazil, under cryopreservation (−80 °C) in tryptic soy broth (TSB; Difco Laboratories, Detroit, MI), containing glycerol at 20% (v/v). The bacteria used were Enterococcus faecalis (ATCC 4082 and CI), Streptococcus salivarius (ATCC 25975 and CI), St. sanguinis (ATCC 10556 and CI), St. oralis (ATCC 5529 and CI), St. sobrinus (ATCC

■

RESULTS AND DISCUSSION Fraction F16−26 of the n-butanol extract obtained from the liquid−liquid extraction of the 70% ethanolic extract derived from the leaves of S. paniculatum L., yielded eight pure compounds 1−8 (Figure 1). The identification of the related compounds was performed using optical rotation along with 1H, 13C, DEPT 135°, gHMBC, gHSQC, 1 H− 1 H gCOSY, TOCSY-1D, and NOESY-1D NMR spectra analyses and mass spectra via the HRESIMS technique. Compound 1 was obtained as a yellow amorphous solid and exhibited a molecular ion [M + H]+ at m/z 741.4410 (calcd for 8707


Article


Figure 1. Isolated compounds from S. paniculatum L. leaves.

displayed the entire spin system of a quinovopyranosyl residue (Table 1). Irradiation of the anomeric proton at δH 5.13 (d, J = 1.6 Hz) exhibited only the signal belonging to H-2′′ at δH 3.92 (dd, J = 1.8 Hz) of a rhamnopyranosyl unit, which is also consistent with the presence of the methyl doublet H-6′′ at δH 1.23 (d, J = 6.2 Hz). The other 27 carbons belonging to the sapogenol moiety were attributed to eight methylene carbons, seven methine carbons, two quaternary carbons, four methyl groups, four oxygen-bearing methine carbons, one oxygenbearing methylene carbon, and one acetal carbon (see Tables 1 and 2). The gHMBC spectrum (Figures S9 and S10) analysis was conducted aiming at demonstrating the connectivity of the respective protons and carbons (J3). Through this analysis, long-range correlations were observed from H3-19 to C-10/C9/C-5, H3-18 to C-12/C-13/C-14, H3-21 to C-17/C-20/C-22, H3-27 to C-24/C-25/C-26, and H-25 to C-23. The correlation in the gHMBC spectrum between H-25 (δH 1.66) and C-23 (δC 69.7) indicates the present of a hydroxyl group at C-23. NOESY-1D spectrum analyses (Figure S11) enabled us to establish the configuration at C-23 (C-23R configuration) since an interaction was observed between the signal at δH 3.56 (H-23, t, J = 3.6 Hz) and the signal at δH 2.34 (H-20, m). In

C39H65O13, 741.4420) in the positive HRESIMS mode (Figures S2 and S3) corresponding to the molecular formula C39H64O13. On the basis of the analysis of the 1H NMR signals (in CD3OD) of compound 1 (Table 1, Figure S4) the conclusion drawn here is that the compound consisted of two tertiary methyl groups at δH 0.81 (3H, s, H3-18), δH 0.87 (3H, s, H3-19), four secondary methyl groups at δH 1.11 (3H, d, J = 7.0 Hz, H3-21), δH 1.20 (3H, d, J = 7.2 Hz, H3-27), δH 1.27 (3H, d, J = 6.2 Hz, H3-6′), δH 1.23 (3H, d, J = 6.2 Hz, H3-6′),́ two protons related to a methylene group at δH 3.31 (overlapped, H-26a), δH 3.95 (d, J = 3.7 Hz, H-26b) linked to an oxygen, three protons at δH 3.37 (overlapped, H-6), δH 3.45 (1H, overlapped, H-3), and δH 3.56 (1H, t, J = 3.6 Hz, H23) assigned to a methine group linked to carbinol carbons, and two protons attached to anomeric carbon at δH 4.26 (1H, d, J = 7.9 Hz, H-1′) and δH 5.13 (1H, d, J = 1.6 Hz, H-1′).́ 13 C NMR and DEPT 135° data of compound 1 (shown in Table 2, Figures S5 and S6) exhibited 39 carbon signals. The comparison between these chemical shifts with those of torvoside K(2)21 helped to identify 12 of these signals which correspond to α-L-rhamnopyranosyl and β-D-quinovopyranosyl units. The TOCSY-1D spectrum (Figures S7 and S8) with irradiation of the anomeric proton at δH 4.26 (d, J = 6.2 Hz) 8708


Article

Journal of Agricultural and Food Chemistry addition, the methyl group H3-18 at δH 0.81 (s) (Figure S12) interacts with H-20 at δH 2.34 (m), H-12 at δH 1.74 (m), H-8 at δH 1.64 (m), and H-11 at δH 1.30 (m). This suggests the existence of β-orientation for H3-18 and for H-20, αorientation for H3-21 (C-20S configuration), trans fusion between CD rings, and cis fusion between DE rings. The longrange interactions involving H3-19 at δH 0.87 (s) (Figure S13) with H-1 at δH 1.71 (m), H-2 at δH 1.15 (m), H-4 at δH 1.38 (m), H-8 δH 1.64 (m), and H-11 δH 1.30 (m) indicate the occurrence of trans fusion between AB and BC rings with H319 occupying a β-position in the sapogenol moiety. The 1H−1H gCOSY spectrum (Figure S14) exhibited a correlation between H-3 at δH 3.45 and H-2 at δH 1.15, H-6 at δH 3.38 and H-7 at δH 0.98, and H-23 at δH 3.56 and H-24 at δH 1.53, thus indicating the presence of hydroxyl groups at C3, C-6, and C-23. For the determination of the configuration of C-25, it was regarded essentially relevant to consider the chemical shift of the H3-27. Reports in the literature show the occurrence of a higher proton chemical shift (δH 1.10−1.53) for the 23-hydroxylspirostanols when the methyl group H3-27 is in the axial position. This is attributed to the 1.3-diaxial interactions. When the methyl group H3-27 is in equatorial position the proton chemical shift occurs between δH 0.73− 0.80.21 The configuration of C-25 was determined as S given that the signal of H3-27 in compound 1 occurs at δH 1.20 (which implies that it is in the axial position). For the determination of the configuration of C-22, an evaluation was conducted on the 1H NMR chemical shifts of the hydrogen related to the methyl group H3-21 and H-16 along with the 13C NMR chemical shifts of C-20 in comparison to other 23hydroxylspirostanols. With regard to 22-α-O-spirostanol glycosides, reports in the literature show that H3-21 and H-16 absorb frequency in the range between δH 1.17−1.26 and δH 4.49−4.56, respectively, and the C-20 absorbs frequency at δC 35.0−36.2.22,23 In the case of 22-β-O-spirostanol, the H3-21 absorbs frequency at δH 1.52−1.54, H-16 at δH 5.18−5.20, and C-20 at δC 43.0−44.1.23,24 Compound 1 exhibited a chemical shift of H3-21 at δH 1.11, H-16 at δH 4.46 and C-20 at δC 39.6, thus suggesting the 22-α-O-configuration for the sapogenol. By virtue of that, the aglycone of compound 1 aglycone was characterized as (22S,23R,25S)-3β,6α,23-trihydroxy-5α-spirostane. For the determination of the sugar linkage, the gHMBC correlations were observed between the quinovopyranosyl H1′ δH 4.26 (d, J = 7.9 Hz) and the sapogenol C-6 at δC 78.9 in addition to the correlation between rhamnopyranosyl C-1 at δC 101.4 and the inner quinovopyranosyl H-3′ at δH 3.41(m). This led us to the unambiguous confirmation of the occurrence of the linkage (1′′→3′) between the rhamnopyranosyl and quinovopyranosyl moieties. In view of that, the structure of compound 1 was determined as the new 6-O-α-L-rhamnopyranosyl-(1′′→3′)-β- D -quinovopyranosyl-(22S,23R,25S)3β,6α,23-trihydroxy-5α-spirostane. Compound 2 was obtained as a yellow amorphous solid. The compound exhibited a molecular ion [M + H]+ at m/z 741.4421 (calcd for C39H65O13, 741.4420) in the positive HRESIMS mode (Figure S15) compatible with the molecular formula C39H64O13. These data demonstrate the presence of a possible isomer of the compound 1. The pattern of the 13C NMR signals of compound 2 (Table 2) is found to be similar to those of compound 1 except for the signal of the methyl group H3-27 δH 0.76 (d. J = 6.6 Hz). This implies that the position occupied by the H3-27 is equatorial

and the configuration of C-25 is R.21 The comparison of the spectroscopic data of compound 2 with the steroidal saponin isolated from S. chrysotrichum leaves25 showed similar chemical shifts in the 1H NMR and 13C NMR. As such, compound 2 was identified as 6-O-α-L-rhamnopyranosyl-(1′′→3′)-β-Dquinovopyranosyl-(23R,25R)-3β,6α,23-trihydroxy-5α-spirostane. Compound 3 was obtained as a yellow amorphous solid. It exhibited a molecular ion at m/z 727.4263 (calcd for C38H63O13, 727.4263) in the positive HRESIMS mode (Figure S16) compatible with the molecular formula C38H62O13. The 1 H NMR chemical shifts (Table 1, Figure S17) and 13C NMR spectra for compound 3 (Table 2, Figure S18) were seen to be in good agreement with those of the spirostanic saponin isolated from S. hispidum.26 Thus, on the basis of these evidence, compound 3 was identified as 6-O-β-D-xylopyranosyl-(1′′→3′)-β-D-quinovopyranosyl-(23R,25S)-3β,6α,23-trihydroxy-5α-spirostane, previously isolated from S. paniculatum.27 Compound 4 was obtained as a yellow amorphous solid. This compound exhibited a molecular ion [M + H]+ at m/z 727.4262 (calcd for C38H63O13, 727.4263) in the positive HRESIMS mode (Figures S19 and S20) compatible with the molecular formula C38H62O13. The 1H, 13C (Tables 1 and 2, Figures S21 and 22), DEPT 135°, gHSQC, and gHMBC (Figures S23−25) spectroscopic data for compound 4 exhibited close similarity with those of compound 3 except for the proton signal of H3-27 at δH 0.76 (d, J = 6.7 Hz), which indicated the equatorial position of the methyl group H3-27 and the C-25R configuration. As a result, the structure of 4 was identified as the new 6-O-β-D-xylopyranosyl-(1′′→3′)-β-Dquinovopyranosyl-(22S,23R,25R)-3β,6α,23-trihydroxy-5α-spirostane, an epimer of compound 3. Compound 5 was obtained as a yellow amorphous solid. The molecular formula C39H64O13 is in agreement with the observed molecular ion [M + H]+ at m/z 741.4407 (calcd for C39H65O13, 741.4420) obtained in the HRESIMS analyses (Figures S26 and 27). The 1H and 13C NMR spectra (Tables 1 and 2, Figures S28 and 29) were similar to those of spirotorvoside.28 The main difference between spirotorvoside and compound 5 is found in the configuration in the configuration of the C-25. The proton NMR resonance of the methyl group H3-27 at δH 0.81 (d, J = 6.5 Hz) in compound 5 indicates that the position occupied by the H3-27 is equatorial and the configuration of C-25 is R. The NOESY1D spectra (Figure S30) showed the interaction between H-23 at δH 3.62 (dd, J = 2.76 Hz) and H3-21 at δH 1.15 (d, J = 7.3 Hz), indicating that H-23 occupied an equatorial position; as such, a C-23S configuration was assigned. On the basis of the data above, compound 5 was characterized as the new 3-O-α-Lr h a m n o p y r a n o s y l - ( 1 ′ ′ → 3 ′ )- β - D - q u i n o v o p y r a n o s y l (22S,23S,25R)-3β,6α,23-trihydroxy-5α-spirostane. Compound 6 was obtained as a yellow amorphous solid. The compound exhibited a molecular ion [M + H]+ at m/z 727.4262 (calcd for C38H63O13, 727.4263) derived from HRESIMS (Figures S31 and 32) analyses compatible with the molecular formula C38H62O13. The 1H and 13C NMR data in CD3OD (Tables 1 and 2, Figures S33 and 34) of the aglycone of compound 6 are similar to those of compound 5 (Tables 1 and 2). A diagnostic difference was observed in the chemical shifts corresponding to the sugar units. The saccharide part of compound 6 exhibited a close similarity to those of compounds 3 and 4; this led to its identification as βD-xylopyranosyl-(1′′→3′)-O-β-D-quinovopyranosyl. Hence, 8709


Article


and H-4 at δH 2.37 (m); H-1 at δH 3.65 (m) with H-2 at δH 1.74 (m) and between H-6 at δH 3.37 (m) with a methylene group H2-7 at δH 0.94, 2.17 (m), thus demonstrating the presence of the hydroxyl groups at C-1, C-3, and C-6. The ketal carbon C-22 was determined as “S” due to the NOESY1D correlation established solely between H-20 at δH 2.53 (q, J = 7.0 Hz) and the methyl group H3-18 at δH 0.85 (s) (Figure S45). By contrast, when the stereocenter C-22 occupies an R configuration, the NOESY-1D spectra exhibit the correlation of the H-20 with the methyl group CH3-18 and the methylene group CH2-23 in the ring F.30 The remaining signals in compound 7 were assigned using the spectroscopic data obtained through gHMBC, gHSQC (Figure S46), and 1H−1H gCOSY analyses, which allowed assigning all signals of this compound for the first time in the literature. In view of that, compound 7 was characterized as the new 6-O-α- L rhamnopyranosyl-(1′′→3′)-β-D-quinovopyranosyl-(22S,25S)1β,3β,6α-trihydroxy-5α-spirostane. Compound 8 was isolated as a yellow amorphous solid with molecular formula C38H62O13 derived from HRESIMS analyses in the positive mode (Figures S47 and S48), which showed an ion peak [M + H]+ at m/z 727.4254 (calcd for C38H63O13, 727.4263). The 1H NMR and 13C NMR (Tables 1 and 2, Figures S49 and S50) spectroscopic signals of the aglycone displayed a pattern similar to that of (22R,25S)-1β,3β,6αtrihydroxy-5α-spirostane (compound 7), which has been previously described. However, differences in 1H−1H gCOSY spectra were detected (Figures S51 and S52). The 1H−1H gCOSY spectra showed correlations between H-3 at δH 3.67 (m) and H-2 at δH 1.92 (m), H-4 at δH 3.45 (m), and H-5 at δH 1.16 (m), and between H-6 δH at 3.40 (m) with H-5 at δH 1.16 (m) and H-7 at δH 0.96 (m). These correlations indicate the presence of hydroxyl groups at C-3, C-4, and C-6. On the other hand, the 1H NMR spectra exhibited two anomeric proton resonances for sugar moieties at δH 4.30 (d, J = 8.0 Hz, 1H) and δH 4.47 (d, J = 7.0 Hz, 1H); these differ from those of the saccharide unit present in compound 7. The TOCSY-1D experiments with selective irradiation of the anomeric proton at δH 4.30 (d, J = 8.0 Hz, 1H) (Figure S53) displayed the proton spin systems of a β-D-quinovose unit [δH 3.34 (m, H2′), δH 3.42 (m, H-3′1H), δH 3.05 (t, J = 9.1 Hz, H-4′, 1H), δH 3.31 (m, H-5′, 1H), and a methyl group at δH 1.26 (d, J = 6.0 Hz, H3-6′, 3H)], whereas irradiation of the anomeric proton at δH 4.47 (d, J = 7.0 Hz 1H) (Figure S54) exhibited the proton ́ spin systems of a β-D-xylose unit [δH 3.26 (m, H-2′,1H), δH ́ ́ 3.33 (m, H-3′,1H), δH 3.49 (m, H-4′,1H), and a methylene at ́́ 1 ́ δH 3.23 (m, H-5á,1H), δH 3.89 (m, H-5b,1H)]. H NMR and 13 C NMR resonances relative to the sugar part of compound 8 were similar to those of compounds 3, 4, and 6 sugar units. This confirms the identity of the sugar units as β-D-quinovose and β-D-xylose. The linkage between the units was determined as (1′′→3′) by the gHMBC correlation (Figure S55) between the anomeric proton of xylose at δH 4.47 (d, J = 7.0 Hz, 1H) and carbon at δC 86.3 which corresponds to C-3′ of quinovose. The site of the sugar-chain bond at the aglycone was also derived from the gHMBC correlation between the anomeric proton of quinovose at δH 4.30 (d, J = 8.0 Hz, 1H) and C-3 at δC 78.9 of the aglycone. The analyses of the interactions in the NOESY-1D spectrum for compound 8 (Figures S56 and 57) revealed the same interactions observed in compound 7. This result confirms the existence of trans fusion between AB and BC, CD rings, a cis fusion between DE rings, a configuration “S” for a ketal C-22

compound 6 was identified as the new 3-O-β-D-xylopyranosyl(1′′→3′)-β-D-quinovopyranosyl-(22S,23S,25R)-3β,6α,23-trihydroxy-5α-spirostane. Compound 7 was obtained as a yellow amorphous solid. The molecular formula was assigned as C39H64O13 based on data from HRESIMS analyses (molecular ion [M + H]+ at m/z 741.4414 (calcd for C39H65O13, 741.4420) (Figures S35 and 36). The 1H NMR spectroscopic signals for compound 7 (Table 1, Figure S37) displayed two tertiary methyl groups at δH 0.85 (s, H-18, 3H) and δH 0.87 (s, H-19, 3H), four secondary methyl groups at δH 1.09 (d, J = 7.1 Hz, H-27, 3H), δH 0.95 (d, J = 7.0 Hz, H-21, 3H), δH 1.26 (d, J = 6.0 Hz, H-6′, 3H), δH 1.23 (d, J = 6.0 Hz, H-6′′, 3H), and two protons attached to anomeric carbons at δH 4.26 (d, J = 7.8 Hz, H-1′) and δH 5.13 (d, J = 1.2 Hz, H-1′′). The 1H NMR and 13C NMR resonances (Table 2, Figure S38) for the sugar moieties of compound 7 were found to be similar to those of the saccharide part of compounds 1, 2, and 5 (Tables 1 and 2), which were also supported by TOCSY-1D experiments (Figures S39 and S40) through the irradiation of the anomeric protons at δH 4.26 (d, J = 7.8 Hz H-1′) and δH 5.13 (d, J = 1.2 Hz, H-1″). Hence, the sugar units were identified as β-Dquinovose and α-L-rhamnose. The linkage between the saccharide units was determined as (1′′→3′) due to the gHMBC correlation (Figure S41) between the anomeric proton of rhamnose at δH 5.13 (d, J = 1.2 Hz) and the C-3′ at δC 82.8 of the quinovose unit. The site of the sugar-chain bond at the aglycone was also derived from the gHMBC correlation between the anomeric proton of quinovose at δH 4.26 (d, J = 7.8 Hz) and the C-3 at δC 79.0 of aglycone. The gHMBC spectrum analysis was conducted in order to demonstrate the connectivity of the respective protons and carbons. The crosspeaks between H-26ax at δH 3.84 (m) and C-27 at δC 16.2, H26ec at δH 3.18 (broad d, J = 10.8 Hz) and C-27 at δC 16.2, and C-24 at δC 34.5 and C-22 at δC 111.2 are indicative of the presence of an unsubstituted F ring (Figure S42). The stereochemistry of the stereocenter C-25 was determined using the Agrawal method, which is based on differences of the chemical shifts between the geminal protons of the H2-26 metilene and the unsubstituted 22, 23, 24, and 25 positions.29 These author observed that, for compounds consisting of stereocenter 25S, the difference between the 1H NMR chemical shifts of the geminal protons of the H2-26 is usually higher than 0.35, whereas for compounds with stereocenter 25R, the difference between these chemical shifts is lower than 0.20. As the difference observed in compound 7 was 0.66 (δHa 3.84 for H26a and δHb 3.18 for H26b), the C-25S configuration was assigned. Furthermore, H3-27 methyl group absorbs frequency within the range between δH 0.95 and 1.13 in spirostanols with 25S configuration, and between δH 0.71 and 0.83 in 25R spirostanols.29 One will note that the 1H NMR spectra of compound 7 displays a signal at δH 1.09 (d, J = 7.1 Hz, 3H); this provides a further evidence of the axial (β) orientation of the H3-27 methyl group, which is typically associated with 25S-spirostanol saponins in compound 7. The NOESY-1D spectra for compound 7 are in good agreement with the NOESY interactions observed in compounds 1, 4, and 5. This is clearly indicative of trans fusion between AB and BC, CD rings, cis fusion between DE rings, as well as the βorientation for the methyl groups H3-18 and H3-19. Other important correlations were observed in 1H−1H gCOSY spectra (Figures S43 and S44). These included the correlations of H-3 at δH 3.45 (m) with H-2 at δH 1.74 (m) 8710


Article


Figure 2. Determination of the minimum inhibitory concentration (MIC), in μg/mL, of the 70% ethanolic extract and fraction F26−16 of S. paniculatum against oral pathogens. Legend: E. Enterococcus; S., Streptococcus; L., Lactobacillus; ATCC, American Type Culture Collection; CI, culture isolate chlorohexidine positive control.

carbon, as well as the β-orientation for the methyl groups H318 and H3-19. The remaining signals in compound 8 were determined using the spectroscopic data obtained through HMBC, HSQC, and 1H−1H COSY analyses. These enabled us to identify all the signals associated with this compound. The structure of compound 8 was thus identified as the new 6-O-β-Dxylopyranosyl-(1′′→3′)-β- D -quinovopyranosyl-(22S,25S)3β,4β,6α-trihydroxy-5α-spirostane. Antibacterial Activity. Numerous studies have been published in the literature focusing on the evaluation of the anticariogenic potential of medicinal plants.31−33 None of these studies has, however, been devoted to investigating the correlation between 70% alcohol tincture of S. paniculatum and antibacterial activity in the dentistry area. The antibacterial assay of the plant extracts employed in this study was performed through the determination of the minimum inhibitory concentration (MIC). According to Rios and Recio,34 a result relative to antibacterial activity can be considered promising in the case of natural products, such as plant extracts, essential oil, and pure substances, by analyzing the results of the MIC assay. These authors consider MIC values lower than 100 μg/mL for extract or 10 μg/mL for pure substances as active. In the present study, the use of 70% alcohol tincture yielded lower MIC values for St. mutans (50 μg/mL, ATCC), St. sanguinis (50 μg/mL, ATCC), L. casei (50 μg/mL, CI), St. mitis (25 μg/mL, ATCC), and St. oralis (12.5 and 6.25 μg/mL, CI and ATCC, respectively). The F26−16 fraction showed a lower MIC value for St. oralis (100 μg/mL, ATCC) (Figure 2), so based on these criteria, the 70% alcohol tincture was considered an effective anticariogenic agent.35 These results imply that other compounds present in the 70% alcohol tincture may be playing an important role in the in vitro antibacterial activity. The results obtained from our investigation provided evidence regarding the chemical composition of the hydro-

alcoholic (70%) tincture of S. paniculatum leaves using as phytomedicine and nutritional supplements in Brazil. This shows that polyhydroxylated steroidal saponins with spirostane skeleton are the main constituents of S. paniculatum leaves. Some of the compounds obtained in this work (1, 4−8) have never been isolated before and are regarded new natural compounds. Furthermore, the tincture displayed a powerful ability to prevent growth of different oral pathogens. This ability is seen to be stronger than that exerted by the isolated fraction of saponins. These results may be attributed to the presence of a series of other components that act synergistically in the hydroalcoholic tincture.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b01262. Chromatographic profile of semipreparative F16−26 fraction by HPLC-RI for the isolation of compounds 1− 8, HRESIMS, 1H, 13C, DEPT 135°, gHMBC, gHSQC, 1 H−1H gCOSY, TOCSY-1D, and NOESY-1D NMR spectra for the characterization of compounds 1−8 (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +55 16 3301-9657. Fax: +55 16 3322-2308. E-mail: [email protected]. ORCID

Alexander B. Valerino-Díaz: 0000-0002-6368-3086 Carlos Henrique Gomes Martins: 0000-0001-8634-6878 Lourdes Campaner dos Santos: 0000-0001-6554-7689 Author Contributions

The listed authors contributed toward the development and drafting of this work. Their respective contributions are described as follows: A.B.V.-D. was the one responsible for the 8711


Article


(11) Wink, M. Evolution of secondary metabolites from an ecological and molecular phylogenetic perspective. Phytochemistry 2003, 64, 3−19. (12) Corrêa, M. Dicionário das Plantas Ú teis do Brasil; Instituto Brasileiro de Desenvolvimento Florestal, Ministério da Agricultura: Rio de Janeiro, Brazil, 1984; Vol. III, p 395. (13) Farmacopéia Brasileira, 4th ed; Atheneu: São Paulo, Brazil, 1988; p 567. (14) Agra, M. F.; Baracho, G. S.; Nurit, K.; Basilio, I. J. L. D.; Coelho, V. P. M. Medicinal and poisonous diversity of the flora of ″Cariri Paraiano″, Brazil. J. Ethnopharmacol. 2007, 111 (2), 383−395. (15) Mesia-Vela, S.; Santos, M. T.; Souccar, C.; Lima-Landman, M. T. R.; Lapa, A. J. Solanum paniculatum L. (jurubeba): Potent inhibitor of gastric acid secretion in mice. Phytomedicine 2002, 9, 508−514. (16) Vieira Júnior, G. M.; da Rocha, C. Q.; de Souza Rodrigues, T.; Hiruma-Lima, C. A.; Vilegas, W. New steroidal saponins and antiulcer activity from Solanum paniculatum L. Food Chem. 2015, 186, 160− 167. (17) Ripperger, H.; Budzikiewicz, H.; Schreiber, K. Jurubin ein stickstoffhaltiges Steroidsaponin neuartigen Struckturtyps aus Solanum paniculatum L. Chem. Ber. 1967, 100, 1725−1740. (18) Hara, S.; Okabe, K.; Mihashi, K. Gas-liquid chromatographic separation of aldose enantiomers as trimethylsilyl ethers of methyl 2(polyhydroxialkyl)-thiazolidine-4(R)-carboxilates. Chem. Pharm. Bull. 1987, 35, 501−506. (19) M07-A9. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Clinical and Laboratory Standards Institute: Wayne, PA, 2012. (20) Sarker, S. D.; Nahar, L.; Kumarasamy, Y. Microtitre platebased antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods 2007, 42, 321−324. (21) Iida, Y.; Yanai, Y.; Ono, M.; Ikeda, T.; Nohara, T. Three unusual 22-β-O-23-Hydroxi-(5α)-spirostanol glycosides from the fruits of Solanum torvum. Chem. Pharm. Bull. 2005, 53, 1122−1125. (22) Honbu, T.; Ikeda, T.; Zhu, X.-H.; Yoshihara, O.; Okawa, M.; Nafady, A. M.; Nohara, T. New Steroidal Glycosides from the Fruits of Solanum anguivi. J. Nat. Prod. 2002, 65, 1118−1120. (23) Mahmood, U.; Thakur, R. S.; Blunden, G. Neochlorogenin, neosolaspigenin and solaspigenin from Solanum torvum leaves. J. Nat. Prod. 1983, 46, 427−428. (24) Matsushita, S.; Yanai, Y.; Fusyuku, A.; Ikeda, T.; Ono, M.; Nohara, T. Distinction of absolute configuration at C-22 of C-23 hydroxyspirostane and C-23-hydroxyspirosolane glycosides. Chem. Pharm. Bull. 2007, 55, 1079−1081. (25) Zamilpa, A.; Tortoriello, J.; Navarro, V.; Delgado, G.; Alvarez, L. Five new steroidal saponins from Solanum chrysotrichum leaves and their antimicotic activity. J. Nat. Prod. 2002, 65, 1815−1819. (26) González, M.; Zamilpa, A.; Marquina, S.; Navarro, V.; Alvarez, L. Antimicotic spirostanol saponins from Solanum hispidum leaves and their structure-activity relationships. J. Nat. Prod. 2004, 67, 938−941. (27) Ripperger, H.; Schreiber, K. Estructure of Paniculonin A and B, two new spirostane glycosides from Solanum Paniculatum L. Chem. Ber. 1968, 101, 2450−2458. (28) Shaiq Ali, M.; Tabbasum, S.; Ahmed, S. Spirotorvoside: A new steroidal-glycoside from Solanum torvum (Solanaceae). J. Chem. Soc. Pak. 2008, 30, 494−498. (29) Agrawal, K. P. Spectral assignment and reference data: 25R/25S stereochemistry of spirotane-type steroidals sapogenins and steroidals saponins via chemical shift of geminals protons of ring F. Magn. Reson. Chem. 2003, 41, 965−968. (30) Perez, J. A.; Calle, M. J.; Simonet, M. A.; Guerra, O. J.; Stochmal, A.; Macias, F. A. Bioactive Steroidal Saponins from Agave of foyana flowers. Phytochemistry 2013, 95, 298−307. (31) Devienne, K. F.; Raddi, M. S. G. Screening for antimicrobial activity of natural products using a microplatephotometer. Braz. J. Microbiol. 2002, 33, 166−168. (32) Cunha, L. C. S.; de Morais, S. A. L.; Martins, C. H. G.; Martins, M. M.; Chang, R.; de Aquino, F. J. T.; de Oliveira, A.; Moraes, T. S.;

acquisition, interpretation of the experimental data besides writing the manuscript, D.G.-T. helped in the analysis and interpretation of the spectral data, A.C.Z. assisted in the experimental design for the compound isolation, C.H.G.M. supervised the experimental design of the antimicrobial activity, T.S.S. conducted the experiments related to antimicrobial activity, L.R. helped in editing the manuscript, W.V. also helped in the editing of the manuscript and in conducting mass analyses, and L.C.S is a corresponding author who supervised the chemical study, conducted the experimental design, and helped to edit the manuscript. All authors have given their approval to the final version of the manuscript. Funding

The authors thank the São Paulo State Research Foundation (FAPESP) (Grant No. 2015/04899-3) led by Lourdes Campaner dos Santos, the Coordination for the Improvement of Higher Education Personnel (CAPES, Grant No. 1877151), and the BIOTA/FAPESP project (Grant No. 2009/522379) led by Wagner Villegas for supporting and funding this research. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors are grateful to Nivaldo Boralle for the acquisition of the 1D and 2D spectra of the isolated compounds and to Luis Vitor Sacramento for supplying the leaves of S. paniculatum L.

■

REFERENCES

(1) D’Arcy, W. G. The Solanaceae since 1976, with a review of its biogeography. In Solanaceae III: Taxonomy, Chemistry, Evolution; Hawkes, J. G., Lester, R. N., Nee, M., Estrada, N., Eds.; Kew Royal Botanical Gardens: London, 1991; pp 75−137. (2) Kodamatani, H.; Saito, K.; Niina, N.; Yamazaki, S.; Tanaka, Y. Simple and sensitive method for determination of glycoalkaloids in potato tubers by high performance liquid chromatography with chemiluminescence detection. J. Chromatogr. A 2005, 1100, 26−31. (3) Friedman, M. Analysis of biologically active compounds in potatoes (Solanum tuberosum), tomatoes (Lycopersicom esculentum), and jimson weed (Datura stamonium) seeds. J. Chromatog. A 2004, 1054, 143−155. (4) Van Eck, J.; Snyder, A. Eggplant (Solanum melongena L.). Methods Mol. Biol. 2006, 343, 439−447. (5) Lorenzi, H.; Matos, F. J. A. Plantas Medicinais no Brasil, Plantas Nativas e Exóticas; Instituto Plantarum de Estudos da Flora Ltda: Nova Odessa, Brazil, 2002; p 129. (6) Saez, J.; Cardona, W.; Espinal, D.; Blair, S.; Mesa, J.; Bocar, M.; Jossang, A. Five New Steroids from Solanum nudum. Tetrahedron 1998, 54, 10771−10778. (7) Zhou, X.; He, X.; Wang, G.; Gao, H.; Zhou, G.; Ye, W.; Yao, X. Steroidal saponins from Solanum nigrum. J. Nat. Prod. 2006, 69, 1158−1163. (8) Esteves-Souza, A.; Silva, T. M. S.; Alves, C. C. F.; Carvalho, M. G.; Braz-Filho, R.; Echevarria, A. Cytotoxic activities against Ehrlich Carcinoma and Human K562 Leukaemia of alkaloids and flavonoid from two Solanum species. J. Braz. Chem. Soc. 2002, 13, 838−842. (9) Cornelius, M. T. F.; Alves, C. C. F.; Silva, T. M. S.; Alves, K. Z.; Carvalho, M. G.; Braz-Filho, R.; Agra, M. F. Solasonina e flavonóides isolados de Solanum crinitum Lam. Rev. Bras. Farmacogn 2004, 85, 57−59. (10) Dinan, L.; Harmatha, J.; Lafont, R. Chromatographic procedures for the isolation of plant steroids. J. Chromatog. A 2001, 935, 105−123. 8712


Article

Journal of Agricultural and Food Chemistry Machado, F. C.; da Silva, C. V.; do Nascimento, E. A. Chemical composition, cytotoxic and antimicrobial activity of essential oils from Cassia bakeriana Craib against aerobic and anaerobic oral pathogens. Molecules 2013, 18, 4588−4598. (33) Moreira, M. R.; Souza, A. B.; Soares, S.; Bianchi, T. C.; de Souza Eugênio, D.; Lemes, D. C.; Martins, C. H. G.; da Silva Moraes, T.; Tavares, D. C.; Ferreira, N. H.; Ambrósio, S. R.; Veneziani, R. C. S. ent-Kaurenoic acid-rich extract from Mikania glomerata: In vitro activity against bacteria responsible for dental caries. Fitoterapia 2016, 112, 211−216. (34) Rios, J. L.; Recio, M. C. Medicinal Plants and Antimicrobial Activity. J. Ethnopharmacol. 2005, 100, 80−84. (35) Moura, C. L.; Casemiro, L. A.; Martins, G. C. H.; Cunha, W. R.; Luis de Andrade e Silva, M.; Cury, V. A. H. Avaliaçaõ da atividade antimicrobiana da espécie vegetal Pfaff ia glomerata frente a patógenos bucais. Investigaçaõ 2011, 11, 24−28.

8713


New Polyhydroxylated Steroidal Saponins from Solanum paniculatum

Recommend Documents