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Bioactive Constituents, Metabolites, and Functions

New polyhydroxylated steroidal saponins from Solanum paniculatum L. leaf alcohol tincture with antibacterial activity against oral pathogens. Alexander Bárbaro Valerino-Diaz, Daylin Gamiotea-Turro, Ana Caroline Zanatta, Wagner Vilegas, Carlos H. Gomes Martins, Thayná de Souza Silva, Luca Rastrelli, and Lourdes Campaner dos Santos J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01262 • Publication Date (Web): 26 Jul 2018 Downloaded from http://pubs.acs.org on July 27, 2018

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Page 1 of 36

Journal of Agricultural and Food Chemistry

1

New

polyhydroxylated

steroidal

saponins

from

Solanum

2

paniculatum L. leaf alcohol tincture with antibacterial activity

3

against oral pathogens.

4

Alexander B. Valerino-Díaza, Daylin Gamiotea-Turroa, Ana C. Zanattaa, Wagner Vilegasb, Carlos

5

Henrique Gomes Martinsc, Thayná de Souza Silvac, Luca Rastrellid and Lourdes Campaner dos

6

Santosa*.

7

8

a

9

14800-060, Araraquara, São Paulo, Brazil.

UNESP - São Paulo State University, Institute of Chemistry. Rua Prof. Francisco Degni, 55,

10

b

11

s/n, 11330-900, São Vicente, São Paulo, Brazil.

12

c

13

Armando Salles Oliveira, 201, 14404-600, Franca, São Paulo, Brazil.

14

d

15

Salerno, Italy.

UNESP - São Paulo State University, Institute of Biosciences. Praça Infante Dom Henrique,

UNIFRAN – University of Franca, Laboratory of Research in Applied Microbiology. Av. Dr.

Dipartimento di Farmacia – University of Salerno, Via Giovanni Paolo II, 84084 Fisciano,

16

a*

17

Brazil. Telefone: +55 16 3301-9657. Fax: +55 16 3322-2308. E-mail: [email protected]

Rua Prof. Francisco Degni, 55 Bairro: Quitandinha, 14800-060, Araraquara, São Paulo,

ACS Paragon Plus Environment

1


Page 2 of 36

18

Abstract.

19

Solanum paniculatum L. is widely used in Brazilian folk medicine for the treatment of liver and

20

gastrointestinal disorders as well as for culinary purposes and beverage production. Fractionation

21

of hydroalcoholic (EtOH 70%) tincture from S. paniculatum leaves, led to the isolation of six

22

new

23

quinovopyranosyl-(22S,23R,25S)-3β,6α,23-trihydroxy-5α-spirostane

24

xylopyranosyl-(1’’→3’)-β-D-quinovopyranosyl-(22S,23R,25R)-3β,6α,23-trihydroxy-5α-

25

spirostane

26

3β,6α,23-trihydroxy-5α-spirostane (5), 3-O-β-D-xylopyranosyl-(1’’→3’)-β-D-quinovopyranosyl-

27

(22S,23S,25R)-3β,6α,23-trihydroxy-5α-spirostane (6), 6-O-α-L-rhamnopyranosyl-(1’’→3’)-β-D-

28

quinovopyranosyl-(22S,25S)-1β,3β,6α-trihydroxy-5α-spirostane (7) and 6-O-β-D-xylopyranosyl-

29

(1’’→3’)-β-D-quinovopyranosyl-(22S,25S)-3β,4β,6α-trihydroxy-5α-spirostane (8) together with

30

two known spirostanic saponins (2, 3). The structure of these compounds was determined by 1D

31

and 2D NMR experiments in addition to HRESIMS analyses. The 70% alcohol tincture, used as

32

phytomedicine, exhibited promising activities against oral pathogens, including, Steptococcus

33

sanguinis, Steptococcus oralis, Steptococcus mutans, Steptococcus mitis and Lactobacillus casei

34

with MIC values ranging from 6.25 to 50 µg/mL. The saponin fraction, nonetheless, showed

35

lower activity against all the strains tested (from 100 to > 400 µg/mL).

36

Keywords: Solanum paniculatum, Spirostanic saponins, Steroidal saponins, Antibacterial

37

activity, Oral pathogens.

spirostanic

(4),

saponins

which

included

6-O-α-L-rhamnopyranosyl-(1’’→3’)-β-D(1),

6-O-β-D-

3-O-α-L-rhamnopyranosyl-(1’’→3’)-β-D-quinovopyranosyl-(22S,23S,25R)-

38 39 40


2

Page 3 of 36


41

Introduction.

42

The Solanaceae family is known to have about 3000 species. The Solanum genus, which belongs

43

to this family, is considered one of the most complex and numerous genera with about 1500

44

species. These species are part of the ecological systems typically prevalent in the tropical and

45

subtropical regions, with their center of diversity and distribution in South America estimated to

46

be between 1000-1100 species of the genus.1 The genus is found to be essentially unique by

47

virtue of the economic importance of many of its species which include potato (S. tuberosum),2

48

tomato (S. lycopersum)3 and egg-plant (S. melongena).4 Many other species belonging to the

49

genus Solanum, including S. americanum, are used in Brazilian popular medicine for the

50

treatment of gastric dysfunctions, such as gastric ulcer.5 Solanum plants have the ability to

51

biosynthesize steroids,6 saponins,7 alkaloids,3 glycosylated flavonoids,8,9 as well as other

52

structurally diversified and complex secondary metabolites.10,11 Solanum paniculatum L.

53

popularly referred to as jurubeba,

54

(with a bitter taste). Remarkably, it is the only species of the genus Solanum that has been

55

included in the Brazilian Ministry of Health list of medicinal plants of interest for phytotherapic

56

formulation13. S. paniculatum is used in the Brazilian folk medicine for the treatment of

57

respiratory tract diseases, for the mitigation of fever and as tonic.14 It is also used for the

58

treatment of liver and gastric dysfunctions.15 In traditional culinary, there are recipes that involve

59

the consumption of S. paniculatum fruits after cooking accompanied with rice, pickles and other

60

cereals. Moreover, fruits from the S. paniculatum plant are used in manufacturing a commercial

61

wine beverage with antioxidant properties.16 The plant is used as a component of various

62

phytotherapic formulations including infusion, decoctions and 70% alcohol tincture.16 The

63

ethanolic extract exhibits antiulcerogenic effect.16 Indeed, numerous works have been published

12

is commonly consumed both as food and medicinal plant


3


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64

in the literature related to the chemical studies conducted on S. paniculatum and many steroidal

65

compounds are reported to have been isolated from this species.17 As part of our contribution

66

towards understanding the uses and benefits derived from this species, the present work aims to

67

better investigate the hydroalcoholic leaf tincture (70%) of S. paniculatum, used in Brazil as a

68

nutritional supplement. The phytochemical study carried out in this work allowed the isolation

69

and identification of eight saponins (1-8) with polyhydroxylated spirostane skeleton. Among the

70

isolated substances, six new natural products were discovered (1, 4-8). Considering the

71

traditional use of S. paniculatum, we also evaluated the antibacterial activity of the alcoholic

72

tincture (70 %) and saponin fraction (F16-26) against oral pathogens.

73 74

Materials and Methods.

75

Chemicals. Methanol HPLC grade, was purchased from Tedia Company (Fairfield, OH,

76

U.S.A). The water used for all HPLC mobile phases in the experiments was purified using a

77

Milli-Q system (Millipore, Billerica, MA, and U.S.A). Other organic solvents (n-butanol and

78

chloroform) used in the experiments were of analytical grade (acquired from Synthlab, São

79

Paulo, Brazil). All solutions prepared for HPLC were filtered with a 0.22-µm GHP filter

80

(Waters, Milford, MA, U.S.A) before use.

81

Plant material. Solanum paniculatum L. leaves were collected from the orchard of

82

medicinal plants of the UNESP-São Paulo State University, situated at the Faculty of

83

Pharmaceutical Sciences of Araraquara, São Paulo State, Brazil, located on GPS 21° 48’

84

52.44’’S and 48°12’ 07.13. The leaves were identified by Dr. Luis Vitor Sacramento. A voucher

85

specimen (HRCB 60754) was deposited at the herbarium of UNESP - São Paulo State

86

University, Institute of Biosciences, Rio Claro, São Paulo State, Brazil.


4

Page 5 of 36


87

General Apparatus. The optical rotations were carried out using a Perkin Elmer

88

Instruments Model 341LC polarimeter (l=10 cm, 589 nm). 1D and 2D NMR spectra were

89

collected from a Bruker Advance III HD 600 spectrometer (14.1 T) using an inverse detection 5

90

mm (1H,

91

Sigma-Aldrich.TM Tetramethylsilane (TMS) was used as reference. HRESIMS data analysis was

92

conducted in the positive ion mode using a Bruker Maxis Impact mass spectrometer with the

93

configuration ESI-QqTOF-MS. HPLC analysis in isocratic mode was performed using a Knauer

94

Azura apparatus equipped with smart line 2300 refractive index detector. Synergy Hydro-RP C18

95

column (4µ, 80Å, 250 × 4,6mm, i.d) was employed for analytical purposes while Synergy

96

Hydro-RP C18 column (4µ, 80Å, 250 × 10 mm, i.d) was used to conduct the semi-preparative

97

analyses. Size exclusion column chromatographic was carried out using Sephadex LH-20

98

(Pharmacia) in order to purify the n-butanol extract. Thin layer chromatography (TLC) analysis

99

was performed using Merck silica gel 60 plates (>230 mesh). The spots on the TLC plates were

100

revealed by sprinkling the plates with anisaldehyde-H2SO4 reagent, followed by heating at 120

101

°C. The determination of absolute configuration of monosaccharides was performed using a

102

CG/MS analysis under the following conditions: Agilent 7890 B Gas Chromatograph equipped

103

with a 5977 A Mass detector (detection temperature 220 °C). Column: HP-5MS capillary

104

column (50 m, 0.25 mm i.d., 0.25 µm). Column temperature: 150-260 °C at the rate of 8 °C/min,

105

the carrier gas was He (0.8 mL/min), split ratio of 1/10, injection temperature of 250 °C and

106

injection volume of 0.5µL.

13

C,

15

N) cryoprobe. The samples were dissolved in CD3OD (≥99.8) acquired from

107

Preparation of tincture. Based on the traditional Brazilian tincture preparation, dried

108

leaves (250 g) of S. paniculatum were extracted by percolation at room conditions with 70%


5


Page 6 of 36

109

EtOH (2.5 L). The tincture obtained 1:10 was a homogeneous brown liquid. The

110

concentration of solid material was 21.1 mg mL−1.

111

Extraction and isolation. Beginning with the tincture, the solvent was evaporated to

112

dryness under a low pressure. This yielded 52.7 g of the crude ethanol extract (EE) (21.1%). The

113

EE (45 g) was dissolved in 2.25 L of water and a liquid-liquid partition with 0.75 L of n-butanol

114

(three times) was carried out. After removing the solvent, n-butanol extract (21.9 g, 48.7%) and

115

aqueous extract (15 g, 33.3%) were derived from the extraction. The n-butanol extract (3g) was

116

fractionated using Sephadex LH-20 gel, column (85 x 2.5 cm; H x d.i.) and eluted with

117

methanol. The fractionation of this extract yielded one hundred and twenty fractions (5 mL each)

118

which were combined into six major fractions (F1-15, F16-26, F27-43, F44-52, F53-59 and F60-

119

76) according to their TLC profiles. The fraction F16-26 (250 mg) was analyzed by HPLC with

120

refractive index detector (HPLC-RI) using analytical C18 column with MeOH-H2O (7:3 v/v, 1

121

mL/min) as mobile phase. This enabled us to establish a better chromatographic condition for the

122

isolation of the compounds in the fraction. After that, the fraction F16-26 was subjected to

123

HPLC-RI with the aid of a semi preparative C18 column (Figure S1, Supporting Information)

124

using MeOH ̶ H2O (7:3 v/v, 2 mL/min) as mobile phase; this gave rise to compounds 1 (13,6

125

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).

126 127

6-O-α-L-rhamnopyranosyl-(1’’→3’)-β-D-quinovopyranosyl-(22S,23R,25S)-3β,6α,23-

128

trihydroxy-5α-spirostane (1):


6

Page 7 of 36


129

Yellow amorphous solid: [α]D25 -34° (c 0.1, MeOH); 1H and 13C NMR data (CD3OD), see Tables

130

1 and 2; HRESIMS m/z 741.4410 [M + H]+ (calcd. for C39H65O13, 741.4420), ESIMS/MS m/z:

131

723.4315 [M + H  H2O]+, 595.3829 [M + H  Rha]+, 431.3150 [M + H  Rha  H2O]+.

132

6-O-α-L-rhamnopyranosyl-(1’’→3’)-β-D-quinovopyranosyl-(23R,25R)-3β,6α,23-trihydroxy-5α-

133

spirostane (2):

134

Yellow amorphous solid: [α]D25 -26° (c 0.1, MeOH); 1H NMR (CD3OD); Aglycone: δH 1.00-1.69

135

(2H, m, H-1), 1.15- 2.37 (2H, m, H-2), 3.45 (1H, m, H-3), 1.40-1.74 (2H, m, H-4), 1.15 (1H, m,

136

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-

137

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

138

(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

139

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,

140

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 =

141

7.9 Hz, H-1’), 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,

142

m, H-5’), 1.27 (3H, d, J = 6.1 Hz, H-6’); α-L-rhamnose: 5.13 (1H, d, J = 1.3 Hz, H-1’’), 3.92

143

(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

144

(3H, d, J = 6.2 Hz, H-6’’); 13C NMR (CD3OD); Aglycone: δc 37.1 (CH2, C-1), 31.3 (CH2, C-2),

145

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-

146

8), 53.7 (CH, C-9), 36.1 (C, C-10), 20.6 (CH2, C-11), δC 39.3 (CH2, C-12), 40.7 (C, C-13), 56.1

147

(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,

148

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),

149

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

150

(CH, C-2’), 82.7 (CH, C-3’), 74.3 (CH, C-4’), 71.6 (CH, C-5’), 17 (CH3, C-6’); α-L-rhamnose:

151

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


7


Page 8 of 36

152

(CH3, C-6’’); HRESIMS m/z 741.4421 [M + H]+ (calcd. for C39H65O13, 741.4420), 763.4240 [M

153

+ Na]+, 723.4316 [M + H  H2O]+.

154

6-O-β-D-xylopyranosyl-(1’’→3’)-β-D-quinovopyranosyl-(23R,25S)-3β,6α,23-trihydroxy-5α-

155

spirostane (3):

156

Yellow amorphous solid: [α]D25 -34° (c 0.1, MeOH); 1H NMR (CD3OD); Aglycone: δH 1.02-

157

1.73 (2H, m, H-1), 1.16-2.39 (2H, m, H-2), 3.46 (1H, m, H-3), 1.40-1.75 (2H, m, H-4), 1.16 (1H,

158

m, H-5), δH 3.40 (1H, m, H-6), 0.95-2.16 (2H, m, H-7), 1.66 (1H, m, H-8), 0.68 (1H, td, J = 2.1

159

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-

160

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

161

(3H, s, 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

162

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,

163

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,

164

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

165

(3H, d, J = 6.1 Hz, H-6’); β-D-xylose: 4.47 (1H, d, J = 7.6 Hz, H-1’’), 3.26 (1H, m, H-2’’), 3.33

166

(1H, m, H-3’’), 3.50 (1H, m, H-4’’), 3.23-3.89 (2H, m, dd, J = 5.4 Hz, H-5’’);

167

(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

168

(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),

169

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

170

(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,

171

C-21), 109.9 (C, C-22), 69.6 (CH, C-23), 33.2 (CH2, C-24), 26.6 (CH, C-25), 64.8 (CH2, C-26),

172

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,

173

C-4’), 71.3 (CH, C-5’), 16.9 (CH3, C-6’); β-D-xylose: 104.6 (CH, C-1’’), 73.8 (CH, C-2’’), 76.3


13

C NMR

8

Page 9 of 36


174

(CH, C-3’’), 69.7 (CH, C-4’’), 65.7 (CH2, C-5’’); HRESIMS m/z 727.4263 [M + H]+ (calcd. for

175

C38H63O13, 727.4263), 749.4080 [M + Na]+, 709.4160 [M + H  H2O]+.

176

6-O-β-D-xylopyranosyl-(1’’→3’)-β-D-quinovopyranosyl-(22S,23R,25R)-3β,6α,23-trihydroxy-

177

5α-spirostane (4):

178


179


180

749.4083 [M + Na]+, 709.4156 [M + H  H2O]+, 595.3843 [M + H  Xyl]+, 431.3150 [M + H 

181

Xyl  Qui  H2O]+, 413.3037 [M + H  Xyl  Qui  2H2O]+.

182

3-O-α-L-rhamnopyranosyl-(1’’→3’)-β-D-quinovopyranosyl-(22S,23S,25R)-3β,6α,23-trihydroxy-

183

5α-spirostane (5):

184

Yellow amorphous solid: [α]D25 -14° (c 0.1, MeOH); 1H and

185

Tables 1 and 2; HRESIMS m/z 741.4407 [M + H]+ (calcd. for C39H65O13, 741.4420), ESIMS/MS

186

m/z: 723.4300 [M + H  H2O]+, 595.3827 [M + H  Rha]+, 431.3148 [M + H  Rha  Qui 

187

H2O]+.

188

3-O-β-D-xylopyranosyl-(1’’→3’)-β-D-quinovopyranosyl-(22S,23S,25R)-3β,6α,23-trihydroxy-5α-

189

spirostane (6):

190

Yellow amorphous solid: [α]D25 +21° (c 0.1, MeOH); 1H and

191

Tables 1 and 2; HRESIMS m/z 727.4262 [M + H]+ (calcd. for C38H63O13, 727.4263), ESIMS/MS

192

m/z: 749.4083 [M + Na]+, 709.4160 [M + H  H2O]+, 431.3150 [M + H  Xyl  Qui  H2O]+.

193

6-O-α-L-rhamnopyranosyl-(1’’→3’)-β-D-quinovopyranosyl-(22S,25S)-1β.3β,6α-trihydroxy-5α-

194

spirostane (7):


13

C NMR data (in CD3OD), see

13

C NMR data (CD3OD), see

9


Page 10 of 36

195


196


197

763.4231 [M + Na]+, 723.4307 [M + H  H2O]+, 595.3835 [M + H  Rha]+, 431.3155 [M + H 

198

Rha  Qui  H2O]+, 413.3042 [M + H  Rha  Qui  2H2O]+.

199

6-O-β-D-xylopiranosyl-(1’’→3’)-β-D-quinovopyranosyl-(22S,25S)-3β,4β,6α-trihydroxy-5α-

200

spirostane (8):

201


202


203

749.4071 [M + Na]+, 709.4150 [M + H  H2O]+, 595.3838 [M + H  Xyl]+, 431.3150 [M + H 

204

Xyl  Qui  H2O]+.

205

Acid Hydrolysis of Saponins 1-8 and determination of absolute configuration of

206

monosaccharides. To determine the absolute configuration of saponins 1-8, acid hydrolysis was

207

performed as described by Hara et al.18 For the determination of the Rf value of the hydrolyzed

208

sugars, TLC analysis was performed using the solvent system EtOAc ̶ MeOH ̶ H2O ̶ AcOH (11:

209

2: 2: 2). The Rf values obtained for the hydrolyzed sugars were compared to those of sugar

210

standards. The following Rf values were obtained for D-quinovose, L-rhamnose and D-xylose:

211

0.65, 0.50 and 0.75 respectively.

212

The aqueous layer was concentrated and analyzed by GC/MS as described by Hara et al.18The

213

absolute configurations of D-quinovose, L-rhamnose and D-xylose were confirmed through the

214

analyses of their retention times and by comparison with standard samples. The retention times


10

Page 11 of 36


215

of monosacccarides derivatives are as follows: D-quinovose (12.51 min), L-rhamnose (12.67

216

min) and D-xylose (11.89 min) respectively.

217

Antibacterial activity evaluation. The evaluation of the antimicrobial activity and the

218

Minimal Inhibitory Concentration (MIC) was performed in triplicate by the microdilution

219

method, as described in the Clinical and Laboratory Standards Institute (CLSI)19 and by Sarker et

220

al.20 The bacterial strains employed were maintained in the culture collection of the Research

221

Laboratory of Applied Microbiology (LaPeMA), University of Franca, São Paulo Brazil, under

222

cryopreservation (-80 ºC) in Tryptic Soy broth (TSB; Difco Laboratories, Detroit, MI, U.S.A),

223

containing glycerol at 20% (v/v). The bacteria used were: Enterococcus faecalis (ATCC 4082

224

and CI), Streptococcus salivarius (ATCC 25975 and CI), Steptococcus sanguinis (ATCC 10556

225

and CI), Steptococcus oralis (ATCC 5529 and CI), Steptococcus sobrinus (ATCC 33478),

226

Steptococcus mutans (ATCC 25175), Steptococcus mitis (ATCC 49456) and Lactobacillus casei

227

(ATCC 11578 and CI).

228 229

Results and Discussion.

230

Fraction F16-26 of the n-butanol extract obtained from the liquid-liquid extraction of the 70%

231

ethanolic extract derived from the leaves of Solanum paniculatum L., yielded eight pure

232

compounds 1-8, (Figure 1).

233

The identification of the related compounds was performed using optical rotation along with 1H,

234

13

235

analyses and mass spectra via the HRESIMS technique.

C, DEPT 135o, gHMBC, gHSQC, 1H‒1H gCOSY, TOCSY-1D, NOESY-1D NMR spectra

236

Compound 1 was obtained as a yellow amorphous solid and exhibited a molecular ion [M

237

+ H]+ at m/z 741.4410 (calcd. for C39H65O13, 741.4420) in the positive HRESIMS mode (Figures


11


Page 12 of 36

238

S2-S3, Supporting Information) corresponding to the molecular formula C39H64O13. Based on the

239

analysis of the 1H NMR signals (in CD3OD) of compound 1 (Table 1, Figure S4, Supporting

240

Information) the conclusion drawn here is that the compound consisted of two tertiary methyl

241

groups at δH 0.81 (3H, s, H3-18), δH 0.87 (3H, s, H3-19), four secondary methyl groups at δH 1.11

242

(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´),

243

δH 1.23 (3H, d, J= 6.2 Hz, H3-6´´), two protons related to a methylene group at δH 3.31

244

(overlapped, H-26a), δH 3.95 (d, J = 3.7 Hz, H-26b) linked to an oxygen, three protons at δH 3.37

245

(overlapped, H-6), δH 3.45 (1H, overlapped, H-3) and δH 3.56 (1H, t, J = 3.6 Hz, H-23) assigned

246

to a methine group linked to carbinol carbons and two protons attached to anomeric carbon at δH

247

4.26 (1H, d, J = 7.9 Hz, H-1´) and δH 5.13 (1H, d, J = 1.6 Hz, H-1´´).

248

13

249

Information) exhibited 39 carbon signals. The comparison between these chemical shifts with

250

those of Torvoside K(2)21 helped to identify 12 of these signals which correspond to α-L-

251

rhamnopyranosyl and β-D-quinovopyranosyl units. The TOCSY-1D spectrum (Figures S7-S8,

252

Supporting Information) with irradiation of the anomeric proton at δH 4.26 (d, J = 6.2 Hz)

253

displayed the entire spin system of a quinovopyranosyl residue (Table 1). Irradiation of the

254

anomeric proton at δH 5.13 (d, J = 1.6 Hz) exhibited only the signal belonging to H-2’’ at δH 3.92

255

(dd, J = 1.8 Hz) of a rhamnopyranosyl unit, which is also consistent with the presence of the

256

methyl doublet H-6’’ at δH 1.23 (d, J = 6.2 Hz). The others 27 carbons belonging to the

257

sapogenol moiety were attributed to eight methylene carbons, seven methine carbons, two

258

quaternary carbons, four methyl groups, four oxygen-bearing methine carbons, one oxygen-

259

bearing methylene carbon and one acetal carbon (see Tables 1 and 2).

C NMR and DEPT 135o data of compound 1, (shown in Table 2, figures S5-S6, Supporting


12

Page 13 of 36


260

The gHMBC spectrum (Figures S9-S10, Supporting Information) analysis was conducted aiming

261

at demonstrating the connectivity of the respective protons and carbons (J3). Through this

262

analysis, long-range correlations were observed from H3-19 to C-10/C-9/ C-5, H3-18 to C-12/C-

263

13/C-14, H3-21 to C-17/C-20/C-22, H3-27 to C-24/C-25/C-26, H-25 to C-23. The correlation in

264

the gHMBC spectrum between H-25 (δH 1.66) and C-23 (δC 69.7) indicates the present of a

265

hydroxyl group at C-23. NOESY-1D spectrum analyses (Figure S11, Supporting Information)

266

enabled to establish the configuration at C-23, (C-23R configuration) since an interaction was

267

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).

268

In addition, the methyl group H3-18 at δH 0.81 (s) (Figure, S12, Supporting Information)

269

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

270

(m). This suggests the existence of β-orientation for H3-18 and for H-20, α-orientation for H3-21

271

(C-20S configuration), trans fusion between CD rings and cis fusion between DE rings. The

272

long-range interactions involving H3-19 at δH 0.87 (s) (Figure S13, Supporting Information) with

273

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

274

(m) indicate the occurrence of trans fusion between AB and BC rings with H3-19 occupying a β-

275

position in the sapogenol moiety.

276

The 1H-1H gCOSY spectrum (Figure S14, Supporting Information) exhibited a correlation

277

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, H-23 at δH 3.56

278

and H-24 at δH 1.53, thus indicating the presence of hydroxyl groups at C-3, C-6 and C-23. For

279

the determination of the configuration of C-25, it was regarded essentially relevant to consider

280

the chemical shift of the H3-27. Reports in the literature show the occurrence of a higher proton

281

chemical shift (δH 1.10-1.53) for the 23-hidroxylspirostanols when the methyl group H3-27 is in

282

the axial position. This is attributed to the 1.3-diaxial interactions. When the methyl group H3-27


13


Page 14 of 36

283

is in equatorial position the proton chemical shift occurs between δH 0.73-0.80.21 The

284

configuration of C-25 was determined as S given that the signal of H3-27 in compound 1 occurs

285

at δH 1.20 (which implies that it is in the axial position). For the determination of the

286

configuration of C-22, an evaluation was conducted on the 1H NMR chemical shifts of the

287

hydrogen related to the methyl group H3-21 and H-16 along with the

288

of C-20 in comparison to other 23-hydroxylspirostanols. With regard to 22-α-O-spirostanol

289

glycosides, reports in the literature show that H3-21 and H-16 absorb frequency in the range

290

between δH 1.17-1.26 and δH 4.49-4.56 respectively, and the C-20 absorbs frequency at δC 35.0-

291

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

292

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

293

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

294

sapogenol. By virtue of that, the aglycone of compound 1 aglycone was characterized as (22S,

295

23R, 25S)-3β, 6α, 23-trihydroxy-5α-spirostane.

296

For the determination of the sugar linkage, the gHMBC correlations were observed between the

297

quinovopyranosyl H-1' δH 4.26 (d, J = 7.9 Hz) and the sapogenol C-6 at δC 78.9 in addition to the

298

correlation between rhamnopyranosyl C-1 at δC 101.4 and the inner quinovopyranosyl H-3' at δH

299

3.41(m). This led us to the unambiguous confirmation of the occurrence of the linkage (1’’→3’)

300

between the rhamnopyranosyl and quinovopyranosyl moieties. In view of that, the structure of

301

compound

302

quinovopyranosyl-(22S,23R,25S)-3β,6α,23-trihydroxy-5α-spirostane.

1

was

determined

as

the

new

13

C NMR chemical shifts

6-O-α-L-rhamnopyranosyl-(1’’→3’)-β-D-

303

Compound 2 was obtained as a yellow amorphous solid. The compound exhibited a

304

molecular ion [M + H]+ at m/z 741.4421 (calcd. for C39H65O13, 741.4420) in the positive


14

Page 15 of 36


305

HRESIMS mode (Figure S15, Supporting Information) compatible with the molecular formula

306

C39H64O13.These data demonstrate the presence of a possible isomer of the compound 1.

307

The pattern of the 13C NMR signals of compound 2 (Table 2) is found to be similar to those of

308

compound 1 except for the signal of the methyl group H3-27 δH 0.76 (d. J = 6.6 Hz). This implies

309

that the position occupied by the H3-27 is equatorial and the configuration of C-25 is R.21 The

310

comparison of the spectroscopic data of compound 2 with the steroidal saponin isolated from S.

311

chrysotrichum leaves

312

compound 2 was identified as 6-O-α-L-rhamnopyranosyl-(1’’→3’)-β-D-quinovopyranosyl-

313

(23R,25R)-3β,6α,23-trihydroxy-5α-spirostane.

25

showed similar chemical shifts in the 1H NMR and 13C NMR. As such,

314

Compound 3 was obtained as a yellow amorphous solid. It exhibited a molecular ion at

315

m/z 727.4263 (calcd. for C38H63O13, 727.4263) in the positive HRESIMS mode (Figure S16,

316

Supporting Information) compatible with the molecular formula C38H62O13. The 1H NMR

317

chemical shifts (Table 1, Figure S17, Supporting Information) and

318

compound 3, (Table 2, Figure S18, Supporting Information) were seen to be in good agreement

319

with those of the spirostanic saponin isolated from S. Hispidum.26 Thus, on the basis of these

320

evidences,

321

quinovopyranosyl-(23R,25S)-3β,6α,23-trihydroxy-5α-spirostane, previously isolated from S.

322

paniculatum.27

compound

3

was

identified

as

13

C NMR spectra for

6-O-β-D-xylopyranosyl-(1’’→3’)-β-D-

323

Compound 4 was obtained as a yellow amorphous solid. This compound exhibited a

324

molecular ion [M + H]+ at m/z 727.4262 (calcd. for C38H63O13, 727.4263) in the positive

325

HRESIMS mode (Figures S19-S20, Supporting Information) compatible with the molecular

326

formula C38H62O13. The 1H, 13C (Tables 1 and 2, Figures S21-22 Supporting Information), DEPT

327

135o, gHSQC and gHMBC (Figures S23-25, Supporting Information) spectroscopic data for


15


Page 16 of 36

328

compound 4 exhibited close similarity with those of compound 3 except for the proton signal of

329

H3-27 at δH 0.76 (d, J = 6.7 Hz), which indicated the equatorial position of the methyl group H3-

330

27 and the C-25R configuration. As a result, the structure of 4 was identified as the new 6-O-β-

331

D-xylopyranosyl-(1’’→3’)-β-D-quinovopyranosyl-(22S,23R,25R)-3β,6α,23-trihydroxy-5α-

332

spirostane, an epimer of compound 3.

333

Compound 5 was obtained as a yellow amorphous solid. The molecular formula

334

C39H64O13 is in agreement with the observed molecular ion [M + H]+ at m/z 741.4407 (calcd. for

335

C39H65O13, 741.4420) obtained in the HRESIMS analyses (Figures S26-27, Supporting

336

Information). The 1H and

337

Information) were similar to those of Spirotorvoside.28 The main difference between

338

Spirotorvoside and compound 5 is found in the configuration in the configuration of the C-25.

339

The proton NMR resonance of the methyl group H3-27 at δH 0.81 (d, J = 6.5 Hz) in compound 5

340

indicates that the position occupied by the H3-27 is equatorial and the configuration of C-25 is R.

341

The NOESY-1D spectra (Figure S30, Supporting Information) showed the interaction between

342

H-23 at δH 3.62 (dd, J = 2.76 Hz) and H3-21 at δH 1.15 (d, J = 7,3Hz), indicating that H-23

343

occupied an equatorial position; as such, a C-23S configuration was assigned. Based on the data

344

above, compound 5 was characterized as the new 3-O-α-L-rhamnopyranosyl-(1’’→3’)-β-D-

345

quinovopyranosyl-(22S,23S,25R)-3β,6α,23-trihydroxy-5α-spirostane.

13

C NMR spectra (Tables 1 and 2, Figures S28-29, Supporting

346

Compound 6 was obtained as a yellow amorphous solid. The compound exhibited a

347

molecular ion [M + H]+ at m/z 727.4262 (calcd. for C38H63O13, 727.4263) derived from

348

HRESIMS (Figures S31-32, Supporting Information) analyses compatible with the molecular

349

formula C38H62O13. The 1H and

350

Supporting Information) of the aglycone of compound 6 are similar to those of compound 5

13

C NMR data in CD3OD (Tables 1 and 2, Figures S33-34,


16

Page 17 of 36


351

(Tables 1 and 2). A diagnostic difference was observed in the chemical shifts corresponding to

352

the sugar units. The saccharide part of compound 6 exhibited a close similarity to those of

353

compounds 3 and 4, this led to its identification as β-D-xylopyranosyl-(1’’→3’)-O-β-D-

354

quinovopyranosyl. Hence, compound 6 was identified as the new 3-O-β-D-xylopyranosyl-

355

(1’’→3’)-β-D-quinovopyranosyl-(22S,23S,25R)-3β,6α,23-trihydroxy-5α-spirostane.

356

Compound 7 was obtained as a yellow amorphous solid. The molecular formula was

357

assigned as C39H64O13 based on data from HRESIMS analyses (molecular ion [M + H]+ at m/z

358

741.4414, (calcd. for C39H65O13, 741.4420) (Figures S35-36, Supporting Information). The 1H

359

NMR spectroscopic signals for compound 7, (Table 1, Figure S37, Supporting Information)

360

displayed two tertiary methyl groups at δH 0.85 (s, H-18, 3H) and δH 0.87 (s, H-19, 3H), four

361

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),

362

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

363

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

364

and

365

of compound 7, were found to be similar to those of the saccharide part of compounds 1, 2 and 5

366

(tables 1 and 2), which were also supported by TOCSY-1D experiments (Figures S39-S40,

367

Supporting Information) through the irradiation of the anomeric protons at δH 4.26 (d, J = 7.8 Hz

368

H-1') and δH 5.13 (d, J = 1.2 Hz, H-1"). Hence, the sugar units were identified as β-D-quinovose

369

and α-L-rhamnose. The linkage between the saccharide units was determined as (1’’→3’) due to

370

the gHMBC correlation (Figure S41, Supporting Information) between the anomeric proton of

371

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

372

sugar-chain bond at the aglycone, was also derived from the gHMBC correlation between the

373

anomeric proton of quinovose at δH 4.26 (d, J = 7.8 Hz) and the C-3 at δC 79.0 of aglycone. The

13

C NMR resonances (Table 2, Figure S38, Supporting Information) for the sugar moieties


17


Page 18 of 36

374

gHMBC spectrum analysis was conducted in order to demonstrate the connectivity of the

375

respective protons and carbons. The cross-peaks between H-26ax at δH 3.84 (m) and C-27 at δC

376

16.2, H-26ec at δH 3.18 (broad d, J = 10.8 Hz) and C-27 at δC 16.2, C-24 at δC 34.5 and C-22 at

377

δC 111.2, are indicative of the presence of an unsubstituted F ring (Figure S42, Supporting

378

Information). The stereochemistry of the stereocenter C-25 was determined using the Agrawal

379

method, which is based on differences of the chemical shifts between the geminal protons of the

380

H2-26 metilene and the unsubstituted 22, 23, 24 and 25 positions.29 These author observed that,

381

for compounds consisting of stereocenter 25S, the difference between the 1H NMR chemical

382

shifts of the geminal protons of the H2-26 is usually higher than 0.35, whereas for compounds

383

with stereocenter 25R, the difference between these chemical shifts is lower than 0.20. As the

384

difference observed in compound 7 was 0.66 (δHa 3.84 for H26a and δHb 3.18 for H26b), the C-25S

385

configuration was assigned. Furthermore, H3-27 methyl group absorbs frequency within the

386

range between δH 0.95-1.13 in spirostanols with 25S configuration, and between δH 0.71-0.83 in

387

25R spirostanols.29 One will note that the 1H NMR spectra of compound 7 displays a signal at δH

388

1.09 (d, J = 7.1 Hz, 3H), this provides a further evidence of the axial (β) orientation of the H3-27

389

methyl group, which is typically associated with 25S-spirostanol saponins in compound 7. The

390

NOESY-1D spectra for compound 7 are in good agreement with the NOESY interactions

391

observed in compounds 1, 4 and 5. This is clearly indicative of trans fusion between AB and BC,

392

CD rings, cis fusion between DE rings, as well as the β-orientation for the methyl groups H3-18

393

and H3-19.

394

Other important correlations were observed in 1H‒1H gCOSY spectra (Figures S43-S44,

395

Supporting Information). These included the correlations of H-3 at δH 3.45 (m) with H-2 at δH

396

1.74 (m) 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


18

Page 19 of 36


397

δH 3.37 (m) with a methylene group H2-7 at δH 0.94, 2.17 (m), thus demonstrating the presence

398

of the hydroxyl groups at C-1, C-3 and C-6. The ketal carbon C-22 was determined as “S” due to

399

the NOESY-1D correlation established solely between H-20 at δH 2.53, (q, J = 7.0 Hz) and the

400

methyl group H3-18 at δH 0.85 (s) (Figure S45, Supporting Information). By contrast, when the

401

stereocenter C-22 occupies an R configuration, the NOESY-1D spectra exhibit the correlation of

402

the H-20 with the methyl group CH3-18 and the methylene group CH2-23 in the ring F.30 The

403

remaining signals in compound 7 were assigned using the spectroscopic data obtained through

404

gHMBC, gHSQC (Figure S46, Supporting Information) and 1H‒1H gCOSY analyses, which

405

allowed assigning all signals of this compound for the first time in the literature. In view of that,

406

compound 7 was

407

quinovopyranosyl-(22S,25S)-1β,3β,6α-trihydroxy-5α-spirostane.

characterized as the new 6-O-α-L-rhamnopyranosyl-(1’’→3’)-β-D-

408

Compound 8 was isolated as a yellow amorphous solid with molecular formula

409

C38H62O13 derived from HRESIMS analyses in the positive mode (Figures S47, S48, Supporting

410

Information), which showed a ion peak [M + H]+ at m/z 727.4254 (calcd. for C38H63O13,

411

727.4263). The 1H NMR and

412

Information) spectroscopic signals of the aglycone, displayed a pattern similar to that of

413

(22R,25S)-1β,3β,6α-trihydroxy-5α-spirostane (compound 7), which has been previously

414

described. However, differences in 1H‒1H gCOSY spectra were detected (Figures S51, S52,

415

Supporting Information). The 1H‒1H gCOSY spectra showed correlations between H-3 at δH 3.67

416

(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

417

3.40 (m) with H-5 at δH 1.16 (m) and H-7 at δH 0.96 (m). These correlations indicate the presence

418

of hydroxyl groups at C-3, C-4 and C-6. On the other hand, the 1H NMR spectra exhibited two

419

anomeric proton resonances for sugar moieties at δH 4.30 (d, J = 8.0Hz, 1H) and δH 4.47 (d, J =

13

C NMR (Tables 1 and 2, Figures S49, S50, Supporting


19


Page 20 of 36

420

7.0 Hz, 1H), these differ from those of the saccharide unit present in compound 7. The TOCSY-

421

1D experiments with selective irradiation of the anomeric proton at δH 4.30 (d, J = 8.0 Hz, 1H)

422

(Figure S53, Supporting Information), displayed the proton spin systems of a β-D-quinovose unit

423

[δH 3.34 (m, H-2´), 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)

424

and a methyl group at δH 1.26 (d, J = 6.0 Hz, H3-6´, 3H)], whereas irradiation of the anomeric

425

proton at δH 4.47 (d, J = 7.0 Hz 1H) (Figure S54, Supporting Information), exhibited the proton

426

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-

427

4´´,1H) and a methylene at δH 3.23 (m, H-5a´´,1H), δH 3.89 (m, H-5b´´,1H)]. 1H NMR and

428

NMR resonances relative to the sugar part of compound 8 were similar with those of compounds

429

3, 4 and 6 sugar units. This confirms the identity of the sugar units as β-D-quinovose and β-D-

430

xylose. The linkage between the units was determined as (1’’→3’) by the gHMBC correlation

431

(Figure S55, Supporting Information), between the anomeric proton of xylose at δH 4.47 (d, J =

432

7.0 Hz, 1H) and carbon at δC 86.3 which corresponds to C-3´ of quinovose. The site of the sugar-

433

chain bond at the aglycone, was also derived from the gHMBC correlation between the anomeric

434

proton of quinovose at δH 4.30 (d, J = 8.0 Hz, 1H) and C-3 at δC 78.9 of the aglycone.

435

The analyses of the interactions in the NOESY-1D spectrum for compound 8 (Figures S56-57,

436

Supporting Information), revealed the same interactions observed in compound 7. This result

437

confirms the existence of trans fusion between AB and BC, CD rings, a cis fusion between DE

438

rings, a configuration “S” for a ketal C-22 carbon, as well as the β-orientation for the methyl

439

groups H3-18 and H3-19.

440

The remaining signals in compound 8 were determined using the spectroscopic data obtained

441

through HMBC, HSQC and 1H‒1H COSY analyses. These enabled us to identify all the signals

442

associated with this compound. The structure of compound 8 was, thus, identified as the new 6-


13

C

20

Page 21 of 36


443

O-β-D-xylopyranosyl-(1’’→3’)-β-D-quinovopyranosyl-(22S,25S)-3β,4β,6α-trihydroxy-5α-

444

spirostane.

445 446

Antibacterial activity. Numerous studies have been published in the literature focusing

447

on the evaluation of the anticariogenic potential of medicinal plants.31-33 None of these studies

448

has, however, been devoted to investigating the correlation between 70 % alcohol tincture of S.

449

paniculatum and antibacterial activity in the dentistry area. The antibacterial assay of the plant

450

extracts employed in this study was performed through the determination of the Minimum

451

Inhibitory Concentration (MIC). According to Rios & Recio,34 a result relative to antibacterial

452

activity can be considered promising in the case of natural products, such as plant extracts,

453

essential oil and pure substances, by analyzing the results of the MIC assay. These authors

454

consider MIC values lower than 100 µg/mL for extract or 10 µg/mL for pure substances as

455

active.

456

In the present study, the use of 70% alcohol tincture yielded lower MIC values for S.

457

mutans (50 µg/mL, ATCC), S. sanguinis (50 µg/mL, ATCC), L. casei (50 µg/mL, CI), S. mitis

458

(25 µg/mL, ATCC), and S. oralis (12.5 and 6.25 µg/mL, CI and ATCC, respectively). The F26-

459

16 fraction showed a lower MIC value for S. oralis (100 µg/mL, ATCC) (Figure 2), so based on

460

these criteria, the 70% alcohol tincture was considered an effective anticariogenic agent.35 These

461

results imply that other compounds present in the 70 % alcohol tincture may be playing an

462

important role in the in vitro antibacterial activity.

463

The results obtained from our investigation provided evidence regarding the chemical

464

composition of the hydroalcoholic (70%) tincture of Solanum paniculatum leaves using as

465

phytomedicine and nutritional supplements in Brazil. This shows that polyhydroxylated steroidal


21


Page 22 of 36

466

saponins with spirostane skeleton are the main constituents of Solanum paniculatum leaves.

467

Some of the compounds obtained in this work (1, 4-8) have never been isolated before and are

468

regarded new natural compounds. Furthermore, the tincture displayed a powerful ability to

469

prevent growth of different oral pathogens. This ability is seen to be stronger than that exerted by

470

the isolated fraction of saponins. These results may be attributed to the presence of a series of

471

other components that act synergistically in the hydroalcoholic tincture.

472 473

Author contributions.

474

The listed authors contributed toward the development and drafting of this work. Their

475

respective contributions are described as follows: A.B.V.D was the one responsible for the

476

acquisition, interpretation of the experimental data besides writing the manuscript; D.G.T helped

477

in the analysis and interpretation of the spectral data; A.C.Z assisted in the experimental design

478

for the compound isolation; C.H.G.M supervised the experimental design of the antimicrobial

479

activity; T.S.S conducted the experiments related to antimicrobial activity; L.R helped in editing

480

the manuscript; W.V also helped in the editing of the manuscript and in conducting mass

481

analyses; L.C.S is a corresponding author who supervised the chemical study, conducted the

482

experimental design and helped to edit the manuscript. All authors have given their approval to

483

the final version of the manuscript.

484 485 486 487 488


22

Page 23 of 36


489

Supporting Information.

490

The chromatographic profile of semipreparative F16-26 fraction by HPLC-RI for the isolation of

491

compounds (1-8) is described in the figure S1; HRESIMS, 1H,

492

gHSQC, 1H‒1H gCOSY, TOCSY-1D and NOESY-1D NMR spectrum for the characterization of

493

the compounds (1-8) are showed in the Figures S2-S57.

494

References.

13

C, DEPT 135o, gHMBC,

495

(1) D´Arcy, W. G. The Solanaceae since 1976, with a review of its biogeography: In

496

Solanaceae III: taxonomy, chemistry, evolution, Eds: Hawkes, J. G., Lester, R. N., Nee,

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M. and Estrada, N, 1991, London: Kew Royal Botanical Gardens, pp. 75-137.

498

(2) Kodamatani, H.; Saito, K.; Nina, N.; Yamazaki, S.; Tanaka, Y. Simple and sensitive

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method for determination of glycoalkaloids in potato tubers by high performance liquid

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chromatography with chemiluminescence detection. J. Chromatog. A. 2005, 1100, 26–31.

501

(3) Friedman, M. Analysis of biologically active compounds in potatoes (Solanum

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tuberosum), tomatoes (Lycopersicom esculentum), and jimson weed (Datura stamonium)

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seeds. J. Chromatog. A. 2004, 1054, 143–155.

504 505

(4) Van Eck, J. & Snyder, A. Eggplant (Solanum melongena L.). Methods. Mol. Biol. 2006, 343, 439-447.

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(5) Lorenzi, H.; Matos, F. J. A. Plantas medicinais no Brasil, Plantas nativas e exóticas, Ed:

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Instituto Plantarum de Estudos da Flora Ltda, 2002, Nova Odessa, Brazil, ISBN:

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8586714186, 9788586714184, pp. 129.

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(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.


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(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.

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(8) Esteves-Souza, A.; Silva, T. M. S.; Alves, C. C. F.; Carvalho, M. G.; Braz-Filho, R.;

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Echevarria, A. Cytotoxic activities against Ehrlich Carcinoma and Human K562

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Leukaemia of alkaloids and flavonoid from two Solanum species. J. Braz. Chem. Soc.

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2002, 13, 838842.

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(9) Cornelius, M. T. F.; Alves, C. C. F.; Silva, T. M. S.; Alves, K. Z.; Carvalho, M. G.; Braz-

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Filho, R.; Agra, M. F. Solasonina e flavonóides isolados de Solanum crinitum Lam.

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Rev. Bras. Farmacogn. 2004, 85, 5759.

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(10) Dinan, L.; Harmatha, J.; Lafont, R. Chromatographic procedures for the isolation of plant steroids. J. Chromatog. A. 2001, 935, 105123. (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 Úteis do Brasil. Ed: Instituto Brasileiro de Desenvolvimento Florestal, Ministério da Agricultura, 1984, Brazil, Vol. III, pp. 395.

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(13) Farmacopéia Brasileira. Ed: Atheneu, 1988, 4.ed. São Paulo,Brazil, pp.567.

527

(14) Agra, M. F.; Baracho, G. S.; Nurit, K.; Basilio, I. J. L. D.; Coelho, V. P. M. Medicinal

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and poisonous diversity of the flora of "Cariri Paraiano", Brazil. J. Etnopharmacol. 2007,

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111 (2), 383395.


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(15) Mesia-Vela, S.; Santos, M. T.; Souccar, C.; Lima-Landman, M. T. R.; Lapa, A. J.

531

Solanum paniculatum L. (jurubeba): Potent inhibitor of gastric acid secretion in mice.

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Phytomedicine. 2002, 9, 508514.

533

(16) Vieira Júnior, G. M.; Quintinho da Rocha, C.; Rodrigues, T de S.; Hiruma-Lima, C. A.;

534

Vilegas, W. New steroidal saponins and antiulcer activity from Solanum paniculatum L.

535

Food Chem. 2015, 186, 160167.

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(17) Rippenger, H.; Budzikiewicz, H.; Schreiber, K. Jurubin ein stickstoffhaltiges

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Steroidsaponin neuartigen Struckturtyps aus Solanum paniculatum L. Chem. Ber. 1967,

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100, 17251740.

539

(18) Hara, S.; Okabe, K.; Mihashi, K. Gas-liquid chromatographic separation of aldose

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enantiomers as trimethylsilyl ethers of methyl 2-(polyhydroxialkyl)-thiazolidine-4(R)-

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carboxilates. Chem Pharm Bull. 1987, 35, 501-506.

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(19) Clinical and Laboratory Standards Institute (CLSI). Methods for dilution antimicrobial

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susceptibility tests for bacteria that grow aerobically. Approved standard. M07–A9,

544

2012, Wayne, PA: USA.

545

(20) Sarker, S. D.; Nahar, L.; Kumarasamy, Y. Microtitre platebased antibacterial assay

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incorporating resazurin as an indicator of cell growth, and its application in the in vitro

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antibacterial screening of phytochemicals. Methods. 2007, 42, 321–324.

548

(21) Lida, Y.; Yanai, Y.; Ono, M.; Ikeda, T.; Nohara, T. Three unusual 22-β-O-23-Hydroxi-

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(5α)-spirostanol glycosides from the fruits of Solanum torvum. Chem. Pharm. Bull. 2005,

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53, 11221125.


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(22) Honbu, T.; Ikeda, T.; Zhu, X-H.; Yoshibara, O.; Okawa, M.; Nafady, A. M.; Nohara, T.

552

New Steroidal Glycosides from the Fruits of Solanum anguivi. J. Nat. Prod. 2002, 65,

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11181120.

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(23) Mahmood, U.; Thakur, R. S.; & Blunden, G. Neochlorogenin, neosolaspigenin and solaspigenin from Solanum torvum leaves. J. Nat. Prod. 2003, 46, 427428.

556

(24) Matsushita, S.; Yanai, Y.; Fusyuku, A.; Ikeda, T.; Nohara, T. Distinction of absolute

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configuration at C-22 of C-23 hydroxyspirostane and C-23-hydroxyspirosolane

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glycosides. Chem. Pharm. Bull. 2007, 55, 10791081.

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(25) Zamilpa, A.; Tortoriello, J.; Navarro, V.; Delgado, G.; Alvarez, L. Five new steroidal

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saponins from Solanum chrysotrichum leaves and their antimicotic activity. J. Nat.

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Prod. 2002, 65, 18151819.

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(26) González, M.; Zamilpa, A.; Marquina, S.; Navarro, V.; Alvarez, L. Antimicotic

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spirostanol saponins from Solanum hispidum leaves and their structure-activity

564

relationships. J. Nat. Prod. 2004, 67, 938941.

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(27) Rippenger, 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.


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569

(29) Agrawal, K. P. Spectral assignment and reference data: 25R/25S stereochemistry of

570

spirotane-type steroidals sapogenins and steroidals saponins via chemical shift of

571

geminals protons of ring F. Magn. Reson. Chem. 2003, 41, 965-968.

572 573 574 575

(30) Perez, J. A.; Calle, M. J.; Simonet, M. A.; Guerra, O. J.; Stochmal, A. Bioactive Steroidal Saponins from Agave offoyana 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.

576

(32) Cunha, L. C. S.; de Morais, S. A. L.; Martins, C. H. G.; Martins, M. M.; Chang, R.; de

577

Aquino, F. J. T.; de Oliveira, A.; Moraes, T. S.; Machado, F. C.; da Silva, C. V.; do

578

Nascimento, E. A. Chemical composition, cytotoxic and antimicrobial activity of

579

essential oils from Cassia bakeriana Craib against aerobic and anaerobic oral

580

pathogens. Molecules. 2013, 18, 4588-4598.

581

(33) Moreira, M. R.; Souza, A. B.; Soares, S.; Bianchi, T. C.; Eugênio, D. S.; Lemes, D. C.;

582

Martins, C. H. G.; Moraes, T. S.; Tavares, D. C.; Ferreira, N. H.; Ambrósio, S. R.;

583

Veneziani, R. C. S. ent-Kaurenoic acid-rich extract from Mikania glomerata: In vitro

584

activity against bacteria responsible for dental caries. Fitoterapia. 2016, 112, 211-216.

585 586

(34) Rios, J. L.; Recio M. C. Medicinal Plants and Antimicrobial Activity. J Ethnopharmacol. 2005, 100, 80-84.

587

(35) Moura, C. L.; Casemiro, L. A.; Martins, G. C. H.; Cunha, W. R.; Luis de Andrade e

588

Silva, M.; Cury, V. A. H. Avaliação da atividade antimicrobiana da espécie vegetal


27


Page 28 of 36

589

Pfaffia glomerata frente a patógenos bucais. Investigação, 2011, 11, 24-28, (ISSN

590

2177-4080 (on-line)).

591 592

Acknowledgments.

593

The authors thanks to São Paulo State Research Foundation (FAPESP) (grant  2015/04899-3)

594

leading by Lourdes Campaner dos Santos, the Coordination for the Improvement of Higher

595

Education Personnel (CAPES, grant 187715-1) and the BIOTA/FAPESP project (grant

596

2009/52237-9) leading by Wagner Villegas for supporting and funding this research. The

597

author is also grateful to Nivaldo Boralle for the acquisition of the 1D and 2D spectra of the

598

isolated compounds and to Luis Vitor Sacramento for supplying the leaves of S. paniculatum L.

599 600

Notes.

601

The authors declare no competing financial interest.

602 603

Figure Captions.

604

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

605

Figure 2. Determination of the Minimum Inhibitory Concentration (MIC), in µg/mL, of the

606

70 % ethanolic extract and fraction F26-16 of S. paniculatum against oral pathogens.

607 608


28

Page 29 of 36


Table 1. 1H NMR spectroscopy data (14.1 T, CD3OD) of compounds 1, 4-8. a 1 H (J in Hz) 1.13 1.74,m 1.15 2.35, m 3.45, m

4 H (J in Hz) 1.01 1.71, m 1.16 2.39, m 3.45, m

5 H (J in Hz) 1.15 1.79, m 1.15 2.37, m 3.38, m

6 H (J in Hz) 1.02 1.78, m 1.16 2.38, m 3.39, m

7 H (J in Hz)

1.38 1.74, m

1.40 1.74, m

0.93 2.15, m

0.94 2.16, m

1.95 2.37, m

3.45, m

1.15, m 3.37, m 0.94 2.16, m 1.64, m

1.16, m 3.39, m 0.95 2.16, m 1.65, m

1.15, m 3.44, m 1.40 1.76, m 1.65, m

1.16, m 3.45, m

1.65, m

1.16, m 3.37, m 0.94 2.17, m 1.66, m

1.15, m 3.40, m 0.96 2.17, m 1.69, m

0.68, td (2.5, 3.9, 4.0)

0.68, td (2.3, 3.8, 3.8)

0.68, td (2.0, 3.8, 4.1)

0.68, td (2.1, 3.8, 4.0)

0.68, td 0.69, td (2.4, 3.8, 3.9) (2.2, 3.8, 3.9)

16

1.30 1.53, m 1.01 1.74, m 1.15, m 1.27 1.96, m 4.46, m

1.31 1.54, m 1.13 1.74, m 1.16, m 1.25 1.97, m 4.45, m

1.32 1.54, m 1.01 1.70, m 1.17, m 1.40 1.75, m 4.43, m

1.69, m

1.68, m

1.74, m

1.75, m

18 19 20

0.81, s 0.87, s 2.34, m

0.81, s 0.87, s 2.25, m

1.33 1.54, m 1.15 1.74, m 1.10, m 1.95 2.30, m 4.71, m 1.83, dd (6.3) 0.86, s 0.87, s 2.18, m

1.31 1.54, m 1.02 1.67, m 1.16, m 1.40 1.75, m 4.42, m

17

1.33 1.54, m 1.15 1.73, m 1.10, m 1.95 2.30, m 4.70, m 1.83,dd (6.4, 9.1) 0.87, s 0.86, s 2.19, m

0.85, s 0.87, s 2.53,q (7.0)

0.84, s 0.87, s 2.52,q (7,0)

21

1.11,d (7.0)

1.09,d (7.0)

1.15,d (7.3)

1.15,d (7.3)

0.95,d (7.0)

0.95,d (7.0)

22

-

-

-

-

-

-

23

3.56,t (3.6)

3.52, m

3.62, dd (2.8, 4.7)

3.62, m

1.15 1.39, m

Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

b

1.39, m


3.65, m 1.16 1.74, m 3.45, m

8 H (J in Hz) 1.15 1.75, m 1.92 2.39, m 3.67, m

1.15 1.39, m

29


1

4

5

6

Page 30 of 36

7

Position

H (J in Hz)

H (J in Hz)

H (J in Hz)

24

1.52 2.06, m

1.60, m

1.47, d (11.9) 1.70 m

25

1.66, m

2.03, m

1.70, m

1.46, dd (11.9) 1.70 m 1.72, m

26

3.31, m 3.95,d (3.7)

3.35 3.46, m

3.29 3.43, m

3.30 3,43, m

3.18,d (10.8) 3.84, m

27

1.20,d (7.2)

0.76,d (6.7)

0.80,d (6.54)

0.81, d ( 6,5) Qui

1.09, d ( 7.1) Qui

c

Qui

Qui

Qui

H (J in Hz)

8

H (J in Hz)

H (J in Hz)

1.64 1.89, m

1.66 1.89, m

1.89, m

1.89, m 3.20, m 3.84, dd (1.9) 1.10,d (7.0) Qui

1’

4.26,d (7.9)

4.31,d (7.8)

4.26,d (7.9)

4.30,d (7.8)

4.26,d (7.8)

4.30,d(8.0)

2’

3.25, m

3.34, m

3.26, m

3.34, m

3.25,dd (8.0, 8.94)

3.34, m

3’

3.41, m

3.42, m

3.41, m

3.42, m

3.41, m

3.42, m

4’

3.00,t (9.2)

3.04, t (9.1)

3.00, t (9.2)

3.05, t (9.1)

3.00, t (9.0)

3.05,t (9.1)

5’

3.30, m

3.31, m

3.30, m

3.32, m

3.30, m

3.31, m

6’

1.27,d (6.2)

1.27, d (6.1)

1.27, d (6.3)

1.27, d (6.2)

1.26,d (6.0)

1.26,d (6.0)

d

e

Rha

Xyl

Rha

Xyl

Rha

Xyl

1’’

5.13,d (1.6)

4.47, d (7.6)

5.12, d (1.5)

4.47, d (7.0)

5.13,d (1.2)

4.47,d (7.6)

2’’

3.92, dd (1.8, 3.3)

3.25, m

3.92,dd (1.8)

3.26, m

3.92, dd (1.7, 3.2)

3.26, m

3’’

3.68,dd (3.4)

3.33, m

3.68, dd (3.4, 9.5)

3.33, m

3.67, m

3.33, m

4’’

3.38, m

3.50, m

3.37, m

3.49, m

3.37, m

3.49, m

5’’

3.99, m

3.23, m 3.89,dd (5.4)

3.99, m

3.23,d (2.6) 3.88,dd (5.4)

3.98, m

3.23, m 3.89, dd (5.3)

6’’

1.23,d (6.2)

-

1.23, d (6.2)

-

a

Assignments were made by

H ̶

1

1

1.23,d (6.0)

H gCOSY, gHSQC, gHMBC, TOCSY-1D, and NOESY- 1D data.

b 1

H-1H

coupling constants were measured from 1H NMR spectra in Hz. cβ-D-quinovopyranoside. dα-L-rhamnopiranoside. e

β-D-xylopyranoside.


30

Page 31 of 36


Table 2.

Position 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 1' 2' 3' 4' 5' 6'

13

C NMR spectroscopy data (14.1T, CD3OD) of compounds 1, 4-8.a

1 δC, type 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 b Qui 103.7,CH 75.0, CH 82.8, CH 74.3, CH 71.6, CH 16.9, CH3

4 δC, type 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

5 δC, type 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

6 δC, type 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


7 δC, type 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

8 δC, type 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 31


Position 1" 2" 3" 4" 5" 6"

1 δC, type c Rha 101.4,CH 71.0, CH 70.8, CH 72.6, CH 68.6, CH 16.5, CH3

4 δC, type d Xyl 104.6,CH 73.8, CH 76.3, CH 69.7, CH 65.7, CH2 -

5 δC, type Rha 101.4,CH 71, CH 70.8, CH 72.6, CH 68.6, CH 16.5, CH3

6 δC, type Xyl 104.0,CH 73.8, CH 76.3, CH 69.6, CH 65.8, CH2 -

Page 32 of 36

7 δC, type Rha 101.4,CH 71.0, CH 70.8, CH 72.6, CH 68.6, CH 16.5, CH3

8 δC, type Xyl 104.6,CH 73.8, CH 76.3, CH 69.6, CH 65.7, CH2 -

a

Assignments were made by DEPT-135o, TOCSY-1D, HSQC, and HMBC data. bβ-D-quinovopyranoside.

c

α-L-rhamnopiranoside. dβ-D-xylopyranoside.


32

Page 33 of 36


Figure 1


33


Page 34 of 36

Figure 2.

Legend. E. S. L. ATCC CI Chlorhexidine

Enterococcus Streptococcus Lactobacillus American Type Culture Collection Culture Isolate

Positive control


34

Page 35 of 36


Graphical Abstracts.

TOC graphic 85x47mm (300 x 300 DPI)


35


85x47mm (300 x 300 DPI)


Page 36 of 36

New Polyhydroxylated Steroidal Saponins from ... - ACS Publications

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