Anti-Inflammatory and Antimicrobial Evaluation of Neovestitol and Vestitol Isolated from Brazilian...

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Article

Anti-inflammatory and antimicrobial evaluation ofneovestitol and vestitol isolated from Brazilian red propolis

Bruno Bueno-Silva, Severino Matias Alencar, Hyun Koo, MasaharuIkegaki, Gil V.J.D. Silva, Marcelo H Napimoga, and Pedro L. Rosalen

J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf305468f • Publication Date (Web): 23 Apr 2013

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Anti-inflammatory and antimicrobial evaluation of neovestitol and vestitol isolated 1

from Brazilian red propolis 2

3

Bruno Bueno-Silvaa,g

; Severino M. Alencar b*

; Hyun Kooc,g

; Masaharu Ikegakid; Gil V. J. 4

Silvae; Marcelo H. Napimogaf; Pedro L. Rosalena,g. 5

6

a Piracicaba Dental School, University of Campinas – UNICAMP, Department of 7

Physiological Science, C.P. 52; Zip Code: 13414-903 – Piracicaba – SP – Brazil. Phone: 8

+55 1921065313. Fax: +55 1921065308 9

b College of Agriculture “Luiz de Queiroz” (ESALQ/USP), C.P. 9; 10

Zip Code: 13418-900 – Piracicaba – SP – Brazil 11

c Center for Oral Biology and Eastman Department of Dentistry, and Department of 12

Microbiology and Immunology, University of Rochester Medical Center, Rochester, 13

N.Y. , USA 14

d Federal University of Alfenas; Zip Code 37130-000 – Alfenas – MG – Brazil 15

eDepartment of Chemistry, FFCLRP-USP, University of São Paulo, Zip Code 14040-16

901 Ribeirão Preto, SP, Brazil 17

f Laboratory of Immunology and Molecular Biology, São Leopoldo Mandic Institute 18

and Research Center, Campinas/SP, Brazil. 19

g NatPROB (Natural Product Research Group in Oral Biology's) 20

* Corresponding author: Phone: +55 1934294150 - Fax: +55 1934294288 - Email: 21

[email protected] 22

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Abstract 39

The objective of this study was to evaluate anti-inflammatory and antimicrobial 40

activities of neovestitol and vestitol isolated from Brazilian red propolis (BRP). BRP 41

ethanolic extract (EEP), neovestitol and vestitol were evaluated by anti-inflammatory 42

properties using a neutrophil migration assays. The antimicrobial activity was evaluated 43

by minimal inhibitory and bactericidal concentrations (MIC and MBC) against 44

Sreptococcus mutans, Sreptococcus sobrinus, Sthaphylococcus aureus, and 45

Actinomyces naeslundii. Neovestitol, vestitol and EEP inhibited neutrophil migration at 46

dose of 10 mg/Kg. Regarding antimicrobial activity, neovestitol showed MIC ranging 47

from <6.25 to 25-50 µg/mL and MBC ranging from 25-50 to 50-100 µg/mL, while 48

vestitol showed MIC ranging from 25-50 to 50-100 µg/mL and MBC ranging from 25-49

50 to 50-100 µg/mL. Both isoflavonoids, neovestitol and vestitol, are consistent 50

bioactive compounds displaying anti-inflammatory and antimicrobial activities that can 51

strongly act in low dose and concentration and have a promising potential to be applied 52

on pharmaceutical and food industries. 53

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Keywords: anti-inflammatory, antimicrobial, isoflavonoids, Neovestitol, Vestitol, red 56

propolis. 57

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Introduction 64

Brazilian propolis has attracted scientific interest due to the discovery of new 65

drugs, such as CAPE (caffeic acid phenyl ester)1, apigenin, tt-farnesol

2,3, artepillin C 66

(3,5-diprenyl-4-hydroxycinnamic acid), as well as more than 250 patents related to its 67

applications and therapeutic and nutrition uses4. Since chemical composition of propolis 68

is dependent of vegetation around the beehive, Brazilian propolis was classified into 13 69

different types according to its physical-chemical properties and geographical 70

location5,6

. The most recent one, red propolis, was classified as type 13 based on its 71

unique chemical composition, particularly rich in isoflavonoids. Recently, its botanical 72

origin was determined as Dalbergia ecatosphyllum, from leguminosae family6, which is 73

known for high content of isoflavonoids, with particular interest on neovestitol and 74

vestitol by the antioxidant activity7. 75

Recent studies have shown that red propolis contains elevated amounts of certain 76

type of isoflavonoids, i.e. isoflavones. This particular group of polyphenols exhibits a 77

wide range of biological properties, including antiviral, antimicrobial, anti-78

inflammatory, and even anti-cancer activities. Furthermore, isoflavonoids have been 79

used as food additives due to low toxicity and also as a health food supplement8,9

. 80

Although a previous study have shown the biological activity of crude extracts of 81

red propolis10

, these authors did not find antimicrobial activity for neovestitol because 82

the yield was insufficient, so this search activity was neglected on that time. Whether 83

the isoflavonoids are associated with bioactivity remains to be elucidated because other 84

studies concerning red propolis such as Piccinelli et al.11

and Righi et al.12

did not 85

correlate the isolated isoflavonoids with biological activity. Moreover, it is important to 86

note that there are no reports in the literature about anti-inflammatory properties of 87

Brazilian red propolis and also, its bioactive compounds as neovestitol and vestitol. In 88

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this way, as isoflavonoids has recognized pharmacological activities13-18

, we 89

hypothesize that the specific isoflavonoids neovestitol and vestitol present in this 90

distinctive propolis are associated with antimicrobial and anti-inflammatory effects. 91

Therefore, the aim of this study was to evaluate the anti-inflammatory and 92

antimicrobial activities of neovestitol and vestitol isolated from Brazilian red propolis. 93

Material and Methods 94

Brazilian Red propolis samples were collected from Maceio, Alagoas State, 95

Northeast of Brazil SL 09.40 and WL 35.41 at the end of summer, during the month of 96

March. Three samples from three different box (beehive) were collected to be used in 97

this study. 98

Extraction and isolation of bioactive compounds 99

Propolis was ground to a fine powder and 2 g (dry weight) were mixed with 25 100

mL of 80% (v/v) ethanol and shaken at 70 °C for 30 min. After extraction, the mixture 101

was centrifuged and the supernatant was evaporated under low pressure to produce the 102

ethanolic extract of propolis (EEP)6. 103

A bioassay-guided fractionation was designed to focus on the isolation and 104

identification of compounds with anti-inflammatory and antimicrobial activities19

as 105

shown in Figure 1. 106

Based on our previous results, reported by Oldoni et al.10

, we modified the 107

extraction method in order to obtain a larger amount of neovestitol and vestitol. These 108

results include the biological inactivity of fractions from the beginning of the open dry 109

column which led us to work only with the second half of the column (the last 10 cm 110

length). Furthermore, the division criteria to obtain the fractions were not only by color 111

but also per size, different from Oldoni et al.10

over again. The maximum size of one 112

fraction was 2 cm length. If the fraction (with the same color) had more than 2 cm, we 113

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divided it into 2 different fractions. Therefore, the maximum volume of each fraction 114

was 3.5 cm3. 115

The fractions obtained were monitored by thin layer chromatography (TLC) using 116

the anisaldehyde reagent (4-methoxybenzaldehyde, acetic acid, sulphuric acid: 117

1.0:48.5:0.5), followed by heating at 100 ºC for 5 min. Fluorescent substances were 118

visualized under ultraviolet (UV) light at the wavelengths of 254 and 366 nm. 119

In order to select the bioactive sub-fraction and consequently, the bioactive 120

compounds, neovestitol and vestitol, we focused to isolate the red-orange band (in 121

TLC). Sub fractions 1 and 5 showed no biological activity and negligible biological 122

activities were found in fractions 2 and 3. The most bioactive fraction (4) was 123

chromatographed over a Sephadex LH-20 column (5x30cm) using methanol to yield 124

three bioactive fractions. Fractions 4.2 and 4.3 were active; however, fraction 4.3 was 125

rejected because it was impossible to be dissolved in buffer solution (AB buffer and 126

phosphate buffer) routinely used to perform anti-inflammatory and antimicrobial tests. 127

Thus, fraction 4.2 (200 mg) was purified by semi-preparative reverse-phase HPLC 128

[Shimadzu PREP-ODS (H) 250X20 mm column eluted with a gradient starting with 129

CH3OH:H2O (65:35) to CH3OH:H2O (95:5) in 35 min, flow rate 3 mL/min] and yielded 130

two active compounds. 131

Identification of bioactive compounds 132

Neovestitol and vestitol were identified using high resolution mass spectrometry 133

(Q-TOF-MS/MS) and Nuclear Magnetic Resonance (NMR) based on findings of 134

Oldoni et al.10

. The isolated neovestitol and vestitol was highly purified (<99.8% pure). 135

136

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Anti-inflammatory test 139

Anti-inflammatory test consisted of evaluating neutrophil migration to peritoneal 140

cavity after treatment with EEP, neovestitol and vestitol. Male Balb/c mice (20–25 g) 141

were housed in temperature controlled rooms (22–25 °C) with ad libitum access to 142

water and food. All experiments were conducted in accordance with National Institutes 143

of Health guidelines for the welfare of experimental animals and with the approval of 144

the institutional Committee for Ethics in Animal Research (protocol number: 1484-1). 145

The animals were used only in a single experimental group. For the determination of 146

neutrophil migration to peritoneal cavity, EEP, neovestitol and vestitol were 147

administered by subcutaneous injection 15 min before (1, 3, or 10 mg/kg) the 148

administration of inflammatory stimuli by intraperitoneal injection of carrageenan at 149

500 µg/cavity in naive mice. Mice were killed 4 h after the challenge (carrageenan) 150

administration and the peritoneal cavity cells were harvested by washing the cavity with 151

3 mL of phosphate buffered saline (PBS) containing 1 mM EDTA. The volumes 152

recovered were similar in all experimental groups and equated to approximately 95% of 153

the injected volume. Total counts were performed in a cell counter (COULTER A CT; 154

Coulter, Miami, FL, USA), and differential cell counts (100 cells total) were carried out 155

on cytocentrifuge (Cytospin 3; Shandon Lipshaw, Pittsburgh, Pennsylvania, USA) 156

slides stained with Rosenfeld. The results are presented as the number of neutrophils per 157

cavity20

. 158

Antimicrobial tests 159

Antimicrobial tests consisted in the determination of minimal inhibitory 160

concentration (MIC) and minimal bactericidal concentration (MBC) of the EEP, 161

neovestitol and vestitol. The tested microorganisms were Streptococcus mutans UA159, 162

Streptococcus sobrinus 6715, Staphylococcus aureus ATCC25923, and Actinomyces 163

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naeslundii ATCC 12104. The methodology described by Koo et al.21

was modified into 164

a micro technique, in which 190 µL of BHI broth with inoculum (1-2 x 105 UFC/mL) 165

and 10 µL of the EEP, neovestitol, vestitol, and control solution (ethanol 4%, v/v) were 166

dispensed onto a microplate. Concentrations of the tested extracts for MIC ranged from 167

12.5 to 800 µg/mL, and bacterial growth was assessed by adding 0.01% resazurin stain 168

(Aldrich). MIC values were defined as the lowest concentration of a given extract that 169

could inhibit bacterial growth. An aliquot (30 µL) of concentrations higher than MIC 170

was cultured on BHI agar supplemented with 5% defibrinated sheep blood for 18-24 h , 171

at 37 ºC, with 10% CO2 to determine MBC. MBC was the lowest concentration that 172

allowed no visible bacterial growth on agar21

. 173

Results and Discussion 174

The inflammation process is a human body response of any damage or infection, 175

such as those associated with oral inflammation such as periodontal complications 176

(gingivitis and periodontitis) and the anti-inflammatory agent could act locally. This 177

process involves a cascade of successively events that will result in neutrophil migration 178

to the inflammatory focus22,23

. The maintenance of the inflammatory process (chronic 179

inflammation) in the tissues may promote its destruction and bone loss, thus the 180

modulation of the host response, reducing the severity of inflammation is somehow 181

necessary to avoid the deleterious effects 20,24-27

. 182

Thus, we used bioassay-guided fractionation, which is a proven method for the 183

discovery of active principles from natural products, to isolated bioactive compounds 184

from Brazilian red propolis ethanolic extract (EEP). Based on Oldoni et al., we used 185

different chromatographic methods combined with anti-inflammatory and antimicrobial 186

assays to obtain neovestitol and vestitol. It is important to note that the main 187

achievement of our research at this step (bioguided fractionation) was the modification 188

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of the method of extraction to obtain neovestitol and vestitol, which improved the yield 189

of neovestitol from 0.04%10

to 0.2% (present data) and vestitol yield was not changed10

. 190

In this way, animal treated with EEP, neovestitol and vestitol showed inhibitory 191

activity against neutrophil migration at 10 mg/Kg dose (Figure 3). It is important to note 192

that the three substances were as effective as the gold standard Dexamethasone at 10 193

mg/Kg (Figure 3) in preventing neutrophil migration to peritoneal cavity, demonstrating 194

that the compounds have a noteworthy anti-inflammatory potential that should be 195

explore in the future. 196

Thus, both isolated compounds presented anti-inflammatory effects and to best of 197

our knowledge, this is the first report of anti-inflammatory properties of Brazilian red 198

propolis extract and its isolated compounds, such as neovestitol and vestitol, and also 199

they may act as a synergist association. 200

Natural products are recognized as an important source of new therapeutic agents. 201

Over 70% of all new drugs approved between 1981 and 2012 were obtained from 202

natural products, which demonstrate their value as sources for the discovery of novel 203

bioactive agents28

. Canavalia grandiflora seeds lectin, a literature recognized natural 204

product with anti-inflammatory properties7,20

, inhibited neutrophil migration at dose of 205

10 mg/Kg31

as determined using the exact same anti-inflammatory model used in this 206

study. It is apparent that the bioactive compounds of Brazilian red propolis are either as 207

effective as a well-known naturally occurring anti-inflammatory agent (Figure 3). The 208

isoflavones, particularly neovestitol and vestitol, may have potential therapeutic 209

application to modulate inflammation processes, such as those involved with 210

periodontal diseases. 211

Regarding antimicrobial activity, EEP showed MIC values ranged from <6.25 212

µg/mL (S. sobrinus) to 100-200 µg/mL (S. mutans and S. aureus), and MBC ranged 213

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from 50-100 µg/mL (S. sobrinus) to 200-400 µg/mL (S. aureus) (Table 1). In contrast 214

with anti-inflammatory results, neovestitol displayed better antimicrobial effect than 215

EEP and vestitol (Table 1). Neovestitol showed potent antibacterial activity with MIC 216

ranging from <6.25 µg/mL (S. sobrinus) to 25-50 µg/mL. Vestitol was less effective 217

than neovestitol, showing MIC ranging 25-50 µg/mL (S. sobrinus) to 50-100 µg/mL 218

(other microorganisms). Regarding MBC, neovestitol showed results from 25-50 µg/mL 219

(S.sobrinus) to 50-100 µg/mL (S. mutans, A.naeslundii, and S. aureus) while vestitol 220

showed values in accordance with Oldoni et al.10

(Table 1). Therefore, both neovestitol 221

and vestitol are the main bioactive compounds associated with antimicrobial activity of 222

Brazilian red propolis since they have same or better MIC and MBC values 223

(antimicrobial parameters used in this study) than EEP. 224

Other antimicrobial compounds were isolated from Brazilian propolis, such as tt-225

farnesol and pinocembrin 3,5

. The MIC of tt-farnesol against S. mutans and S. sobrinus, 226

two microorganisms associated with oral biofilm disorders, was 28 and 14 µg/mL, 227

respectively while pinocembrim showed 64 µg/mL for both microorganisms3. Thus, 228

neovestitol appears to be more effective against mutans streptococci than the previously 229

identified compounds. 230

Although several compounds, including 7-O-vestitol, have been identified in red 231

propolis32,33

, there is no report in literature about any pharmacological properties or use 232

of this isolated compounds. In addition, to the best of our knowledge, this is the first 233

report in the literature about antimicrobial activity of neovestitol. 234

Recently, a methanolic fraction of Brazilian red propolis showed antibacterial 235

activity against Pseudomonas aeruginosa, Bacillus subtillis, Candida albicans, 236

Salmonella typhimurium, Klebsiella pnemoniae, Enterococcus faecallis, Escherichia 237

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coli, Proteus mirabilis, and Streptococcus pyogenes34

. However, there were no attempts 238

to precisely isolate and identify the active individual compounds. 239

The major challenge in the discovery of new natural agents is the isolation and 240

purification of sufficient amounts of active principles (with high purity) from 241

chemically-complex crude extracts, such as propolis. The biological effect must be 242

correlated to effector compound and preparative separation methods should be refined 243

to yield the necessary quantity of the agent23

as we did in the present study, attesting 244

that our modification on Odoni et al.10

methodology improved the benefit to extract 245

neovestitol. 246

Therefore, our study confirms the antimicrobial activity of red propolis and 247

isolated compounds. Clearly, neovestitol and vestitol could be potentially antimicrobial 248

agents against infectious diseases (such as dental caries) pending further investigation 249

using in vitro and in vivo biofilm models and elucidation of their mechanism of action. 250

Our data support the hypothesis that the antimicrobial and anti-inflammatory 251

activities of Brazilian red propolis are associated with the presence of at least two 252

bioactive isoflavonoids, neovestitol and vestitol. Interestingly, neovestitol (a major 253

component) displayed better antimicrobial activity than vestitol (a minor component) 254

whereas the latter was an equal anti-inflammatory compound. We are currently further 255

characterizing its anti-inflammatory mechanisms of action as well evaluating their 256

effectiveness using in vitro biofilm systems and in vivo model of dental caries disease. 257

Furthermore, it is plausible that neovestitol and vestitol , as local bucal bioactive agents, 258

can be exploited commercially as they appear to have no cellular toxicity because they 259

have been used as food additives8,9

or nutraceuticals in preventing the oral biofilm 260

disorders dependent. Clearly, neovestitol and vestitol are a nutraceutical since its 261

supported by the definition “A nutraceutical is a product isolated or purified from foods 262

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that is generally sold in medicinal forms not usually associated with food. A 263

nutraceutical is demonstrated to have a physiological benefit or provide protection 264

against chronic disease”.35 265

The isoflavonoids neovestitol (2',4'-dihydroxy-7-methoxyisoflavan) and vestitol 266

(2',7-dihydroxy- 4'metoxyisoflavan), both isolated from Brazilian red propolis, showed 267

in vivo anti-inflammatory and in vitro antimicrobial properties and also, this is the first 268

literature report about anti-inflammatory properties of Brazilian red propolis, neovestitol 269

and vestitol and also, antimicrobial activity of neovestitol. Therefore, BRP is a 270

promising natural source of bioactive compounds such neovestitol and vestitol that both 271

deserve a further investigations to improve the production process, evaluate 272

effectiveness, elucidate the mechanisms of action, and investigate other possible 273

pharmacological and/or nutraceutical properties. 274

Acknowledgements 275

The authors are grateful to Mr. Alessandro Esteves for providing the Brazilian red 276

propolis samples. 277

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characterization of Italian propolis obtained by different harvesting methods. J. Agric. 388

Food Chem. 2012, 60, 2852-62.389

1 This research was supported by FAPESP (#2008/58492-8) and CNPq (CNPq 200174/2009-6).

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Figures Captions 390

Figure 1: Bioassay-guided fractionation focused on identification of antimicrobial 391

and anti-inflammatory compounds. Antimicrobial tests: MIC and MBC against S. 392

mutans, S. sobrinus, S. aureus, and A. naeslundii. Anti-inflammatory test: evaluation of 393

neutrophil migration. 394

* Best antimicrobial and anti-inflammatory activities when compared with others 395

from same line; # Easier to dissolve when compared with others from the same line. 396

397

Figure 2: Chemical Structure of bioactive compounds: A: neovestitol and B: 398

vestitol. 399

400

Figure 3: Recruitment of leukocytes into the peritoneal cavity induced by 401

carrageenan. The neutrophil migration was determined 4 h after injection of carrageenan 402

500 ug/cavity. Mice were previously treated with vehicle (saline), ethanolic extract of 403

propolis (EEP), neovestitol and vestitol, followed by carrageenan injection. The results 404

are expressed as mean ± S.E.M., n = 5-6. The symbol (#) indicates statistical difference 405

compared to saline group. The symbol (*) indicates statistical difference compared to 406

carrageenan group (one-way ANOVA followed by Bonferroni test, p <0.05). 407

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Table 1: Results of Antimicrobial Tests (MIC and MBC) of Isolated Compounds.

Comp. MIC (µg/mL) MBC (µg/mL)

S. mutans S. sobrinus S. aureus A. naeslundii S. mutans S. sobrinus S. aureus A. naeslundii

EEP 100-200 <6.25 100-200 25-50 100-200 50-100 200-400 50-100

Neovestitol 25-50 <6.25 25-50 25-50 50-100 25-50 50-100 50-100

Vestitol 50-100 25-50 50-100 50-100 100-200 200-400 100-200 >1600

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Figure 1

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Figure 2

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Figure 3

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Graphic for table of contents

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