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Title: Seasonal effect on chemical composition of green propolis from Minas Gerais (Brazil) over six
years

Article Type: Research Article (max 7,500 words)

Keywords: Green Propolis, Seasonal Effects, Baccharis dracunculifolia, Seasonality.

Corresponding Author: Prof. Sônia Maria de Figueiredo, M.

Corresponding Author's Institution: IEP/SCBH Instituto de Ensino e Pesquisa da Santa Casa de Belo
Horizonte

First Author: Sônia Maria de Figueiredo, M.

Order of Authors: Sônia Maria de Figueiredo, M.; Nancy S Binda, Dr; Bruno M Almeida, graduate; Sheila
R Abreu, graduate; Jose Alexandre S Abreu, graduate; Sidney B Vieira-Filho, Dr; Rachel B caligiorne, Dr

Abstract: Propolis is widely used in folk medicine and as a component of health foods in many parts of
the world. The main objective of this study was to evaluate the seasonal effect on the chemical
composition of propolis from Minas Gerais (Brazil) in a period of six years. The propolis was collected
during the months of February , March, May, July, September and October by six years (2008 to 2013).
The HPLC analysis of the chemical composition of thirteen chemical constituents was statistica lly
evaluated. The relative amounts of the majority of propolis chemical components were similar in most
samples analyzed by HPLC. On the other hand, p-coumaric acid, chrysin, galangin and kaempferide
showed statistically significant differences in the certain months analyses, through those six years. Our
results indicated that green propolis maintained similar and stable characteristics through the six
years of study.
Cover Letter

Belo Horizonte, July, 03 2014

John Van Camp

Food Chemistry

Dear Dr. John Van Camp,

We would like to submit our manuscript entitled “Seasonal effect on chemical

composition of green propolis from Minas Gerais (Brazil) over six years” by S.M.
Figueiredo, N.S. Binda, B.M. Almeida, S.R.L. Abreu, J.A.S. Abreu, S.A.Vieira-Filho
and R.B. Caligiorne to your Journal.
This study evaluates the seasonal effect on the chemical composition of propolis
from Minas Gerais (Brazil) in a period of six years.
The authors have no conflicts of interest to declare and are agree to publish the
data in your journal, as we believe that these findings are within its scope.
We believe that our work will be of great interest to your readers, because this
data will contribute greatly to the advancement of studies about the propolis.

Sincerely,

Sonia Maria de Figueiredo

Instituto de Ensino e Pesquisa da Santa Casa de Belo Horizonte (IEP/SCBH)
Rua Domingos Vieira, 590, Belo Horizonte 30150-240, Minas Gerais, Brazil
smfigue@gmail.com
*Highlights (for review)

Highlights

 The present study aimed to identify some of the chemical compounds of green propolis.

 Investigate the seasonal variation in samples of green propolis though six years.

 The extracts of green propolis from Minas Gerais, Brazil were analyzed by HPLC.

 The results suggest the main chemical constituents of green propolis are very stable.

 The propolis components can be used as functional food, drug and cosmetics products.
*Manuscript
Click here to view linked References

1 Seasonal effect on chemical composition of green propolis from Minas Gerais (Brazil)

2 over six years

3
a,b a c c d
4 S.M. Figueiredo *, N.S. Binda , B.M. Almeida , S.R.L. Abreu , J.A.S. Abreu , S.A. Vieira-
e a
5 Filho , R.B. Caligiorne

6
a
7 Instituto de Ensino e Pesquisa da Santa Casa de Belo Horizonte (IEP/SCBH), rua Domingos

8 Vieira, 590, Belo Horizonte 30150-240, Minas Gerais, Brazil

b
9 Departamento de Alimentos, Escola de Nutrição, Universidade Federal de Ouro Preto ‒

10 Campus Morro do Cruzeiro, Ouro Preto 35400-000, Minas Gerais, Brazil

c
11 Pharmanectar, Rua Pernambuco 1066, Belo Horizonte 30130-151, Minas Gerais, Brazil

d
12 Nectar Pharmaceuticals, MG 435 - 2.5 Km. Caeté, Minas Gerais 34800-000, Brazil;

e
13 DEFAR, Escola de Farmácia, Universidade Federal de Ouro Preto ‒ Campus Morro do
14 Cruzeiro, Ouro Preto 35400-000, Minas Gerais, Brazil

15

16 *Address correspondence to this author at the Núcleo de Pós-Graduação e Pesquisa da

17 Santa Casa de Belo Horizonte, Rua Domingos Vieira, 590, Belo Horizonte, Minas Gerais,

18 Brazil. CEP 30.150-240; Telephone/fax: 55 31 32388677; Mobile: +55 31 8896 4089; E-mail:

19 smfigue@gmail.com

20 Running head: Seazonal effect on green propolis

21

22 Email addresses:

23 SMF: smfigue@gmail.com

24 BMA: brunogarantia@yahoo.com.br

25 SRLA: sheila@pharmanectar.com.br

26 JASA: abreu@pharmanectar.com.br

27 NSB: nancysbinda@yahoo.com.br

28 RBC:rachelbc@santacasabh.org.br

29 SAVF: bibo@ef.ufop.br

1
30 Abstract

31 Propolis is widely used in folk medicine and as a component of health foods in many parts of

32 the world. The main objective of this study was to evaluate the seasonal effect on the chemical

33 composition of propolis from Minas Gerais (Brazil) in a period of six years. The propolis was

34 collected during the months of February, March, May, July, September and October by six years

35 (2008 to 2013). The HPLC analysis of the chemical composition of thirteen chemical

36 constituents was statistically evaluated. The relative amounts of the majority of propolis

37 chemical components were similar in most samples analyzed by HPLC. On the other hand, p-

38 coumaric acid, chrysin, galangin and kaempferide showed statistically significant differences in

39 the certain month’s analyses, through those six years. Our results indicated that green propolis

40 maintained similar and stable characteristics through the six years of study.

41

42 Keywords: Green Propolis, Seasonal Effects, Baccharis dracunculifolia, Seasonality.

2
43 INTRODUCTION

44

45 Propolis, also known as bee glue, is a greenish resinous substance collected by bees,

46 mainly from plants around their habitat (Burdock, 1998). Propolis is a Greek word that comes

47 from the combination of "pro" (preservation or defense) and "polis" (hive or community or

48 cluster) (Toreti, Sato, Pastore, & Park, 2013)

49 According to some authors (Bastos, Santana, Calaca-Costa, & Thiago, 2011), the bees

50 (Apis mellifera) cut the shoot apices of Baccharis dracunlifolia (Rosemary field), and with their

51 paws they create "green acorns" full of glandular and non-glandular trichomes, and fragments of

52 epidermis, which are accumulated in the corbicula, transported to the hive and then used in the

53 production of the propolis called "green"(Park, Fukuda, Ashida, Nishiumi, Guzman, Sato, et al.,

54 2004).

55 The Honeybees (Apis mellifera) use propolis to protect the hive against pests, invasive

56 insects, microorganisms and other contaminants. On its inside insects can be found in perfect

57 condition, involved in propolis, thanks to its antimicrobial action. It is also used to repair open

58 cracks and damages to the hive and in the preparation of sites for aseptic posture of the queen

59 bee (Bankova & Marcucci, 2000; Toreti et al., 2013).

60 The different kinds of propolis in the world, have diverse characteristics depending on

61 the climate, the seasons and its botanical origin. Included in its composition are 55% of resins

62 and balms, 30% wax, 10% pollen and 5% of other substances (Kaškonienė, Kaškonas,

63 Maruška, & Kubilienė, 2014)

64 In Brazil, 13 types of propolis have been cataloged and classified by region, each

65 sample seems to have a different chemical profile, according to their geographical localization

66 (Alencar, Oldoni, Castro, Cabral, Costa-Neto, Cury, et al., 2007; Park, Alencar, & Aguiar, 2002).

67 This variation is easily explained by the great Brazilian biodiversity (Nunes, Galindo, Lustosa,

68 Brasileiro, Egito, Freitas, et al., 2013; Pereira, Nascimento, & Neto, 2002). In addition, some

69 influences as climatic factors and soil quality, among others can change their composition

70 seasonally (Valencia, Alday, Robles-Zepeda, Garibay-Escobar, Galvez-Ruiz, Salas-Reyes, et

71 al., 2012)

3
 72 The Brazilian propolis, internationally known as green propolis, shows its characteristic

73 color due to the high concentration of chlorophyll and its color ranges from greenish yellow to

74 dark brown, having differences in their chemical composition according to their origin, age and

75 harvest time (Bankova et al., 2000; Chang, Pilo-Veloso, Morais, & Nascimento, 2008; Lustosa,

76 Galindo, Nunes, Randau, & Neto, 2008).

77 Propolis can be considered one of the most heterogeneous mixtures found in natural

78 sources. The main chemical constituents are the phenolic compounds: p-coumaric acid,

79 cinnamic acid derivatives, flavonoids, benzoic acid, aliphatic acids and esters (Kasote,

80 Suleman, Chen, Sandasi, Viljoen, & van Vuuren, 2014).

81 The phenolic compounds are characterized by the presence of at least one hydroxyl

82 group attached directly to an aromatic ring (Toreti et al., 2013). According to studies, these

83 substances represented by the flavonoid aglycones, phenolic acids and bioactive esters can

84 prevent the growth and proliferation of pathogenic microorganisms in vivo or in vitro (Banskota,

85 Tezuka, Prasain, Matsushige, Saiki, & Kadota, 1998; Burdock, 1998). In figure 1 it is possible to

86 observe some typical chemical constituents present in green propolis.

87 Propolis is remarkable for both its therapeutic properties resulting from their biological

88 activities as for the possibility of application in the pharmaceutical and food industry as

89 functional foods (Park, Fukuda, Ashida, Nishiumi, Yoshida, Daugsch, et al., 2005; Paulino,

90 Dantas, Bankova, Longhi, Scremin, de Castro, et al., 2003; Sforcin, Fernandes, Lopes,

91 Bankova, & Funari, 2000; Santos, 2012).

92 Several studies have shown that green propolis has various biological activities, such as

93 antibacterial (Alencar et al., 2007; Loureiro & Galbiati, 2013; Sawaya, Souza, Marcucci, Cunha

94 & Shimizu, 2004; Szliszka, Czuba, Domino, Mazur, Zydowicz, & Krol, 2009; Sforcin et al.,

95 2000); antifungal (Murad, Calvi, Soares, Bankova, & Sforcin, 2002); (Araujo, Mattar, Reis,

96 Serra, Fialho, Assuncao, et al., 2011; Szliszka et al., 2009); anti-inflammatory (Park & Ikegaki,

97 1998); antioxidant (Simoes, Gregorio, Da Silva, de Souza, Azzolini, Bastos, et al., 2004),

98 immunomodulatory (Araujo, et al., 2011; Kasote et al., 2014; Szliszka et al., 2009), antitumor

99 (Bazo, Rodrigues, Sforcin, de Camargo, Ribeiro, & Salvadori, 2002) and antiulcer (de Barros,

100 Lemos, Maistro, Leite, Sousa, Bastos, et al., 2008).

4
101 Considering the wide variety of bioactive compounds in propolis, the present study

102 aimed to identify some of the chemical compounds of pharmacological importance and

103 investigate the seasonal variation in samples of extracts of green propolis collected in Minas

104 Gerais state, during four seasons in a six-year study (2008 -2013).

105

106

107 MATERIALS AND METHODS

108

109 To study the seasonality, the data of HPLC analyzes were collected from the data base

110 of Nectar Pharmaceuticals Company, for the months from February to October, between the

111 years 2008 - 2013. Analyses are routinely performed by the Quality Control Division of the

112 company, always immediately after collection and processing of propolis in natura.

113 Ethanol extracts of green propolis are subjected to analysis by HPLC with UV detection

114 for the determination of the qualitative profile and quantification of major compounds, according

115 to previous studies (Park et al., 2002).

116 For comparison between the levels of the propolis compounds the readings that

117 corresponded to the four seasons of the year were considered (spring, summer, autumn and

118 winter).

119 After checking for normality (Kolmogorov-Smirnov test) and homogeneity of variances

120 (Bartlett’s test), the intergroup variation of different parameters were estimated by the analysis

121 of variance (ANOVA). These ANOVA analyses were then completed by Newman Keuls multiple

122 range test, in order to locate the differences. Thus, qualitative treatments were compared by

123 Newman Keuls test, with a confidence interval of 95% and significance level of 5% (p < 0.05)

124 for comparative studies among populations. It was also considered the confidence interval of

125 99% and significance level of 1% (P < 0.01) for comparison among months and considering

126 each population as well. Statistics calculations, as well as graphic representation were prepared

127 by using Graph Pad Prim (v. 5.0).

128

129

130 RESULTS

5
131

132 The HPLC method used for this study allowed the analysis of thirteen chemical

133 constituents that are present in green propolis extract, showing that there is no variations in

134 concentration of most of the constituents through the six years of study (2008 - 2013) as well as

135 between different months each year. The concentrations of the compounds identified in this

136 analysis are shown in Table 1.

137 The average values of the chemical constituents concentrations during the months of

138 the study, ie., February, March, May, July, September and October, through the six years, were:

139 artepillin C (81.36 ± 13.45 mg/g); rutin (16.87 ± 9.36 mg/g); pinocembrin (15.28 ± 10.45 mg/g);

140 pinobancksin-3-acetate (6.86 ± 2.28 mg/g); kaempferide (8.60 ± 2.1 mg/g), pinobanksin (6.86 ±

141 2.28 mg/g), p-coumaric acid (3.91 ± 0.86 mg/g); quercetin (2.69 ± 1.06 mg/g); Chrysin (2.15 ±

142 0.45 mg/g); galangin (1.95 ± 0.63 mg/g); apigenin (1.74 ± 0.53 mg/g); tectochrysin (1.65 ± 0.82

143 mg/g) and kaempferol (0.81 ± 0.33 mg/g).

144 In this study, it was observed that after quantification of the thirteen components, the

145 major constituent was artepillin C (81.36 ± 13.45 mg/g). Considering the six years of study, the

146 concentration of this compound compared to the other was relatively high.

147 Among the compounds analyzed, four of them showed statistically significant seasonal

148 differences: p-coumaric acid (3.91 ± 0.86 mg/g); chrysin, (2.15 ± 0, 45 mg/g); galangin (1.95 ±

149 0.63 mg/g) and kaempferide (8.60 ± 2.1 mg/g) (Figure 2).

150 There was a statistically significant difference (p < 0.05) in the concentration of p-

151 coumarin acid between February and July (3.10 ± 0.20mg/g and 4.39 ± 0.61mg/g).The same

152 was observed to chrysin (p < 0.05) between March and October (1.50 ± 0.30 mg/g and 2.43 ±

153 0.14 mg/g) and between March and July (1.50 ± 0.30 mg/g and 2.51 g ± 0.23 mg/g).

154 Additionally, there was statistically significant difference (p < 0.05) in kaempferide

155 concentrations between February and the months of March (11.66 ± 1.90 mg/g and 6.61 ± 0.55

156 mg/g) and September (11.66 ± 1.90 mg/g and 7.45 ± 0.76mg/g). The galangin concentrations

157 showed differences (p < 0.05) between February and September (2.41 ± 0.40 mg/g and 0.92 ±

158 0.26 mg/g) and May and September (2.39 ± 0.21 mg/g and 0.92 ± 0.26 mg/g). The graphs of

159 the concentration of the chemical constituents of green propolis are shown in figure 2 (a, b, c,

160 d).

6
161

162 Despite the differences on the compound concentrations in some months, as mentioned

163 above, it did not alter the total amount of flavonoids. Also the flavonoids showed low standard

164 deviation compared to the other chemical constituents analyzed indicating that the

165 concentrations tend to be near average. The graphs of the green propolis chemical constituents

166 concentration, which showed no statistically significant difference, are shown in figure 3 (a, b, c,

167 d, e, f, g, h, i, j). The Table 1 presents the mean and standard error of the mean of the thirteen

168 chemical constituents and total flavonoids found in extracts of propolis evaluated in the six

169 months during the years 2008 to 2013.

170

171

172 DISCUSSION

173

174 The HPLC method is an important analysis tool to assess the influence of seasonality in

175 the chemical compounds concentration. This study demonstrated the high quality of Brazilian

176 green propolis ethanolic extract, which has been considered the “gold standard” when

177 compared to other propolis from several countries (Kasote et al., 2014). The most biologically

178 active fraction of propolis are flavonoids and some authors reported that some of the biological

179 properties of the ethanol extract of green propolis, particularly the antioxidant activity, are due to

180 its high content of flavonoids. This suggest that the flavonoids have an important role in

181 antioxidant activity in extracts of Brazilian green propolis, but that other factors may be involved

182 (Toreti et al., 2013; Nunes, Galindo, de Deus, Rufino, Randau, Xavier, et al., 2009).

183 The flavonoids and cinamic acids are important markers to assess the quality of

184 Brazilian green propolis. According to the results, it is significant that the total flavonoid

185 concentration, evaluated by HPLC, was stable during the six years analyzed (Table 1). This

186 suggests that the decrease in some biologically active components may be balanced by others,

187 in agreement with other authors findings (de Oliveira, Lima, Munari, Bastos, Filho, & Tavares,

188 2014; Kumazakia, Shinoharaa, Taniguchia, Yamada, Ohta, Ichihara, et al., 2014).

189 In a study realized by Sforcin, it was demonstrated that different extracts of Brazilian

190 propolis collected in different seasons of the year were not able to change the values of

7
191 minimum inhibitory concentration on Candida spp (Sforcin et al., 2000). In another study

192 realized by the same group, it was observed that the effect of propolis on antibody production

193 occurs regardless of time and geographical origin (Sforcin, Orsi, & Bankova, 2005).

194 It this work there was an apparent increase in all chemical components concentration in

195 the months of May and July, but it was not statistically significant (p>0.05). This little variation

196 suggests that due the low relative humidity of the dry season there is a consequent

197 concentration of the compounds in the extracted propolis.

198 Overall, our results indicate that green propolis extracts did not show large seasonal

199 variations, maintaining the chemical characteristics throughout the years. This observation is in

200 agreement with the study of Valencia et al. which showed that seasonality does not significantly

201 change the chemical composition of propolis (Valencia et al., 2012).

202 The spectrum of these compounds showed a very similar mean over the six years of

203 study. However, there were statistically significant variations (p<0.05) in the concentrations the

204 compounds p-coumaric acid, chrysin, galangin and kaempferide (Figure 2).

205 It is already observed in several studies that the biological activities of green propolis

206 are similar even when being collected in different months or seasons (Araujo et al., 2011;

207 Loureiro et al., 2013; Missima et al., 2007; Nunes et al., 2009; Szliszka et al., 2009). Some

208 authors have described the biological activity of galangin and that this compound is capable of

209 stimulating enzymes like the P450 isoforms or to act in synergism even at lower concentrations.

210 Despite being found in varying quantities throughout the year, it is believed that the

211 pharmacological properties of propolis were not changed (Borrelli, Izzo, Di Carlo, Maffia, Russo,

212 Maiello, et al., 2002; Borrelli, Maffia, Pinto, Ianaro, Russo, Capasso, et al., 2002; Russo, Longo,

213 & Vanella, 2002).

214 The anti-inflammatory capacity can be allocated to chrysin and Kaempferol as well as a

215 derivative of Kaempferide (Santos, 2012). The cinnamic acid, artepillin C, coumarin acid,

216 pinocembrim may be involved with this function too (Toreti et al., 2013). Thus, we can infer that

217 the difference in concentration found in chrysin and kaempferol would not alter the anti-

218 inflammatory property of propolis, because other compounds will be involved in this activity,

219 preserving the anti-inflammatory capacity of the propolis.

8
220 In the same manner, the significantly differences in the Kaempferide concentration,

221 seems to be related with antifungal activity, do not alter this property of propolis, because others

222 compounds, like Pinobanskin-3-acetate, isosakuranetin, ferulic and caffeic acid, also present

223 antifungal activity (Santos, 2012), maintaining this activity of the propolis.

224 The pinocembrin components, derivatives of caffeic acid and chrysin have antimicrobial

225 activity (Jug, Koncic, & Kosalec, 2014). Also, various constituents in propolis (eg. flavonoids,

226 ferulate and caffeate esters, prenylated coumaric acid and benzophenone derivatives,

227 diterpenic acids) have antimicrobial activity. On the other hand, Toreti et al. (2013) reported that

228 caffeic acid, p-coumaric acid, ferulic acid, rutin glycosylated flavonols, quercetin and isoquercitin

229 exert antioxidant activity.

230 All compounds evaluated, beside with flavonoids, are responsible for various

231 pharmacological activities previously described for propolis, highlighting the major constituent,

232 the artepillin C (marker of ethanol extract of propolis), this constituent is responsible for about

233 80% to 93% of the concentration in relation to the other twelve compounds evaluated during the

234 six years of this research (Table 1). The artepillin C presents several proven activities, such as

235 antioxidant and antitumor (Valencia, et al., 2012; Bankova, 2005; Sforcin et al., 2000). Studies

236 suggest that it could be linked to multiple targets with various substances acting in synergy

237 (Bankova, M. Popova, & B. Trusheva, 2014).

238 Artepilin C showed genotoxic and chemopreventive activity because it affects cancer

239 cells by inhibiting cell growth, exhibits potent cytocidal effects and induces marked levels of

240 apoptosis (de Oliveiraet al., 2014; Kumazakia, et al., 2014). In this study there are no

241 differences in the artepilin C concentrations though the six years analyzed, indicating be an

242 important markers to the propolis quality control.

243 According to Szliszka, Zydowicz, Mizgala & Krol (2012), artepillin C possesses

244 antioxidant, antimicrobial, anti-inflammatory, antigenotoxic, anti-angiogenic and anticancer

245 properties through the modulation of tumour necrosis factor related apoptosis-inducing ligand

246 (TRAIL)-mediated apoptotis signalling pathways. The structure of this hydroxycinnamic acid

247 derivative is presented in Figure 1. Akao, Maruyama, Matsumoto, Ohguchi, Nishizawa,

248 Sakamoto, Araki, Mishima & Nozawa, 2003 also demonstrated the suppression of tumour cell

9
249 growth by artepillin C and two other cinnamic acid derivatives detected in propolis, drupanin and

250 baccharin.

251 According to the review of Santos (2012), the biological activities of isolated propolis

252 substances do not have the same effect as when they are combined in the extract. Together,

253 these propolis constituents have a much greater activity due to the complexity of its composition

254 and the synergistic effect that occurs between its compounds (de Oliveira et al., 2014).

255 The data on chemical composition of extracts of Brazilian propolis and seasonal

256 variations of its main constituents gave us useful information about the origin of the plant and

257 the quality of the investigated propolis extracts. Comparing the results obtained in this study

258 with the literature data, we can infer that the plant source of the samples from the ethanol

259 extracts of propolis analyzed is Baccharis dracunculifolia, since chemical markers founded in

260 this study are the same as those mentioned for this plant ( de Oliveira et al., 2014; Park et al.,

261 2002).

262 Additionally, some authors suggest that B. dracunculifolia the main botanical source of

263 Brazilian green propolis, exhibited in most analyzes similar levels of compounds when

264 compared with the green propolis (Bankova, Boudourova-Krasteva, Sforcin, Frete, Kujumgiev,

265 Maimoni-Rodella, et al., 1999; Park et al., 2002).

266 Studies that examine the effect of seasonality on chemical profile and biological

267 activities of ethanol extracts of propolis are very important for the standardization of the raw

268 material. Such studies provide information not only to the definition of the best regions for the

269 production of green propolis extracts, but also to the selection of the best periods for collection

270 in apiaries (Bankova, 2005). Therefore, green propolis has potential application for the

271 development of products because it keeps the markers pattern over the years.

272 Bankova et al. (2014) investigated seasonal variations in the composition of volatile oils

273 of green propolis from Minas Gerais state, this group perceived that there were no significant

274 variations, and that those were mostly qualitative, they also report that honeybees can be

275 attracted by volatile components of plants (aroma). These data are very similar to those found in

276 the present study using the green propolis.

277 To obtain a extract with good quality it is required to develop a standardized and

278 validated analytical method, so that the concentration of each metabolite of interest can be well

10
279 determined (Souza, Tacon, Correia, Bastos, & Freitas, 2007). Since the propolis has a complex

280 mixture of different classes of secondary compounds, which can vary depending on local

281 vegetation and biology of bees, technique used for the production as type of collectors, and

282 analysis, as the eluent choice, are important tools for the development of the extraction process

283 and formulation of the final products. These factors exert an enormous importance in physical,

284 chemical and biological properties. This is the main challenge to the use of propolis as a

285 functional food and drug.

286 Understanding how to obtain this high quality standardized extract offsetting the

287 chemical composition of the crude extract of green propolis by bees in B. dracunculifolia, its

288 main botanical source, is something yet to be studied.

289 These results suggest that the main chemical constituents of Brazilian green propolis

290 are very stable and also indicate that its main botanical source was available during all four

291 seasons during the six years of the study, allowing its use as a functional food. Future studies

292 "in vivo" to evaluate the biological activity of propolis components in humans should be

293 conducted to supplement the still deficient information about the propolis as functional a food,

294 drugs and cosmetics products.

295

296 Acknowledgments

297 This work was supported by the National Council for Research and Development -

298 CNPq and Research Foundation of the State of Minas Gerais – FAPEMIG.

299

300 Conflicts of interest:

301 The authors have no conflicts of interest to declare.

11
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303

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331 Bazo, A. P., Rodrigues, M. A. M., Sforcin, J. M., de Camargo, J. L. V., Ribeiro, L. R., &

332 Salvadori, D. M. F. (2002). Protective action of propolis on the rat colon carcinogenesis.

333 Teratogenesis Carcinogenesis and Mutagenesis, 22(3), 183-194.

334 Borrelli, F., Izzo, A. A., Di Carlo, G., Maffia, P., Russo, A., Maiello, F. M., Capasso, F., &

335 Mascolo, N. (2002). Effect of a propolis extract and caffeic acid phenethyl ester on

336 formation of aberrant crypt foci and tumors in the rat colon. Fitoterapia, 73, S38-S43.

337 Borrelli, F., Maffia, P., Pinto, L., Ianaro, A., Russo, A., Capasso, F., & Ialenti, A. (2002).

338 Phytochemical compounds involved in the anti-inflammatory effect of propolis extract.

339 Fitoterapia, 73, S53-S63.

340 Burdock, G. A. (1998). Review of the biological properties and toxicity of bee propolis (propolis).

341 Food and Chemical Toxicology, 36(4), 347-363.

342 Chang, R., Pilo-Veloso, D., Morais, S. A. L., & Nascimento, E. A. (2008). Analysis of a Brazilian

343 green propolis from Baccharis dracunculifolia by HPLC-APCI-MS and GC-MS. Revista

344 Brasileira De Farmacognosia-Brazilian Journal of Pharmacognosy, 18(4), 549-556.

345 de Barros, M. P., Lemos, M., Maistro, E. L., Leite, M. F., Sousa, J. P. B., Bastos, J. K., & de

346 Andrade, S. F. (2008). Evaluation of antiulcer activity of the main phenolic acids found

347 in Brazilian Green Propolis. Journal of Ethnopharmacology, 120(3), 372-377.

348 de Oliveira, P. F., Lima, I. M. S., Munari, C. C., Bastos, J. K., Filho, A. A. S., & Tavares, D. C.

349 (2014). Comparative Evaluation of Antiproliferative Effects of Brazilian Green Propolis,

350 Its Main Source Baccharis dracunculifolia, and Their Major Constituents Artepillin C and

351 Baccharin. Planta Med, 80, 490–492.

352 Jug, M., Koncic, M. Z., & Kosalec, Ivan. (2014). Modulation of antioxidant, chelating and

353 antimicrobial activity of poplar chemo-type propolis by extraction procures. LWT-Food

354 Science and Technology, 57, 530-537.

355 Kaškonienė, V., Kaškonas, P., Maruška, A., & Kubilienė, L. (2014). Chemometric analysis of

356 volatiles of propolis from different regions using static headspace GC-MS. Central

357 European Journal of Chemistry, 12(6), 736-746.

358 Kasote, D., Suleman, T., Chen, W., Sandasi, M., Viljoen, A., & van Vuuren, S. (2014). Chemical

359 profiling and chemometric analysis of South African propolis. Biochemical Systematics

360 and Ecology, 55(0), 156-163.

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361 Kumazakia, M., Shinoharaa, H., Taniguchia, K., Yamada, N., Ohta, S., Ichihara, K., & Akao, Y.

362 (2014). Propolis cinnamic acid derivatives induce apoptosis through both extrinsic and

363 intrinsic apoptosis signaling pathways and modulate of miRNA expression.

364 Phytomedicine, 21, 1070–1077.

365 Loureiro, E. M., & Galbiati, C. (2013). Evaluation of the influence of seasonality and landscape

366 on the physicochemical characteristics of propolis. Food Science and Technology

367 (Campinas), 33(4), 790-795.

368 Lustosa, S. R., Galindo, A. B., Nunes, L. C. C., Randau, K. P., & Neto, P. J. R. (2008). Propolis:

369 updates on chemistry and pharmacology. Revista Brasileira De Farmacognosia-

370 Brazilian Journal of Pharmacognosy, 18(3), 447-454.

371 Missima, F., da Silva, A. A., Nunes, G. A., Bueno, P. C. P., de Sousa, J. P. B., Bastos, J. K., &

372 Sforcin, J. M. (2007). Effect of Baccharis dracunculifolia DC (Asteraeeae) extracts and

373 its isolated compounds on macrophage activation. Journal of Pharmacy and

374 Pharmacology, 59(3), 463-468.

375 Murad, J. M., Calvi, S. A., Soares, A. M. V. C., Bankova, V., & Sforcin, J. M. (2002). Effects of

376 propolis from Brazil and Bulgaria on fungicidal activity of macrophages against

377 Paracoccidioides brasiliensis. Journal of Ethnopharmacology, 79(3), 331-334.

378 Nunes, L. C. C., Galindo, A. B., de Deus, A. D. O., Rufino, D. A., Randau, K. P., Xavier, H. S.,

379 Cito, A. M. D. L., & Neto, P. J. R. (2009). "Seasonal variability of red propolis

380 constituents and brine shrimp bioassay". Revista Brasileira De Farmacognosia-Brazilian

381 Journal of Pharmacognosy, 19(2B), 524-529.

382 Park, Y. K., Alencar, S. M., & Aguiar, C. L. (2002). Botanical origin and chemical composition of

383 Brazilian propolis. Journal of Agricultural and Food Chemistry, 50(9), 2502-2506.

384 Park, Y. K., Fukuda, I., Ashida, H., Nishiumi, S., Guzman, J. P., Sato, H. H., & Pastore, G. A.

385 (2004). Suppression of dioxin mediated aryl hydrocarbon receptor transformation by

386 ethanolic extracts of propolis. Bioscience Biotechnology and Biochemistry, 68(4), 935-

387 938.

388 Park, Y. K., Fukuda, I., Ashida, H., Nishiumi, S., Yoshida, K. I., Daugsch, A., Sato, H. H., &

389 Pastore, G. M. (2005). Suppressive effects of ethanolic extracts from propolis and its

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390 main botanical origin on dioxin toxicity. Journal of Agricultural and Food Chemistry,

391 53(26), 10306-10309.

392 Park, Y. K., & Ikegaki, M. (1998). Preparation of water and ethanolic extracts of propolis and

393 evaluation of the preparations. Bioscience Biotechnology and Biochemistry, 62(11),

394 2230-2232.

395 Paulino, N., Dantas, A. P., Bankova, V., Longhi, D. T., Scremin, A., de Castro, S. L., & Calixto,

396 J. B. (2003). Bulgarian propolis induces analgesic and anti-inflammatory effects in mice

397 and inhibits in vitro contraction of airway smooth muscle. Journal of Pharmacological

398 Sciences, 93(3), 307-313.

399 Pereira, A. S., Nascimento, E. A., & Neto, F. R. D. (2002). Lupeol alkanoates in Brazilian

400 propolis. Zeitschrift Fur Naturforschung C-a Journal of Biosciences, 57(7-8), 721-726.

401 Pereira, O. R., Macias, R. I. R., Perez, M. J., Marin, J. J. G., & Cardoso, S. M. (2013).

402 Portuguese propolis enriched phenolic extract: reactive oxygen scavenging and

403 cytoprotective activities. European Journal of Clinical Investigation, 43, 63-63.

404 Russo, A., Longo, R., & Vanella, A. (2002). Antioxidant activity of propolis: role of caffeic acid

405 phenethyl ester and galangin. Fitoterapia, 73, S21-S29.

406 Santos, V. R. (2012). Propolis: Alternative Medicine for the Treatment of Oral Microbial

407 Diseases. Alternative Medicine, 1(DOI: 10.5772/46204), 38.

408 Sforcin, J. M., Fernandes, A., Lopes, C. A. M., Bankova, V., & Funari, S. R. C. (2000). Seasonal

409 effect on Brazilian propolis antibacterial activity. Journal of Ethnopharmacology, 73(1-2),

410 243-249.

411 Sforcin, J. M., Orsi, R. O., & Bankova, V. (2005). Effect of propolis, some isolated compounds

412 and its source plant on antibody production. Journal of Ethnopharmacology, 98(3), 301-

413 305.

414 Simoes, L. M. C., Gregorio, L. E., Da Silva, A. A., de Souza, M. L., Azzolini, A. E. C. S., Bastos,

415 J. K., & Lucisano-Valim, Y. M. (2004). Effect of Brazilian green propolis on the

416 production of reactive oxygen species by stimulated neutrophils. Journal of

417 Ethnopharmacology, 94(1), 59-65.

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418 Souza, J. P. B., Tacon, L. A., Correia, C. C., Bastos, J. K., & Freitas, L. A. P. (2007). Spray-

419 dried propolis extract, II: Prenylated components of green propolis. Pharmazie, 62(7),

420 488-492.

421 Szliszka, E., Czuba, Z. P., Domino, M., Mazur, B., Zydowicz, G., & Krol, W. (2009). Ethanolic

422 Extract of Propolis (EEP) Enhances the Apoptosis-Inducing Potential of TRAIL in

423 Cancer Cells. Molecules, 14(2), 738-754.

424 Szliszka, E., Zydowicz, G. Mizgala, E. & Krol, W. (2012). Artepillin C (3,5-diprenyl-4-

425 hydroxycinnamic acid) sensitizesLNCaP prostate cancer cells to TRAIL-induced

426 apoptosis. International Journal Of Oncology, 41, 818-828.

427 Toreti, V. C., Sato, H. H., Pastore, G. M., & Park, Y. K. (2013). Recent Progress of Propolis for

428 Its Biological and Chemical Compositions and Its Botanical Origin. Evidence-Based

429 Complementary and Alternative Medicine,13 (1) 1-14.

430 Valencia, D., Alday, E., Robles-Zepeda, R., Garibay-Escobar, A., Galvez-Ruiz, J. C., Salas-

431 Reyes, M., Jiménez-Estrada, M., Velazquez-Contreras, E., Hernandez, J., & Velazquez,

432 C. (2012). Seasonal effect on chemical composition and biological activities of Sonoran

433 propolis. Food Chemistry, 131, 645–651.

434

435

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436 Captions of the Figures and Table:

437

438 Figure 1: Chemical structures of compounds isolated from green propolis.

439

440 Figure 2: Graphs of the concentration of the chemical constituents of green propolis: (a)

441 Kaempferide; (b) galangine; (c) Chrysin; (d) p-coumarin.

442

443

444 Figure 3: Graphs of the concentration of the chemical constituents of green propolis: (a)total

445 amount of flavonoids; (b)artepillin C; (c)quercetin; (d)apigenin; (e)tectochrysin; (f)rutin;

446 (g)pinobanksin; (h)kaempferol; (i)pinocembrin; (j)pinobancksin-3-acetate.

447

448

449 Table 1: Mean and standard error of the mean of the thirteen chemical constituents and total

450 flavonoids found in extracts of propolis evaluated in six months during the years 2008 to 2013.

451

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Figure(s)
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Figure(s)
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Table(s)
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