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Phenolic contents, antioxidant and antiacetylcholinesterase properties of honeys from different floral origins

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Maria da Conceição Tavares Cavalcanti Liberato*, Selene Maia de Morais, Sônia Maria Costa Siqueira, Jane Eire Silva Alencar de Menezes, Denise Nogueira Ramos, Lyeghyna Karla Andrade Machado, Islay Lima Magalhães

Curso de Licenciatura em Química, Universidade Estadual do Ceará – UECE, Av. Paranjana 1700, CEP 60740-000, Fortaleza, Ceará, Brasil

Running Title: Phenolic contents and properties of honey

*Corresponding author: Curso de Licenciatura em Química, Universidade Estadual do Ceará – UECE, Av. Paranjana 1700, CEP 60740-000, Fortaleza, Ceará, Brasil Tel: 55 85 3101 9933. Fax number: 55 85 3252 2328. E-mail address: liberato@secrel.com.br

forged on plants of the Northeastern Brazil were analyzed to determine total phenolics content.41 mg/mL) showed better antioxidant activity and presented higher values of total phenols (108.52 mg GAE/100g for L. Keywords: Honey. Free radical scavenging. The antioxidant activity was evaluated using the DPPH scavenging test.27 ± 1. urundeuva). flavonoids content.07 mg/mL) and Myracrodruon urundeuva Fr. Inhibition . All. antioxidant activity and antiacetylcholinesterase activity. Bioactivecompounds. (IC50 4. Honey samples from Lippia sidoides Cham.50 ± 3. The total phenol content was determined by the Folin-Ciocalteu method and the flavonoid content was analyzed by the AlCl3 method. In antiacetylcholinesterase assay several honey samples presented relevant results. (IC50 28. sidoides and 68. Flavonoids.20 ± 1.55 ± 1.01 mg GAE/100g for M. The biological activity of honeys is related to floral origin and medicinal plants constitute a useful resource for the generation of functional foods.2 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 Abstract Twenty-three honey samples of Apis mellifera L.

aromatic carboxylic acids and esters. whereas proteins. asthma. Several studies have indicated that phenolic substances such as flavonoids and phenolic acids. heat and some metals. carotenoids. terpenoid derivatives. Honey has been used by humans since ancient times. there has been an increasing attention on the use of “functional foods. both in traditional medicine as well as for preserving food by retarding deterioration. the most widely studied group is antioxidants. rancidity or discoloration caused by light. The functionality of a food is usually related to some of the ingredients that it contains naturally or additives. are considerably more powerful antioxidants than vitamin C and vitamin E1. Many compounds in this wide assortment of smaller constituents show antioxidant properties. Studies have indicated that honey contains about 200 substances2 and it is the only concentrated sugar form available in the world3. the flower type used in gathering nectar and pollen and on the climatic conditions. including the phenolic compounds that also contribute to the sensory qualities of honey. Honey is one of the most complex mixtures of carbohydrates and other smaller components produced in nature. Sugars represent the largest portion of the composition of honey (95-99% of honey solids).” which can be defined as foods that produces a beneficial effect on one or more physiological functions. aromatic aldehydes. flavonoids and other compounds appear in smaller proportions. Phenolic compounds are one of the most important groups of compounds that occur in plants. infected wounds and skin ulcers4. The composition of honey depends on the honeybee species. Popularly. gastrointestinal problems. honey has been used in the treatment of burns.3 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 1) Introduction In recent years. . Among the known functional ingredients.

including catechic and pyrogallic tannins. acetylcholinesterase inhibitors have demonstrated efficiency in the . which contains two phenolic terpenes.4 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 Honey has many antioxidant properties that make it beneficial to human health by fighting damage caused by oxidizing agents. urundeuva (Anacardiaceae) is native to the Brazilian Northeastern extending up to São Paulo and Mato Grosso do Sul. The effectiveness of this therapy has been demonstrated by studies performed with the essential oil of the leaves. and Myracrodruon urundeuva Fr. An increase in the acetylcholine level should be helpful to combat this disease15. free and glycosylated sterols and organic acids have all been found in organic solvent extracts of the plant leaves10-12. It is related with reduction of acetylcholine (ACh) levels in the cells. Alzheimer disease (AD) has been responsible for 50-60% of total number of cases of diseases among persons above 65 years old. Flavonoids. and other flavonoids with biological activity14. urundeuva have shown the presence of various phenolic compounds. thymol and carvacrol. L. This study investigated several honeys of different floral origins including honey from important Brazilian medicinal plants such as Lippia sidoides Cham. The plant is indicated for use as an antiinflammatory and anti-scarring agent6. sidoides (Verbenaceae) is a shrub native to the semi-arid Brazilian Northeastern that is widely used in folk medicine as an antiseptic6. It is one of the main plants of Brazilian Northeastern traditional medicine. Additionally. (Table 1). as major constituents. Nowadays. It occurs widely in semi-arid and also in dry and subhumid forests13. naphthoquinones. The acetylcholinesterase (AChE) is responsible for reduction of acetylcholine in the nervous synapse. since honey contains both hydrophilic and lipophilic antioxidants. M. dimeric chalcones. All. its compounds can exhibit antioxidant activity in different areas of the cell5. causing the loss of cholinergic neurons. Phytochemical analysis of M. with bactericidal and fungicidal activities7-9.

5 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 clinical treatment of Alzheimer disease. Anacardium occidentale L. Serjania sp. Piptadenia moniliformis Benth. stored in amber flasks and kept at 4-5°C until analysis. and Ziziphus joazeiro Mart. Mimosa verrucosa Benth. Honey samples were collected between July 2007 and April 2009.. Total phenolic content .. Licania rigida Benth.. Spermacoce verticillata L.. Myracrodruon urundeuva Fr. it is important to perform more studies to elucidate the potential of protective activities of honey.. Lippia sidoides Cham. such as AD and cancer16. All samples were transferred to the laboratory.2. including: Hyptis suaveolens Poit.... New natural inhibitors of AChE in foods could be a great deal for treatment of AD. 2) Materials and Methods 2. The use of foods with therapeutic benefits is a growing goal for achieving a healthy lifestyle. The other honey samples were heteroflorals (Table 1). through its antioxidant and antiacetylcholinesterase activities for reducing dietary-related chronic diseases. All. The honey samples were from different botanical sources.1 Honey samples Twenty-three honey samples of Apis mellifera from Brazilian Northeastern forged on different plants were obtained from apiarists and beekeeper associations from collection sites. Therefore. 2.

6 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 The Folin-Ciocalteau method17 was used to determine the total phenolic content of the honeys. and 2 mL of 75 g/L sodium carbonate (Na2CO3) was added. The mean of three readings was used and the total phenolic content was expressed in mg of gallic acid equivalents (GAE)/100 g of honey. The radical scavenging activity of honey in the presence of the stable free . The honey sample (5 g of each) was diluted to 50 mL with distilled water and then filtered. After incubation at room temperature for 2 h. Steinheim.5 mL of this solution was then mixed with 2. The mean of three readings was used and expressed as mg of quercetin equivalents (QE)/100 g of honey.5 mL of Folin-Ciocalteau reagent (Sigma-Aldrich Chemie.02 mg/mL). Germany) was used as a standard to produce the calibration curve. Absorption readings at 415 nm were taken after 10 min against a blank sample consisting of a 5 mL honey solution with 5 mL methanol without AlCl3. 2. The total flavonoid content was determined using a standard curve with quercetin as the standard. Germany) for 5 min. Antioxidant Activity The free antiradical activity of the honey samples was measured using the DPPH method. 2. Gallic acid (Sigma-Aldrich Chemie. A 5 mL aliquot of 2% aluminum chloride (AlCl3) in methanol was mixed with the same volume of a honey solution (0. One aliquot of 0. the absorbance of the reaction mixture was measured at 760 nm against a methanol blank (Biomate Spectrophotometer).4. Total flavonoid content The total flavonoid content was determined using the Dowd method with adaptations18.3. Steinheim.

5 '-dithiobis-[2-nitrobenzoic acid] (DTNB or Ellman's reagent) and 1 mM Acetylcholine iodide (ACTI). Steinheim. The mean of three IC50 (concentration causing 50% inhibition) for each honey sample was determined graphically2. 2.5 mg (Table 2). After 15 min of incubation. Sigma-Aldrich Chemie.5 mL) were applied to TLC plate. 3) Results The total phenolic content of honey samples from Brazilian Northeastern varied from 10.025g/mL) was mixed with 1. 0. Where there is inhibition of the enzyme. and then it was left to rest for 3 min.19. the plate was sprayed with 3 U enzyme / mL. as determined using the standard curve of gallic acid . Ascorbic acid was used as a positive control.5 to 2. The radical scavenging activity was calculated as follows: % Inhibition = [(blank absorbance – sample absorbance)/blank absorbance] x 100. A 1. the absorbance was read at 517 nm against a water/methanol (1:1) blank.5. DCAlufolien. The plate was sprinkled with the solutions: 1 mM of 5.21 to 108.25 mL aliquot of a honey solution (0. After drying. Antiacetylcholinestarase Thin Layer Chromatography Test The antiacetylcholynesterase activity assay was based on the Ellman method adapted by Rhee et al. Silica gel 60 F254.5 mL of a 90 mg/L solution of DPPH in methanol.7 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 radical DPPH (95%. Germany) was determined spectrophotometrically (Biomate Spectrophotometer). a white spot appears. Samples (1.2 mm Merck. Physostigmine was used as control. and after 10 min the yellow color appeared.

25 to 8. genistein. flavonoids. Serjania sp. suaveolens and a heterofloral honey sample (S19) tested in the assay for acetylcholinesterase inhibitors. such as allyl sulfides. The total flavonoid content of the honey samples varied from 0. The IC50 values for ascorbic acid and BHT (a synthetic antioxidant) were 0.72 mg/mL. The highest value for DPPH RSAs was found for L. The IC50 values ranged from 4. The results were displayed in the Table 2. H. sidoides presented smaller antioxidant activity than either of these two known antioxidant compounds. . The results of the analysis of the radical scavenging activity (RSA) of the honey samples are displayed in Table 2. 4) Discussion Functional food is defined as a food or food ingredient that can provide beneficial health effects in addition to the traditional nutrients that it contains21. indoles.2 to 106. catechins. another heterofloral honey sample (S03). The highest values were observed for the monofloral honey samples of L. sidoides. and by a honey sample from flowers of M. urundeuva. Phytochemicals are among the most important functional food components. urundeuva (S08 and S22). A number of phytochemicals.255 mg/mL and 0. The honey samples from flowers of M. followed by that of M. Thus. urundeuva. sidoides followed by heterofloral honey sample (S03). presented inhibition spots with sizes near or identical to those of the standard physostigmine. The heterofloral honey sample (S19) showed the highest value. followed by the honey of the flowers of L. the honey sample of L. rigida.8 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 (R2 = 0.307 mg/mL respectively.9912).9995).38 mg (Table 2) using the quercetin standard curve (R2 = 0.

Total phenolic contents of 10 honey samples of different floral origin from Poland have been shown to vary between 21.5 mg of GAE/100 g honey) are similar to those described for Burkina Faso honey samples2 with values in the range of 32.9 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 limonoids. have been thoroughly studied for their potential to prevent cancer. honey samples in this study. and phytosterols.2 have also described a low correlation (R = 0. However.15) between the total flavonoid and total phenolic content was observed. a low correlation (R = 0. the data observed for Northeastern Brazil honey samples (10. Although different plants present different phenolic compounds and therefore present variations in the total phenolic content21.75 (mg of GAE/100 g honey) using the same method. tocopherols and flavonoids are known to prevent oxidative damage by eliminating free radicals20. Meda et al. while carotenoids.2 have been shown to present a variation range between 0.59 to 114. coumarins and triterpenoids are thought to be inhibitors of tumor initiation.0 to 8. . Honey samples from Yemen have been shown to present phenol content ranging from 75. Honey samples from Chile presented flavonoid content from 0.7 to 75. monoterpenes. As suggested by in vitro studies.13 to 246. Flavonoid content in Burkina Fasan honey samples studied by Meda et al.8 mg QE/100 g of honey22. phenolic acids.21 mg CE/100 g of honey25.014 to 13.8 mg and 241 mg GAE/kg of honey24. presented significant differences with respect to honey samples of Chile with total phenolic content varying from 0.35 mg QE/100 g of honey. terpenes.17 and 8.11) between the total amount of flavonoids and the total amount of phenolic compounds. In the analysis of the Brazilian Northeastern honey samples.21 to 108.3 mg GAE/100 g of honey23 whereas honey samples from Slovenia varied between 44. phenolic compounds.83 mg/100 g of honey22.

13 mg/mL for honey samples.2 mg GAE/kg. necessarily. Liviu et al.9 mg GAE/kg and 215. the antioxidant capacity in honey samples is the result of the combined activity of a wide range of compounds including phenolics. peptides. and possibly other minor components. A linear correlation (R2 = 0. organic acids. Meda et al. urundeuva are due probably to seasonal variation of phenolic compounds: S08 was collected in November (dry period) and S22 in June (rainy period).10 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 A linear correlation was observed (R2 = 0. suggesting that phenolic compounds are directly responsible for the antioxidant properties of honey. The effects of .2 have described values of IC50 varying from 1. Differences in the activities and phenol content of two honey samples of M. Atoui et al. They have used the Folin-Ciocalteau method to determine the total phenolic content and have reported values between 89.583) between the DPPH RSA results and the total phenolic levels of Brazilian Northeastern honey samples.63 to 29. According to Gheldof et al. enzymes. Lachman et al.29.852) has been observed between the total phenolic content and the antioxidant activity.28 have suggested that similar results of phenolic levels do not. suggesting that phenolic compounds correlate better to the RSA of these honeys (Figure 1). Maillard reaction products. correspond to the same antioxidant responses because the amount of phenolics found in the Folin-Ciocalteau assay also depends on their chemical structure. However. They have suggested that the differences depend on the geographic location and period of honey collection.26 have studied 23 honey samples collected in different Romania regions and have confirmed a variation in the antioxidant properties and total phenolic contents in honey samples depending on their botanic or geographic source.27 have analyzed 40 honey samples mainly from Northern Moravia and found a great variation.

Northeastern Brazilian honeys may have therapeutic potential as functional foods due to their antioxidant and antiacetylcholinesterase activities. .11 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 seasonal climate changes in the caatinga biome on tannin levels of M. Free Radical Biol Med 1997. Thus. L. urundeuva are widely used medicinal plants in Northeastern Brazil and honeys from their flowers showed high values of total phenolic content and antioxidant activity. However. sidoides and M. 22: 749-760. the importance of medicinal plants in furnishing nectar for Apis mellifera is often overlooked. as indicated by this study of honey samples from Brazilian Northeastern. urundeuva and Anadenanthera colubrina had been previously reported by Monteiro et al. Sofic E.30 Honey is an ideal energetic food due to its sugar content. Authors Disclosure Statement “No competing financial interests exist” References 1) Cao G. Aknowledgements The authors thank Ceará State Support to Micro and Small Companies Service (SEBRAE) for the honey samples. Prior RL: Antioxidant and prooxidant behavior of flavonoids: structure-activity relationships.

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