3 - Cap+tulo I - PHENOLIC CONTENTS, ANTIOXIDANT AND ANTIACETYLCHOLINESTERASE PROPERTIES OF HONEYS

7/31/2019 3 - Cap+tulo I - PHENOLIC CONTENTS, ANTIOXIDANT AND ANTIACETYLCHOLINESTERASE PROPERTIES OF HONEYS

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1) Introduction5253

In recent years, there has been an increasing attention on the use of functional54

foods, which can be defined as foods that produces a beneficial effect on one or more55

physiological functions. The functionality of a food is usually related to some of the56

ingredients that it contains naturally or additives. Among the known functional57

ingredients, the most widely studied group is antioxidants. Phenolic compounds are one58

of the most important groups of compounds that occur in plants. Several studies have59

indicated that phenolic substances such as flavonoids and phenolic acids, are60

considerably more powerful antioxidants than vitamin C and vitamin E1.61

Honey has been used by humans since ancient times, both in traditional medicine as62

well as for preserving food by retarding deterioration, rancidity or discoloration caused63

by light, heat and some metals. Honey is one of the most complex mixtures of64

carbohydrates and other smaller components produced in nature. Studies have indicated65

that honey contains about 200 substances2 and it is the only concentrated sugar form66

available in the world3. Popularly, honey has been used in the treatment of burns,67

gastrointestinal problems, asthma, infected wounds and skin ulcers4.68

The composition of honey depends on the honeybee species, the flower type used in69

gathering nectar and pollen and on the climatic conditions. Sugars represent the largest70

portion of the composition of honey (95-99% of honey solids), whereas proteins,71

aromatic aldehydes, aromatic carboxylic acids and esters, carotenoids, terpenoid72

derivatives, flavonoids and other compounds appear in smaller proportions. Many73

compounds in this wide assortment of smaller constituents show antioxidant properties,74

including the phenolic compounds that also contribute to the sensory qualities of honey.75


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Honey has many antioxidant properties that make it beneficial to human health by76

fighting damage caused by oxidizing agents. Additionally, since honey contains both77

hydrophilic and lipophilic antioxidants, its compounds can exhibit antioxidant activity78

in different areas of the cell5. This study investigated several honeys of different floral79

origins including honey from important Brazilian medicinal plants such as Lippia80

sidoides Cham. and Myracrodruon urundeuva Fr. All. (Table 1). L. sidoides81

(Verbenaceae) is a shrub native to the semi-arid Brazilian Northeastern that is widely82

used in folk medicine as an antiseptic6. The effectiveness of this therapy has been83

demonstrated by studies performed with the essential oil of the leaves, which contains84

two phenolic terpenes, thymol and carvacrol, as major constituents, with bactericidal85

and fungicidal activities7-9. Flavonoids, naphthoquinones, free and glycosylated sterols86

and organic acidshave all beenfound in organic solvent extractsof the plantleaves10-12.87

M. urundeuva (Anacardiaceae) is native to the Brazilian Northeastern extending up to88

So Paulo and Mato Grosso do Sul. It occurs widely in semi-arid and also in dry and89

subhumid forests13. It is one of the main plants of Brazilian Northeastern traditional90

medicine. Phytochemical analysis ofM. urundeuva have shown the presence of various91

phenolic compounds, including catechic and pyrogallic tannins, dimeric chalcones, and92

other flavonoids with biological activity14. The plant is indicated for use as an anti-93

inflammatory and anti-scarring agent6.94

Alzheimer disease (AD) has been responsible for 50-60% of total number of cases95

of diseases among persons above 65 years old. It is related with reduction of96

acetylcholine (ACh) levels in the cells. The acetylcholinesterase (AChE) is responsible97

for reduction of acetylcholine in the nervous synapse, causing the loss of cholinergic98

neurons. An increase in the acetylcholine level should be helpful to combat this99

disease15

. Nowadays, acetylcholinesterase inhibitors have demonstrated efficiency in the100


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clinical treatment of Alzheimer disease. New natural inhibitors of AChE in foods could101

be a great deal for treatment of AD. The use of foods with therapeutic benefits is a102

growing goal for achieving a healthy lifestyle. Therefore, it is important to perform103

more studies to elucidate the potential of protective activities of honey, through its104

antioxidant and antiacetylcholinesterase activities for reducing dietary-related chronic105

diseases, such as AD and cancer16.106

107

108

2) Materials and Methods109110

2.1 Honey samples111

112

Twenty-three honey samples ofApis mellifera from Brazilian Northeastern forged on113

different plants were obtained from apiarists and beekeeper associations from collection114

sites. The honey samples were from different botanical sources, including: Hyptis115

suaveolens Poit., Anacardium occidentale L., Spermacoce verticillata L., Mimosa116

verrucosa Benth., Piptadenia moniliformis Benth., Myracrodruon urundeuva Fr. All.,117

Licania rigida Benth., Lippia sidoides Cham.,Serjania sp, and Ziziphus joazeiro Mart.118

The other honey samples were heteroflorals (Table 1). Honey samples were collected119

between July 2007 and April 2009. All samples were transferred to the laboratory, stored120

in amber flasks and kept at 4-5C until analysis.121

122

2.2. Total phenolic content123

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The Folin-Ciocalteau method17 was used to determine the total phenolic content of125

the honeys. The honey sample (5 g of each) was diluted to 50 mL with distilled water126

and then filtered. One aliquot of 0.5 mL of this solution was then mixed with 2.5 mL of127

Folin-Ciocalteau reagent (Sigma-Aldrich Chemie, Steinheim, Germany) for 5 min, and128

2 mL of 75 g/L sodium carbonate (Na2CO3) was added. After incubation at room129

temperature for 2 h, the absorbance of the reaction mixture was measured at 760 nm130

against a methanol blank (Biomate Spectrophotometer). Gallic acid (Sigma-Aldrich131

Chemie, Steinheim, Germany) was used as a standard to produce the calibration curve.132

The mean of three readings was used and the total phenolic content was expressed in133

mg of gallic acid equivalents (GAE)/100 g of honey.134

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2.3. Total flavonoid content136

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The total flavonoid content was determined using the Dowd method with138

adaptations18. A 5 mL aliquot of 2% aluminum chloride (AlCl3) in methanol was mixed139

with the same volume of a honey solution (0.02 mg/mL). Absorption readings at 415140

nm were taken after 10 min against a blank sample consisting of a 5 mL honey solution141

with 5 mL methanol without AlCl3. The total flavonoid content was determined using a142

standard curve with quercetin as the standard. The mean of three readings was used and143

expressed as mg of quercetin equivalents (QE)/100 g of honey.144

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2.4. Antioxidant Activity146

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The free antiradical activity of the honey samples was measured using the DPPH148

method. The radical scavenging activity of honey in the presence of the stable free149


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radical DPPH (95%, Sigma-Aldrich Chemie, Steinheim, Germany) was determined150

spectrophotometrically (Biomate Spectrophotometer). A 1.25 mL aliquot of a honey151

solution (0.025g/mL) was mixed with 1.5 mL of a 90 mg/L solution of DPPH in152

methanol. After 15 min of incubation, the absorbance was read at 517 nm against a153

water/methanol (1:1) blank. Ascorbic acid was used as a positive control. The radical154

scavenging activity was calculated as follows: % Inhibition = [(blank absorbance 155

sample absorbance)/blank absorbance] x 100. The mean of three IC50 (concentration156

causing 50% inhibition) for each honey sample was determined graphically2.157

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2.5. Antiacetylcholinestarase Thin Layer Chromatography Test159

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The antiacetylcholynesterase activity assay was based on the Ellman method161

adapted by Rhee et al.19. Samples (1.5 to 2.5 mL) were applied to TLC plate, DC-162

Alufolien, Silica gel 60 F254, 0.2 mm Merck. The plate was sprinkled with the163

solutions: 1 mM of 5,5 '-dithiobis-[2-nitrobenzoic acid] (DTNB or Ellman's reagent)164

and 1 mM Acetylcholine iodide (ACTI), and then it was left to rest for 3 min. After165

drying, the plate was sprayed with 3 U enzyme / mL, and after 10 min the yellow color166

appeared. Where there is inhibition of the enzyme, a white spot appears. Physostigmine167

was used as control.168

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3) Results171

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The total phenolic content of honey samples from Brazilian Northeastern varied173

from 10.21 to 108.5 mg (Table 2), as determined using the standard curve of gallic acid174


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(R2 = 0.9912). The highest values were observed for the monofloral honey samples ofL.175

sidoides, followed by that ofM. urundeuva. The total flavonoid content of the honey176

samples varied from 0.25 to 8.38 mg (Table 2) using the quercetin standard curve (R2 =177

0.9995). The heterofloral honey sample (S19) showed the highest value, followed by the178

honey of the flowers ofL. rigida, another heterofloral honey sample (S03), and by a179

honey sample from flowers ofM. urundeuva.180

The results of the analysis of the radical scavenging activity (RSA) of the honey181

samples are displayed in Table 2.The IC50 values ranged from 4.2 to 106.72 mg/mL.182

The highest value for DPPH RSAs was found forL. sidoides followed by heterofloral183

honey sample (S03). The IC50 values for ascorbic acid and BHT (a synthetic184

antioxidant) were 0.255 mg/mL and 0.307 mg/mL respectively. Thus, the honey sample185

ofL. sidoides presented smaller antioxidant activity than either of these two known186

antioxidant compounds.187

The honey samples from flowers ofM. urundeuva (S08 and S22), Serjania sp, H.188

suaveolens and a heterofloral honey sample (S19) tested in the assay for189

acetylcholinesterase inhibitors, presented inhibition spots with sizes near or identical to190

those of the standard physostigmine. The results were displayed in the Table 2.191

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4) Discussion194195

Functional food is defined as a food or food ingredient that can provide beneficial196

health effects in addition to the traditional nutrients that it contains21. Phytochemicals197

are among the most important functional food components. A number of198

phytochemicals, such as allyl sulfides, catechins, flavonoids, genistein, indoles,199


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limonoids, monoterpenes, phenolic acids, and phytosterols, have been thoroughly200

studied for their potential to prevent cancer. As suggested by in vitro studies, coumarins201

and triterpenoids are thought to be inhibitors of tumor initiation, while carotenoids,202

phenolic compounds, terpenes, tocopherols and flavonoids are known to prevent203

oxidative damage by eliminating free radicals20.204

Although different plants present different phenolic compounds and therefore205

present variations in the total phenolic content21, the data observed for Northeastern206

Brazil honey samples (10.21 to 108.5 mg of GAE/100 g honey) are similar to those207

described for Burkina Faso honey samples2 with values in the range of 32.59 to 114.75208

(mg of GAE/100 g honey) using the same method. However, honey samples in this209

study, presented significant differences with respect to honey samples of Chile with210

total phenolic content varying from 0.0 to 8.83 mg/100 g of honey22. Total phenolic211

contents of 10 honey samples of different floral origin from Poland have been shown to212

vary between 21.7 to 75.3 mg GAE/100 g of honey23 whereas honey samples from213

Slovenia varied between 44.8 mg and 241 mg GAE/kg of honey24.214

Honey samples from Yemen have been shown to present phenol content ranging215

from 75.13 to 246.21 mg CE/100 g of honey25. Honey samples from Chile presented216

flavonoid content from 0.014 to 13.8 mg QE/100 g of honey22. Flavonoid content in217

Burkina Fasan honey samples studied by Meda et al.2 have been shown to present a218

variation range between 0.17 and 8.35 mg QE/100 g of honey. In the analysis of the219

Brazilian Northeastern honey samples, a low correlation (R = 0.15) between the total220

flavonoid and total phenolic content was observed. Meda et al.2 have also described a221

low correlation (R = 0.11) between the total amount of flavonoids and the total amount222

of phenolic compounds.223


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A linear correlation was observed (R2 = 0.583)between the DPPH RSA results and224

the total phenolic levels of Brazilian Northeastern honey samples, suggesting that225

phenolic compounds correlate better to the RSA of these honeys (Figure 1).Meda et al.2226

have described values of IC50 varying from 1.63 to 29.13 mg/mL for honey samples.227

Liviu et al.26 have studied 23 honey samples collected in different Romania regions and228

have confirmed a variation in the antioxidant properties and total phenolic contents in229

honey samples depending on their botanic or geographic source. Lachman et al.27have230

analyzed 40 honey samples mainly from Northern Moravia and found a great variation.231

They have suggested that the differences depend on the geographic location and period232

of honey collection. They have used the Folin-Ciocalteau method to determine the total233

phenolic content and have reported values between 89.9 mg GAE/kg and 215.2 mg234

GAE/kg. A linear correlation (R2 = 0.852) has been observed between the total phenolic235

content and the antioxidant activity, suggesting that phenolic compounds are directly236

responsible for the antioxidant properties of honey.237

However,Atoui et al.28 have suggested that similar results of phenolic levels do not,238

necessarily, correspond to the same antioxidant responses because the amount of239

phenolics found in the Folin-Ciocalteau assay also depends on their chemical structure.240

According to Gheldofet al.29,the antioxidant capacity in honey samples is the result of241

the combined activity of a wide range of compounds including phenolics, peptides,242

organic acids, enzymes, Maillard reaction products, and possibly other minor243

components.244

Differences in the activities and phenol content of two honey samples of M.245

urundeuva are due probably to seasonal variation of phenolic compounds: S08 was246

collected in November (dry period) and S22 in June (rainy period). The effects of247


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seasonal climate changes in the caatinga biome on tannin levels ofM. urundeuva and248

Anadenanthera colubrina had been previously reported by Monteiro et al.30249

Honey is an ideal energetic food due to its sugar content. However, the importance250

of medicinal plants in furnishing nectar forApis mellifera is often overlooked, as251

indicated by this study of honey samples from Brazilian Northeastern. L. sidoides and252

M. urundeuva are widely used medicinal plants in Northeastern Brazil and honeys from253

their flowers showed high values of total phenolic content and antioxidant activity.254

Thus, Northeastern Brazilian honeys may have therapeutic potential as functional foods255

due to their antioxidant and antiacetylcholinesterase activities.256

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Aknowledgements258

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The authors thank Cear State Support to Micro and Small Companies Service260

(SEBRAE) for the honey samples.261

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Authors Disclosure Statement264

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No competing financial interests exist266

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References269

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1)Cao G, Sofic E, Prior RL: Antioxidant and prooxidant behavior of flavonoids:271

structure-activity relationships. Free Radical Biol Med 1997; 22: 749-760.272

273


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2)Meda A, Lamien CE, Romito M, Millogo J, Nacoulma OG: Determination of274

the Total Phenolic, Flavonoid and Proline Contents in Burkina Fasan Honey, as275

well as their Radical Scavenging activity. Food Chem 2005; 91: 571-577.276

277

3)FAO: Value-added products from beekeeping. FOA Agric Ser Bull 1996.278

Rome, Italy. FAO.279

280

4)Kk M, Kolayh S, Karaolu , Ulusoy E, Baltaci C, Candan F: Biological281

activities and chemical composition of three honeys of different types from282

Anatolia. Food Chem 2007; 100: 526-534.283

284

5)Aljadi AM, Kamaruddin MY: Evaluation of the phenolic contents and285

antioxidant capacities of two Malaysian floral honeys. Food Chem 2004; 85:286

513-518.287

288

6)Matos FJA: Lippia sidoides Cham. farmacognosia, qumica e farmacologia.289

Rev Bras Farm 1998; 79: 84-87.290

291

7)Lemos TLG, Craveiro AA, Alencar JW, Matos FJ, Clarck AM, Mac-Chesney292

JD: Antimicrobial activity of essential oil of Brazilian plants. Phytotherapy Res293

1990; 4: 82-84.294

295

8)Lacoste E, Chaumont JP, Mandin D, Plumel MM, Matos FJ: Antiseptic296

properties of essential oil ofLippiasidoides Cham. Application to the cutaneous297

microflora. Ann Pharm Fr 1996; 54: 228-230.298


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13

299

9)Fontenelle ROS, Morais SM, Brito EHS, Kerntopf MR, Brilhante RSN,300

Cordeiro RA, Tom AR, Queiroz MGR, Nascimento NRF, Sidrim JJC, Rocha301

MFG: Chemical composition, toxicological aspects and antifungal activity of302

essential oil from Lippia sidoides Cham. J Antimicrob Chemother2007; 59, 5:303

934-940.304

305

10)Macambira LMA, Andrade CHS, Matos FJA: Naphtoquinoids from Lippia306

sidoides. J Nat Prod1986; 49: 310-312.307

308

11)Costa SMO, Lemos TLG, Pessoa ODL, Pessoa C, Montenegro RC, Braz-Filho309

R: Chemical constituents fromLippia sidoides and cytotoxic activity. J Nat Prod310

2001; 64, 6: 792-795.311

312

12)Costa SMO, Lemos TLG, Pessoa ODL, Assuno JC, Braz-Filho R:313

Constituintes qumicos de Lippia sidoides (Cham.) Verbenaceae. Rev Bras314

Farmacog 2002; 12: 66 - 67.315

316

13)Lorenzi H, Matos FJA: Myracrodruon urundeuva Allemo; Lippia sidoides317

Cham. In: Plantas Medicinais no Brasil Nativas e Exticas. Instituto Plantarum318

de Estudos da Flora Ltda, So Paulo, 2002, pp. 52-53; 494-495.319

320

14)Sousa, MP, Matos, MEO, Matos, FJA, Machado, MIL, Craveiro, AA: Alecrim-321

pimenta; Aroeira. In: Constituintes qumicos ativos e propriedades biolgicas de322


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14

plantas medicinais brasileiras, Editora Universidade Federal do Cear - UFC,323

Fortaleza, 2004, pp. 51-58; 86-90.324

325

15)George NM, Cutting KF: Antibacterial Honey (Medihoney TM): in-vitro Activity326

Against Clinical Isolates of MRSA, VRE, and Other Multiresistant Gram-327

negative Organisms Including Pseudomonas aeruginosa. Wounds 2007; 19, 9:328

231-236.329

330

16)Nordberg A, Carlson LA, Winblad B: Biological markers and the cholinergic331

hypothesis in Alzheimers disease. Acta Neurol Scand1992; 85:54-58.332

333

17)Singleton VL, Orthofer R, Lamuela-Raventos RM: Analysis of total phenols and334

other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent.335

Methods Enzymol 1999; 299: 152-178.336

337

18)Arvouet-Grand A, Vennat B, Pourrat A, Legret P: Standardisation dun extrait de338

propolis et identification des principaux constituants. J Pharm Belgique 1994; 49:339

462-468.340

341

19)Rhee IK, Meent M, Ingkaninan K, Verpoorte R: Screening for342

acetylcholinesterase inhibitors from Amaryllidaceae using silica gel thin-layer343

chromatography in combination with bioactivity staining. J Chromatogr A2001;344

915: 217-223.345

346


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20)Darrington H: Ten years of food technology. Intern Food Ingredients1995; 4: 59-347

62.348

349

21)Blum M: Designing foods for better health. Intern Food Ingredients 1996; 3: 25-350

29.351

352

22)Muoz O, Copaja S: Contenido de Flavonoides y Compuestos fenlicos de353

Mieles Chilenas e ndice Antioxidante. Qum Nova, 2007; 30, 4: 848-851.354

355

23)Socha R, Juszczak L, Pietrzyk S, Fortuna T: Antioxidant activity and phenolic356

composition of herbhoneys. Food Chem 2009; 113, 2: 568-574.357

358

24)Bertoncelj J, Doberek U, Jamnik, M, Golob T: Evaluation of the phenolic359

content, antioxidant activity and colour of Slovenian honey. Food Chem 2007;360

105, 2: 822-828.361

362

25)Al-Mamary M, Al-Meeri A, Al-Habori M: Antioxidant activities and total363

phenolics of different types of honey. Nut Res 2002; 22: 1041-1047.364

365

26)Liviu Al M, Daniel D, Moise A, Bobis O, Laslo L, Bogdanov S: Physico -366

chemical and bioactive properties of different floral origin honeys from Romania.367

Food Chem 2009; 112,4: 863-867.368

369


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16

27)Lachman J, Orsk M, Hejtmnkov A, Kovova E: Evaluation of Antioxidant370

Activity and Total Phenolics of Selected Czech Honeys. LWT- Food S Tech371

2009.(Article in Press, Corrected Proof).372

373

28)Atoui AK, Mansouri A, Boskou G, Kefalas P: Tea and herbal infusions: their374

antioxidant activity and phenolic profile. Food Chem 2005; 89: 27-36.375

376

29)Gheldof N, Wang X, Engeseth NJ: Identification and quantification of377

antioxidant components of honeys from various floral sources. J Agric Food378

Chem 2002; 50: 5870-5877.379

380

30)Monteiro JM, Albuquerque UP, Lins Neto EMF, Arajo EL, Albuquerque MM,381

Amorim ELC: The effects of seasonal climate changes in the caatinga on tannin382

levels inMyracrodruon urundeuva (Engl.) Fr. All. andAnadenanthera colubrina383

(Vell.) Brenan. Rev Bras Farmacogn 2006; 16: 338-344.384

385

386

387

388

389

390

391

392

393

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3 - Cap+tulo I - PHENOLIC CONTENTS, ANTIOXIDANT AND ANTIACETYLCHOLINESTERASE PROPERTIES OF HONEYS

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