The contribution of phenolic acids to the anti-inflammatory activity of mushrooms: Screening in phenolic extracts, individual parent molecules and synthesized glucuronated and methylated derivatives

1 The contribution of phenolic acids to the anti-inflammatory activity of 1 mushrooms: screening in phenolic extracts, individual parent molecules 2 and synthesized glucuronated and methylated derivatives 3 4 Oludemi Taofiq a,b , Ricardo C. Calhelha a,c , Sandrina Heleno a,c , Lillian Barros a , Anabela 5 Martins a , Celestino Santos-Buelga b , Maria João R.P. Queiroz c , Isabel C.F.R. Ferreira a,* 6 7 a Mountain Research Centre (CIMO), ESA, Polytechnic Institute of Bragança, Campus de 8 Santa Apólónia, 1152, 5301-855 Bragança, Portugal 9 b GIP-USAL, Faculty of Pharmacy, University of Salamanca, Campus Miguel de Unamuno, 10 37007 Salamanca, Spain 11 c Center of Chemistry, University of Minho, Campus de Gualtar 4710-057 Braga, Portugal 12 2 13 ABSTRACT 14 In the present study, the ethanolic extracts of fourteen edible mushrooms were investigated 15 for their anti-inflammatory potential in LPS (lipopolysaccharide) activated RAW 264.7 16 macrophages. Furthermore the extracts were chemically characterized in terms of phenolic 17 acids and related compounds. The identified molecules (p-hydroxybenzoic, p-coumaric and 18 cinnamic acids) and their glucuronated and methylated derivatives obtained by chemical 19 synthesis were also evaluated for the same bioactivity, in order to establish structure-activity 20 relationships and to comprehend the effects of in vivo metabolism reactions in the activity of 21 the compounds. The extracts of Pleurotus ostreatus, Macrolepiota procera, Boletus impolitus 22 and Agaricus bisporus revealed the strongest anti-inflammatory potential (EC 50 values 96 ± 1 23 to 190 ± 6 µg/mL, and also the highest concentration of cinnamic acid (656 to 156 µg/g), 24 which was also the individual compound with the highest anti-inflammatory activity. The 25 derivatives of p-coumaric acid revealed the strongest properties, specially the derivative 26 methylated in the carboxylic group (CoA-M1) that exhibited similar activity to the one 27 showed by dexamethaxone used as anti-inflammatory standard; by contrast, the derivatives of 28 p-hydroxybenzoic revealed the lowest inhibition of NO production. All in all, whereas the 29 conjugation reactions change the chemical structure of phenolic acids and may increase or 30 decrease their activity, the glucuronated and methylated derivatives of the studied compounds 31 are still displaying anti-inflammatory activity. 32 33 Keywords: Edible Mushrooms; Phenolic acids; glucuronated and methylated derivatives; 34 Anti-inflammatory; Nitric oxide production; HPLC-PDA 35 3 36 1. INTRODUCTION 37 Inflammation is considered to be part of the complex biological response to remove injury or 38 harmful stimuli such as pathogens, damaged cells, or irritation and this is a central feature of 39 many pathophysiological conditions such as atherosclerosis, obesity, metabolic syndrome, 40 diabetes (Pradhan, 2007) and even several types of cancers (Moro et al., 2012). 41 When cells are exposed to immune stimulants, the pro-inflammatory cells, such as 42 macrophages, monocytes, or other host cells, start to produce cytokines and other mediators, 43 which initiate the inflammation process. Among the various inflammatory mediators, the 44 most common are interleukins (IL-1β, IL-6, IL-8), tumour necrosis factor (TNF-α), nuclear 45 factor-κB (NF-κB), intercellular adhesion molecule-1 (ICAM-1), inducible type 46 cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), 5-lipooxygenase (5-LOX), and 47 inducible nitric oxide synthase (iNOS) that leads to the production of reactive nitrogen 48 species such as nitric oxide (NO). Overproduction of these inflammatory mediators leads to 49 different kinds of cell damage (Kanwar, Kanwar, Burrow, & Baratchi, 2009). 50 Recently, the nonsteroidal anti-inflammatory drugs (NSAIDs) are usually the most 51 commonly administered drugs to reduce inflammation in the body. Many studies, however, 52 have shown that the long-term administration of NSAIDs has the potential for significant side 53 effects on the gastrointestinal tract with numerous harmful effects such as mucosal lesions, 54 bleeding, peptic ulcers, and intestinal perforation (Dugowson & Gnanashanmugam, 2006). 55 Recent studies show that NSAIDs are also associated with a relatively high incidence of renal 56 adverse drug reactions, nephrotic syndrome, high blood pressure, acute tubular necrosis and 57 cardiovascular toxicity (Elsayed, Hesham, Mohammad, & Ramlan, 2014). 58 As a result, research studies are now being channelled towards discovery of bioactive 59 compounds with ability to suppress the production of inflammatory mediators. A good model 60 to test potential anti-inflammatory drugs are macrophages, which are large specialized cells 61 4 that engulf and digest cellular debris, microbes or cancer cells in a process called 62 phagocytosis. They play an important role in non-specific host defence mechanisms and help 63 to initiate other defence mechanisms. Beyond stimulating the immune system, macrophages 64 play a crucial role in the inflammatory response through the release of a variety of factors, 65 such as NO, TNF-α, IL-1β, IL-6, in response to an activating stimulus, e.g. 66 lipopolysaccharide (Moro et al., 2012). 67 Mushrooms are widely appreciated all over the world not only for their culinary and 68 nutritional properties (Kalac, 2009), but also for their pharmacological value as sources of 69 important bioactive molecules, such as antioxidant (Puttaraju, Venkateshaiah, Dharmesh, 70 Urs, & Somasundaram, 2006; Ferreira, Barros, & Abreu, 2009; Heleno, Martins, Queiroz, & 71 Ferreira, 2015), antitumor (Moradali, Mostafavi, Ghods, & Hedjaroude, 2007; Ferreira, Vaz, 72 Vasconcelos, & Martins, 2010, Carocho & Ferreira, 2013), antimicrobial (Alves, Ferreira, 73 Dias, Teixeira, Martins, & Pintado, 2012; Alves, Ferreira, Froufe, Abreu, Martins, & Pintado, 74 2013), immunomodulator (Borchers, Krishnamurthy, Keen, Meyers, & Gershwin, 2008), 75 antiatherogenic (Mori, Kobayashi, Tomita, Inatomi, & Ikeda, 2008) and hypoglycemic 76 compounds (Hu, Wang, Lien, Liaw, & Lee, 2006). Due to these properties, they have been 77 recognized as functional foods, as well as valuable sources of natural medicines and 78 nutraceuticals (Lindequist, Niedermeyer, Jülich, 2005; Guillamón et al., 2010). 79 Moreover, mushrooms have also demonstrated some anti-inflammatory potential based on 80 their ability to reduce the production of inflammatory mediators (Padilha, Avila, Sousa, 81 Cardoso, Perazzo, & Carvalho, 2009; Elsayed et al., 2014). Previous research studies have 82 been carried out on several mushroom species, mainly in methanolic and ethanolic extracts 83 (Table 1). Different compounds have been pointed out as the responsible for the anti84 inflammatory activity such as β-glucans (Nosálóva, Bobek, Cerna, Galbavy, & Tvrtina, 85 2001), triterpenes (Ma, Chen, Dong, & Lu, 2013), glycoproteins (Gunawardena et al., 2014) 86 5 and even phenolic compounds (Moro et al., 2012). However, not much is known about the 87 phenolic bioactive forms in vivo; these compounds are metabolized and circulate in the 88 organism as glucuronated, sulfated and methylated metabolites, displaying higher or lower 89 bioactivity (Heleno et al., 2015). 90 Therefore, the present study aimed to investigate the anti-inflammatory activity of selected 91 mushroom species from the Northeast of Portugal, using ethanolic extracts in LPS activated 92 RAW 264.7 macrophages. Furthermore, after characterization of the extracts in terms of 93 phenolic acids and related compounds, the identified individual parent molecules and their 94 synthesised glucuronated and methylated derivatives were evaluated for the same bioactivity, 95 in order to establish structure-activity relationships. 96 97 2. MATERIALS AND METHODS 98 2.1. Mushroom species and extracts preparation 99 Ten wild mushroom species (Amanita caesaria (Scop.) Pers., Boletus aereus Bull., B. edulis 100 Bull., B. flagrans Vittad., B. impolitus Fr., B. reticulatus Schaeff., Cantharellus cibarius Fr., 101 Lactarius deliciosus (L. ex Fr.) S.F. Gray, Macrolepiota procera (Scop.) Singer and 102 Morchella esculenta Fr.), collected in the Northeast of Portugal, and four cultivated species 103 (Agaricus bisporus (J.E. Lange) Emil J. Imbach, A. bisporus Portobello (J.E. Lange) Emil J. 104 Imbach, Pleurotus eryngii (DC.) Quél. and Pleurotus. ostreatus (Jacq. ex Fr.) P. Kumm.) 105 were used in the present study. All species were deposited in the herbarium of the School of 106 Agriculture in Polytechnic Institute of Bragança, and were previously characterized by the 107 research group in terms of nutritional value and chemical composition (including primary and 108 secondary metabolites) (Barros, Dueñas, Ferreira, Baptista, & Santos-Buelga, 2009, 109 Grangeia, Heleno, Barros, Martins, & Ferreira, 2011; Heleno, Barros, Sousa, Martins, Santos110 Buelga, & Ferreira., 2011; Reis et al., 2011; Pereira, Barros, Martins, & Ferreira, 2012; Reis, 111 6 Barros, Martins, & Ferreira, 2012; Heleno et al., 2013b). Their antioxidant, antimicrobial and 112 antitumor properties were also previously evaluated by the group 113 (http://esa.ipb.pt/biochemcore/index.php/studied-mushrooms). In the present work, the in 114 vitro anti-inflammatory activity was evaluated in ethanolic extracts prepared as follows. 115 Lyophilized (Ly-8-FM-ULE, Snijders, Holland) mushroom powder (20 mesh) of each species 116 (0.5 g) was extracted with ethanol (15 mL), by maceration with stirring for 1 h. Then, the 117 extract was filtered through Whatman no 4 filter paper and the extraction procedure was 118 repeated one more time. The filtrate was rotary evaporated to remove ethanol and the 119 extraction yield was calculated by measuring the extract weight. 120 121 2.2. Reagents 122 Acetonitrile 99.9% was of high-performance liquid chromatography (HPLC) grade from Lab123 Scan (Lisbon, Portugal). p-Hydroxybenzoic acid, p-coumaric acid, cinnamic acid, Dulbecco’s 124 modified Eagle’s minimum essential medium (DMEM), fetal bovine serum (FBS), penicillin, 125 streptomycin, Griess reagent system (Promega), DMSO, sulphorodamine B (SRB) and 126 lipopolysaccharide (LPS) were obtained from Sigma-Aldrich Co. (Saint Louis, MO, USA). 127 All other chemicals and solvents were of analytical grade and purchased from common 128 suppliers. 129 130 2.3. Chemical characterization of the extracts 131 The dry mass of each mushroom extract was re-dissolved in water/ethanol (50:50, v/v) and 132 filtered through a 0.22 µm nylon disposable filter for HPLC analysis. The analysis was 133 performed using a Shimadzu 20A series ultra-fast liquid chromatograph (UFLC, Shimadzu 134 Coperation, Kyoto, Japan). Separation was achieved on a Waters Spherisorb S3 ODS2 C 18 135 column (3 µm, 150 mm x 4.6 mm) column thermostatted at 35 ºC. The solvents used were: 136 7 (A) 0.1% formic acid in water, (B) acetonitrile. The elution gradient established was: 10% B 137 to 15% B over 5 min, 15–25% B over 5 min, 25–35% B over 10 min, isocratic 50% B for 10 138 min, and re-equilibration of the column, using a flow rate of 0.5 mL/min. Detection was 139 carried out in a photodiode array detector (PDA), using 280 nm as the preferred wavelength. 140 The phenolic acids (group of phenolic compounds identified in the samples) were quantified 141 by comparison of the area of their peaks recorded at 280 nm with calibration curves obtained 142 from commercial standards of each compound: protocatechuic acid (y = 164741x, R 2 =0.999), 143 p-hydroxybenzoic acid (y = 113523x, R 2 =0.999), p-coumaric acid (y = 433521x, R 2 =0998) 144 and cinnamic acid (y = 583527x, R 2 =0.998), 5 to 80 µg/mL. The results were expressed as µg 145 per g of extract. 146 147 2.4. Phenolic acids and synthesised derivatives 148 p-Hydroxybenzoic acid, p-coumaric acid and cinnamic acid, identified in the mushroom 149 extracts, were used as staring reagents for the synthesis of methylated and glucuronated 150 derivatives of the identified compounds (Figure 1); these compounds were previously 151 synthesized and completely characterized by the authors (Heleno et al., 2013a; Heleno et al., 152 2014b). Briefly, the glucuronated derivatives (HA-GP, CoA-GP and CA-GP) were obtained 153 by reacting the parent molecules with acetobromo-α-D-glucuronic acid methyl ester under 154 argon and using DMSO (dimethylsulfoxide) as solvent at room temperature. The methylated 155 derivatives (HA-M1, CoA-M1 and CA-M) were prepared using methanol and sulphuric acid 156 at room temperature; HA-M2 and CoA-M2 were synthesised using dimethyl sulphate in 157 acetone at room temperature. Finally, HA-M3 and CoA-M3 were obtained by the hydrolysis 158 of compounds HA-M2 and CoA-M2 using ethanol at 65ºC and adjusting the pH to 3. All the 159 synthesised compounds were fully characterized by 1 H NMR, 13 C NMR, HRMS (high 160 resolution mass spectrometry) and melting point. 161 162 8 2.5. Evaluation of the anti-inflammatory activity 163 2.5.1. Cells treatment 164 The anti-inflammatory activity was carried out according to Moro et al. (2012) and García165 Lafuente et al. (2014) with some modifications. The mouse macrophage-like cell line RAW 166 264.7 was cultured in DMEM medium supplemented with 10% heat-inactivated foetal bovine 167 serum, glutamine and antibiotics at 37 °C under 5% CO 2 , in humidified air. For each 168 experiment, cells were detached with a cell scraper. A cell density of 5 x 10 5 cells/mL was 169 used, and the proportion of dead cells was less than 5%, according to Trypan blue dye 170 exclusion test. 171 Cells were seeded in 96-well plates at 150,000 cells/well and allowed do attach to the plate 172 overnight. Then, cells were treated with the different concentrations of each one of the 173 extracts for 1h. Dexamethasone (50 µM) was used as a positive control for the experiment. 174 The following step was the stimulation with LPS (1 µg/mL) for 18h. The effect of all the 175 tested samples in the absence of LPS was also evaluated, in order to observe if they induced 176 changes in nitric oxide (NO) basal levels. In negative controls, no LPS was added. Both 177 extracts and LPS were dissolved in supplemented DMEM. 178 179 2.5.2. Nitric oxide determination 180 Both the extracts, and the pure identified compounds and their synthezised derivatives were 181 submitted to the anti-inflammatory activity assay. The ethanolic extracts were dissolved in 182 water (non cytotoxic solvent) at 8 mg/mL, while the identified individual compounds and 183 their synthesised methylated and glucuronated derivatives were dissolved in DMSO at 50% 184 concentration in stock solutions. These solutions were then submitted to further dilutions 185 (400 µg/mL to 50 µg/mL and 2500 µM to 39 µM, for the extracts and compounds, 186 9 respectively) in order to determine effective concentrations (Moro et al., 2012; García187 Lafuente et al., 2014). 188 For the determination of nitric oxide, Griess Reagent System kit was used, which contains 189 sulphanilamide, N-(1-napthyl)ethylenediamine hydrochloride (NED) and nitrite solutions. A 190 reference curve of the nitrite (sodium nitrite 100 µM to 1.6 µM; y=0.0066x+0.1349; 191 R 2 =0.9986) was prepared in a 96-well plate. The cell culture supernatant (100 µL) was 192 transferred to the plate and mixed with Sulphanilamide and NED solutions, 5-10 minutes 193 each, at room temperature. The nitric oxide produced was determined by measuring the 194 absorbance at 540 nm (microplate reader ELX800 Biotek), and by comparison with the 195 standard calibration curve. 196 197 2.6. Statistical analysis 198 For all the experiments three samples were analyzed and all the assays were carried out in 199 triplicate. The results are expressed as mean values ± standard deviation (SD). The 200 differences between the different samples were analyzed using one-way analysis of variance 201 (ANOVA) followed by Tukey’s honestly significant difference post hoc test with α = 0.05, 202 coupled with Welch’s statistic. This treatment was carried out using SPSS v. 22.0 program. 203 204 3. RESULTS AND DISCUSSION 205 3.1. Chemical characterization of the extracts 206 Ethanolic extracts were prepared from fourteen different edible mushroom species: Agaricus 207 bisporus, A. bisporus Portobello, Amanita caesaria, Boletus aereus, B. edulis, B. flagrans, B. 208 impolitus, B. reticulatus, Cantharellus cibarius, Lactarius deliciosus, Macrolepiota procera, 209 Pleurotus eryngii, Pleurotus ostreatus and Morchella esculenta. One phenolic acid (p210 hydroxybenzoic acid) and two cinnamic acids (p-coumaric and cinnamic acids) were detected 211 by HPLC-PDA in the different extracts; their concentrations are presented in Table 2. The 212 16 Elsayed, A. E., Hesham, E.L.E., Mohammad, A.M.W., & Ramlan Aziz. (2014). Mushrooms: 365 A Potential Natural Source of Anti-Inflammatory Compounds for Medical Applications. 366 Mediators of Inflammation, 2014, 1-15. 367 Ferreira, I.C.F.R., Barros, L., & Abreu, R.M.V. (2009). 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Species Country Extract Inhibition of NO production References Spain Methanol 30% at 0.5 mg/mL Moro et al., 2012 Agaricus bisporus Australia Ethanol 50% at 0.032 mg/mL Gunawardena et al., 2014 Boletus edulis Spain Methanol 10% at 0.5 mg/mL Moro et al., 2012 Cantharellus cibarius Spain Methanol 70% at 0.5 mg/mL Moro et al., 2012 Caripia montagnei Brazil Acetone 80%, methanol 43% at 10 mg/kg 54% at 30 mg/kg 49% at 50 mg/kg Queiroz et al., 2010 Cratarellus cornucopoides Spain Methanol 55% at 0.5 mg/mL Moro et al., 2012 Flammulina velutipes Australia Ethanol 50% at 0.024 mg/mL Gunawardena et al., 2014 China Ethanol 65% at 40 µg/mL Ma et al., 2013 Inonotus obliquus South Korea Ethanol 50% at 89 µg/mL Park et al., 2005 Lactarius deliciosus Spain Methanol 40% at 0.5 mg/mL Moro et al., 2012 Lentinus edodes Australia Ethanol 50% at 0.027 mg/mL Gunawardena et al., 2014 Australia Ethanol 50% at 0.077 mg/mL Gunawardena et al., 2014 Pleurotus ostreatus Spain Methanol 15% at 0.5 mg/mL Moro et al., 2012 Pleurotus tuber-regium Belgium Ethanol 70% at 0.5 mg/mL Liu et al., 2014 Tricholoma matsutake South Korea Dichloromethane 47% at 2 mg/mL Lim et al., 2007 21 Table 2. Cinnamic and phenolic acids identified and quantified by HPLC-PDA in the ethanolic extracts of the analysed mushrooms. Mushroom species Extraction yield (%) p-Hydroxybenzoic acid (µg/g) p-Coumaric acid (µg/g) Cinnamic acid (µg/g) Agaricus bisporus 6.9 ± 0.7 abcd nd nd 149 ± 2 d Agaricus bisporus Portobello 6.0 ± 0.1 bcd nd nd 11 ± 1 hi Amanita caesaria 5.4 ± 0.5 cd 57 ± 3 e nd 156 ± 3 d Boletus aereus 7.7 ± 0.6 abc 43 ± 1 f 74 ± 1 b 50 ± 3 f Boletus edulis 10.4 ± 0.5ª nd nd 14.2 ± 0.4 gh Boletus flagrans 4.9 ± 0.3 cd nd nd 6.1 ± 0.3 i Boletus impolitus 4.1 ± 0.5 d 125 ± 9 c 45 ± 2 c 505 ± 12 c Boletus reticulatus 9.5 ± 0.3 ab nd nd 20.3 ± 0.1 g Cantharellus cibarius 4.0 ± 0.3 d 151 ± 2 b nd 71 ± 1 e Lactarius deliciosus 3.7 ± 0.4 d 108 ± 5 d nd 67 ± 1 e Morchella esculenta 4.0 ± 0.8 d nd nd 71 ± 3 e Macrolepiota procera 3.7 ± 0.4 d nd nd 522 ± 1 b Pleurotus eryngii 10.1 ± 0.6 a nd nd 16 ± 1 gh Pleurotus ostreatus 4.0 ± 0.6 d 297 ± 5 a 171 ± 1 a 619 ± 3 a ndnot detected. ANOVA In each column, different letters mean statistical significant differences (p<0.05) between samples. 22 Table 3. Extract concentrations responsible for 50% of reduction of NO production (EC 50 values) in RAW 264.7 cell line. EC 50 values correspond to 50% of inhibition of the NO production in comparison with the negative control (100% of NO production). In the columns, different letters mean statistical significant differences (p<0.05) between samples. Dexamethaxone EC 50 value = 16 ± 2 µg/mL. Mushroom species EC 50 values (µg/mL) Mushroom species EC 50 values (µg/mL) Agaricus bisporus 190 ± 6 ef Boletus reticulatus 378 ± 28 ab Agaricus bisporus portobelo >400ª Cantharellus cibarius 202 ± 17 e Amanita caesaria 186 ± 7 ef Lactarius deliciosus 253 ± 14 d Boletus aereus 357 ± 3 b Macrolepiota procera 162 ± 2 g Boletus edulis >400ª Morchella esculenta 287 ± 9 c Boletus flagrans >400ª Pleurotus eryngii 388 ± 17 a Boletus impolitus 166 ± 10 fg Pleurotus ostreatus 96 ± 1 h 23 Table 4. Concentrations of the studied acids and their glucuronated and methylated derivatives responsible for 50% of reduction of NO production (EC 50 values, µM) in RAW 264.7 cell line. p-Hydroxybenzoic acid and derivatives p-Coumaric acid and derivatives Cinnamic acid and derivatives HA 239 ± 29 c CoA 442 ± 33 a CA HA-GP 1901 ± 104 a CoA-GP 58 ± 5 c CA-GP HA-M1 1825 ± 120 a CoA-M1 35 ± 2 c CA-M 182 ± 16 b 179 ± 17 b 224 ± 16 a HA-M2 526 ± 26 b CoA-M2 128 ± 10 b HA-M3 509 ± 47 b CoA-M3 129 ± 6 b EC 50 values correspond to 50% of inhibition of the NO production in comparison with the negative control (100% of NO production). In each column, different letters mean statistical significant differences (p<0.05) between compounds. Dexamethaxone EC 50 value = 40 ± 4 µM. 24 CA H CC HC O OH CAM H CC HC O OMe CA-GP O O AcO OAc OAc COOMe O i) K2CO31.5 equivs, DMSO, Ar, RT, 24h; ii) MeOH; H2SO4, 5 days, R.T. i) ii) O Br AcO AcO AcO MeOOC 1equiv. A B 25 HO C O OH HO C O OMe MeO C O OMe MeO C O OH HA-M1 HA-M2 HA-M3 SO O OMe MeO 1equiv. i) K2CO31.5 equivs, DMSO, Ar, RT, 24h; ii) MeOH; H2SO4, 5 days, R.T. iii) K2CO3, 2 equivs, acetone, 50ºC, 24h; iv) NaOH, 3 equivs, EtOH, 65ºC, 3h i) ii) O Br AcO AcO AcO MeOOC 1.5equiv. iii) iv) HA HA-GP O HO O AcO OAc OAc COOMe O C Figure 1. Synthesis of methylated and glucuronated derivatives of p-hydroxybenzoic, pcoumaric and cinnamic acids. A) i) Glucuronidation of cinnamic acid (CA). CA-GPcinnamic acid glucuronide protected form, 2,3,4-tri-O-acetyl-1-cinnamoyl-D-glucuronic acid methyl ester (Heleno et al., 2013a); ii) Methylation of CA. CAMmethyl 3-phenylacrylate (Heleno et al., 2014b). B ) i) Glucuronidation of p-coumaric acid (CoA). CoA-GPp-Coumaric acid glucuronide protected form, 2,3,4-tri-O-acetyl-1-p-coumaroyl-D-glucuronic acid methyl ester (Heleno et al., 2014b); ii)-iv) Methylations of CoA. CoA-M13-(4-hydroxyphenyl) acrylate, CoA-M2methyl-(4-methoxyphenyl) acrylate, CoA-M33-(4-methoxyphenyl) acrylic acid (Heleno et al. 2014b). C) i) Glucuronidation of p-hydroxybenzoic acid (HA). HA-GPp-hydroxybenzoic acid protected form, 2,3,4-tri-O-acetyl-1-phydroxybenzoyl-D-glucuronic acid methyl ester (Heleno et al., 2013a); ii-iv) Methylations of phydroxybenzoic acid, HA-M1methyl 4-hydroxybenzoate, HA-M2methyl-p-anisate, HA-M34methoxybenzoic acid (Heleno et al., 2014b).