Effect of light, oxygen and temperature on the stability of artepillin C and p-coumaric acid from Brazilian green propolis

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Effect of light, oxygen and temperature on the stability of artepillin C and p-coumaric acid from Brazilian green propolis Caroline Arruda, Victor Pena Ribeiro, Marília Oliveira Almeida, Jennyfer Andrea Aldana Mejía, Rosana Casoti, Jairo Kenupp Bastos ∗ School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café S/N, 14040-930, Ribeirão Preto, SP, Brazil

a r t i c l e

i n f o

Article history: Received 17 April 2019 Received in revised form 5 October 2019 Accepted 9 October 2019 Available online xxx Keywords: Green propolis Artepillin C p-Coumaric acid Stability studies RP-HPLC method Quality control

a b s t r a c t Brazilian Green Propolis (BGP) is an important bee product, which displays important biological activities, making it valuable in the international market. The major prenylated phenolic compound in BPG is (E)artepillin C, along with its precursor (E)-p-coumaric acid, both contributing to the biological effects of BGP. Taking that into account, it was evaluated the effect of light, temperature and air oxygen in their content to establish the best storage and transport conditions for crude BGP and the pure compounds. For that, (E)-artepillin C and (E)-p-coumaric acid were initially submitted to degradation for five days under sunlight and high temperature (50 ◦ C), furnishing three major (E)-Artepillin C isomers and one from (E)-p-coumaric acid. Then, it was developed and validated a Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method for quantifying these compounds in crude BGP and in its extracts. In the stability studies, it was used a Full Factorial and Central Composite Design to establish the desirable storage conditions. (E)-Artepillin C, both pure and in BGP should be kept protected from light and storage below -2.5 ◦ C. (E)-p-Coumaric acid can be stored at room temperature. Therefore, the best storage and transport conditions to keep the content of both compounds in BGP are protection from light at low temperatures. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Propolis is a natural resinous product formed by plant material collected by bees that are deposited in the hives along with their saliva. Its chemical composition is complex and depends on the botanical sources available in the geographic region of propolis production [1]. One of its important type is Brazilian green propolis (BGP): the main plant used for BGP production is Baccharis dracunculifolia DC, found mainly in the southeast of Brazil, in the states of São Paulo and Minas Gerais [2]. BGP has a characteristic chemical composition, containing mainly prenylated phenolic compounds, such as artepillin C, baccharin and drupanin, as well as phenolic acids such as their biosynthetic precursor p-coumaric acid. Phenolic compounds, including the prenylated ones, are known for displaying many biological effects [2–7]. Therefore, due to the presence of these compounds, green propolis, besides its important functions in the hives by filling the holes to keep an adequate temperature for the bees, displays biological activities, which are interesting from the pharmacological point

∗ Corresponding author. E-mail address: [email protected] (J. Kenupp Bastos).

of view [8]. Some of the biological activities reported for BGP are cytotoxicity against cancer cell lines [9], anti-inflammatory [3], gastroprotective [10], antimicrobial [11] and antiparasitic [12]. From the prenylated phenolics found in BGP, artepillin C is the most abundant one [13] and it probably contributes to the biological activities displayed by green propolis extract, since the isolated artepillin C displays gastroprotective [2], cytotoxic [14], anti-inflammatory [15], antidiabetes [16] and antigenotoxic [17] activities. p-Coumaric acid has also potential to treat different injuries by acting as antiulcerogenic [18], antimicrobial, anticancer and antimutagenic [19]. Artepillin C is found in BGP in large amounts, which contributes to the high commercial value of Brazilian propolis: while the price of one kilogram of propolis is nowadays sold by approximately U$ 120.00, the same quantity of honey is sold by approximately U$ 5.00 [20]. Because of that, approximately 75% of the Brazilian production of green propolis are exported, mainly to Japan [21], and it takes several days from the site of production to the companies in Japan under different environmental conditions. Therefore, its artepillin C content should be kept stable during storage and transportation, making it mandatory to perform quality control of this important raw material. The stability of natural raw materials ought to be evaluated, especially facing high temperature, light and oxygen naturally

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Please cite this article as: C. Arruda, V. Pena Ribeiro, M. Oliveira Almeida et al., Effect of light, oxygen and temperature on the stability of artepillin C and p-coumaric acid from Brazilian green propolis, J. Pharm. Biomed. Anal., https://doi.org/10.1016/j.jpba.2019.112922

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present in the air, which are well known factors able to cause degradation of compounds bearing double bonds: Daskalaki et al. [22] evaluated the influence of the presence of oxygen in virgin olive oil glass bottle headspace and showed that at room temperature in the dark, with low oxygen available, approximately 30% of hydroxytyrosol derivatives were lost after 15 months of storage. Likewise, these compounds content decreased by 60% and 90% after 30 and 90 min of the oil high temperature exposure, respectively. UV light, emitted by sunlight and lamps used to illuminate internal storage facilities can also induce chemical structural changes, especially in the double bonds: The UV light exposure (365 nm) caused modification in the double bond of (E)-2,2-dimethyl-8(3-methyl-2-butenyl)-benzopyran-6-propenoic acid, a compound isolated from Brazilian propolis, furnishing its Z isomer [23]. Taking that into account, it was evaluated the effect of light, different temperatures and air oxygen presence on artepillin C and p-coumaric acid stabilities. Besides, it was identified their main degradation products, allowing the development of a validated RPHPLC method to perform BGP quality control and to establish the best storage and transport conditions for green propolis raw material and its products. 2. Material and methods 2.1. General experimental procedures Preparative procedures were performed in Shimadzu HPLC equipment, coupled to a LC-6A mobile phase pump, to a CBM-20A controller and to a UV-VIS SPD-20 detector. The software used for data processing and acquisition was LC solution version 1.25. The preparative column used for artepillin C isolation was a RP Shimadzu Shim- Pack (20 × 250 mm, 5 ␮m) at a flow rate of 8 mL/min and detection at 275 and 300 nm. For purification of artepillin C, the fractions containing this compound in high amounts were injected at 200 mg/mL (volume of injection 500 ␮L) in each run. For the degradation products, a Kromasil® C18 (250 mm × 10 mm-5 ␮m) column was selected with flow rate of 4.6 mL/min, 50 ␮L volume injection (200 mg/mL) and detection at 275 and 300 nm. A Waters® HPLC with a 1525 binary pump, automatic injector and a 2998 PDA detector was used for the analytical measures. Chromatographic data was acquired by Empower® software. The analytical flow rate and injection volume were 1 mL/min and 15 ␮L, respectively. The reading wavelengths were the same as for preparative analyses. As stationary phase, the selected chromatographic column was a Phenomenex C-18 LUNA (100, 250 × 4.6 mm, 5 ␮m). A Jasco P-2000 polarimeter at 25 ◦ C and 589 nm was used for specific rotation 25

([␣] D ) readings. The pure compounds were dissolved in HPLCgrade chloroform for the specific rotation analyses. NMR spectra were recorded in CDCl3 on a Bruker spectrometer (DRX-500) at 500 MHz and 125 MHz for 1 H and 13 C, respectively. High resolution mass spectrometry analyses were acquired by direct injection on a Thermo Scientific Exactive Plus OrbitrapTM mass spectrometer coupled to an electrospray probe (H-ESI II) and acquiring data in both positive and negative ionization modes in the full scan with m/z ranging between 120-1200. For the experimental designs obtainment and processing, the STATSOFT StatisticaTM software was used. The light used in the stability experiments was a cold daylight Taschibra® LED 500 lumens, 6 W 6500 K, 83 lm/W lamp [23]. p-Coumaric acid was purchased from Sigma-Aldrich® . 2.2. Isolation of artepillin C from Brazilian Green Propolis Artepillin C was isolated from BGP: two hundred grams of BGP, provided by Apis Flora ® Ribeirão Preto, SP, Brasil, were ground and macerated at 30 ◦ C and 120 rpm three times with ethanol-water

7:3 for 24 h each time. After filtration, concentration under vacuum and lyophilization, the crude extract yielded 87 g. The crude extract was fractionated by vacuum liquid chromatography (13.5 diameter sintered glass column) using 5.5 cm height silica gel 60 H (60–200 ␮m- Across organics® ) as stationary phase. Hexane and ethyl acetate (95:05) were used as mobile phase. It was collected 60 fractions of 1 L each, and artepillin C was found in fractions 12–60 (AF-9.65 g.). Artepillin C was purified by preparative RP-HPLC by using C-18 as stationary phase, as described in the previous section, and a gradient of methanol (B) and acid water (with 0.1% of formic acid) (A) as elution solvents. The gradient was as following: 0.00–15.00 min. 80–95% B; 15.00–18.00 min. 95%-95% B; 18.00–19.00 min 95-80% B, 19.00–23.00 min. 80%-80%B. Artepillin C’s retention time in this method was at 14.3 min at 275 nm [2]. From the 9.65 g. of AF, it was isolated 1.6 g of pure artepillin C. 2.3. Obtainment and isolation of the major degradation products To induce the degradation of artepillin C and p-coumaric acid, they were diluted in ethyl acetate (2 mg/mL) separately and exposed to sunlight or to a temperature of 50 ◦ C in the dark for five days. The major degradation products were isolated by semi-preparative HPLC. The equipment and column used are in the general experimental procedure section. The mobile phase used for the isolation of p-coumaric acid degradation products of the experiment under sunlight exposure was methanol as organic solvent (B) and acid water (with 0.1% of formic acid) as aqueous phase (A) in a gradient of 0.00–20.00 min.: 25–50% B; 20.00–22.00 min.: 50-50% B; 22.00–24.00 min.: 50-25% B. In this experiment, from 200 mg of the standard, it was obtained 51 mg of the main degradation product, which corresponded to (Z)-p-coumaric acid (CA1). From the sunlight degradation of artepillin C (from 200 mg), it also furnished one major compound, which corresponded to the (Z)-artepillin C (ART 4–14.6 mg). It was isolated by using acetonitrile (B) and water+0.1% of formic acid+5% methanol (A): 0.00–60.00 min. 20–95% B; 60.00–63.00 min. 95-95% B; 63.00–65.00 min. 95-20% B. After five days of heat exposure of 200 mg of artepillin C, three other derivatives were isolated by the following method: acetonitrile (B) and water+0.1% of formic acid+5% methanol (A): 0.00–20.00 min. 55–82% B; 20.00–22.00 min. 82-82% B; 22.00–24.00 min. 82-55% B. They were identified as 2-propenoic (E)-3-[2,3dihydro-2-(1-methylethyl)-7-prenyl-5-benzofuranyl] acid (ART 1- 6.4 mg), 2-propenoic (E)-3-(2,2-dimethyl-3,4-dihydro-3hydroxi-8-prenyl-2H-1-benzopyran-6-yl) acid (ART 2–7.8 mg) and 3-[2-dimethyl-8-(3-methyl-2-butenyl) benzopiran]-6-propenoic acid (ART 3–7.8 mg). 2.4. Development and validation of the RP-HPLC method For the analytical method development, it was used the Waters® HPLC and the experimental conditions previously described. The mobile phase consisted of acetonitrile + 2% isopropanol as organic solvent (B) and water + 2% isopropanol + 5% methanol + 0.4% formic acid as the aqueous solvent (A) in gradient mode: 0.00–3.00 min. 20-20% B; 3.00–4.00 min. 20–25% B: 4.00–15.00 min. 25-25% B; 15.00–20.00 min. 25–45% B; 20.00–40.00 min. 45-45% B; 40.00–45.00 min. 45–60% B; 45.00–68.83 min. 60–80% B; 68.83–70.00 min. 80-20% B; 70.00–80.00 min. 20-20% B. In the validation experiments the parameters selectivity, linearity, accuracy, precision and robustness were evaluated and all the experiments were performed in triplicate. All the standards and BGP solutions were prepared in methanol HPLC grade. The selectivity was considered for BGP hydroalcoholic extract in the 300 nm wavelength and established according to the separation

Please cite this article as: C. Arruda, V. Pena Ribeiro, M. Oliveira Almeida et al., Effect of light, oxygen and temperature on the stability of artepillin C and p-coumaric acid from Brazilian green propolis, J. Pharm. Biomed. Anal., https://doi.org/10.1016/j.jpba.2019.112922

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efficiency between the chromatographic peaks measured using the equation Rs = 2(dR2 − dR1)/(wb2 + wb1). It was also considered the relative retention times and UV spectra of the peaks corresponding to the standards. Veratraldehyde at 25 ␮g/mL was used as the internal standard (IS). Then, the standard concentrations were determined for construction of the analytical curve at five different concentrations: for Z-artepillin C, solutions at 3.6, 6.0, 24, 60 and 90 ␮g/mL; for Eartepillin C and ART2, solutions at 6.0, 15, 42, 60 and 90 ␮g/mL; and for E-p-coumaric acid, Z-p-coumaric acid, ART1 and ART3 solutions at 6.0, 24, 60, 90 and 105 ␮g/mL. The solutions of the standards were obtained by stock solution dilutions and addition of the IS. Limits of detection and quantification were estimated by the calibration curve equation and confirmed by construction of another analytical curve with five lower concentrations ranging from 0.36 to 6 ␮g/mL. Precision of the method was assessed by measuring the repeatability of the results at three concentrations at high (60 ␮g/mL), medium (42 ␮g/mL for E-artepillin C and ART2 and 24 ␮g/mL for the other compounds) and low (6.0 ␮g/mL) levels of the calibration curve performed in the same day (intraday precision) and in three different consecutive days (interday precision). Solutions at the same three concentrations were prepared and the comparison between the theoretical and real values furnished accuracy results. Since it was used an extraction method to obtain BGP extract in the stability studies, it was measured the recovery of this extraction method as well: 100 ␮L of the standards solution at 105, 60 and 30 ␮g/mL (high, medium and low levels) were added to 2.5 mg of raw powdered BGP. After solvent drying, three milliliters of a solution of methanol + IS were added to the sample, macerated for 1 h, followed by analysis. The samples concentration values were compared to the control (BGP extract without standards addition) and to the theoretical concentrations to calculate the percentage of recovery of the method. Finally, the robustness was determined by Plackett-Burman design for seven factors and eight experiments [5]: the nominal values of the wavelength, oven temperature, injection volume and flow rate were slightly changed to higher and lower levels, as: 295 and 300 nm; 25 and 35 ◦ C; 14.5 and 15.5 ␮L; 0.95 and 1.05 mL/min., respectively. In these experiments, it was taken into account the relative retention time and the concentration of the standards.  To calculate  the effects, the following equation was used Ex =

y(+)−

n/2

y(−)

[5] and they were expressed as variation

coefficients in percentage. Three concentrations were evaluated regarding the robustness in a low, medium and high level: 6, 42 and 90 ␮g/mL, respectively.

2.5. Stability studies As for the validation tests, all the experiments were performed in triplicate. The effects of light, temperature and oxygen in artepillin C and p-coumaric acid’s concentration were initially evaluated by a set of experiments varying these conditions, with presence or absence of light and oxygen, at 40 ◦ C or -20 ◦ C. The experiments were performed in accordance to the Box, Hunter & Hunter experimental design obtained by Statistica® . Four batches of experiments were performed with times of 7, 14, 21 and 30 days. In each assay, 50 ␮g of the pure compounds were placed in transparent glass flasks and stored in the conditions established by the experimental design. For the ones without oxygen, nitrogen was added to each flask, then closed and sealed with paraffin. After the set period of time, artepillin C, p-coumaric acid and their degradation products were quantified by the validated analytical method.

3

After determining the factors responsible for degradation of artepillin C and p-coumaric acid, another set of experiments was performed using a Central Composite Design for two factors: storage temperature (X1 ) and time (X2 ). In these assays, the samples were protected from the light and both pure compounds and the raw BGP were studied by placing 50 ␮g of the pure compounds and 2.5 mg of BGP in the flasks and exposing them to the condition set for each experiment. After that, BGP underwent the extraction procedure described for the recovery tests. For establishment of the number of experiments (N), the Central Composite Design uses the equation N = 2k + 2k + C; where k corresponds to the number of factors and C to the amount of central points. Therefore, a total of 10 experiments were conducted in one block. X1 and X2 were tested at low, medium and high levels, being -10, 20 and 40 ◦ C for X1 , and 10, 20 and 30 days for X2 . Two star points for each factor (-␣ and +␣ with ␣ value for rotatability of 1.414 [24]) were also evaluated: -20 and 50 ◦ C for X1 ; 6 and 34 days for X2 . The pure error was determined by four replicates. According et al. second-order polynomial equation to Albuquerque  [25], the  Y = ˇ0 + ˇi i + ˇii i 2 + ˇij i j explains response surface methodology experimental designs, where Y corresponds to the response and ␤0 , ␤i , ␤ii and ␤ij to the coefficients in the constant, linear and quadratic models; and the interaction coefficients, respectively. Xi and Xj represent the independent variables. The equations and other statistical parameters, such as the determination coefficient (R), and model consistency by the lack of fit analyses were obtained by Statistica ® software. To find an optimum storage condition, it was used the Statistica ® desirability function: the concentration of artepillin C and p-coumaric acid ranged from undesirable (0.0) to desirable (1.0), their lower and higher experimental concentrations, respectively.

3. Results and discussion 3.1. Obtainment of artepillin C, p-coumaric acid and their degradation products Artepillin C was isolated by preparative HPLC furnishing a white powder, which was identified by NMR means in comparison with published data [2]. The exposure of (E)-artepillin C and (E)-p-coumaric acid to sunlight for five days lead to the formation of their Z isomers as the major degradation products. (Z)-p-Coumaric acid and (Z)-artepillin C were coded as CA1 and ART4, ([M−H]- m/z 399,09; [M+H]+ m/z 301,18), respectively. According to Metternich et al. [26] (-)riboflavin had one of its double bond isomerized from E to Z by exposing the E isomer to a UV light that emits electromagnetic waves at 402 nm. They proposed that the reaction mechanism takes place by radical intermediates formation. Taking that into account and considering that natural sunlight spectrum is composed of 43% wavelength in the visible region (400–700 nm) and 4% in the ultraviolet region (320–400 nm) [27], we proposed the E–Z isomerization mechanism for artepillin C and p-coumaric acid (Fig. 1). The yield of CA1 was considerably high (25.6%) in comparison to ART4 (7.3%). One of the possible reasons is that artepillin C degraded into several other compounds, while p-coumaric acid furnished only one degradation product. The exposure of artepillin C to sunlight for more than five days leads to its continuous degradation over time. After 20 days of exposure, (E) or (Z)-artepillin C were no longer found in the samples, while (E) and (Z)-p-coumaric acids did not undergo additional degradation. Exposure of p-coumaric acid to high and temperature (50 ◦ C) for five days did not lead to significant degradation. On the other hand, for artepillin C this temperature led to the formation of many derivatives, being the major ones identified

Please cite this article as: C. Arruda, V. Pena Ribeiro, M. Oliveira Almeida et al., Effect of light, oxygen and temperature on the stability of artepillin C and p-coumaric acid from Brazilian green propolis, J. Pharm. Biomed. Anal., https://doi.org/10.1016/j.jpba.2019.112922

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Fig. 1. Proposed mechanism for artepillin C and p-coumaric acid degradation by sunlight exposure.

Fig. 2. Proposed mechanism for the formation of artepillin C degradation products after heat and air exposure.

as 2-propenoic (E)-3-[2,3-dihydro-2-(1-methylethyl)-7-prenyl-5benzofuranyl] acid (ART 1) ([M + Na]+ m/z 339,15; [M−H]- m/z 315,16), 2-propenoic (E)-3-(2,2-dimethyl-3,4-dihydro-3-hydroxi8-prenyl-2H-1-benzopyran-6-yl) acid (ART 2) ([M + Na]+ m/z 339,16; [M−H]- m/z 315,16) and 3-[2-dimethyl-8-(3-methyl-2butenyl) benzopiran]-6-propenoic acid (ART 3). Considering that ART1 and ART2 are isomers, it was undertaken a high resolution mass spectrometry analysis, in addition to the NMR analyses, to assure their identity. Their yields were 3.2, 3.9 and 3.9% respectively. The formation of these compounds are probably due to the presence of unsaturated carbons, which can undergo autoxidation when exposed to air, usually by radical mechanism, through formation of hydroperoxides: the reaction begins with an allylic hydrogen withdrawing, furnishing the respective radical and its resonance forms, which are able to react with triplet state oxygen, giving peroxyl radicals. After a hydrogen atom withdrawing, hydroperoxides are formed, which can give rise to secondary oxidized compounds [28]. Therefore, taking that into account, we have proposed the degradation mechanism (Fig. 2), in which occurs the formation of furan and pyranoxides artepillin C derivatives from a tertiary hydroperoxide that gives an epoxide, which further undergo hydroxyl group attack in one of the two carbons from

the epoxide, furnishing the degradation products ART1 and ART2. After dehydration of ART2, the compound ART3 can be formed. The reactions are apparently dependent of the temperature. The first two compounds (ART1 and ART2) have one asymmetric carbon each and are probably a mixture of their enantiomers because these compounds were produced by degradation, which is a process that usually furnishes compounds that are not enantiomeric pure. We had measured their optical rotations and we found that there’s an enantiomeric excess of the dextrorotatory enantiomer 25

25

([␣] D : +3.9 (c 0.5, CHCl3 ) for ART 1 and ([␣] D : +18.67 (c .0, CHCl3 ) for ART 2). 3.2. Development and validation of a RP-HPLC method to quantify artepillin C, p-coumaric acid and their degradation products To quantify the major degradation products artepillin C and p-coumaric acids, a RP-HPLC method was developed for quantification of these standards in both Brazilian green propolis crude raw material and its extract. The development of a validated and reliable RP-HPLC analytical method was undertaken to perform the analyses of the artepillic C, p-coumaric acid and their degradation

Please cite this article as: C. Arruda, V. Pena Ribeiro, M. Oliveira Almeida et al., Effect of light, oxygen and temperature on the stability of artepillin C and p-coumaric acid from Brazilian green propolis, J. Pharm. Biomed. Anal., https://doi.org/10.1016/j.jpba.2019.112922

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Inter day

99.31 ± 1.38 97.69 ± 2.36 100.13 ± 0.85 91.09 ± 0.53 92.06 ± 0.93 86.36 ± 1.14 – 100.63 ± 1.47 100.75 ± 0.86 102.86 ± 0.38 97.55 ± 0.94 94.94 ± 0.31 91.60 ± 0.85 97.94 ± 0.83 98.03 ± 0.77 96.96 ± 0.69 99.75 ± 1.38 98.63 ± 0.25 96.95 ± 0.94 101.01 ± 0.48 114.03 ± 1.17 105.39 ± 0.07 0.71 0.13 0.45 0.32 0.87 0.58 – 1.72 0.61 2.50 1.64 2.60 0.83 1.11 1.52 1.53 0.45 0.38 1.62 0.17 0.74 2.73 0.88 0.16 0.53 0.43 0.56 0.38 – 0.82 0.73 3.43 2.11 1.41 1.04 0.87 1.90 1.23 0.45 0.52 0.34 0.05 0.77 3.84 0.6 0.36 0.0313 −0.0144 0.9998 y = 0.0002 + 0.0177x 275.8; 241.4; 319.8

51.1

58.9

Z-Artepillin C

ART3

0.9997

1.20 0.0185 0.9998 y = 0.0037 + 0.0287x 304.3

50.6 E-Artepillin C

0.9999

−0.0355

0.36

0.60 0.38 0.0277 −0.0547 0.9997 y = 0.0116 + 0.0383x 311.5; 239.0; 222.5

41.2 ART2

0.9998

1.20 0.31 0.0423 −0.0673 0.9994 y=0.0026 + 0.0412x 319.8; 239.0; 222.5

36.4 ART1

0.9997

1.20 0.35 0.0727 −0.0818 0.9989 y = 0.0023 + 0.0242x 323.4; 239.0; 222.5

0.9994

–

0.60 0.38

– –

0.0460 −0.0457

– –

0.9999 y = 0.0138 + 0.0494x 297.2; 224.9

– IS (Veratraldehyde)

0.9998

– 230.8; 276.9; 309.1

9.4

13.1

Z-p-Coumaric acid

8.6 E-p-Coumaric acid

310.3; 227.2

y = 0.0193 + 0.1073x

0.9999

0.9999

−0.0436

0.0704

0.34

1.20

100.49 98.55 101.40 98.86 101.87 99.40 – 98.64 100.47 108.85 99.40 98.35 98.80 100.16 99.00 94.51 100.77 97.99 102.22 99.30 98.68 109.55 High Medium Low High Medium Low – High Medium Low High Medium Low High Medium Low High Medium Low High Medium Low

Intraday

Accuracy (%)

Precision (RSD)

5

Level Limits of Detection and quantification (␮g/mL) Maximum Observed residual value Minimum Observed residual value R R2 Equation Max UV absorptions Retention Time (min.)

After development and validation of the analytical method, it was performed four batches of experiments (after 7, 14, 21 and 30 days) to find out which factors affect significantly the concentration of stored artepillin C and p-coumaric acid overtime. It was used a Full Factorial Design for establishing the experiments and the 2way interaction model for processing the results, since the R value, which indicates the mathematical model adjustment, was higher than 0.95 for all batches. In the experiments without air oxygen, the samples were stored under nitrogen. Considering a p-value of 0.05, the presence of light, high temperature (40 ◦ C), air oxygen and the combination of them did not affect p-coumaric acid’s content. Although, after 30 days of exposure to combined light and high temperature, approximately 15% of its content underwent degradation. Regarding artepillin C, both the combination of light and temperature and these factors separately influenced its concentration in a significant manner: after seven days of exposure to light 22.5% of its content underwent degradation and after 14, 21 and 30 days it increased to 52.9, 54.9 and 86.5%, respectively (Table 2). In a similar way, the presence of light at 40 ◦ C caused degradation of 94.0, 98.9, 98.6 and 98.1% after 7, 14, 21 and 30 days, which means that in a short period of time almost all the amount of artepillin C underwent degradation. High temperature itself was also able to decrease artepillin C content, but in less extension: 19.97, 20.01 and 37.12% were degraded after 14, 21 and 30 days of exposure. The presence or absence of air oxygen did not affect the concentration

Compound

3.3. Stability studies

Table 1 Parameteres evaluated in the method validation: linearity, limits of detection and quantification, accuracy, precision and recovery.

products in the stability studies. To assure the reliability of the method, validation parameters such as linearity, limits of detection and quantification, accuracy, precision, extraction method recovery and robustness were evaluated. All the validation experiments were performed in triplicate. For linearity assessment, solutions of the standards at five different concentrations were analyzed by the RP-HPLC method and an analytical curve of each standard was drawn and statistically analyzed by minimum square method, furnishing the correlation (R) and determination (R2 ) coefficients values, along with the equation for their concentration calculation. The R values of all analytical curves were above 0.999 giving the linearity according to ANVISA [29] and ICH [30] validation guidelines. Besides, residual analyzes was performed, confirming homoscedasticity of the data. Also, the analytical curves do not have lack of fit (p > 0.05) and their angular coefficients are statistically different from zero (p < 0.05). For establishing the limits of detection and quantification, new solutions of the standards in lower concentrations were analyzed and for the limit of detection it was considered the concentration which was possible to distinguish the peak of the analyte from the base line. Regarding the limit of quantification, it was considered the concentration that the method was able to quantify with accuracy and precision, considering a 5% limit (Table 1). The accuracy of all samples was above 97.9%, except for (E)-artepillin C in the lower level, which displayed accuracy of 94.51%. Intra-day and inter-day precision demonstrated that the relative standard deviation of the responses were less than 4% for all standards. Therefore, both precision and accuracy are suitable according to ANVISA [29] and ICH [30]. The extraction method was adequate as well, since it was possible to extract more than 88% of (Z)-p-coumaric acid and more than 90% of the other standards from the crude green propolis matrix. Regarding the robustness, small variations in the wavelength, oven temperature, injection volume and flow rate did not have a significant effect (less than 20%) neither in the standards areas nor in the relative retention times. Even though, the oven temperature has caused a deviation of -17.59% in the concentration of ART2 at the higher level. Therefore, this parameter should be carefully adjusted to furnish a reliable response for this compound.

Extraction method Recovery (%)

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0.999 0.983 0.999 0.983 0.999 0.983 0.0497 0.2354 0.5018 0.5377 0.6281 0.9158

0.999 0.983 0.999 0.983 0.999 0.983 0.0079 0,1572 0.0248 0.2706 0.4096 0.6044

0.999 0.953 0.999 0.953 0.999 0.953 0.0455 0.4397 0.1361 0.6173 0.1297 0.3282

0.999 0.953 0.999 0.953 0.999 0.953 0.0091 0.4028 0.0159 0.4583 0.1641 0.4119

p-value

0.999 0.972 0.999 0.972 0.999 0.972

0.0332 0.2682 0.0998 0.4582 0.5516 0.5030

0.0066 0.3652 0.0122 0.2308 0.0839 0.6607

0.999 0.972 0.999 0.972 0.999 0.972

52.53 ± 6.28 43.20 ± 0.77 24.75 ± 8.54 43.88 ± 0.16 42.05 ± 3.66 42.70 ± 2.11 53.56 ± 2.30 42.52 ± 1.53 5.44 ± 7.63 39.95 ± 0.79 0.74 ± 1.04 35.30 ± 2.66 29.35 ± 6.93 43.55 ± 0.17 41.83 ± 4.88 42.00 ± 1.10 0.999 0.989 0.999 0.989 0.999 0.989 0.0055 0.1370 0.0094 0.1949 0.8004 0.4505

0.999 0.989 0.999 0.989 0.999 0.989

Concentration (␮g/mL) R value p-value

50.55 ± 6.31 42.82 ± 1.58 39.17 ± 6.77 41.94 ± 3.48 50.36 ± 2.25 42.49 ± 0.42 51.22 ± 7.71 42.94 ± 1.20 2.70 ± 0.80 35.24 ± 2.38 3.02 ± 0.02 38.68 ± 1.58 37.57 ± 7.52 41.28 ± 0.35 51.26 ± 1.19 42.89 ± 0.96

Concentration (␮g/mL) R value p-value

2 by 3

1 by 3

1 by 2

1+2 + 3

(3)Oxygen

(2)Temperature

Nominal value (no presence of 1, 2 or 3)

(1)Light

Compound

Artepillin C p-Coumaric acid Artepillin C p-Coumaric acid Artepillin C p-Coumaric acid Artepillin C p-coumaric acid Artepillin C p-Coumaric acid Artepillin C p-Coumaric acid Artepillin C p-Coumaric acid Artepillin C p-Coumaric acid

Factor

0.0094 0.2103 0.1858 0.3722 0.3876 0.5639

Concentration (␮g/mL) R value p-value

42.69 ± 0.01 43.37 ± 1.48 19.25 ± 0.02 41.92 ± 0.16 34.15 ± 1.01 42.08 ± 0.85 43.85 ± 2.96 41.03 ± 0.96 1.73 ± 2.84 45.02 ± 3.46 0.75 ± 1.06 38.72 ± 1.04 24.71 ± 0.01 41.52 ± 0.74 32.36 ± 8.60 57.31 ± 0.87

R value

Concentration (␮g/mL)

30 days 21 days 14 days 7 days

Table 2 p-values of ANOVA analyses of the full factorial experimental designs considering the presence or absence of light, high temperature and air oxygen on the concentration of (E)-artepillin C and (E)-p-coumaric acid.

53.37 ± 1.45 41.35 ± 2.48 7.20 ± 0.92 41.90 ± 0.54 33.56 ± 2.17 42.99 ± 0.51 53.69 ± 0.76 42.77 ± 1.02 1.00 ± 0.02 34.93 ± 5.49 1.00 ± 0.02 35.73 ± 3.41 7.53 ± 7.06 39.20 ± 3.11 35.53 ± 4.98 41.94 ± 1.06

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of the evaluated compounds. Therefore, light was the major factor responsible for the degradation of artepillin C at higher extension and, when combined with high temperature, it caused significant degradation not only of this compound but also of p-coumaric acid. ART1, ART2, ART3, (Z)-artepillin C and (Z)-p-coumaric acid were detected in a few experiments at low concentrations (below 1.24 ␮g/mL). Furthermore, they kept being formed and degraded overtime, which indicates that they are not stable either. Taking that into account, a central composite design was used to establish the best storage condition for (E)-Artepillin C, (E)-pcoumaric acid and Brazilian green propolis. In these experiments, two factors were evaluated: temperature (X1 ) and time (X2 ). The samples were protected from light in all experiments. One of the advantages of using an experimental design instead of performing experiments that vary one factor at a time is the assessment of the effect of the combination of the factors, besides the effect of each factor separately, which leads to more accurate results [25]. The quadratic model was selected to process the results because the R-squared value is considerably close to 1.0 for all the analyses (Table 3), which indicates that the dependent variable is well explained by this model. Moreover, the lack of fit value over 0.05 shows good model adequacy and predictability considering the pure error. From these data, it was obtained the empirical polynomial equation for each analysis, that furnishes the predictive response, where y corresponds to the concentration in ␮g/mL (dependent variable response); and X1 and X2 to the temperature in o C and time in days, respectively (independent variables). Considering the concentration of pure (E)-artepillin C, the temperature was statistically significant for both linear and quadratic terms and the time for linear term. Also, the interaction between them showed a p-value<0.05 as well (Table 3). Likewise, the temperature (linear terms) and the combination of temperature and time affected the content of (E)-artepillin C in BGP. On the other hand, the temperature did not significantly affect the concentration of isolated p-coumaric acid in the BGP, which means that this compound should be stable at high storage temperatures. By analyzing the 3D plots of the effects of X1 and X2 , as well as the interaction between them (Fig. 3) on the concentration of (E)-artepillin C, it was possible to observe that as the temperature and time increases, the concentration decreases, even at room temperature (20 ◦ C), in which there was a significant degradation of this compound. After 30 days at 50 ◦ C all content of (E)-artepillin C underwent degradation. By using the desirability function aiming at keep the highest concentration, it was established as the best storage temperature for pure (E)-artepillin C -2.5 ◦ C. On the other hand, (E)-p-coumaric acid after 30 days of storage at temperatures between 40 and 50 ◦ C did not undergo significant degradation. However, after 30 days at 50 ◦ C, approximately 13% of this compound underwent degradation. The desirability showed that at 32.5 ◦ C, the concentration of (E)-p-coumaric acid was maintained, when the sample was protected from light. Artepillin C in BGP underwent degradation when exposed to high temperature over time: above 50 ◦ C after 30 days approximately 50% of the initial amount underwent degradation. The desirability showed that the best storage temperature is -2.5 ◦ C for the raw material, as well. Regarding p-coumaric acid content in BPG, despite of the desirability establishing that the best storage temperature was ≤ 20 ◦ C, the 3D plots showed that the concentrations of this compound up to 50 ◦ C remained between 4.6 and 5.8 ␮g/mL, even after 30 days. Therefore, for p-coumaric acid, storage at room temperatures did not lead to significant degradation. Thus, the ideal conditions for storage and transportation of BGP, considering both (E)-artepillin C and (E)-p-coumaric acid content should be protection from light and at temperatures below -2.5 ◦ C. This condition shall be also ideal for storage of the isolated compounds, although (E)-p-coumaric acid, pure or in BPG, was stable at room temperature protected

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Table 3 o Statistical data from ANOVA of Lack of Fit and Regression of the Quadratic Model analyses for the Response concentration. Variables: X1 : Temperature ( C) and X2 : Time (days). Response

R2

R

Predictive response equation

y = 68.89554 + 0.40847*X1 -0.01471*X1 2 0.18603*X2 +0.00262*X2 2 -0.03062*X1 *X2 y = 48.82206 + 0.24541*X1 0.00404*X1 2 +0.07012*X2 -0.00098*X2 2 0.01336*X1 *X2 y = 60.19528 + 0.03078*X1 -0.00070*X1 2 0.36096*X2 +0.00555*X2 2 -0.00128*X1 *X2 y = 5.77492-0.00987*X1 -0.00012*X1 2 0.01607*X2 +0.00035*X2 2 -0.00008*X1 *X2

Lack-of-fit (p-value)

Linear (L)/quadratic (Q) main effects (p < 0.05)

(E)-Artepillin C

0.222890

X1 (L/Q), X2(L), X1/X2

0.98601

0.99298

(E)-Artepillin C in BGP

0.123302

X1(L), X1/X2

0.89532

0.94621

(E)-p-Coumaric acid

0.155413

X2(L)

0.92337

0.96092

(E)-p-Coumaric acid in BGP

0.495247

–

0.85478

0.92454

Fig. 3. Response surface (3D) plots of the effects of X1 (temperature) and X2 (time) on the concentration of (E)-artepillin C and (E)-p-coumaric acid in BGP and pure.

from light. Therefore, the storage of (E)-p-coumaric acid in plastic commercial bottles is suitable for preventing its degradation. After six months stored in a freezer at-10 ◦ C protected from light, there were 98% of the initial amounts of the pure compounds, and in BGP there were 100% of (E)-p-coumaric acid and 91% of (E)-artepillin C remaining. The producers and companies involved in the production and commercialization of this important bee product should also take measures to protect this product from degradation by mainly incidence of light and high environmental temperatures both in transportation and storage of green propolis.

4. Conclusions When exposed to sunlight both (E)-artepillin C and (E)-pcoumaric acid furnished their Z isomers as major degradation products, and at higher temperatures, (E)-artepillin C furnished, ART1, ART2 and ART3. Additionally, the RP-HPLC developed method to quantify these compounds displayed linearity, accuracy, precision and robustness according to ANVISA and ICH guidelines. Finally, the stability studies demonstrated that (E)-artepillin C is sensitive to light and temperature, and that (E)-p-coumaric acid is stable, unless exposed simultaneously to high temperature and light. The best storage condition for artepillin C pure or in BGP is at temperatures lower than -2.5 ◦ C and protected from light. (E)-pCoumaric acid can be stored at room temperature in plastic bottles commonly used by industries, without significant loss in its content.

Acknowledgements Authors acknowledge the São Paulo Research Foundation (FAPESP) grant number 2017/04138-8 for funding this work, CNPq and CAPES for both financial support and scholarships, as well as to the University of São Paulo for providing the infrastructure. We are also thankful to Cézar Ramos Junior from Natucentro and Bee Propolis Companies for providing propolis samples and for enriching the discussions and ideas during the development of this project. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jpba.2019. 112922. References [1] F. Pellati, G. Orlandini, D. Pinetti, S. Benvenutia, HPLC-DAD and HPLC-ESI-MS/MS methods for metabolite profiling of propolis extracts, J. Pharm. Biomed. Anal. 55 (2011) 934–948, http://dx.doi.org/10.1016/j.jpba. 2011.03.024. [2] P. Costa, M.O. Almeida, M. Lemos, C. Arruda, R. Casoti, L.B. Somensi, T. Boeing, M. Mariott, R. de C.M.V. de A.F. da Silva, B.D.P. Stein, P. de Souza, A.C. dos Santos, J.K. Bastos, L.M. da Silva, S.F. de Andrade, Artepillin C, drupanin, aromadendrin-4 -O-methyl-ether and kaempferide from Brazilian green propolis promote gastroprotective action by diversified mode of action, J. Ethnopharmacol. 226 (2018) 82–89, http://dx.doi.org/10.1016/j.jep.2018.08. 006.

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