Development of a Validated High-Performance Liquid

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/JAFC

Development of a Validated High-Performance Liquid Chromatography Method and Optimization of the Extraction of Lignans from Piper cubeba Caroline Arruda,† Jennyfer Andrea Aldana Mejía,† Victor Pena Ribeiro,† Larissa Costa Oliveira,‡ Márcio Luis Andrade e Silva,‡ and Jairo Kenupp Bastos*,†

Downloaded via UNIV OF LOUISIANA AT LAFAYETTE on January 8, 2019 at 17:56:11 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Avenida do Café s/n, Ribeirão Preto, São Paulo 14040-930, Brazil ‡ Grupo de Pesquisas em Produtos Naturais, Núcleo de Ciências Exatas e Tecnológicas, Universidade de Franca, Avenida Dr. Armando Salles de Oliveira, 2001, Franca, São Paulo 14404-600, Brazil S Supporting Information *

ABSTRACT: Piper cubeba L. f. is a food seasoning, which contains secondary metabolites displaying several biological properties, such as cytotoxic, anti-inflammatory, and antiparasitic activities. The lignans (+)-dihydroclusin, (−)-clusin, (−)-cubebin, (−)-yatein, and (−)-haplomyrfolin were isolated, with (−)-haplomyrfolin reported for the first time in P. cubeba seeds. Chromatographic standards were used to develop a reliable reverse-phase high-performance liquid chromatography analytical method according to the Agência Nacional de Vigilância Sanitária and International Conference on Harmonization guidelines to quantitate these lignans in both P. cubeba seeds and their extracts. The extraction of the lignans was also optimized, with the best conditions being ultrasound-assisted extraction, with 84% aqueous ethanol for 38 min in a single extraction. This procedure allows for the extraction of more than 80% of the total lignans, which is better in comparison to other techniques, such as maceration and Soxhlet extraction. KEYWORDS: (−)-cubebin, Piper cubeba, RP-HPLC, lignans, extraction optimization

■

INTRODUCTION Plants from the Piper genus are found worldwide and belong to the Piperaceae family.1,2 Piper cubeba L. f. occurs mainly in Europe and Asia, especially in India, Morocco, China, and Indonesia.1 Traditionally, this plant has been used as a culinary spice as a result of its flavor, smell, and color. In ancient times, this plant was also used for several medicinal purposes, including for the treatment of dysentery, syphilis, and fever as well as a pain killer.1 Several of its medicinal properties have been proven by scientific studies, including anti-inflammatory and analgesic,3 antimicrobial,1 antiestrogenic,4 hepatoprotective,5 antiparasitic,6 and cytotoxic7 activities. These biological activities are mainly due to the presence of bioactive lignans, such as (−)-cubebin, (−)-hinokinin, clusin, (−)-dihydrocubebin, (−)-dihydroclusin, and (−)-cubebinin.8−10 Considering that lignans are the main compounds in P. cubeba responsible for the reported biological activities, the development of validated analytical methods is necessary for quality control. The use of analytical methods can ensure the identity of the plant material and the quantitation of its major compounds, for which the validation process proves and documents the application and reliability of the method.11 Different agencies, such as the U.S. Food and Drug Administration (FDA), Agência Nacional de Vigilância Sanitária (ANVISA), and International Conference on Harmonization (ICH), provide guidelines for validation, which can be selected according to the type of analyses to be performed. Thus, considering that there is no analytical method reported for P. cubeba seeds, we report the © XXXX American Chemical Society

development of a reliable reverse-phase high-performance liquid chromatography (RP-HPLC) method for the quantitation of the main lignans in both P. cubeba seeds and their extracts. Additionally, the validation of the analytical method was performed according to ICH12 and ANVISA.13 With regard to the extraction of lignans from this plant, it has usually been accomplished by maceration with 96 or 70%1,5 ethanol. Graidist et al.7 used methanol and dichloromethane as extraction solvents for 72 h to obtain two fractions rich in lignans. Using a Soxhlet apparatus, Haribabu et al.14 performed extraction with methanol under heat for 12 h. In the comparison between these methods, ethanol is often chosen as the extraction solvent because it is allowed to be used in the supplement industry and is not harmful to the environment and animals. Additionally, it is capable of extracting phenolic compounds, such as lignans, at low temperatures, and no data regarding the stabilities of the main P. cubeba lignans are available. The development of a validated high-performance liquid chromatography (HPLC) analytical method and the optimization of the extraction process have been performed for the first time. The optimum extraction time and ethanol/water ratio were evaluated using a central composite experimental design to determine the best conditions to furnish a better yield of lignans from the seeds of P. cubeba. Received: Revised: Accepted: Published: A

September 30, 2018 December 24, 2018 December 25, 2018 December 25, 2018 DOI: 10.1021/acs.jafc.8b05359 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

■

Selectivity. To determine the selectivity, the wavelength was set according to the UV spectra of the lignans, which were recorded using a photodiode array (PDA) detector, and the separation of the chromatographic peaks was measured according to its resolution. The purity of each peak was verified by comparison of the retention time and UV spectrum to authentic standards previously isolated. Additionally, the extract was analyzed by LC−MS to confirm that each peak corresponded to one single compound because the lignans found in the P. cubeba extract show similar UV spectra. Linearity, Limit of Detection, and Limit of Quantitation. These parameters were assessed by the construction of an analytical curve of each target compound from 15 to 200 μg/mL in methanol. For (+)-dihydroclusin, the curve concentration range was from 25 to 200 μg/mL. In each solution, veratraldehyde was added as the internal standard at 50 μg/mL. For each calibration experimental level, three genuine replicates were performed. To plot the analytical curve, the ratio between the area of the standard and the internal standard was the response on the y axis and the respective concentrations were plotted on the x axis. Statistica 8 (StatSoft, Inc.) was used to plot the analytical curve as well as to obtain the equation to calculate the concentration and R value, which is a parameter to measure the linearity of the responses. The lack of fit and residual dispersion of the analytical curve were obtained as well. From the analytical curve, the limits of detection and quantitation of the method were calculated according to the following equations: LD = 3.3SD/IC and 10SD/IC, respectively, in which SD is the standard deviation of the intercept of the y axis when x is equal to 0 obtained from three analytical curves and IC is the slope of the analytical curve. Precision and Accuracy. The intraday precision was evaluated at high, medium, and low concentrations of 200, 75, 50, and 25 μg/mL by injecting solutions of the standards at these concentrations on the same day. The interday precision was evaluated by injecting the same samples on three different days. The accuracy was determined by comparison between the real concentrations and the theoretical concentrations of the solutions at the same levels considered for precision. Robustness. A full standard design15 was selected to measure the capacity of the method to quantitate the standards by deliberately making small changes in the run conditions within three different levels. The parameters measured were the wavelength, flow rate, and mobile phase composition. The low, medium, and high levels were as follows: wavelength, 277, 280, and 283 nm; flow rate, 0.9, 1.0, and 1.1 mL/min; mobile phase composition, 39−55, 40−56, and 41−57% of acetonitrile. The effect of changing the column to a 150 × 4.60 mm inner diameter, 4 μm, Synergi Fusion-RP (Phenomenex, Torrance, CA, U.S.A.) on the method robustness was also evaluated. Recovery. A total of 5 g of ground P. cubeba seeds was exhaustively extracted with 400 mL of 96% ethanol for 12 h using a Soxhlet apparatus. The plant material was then dried in an aircirculating oven at 40 °C for 3 h. Both the plant material and dried extract were analyzed using the developed HPLC method. After that, 250 mg of this plant biomass was spiked with (−)-cubebin by adding 1.5 mL of an ethanolic solution of the standard at 500 μg/mL (high level). Likewise, the same amount of plant material was spiked with 1.0 mL (medium level) and 0.50 mL (low level) of the same solution. The spiked matrix was then dried at room temperature. It was extracted using the optimized ultrasound-assisted method as follows: in each flask, 5 mL of ethanol/water (84:16, v/v) was sonicated for 38 min. The samples were filtered and analyzed by HPLC to quantitate the standard compounds. Veratraldehyde was added to the extraction solvent as the internal standard, and benzophenone was used as the secondary internal standard at 50 μg/mL. The experiments were performed in quadruplicate, and the recovery percentage was calculated considering the theoretical and real concentration values. Matrix Effect. To seven different flasks each containing 250 mg the dried plant biomass, 5 mL of a solution of (−)-cubebin at 15, 25, 50, 75, 125, 150, and 200 μg/mL was added to the respective flasks. One additional flask containing the plant biomass without spiking with (−)-cubebin was used to determine the concentration of this compound in the seeds. Thereafter, the extraction was performed in

MATERIALS AND METHODS

Chemicals. Analytical ethanol was obtained from Labshynth (Diadema, Brazil). HPLC-grade methanol and acetonitrile were purchased from J.T. Baker (Ecatepec, Mexico). HPLC-grade chloroform was obtained from Tedia (Fairfield, OH, U.S.A.). Isopropanol was obtained from Honeywell (Morris Plains, NJ, U.S.A.). Formic acid, hydrochloric acid, and hydrogen peroxide were obtained from Synth (São Paulo, Brazil). Water was purified using a Milli-Q system from Millipore (Bedford, MA, U.S.A.). Veratraldehyde and benzophenone standards were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Isolation of the Major Lignans from P. cubeba. A total of 15 g of dried and powdered P. cubeba seeds bought from Floral Seeds (New Delhi, India) was extracted with 400 mL of 96% ethanol for 24 h in an Innova 4300 incubator shaker (New Brunswick Scientific, Edison, NJ, U.S.A.) at 35 °C and 120 rpm, followed by filtration and concentration under vacuum using a Buchi rotary evaporator. The procedure was repeated 3 times in sequence, and the extract was lyophilized using a LIOTOP-K105 freeze-dryer, furnishing 2.9 g of crude extract. Then, 500 mg of the crude extract was solubilized in 1.5 mL of methanol, filtered through 0.45 μm filters (Millipore, Burlington, MA, U.S.A.), and then injected into Prominence preparative HPLC (Shimadzu, Kyoto, Japan) interfaced with a SPD-20 ultraviolet (UV) detector and a FCR-10A automatic sample collector. The column used was a 250 × 10 mm inner diameter, 4 μm, semi-preparative Polar-RP (Phenomenex, Torrance, CA, U.S.A.). The injection volume was 100 μL in each run. The mobile phase consisted of 97.7% water, 0.3% formic acid and 2% isopropanol (v/v/v) (solvent A) and acetonitrile (solvent B). The flow was adjusted to 4 mL/min, and the UV detector was set at 280 nm. The gradient used was as follows: 0.01−19.00 min, 40−56% B; 19.00− 20.00 min, 56−40% B; and 20.00−23.00 min, 40−40% B. The chemical structures of the purified compounds were determined by analyses of the 1H and 13C nuclear magnetic resonance (NMR), heteronuclear single quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC), and mass spectrometry (MS) data. NMR analyses were recorded using a DRX 500 spectrometer at 500 MHz (Bruker, Billerica, MA, U.S.A.). The solvent used was CDCl3 without tetramethylsilane (TMS) from Cambridge Isotope Laboratories (Tewksbury, MA, U.S.A.). As a reference, it was used as the solvent signal. Liquid chromatography− mass spectrometry (LC−MS) used for the analyses was a H-Class Acquity UPLC system (Waters, Milford, MA, U.S.A.) coupled to a quadrupole tandem Xevo TQ-S with a Z-spray source working in the negative mode under the following parameters: capillary voltage, −2.5 kV; cone voltage, −40 V; Z-spray temperature, 150 °C; desolvation temperature (N2), 350 °C; desolvation gas flow rate (N2), 600 L/h; mass range, m/z 100−1000; and collision gas for the selected precursor ions, argon. Data were processed using Mass Lynx V4.1 software. The specific rotation ([α]25 D ) was determined using a Jasco P-2000 polarimeter (serial number A104161232, MD) at 25 °C and a wavelength of 589 nm. All samples were dissolved in HPLC-grade chloroform, and three readings were recorded. Development and Validation of the RP-HPLC Method. The method was developed and validated using a HPLC 1525 binary pump system coupled to a 2998 ultraviolet−visible (UV−vis) detector and a 2707 auto collector (Waters, Milford, MA, U.S.A.) using EMPOWER 3 processing software. All validation and method development experiments were performed in triplicate. The column used was a 150 × 4.6 mm inner diameter, 4 μm, Synergi Polar-RP (Phenomenex, Torrance, CA, U.S.A.), and the mobile phase consisted of 97.7% water, 0.3% formic acid and 2% isopropanol (v/v/v) (solvent A) and acetonitrile (solvent B). The gradient was as follows: 0.01−19.00 min, 40−56% B; 19.00−20.00 min, 56−40% B; and 20.00−23.00 min, 40−40% B. Veratraldehyde was chosen as the internal standard. The set injection volume and oven temperature were 20 μL and 35 °C, respectively. The wavelength read was 280 nm, and the flow rate was 1 mL/min. B

DOI: 10.1021/acs.jafc.8b05359 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article


Figure 1. Chromatograms of P. cubeba fruit crude extract (red) and chromatographic standards (blue). Internal standard, veratraldehyde, 1; (+)-dihydroclusin, 2; (−)-clusin, 3; (−)-haplomyrfolin, 4; (−)-cubebin, 5; and (−)-yatein, 6. where k is the number of factors and C0 is the number of central points. A total of 16 experiments (runs) were conducted in two blocks. The ethanol percentage, time of extraction, and number of extractions, coded as X1, X2, and X3, respectively, were evaluated at low, medium, and high levels as follows: X1, 50, 70, and 90%; X2(USAE), 10, 20, and 30 min; X2(MAE), 1, 2, and 3 h; and X3, 2, 3, and 4 times. Besides the factorial points, two star points denoted as −α and +α were considered (α value for the rotatability of 1.6818): X1, 35 and 100%; X2(USAE), 3 and 38 min; X2(MAE), 0.3 and 3.7 h; and X3, 1 and 5 times. Six replicates were used to evaluate the pure error. Data obtained by the USAE experiments were analyzed by a general model for the response value (Y) explained by a second-order polynomial equation, which was used to predict the optimum conditions of the extraction process

the same way as described in the recovery experiments, followed by analysis. Using the areas of the (−)-cubebin peak and the internal standard at 50 μg/mL, the analytical curve in the matrix was plotted, which was compared to the analytical curve of (−)-cubebin in solution to verify if the plant matrix influences the quantitation of the lignans. The angular coefficients of the two analytical curves were compared to verify the parallelism between them. Stability. As in the matrix effect and recovery experiments, (−)-cubebin was chosen as the representative of the lignans from P. cubeba. Stability toward acid, alkaline, oxidative, and high-temperature conditions was assessed. For that assessment, 120 μg of the sample in 120 μL of methanol was mixed with 1 mL of 0.1 M hydrochloric acid, reaching pH 1. The assessment was performed in the same way with sodium hydroxide, reaching pH 13. The same amount of standard was exposed to a temperature of 70 °C in an air-circulating oven. The stability in an oxidative environment was assessed by adding 0.1 mL of 30% hydrogen peroxide to a small flask containing 120 μg of (−)-cubebin. Additionally, the standard stability in hydroalcoholic solution stability in 84% ethanol after ultrasound exposure was evaluated. The experiments were kept in these conditions for 4 h. Additionally, the (−)-cubebin stability after light exposure was determined: flasks with 120 μg of the (−)-cubebin were kept in a 15 cm metallic sealed container under light (Daylight Taschibra, Indaial, Brazil, LED 500 lm, 6 W, 6500 K, and 83 lm/W). The experiments were conducted for 4 days. After that, the acid and alkaline samples were neutralized to pH 7, frozen, and lyophilized. The samples with hydrogen peroxide were also frozen and lyophilized. The samples exposed to ultrasound were dried. Then, 1 mL of methanol with the internal standard at the determined concentration was added to all samples, and 20 μL of each was injected into HPLC using the developed method. Optimization of Lignan Extraction from P. cubeba Using Ultrasonication and Maceration. Plant Material. The dried seeds of P. cubeba were ground, and the particle size of the samples was standardized using a sieve with a mesh size of 42 (pore size of 0.354 mm). The powder was stored in airtight bags until use. Experimental Design: Central Composite Design (CCD) and Three-Dimensional (3D) Surface Plots. To compare the traditional maceration extraction (ME) of the lignans of P. cubeba with the ultrasound-assisted extraction (USAE), two different batches of experiments were carried out considering the following three variables: ethanol percentage, time of extraction, and number of extractions. The ME was performed at 30 °C and 120 rpm using an incubator shaker. The USAE was performed using an ultrasonicator. The volume of solvent used to perform the extraction of 250 mg of plant biomass was 5 mL. The design of both experimental studies was established using the software STATISTICA 8 (StatSoft, Inc.), which consisted of a central composite rotatable design with three factors studied at three levels. The number of experiments (N) required was defined according to the following equation:

N = 2k(k − 1) + C0

Y = β0 +

∑ βi Xi + ∑ βiiXi 2 + ∑ βijXiXj

(2)

where Y is the response function, β0, βi, βii, and βij represent the model constant, linear, quadratic, and interaction coefficients, respectively, and Xi and Xj are the independent variables. The same software was used for statistical data processing, performing the analysis of variance (ANOVA), and determination of the regression coefficients of individual linear, interaction, and quadratic terms. The interactive effects were represented by 3D surface plots from the fitted polynomial equation. Data related to the maceration process were used for comparison of the responses of each equivalent experiment in USAE optimization. Analytical Determinations of the Extracts: Concentrations of the Seed Lignans and Yield Percentages. The response of the model was expressed as a function of the concentrations of the compounds and the yield percentage of each USAE experiment. The yield percentage obtained in each experiment was calculated considering the mean of the dried mass of the triplicates (mg) and the initial mass of the ground P. cubeba seeds used (250 mg). The concentrations of the analytes were determined by the area ratio between the analyte and the internal standard (veratraldehyde, 50 μg/mL) using the validated RP-HPLC method. The concentrations of the lignans obtained from the extraction of the seeds with the optimized method were compared to the conventional Soxhlet extraction. Desirability Function. STATISTICA 8 software was used to obtain the profile of desirable responses using a scale in the range from 0.0 (undesirable) to 1.0 (very desirable) to obtain a global function (D), in which their maximum and minimum values were obtained from both the dried seed extract mass and the concentration obtained from each experiment.

■

RESULTS AND DISCUSSION Isolation of the Major Lignans from P. cubeba. From the crude ethanolic extract of P. cubeba, six compounds were isolated, 1−6, numbered according to the order of elution observed in the chromatogram (Figures 1 and 2).

(1) C


Article


Figure 2. Chemical structures of the lignans isolated from P. cubeba.

Table 1. Linearity Data, Retention Time, and Maximum UV Absorptions of the Lignans from P. cubeba

compound

retention time (min)

(+)-dihydroclusin (−)-clusin (−)-haplomyrfolin (−)-cubebin (−)-yatein

6.0 7.9 9.5 10.2 12.5

maximum UV absorptions 321.9 233.1 241.4 236.7 234.6

and and and and and

284.1 286.5 284.1 286.5 285.3

equation y y y y y

= = = = =

−0.0006 + 0.0015x −0.0012 + 0.0024x 0.0008 + 0.0011x 0.0025 + 0.0047x 0.002 + 0.0024x

R2

R

minimum observed residual value

0.9993 0.9995 0.9998 0.9997 0.9999

0.9996 0.9997 0.9999 0.9998 0.9999

0.034965 0.032621 0.014700 0.075133 0.035301

maximum observed residual value 0.296438 0.474205 0.202176 1.028757 0.480455

limits of detection and quantitation (μg/mL) 3.24 1.96 2.27 2.14 2.15

9.82 5.96 6.88 6.47 6.51

following peak correlations were found: peak 1, tR of 6.08 min, corresponded to (+)-dihydroclusin, 1; peak 2, tR of 8.02 min, corresponded to (−)-clusin, 2; peak 3, tR of 9.5 min, corresponded to (−)-haplomyrfolin, 3; peak 4, tR of 10.2 min, corresponded to (−)-cubebin, 4; and peak 5, tR of 12.5 min, corresponded to (−)-yatein, 5 (Figures 1 and 2). The UV spectra of each peak and its respective standard matched perfectly, indicating that each peak corresponds to the respective chromatographic standard. The first peak in the chromatogram at 4.3 min corresponds to veratraldehyde, the internal standard. Considering that these lignans show similar UV spectra, the extract was also analyzed by LC−MS to unequivocally identify the compounds. The obtained mass spectrometric data of the compounds in the extract and the data of the standards also matched, confirming the identity of the compounds and that there was no co-elution with other compounds. The wavelengths of maximum UV absorption of each standard along with their respective retention times are displayed in Table 1. Linearity, Limit of Detection, and Limit of Quantitation. The peak areas displayed a proportional response according to

Among the isolated compounds, compounds 4 (57.2 mg) and 2 (46.0 mg) had the highest yields of 9.2 and 11.5%, respectively. Compounds 1 (13.9 mg), 6 (23.0 mg), and 5 (18.5 mg) furnished between 2.7 and 4.6%, and the lowest yield was obtained for compound 3 (9.4 mg), with 1.8%. With regard to their chemical structures, the lignans from P. cubeba were identified as (+)-dihydroclusin, (−)-clusin, (−)-haplomyrfolin, (−)-cubebin, (−)-yatein, and (−)-hinokinin (Figure 2). Their identities were established according to NMR, MS, and specific rotation measurements in comparison to the literature.16−19 Compound 3, identified as (−)-haplomyrfolin, is reported in P. cubeba seeds for the first time. Development and Validation of the RP-HPLC Method. Selectivity. The isolated lignans with estimated purities higher than 98% were used as standards for the validation of the analytical method. Peak 6, with retention time (tR) of 15.4 min, identified as (−)-hinokinin, 6, did not display suitable peak purity and was not used for quantitation. The other compounds showed adequate peak purities. The UV spectrum and retention time of each peak in the extract were compared to those of the authentic standards, and the D


Article


Robustness. The wavelength, mobile phase composition, and flow rate were selected because they could possibly influence in the quantitation of the lignans.8,22,23 It was found that the modification of both the mobile phase and flow rate did not significantly change the concentration (p > 0.05) of the compounds after analysis. However, when the wavelength was slightly changed, all lignans displayed significant divergence at the high, medium, and low points of their analytical curves. This indicates that the exact wavelength is necessary for an accurate analysis with the goal of quantitating P. cubeba lignans. This could be foreseen as a possible result, because at different wavelengths, the absorption of these lignans changes significantly, as proven by the experiments undertaken. At high concentrations of the compounds, besides the wavelength, the mobile phase composition also caused significant disparity in (+)-dihydroclusin and (−)-cubebin concentration measurements. The (−)-cubebin concentration was affected by the flow rate change as well. This indicates that, when the analyzed compounds are present in concentrations above the concentrations set at the analytical curve middle point, the wavelength, flow rate, and mobile phase composition must be adjusted for accurate quantitation results. In addition, there were no significant interactions between these factors. Therefore, the developed method presents robustness for most compounds with small variations in the flow rate and mobile phase composition, but the wavelength should be carefully adjusted for reliable results. With regard to column changes, by replacing the Polar-RP stationary phase with a Fusion-RP column, the ratios between the areas of the standards and internal standard, which are proportional to their concentrations, varied less than 20%. For (+)-dihydroclusin, (−)-cubebin, and (−)-yatein, the percentage of variation was from 3.2 to 9.3%, and for (−)-clusin and (−)-haplomyrfolin, it was from 12.4 to 19.9%. These data show that changing the column affects the quantitation of (−)-clusin and (−)-yatein more in comparison to the other lignans. On the other hand, because the variations are less than 20%, the method is considered robust using another column stationary phase.24 Stability. As in the recovery and matrix effect studies, (−)-cubebin was used as a representative for the stability studies of lignans from P. cubeba. After cubebin was exposed to ultrasound, light, and 30% hydrogen peroxide oxidation, no significant degradation was observed (0, 0, and 3.75% loss, respectively), which indicates that cubebin is stable in these conditions. In the same manner, temperatures up to 70 °C did not promote a significant decrease in the (−)-cubebin concentration (1.2% loss). On the other hand, acid and alkali have caused a significant degradation of (−)-cubebin by 88.4% at pH 1.0 and 50.8% at pH 13. Optimization. Lignan Extraction: Comparison between ME and USAE. The traditional ME of P. cubeba compounds is generally performed using 96% aqueous ethanol in sequential extractions, as reported in the literature.1,8 Although this process is efficient for the extraction of these compounds, it involves long extraction times and high volumes of organic solvents, leading to interest in the study of other extraction processes that can be optimized. USAE is a simple and inexpensive method that, as a result of its effect of acoustic cavitation, can disrupt cell walls, enhancing the extraction efficiency.25,26 A paired-sample t test was conducted to compare the responses obtained in each ME experiment to the equivalent

the concentrations of the standards, which is shown by the correlation (R) and determination (R2) coefficients calculated by regression analysis. From the analytical curves, the equations for the calculation of the concentrations in P. cubeba extract samples as well as the correlation coefficients and limits of detection and quantitation were also obtained (Table 1). These results demonstrated that the method has a linear response according to ICH12 and ANVISA,13 considering that the correlation coefficient is higher than 0.99. Additionally, the results of residual values and dispersion of the residues confirm the linearity by showing the random distribution of residues and homoscedasticity of data. Residual minimum and maximum values are depicted in Table 1 as well. The plotted curves did not show a significant lack of fit because the p values for the lack of fit analyses are >0.05. Precision and Accuracy. According to ANVISA13 and ICH12 guidelines, the precision should be evaluated regarding the repeatability (intraday precision) and intermediate precision (interday precision) considering the standard deviation between the samples. Both the intra- and interday precision values showed relative standard deviations (RSDs) lower than 5%, which demonstrates low variation between the analysis at low, medium, and high concentrations considering the concentration range of the analytical curve. With regard to the accuracy, it consists of the proximity between the theoretical and experimental values of the concentration. The obtained accuracy results indicated that the analytical curves of the method are able to furnish accurate results because the recovery percentages were approximately 100%, with variations from 96.6 to 100.9%. Therefore, the developed method can be considered accurate and precise. Recovery. A Soxhlet apparatus was used to perform the extraction of lignans from P. cubeba seeds because this technique allows for the total extraction of phenolic compounds.20,21 The extract yield was 17.6% (882 mg of extract from 5 g of seeds). The total amount of (−)-cubebin in the seeds was 321.93 mg/100 g, which corresponded to 0.3%. Then, the plant matrix without lignans was spiked with (−)-cubebin and extracted using the optimized ultrasound extraction method. It was found that recovery was very good, between 93 and 107% at low, medium, and high concentrations. Thus, the optimized method can be considered reliable for performing the extraction of (−)-cubebin and other chemically similar lignans. Because (−)-cubebin is the major compound in the extract of P. cubeba seeds and belongs to the same lignan subclass as the other major compounds, it was used as a representative lignan for recovery studies. Furthermore, the recovery of the internal standard was also high, between 95 and 105%. Matrix Effect. To evaluate the matrix effect in the quantitation of lignans, the parallelism between the lines obtained from analytical curves of (−)-cubebin in solution and in the crude extract after spiking (−)-cubebin in the same concentrations as those of the analytical curve was verified. To be parallel, two lines should present similar angular coefficients,15 which was observed statistically after applying the t test between the angular coefficients of the obtained analytical curves. The p value was >0.05. Therefore, no significant difference was observed between them, which confirms the parallelism between the lines and that there was no significant matrix effect in the quantitation of the samples, which is in accordance with ANVISA.13 E


Article


maximum in the desirability function, were 84% ethanol, 38 min, and one single extraction. Comparison of USAE to the Traditional Soxhlet Extraction Method. To determine the efficiency of the USAE method for the extraction of lignans of P. cubeba, a comparison was performed between the concentrations found using the USAE optimized method and the concentrations found using the Soxhlet recovery technique. The results (Table 2) showed that the concentrations furnished by the optimized

experiment using USAE. The ultrasound method displayed similar results to the traditional ME with no statistical differences in the responses, which were expressed in terms of the concentrations of the five identified lignans and yield percentages. Only experiment 6 (90% EtOH, 1 h on ME/10 min on USAE, and 4 extraction cycles) and experiment 9 (70% EtOH, 2 h on ME/20 min on USAE, and 3 extraction cycles) showed statistical significance for the concentrations of (+)-dihydroclusin and (−)-clusin, respectively, exhibiting higher concentrations of these compounds in the maceration experiments. With regard to the yield percentage, the statistical analysis showed differences only for the first four experiments, all of which were performed with 50% ethanol. In these four experiments, the yield was lower for the USAE extraction, indicating the important effect of the ethanol proportion on the extraction process, even for different techniques. Optimization of the Extraction of Lignans by USAE: CCD Model Fitting. Because the USAE method furnished similar responses in shorter periods, it was an advantageous method in comparison to the maceration technique. Therefore, the CCD data of the ultrasound extraction was analyzed to be fitted to a quadratic model, with the goal of explaining the behavior of the responses under the evaluated conditions. ANOVA was carried out considering significance for p values lower than 0.05. All of the responses were fitted to the quadratic models (p < 0.05), which were reliable to be used in the optimization of the extraction process while taking into account the selected variables. Additionally, the non-statistical significance of the lack-of-fit test (p > 0.05) reflects that the models were adequate to describe the experimental data corresponding to concentration terms for the five compounds and yield percentages of the runs. Furthermore, the R2 coefficients (between 0.77 and 0.92) were reasonably close to 1, indicating a good degree of correlation between the observed and predicted values. The empirical second-order polynomial equations were also obtained. The 3D plots representing the effects of the studied variables and their mutual interactions on the yields and concentrations of the five compounds showed that the percentage yield increased at medium points of the time and ethanol proportion variables (20 min and 70%). However, under these conditions, the number of extractions would be increased to optimize the process. With regard to the extraction of the lignans, by increasing the ethanol percentage along with the time of ultrasound exposure, the concentrations of the lignans increase. The analysis of the linear and quadratic main effects of the responses showed that, at a 95% confidence level, the linear terms of the percentage of ethanol and the number of extractions affected the concentration of (−)-clusin and there were no interactions between these factors that would influence the concentration. Additionally, the linear term of the ethanol proportion influenced the concentrations of (−)-haplomyrfolin and (−)-cubebin. The interactions between the variables did not reflect a significant effect on the concentrations of the compounds. Optimization of the Extraction Process by the CCD Desirability Function. Using desirability function analysis, the six responses of the USAE were simultaneously optimized considering the same level of importance for all of the responses (mass yield and concentration of each compound) and the lower and upper limits obtained for each response. The experimental conditions corresponding to 1, which is the

Table 2. Comparison of USAE Method to Soxhlet Extraction lignan concentration (μg/mL)

compound

Soxhlet

USAE desirable method

(+)-dihydroclusin (−)-clusin (−)-haplomyrfolin (−)-cubebin (−)-yatein

36.53 146.30 37.77 146.45 52.71

30.18 138.57 31.33 139.08 47.05

percentage of extraction of USAE desirable method (%) 82.60 94.71 82.95 94.96 89.25

USAE extraction method are similar to those obtained using the Soxhlet apparatus, despite the differences in the extraction principles (acoustic cavitation versus continuous liquid/solid extraction) and temperature conditions. USAE was performed at room temperature, avoiding the risk of possible thermal degradation for bioactive compounds that could occur using methods that require temperatures equal to the boiling temperatures of the extraction solvent, such as Soxhlet.27 Considering that Soxhlet extraction is a technique that allows for the total extraction of lignans, the concentration values furnished by extraction using such an apparatus were appraised as 100%. This allowed for the calculation of the percentage of extraction of each compound by the optimized method. The results showed that the optimized USAE is capable of extracting more than 82% of all of the studied lignans, highlighting clusin and cubebin, which can reach 94% extraction of their total amount from the P. cubeba seeds. Therefore, the optimized USAE has the potential of extracting almost all of the compounds found in the plant material in a shortened extraction time (38 min) in comparison to the Soxhlet method (12 h). In conclusion, the developed RP-HPLC analytical method can be considered to be reliable according to ICH12 and ANVISA13 guidelines for the quantitative analysis of lignans in both P. cubeba seeds and their extracts as well as any products. The use of USAE is advantageous for the extraction of P. cubeba lignans in comparison to traditional techniques, such as ME and Soxhlet apparatus extraction, because it is an easier procedure in a shorter time and allows for the use of ethanol and water, which are considered green solvents.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b05359. Additional information on the statistical analysis of the analytical method validation and optimization extraction process, 3D response surface plots, and profiles for predicted values and desirability function (PDF) F


Article


■

(12) International Conference on Harmonization (ICH). Q2(R1) Validation of Analytical Procedures: Text and Methodology; ICH: Geneva, Switzerland, 2005; pp 13. (13) Agência Nacional de Vigilância Sanitária (ANVISA). Resoluçaõ Da Diretoria ColegiadaRCD No. 166, de 24 de Julho de 2017; ́ Brazil, 2017; pp 21. Ministério da Saúde (MS): Brasilia, (14) Haribabu, K.; Ajitha, M.; Mallavadhani, U. V. Quantitative estimation of (−)-hinokinin, a trypanosomicidal marker in Piper cubeba and some of its commercial formulations using HPLC-PDA. J. Pharm. Anal. 2015, 5, 130−136. (15) Box, G. E. P.; Hunter, W. G.; Hunter, J. S. Statistics for Experimenters an Introduction to Design, Data Analysis, and Model Building; Wiley: New York, 1978. (16) Enders, D.; Lausberg, V.; Del Signore, G.; Berner, O. M. A general approach to the asymmetric synthesis of lignans: (−)-Methyl piperitol, (−)-sesamin, (−)-aschantin, (+)-yatein, (+)-dihydroclusin, (+)-burseran, and (−)-isostegane. Synthesis 2002, 2002 (4), 515−522. (17) Koul, S. K.; Taneja, S. C.; Dhar, K. L.; Atal, C. K. Lignans of Piper clusii. Phytochemistry 1983, 22, 999−1000. (18) Yen, P.-L.; Wu, C.-L.; Chang, S.-T.; Huang, S.-L.; Chang, H.-T. Antioxidative lignans from phytochemical extract of Calocedrus formosana florin. BioResources 2012, 7 (3), 4122−4131. (19) Messiano, G. B.; Vieira, L.; Machado, M. B.; Lopes, L. M. X.; de Bortoli, S. A.; Zukerman-Schpector, J. Evaluation of insecticidal activity of diterpenes and lignans from Aristolochia malmeana against Anticarsia gemmatalis. J. Agric. Food Chem. 2008, 56, 2655−2659. (20) Sousa, J.; Brancalion, A.; Júnior, M.; Bastos, J. A validated chromatographic method for the determination of flavonoids in Copaifera langsdorff ii by HPLC. Nat. Prod. Commun. 2012, 7 (1), 25− 28. (21) de Sousa, J. P. B.; Bueno, P. C. P.; Gregório, L. E.; da Silva Filho, A. A.; Furtado, N. A. J. C.; de Sousa, M. L.; Bastos, J. K. A reliable quantitative method for the analysis of phenolic compounds in brazilian propolis by reverse phase high performance liquid chromatography. J. Sep. Sci. 2007, 30, 2656−2665. (22) Lu, N.; An, Q.; Li, N.; Dong, Y. RP-HPLC method using one marker for quantification of four Podophyllum lignans in medicinal plants. J. Chromatogr. Sci. 2014, 52, 514−519. (23) Zhao, B. T.; Jeong, S. Y.; Kim, T. I.; Seo, E. K.; Min, B. S.; Son, J. K.; Woo, M. H. Simultaneous quantitation and validation of method for the quality evaluation of Eucommiae cortex by HPLC/UV. Arch. Pharmacal Res. 2015, 38, 2183−2192. (24) da Silva, J. J. M.; Crevelin, E. J.; Carneiro, L. J.; Rogez, H.; Veneziani, R. C. S.; Ambrósio, S. R.; Beraldo Moraes, L. A.; Bastos, J. K. Development of a validated ultra-high-performance liquid chromatography tandem mass spectrometry method for determination of acid diterpenes in Copaifera oleoresins. J. Chromatogr. A 2017, 1515, 81−90. (25) Chen, S.; Zeng, Z.; Hu, N.; Bai, B.; Wang, H.; Suo, Y. Simultaneous optimization of the ultrasound-assisted extraction for phenolic compounds content and antioxidant activity of Lycium ruthenicum Murr. fruit using response surface methodology. Food Chem. 2018, 242, 1−8. (26) Xu, D. P.; Zheng, J.; Zhou, Y.; Li, Y.; Li, S.; Li, H. B. Ultrasound-assisted extraction of natural antioxidants from the flower of Limonium sinuatum optimization and comparison with conventional methods. Food Chem. 2017, 217, 552−559. (27) Tian, Y.; Xu, Z.; Zheng, B.; Martin Lo, Y. Optimization of ultrasonic-assisted extraction of pomegranate (Punica granatum L.) seed oil. Ultrason. Sonochem. 2013, 20, 202−208.

AUTHOR INFORMATION

Corresponding Author

*Telephone: +55-16-3315-4230. E-mail: [email protected]. br. ORCID

Jairo Kenupp Bastos: 0000-0001-8641-9686 Funding

The authors are thankful to the São Paulo Research Foundation (FAPESP), CNPq, and CAPES for both financial support and scholarships and the University of São Paulo for infrastructure. Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS USED CCD, central composite design; ME, maceration extraction; USAE, ultrasound-assisted extraction

■

REFERENCES

(1) Silva, M. L. A.; Coímbra, H. S.; Pereira, A. C.; Almeida, V. A.; Lima, T. C.; Costa, E. S.; Vinhólis, A. H. C.; Royo, V. A.; Silva, R.; Filho, A. A. S.; Cunha, W. R.; Furtado, N. A. J. C.; Martins, C. H. G.; Carvalho, T. C.; Bastos, J. K. Evaluation of Piper cubeba extract, (−)-cubebin and its semi-synthetic derivatives against oral pathogens. Phytother. Res. 2007, 21, 420−422. (2) Ribeiro, V. P.; Arruda, C.; Abd El-Salam, M.; Bastos, J. K. Brazilian medicinal plants with corroborated anti-inflammatory activities: A review. Pharm. Biol. 2018, 56, 253−268. (3) Nahak, G.; Sahu, R. K. Phytochemical evaluation and antioxidant activity of Piper cubeba and Piper nigrum. J. Appl. Pharm. Sci. 2011, 1 (8), 153−157. (4) Yam, J.; Schaab, A.; Kreuter, M.; Drewe, J. Piper cubeba demonstrates anti-estrogenic and anti-inflammatory properties. Planta Med. 2008, 74, 142−146. (5) AlSaid, M.; Mothana, R.; Raish, M.; Al-Sohaibani, M.; Al-Yahya, M.; Ahmad, A.; Al-Dosari, M.; Rafatullah, S. Evaluation of the effectiveness of Piper cubeba extract in the amelioration of CCl4induced liver injuries and oxidative damage in the rodent model. BioMed Res. Int. 2015, 2015, 1−11. (6) Esperandim, V.; da Silva Ferreira, D.; Sousa Rezende, K.; Magalhães, L.; Medeiros Souza, J.; Pauletti, P.; Januário, A.; da Silva de Laurentz, R.; Bastos, J.; Símaro, G.; Cunha, W.; Andrade e Silva, M. In vitro antiparasitic activity and chemical composition of the essential oil obtained from the fruits of Piper cubeba. Planta Med. 2013, 79, 1653−1655. (7) Graidist, P.; Martla, M.; Sukpondma, Y. Cytotoxic activity of Piper cubeba extract in breast cancer cell lines. Nutrients 2015, 7, 2707−2718. (8) Arruda, C.; Eugênio, D. de S.; Moreira, M. R.; Símaro, G. V.; Bastos, J. K.; Martins, C. H. G.; Silva, M. L. A.; Veneziani, R. C. S.; Vieira, P. B.; Ambrósio, S. R. Biotransformation of (−)-cubebin by Aspergillus spp. into (−)-hinokinin and (−)-parabenzlactone, and their evaluation against oral pathogenic bacteria. Nat. Prod. Res. 2017, 6419, 1−14. (9) Badheka, L. P.; Prabhu, B. R.; Mulchandani, N. B. Lignans of Piper cubeba. Phytochemistry 1987, 26, 2033−2036. (10) Prabhu, B. R.; Mulchandani, N. B. Lignans from Piper cubeba. Phytochemistry 1985, 24, 329−331. (11) Moreira, M. R.; Souza, A. B.; Moreira, M. A.; Bianchi, T. C.; Carneiro, L. J.; Estrela, F. T.; dos Santos, R. A.; Januário, A. H.; Martins, C. H. G.; Ambrosio, S. R.; Veneziani, R. C. S. RP-HPLC analysis of manool-rich Salvia of ficinalis extract and its antimicrobial activity against bacteria associated with dental caries. Rev. Bras. Farmacogn. 2013, 23, 870−876. G


Development of a Validated High-Performance Liquid

Recommend Documents