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ISSN 1420-3049 www.mdpi.com/journal/molecules Article
Application and Analysis of the Folin Ciocalteu Method for the Determination of the Total Phenolic Content from Limonium Brasiliense L.
Andressa Blainski, Gisely Cristiny Lopes and João Carlos Palazzo de Mello * Programa de Pós Graduação em Ciências Farmacêuticas, Universidade Estadual de Maringá, Av. Colombo, 5790, BR-87020-900, Paraná, Brazil; E-Mails: firstname.lastname@example.org (A.B.) email@example.com (G.C.L.) * Author to whom correspondence should be addressed; E-Mail: firstname.lastname@example.org; Tel.: +55-44-3011-4816; Fax: +55-44-3011-5050. Received: 1 April 2013; in revised form: 1 June 2013 / Accepted: 6 June 2013 / Published: 10 June 2013
Abstract: Limonium brasiliense is a common plant on the southern coast of Brazil. The roots are traditionally used for treatment of premenstrual syndrome, menstrual disturbances and genito-urinary infections. Pharmaceutical preparations obtained from its roots and used for these purposes were marketed in Brazil in the 1980s and 1990s. Currently, the Brazilian Drug Agency (National Health Surveillance Agency, ANVISA) has canceled the registration of these products, and their use was discontinued because of a lack of studies to characterize the plant raw material and ensure the effectiveness and safety of its use. The aim of the present study was to develop and validate an analytical method to determine the content of total polyphenols (TP) in an extract from L. brasiliense roots, by the UV/Vis spectrophotometric method. L. brasiliense roots were extracted in acetone:water (7:3, v/v-10% w/v). The crude extract was used to develop a method for TP assay. The method was validated according to national and international guidelines. The optimum conditions for analysis time, wavelength, and standard substance were 30 min, 760 nm, and pyrogallol, respectively. Under these conditions, validation by UV/Vis spectrophotometry proved the method to be linear, specific, precise, accurate, reproducible, robust, and easy to perform. This methodology complies with the requirements for analytical application and to ensure the reliability of the results.
contributors to plant pigmentation. attractants for pollinators. However. Folin-Ciocalteu 6853 1. methodology optimization. and lignins. Among the many varieties of natural phenolic compounds. Assuming that quantification of individual polyphenols does not adequately reveal the proportion of polymeric procyanidins.9]. stilbenes. flavonoids. and the general or specific value of the method. and on the southern coast of Brazil.14]. antioxidants. menstrual disorders and genitourinary infections [19–21]. which is easy to perform. it is important that colorimetric assay need to use a reference substance. Many studies have discussed the use of the Folin-Ciocalteau reagent to determine polyphenols. The reaction forms a blue chromophore constituted by a phosphotungsticphosphomolybdenum complex [11. However. and low-cost . where the maximum absorption of the chromophores depends on the alkaline solution and the concentration of phenolic compounds . Polyphenols in plant extracts react with specific redox reagents (Folin-Ciocalteu reagent) to form a blue complex that can be quantified by visible-light spectrophotometry . total polyphenols. The flavan-3-ol units can be doubly linked by a C4→C8 bond and/or an additional ether bond from O7→C2 (A-type) [2. Folin and Ciocalteu included lithium salts in the reagent. Pharmaceutical preparations obtained from its roots and used for . These compounds are among the most widely occurring secondary metabolites in the plant kingdom. because some specific details may be modified [9. lignans. This excess can result in precipitates and high turbidity. and protective agents against UV light. The reaction generally provides accurate and specific data for several groups of phenolic compounds. It is popularly known in Brazil as “baicuru” or “guaicuru”. phenolic acids (benzoic and cinnamic acid derivatives).3]. making spectrophotometric analysis impossible.18]. The Folin-Ciocalteu method is described in several pharmacopoeias [12.13]. hydrolyzable and condensed tannins. 18 Keywords: Limonium brasiliense. the procyanidins are an important subgroup. UV/Vis spectrophotometric validation. coumarins. rapid and applicable in routine laboratory use. among others . then this method mensures the total concentration of phenolic hydroxyl groups in the plant extract. this reagent rapidly decomposes in alkaline solutions. Uruguay. several methods have used to determine polyphenols in plant extracts [4–7]. acting mainly as phytoalexins. Limonium brasiliense (Boiss. Although quantitative determination of polyphenols is hampered by their structural complexity and diversity. and is traditionally used to treat premenstrual syndrome.Molecules 2013. which makes it necessary to use an enormous excess of the reagent to obtain a complete reaction.) Kuntze (Plumbaginaceae) is a perennial herb that occurs in Argentina.14. Colorimetric reactions are widely used in the UV/VIS spectrophotometric method. which prevented the turbidity . then spectrophotometry in the ultraviolet region may be a useful tool to help resolve this problem [8. These substances are composed of oligomers and polymers that consist of catechin and/or epicatechin units linked mainly through C4→C8 and/or C4→C6 bonds (B-type). Introduction Phenolics include simple phenols. To solve this problem.17. because many compounds change color differently due to differences in unit mass  and reaction kinetics .
brasiliense describe its biological activities. Results and Discussion 2. Figure 1. the Brazilian Drug Agency (National Health Surveillance Agency. Absorption spectra (400 to 900 nm) of reference compounds (gallic acid. and their use was discontinued because of a lack of studies to characterize the plant raw material and ensure the effectiveness and safety of its use. This study deals with the optimization of a method for determination of total polyphenols and an evaluation of possible variations of the Folin-Ciocalteau method applied to the crude extract. . with uniform phenolic groups present in the same species. leucoanthocyanins.Molecules 2013. This report describes the first of a series of chemical studies of L. each plant has a characteristic chemical composition. The few studies of L. so it is not possible to determine the best reference compound for this extract by means of their spectra (Figure 1). anti-inflammatory and antioxidant. ANVISA) has canceled the registration of these products. All the reference substances used show approximately the same spectra as the CE from L. β-sitosterol. brasiliense. 2. which were related to the presence of condensed and hydrolyzable tannins. 18 6854 these purposes were marketed in Brazil in the 1980s and 90s [22.23]. flavonoids. Currently. brasiliense by Folin-Ciocalteu reaction after 30 min. tannic acid.1. including bacteriostatic. brasiliense. Method Optimization According to Glasl . Similar chemical structures may show the same chemical interactions with specific reagents during the reaction . saponins and coumarin in the extracts evaluated [20–24]. aimed at characterizing the plant drug and standardization and normalization of the content of polyphenols in the extracts. catechin and pyrogallol) and CE from L.
which appeared to be best suited to produce maximum absorption of the substances under study. The statistical analysis indicated a significant increase in absorbance at 760 nm compared to data for 691 and 715 nm for all solutions (gallic acid. A1%catechin = 1. F60. which suggests that this parameter is stable. Chemical structures of reference compounds: (1) gallic acid (C7H6O5). F8. 30 and 40 min was determined.46.3 = 0. pyrogallol.3 = 4.9. P = 0.0 ± 39.033. F8.001.001). Using the wavelength of 760 nm.8. tannic acid.0042.001.001.213. F8.9849. F8. F60. and did not decrease between 30 and 40 min. 20. pyrogallol has .50 and A1%pyrogallol = 1.0483.3 = 1.95.3 = 0. The absorbance at 800 nm was higher than at the other wavelengths.0. CE from L. as determined by the European Pharmacopoeia. the next step was to determine the reaction kinetics with respect to the time period prior to the spectrophotometric measurement. 760.Molecules 2013.337. different specific absorptivities (760 nm.3 = 0.4.9777). F60. since after 30 min the absorbance increased less than 5% of the value at 10 min. catechin.7 ± 62. P < 0. tannic acid. P < 0. in which the order of activity is proportional to the availability of hydroxyl groups present on the aromatic ring and influenced by groups that can decrease or increase the reductive potential of the molecule . catechin. P < 0. These results showed that the intensity of the reaction of phenolic compounds with Folin–Ciocalteu reagent follows the classical principle of structure-activity relationship. and 800 nm. These data showed that the Folin–Ciocalteu reaction was stable during the period analyzed.910. P < 0. The increase in percentage absorbance of each solution relative to the time periods of 10. P = 0. P = 0.0108. (3) pyrogallol (C6H6O3) and (4) tannic acid (C76H52O46). P = 0.551.0630. this increase was not statistically significant for the ratio at 760 nm. P < 0. F60. Figure 2.3 = 0.3 = 1. 18 6855 The percentage increase in absorbance of each solution was compared at the wavelengths of 691. brasiliense.8 ± 93.3 = 1. Of the substances in this study (Figure 2).2.188. pyrogallol. F60. These results suggested a period of 30 min for the spectrophotometric measurement of the CE. 715.9983. With respect to the standard.29.081.9996. 30 min) were observed for each reference compound: A1%gallic acid = 970.3 = 1.001. The statistical analysis indicated no significant diference in relation at the time (gallic acid.8 ± 31. however. A1%tannic acid = 830. (2) catechin (C15H14O6).
and similar results were obtained .38]). as well as the precision and accuracy of the back-fit calculations.5%.00 80.03 [5.00 70. TP of CE from L. brasiliense.550 ± 0.800 0. The specific absorptivity is the absorbance of a solution in a concentration of 1 g/100 mL (1%-10.200 0. 18 6856 the largest number of hydroxyl groups proportional to its molar mass.00 Concentration (µg/mL) .00 20.14073x + 0.407.000 µg/mL). The relative standard deviations (RSDs) of the slopes were ≤5% for the analyte (n = 5). Table 1 shows the back-fit calculations for curve data for the standard used in the validation runs. Therefore. the mean absorbance for CE from L.00 30. brasiliense was (0.100 0.000 0. considering the total availability of hydroxyls linked to the aromatic ring. Pyrogallol therefore has the highest specific absorptivity and is probably the best reference substance for the determination of TP.88 µg/mL)..00 50. pyrogallol indirectly represents the reduction potential of the plant drug.Molecules 2013.000 0.900 0. The method was optimized for an extract of Caesalpinia peltophoroides Benth. The specific absorptivity of pyrogallol was calculated using the linear equation (y = 0.996) for pyrogallol. pyrogallol has only one ring and no substituted groups.700 Absorbnce (nm) 0. Figure 3. In addition.500 0. linearity (6. brasiliense and Calibration Curve of Pyrogallol Standard The calibration equation was y = 0. Linearity Based on 1/x weighted linear regression analysis.00 40. 1. the responses for the CE in related concentration ranges were linear (Figure 3).0044 (n = 5. forming a three-dimensional structure with a hydroxyl that is less influenced by electronic interactions such as steric or resonance effects. Also. and TP expressed as pyrogallol was 23.300 0.2.0044).3. 2. 2.400 0.14073x + 0.3. Absorbance versus concentration curve in the analysis of TP in CE from L. r2 = 0.00 10. The data obtained in the linearity test for CE and pyrogallol appear in Table 1.600 0.00 60.40 to 26. obtaining a value of 1.
0078) 0.996 0. 0.0097 F1.0286.997 1. 0. Statistical data for the regression equations of the calibration curve for pyrogallol.2.00040) 0.0303. 0.029 (0.0284. P < 0.0012 0. CL = confidence limit . and linearity test and specificity test for TP of CE from L.031 (0.1359.71) 0.8.13 = 3999.996 3.001 0.046 (0.1407 (0.0307 0. P < 0.Molecules 2013. P < 0.0044 (0.12 (3. 0. 0.0010 F1.0990 0.71) 0.1173. 0.1456 −0.0652 Specificity test for CE 0.0009 0. brasiliense.1491 6857 SE = standard error.0008) 0.001 0.133 (0.0125.30 (3.0320 0.00030) 0.13 = 3901. 18 Table 1.00010) 0.26 (3. Regression analysis Slope (SE) Intercept (SE) Regression coefficient (R2) Calculated F-value (critical F-value) Sum of pure error Lack of fit error Analysis of variance CL slope CL intercept Calibration curve for pyrogallol 0.8.0008 F1.001 0.13 = 5880.71) 0.0213 Linearity test for CE 0.00225) 0.
with correlation coefficient (R2) and linear equation.105 to 0.600 0.7. 1. the absorbance is not proportional to the concentration . Linearity curve and specificity curve.92 µg/mL. brasiliense. Specificity 6858 Analysis of the results of the specificity test indicated that the conditions were satisfactory (Table 1). Based on the linear equation from the linearity test.400 0.0 µg/mL. The experimental determination may be affected by other interfering factors. such as human manipulation and differences in the standard conditions among laboratories.68 to 26. In the case of complex matrices. see Table 1) were very similar (about 6. we can state that the method is specific .001).88 µg/mL (absorbance from 0.84 µg/mL (absorbance of 0.5. which is directly proportional to both limits.000 y = 0.4.9968 Specificity Linearity 2. because there was a significant difference between this concentration and the next-lower concentration (0. which reduces the standard deviation of the y intercept. the LOQ was 3.200 1. According to the Lambert-Beer law.Molecules 2013.200 0.000 0 5 10 15 20 Concentration (µg/mL) 25 30 y = 0.0297x + 0. the effects of the matrix system can be tested by comparing the slopes of linearity and specificity [26–28]. The LOD and LOQ determined by the linear equation gave different results from those observed experimentally.4 = −5.9978 Absorbance (uA) 0. These differences suggest be by linear regression from linearity curve providing a satisfactory result. for a given concentration of the sample.0468 R² = 0.800 0.34 µg/mL. If the curves are parallel. because the slopes of the linear equations (linearity and specificity. . P < 0.0311x + 0.1332 R² = 0. because linearity was maintained (R2 = 0. LOD was 1.5% difference). Figure 4. 18 2. Limits of Detection and Quantification The quantification range was from 3. for TP of CE from L. The LOD was 1.055). if the matrix without the analyte is not available. The specificity of the method for CE was confirmed by superimposing the analytical curves (Figure 4). t1.996). Based on the linear equation from the test for linearity.834).
8 = 4. Standards and Chemicals All chemicals were analytical-reagent grade and the water was distilled. 05/07/2007).2. anhydrous sodium carbonate (Synth®). 3. Comparison of these results with those obtained in the linearity assay indicated no significant difference between means (F3. Extraction The plant material was washed with water to remove soil. intermediate and highest levels. 18 2. with no significant difference between them (t1. However. These percentages were within the range of 85%–115% established in published reports. dried in a circulating-air oven (37 ± 2 °C) and powdered in a hammer mill (Tigre ASN-5. Diadema.3.75% (w/v)]. brasiliense were collected in Rio Grande.75% in the European Pharmacopoeia . This indicates that the method has good accuracy for determining TP in CE from L. St. 0.003 [0. and catechin (Sigma-Aldrich). without protection from light. A voucher specimen was deposited at the Herbarium of the Federal University of Rio Grande under number HURG-004208. 2. mean diameter 0. USA).8. respectively.01 [1. 126.96.36.199% in the Brazilian Pharmacopeia . USA).531 ± 0.06% (w/v). St. The milled roots (630 g) . Na2CO3 solution 7.530 ± 0. and Na2CO3 solution 14. Brazil). respectively. P = 0.6.552 ± 0. Robustness The change in Na2CO3 concentration in the robustness assay took into account data from the literature: 7. 14:05% in Glasl .5% (w/v).82%]. there was a tendency for the proposed conditions and validated by method [protection from light and Na2CO3 solution 10. brasiliense. Accuracy The result for the accuracy test showed a percentage recovery of 101.027 [4. 2 N Folin-Ciocalteu reagent (Dinâmica®. Brazil).42 mm).530 ± 0.81%].7 = 1.01 [1.005 [0. P = 0.2 and 89. tannic acid (Acros).98%] and 0.0 for both solutions. Brazil (S 32° 09' 22''/W 52° 06' 01'') in May 2006. and 10.530 ± 0. the proposed method is adequately precise. IBAMA-SISBIO gave permission for this collection under a license granted to João Carlos Palazzo de Mello (No.89%].34. and 0. Louis. Diadema. 3. Plant Material Roots of L.2.22).6. state of Rio Grande do Sul.9%. Precision 6859 The repeatability and the intermediate precision for CE were 0. The chemicals included acetone (Synth®. pyrogallol (Fluka.Molecules 2013. MO.65%]. for the lowest. respectively. Experimental 3. MO. gallic acid (Sigma-Aldrich®. pH 11. The values for absorbance measured after changing the Na2CO3 concentration and the natural light incidence in the Folin–Ciocalteu reaction were 0. 3. Therefore.05). 11995-2. 2. Louis.
The kinetic reaction was evaluated (ANOVA) by comparing the percentage increase in absorbance of each solution. 20 µg/mL) and CE (300 µg/mL) were prepared in distilled water. Ika® Works. resulting in final concentrations of 1. Distilled water was used as a blank. using the percentage increase between the reaction times (10. Japan) beginning 10 to 40 min after the addition of the sodium-carbonate solution. and volumetrically diluted to 25 mL with 10. using water . 1 mL of 2 N Folin-Ciocalteu reagent. Kyoto. based on general procedures recommended by the European Pharmacopoeia  for determination of total tannins. brasiliense and Calibration Curve of Pyrogallol Standard All extraction and dilution operations were protected from light. 3.75% anhydrous sodium carbonate (w/v).75% w/v anhydrous sodium carbonate (w/v). 18 6860 were extracted in 6. The volume was completed with 10. 20.6. The specific absorptivity of each standard was calculated based on the Lambert-Beer Law . three parameters were analyzed: (1) reaction kinetics. Wilmington. The method utilized for the test was a colorimetric assay. Method for TP Analysis of CE from L. washed with 7. CE solution: the CE (26.3 L of 7:3 acetone:water (v/v) by turbo-extraction (Ultra-Turrax®. (2) maximum absorption wavelength.30]. After 30 min. the guidelines established by the ICH (International Conference on the Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use) and by Brazilian regulation RE No. and scanning from 400 to 900 nm at intervals of 2 min between each reading. 715 . to determine the spectra. USA. and 5 mL of this stock CE solution was diluted to 25 mL with water. 3. for the wavelengths of 691 .5. Then. stock solutions of each standard (gallic acid. UTC 115KT.6 µg/mL of each standard and 24 µg/mL of CE. 3. and 10 mL of water. 760 . lyophilized (Martin Christ Alpha 1-4) to yield a crude extract (CE.2 L of 7:3 acetone:water (v/v). concentrated in a rotavapor under reduced pressure. catechin and pyrogallol.0 mg) was transferred to a 25 mL volumetric flask and diluted with water. The reaction times were observed and compared statistically. corresponding to 29% sodium carbonate decahydrate. 30 min. and 800 nm. 30. 272 g) and stored at −20 °C. with a quartz cell (1 cm path length).Molecules 2013. tannic acid. 899/2003 of ANVISA were employed [28.75% anhydrous sodium carbonate. <40°C). the absorbance was measured at 760 nm. Method Optimization and Standardization for Determination of Total Polyphenols (TP) For the optimization and standardization of the spectrophotometric method using the Folin-Ciocalteau reagent. North Carolina.4. Two milliliters of each solution was transferred to a 25 mL volumetric flask containing distilled water (10 mL) and Folin-Ciocalteu reagent (1 mL) . Analytical Method Validation For validation of the analytical method. a mixture was prepared with 2 mL of this solution. and 40 min) at 760 nm. The samples were scanned in a UV/Vis spectrophotometer (Shimadzu PC-1650. and (3) the standard that best characterizes the CE of L. For the selection of a reference chemical standard. brasiliense. This percentage increase was calculated by dividing the difference in absorbance between two wavelengths by the mean absorbance of the shorter wavelength and multiplying by 100. Next the extractive solution was filtered. We used a solution of 10. with slight modifications.
0.52.0 mg/mL) to each stock solution described in the linearity test. A1% = Specific absorptivity of pyrogallol.4. Specificity Specificity was determined by adding 1 mL of a pyrogallol solution (1. Aliquots of 1. in three replicates. 2. 3. brasiliense was calculated by Equation (1): TP = 1562.7.0 mg of CE.05 and 0. m = mass of the sample examined. 0.2. 34. Concentrations of 6.0 mg of pyrogallol was dissolved in water.0. 11.9.88 µg/mL were obtained.8. The method is considered specific if the slopes of the linear equations in the tests for linearity and specificity are equal or very similar.0 mg of CE. 3.Molecules 2013.0 mL from these solutions were further diluted to 25 mL (stock solution).25 or 4.0 mL were diluted to 25 mL in water.40. together with the results for linearity. The repeatability was assessed using six samples of 26.10. and aliquots of 5. Calibration curve: In three replicates. A reaction mixture was prepared as described in the TP method. 3. The absorbance was measured under the same conditions as for the CE.0 or 42.0. diluted to 25 mL.20.75. 21. 1562. 3. and diluted to 25 mL. A coefficient of variation over 15% and a significant difference between days were considered unacceptable.16 or 5. 4.5 A/A1% m (1) where A = Absorbance of sample for TP.1. Limits of Detection and Quantification The limits of detection (LOD) and quantification (LOQ) were calculated from the relationship between the standard deviation (SD) of the CE linearity and the slope. were submitted to statistical analysis. and the intermediate level was assessed using three samples and on at least two different days. 18. The LOD was determined as the lowest concentration that was significantly different from the next lowest concentration.0. 50. 0. of CE (4. and aliquots of 5. Linearity To establish the linearity of the proposed method. 2.5. from 10. 0.5 = dilution factor of the sample. 2.0. The specific absorptivity was determined from the linear equation. using the appropriate multiplier.12 µg/mL of pyrogallol.1. 3.64. five stock solutions were prepared. with 2 mL from each solution. in three replicates. 3. Precision Two levels were evaluated: repeatability (intra-day) and intermediate (inter-day). 18 6861 as the compensation liquid and a quartz cell (1 cm path length) in a UV-Vis spectrophotometer. Each stock solution was prepared according to the procedure described above for the CE. The results. immediately before use. The final concentrations were 1.24. 3.025 mL were. . 16.56 mg/mL).76 and 26. The TP expressed as pyrogallol of the CE from L. separately. 26.28. These results were compared with the LOD and LOQ obtained by extension of the linearity curve of CE: a stock solution was prepared. in grams.7. 1. The quantification range was determined based on the lowest and highest concentrations that maintained linearity. 2.
The data were expressed as mean ± standard deviation [relative standard deviation (%)]. following the addition of 1. and the residual sum of squares was evaluated. in three replicates. 4.100 AT (2) where A = absorbance of sample after addition of the standard. The method is considered accurate if the recovery percentages are between 85% and 115%.06% (w/v) anhydrous sodium carbonate solutions). for English revision. The technical assistance provided by A. Accuracy was assessed as the percentage recovery. considering P < 0. CNPq. New York. Acknowledgments The authors are grateful for financial support from CAPES.Molecules 2013. or 3 mL of pyrogallol solution (1 mg/mL) to the CE stock solution. R. all parameters analyzed showed adequate results. . USA. which was calculated from Equation (2): %recovery = A. Accuracy 6862 The accuracy was evaluated using different concentration levels: lowest concentration (LC). and Fundação Araucária.. Robustness The robustness was demonstrated by analyzing the stability of the solution under the influence of natural light and a range of pH changes (7. 18 3. intermediate concentration (IC) and highest concentration (HC). JWR Associates.12. 3. Statistical Analysis Data were analyzed by the program Statistica® 8.13. 1984–2007) by one-way analysis of variance (ANOVA) followed by Tukey’s test. FINEP. The linear correlation tests and residual analyses were performed by simple linear regression. This methodology using the Folin-Ciocalteu reaction complies with the requirements for analytical use and for ensuring the reliability of the results. evaluated in three replicates. Thanks are due to Janet W. Tulsa. Novello is gratefully acknowledged.11. 3. brasiliense and the expected absorbance of pyrogallol. Inc. Arantes and C.98.5 and 14. based on the calibration curve for each level. Reid. INCT_if. 2. OK.05 as significant. brasiliense. Conclusions After optimization of the conditions for the spectrophotometric determination of phenolics using the Folin-Ciocalteu reagent. Trumansburg.0 (StatSoft. considering R2 equal to or higher than 0. AT = theoretical absorbance calculated for the sum of the absorbance of CE from L. The UV-Vis spectrophotometric method described here was successfully validated as suitable for the determination of TP of CE from L.
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