Spectrophotometric determination of the total flavonoid content in Ocimum basilicum L. (Lamiaceae) leaves
Layzon Silva, Bianca Ramos Pezzini, Luciano Soares
Department of Pharmacy, University of Region of Joinville Univille, Rua Paulo Malschitzki, No. 10, Campus Universitário Zona Industrial, Joinville, Santa Catarina, Brazil
Date of Submission  10Mar2014 
Date of Acceptance  19May2014 
Date of Web Publication  20Jan2015 
Correspondence Address: Luciano Soares Department of Pharmacy, University of Region of Joinville Univille, Rua Paulo Malschitzki, No. 10, Campus Universitário Zona Industrial, Joinville, Santa Catarina Brazil
Source of Support: None, Conflict of Interest: None  Check 
DOI: 10.4103/09731296.149721
Abstract   
Background: The chemical characterization is essential to validate the pharmaceutical use of vegetable raw materials. Ultraviolet spectroscopy is an important technique to determine flavonoids, which are important active compounds from Ocimum basilicum. Objective: The objective of this work was to optimize a spectrophotometric method, based on flavonoidaluminum chloride (AlCl _{3} ) complexation to determine the total flavonoid content (TFC) in leaves of O. basilicum (herbal material), using response surface methodology. Materials and Methods: The effects of (1) the herbal material: Solvent ratio (0.02, 0.03, 0.05, 0.07, and 0.08 g/mL), (2) stock solution volume (0.8, 2.3, 4.4, 6.5, and 8.0 mL) and (3) AlCl _{3} volume (0.8, 1.0, 1.2, 1.4, and 1.6 mL) on the TFC were evaluated. The analytical performance parameters precision, linearity and robustness of the method were tested. Results: The herbal material: Solvent ratio and stock solution volume showed an important influence on the method response. After choosing the optimized conditions, the method exhibited a precision (RSD%) lower than 6% for repeatability (RSD%) and lower than 8% for intermediate precision (on the order of literature values for biotechnological methods), coefficient of correlation of 0.9984, and no important influence could be observed for variations of the time of complexation with AlCl _{3} . However, the time and temperature of extraction were critical for TFC method and must be carefully controlled during the analysis. Conclusion: Thus, this study allowed the optimization of a simple, fast and precise method for the determination of the TFC in leaves of O. basilicum, which can be used to support the quality assessment of this herbal material. Keywords: Flavonoids, Ocimum basilicum, response surface, spectrophotometry
How to cite this article: Silva L, Pezzini BR, Soares L. Spectrophotometric determination of the total flavonoid content in Ocimum basilicum L. (Lamiaceae) leaves. Phcog Mag 2015;11:96101 
How to cite this URL: Silva L, Pezzini BR, Soares L. Spectrophotometric determination of the total flavonoid content in Ocimum basilicum L. (Lamiaceae) leaves. Phcog Mag [serial online] 2015 [cited 2021 May 16];11:96101. Available from: http://www.phcog.com/text.asp?2015/11/41/96/149721 
Introduction   
Ocimum basilicum L. (Lamiaceae), known in the traditional medicine as Manjericão and Basil, ^{[1],[2],[3]} has biologically active constituents associated with antiulcerogenic, ^{[4]} antibacterial, antifungal effects. ^{[4],[5],[6],[7]} Flavonoids, including nevadensin, salvigenin, cirsileol, eupatorin, apigenin, acacetin, cirsimaritin, quercetin and ladanein, are among the main secondary metabolites identified in Basil, ^{[8],[9]} besides the high essential oils content. ^{[1],[2],[3],[4],[5],[6]}
The analysis of the content of main active components in raw materials and phytomedicines is an essential step to evaluate the quality of products and validate the efficacy and safety of their therapeutic use. ^{[10]} A widely used technique for the quantification of flavonoids is based on its complexation with aluminum chloride (AlCl _{3} ) and the spectrophotometric determination of the formed complex, which provides a bathochromic displacement and the hyperchromic effect. In this method, several factors are crucial for the formation of the flavonoidAlCl _{3} complexes and must be optimized to improve the method performance such as the reaction time, the complexant concentration, flavonoid: Herbal material ratio and the chemical structure of the polyphenol. Thus, a multivariate approach can be used to evaluate the effect of the critical variables on the selected response. Experimental designs are multivariate tools used for the systematic and effective evaluation of simultaneous modifications of several variables in a specific system. ^{[11]}
Response surface methodology (RSM) is a set of mathematical and statistical techniques useful for designing experiments, building models and analyzing the effects of several independent factors. RSM comprises the evaluation of empirical models fitted to experimental data. The advantages of its application on the optimization of analytical procedures are widely disseminated, since the design of polynomial experiments allow testing, simultaneously, several factors with potential influence on the response, producing large amounts of information from a relatively small number of experiments. ^{[12]}
The objective of this work was to optimize a spectrophotometric method based on flavonoidAlCl _{3} complexation to determine the total flavonoids content in leaves of O. basilicum, using RSM.
Materials and methods   
Material
Plant material
The dried and ground leaves of O. basilicum L. of d90 ≤ 0.500 mm (herbal material) used in the experiments were obtained from specimens collected on August 2011, in Joinville, SC, Brazil. The voucher specimen deposited in the herbarium Joinvillea (number 8234) was taxonomically identified.
Chemicals and reagents
The standard of quercetin dehydrate (≥98% HPLC) was purchased from Sigma Co. (St. Louis, MO, USA). Absolute ethanol from Labmaster (Pinhais, PR, Brazil), methanol from Cromato Produtos Químicos (Diadema, SP, Brazil), AlCl _{3} hexahydrate from Nuclear (Diadema, SP, Brazil) pro analysis grade. Distilled water was prepared with distiller Quimis Q341 (Diadema, SP, Brazil).
Instrumentation
Spectrophotometric measurements were performed on an ultraviolet (UV)1601PC UV/visible (UVVis) spectrophotometer (Shimadzu, Kyoto, Japan) equipped with 1 cm quartz cells.
Methods
Wavelength selection
For the determination of the maximum absorption, the herbal material: 60% ethanol (v/v) extract (0.08 g/mL) was subjected to a general procedure for flavonoid quantification (subsection General method for total flavonoids content [TFC] determination). ^{[13]} The UVVis spectra (300500 nm) were obtained in a spectrophotometer after complexation with AlCl _{3} 2% (w/v). The same sample without AlCl _{3} was used as a blank solution.
Optimizing the determination of the TFC
Conditions for the optimal TFC were determined by RSM, which was performed using the software DesignExpert ^{®} 8.0.6 from StatEase, Inc. (Minneapolis, MN, USA). A central composite design (CCD) was used to investigate the effects of three independent variables, which are coded as (A) the herbal material: Ethanol ratio (w/v), (B) the volume of stock solution and (C) the AlCl _{3} volume, on the dependent variable (TFC in mg/g herbal material). The data obtained from the CCD was fitted to a second order polynomial equation. The generalized nonlinear computergenerated quadratic model is given as Equation 1.
where, Y is the predicted response associated with each factor level combination; β_{} is the intercept; β_{i} is the linear coefficient; β_{ii} is the quadratic coefficient; β_{ij} is the interaction coefficient; and X _{i} and X _{j} are the coded levels of independent variables. The terms X _{i} X _{j} and X _{i} ^{2} represent the interaction and quadratic terms, respectively. From preliminary experiments, the levels of the independent variables were selected: (A) 0.02, 0.03, 0.05, 0.07, and 0.08 g/mL; (B) 0.8, 2.3, 4.4, 6.5, and 8.0 mL; (C): 0.8, 1.0, 1.2, 1.4 and 1.6 mL. The experimental conditions for CCD and the TFC results are presented in [Table 1]. The experimental design required 17 experiments, including five replicates at the center point to estimate the overall error. The experiments were performed in random order to avoid systematic error. All trials were carried out in triplicate and the data were reported as means ± standard deviation (SD).{Table 1}
The adequacy of the model was determined by evaluating the lack of fit, coefficient of regression (R^{2} ) and the Fisher test value (Fvalue) obtained from the analysis of variance (ANOVA) considering P < 0.05 as significant. The software uses the quadratic model equation shown above to generate response surfaces. Threedimensional response surface plots were generated by keeping one response variable at its optimal level and plotting it against two factors (independent variables).
General method for total flavonoid content determination
The herbal material was extracted under reflux conditions (80 °C) with 20.0 mL waterethanol solution 60% (v/v) (pH = 5.06) during 60 min. The extract was cooled to room temperature and filtered. The residue was reextracted under equivalent conditions. Both hydroalcoholic extract and reextract were combined, and the volume was completed to 50 mL of waterethanol solution 60% (v/v), resulting in the stock solution.
An aliquot of the stock solution was transferred to a 10.0 mL volumetric flask and made to volume with methanol, resulting in the blank solution. A second aliquot of the stock solution was transferred to another 10.0 mL volumetric flask, a volume of the 2% AlCl _{3} was added and made to volume with methanol, which was named test solution. After 25 min the absorbance of the test solution was measured at 430 nm against blank solution.
TFC _{herbal material} results were calculated, as quercetin, using Equation 2, and it represents the average of threedeterminations. The results were expressed as the amount of flavonoid (mg)/g of herbal material (corrected for moisture content).
where TFC _{test} _{solution} is the total concentration of flavonoids in the test solution (mg/mL), 1.25 corresponds to the dilution factor, 50 is the volume of the stock solution (mL), w is the mass of herbal material (g), and ld is the loss on drying of herbal material.
Calibration curve and linearity
Standard solution of quercetin in methanol (0.050 mg/mL) was prepared in triplicate and diluted to concentrations of 0.001, 0.006, 0.011, 0.016, and 0.021 mg/mL. The absorbance of diluted solutions was measured at 430 nm 25 min after adding AlCl _{3} against the blank solution. The calibration curve was constructed by plotting the measurements of mean absorbance versus the concentration of the standard solutions. The results were analyzed by linear regression and the correlation coefficient (R^{2} ) was calculated GraphPad Prism 5.00 from GraphPad Software, Inc. (La Jolla, CA, USA). Oneway ANOVA was calculated to compare the replicates of the calibration curve.
Precision
The intraday and interday variabilities were studied to assess the precision of the method. Intraday variability was determined by analyzing within 24 h. TFC was measured 6 times under the optimized method conditions and the relative standard deviation (RSD%) was considered a measure of repeatability. Interday variability was determined by performing the analysis in sextuplicate by two analysts in four different days (analyst A performed the analysis in days 1, 2, and 3, while analyst B performed the analysis in day 4), the RSD% being considered a measure of intermediate precision.
Robustness
Robustness was evaluated by the variation of the following conditions of the TFC method: Time of complexation with AlCl _{3} solution (15 and 35 min), time of extraction of herbal material (50 and 70 min) and temperature of extraction (70 °C and 90 °C). The results were evaluated by twoway ANOVA followed by Bonferroni posthoc test, with a confidence level of 95% (GraphPad Prism ^{®} 5.00).
Results and discussion   
Wavelength selection
The UVspectra for herbal material and standard quercetin after reaction with AlCl _{3} are presented in [Figure 1]. Both UVprofiles observed for the tested samples presented a maximum at 430 nm, defining this wavelength for the TFC analysis.  Figure 1: Spectrum for aluminum chlorideflavonoid complexes: Herbal material (leaves of Ocimum basilicum) and standard (quercetin)
Click here to view 
Optimizing the determination of the total flavonoid content
[Table 1] shows the experimental design employed and the corresponding TFC results. [Figure 2] illustrates response surface plots showing the effect of herbal material: 60% ethanol ratio and volume of stock solution on TFC response. The results of ANOVA for TFC with corresponding coefficients of multiple determination (R^{2} ) for leaves of O. basilicum are shown in [Table 2]. The model was adequate and explained most of the observed variability. For the model fitted, the software generates model coefficients, R^{2} values, Fvalues and significant probabilities and hence one can justify the significance of each experimental variable. The maximum predictable response for TFC was obtained based on a total of 17 experiments required for determining 12 regression coefficients of the model [Table 2]. A plot of experimental and theoretical values indicated an excellent fit of the model (F = 6.93; P = 0.126; R^{2} = 0.9985).  Figure 2: Response surface (threedimension) showing the effect of factors (high aluminum chloride volume as fixed factor; (a) Herbal material: Solvent ratio; (b) Stock solution volume) added on TCF
Click here to view 
 Table 2: Analysis of variance for response surface of reduced quadratic model
Click here to view 
The data listed in [Table 1] were converted into a secondorder polynomial equation with three independent variables. Consequently, the polynomial models describing the correlation between the response (TFC) and the variables were presented as follows:
where A, B and C denoted the herbal material: 60% ethanol ratio (w/v), the volume of stock solution and the AlCl _{3} volume, respectively. The values of adjusted R^{2} ^{(} 0.9940), a measurement for fitness of the regressed model (Equation 3), indicate a high degree of correlation between observed and predicted values. The analysis of variance (ANOVA) of the regression model was performed to determine the significance. The F = 221.74 implies that the model is highly significant (P < 0.0001), that is, there is a chance of only 0.01% of the model occur due to noise, as can be seen in [Table 2]. A lack of fit was not significant (P > 0.05) showing that the reduced quadratic model is valid for the spatial influence of variables on the response. The linear and quadratic terms of the herbal material: 60% ethanol ratio and the volume of stock solution, the cubic term of herbal material: 60% ethanol ratio, the linear interactions between these terms and between them and the AlCl _{3} volume, the interactions between the square of the herbal material: 60% ethanol ratio and the other two factors, the interaction of the square of the volume of stock solution and the herbal material: 60% ethanol ratio had statistically significant effects on TFC (P < 0.05).
The model (Equation 3) clearly demonstrates that A and B have negative effects on TFC, while C has a positive effect on it. The absolute value for the coefficient of A is much greater than those of B and C. This indicates that the linear term influence of A is more significant than those of B and C. The response surface of the TFC model indicates that the lower the herbal material: 60% ethanol ratio (0.02 g/mL or 1:66), the higher the content of flavonoids, independent of the AlCl _{3} volume, when intermediate levels of stock solution are used. These results indicate that the herbal material: Solvent ratio has a negative influence on TFC. The use of extreme volumes (0.8 or 8.0 mL) of the stock solution implies in higher TFC when herbal material: 60% ethanol ratio is higher (0.08 g/mL).
Based on the preceding analysis, and considering that higher herbal material: 60% ethanol ratio provides more precise results, the optimized conditions for the TFC method were: An herbal material: 60% ethanol ratio of 0.08 g/mL, 8.0 mL of stock solution and 1.6 mL of AlCl _{3} solution.
Calibration curve and linearity
Calibration solutions were prepared in triplicate and analyzed under the optimal conditions as described above. The oneway ANOVA calculated to compare the replicates of the calibration curve showed no significant difference between groups (F = 0.00007, P > 0.99). The calibration curve was found to be linear in the range 0.0010.021 mg/mL for quercetin dihydrate (C _{15} H _{10} O7 ) [Figure 3]. The regression equation (y = 80.79 ×  0.0734) and correlation coefficient of 0.9984 revealed a good linearity response for the method developed, since according to the literature a coefficient of determination for standard curves >0.99 indicates that the calculated line could explain more than 99% of the experimental data. ^{[11]}  Figure 3: Calibration curve for standard quercetin aluminum chloride complex spectrophotometrically assayed at 430 nm
Click here to view 
Precision
Due to their complex nature, analytical procedures for biological and biotechnological products can present a higher inherent variation. The results of repeatability (intraday variation) and intermediary precision (interday variation) showed RSD% values below 6% and below 8%, respectively [Table 3]. For intermediary precision, no statistical difference was detected when the same analyst evaluated the method on different days and when different analysts performed it on different days (P < 0.05). Replicates of all performed analysis included the extraction, complexation and quantification steps. Although this approach increases the sources of variation, it allows evaluating the influence of all involved steps on the precision of the method. Therefore, our data indicate an acceptable precision and they are in accordance with those found in other works for samples of the same nature. ^{[14],[15]}  Table 3: Repeatability and intermediate precision of the TFC method for leaves of O. basilicum (herbal material)
Click here to view 
Robustness
Results of TFC obtained under variation of analytical conditions of the method for robustness evaluation are presented in the [Table 4]. The analysis by twoway ANOVA revealed that the response (TFC) was not affected by any significant interference when small changes were inserted in the time of complexation with AlCl _{3} (P > 0.05). However, the statistical analysis points out that variations in the time or temperature of extraction influence the TFC results (P < 0.05) [Figure 4]. These results show that the time and the temperature of extraction are critical for the TFC method and should be carefully controlled during the analysis.  Figure 4: Robustness evaluation of the total flavonoid content (TFC) obtained under variations of analytical conditions. *P < 0.05 and ***P < 0.001 compared with the TFC obtained under general method conditions
Click here to view 
 Table 4: TFC obtained under variations of the analytical conditions of the method for robustness evaluation
Click here to view 
Conclusion   
This study allowed the optimization of a simple, fast and precise method for the determination of the TFC in leaves of O. basilicum, which can be used to support the quality assessment of this herbal material.
Acknowledgments   
The authors wish to acknowledge the Research Support FundFAP/UNIVILLE, and the trainees of the Laboratory of Pharmacognosy of UNIVILLE.
References   
1.  Grayer RJ, Kite GC, Goldstone FJ, Bryan SE, Paton A, Putievsky E. Infraspecific taxonomy and essential oil chemotypes in sweet basil, Ocimum basilicum. Phytochemistry 1996;43:10339. 
2.  Nacar S, Tansi S. Chemical components of different basil ( Ocimum basilicum L.) cultivars grown in Mediterranean regions in Turkey. Isr J Plant Sci 2000;48:10912. 
3.  Deschamps C, Simon J. Agrobacterium tumefaciensmediated transformation of Ocimum basilicum and O. citriodorum. Plant Cell Rep 2002;21:35964. 
4.  Bilal A, Jahan N, Ahmed A, Bilal SN, Habib S, Hajra S. Phytochemical and pharmacological studies on Ocimum basilicum LinnA review. Int J Curr Res Rev 2012;4:7383. 
5.  Opalchenova G, Obreshkova D. Comparative studies on the activity of basil  an essential oil from Ocimum basilicum L.  against multidrug resistant clinical isolates of the genera Staphylococcus, Enterococcus and Pseudomonas by using different test methods. J Microbiol Methods 2003;54:10510. 
6.  Hussain AI, Anwar F, Sherazi ST, Przybylski R. Chemical composition, antioxidant and antimicrobial activities of basil ( Ocimum basilicum) essential oils depends on seasonal variations. Food Chem 2008;108:98695. 
7.  Zhang JW, Li SK, Wu WJ. The main chemical composition and in vitro antifungal activity of the essential oils of Ocimum basilicum Linn. var. pilosum (Willd.) Benth. Molecules 2009;14:2738. 
8.  Lee J, Scagel CF. Chicoric acid found in basil ( Ocimum basilicum L.) leaves. Food Chem 2009;115:6506. 
9.  Grayer RJ, Bryan SE, Veitch NC, Goldstone FJ, Paton A, Wollenweber E. External flavones in sweet basil, Ocimum basilicum, and related taxa. Phytochemistry 1996;43:10417. 
10.  Bara MT, Ribeiro PA, Arantes MC, Amorim L, Paula JR. Determination of active principles in vegetable raw materials. J Pharmacogn 2006;16:2115. 
11.  Fernandes AJ, Ferreira MR, Randau KP, de Souza TP, Soares LA. Total flavonoids content in the raw material and aqueous extractives from Bauhinia monandra kurz (Caesalpiniaceae). Scientific World J 2012;923462. 
12.  Bezerra MA, Santelli RE, Oliveira EP, Villar LS, Escaleira LA. Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta 2008;76:96577. 
13.  France. Pharmacopée Française. Paris: Ministère de la Santé; 1992. 
14.  Butnariu M, Coradini CZ. Evaluation of biologically active compounds from Calendula officinalis flowers using spectrophotometry. Chem Cent J 2012;6:35. 
15.  Motilva MJ, Macià A, Romero MP, Labrador A, Domínguez A, Peiró L. Optimisation and validation of analytical methods for the simultaneous extraction of antioxidants: Application to the analysis of tomato sauces. Food Chem 2014;163:23443. 
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 2], [Table 3], [Table 4]
This article has been cited by  1 
South American herbal extracts reduce food intake through modulation of gastrointestinal hormones in overweight and obese women 

 Marina Monteiro Celestino,Aline Corado Gomes,Patrícia Borges Botelho,Alessandra Gambero,Leonardo Mendes Mesquita,Wagner Vilegas,Marcelo Lima Ribeiro,João Felipe Mota   Journal of Functional Foods. 2017; 35: 555   [Pubmed]  [DOI]   2 
Modulatory Effects of Guarana (Paullinia cupana) on Adipogenesis 

 Natália Lima,Erica Numata,Leonardo Mesquita,Pollyana Dias,Wagner Vilegas,Alessandra Gambero,Marcelo Ribeiro   Nutrients. 2017; 9(6): 635   [Pubmed]  [DOI]   3 
Studies on the protective effect of the artichoke (Cynara scolymus) leaf extract against cadmium toxicityinduced oxidative stress, hepatorenal damage, and immunosuppressive and hematological disorders in rats 

 Mohamed ElBoshy,Ahmad Ashshi,Mazen Gaith,Naeem Qusty,Thalat Bokhary,Nagwa AlTaweel,Mohamed Abdelhady   Environmental Science and Pollution Research. 2017; 24(13): 12372   [Pubmed]  [DOI]   4 
In Vitro Propagation, Phytochemical Analysis, and Evaluation of Free Radical Scavenging Property ofScrophularia kakudensisFranch Tissue Extracts 

 Abinaya Manivannan,Prabhakaran Soundararajan,Yoo Gyeong Park,Byoung Ryong Jeong   BioMed Research International. 2015; 2015: 1   [Pubmed]  [DOI]   5 
Effects of Cydonia oblonga Miller leaf and fruit flavonoids on blood lipids and antioxydant potential in hyperlipidemia rats 

 Anwar Umar,Guldiyar Iskandar,Ainiwaer Aikemu,Wuliya Yiming,Wenting Zhou,Bénedicte Berké,Bernard Begaud,Nicholas Moore   Journal of Ethnopharmacology. 2015; 169: 239   [Pubmed]  [DOI]  



