Anjoo Kamboj et al. / Journal of Pharmacy Research 2015,9(5), Available online through

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1 Research Article ISSN: Anjoo Kamboj et al. / Journal of Pharmacy Research 2015,9(5), Available online through Application and Analysis of the Folin Ciocalteu Method for the Determination of the Total Phenolic Content from Leaves, Stems and Seeds of Cucumis sativus L. Anjoo Kamboj*, Ankita Rana, Ripanjot Kaur, Upendra K Jain Chandigarh College of Pharmacy, Landran, Mohali , Punjab,India. Received on: ; Revised on: ; Accepted on: ABSTRACT Cucumis sativus L. is a annual climber belongs to the family cucurbitaceae. Cucumber is a native to the tropics and is one of the oldest cultivated vegetable crop. C. sativus (CS) is growing widely throughout the Bangladesh, Indian subcontinent, Sri Lanka. It is an important medicinal plant with diverse pharmacological spectrum widely used in Ayurveda, Siddha, Chinese medicine etc. Plant has many important phytoconstituents like glycosides, flavones, terpinoids, phytosterol, saponins etc responsible for many of the pharmacological activities such as antibacterial, antifungal, antidiabetic, cytotoxic, antacid, hepatoprotective activity, wound healing activities. 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 C. sativus leaves, stems and seeds, by the UV/Vis spectrophotometric method. The crude extract was used to develop a method for TP assay. The optimum conditions for analysis time, wavelength and standard substance were 30 min, 760 nm and gallic acid, respectively. Under these conditions, validation as per ICH guidelines 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. KEYWORDS:Cucumis sativus, polyphenols, Folin Ciocalteu method. INTRODUCTION Phenolics include simple phenols, phenolic acids, coumarins, flavonoids, stilbenes, hydrolysable and condensed tannins, lignans, and lignins. These compounds are among the most widely occurring secondary metabolites in the plant kingdom, acting mainly as phytoalexins, attractants for pollinators and contributors to plant pigmentation, antioxidants, and protective agents against UV light, among others. Although quantitative determination of polyphenols is hampered by their structural complexity and diversity, several methods have used to determine polyphenols in plant extracts [1-3]. Spectrophotometry in the UV-Vis region may be a useful tool to help to resolve this problem. Colorimetric reactions are widely used in the UV/VIS spectrophotometric method, which is easy to perform, rapid and applicable in routine laboratory use, and low-cost [4]. However, it is important that colorimetric assay need to use a reference substance, then this method *Corresponding author. Dr. Anjoo Kamboj Professor & Vice Principal Chandigarh College of Pharmacy, Landran,Mohali , Punjab INDIA measures the total concentration of phenolic hydroxyl groups in the plant extract. 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 [5]. The Folin-Ciocalteu method is described in several pharmacopoeias [6,7]. The reaction forms a blue chromophore constituted by a phosphotungstic phosphomolybdenum complex [8], where the maximum absorption of the chromophores depends on the alkaline solution and the concentration of phenolic compounds [5]. However, this reagent rapidly decomposes in alkaline solutions, which makes it necessary to use an enormous excess of the reagent to obtain a complete reaction. This excess can result in precipitates and high turbidity, making spectrophotometric analysis impossible. To solve this problem, Folin and Ciocalteu included lithium salts in the reagent, which prevented the turbidity [9]. The reaction generally provides accurate and specific data for several groups of phenolic compounds, because many compounds change color differently due to differences in unit mass [10] and reaction kinetics [9]. Many studies have discussed the use of the Folin-Ciocalteau reagent to determine polyphenols, and the general or specific value of the method, because some specific details may be modified [8,11,12].

2 Anjoo Kamboj et al. / Journal of Pharmacy Research 2015,9(5), Cucumber (Cucumis sativus L.) belongs to the family cucurbitaceae. Cucumber is a native to the tropics and is one of the oldest cultivated vegetable crop. It is known in the history for over 3,000 years. C. sativus is growing widely throughout the Bangladesh, Indian subcontinent, Sri Lanka. It is an important medicinal plant with diverse pharmacological spectrum widely used in Ayurveda, Siddha, Chinese medicine etc. They were already used in ancient times to dissolve stones caused by uric acid. Their cleansing effect on the intestines, kidneys, lung and skins was also known. People suffering from stomach or liver diseases also benefit from the consumption of cucumbers. They have been known to cure some headaches, bleeding, dizziness, and pale skin. Cucumber juice contains a substance, which promotes blood circulation of the skin. It is for this reason that it is widely used in cosmetics. Plant has many important phytoconstituents like Glycosides, flavones, terpinoids, phytosterol, saponins and anolignan B, tannins, ellagic acid, glucose, and fructose. These compounds were found to be responsible for many of the pharmacological activities such as antibacterial, antifungal, antidiabetic, cytotoxic, antacid & carminative activity, hepatoprotective activity, wound healing activities. Further the plant is used in the treatment of gastric ulcer, constipation, general debility, piles. Hence, this plant provides a significant role in the prevention and treatment of a disease [13-20]. This report describes the chemical studies of C. sativus, aimed at characterizing the plant drug and standardization of the content of polyphenols in the extracts. 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 of C. sativus.. RESULTS AND DISCUSSION Method Optimization Each plant has a characteristic chemical composition, with uniform phenolic groups present in the same species. Similar chemical structures may show the same chemical interactions with specific reagents during the reaction [10]. Both the reference substances used show approximately the same spectra as the methanolic extract (ME) from C. sativus (Figure 1-5). Figure 2: UV spectrum of tannic acid after folin-ciocalteu reaction with lmax at 760nm Figure 3: UV spectrum of ME extract of Leaves part of C. sativus after Folin-ciocalteu reaction with lmax at 760nm. Figure 4: UV spectrum of ME extract of Seeds part of C. sativus after Folin-ciocalteu reaction with lmax at 760nm. Figure 1: UV spectrum of gallic acid after folin-ciocalteu reaction Figure 5: UV spectrum of ME extract of Stems part of C. sativus with lmax at 760nm. after Folin-ciocalteu reaction with lmax at 760nm.

3 The percentage increase in absorbance of each solution was compared at the wavelengths of 645, 675, 745, 760, and 800nm. The statistical analysis indicated a significant increase in absorbance at 760nm compared to 645, 675 and 745nm for all solutions. The absorbance at 800nm was lowered than at other wavelengths; however this decrease was not statistically significant, so 760nm is the most stable wavelength. Using the wavelength of 760nm, which appeared to be best suited to produce maximum absorption of the substances under study, the next step was to determine the reaction kinetics with respect to the time period prior to the spectrophotometric measurement. The increase in percentage absorbance of each solution relative to the time periods of 10, 20, 30 and 40 min was determined. These data showed that the Folin Ciocalteu reaction was stable during the period analyzed, since after 30 min the absorbance increased less than 5% of the value at 10 min, and did not decrease between 30 and 40 min. With respect to the standard, different specific absorptivities (760 nm; 30 min) were observed for each reference compound: A1%,1cm gallic acid = 1130 ± 42.72; A1%, 1cm tannic acid = ± The intensity of reaction of phenolic compounds with Folin- Ciocalteu reagent is proportional to the availability of hydroxyl groups present on the aromatic ring and influences the reductive potential of the molecule. Gallic acid has only one ring and three unsubstituted hydroxyl groups and is less influenced by electronic interactions such as steric or resonance effects. Gallic acid therefore has higher specific absorptivity and is probably best reference substance for determination of TP. The method was optimized for an extract of C. sativus, and similar results were obtained. TP of ME from C. sativus and Calibration Curve of Gallic acid Standard The calibration equation was y=0.1071x (n = 6, r 2 = 0.998) for gallic acid (Figure 6). The relative standard deviations (RSDs) of the slopes were =5% for the analyte (n = 6). Table 2 shows the back-fit calculations for curve data for the standard used in the validation runs, as well as the precision and accuracy of the back-fit calculations. Anjoo Kamboj et al. / Journal of Pharmacy Research 2015,9(5), Table 2: Statistical data of regression equations of calibration curve for gallic acid & linearity and specificity test for TP content from ME of C. sativus by FC (Folin-ciocalteu) reaction method Regression Calibration Curve Linearity of Specificity of Analysis for Gallic acid (FC reaction (FC reaction method) method) Regression equation y = x Leaves y=0.029x y=0.029x Stems y=0.024x y=0.024x Seeds y=0.026x y=0.026x Slope Leaves Stems Seeds Intercept Leaves Stems Seeds Regression coefficient (R2) Leaves Stems Seeds Linear Range (µg/ml) Leaves Stems Seeds The specific absorptivity is the absorbance of a solution in a concentration of 1 g/100 ml (1%-10,000 µg/ml). The specific absorptivity of gallic acid was calculated using the linear equation (y = x ), obtaining a value of Therefore, the mean absorbance for ME from C. sativus was ± [4.0µg/ml] for leaves, 0.138± [4.0µg/ml] for stems and ± [4.0µg/ml]) for seeds, and TP expressed as gallic acid equivalent was g %w/w, g %w/w and g% w/w for leaves, stems and seeds respectively. [Table 1] Determination of Gallic acid Equivalent Content in Leaves, Stems & Seeds of C. sativus by Folin-ciocalteu reaction Method Using validated method the TP content in leaves, stems and seeds of methanolic extracts of C. sativus were determined by Folin-ciocalteu reaction method. [Table 1] Table 1: Content in Leaves, Stems & Seeds of C. sativus by Folinciocalteu reaction Method Folin-ciocalteu Reaction Method GAE Content GAE Content (gm %w/w of dry pwd) (%w/w of Extract) Extract Methanolic Methanolic Leaves Stems Seeds Figure 6: Calibration curve of Gallic acid by Folin-Ciocalteu Reaction Method at 760nm. All values are expressed as mean± SD (n=3), *GAE= Gallic acid Equivalent

4 Anjoo Kamboj et al. / Journal of Pharmacy Research 2015,9(5), Linearity Based on 1/x weighted linear regression analysis, the responses for the ME in related concentration ranges were linear (Figure 7). The data obtained in the linearity test for ME and gallic acid appears in Table 2. Linearity range for ME of leaves, stems and seeds of C. sativus was found to be µg/ml, µg/ml and µg/ml respectively. Specificity Analysis of the results of the specificity test indicated that the conditions were satisfactory (Table 2). In the case of complex matrices, if the matrix without the analyte is not available, the effects of the matrix system can be tested by comparing the slopes of linearity and specificity. If the curves are parallel, we can state that the method is specific [17-19]. The specificity of the method for ME was confirmed by superimposing the analytical curves (Figure 7a-c), because the slopes of the linear equations (linearity and specificity, see Table 2) were very similar. Limits of Detection and Quantification The quantification range was from 0.8 to 7.2 µg/ml, because linearity was maintained (r 2 = 0.999). Based on the linear equation from the test for linearity, the LOQ was 0.317µg/ml (absorbance of 0.032). The LOD was µg/ml (absorbance of 0.026), because there was a significant difference between this concentration and the next-lower concentration (0.8 µg/ml; t1,4 = 1.398, P < 0.5). Based on the linear equation from the linearity test, LOD was 0.117µg/ml for leaves extract. Similarly the LOQ was µg/ml & µg/ml and LOD was 0.137µg/ml & µg/ml for stems and seeds respectively. Precision The repeatability and the intermediate precision for ME were with no significant difference between them Therefore, the proposed method is adequately precise within the prescribed limits of ICH guidelines (%RSD < 2%). (Table 3) Table 3: Intraday and Interday Precision (GAE) Intraday Precision Interday Precision Conc. Absorbance Absorbance (4µg/ml) Leaves Stems Seeds Leaves Stems Seeds a. Stems b. Seeds Mean SD %RSD (t 1,5 ) P t critical Accuracy The result for the accuracy test showed a percentage recovery was in range of %, % and %, for leaves, stems and seeds respectively, for the lowest, intermediate and highest levels (Table 4). These percentages were within the range of 85% 115% established in published reports. This indicates that the method has good accuracy for determining TP in ME from C. sativus. Table 4: Accuracy of Gallic Acid Equivalent (TP) by Recovery Method in methanolic extract by Folin-ciocalteu Reaction Method Levels % Recovery Leaves Stems Seeds c. Leaves Figure 7a-c: Linearity curve & specificity curve, with correlation coefficient (r 2 ) and linear equation for TP in ME from C. sativus by Folin-ciocalteu reaction method. 80% % % *The data is the result of triplicate analysis. Robustness The change in Na 2 concentration in the robustness assay took into account data from the literature: 7.5% in the Brazilian Pharmacopeia [6], 14:05% in Glasl [10], and 10.75% in the European Pharmacopoeia [7]. The values for absorbance measured after changing the Na 2 concentration and the natural light incidence in the Folin

5 Anjoo Kamboj et al. / Journal of Pharmacy Research 2015,9(5), Ciocalteu reaction i.e. without protection from light; Na 2 solution solution 7.5% (w/v);). Comparison of these results with those obtained in the linearity assay indicated no significant difference between means (F1,5=1.33: P=0.38: Fcritical=5.05; F1,5=1.30,1.41: P=0.38,0.35: Fcritical=5.05; F1,5=1.22,1.26: P=0.41, 0.40: Fcritical=5.05) for leaves, stems and seeds respectively. However, there was a tendency for the proposed conditions and validated by method [protection from light and Na 2 solution 10.75% (w/v)] (Table 5). Table 5. Robustness Analysis S. No Leaves Stems Seeds 7.5% Without 7.5% Without 7.5% Without Na 2 light Na 2 light Na 2 light protection protection protection Mean df F P F critical Experimental Standards and Chemicals All chemicals were analytical-reagent grade and the water was distilled. The chemicals included 2 N Folin-Ciocalteu reagent, anhydrous sodium carbonate, gallic acid (Sigma-Aldrich), tannic acid (Sigma- Aldrich). Plant Material Fresh and fully grown plant of C. sativus was collected from fields of Khanna, Punjab (India) in month of May and June. Plant were subjected to first the morphological identification as described in the literature and followed by authentication by Dr. Sunita Garg, Chief Scientist and Head, Raw Materials Herbarium and Museum (RHMF), NISCAIR, New Delhi. procedures recommended by the European Pharmacopoeia [5] for determination of total tannins, with slight modifications. We used a solution of 10.75% anhydrous sodium carbonate. For the selection of a reference standard, stock solutions of each standard (gallic acid, tannic acid 50 µg/ml) and ME (50 µg/ml) were prepared in distilled water. Two milliliters of each solution was transferred to a 25 ml volumetric flask containing distilled water (10 ml) and Folin-Ciocalteu reagent (1 ml) [9]. The volume was completed with 10.75% anhydrous sodium carbonate (w/v), resulting in final concentrations of 4 µg/ml of each standard and 4 µg/ml of ME. The samples were scanned in a UV/Vis spectrophotometer (Shimadzu PC-1800, Kyoto, Japan) beginning 10 to 40 min after the addition of the sodium-carbonate solution, and scanning from 400 to 900 nm at intervals of 2 min between each reading, to determine the spectra. Distilled water was used as a blank, with a quartz cell (1 cm path length). The kinetic reaction was evaluated by comparing the percentage increase in absorbance of each solution, for the wavelengths of 645, 675, 760, 800 nm. 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.The reaction times were observed and compared statistically, using the percentage increase between the reaction times (10, 20, 30, and 40 min) at 760 nm. The specific absorptivity of each standard was calculated based on the Lambert-Beer Law [20]. Analytical Method Validation For validation of the analytical method, the guidelines established by the ICH (International Conference on the Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use) were employed [19, 21-24]. Method for TP Analysis of ME from C. sativus and Calibration Curve of gallic acid Standard Extraction The plant material (Leaves, stems and seeds) was washed with water to remove soil, dried in air under shade at room temperature (25± 2 o C) and reduced to coarse powder in a mixer grinder. Using Soxhlet apparatus successive extraction was carried out with methanol after defatting with petroleum ether. The liquid extracts obtained were concentrated and dried in vacuum desiccators and weighed and stored at -20 C. Method Optimization and Standardization for Determination of Total Polyphenols (TP) For the optimization and standardization of the spectrophotometric method using the Folin-Ciocalteu reagent, three parameters were analyzed: (1) reaction kinetics, (2) maximum absorption wavelength, and (3) the standard that best characterizes the ME of C. sativus. The method utilized for the test was a colorimetric assay, based on general All extraction and dilution operations were protected from light. ME solution: The ME (10 mg) was transferred to a 100 ml volumetric flask and diluted with water, and 1-9 ml of this stock ME solution was diluted to 10 ml with water. Then, a mixture was prepared with 2 ml of this solution, 1 ml of 2 N Folin-Ciocalteu reagent, and 10 ml of water, and volumetrically diluted to 25 ml with 10.75% w/v anhydrous sodium carbonate (w/v). After 30 min, the absorbance was measured at 760 nm, using water as the compensation liquid and a quartz cell (1 cm path length) in a UV-Vis spectrophotometer. Calibration curve: In three replicates, 10.0 mg of gallic acid was dissolved in water, immediately before use, and diluted to 100 ml. A liquots of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6, 7, 8, 9 ml were diluted to 10 ml in water (stock solution). Each stock solution was prepared according to the procedure described above for the ME. The final

6 Anjoo Kamboj et al. / Journal of Pharmacy Research 2015,9(5), concentrations were 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, 4.0, 4.8, 5.6, 6.4, 7.2 µg/ml of gallic acid. The absorbance was measured under the same conditions as for the ME. The specific absorptivity was determined from the linear equation. The TP expressed as gallic acid of the ME from C. sativus was calculated by Equation (1): Accuracy The accuracy was evaluated using different concentration levels: lowest concentration (LC), intermediate concentration (IC) and highest concentration (HC), following the addition of 3.2, 4.0 and 4.8 µg/ ml of gallic acid solution to the 4 µg/ml of ME solution, in three replicates. Accuracy was assessed as the percentage recovery, which was calculated from Equation (2): TP = A/A1% m Where A = Absorbance of sample for TP; A1%, 1cm = Specific absorptivity of gallic acid; m = mass of the sample examined, in grams; = dilution factor of the sample Linearity To establish the linearity of the proposed method, five stock solutions were prepared, in three replicates, from 10.0 mg of CE. Concentrations of 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, 4.0, 4.8, 5.6, 6.4, 7.2 µg/ml were obtained. Specificity Specificity was determined by adding 1 ml of a gallic acid solution (1.0 µg/ml) to each stock solution described in the linearity test. The method is considered specific if the slopes of the linear equations in the tests for linearity and specificity are equal or very similar. 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 ME linearity and the slope, using the appropriate multiplier. These results were compared with the LOD and LOQ obtained by extension of the linearity curve of ME: a stock solution was prepared, in three replicates, of ME (4 µg/ml), and aliquots of 5.0, 3.0, 2.0, 1.5, 1.25, 1.15, 1.0, 0.5, 0.25, 0.2, 0.17, 0.15 and 1.0 ml were, separately, diluted to 10 ml. A reaction mixture was prepared as described in the TP method, with 2 ml from each solution. The results, together with the results for linearity, were submitted to statistical analysis. The quantification range was determined based on the lowest and highest concentrations that maintained linearity. The LOD was determined as the lowest concentration that was significantly different from the next lowest concentration. Precision Two levels were evaluated: repeatability (intra-day) and intermediate (inter-day). The repeatability was assessed using six samples of 4 µg/ ml of ME, and the intermediate level was assessed using three samples and on at least two different days. A coefficient of variation over 15% and a significant difference between days were considered unacceptable. where A = absorbance of sample after addition of the standard; AT = theoretical absorbance calculated for the sum of the absorbance of ME from C. sativus and the expected absorbance of gallic acid, based on the calibration curve for each level. The method is considered accurate if the recovery percentages are between 85% and 115%. Robustness The robustness was demonstrated by analyzing the stability of the solution under the influence of natural light and a range of ph changes (7.5 % (w/v) anhydrous sodium carbonate solution), evaluated in three replicates. Statistical Analysis The data were expressed as mean ± standard deviation [relative standard deviation (%)]. The linear correlation tests and residual analyses were performed by simple linear regression, considering R 2 equal to or higher than Table 6: Method Validation Parameters Parameters Leaves Stems Seeds Detection wavelength (nm) Linearity range (µg/ml) Slope Intercept Correlation coefficient y =0.029x y=0.024x y=0.026x Regression coefficient (r 2 ) Limit of Detection (LOD) µg/ml Limit of Quantitation (LOQ) µg/ml CONCLUSIONS After optimization of the conditions for the spectrophotometric determination of phenolic using the Folin-Ciocalteu reagent, all parameters analyzed showed adequate results. The UV-Vis spectrophotometric method described here was successfully validated as suitable for the determination of TP of methanolic extract leaves, stems and seeds part of C. sativus. This methodology using the Folin-Ciocalteu reaction complies with the requirements for analytical use and for ensuring the reliability of the results.

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