amount using FC reagent Protocol No. & Title: 15.3d ...

Protocol No. & Title: 15.3d Estimation of the phenolic and other oxidation substrate content in extract using Folin-Ciocalteau reagent Version Date: 5 November 2012: Version 1 Author: Dr. Marsha J. Lewis Purpose:

This is known as Folin-Ciocalteau method. Phenolic and antioxidant compounds are commonly present in extracts of natural products from plants and fungi. These compounds may exhibit many biological effects including antioxidant activity. The phenolic compound content of extract can be approximated by F-C reagent and expressed as gallic acid equivalents in mg (or µg or mmol) /g of crude extract. The F–C assay relies on the transfer of electrons in alkaline medium from phenolic/antioxidant compounds to phosphomolybdic and phosphotungstic acid complexes, which can be determined by measuring the absorbance at 765 nm. Owing to the general nature of the F–C chemistry, it is a measure of total phenols and other oxidation substrates. However, the F–C assay is simple and reproducible and has been widely used for studying phenolic antioxidants. (1)

References:

1. 2.

Materials (partial list, these are stock solutions or solutions you need to prep): Safety Notes:

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Ainsworth, E. A.; Gillespie, K. M., Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent. Nature Protocols 2007, 2, (4), 875-877. Everette, J. D.; Bryant, Q. M.; Green, A. M.; Abbey, Y. A.; Wangila, G. W.; Walker, R. B., Thorough study of reactivity of various compound classes toward the FolinCiocalteu reagent. Journal of agricultural and Food Chemistry 58, (14), 8139-81

 700 mM Na2CO3 in water.  25 mM Gallic acid in 95% methanol is C7H6O5 with a molar mass of 170.12 g/mol (stable for 3 weeks, in refrigerator, in dark). Instructions in protocol.  95% (vol/vol) methanol in water 1. Wear gloves, lab coat, and goggles as needed. Work in fume hood as required.

15.3d Natural Product Screening- Estimatin of phenolic compounds and oxidizing substrate

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Prepare extract or purified fraction for testing You need at least 1 mgs (that is milligrams---not grams!) of product for testing.

How do measure 1 mg of extract? You should have a known amount of extract that you weighed after isolation (X mgs) and you should have added a known amount of solvent for

temporary storage (e.g. 0.5 ml (a.k.a. 500 µl) methylene chloride). So, you have a solution that you know is X mgs of extract per ml (in glass vial—hopefully, you did NOT use plastic or the plastic could degrade in your solvent). One word of warning: if you used a very volatile solvent for dissolution of your isolated product, it will evaporate, even in cold storage. To ensure you know the correct concentration of isolated product, make sure you have the volume that you think you have by measuring the volume with a pipettor or mark the bottle when you first dissolve your product so you know the initial volume. As the solvent evaporates, your isolated product may also begin to come out of solution. Add back solvent to completely solubilize or to bring to the concentration you desire. Then, measure 1.0 mgs for this assay preparation:

Use the rotary evaporator to remove the solvent from your 1.0 mgs of extract, unless your product is dissolved in methanol. If your product is dissolved in methanol, you do not need to remove the solvent, add enough autoclaved DIW to bring the solution to 95% (vol/vol) methanol. If necessary, after the solvent is removed, add 500 µl of 95% methanol or whatever you need to dissolve your product up to 1ml. Your extract should be soluble in 95% methanol. If it is not, let me know. You can prepare this a day or two or a week ahead of time and store in cold storage (-20 °C). Prepare Gallic Acid serial dilutions for standard curve You need to prepare a standard curve to relate absorbance to amount of antioxidant substrate of a known (gallic acid in this protocol). You will prepare serial dilutions covering 8 µM–6.0 mM gallic acid [in 95% (vol/vol)methanol]. First, prepare the most concentrated solution (6mM). 1. Prepare Lab Gallic Acid Stock Solution (if necessary, check refrigerator). In a 100-mL volumetric flask, add 0.43 g of dry gallic acid and dissolve and bring to 100 mL with 95% (vol/vol) methanol (this will be 25 mM). Can be opened daily, but to store, keep closed in a refrigerator up to three weeks. If beyond three weeks, make new Gallic Acid Stock solution. 2. To prepare a calibration curve, add 500 µl of the Lab Gallic Acid Stock Solution (25 mM) into a 2 mL micro-centrifuge tube. Label as 6 mM Gallic Acid. 3. Add 1 ml of 95% methanol to each seven additional tubes and label: 2 mM, 0.67 mM, 0.22 mM, 75 µM , 25 µM , and 8 µM, and 0.0. 4. Perform serial dilutions (*if you need to brush up on this concept, see end of protocol*) using the 6mM solution prepared in Step 2 as your starting solution. Pipet 500 µl from the 2/6

15.3d Natural Product Screening- Estimatin of phenolic compounds and oxidizing substrate

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6mM solution into the second tube labeled as 2mM that contains 1 ml of diluent (the 95% methanol), mix well. 5. Next add 500 µl from the 2 mM tube to the third tube (labeled as 0.67 mM) that has 1 ml of diluent, mix well, and repeat until all solutions have been made. Do not add any gallic acid solution to the control labeled 0.0. That tube should only contain 1 ml of 95% methanol. 6. Remember--leave the last tube with 1 ml of 95% methanol only. That means the tube with 8 µM gallic acid solution will contain a total of 1.5 ml and the other tubes will all have a total volume of 1ml. Check this to ensure you executed the serial dilution correctly. NOTE: These solutions will have gallic acid concentrations of 1 mg/ml (or 6 mM), 0.3 mgs/ml (2 mM), 0.1 mg/ml (0.67 mM), 0.04 mg/ ml (0.22 mM), 13 µg/ml (75 µM) , 4.3 µg/ml (25 µM) and 1.4 µg/ml (25 µM) and 0 mg/ml in your 95% methanol sample only. Below is an example of a Gallic Acid Standard curve. You will construct this standard curve with your prepared samples later in this method. Execute F-C Method using Extract samples, Standard samples, and Control samples You will now execute the F-C method to determine the amount of phenolic compounds in your extract in terms of gallic acid. In this section: the “extract sample” is your isolated fungal extract, the “standard samples” refer to the gallic acid samples prepared for your standard curve, the negative control is your methanol blank. Think about which sample serves as your positive control(s)? You will want to perform this method in duplicat or triplicate. Think about what you want to do. You will have to assemble enough 2ml tubes and pre-label accordingly. If you have two extract samples, eight standard samples, and one negative control, that is eleven (11) samples. If you choose to perform the experiment in duplicate (recommended), then you will need 22 sample tubes, pre-labeled. 1. Add 100 µl of each sample extract sample, standard sample, and 95% (vol/vol) methanol blank to duplicate pre-labeled, prepared 2-ml microtubes. 2. Add 200 µl of 10% (vol/vol) F–C reagent and vortex thoroughly. Wait 1 to 2 minutes before adding the sodium carbonate. Mixing well is critical to the success of this assay.

NOTE: CRITICAL STEP The F–C reagent should be added before the alkali to avoid the air-oxidation of phenols. 3. Add 800 µl of 700 mM Na2CO3 into each tube and incubate the assay tubes at ambient room temperature for 2 hours. Total volume in each tube is 1.1 ml.

NOTE: CRITICAL STEP This assay can be incubated at warmer temperatures for shorter times to obtain faster color development, but this can increase the standard error of the assay. This has not yet been optimized for fungal extracts. If you are in a position to investigate different time and temperatures: try 37°C for 30 or 60 minutes.

4. Set up the NanoPhotometer™ for single wavelength UV-vis spectrophotometry. a. Power on NanoPhotometer™. b. Select 3) Functions c. Select 1) Single Wavelength d. Enter 765 nm for the wavelength e. The mode should be “Absorbance” f. The pathlength should be 10mm g. Use the 1 ml disposable cuvettes. Blank with your negative control (the 95% methanol). 3/6

15.3d Natural Product Screening- Estimatin of phenolic compounds and oxidizing substrate

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5. Transfer 1.0 ml from each tube of extract sample or standard sample into a clear disposable 1ml cuvette and read the absorbance of each duplicate sample at 765 nm on the Nanophotmeter™. Record all your data in your lab notebook. It will be easier to set up a table to track the data. See Table 1 for an example of how to organize your data. Standard Sample Data: A765 mM mg/L (1)

A765 (2)

6.00 2.00 0.67 0.22 0.07 0.02 0.01 0.00

1.400 0.467 0.156 0.052 0.017 0.006 0.002 0.001

1000 333 111 37 12 4 1 0

1.500 0.500 0.167 0.056 0.019 0.006 0.002 0.001

Table 1. Example Data Table

Data Analysis You will now analyze the data. First you will need to analyze the absorbance data from the standard samples and negative control. The absorbance is the dependent variable as it is the response to the standard sample concentration, which is the independent variable. Linear regression attempts to model the relationship between two variables by fitting a linear equation to observed data. Before attempting to fit a linear model to observed data, you should first determine whether or not there is a relationship between the variables. A scatterplot can be a helpful tool in determining the strength of the relationship between two variables. If there appears to be no association between variables (i.e., the scatterplot does not indicate any increasing or decreasing trends), then fitting a linear regression model to the data probably will not provide a useful model. An association is expected and if you do not see a trend, then something is wrong in the execution of the experiment. A valuable numerical measure of association between two variables is the correlation coefficient (R), which is a value between -1 and 1 indicating the strength of the association of the observed data for the two variables. The square of the correlation, called "R-squared", measures the "fit" of the regression line to the data. The standard phrase is: r-squared measures the percent of variation in y explained by the variable x. It is easy to use r-squared: if it is low (near 0) then there's still lots of unexplained variability in the data. If it's close to 1, then the regression line does a good job of fitting the data. In the context of prediction: a high r-squared means that if you tell me the value of x, my prediction of y will be pretty close to what we actually observe. But if r-squared is low, my prediction might be pretty far off.

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15.3d Natural Product Screening- Estimatin of phenolic compounds and oxidizing substrate

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A linear regression line has an equation of the form Y = b + mX, where X is the independent variable and Y is the dependent variable. The slope of the line is m, and b is the intercept (the value of y when x = 0). 1. Plot the standard sample data as shown in Table 1 in an Excel or similar program using the “scatterplot” functin. Plot Absorbance (Y axis, dependent variable) versus Molarity (X-axis). Derive the linear regression equation. In Excel, to derive the linear regression equation, follow these simple steps: a. Click on one data set on the graph b. Right click and select “Add trendline…” from the options that appear c. A new dialog box will appear (Figure 1). Select “Linear” and click on “Display Equation on chart” and “Display R-squared value on chart”

Figure 1. Dialog box that appears after selecting “Add trendline…”

d. Your graph should now look like Figure 2. Notice the R-squared is 1.0 in my example, which is a perfect fit. Your R-squared value should be between 0.95 and 1.0. If it is lower than 0.95, you do not have good data.

Figure 2. Excell Scatter plot with linear trendline fit to data

2. Now you can calculate your total phenolic using the regression equation derived from your standard curve (you measured Y, the absorbance of your extract samples, and now calculate X, the gallic acid equivalents of your phenolic compounds). 5/6

15.3d Natural Product Screening- Estimatin of phenolic compounds and oxidizing substrate

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Troubleshooting You may find variability between replicates of the same extract from different extraction preparations. Because the fungus is cultured in a controlled environment, the phenolic content is not expected to vary much. If you see variability, I suggest replicating the experiment. If there is significant variability between replicate samples, typical issues are lack of mixing reagents well with the samples or the incubation time and temperature need to be further optimized. So, ensure the assays solutions are vortexed well after the addition of the F-C reagent and allow it to stand for 1 to 2 minutes before adding the sodium carbonate solution. Serial Dilution Often in experimental work, you need to cover a range of concentrations, so you need to make a bunch of different dilutions. You might think it would be good to dilute 1/2, 1/3, 1/10, 1/100. These seem like nice numbers. There are two problems with this series of dilutions. 1. The dilutions are unnecessarily complicated to make. You need to do a different calculation, and measure different volumes, for each one. It takes a long time, and it is too easy to make a mistake. 2. The dilutions cover the range from 1/2 to 1/100 unevenly. In fact, the 1/2 vs. 1/3 dilutions differ by only 1.5-fold in concentration, while the 1/10 vs. 1/100 dilutions differ by ten-fold. If you are going to measure results for four dilutions, it is a waste of time and materials to make two of them almost the same. Serial dilutions are much easier to make and they cover the range evenly. Serial dilutions are made by making the same dilution step over and over, using the previous dilution as the input to the next dilution in each step (Figure 3). Since the dilution-fold is the same in each step, the dilutions are a geometric series (constant ratio between any adjacent dilutions). For example: 1/3, 1/9, 1/27, 1/81

Figure 3. Different ways of making serial dilutions.

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15.3d Natural Product Screening- Estimatin of phenolic compounds and oxidizing substrate

amount using F-C reagent