AT17526U1 - Method for the colorimetric determination of polyphenols in veterinary or human fluids - Google Patents

Abstract

In a method for the colorimetric determination of polyphenols in veterinary or human fluids, in which the veterinary or human fluid is reacted with Folin-Ciocalteu reagent (FCR) to form a color reaction and the color reaction formed is evaluated quantitatively colorimetrically by comparison with a standard, it comprises the steps: providing the veterinary or human fluid, adding diluent until at least a 7-fold dilution is achieved, removing a quantity of the diluted veterinary or human fluid from the wells and presenting this quantity in a test plate, and presenting it in further wells of the test plate of the standard and a control, addition of diluent, addition of an amount of 0.2 M FCR, addition of an enhancing reagent, mixing of all ingredients, allowing to incubate at room temperature for a period between 1 and 24 hours, colorimetric determination of the color formed breaktion at wavelengths between 600 and 766 nm, correcting a result obtained from polyphenols contained in the veterinary or human fluid and quantitatively determining the phenol content in the biological fluid by comparison with the color reaction of the standard, and use.

Description

description

The present invention relates to a method for the colorimetric determination of polyphenols in veterinary and human fluids, in which the veterinary or human fluid is reacted with Folin-Ciocalteu reagent (FCR) to form a color reaction and the color reaction formed is analyzed colorimetrically Comparison with a standard is evaluated quantitatively and the use of the same.

[0002] In living organisms there is usually a balance between pro-oxidative and anti-oxidative substances. This balance can be disturbed for a variety of reasons, such as an excessive intake of antioxidants, diseases that are promoted or mediated by free radicals or disorders that impede or impede the absorption of antioxidants. It has long been known that the presence of high levels of polyphenols indicates that adequate levels of protective antioxidants are present in biological samples and especially living organisms. These antioxidants have a beneficial effect and also protective effects on diseases caused by free radicals. Since antioxidants can also serve as radical scavengers, among other things, it is assumed that they can even prevent these diseases and in any case have a positive effect on them.

Of course, it is also obvious to a person skilled in the art that excessively high concentrations of polyphenols can also have dangerous effects, so that in principle an equilibrium or as stable a level as possible in living organisms of the same should be maintained. In order to be able to maintain such a uniform level, it is important on the one hand to know or to be able to determine the polyphenol content in food consumed by the individual test persons and on the other hand to determine the polyphenol content in biological samples from human or animal organisms easily and reliably to be able to

A method for determining tyrosine and tryptophan in proteins using a phenolic reagent has already been described by O. Folin and V. Ciocalteu in the Journal of Biological Chemistry, Vol. LXXIII, No. 2, page 627 ff. This colorimetric method has succeeded in solving the turbidity problem, which prevented a halfway decent measurement in even older methods. In order to be able to carry out this reaction, Folin and GCiocalteu have developed a phenol reagent, which has found its way into analysis as the Folin and Ciocalteu reagent and which reagent is based in particular on the fact that polyphenols with transition metals, in the present case with tungstate and molybdate in phosphoric acid, produce a dark blue colored Form a complex which can be measured photometrically or colorimetrically. Depending on the type of sample obtained, these measurements are carried out at different wavelengths, up to a maximum of 766 nm.

A disadvantage of this old method by Folin and Ciocalteu is the fact that for a more or less exact determination of the phenol content, huge amounts of sample have to be processed, which means that this old method no longer proves to be economical.

The original Folin and Ciocalteu method was further developed by Andrew Waterhouse, Department of Viticulture & Enology, University of California, Davis. With this method, it has been possible to significantly reduce the volumes of the reagents and, in particular, to largely avoid an excess of reagent. Despite this miniaturization of the method, work is still carried out on a milliliter scale, so that large quantities of reagents have to be used, which makes the detection economical.

The Folin-Ciocalteu reagent used for the detection of polyphenols, which can be purchased from Sigma-Aldrich, for example, is essentially a solution of phosphomolybdic acid and phosphotungstic acid and its preparation was described, for example, by Singleton and Rossi, AJEV 1965, 16: 144-158. The use of the FolinCiocalteu reagent for the detection of phenols in a wide variety of fluids, including biological

chemical and plant fluids, excellent results, but has not been successfully applied to animal and human samples to date, since these biological fluids sometimes cause violent side reactions due to the interaction of a wide variety of other substances contained in the fluids. In particular, biological fluids foam extremely when mixed with Folin-Ciocalteu serum, so that a meaningful measurement of the phenol content in these fluids did not appear possible due to the disturbing effects and in particular due to the loss that is to be feared in the event of excessive foaming, and thus this method to date cannot be applied to such samples.

The aim of the present invention is to further develop known detection methods for polyphenols using the Folin-Ciocalteu reagent so that it can be used without hesitation on human and veterinary fluids and reproducible, quantitative results can be obtained.

To solve this problem, the method according to the invention is essentially characterized in that it comprises the following steps:

a. Submission of the possibly previously cleaned veterinary or human fluid in wells of a dilution plate,

b. adding diluent until at least 7-fold dilution, in particular at least 10-fold dilution of the veterinary or human fluid is achieved,

c. Taking a quantity of the diluted veterinary or human fluid from the wells of the dilution plate and presenting this quantity in wells of a test plate, as well as presenting the standard and a comparison in further wells of the test plate,

d. adding diluent to the diluted veterinary or human fluid, the standard and the control,

e. adding an amount of 0.2 M FCR corresponding to 3 to 7 times the amount, in particular 5 times the amount of the diluted veterinary or human fluid,

f. adding an amplification reagent,

g. Mixing of all the components, in particular by shaking on a horizontal shaker,

h. Allow to incubate at temperatures between 20 °C and 25 °C for a period between 1 and 24 hours, preferably 2 to 4 hours,

i. colorimetric determination of the color reaction formed at wavelengths between 600 and 766 nm,

j. correcting an obtained result of polyphenols contained in the veterinary or fluid by multiplying by the value of the in step b. chosen dilution and

k. quantitative determination of the phenol content in veterinary or human fluid by comparison with the color reaction of the standard.

Because the biological fluid is diluted with a diluent to at least a seven-fold, in particular ten-fold dilution before being mixed with the Folin-Ciocalteu reagent, it is surprisingly possible to achieve the foaming of the biological that usually occurs when reacting with the FCR reagent To prevent samples and thereby to obtain a reproducible color value in the reaction with the FCR reagent, which can then be determined colorimetrically. For a quantitative determination of the phenol content in biological fluids, it is additionally necessary with such a procedure to multiply the result obtained by multiplying it by the value of the selected dilution in order to quantify the amount of polyphenols contained in the biological fluids. Such a procedure is in clear contradiction with the original view of Folin and Ciocalteu, who believed that excessive dilution

would not lead to reproducible results and would therefore not be usable for detecting the phenol content.

[0022] Surprisingly, a number of problems could be solved with a procedure according to the present invention. On the one hand, exactly reproducible results could be achieved by pre-diluting the biological fluids, such as patient sera from plants or animals, by subjecting the serum to a dilution, in particular at least a seven-fold, preferably ten-fold, dilution prior to the addition of FCR. Furthermore, the Folin-Ciocalteu reagent was not used as a 2 M reagent, but as a 0.2 M reagent, which not only saves on reagent but also completely suppresses the problem of undesired gas formation. Folin and Ciocalteu's fear that a reproducible result could no longer be achieved due to the strong dilution proved to be unfounded and clearly detectable amounts of polyphenols could be found in all the samples examined. Particularly in cases where the result may not be 100% conclusive, a comparison sample could be carried out with undiluted reagent, which in turn confirmed that a reproducible and quantifiable result could be achieved through dilution.

According to a preferred development, the method according to the invention is carried out in such a way that the veterinary or human fluid is cleaned by centrifugation and/or filtration prior to use or prior to diluting and adding FCR. Such a step has proven to be particularly advantageous when the samples contain visible amounts of cryoproteins, which can be separated by centrifugation, so that a clear solution is obtained, which can be used as a basis for further investigation and subsequently clear ones delivers results. Hemolytic specimens should also be removed from experiments as they may affect the results. Finally, microbial contamination and other suspended matter that may be present in the samples can affect the results, so it is beneficial to separate such substances and substances by centrifugation and/or filtration prior to dilution.

Results that can be reproduced particularly well can be achieved by using water, in particular distilled water or deionized water, as the diluent. Conducting the process in aqueous solution has proven to be a favorable and easily reproducible process, with water being usable as a diluent, both in the form of distilled water and deionized water. The choice of the diluent depends not least on the origin of the biological fluid to be examined and in particular on the alcohol content in the sample, which alcohol may have come from the necessary previous extraction.

In order in particular to achieve an improved or intensified color reaction and in particular a clearer coloring of the sample, the method according to the invention is further developed in that at least one or a mixture of amplifying agents selected from the group of bases such as sodium carbonate, sodium bicarbonate , potassium carbonate, potassium hydrocarbonate and the like. By using a basic amplifying agent, it is possible to amplify the color reaction and achieve a clearer coloration, so that not only can a reproducible result be achieved, but in particular a quantification of the polyphenols contained in the biological fluid being examined is possible.

In particular, if it is not known which polyphenols are to be expected in a sample to be examined, an attempt is made to choose a standard which can be used for a plurality of polyphenols or can serve as a substitute for a number of polyphenols, such as gallic acid or ascorbic acid, as has already been described in the prior art. However, if there is any suspicion as to which polyphenol might be present in the fluid to be tested, it is advisable to establish the standard using a serial dilution of the polyphenol expected to be present in the veterinary or human fluid, since then the uncertainty

accuracy in the quantification can be further minimized.

If, as is often the case in practice, solid or pasty substances are present from which the biological fluid cannot be obtained directly, the method according to the invention is further developed in such a way that obtaining the veterinary or human fluid is based on veterinary or human samples to be subjected to at least one pre-treatment selected from drying, grinding, extraction with an alcohol such as ethanol and/or isopropanol, filtration, centrifugation and/or sieving. Especially in the case of plant biological samples, which are known to have a relatively high phenol content, such as grapes, dark-colored fruits such as cherries, blueberries, blackberries, but also coffee, cocoa, asparagus and the like, the plant product cannot be directly used as a basis for an analysis. in which case it must be previously processed in order to make the polyphenols contained in the plant products or plant biological samples accessible to analysis. The usual known methods, such as drying, grinding, extraction with alcohol, in particular pharmacologically acceptable alcohols such as ethanol, isopropanol and the like, filtration, centrifugation or sieving are used here in order to crush and extract the fluid obtained and clean it subject to the same prior to an investigation.

The method according to the invention is preferably used to determine the polyphenol content in veterinary or human fluids. The use of the Folin-Ciocalteu reagent to determine the polyphenol content in human or animal samples has hitherto hardly been possible or only possible when using extremely large volumes of the sera, since a reproducible result could not be achieved due to the strong foaming during the reaction. The method according to the invention, in which the biological fluid is diluted by at least seven times, in particular ten times, before it is used, has made it possible to completely suppress foaming during the reaction and still achieve quantifiable results, so that the method according to the invention now Animal and human fluids can be applied and used.

A further advantage of the method according to the invention is that a further minimization of the amounts of substrate and reagent used could be achieved, so that now a reaction can be carried out in the wells of a microtiter plate or a microtest plate, resulting in significant savings in reagents and reagents as well as to sample material, which not only saves costs but also, in particular, enables the detection method known per se to be used in a significantly more comprehensive manner.

Finally, in the case of hardly or weakly colored samples, the photometric or colorimetric measurement is carried out at wavelengths between 600 nm and 650 nm, with strongly colored samples, as is known per se, being measured at 766 nm.

Finally, if this is necessary and a higher sensitivity than can be achieved with an existing fluid is desired, the procedure can be that the sample volume is increased and instead the dilution in the second dilution step is reduced somewhat, whereupon the sensitivity as detection can be increased. In this case, the comparison with the standard is carried out in an appropriate dilution.

The invention is explained in more detail below using exemplary embodiments.

EXPERIMENT 1 Analysis of asparagus for polyphenol content

Approximately 5 g of white and green asparagus were weighed and treated with a volume in milliliters of a mixture of extractants and diluents (each 50% ethanol and distilled water) corresponding to the weight in grams. The batch was homogenized using a blender and incubated for 24 hours on a 3D mixing platform (Heidolph) at room temperature of 20+/-2°C. At the end of the incubation period, the batches were centrifuged

(15 minutes 2000xg, Heraeus centrifuge) separated into a solid and a liquid phase. If the liquid phase was not clear, clarification was achieved by a further centrifugation step for 5 minutes at 10,000×g. 180 μl of distilled water were added to 20 μl of the clear supernatant (dilution 1:10). The dilution resulted in an ethanol content of the sample of less than 5%, which does not affect the measurement of the polyphenols. 20 μl of this dilution were used in the polyphenol test in the microtiter plate. For this purpose, a further 50 μl of distilled water (aqua dest) were pipetted in and 100 μl of 0.2 M FCR were pipetted into each reaction batch, i.e. each well. Finally, 30 μl of amplification reagent consisting of 7% sodium carbonate solution containing 10% isopropanol were added to each reaction mixture. The plate was incubated at room temperature for 2 hours and then photometrically measured at 766 nm. Serial dilutions of gallic acid from 3mM to 0mM were used as standards. The standard value in mM obtained from photometry was multiplied by 10, giving the exact phenol content in the sample.

Four different asparagus samples were examined using the method according to the invention and the concentration of polyphenols in the asparagus samples was determined. Concentration values of polyphenols in asparagus between 2.74 and 3.09 mM were found. When obtaining these test results, it is also important that when preparing the biological fluid, care must be taken to ensure that the extraction takes a sufficiently long time, since the extraction of the polyphenols from the biological sample was relatively slow and the concentration values for polyphenols determined only after an extraction time of 3 hours remained constant in the asparagus.

Another result of this study showed that asparagus contains measurable amounts of polyphenols, but these were, not unexpectedly, significantly lower than the polyphenol levels in fruit juices, red berries, coffee or the like.

EXPERIMENT 2 Analysis of pressing residues of red grapes

Press residues of red grapes were dried at 60°C in an adjustable oven for 48 hours and separated by sieving into fractions, one of which contained essentially the pips (TC) and the other essentially the skins (TS). Both fractions were homogenized using a percussion mill. The homogenates were weighed in at 2-3 g each, and a volume in ml of extraction agent corresponding to 10 times the weight in g was added. (Aqua dest and ethanol each from 0 to 100%). The batches were extracted for 24, 48, 72, 96, 120 and 144 hours at a room temperature of 23 + 2 °C with constant agitation on a Heidolph 3D shaker. At the end of each extraction time, the corresponding batches were centrifuged at 2000×g for 15 minutes and the clear supernatants were used in the polyphenol determination. For this purpose, they were pre-diluted 1:10 in distilled water, also to minimize the disruptive effects of ethanol. 20 μl of the dilutions were pipetted into the corresponding wells of a microtiter plate and 50 μl of distilled water were pipetted in each case. In a further pipetting step, 100 μl of FCR (0.2 M) were added to each batch. The preparation of the reaction mixture was completed by the addition of 30 µl of enhancement reagent consisting of 7% sodium carbonate solution containing 10% isopropanol. The reaction mixtures were incubated at room temperature for 2 hours and then photometrically measured at 766 nm.

An examination of coffee also showed that the extraction yields increase proportionally to the extraction time and that care must therefore be taken to extract for a sufficiently long time in order to extract the polyphenols as quantitatively as possible from the plant sample before it is determined. Furthermore, it was shown that the polyphenol concentrations also depend on the alcohol concentration of the extraction agent, so that tests were carried out to determine which extraction agents had the highest polyphenol concentrations. It was found that semi-concentrated extractants, i.e. 50% water and 50% alcohol, produced the highest concentrations. In a 1% coffee extract, 3.77 mM

found polyphenols.

In the investigations carried out on asparagus and coffee, there were deviations from the standard during the measurement of the order of 2 to 5%, which appears to be sufficient for a quantification of the method and, in particular, provides an unexpectedly exact result.

EXPERIMENT 3: Polyphenols in human samples

Blood was obtained from the arm vein of the test subjects by puncture and centrifuged after coagulation. The clear supernatant was aliquoted and stored at -20°C until use. 20 μl of the serum were diluted with 180 μl of distilled water (1:10). 20 μl each of this dilution were used for the polyphenol determination. For this purpose, 50 hl of distilled water and 100 ul of 0.2 M FCR were pipetted into the diluted sera. Reactions were completed by the addition of 30 µl of enhancement reagent consisting of 7% aqueous sodium carbonate solution containing 10% isopropanol. After the batches had been incubated at room temperature for at least 2 hours, the batches were photometrically measured at 766 nm. A serial dilution of gallic acid was used as a standard. (0-3mM).

With the method described above, 16 patients were each subjected to two examinations at an interval of one week according to the above method and the sera were subjected to the examination for polyphenols.

[0041] It was found here that none of the patients showed constant levels of polyphenols in the serum over time, but that every serum contained measurable amounts of polyphenols without any problems. The lowest value detected was 3.53 mM polyphenol, the highest value 8.40 mM polyphenol. On average, the values were above 5 mM and below 8 mM polyphenol in the serum, with only one sample of the 32 samples examined giving a meaningful result did not deliver, so that it can be summarized that human sera and plasma from the 1:7, especially 1:10 dilution used also delivered measurable amounts of polyphenols without any problems.

Claims (7)

Expectations 1. Method for the colorimetric determination of polyphenols in veterinary or human fluids, in which the veterinary or human fluid is reacted with Folin-Ciocalteu reagent (FCR) to form a color reaction and the color reaction formed is evaluated quantitatively colorimetrically by comparison with a standard, characterized in that it comprises the following steps: a) placing the previously cleaned veterinary or human fluid in the wells of a dilution plate, b) adding diluent until at least a 7-fold dilution, in particular at least a 10-fold dilution of the veterinary or human fluid is achieved, c) removing a quantity of the diluted veterinary or human fluid from the wells of the dilution plate and presenting this quantity in wells of a test plate, as well as presenting the standard and a comparison in further wells of the test plate, d) adding diluent to the diluted veterinary or human fluid, the standard and the control, e) adding an amount of 0.2 M FCR which corresponds to 3 to 7 times the amount, in particular 5 times the amount of the diluted veterinary or human fluid, f) adding an amplification reagent, g) Mixing of all components, in particular by shaking on a horizontal shaker, h) allowing to incubate at temperatures between 20 °C and 25 °C for a period between 1 and 24 hours, preferably 2 to 4 hours, i) colorimetric determination of the color reaction formed at wavelengths between 600 and 766 nm, ]j) correcting an obtained result of polyphenols contained in the veterinary or human fluid by multiplying by the value of the in step b. chosen dilution and k) Quantitative determination of the phenol content in the veterinary or human fluid by comparison with the color reaction of the standard. 2. Method according to claim 1, characterized in that the veterinary or human fluid is purified by centrifugation and/or filtration. 3. The method according to claim 1 or 2, characterized in that the diluent used is water, in particular distilled water or deionized water. 4. The method according to claim 1, 2 or 3, characterized in that at least one or a mixture of reinforcing agents selected from the group of bases, such as sodium carbonate, potassium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, is used as the reinforcing agent. 5. The method according to any one of claims 1 to 4, characterized in that a serial dilution of a polyphenol expected in the veterinary or human fluid, in particular a serial dilution of an organic acid such as gallic acid or ascorbic acid, is used as the standard. 6. The method according to any one of claims 1 to 5, characterized in that to obtain the veterinary or human fluid to be examined, veterinary or human samples undergo at least one pretreatment selected from drying, grinding, extraction with an alcohol such as ethanol and/or isopropanol, filtration , centrifugation and/or sieving. 7. Use of the method according to any one of claims 1 to 6 for determining the polyphenol content in veterinary or human fluids. No drawings for this 777