CN102183517A - Method for determining general flavone content - Google Patents

Abstract

The invention discloses a method for determining the content of total flavonoids, in particular to a method for determining the content of total flavonoids in blue skyflower or blue skyflower extracts or substances identical to the total flavonoids of blue skyflower. The method is an ultraviolet-visible spectrophotometric method. The rutin reference substance is used to prepare a gradient standard solution, and the medicinal materials or extracts of A. radix are prepared to be a test solution. The AlCl ₃ -HAc-NaAc chromogenic system is used to measure the gradient at a wavelength of 405nm. The absorbance of the standard solution was used to establish a standard curve. Then measure the absorbance of the solution to be tested, find out the concentration of the solution to be tested from the standard curve, and finally calculate the total flavonoid content of the blue skyflower medicinal material or extract according to the dilution factor when preparing the solution. Compared with the prior art, the invention has the advantages of specificity and strong pertinence of the detection method, accurate and reliable results, good precision, good stability, good repeatability and high accuracy. The invention can be used for quality control of medicinal materials or extracts.

Description

A kind of total flavonoid content assay method

technical field

The present invention relates to a method for determining substance content by ultraviolet-visible spectrophotometry, in particular to a method for determining the content of total flavonoids, especially a method for determining the total flavonoids of blue skyflower or blue skyflower extract or the same substance as blue skyflower total flavonoids Methods for total flavonoid content.

Background technique

Many plants in nature contain flavonoids. It is estimated that 20% of traditional Chinese herbal medicines in my country contain flavonoids. Flavonoids have always been a hot research substance in the medicine and food industry, because flavonoids have various physiological effects, such as rutin, quercetin, and quercetin can enhance heart contraction and reduce the number of heart beats; Chrysanthemum, etc. have antitussive and expectorant effects; vitexin, wogonin, etc. have anti-tumor activity; silymarin has hepatoprotective effect, etc. The flavonoids contained in plants generally do not exist as a single component, but exist together in several components with similar or similar structures, and the total amount is called the total flavonoid content.

Qingtiankui is a famous Chinese herbal medicine in Guangdong and Guangxi. Its export volume is relatively large, and the market price of medicinal materials is expensive. There are more and more clinical applications. Current research shows that flavonoids are a major class of phytochemical constituents in tianhua. There are many methods for determining the content of total flavonoids, but there is no report on the method for determining the content of total flavonoids in N. chinensis. Therefore, the establishment of a specific and targeted method for the determination of the total flavonoids content of S. chinensis has important reference significance for the quality control of S. chinensis medicinal materials. In addition, there is no statutory quality standard for A. celery, so it is difficult to effectively supervise and regulate the market. The establishment of this assay method will also be of great help in this regard.

Contents of the invention

The purpose of the present invention is to provide an accurate and fast method for determining the content of total flavonoids in the blue skyflower or the blue skyflower extract or the same substance as the total flavonoids of the blue skyflower.

The technical scheme that the present invention solves the problems of the technologies described above is:

A method for determining the content of total flavonoids, specifically a method for determining the content of total flavonoids in blue skyflower or blue skyflower extracts or substances identical to the total flavonoids of blue skyflower. The method is an ultraviolet-visible spectrophotometric method. The rutin reference substance is used to prepare a gradient standard solution, and the medicinal materials or extracts of A. radix are prepared to be a test solution. The AlCl ₃ -HAc-NaAc chromogenic system is used to measure the gradient at a wavelength of 405nm. The absorbance of the standard solution, establish a standard curve, then measure the absorbance of the test solution, find out the concentration of the test solution from the standard curve, and finally calculate the total flavonoid content of the blue skyflower medicinal material or extract according to the dilution factor when preparing the solution. The specific operation steps are as follows :

1. Pulverize the medicinal material of Qingtiankui, accurately weigh a certain amount of medicinal material, place it in a stoppered conical flask, add a certain amount of petroleum ether, ultrasonicate, filter and remove the filtrate after standing still, repeat three times, evaporate the petroleum ether in the filter residue, add 70% ethanol, weighed, sonicated, cooled to room temperature, supplemented with 70% ethanol, shaken, centrifuged to obtain the sample solution;

2. Heat the rutin reference substance at 110°C to constant weight;

3. Precisely weigh the rutin reference substance, dissolve with methanol and constant volume to obtain the rutin reference substance solution. Precisely measure a certain amount of this solution and place it in a volumetric flask, dilute to the mark with methanol, and shake well to obtain the rutin standard solution;

4. Accurately draw the rutin standard solution with a pipette, place it in a 25mL volumetric flask, add 0.1mol L ^-1 AlCl ₃ solution, add HAc-NaAc buffer solution (pH5.5), add 70% ethanol to dilute to scale and shake well to obtain a series of standard solutions with different concentrations. Take the above solutions respectively, and use the corresponding reagent as a blank to measure the absorbance at a wavelength of 405nm. With the absorbance (A) as the ordinate and the solution concentration (C) as the abscissa, draw a standard curve;

5. Precisely measure 1.0 mL of the sample solution obtained in step (1), and measure the absorbance value according to step (4). Calculate the total flavonoid content of the samples.

The AlCl ₃ -HAc-NaAc color development system is used above, and the required pH is pH=5.5.

When extracting the above-mentioned blue skyflower medicinal material, the amount of sample and the amount of extraction solvent used are 1:75, and the extraction time is 60 minutes.

The present invention has the following advantages:

1. The detection method is specific and targeted, and the result is accurate and reliable. The invention is specially aimed at the determination of the total flavone content in the blue skyflower or the same substance as the blue skyflower total flavonoids. The structure of the flavonoids contained in the blue skyflower generally lacks the ortho-diphenol hydroxyl structural fragment. When the traditional method of detecting the total flavonoid content of Chinese medicinal materials is used, there is no overlap between the blue skyflower sample and the rutin reference substance in the wavelength range of 200-800nm. Maximum absorption wavelength (see Figure 1). However, the flavonoids contained in the blue skyflower and the plants of the same genus basically contain the 5-OH, 4-C=O, in an acidic environment, it is easy to form a complex with Al ³⁺ , and has a maximum absorption around 400nm (See Figure 2), and this reaction is specific. Therefore, we adopt AlCl ₃ -HAc-NaAc as the color development system, and investigate the different pH conditions of the color development system, thus confirming that the color development condition is AlCl ₃ -HAc-NaAc (pH5.5), the detection wavelength 405nm.

2. Good precision. Accurately measure 1.0 mL of the sample solution, measure continuously for 6 times, and measure the absorbance value. The result shows that RSD%=1.30%, indicating that the method has good instrument precision.

3. Good stability. Precisely measure the sample solution, measure the absorbance every 20 minutes, measure the absorbance at 0, 20, 40, 60, 80, and 100 minutes respectively, and measure 6 times in total. The results show that the absorbance of this method basically does not change within 100 minutes, RSD% =1.02%, indicating that the method has good stability.

4. Good repeatability. Weigh 5 samples of the same batch in parallel, about 1 g each, and weigh them accurately. Prepare the sample solution, measure the absorbance, calculate the total flavonoid content in the sample, and get RSD%=2.88%, which shows that the method has good repeatability.

5. High accuracy. Take 6 samples of blue skyflower with known total flavonoid content, each about 0.2g, and weigh them accurately. A certain amount of rutin reference substance was added to each portion to prepare a sample solution, and the absorbance of each sample was measured to calculate the total flavonoid content of the sample, the recovery rate and the RSD value. The results showed that the average recovery rate was 99.78%, and RSD%=1.94%, which indicated that the accuracy of the method was high.

The invention can be used for quality control of medicinal materials or extracts.

Description of drawings

Fig. 1 is the ultraviolet scanning picture when the total flavonoids content of N. chinensis is detected by the traditional method for detecting the total flavonoids content of Chinese herbal medicines.

In the figure: Ⅰ blue skyflower sample, Ⅱ rutin reference substance.

Fig. 2 is the ultraviolet scanning figure when adopting the method of the present invention to detect the total flavonoid content of the blue skyflower.

In the figure: Ⅰ blue skyflower sample, Ⅱ rutin reference substance.

Detailed ways

The NaNO ₂ -Al(NO ₃ ) ₃ -NaOH color development system is usually used for the determination of the total flavonoids content in Chinese medicinal materials by ultraviolet spectrophotometry. Effect. This method is widely used, but it cannot accurately measure the total flavonoids content of A. chinensis or plants of the same genus. This is because the structure of flavonoids contained in A. chinensis generally lacks the ortho-diphenolic hydroxyl structural fragment. NaNO ₂ -Al( In the NO ₃ ) ₃ -NaOH color development system, there is no overlapping maximum absorption wavelength between the blue skyflower sample and the rutin reference substance in the wavelength range of 200-800 nm (see Figure 1). However, the flavonoids contained in the blue skyflower and the plants of the same genus basically contain 5-OH, 4-C=O, in acidic environment, it is easy to complex with Al ³⁺ to form a complex, and there is a maximum absorption at about 400nm ( See Figure 2), and this reaction is specific. Therefore, we adopt AlCl ₃ -HAc-NaAc as the color development system, and investigate the different pH conditions of the color development system, thus confirming that the color development condition is AlCl ₃ -HAc-NaAc (pH5.5), the detection wavelength 405nm.

The present invention will be further elaborated below in conjunction with embodiment.

Example 1

Determination of the total flavonoid content of Qingtiankui medicinal materials, the specific operation steps are as follows:

1. Determination of moisture content of the medicinal material of A. chinensis is determined with a rapid moisture analyzer according to the method of "Chinese Pharmacopoeia" (2010 edition). Crush the medicinal material, weigh 1g accurately, place in a stoppered Erlenmeyer flask, add 50mL of petroleum ether, ultrasonicate for 30min, filter and remove the filtrate after standing still, repeat three times, evaporate the petroleum ether in the filter residue, and precisely add 70% ethanol 75mL, Weigh it, sonicate for 60 minutes, let it cool, make up the weight with 70% ethanol, shake well, and centrifuge for 15 minutes (3000r·min ^-1 ) to obtain the sample solution.

2. Heat the rutin reference substance at 110°C to constant weight.

3. Precisely weigh 35.00 mg of rutin reference substance, dissolve it in methanol and dilute to 20 mL to obtain 1.75 mg·mL ^-1 rutin reference substance solution. Precisely measure 4mL of 1.75mg·mL ^-1 rutin reference solution and place it in a 50mL volumetric flask, dilute to the mark with methanol, shake well, and obtain 0.1400mg·mL ^-1 rutin standard solution.

4. Precisely pipette 0.1400 mg·mL ^-1 rutin reference substance solution 0.0, 1.3, 2.5, 3.5, 4.5 and 5.5mL into 25mL volumetric flask respectively, add 0.1mol·L ^-1 AlCl ₃ solution 1.5 mL, add 1 mL of HAc-NaAc buffer solution (pH5.5), add 70% ethanol to dilute to the mark, and shake well to obtain the control with concentrations of 0.0, 7.28, 14, 19.6, 25.2, and 30.8 μg·mL ^-1 product solution. Take respectively

For the above solution, use the corresponding reagent as a blank, and measure the absorbance at a wavelength of 405 nm using a Shanghai Phoenix Optical 72 series digital display 722 visible spectrophotometer (or a spectrophotometer with the same function). Draw the standard curve with the absorbance (A) as the ordinate and the solution concentration (C) as the abscissa.

5. Precisely measure 1.0 mL of the sample solution obtained in step 1, and measure the absorbance value according to step 4.

The calculated total flavonoids content of A. chinensis medicinal material is 1.8%.

Example 2

Determination of the total flavonoid content of the extract of Tiankuai, the specific operation steps are as follows:

1. Accurately weigh 0.01g of the extract, accurately add 10mL of 70% ethanol, weigh the weight, sonicate for 60min, let it cool, make up the weight with 70% ethanol, shake well, and centrifuge for 15min (3000r·min ^-1 ), namely to obtain the sample solution.

2. The processing of rutin reference substance and the preparation of rutin reference substance solution are the same as example 1.

3. Accurately measure 1.0mL of the sample solution obtained in step 1, operate according to step 4 of example 1, and measure the absorbance value. The total flavonoid content of the extract of Tiankuai was calculated to be 86.2%.

Claims (3)

1. a method for measuring total flavonoids content, characterized in that, is the method for measuring total flavonoids content in blue skyflower or blue skyflower extract or the same material as blue skyflower total flavonoids, the method is ultraviolet-visible spectrophotometry, using aloe The reference substance was prepared into a gradient standard solution, and the medicinal materials or extracts of N. radiata were prepared into a test solution. Using the AlCl ₃ -HAc-NaAc chromogenic system, the absorbance of the gradient standard solution was measured at a wavelength of 405 nm, and a standard curve was established, and then the test solution was determined. Measure the absorbance of the solution, find out the concentration of the solution to be tested from the standard curve, and finally calculate the total flavonoid content of the blue skyflower medicinal material or extract according to the dilution factor when preparing the solution. The specific operation steps are as follows: 1) Pulverize the medicinal material of Qingtiankui, accurately weigh a certain amount of medicinal material, place it in a stoppered conical flask, add a certain amount of petroleum ether, ultrasonicate, filter and remove the filtrate after standing still, repeat three times, evaporate the petroleum ether in the filter residue, add 70% ethanol, weighed, sonicated, cooled to room temperature, supplemented with 70% ethanol, shaken, centrifuged to obtain the sample solution; 2) Heat the rutin reference substance at 110°C to constant weight; 3) Precisely weigh the rutin reference substance, and dissolve it in methanol to obtain the rutin reference substance solution. Precisely measure a certain amount of this solution and place it in a volumetric flask, dilute to the mark with methanol, and shake well to obtain the rutin standard solution; 4) Accurately absorb the standard solution of rutin with a pipette, place it in a 25mL volumetric flask, add 0.1mol L ^-1 AlCl ₃ solution, add HAc-NaAc buffer solution (pH5.5), add 70% ethanol to dilute to Scale and shake well to get a series of standard solutions with different concentrations; take the above solutions respectively, take the corresponding reagent as a blank, and measure the absorbance at a wavelength of 405nm; take the absorbance (A) as the ordinate, and the solution concentration (C) as the abscissa , to draw a standard curve; 5) Precisely measure 1.0 mL of the sample solution obtained in step (1), and measure the absorbance value according to step (4). Calculate the total flavonoid content of the samples. 2. A method for determining the content of total flavonoids according to claim 1, characterized in that: the AlCl ₃ -HAc-NaAc color development system is used, and the required pH is pH=5.5. 3. A method for determining the content of total flavonoids according to claim 1, characterized in that: when the blue skyflower medicinal material is extracted, the amount of sample and the amount of extraction solvent are 1:75, and the extraction time is 60 min.

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