CN107478649A - Use of phenolic compounds as alpha-cardiac glycoside chromogen and determination method for alpha-cardiac glycoside content - Google Patents

Abstract

The invention provides application of a phenol compound as a first type cardiac glycoside developer and a method for measuring the content of the first type cardiac glycoside, which relate to the technical field of a method for measuring the content of the first type cardiac glycoside, and comprise the following steps: dissolving the alpha-cardiac glycoside in an alcohol solvent to prepare an alcohol solution of the alpha-cardiac glycoside, mixing the alcohol solution with a phenol compound, carrying out a color development reaction, measuring the absorbance of the color development solution at 485nm-500nm to obtain the content of the alpha-cardiac glycoside, relieving the technical problem that the measurement of the content of the alpha-cardiac glycoside is always a difficult point in the aspect of application research of the alpha-cardiac glycoside, achieving the purposes of providing a simple, convenient, fast, accurate and efficient method for measuring the content of the alpha-cardiac glycoside, solving the problem of restricting the application research of the alpha-cardiac glycoside, and effectively promoting the technical effect of the application of the alpha-cardiac glycoside in the aspect of tumor and cancer treatment.

Description

Use of phenolic compounds as alpha-cardiac glycoside chromogenic agent and alpha-cardiac glycoside content Quantitative method

technical field

The present invention relates to the technical field of methods for determining the content of alpha cardiac glycosides, in particular to the use of phenolic compounds as a color developer for alpha cardiac glycosides and a method for determining the content of alpha cardiac glycosides.

Background technique

Cardiac glycosides are steroidal compounds that exist in plants and have cardiotonic effects. They are formed by condensation of cardiac aglycones and sugars. Cardiac glycosides are divided into type A cardiac glycosides and type B cardiac glycosides according to different substituent groups. There are two types of glycosides, among which alpha cardiac glycosides are the most common, such as digitoxin and ouabain. Alpha-cardiac glycosides not only have a wide range of pharmacological effects, but also have cardiotonic, anti-tumor, diuretic, and anti-cancer activities, making more and more scholars pay attention to alpha-cardiac glycosides. However, the determination of the content of alpha cardiac glycoside has always been a difficult point in the application research of alpha cardiac glycoside. The 2015 edition of "Chinese Pharmacopoeia" lacks the records of the method for the determination of alpha cardiac glycoside, and there is no current literature report about alpha cardiac glycoside. Determination method of cardiac glycoside content.

In view of this, the present invention is proposed.

Contents of the invention

One of the purposes of the present invention is to provide a kind of phenolic compound as the purposes of alpha cardiac glycoside chromogenic agent, to alleviate the technical problem that the mensuration of alpha cardiac glycoside content has always been a difficult point in the aspect of alpha cardiac glycoside application research .

The present invention provides the purposes of phenolic compound as alpha-cardiac glycoside chromogenic agent, and the general formula of described phenolic compound is:

Wherein, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are all independently selected from hydrogen, halogen, carboxyl, nitro, C _1-6 alkyl, C _3-5 alkenyl, C _3-5 alkyne One of group, acyl group, ether group and ester group, and R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are not hydrogen at the same time, where the general formula of acyl group is COR ₆ , and the general formula of ether group is OR ₇ , the general formula of the ester group is COOR ₈ , and the R ₆ , R ₇ and R ₈ are all independently selected from C _1-6 alkyl, C _1-6 alkoxy or C _1-6 alkoxy ester.

Further, the phenolic compound is selected from 3,5-dinitrophenol, m-nitrophenol, 2,6-dinitrophenol, p-cresol, p-chlorophenol, p-bromophenol, p-acetylphenol , p-nitrophenol, 3,5-dibromophenol, 3,5-dimethylphenol, 2,6-dimethylphenol, 3,5-difluorophenol, m-fluorophenol, p-tert-butylphenol, One of p-ethylphenol, p-methoxyphenol and 2-nitrophenol, preferably 3,5-dinitrophenol or 3,5-dibromophenol.

The second object of the present invention is to provide a method for measuring the content of alpha cardiac glycoside, so as to alleviate the technical problem that the determination of alpha cardiac glycoside content has always been a difficult technical problem in the application research of alpha cardiac glycoside.

The assay method of alpha cardiac glycoside content provided by the invention comprises the steps:

Dissolve alpha-cardiac glycoside in an alcohol solvent, configure an alcohol solution of alpha-cardiac glycoside, mix it with a phenolic compound, carry out a color reaction, measure the absorbance of the color-developing solution at 485nm-500nm, and obtain alpha-cardiac glycoside Cardiac glycoside content.

Further, the assay method of described alpha cardiac glycoside content, comprises the steps:

(a) Alcoholic solutions of alpha-cardiac glycosides of different concentrations are configured to carry out color reaction with phenolic compounds respectively, and the absorbance after the color development of the alcohol solutions of alpha-cardiac glycosides of different concentrations is measured to obtain a standard curve and its regression equation;

(b) dissolving the alpha cardiac glycoside to be tested in an alcohol solvent, configuring it into an alpha cardiac glycoside alcohol solution, and then carrying out a color reaction with a phenolic compound to measure the absorbance;

(c) Substituting the measured absorbance into the regression equation to obtain the content of alpha cardiac glycoside in the alcohol solution of alpha cardiac glycoside to be tested.

Further, the phenolic compound is firstly dissolved in water to form an alpha-cardiac glycoside color developer solution, and then mixed with an alpha-cardiac glycoside alcohol solution to carry out a color reaction.

Further, the concentration of alpha cardiac glycoside in the alcohol solution of alpha cardiac glycoside is 16-32 mg/L, and in the alpha cardiac glycoside developer solution, the molar concentration of phenolic compound is 0.01-0.1mol/L .

Further, the volume ratio of the alpha-cardiac glycoside alcohol solution to the alpha-cardiac glycoside developer solution is (2-4):1, preferably 3:1.

Further, the time for the color reaction is 5-40 minutes, preferably 10-30 minutes, more preferably 20 minutes.

Further, the alcohol solvent is a lower alcohol, and the lower alcohol is preferably methanol, ethanol or propanol, more preferably ethanol.

Further, the alpha cardiac glycoside is extracted from plants, and the extraction method includes the following steps: first crushing the plants, and then using ethanol as the extraction liquid, and performing ultrasonic extraction to obtain an ethanol solution of alpha cardiac glycosides.

The alpha-cardiac glycoside chromogen provided by the present invention can carry out color reaction with alpha-cardiac glycoside to generate an orange-yellow solution, so that it can obtain the alpha-cardiac glycoside by measuring the absorbance of the orange-yellow solution at 485nm-500nm. The content of cardiac glycosides provides a simple, fast, accurate and efficient method for the determination of cardiac glycosides, which solves the problem that restricts the application of cardiac glycosides and can effectively promote Applications in tumor and cancer therapy.

The determination method of the content of alpha cardiac glycoside provided by the present invention, produces orange-yellow solution through color development reaction between alpha-cardiac glycoside and alpha-cardiac glycoside, and then by measuring the absorbance of orange-yellow solution at 485nm-500nm, Obtaining the content of alpha cardiac glycoside, thus providing a simple, fast, accurate and efficient method for the determination of alpha cardiac glycoside content, solving the difficult problem that restricts the application research of alpha cardiac glycoside, and can effectively promote the development of alpha cardiac glycoside Application of type cardiac glycosides in tumor and cancer therapy.

Description of drawings

Fig. 1 is the relationship curve of the volume ratio and the absorbance value of the ethanol solution of alpha cardiac glycoside and alpha cardiac glycoside developer solution;

Fig. 2 is the relational curve of the reaction time and the absorbance value of the ethanol solution of alpha cardiac glycoside and alpha cardiac glycoside developer solution;

Fig. 3 is the relationship curve between the concentration of alpha cardiac glycoside and the absorbance.

detailed description

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only for illustrating the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

According to one aspect of the present invention, the present invention provides the purposes of phenolic compound as alpha cardiac glycoside chromogenic agent, and the general formula of described phenolic compound is:

R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are all independently selected from hydrogen, halogen, carboxyl, nitro, C _1-6 alkyl, C _3-5 alkenyl, C _3-5 alkynyl, One of acyl group, ether group and ester group, and R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are not hydrogen at the same time, wherein the general formula of acyl group is COR ₆ , and the general formula of ether group is OR ₇ , The general formula of the ester group is COOR ₈ , and the R ₆ , R ₇ and R ₈ are all independently selected from a C _1-6 alkyl group, a C _1-6 alkoxy group or a C _1-6 alkoxy ester group.

The phenolic compound provided by the present invention can carry out color reaction with alpha cardiac glycoside to generate an orange-yellow solution, so that it can obtain the content of alpha-cardiac glycoside by measuring the absorbance of the orange-yellow solution at 485nm-500nm, Thereby providing a simple, fast, accurate and efficient method for the determination of the content of alpha cardiac glycoside, which solves the difficult problem restricting the application research of alpha cardiac glycoside, and can effectively promote the use of alpha cardiac glycoside in the treatment of tumors and cancers. aspects of application.

In the present invention, typical but non-limiting wavelengths for measuring chromogenic solutions are 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498 or 499 nm.

In the present invention, halogen is Cl, Br or F; C _1-6 alkyl is selected from one of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl C _3-5 alkenyl is selected from one of propylene, butene and pentene; C _3-5 alkynyl is selected from one of propynyl, butynyl and pentynyl; acyl is selected from methyl One of acyl, acetyl and propionyl; one of ether group selected from methyl ether group, ethyl ether group and propyl ether group; one of ester group selected from methyl carboxyl group, ethyl carboxyl group and propyl carboxyl group C _1-6 alkyl is selected from one of methyl, ethyl, propyl, butyl, pentyl and hexyl, and C _1-6 alkoxy is selected from methoxy, ethoxy, propoxy One of , butoxy, pentyloxy and hexyloxy; C _1-6 alkoxy ester group is selected from methoxy ester group, ethoxy ester group, propoxy ester group, butoxy ester group, pentyloxy ester group and one of the hexyloxyester groups.

In a preferred embodiment of the present invention, the phenolic compound is selected from 3,5-dinitrophenol, m-nitrophenol, 2,6-dinitrophenol, p-cresol, p-chlorophenol, p-bromo Phenol, p-acetylphenol, p-nitrophenol, 3,5-dibromophenol, 3,5-dimethylphenol, 2,6-dimethylphenol, 3,5-difluorophenol, m-fluorophenol, One of p-tert-butylphenol, p-ethylphenol, p-methoxyphenol and 2-nitrophenol, preferably 3,5-dinitrophenol or 3,5-dibromophenol.

According to another aspect of the present invention, the present invention provides a kind of assay method of alpha cardiac glycoside content, comprising the steps:

Dissolve alpha-cardiac glycoside in an alcohol solvent, configure an alcohol solution of alpha-cardiac glycoside, mix it with a phenolic compound, carry out a color reaction, measure the absorbance of the color-developing solution at 485nm-500nm, and obtain alpha-cardiac glycoside Cardiac glycoside content.

The determination method of the content of alpha cardiac glycoside provided by the present invention, generates orange-yellow solution through color reaction between alpha-cardiac glycoside and phenolic compound, and then obtains formazan by measuring the absorbance of the orange-yellow solution at 485nm-500nm. Type A cardiac glycoside content, thus providing a simple, fast, accurate and efficient method for the determination of Type A cardiac glycoside content, which solves the difficult problem restricting the application research of Type A cardiac glycoside, and can effectively promote the development of Type A cardiac glycoside. Application of cardioglycosides in tumor and cancer therapy.

In a preferred embodiment of the present invention, the assay method of alpha-cardiac glycoside content comprises the following steps:

(a) Alcoholic solutions of alpha-cardiac glycosides of different concentrations are configured to carry out color reaction with phenolic compounds respectively, and the absorbance after the color development of the alcohol solutions of alpha-cardiac glycosides of different concentrations is measured to obtain a standard curve and its regression equation;

(b) dissolving the alpha cardiac glycoside to be tested in an alcohol solvent, configuring it into an alpha cardiac glycoside alcohol solution, and then carrying out a color reaction with a phenolic compound to measure the absorbance;

(c) Substituting the measured absorbance into the regression equation to obtain the content of alpha cardiac glycoside in the alcohol solution of alpha cardiac glycoside to be tested.

In a preferred embodiment of the present invention, in step (a), first configure alpha cardiac glycoside alcohol solutions of different concentrations, and then add phenolic compounds in the alcohol solutions of alpha cardiac glycosides of different concentrations, so that formazan Alpha-cardiac glycoside and phenolic compounds undergo color development to generate orange-yellow solutions with different absorbances. The absorbance of the orange-yellow solutions with different absorbances is measured by a spectrophotometer, and the standard curve and regression of alpha-cardiac glycoside concentration and absorbance are drawn up. Equation; preferably, no less than 5 alcohol solutions of alpha cardiac glycosides with different concentrations are configured to ensure the accuracy of the standard curve and regression equation of alpha cardiac glycoside concentration and absorbance.

In step (b), the phenolic compound is added into the alcohol solution of alpha cardiac glycoside to be tested, a color reaction occurs to generate an orange-yellow solution, and the absorbance of the orange-yellow solution is measured by a spectrophotometer.

In step (c), the measured absorbance is substituted into the regression equation to obtain the content of alpha cardiac glycoside in the alcohol solution of alpha cardiac glycoside to be tested.

In a preferred embodiment of the present invention, the phenolic compound is firstly dissolved in water to form an alpha-cardiac glycoside developer solution, and then mixed with an alpha-cardiac glycoside alcohol solution for color reaction.

By first dissolving the phenolic compound in water, it is configured into a chromogenic agent solution of alpha cardiac glycoside, and when it is mixed with alpha cardiac glycoside solution for color reaction, the color development is more sensitive, and it is also easier to make alpha cardiac glycoside The cardiac glycoside chromogen solution is mixed more evenly with the alcohol solution of alpha cardiac glycoside, the color reaction is carried out more fully, and the measured absorbance is more accurate.

In a preferred embodiment of the present invention, in the alcohol solution of alpha cardiac glycoside, the concentration of alpha cardiac glycoside is 16-32 mg/L; in the alpha cardiac glycoside developer solution, the molar concentration of phenolic compound 0.01-0.1mol/L.

In order to make the content determination of alpha-cardiac glycosides more accurate and the color reaction more sensitive, the alpha-cardiac glycosides are configured into an alcohol solution with a concentration of 16-32 mg/L, and the phenolic compounds are configured into a concentration of 0.01-0.1 The alpha cardiac glycoside chromogenic agent solution of mol/L, especially in the alcoholic solution of alpha cardiac glycoside, the concentration of alpha cardiac glycoside is 24mg/L, in the alpha cardiac glycoside chromogenic agent solvent, When the molar concentration of phenolic compounds is 0.05mol/L, the color reaction is the most sensitive, and the concentration of alpha-cardiac glycoside is the most accurate.

In a preferred embodiment of the present invention, the volume ratio of the alcohol solution of alpha cardiac glycoside to the developer solution of alpha cardiac glycoside is (2-4):1, preferably 3:1.

In order to verify the optimal volume ratio of the alcohol solution of alpha cardiac glycoside and the alpha cardiac glycoside chromogen solution for color reaction, the ethanol solution of alpha cardiac glycoside with a molar concentration of 24 mg/L and the molar concentration of The volume ratio of the 0.5mol/L alpha cardiac glycoside developer solution is set to 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1 , 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 and then measure the absorbance after 20 minutes of color reaction, the results are shown in Figure 1, as can be seen from Figure 1, the concentration is 24mg When the volume ratio of the ethanol solution of the alpha cardiac glycoside of /L and the alpha cardiac glycoside chromogen solution of 0.5mol/L was changed from 8:1 to 1:7, the absorbance value first gradually increased, Then gradually decrease, when the volume ratio of the two is (2-4):1, the absorbance of the solution is higher, especially when it is 3:1, the absorbance of the solution is the largest.

In addition, the volume ratio of the methanol solution of alpha-cardiac glycoside and the alpha-cardiac glycoside developer solution of 0.5mol/L with a molar concentration of 28 mg/L was also determined to be 8:1, 7:1. , 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 The absorbance was measured after 20 minutes of the color reaction, and the law of the curve is the same as that in Figure 1, so it will not be repeated here. Also, when the volume ratio of the two is (2-4):1, the absorbance of the solution is higher, especially 3:1 , the absorbance of the solution is at its maximum.

In addition, the volume ratio of the alpha-cardiac glycoside chromogen solution that the concentration is 30mg/L and the molar concentration of the alpha-cardiac glycoside developer solution of 0.5mol/L is set to 8:1, 7: 1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 The absorbance was measured after 20 minutes of color reaction, and the law of the curve is the same as that in Figure 1, so I won’t go into details here, and the volume ratio of the two is (2-4):1, and the absorbance of the solution is relatively high, especially 3: 1, the absorbance value of the solution is maximum.

This shows that the volume ratio of the alcohol solution of alpha cardiac glycoside to the developer solution of alpha cardiac glycoside is (2-4): 1, and when it is preferably 3: 1, its color development is more sensitive, and alpha cardiac glycoside The content determination is more accurate.

In a preferred embodiment of the present invention, the color reaction time is 5-40 min, preferably 10-30 min, more preferably 20 min.

In order to verify the relationship between the color reaction time and the absorbance value, the ethanol solution of alpha cardiac glycoside with a concentration of 24mg/L and the alpha cardiac glycoside chromogenic agent solution with a molar concentration of 0.5mol/L were mixed according to the volume ratio 3:1 for color reaction, measure the absorbance when the color time is 2min, 4min, 6min, 8min, 10min, 15min, 20min, 25min, 30min, 40min, 50min, 60min, and draw up the relationship between color time and absorbance value The curve, as shown in Figure 2, can be seen from Figure 2, with the continuous increase of the reaction time, the absorbance value continues to increase, when the reaction time is 20min, the absorbance value reaches the maximum, after 20min with the increase of time, The absorbance value is basically unchanged, and can be stable for more than 1 hour. Therefore, in order to improve the assay efficiency of alpha-cardiac glycoside content, when the color reaction time is 5-40min, especially when the color reaction time is 10-30min, The effect of absorbance measurement is better. When the color reaction is 20 minutes, the stability of absorbance measurement is the best, and the effect is the best.

In addition, the color development time of the methanol solution of alpha cardiac glycoside and the propanol solution of alpha cardiac glycoside were also measured respectively, and the color development time of the ethanol solution of alpha cardiac glycoside was the same rule, and it is not mentioned here Let me repeat.

In a preferred embodiment of the present invention, alpha cardiac glycoside is extracted from plants, and the extraction method includes the following steps: first crushing the plants, then using ethanol as the extracting solution, and performing ultrasonic extraction to obtain ethanol of alpha cardiac glycoside solution.

In a preferred embodiment of the present invention, the cavitation effect, mechanical effect and thermal effect of ultrasonic waves are used to enhance the penetrating power of ethanol solution to plants, which contributes to the stripping of active ingredients in plants, thereby improving the extraction of alpha cardiac glycosides The extraction rate is shortened, the extraction temperature is lowered, the energy consumption is reduced, and the extraction efficiency is improved.

In a preferred embodiment of the present invention, during the process of crushing the plants, the plants are crushed into 40-mesh plant powder.

By crushing the plants into 40-mesh plant powder, the alpha-cardiac glycosides in the plants can be completely extracted.

The above-mentioned plants are angiosperms, such as Scrophulariaceae, Apocynaceae, Ascariaceae, Liliaceae, Cruciferae, and Ranunculaceae.

The technical solutions provided by the present invention will be further described below in conjunction with examples and comparative examples.

Example 1

The present embodiment provides a method for drawing a standard curve of alpha-cardiac glycoside content and absorbance, which is carried out according to the following steps:

(a) Accurately weigh 10 mg of alpha-cardiac glycoside, place it in a 25 mL volumetric flask, dilute to volume with ethanol, and configure it into a standard mother solution with a concentration of 400 mg/L;

(b) Weigh 0, 0.4, 0.6, 0.8, 1.0, 1.2mL of the mother liquor of the standard product respectively, place them in six 10mL stoppered test tubes, and dilute to volume with ethanol;

(c) 3,5-dinitrophenol is dissolved in water to make a 0.5mol/L alpha-cardiac glycoside developer solution

(d) Take 3ml of the ethanol solution of alpha-cardiac glycoside prepared in step (b) respectively and place them in 6 test tubes, add 1 mL of alpha-cardiac glycoside chromogen solution with a concentration of 0.5mol/L in turn, and react for 20min. At 495nm, the absorbance of the solution in six test tubes was measured respectively, and the results are shown in Table 1:

Table 1

The data in Table 1 is drawn up the relation curve of alpha cardiac glycoside concentration and absorbance, as shown in Figure 3, as can be seen from this curve, the content of alpha cardiac glycoside and absorbance are linearly correlated, and according to its linear correlation Obtain linear regression equation Y=15.536X+0.0027, and its linear correlation R ² is 0.999, this shows that the linear fitting degree of this linear regression equation is good, and this linear regression equation can accurately calculate the content of alpha cardiac glycoside.

Example 2

This embodiment provides a method for drawing a standard curve of alpha-cardiac glycoside content and absorbance, which differs from Example 1 in that in step (c), 3,5-dibromophenol is dissolved in water, make the alpha cardiac glycoside chromogen solution of 0.5mol/L, and the absorbance of its 6 test tubes is as shown in table 2:

Table 2

As can be seen from the data in Table 2, the content of alpha-cardiac glycoside is linearly correlated with the absorbance, and according to its linear correlation, the linear regression equation Y=15.323X+0.0015 is obtained, and its linear correlation R ² is 0.999, This shows that the linear fitting degree of the linear regression equation is good, and the linear regression equation can accurately calculate the content of alpha-cardiac glycoside.

Example 3

This embodiment provides a method for drawing a standard curve of alpha cardiac glycoside content and absorbance, which differs from Example 1 in that in step (c), p-cresol is dissolved in water to prepare Become the alpha cardiac glycoside chromogen solution of 0.5mol/L, the absorbance of its 6 test tubes is as shown in table 3:

table 3

As can be seen from the data in Table 3, the content of alpha cardiac glycoside is linearly correlated with the absorbance, and according to its linear correlation, the linear regression equation Y=14.235X+0.0019 is obtained, and its linear correlation R ² is 0.999, This shows that the linear fitting degree of the linear regression equation is good, and the linear regression equation can accurately calculate the content of alpha-cardiac glycoside.

Example 4

This embodiment provides a method for drawing a standard curve of alpha cardiac glycoside content and absorbance, which differs from Example 1 in that in step (c), p-chlorophenol is dissolved in water to prepare The alpha cardiac glycoside chromogen solution of 0.5mol/L, the absorbance of its 6 test tubes is as shown in table 4:

Table 4

As can be seen from the data in Table 4, the content of alpha cardiac glycoside is linearly correlated with the absorbance, and according to its linear correlation, the linear regression equation Y=14.525X+0.0016 is obtained, and its linear correlation R ² is 0.999, This shows that the linear fitting degree of the linear regression equation is good, and the linear regression equation can accurately calculate the content of alpha-cardiac glycoside.

Example 5

This embodiment provides a method for drawing a standard curve of alpha cardiac glycoside content and absorbance, which differs from Example 1 in that in step (c), p-acetylphenol is dissolved in water to prepare Become the alpha cardiac glycoside chromogen solution of 0.5mol/L, and the absorbance of its 6 test tubes is as shown in table 5:

table 5

It can be seen from the data in Table 5 that the content of alpha cardiac glycoside is linearly correlated with the absorbance, and according to its linear correlation, the linear regression equation Y=14.226X+0.0021 is obtained, and its linear correlation R ² is 0.999, which It shows that the linear fitting degree of the linear regression equation is good, and the linear regression equation can accurately calculate the content of alpha-cardiac glycoside.

Example 6

This embodiment provides a method for drawing a standard curve of alpha-cardiac glycoside content and absorbance, which differs from Example 1 in that in step (c), p-tert-butylphenol is dissolved in water, Make the alpha cardiac glycoside chromogen solution of 0.5mol/L, the absorbance of its 6 test tubes is as shown in table 6:

Table 6

It can be seen from the data in Table 6 that the content of alpha cardiac glycoside is linearly correlated with the absorbance, and according to its linear correlation, the linear regression equation Y=14.823X+0.0024 is obtained, and its linear correlation R ² is 0.999, which It shows that the linear fitting degree of the linear regression equation is good, and the linear regression equation can accurately calculate the content of alpha-cardiac glycoside.

Example 7

This embodiment provides a method for drawing a standard curve of alpha cardiac glycoside content and absorbance, which differs from Example 1 in that in step (c), p-methoxyphenol is dissolved in water, Make the alpha cardiac glycoside chromogen solution of 0.5mol/L, the absorbance of its 6 test tubes is as shown in table 7:

Table 7

It can be seen from the data in Table 7 that the content of alpha cardiac glycoside is linearly correlated with the absorbance, and according to its linear correlation, the linear regression equation Y=14.458X+0.0018 is obtained, and its linear correlation R ² is 0.999, which It shows that the linear fitting degree of the linear regression equation is good, and the linear regression equation can accurately calculate the content of alpha-cardiac glycoside.

Example 8

This embodiment provides a method for drawing a standard curve of alpha cardiac glycoside content and absorbance, which differs from Example 1 in that in step (c), p-propynylphenol is dissolved in water, Make the alpha cardiac glycoside chromogen solution of 0.5mol/L, the absorbance of its 6 test tubes is as shown in table 8:

Table 8

It can be seen from the data in Table 8 that the content of alpha cardiac glycoside is linearly correlated with the absorbance, and according to its linear correlation, the linear regression equation Y=14.027X+0.0023 is obtained, and its linear correlation R ² is 0.999, which It shows that the linear fitting degree of the linear regression equation is good, and the linear regression equation can accurately calculate the content of alpha-cardiac glycoside.

Example 9

In order to verify the accuracy of the linear regression equation provided in Example 1, the ethanol solution of alpha-cardiac glycoside with a concentration of 35 mg/L was configured, and 3 mL was placed in a test tube, and 1 mL of 3,5-dinitrophenol was added. Concentration is the alpha cardiac glycoside chromogen solution of 0.5mol/L, after reacting for 20min, measure the absorbance of the solution in the test tube at 495nm, its absorbance value is 0.5465, coincides with the linear regression equation Y=15.536X+0.0027 , which shows that when 3,5-dinitrophenol is used as the alpha-cardiac glycoside developer, the method for determining the alpha-cardiac glycoside content provided in Example 1 of the present invention can accurately measure the alpha-cardiac glycoside content.

Example 10

In order to verify the accuracy of the linear regression equation provided in Example 2, the ethanol solution of alpha-cardiac glycoside with a concentration of 30mg/L was configured, and 3mL was placed in a test tube, and 1mL concentration of 3,5-dibromophenol was added. Be the alpha cardiac glycoside chromogen solution of 0.5mol/L, react after 20min, at 495nm place, measure the absorbance of solution in test tube, its absorbance value is 0.4612, matches with linear regression equation Y=15.323X+0.0015, This shows that when 3,5-dinitrophenol is used as the alpha-cardiac glycoside developer, the method for determining the alpha-cardiac glycoside content provided in Example 2 of the present invention can accurately measure the alpha-cardiac glycoside content.

Example 11

In order to verify the accuracy of the linear regression equation provided in Example 3, the configuration concentration is 20mg/L of alpha-cardiac glycoside ethanol solution, and get 3mL and place it in a test tube, add 1mL concentration of p-cresol configuration to be 0.5mol/L The alpha cardiac glycoside chromogen solution of L, after reacting 20min, at 495nm place, measure the absorbance of solution in test tube, its absorbance value is 0.2866, coincides with linear regression equation Y=14.235X+0.0019, this shows that adopts When methyl phenol is used as the developer of alpha-cardiac glycoside, the method for determining the content of alpha-cardiac glycoside provided in Example 3 of the present invention can accurately measure the content of alpha-cardiac glycoside.

Example 12

In order to verify the accuracy of the linear regression equation provided in Example 4, the ethanol solution of alpha-cardiac glycoside with a concentration of 28 mg/L was configured, and 3 mL was placed in a test tube, and 1 mL of p-chlorophenol was added with a concentration of 0.5 mol/L. The alpha cardiac glycoside chromogen solution of L, after reacting for 20min, at 495nm, measure the absorbance of the solution in the test tube, its absorbance value is 0.4083, coincides with the linear regression equation Y=14.525X+0.0016, this shows that adopts When chlorophenol is used as the developer of alpha-cardiac glycoside, the method for determining the content of alpha-cardiac glycoside provided in Example 4 of the present invention can accurately measure the content of alpha-cardiac glycoside.

Example 13

In order to verify the accuracy of the linear regression equation provided in Example 5, the ethanol solution of alpha-cardiac glycoside with a concentration of 22 mg/L was configured, and 3 mL was placed in a test tube, and the concentration of 1 mL of p-acetylphenol configuration was 0.5 mol /L of the alpha cardiac glycoside chromogenic agent solution, after reacting for 20min, measure the absorbance of the solution in the test tube at 495nm, and its absorbance value is 0.3151, which coincides with the linear regression equation Y=14.226X+0.0021, which shows that using When p-acetylphenol is used as the developer of alpha cardiac glycoside, the method for determining the content of alpha cardiac glycoside provided in Example 5 of the present invention can accurately measure the content of alpha cardiac glycoside.

Example 14

In order to verify the accuracy of the linear regression equation provided in Example 5, the ethanol solution of alpha-cardiac glycoside with a concentration of 26mg/L was configured, and 3mL was placed in a test tube, and the concentration of 1mL of p-acetylphenol configuration was 0.5mol /L of the alpha cardiac glycoside chromogen solution, after reacting for 25min, measure the absorbance of the solution in the test tube at 495nm, and its absorbance value is 0.3878, which coincides with the linear regression equation Y=Y=14.823X+0.0024, which It shows that when p-acetylphenol is used as the developer of alpha cardiac glycoside, the method for determining the content of alpha cardiac glycoside provided in Example 6 of the present invention can accurately measure the content of alpha cardiac glycoside.

Example 15

In order to verify the accuracy of the linear regression equation provided in Example 5, the ethanol solution of alpha-cardiac glycoside with a concentration of 35mg/L was configured, and 3mL was placed in a test tube, and the concentration of 1mL of p-acetylphenol configuration was 0.5mol /L of the alpha cardiac glycoside chromogenic agent solution, after reacting for 30min, at 495nm, measure the absorbance of the solution in the test tube, and its absorbance value is 0.5078, which coincides with the linear regression equation Y=14.458X+0.0018, which shows that using When p-acetylphenol is used as the developer of alpha cardiac glycoside, the method for determining the content of alpha cardiac glycoside provided in Example 7 of the present invention can accurately measure the content of alpha cardiac glycoside.

Example 16

In order to verify the accuracy of the linear regression equation provided in Example 5, the ethanol solution of alpha-cardiac glycoside with a concentration of 27 mg/L was configured, and 3 mL was placed in a test tube, and the concentration of 1 mL of p-acetylphenol configuration was 0.5 mol /L of the alpha cardiac glycoside chromogen solution, after reacting for 10min, measure the absorbance of the solution in the test tube at 495nm, and its absorbance value is 0.381, which coincides with the linear regression equation Y=14.027X+0.0023, which shows that using When p-acetylphenol is used as the developer of alpha cardiac glycoside, the method for determining the content of alpha cardiac glycoside provided in Example 8 of the present invention can accurately measure the content of alpha cardiac glycoside.

In addition, it can be seen from the linear regression equation of the content and absorbance of the alpha cardiac glycosides provided by Examples 1-8 that when 3,5-dinitrophenol and 3,5-dibromophenol are used as the alpha cardiac glycoside When the glycoside color reagent is used, the absorbance value is higher, the measurement error is smaller, and the measurement result is more accurate.

In summary, the assay method for the content of alpha cardiac glycoside provided by the present invention, generates an orange-yellow solution through a color reaction between alpha-cardiac glycoside and a phenolic compound, and then measures the absorbance of the orange-yellow solution at 485nm-500nm. , to obtain the content of alpha cardiac glycoside, thus providing a simple, quick, accurate and efficient method for the determination of alpha cardiac glycoside, which solves the problem that restricts the application research of alpha cardiac glycoside, and can effectively promote Use of alphacardiac glycosides in tumor and cancer therapy.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. scope.

Claims (10)

1. purposes of the phenol compound as A type cardiac glycoside developer, the formula of the phenol compound are：

Wherein, R₁、R₂、R₃、R₄And R₅Independently selected from hydrogen, halogen, carboxyl, nitro, C_1-6Alkyl, C_3-5Alkenyl, C_3-5Alkynes One kind in base, acyl group, ether and ester group, and R₁、R₂、R₃、R₄And R₅It is asynchronously hydrogen, the wherein formula of acyl group is COR₆, ether The formula of base is OR₇, the formula of ester group is COOR₈, the R₆、R₇And R₈Independently selected from C_1-6Alkyl, C_1-6Alkoxy or C_1-6 Alcoxyl ester group.

2. purposes of the phenol compound according to claim 1 as A type cardiac glycoside developer, it is characterised in that institute State phenol compound be selected from 3,5- dinitrophenol, metanitrophenol, 2,6- dinitrophenol, p-methyl phenol, to chlorobenzene Phenol, p bromophenol, to acetyl phenol, p-nitrophenol, 3,5- dibromophenols, 3,5- xylenols, 2,6- dimethyl benzenes In phenol, 3,5- difluorophenols, m fluorophenol, p-t-butyl phenol, paraethyl phenol, p methoxy phenol and 2- nitrophenols One kind, preferably 3,5- dinitrophenol or 3,5- dibromophenol.

3. a kind of assay method of A type cardiac glycoside content, it is characterised in that comprise the following steps：

A type cardiac glycoside is dissolved in alcoholic solvent, is configured to the alcoholic solution of A type cardiac glycoside, then carried with claim 1 or 2 The phenol compound mixing of confession, chromogenic reaction is carried out, absorbance of the measure chromophoric solution at 485nm-500nm, obtains first The content of type cardiac glycoside.

4. the assay method of A type cardiac glycoside content according to claim 3, it is characterised in that comprise the following steps：

(a) alcoholic solution of the A type cardiac glycoside of various concentrations is configured, is allowed to carry out chromogenic reaction with phenol compound respectively, surveys Determine the absorbance after the alcoholic solution colour developing of the A type cardiac glycoside of various concentrations, obtain standard curve and its regression equation；

(b) A type cardiac glycoside to be measured is dissolved in alcoholic solvent, is configured to A type cardiac glycoside alcoholic solution, then with phenol compound Chromogenic reaction is carried out, determines absorbance；

(c) absorbance of measure is substituted into regression equation, obtains containing for A type cardiac glycoside in the alcoholic solution of A type cardiac glycoside to be measured Amount.

5. the assay method of the A type cardiac glycoside content according to claim 3 or 4, it is characterised in that first by phenol Compound is dissolved in water, is configured to A type cardiac glycoside chromogenic reagent solution, then is mixed with the alcoholic solution of A type cardiac glycoside, develop the color instead Should.

6. the assay method of A type cardiac glycoside content according to claim 5, it is characterised in that the alcohol of A type cardiac glycoside is molten In liquid, the concentration of A type cardiac glycoside is 16-32mg/L；In A type cardiac glycoside chromogenic reagent solution, phenol compound it is mole dense Spend for 0.01-0.1mol/L.

7. the assay method of A type cardiac glycoside content according to claim 6, it is characterised in that the alcohol of A type cardiac glycoside is molten The volume ratio of liquid and A type cardiac glycoside chromogenic reagent solution is (2-4)：1, preferably 3：1.

8. the assay method of the A type cardiac glycoside content according to claim 3 or 4, it is characterised in that carry out chromogenic reaction Time be 5-40min, preferably 10-30min, more preferably 20min.

9. the assay method of the A type cardiac glycoside content according to claim 3 or 4, it is characterised in that the alcoholic solvent is Low-carbon alcohols, preferably methanol, ethanol or propyl alcohol, more preferably ethanol.

10. the assay method of the A type cardiac glycoside content according to claim 3 or 4, it is characterised in that the A type cardiac stimulant Glycosides is extracted from plant, and its extracting method comprises the following steps：First plant is crushed, then entered using ethanol as extract solution Row ultrasonic extraction, that is, obtain the ethanol solution of A type cardiac glycoside.