Method for measuring flavonoid amount in choleretic tea № 3

FIELD: medicine.

SUBSTANCE: method is based on measuring a flavonoid amount by differential spectral photometry, calculated as Cinaroside at wave length 400 nm, extracting in water and alcohol and using 70% ethanol as an extraction fluid; the total flavonoid amount calculated as Cinaroside and an absolutely dry raw material in percentage (X) is calculated by formula.

EFFECT: method enables measuring the total flavonoid amount as biologically active components having a basic therapeutic action.

3 ex, 14 dwg, 2 tbl

 

The invention relates to chemical-pharmaceutical industry and can be used in the centers of drug quality control and analytical laboratories in the analysis of flavonoids in Choleretic collection No. 3" ("Hitogata No. 3").

The current system of quality control requires continuous improvement approaches to standardization of biologically active compounds using modern methods of analysis and relevant data about their properties, allowing to identify differentially specific group of biologically active compounds.

"Choleretic collection №3" is widely used in modern medicine in the treatment of diseases of the liver and biliary tract (2, 5, 6). The pharmacological action of this drug collection caused by different groups of biologically active compounds. Part of the collection presents chamomile flowers, peppermint leaves, marigold flowers, grass, yarrow, tansy flowers. Designation in the chamomile, peppermint, yarrow and tansy the two most important groups of active ingredients - essential oils and flavonoids suggests the prediction (8). It is known that antispasmodic and anti-inflammatory action of the drug is due to the presence of essential�about oil this choleretic effect is due to a second group of biologically active compounds flavonoids. The presence in the collection of the flowers of calendula enhances anti-inflammatory drug collection (5, 6, 8). According to the normative documentation on "Cholagogue collection No. 3" the definition of quality is the essential oil content (1). Taking into account the fact that the components of essential oils practically insoluble in water while getting the infusion, the content of essential oils in "Cholagogue collection No. 3 does not allow to assess the degree of effectiveness of this drug. In our opinion, in the specified collection is expedient to determine the content of water-soluble active substances - flavonoids, because they determine the main pharmacological action, namely choleretic effect collection (9, 10).

Thus, the aim of the invention is to develop a method of analysis of flavonoids in Choleretic collection No. 3".

The technical result of the proposed method is an improvement of a method of quantitative analysis "Cholagogue collection No. 3", by identification of flavonoids as bioactive compounds, determine the main pharmacological properties of the collection.

The technical result is achieved in that conduct quantitative determination of total flavonoids by the method of differential SPECT�optometrie, in terms of cynaroside, at a wavelength of 400 nm, aqueous-alcoholic extraction and use as an extractant 70% ethanol, the content of total flavonoids in terms of cynaroside and absolutely dry raw materials in percentage (X) is calculated by the formula:

X=Dm0301025100100D0m25125(100W),

where D is the optical density of the test solution;

D0optical density of the solution of the state standard sample of cynaroside;

m is the mass of raw material, g;

m0is the mass of the state standard sample of cynaroside, g;

W is the loss in weight on drying as a percentage;

in the absence of a standard sample of cynaroside use theoretical value of the specific absorbance of its alcoholic solution, is 350, and is absolutely dry raw materials in percentage (X) is calculated by the formula:

X=D3025100m350(100W )

where D is the optical density of the test solution;

m is the mass of raw material, g;

350 - specific absorption coefficient(E1Cm%a)alcohol solution of the state standard sample of cynaroside at 400 nm;

W is the loss in weight of the raw material during drying, in percent.

In the analysis measure the optical density of the complex of flavonoids with aluminum chloride of the analyzed solution in the background of the initial solution. In this case, the observed bathochromic shift of the long wavelength bands of flavonoids, which is found in the UV spectrum as the absorption maximum at a wavelength of 406 nm. This is confirmed in terms of differential spectrophotometry: absorption maximum at 406 nm (Fig. 1). The study of UV spectra of the state standard sample of cynaroside showed (Fig. 2) that the solution of this standard in the presence of aluminum chloride is close to the maximum absorption (400 nm). Consequently, cynaroside can be used in the method of analysis as a standard sample.

We studied the spectral characteristics of water-alcohol extraction from raw plant components "Geelong�nny collection No. 3" (Fig. 3), to determine the content of total flavonoids in each component of the collection (Fig. 4).

Based on the spectral characteristics of components of data collection and model compound (Fig. 1, 3), to determine the optimal extraction conditions "Cholagogue collection No. 3" has developed a method for determination of total flavonoids in the Choleretic collection No. 3 by the method of differential spectrophotometry in terms of cynaroside.

The method is implemented as follows.

An analytical sample of the raw material is ground to a particle size passing through a sieve with holes of diameter 1 mm. to About 1 g of the crushed raw material (a precisely weighed) was placed in a flask with a ground joint with a capacity of 50 ml, add 30 ml of 70% ethanol. The flask is closed with a stopper and weighed on terimah scales with an accuracy of ±0.01 g. the Flask is attached to the reverse refrigerator and heated in a boiling water bath (moderate boiling) for 90 min, Then the flask close to the same tube, weighed again and fill the missing extractant to the initial weight. The extract was filtered through filter paper and cooled for 30 min. the Test solution was prepared as follows: 1 ml of extract obtained is placed in a volumetric flask with a capacity of 25 ml, add 1 ml of a 3% alcohol solution of aluminum chloride and bring the volume of solution up to the mark with 95% ethyl alcohol (test solution a). In CA�ETS of the reference solution using a solution prepared under the same conditions, but without addition of aluminum chloride solution (reference solution A). Optical density measurements carried out on the spectrophotometer at a wavelength of 400 nm. In parallel, measure the optical density of the solution of the state standard sample of cynaroside at a wavelength of 400 nm, prepared by analogy with the test solution (see note).

Note. Preparation of solution of cynaroside-standard sample. About 0.02 (a precisely weighed) of cynaroside placed in a volumetric flask with a capacity of 25 ml, was dissolved in 15-20 ml of 70% ethyl alcohol by heating on a water bath. After cooling the contents of the flask to room temperature the volume was adjusted solution of 70% ethanol to the mark (solution A). 1 ml of a solution of cynaroside placed in a volumetric flask of 25 ml, add 1 ml of a 3% alcohol solution of aluminum chloride and the volume was adjusted solution of 95% ethanol to the mark and mix (test solution B). As of the reference solution use a solution prepared as follows: 10 ml of solution A of cynaroside placed in a volumetric flask of 25 ml and the volume was adjusted solution up to the mark with 95% ethanol (reference solution B).

The content of total flavonoids in terms of cynaroside and absolutely dry raw materials in percentage (X) is calculated by the formula:

X=D m0301025100100D0m25125(100W),

where D is the optical density of the test solution;

D0optical density of the solution of the state standard sample of cynaroside;

m is the mass of raw material, g;

m0is the mass of the state standard sample of cynaroside, g;

W is the loss in weight on drying as a percentage;

in the absence of a standard sample of cynaroside use theoretical value of the specific absorbance of its alcoholic solution, is 350, and is absolutely dry raw materials in percentage (X) is calculated by the formula:

X=D3025100m350(100W)

where D is the optical density of the test solution;

m is the mass of raw material, g;

350 - specific absorption coefficient(E1Cm%a) alcohol solution of the state standard sample of cynaroside at 400 nm;

W is the loss in weight of the raw material during drying, in percent.

The proposed method is illustrated by the following examples.

Example 1.

An analytical sample of the raw material (collection "Hitogata No. 3" ("Cholagogue collection No. 3"), OAO "Krasnogorskleksredstva", series 60912) is ground to a particle size passing through a sieve with holes of diameter 1 mm. to About 1 g of the crushed raw material (a precisely weighed) was placed in a flask with a ground joint with a capacity of 50 ml, add 30 ml of 70% ethanol. The flask is closed with a stopper and weighed on terimah scales with an accuracy of ±0.01 g. the Flask is attached to the reverse refrigerator and heated in a boiling water bath (moderate boiling) for 90 min, Then the flask close to the same tube, weighed again and fill the missing extractant to the initial weight. The extract was filtered through filter paper and cooled for 30 min. the Test solution was prepared as follows: 1 ml of extract obtained is placed in a volumetric flask with a capacity of 25 ml, add 1 ml of a 3% alcohol solution of aluminum chloride and bring the volume of solution up to the mark with 95% ethyl alcohol (test solution a). As of the reference solution use the solution prepared under the same conditions, but without addition of aluminum chloride (solution CPA�tion A). Optical density measurements carried out on the spectrophotometer at a wavelength of 400 nm. In parallel, measure the optical density of the solution of the standard sample of cynaroside at a wavelength of 400 nm, prepared by analogy with the test solution (see note).

The content of total flavonoids in terms of cynaroside and absolutely dry raw materials in percentage (X) is calculated by the formula:

X=0,4135690,0014301251001000,95620,997325125(10010)

The optical density D=0,413569, the raw material mass m=0,9973.

The content of total flavonoids = 1,05%.

All results were statistically processed. A single error quantification is ±3,12%.

Example 2.

Open 10 filter bags (collection "Hitogata No. 3" ("Cholagogue collection No. 3"), OAO "Krasnogorskleksredstva", filter bags, series bearings 30213). About 1 g of the crushed material (a precisely weighed) was placed in a flask with a ground joint with a capacity of 50 ml. Then the process is carried out in accordance with example 1.

The content of total flavonoids in terms of cynaroside and absolutely dry raw materials in percent� (X) is calculated by the formula:

X=0,5972480,0014301251001000,95621,001625125(10010)

The optical density D=0,597248, the raw material mass m=1,0016.

The content of total flavonoids = 1,16%.

All results were statistically processed. A single error quantification ±or 3.28%.

Example 3.

An analytical sample model compound, the structure represented in Fig. 5, crushed to a particle size passing through a sieve with holes of diameter 1 mm. to About 1 g of the crushed raw material (a precisely weighed) was placed in a flask with a ground joint with a capacity of 50 ml. Then the process is carried out in accordance with example 1.

The content of total flavonoids in terms of cynaroside and absolutely dry raw materials in percentage (X) is calculated by the formula:

X=0,5264870,0014301251001000,95620,996325125(10010)

The optical density D=0,526487, the raw material mass m=0,9963.

The content of total flavonoids = 1,03%.

All results were statistically processed. A single error quantification is ±3,19%.

Thus, the proposed method for quantitative determination of total flavonoids in the Choleretic collection No. 3" first developed for the analysis of this group of biologically active compounds collection and has the following advantages over the methods described in Pharmacopoeial article:

1. The definition is the content of water-soluble substances - flavonoids, instead of essential oils, the components of which are virtually insoluble in water.

2. This method allows to evaluate the content of total flavonoids as bioactive components have a major therapeutic effect choleretic effect.

3. The developed method is specific, because it allows to eliminate the contribution of other compounds (taking into account the ability of flavonoids to form complexes with aluminum chloride).

SOURCES of INFORMATION:

1. VFS 42-2558-95. Cholagogue collection No. 3 / state Pharmacopoeia Committee. Introduction. 09.07.1996. - M., 1996. - 12 p.

2. The state register of medicines. Official edition as of April 1, 2009: in 2 t. T. 1. - M.: Publishing house "Medical Council�, 2009. - 1359 S.

3. State Pharmacopoeia of the USSR. - Eleventh edition. - Vol. 1. - M.: Medicine, 1987. - 336.

4. State Pharmacopoeia of the USSR. - Eleventh edition. - Vol. 2. - M.: Medicine, 1990. - 400 p.

5. Kurkin V. A. Fundamentals of herbal medicine: a textbook for students of pharmaceutical universities. - Samara: LLC "Etching", GOU VPO "Samara state medical University of Roszdrav", 2009. - 963 S.

6. Kurkin V. A. Pharmacognosy: a Textbook for students of pharmaceutical universities / Ed. 2nd, revised and enlarged. - Samara: LLC "Etching", GOU VPO "Samara state medical University", 2007. - S. 1239

7. Kurkin V. A. Prospects for the creation and implementation of import-substituting medicinal herbal remedies / V. A. Kurkin, E. V. Avdeeva, V. B. Braslavsky, O. Pravdivtseva, A. V. Kurkina et al. / / XVIII Russian national Congress "Man and medicine: abstracts. - Moscow, 2011. - P. 507.

8. Kurkin V. A. Current aspects of standardization of medicinal plants containing flavonoids / V. A. Kurkin // materials of the Interregional scientific conference with international participation, dedicated to the 70th anniversary of pharmaceutical faculty, Siberian state medical University. - Tomsk, 2011. - Pp. 86-90.

9. Kurkin V. A. New approaches to the standardization of medicinal plants containing flavonoids. 1. Common tansy / V. A. Kurkin // 65-th international conference on pharmacy and pharmacology "Development�, research and marketing of new pharmaceutical products": collection of scientific works. - Pyatigorsk, 2011. - Vol. 66. - Pp. 134-137.

10. Kurkin V. A. Flavonoids as a source of medicinal preparations on the basis of raw materials of pharmacopoeial plants / V. A. Kurkin // 3rd international Congress of herbalists and herbalists "Modern problems of herbal medicine and Travnikova: proceedings of scientific works. - M., 2013. - P. 111-114.

Method of quantitative determination of flavonoids in Choleretic collection No. 3" performed by the method of differential spectrophotometry, based on cynaroside, at a wavelength of 400 nm, aqueous-alcoholic extraction and use as an extractant 70% ethanol, the content of total flavonoids in terms of cynaroside and absolutely dry raw materials in percentage (X) is calculated by the formula:

where D is the optical density of the test solution;
Dooptical density of the solution of the state standard sample of cynaroside;
m is the mass of raw material, g;
mois the mass of the state standard sample of cynaroside, g;
W is the loss in weight on drying as a percentage;
in the absence of a standard sample of cynaroside use theoretical value of the specific absorbance of its alcoholic solution, is 350, and is absolutely dry raw materials in percentage (X) is calculated according to forms�Le:

where D is the optical density of the test solution;
m is the mass of raw material, g;
350 - specific absorption coefficientalcohol solution of the state standard sample of cynaroside at 400 nm;
W is the loss in weight of the raw material during drying, in percent.



 

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