Quantitative method of determining thymol and carvacrol in medicinal plant raw material

FIELD: chemistry.

SUBSTANCE: invention pertains to the quantitative method of determining thymol and carvacrol in medicinal plant raw material, in extracts and infusions of plant raw material using high-performance liquid chromatography. The result is attained by that, in the quantitative measurement of thymol and carvacrol present in the extract, a column with dimensions 250×4.6 mm is used. The column is filled with silica gel with grafted straight alkyl groups, in which the number of atoms equals sixteen (C 16), with particle size of 7 mcm. The mobile phase used is a methanol-water solution with ratio of 62:38. The speed of the mobile phase is 1 ml/min. Detection is done using a UV-detector with wavelength of 277 nm, with subsequent calculation of thymol and carvacrol relative to the area of peaks of the analysed and standard substance. The standard substance used is a standard solution of thymol-carvarol.

EFFECT: method allows for separation and quantitative measurement of content of separate thymol and carvacrol in medicinal raw material, in extracts and infusions of plant raw material, simplifies the sample preparation process, reduces the time of analysing and simplifies identification of the substance.

8 ex, 1 tbl

 

The invention relates to analytical chemistry and can be used for the quantitative determination of thymol and carvacrol in their joint presence in medicinal plant raw materials, extracts and tinctures of vegetable raw materials.

There is a method of quantitative determination of thymol in ethanol extracts of medicinal raw spectrophotometric (Patent 2025717, Russia, C15 G01N 21/33. The method of quantitative determination of thymol in medicinal plant raw materials. / Musulin A.V., V.V. Petrenko, Kaloshina N.A.; Musulin AV; 4906687/25; Appl. 31.01.1991; Publ. 1994.12.30). The disadvantage of this method is the presence in the extract of plant materials related substances that interfere with the spectrophotometric determination of, inability to determine thymol, bypassing the stage of precipitation of interfering substances 10%solution of lead acetate.

There is a method of quantitative determination in the essential oil thymol and carvacrol spectrophotometric (Markov O.M., Klochkov, S. C. environmental Protection, environmental issues and quality control. M: niitekhim, 1994, no 2, p.6-7). The disadvantage of this method is the necessity of selection of plant material or plant extracts essential oils by distillation with water vapor and the ability to determine only the total content of thymol and carvacrol.

<> The known method the qualitative determination of thymol using thin-layer chromatography (Markov O.M., Karpenko, VA, Shushkina A.S., Lichota TT Using physico-chemical methods in the analysis of herbal medicines // Herald of the Voronezh state University. Series chemistry, biology, pharmacy. 2003. No. 1. S-100). The disadvantage of this method is the impossibility of quantitative determination of thymol, complexity and duration of the experiment.

Known methods of determination of thymol and carvacrol gas chromatography-mass spectrometry and gas chromatography (Badaeva Y.A., L. Pokrovsky, Tkachev A.V. Investigation of the chemical composition of the essential oils of the genus Thymus L. growing in Altai // Chemistry of plant raw materials. 1999. No. 3. P.41-48). The disadvantage of this method is the necessity of large quantities of plant material, the necessity of selection of plant material or extracts essential oils by distillation with water vapor, the duration of the steam distillation, the duration of the analysis, a large number of related components of essential oils and their similar retention time and consequently the difficulty of identification.

There is a method of determining the total content of thymol and carvacrol high-performance liquid chromatography using a colon and Silsor SPH and eluent acetonitrile - phosphate buffer (I.G. Zenkevich, the head V.M., Tkachev KG Some features of the quantitative analysis of components of essential oils in high-performance liquid chromatography // Plant resources. 1999. No. 1. S-137), is selected as the closest analogue of the definition of thymol and carvacrol in vegetable raw materials. Silsor SPH is unmodified silica gel production "Lachema" (the Companion of chromatographica. Methods liquid chromatography / Rudakov O., Vostrov I.A., Fedorov S.V. and others Voronezh: Publishing house. Aquarius, 2004, PP 156). The disadvantage of this method is the impossibility of the separation of thymol and carvacrol.

In medicine and perfumes are widely used Thymus serpillum (creeping thyme, creeping thyme)Thymus vulgaris (thyme), Origanum vulgare (oregano ordinary), Ledum palustre (marsh Labrador tea), Ledum decumbens (Ledum creeping), Ledum hypoleucum (Ledum-croup), Ledum macrophyllum (large-leaved Labrador tea), Paeonia anomala (peony), Ruta graveolens (Ruta fragrant), Mentha piperita (peppermint), their extracts and tinctures and preparations on their basis (Pharmacognosy. Atlas: Educational. the allowance. M.: Medicine, 1989. 512 C.). For these plants and products made on their basis, are shown antimicrobial, anti-inflammatory, antisemitica and antioxidant activity (Pharmacognosy, 1989)due to its high content of thymol and carvacrol (Couldis M, Tzakou O., Kujundzic S., Sokovic M., Mimica N. Chemical analysis and antifungal activity of Thymus striatus // Phytother. Research. 2004. Vol.18, No. 1. P.40-42; Kahkonen M.P., Hopia, A.I., Vuorela H.J. Rauha, J.-P, Pihiaja K., Kujala T.S, Heinonen M. Antioxidant activity of plant extracts containing phenolic // J.Agric. Food Chem. 1999. No. 47. P.3954-3962; M. Korayem, Hasabo S., Ameen H. Effects and mode of action of some plant extracts on certain plant parasitic nematodes // J.Pest Science. 1999. Vol.66, No. 2. P.32-36). Plants of the same species but collected at different times and different chemotype can vary dramatically content of thymol and carvacrol. For example, Thymus vulgaris (thyme), it was shown that plants dominated thymol and carvacrol, have a high antioxidant activity in comparison with plants dominated linalool (M. Jukic, Milos M. Catalytic Oxidation and Antioxidant Properties of Thyme Essential Oils (Thymus vulgarae L.) // Croatica Chemica Acta. 2005. Vol.78, No.1. P.105-110).

The task on which the invention is directed, is the determination of thymol and carvacrol in vegetable raw materials, extracts and tinctures of vegetable raw materials.

The applied method allows to extend the range of applications, to share and to determine the quantitative content of the individual compounds thymol and carvacrol in medicinal plant raw materials, extracts and tinctures of plant material, to simplify the sample preparation process, to reduce the analysis time and to simplify the identification of substances.

The technical result is achieved by the fact that the quantitative method defined who I thymol and carvacrol at their joint presence in medicinal plant raw materials, which pre-extracted, extracts and tinctures of plant material, including high-performance liquid chromatography, according to the invention using a column Packed with silica gel with grafted straight alkyl groups, the number of atoms that is equal to sixteen (C16), with a particle size of 7 μm, as the mobile phase, use a solution of methanol - water at a ratio of 62:38 with the velocity of the mobile phase, 1 ml /min, detection is performed on UV-detecter at a wavelength of 277 nm, with subsequent calculation of the concentration of thymol and carvacrol against peak areas analyzed and a standard substance, which is used standard Rasbora thymol and carvacrol, respectively, given its concentration, volume of the extract samples of raw materials or the dilution of the investigational product.

Example 1.

Control. Standard solutions of thymol and carvacrol concentration 0.001 mg/ml were analyzed on column size 250×4.6 mm, filled with silica gel grafted straight alkyl groups, the number of atoms that is equal to sixteen (C16), with a particle size of 7 μm. As the mobile phase used a solution of methanol - water in different ratios, the velocity of the mobile phase, 1 ml /min Detection was carried out by UV detector at a wavelength of 277 nm.

Table 1
EluentThe retention time of carvacrol, minThe retention time of thymol, min
methanol 1002.52.5
methanol - water 90:103.23.2
methanol - water 70:306.87.1
methanol - water 65: 358.38.9
methanol - water 62:3811.012.7

Example 2.

A portion of the herb thyme (LLC "Medical company "folk medicine"") 0.15 g was extracted with 10 ml of 96%methanol by heating on a water bath at 60°With 30 minutes, the Extract was analyzed on a column size 250×4.6 mm, filled with silica gel grafted straight alkyl groups, the number of atoms that is equal to sixteen (C16), with a particle size of 7 μm. As the mobile phase used a solution of methanol - water in a ratio of 62:38 (by volume), the velocity of the mobile phase, 1 ml /min Detection was carried out by UV detector at a wavelength of 277 nm. Carvacrol in the extract of the herb thyme is not detected. The retention time of thymol is 12.7 minutes Quantitative content of thymol in thyme herb (mg/g) was calculated by the formula:

where S1- value is the peak area of thymol in the extract sample thyme herb;

c0- the concentration of a standard solution of carvacrol, mg/ml;

V - volume of 96%methanol, which was extracted with a portion of the herb thyme, in this example equal to 10 ml;

S0- the value of the peak area of thymol standard solution;

m - hitch thyme herbs, in this example equal to 0.15,

The content of thymol in thyme herb is 0.08 mg/g

Example 3.

A portion of the herb thyme (LLC "Medical company "folk medicine"") 0.15 g was extracted with 10 ml of 96%methanol by heating on a water bath at 60°With 30 minutes Extract of the herb thyme before analysis was pre-purified by concentrating the cartridge, Deepak C16 (JSC Biohimik", Russia). To 1 ml of extract was added to 3 ml of water was sampled aliquot of 1 ml was passed through the concentrating cartridge, Deepak C16 (JSC Biohimik", Russia), previously activated with methanol and rinsed with distilled water. Then the cartridge was washed sequentially with 1 ml water and 1 ml of methanol. The fraction obtained by elution with methanol, and analyzed on column size 250×4.6 mm, filled with silica gel grafted pryamimi alkyl groups, the number of atoms that is equal to sixteen (C16), with a particle size of 7 μm. As the mobile phase used a solution of methanol - water in a ratio of 62:38 (by volume), the velocity of the mobile phase, 1 ml/min Detection on Westlake on UV-detector at a wavelength of 277 nm. Carvacrol in the extract of the herb creeping thyme is not detected. The quantitative content of thymol in thyme herb (mg/g) was calculated by the formula:

where S1- the value of the peak area of thymol in the extract sample thyme herbs;

with0- the concentration of a standard solution of carvacrol, mg/ml;

V - volume of 96%methanol, which was extracted with a portion of the herb thyme, in this example equal to 10 ml;

n is the dilution of the extract before cleaning concentrating on the cartridge equals 4;

S0- the value of the peak area of thymol standard solution;

m - hitch thyme herbs, in this example equal to 0.15,

The content of thymol in thyme herb is 0.08 mg/g

Example 4.

The drug Pinosol" (Zentiva, Slovak Republic) is a combination drug of plant origin, containing 0.0032 g of thymol in 10 ml (0.32 mg/ml), and oil of pine, peppermint oil and eucalyptus oil. To 0.1 ml of pinosol was added 9.9 ml of methanol and heated in a water bath at 60°30 minutes was Collected and 1 ml of sample was added to 3 ml of water. An aliquot of 1 ml was passed through the concentrating cartridge, Deepak C16 (JSC Biohimik", Russia), previously activated with methanol and rinsed with distilled water. Then the cartridge was washed sequentially with 1 ml water and 1 ml of the of ethanol. The fraction obtained by elution with methanol, and analyzed on column size 250×4.6 mm, filled with silica gel grafted straight alkyl groups, the number of atoms that is equal to sixteen (C16), with a particle size of 7 μm. As the mobile phase used a solution of methanol - water in a ratio of 62:38 (by volume), the velocity of the mobile phase, 1 ml/min Detection was carried out by UV detector at a wavelength of 277 nm. The retention time of carvacrol is 11.0 min, thymol - 12.7 minutes the Concentration of carvacrol (mg/ml) in the preparation Pinosol" was calculated by the formula:

where S1- the value of the peak area of carvacrol researched solution;

c0- the concentration of a standard solution of carvacrol, mg/ml;

n1- thinning drug, in this example equal to 10;

n2- dilution before cleaning concentrating on the cartridge equals 4;

S0- the value of the peak area of carvacrol standard solution.

The concentration of carvacrol in the preparation Pinosol" is 0.029 mg/ml

The concentration of thymol (mg/ml) in the preparation Pinosol" was calculated by the formula:

where S1- the value of the peak area of thymol researched solution;

c0- the concentration of a standard solution of thymol, mg/ml;

n1- dilution prep the rata, in this example, is 10;

n2- dilution before cleaning concentrating on the cartridge equals 4;

S0- the value of the peak areas of thymol standard solution.

The content of thymol in the preparation Pinosol" is 0.420 mg/ml

Example 5.

The drug Pertussin" (CJSC "Ecolab") is the drug of plant origin containing 12 g of the extract of thyme or liquid extract of thyme liquid in 100 ml of the drug. To 1 ml of the drug "Pertussis" was added 3 ml of water was sampled aliquot of 1 ml was passed through the concentrating cartridge with Deepak 16 (JSC Biohimik", Russia), previously activated with methanol and rinsed with distilled water. Then the cartridge was washed sequentially with 1 ml water and 1 ml of methanol. The fraction obtained by elution with methanol, and analyzed on column size 250×4.6 mm, filled with silica gel grafted straight alkyl groups, the number of atoms that is equal to sixteen (C16), with a particle size of 7 μm. As the mobile phase used a solution of methanol - water in a ratio of 62:38 (by volume), the velocity of the mobile phase, 1 ml/min Detection was carried out by UV detector at a wavelength of 277 nm. The retention time of carvacrol is 11.0 minutes Thymol not detected. The concentration of carvacrol (mg/ml) in the preparation Pertussin" was calculated by the formula:

,

where S1- the value of the peak area of carvacrol researched solution;

c0- the concentration of a standard solution of carvacrol, mg/ml;

n - thinning before cleaning concentrating on the cartridge equals 4;

S0- the value of the peak area of carvacrol standard solution.

The concentration of carvacrol (mg/ml) in the preparation Pertussin" is 0.0015 mg/ml

Example 6.

It shoots Ledum palustre (CJSC "Company "Health") of 0.15 g was extracted with 10 ml of 96%methanol by heating on a water bath at 60°With 30 minutes, the Extract was filtered and analyzed on column size 250×4.6 mm, filled with silica gel grafted straight alkyl groups, the number of atoms that is equal to sixteen (16), with a particle size of 7 μm. As the mobile phase used a solution of methanol - water in a ratio of 62:38 (by volume), the velocity of the mobile phase, 1 ml /min Detection was carried out by UV detector at a wavelength of 277 nm. Carvacrol is not detected. The retention time of thymol is 12.7 minutes Quantitative content of thymol in shoots of wild rosemary was calculated analogously to example 2. The content of thymol in shoots of wild rosemary is 0.156 mg/g

Example 7.

A portion of marjoram (LLC "Medical company "folk medicine"") 0.15 g was extracted with 10 ml of 96%methane is and when heated in a water bath at 60° With 30 minutes, the Extract was filtered and analyzed on column size 250×4.6 mm, filled with silica gel grafted straight alkyl groups, the number of atoms that is equal to sixteen (C16), with a particle size of 7 μm. As the mobile phase used a solution of methanol - water in a ratio of 62:38 (by volume), the velocity of the mobile phase, 1 ml/min Detection was carried out by UV detector at a wavelength of 277 nm. The retention time of carvacrol is 11.0 min, thymol - 12.7 minutes Quantitative content of carvacrol and thymol in the grass oregano was calculated analogously to example 2. The content of carvacrol in the herb oregano is 0.035 mg/g Content of thymol in the herb oregano is 0.132 mg/g

Example 8.

The hinge leaf peppermint (000 "Medical company "folk medicine"") 0.15 g was extracted with 10 ml of 96%methanol by heating on a water bath at 60°With 30 minutes, the Extract was filtered and analyzed on column size 250×4.6 mm, filled with silica gel grafted straight alkyl groups, the number of atoms that is equal to sixteen (C16), with a particle size of 7 μm. As the mobile phase used a solution of methanol - water in a ratio of 62:38 (by volume), the velocity of the mobile phase, 1 ml /min Detection was carried out by UV detector at a wavelength of 277 nm. The retention time of carvacrol is 11.0 min, t is Ola - 12.7 minutes Quantitative content of carvacrol and thymol in the leaves of peppermint was calculated analogously to example 2. The content of carvacrol in the leaves of peppermint is 0.093 mg/g Content of thymol in the leaves of peppermint is 0.110 mg/g

Thus, the application of the proposed method allows to determine quantitatively the content of thymol and carvacrol in medicinal plant raw materials, extracts and tinctures of vegetable raw materials.

The method of quantitative determination of thymol and carvacrol in their joint presence in medicinal plant raw materials, which is preliminarily extracted, extracts and tinctures of plant material, including high-performance liquid chromatography, characterized in that using a column Packed with silica gel with grafted straight alkyl groups, the number of atoms that is equal to sixteen (C16), with a particle size of 7 μm, as the mobile phase, use a solution of methanol - water at a ratio of 62:38 with the velocity of the mobile phase, 1 ml/min, detection at UV detector at a wavelength of 277 nm, with subsequent calculation of the concentration thymol and carvacrol against peak areas analyzed and a standard substance, which is used as the standard solutions of thymol and carvacrol, respectively, given its concentration is then, the volume of the extract samples of raw materials or the dilution of the investigational product.



 

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FIELD: analytical methods.

SUBSTANCE: to determine methyl alcohol in water, sample to be assayed is preliminarily subjected to distillation with sulfuric acid added in amount required to provide its concentration in mixture to be distilled c(1/2 H2SO4) = 0.002 M, while strippings constitute 6-7% of the volume of sample. Stripped liquid is thrice rinsed with hexane or Nefras at 1:1 hexane (Nefras)-to-strippings ratio. Rinsed material is then introduced into packed column filled with diatomite modified with 1,2,3-tris(β-cyanoethoxy)propane having deposited fixed phase thereon, which phase is prepared by way of consecutively keeping glycerol each time for 4 h at ambient temperature, 100°C, 130°C, 160°C, and 200°C, and then for 8 h at 230°C and for 40 h at 200°C under nitrogen bubbling conditions. Calculation of methanol content is performed taking into consideration calibrating coefficient.

EFFECT: enabled determination of small concentrations of methyl alcohol in water with sufficient selectivity and reliability.

2 cl, 2 tbl, 6 ex

FIELD: chemical engineering; medical engineering.

SUBSTANCE: method involves plotting two chromatograms one of which is based on radioactivity (No 1) and the other one on ultraviolet absorption (No 2) or on radioactivity (No 1) and on fluorescence (No 2) and chromatogram specific relative to ultraviolet absorption (No 3) or relative to fluorescence (No 3). Material quality is estimated to be the more high the more close studied labeled compound peak shape is to trapezoid shape on the third chromatogram.

EFFECT: high accuracy of the method.

8 dwg

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