CN114324724A - Method for identifying authenticity of liver essence paste based on thin-layer chromatography - Google Patents
Method for identifying authenticity of liver essence paste based on thin-layer chromatography Download PDFInfo
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Abstract
The invention relates to a method for identifying the authenticity of a liver extract based on thin-layer chromatography, which comprises the following specific steps: preparing a test solution; preparing a pork liver reference medicinal material solution: drying fresh pork liver at low temperature, pulverizing to obtain pork liver powder, adding 1g pork liver powder, adding 4ml water, adding 1 drop of 2% hydrochloric acid, shaking, keeping the temperature at 90-100 deg.C for 40min, centrifuging, collecting supernatant, concentrating to near dryness, adding 4ml ethanol for dissolving, centrifuging again, and collecting supernatant as pork liver control medicinal material solution; preparing a reference substance solution; and (5) detecting by thin layer chromatography. The invention aims to fill the blank of the liver extract identification method, solve or at least reduce the problems of poor specificity and inaccurate result of the existing liver extract identification method, and provide a method for identifying the authenticity of the liver extract based on thin-layer chromatography.
Description
Technical Field
The invention relates to the technical field of liver extract identification, in particular to a method for identifying the authenticity of liver extract based on thin-layer chromatography.
Background
The liver extract is a liver extract prepared by extracting, concentrating and refining livers of animals such as pigs, cattle, sheep and the like. The liver essence ointment prepared by taking pork liver as a raw material is an important intermediate of a medicinal preparation, namely the liver essence hematotonin oral liquid. Pig liver, recorded in Zhonghua Bencao, has the actions of tonifying liver, improving vision, nourishing spleen and strengthening blood. Can be used for treating liver deficiency, blurred vision, and night blindness. Sallow complexion due to blood deficiency, etc. The liver essence paste is extracted and refined from pig liver and other raw materials, and also has the efficacy. In particular, the liver essence paste made from pork liver is the main component of the medicine such as liver essence hematinic oral liquid. The execution standard is the Chinese patent medicine preparation standard in the ministry of health, wherein the preparation method and the quality standard of the liver essence ointment are described more sparsely, and no proper identification method is available for the chemical characteristics of the liver essence ointment in the standard. The quality standards aiming at chemical properties are not collected in the Chinese pharmacopoeia edition and local standards all the year round, and only physical indexes are available, so that the main properties of the Chinese pharmacopoeia edition and the local standards cannot be reflected.
According to the existing literature, the main components in the pork liver are: protein, fat, vitamin A, vitamin D, and vitamin B1Vitamin B2Vitamin B6Vitamin B12Nicotinamide, vitamin C, folic acid, vitamin E, phosphorus, potassium, magnesium, iron, zinc, calcium, selenium and the like. After extraction, concentration and refining, the components of the liver essence paste mainly comprise the above parts, the content of some components is greatly improved, and some components are reduced or even lost.
The current state of quality standard for load collection:
(1) the ministerial standard Chinese medicine prescription preparation, WS3-B-2530-97, in the thirteenth volume, contains liver extract standard, which is a pre-refining process of pork liver extract, and has only examination items and no identification items;
(2) the Jiangsu province drug standard is the liver extract loading standard in 1997 edition, only has inspection items and no identification items;
(3) the national standard of chemical medicine is upgraded to liver extract standard WS-10001- (HD-0812) -2002 in volume 9 of the national standard, and the identification item is chemical identification which is the color reaction of amino acid to ninhydrin and has poor specificity.
In summary, the existing standard is that there is no differentiation item or simple chemical differentiation with poor specificity for liver extract of liver paste animals. The standard can not reflect the due components in the pork liver and can not reflect the quality characteristics of the liver essence paste. Therefore, the existing method has weak specificity and inaccurate result.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, fill the blank of the liver extract identification method, solve or at least alleviate the problems of poor specificity and inaccurate result of the existing liver extract identification method, and provide a method for identifying the authenticity of the liver extract based on thin-layer chromatography.
The invention is realized by the following technical scheme:
a method for identifying the authenticity of liver essence paste based on thin-layer chromatography comprises the following specific steps:
preparation of a test solution: taking 1g of a to-be-detected product, adding 20ml of ethanol, carrying out ultrasonic treatment for 30min, and then filtering, wherein the filtered filtrate is used as a test solution;
preparing a pork liver reference medicinal material solution: drying fresh pork liver at low temperature, pulverizing to obtain pork liver powder, adding 1g pork liver powder, adding 4ml water, adding 1 drop of 2% hydrochloric acid, shaking, keeping the temperature at 90-100 deg.C for 40min, centrifuging, collecting supernatant, concentrating to near dryness, adding 4ml ethanol for dissolving, centrifuging again, and collecting supernatant as pork liver control medicinal material solution;
preparation of control solutions: respectively taking valine, alanine, glycine and citrulline reference substances, and adding methanol to obtain solutions each containing 0.5mg per 1ml as reference substance solutions;
and (3) detection by thin-layer chromatography: sucking 2-4 mul of a test solution, 2-4 mul of a pork liver control medicinal material solution and 2 mul of a control solution respectively, respectively dropping the solutions on the same silica gel G thin layer plate, developing by using n-butyl alcohol-glacial acetic acid-water as a developing agent, taking out the silica gel G thin layer plate, drying in the air, uniformly spraying a layer of ninhydrin solution on the silica gel G thin layer plate, and heating the silica gel G thin layer plate in a drying oven at the constant temperature of 105 ℃ until spots are clearly developed; observing the color and the quantity of main spots at the positions where the spots appear in the chromatogram of the test solution, wherein the spots with the same color and quantity appear at the corresponding positions in the chromatogram of the pork liver reference medicinal material, and four red spots also appear at the corresponding positions in the chromatogram of the reference solution, wherein the positions of the four red spots are valine, alanine, glycine and citrulline from top to bottom in sequence, and when the spots with the same color and quantity appear at the corresponding positions in the chromatogram of the test solution, the to-be-tested sample can be determined to be the liver extract. When the spots are weak and difficult to distinguish, the color can be deepened after the product is placed for 1 day and observed.
In order to further implement the present invention, the following technical solutions may be preferably selected:
preferably, the n-butanol-glacial acetic acid-water is n-butanol, and the glacial acetic acid and the water are prepared according to the volume ratio of 6-8:2-3: 1.
Preferably, the ninhydrin solution is prepared by diluting 1-2g of ninhydrin with ethanol to 100 ml.
Preferably, the drying temperature of the fresh pork liver is not higher than 60 ℃, and the water content after drying is not more than 5%.
Through the technical scheme, the invention has the beneficial effects that:
the method has strong specificity, can detect the specific components in the liver essence paste, has good reproducibility, short detection time, high speed, intuition, simple and convenient steps, simple instrument and low detection cost. The main components of the pork liver after extraction, concentration and refining are amino acid or polypeptides, vitamins and minerals. The thin-layer map of amino acid components has specificity, and the combination of color depth, number and position of each spot is unique and different from other substances. The liver essence cream contains at least four amino acid components of valine, alanine, glycine, citrulline and the like, has a certain proportion relation, and can be identified by taking the four amino acid components as the characteristics. The pork liver powder prepared from the fresh pork liver is used as a reference medicinal material to replace a fresh pork liver reference medicinal material as a standard substance, the map of the pork liver powder is consistent with that of the fresh pork liver, the pork liver powder can be stored for a long time, the use is convenient, and the feasibility of the method is greatly improved.
Drawings
FIG. 1 is a chromatogram of a liver extract sample of the present invention and a citrulline and glycine control solution;
FIG. 2 is a chromatogram of a sample of liver extract of the present invention and a control solution of alanine and valine;
FIG. 3 is a chromatogram of a liver extract sample and a pork liver control solution of the present invention;
FIG. 4 is a comparison chromatogram of a pork liver control solution prepared from a pork liver paste sample and fresh pork liver and a pork liver powder sample of the present invention;
FIG. 5 is a chromatogram of a sample of liver essence cream of the present invention and a reference solution of citrulline, glycine, alanine, and valine;
FIG. 6 is one of chromatograms of the porcine liver control drug solution and valine, alanine, glycine and citrulline control solution of the present invention;
FIG. 7 is a second chromatogram of the control solution of pork liver of the present invention and the control solution of valine, alanine, glycine and citrulline;
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
a method for identifying the authenticity of liver essence paste based on thin-layer chromatography comprises the following specific steps:
preparation of a test solution: taking 1g of a sample to be detected, adding 20ml of ethanol, carrying out ultrasonic treatment for 30min, and then filtering, wherein the filtered filtrate is used as a sample solution;
preparing a pork liver reference medicinal material solution: drying fresh pork liver at low temperature, pulverizing to obtain pork liver powder, adding 1g pork liver powder, adding 4ml water, adding 1 drop of 2% hydrochloric acid, shaking, keeping the temperature at 100 deg.C for 40min, centrifuging, collecting supernatant, concentrating to near dryness, adding 4ml ethanol to dissolve, centrifuging again, and collecting supernatant as pork liver control medicinal material solution;
preparation of control solutions: respectively taking valine, alanine, glycine and citrulline reference substances, and adding methanol to obtain solutions each containing 0.5mg per 1ml as reference substance solutions;
and (3) detection by thin-layer chromatography: sucking 2-4 mul of a test solution, 2-4 mul of a pork liver control medicinal material solution and 2 mul of a control solution respectively, respectively dropping the solutions on the same silica gel G thin layer plate, developing by using n-butyl alcohol-glacial acetic acid-water as a developing agent, taking out the silica gel G thin layer plate, drying in the air, uniformly spraying a layer of ninhydrin solution on the silica gel G thin layer plate, and heating the silica gel G thin layer plate in a drying oven at the constant temperature of 105 ℃ until spots are clearly developed; observing the color and the quantity of main spots at the positions where the spots appear in the chromatogram of the test solution, wherein the spots with the same color and quantity appear at the corresponding positions in the chromatogram of the pork liver reference medicinal material, and four red spots also appear at the corresponding positions in the chromatogram of the reference solution, wherein the positions of the four red spots are valine, alanine, glycine and citrulline from top to bottom in sequence, and when the spots with the same color and quantity appear at the corresponding positions in the chromatogram of the test solution, the to-be-tested sample can be determined to be the liver extract. When the spots are weak and difficult to distinguish, the color can be deepened after the product is placed for 1 day and observed.
In the embodiment, the n-butanol-glacial acetic acid-water is n-butanol, glacial acetic acid and water are prepared according to the volume ratio of 8:3:1, and the ninhydrin test solution is prepared by diluting 2g of ninhydrin with ethanol to 100 ml.
In order to ensure the identification accuracy, the drying temperature of the fresh pork liver is not higher than 60 ℃, and the moisture content after drying is not more than 5%.
Example 2:
a method for identifying the authenticity of liver essence paste based on thin-layer chromatography comprises the following specific steps:
preparation of a test solution: taking 1g of a sample to be detected, adding 20ml of ethanol, carrying out ultrasonic treatment for 30min, and then filtering, wherein the filtered filtrate is used as a sample solution;
preparing a pork liver reference medicinal material solution: mashing fresh pork liver, weighing 2-3g, adding 4ml of water, adding 1 drop of 2% hydrochloric acid, shaking, keeping the temperature at 90 ℃ for 40min, centrifuging, collecting supernatant, concentrating to near dryness, adding 4ml of ethanol for dissolving, centrifuging again, and collecting supernatant as pork liver control solution;
preparation of control solutions: respectively taking valine, alanine, glycine and citrulline reference substances, and adding methanol to obtain solutions each containing 0.5mg per 1ml as reference substance solutions;
and (3) detection by thin-layer chromatography: sucking 2-4 mul of a test solution, 2-4 mul of a pork liver control medicinal material solution and 2 mul of a control solution respectively, respectively dropping the solutions on the same silica gel G thin layer plate, developing by using n-butyl alcohol-glacial acetic acid-water as a developing agent, taking out the silica gel G thin layer plate, drying in the air, uniformly spraying a layer of ninhydrin solution on the silica gel G thin layer plate, and heating the silica gel G thin layer plate in a drying oven at the constant temperature of 105 ℃ until spots are clearly developed; observing the color and the quantity of main spots at the positions where the spots appear in the chromatogram of the test solution, wherein the spots with the same color and quantity appear at the corresponding positions in the chromatogram of the pork liver reference medicinal material, and four red spots also appear at the corresponding positions in the chromatogram of the reference solution, wherein the positions of the four red spots are valine, alanine, glycine and citrulline from top to bottom in sequence, and when the spots with the same color and quantity appear at the corresponding positions in the chromatogram of the test solution, the to-be-tested sample can be determined to be the liver extract. When the spots are weak and difficult to distinguish, the color can be deepened after the product is placed for 1 day and observed.
In the embodiment, the n-butanol-glacial acetic acid-water is n-butanol, glacial acetic acid and water are prepared according to the volume ratio of 6:2:1, and the preparation method of the ninhydrin test solution is to dilute 2g of ninhydrin to 100 ml.
As shown in fig. 1, the liver extract sample has spots with the same color as citrulline and glycine, which indicates that the liver extract can contain the above components, and the liver extract can be identified.
As shown in fig. 2, the same color spots of the liver extract sample as alanine and valine showed that the liver extract can contain the above components, and the liver extract can be identified. The corresponding spots of arginine are not obvious and the positions are too low, which is not used as a judgment basis.
As shown in fig. 3 and 4, the liver essence cream sample and the pork liver control solution have the same color spots, which indicates that the liver essence cream can contain the above components, and the liver essence cream can be identified by the same; the deployment conditions of fig. 3 and 4 are slightly different. The fresh pork liver has the same atlas with pork liver powder, which shows that the pork liver powder can replace the fresh pork liver as a reference medicine. The pork liver powder prepared from the fresh pork liver is used as a reference medicinal material to replace a fresh pork liver reference medicinal material to be used as a standard substance, can be stored for a long time, is convenient to use, and greatly improves the feasibility and the practicability of the method.
As shown in fig. 5, the same color spots of the liver extract samples as valine, alanine, glycine and citrulline indicate that the liver extract can contain the above components, and the liver extract can be identified.
As shown in fig. 6 and 7, the pork liver control solution and the valine, alanine, glycine and citrulline control solution have the same color spots, and no other spots are generated in the mixed sample, which indicates that the above components can be contained in the liver extract, and the liver extract can be identified; FIG. 7 is a graph of FIG. 6 after 1 day of storage, and it was found that the spots were more visible, the glycine spots were not visible in FIG. 6 but were barely visible, and the glycine spots were more visible in FIG. 7.
The method has strong specificity, can detect the specific components in the liver essence paste, has good reproducibility, short detection time, high speed, intuition, simple and convenient steps, simple instrument and low detection cost. The main components of the pork liver after extraction, concentration and refining are amino acid or polypeptides, vitamins and minerals. The thin-layer map of amino acid components has specificity, and the combination of color depth, number and position of each spot is unique and different from other substances. The liver essence cream contains at least four amino acid components of valine, alanine, glycine, citrulline and the like, has a certain proportion relation, and can be identified by taking the four amino acid components as the characteristics. The pork liver powder prepared from the fresh pork liver is used as a reference medicinal material to replace a fresh pork liver reference medicinal material as a standard substance, the map of the pork liver powder is consistent with that of the fresh pork liver, the pork liver powder can be stored for a long time, the use is convenient, and the feasibility of the method is greatly improved.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.
Claims (4)
1. A method for identifying the authenticity of liver essence paste based on thin-layer chromatography is characterized by comprising the following specific steps:
preparation of a test solution: taking 1g of a sample to be detected, adding 20ml of ethanol, carrying out ultrasonic treatment for 30min, and then filtering, wherein the filtered filtrate is used as a sample solution;
preparing a pork liver reference medicinal material solution: drying fresh pork liver at low temperature, pulverizing to obtain pork liver powder, adding 1g pork liver powder, adding 4ml water, adding 1 drop of 2% hydrochloric acid, shaking, keeping the temperature at 90-100 deg.C for 40min, centrifuging, collecting supernatant, concentrating to near dryness, adding 4ml ethanol for dissolving, centrifuging again, and collecting supernatant as pork liver control medicinal material solution;
preparation of control solutions: respectively taking valine, alanine, glycine and citrulline reference substances, and adding methanol to obtain solutions each containing 0.5mg per 1ml as reference substance solutions;
and (3) detection by thin-layer chromatography: sucking 2-4 mul of a test solution, 2-4 mul of a pork liver control medicinal material solution and 2 mul of a control solution respectively, respectively dropping the solutions on the same silica gel G thin layer plate, developing by using n-butyl alcohol-glacial acetic acid-water as a developing agent, taking out the silica gel G thin layer plate, drying in the air, uniformly spraying a layer of ninhydrin solution on the silica gel G thin layer plate, and heating the silica gel G thin layer plate in a drying oven at the constant temperature of 105 ℃ until spots are clearly developed; observing the color and the quantity of main spots at the positions where the spots appear in the chromatogram of the test solution, wherein the spots with the same color and quantity appear at the corresponding positions in the chromatogram of the pork liver reference medicinal material, and four red spots also appear at the corresponding positions in the chromatogram of the reference solution, wherein the positions of the four red spots are valine, alanine, glycine and citrulline from top to bottom in sequence, and when the spots with the same color and quantity appear at the corresponding positions in the chromatogram of the test solution, the to-be-tested sample can be determined to be the liver extract. When the spots are weak and difficult to distinguish, the color can be deepened after the product is placed for 1 day and observed.
2. The method for identifying the authenticity of the liver extract based on the thin-layer chromatography as claimed in claim 1, wherein the n-butanol-glacial acetic acid-water is n-butanol, and the glacial acetic acid and the water are prepared according to a volume ratio of 6-8:2-3: 1.
3. The method for identifying the authenticity of the liver extract based on the thin layer chromatography as claimed in claim 1, wherein the ninhydrin solution is prepared by diluting 1-2g of ninhydrin with ethanol to 100 ml.
4. The method for identifying the authenticity of the liver extract based on the thin layer chromatography as claimed in claim 1, wherein the drying temperature of the fresh pork liver is not higher than 60 ℃, and the moisture content after drying is not more than 5%.
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