Quality detection method of compound phyllanthus emblica ginseng schisandra preparation

Abstract

The application discloses a quality detection method for a compound phyllium panax schisandra preparation, and belongs to the technical field of drug detection and analysis. The traditional Chinese medicine is composed of phyllium, panax and schisandra. Qualitative analysis is conducted on the phyllium, panax and schisandra in the compound by using a thin layer chromatography method; and the contents of gallic acid, ginsenoside Rg1 and schisandrin A in the compound are determined by using a high performance liquid chromatography method. The thin layer chromatography method is one of classical traditional Chinese medicine analysis methods, and has the advantages of rapidness, convenience and the like; the high performance liquid chromatography method is also one of classical traditional Chinese medicine analysis methods, and has the advantages of mature technology, good stability, high universality and the like, can be used for widely and accurately detecting chemical components in a complex traditional Chinese medicine compound, the content determination method passes the method validation, has good linear relationship, and the precision, stability, repeatability and recovery rate meet the regulations, and the combination of qualitative analysis and quantitative analysis lays a foundation for establishing the quality standard of the compound preparation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of drug detection and analysis, and particularly relates to a quality detection method of a compound preparation. BACKGROUND

[0002] The compound preparation is based on a classic composition for protecting liver in a classic composition for protecting liver recorded in a classic book of Tibetan medicine, Four-Part Medical Classics, and is improved according to the three-cause theory of Tibetan medicine through years of clinical research. Emblica, ginseng and schisandra are scientifically matched, and the modern biological extraction technology and ultra-nano purification process are used to condense the composition of the medicinal materials to the greatest extent. The compound preparation can quickly form a protective film in the stomach, share or relieve the direct damage of alcohol metabolic toxins to liver cells, improve the detoxification function of the liver, play a role in protecting the liver, and effectively inhibit the deposition of fat cells in the liver, reduce the formation of fatty liver, and have the effects of calming the heart, soothing the nerves, detoxifying, improving immunity, etc.

[0003] In order to be more in line with the modern pace of life, the compound preparation is made into tablets, capsules and other preparations by using modern preparation technology, which is more convenient for taking and carrying.

[0004] The main effective components of the compound preparation are derived from three kinds of medicinal materials, namely, emblica, ginseng and schisandra. Therefore, the quality standard of the compound preparation is established by researching the compound preparation from the aspects of qualitative analysis and quantitative analysis of the above three kinds of medicinal materials, which lays a foundation for ensuring the quality of the preparation. SUMMARY

[0005] The application aims to extract a quality detection method of a compound preparation.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows:

[0007] A quality detection method of a compound emblica-ginseng-schisandra preparation, which comprises the following steps: firstly, using thin layer chromatography to qualitatively analyze emblica, ginseng and schisandra in the compound preparation; and then using high performance liquid chromatography to determine the contents of gallic acid, ginsenoside Rg1 and schisandrin in the compound preparation.

[0008] The preparation is composed of three kinds of medicinal materials, namely, emblica, ginseng and schisandra.

[0009] The specific detection method is as follows:

[0010] (1) using thin layer chromatography to qualitatively analyze emblica, ginseng and schisandra in the compound preparation

[0011] Take compound Phyllanthus emblica ginseng Schisandra preparation 6g, grind, add methanol 30ml, heat reflux extraction 2h, filter, filter, dry, add water 30ml, make dissolution, filter, filter, extract with ethyl acetate 2 times, 30ml each time, combine ethyl acetate layer, dry, add methanol 1ml, make dissolution, as test solution; Take Phyllanthus emblica control drug 1g, ginseng control drug 2g, Schisandra chinensis control drug 0.5g, add methanol 20ml, heat reflux extraction 2h, filter, filter, dry, add water 20ml, make dissolution, filter, extract with ethyl acetate 2 times, 20ml each time, combine ethyl acetate extract, dry, add methanol 1ml, make dissolution, take 1ul of the above test solution and control drug solution, respectively, point on the same silica gel GF254 thin layer plate, with chloroform-ethyl acetate-formic acid 6:6:1 as developing agent, develop, take out, dry, place under UV light 254nm, view; Then spray with 10% sulfuric acid ethanol solution, hot air blow until the spots are clear. The test sample shows the same color fluorescent spots or spots as the Phyllanthus emblica control drug, ginseng control drug and Schisandra chinensis control drug in the chromatographic position;

[0012] (2) Quantitative analysis of gallic acid, ginsenoside Rg1 and schisandrin in compound preparation by high performance liquid chromatography

[0013] Chromatographic conditions and system suitability test With octadecylsilane bonded silica gel as filler; With acetonitrile (A)-0.2% phosphoric acid solution (B) as mobile phase gradient elution: 0-10min, 3%-5%A, 10-30min, 5%-35%A, 30-35min, 35%A, 35-40min, 35%-40%A, 40-65min, 40%-75%A; Detection wavelength: 203nm; Column temperature: 25℃; Theoretical plate number should not be less than 3000 calculated by gallic acid peak;

[0014] Preparation of control solution Take gallic acid, ginsenoside Rg1 and schisandrin control solution, mix, prepare mixed control solution containing gallic acid 171.6ug, ginsenoside Rg1 232.3ug, schisandrin 178.5ug per 1ml;

[0015] Preparation of test solution Take compound Phyllanthus emblica ginseng Schisandra preparation, grind, take 2g, accurately weigh, put in a conical flask with plug, accurately add methanol 20ml, tightly plug, weigh, ultrasonic treat with power 500w, frequency 40khz for 30min, cool, weigh again, make up the weight loss with methanol, shake, filter, as test solution, prepare negative sample solution by the same method;

[0016] The determination method is that 10 muL of the control sample solution, the test sample solution and the negative sample solution are respectively precisely sucked, injected into the liquid chromatograph, and determined, and then the determination is obtained.

[0017] The qualitative analysis method is a thin layer identification method, using the same development condition and color development condition, and using the control medicinal material as a reference object to identify each medicinal material in the compound.

[0018] The quantitative method is a high performance liquid chromatography method, and the mobile phase gradient elution is as follows: 0-10 min, 3%-5% A, 10-30 min, 5%-35% A, 30-35 min, 35% A, 35-40 min, 35%-40% A, 40-65 min, 40%-75% A; the ultraviolet detector is used to quantitatively analyze the index components, i.e., gallic acid, ginsenoside Rg1 and schisandrin in the three medicinal materials in the compound.

[0019] The contents of gallic acid, ginsenoside Rg1 and schisandrin in 1 g of the phyllanthus emblica ginseng schisandra preparation are 4.87 mg, 12.84 mg and 0.76 mg respectively.

[0020] The TLC method adopted in the application has simple sample pretreatment, and only needs to be ultrasonically extracted by methanol, and the same development system can be used to identify phyllanthus emblica, ginseng and schisandra, compared with the conventional TLC identification in the pharmacopoeia, the method has the advantages of high efficiency and rapidness; the HPLC method can use the same chromatographic condition, and the content determination of the index components in the compound can be realized by using the ultraviolet detector at the same wavelength.

[0021] The conventional TLC method and the HPLC method are adopted in the application, so that the control of the effective components in the compound preparation is realized, and the detection technology has universality and is suitable for popularization and application.

[0022] The principle of the application is as follows:

[0023] 1. According to the different polarities of the chemical components of traditional Chinese medicines, the chemical components are adsorbed with silica gel in different regions of the silica gel plate with the movement of the developing agent, and finally a series of spots are presented. Under different viewing conditions, the spots present different colors, are overlapped with each other, but do not interfere with each other, a plurality of medicinal materials can be identified on the same thin layer plate at the same time, the same sample solution is used, and the method is suitable for multiple identification applications.

[0024] 2. By adjusting the components and proportion of the mobile phase, each chemical component can present a well-separated chromatographic peak without interference under the same mobile phase and the same detection wavelength, and the suitable chromatographic peak area and the content have a good linear relationship, so that quantitative determination can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1TLC chromatogram of Phyllanthus emblica, Panax ginseng and Schisandra chinensis in the compound preparation under 254 nm; wherein, 1-Phyllanthus emblica reference material; 2-sample; 3-Phyllanthus emblica negative control; 4-Panax ginseng reference material; 5-sample; 6-Panax ginseng negative control; 7-Schisandra chinensis reference material; 8-sample; 9-Schisandra chinensis negative control;

[0026] Figure 2 TLC chromatogram of Phyllanthus emblica, Panax ginseng and Schisandra chinensis in the compound preparation under sunlight; wherein, 1-Phyllanthus emblica reference material; 2-sample; 3-Phyllanthus emblica negative control; 4-Panax ginseng reference material; 5-sample; 6-Panax ginseng negative control; 7-Schisandra chinensis reference material; 8-sample; 9-Schisandra chinensis negative control;

[0027] Figure 3 Gallic acid sample amount vs. peak area, wherein, the horizontal coordinate is sample amount μg, and the vertical coordinate is peak area;

[0028] Figure 4 Panax ginseng saponin Rg1 sample amount vs. peak area, wherein, the horizontal coordinate is sample amount μg, and the vertical coordinate is peak area;

[0029] Figure 5 Schisandra chinensis alcohol A sample amount vs. peak area, wherein, the horizontal coordinate is sample amount μg, and the vertical coordinate is peak area;

[0030] Figure 6 Gallic acid reference substance chromatogram;

[0031] Figure 7 Compound preparation chromatogram;

[0032] Figure 8 Blank sample chromatogram without Phyllanthus emblica;

[0033] Figure 9 Panax ginseng saponin Rg1 reference substance chromatogram;

[0034] Figure 10 Compound preparation chromatogram;

[0035] Figure 11 Blank sample chromatogram without Panax ginseng;

[0036] Figure 12 Schisandra chinensis alcohol A reference substance chromatogram;

[0037] Figure 13 Compound preparation chromatogram;

[0038] Figure 14 Blank sample chromatogram without Schisandra chinensis.

[0039] Specific embodiment mode

[0040] The application will be further described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are only used to illustrate the application and not to limit the scope of the application.

[0041] A quality detection method of the compound phyllanthus emblica ginseng schisandra preparation, comprising the following steps:

[0042] (1) Rapid qualitative identification method

[0043] Take 6g of the compound phyllanthus emblica ginseng schisandra preparation, grind finely, add 30mL of methanol, heat reflux extraction for 2h, filter, evaporate the filtrate to dryness, add 30mL of water to the residue to dissolve, filter, extract the filtrate with ethyl acetate twice, 30mL each time, combine the ethyl acetate layers, evaporate to dryness, add 1mL of methanol to the residue to dissolve, as the test solution; take 1g of phyllanthus emblica control drug, 2g of ginseng control drug, and 0.5g of schisandra control drug, add 20mL of methanol respectively, heat reflux extraction for 2h, filter, evaporate the filtrate to dryness, add 20mL of water to the residue to dissolve and filter, extract the filtrate with ethyl acetate twice by shaking, 20mL each time, combine the ethyl acetate extracts, evaporate to dryness, add 1mL of methanol to the residue to dissolve, take 1μL of the test solution and the control drug solution respectively, and point them on the same silica gel GF254 thin layer plate, use chloroform-ethyl acetate-formic acid (6:6:1) as the developing agent, develop, take out, dry, and observe under ultraviolet light (254nm); then spray with 10% sulfuric acid ethanol solution, and blow with hot air until the spots are clear. The test sample shows the same color fluorescent spots or spots as the phyllanthus emblica control drug, ginseng control drug, and schisandra control drug at the chromatographic position;

[0044] As shown in Figure 1 , which is the TLC diagram of phyllanthus emblica, ginseng, and schisandra, wherein, 1- phyllanthus emblica control drug; 2- sample; 3- phyllanthus emblica negative control; 4- ginseng control drug; 5- sample; 6- ginseng negative control; 7- schisandra control drug; 8- sample; 9- schisandra negative control.

[0045] (2) One measurement multiple evaluation rapid quantitative method

[0046] The chromatographic conditions and system suitability test use octadecylsilane-bonded silica gel as the filler; use acetonitrile (A)-0.2% phosphoric acid solution (B) as the mobile phase for gradient elution: 0-10min, 3%-5% A, 10-30min, 5%-35% A, 30-35min, 35% A, 35-40min, 35%-40% A, 40-65min, 40%-75% A; the detection wavelength is 203nm; the column temperature is 25℃; the theoretical plate number calculated according to the gallic acid peak should not be less than 3000;

[0047] The control solution was prepared by mixing gallic acid, ginsenoside Rg1 and schisandrin C control solution, and then 171.6 μg of gallic acid, 232.3 μg of ginsenoside Rg1 and 178.5 μg of schisandrin C were contained in 1 mL of the mixed control solution.

[0048] The test solution was prepared by taking a certain amount of compound phyllanthus emblica ginseng schisandra preparation, grinding it, taking 2 g, accurately weighing it, placing it in a conical flask with a plug, accurately adding 20 mL of methanol, tightly sealing it, weighing it, ultrasonically treating it at a power of 500 W and a frequency of 40 kHz for 30 minutes, cooling it, re-weighing it, making up the weight loss with methanol, shaking it, and filtering it as the test solution.

[0049] The determination method was as follows: 10 μL of the control solution, the test solution and the negative sample solution were precisely taken and injected into the liquid chromatograph for determination.

[0050] The rapid qualitative method refers to the thin layer identification method, and the rapid quantitative method refers to the high performance liquid chromatography method. The three components can be quantified by using only a UV detector, which has a lower cost than a DAD detector.

[0051] Through the methodological investigation, the injection amount of gallic acid was in the range of 0.5148-5.148 μg, which had a good linear relationship with the peak area, the regression equation was Y=5,230,302X-481794, r=0.9994 (Table 1, Figure 2 ); the injection amount of ginsenoside Rg1 was in the range of 1.358-12.58 μg, which had a good linear relationship with the peak area, the regression equation was Y=386811X-44626, r=0.9995 (Table 2, Figure 3 ); the injection amount of schisandrin C was in the range of 0.5355-5.355 μg, which had a good linear relationship with the peak area, the regression equation was Y=6,358,918X-1,240,922, r=0.9999 (Table 3, Figure 4 ); the results of the addition recovery experiment showed that the average recovery rate of gallic acid was 99.51% in 6 determinations, and the RSD was 1.66% (Table 4); the average recovery rate of ginsenoside Rg1 was 101.85% in 6 determinations, and the RSD was 1.89% (Table 5); the average recovery rate of schisandrin C was 100.89% in 6 determinations, and the RSD was 2.70% (Table 6); the precision (Table 7), the stability (Table 8), the repeatability (Table 9), the specificity (Table 10) and the accuracy (Table 11) were good. Figure 5 6 7、 Figure 8 9 10、 Figure 11 12 ​​​, 13) The data were in accordance with the methodological requirements. It was applicable to the determination of gallic acid, ginsenoside Rg1 and schisandrin in compound preparation by one method for multiple quantitative determination. The detection structure was shown in Table 10, Figure 14 The chromatogram of blank sample without schisandra.

[0052] Table 1 Gallic acid injection amount and peak area

[0053]

[0054] Table 2 Ginsenoside Rg1 injection amount and peak area

[0055]

[0056] Table 3 Schisandrin injection amount and peak area

[0057]

[0058] Table 4 Gallic acid recovery rate test results in samples

[0059]

[0060] Table 5 Ginsenoside Rg1 recovery rate test results in samples

[0061]

[0062] Table 6 Schisandrin recovery rate test results in samples

[0063]

[0064] Table 7 Precision test results

[0065]

[0066] Table 8 Stability test results

[0067]

[0068]

[0069] Table 9 Reproducibility test results

[0070]

[0071] Table 10 Determination results of three components in compound preparation (mg / g)

[0072]

[0073] The above merely describes the preferred embodiments of the present application, and it should be pointed out that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A quality detection method of a compound phyllanthus emblica ginseng schisandra preparation, characterized by: The preparation is composed of three medicinal materials, i.e. Phyllanthus emblica, Panax ginseng and Schisandra chinensis. The specific testing method is as follows: (1) Qualitative analysis of Phyllanthus emblica, Panax ginseng and Schisandra chinensis in the compound preparation by thin layer chromatography Take 6 g of the compound Phyllanthus emblica, Panax ginseng and Schisandra chinensis preparation, grind finely, add 30 mL of methanol, heat reflux extraction for 2 h, filter, evaporate the filtrate, add 30 mL of water to the residue to dissolve, filter, extract the filtrate with ethyl acetate for 2 times, 30 mL each time, combine the ethyl acetate layers, evaporate, add 1 mL of methanol to the residue to dissolve, as the test solution; take 1 g of Phyllanthus emblica control drug, 2 g of Panax ginseng control drug and 0.5 g of Schisandra chinensis control drug, add 20 mL of methanol respectively, heat reflux extraction for 2 h, filter, evaporate the filtrate, add 20 mL of water to the residue to dissolve, filter, extract the filtrate with ethyl acetate for 2 times, 20 mL each time, combine the ethyl acetate extracts, evaporate, add 1 mL of methanol to the residue to dissolve, take 1 μL of the test solution and the control drug solution respectively, point on the same silica gel GF254 thin layer plate, develop with trichloromethane-ethyl acetate-formic acid 6:6:1 as the developing agent, take out, dry, observe under UV light 254 nm; then spray with 10% sulfuric acid ethanol solution, blow with hot air until the spots are clear; the test solution shows the same color fluorescent spots or spots as the Phyllanthus emblica control drug, Panax ginseng control drug and Schisandra chinensis control drug at the chromatographic position; (2) Quantitative analysis of gallic acid, ginsenoside Rg1 and schisandrin C in the compound preparation by high performance liquid chromatography Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filling agent; acetonitrile as the mobile phase A, 0.2% phosphoric acid solution as the mobile phase B, gradient elution: 0~10 min, 3%~5% A, 10~30 min, 5%~35% A, 30~35 min, 35% A, 35~40 min, 35%~40% A, 40~65 min, 40%~75% A; detection wavelength: 203 nm; column temperature: 25 ℃; the theoretical plate number calculated according to the gallic acid peak should not be less than 3000; Preparation of the control solution: take gallic acid, ginsenoside Rg1 and schisandrin C control solutions, mix, prepare the mixed control solution containing 171.6 μg of gallic acid, 232.3 μg of ginsenoside Rg1 and 178.5 μg of schisandrin C per 1 mL; Preparation of the test solution: take the compound Phyllanthus emblica, Panax ginseng and Schisandra chinensis preparation, grind finely, take 2 g, accurately weigh, put in a conical flask with a plug, accurately add 20 mL of methanol, tightly plug, weigh, ultrasonic treat for 30 minutes with the power of 500 W and the frequency of 40 kHz, cool, weigh again, make up the weight loss with methanol, shake, filter, as the test solution, prepare the negative sample solution by the same method; Determination: accurately take 10 μL of the control solution, the test solution and the negative sample solution respectively, inject into the liquid chromatograph, determine, and the result is obtained. ​ 2. The quality detection method according to claim 1, characterized in that, Qualitative analysis method is to use thin layer identification method, with the same development conditions and color conditions, to realize the identification of each medicinal material in the compound.

3. The quality detection method of claim 1, wherein, Quantitative method refers to high performance liquid chromatography method, using ultraviolet detector, to quantitatively analyze the three kinds of medicinal materials in the compound, namely gallic acid, ginsenoside Rg1 and schisandrin.

4. The quality detection method of claim 1, wherein The contents of gallic acid, ginsenoside Rg1 and schisandrin in each 1 g of Phyllanthus emblica ginseng schisandra preparation are 4.87 mg, 12.84 mg and 0.76 mg, respectively.

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