Quality detection method of compound phyllanthus emblica, ginseng and schisandra chinensis preparation

Abstract

The invention discloses a quality detection method of a compound phyllanthus emblica, ginseng and schisandra chinensis preparation, and belongs to a medicine detection and analysis technology. The traditional Chinese medicine is composed of emblic leafflower fruit, ginseng and Chinese magnoliavine fruit. Carrying out qualitative analysis on the emblic leafflower fruit, the ginseng and the Chinese magnoliavine fruit in the compound by using a thin layer chromatography; and determining the contents of gallic acid, ginsenoside Rg1 and schizandrin in the compound by using a high performance liquid chromatography. The thin-layer chromatography is one of classical traditional Chinese medicinal material analysis methods, and has the advantages of rapidness, convenience and the like; the high performance liquid chromatography is also one of classical traditional Chinese medicinal material analysis methods, has the advantages of mature technology, good stability, high universality and the like, can be used for widely and accurately detecting the chemical components in the complex traditional Chinese medicinal compound, passes methodology verification, and is good in linear relation and high in accuracy. The precision, the stability, the repeatability, the recovery rate and the like all meet regulations, and the combination of qualitative analysis and quantitative analysis lays a foundation for the establishment of the quality standard of the compound preparation.

Description

Technical Field

[0001] The invention belongs to drug detection and analysis technology, and specifically relates to a quality detection method for a compound preparation. Background Art

[0002] A compound preparation is based on a classic formula for liver protection included in the Tibetan medical classic "Four Medical Classics". After years of clinical research by Professor Sangji Qunpei, a disciple of Master Cuo Ru Cailang, the old president of the Tibetan Medical College, the original formula was improved according to the Three Factors Theory of Tibetan Medicine, and the emblica, ginseng and schisandra chinensis were scientifically combined. It was condensed through modern bio-extraction technology and ultra-nano purification technology to retain the ingredients of the medicinal materials to the greatest extent. It can quickly form a protective film in the stomach, share or relieve the direct damage of alcohol metabolic toxins to liver cells, improve the liver's detoxification function, and play a role in protecting the liver. It can also effectively inhibit the deposition of fat cells in the liver and reduce the formation of fatty liver. It also has the effects of calming the mind, soothing the nerves, detoxifying and improving immunity.

[0003] In order to better suit the pace of modern life, Professor Sangji Qunpei used modern preparation technology to make the compound into tablets, capsules and other preparations, which are easier to take and carry.

[0004] The main active ingredients of this compound preparation come from three medicinal materials: emblica, ginseng and schisandra. Therefore, the compound preparation was studied from both qualitative and quantitative aspects based on the above three medicinal materials, and its quality standards were established, laying the foundation for ensuring the quality of the preparation. Summary of the invention

[0005] The invention relates to a quality detection method for extracting a compound preparation.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

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

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

[0009] The specific detection methods are as follows:

[0010] (1) Qualitative analysis of Phyllanthus emblica, Ginseng and Schisandra chinensis in compound preparations by TLC

[0011] Take 6g of compound emblica ginseng and schisandra preparation, grind it, add 30mL of methanol, heat and reflux to extract for 2h, filter, evaporate the filtrate to dryness, add 30mL of water to the residue to dissolve it, filter, extract the filtrate with ethyl acetate twice, 30mL each time, combine the ethyl acetate layers, evaporate to dryness, add 1mL of methanol to dissolve the residue as the test solution; take 1g of emblica ginseng control medicinal material, 2g of ginseng control medicinal material, and 0.5g of schisandra control medicinal material, add 20mL of methanol respectively, heat and reflux to extract for 2h, filter, and evaporate the filtrate to dryness , add 20mL of water to the residue to dissolve and filter, shake and extract the filtrate with ethyl acetate twice, 20mL each time, combine the ethyl acetate extracts, evaporate to dryness, add 1mL of methanol to the residue to dissolve, take 1μL of the above test solution and control medicinal material solution, respectively, and spot 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 inspect 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 fluorescent spots or spots of the same color at the chromatographic position as the control medicinal materials of Phyllanthus emblica, ginseng, and Schisandra chinensis;

[0012] (2) Quantitative analysis of gallic acid, ginsenoside Rg1 and schisandrin A in the compound preparation by HPLC

[0013] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; acetonitrile (A)-0.2% phosphoric acid solution (B) was used as mobile phase for 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 was 25°C; the number of theoretical plates calculated based on the gallic acid peak should be no less than 3000;

[0014] Preparation of reference solution: Take appropriate amounts of gallic acid, ginsenoside Rg1, and schisandrin alcohol A reference solution, mix them, and prepare a mixed reference solution containing 171.6 μg of gallic acid, 232.3 μg of ginsenoside Rg1, and 178.5 μg of schisandrin alcohol A per 1 mL;

[0015] Preparation of test solution Take an appropriate amount of compound Phyllanthus emblica, ginseng and Schisandra chinensis preparation, grind it into powder, take 2g, weigh it accurately, put it in a stoppered conical bottle, add 20mL of methanol accurately, stopper it tightly, weigh it, treat it with ultrasound at a power of 500W and a frequency of 40kHz for 30 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and use it as the test solution. Prepare the negative sample solution in the same way;

[0016] The determination method is to accurately aspirate 10 μL of the reference solution, the test solution, and the negative sample solution, inject them into the liquid chromatograph, and determine the result.

[0017] The qualitative analysis method is to adopt the thin layer identification method, use the control medicinal materials as the reference, and use the same development conditions and color development conditions to achieve the identification of each medicinal material in the compound.

[0018] The quantitative method refers to 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; an ultraviolet detector is used to quantitatively analyze the index components in the three medicinal materials in the compound, namely gallic acid, ginsenoside Rg1 and schisandrin A.

[0019] The contents of gallic acid, ginsenoside Rg1 and schisandrin A in each 1g sample of the emblica ginseng and schisandra chinensis preparation were 4.87mg, 12.84mg and 0.76mg respectively.

[0020] The TLC method adopted by the present invention has simple sample pretreatment and only requires methanol ultrasonic extraction. The same development system can be used to identify the emblica, ginseng and schisandra chinensis. Compared with the conventional TLC identification in the pharmacopoeia, the method has the advantages of high efficiency and rapidity. The HPLC method can adopt the same chromatographic conditions and use an ultraviolet detector to achieve the content determination of the index components in the compound at the same wavelength.

[0021] The present invention adopts conventional TLC method and HPLC method to realize the control of the effective ingredients in the compound preparation, and the detection technology has universal applicability and is suitable for popularization and application.

[0022] The principle of the present invention is as follows:

[0023] 1. According to the different polarities of the chemical components of traditional Chinese medicine, as the developing agent moves, the chemical components are adsorbed on the silica gel in different areas of the silica gel plate, and finally present a series of spots. Under different inspection conditions, the spots present different colors, overlap each other, but do not interfere with each other. Several medicinal materials can be identified on the same thin layer plate at the same time, and the same sample solution can be used for multiple identification applications.

[0024] 2. By adjusting the composition and proportion of the mobile phase, each chemical component can present well-separated chromatographic peaks without interference in the same mobile phase and at the same detection wavelength, and the appropriate chromatographic peak area and content show a good linear relationship, so that quantitative determination can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0026] Figure 2 The TLC chart of Phyllanthus emblica, Ginseng and Schisandra chinensis in the compound preparation under sunlight; 1-Phyllanthus emblica control medicinal material; 2-sample; 3-Phyllanthus emblica negative control; 4-Ginseng control medicinal material; 5-sample; 6-Ginseng negative control; 7-Schisandra chinensis control medicinal material; 8-sample; 9-Schisandra chinensis negative control;

[0027] Figure 3 is the injection amount and peak area of ​​gallic acid, where the horizontal axis is the injection amount μg and the vertical axis is the peak area;

[0028] Figure 4 The injection amount and peak area of ​​ginsenoside Rg1, where the horizontal axis is the injection amount μg and the vertical axis is the peak area;

[0029] Figure 5 is the injection amount and peak area of ​​Schisandrin A, where the abscissa is the injection amount μg and the ordinate is the peak area;

[0030] Figure 6 This is the chromatogram of gallic acid reference substance;

[0031] Figure 7 This is the chromatogram of the compound preparation;

[0032] Figure 8 This is the chromatogram of the blank sample lacking Phyllanthus emblica;

[0033] Fig. 9 This is the chromatogram of ginsenoside Rg1 reference substance;

[0034] Fig.10 This is the chromatogram of the compound preparation;

[0035] Fig.11 This is the chromatogram of the blank sample lacking ginseng;

[0036] Fig.12 This is the chromatogram of Schisandrin A reference substance;

[0037] Fig.13 This is the chromatogram of the compound preparation;

[0038] Fig.14 This is the chromatogram of the blank sample lacking Schisandra chinensis.

[0039] Specific embodiment

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

[0041] A quality detection method for a compound emblica chinensis and schisandra chinensis preparation comprises the following steps:

[0042] (1) Rapid qualitative identification method

[0043] Take 6g of compound emblica ginseng and schisandra preparation, grind it, add 30mL of methanol, heat and reflux to extract for 2h, filter, evaporate the filtrate to dryness, add 30mL of water to dissolve the residue, filter, extract the filtrate with ethyl acetate twice, 30mL each time, combine the ethyl acetate layers, evaporate to dryness, add 1mL of methanol to dissolve the residue as the test solution; take 1g of emblica ginseng control medicinal material, 2g of ginseng control medicinal material, and 0.5g of schisandra control medicinal material, add 20mL of methanol respectively, heat and reflux to extract for 2h, filter, evaporate the filtrate to dryness, and the residue Add 20mL of water to the residue to dissolve and filter. Shake and extract the filtrate with ethyl acetate twice, 20mL each time. Combine the ethyl acetate extracts and evaporate to dryness. Add 1mL of methanol to the residue to dissolve. Take 1μL of the test solution and the control medicinal material solution, respectively, and spot 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 inspect 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 at the chromatographic position as the control medicinal materials of Phyllanthus emblica, ginseng, and Schisandra chinensis;

[0044] like Figure 1 Shown are TLC graphs of Phyllanthus emblica, ginseng and Schisandra chinensis, where 1-Phyllanthus emblica control medicinal material; 2-sample; 3-Phyllanthus emblica negative control; 4-Ginseng control medicinal material; 5-sample; 6-Ginseng negative control; 7-Schisandra chinensis control medicinal material; 8-sample; 9-Schisandra chinensis negative control.

[0045] (2) Rapid quantitative method of one test and multiple evaluations

[0046] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; acetonitrile (A)-0.2% phosphoric acid solution (B) was used as mobile phase for 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 was 25°C; the number of theoretical plates calculated based on the gallic acid peak should be no less than 3000;

[0047] Preparation of reference solution: Take appropriate amounts of gallic acid, ginsenoside Rg1, and schisandrin alcohol A reference solution, mix them, and prepare a mixed reference solution containing 171.6 μg of gallic acid, 232.3 μg of ginsenoside Rg1, and 178.5 μg of schisandrin alcohol A per 1 mL.

[0048] Preparation of test solution Take an appropriate amount of compound Phyllanthus emblica, ginseng and Schisandra chinensis preparation, grind it into powder, take 2g, weigh it accurately, put it in a stoppered conical bottle, add 20mL of methanol accurately, stopper it tightly, weigh it, treat it with ultrasound at a power of 500W and a frequency of 40kHz for 30 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and use it as the test solution. Prepare the negative sample solution in the same way;

[0049] Determination method: Accurately pipette 10 μL of reference solution, test solution, and negative sample solution, inject into liquid chromatograph, and determine;

[0050] The rapid qualitative method refers to a thin layer identification method; the rapid quantitative method refers to a high performance liquid chromatography method, which only requires an ultraviolet detector to quantify the three components, and has a lower cost than a DAD detector.

[0051] Through methodological investigation, the injection amount of gallic acid was 0.5148-5.148 μg, which showed 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 1.358-12.58 μg, which showed a good linear relationship with the peak area, and the regression equation was: Y=386811X-44626, r=0.9995 (Table 2, Figure 3 ); the injection amount of schisandrae alcohol A was 0.5355-5.355 μg, which showed a good linear relationship with the peak area, and the regression equation was: Y = 6,358,918X-1,240,922, r = 0.9999 (Table 3, Figure 4 ); The results of the sample recovery experiment showed that the average recovery rate of gallic acid was 99.51% for 6 determinations, and the RSD was 1.66% (Table 4); the average recovery rate of ginsenoside Rg1 was 101.85% for 6 determinations, and the RSD was 1.89% (Table 5); the average recovery rate of schisandrin A was 100.89% for 6 determinations, and the RSD was 2.70% (Table 6); the precision (Table 7), stability (Table 8), repeatability (Table 9), specificity ( Figure 5 , 6 7. Figure 8 , 9 、10、 Fig.11 , 12, 13) The data all meet the methodological requirements. It is applicable to the quantitative determination requirements of gallic acid, ginsenoside Rg1 and schisandrin A in compound preparations. The detection structure is shown in Table 10. Fig.14 This is the chromatogram of the blank sample lacking Schisandra chinensis.

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

[0053]

[0054] Table 2 Injection amount and peak area of ​​ginsenoside Rg1

[0055]

[0056] Table 3 Schisandrin A injection volume and peak area

[0057]

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

[0059]

[0060] Table 5 Results of the recovery test of ginsenoside Rg1 in samples

[0061]

[0062] Table 6 Results of the recovery test of Schisandrin A in samples

[0063]

[0064] Table 7 Precision test results

[0065]

[0066] Table 8 Stability test results

[0067]

[0068]

[0069] Table 9 Repeatability test results

[0070]

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

[0072]

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A quality detection method for a compound emblica ginseng schisandra chinensis preparation, characterized in that: The preparation is composed of three medicinal materials: Phyllanthus emblica, Ginseng and Schisandra chinensis. The specific detection method is as follows: (1) Qualitative analysis of Phyllanthus emblica, Ginseng and Schisandra chinensis in compound preparations by TLC Take 6g of compound emblica ginseng and schisandra preparation, grind it, add 30mL of methanol, heat and reflux to extract for 2h, filter, evaporate the filtrate to dryness, add 30mL of water to the residue to dissolve it, filter, extract the filtrate with ethyl acetate twice, 30mL each time, combine the ethyl acetate layers, evaporate to dryness, add 1mL of methanol to dissolve the residue as the test solution; take 1g of emblica ginseng control medicinal material, 2g of ginseng control medicinal material, and 0.5g of schisandra control medicinal material, add 20mL of methanol respectively, heat and reflux to extract for 2h, filter, and evaporate the filtrate to dryness , add 20mL of water to the residue to dissolve and filter, shake and extract the filtrate with ethyl acetate twice, 20mL each time, combine the ethyl acetate extracts, evaporate to dryness, add 1mL of methanol to the residue to dissolve, take 1μL of the above test solution and control medicinal material solution, respectively, and spot 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 inspect 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 fluorescent spots or spots of the same color at the chromatographic position as the control medicinal materials of Phyllanthus emblica, ginseng, and Schisandra chinensis; (2) Quantitative analysis of gallic acid, ginsenoside Rg1 and schisandrin A in the compound preparation by HPLC Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; acetonitrile (A)-0.2% phosphoric acid solution (B) was used as mobile phase for 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 was 25°C; the number of theoretical plates calculated based on the gallic acid peak should be no less than 3000; Preparation of reference solution: Take appropriate amounts of gallic acid, ginsenoside Rg1, and schisandrin alcohol A reference solution, mix them, and prepare a mixed reference solution containing 171.6 μg of gallic acid, 232.3 μg of ginsenoside Rg1, and 178.5 μg of schisandrin alcohol A per 1 mL; Preparation of test solution Take an appropriate amount of compound Phyllanthus emblica, ginseng and Schisandra chinensis preparation, grind it into powder, take 2g, weigh it accurately, put it in a stoppered conical bottle, add 20mL of methanol accurately, stopper it tightly, weigh it, treat it with ultrasound at a power of 500W and a frequency of 40kHz for 30 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and use it as the test solution. Prepare the negative sample solution in the same way; The determination method is to accurately aspirate 10 μL of the reference solution, the test solution, and the negative sample solution, inject them into the liquid chromatograph, and determine the result.

2. The quality inspection method according to claim 1, characterized in that: The qualitative analysis method is to adopt the thin layer identification method, use the control medicinal materials as the reference, and use the same development conditions and color development conditions to achieve the identification of each medicinal material in the compound.

3. The quality inspection method according to claim 1, characterized in that: The quantitative method refers to 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; an ultraviolet detector is used to quantitatively analyze the index components in the three medicinal materials in the compound, namely gallic acid, ginsenoside Rg1 and schisandrin A.

4. The quality inspection method according to claim 1, characterized in that: The contents of gallic acid, ginsenoside Rg1 and schisandrin A in each 1g sample of Phyllanthus emblica, ginseng and Schisandra chinensis preparation were 4.87mg, 12.84mg and 0.76mg respectively.

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