Thin-layer chromatography detection method for fructus aurantii in slow kidney rehabilitation granules

Abstract

The invention belongs to the field of traditional Chinese medicinal material preparation testing, and particularly relates to a thin-layer chromatography detection method for fructus aurantii in slow kidney-rehabilitation granules, and the slow kidney-rehabilitation granules are raw medicinal materials containing salvia miltiorrhiza, rheum officinale, codonopsis pilosula, fructus aurantii, steamed polygonatum kingianum, rhizoma smilacis glabrae, oyster and liquorice and extracts of the raw medicinal materials. The method comprises the following steps: respectively carrying out alcohol extraction, water dissolution, hydrophobic solvent extraction and concentration and alcohol dissolution treatment on a sample of the Shenshenkang granules, the fructus aurantii or the Shenshenkang granules without the fructus aurantii to respectively obtain a solution to be detected, a standard solution and a negative control solution; performing thin-layer chromatography treatment on the solution to be detected, the standard solution and the negative control solution, and then performing color development and ultraviolet detection; wherein a developing solvent in the thin-layer chromatography stage is prepared from trichloromethane, methanol and water in a volume ratio of (10-15): (6-8): 1; the temperature in the expansion stage is 4 DEG C or above, and the humidity is 40% or above. The method provided by the invention can adapt to the characteristics of the slow-kidney-rehabilitation granules, can effectively resist interference of other medicinal materials in the slow-kidney-rehabilitation granules on the fructus aurantii, and can obtain an excellent test effect.

Description

Technical Field

[0001] The invention belongs to the field of analysis of traditional Chinese medicine preparations, and in particular to the field of detection of components in traditional Chinese medicine preparations. Background Art

[0002] Chronic renal failure, also known as uremia, is caused by various causes of kidney damage and progressive deterioration. When it develops to the terminal stage, when renal function is close to 10% to 15% of normal, a series of clinical symptoms appear. With the increasing incidence of chronic kidney disease, chronic renal failure has become one of the major diseases threatening human health and a global public health issue.

[0003] TCM believes that chronic renal failure is the result of prolonged edema, stranguria, thirst, hematuria, dizziness and other diseases. After the symptoms subside, the patient will show symptoms of long-term weakness. In the later stage, vomiting is often accompanied by urinary retention. Combined with the serious prognosis of this disease, it should be classified as "guange". Its pathogenesis is very complicated, but the deficiency of the body and the excess of evil are the basic factors throughout the course of the disease. In terms of its evolution, the early stage is mainly deficiency, although there is evil but it is not very serious; in the later stage, it is mostly mixed with deficiency and excess, the spleen and kidney are more deficient, and the excess evil is more prominent. In the whole treatment process, strengthening the spleen and kidney, removing dampness and turbidity, promoting blood circulation and removing blood stasis, and combining tonification and purgation are the common methods. At the same time, attention should be paid to strengthening the body without leaving the evil, and removing the evil without hurting the body. The understanding of traditional Chinese medicine on chronic renal failure has been relatively mature and perfect, and Chinese medicine has great advantages in treating chronic renal failure. Therefore, the use of traditional Chinese medicine to prevent and delay the development of chronic renal failure has attracted more and more attention.

[0004] Manshenkang granules are preparations developed by the Second Xiangya Hospital of Central South University, which contain salvia miltiorrhiza, rhubarb, codonopsis, fructus aurantii, steamed polygonatum, smilax glabra, oyster and liquorice. The medicine has the effects of strengthening the spleen and kidney, clearing the bowels and relieving turbidity, removing blood stasis and detoxifying, and is mainly used for chronic renal insufficiency and chronic kidney disease, and the syndrome belongs to spleen and kidney deficiency and dampness and toxicity internal obstruction syndrome. However, the prior art does not have a rapid detection method for fructus aurantii in Manshenkang granules. Although, the prior art has some detection methods for fructus aurantii in other Chinese medicine preparations, such as the Chinese patent literature with publication number CN102091168A reports the thin layer detection method of fructus aurantii in Xuefu Zhuyu capsule. The Chinese patent literature with publication number CN102048941A discloses the detection method of fructus aurantii in Xiaoer Jindan. The Chinese patent literature with publication number CN100363048A discloses the detection method of fructus aurantii in Chinese medicine Huazhi tablets. The above technologies all involve the determination of Fructus Aurantii Immaturus in different preparations. However, different Chinese medicine preparations have different compositions of Chinese medicinal materials, different active ingredients, different components that require thin layer separation, and different separation selectivity issues between components. It is impossible to simply transfer the inspection methods of Fructus Aurantii Immaturus in different Chinese medicine preparations, nor is it possible to clearly predict the effect of technology transfer. Summary of the invention

[0005] In view of the blank in the inspection of Citrus aurantium in Manshenkang Granules, the present invention provides a thin layer chromatography detection method of Citrus aurantium in Manshenkang Granules, aiming to solve the problem of interference of other medicinal ingredients in Manshenkang Granules on the separation of Citrus aurantium, thereby improving the thin layer chromatography detection effect of Citrus aurantium in Manshenkang Granules.

[0006] Manshenkang granules contain salvia miltiorrhiza, rhubarb, codonopsis pilosula, fructus aurantii, steamed polygonatum, smilax glabra, oyster and liquorice. These ingredients and their active ingredients will interfere with the separation of the effective ingredients in fructus aurantii. In view of this problem, the present invention has provided the following solutions after in-depth research:

[0007] A thin layer chromatography detection method for Citrus aurantium in Manshenkang granules, wherein the Manshenkang granules are raw medicinal materials and extracts thereof including Salvia miltiorrhiza, Rhubarb, Codonopsis pilosula, Citrus aurantium, steamed Polygonatum sibiricum, Smilax glabra, oyster and licorice; samples of Manshenkang granules, Citrus aurantium or Manshenkang granules lacking Citrus aurantium are respectively subjected to alcohol extraction, water dissolution, hydrophobic solvent extraction and concentration, and alcohol dissolution treatment to obtain a test solution, a standard solution and a negative control solution, respectively;

[0008] The test solution, standard solution and negative control solution are subjected to thin layer chromatography, followed by color development and ultraviolet detection; wherein the developing agent in the thin layer chromatography stage is chloroform, methanol and water in a volume ratio of 10-15:6-8:1; the temperature in the developing stage is above 4°C and the humidity is above 40%.

[0009] In response to the problem of interference of other medicinal materials in Manshenkang Granules on the thin layer of Fructus Aurantii Immaturus, the present invention innovatively solves the problem of interference of other medicinal materials in Manshenkang Granules on the thin layer of Fructus Aurantii Immaturus by combining the developing agent, developing temperature and humidity, and can efficiently separate the Fructus Aurantii Immaturus components in Manshenkang Granules, thereby improving the detection effect.

[0010] In the present invention, the weight ratios of salvia miltiorrhiza, rhubarb, codonopsis pilosula, fructus aurantii, steamed polygonatum, smilax glabra, oyster and liquorice in the Manshenkang granules are 300-400:200-300:150-250:150-250:150-250:200-350:50-150:150-250 respectively; further can be 360-380:270-280:180-190:180-190:180-190:270-280:80-100:180-190.

[0011] The Manshenkang granules contain the water extract of the original medicinal material. Furthermore, the Manshenkang granules also contain auxiliary materials.

[0012] In the present invention, the raw medicinal material can be subjected to water extraction treatment and then dried to obtain the raw medicinal material water extract, which is then mixed with auxiliary materials to obtain the Manshenkang granules.

[0013] In the present invention, the amount of water used for water extraction and the number of times can be adjusted as needed. For example, the water extraction can be performed twice, and the amount of water used for a single extraction can be 5 to 10 times the weight of the medicinal material. After the water extraction, the water can be concentrated and dried as needed.

[0014] In the present invention, the auxiliary material may be a conventional component capable of preparing granules, for example, at least one of dextrin, starch, sucrose, microcrystalline cellulose, and calcium sulfate;

[0015] In the present invention, there is no special requirement for the content of the auxiliary materials in the Manshenkang granules, for example, it can be 25-55%.

[0016] In the present invention, the alcohol selected in the alcohol extraction stage is methanol and / or ethanol.

[0017] In the present invention, the liquid-to-solid ratio in the alcohol extraction stage may be 5 to 15 ml / g.

[0018] The liquid-to-solid ratio in the water extraction stage can be 1 to 10 ml / g.

[0019] In the present invention, the hydrophobic solvent in the hydrophobic solvent extraction stage includes at least one of dichloromethane and ethyl acetate. The O / A ratio in the hydrophobic solvent extraction stage can be 1 to 10:1.

[0020] There is no particular requirement for the amount of solvent used in the alcohol dissolution stage.

[0021] In the present invention, a silica gel plate is used to carry out the thin layer chromatography treatment.

[0022] The research of the present invention shows that, in response to the problem of thin-layer interference of other medicinal ingredients in Manshenkang Granules on Fructus Aurantii Immaturus, the research of the present invention innovatively shows that the combined control of the composition and proportion of the developing agent, and the temperature and humidity in the developing stage can unexpectedly solve the interference of other medicinal ingredients in Manshenkang Granules on Fructus Aurantii Immaturus, and can facilitate high-selective separation testing of the ingredients therein.

[0023] In the present invention, in the developing solvent, the volume ratio of chloroform, methanol and water is 13:7:1.

[0024] In the present invention, the developing agent is a developing agent that is prepared or sealed and stored for 1 to 2 hours at a temperature below 10°C (or further 1 to 10°C) after preparation. The present invention also unexpectedly shows that the use of the developing agent after low-temperature storage, combined with the control of storage time, is more suitable for the characteristics of Manshenkang Granules, and can further solve the interference of other medicinal ingredients in Manshenkang Granules on Fructus Aurantii Immaturus.

[0025] In the present invention, the temperature in the development stage is 20°C or higher, further 30 to 40°C, and further 35 to 40°C.

[0026] In the present invention, the humidity in the development stage is 60% or more, further 90% or more, and further 90 to 100%.

[0027] The present invention unexpectedly discovered that the use of high temperature and high humidity conditions can unexpectedly further adapt the characteristics of Manshenkang Granules, and can further resolve the interference of other medicinal ingredients in Manshenkang Granules on Fructus Aurantii Immaturus.

[0028] In the present invention, the color developing agent in the color developing stage is 1-5% aluminum chloride ethanol solution.

[0029] In the present invention, ultraviolet lamp is used for irradiation after color development, wherein the wavelength of the ultraviolet lamp is 360-370nm.

[0030] In the present invention, the fluorescent spots of the test solution, the standard solution and the negative control solution can be used to determine whether the spots in the standard sample exist in the test solution, thereby determining the components of the Fructus Aurantii Immaturus in the Manshenkang Granules.

[0031] Beneficial Effects

[0032] The present invention innovatively solves the problem of interference of other medicinal materials in Manshenkang granules with the thin layer of Fructus Aurantii Immaturus by combining the developing agent, developing temperature and humidity, and can efficiently separate and effectively test the Fructus Aurantii Immaturus components in Manshenkang granules. The research of the present invention also shows that the problem of interference of other medicinal materials in Manshenkang granules with the thin layer of Fructus Aurantii Immaturus can be further solved by combining the developing agent, developing high temperature and high humidity.

[0033] The method of the present invention can effectively identify the Fructus Aurantii Immaturus in the prescription, thereby being more conducive to ensuring the clinical efficacy of the preparation. The detection method is simple to operate, has a low detection cost, is conducive to quality control of the preparation, and is intuitive and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Example 1 Thin layer chromatogram of Fructus Aurantii Immaturus in Manshenkang Granules;

[0035] Figure 2 Comparative Example 1: Thin layer chromatogram of Fructus Aurantii Immaturus in Manshenkang Granules;

[0036] Figure 3 Example 2 Thin layer chromatogram of Fructus Aurantii Immaturus in Manshenkang Granules;

[0037] Figure 4 Example 3 Thin layer chromatogram of Fructus Aurantii Immaturus in Manshenkang Granules. DETAILED DESCRIPTION

[0038] The Manshenkang granules described in the present invention can be purchased publicly through well-known commercial channels.

[0039] For example, as an optional solution, the Manshenkang granules can be prepared by the following enumerated methods:

[0040] Prescription: Danshen 370g, Rhubarb 278g, Codonopsis pilosula 185g, Citrus aurantium 185g, Steamed Polygonatum 185g, Smilax glabra 278g, Oyster 93g, Licorice 185g;

[0041] Preparation method: The above eight ingredients are fried with oysters, and the other seven ingredients such as Danshen are decocted twice with water. The first time, 8 times the amount of water is added, decocted for 2 hours, and filtered; the second time, 6 times the amount of water is added, decocted for 1.5 hours, and filtered. The filtrate is concentrated under reduced pressure to a clear paste with a relative density of 1.05-1.10 (60°C), refrigerated for 12-18 hours, centrifuged, and the centrifuged liquid is concentrated under reduced pressure to a thick paste with a relative density of 1.20-1.25 (60°C), dried under reduced pressure, added with an appropriate amount of dextrin, crushed, mixed, made into granules, dried, and the granules are about 1000g.

[0042] Examples of thin layer testing methods include:

[0043] Taking the Fructus Aurantii Immaturus control medicinal material as a reference substance, thin layer chromatography was used to identify whether the Manshenkang granules contained Fructus Aurantii Immaturus components, wherein the upper layer solution obtained by mixing chloroform-methanol-water was used as a developing agent, and the volume ratio of chloroform, methanol, and water was 10-15:6-8:1.

[0044] Furthermore, the quality detection method for Citrus aurantium includes: preparation of the test solution: take 5g of the product, add 50ml of ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 10ml of water to the residue to dissolve, shake and extract with ethyl acetate twice, 15ml each time, combine the ethyl acetate solution, evaporate to dryness, add 1ml of anhydrous ethanol to the residue to dissolve, as the test solution. Preparation of the reference solution: take 0.5g of Citrus aurantium reference medicinal material, add 10ml of ethyl acetate, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 1ml of anhydrous ethanol to the residue to dissolve, as the control medicinal material solution. Preparation of the negative sample solution: take 5g of the negative Manshenkang granules lacking Citrus aurantium, and prepare the negative sample solution in the same way.

[0045] The following is a more specific implementation method:

[0046] Example 1: A thin layer detection method for the quality of Fructus Aurantii Immaturus in Manshenkang Granules

[0047] The quality detection method for Fructus Aurantii Immaturus comprises: taking an appropriate amount of the product and crushing it, taking 5 g, adding 50 mL of ethanol, ultrasonicating for 30 minutes, filtering, evaporating the filtrate to dryness, adding 10 mL of water to the residue to dissolve it, shaking and extracting with ethyl acetate twice, 15 mL each time, combining the ethyl acetate liquid, evaporating to dryness, adding 1 mL of anhydrous ethanol to the residue to dissolve it, and using it as the test solution.

[0048] Take 5g of negative Manshenkang granules without Fructus Aurantii Immaturus and prepare negative sample solution in the same way.

[0049] Take another 0.5 g of Citrus aurantium reference medicinal material, add 10 mL of ethyl acetate, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 1 mL of anhydrous ethanol to dissolve the residue, and use it as the reference medicinal material solution.

[0050] Identification: Take 2 μL of each of the above solutions and spot them on the same prefabricated silica gel G thin layer plate (Qingdao Ocean or Shanghai Shengya), use chloroform-methanol-water (13:7:1) solution (the time from preparation to use should be controlled within 5 min) as the developing agent, develop at 37°C and 66% humidity, take out, dry, spray with 2% aluminum chloride ethanol solution, and inspect under ultraviolet light (365 nm).

[0051] like Figure 1 As shown in the figure, the homemade silica gel G plate has poor separation effect. Commercially available prefabricated silica gel G plate should be used to identify the Fructus Aurantii Immaturus in Manshenkang Granules.

[0052] Comparative Example 1

[0053] Compared with Example 1, the only difference is that the developing solvent is replaced with a freshly prepared 13:7:1 dichloromethane-methanol-water. Other operations and parameters are the same as in Example 1.

[0054] The results are as follows Figure 2 It can be seen that this method is not suitable for the identification of Fructus Aurantii Immaturus in Manshenkang Granules, and the fluorescent spots are not clear. From left to right are the negative control sample lacking Fructus Aurantii Immaturus, the control medicinal material of Fructus Aurantii Immaturus, Manshenkang Granules, Manshenkang Granules, and Manshenkang Granules.

[0055] Embodiment 2:

[0056] Compared with Example 1, the only difference is that the storage conditions of the developing agent are changed, specifically:

[0057] Group A: Store the freshly prepared developer at 4°C for 4 h;

[0058] Group B: Store the freshly prepared developer at 4°C for 1 h;

[0059] Group C: the developer is a freshly prepared developer (that is, the freshly prepared developer is stored at 4°C for less than 5 minutes);

[0060] Other operations and parameters are the same as in Example 1.

[0061] Results Figure 3(1 to 5 are negative control samples, Zhi Fructus control medicinal materials, and three parallel groups of Man Shen Kang granules, respectively). From left to right are the results of Group A, B, and C, respectively. It can be seen that storing the developer at low temperature for 1 hour can unexpectedly effectively avoid the interference of other components in Man Shen Kang granules on the development of the components in Zhi Fructus, and can obtain a better development effect.

[0062] Embodiment 3:

[0063] Compared with Example 1, the only difference is that the temperature and humidity of the unfolding are changed. The experimental groups are:

[0064] Group A: development temperature was 4°C, humidity was 49%, 66%, and 94%, respectively;

[0065] Group B: development temperature was 25°C and humidity was 49%, 66% and 94% respectively;

[0066] Group C: development temperature was 35°C and humidity was 49%, 66%, and 94% respectively;

[0067] Test results see Figure 4 (The stripes in each sub-figure from left to right are negative control sample, Fructus Aurantii Immaturus control medicinal material, and three parallel groups of Manshenkang granules). Figure 4 It can be seen that for the determination of Fructus Aurantii Immaturus in the Manshenkang Granules involved in the present invention, in order to avoid the interference of other ingredients in the Manshenkang Granules on Fructus Aurantii Immaturus, an excellent anti-interference effect can be obtained under ultra-high humidity. On this basis, further anti-interference effect can be obtained by further combining with a development temperature above 35°C.

Claims

1. A thin layer chromatography detection method for Fructus Aurantii Immaturus in Manshenkang Granules, wherein the Manshenkang Granules are raw medicinal materials and extracts thereof including Salvia miltiorrhiza, Rhubarb, Codonopsis pilosula, Fructus Aurantii Immaturus, Steamed Polygonatum sibiricum, Smilax glabra, Oyster and Licorice; characterized in that, The samples of Manshenkang granules, Fructus Aurantii Immaturus or Manshenkang granules without Fructus Aurantii Immaturus were respectively subjected to alcohol extraction, water dissolution, hydrophobic solvent extraction and concentration, and alcohol dissolution treatment to obtain a test solution, a standard solution and a negative control solution respectively; The test solution, standard solution and negative control solution are subjected to thin layer chromatography, followed by color development and ultraviolet detection; wherein the developing agent in the thin layer chromatography stage is chloroform, methanol and water in a volume ratio of 10-15:6-8:1; the developing temperature in the thin layer chromatography stage is above 4°C and the humidity is above 40%.

2. The thin layer chromatography detection method of Fructus Aurantii Immaturus in Manshenkang Granules according to claim 1, characterized in that: The weight ratios of salvia miltiorrhiza, rhubarb, codonopsis pilosula, fructus aurantii, steamed polygonatum sibiricum, smilax glabra, oyster and liquorice are 300-400:200-300:150-250:150-250:150-250:200-350:50-150:150-250 respectively; Preferably, the Manshenkang granules contain the water extract of the original medicinal material; Preferably, the Manshenkang granules further comprise auxiliary materials; Preferably, the auxiliary material is at least one of dextrin, starch, sucrose, microcrystalline cellulose and calcium sulfate; Preferably, in the Manshenkang granules, the content of the auxiliary materials is 25-55%.

3. The thin layer chromatography detection method of Fructus Aurantii Immaturus in Manshenkang Granules according to claim 1, characterized in that: The alcohol selected in the alcohol extraction stage is methanol and / or ethanol; Preferably, the hydrophobic solvent in the hydrophobic solvent extraction stage comprises at least one of dichloromethane and ethyl acetate.

4. The thin layer chromatography detection method of Fructus Aurantii Immaturus in Manshenkang Granules according to claim 1, characterized in that: The thin layer chromatography was performed using silica gel plates.

5. The thin layer chromatography detection method of Fructus Aurantii Immaturus in Manshenkang Granules according to claim 1, characterized in that: In the developing solvent, the volume ratio of chloroform, methanol and water is 13:7:

1.

6. The thin layer chromatography detection method of Fructus Aurantii Immaturus in Manshenkang Granules according to claim 1, characterized in that: The developing agent is a developing agent that is prepared fresh or sealed and stored at a temperature below 10° C. for 1 to 2 hours after preparation.

7. The thin layer chromatography detection method of Fructus Aurantii Immaturus in Manshenkang Granules according to claim 1, characterized in that: The temperature in the development stage is 20°C or higher, further 30 to 40°C, and further 35 to 40°C.

8. The thin layer chromatography detection method of Fructus Aurantii Immaturus in Manshenkang Granules according to claim 1, characterized in that: The humidity in the development stage is 60% or more, further 90% or more, and further 90 to 100%.

9. The thin layer chromatography detection method of Fructus Aurantii Immaturus in Manshenkang Granules according to claim 1, characterized in that: The color developing agent in the color developing stage is 1-5% aluminum chloride ethanol solution.

10. The thin layer chromatography detection method of Fructus Aurantii Immaturus in Manshenkang Granules according to claim 1, characterized in that: The wavelength of the UV detection stage is 360-370nm.

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