Smilax reference extract as well as quality control method and application thereof

Abstract

The invention discloses a chinaroot greenbrier reference extract as well as a quality control method and application thereof. The chinaroot greenbrier reference extract is prepared by the following steps: performing water extraction on chinaroot greenbrier raw medicinal materials to obtain a chinaroot greenbrier extracting solution, concentrating, centrifuging and purifying the chinaroot greenbrier extracting solution, recovering a solvent, and freeze-drying to obtain the chinaroot greenbrier reference extract. The chinaroot greenbrier reference extract provided by the invention can be used as a reference substance for detecting the quality of a Sanjin preparation, so that the problem of lack of reference substances is solved; meanwhile, the preparation steps of the reference substance are greatly simplified, convenience is brought to a user, and the detection efficiency is improved; quality control conditions are provided and optimized for the chinaroot greenbrier reference extract, and production and use of the chinaroot greenbrier reference extract are facilitated; the chinaroot greenbrier reference extract is applied to quality detection of Sanjin preparations such as Sanjin tablets and Sanjin particles, the detection blank of indexes in chinaroot greenbrier is filled, the quality control standard of the Sanjin preparations is further perfected, and the overall quality of the Sanjin preparations is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to a smilax china control extract and a quality control method and application thereof. Background Art

[0002] Smilax china is a plant of the lily family, mainly containing saponins and alkaloids. Sanjin preparations are composed of five Chinese medicinal herbs, including golden cherry root, Smilax china, sheep's mouth, golden sand vine, and Centella asiatica. They have the effects of clearing away heat and detoxification, promoting dampness and relieving stranguria, and benefiting the kidneys. They are mainly used to treat acute and chronic pyelonephritis, cystitis, and urinary tract infections. In recent years, due to its excellent quality and significant efficacy, Sanjin preparations have always been the top brand of similar Chinese patent medicines for the treatment of urinary tract infections.

[0003] The current quality control method for Smilax china medicinal materials requires the use of multiple single ingredients as reference substances to identify and measure the quality of Smilax china medicinal materials. The steps are relatively cumbersome and cannot fully reflect the true quality of Smilax china medicinal materials.

[0004] The Chinese patent application with application number 200510200471.8 relates to a Sanjin drug preparation for treating diseases of the urinary system, as well as a preparation method and a quality control method. The quality control method involved in the application includes a thin layer chromatography identification test method for diosgenin.

[0005] However, diosgenin is a hydrolysis product derived from the medicinal ingredients of Smilax china. It is affected by many factors during the detection process, has poor reproducibility, and requires high standards from the testers. Therefore, in order to more effectively control the quality of Sanjin preparations, it is necessary to re-study the detection methods of the physical and chemical components in Smilax china.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a Smilax china control extract and a quality control method and application thereof. The Smilax china control extract provided by the present invention can be used as a reference substance to detect the quality of Sanjin preparations, which solves the problem of lack of reference substances and improves the quality control standards of Sanjin preparations, which is beneficial to improving the overall quality of the preparations.

[0008] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:

[0009] A Smilax china control extract, which is prepared by the following method:

[0010] The raw medicinal material of Smilax china is subjected to water extraction to obtain a Smilax china extract, and the Smilax china extract is concentrated, centrifuged, purified, the solvent is recovered, and freeze-dried to obtain a Smilax china control extract.

[0011] The Smilax china control extract provided by the present invention can be used as a control substance for detecting the quality of the Sanjin preparation, thus solving the problem of lack of control substances; at the same time, the preparation steps of the control substances are greatly simplified, which is convenient for users and is also conducive to improving detection efficiency.

[0012] Furthermore, the purification includes: taking the centrifugal liquid after centrifugation and adding it to a polyamide or macroporous resin column for elution purification;

[0013] Preferably, the mass ratio of the polyamide or macroporous resin to the raw medicinal material of Smilax china is 1:1-4; preferably, 1:2;

[0014] Preferably, the preparation of the polyamide or macroporous resin column comprises: soaking the polyamide powder or macroporous resin with ethanol, wet packing the column, washing with ethanol until no white matter is produced when the ethanol drips into water, and / or washing with drinking water until the ethanol content is less than 5%.

[0015] Further, the elution solvent used for elution purification includes one or two of ethanol solution and ethyl acetate;

[0016] Preferably, the elution solvent is an ethanol solution with a mass fraction of 20% to 70%;

[0017] Preferably, the elution solvent is an ethanol solution with a mass fraction of 50%;

[0018] Preferably, the volume ratio of the elution solvent to the centrifuge liquid is 6 to 10:1.

[0019] Furthermore, the Smilax china extract is concentrated to obtain a concentrated solution, and the weight ratio of the concentrated solution to the Smilax china raw medicinal material is 0.5 to 5:1; preferably, 5:1.

[0020] Furthermore, the water extraction comprises: dissolving the raw medicinal material of Smilax china in water, filtering the obtained filtrate to obtain the Smilax china extract;

[0021] Preferably, the raw medicinal material of Smilax china is extracted twice with water;

[0022] Preferably, the amount of water used in the first step is 8 to 14 times the mass of the raw material of Smilax china, and the amount of water used in the second step is 6 to 10 times the mass of the raw material of Smilax china;

[0023] Preferably, the amount of water used for the first time is 10 times the quality of the raw material of Smilax china, and the amount of water used for the second time is 8 times the quality of the raw material of Smilax china.

[0024] Furthermore, the Smilax china control extract is a brownish yellow to reddish brown powder and is hygroscopic.

[0025] The invention detects and clarifies the shape and properties of the Smilax china control extract, which is helpful to provide a theoretical basis for determining storage and packaging conditions. According to the water content and hygroscopicity of the Smilax china control extract, a fully enclosed brown ampoule can be used, which can prevent deliquescence and moisture absorption during storage and is convenient for users to use. The storage condition is 4° C. and stored away from light. The sample should be weighed quickly when used.

[0026] The invention discloses a quality control method for a Smilax china reference extract, and adopts thin layer chromatography to identify the Smilax china reference extract.

[0027] Further, a Smilax china control extract solution is prepared as a test solution, and a Smilax china control medicinal material solution is prepared, and the test solution and the control medicinal material solution are aspirated and spotted on the same thin layer plate, and a butyl acetate-methanol-formic acid-water system or toluene-ethyl acetate-formic acid system is used as a developing agent to perform thin layer identification;

[0028] Preferably, the volume ratio of butyl acetate-methanol-formic acid-water is 16:3:1:1, and the volume ratio of toluene-ethyl acetate-formic acid is 4:10:1;

[0029] When the above-mentioned developing agent is used and the proportions of the components in the developing agent are as above, the developing effect is good, the difference in development on different thin layer plates is small, it is more stable, and the developing agent is non-toxic and easy to use.

[0030] Preferably, the developer is a 9% ferric chloride ethanol solution or a mixed solution of 1% potassium ferrocyanide-2% ferric chloride;

[0031] Preferably, the thin layer plate is a high-efficiency silica gel G thin layer plate.

[0032] A method for detecting the quality of Sanjin preparations using the above-mentioned Smilax china control extract, comprising preparing the Smilax china control extract into a reference solution, and preparing a test solution, and taking the Smilax china reference solution and the test solution on the same thin layer plate for determination using thin layer chromatography.

[0033] The present invention applies the Smilax china control extract to the quality detection of Sanjin preparations such as Sanjin tablets, fills the detection gap of indicators in Smilax china, further improves the quality control standard of Sanjin preparations, and is beneficial to improving the overall quality of Sanjin preparations.

[0034] Further, in the thin layer chromatography method, butyl acetate-methanol-formic acid-water system or toluene-ethyl acetate-formic acid system is used as a developing solvent;

[0035] Preferably, the volume ratio of butyl acetate-methanol-formic acid-water is 16:3:1:1, and the volume ratio of toluene-ethyl acetate-formic acid is 4:10:1;

[0036] When the above-mentioned developing agent is used and the proportions of the components in the developing agent are as above, the developing effect is good, the difference in development on different thin layer plates is small, and it is more stable.

[0037] Preferably, the color developer is a 9% ferric chloride ethanol solution or a mixed solution of 1% potassium ferrocyanide-2% ferric chloride.

[0038] Furthermore, in the thin layer chromatography method, the spotting amount of the Smilax china reference solution and the test solution is 5 to 10 μl, the temperature during development is 5 to 30° C., and the humidity is 30% to 90%;

[0039] Preferably, the spotting volume is 10 μl, the temperature during development is 25° C., and the humidity is 60%.

[0040] Further, the preparation of the test solution includes: adding the extract of the three gold preparations to a polyamide or macroporous resin column, eluting and purifying, collecting the eluate, evaporating the eluate to dryness, and then adding methanol to dissolve the eluate to obtain the test solution;

[0041] Preferably, the elution purification comprises: first eluting with water to purify until colorless, draining the water washing liquid, then eluting with ethanol, and collecting the eluate;

[0042] Preferably, the mass fraction of the ethanol is 50%;

[0043] Preferably, the preparation of the polyamide or macroporous resin column comprises: soaking the polyamide powder or macroporous resin in ethanol and stirring to remove bubbles, and then loading the polyamide powder or macroporous resin into a chromatography column;

[0044] Preferably, the polyamide powder or macroporous resin is soaked in 90-95% by mass ethanol;

[0045] Preferably, the Sanjin preparation is placed in methanol, sonicated, filtered, the filtrate after filtration is evaporated to dryness, dissolved in water, centrifuged, and the supernatant obtained by centrifugation is used as the Sanjin preparation extract.

[0046] After adopting the above technical scheme, the present invention has the following beneficial effects compared with the prior art.

[0047] The Smilax china control extract provided by the present invention can be used as a control substance for detecting the quality of the Sanjin preparation, thus solving the problem of lack of control substances; at the same time, the preparation steps of the control substances are greatly simplified, which is convenient for users and is also conducive to improving detection efficiency.

[0048] The invention proposes and optimizes the quality control conditions for the Smilax china control extract, which is beneficial to the production and use of the Smilax china control extract. In addition, a draft quality standard is proposed, which is beneficial to product quality control.

[0049] The present invention applies the Smilax china control extract to the quality detection of Sanjin preparations such as Sanjin tablets and Sanjin granules, fills the detection gap of indicators in Smilax china, further improves the quality control standard of Sanjin preparations, and is beneficial to improving the overall quality of Sanjin preparations.

[0050] The determination process of the present invention is not easily disturbed by the outside world or other conditions, and the determination result has clear spots, good separation and good reproducibility; the determination process is simple and convenient, which is convenient for detection personnel to determine; the determination result is highly specific, accurate and reliable, and the quality of the three gold preparations can be more effectively controlled by accurately determining the components of Smilax china.

[0051] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The accompanying drawings are part of the present invention and are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an improper limitation of the present invention. Obviously, the drawings described below are only some embodiments. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the accompanying drawings:

[0053] Figure 1 , is the Smilax china thin layer identification chromatogram of the Smilax china control extract in Example 1.2; (In the figure, 1 is the Smilax china control medicinal material, 2 is the Smilax china negative control, 3 is the Smilax china control extract 210801, 4 is the Smilax china control extract 210802, and 5 is the Smilax china control extract 210901);

[0054] Figure 2 , are the thin layer identification chromatograms of Smilax china corresponding to different purification methods in Example 1.3; (in the figure, 1 is water extraction of Smilax china, 2 is Smilax china (water extraction, HPD100 50% ethanol elution), 3 is Sanjin tablets (methanol reflux through polyamide ethyl acetate elution), 4 is Smilax china (water extraction, alcohol precipitation through HPD100 + polyamide 50% ethanol elution), 5 is Smilax china paste powder (alcohol precipitation through HPD100 + polyamide + dextran gel methanol elution), 6 is Smilax china negative methanol ultrasound);

[0055] Figure 3 , is the thin layer identification chromatogram of Smilax china corresponding to different eluents in Example 1.3; (in the figure, 1 is gallic acid, 2 is resveratrol, 3 is Sanjin tablets (50% methanol reflux ethyl acetate extraction), 4 is Smilax china paste powder (alcohol precipitation D101 + polyamide water washing solution), 5 is Smilax china paste powder (alcohol precipitation D101 + polyamide 50% ethanol elution), 6 is Smilax china paste powder (alcohol precipitation D101 + polyamide 70% ethanol elution); 7 is Smilax china paste powder (alcohol precipitation D101 + polyamide 95% ethanol elution));

[0056] Figure 4 , are the thin layer identification chromatograms of Smilax china corresponding to different eluents in Example 1.3; (in the figure, 1 is Smilax china water-extracted alcohol precipitation through HPD100 (20% ethanol elution), 2 is Smilax china water-extracted alcohol precipitation through HPD100 (50% ethanol elution), 3 is Smilax china water-extracted alcohol precipitation through HPD100 (80% ethanol elution), 4 is Smilax china water-extracted alcohol precipitation through HPD100 (95% ethanol elution), 5 is Sanjin tablets (50% methanol reflux ethanol elution) ethyl ester extraction), 6 is water extraction of Smilax china followed by alcohol precipitation with D101 + polyamide (eluted with 50% ethanol); 7 is water extraction of Smilax china followed by alcohol precipitation with HPD100 + polyamide (eluted with water); 8 is water extraction of Smilax china followed by alcohol precipitation with HPD100 + polyamide (eluted with 50% ethanol); 9 is water extraction of Smilax china followed by alcohol precipitation with HPD100 + polyamide (eluted with 70% ethanol); 10 is water extraction of Smilax china followed by alcohol precipitation with HPD100 + polyamide (eluted with 95% ethanol));

[0057] Figure 5 , are thin layer identification chromatograms of Smilax china corresponding to different eluents in Example 1.3; (in the figure, 1 is 50% ethanol elution of Smilax china extract HPD100 column, 2 is 50% methanol elution of Smilax china extract polyamide column, 3 is ethyl acetate elution of Sanjin tablet polyamide column, 4 is 50% ethanol elution of Smilax china extract HPD100-polyamide column, 5 is 50% ethanol elution of Smilax china extract polyamide column, 6 is methanol ultrasonic extraction of Smilax china negative sample lacking Sanjin tablet; 7 is ethyl acetate elution of Smilax china extract polyamide column; 8 is water washing liquid of Smilax china extract polyamide column);

[0058] Figure 6 , is the thin layer identification chromatogram of Smilax china corresponding to Group A in Example 1.4; the first figure on the left is the inspection under ultraviolet lamp (254nm) before color development, the second figure on the left is the inspection under ultraviolet lamp (365nm) before color development, and the third figure on the left is the inspection under sunlight after color development; (in the figure, 1 is astilbin, 2 is cyperoside, 3 is Sanjin tablets (water extraction, n-butanol extraction, alkali precipitation, and then n-butanol extraction), 4 is resveratrol, 5 is Sanjin tablets (50% methanol extraction, ethyl acetate extraction), 6 is Smilax china (water extraction, alcohol precipitation, D101-polyamide column water washing solution); 7 is Smilax china (water extraction, alcohol precipitation, D101-polyamide column 50% ethanol eluent); 8 is Smilax china (water extraction, alcohol precipitation, D101-polyamide column 70% ethanol eluent); 9 is Sanjin tablets (acetone ultrasound);

[0059] Figure 7, is the thin layer identification chromatogram of Smilax china corresponding to Group B in Example 1.4; (In the figure, 1 is Smilax china (water extraction and alcohol precipitation through HPD100-20% ethanol eluent), 2 is Smilax china (water extraction and alcohol precipitation through HPD100-50% ethanol eluent), 3 is Smilax china (water extraction and alcohol precipitation through HPD100-80% ethanol eluent), 4 is Smilax china (water extraction and alcohol precipitation through HPD100-95% ethanol eluent), 5 is Sanjin tablets (50% methanol extraction and ethyl acetate eluent), 6 is Sanjin tablets (50% methanol extraction and ethyl acetate eluent), 7 is Sanjin tablets (water extraction and alcohol precipitation through HPD100-80% ethanol eluent), 8 is Sanjin tablets (water extraction and alcohol precipitation through HPD100-95% ethanol eluent), 9 is Sanjin tablets (water extraction and ethyl acetate eluent), 10 is Sanjin tablets (water extraction and ethyl acetate eluent), 11 is Sanjin tablets (water extraction and ethyl acetate eluent), 12 is Sanjin tablets (water extraction and ethyl acetate eluent), 13 is Sanjin tablets (water extraction and ethyl acetate eluent), 14 is Sanjin tablets (water extraction and ethyl acetate eluent), 15 is Sanjin tablets (water extraction and ethyl acetate eluent), 16 is Sanjin tablets (water extraction and ethyl acetate eluent), 17 is Sanjin tablets (water extraction and ethyl acetate eluent), 18 is Sanjin tablets (water extraction and ethyl acetate eluent), 19 is Sanjin tablets (water extraction and ethyl acetate eluent), 20 is Sanjin tablets ester extraction), 6 is Smilax china (water extraction and alcohol precipitation through D101-polyamide column 50% ethanol eluent); 7 is Smilax china (water extraction and alcohol precipitation through HPD100-polyamide column water eluent); 8 is Smilax china (water extraction and alcohol precipitation through HPD100-polyamide column 50% ethanol eluent); 9 is Smilax china (water extraction and alcohol precipitation through HPD100-polyamide column 70% ethanol eluent); 10 is Smilax china (water extraction and alcohol precipitation through HPD100-polyamide column 95% ethanol eluent);

[0060] Figure 8 , are the thin layer identification chromatograms of Smilax china corresponding to different color developers in Example 1.4; the color developer in Figure 1 on the left is a mixed solution of 1% potassium ferricyanide-2% ferric chloride; the color developer in Figure 2 on the left is a 9% ferric chloride ethanol solution; (in the figure, 1 is a negative sample lacking Smilax china (extracted with methanol and passed through a polyamide column with ethyl acetate eluent), 2 is Smilax china (extracted with water with 80% ethanol and precipitated with HPD100-polyamide column with ethyl acetate eluent), 3 is Sanjin tablets (extracted with methanol and passed through a polyamide column with ethyl acetate eluent), 4 is Sanjin tablets (extracted with methanol and passed through a polyamide column with ethyl acetate eluent), 5 is Sanjin tablets (extracted with methanol and passed through a polyamide column with 50% ethanol eluent), 6 is Sanjin tablets (extracted with methanol and passed through a polyamide column with ethyl acetate eluent); 7 is Smilax china (extracted with water with 80% ethanol and precipitated with HPD100-polyamide column with ethyl acetate eluent);

[0061] Fig. 9 , is the Smilax china thin layer identification chromatogram of 11 batches of Sanjin Tablets in Example 2; (in the figure, the batch numbers of 1 to 5, 8 to 13 Sanjin Tablets are: 1307043, 1507002, 1610001, 180805, 181209, 190433, 191003, 200603, 200615, 201128, 210302; 6 is the Smilax china control extract; 7 is the Smilax china negative control lacking);

[0062] Fig.10 , is the thin layer identification chromatogram of Smilax china of the other 11 batches of Sanjin Tablets in Example 2; (in the figure, the batch numbers 1 to 5 and 8 to 13 are: 210912, 210932, 210934, 211002, 211031, 211038, 211109, 211119, 211123, 211205, 211217; 6 is the Smilax china control extract; 7 is the Smilax china negative control lacking);

[0063] Fig.11 , are the thin layer identification chromatograms of Smilax china in different development systems in Example 2;

[0064] Left figure: The developing solvent is butyl acetate-methanol-formic acid-water (volume ratio: 16:3:1:1); 1 is the negative control lacking Smilax china, 2 is the control extract of Smilax china, 3-5 are the batch numbers of the test samples Sanjin Tablets are 220125, 220210, 220222 respectively;

[0065] The second figure from the left: the developing solvent is ethyl acetate-methanol-formic acid (volume ratio: 10:1:1); 1 is the negative control lacking Smilax china, 2 is the control extract of Smilax china, 3-5 are the batch numbers of the test samples Sanjin Tablets are 200615, 210932, 211217 respectively;

[0066] Left three figures: the developing solvent is toluene-ethyl acetate-formic acid (volume ratio: 4:10:1); 1 is the negative control lacking Smilax china, 2 is the control extract of Smilax china, 3-5 are the batch numbers of the test samples Sanjin Tablets are 200615, 210932, 211217 respectively;

[0067] Fig.12 , are the thin layer identification chromatograms of Smilax china with different sample amounts in Example 2;

[0068] In the figure: 1-4 are Smilax china control extracts, with the sample volumes being 3, 5, 10, and 15 μl, respectively; 5-8 are Sanjin tablets, with the batch number being 210932, with the sample volumes being 3, 5, 10, and 15 μl, respectively;

[0069] Fig.13 , are the identification chromatograms of Smilax china on different thin layer plates in Example 2; in the figure, 1 is the negative control lacking Smilax china, 2 is the control extract of Smilax china, and the batch numbers of 3-5 test samples Sanjin Tablets are 200615, 210932, and 211217 respectively;

[0070] Left: The thin layer plate is a thin layer chromatography silica gel prefabricated plate (model: HSG, batch number: 20210402, Yantai Yinlong);

[0071] The second figure from the left: The thin layer plate is a thin layer chromatography silica gel prefabricated plate (model: HPTLC, batch number: 812011, Merck);

[0072] The third picture from the left: The thin layer plate is a thin layer chromatography silica gel prefabricated plate (model: high efficiency, batch number 20210503, Qingdao Ocean);

[0073] Fig.14, are the thin layer identification chromatograms of Smilax china at different temperatures in Example 2; in the figure, 1 is the negative control lacking Smilax china, 2 is the control extract of Smilax china, and the batch numbers of 3-5 test samples Sanjin Tablets are 200615, 210932, and 211217 respectively; the first figure on the left: 5°C; the second figure on the left: 25°C; the third figure on the left: 30°C;

[0074] Fig.15 , are the thin layer identification chromatograms of Smilax china at different temperatures in Example 2;

[0075] Left: Humidity is 30%; 1 is the Smilax china control extract, 2 is the Smilax china negative control, 3-5 are the batch numbers of the test samples Sanjin Tablets, which are 200615, 210932, and 211217, respectively;

[0076] The second figure from the left: humidity is 60%; 1 is the negative control lacking Smilax china, 2 is the Smilax china control extract, 3-5 are the batch numbers of the test samples Sanjin Tablets, which are 200615, 210932, and 211217 respectively;

[0077] The left three pictures: humidity is 90%; 1 is the negative control lacking Smilax china, 2 is the Smilax china control extract, 3-5 are the batch numbers of the test samples Sanjin Tablets, which are 200615, 210932, and 211217 respectively;

[0078] It should be noted that these drawings and textual descriptions are not intended to limit the conceptual scope of the present invention in any way, but are intended to illustrate the concept of the present invention for those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0079] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0080] Equipment: multifunctional extraction tank, vacuum concentration tank, tripod centrifuge, polyamide purification column, freeze dryer.

[0081] Solvents and reagents: drinking water, ethanol, polyamide (30-60 mesh), high-efficiency silica gel G prefabricated thin layer plate, Smilax china slices (batch numbers: 200601, 200602, 210401, 210501, 210602), Smilax china control extract (batch numbers: 210801, 210802, 210901, 220301).

[0082] Embodiment 1,

[0083] 1.1a. A method for preparing a control extract of Smilax china

[0084] Water extraction: Take the Smilax china slices, place them in an extraction container, add water to extract twice, the first time the amount of water is 10 times the mass of the Smilax china slices for 2 hours, the second time the amount of water is 8 times the mass of the Smilax china slices for 1 hour, filter, and combine the filtrates to obtain the extract;

[0085] Concentration: Concentrate the extract to 5 times the weight of the Smilax china slices;

[0086] Centrifugation: Use canvas bag as centrifugal medium, remove particulate impurities by centrifugation, and take centrifugal liquid;

[0087] Polyamide purification: take polyamide (30-60 mesh), the mass ratio of polyamide to Smilax china slices is 1:2, soak with ethanol overnight, wet-load column, wash with ethanol until no white matter is produced when the ethanol drips into water, wash with drinking water until the alcohol content is less than 5%; apply the centrifuge liquid to the treated polyamide column at a uniform speed, wash with water until colorless, elute with 50% ethanol as elution solvent, the volume ratio of elution solvent to centrifuge liquid is 6:1, and collect 50% ethanol eluate;

[0088] Recovering ethanol and concentrating: recovering the ethanol eluate under reduced pressure and concentrating it into a concentrated solution with a mass fraction of ethanol less than 5%;

[0089] Freeze-drying: The concentrated solution is freeze-dried to obtain the Smilax china control extract.

[0090] In this example, four batches of Smilax china control extracts were prepared according to the preparation method described in 1.1 above, as shown in Table 1 below:

[0091] Table 1:

[0092]

[0093]

[0094] It can be seen that the Smilax china control extract prepared in this example is a brown to reddish brown powder and has hygroscopicity.

[0095] 1.1b. A method for preparing a control extract of Smilax china

[0096] Water extraction: Take the Smilax china slices, place them in an extraction container, add water to extract twice, the first time the amount of water is 8 times the mass of the Smilax china slices for 2 hours, the second time the amount of water is 6 times the mass of the Smilax china slices for 1 hour, filter, and combine the filtrates to obtain the extract;

[0097] Concentration: Concentrate the extract to 0.5 times the weight of the Smilax china slices;

[0098] Centrifugation: Use canvas bag as centrifugal medium, remove particulate impurities by centrifugation, and take centrifugal liquid;

[0099] Polyamide purification: take polyamide (30-100 mesh), the mass ratio of polyamide to Smilax china slices is 1:1, soak with ethanol overnight, wet-load column, wash with ethanol until no white matter is produced when the ethanol drips into water, wash with drinking water until the alcohol content is less than 5%; apply the centrifuge liquid to the treated polyamide column at a uniform speed, wash with water until colorless, elute with 20% ethanol as elution solvent, the volume ratio of elution solvent to centrifuge liquid is 10:1, and collect 20% ethanol eluate;

[0100] Recovering ethanol and concentrating: recovering the ethanol eluate under reduced pressure and concentrating it into a concentrated solution with a mass fraction of ethanol less than 5%;

[0101] Freeze-drying: The concentrated solution is freeze-dried to obtain the Smilax china control extract.

[0102] 1.1c. A method for preparing a control extract of Smilax china

[0103] Water extraction: Take the Smilax china slices, place them in an extraction container, add water to extract twice, the first time the amount of water is 14 times the mass of the Smilax china slices for 2 hours, the second time the amount of water is 10 times the mass of the Smilax china slices for 1 hour, filter, and combine the filtrates to obtain the extract;

[0104] Concentration: Concentrate the extract to 3 times the weight of the Smilax china slices;

[0105] Centrifugation: Use canvas bag as centrifugal medium, remove particulate impurities by centrifugation, and take centrifugal liquid;

[0106] Polyamide purification: take polyamide (30-100 mesh), the mass ratio of polyamide to Smilax china slices is 1:4, soak with ethanol overnight, wet-load column, wash with ethanol until no white matter is produced when the ethanol drips into water, wash with drinking water until the alcohol content is less than 5%; apply the centrifuge liquid to the treated polyamide column at a uniform speed, wash with water until colorless, elute with 70% ethanol as elution solvent, the volume ratio of elution solvent to centrifuge liquid is 8:1, and collect 70% ethanol eluate;

[0107] Recovering ethanol and concentrating: recovering the ethanol eluate under reduced pressure and concentrating it into a concentrated solution with a mass fraction of ethanol less than 5%;

[0108] Freeze-drying: The concentrated solution is freeze-dried to obtain the Smilax china control extract.

[0109] 1.2. Identification of the Smilax china reference extract prepared in 1.1a by thin layer chromatography

[0110] Take 100 mg of Smilax china control extract, add 5 ml of methanol to dissolve it, and obtain the Smilax china control extract solution as the test solution.

[0111] Take 2g of Smilax china reference medicinal material, add 50ml of water, heat and reflux for 2 hours, filter, evaporate to dryness, add 25ml of water to the residue to dissolve, centrifuge (speed is 4000 revolutions per minute) for 10 minutes, take the supernatant and add it to the treated polyamide column (30-100 mesh, 5g, inner diameter is 1.5cm, wet column), wash with water until colorless, discard the water washing liquid, and then elute with 100ml50% ethanol, collect the effluent and eluent, evaporate to dryness, add 1ml of methanol to dissolve the residue as the reference medicinal material solution.

[0112] According to the thin layer chromatography method (General Rule 0502), 5-10 μl of each of the above two solutions were taken and spotted on the same high-efficiency silica gel G thin layer plate (model: HSG, batch number: 20210402, Yantai Yinlong), and butyl acetate-methanol-formic acid-water (volume ratio of 16:3:1:1) was used as the developing solvent. After development, the plate was taken out, dried, sprayed with 9% ferric chloride ethanol solution, and examined under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding position of the chromatogram of the control medicinal material. Figure 1 .

[0113] Comprehensive analysis of the development results under multiple conditions showed that the chromatographic results obtained by the above development system and color developer were the clearest, so it was selected as the identification method of the extract. This method was used to detect the three batches of extracts prepared, and the results showed clear spots, and there was no interference at the target spots for the Smilax china negative samples in Sanjin Tablets.

[0114] 1.3 Investigation of process conditions for preparing Smilax china control extract

[0115] (1) Extraction: Since the Smilax china control extract is mainly used for TLC identification of Sanjin Tablets, the extraction solvent and extraction time of Smilax china medicinal materials are consistent with those of Sanjin Tablets, and the amount of water added is determined to cover the medicinal materials. Therefore, the extraction process is to boil Smilax china medicinal materials with water twice, the first time adding 8 to 14 times the amount of water and extracting for 2 hours, and the second time adding 6 to 10 times the amount of water and extracting for 1 hour.

[0116] (2) Concentration: The actual effects corresponding to the weight ratio of Smilax china slices to concentrated solution of 1:5, 2:5 and 2:1 are verified, as shown in Table 2 below:

[0117] Table 2:

[0118] Smilax china slices-concentrate Concentrate Adsorption 1∶5 Good fluidity, less granular impurities The adsorption process is smooth 2∶5 Good fluidity, more granular impurities The chromatography column is clogged during the adsorption process 2∶1 Good fluidity, more granular impurities The column is prone to clogging during the adsorption process

[0119] It can be seen from the above table that when the weight ratio of Smilax china slices to concentrated solution is 1:5, the concentrated solution has good fluidity, less granular impurities, and the adsorption process is smooth, so it is better to concentrate the extract to about 5 times the weight of Smilax china slices.

[0120] (3) Purification:

[0121] Compare the various purification routes of Smilax china centrifuge liquid, such as alcohol precipitation, organic solvent extraction, column chromatography (macroporous resin D101, macroporous resin HPD100, polyamide, etc.). Specifically, the following purification methods were verified:

[0122] Smilax china: water extraction, macroporous resin HPD100, elution with 50% ethanol;

[0123] Three gold sheets: methanol refluxed through polyamide ethyl acetate to elute;

[0124] Smilax china: water extraction, alcohol precipitation, macroporous resin HPD100, polyamide column, elution with 50% ethanol;

[0125] Smilax china powder: alcohol precipitation through macroporous resin HPD100, polyamide column, dextran gel methanol elution;

[0126] The prepared Smilax china control extract was measured by thin layer chromatography, and the measurement conditions included:

[0127] Thin layer plate: Thin layer chromatography silica gel precast plate (model: G, batch number: HX98383926, Merck);

[0128] Developing solvent: butyl acetate-formic acid-methanol (volume ratio of 6:1:1);

[0129] Color development and inspection: 1% potassium ferrocyanide-2% ferric chloride mixed aqueous solution, inspect under sunlight. Figure 2 The results showed that the background interference of the thin layer chromatography spectrum of the aqueous extract was less after purification by column chromatography, and the purification effect of polyamide column was the best.

[0130] (4) Selection of eluent:

[0131] The effects of different eluents such as 20% ethanol, 50% ethanol, 70% ethanol, and ethyl acetate on the determination results were investigated. Specifically, the following elution methods were verified:

[0132] Group A,

[0133] Sanjin tablets: 50% methanol reflux ethyl acetate extraction;

[0134] Smilax paste powder: alcohol-precipitated macroporous resin D101, polyamide, water washing liquid;

[0135] Smilax china powder: alcohol precipitation over macroporous resin D101, polyamide, eluted with 50% ethanol;

[0136] Smilax china powder: alcohol precipitation over macroporous resin D101, polyamide, eluted with 70% ethanol;

[0137] Smilax china paste powder: alcohol precipitation over macroporous resin D101, polyamide, elution with 95% ethanol;

[0138] The prepared Smilax china control extract was measured by thin layer chromatography, and the measurement conditions included:

[0139] Thin layer plate: Thin layer chromatography silica gel prefabricated plate (model: G254, batch number: 20161222, Qingdao Ocean Chemical Co., Ltd.)

[0140] Developing solvent: butyl acetate-formic acid-methanol (volume ratio is 6:1:1)

[0141] Color development and inspection: 1% potassium ferrocyanide-2% ferric chloride mixed aqueous solution, inspect under sunlight. Figure 3 .

[0142] Group B,

[0143] Smilax china: water extraction, alcohol precipitation through macroporous resin HPD100, elution with 20% ethanol;

[0144] Smilax china: water extraction, alcohol precipitation through macroporous resin HPD100, elution with 50% ethanol;

[0145] Smilax china: water extraction, alcohol precipitation through macroporous resin HPD100, elution with 80% ethanol;

[0146] Smilax china: water extraction, alcohol precipitation through macroporous resin HPD100, elution with 95% ethanol;

[0147] Sanjin tablets: 50% methanol reflux ethyl acetate extraction;

[0148] Smilax china: water extraction, alcohol precipitation through macroporous resin D101, polyamide, elution with 50% ethanol;

[0149] Smilax china: water extraction, alcohol precipitation with macroporous resin HPD100, polyamide, water elution;

[0150] Smilax china: water extraction, alcohol precipitation through macroporous resin HPD100, polyamide, elution with 50% ethanol;

[0151] Smilax china: water extraction, alcohol precipitation through macroporous resin HPD100, polyamide, elution with 70% ethanol;

[0152] Smilax china: water extraction, alcohol precipitation, macroporous resin HPD100, polyamide, elution with 95% ethanol.

[0153] The prepared Smilax china control extract was measured by thin layer chromatography, and the measurement conditions included:

[0154] Thin layer plate: polyamide film (batch number: 20191105, Luqiao Sijia Biochemical Plastic Factory, Taizhou City, Zhejiang Province);

[0155] Developing solvent: butyl acetate-formic acid-methanol (volume ratio 8:1:1);

[0156] Color development and inspection: Aluminum chloride solution is heated at 105°C until the spots are clear, and then inspected under ultraviolet light (365nm). Figure 4 .

[0157] Group C,

[0158] Smilax china extract: passed through macroporous resin HPD100, eluted with 50% ethanol;

[0159] Smilax china extract: passed through polyamide column, eluted with 50% methanol;

[0160] Three gold tablets: pass through a polyamide column and elute with ethyl acetate;

[0161] Smilax china extract: passed through a macroporous resin HPD100-polyamide column, eluted with 50% ethanol;

[0162] Smilax china extract: passed through polyamide column, eluted with 50% ethanol;

[0163] Sanjin Tablets: Methanol ultrasonic extraction of negative samples lacking Smilax china;

[0164] Smilax china extract: polyamide column eluted with ethyl acetate;

[0165] Sarsaparilla extract: polyamide column water wash.

[0166] The prepared Smilax china control extract was measured by thin layer chromatography, and the measurement conditions included:

[0167] Thin layer plate: Thin layer chromatography silica gel precast plate (model: G, batch number: HX98383926, Merck)

[0168] Developing agent: butyl acetate-methanol-formic acid (volume ratio is 8:0.5:0.5)

[0169] Color development and inspection: Spray with a mixed aqueous solution of 1% potassium ferrocyanide and 2% ferric chloride, and inspect the results under sunlight. Figure 5 .

[0170] It can be seen that the thin layer identification spots of ethyl acetate eluent are obvious and the background interference is less, but the extract is difficult to dry and the yield is low, so 50% ethanol is selected as the elution solvent.

[0171] 1.4 Research on thin layer chromatography identification method

[0172] (1) Test solution treatment method:

[0173] After comparing extractions with different solvents such as water, methanol, and acetone, extraction methods such as ultrasound and reflux, and purification methods such as extraction and column chromatography, the experimental results determined that the treatment method for Sanjin tablets is methanol extraction, passing through a polyamide column, and eluting with 50% ethanol.

[0174] (2) Thin layer chromatography conditions: The effects of different thin layer plates, developing agents, and colorants on the determination results were investigated, including:

[0175] Group A,

[0176] Samples: astilbin, huangqi glycoside, Sanjin tablets (extracted with water, extracted with n-butanol, alkali precipitated, and then extracted with n-butanol), resveratrol, Sanjin tablets (extracted with 50% methanol and extracted with ethyl acetate), Smilax china (extracted with water, precipitated with alcohol, and washed with D101-polyamide column with water), Smilax china (extracted with water, precipitated with alcohol, and washed with 50% ethanol, and eluent of D101-polyamide column), Smilax china (extracted with water, precipitated with alcohol, and washed with 70% ethanol, and eluent of D101-polyamide column), Sanjin tablets (acetone ultrasound).

[0177] The prepared Smilax china control extract was measured by thin layer chromatography, and the measurement conditions included:

[0178] Thin layer plate: Thin layer chromatography silica gel prefabricated plate (model: GF254, batch number: 20161222, Qingdao Ocean Chemical Co., Ltd.);

[0179] Developing solvent: toluene-ethyl acetate-formic acid (5:5:0.2);

[0180] Color developer: spray with a mixed solution of 1% potassium ferrocyanide and 2% ferric chloride;

[0181] Inspection: Inspect under UV light (254nm) and UV light (365nm) before color development, and inspect under sunlight after color development.

[0182] The results are as follows Figure 6 Group B,

[0183] Samples: Smilax china (water extraction, alcohol precipitation, HPD 100-20% ethanol eluent), Smilax china (water extraction, alcohol precipitation, HPD 100-50% ethanol eluent), Smilax china (water extraction, alcohol precipitation, HPD 100-80% ethanol eluent), Smilax china (water extraction, alcohol precipitation, HPD 100-95% ethanol eluent), Sanjin tablets (50% methanol extraction, ethyl acetate extraction), Smilax china (water extraction, alcohol precipitation, D 101-polyamide column 50% ethanol eluate), Smilax china (water extraction and alcohol precipitation through HPD100-polyamide column water eluate), Smilax china (water extraction and alcohol precipitation through HPD100-polyamide column 50% ethanol eluate), Smilax china (water extraction and alcohol precipitation through HPD100-polyamide column 70% ethanol eluate), Smilax china (water extraction and alcohol precipitation through HPD100-polyamide column 95% ethanol eluate).

[0184] The prepared Smilax china control extract was measured by thin layer chromatography, and the measurement conditions included:

[0185] Thin layer board: polyamide film (batch number: 20191105, Luqiao Sijia Biochemical Plastic Factory, Taizhou City, Zhejiang Province)

[0186] Developing solvent: toluene-ethyl acetate-formic acid (5:5:0.2)

[0187] Color development and inspection: Spray with aluminum chloride solution, heat at 105℃ until the spots are clear, and inspect under ultraviolet light (365nm). Figure 7 shown.

[0188] Group C,

[0189] sample:

[0190] The negative sample lacking Smilax china (ethyl acetate eluate after methanol extraction and polyamide column);

[0191] Smilax china (water extraction, 80% ethanol precipitation, HPD100-polyamide column, ethyl acetate eluent);

[0192] Three gold tablets (methanol extraction polyamide column ethyl acetate eluent);

[0193] Three gold tablets (methanol extraction polyamide column ethyl acetate eluent);

[0194] Three gold tablets (methanol extraction polyamide column 50% ethanol eluent);

[0195] Three gold tablets (methanol extraction polyamide column ethyl acetate eluent);

[0196] Smilax china (water extraction, 80% ethanol precipitation, HPD100-polyamide column, ethyl acetate eluent);

[0197] The prepared Smilax china control extract was measured by thin layer chromatography, and the measurement conditions included:

[0198] Thin layer plate: Thin layer chromatography silica gel prefabricated plate (model: G, batch number: 20190606, Qingdao Ocean Chemical Co., Ltd.)

[0199] Developing solvent: butyl acetate-methanol-formic acid (8:0.5:0.5)

[0200] Color development: ① Spray with a mixed solution of 1% potassium ferrocyanide and 2% ferric chloride; ② Spray with a 9% ferric chloride ethanol solution;

[0201] Inspection: Inspection under daylight. Figure 8 shown.

[0202] In this embodiment, Figure 6 It is measured when the thin layer plate is GF254 thin layer chromatography silica gel prefabricated plate. Figure 7 It is measured when the thin layer plate is a polyamide film. Figure 8It is measured when the thin layer plate is a silica gel G type prefabricated plate, and Figure 1 It was measured when the thin layer plate was a thin layer chromatography silica gel prefabricated plate (model: HSG, batch number: 20210402, Yantai Yinlong), and the development effects of silica gel GF254 thin layer plate, silica gel G thin layer plate, polyamide film, high-efficiency silica gel G thin layer plate, etc. were compared. The results showed that the high-efficiency silica gel G thin layer plate had a better development effect. Therefore, the high-efficiency silica gel G thin layer plate is preferably selected as the thin layer plate for the thin layer chromatography identification method of the present invention.

[0203] In this embodiment, Figure 6 The corresponding developing solvent is toluene-ethyl acetate-formic acid (volume ratio is 5:5:0.2). Figure 7 The corresponding developing solvent is toluene-ethyl acetate-formic acid (volume ratio is 5:5:0.2), Figure 8 The corresponding developing solvent is butyl acetate-methanol-formic acid (volume ratio is 8:0.5:0.5), and Figure 1 The corresponding developing agent is butyl acetate-methanol-formic acid-water (volume ratio is 16: 3: 1: 1). This embodiment compares various developing agents such as toluene-ethyl acetate-formic acid (volume ratio is 5: 5: 0.2), butyl acetate-methanol-formic acid (volume ratio is 8: 0.5: 0.5), butyl acetate-methanol-formic acid-water (volume ratio is 16: 3: 1: 1), and the result shows that the developing effect is better with butyl acetate-methanol-formic acid-water (volume ratio is 16: 3: 1: 1) as the developing agent, and the spots are clear. Therefore, butyl acetate-methanol-formic acid-water (volume ratio is 16: 3: 1: 1) is preferably selected as the developing agent.

[0204] This example investigates the color development effects of a mixed solution of 1% potassium ferrocyanide and 2% ferric chloride and a 9% ferric chloride ethanol solution as color developers. Figure 8 As shown in the results, 9% ferric chloride ethanol solution has better color development effect, less background interference and good reproducibility. Therefore, 9% ferric chloride ethanol solution is preferably selected as the color developer.

[0205] Embodiment 2,

[0206] 2.1. Methods for testing the quality of Sanjin preparations

[0207] 22 batches of commercial production scale Sanjin tablets were taken and measured using the method of this embodiment:

[0208] (1) Preparation of test solution:

[0209] Take 15 pieces (small pieces) or 10 pieces (large pieces) of Sanjin Tablets, grind them into powder, add 50 ml of methanol, ultrasonically treat for 45 minutes, filter, evaporate to dryness, add 25 ml of water to the residue to dissolve it, centrifuge (speed is 4000 revolutions per minute) for 10 minutes, take the supernatant and add it to the treated polyamide column (30-100 mesh, 5 g, column inner diameter is 1.5 cm, wet column loading), wash with water until colorless, discard the water washing liquid, and then elute with 100 ml of 50% ethanol, collect the eluate, evaporate to dryness, add 2 ml of methanol to the residue to dissolve it, and use it as the test solution;

[0210] The preparation of the polyamide column includes: soaking the polyamide powder in 90-95% ethanol, stirring continuously, removing bubbles and then loading into the chromatography column, washing with 3-4 times the volume of 90-95% ethanol until the washing liquid is transparent and there is no residue (or very little residue) after evaporation, and setting aside.

[0211] In this step, the polyamide column can be replaced with a macroporous resin column. The preparation of the macroporous resin column includes: soaking the macroporous resin with ethanol, wet-packing the column, and washing with ethanol until no white matter is produced when the ethanol drips into water.

[0212] (2) Preparation of Smilax china reference solution:

[0213] Take the Smilax china control extract prepared in 1.1 of Example 1, add 1 ml of methanol to dissolve the Smilax china control extract, and use it as the reference solution.

[0214] (3) Preparation of negative control sample solution lacking Smilax china:

[0215] Prepare the negative control sample solution in the same manner as the test sample solution in (1).

[0216] Specifically, remove the medicinal material Smilax china and take the medicinal materials of Chrysanthemum root, Ophiopogon japonicus, Rhizoma Cibotii and Centella asiatica in Sanjin Tablets, extract, concentrate and dry them according to the method of step (1), add appropriate amount of auxiliary materials, and obtain a negative control sample lacking Smilax china.

[0217] (4) Determination by thin layer chromatography:

[0218] Chromatographic conditions:

[0219] Temperature: 25℃;

[0220] Relative humidity: 60%;

[0221] Spread: 8.0cm;

[0222] Spotting volume: 10μ1;

[0223] Thin layer plate: Thin layer chromatography silica gel precast plate (model: HSG, batch number: 20210402, Yantai Yinlong);

[0224] Developing solvent: butyl acetate-methanol-formic acid-water (volume ratio: 16:3:1:1);

[0225] Color development and inspection: Spray with 9% ferric chloride ethanol solution and inspect under sunlight. Fig. 9 , Fig.10 .

[0226] In particular, in this embodiment, the developer may also be a mixed solution of 1% potassium ferrocyanide and 2% ferric chloride.

[0227] 2.2. Investigation of the development conditions and specificity of thin layer chromatography:

[0228] 2.2.1. This example investigates the effects of three development systems on the determination results: butyl acetate-methanol-formic acid-water (volume ratio: 16:3:1:1), ethyl acetate-methanol-formic acid (volume ratio: 10:1:1), and toluene-ethyl acetate-formic acid (volume ratio: 4:10:1).

[0229] Specifically, the samples include: negative control lacking Smilax china, Smilax china control extract, and test samples with batch numbers 220125, 220210, 220222, 200615, 210932, and 211217. The assay conditions include:

[0230] Chromatographic conditions:

[0231] Temperature: 25℃;

[0232] Relative humidity: 60%;

[0233] Spread: 8.0cm;

[0234] Spotting volume: 10 μl;

[0235] Thin layer plate: Thin layer chromatography silica gel prefabricated plate (model: HSG, batch number: 20210402, Yantai Yinlong)

[0236] Developing agent: ①Butyl acetate-methanol-formic acid-water (16:3:1:1), ②Ethyl acetate-methanol-formic acid (10:1:1), ③Toluene-ethyl acetate-formic acid (4:10:1)

[0237] Color development and inspection: Spray with 9% ferric chloride ethanol solution and inspect under sunlight. Fig.11

[0238] Depend on Fig.11It can be seen that, firstly, butyl acetate-methanol-formic acid-water (volume ratio: 16:3:1:1) has the best development effect, with clear spots, no interference with the negative control, and strong specificity; secondly, compared with ethyl acetate-methanol-formic acid (volume ratio: 10:1:1), toluene-ethyl acetate-formic acid (volume ratio: 4:10:1) has a better development effect. When the developing agent is toluene-ethyl acetate-formic acid (volume ratio: 4:10:1), the spots are relatively clearer, and the negative control has less interference. Therefore, toluene-ethyl acetate-formic acid (volume ratio: 4:10:1) can also be used as an alternative to the developing agent of the present application.

[0239] 2.2.2 This example investigates the effect of sample volume on the results of thin layer chromatography.

[0240] 3, 5, 10, and 15 μl of the control extract solution and the test solution were respectively spotted on the same high-efficiency silica gel G thin layer plate (thin layer chromatography silica gel prefabricated plate (model: HSG, batch number: 20210402, Yantai Yinlong)), developed with butyl acetate-methanol-formic acid-water (volume ratio of 16:3:1:1), taken out, dried, sprayed with 9% ferric chloride ethanol solution, and examined under sunlight. The results are shown in Table 1. Fig.12 .

[0241] Depend on Fig.12 It can be seen that corresponding spots can be detected on the thin layer plate with different spotting amounts. When the spotting amount is 15 μl, the spots have tailing phenomenon and large background interference. When the spotting amount is 3 μl, the spots are lighter. Taking into account many influencing factors in the work, the spotting amount of the control extract solution and the test solution should be 5-10 μl.

[0242] 2.2.3 This example investigates the effects of different thin layer plates on the results of thin layer chromatography;

[0243] This example investigates the thin layer high-efficiency silica gel prefabricated plates of three manufacturers: Yantai Yinlong, Merck, and Qingdao Ocean, specifically including:

[0244] ① Thin layer chromatography silica gel prefabricated plate (model: HSG, batch number: 20210402, Yantai Yinlong);

[0245] ② Thin layer chromatography silica gel prefabricated plate (model: HPTLC, batch number: 812011, Merck);

[0246] ③ Thin layer chromatography silica gel prefabricated plate (model: high efficiency, batch number 20210503, Qingdao Ocean);

[0247] Developing solvent: butyl acetate-methanol-formic acid-water ((16:3:1:1));

[0248] Color development and inspection: Spray with 9% ferric chloride ethanol solution and inspect under sunlight. Fig.13 .

[0249] The results of the test are shown in Fig.13 .Depend on Fig.13 It can be seen that in the test sample chromatogram, two spots of the same color appear at the corresponding positions in the control extract chromatogram. The spots are clear and have good separation, indicating that the durability of thin layer plates from different manufacturers is good.

[0250] 2.2.4 This example investigates the effect of different temperatures on the results of thin layer chromatography;

[0251] The chromatographic detection of this embodiment is carried out at 5°C, 25°C, and 30°C respectively; specifically, the measurement conditions also include:

[0252] Samples: negative control lacking Smilax china, Smilax china control extract, test products (lot numbers: 200615, 210932, 211217);

[0253] Spread: 8.0cm;

[0254] Relative humidity: 60%;

[0255] Spotting volume: 10 μl;

[0256] Thin layer plate: Thin layer chromatography silica gel precast plate (model: HSG, batch number: 20210402, Yantai Yinlong);

[0257] Developing solvent: butyl acetate-methanol-formic acid-water (16:3:1:1);

[0258] Color development and inspection: Spray with 9% ferric chloride ethanol solution and inspect under sunlight. Fig.14 .

[0259] Depend on Fig.14 It can be seen that under different temperature conditions, in the chromatogram of the test sample, two spots of the same color appear at the corresponding positions of the chromatographic peaks of the control extract. The spots are clear and have good separation, indicating that this identification method is suitable for temperature conditions of 5 to 30°C.

[0260] 2.2.5 This example investigates the effect of different humidity on the results of thin layer chromatography;

[0261] The chromatographic detection in this embodiment is carried out under the conditions of relative humidity of 30%, 60% and 90% respectively; specifically, the measurement conditions also include:

[0262] Samples: Smilax china control extract, Smilax china negative control, test products (lot numbers: 200615, 210932, 211217);

[0263] Spread: 8.0cm;

[0264] The sample volume was: 10 μl;

[0265] Temperature: 25℃;

[0266] Thin layer plate: Thin layer chromatography silica gel precast plate (model: HSG, batch number: 20210402, Yantai Yinlong);

[0267] Developing solvent: butyl acetate-methanol-formic acid-water (16:3:1:1);

[0268] Color development and inspection: Spray with 9% ferric chloride ethanol solution and inspect under sunlight. Fig.15 .Depend on Fig.15 It can be seen that under different relative humidity conditions, in the chromatogram of the test sample, two spots of the same color appear at the corresponding positions of the chromatographic peak of the reference sample. The spots are clear and have good separation, indicating that this identification method is suitable for humidity conditions of 30% to 90% relative humidity.

[0269] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A control extract of Smilax china, characterized in that: The Smilax china control extract was prepared by the following method: The raw medicinal material of Smilax china is subjected to water extraction to obtain a Smilax china extract, and the Smilax china extract is concentrated, centrifuged, purified, the solvent is recovered, and freeze-dried to obtain a Smilax china control extract.

2. The Smilax china control extract according to claim 1, characterized in that: The purification comprises: taking the centrifugal liquid after centrifugation and adding it to a polyamide column or a macroporous resin column for elution purification; Preferably, the mass ratio of the polyamide or macroporous resin to the raw medicinal material of Smilax china is 1:1-4; preferably, 1:2; Preferably, the preparation of the polyamide or macroporous resin column comprises: soaking the polyamide powder or macroporous resin with ethanol, wet packing the column, washing with ethanol until no white matter is produced when the ethanol drips into water, and / or washing with drinking water until the ethanol content is less than 5%.

3. The Smilax china control extract according to claim 2, characterized in that: The elution solvent used for elution purification includes one or two of ethanol solution and ethyl acetate; Preferably, the elution solvent is an ethanol solution with a mass fraction of 20% to 70%; Preferably, the elution solvent is an ethanol solution with a mass fraction of 50%; Preferably, the volume ratio of the elution solvent to the centrifuge liquid is 6 to 10:

1.

4. A Smilax china control extract according to any one of claims 1 to 3, characterized in that: The Smilax china extract is concentrated to obtain a concentrated solution, wherein the weight ratio of the concentrated solution to the Smilax china raw medicinal material is 0.5 to 5:1, preferably 5:

1.

5. A Smilax china control extract according to any one of claims 1 to 4, characterized in that: The water extraction comprises: dissolving the raw medicinal material of Smilax china in water, filtering the obtained filtrate to obtain the Smilax china extract; Preferably, the raw medicinal material of Smilax china is extracted twice with water; Preferably, water is added for extraction twice, the amount of water used for the first extraction is 8 to 14 times the mass of the raw material of Smilax china, and the amount of water used for the second extraction is 6 to 10 times the mass of the raw material of Smilax china.

6. A Smilax china control extract according to any one of claims 1 to 5, characterized in that: The Smilax china control extract is brownish yellow to reddish brown powder and is hygroscopic.

7. A quality control method for the Smilax china control extract according to any one of claims 1 to 6, characterized in that: Thin layer chromatography was used to identify the reference extract of Smilax china.

8. The quality control method according to claim 7, characterized in that: Prepare a Smilax china control extract solution as a test solution and a Smilax china control medicinal material solution, draw the test solution and the control medicinal material solution and spot them on the same thin layer plate, use butyl acetate-methanol-formic acid-water system or toluene-ethyl acetate-formic acid as a developing agent, and perform thin layer identification; Preferably, the volume ratio of butyl acetate-methanol-formic acid-water is 16:3:1:1, and the volume ratio of toluene-ethyl acetate-formic acid is 4:10:1; Preferably, the developer is a 9% ferric chloride ethanol solution or a mixed solution of 1% potassium ferrocyanide-2% ferric chloride; Preferably, the thin layer plate is a high-efficiency silica gel G thin layer plate.

9. A method for detecting the quality of Sanjin preparations using the Smilax china control extract according to any one of claims 1 to 6, characterized in that: The Smilax china reference extract is prepared into a reference solution, and a Sanjin preparation test solution is prepared, and the Smilax china reference solution and the Sanjin preparation test solution are respectively taken on the same thin layer plate for determination by thin layer chromatography; Preferably, in the thin layer chromatography method, butyl acetate-methanol-formic acid-water system or toluene-ethyl acetate-formic acid system is used as a developing solvent; Preferably, the volume ratio of butyl acetate-methanol-formic acid-water is 16:3:1:1, and the volume ratio of toluene-ethyl acetate-formic acid is 4:10:1; Preferably, the developer is a 9% ferric chloride ethanol solution or a mixed solution of 1% potassium ferrocyanide-2% ferric chloride; Preferably, the spotting amount of the Smilax china reference solution and the test solution in the thin layer chromatography method is 5 to 10 μl, the temperature during development is 5 to 30° C., and the humidity is 30% to 90%; Preferably, the spotting volume is 10 μl, the temperature during development is 25° C., and the humidity is 60%.

10. The method for detecting the quality of three gold preparations according to claim 9, characterized in that: The preparation of the Sanjin preparation test solution includes: adding the Sanjin preparation extract to a polyamide or macroporous resin column, eluting and purifying, collecting the eluate, evaporating the eluate to dryness, and then adding methanol to dissolve the eluate to obtain the test solution; Preferably, the elution purification comprises: first eluting with water to purify until colorless, draining the water washing liquid, then eluting with ethanol, and collecting the eluate; Preferably, the mass fraction of the ethanol is 50%; Preferably, the preparation of the polyamide column or macroporous resin column comprises: soaking the polyamide powder or macroporous resin in ethanol and stirring to remove bubbles, and then loading the polyamide powder or macroporous resin into a chromatography column; Preferably, the polyamide powder or macroporous resin is soaked in 90-95% by mass ethanol; Preferably, the Sanjin preparation is placed in methanol, sonicated, filtered, the filtrate after filtration is evaporated to dryness, dissolved in water, centrifuged, and the supernatant obtained by centrifugation is used as the Sanjin preparation extract.

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