Method for identifying rhizoma drynariae in glucosamine chondroitin calcium tablets
By optimizing the sample extraction and preparation process and selecting appropriate developer, the problem of unsatisfactory identification and separation effect of bone chondroitin calcium tablets in the prior art was solved, and the identification effect of high specialization, repeatability and durability was achieved.
Patent Information
- Application Number
- CN202411910637.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-03-28
AI Technical Summary
When existing thin-layer chromatography is used to identify bone granule extracts in glucosamine chondroitin calcium tablets, the separation effect is not ideal, the reproducibility is poor, and it is difficult to achieve accurate identification.
By optimizing the sample extraction and preparation process and selecting appropriate developer, the interference of turmeric extract was removed, and thin-layer chromatography was used to identify the bone granule extract. The specific steps include preparing a control reagent solution, a test sample solution and a control medicinal material solution, performing sampling, unfolding, developing and examining to determine whether the calcium tablets of glucosamine chondroitin contain bone granules extract.
The accurate identification of bone graft extracts in glucosaminochondroitin calcium tablets has been achieved, the interference of turmeric extract was removed, the specificity, repetition and durability of the detection were improved, and the quality control of glucosaminochondroitin calcium tablets was provided with a scientific and effective method.
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Figure CN119715898A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of detection technology, and in particular to an identification method for a rhizoma drynariae extract in glucosamine chondroitin calcium tablets. Background Art
[0002] According to the new registration system health food application requirements, health foods containing medicinal material extracts need to develop medicinal material identification and testing methods to monitor the quality of health foods. Thin layer chromatography identification method is the most common identification method in the current standards for Chinese medicinal materials and Chinese medicine preparations due to its advantages such as simple operation, rapid analysis and wide application.
[0003] Glucosamine chondroitin calcium tablets are products for bone and joint health, and common product categories include medicines and health foods. This type of product often contains the Chinese medicinal material bone drynaria, and its identification and detection methods need to be established according to relevant regulations. The "Chinese Pharmacopoeia" and the "National Drug Standard for Chinese Medicine Formula Granules-Bosu Formula Granules" both use thin layer chromatography to identify bone drynaria. Since naringin is used as a reference substance in the thin layer identification of bone drynaria, when the formula of glucosamine chondroitin tablets contains polyphenols, it will interfere with the thin layer identification of bone drynaria, affecting the identification of bone drynaria. The master's thesis of Nanjing University of Chinese Medicine, "Pharmaceutical Research on Xianling Strong Bone Oral Liquid", mentioned that due to the large number of flavonoids contained in the prescription, the thin layer identification of naringin in bone drynaria was greatly interfered, the separation effect was not ideal, and the reproducibility was poor.
[0004] Therefore, it is of great significance to develop a thin layer identification method that can effectively eliminate the interference of polyphenol components and achieve accurate identification of Drynaria fortunei in glucosamine chondroitin calcium tablet products. It also has important reference value for the health food industry and even the pharmaceutical field. Summary of the invention
[0005] Glucosamine chondroitin calcium tablets are a health food for bone and joint health, and its ingredients include turmeric extract and bone drynaria extract. When using the existing thin layer chromatography method to identify bone drynaria, the inventors noticed that the curcumin components interfered seriously, making it difficult to accurately identify bone drynaria.
[0006] After extensive research and practice, the present invention provides a method for identifying Rhizoma Drynariae in Glucosamine Chondroitin Calcium Tablets, which has the characteristics of strong specificity and good separation. The specific technical scheme of the present invention is as follows:
[0007] The invention discloses a method for identifying osteoporosis in glucosamine chondroitin calcium tablets. The ingredients of the glucosamine chondroitin calcium tablets include D-glucosamine potassium sulfate, sodium chondroitin sulfate, turmeric extract and osteoporosis extract. The osteoporosis extract is identified by thin layer chromatography, and the specific steps include:
[0008] S1, preparation of control reagent solution: taking naringin, adding organic solvent A to dissolve it, and preparing a control reagent solution;
[0009] S2, preparation of test solution: take glucosamine chondroitin calcium tablets, grind into fine powder, add extraction reagent, perform ultrasonic assisted extraction for 10-30 minutes, then centrifuge to obtain extract, concentrate the extract into solid extract, then add organic solvent B to dissolve the solid extract to obtain the test solution;
[0010] S3, preparation of control medicinal material solution:
[0011] S3.1, taking Drynaria officinalis, adding water, and then extracting in boiling water bath under reflux, and concentrating the extract to obtain a solid extract;
[0012] S3.2, replace the glucosamine chondroitin calcium tablets in step S2 with the solid extract prepared in step S3.1, and then prepare a control medicinal material solution using the same method as step S2;
[0013] S4, spotting, developing, developing, and inspecting:
[0014] S4.1, aspirate the control reagent solution, control medicinal material solution, and test sample solution and spot them on the same silica gel G thin layer plate, and develop them with a developing agent;
[0015] S4.2, take out the developed silica gel G thin layer plate, dry it, spray it with a developing reagent, and then heat it at 90-120°C for 1-3 minutes;
[0016] S4.3. Inspect under ultraviolet light at 365 nm and compare the spots formed by the test solution on the silica gel G thin layer plate with the spots formed by the control reagent and control medicinal material solutions. Determine whether the glucosamine chondroitin calcium tablets are added with osteopyralis extract based on the positions of the spots.
[0017] Those skilled in the art will understand that when the control reagent solution, the control medicinal material solution, and the test sample solution are spotted on the same silica gel G thin layer plate, it should be ensured that the spots are on the same straight line, and the straight line is parallel or perpendicular to the long side of the G thin layer plate.
[0018] Those skilled in the art will understand that if the control reagent solution and the control medicinal material solution are spotted on the G thin layer plate at the same horizontal position where the spots are formed, and the test solution also forms corresponding spots, then it can be determined that the glucosamine chondroitin calcium tablets are added with osteopyralis extract.
[0019] In certain specific embodiments, the ingredients of the glucosamine chondroitin calcium tablets also include eucommia extract, sodium hyaluronate, and vitamin D3 powder.
[0020] In certain specific embodiments, organic solvent A and organic solvent B are the same, namely methanol.
[0021] In certain specific embodiments, the concentration of naringin in step S1 is 0.5-1 mg / mL.
[0022] In certain specific embodiments, the extraction reagent in step S2 comprises methanol.
[0023] In certain specific embodiments, before the extract is concentrated into a solid extract in step S2, chloroform is added to the extract and shaken for 1-3 minutes, and then water is added and shaken for 1-3 minutes. After the solution is separated, the upper layer is taken and concentrated into a solid extract. The volume ratio of the extraction reagent, chloroform and water is 4:3-4:3-4. The obtained solid extract is dissolved by adding methanol to obtain a test solution.
[0024] Those skilled in the art will understand that, for a solution system composed of two or more solvents, if all or some of the solvents are immiscible and have different densities, the solution will usually show stratification. The stratification of the solution can be naturally formed by leaving the solution to stand for a long time, or it can be accelerated by centrifugation or the like.
[0025] Preferably, the upper layer solution is extracted with chloroform for 1-3 times, the chloroform extract is removed after each extraction, and the upper layer solution of the last extraction is concentrated into a solid extract.
[0026] In certain specific embodiments, the mass volume ratio of Drynaria fortunei medicinal material to water in step S3.1 is 1:10-40.
[0027] In certain specific embodiments, the spotting volume in step S4.1 is 3-6 μL.
[0028] In certain specific embodiments, the developing agent in step S4.1 is a mixed solvent of methanol, butanone, chloroform, water and formic acid in a volume ratio of 2-3:5-6:5-6:2-4:0.5-1.
[0029] In certain specific embodiments, the developing agent in step S4.1 is a lower layer solution of a mixed solvent of methanol, butanone, chloroform, water, and formic acid in a volume ratio of 2-3:5-6:5-6:2-4:0.5-1 placed at 4-15°C for 0.5-2h.
[0030] In certain specific embodiments, the developer in step S4.2 is a 5% aluminum chloride ethanol solution.
[0031] Advantageous Effects of the Invention
[0032] The present invention establishes a thin layer chromatography detection method for the osteomyelitis extract in glucosamine chondroitin calcium tablets. Compared with the thin layer chromatography detection method of osteomyelitis in the prior art, the detection method of the present application has at least the following beneficial effects: (1) Based on the component characteristics of the glucosamine chondroitin calcium tablets and the existing identification and detection technology of the osteomyelitis extract, the present invention optimizes the sample extraction preparation process and the developing agent, which can remove the interference of the turmeric extract in the product, and solves the problems of separation difficulty, severe tailing, and difficulty in judgment caused by the complex composition of the product; (2) The thin layer chromatography detection method established by the present invention has good detection specificity, repeatability and durability when detecting the osteomyelitis extract in glucosamine chondroitin, and provides a scientific and effective method for the quality control of glucosamine chondroitin calcium tablets. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1 The thin layer chromatogram of Example 1 is shown, wherein S is the chromatographic band of the control reagent solution, D is the chromatographic band of the control medicinal material solution, and 1 and 2 are the chromatographic bands of the test solution.
[0034] Figure 2 The thin layer chromatogram of Example 2 is shown, wherein S is the chromatographic band of the control reagent solution, D is the chromatographic band of the control medicinal material solution, and 1 and 2 are the chromatographic bands of the test solution.
[0035] Figure 3 The thin layer chromatogram of Example 3 is shown, wherein S is the chromatographic band of the control reagent solution, D is the chromatographic band of the control medicinal material solution, and 1 and 2 are the chromatographic bands of the test solution.
[0036] Figure 4 The thin layer chromatogram of Comparative Example 1 is shown, wherein S is the chromatographic band of the control reagent solution, D is the chromatographic band of the control medicinal material solution, and 1 and 2 are the chromatographic bands of the test solution.
[0037] Figure 5 The thin layer chromatogram of Comparative Example 2 is shown, wherein S is the chromatographic band of the control reagent solution, D is the chromatographic band of the control medicinal material solution, and 1 and 2 are the chromatographic bands of the test solution.
[0038] Figure 6 The thin layer chromatogram of Comparative Example 3 is shown, wherein S is the chromatographic band of the control reagent solution, D is the chromatographic band of the control medicinal material solution, and 1 and 2 are the chromatographic bands of the test solution.
[0039] Figure 7 The thin layer chromatogram of Comparative Example 4 is shown, wherein S is the chromatographic band of the control reagent solution, D is the chromatographic band of the control medicinal material solution, and 1 and 2 are the chromatographic bands of the test solution.
[0040] Figure 8The thin layer chromatogram of Comparative Example 5 is shown, wherein S is the chromatographic band of the control reagent solution, D is the chromatographic band of the control medicinal material solution, and 1 and 2 are the chromatographic bands of the test solution.
[0041] Fig. 9 The thin layer chromatogram of Comparative Example 6 is shown, wherein S is the chromatographic band of the control reagent solution, D is the chromatographic band of the control medicinal material solution, and 1 and 2 are the chromatographic bands of the test solution.
[0042] Fig.10 The thin layer chromatogram of the specificity investigation is shown, wherein S is the chromatographic band of the control reagent solution, D is the chromatographic band of the control medicinal material solution, Y is the chromatographic band of the glucosamine chondroitin calcium tablet (without bone drynaria extract) sample, T is the chromatographic band of the bone drynaria extract sample, and 1 and 2 are the chromatographic bands of the glucosamine chondroitin calcium tablet (containing bone drynaria extract) sample.
[0043] Fig.11 The re-examined chromatographic bands are shown, wherein S is the chromatographic band of the control reagent solution, D is the chromatographic band of the control medicinal material solution, Y is the chromatographic band of the glucosamine chondroitin calcium tablet (without bone-healing extract) sample, and 1, 2 and 3 are the chromatographic bands of the glucosamine chondroitin calcium tablet (with bone-healing extract) sample. Specific implementation methods
[0045] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.
[0046] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references.
[0047] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.
[0048] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as every value between the minimum and maximum values. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.
[0049] The percentage contents involved in this application, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.
[0050] The percentage concentrations mentioned in this application, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.
[0051] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range of instrument control.
[0052] The reagents and materials used in the examples of the present application are conventional reagents and materials in the field of thin layer detection and can be purchased on the market.
[0053] Example 1
[0054] 1. Preparation of control reagent solution:
[0055] Take naringin and dissolve it in methanol to prepare a 0.5 mg / mL solution.
[0056] 2. Preparation of test solution:
[0057] Take 2g of glucosamine chondroitin calcium tablets, grind into fine powder, add 20mL of methanol, ultrasonically treat at room temperature for about 15min, centrifuge to obtain the extract, add 20mL of chloroform to the extract and shake for 1min, then add 15mL of water and shake for 1min, centrifuge and take the upper solution.
[0058] The upper solution was extracted twice with chloroform, 20 mL each time, and the lower chloroform liquid was discarded each time. The upper solution was concentrated into a solid after the last extraction, and 4 mL of methanol was added to dissolve it to obtain the test solution.
[0059] 3. Preparation of control medicinal material solution:
[0060] Take 1 g of Drynaria fortunei, add 20 ml of water and extract under reflux in a boiling water bath for 30 min, and concentrate the extract to obtain a solid extract; replace the glucosamine chondroitin calcium tablets with the solid extract, and prepare a control medicinal solution in the same manner as the test solution in this example.
[0061] 4. Spotting, unfolding, developing, and inspecting:
[0062] 4uL of control reagent solution, 4uL of control medicinal material solution and 4uL of test sample solution (spotted twice) were respectively spotted on the same silica gel G thin layer plate, and developed with the lower layer solution which was placed at 10℃ for 1h in a mixed solvent of methanol, butanone, chloroform, water and formic acid in a volume ratio of 3:5:5:2:0.5 as the developing agent.
[0063] The developed silica gel G thin layer plate was taken out, air-dried, sprayed with a developing agent (5% aluminum chloride ethanol solution), and then heated at 105° C. for 2-3 minutes.
[0064] The plates were examined under ultraviolet light (365 nm), and the spots formed by spotting the test sample solution on the silica gel G thin layer plate were compared with the spots formed by spotting the control reagent and the control medicinal material solution.
[0065] 5. Test results
[0066] The results are as follows Figure 1 As shown, in the chromatographic band of the test solution, very clear spots appear at the corresponding positions of the spots of the chromatographic band of the control reagent solution and the chromatographic band of the control medicinal material solution, the corresponding positions are clear, the bands are clear, and the separation effect is good. This shows that the detection method used in this embodiment can effectively identify the bone drynaria extract in the glucosamine chondroitin calcium tablets.
[0067] Example 2
[0068] The difference between this embodiment and embodiment 1 in the operation method is only the preparation of the test solution and the control medicinal material solution, which is as follows:
[0069] 1. Preparation of test solution:
[0070] Take 2g of glucosamine chondroitin calcium tablets, grind into fine powder, add 20mL of methanol, ultrasonically treat at room temperature for about 15min, centrifuge to obtain the extract, add 15mL of chloroform to the extract and shake for 1min, then add 20mL of water and shake for 1min, and take the upper solution after the solution is separated.
[0071] The upper solution was extracted twice with chloroform, 15 mL each time, and the lower chloroform liquid was discarded each time. The upper solution was concentrated into a solid after the last extraction, and 4 mL of methanol was added to dissolve it to obtain the test solution.
[0072] 2. Preparation of reference medicinal material solution:
[0073] Take 1 g of Drynaria fortunei, add 40 ml of water and extract under reflux in a boiling water bath for 20 min, and concentrate the extract to obtain a solid extract; replace the glucosamine chondroitin calcium tablets with the solid extract, and prepare a control medicinal solution in the same manner as the test solution in this example.
[0074] 3. Test results
[0075] The results are as follows Figure 2As shown, in the chromatographic band of the test solution, very clear spots appear at the corresponding positions of the chromatographic band of the control reagent solution and the chromatographic band of the control medicinal material solution, the corresponding positions are clear, the bands are clear, and the separation effect is good. This shows that the detection method used in this embodiment can effectively identify the bone drynaria extract in the glucosamine chondroitin calcium tablets.
[0076] Example 3
[0077] The difference between this embodiment and embodiment 1 in the operation method is only the volume ratio of each solvent in the developing agent. In this embodiment, the developing agent is a lower layer solution of methanol, butanone, chloroform, water, and formic acid in a volume ratio of 2:6:6:4:1 placed at 10°C for 1 hour.
[0078] 1. Test results
[0079] The results are as follows Figure 3 As shown, in the chromatographic band of the test solution, very clear spots appear at the corresponding positions of the spots of the chromatographic band of the control reagent solution and the chromatographic band of the control medicinal material solution, the corresponding positions are clear, the bands are clear, and the separation effect is good. This shows that the detection method used in this embodiment can effectively identify the bone drynaria extract in the glucosamine chondroitin calcium tablets.
[0080] Comparative Example 1
[0081] Referring to the method of the National Drug Standard for Chinese Medicine Formula Granules - Drynaria Formula Granules, the control drug material of this method is extracted by water, which is similar to the extraction process of Drynaria in this product. The specific method is as follows:
[0082] 1. Preparation of control reagent solution:
[0083] Take naringin reference substance and add methanol to make a solution containing 0.5 mg per 1 mL to obtain the reference substance solution.
[0084] 2. Preparation of test solution:
[0085] Take 2 g of glucosamine chondroitin calcium tablets, grind into fine powder, add 20 mL of methanol, ultrasonically treat at room temperature for about 15 min, centrifuge to obtain the extract and concentrate it into a solid, add 4 mL of methanol to dissolve it, and the test solution is obtained.
[0086] 3. Preparation of control medicinal material solution:
[0087] Take 1 g of Drynaria fortunei, add 20 mL of water and extract under reflux in a boiling water bath for 30 min, filter, evaporate the filtrate to dryness, add 20 mL of methanol to the residue, ultrasonically treat at room temperature for 15 min, centrifuge to obtain the extract and concentrate it into a solid, add 4 mL of methanol to dissolve it, and obtain the control medicinal solution.
[0088] 4. Spotting, unfolding, developing, and inspecting:
[0089] According to the thin layer chromatography method (General Rule 0502), 4 μL of the control reagent solution and the control medicinal material solution were taken, and the test solution was spotted in an amount of 4 μL (spotted twice), and they were spotted on the same silica gel G thin layer plate. The upper layer solution of toluene-ethyl acetate-formic acid-water (1:12:2.5:3) was used as the developing agent. The plate was developed, taken out, dried, sprayed with aluminum chloride test solution, and examined under ultraviolet light (365 nm).
[0090] 5. Test results
[0091] The results are as follows Figure 4 As shown, using the national drug standard identification method for Chinese herbal formula granules, the chromatographic bands of the test sample solution had serious matrix interference, and the main spot of the test sample was masked by the pigment of the turmeric extract in the product, resulting in the inability to distinguish the main spot and to identify whether Rhizoma Drynariae extract was added to the Glucosamine Chondroitin Calcium Tablets.
[0092] Comparative Example 2
[0093] In the thin layer identification method, a neutral alumina column is often used to adsorb interfering pigments in the sample, so this method is tried to remove the pigment interference caused by the turmeric extract in the sample. The difference between this comparative example and Example 1 in the operation method is only the preparation method of the test solution and the control medicinal material solution, which is as follows:
[0094] 1. Preparation of test solution:
[0095] Take 2g of glucosamine chondroitin calcium tablets, grind them into fine powder, add 20mL of methanol, ultrasonically treat at room temperature for about 15min, centrifuge for 5min, take the supernatant and evaporate to dryness, add 5mL of water to the residue to dissolve it, add the solution to a neutral alumina column, discard the effluent, elute with 40% methanol, collect the eluate, evaporate to dryness in a water bath, dissolve the residue with 4mL of methanol to obtain the test solution.
[0096] 2. Preparation of control medicinal material solution:
[0097] Take 1 g of Drynaria fortunei, add 20 mL of water and extract under reflux in a boiling water bath for 30 min, and concentrate the extract to obtain a solid extract; replace the glucosamine chondroitin calcium tablets with the solid extract, and prepare a control medicinal solution in the same manner as the test solution of this comparative example.
[0098] 3. Test results
[0099] The results are as follows Figure 5As shown, there is no interference of turmeric extract pigment in the chromatographic band of the test solution, but there is no spot corresponding to the chromatographic band of the control reagent in the chromatographic band of the test solution and the chromatographic band of the control medicinal material. This shows that although the use of a neutral alumina column can effectively remove the interference of turmeric extract pigment, the characteristic components in Drynaria Rhizoma are also removed at the same time, which cannot achieve the identification effect of Drynaria Rhizoma extract in Glucosamine Chondroitin Calcium Tablets.
[0100] Comparative Example 3
[0101] Taking advantage of the difference in solubility of substances in different solvents, extraction with specific solvents is a common method to remove interfering substances. Since curcumin is slightly soluble in water and the bone drynaria extract is easily soluble in water, water is used to further extract the crude extract. Since most pigments cannot be dissolved, the interference of curcumin can theoretically be removed by centrifugation. The difference in the operation method between this comparative example and Example 1 is only the preparation method of the test solution and the control medicinal material solution, which is as follows:
[0102] 1. Preparation of test solution:
[0103] Take 2g of glucosamine chondroitin calcium tablets, grind into fine powder, add 20mL of methanol, ultrasonically treat at room temperature for about 15min, centrifuge to obtain the extract and concentrate it into solid. Add 15mL of water to the solid to fully dissolve it, centrifuge and take the supernatant.
[0104] The supernatant was extracted twice with chloroform, 20 mL each time, and the lower layer of chloroform was discarded each time. The upper layer solution was concentrated into a solid after the last extraction, and 4 mL of methanol was added to dissolve it to obtain the test solution.
[0105] 2. Preparation of control medicinal material solution:
[0106] Take 1 g of Drynaria fortunei, add 20 mL of water and extract under reflux in a boiling water bath for 30 min, and concentrate the extract to obtain a solid extract; replace the glucosamine chondroitin calcium tablets with the solid extract, and prepare a control medicinal solution in the same manner as the test solution of this comparative example.
[0107] 3. Test results
[0108] The results are as follows Figure 6 As shown, on the one hand, there is a serious interference of turmeric extract pigment in the chromatographic band of the test solution; on the other hand, although spots appear in the chromatographic band of the test solution at the corresponding position of the spots in the chromatographic band of the control reagent, the main spots are unclear or missing at the corresponding position of the spots in the chromatographic band of the control medicinal material solution, and the corresponding position is unclear. Analysis believes that this may be because the glucosamine chondroitin calcium tablets contain more flavonoid interfering substances such as curcumin, which are similar to the structure of Drynaria fortunei, and cannot be removed by the pretreatment method of the previous comparative example. The extraction and purification efficiency is not high, and it is impossible to identify whether Drynaria fortunei extract is added to the product.
[0109] Comparative Example 4
[0110] The difference between this comparative example and Example 1 in the operation method is only the preparation method of the test solution and the control medicinal material solution, which is as follows:
[0111] 1. Preparation of test solution:
[0112] Take 2g of glucosamine chondroitin calcium tablets, grind into fine powder, add 20mL of methanol, ultrasonically treat at room temperature for about 15min, centrifuge to obtain the extract, add 15mL of water to the extract and shake for 1min, then add 20mL of chloroform and shake for 1min, centrifuge and take the upper solution.
[0113] The upper solution was extracted twice with chloroform, 20 mL each time, and the lower chloroform liquid was discarded each time. The upper solution was concentrated into a solid after the last extraction, and 4 mL of methanol was added to dissolve it to obtain the test solution.
[0114] 2. Preparation of control medicinal material solution:
[0115] Take 1 g of Drynaria fortunei, add 20 mL of water and extract under reflux in a boiling water bath for 30 min, and concentrate the extract to obtain a solid extract; replace the glucosamine chondroitin calcium tablets with the solid extract, and prepare a control medicinal solution in the same manner as the test solution of this comparative example.
[0116] 3. Test results
[0117] The results are as follows Figure 7 As shown, in the preparation of the test solution and the control medicinal material solution, when water was first added to the extraction solvent and then chloroform was added, the fluorescent spots of the chromatographic band of the test solution were missing at the corresponding positions of the spots of the chromatographic band of the control medicinal material solution. This shows that the extraction effect of the Drynaria extract using the test solution preparation scheme of this detection method is poor, and the thin layer identification effect of the Drynaria extract in the Glucosamine Chondroitin Calcium Tablets cannot be achieved.
[0118] Comparative Example 5
[0119] The difference between this comparative example and Example 1 in the operation method is only the preparation method of the test solution and the control medicinal material solution, which is as follows:
[0120] 1. Preparation of test solution:
[0121] Take 2g of glucosamine chondroitin calcium tablets, grind into fine powder, add 20mL of methanol, ultrasonically treat at room temperature for about 15min, centrifuge to obtain the extract, add 10mL of chloroform to the extract and shake for 1min, then add 5mL of water and shake for 1min, centrifuge and take the upper solution.
[0122] The upper solution was extracted twice with chloroform, 20 mL each time, and the lower chloroform liquid was discarded each time. The upper solution was concentrated into a solid after the last extraction, and 4 mL of methanol was added to dissolve it to obtain the test solution.
[0123] 2. Preparation of control medicinal material solution:
[0124] Take 1 g of Drynaria fortunei, add 20 mL of water and extract under reflux in a boiling water bath for 30 min, and concentrate the extract to obtain a solid extract; replace the glucosamine chondroitin calcium tablets with the solid extract, and prepare a control medicinal solution in the same manner as the test solution of this comparative example.
[0125] 3. Test results
[0126] The results are as follows Figure 8 As shown, in the preparation of the test solution and the control medicinal material solution, when the ratio of the extraction solvent, chloroform and water was 4:2:1, the interfering impurities such as the Rhizoma Drynariae extract and the curcumin extract in the test solution could not be separated at all, and the sample chromatogram formed a band without obvious spots, making it impossible to identify the Rhizoma Drynariae extract in the Glucosamine Chondroitin Calcium Tablets.
[0127] Comparative Example 6
[0128] The difference between this comparative example and Example 1 in the operation method is only the volume ratio of the developing agent. In this comparative example, the developing agent is a mixed solvent of methanol, butanone, chloroform, water and formic acid in a volume ratio of 3:5:5:2:0.5.
[0129] 1. Test results
[0130] A mixed solvent of methanol, butanone, chloroform, water and formic acid in a volume ratio of 3:5:5:2:0.5 was used as a developing agent to separate the extract of Drynariae Rhizoma and interfering substances.
[0131] The results are as follows Fig. 9 As shown, in the chromatographic band of the test solution, there are spots at the corresponding positions of the spots of the chromatographic band of the control reagent and the chromatographic band of the control medicinal material, but the spots are blurred and tailing, and the band boundaries are not clear. This shows that the development system cannot effectively develop the test solution, resulting in the inability of the detection method to achieve the thin layer identification effect of the bone drynaria extract in the glucosamine chondroitin calcium tablets.
[0132] Exclusive inspection
[0133] In order to ensure the detection effect of the present invention, the thin layer detection method of the present application was investigated for specificity, and the specific test method is as follows:
[0134] Tested samples: 1. Glucosamine chondroitin calcium tablets (without osteopyralis extract) 2g; 2. Glucosamine chondroitin calcium tablets 2g (containing osteopyralis extract); 3. osteopyralis extract (product raw material) 0.5g.
[0135] Detection method: The specific detection method is the same as that in Example 1, and the above-mentioned samples to be detected all adopt the preparation method of the test solution in Example 1.
[0136] The results are as follows Fig.10 As shown, at the corresponding positions where spots appeared on the control medicinal material chromatographic band, the control reagent chromatographic band, and the Drynaria extract chromatographic band, corresponding spots appeared on the chromatographic band of the glucosamine chondroitin calcium tablet (containing the Drynaria extract) sample, while the chromatographic band of the glucosamine chondroitin calcium tablet (not containing the Drynaria extract) sample had no corresponding fluorescent spots, indicating that the method has good specificity.
[0137] Repeatability test
[0138] In order to ensure the detection effect of the present invention, the thin layer detection method of the present application was investigated for repeatability, and the specific test method is as follows:
[0139] Weigh 3 portions of glucosamine chondroitin calcium tablets and 1 portion of glucosamine chondroitin tablet negative sample, each weighing 2 g. The specific detection method is the same as in Example 1.
[0140] The results are as follows Fig.11 As shown, in the chromatographic bands of the three glucosamine chondroitin calcium tablets, fluorescent spots of the same color were displayed at the corresponding positions of the control medicinal material chromatographic band and the control reagent chromatographic band, indicating that the method has good repeatability.
[0141] Durability test
[0142] In order to ensure the detection effect of the present invention, the durability of the thin layer detection method of the present application was investigated. According to Table 1, after the treatment conditions of ultrasonic treatment for 15 minutes in Example 1 were changed to ultrasonic treatment for 20 minutes, ultrasonic treatment for 30 minutes, and ultrasonic treatment for 40 minutes, the average ratio shift value and separation degree were 0.324 and 3.333, respectively, and the standard deviations were 0.029 and 0.152, respectively; the development temperature was changed from room temperature to 10°C, 25°C, and 30°C, respectively, and the average ratio shift value and separation degree were 0.319 and 3.325, respectively, and the standard deviations were 0.031 and 0.241, respectively. The ratio shift value (Rf) and the separation degree (R) both meet the requirements of the thin layer chromatography identification method (0.2<Rf<0.8, R>1). According to the above results, it can be seen that no matter whether the sample processing time or the development temperature is changed, the standard deviation is small, indicating that the thin layer identification method has good durability.
[0143] Table 1 Results of method durability investigation
[0144]
Claims
1. A method for identifying Rhizoma Drynariae in Glucosamine Chondroitin Calcium Tablets, wherein the ingredients of the Rhizoma Drynariae include D-glucosamine sulfate potassium salt, chondroitin sulfate sodium, turmeric extract and Rhizoma Drynariae extract, characterized in that: Thin layer chromatography was used to identify Rhizoma Drynariae, and the specific steps included: S1, preparation of control reagent solution: taking naringin, adding organic solvent A to dissolve it, and preparing a control reagent solution; S2, preparation of test solution: take glucosamine chondroitin calcium tablets, grind into fine powder, add extraction reagent, perform ultrasonic assisted extraction for 10-30 minutes, then centrifuge to obtain extract, concentrate the extract into solid extract, then add organic solvent B to dissolve the solid extract to obtain the test solution; S3, preparation of control medicinal material solution: S3.1, taking Drynaria officinalis, adding water, and then extracting in boiling water bath under reflux, and concentrating the extract to obtain a solid extract; S3.2, replace the glucosamine chondroitin calcium tablets in step S2 with the solid extract prepared in step S3.1, and then prepare a control medicinal material solution using the same method as step S2; S4, spotting, developing, developing, and inspecting: S4.1, aspirate the control reagent solution, control medicinal material solution, and test sample solution and spot them on the same silica gel G thin layer plate, and develop them with a developing agent; S4.2, take out the developed silica gel G thin layer plate, dry it, spray it with a developing reagent, and then heat it at 90-120°C for 1-3 minutes; S4.
3. Inspect under ultraviolet light at 365 nm and compare the spots formed by the test solution on the silica gel G thin layer plate with the spots formed by the control reagent and control medicinal material solutions. Determine whether the glucosamine chondroitin calcium tablets are added with osteopyralis extract based on the positions of the spots.
2. The identification method of Rhizoma Drynariae according to claim 1, characterized in that: The ingredients of the glucosamine chondroitin calcium tablets also include eucommia extract, sodium hyaluronate, and vitamin D3 powder.
3. The identification method of Rhizoma Drynariae according to claim 1, characterized in that: The organic solvent A and the organic solvent B are the same, both are methanol.
4. The identification method of Rhizoma Drynariae according to claim 1, characterized in that: The concentration of naringin in step S1 is 0.5-1 mg / mL.
5. The identification method of Rhizoma Drynariae according to claim 1, characterized in that: The extraction reagent in step S2 includes methanol.
6. The identification method of Rhizoma Drynariae according to claim 1, characterized in that: Before concentrating the extract into a solid extract in step S2, chloroform is added to the extract and shaken for 1-3 minutes, and then water is added and shaken for 1-3 minutes. After the solution is separated into layers, the upper layer of the solution is taken and concentrated into a solid extract; wherein the volume ratio of the extraction reagent, chloroform and water is 4:3-4:3-4.
7. The identification method of Rhizoma Drynariae according to claim 6, characterized in that: The upper layer solution is extracted with chloroform for 1-3 times, and the chloroform extract is removed after each extraction. The upper layer solution of the last extraction is concentrated into a solid extract.
8. The identification method of Rhizoma Drynariae according to claim 1, characterized in that: In step S3.1, the mass volume ratio of Drynaria fortunei medicinal material to water is 1:10-40.
9. The identification method of Rhizoma Drynariae according to claim 1, characterized in that: The developing agent in step S4.1 is a mixed solvent of methanol, butanone, chloroform, water and formic acid in a volume ratio of 2-3:5-6:5-6:2-4:0.5-1; Preferably, the developing agent in step S4.1 is a lower layer solution of a mixed solvent of methanol, butanone, chloroform, water and formic acid in a volume ratio of 2-3:5-6:5-6:2-4:0.5-1 placed at 4-15°C for 0.5-2h.
10. The identification method of Rhizoma Drynariae according to claim 1, characterized in that: The sample spotting volume in step S4.1 is 3-6 μL, and the developer in step S4.2 is 5% aluminum chloride ethanol solution.