Rapid detection method for medicinal material components of ginseng and tortoise foundation strengthening wine based on thin-layer chromatography technology

Abstract

The invention relates to the technical field of quality control of traditional Chinese medicines, in particular to a thin-layer chromatography technology-based rapid detection method for medicinal material components of ginseng and tortoise foundation strengthening wine, which comprises the following steps: preparing a test solution, a reference medicinal material solution and a reference substance solution, and performing thin-layer chromatography analysis by adopting a specific developing solvent system and developing conditions. The method can effectively separate and qualitatively analyze various key medicinal material components in the ginseng and tortoise foundation strengthening wine, and has the advantages of clear spots, stable ratio shift value and good reproducibility. The method is easy and convenient to operate, low in equipment cost and suitable for being popularized and used in basic laboratories, has good durability and stability under different environments and operation conditions, provides a scientific basis for quality control of the ginseng and tortoise foundation strengthening wine, fills the technical blank of rapid detection of medicinal material components of the preparation based on the thin-layer chromatography technology, and has a wide application prospect. The product quality and the medication safety are ensured.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection of traditional Chinese medicine components, and specifically relates to a rapid detection method for the components of Shengui Guben Wine based on thin-layer chromatography technology. Background Art

[0002] Shengui Guben Wine is a traditional Chinese medicine health wine mainly made from angelica sinensis, astragalus membranaceus, codonopsis pilosula, poria cocos, cornelian cherry fruit, ligusticum wallichii, atractylodes macrocephala, schizandra chinensis, tangerine peel, wolfberry fruit, ledebouriella seseloides, notopterygium incisum, poria with hostwood, ophiopogon japonicus, rehmannia glutinosa, prepared rehmannia root, Chinese date, and tortoise plastron glue. It has the effects of replenishing qi and nourishing blood, nourishing yin and boosting yang, and is widely used in the fields of health care and adjuvant treatment. The "Pharmacopoeia of the People's Republic of China" and relevant traditional Chinese medicine literature have detailed records and explanations on its functions, prescription principles, and scope of application. The components of the medicinal materials in Shengui Guben Wine are complex and contain various active substances, and its quality control is crucial for ensuring the efficacy and safety of the product. At present, the detection methods for such compound traditional Chinese medicine preparations mainly rely on modern analytical techniques such as high-performance liquid chromatography (HPLC) and gas chromatography (GC). Although these methods can provide relatively accurate quantitative analysis results, they have high requirements for equipment and cumbersome operation steps, and it is difficult to meet the needs of rapid detection. In contrast, thin-layer chromatography technology has been widely used in the qualitative analysis of traditional Chinese medicine components due to its simple operation, low cost, and strong intuitiveness. However, there are few reports on the rapid detection of the components of Shengui Guben Wine medicinal materials based on thin-layer chromatography technology in existing research, and a systematic detection scheme has not been formed. Therefore, developing a rapid detection method for the components of Shengui Guben Wine medicinal materials based on thin-layer chromatography technology is of great significance for improving the efficiency of product quality control. Summary of the Invention

[0003] The purpose of the present invention is to provide a rapid detection method for the components of Shengui Guben Wine medicinal materials based on thin-layer chromatography technology in view of the deficiencies of the prior art. Shengui Guben Wine is prepared from angelica sinensis, astragalus membranaceus, codonopsis pilosula, poria cocos, cornelian cherry fruit, ligusticum wallichii, atractylodes macrocephala, schizandra chinensis, tangerine peel, wolfberry fruit, ledebouriella seseloides, notopterygium incisum, poria with hostwood, ophiopogon japonicus, rehmannia glutinosa, prepared rehmannia root, Chinese date, and tortoise plastron glue, and its medicinal material components are complex and there are many types of active substances. The existing quality control methods mainly rely on high-performance liquid chromatography (HPLC) or gas chromatography (GC). Although these methods can provide accurate quantitative analysis results, the equipment is expensive and the operation is cumbersome, and it is difficult to meet the needs of rapid detection. The present invention uses thin-layer chromatography technology, combined with specific sample treatment and development conditions, to qualitatively analyze various key medicinal material components in Shengui Guben Wine, with good separation effect, clear and distinct spots, moderate development rate, and intuitive and easy-to-judge results.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] A rapid detection method for medicinal components in Ginseng and Tortoise Tonic Wine based on thin-layer chromatography technology, the method comprising the following steps:

[0006] (1) Preparation of test solution: Take 5 ml of Ginseng and Turtle Tonic Wine sample, add 20 ml of ethyl acetate, shake thoroughly for 3 minutes, let stand for layering, separate the organic phase, concentrate the organic phase under reduced pressure in a 40℃ water bath to dryness, dissolve the residue in 1 ml of methanol, and use it as test solution.

[0007] (2) Preparation of control medicinal material solutions: Take 1g each of Angelica sinensis, Astragalus membranaceus, Codonopsis pilosula, Poria cocos, Cornus officinalis, Ligusticum chuanxiong, Atractylodes macrocephala, Schisandra chinensis, Citrus reticulata, Lycium barbarum, Saposhnikovia divaricata, Notopterygium incisum, Poria cocos, Ophiopogon japonicus, Rehmannia glutinosa, Rehmannia glutinosa (processed), Ziziphus jujuba, and tortoise shell glue. Add 25ml of ethanol to each, sonicate for 30 minutes, filter, concentrate the filtrate under reduced pressure to dryness in a 40℃ water bath, dissolve the residue in 1ml of methanol, and prepare the corresponding control medicinal material solutions.

[0008] (3) Preparation of reference solutions: Ferulic acid from Angelica sinensis, astragaloside A from Astragalus membranaceus, codonopsis glycoside from Codonopsis pilosula, poria cocos acid from Poria cocos, loganin from Cornus officinalis, ligusticum chuanxiong lactone from Ligusticum chuanxiong, atractylone from Atractylodes macrocephala, schisandrin A from Schisandra chinensis, hesperidin from Citrus reticulata peel, betaine from Lycium barbarum, cimicifuga glycoside from Saposhnikovia divaricata, notopterygium incisum, poria cocos acid from Poria cocos, ophiopogonin from Ophiopogon japonicus, catalpol from Rehmannia glutinosa, verbascoside from Rehmannia glutinosa (processed), cyclic adenosine monophosphate from Ziziphus jujuba, and amino acids from tortoise shell glue were selected as reference standards and prepared into solutions containing 1 mg per ml with methanol as reference solutions.

[0009] (4) Detection: Perform the test according to the thin-layer chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Take 2 μl of each of the above test sample solution, reference medicinal material solution and reference solution, and spot them separately on the same silica gel GF254 thin-layer plate. Use petroleum ether-ethyl acetate-methanol-glacial acetic acid with a volume ratio of 6:8:2:1 as the developing solvent. Develop, remove, air dry, and examine under a UV lamp at 254 nm. Record the position and color of the spots. Then spray with 10% sulfuric acid ethanol solution and heat at 105℃ until the spots are clearly visible. Examine under sunlight. In the chromatogram of the test sample, spots of the same color should appear at the corresponding positions as in the chromatograms of the reference medicinal material and the reference solution.

[0010] The above-mentioned rapid detection method for medicinal components of Shengui Guben Wine based on thin-layer chromatography requires that the ethyl acetate extraction process described in step (1) be carried out at room temperature, with a shaking frequency of 200 times per minute and a shaking time of 3 minutes.

[0011] The ultrasonic treatment described in step (2) must be performed at a power of 250W and a frequency of 40kHz for 30 minutes.

[0012] In the preparation process of the reference substance solution described in step (3), methanol of analytical purity grade is required and it is filtered through a 0.45 μm microporous filter membrane for standby.

[0013] The beneficial effects of the present invention are as follows: The present invention provides a rapid detection method for the medicinal material components of Shengui Guben Wine based on thin-layer chromatography technology. Through the preparation of the test solution, the reference medicinal material solution and the reference substance solution, combined with a specific developing agent system and color development conditions, effective separation and qualitative analysis of various key medicinal material components in Shengui Guben Wine are achieved. The thin-layer chromatogram obtained by using this method has uniform spot distribution, regular shape, obvious color contrast, stable retention factor, and good reproducibility. In addition, the operation process of this method is simple, the required equipment cost is low, and it is suitable for popularization and use in grass-roots laboratories.

[0014] At the same time, the present invention has carried out a durability investigation on Shengui Guben Wine samples of different batches. The results show that this method has good adaptability under different environmental humidities, different brands of thin-layer plates and different experimental personnel operating conditions, and the separation effect is stable and reliable. Using the method of the present invention can provide a scientific basis for the quality control of Shengui Guben Wine, fill the technical blank of rapid detection of the medicinal material components of this preparation based on thin-layer chromatography technology, and lay a solid foundation for ensuring product quality and medication safety. Specific Embodiments

[0015] The present invention provides a rapid detection method for the medicinal material components of Shengui Guben Wine based on thin-layer chromatography technology, and its specific embodiments are as follows.

[0016] A rapid detection method for the medicinal material components of Shengui Guben Wine based on thin-layer chromatography technology, the detection method includes the following steps:

[0017] (1) Take 5 ml of the Shengui Guben Wine sample and place it in a separatory funnel, add 20 ml of ethyl acetate as the extraction solvent, control the shaking frequency at 200 times per minute, and the shaking time is 3 minutes. After shaking, let it stand for natural stratification, separate the organic phase and transfer it to a rotary evaporator, and concentrate it to dryness under reduced pressure in a water bath at 40°C. The obtained residue is dissolved with 1 ml of methanol to finally obtain the test solution. In this process, the selection of ethyl acetate is based on its good extraction ability for various active components in Shengui Guben Wine, and the 40°C water bath condition ensures the solvent evaporation efficiency while avoiding the degradation of thermosensitive components. The shaking frequency and time have been optimized to ensure sufficient extraction and no damage to the structural integrity of the target components.

[0018] (2) The preparation steps of the control herbal solutions are as follows. Weigh 1g each of Angelica sinensis, Astragalus membranaceus, Codonopsis pilosula, Poria cocos, Cornus officinalis, Ligusticum chuanxiong, Atractylodes macrocephala, Schisandra chinensis, Citrus reticulata peel, Lycium barbarum, Saposhnikovia divaricata, Notopterygium incisum, Poria cocos sclerotium, Ophiopogon japonicus, Rehmannia glutinosa, Rehmannia glutinosa (processed), Ziziphus jujuba, and tortoise shell glue. Add 25ml of ethanol to each, and place the mixture in an ultrasonic cleaner for ultrasonic treatment. The ultrasonic power is set to 250W, the frequency to 40kHz, and the treatment time to 30 minutes. After ultrasonic treatment, filter the obtained filtrate and transfer it to a rotary evaporator. Concentrate to dryness under reduced pressure in a 40℃ water bath. Dissolve the residue in 1ml of methanol to prepare the corresponding control herbal solutions. The ultrasonic treatment conditions have been experimentally verified to effectively extract the target components from the herbs while avoiding component degradation due to excessive time or high power.

[0019] (3) The preparation of the reference solution is carried out using the following steps. Ferulic acid from Angelica sinensis, astragaloside A from Astragalus membranaceus, codonopsis pilosula glycoside from Codonopsis pilosula, poria cocos acid from Poria cocos, loganin from Cornus officinalis, ligustilide from Ligusticum chuanxiong, atractylone from Atractylodes macrocephala, schisandrin A from Schisandra chinensis, hesperidin from Citrus reticulata peel, betaine from Lycium barbarum, cimicifuga oleracea var. sarcodactylis, notopterygium incisum, poria cocos acid from Poria cocos, ophiopogonin from Ophiopogon japonicus, catalpol from Rehmannia glutinosa, verbascoside from Rehmannia glutinosa (processed), cyclic adenosine monophosphate from Ziziphus jujuba, and amino acids from tortoise shell glue are selected as reference standards.

[0020] (4) Prepare solutions of the above reference standards with analytical grade methanol to a concentration of 1 mg / ml, and filter them through a 0.45 μm microporous membrane for later use. In this step, the reference standards are selected based on their representativeness in their respective medicinal materials, which can effectively reflect the key component characteristics of the medicinal materials. The use of microporous membranes ensures the purity of the solution and avoids interference from impurities in subsequent detection. The detection process is carried out according to thin-layer chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0502). Place an appropriate amount of developing solvent in the developing tank. The developing solvent is prepared by mixing petroleum ether, ethyl acetate, methanol, and glacial acetic acid in a volume ratio of 6:8:2:1. Place the silica gel GF254 thin-layer plate in the developing tank, and take 2 μl each of the test solution, reference medicinal material solution, and reference standard solution, and spot them on the same thin-layer plate. The spotting positions should be evenly distributed, and the spotting volume should be consistent to ensure the comparability of the detection results. During the development process, the developing solvent moves upward along the thin-layer plate. When it reaches an appropriate height, the plate is removed, dried, and then examined under a 254nm UV lamp. The position and color of the spots are recorded. Subsequently, it is sprayed with a 10% sulfuric acid ethanol solution and heated at 105℃ until the spots are clearly visible. It is then examined again under sunlight.

[0021] In this step, the ratio of the developing solvent has been optimized through multiple experiments to achieve a balance between separation effect and development rate, ensuring that the spots of the target components are clear and easy to identify.

[0022] The color development results showed that the spots' positions and color contrasts of the test sample, control crude drug, and reference substance after development under the same conditions were clearly visible. In the chromatogram of the test sample, spots of the same color should appear at the corresponding positions of the chromatograms of the control crude drug and the reference substance. This result indicated that the components in the test sample solution had a good corresponding relationship with the control crude drug and the reference substance, enabling effective qualitative analysis of multiple key crude drug components in Shengui Guben Wine. The consistency of the spots' color and position reflected good separation effect and high reproducibility of the target components.

[0023] The operation process of the entire detection method was simple, and the required equipment cost was low, making it suitable for promotion and use in grass-roots laboratories. By detecting multiple batches of Shengui Guben Wine samples, the enterprise verified the reproducibility and reliability of this method. These characteristics enabled this method to provide a scientific basis for the quality control of Shengui Guben Wine, filling the technical gap in the rapid detection of the crude drug components of this preparation based on thin-layer chromatography technology, and laying a solid foundation for ensuring product quality and medication safety. The content not described in detail in the specification belongs to the prior art well-known to those skilled in the art, and the model parameters of each device are not specifically limited, and conventional devices can be used. In this technical solution, the experimental conditions not mentioned are not shown because they belong to the prior art and will not be described here either. The above are only the preferred embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A rapid detection method for medicinal components in a tortoise shell and ginseng tonic wine based on thin-layer chromatography, characterized in that, The detection method includes the following steps: (1) Preparation of test solution: Take the sample of Shengui Gubenjiu, add ethyl acetate, shake and extract at room temperature, let stand and separate the organic phase, concentrate the organic phase to dryness under reduced pressure in a water bath, dissolve the residue in methanol and use it as test solution. (2) Preparation of control medicinal material solutions: Take 1g each of Angelica sinensis, Astragalus membranaceus, Codonopsis pilosula, Poria cocos, Cornus officinalis, Ligusticum chuanxiong, Atractylodes macrocephala, Schisandra chinensis, Citrus reticulata, Lycium barbarum, Saposhnikovia divaricata, Notopterygium incisum, Poria cocos, Ophiopogon japonicus, Rehmannia glutinosa, Rehmannia glutinosa (processed), Ziziphus jujuba, and tortoise shell glue. Add 25ml of ethanol to each, sonicate for 30 minutes at a power of 250W and a frequency of 40kHz, filter, concentrate the filtrate under reduced pressure to dryness in a 40℃ water bath, dissolve the residue in 1ml of methanol, and prepare the corresponding control medicinal material solutions. (3) Preparation of reference solutions: Ferulic acid, astragaloside A, codonopsis glycoside, poria cocos acid, loganin, ligustrazine lactone, atractylone, schisandrol A, hesperidin, betaine, cimicifugain glycoside, notopterygium alcohol, ophiopogonin, catalpol, verbascoside, cyclic adenosine monophosphate, and amino acids were selected and prepared into reference solutions with analytical grade methanol, and filtered through a microporous membrane for later use as reference solutions; (4) Detection: According to the thin-layer chromatography method, take 2 μl of each of the above test solution, reference medicinal material solution and reference solution and spot them on the same silica gel GF254 thin-layer plate. Use petroleum ether, ethyl acetate, methanol and glacial acetic acid as developing solvents, develop, remove, dry and examine under ultraviolet light, record the spot position and color, then develop color and examine under sunlight.

2. The detection method according to claim 1, characterized in that, Step (1) Preparation of test solution: Take 5 ml of Ginseng and Turtle Tonic Wine sample, add 20 ml of ethyl acetate, and shake to extract for 3 minutes at room temperature. The shaking frequency is 200 times per minute. After standing and separating the layers, separate the organic phase and concentrate the organic phase to dryness under reduced pressure in a 40℃ water bath. Dissolve the residue in 1 ml of methanol to obtain the test solution.

3. The detection method according to claim 1, characterized in that, Step (2) Preparation of control medicinal material solutions: Take 1g each of Angelica sinensis, Astragalus membranaceus, Codonopsis pilosula, Poria cocos, Cornus officinalis, Ligusticum chuanxiong, Atractylodes macrocephala, Schisandra chinensis, Citrus reticulata peel, Lycium barbarum, Saposhnikovia divaricata, Notopterygium incisum, Poria cocos, Ophiopogon japonicus, Rehmannia glutinosa, Rehmannia glutinosa (processed), Ziziphus jujuba, and tortoise shell glue. Add 25ml of ethanol to each, sonicate for 30 minutes at a power of 250W and a frequency of 40kHz, filter, concentrate the filtrate under reduced pressure to dryness in a 40℃ water bath, dissolve the residue in 1ml of methanol, and prepare the corresponding control medicinal material solutions.

4. The detection method according to claim 1, characterized in that, In step (3), solutions containing 1 mg per 1 ml were prepared using analytical grade methanol and filtered through a 0.45 μm microporous membrane for later use as reference solutions.

5. The detection method according to claim 1, characterized in that, The developing agent mentioned in step (4) is prepared by mixing petroleum ether, ethyl acetate, methanol and glacial acetic acid in a volume ratio of 6:8:2:

1.

6. The detection method according to claim 1, characterized in that, The thin-layer plate mentioned in step (4) is a silicone GF254 thin-layer plate.

7. The detection method according to claim 1, characterized in that, The sample quantity mentioned in step (4) is 2 μl for each solution, and the sample locations are evenly distributed.

8. The detection method according to claim 1, characterized in that, The color development operation described in step (4) includes spraying with a 10% sulfuric acid ethanol solution and heating at 105°C until the spots are clearly visible.

9. The detection method according to claim 1, characterized in that, The inspection conditions described in step (4) include inspection under a 254nm ultraviolet lamp and inspection under sunlight.