Rapid detection method for adenosine in artificial ganoderma lucidum mycelium powder and application

Abstract

The invention relates to a method for rapidly detecting adenosine in artificial ganoderma lucidum mycelium powder and application, and belongs to the technical field of medicine component extraction and detection. The invention discloses a method for rapidly detecting adenosine in artificial ganoderma lucidum mycelium powder, and the method is characterized in that the ganoderma lucidum mycelium powder is prepared by processing a dried mycelium product obtained by performing liquid submerged fermentation culture on porous fungus Ganoderma lucidum (Leyss.ex Fr.) Karst. The method for rapidly detecting adenosine in the artificial ganoderma lucidum mycelium powder comprises the following steps: (a) taking about 0.25 g of a sample, and adding 50 ml of 20% ethanol; (b) heating and refluxing at 100 DEG C for 1 hour; (c) performing microfiltration to obtain a test solution; and (d) injecting 5 [mu] l of the test solution into high performance liquid chromatography, separating by using an octadecylsilane chemically bonded silica chromatographic column and 7% acetonitrile as a mobile phase, and inspecting under the wavelength of a 260nm ultraviolet lamp. The method can be used for rapidly detecting the adenosine content in the artificial ganoderma lucidum mycelium powder.

Description

Technical Field

[0001] This invention relates to a method for the extraction and rapid detection of adenosine in artificial Ganoderma lucidum mycelium powder, belonging to the field of drug component extraction and detection technology. Background Technology

[0002] The 2025 edition of the Chinese Pharmacopoeia records medicinal Ganoderma as the dried fruiting body of the fungus *Ganoderma lucidum* (Leyss. ex Fr.) Karst. or *Ganoderma sinense* Zhao, Xu et Zhang, belonging to the Polyporaceae family. Referred to as the "immortal herb," ​​it holds a pivotal position in traditional Chinese medicine, with records dating back to the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). It possesses the effects of tonifying qi and calming the mind, relieving cough and asthma. It is used for restlessness, insomnia, palpitations, cough and asthma due to lung deficiency, weakness and shortness of breath, and loss of appetite. Modern research shows that Ganoderma polysaccharides have immunomodulatory, hypoglycemic, hypolipidemic, antioxidant, anti-aging, and antitumor effects; triterpenoids can purify the blood and protect liver function; various Ganoderma preparations have sedative, anticonvulsant, cardiotonic, antiarrhythmic, hypotensive, and antitussive / asthmatic effects; in addition, Ganoderma also has anticoagulant, platelet aggregation inhibitory, and anti-allergic effects. In recent years, the Ganoderma industry in China has shown rapid development. With increasing awareness of health and wellness and the revival of traditional Chinese medicine, the market demand for Ganoderma lucidum (Reishi mushroom) continues to grow. Accompanying this growing demand and the shortage of Ganoderma lucidum resources, artificially produced Ganoderma lucidum mycelium powder has emerged. This powder is made from the dried mycelium of the fungus Ganoderma lucidum (Leyss. ex Fr.) Karst, cultivated through deep liquid fermentation, and has the same effects as Ganoderma lucidum. Currently, its quality control relies solely on the nitrogen content determination standard stipulated in the Twelve Volumes of Traditional Chinese Medicine issued by the Chinese Ministry of Health, which is insufficient to meet modern needs. To further improve the quality standard system of artificially produced Ganoderma lucidum mycelium powder, this invention proposes a rapid detection method for adenosine in artificially produced Ganoderma lucidum mycelium powder. Summary of the Invention

[0003] The present invention aims to provide a rapid detection method for adenosine in artificial Ganoderma lucidum mycelium powder, which is simple, fast, efficient and low cost.

[0004] To achieve the above objectives, the technical solution of this application is implemented as follows:

[0005] A method for detecting adenosine in artificially produced Ganoderma lucidum mycelium powder, the detection method comprising:

[0006] Take an appropriate amount of artificial Ganoderma lucidum mycelium powder, add a certain proportion of ethanol aqueous solution, and heat under reflux to extract;

[0007] After the heat reflux treatment, the extract was cooled to room temperature, the weight was made up with the extraction solvent, and the solution was microfiltered to obtain the test solution.

[0008] Furthermore, the ethanol aqueous solution is an ethanol aqueous solution with a volume fraction of 10-30%.

[0009] Furthermore, the heating reflux temperature is 95-105℃.

[0010] Furthermore, the heating reflux time is 0.5-1.5h.

[0011] Furthermore, the adenosine content in the artificial Ganoderma lucidum mycelium powder was determined by high performance liquid chromatography using 5-9% acetonitrile as the mobile phase.

[0012] Compared with existing methods for detecting artificial Ganoderma lucidum mycelium powder, this invention has the following advantages:

[0013] The detection method of this application only requires an ultraviolet detector, the extraction solvent is an readily available aqueous ethanol solution, and the mobile phase is a common aqueous acetonitrile solution. Compared with other quality control detection methods for artificial Ganoderma lucidum mycelium powder, this invention has the characteristics of being simple, fast, efficient, and low-cost.

[0014] Another objective of this invention is to provide an application of the above-mentioned testing method, specifically the application of the rapid detection method for adenosine in the quality control of artificial Ganoderma lucidum mycelium powder. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of this application. In the drawings:

[0016] Figure 1 This is a schematic diagram of the linear regression equation between the adenosine injection volume and the peak area corresponding to Table 1 in the example.

[0017] Figure 2 The image shows the chromatogram of adenosine reference standard at 260 nm in the example.

[0018] Figure 3 The image shows the chromatogram of the artificial Ganoderma lucidum mycelium powder extract at 260 nm in the example. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments.

[0020] Example 1

[0021] To make the technical solution and advantages of this patent application clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0022] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0023] For items not specified in this invention, conventional conditions or conditions recommended by the manufacturer of the equipment used shall be followed. For reagents or instruments whose manufacturers are not specified, conventional products purchased from the market shall be used. For technical means or methods involved, if specific conditions are not specified, they shall be carried out in accordance with existing methods in the field.

[0024] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0025] An embodiment of the first aspect of the present invention provides a rapid detection method for adenosine in artificial Ganoderma lucidum mycelium powder, which can be used to improve the quality standards of artificial Ganoderma lucidum mycelium powder, improve product quality, and reduce detection costs.

[0026] In current technology, the quality control standard for artificial Ganoderma lucidum mycelium powder still relies on testing the nitrogen content of a single element. This quality control method is relatively outdated. The following analysis uses the determination of nitrogen content in the twelve volumes of Chinese herbal medicine issued by the Ministry of Health as an example:

[0027] Nitrogen content detection

[0028] Take an appropriate amount of the test sample (approximately equivalent to 25-30 mg of nitrogen) and place it in a 500 ml Kjeldahl flask. Add 10.0 g of anhydrous sodium sulfate and 0.5 g of copper sulfate. Slowly add 20 ml of sulfuric acid along the flask wall. Place a small funnel at the mouth of the flask and tilt it at a 45° angle. Heat slowly with a direct flame to keep the temperature of the solution below the boiling point. When the bubbling stops, heat strongly to boiling. When the solution turns clear green, continue heating for 30 minutes. Let it cool, then slowly add 250 ml of water along the flask wall, shake well, and let it cool. After cooling, add 75 ml of 40% sodium hydroxide solution, ensuring it flows along the flask wall to the bottom to form a separate liquid layer. Add a few zinc granules. Connect the Kjeldahl flask to the condenser using a nitrogen balloon. Separately, take 50 ml of 2% boric acid solution and place it in a 500 ml Erlenmeyer flask. Add 10 drops of methyl red-bromocresol green mixed indicator. Insert the lower end of the condenser below the surface of the boric acid solution and gently rotate the flask to mix the solution thoroughly. Heat and distill until the total volume of the receiving liquid is approximately 250 ml. Remove the tip of the condenser from the liquid surface and allow it to be rinsed with steam for about 1 minute. Rinse the tip with water and then stop distillation. Titrate the distillate with sulfuric acid titrant (0.05 mol / L) until the solution changes from blue-green to gray-purple. Correct the titration result with a blank test. Each 1 ml of sulfuric acid titrant (0.05 mol / L) is equivalent to 1.401 mg of N. The total nitrogen (N) content of this product, calculated on a dried basis, shall not be less than 5.5%.

[0029] The examples above involve the use of corrosive liquids and toxic reagents, and the processes are cumbersome and costly. Against this backdrop, research into improving the quality standards for artificially cultivated Ganoderma lucidum mycelium powder products is urgently needed.

[0030] In view of this, in order to overcome the shortcomings of the existing technology, the rapid detection method for adenosine in artificial Ganoderma lucidum mycelium powder in this embodiment is specifically designed to include the following steps:

[0031] Step S1 involves extracting artificial Ganoderma lucidum mycelium powder using a specific ratio of ethanol-water solution and heating under reflux. The extracted components are then filtered to prepare a test solution.

[0032] It is worth noting that the ethanol-water solution in the specific ratio in step S1 above can extract adenosine from the artificial Ganoderma lucidum mycelium powder more effectively than other organic solvents.

[0033] Specifically, in S1 above, adenosine is extracted from artificial Ganoderma lucidum mycelium powder. The extracted components are filtered to prepare a test solution. As a preferred embodiment, this may include heating and refluxing for a specific time, cooling the solution after heating and refluxing to room temperature, replenishing the weight, filtering, and obtaining the test solution.

[0034] It should be noted that during the preparation of the test solution, since the extraction method is heating and reflux, there will be a loss of extraction solvent after cooling to room temperature. Therefore, it is necessary to replenish the weight with extraction solvent and then filter to obtain the test solution.

[0035] The methanol aqueous solution is preferably an ethanol aqueous solution with a volume fraction of 10-30%.

[0036] The heating reflux temperature is 95-105℃.

[0037] During this period, the heating reflux time is 0.5-1.5h.

[0038] Step S2 involves dissolving the reference standard to prepare a reference standard solution.

[0039] In step S2 above, the reference standard is dissolved. As a preferred embodiment, the reference standard can be dissolved in 50% ethanol-water.

[0040] Step S3: Inject the reference solution and the test solution into the high-performance liquid chromatograph and perform detection according to the preset chromatographic conditions.

[0041] Specifically, the preset chromatographic conditions in step S3 above may preferably include using acetonitrile as the mobile phase. Acetonitrile is widely used as an organic phase, has low toxicity, is suitable for UV detector analysis, and has high separation efficiency.

[0042] This invention uses acetonitrile-water as the mobile phase, which enables the extracted components to have reasonable peak elution times, meet the required separation degree, and have normal peak shapes.

[0043] It is worth noting that the preferred mobile phase is, for example, an aqueous solution of acetonitrile with a volume fraction of 5%-9%.

[0044] The above-mentioned preset chromatographic conditions, as a preferred embodiment, may also include, for example, using a chromatographic column packed with octadecyl bonded silica gel, using an ultraviolet detector with a detection wavelength of 258-262 nm, and using a column temperature between 28-32 °C.

[0045] Specifically, chromatographic columns packed with octadecyl-bonded silica gel, commonly referred to as C18 columns, are a very common type of reverse-phase chromatographic column. They possess a high carbon content and good hydrophobicity, enabling good separation performance. This invention utilizes an octadecyl-bonded silica gel column to achieve rapid separation of adenosine.

[0046] Ultraviolet (UV) detectors are among the most commonly used detectors in liquid chromatography systems and are highly cost-effective detection tools in liquid chromatography.

[0047] As described above, the rapid adenosine detection method of this embodiment only requires an ultraviolet detector to detect adenosine in multiple components within a short time. Furthermore, the mobile phase is a commonly used acetonitrile aqueous solution. Compared with the nitrogen content detection method described above, this method is simpler and more convenient. Therefore, the detection method of this invention not only improves the quality controllability of artificially prepared mycelium powder but also increases detection efficiency, saves detection time, reduces detection costs, and minimizes environmental pollution.

[0048] Furthermore, embodiments of the second aspect of the present invention provide the use of the above-described adenosine detection method. In general, the rapid adenosine detection method of this embodiment can be used for the quality control of artificial Ganoderma lucidum mycelium powder.

[0049] It is worth noting that, regarding the adenosine rapid detection method and its application in this embodiment, based on the above preferred embodiments, the following examples can be referred to in specific implementation.

[0050] Example

[0051] This example demonstrates the rapid detection method for adenosine in artificially cultivated Ganoderma lucidum mycelium powder. The specific detection steps are as follows:

[0052] Chromatographic conditions and system suitability tests were performed using octadecyl-bonded silica gel as the packing material and isocratic elution with 7% acetonitrile aqueous solution; detection wavelength: 260 nm; flow rate: 1.00 ml / min; column temperature: 30 °C; theoretical plate number calculated based on adenosine peak should not be less than 3000.

[0053] Preparation of the reference solution: Take an appropriate amount of adenosine reference standard, accurately weigh it, place it in a suitable volumetric flask, add 50% ethanol to prepare a solution containing 20 μg per ml, and use it as the reference solution.

[0054] Preparation of the test solution: Accurately measure 0.25g of artificial Ganoderma lucidum mycelium powder, add 20% ethanol aqueous solution, heat to reflux at 100℃ for 1h, cool to room temperature, make up the weight with 20% ethanol water, filter, discard the initial filtrate, and take the subsequent filtrate as the test solution.

[0055] The assay involves precisely pipetting 5 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0056] The applicability of the above detection methods was investigated, and the results of method specificity, repeatability, precision, and stability are shown in Table 1-. Figure 1 -As shown.

[0057] Table 1. Results of linear experiments

[0058] name Adenosine content (μg) Peak area Linear 1 0.0744 186.859 Linear 2 0.0837 212.423 Linear 3 0.093 234.483 Linear 4 0.1023 257.631 Linear 5 0.1116 283.516 Linear equations Y = 0.0093x + 0.0651 r

[0059] Table 2. Experimental results of adenosine recovery rate

[0060]

[0061] Table 3 Precision Experiment Results

[0062]

[0063]

[0064] Table 4. Stability test results

[0065] Time (h) Reference peak area Change (%) Time (h) Peak area of ​​test sample Change (%) 0h 234.328 / 0h 234.850 / 4h 234.966 0.27 4h 235.030 0.08 8h 234.961 0.27 8h 235.565 0.30 12h 236.480 0.92 12h 234.845 0.00

[0066] Table 5 Results of Repeatability Experiments

[0067]

[0068] Table 63 Results of Adenosine Content Determination in Artificial Ganoderma Lucidum Mycelium Powder

[0069] batch Sample 1 Sample 2 Sample 3 Adenosine content (mg / g) 3.05 3.37 3.52

[0070] The methodological results of the above examples show that, using this detection method, the injection amount of adenosine reference standard and the peak area both exhibit a good linear relationship.

[0071] Specifically, within the adenosine injection range of 0.0744 μg–0.1116 μg, the peak area showed a good linear relationship, with the regression equation being: Y = 0.0093x + 0.0651, r = 1.00 (results are shown in Table 1). Figure 1 ).

[0072] Similarly, based on the results of the above-mentioned spiking recovery experiment, the average recovery rate of adenosine in 6 determinations was 99.54%, and the RSD value was 0.39 (see Table 2 for results).

[0073] Therefore, the detection method in this example is suitable for the rapid determination of adenosine in artificial Ganoderma lucidum mycelium powder. This method is simple, fast, efficient and low cost.

[0074] It should be understood that, in order to simplify this disclosure and aid in understanding one or more of the various aspects of the invention, features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof in the above description of exemplary embodiments of the invention. However, this method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than expressly recited in each claim. Rather, as reflected in the claims, inventive aspects lie in fewer than all the features of the foregoingly disclosed embodiments. Therefore, the claims, following the detailed description, are hereby expressly incorporated into that detailed description, wherein each claim itself is a separate embodiment of the invention.

[0075] Although the invention has been described with reference to a limited number of embodiments, those skilled in the art will understand from the foregoing description that other embodiments are conceivable within the scope of the invention described herein. Furthermore, it should be noted that the language used in this specification has been chosen primarily for readability and edibility purposes, and not for the purpose of interpreting or limiting the subject matter of the invention. Therefore, many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the appended claims. The disclosure of the invention is illustrative and not restrictive, and the scope of the invention is defined by the appended claims.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A rapid detection method for artificial Ganoderma lucidum mycelium powder, characterized in that, The method involves extracting the material using a 10%-30% ethanol solution, heating it under reflux at 95-105℃ for 0.5-1.5 hours, and then determining the adenosine content in the artificial Ganoderma lucidum mycelium powder using a high-performance liquid chromatograph with 5-9% acetonitrile as the mobile phase.

2. A rapid detection method for artificial Ganoderma lucidum mycelium powder, characterized in that, The extraction solvent was 10%-20% ethanol solution, and the mixture was heated to reflux at 95-100℃ for 0.5-1.0 h. The content of adenosine in the artificial Ganoderma lucidum mycelium powder was determined by high performance liquid chromatography using 5-7% acetonitrile as the mobile phase.

3. A rapid detection method for artificial Ganoderma lucidum mycelium powder, characterized in that, The extraction solvent was 20%-30% ethanol solution. The mixture was heated to reflux at 100-105℃ for 1.0-1.5h. The adenosine content in the artificial Ganoderma lucidum mycelium powder was determined by high performance liquid chromatography using 7-9% acetonitrile as the mobile phase.

4. A rapid detection method for artificial Ganoderma lucidum mycelium powder as described in any one of claims 1-4, characterized in that, Includes the following steps: Step 1: Take about 0.25g of sample and add 50ml of 20% ethanol. Step 2: Heat to 100℃ and reflux for 1 hour. Step 3: Inject 5 µl of the test solution into a high-performance liquid chromatograph and use an octadecylsilane-bonded silica gel column with 7% acetonitrile as the mobile phase for separation.

5. The detection method according to claim 4, characterized in that, The extraction solvent is a 10%-30% ethanol solution.

6. The detection method according to claim 4, characterized in that, Extract by reflux at 95-105℃ for 0.5-1.5 hours.

7. The detection method according to claim 4, characterized in that, The content of adenosine in artificial Ganoderma lucidum mycelium powder was determined by high performance liquid chromatography using 5-9% acetonitrile as the mobile phase.

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