Preparation method and application of ganoderma lucidum nano powder, ganoderma lucidum polysaccharide extract or ganoderma lucidum triterpenoid extract

Abstract

The invention provides a preparation method and application of lucid ganoderma nano powder, a lucid ganoderma polysaccharide extract or a lucid ganoderma triterpenoid extract, and belongs to the technical field of lucid ganoderma processing and preparation. The preparation method of the lucid ganoderma nano powder comprises the following steps: airing lucid ganoderma sporocarp, cutting into lucid ganoderma particles, and crushing the lucid ganoderma particles to obtain lucid ganoderma coarse powder; drying the ganoderma lucidum coarse powder at constant temperature, and performing superfine grinding on the dried ganoderma lucidum coarse powder to obtain the ganoderma lucidum powder. The ganoderma lucidum nano powder is successfully prepared through superfine grinding, the content of heavy metal in the ganoderma lucidum nano powder is low, the content of polysaccharide and the content of triterpenes measured in the ganoderma lucidum polysaccharide extract and the ganoderma lucidum triterpenes extract obtained through extraction of the ganoderma lucidum nano powder are high, and the antioxidant activity of the ganoderma lucidum polysaccharide extract and the antioxidant activity of the ganoderma lucidum triterpenes extract are high; further research finds that the ganoderma triterpenoid extract has remarkable activity of inhibiting brain glioma. Various functional products are prepared from the ganoderma lucidum nano powder, and the ganoderma lucidum nano powder has important significance and application value.

Description

Technical Field

[0001] The invention belongs to the technical field of ganoderma lucidum processing and preparation, and in particular relates to a preparation method and application of ganoderma lucidum nano powder, ganoderma lucidum polysaccharide extract or ganoderma lucidum triterpene extract. Background Art

[0002] Ganoderma lucidum, as a traditional Chinese medicinal material, has attracted widespread attention due to its rich nutritional ingredients and unique medicinal value. In recent years, studies have found that the selenium (Se) content in Ganoderma has a significant effect on its biological activity, which has triggered a wave of research on selenium-enriched Ganoderma. Selenium-enriched Ganoderma refers to Ganoderma that is enriched with selenium through specific culture conditions or the addition of selenium sources during its growth process, thereby enhancing its health and medical value. The main components of selenium-enriched Ganoderma include polysaccharides, triterpenoids, proteins, amino acids and trace elements such as selenium. As an emerging product, selenium-enriched Ganoderma has attracted much attention due to its rich nutritional ingredients and significant biological activity. Its efficacy in immunomodulation, anti-tumor and anti-oxidation provides a scientific basis for its clinical application.

[0003] Selenium-enriched Ganoderma lucidum powder obtained by traditional crushing process is widely used, but it has the following disadvantages. (1) Low bioavailability. The particles of traditional selenium-enriched Ganoderma lucidum powder are generally large, which may lead to reduced solubility and absorption rate of its active ingredients (such as polysaccharides, triterpenoids, etc.) in the gastrointestinal tract, thereby affecting its bioavailability. A larger dose may be required to achieve the desired effect. (2) The powder size is large and the particles are coarse. The crushing process of traditional selenium-enriched Ganoderma lucidum powder may not be able to grind it finely enough. The powder particles are coarse, which affects its mixing effect in food or beverages and makes consumers feel uncomfortable during use. (3) Unpleasant taste. Because the particles of traditional selenium-enriched Ganoderma lucidum powder are large and taste bitter, it results in a poor taste when used. This makes some consumers feel uncomfortable when drinking or eating products containing Ganoderma lucidum, affecting the acceptance of the product. (4) It is not easy to be compatible with other ingredients. The larger particles of selenium-enriched Ganoderma lucidum powder are not easy to mix evenly with other food ingredients (such as beverages, food additives, etc.), affecting the overall flavor and effect of the product. (5) It is difficult to meet personalized needs. As consumers pay more and more attention to health products, the demand for personalized and functional products continues to increase. Traditional selenium-rich Ganoderma lucidum powder may have limitations in meeting these needs. For this reason, researchers and enterprises are encouraged to explore more advanced technologies, such as nanotechnology, to improve the quality and efficacy of selenium-rich Ganoderma lucidum powder.

[0004] Nanoprocessing technology is an emerging technology that uses nanotechnology to apply in the production and processing of products. By processing and controlling materials and ingredients at the nanoscale, the performance and characteristics of the product are enhanced, and the quality, functionality and safety of the product are significantly improved. As consumers' requirements for food safety, nutrition and flavor continue to increase, product nano-ization has gradually become the development direction of food processing technology. The patent "A kind of Ganoderma lucidum ultrafine powder with high activity and low heavy metal content and its production method, application number 202111606551.9" uses conventional Ganoderma lucidum fruiting bodies as raw materials, and uses liquid nitrogen to ultra-low temperature freeze the Ganoderma lucidum coarse particles to be crushed, and then crushes them with ultra-low temperature freezing equipment. The particle size distribution of the prepared Ganoderma lucidum ultrafine powder is in the micron level (ranging from 10 to 30 microns), which has not yet reached the nanometer level. At the same time, since the temperature of the Ganoderma lucidum coarse particles must be lowered to -100℃~-150℃ for processing, the energy consumption is large. The patent "An ultrafine broken wall Ganoderma lucidum composition and its preparation method" (CN201410316806.1) proposes to cook Ganoderma lucidum at 110℃~120℃ for 6h, then cut Ganoderma lucidum into slices, segments, shreds and blocks, and then use mechanical impact crusher, air flow crusher, ball mill, vibration mill or stirring mill to prepare. These crushing methods will inevitably cause chromium and nickel pollution of harmful heavy metals in ultrafine powder, and high cooking will cause loss of effective ingredients. At present, there are few studies on the preparation of Ganoderma lucidum nanopowder, especially selenium-enriched Ganoderma lucidum nanopowder, the preparation method of extract and the extract in inhibiting tumor growth.

[0005] Gliomas are a common primary brain tumor that originates from glial cells in the brain. Based on their histological characteristics, gliomas can be divided into several types, including astrocytomas, oligodendrogliomas, and ependymomas. The harm of this type of tumor is mainly reflected in its rapid growth and invasiveness, which often causes patients to experience symptoms such as neurological dysfunction, epilepsy, and headaches, which can be life-threatening in severe cases. Although existing treatments include surgery, radiotherapy, and chemotherapy, the prognosis is still poor due to the high heterogeneity of gliomas and their resistance to treatment. There is an urgent need to find an effective drug for the treatment of gliomas. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a method for preparing and using a Ganoderma lucidum nanopowder, a Ganoderma lucidum polysaccharide extract or a Ganoderma lucidum triterpene extract. The Ganoderma lucidum nanopowder successfully prepared by ultrafine grinding of the present invention has significantly increased polysaccharide content and triterpene content in the extract further extracted from the Ganoderma lucidum nanopowder, and significantly reduced heavy metal content, and the functional activity of the Ganoderma lucidum polysaccharide extract or Ganoderma lucidum triterpene extract prepared using the Ganoderma lucidum nanopowder is significantly improved.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing ganoderma lucidum nano powder, comprising the following steps:

[0009] Dry the ganoderma lucidum fruiting bodies for 1 to 2 days, cut them into ganoderma lucidum particles, and then dry them at a constant temperature of 50 to 65° C. until the water content is 10 to 15% to obtain dried ganoderma lucidum particles, and grind them at 20 to 30° C. for 1 to 3 minutes to obtain ganoderma lucidum coarse powder;

[0010] The ganoderma lucidum powder is dried at a constant temperature of 50-65° C. until the water content is below 7% to obtain dry ganoderma lucidum powder; 250-650 g of the dry ganoderma lucidum powder is ultrafinely ground at -20-0° C. for 15-55 minutes to obtain ganoderma lucidum nanopowder.

[0011] Preferably, the Ganoderma lucidum is selenium-enriched Ganoderma lucidum.

[0012] Preferably, the ultrafine grinding method is to ultrafine grind 500-600g of coarse Ganoderma lucidum powder at -20--10°C for 40-50min.

[0013] Preferably, the ultrafine grinding method is to ultrafine grind 600g of coarse Ganoderma lucidum powder at -20 to -10°C for 50min.

[0014] The present invention provides a method for preparing a ganoderma lucidum polysaccharide extract, comprising the following steps:

[0015] The above-mentioned Ganoderma lucidum nanopowder is mixed with water, heated under reflux for extraction, filtered to collect the filtrate, concentrated under reduced pressure and dried to obtain the product.

[0016] Preferably, the mass volume ratio of the Ganoderma lucidum nanopowder to water is 1 g: 25-32 mL; the number of reflux extractions is 1-3 times, and the time for each extraction is 1.5-2.5 h.

[0017] The present invention provides a method for preparing a ganoderma triterpene extract, comprising the following steps:

[0018] The above-mentioned Ganoderma lucidum nano powder is mixed with 65% to 75% ethanol solution, subjected to ultrasonic extraction, filtered and collected the filtrate, concentrated under reduced pressure and dried to obtain the product.

[0019] Preferably, the mass volume ratio of the Ganoderma lucidum nanopowder to the 65%-75% ethanol solution is 1g:20-27mL; the ultrasonic extraction is performed 1-3 times at room temperature and a power of 250-350W, and the time of each ultrasonic extraction is 55-65min.

[0020] The present invention provides an application of the ganoderma lucidum polysaccharide extract or ganoderma lucidum triterpene extract in preparing an antioxidant product.

[0021] The present invention provides an application of the above-mentioned ganoderma triterpene extract in preparing an anti-glioma product.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a preparation method and application of ganoderma lucidum nanopowder, ganoderma lucidum polysaccharide extract or ganoderma lucidum triterpene extract. The present invention successfully prepares ganoderma lucidum nanopowder by ultrafine grinding. The ganoderma lucidum nanopowder has low heavy metal content. The ganoderma lucidum polysaccharide extract and ganoderma lucidum triterpene extract obtained by extracting ganoderma lucidum nanopowder have high polysaccharide and triterpene contents respectively measured. The ganoderma lucidum polysaccharide extract and ganoderma lucidum triterpene extract have high antioxidant activity. Further research has found that the ganoderma lucidum triterpene extract has significant activity in inhibiting brain glioma. The present invention prepares a variety of functional products using ganoderma lucidum nanopowder, which has important significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The particle size distribution diagram of the selenium-enriched Ganoderma lucidum nanopowder prepared in Experiment Nos. 1 to 16, where 1 # ~16 # They represent the selenium-enriched Ganoderma lucidum nanopowders prepared in Experiment Nos. 1 to 16 respectively;

[0025] Figure 2 The electron microscope images are of selenium-enriched Ganoderma lucidum coarse powder (left) and selenium-enriched Ganoderma lucidum nanopowder a1 (right);

[0026] Figure 3 The results of polysaccharide and triterpenoid contents of selenium-enriched Ganoderma lucidum coarse powder before and after ultrafine grinding;

[0027] Figure 4 The results are as follows: the DPPH free radical scavenging ability of polysaccharide and triterpenoid extracts of selenium-enriched Ganoderma lucidum coarse powder before and after ultrafine grinding;

[0028] Figure 5 The results are as follows: the ABTS free radical scavenging ability of polysaccharide and triterpene extracts of selenium-enriched Ganoderma lucidum coarse powder before and after ultrafine grinding;

[0029] Figure 6 The results of the effects of different Ganoderma lucidum polysaccharide extracts on the proliferation activity of U251 cells;

[0030] Figure 7 These are the results of the effects of different Ganoderma triterpenoid extracts on the proliferation activity of U251 cells. DETAILED DESCRIPTION

[0031] The present invention provides a method for preparing ganoderma lucidum nano powder, comprising the following steps:

[0032] Dry the ganoderma lucidum fruiting bodies for 1 to 2 days, cut them into ganoderma lucidum particles, and then dry them at a constant temperature of 50 to 65° C. until the water content is 10 to 15% to obtain dried ganoderma lucidum particles, and grind them at 20 to 30° C. for 1 to 3 minutes to obtain ganoderma lucidum coarse powder;

[0033] The ganoderma lucidum powder is dried at a constant temperature of 50-65° C. until the water content is below 7% to obtain dry ganoderma lucidum powder; 250-650 g of the dry ganoderma lucidum powder is ultrafinely ground at -20-0° C. for 15-55 minutes to obtain ganoderma lucidum nanopowder.

[0034] In the present invention, the ganoderma lucidum fruiting body is dried for 1 to 2 days, and the step of cleaning the ganoderma lucidum fruiting body before drying is also included. After drying for 1 to 2 days, it is cut into ganoderma lucidum particles, and the size of the ganoderma lucidum particles is preferably 2×2 cm. Then the ganoderma lucidum particles are dried at a constant temperature of 50 to 65° C. to a water content of 10 to 15%, to obtain dried ganoderma lucidum particles, and crushed at 20 to 30° C. for 1 to 3 minutes to obtain ganoderma lucidum coarse powder. Further preferably, it is dried at a constant temperature of 55 to 60° C. to a water content of 11 to 14%. The present invention uses a conventional pulverizer to crush the ganoderma lucidum into coarse powder with a length of less than 20 mm and a diameter of 1 to 6 μm, which is convenient for ultrafine grinding and feeding, and improves the crushing effect. After obtaining the ganoderma lucidum coarse powder, the ganoderma lucidum coarse powder is dried at a constant temperature of 50 to 65° C. to a water content of less than 7%, to obtain dry ganoderma lucidum coarse powder. The present invention adopts a double drying process, which can effectively reduce the water content, not only helps to improve the grinding fineness, but also reduces the adhesion in the low-temperature ultrafine grinder, making it easy to discharge. At the same time, low-temperature grinding (i.e. grinding at -20 to 0°C) can increase the brittleness of selenium-rich Ganoderma lucidum, making it easier to grind. As a preferred method, the ultrafine grinding method is to ultrafine grind 500 to 600g of Ganoderma lucidum coarse powder at -20 to -10°C for 40 to 50min. It is further preferred that the ultrafine grinding method is to ultrafine grind 600g of Ganoderma lucidum coarse powder at -20 to -10°C for 50min. In the present invention, the Ganoderma lucidum is preferably selenium-rich Ganoderma lucidum. The selenium content of the selenium-rich Ganoderma lucidum nanopowder prepared by the present invention using selenium-rich Ganoderma lucidum is high.

[0035] The present invention provides a method for preparing a ganoderma lucidum polysaccharide extract, comprising the following steps:

[0036] The above-mentioned Ganoderma lucidum nanopowder is mixed with water, heated under reflux for extraction, filtered to collect the filtrate, concentrated under reduced pressure and dried to obtain the product.

[0037] In the present invention, the mass volume ratio of the Ganoderma lucidum nanopowder to water is 1g:25-32mL, more preferably 1g:27-30mL; the number of reflux extractions is preferably 1-3 times, more preferably 2 times, and the time for each extraction is preferably 1.5-2.5h, more preferably 2h. The Ganoderma lucidum polysaccharide extract prepared by water extraction using Ganoderma lucidum nanopowder in the present invention has a high polysaccharide content. In the present invention, the Ganoderma lucidum polysaccharide extract contains not only polysaccharide active ingredients, but also other active ingredients, that is, the Ganoderma lucidum polysaccharide extract of the present invention is a mixture composed of multiple active ingredients including polysaccharides.

[0038] In the present invention, by adjusting the ultrafine grinding feed amount, grinding temperature and grinding time, the particle size of the selenium-rich Ganoderma lucidum nano powder can be effectively controlled, the grinding time and the heavy metal content can be reduced, and the selenium content in the selenium-rich Ganoderma lucidum can be retained at 85%. The preparation method of the Ganoderma lucidum nano powder provided by the present invention has simple process and convenient operation, and the particle size of the selenium-rich Ganoderma lucidum nano powder can be controlled by adjusting the grinding temperature, grinding time and feeding amount of the low-temperature ultrafine grinder to make it completely reach the nanometer level.

[0039] The present invention provides a method for preparing a ganoderma triterpene extract, comprising the following steps:

[0040] The above-mentioned Ganoderma lucidum nano powder is mixed with 65%-75% ethanol solution, subjected to ultrasonic extraction, filtered and collected the filtrate, concentrated under reduced pressure and dried to obtain the product.

[0041] In the present invention, the mass volume ratio of the Ganoderma lucidum nanopowder to the 65% to 75% ethanol solution is 1g:20 to 27mL, more preferably 1g:22 to 26mL; the ultrasonic extraction is 1 to 3 times of ultrasonic extraction at room temperature and a power of 250 to 350W, more preferably 2 times of ultrasonic extraction at room temperature and a power of 280 to 320W, and the time of each ultrasonic extraction is preferably 55 to 65min, more preferably 58 to 62min. In the present invention, 65% to 75% of the 65% to 75% ethanol solution is a volume percentage, that is, 65 to 75mL of anhydrous ethanol is contained in 35 to 25mL of water. In the present invention, the Ganoderma lucidum triterpene extract contains not only triterpene active ingredients, but also other active ingredients, that is, the Ganoderma lucidum triterpene extract of the present invention is a mixture composed of multiple active ingredients including triterpene.

[0042] The ganoderma lucidum polysaccharide extract and ganoderma lucidum triterpene extract prepared by the present invention have significant antioxidant activity. Based on this, the present invention provides an application of the ganoderma lucidum polysaccharide extract or ganoderma lucidum triterpene extract in the preparation of antioxidant products, including medicines, foods or health products.

[0043] The present invention provides an application of the above-mentioned ganoderma triterpene extract in preparing an anti-glioma product.

[0044] In the present invention, it is found that the triterpene extract of Ganoderma lucidum has high anti-glioma activity. The product includes a drug. The triterpene extract of Ganoderma lucidum can be used as the only active ingredient for treating glioma, or it can be used in combination with other active ingredients for treating glioma to treat glioma. The drug preferably includes pharmaceutically acceptable excipients, including adjuvants, excipients, glidants, sweeteners, diluents, preservatives, colorants, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers.

[0045] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0046] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0047] In the following examples, the selenium-rich Ganoderma lucidum is selenium-rich purple Ganoderma lucidum.

[0048] Example 1

[0049] A method for preparing selenium-enriched Ganoderma lucidum nanopowder, the specific steps are as follows:

[0050] (1) Take 2 kg of fresh selenium-enriched Ganoderma lucidum fruiting bodies (common Ganoderma lucidum enriched with selenium, purchased from Ganzhou Zhensheng Agricultural Development Co., Ltd., which has obtained selenium-enriched certification, certificate number: 752SEP2200135), clean them, dry them in natural sunlight for 24 hours, cut them into 2×2 cm Ganoderma lucidum particles, dry them in a temperature-controlled forced air drying oven at 65°C to a moisture content of 13%, and grind them with a YB-type high-speed multifunctional pulverizer (purchased from Yongkang Sufeng Industry and Trade Co., Ltd.) at 25°C for 2 minutes to obtain selenium-enriched Ganoderma lucidum coarse powder.

[0051] (2) The selenium-enriched Ganoderma lucidum coarse powder was dried in a temperature-controlled forced air drying oven at a constant temperature of 65°C to a moisture content of 7%, and further pulverized using a BW-type low-temperature ultrafine pulverizer to obtain selenium-enriched Ganoderma lucidum nanopowder. At the same time, an orthogonal experiment was used to optimize the pulverization time, pulverization temperature, feed ratio and feed amount when pulverizing using the BW-type low-temperature ultrafine pulverizer. The specific parameters are shown in Table 1. Selenium-enriched Ganoderma lucidum nanopowder was prepared according to the pulverization time, pulverization temperature, feed ratio and feed amount given in Experiment Nos. 1 to 16.

[0052] The feed ratio refers to the weight ratio of the selenium-rich ganoderma lucidum coarse powder to the volume ratio of the feed sample bin of the low-temperature ultrafine pulverizer.

[0053] The water content refers to the percentage of the weight of water in Ganoderma lucidum to the total weight of Ganoderma lucidum after drying.

[0054] Table 1 Selenium-enriched Ganoderma lucidum nano-crushing process parameters

[0055]

[0056]

[0057] Selenium-enriched Ganoderma lucidum nanopowders prepared in Experiment Nos. 1 to 16 were respectively poured into glass test tubes containing 6 mL of water and dispersed in the water by ultrasound. The hydrated particle size (HD) was measured by Zeta potential and particle size analyzer (90PlusPLAS, Brookhaven, USA). Each sample was measured six times and the average value was taken. The results are shown in Table 2.

[0058] Table 2 Hydration particle size results (spherical diameter) of selenium-enriched Ganoderma lucidum nanopowder prepared by different nano-crushing processes

[0059]

[0060] From the results in Table 2, we can see that the results can be analyzed using the intuitive analysis method, and the extreme difference R crushing time>R sample volume>R crushing temperature can be obtained. It can be seen that the factors affecting the ultrafine crushing effect are: crushing time> sample volume> crushing temperature. Since the smaller the size, the better, the smaller the K value, the better the condition. Therefore, the optimal conditions are crushing time 50min, crushing temperature -20℃, and sample volume 600g. Because the difference between the results at -10℃ and -20℃ is very small, both conditions are verified.

[0061] Furthermore, G(d) and C(d) were used to analyze in detail the particle size distribution of the selenium-enriched Ganoderma lucidum nanopowders prepared in Experiment Nos. 1 to 16.

[0062] G(d) represents the number distribution of particle size, which generally shows whether the distribution of particle size conforms to the normal distribution. If it conforms to the normal distribution, its highest point can be used as the average particle size; C(d) represents the cumulative percentage of particle size distribution, which represents the total proportion of all powder particle sizes below this particle size.

[0063] Figure 1 From the results, it can be seen that the number distribution of selenium-rich Ganoderma lucidum nanopowders prepared in Experimental Numbers 1 to 16 all presents a good normal distribution, so the highest point is taken as the average particle size. According to the value of the highest point, it can be seen that the particle size of selenium-rich Ganoderma lucidum nanopowders prepared in Experimental Numbers 1 to 16 is all nanometer-scale.

[0064] Statistical analysis of D in selenium-enriched Ganoderma lucidum nanopowders prepared in Experiment Nos. 1 to 16 0.1 , D 0.5 , D 0.9 Value. 0.1 , D 0.5 , D 0.9The values ​​represent the particle size values ​​corresponding to the cumulative distribution percentages of 10%, 50% and 90%. The particle size distribution width (SPAN) indicates the width of the particle size distribution. The larger the SPAN value, the wider the particle size distribution. The calculation formula is as follows:

[0065] SPAN=(D 0.9 -D 0.1 )÷D 0.5 .

[0066] Table 3 D of selenium-enriched Ganoderma lucidum nanopowder prepared in Experiment No. 1 to 16 0.1 , D 0.5 , D 0.9 , SPAN value

[0067] Experiment number <![CDATA[D 0.1 (nm)]]> <![CDATA[D 0.5 (nm)]]> <![CDATA[D 0.9 (nm)]]> SPAN SD 1 444.39 696.37 1123.08 1.135 0.393 2 320.05 595.72 1088.90 1.312 0.135 3 232.88 440.89 842.94 1.386 0.068 4 311.70 594.60 1128.30 1.413 0.180 5 236.29 445.42 832.67 1.352 0.095 6 262.18 509.79 980.28 1.437 0.183 7 266.07 495.68 931.98 1.395 0.194 8 232.63 451.67 868.91 1.415 0.099 9 301.43 522.98 913.57 1.256 0.299 10 240.47 448.28 830.37 1.388 0.223 11 284.62 522.78 955.27 1.309 0.158 12 128.32 273.59 591.72 1.700 0.182 13 255.03 455.32 824.42 1.273 0.110 14 169.77 336.93 659.28 1.463 0.113 15 211.60 414.77 803.95 1.448 0.138 16 225.50 427.99 821.36 1.441 0.224

[0068] The results in Table 3 show that the SPAN values ​​of the selenium-rich Ganoderma lucidum nanopowders prepared in Experiment Nos. 1 to 16 are mostly below 1.5, and the difference is below 0.4, indicating that the particle size distribution of the selenium-rich Ganoderma lucidum nanopowders prepared in Experiment Nos. 1 to 16 under different grinding conditions is relatively concentrated. 0.9 Most of them are below 1000nm, which shows that selenium-rich Ganoderma lucidum powder can reach nanometer level after ultrafine grinding.

[0069] Verification experiment: The selenium-rich Ganoderma lucidum coarse powder prepared according to the above step (1) was dried in a temperature-controlled forced air drying oven at a constant temperature of 65°C to a moisture content of 7%. 600 g of the selenium-rich Ganoderma lucidum coarse powder was further crushed using a BW low-temperature ultrafine grinder at -10°C or -20°C for 50 min to obtain selenium-rich Ganoderma lucidum nanopowder a1 and selenium-rich Ganoderma lucidum nanopowder a2, respectively.

[0070] Selenium-enriched Ganoderma lucidum nanopowder a1 and selenium-enriched Ganoderma lucidum nanopowder a2 were poured into glass test tubes containing 6 mL of water, and dispersed in the water by ultrasound. The hydrated particle size (HD) was measured by Zeta potential and particle size analyzer, and each parallel sample was measured 3 times. The results are shown in Table 4.

[0071] Table 4 Hydration particle size of verification experiment

[0072]

[0073] The results in Table 4 show that at -10℃, the size of nanopowder is slightly smaller than that at -20℃, and -10℃ is also more energy-efficient, so the optimal conditions for ultrafine grinding are confirmed to be 50min of grinding time, -10℃ of grinding temperature, and 600g of sample volume.

[0074] Electron microscopic measurement: Take appropriate amounts of the selenium-enriched Ganoderma lucidum coarse powder and selenium-enriched Ganoderma lucidum nanopowder a1 prepared in Example 1, respectively, and perform electron microscopic measurement, and use Nano Measurer software to measure the particle size in the figure, and use origin to draw the figure (see Figure 2 ).

[0075] Figure 2 The results show that the particle size of selenium-rich Ganoderma lucidum nanopowder a1 is 330nm, and its Ganoderma lucidum fiber structure is destroyed to varying degrees, while the average diameter of selenium-rich Ganoderma lucidum coarse powder is 2.52μm and the length is less than 20mm, indicating that the ultrafine grinding process of the present invention can obtain selenium-rich Ganoderma lucidum powder with a particle size of nanometer scale.

[0076] Example 2

[0077] S1. Preparation of selenium-enriched Ganoderma lucidum polysaccharide extract: Weigh 4 g of the selenium-enriched Ganoderma lucidum nanopowder a1 prepared in Example 1 into a round-bottom flask, add 120 mL of water, shake the flask to mix, let stand for 30 min, heat to reflux and keep it slightly boiling for 2 h, filter, collect the first filtrate and the first filter residue, put the first filter residue back into the flask, add 112 mL of water, shake the flask to mix, heat to reflux and keep it slightly boiling for 2 h, filter, collect the second filtrate, combine the first filtrate and the second filtrate to obtain a total filtrate, concentrate the total filtrate under reduced pressure, and finally freeze-dry to obtain a selenium-enriched Ganoderma lucidum polysaccharide extract.

[0078] Ganoderma lucidum polysaccharide content determination:

[0079] Preparation of reference solution: Take an appropriate amount of anhydrous glucose reference, weigh accurately, add water to make a solution containing 0.12 mg per 1 mL to obtain the reference solution.

[0080] Preparation of standard curve: Accurately measure 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, and 1.2mL of the reference solution and place them in 10mL stoppered test tubes respectively. Add water to 2.0mL each, quickly and accurately add 6mL of anthrone sulfate solution (accurately weigh 0.1g of anthrone, add 100mL of sulfuric acid to dissolve, and shake well), shake well immediately, let it stand for 15min, then immediately cool it in an ice bath for 15min, take it out, use the corresponding reagent as blank, use a UV-visible spectrophotometer to measure the absorbance at a wavelength of 625nm, and draw a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0081] Preparation of the test solution: Take the selenium-enriched Ganoderma lucidum polysaccharide extract prepared in step S1, add 100 mL of water to the scale, and shake well.

[0082] Weigh 4 g of the selenium-enriched Ganoderma lucidum powder prepared in step (1) of Example 1 into a round-bottom flask, add 120 mL of water, shake the flask to mix, let stand for 30 min, heat to reflux and keep it slightly boiling for 2 h, filter, collect the first filtrate 1 and the first filter residue 1, put the first filter residue 1 back into the flask, add 112 mL of water, shake the flask to mix, heat to reflux and keep it slightly boiling for 2 h, filter, collect the second filtrate 1, combine the first filtrate 1 and the second filtrate 1 to obtain a total filtrate 2, concentrate the total filtrate 2 under reduced pressure, and finally freeze-dry to obtain a selenium-enriched Ganoderma lucidum polysaccharide extract a, add 100 mL of water, shake well, and obtain a control sample solution 1 (recorded as selenium-enriched Ganoderma lucidum powder).

[0083] Determination method: Accurately measure 2 mL of the test solution and place it in a 10 mL stoppered test tube. Follow the method under the standard curve preparation, starting from "rapidly and accurately add 6 mL of anthracene sulfate solution", and operate in the same way to determine the absorbance. Read the content of anhydrous glucose in the test solution or control sample solution 1 from the standard curve and calculate.

[0084] Standard curve: Use concentration as the horizontal axis and absorbance as the vertical axis to draw a standard curve, and the regression equation is: y = 38.306x-0.0119, and the correlation coefficient R 2 =0.9985.

[0085] The polysaccharide content in the sample is calculated according to the following formula:

[0086] Calculation formula: ω=m1÷m2×2.5×100%.

[0087] The polysaccharide content in the sample is calculated according to the following formula:

[0088] Where: ω—the percentage of polysaccharide in the sample;

[0089] m1—the mass of anhydrous glucose read from the standard curve in the test method, in grams (g);

[0090] m2—the mass of the selenium-enriched Ganoderma lucidum polysaccharide extract after drying, in grams (g);

[0091] 2.5—Conversion factor.

[0092] S2. Preparation of selenium-enriched Ganoderma lucidum triterpene extract: Take 5 g of the selenium-enriched Ganoderma lucidum nanopowder a1 prepared in Example 1 and put it into a conical flask, add 125 mL of 70% ethanol solution, seal and let it stand for 30 minutes, then perform ultrasound, after ultrasound at room temperature with a power of 300 W for 60 minutes, filter, collect filtrate 1 and residue 1, put the residue 1 back into the conical flask, add 115 mL of 70% ethanol solution, seal and perform ultrasound, after ultrasound at room temperature with a power of 300 W for 60 minutes, filter, collect filtrate 2, combine filtrate 1 and filtrate 2 to obtain total filtrate a, concentrate the total filtrate a under reduced pressure, and finally freeze-dry to obtain the selenium-enriched Ganoderma lucidum triterpene extract.

[0093] Determination of triterpenoid content in Ganoderma lucidum:

[0094] Preparation of reference solution: Take an appropriate amount of oleanolic acid reference substance, weigh accurately, and add methanol to make a solution containing 0.2 mg per 1 mL to obtain the reference solution.

[0095] Preparation of standard curve: Accurately measure 0.1mL, 0.2mL, 0.3mL, 0.4mL, and 0.5mL of reference solution, place them in 15mL stoppered test tubes respectively, evaporate to dryness, let cool, accurately add 0.2mL of newly prepared vanillin glacial acetic acid solution (accurately weigh 0.5g of vanillin, add glacial acetic acid to dissolve it into 10mL), 0.8mL of perchloric acid, shake well, heat in a 70℃ water bath for 15min, immediately cool in an ice bath for 5min, take out, accurately add 4mL of ethyl acetate, shake well, use the corresponding reagent as blank, measure the absorbance at a wavelength of 546nm by UV-visible spectrophotometry, and draw a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0096] Preparation of the test solution: Take the selenium-enriched Ganoderma lucidum triterpene extract prepared in step S2, place it in a 100mL volumetric flask, add 100mL of ethanol to the scale, shake well, and obtain.

[0097] 5 g of the selenium-enriched Ganoderma lucidum powder prepared in step (1) of Example 1 was added to 125 mL of 70% ethanol solution, the mixture was sealed and allowed to stand for 30 min, then ultrasonicated. After ultrasonicating at room temperature with a power of 300 W for 60 min, the mixture was filtered, and the filtrate a and the residue a were collected. The residue a was put back into a conical flask, 115 mL of 70% ethanol solution was added, the mixture was sealed and ultrasonicated. After ultrasonicating at room temperature with a power of 300 W for 60 min, the mixture was filtered, and the filtrate b was collected. The filtrate a and the filtrate b were combined to obtain a total filtrate A. The total filtrate A was concentrated under reduced pressure, and finally freeze-dried to obtain a selenium-enriched Ganoderma lucidum triterpene extract a. 100 mL of ethanol was added and the mixture was shaken to obtain a control sample solution (referred to as the selenium-enriched Ganoderma lucidum powder group).

[0098] Determination method: Accurately measure 0.2 mL of the control sample solution or the test solution and place it in a 15 mL stoppered test tube. According to the method under the standard curve preparation, from "evaporation to dryness", operate in the same way to determine the absorbance, read the content of oleanolic acid in the control sample solution or the test solution from the standard curve, and calculate.

[0099] Standard curve: Use concentration as the horizontal axis and absorbance as the vertical axis to draw a standard curve. The regression equation of the curve is: y = 34.443x-0.0621, and the correlation coefficient R 2 is 0.9983.

[0100] The triterpene content in the sample is calculated according to the following formula:

[0101] Calculation formula: ω=m3÷m4×2.5×100%.

[0102] The polysaccharide content in the sample is calculated according to the following formula:

[0103] Where: ω—the percentage of triterpenes in the sample;

[0104] m3—the mass of oleanolic acid read from the standard curve in the test method, in grams (g);

[0105] m4—the mass of the selenium-enriched Ganoderma triterpenoid extract after the sample is dried, in grams (g);

[0106] 2.5—Conversion factor.

[0107] The results are shown in Table 5 and Table 5. Figure 3 .

[0108] Table 5 Results of polysaccharide and triterpenoid contents of selenium-enriched Ganoderma lucidum coarse powder before and after nano-crushing

[0109] sample Polysaccharide content / % Triterpene content / % Selenium-enriched Ganoderma lucidum powder 0.962 1.167 Selenium-enriched Ganoderma lucidum nanopowder 6.257 1.285

[0110] Table 5 and Figure 3 The results showed that compared with selenium-enriched Ganoderma lucidum crude powder, the polysaccharide and triterpenoid contents extracted using selenium-enriched Ganoderma lucidum nanopowder were significantly increased.

[0111] S3. Preparation of selenium-rich Ganoderma lucidum polysaccharide extract: Weigh 200 g of the selenium-rich Ganoderma lucidum nanopowder a1 prepared in Example 1 into a round-bottom flask, add 6000 mL of water, shake the flask to mix, let stand for 30 min, heat to reflux and keep it at a slight boiling point for 2 h, filter, collect filtrate I and residue I, put the residue I back into the flask, add 5600 mL of water, shake the flask to mix, heat to reflux and keep it at a slight boiling point for 2 h, filter, collect filtrate II, combine filtrate I and filtrate II to obtain total filtrate I, concentrate total filtrate I under reduced pressure, and finally freeze-dry to obtain a selenium-rich Ganoderma lucidum polysaccharide extract.

[0112] Preparation of control polysaccharide extract: Weigh 200 g of the selenium-enriched Ganoderma lucidum crude powder prepared in step (1) of Example 1 into a round-bottom flask, add 6000 mL of water, shake the flask to mix, let stand for 30 min, heat to reflux and keep it at a slight boiling point for 2 h, filter, collect the filtrate c and the residue c, put the residue c back into the flask, add 5600 mL of water, shake the flask to mix, heat to reflux and keep it at a slight boiling point for 2 h, filter, collect the filtrate d, combine the filtrate c and the filtrate d to obtain the total filtrate B, concentrate the total filtrate B under reduced pressure, and finally freeze-dry to obtain a selenium-enriched Ganoderma lucidum crude powder polysaccharide extract.

[0113] S4. Preparation of selenium-enriched Ganoderma lucidum triterpene extract: Take 200g of the selenium-enriched Ganoderma lucidum nanopowder a1 prepared in Example 1 and put it into a conical flask, add 5000mL of 70% ethanol solution, seal and let it stand for 30min, then perform ultrasound, after ultrasound at room temperature with a power of 300W for 60min, filter, collect filtrate 3 and residue 3, put the residue 3 back into the conical flask, add 4600mL of 70% ethanol solution, seal and perform ultrasound, after ultrasound at room temperature with a power of 300W for 60min, filter, collect filtrate 4, combine filtrate 3 and filtrate 4 to obtain total filtrate C, concentrate the total filtrate C under reduced pressure, and finally freeze-dry to obtain the selenium-enriched Ganoderma lucidum triterpene extract.

[0114] Preparation of control polysaccharide extract: Weigh 200g of the selenium-enriched Ganoderma lucidum crude powder prepared in step (1) of Example 1 and put it into a conical flask, add 5000mL of 70% ethanol solution, seal and let it stand for 30min, then perform ultrasound, after ultrasound at room temperature with a power of 300W for 60min, filter, collect filtrate 3 and residue 3, put residue 3 back into the conical flask, add 4600mL of 70% ethanol solution, seal and perform ultrasound, after ultrasound at room temperature with a power of 300W for 60min, filter, collect filtrate 4, combine filtrate 3 and filtrate 4 to obtain total filtrate C, concentrate total filtrate C under reduced pressure, and finally freeze-dry to obtain a triterpene extract of selenium-enriched Ganoderma lucidum crude powder.

[0115] The selenium-rich Ganoderma lucidum polysaccharide extract, selenium-rich Ganoderma lucidum crude powder polysaccharide extract, selenium-rich Ganoderma lucidum triterpene extract and selenium-rich Ganoderma lucidum crude powder triterpene extract prepared in steps S3 and S4 were added with 100 mL of water, 100 mL of water, 100 mL of ethanol and 100 mL of ethanol, respectively, to prepare selenium-rich Ganoderma lucidum polysaccharide test solution, selenium-rich Ganoderma lucidum polysaccharide reference solution, selenium-rich Ganoderma lucidum triterpene test solution and selenium-rich Ganoderma lucidum triterpene reference solution respectively.

[0116] Then, the polysaccharide contents of the selenium-enriched Ganoderma lucidum polysaccharide test solution and the selenium-enriched Ganoderma lucidum polysaccharide reference solution were determined according to the above-mentioned Ganoderma lucidum polysaccharide determination method, and the polysaccharide contents of the selenium-enriched Ganoderma lucidum triterpenoid test solution and the selenium-enriched Ganoderma lucidum triterpenoid reference solution were determined according to the Ganoderma lucidum triterpenoid determination method. The Ganoderma lucidum extracts prepared in steps S3 and S4 were weighed, and the extraction rate was calculated.

[0117] Extraction rate calculation formula: ω=m5÷200×100%.

[0118] Where: ω—extraction rate of sample extract;

[0119] m5—extract mass, in grams (g);

[0120] 200—The mass of the sample after drying, in grams (g).

[0121] Table 6 Results of polysaccharide and triterpene extraction rates of selenium-enriched Ganoderma lucidum coarse powder before and after nano-crushing

[0122] sample Triterpene extraction rate / % Polysaccharide extraction rate / % Selenium-enriched Ganoderma lucidum powder 4.55 7.77 Selenium-enriched Ganoderma lucidum nanopowder 5.28 12.2

[0123] The results in Table 6 show that compared with selenium-enriched Ganoderma lucidum crude powder, the extraction rate of Ganoderma lucidum polysaccharides extracted using selenium-enriched Ganoderma lucidum nanopowder increased by about 56%, and the extraction rate of triterpenes also increased by about 16%.

[0124] Example 3

[0125] A1. Determination of selenium content: The selenium content in the selenium-enriched Ganoderma lucidum nanopowder a1 prepared in Example 1 was determined according to the method of GB5009.93-2017. The specific results are shown in Table 7.

[0126] Table 7 Selenium content results of selenium-enriched Ganoderma lucidum coarse powder before and after nano-crushing

[0127]

[0128] The results in Table 7 show that the selenium content in the selenium-enriched Ganoderma lucidum nanopowder can retain 85% of that in the selenium-enriched Ganoderma lucidum coarse powder.

[0129] A2. Determination of heavy metal content:

[0130] Determination standards: lead-GB5009.12, mercury-GB 5009.17, arsenic-GB 5009.11, cadmium-GB 5009.15. The contents of heavy metals (lead, mercury, arsenic and cadmium) in the selenium-enriched Ganoderma lucidum nanopowder a1 prepared in Example 1 and the selenium-enriched Ganoderma lucidum coarse powder prepared in step (1) were respectively determined. The results are shown in Table 8.

[0131] The standard limit adopts the "Announcement on 9 New Substances Including Codonopsis Pilosula That Are Traditionally Both Food and Chinese Medicinal Materials".

[0132] Table 8 Results of heavy metal content in selenium-enriched Ganoderma lucidum coarse powder before and after nano-crushing

[0133]

[0134] The results in Table 8 show that the contents of lead, mercury, arsenic and cadmium in the selenium-enriched Ganoderma lucidum nanopowder a1 and the selenium-enriched Ganoderma lucidum coarse powder prepared by the present invention are all below the standard limits, which meets the requirements of the Announcement on 9 Newly Added Substances That Are Traditionally Both Food and Chinese Medicinal Materials, Including Codonopsis Pilosula.

[0135] Example 4

[0136] Example 2 Determination of the free radical scavenging ability of the selenium-enriched Ganoderma lucidum polysaccharide extract prepared in step S1 and the selenium-enriched Ganoderma lucidum triterpene extract prepared in step S2.

[0137] Polysaccharide test solution: 4 mg of the selenium-enriched Ganoderma lucidum polysaccharide extract or the selenium-enriched Ganoderma lucidum polysaccharide extract a prepared in step S1 of Example 2 was dissolved in 20 mL of water to prepare polysaccharide test solution 1 or polysaccharide test solution 2, respectively.

[0138] Triterpene test solution: 4 mg of the selenium-enriched Ganoderma triterpene extract or the selenium-enriched Ganoderma triterpene extract a prepared in step S2 of Example 2 was dissolved in 20 mL of 95% ethanol solution to prepare triterpene test solution 1 or triterpene test solution 2.

[0139] DPPH free radical scavenging activity test:

[0140] First, 0.1 mmol·L was prepared using 95% ethanol solution. -1 DPPH free radical solution.

[0141] For the sample group, 1 mL of the above polysaccharide test solution 1, polysaccharide test solution 2, triterpene test solution 1 or triterpene test solution 2 (0.2 mg mL -1 ) was mixed with 2 mL of DPPH free radical solution, shaken, and incubated at room temperature in the dark for 30 min. The above reaction solutions were taken separately and their absorbance at 517 nm was measured using a UV-visible spectrophotometer.

[0142] The control group used a sample dissolving solvent (water for polysaccharide test solution and 95% ethanol solution for triterpenoid test solution) instead of DPPH free radical solution. That is, 1 mL of the above polysaccharide test solution 1, polysaccharide test solution 2, triterpenoid test solution 1 or triterpenoid test solution 2 (0.2 mg mL -1 ) was mixed with 2 mL of 95% ethanol solution, shaken, and incubated at room temperature in the dark for 30 min. The above reaction solutions were taken separately and their absorbance at 517 nm was measured using a UV-visible spectrophotometer.

[0143] The blank group was replaced with a sample solvent (water for polysaccharide test solution and 95% ethanol solution for triterpenoid test solution). That is, 1 mL of water or 95% ethanol solution was mixed with 2 mL of DPPH free radical solution, shaken well, and incubated at room temperature in the dark for 30 minutes. The above reaction solutions were taken separately and their absorbance at 517 nm was measured using a UV-visible spectrophotometer.

[0144] Each sample was measured three times, and the DPPH free radical scavenging rate (E1, %) was calculated as follows:

[0145] E1 = [1-(A1-A) / A0] × 100%)

[0146] Where: A1 is the absorbance of the sample group; A is the absorbance of the control group; A0 is the absorbance of the blank group.

[0147] ABTS free radical scavenging activity test:

[0148] 7mmol·L -1 ABTS and 2.45mmol·L -1 After mixing 10 mL of potassium persulfate aqueous solution, react at room temperature in the dark for 12 to 16 hours to obtain ABTS free radical solution. Before use, dilute the ABTS free radical solution with distilled water to make its initial absorbance (734 nm) 0.70 ± 0.02 to prepare ABTS free radical working solution for use.

[0149] For the sample group, 700 μL of ABTS free radical working solution was mixed with 100 μL of polysaccharide test solution 1, polysaccharide test solution 2, triterpene test solution 1 or triterpene test solution 2 (0.2 mg mL -1 ) and shake well, incubate in dark for 30 min, and then measure the absorbance at 734 nm.

[0150] The control group used the sample dissolving solvent (water for polysaccharide test solution and 95% ethanol solution for triterpenoid test solution) instead of the ABTS free radical working solution, that is, 700 μL of distilled water was mixed with 100 μL of polysaccharide test solution 1, polysaccharide test solution 2, triterpenoid test solution 1 or triterpenoid test solution 2 (0.2 mg mL-1 ) and shake well, incubate in dark for 30 min, and then measure the absorbance at 734 nm.

[0151] The blank group was replaced with the sample solution by the sample dissolving solvent (water for polysaccharide test solution and 95% ethanol solution for triterpenoid test solution). That is, 1 mL of water or 95% ethanol solution was mixed with 700 μL of ABTS free radical working solution, shaken well, incubated in the dark for 30 minutes, and then the absorbance at 734 nm was measured.

[0152] Each sample was measured three times, and the ABTS free radical scavenging rate (E2, %) was calculated as follows:

[0153] E2 = [1-(A2-A') / A0'] × 100%)

[0154] Where: A2 is the absorbance of the sample group; A' is the absorbance of the control group; A0' is the absorbance of the blank group.

[0155] Table 9 Results of free radical scavenging ability of polysaccharide and triterpene extracts before and after ultrafine grinding of selenium-enriched Ganoderma lucidum coarse powder

[0156]

[0157] Figure 4-5 The results in Table 9 show that after ultrafine grinding, the DPPH and ABTS free radical scavenging abilities of the selenium-rich Ganoderma polysaccharide extract and the selenium-rich Ganoderma triterpene extract extracted by selenium-rich Ganoderma nanopowder were improved, among which the ABTS free radical scavenging ability of the selenium-rich Ganoderma polysaccharide extract extracted by selenium-rich Ganoderma nanopowder was the highest, which was about 32.2% higher than that of the ABTS free radical scavenging ability of the selenium-rich Ganoderma triterpene extract of selenium-rich Ganoderma crude powder.

[0158] Example 5

[0159] 1. Anti-cancer experiment of selenium-rich Ganoderma lucidum extract

[0160] The common ganoderma lucidum of this example was purchased from Ganzhou Zhensheng Agricultural Development Co., Ltd. The common ganoderma lucidum refers to conventional ganoderma lucidum that has not been cultivated fortified with selenium nutrition.

[0161] The preparation method of ordinary Ganoderma lucidum nano powder comprises the following steps:

[0162] (1) 2 kg of fresh common Ganoderma lucidum fruiting bodies were taken, cleaned, dried in natural sunlight for 24 hours, cut into 2×2 cm Ganoderma lucidum particles, dried in a temperature-controlled forced air drying oven at 65° C. until the moisture content reached 13%, and pulverized with a YB-type high-speed multifunctional pulverizer (purchased from Yongkang Sufeng Industry and Trade Co., Ltd.) at 25° C. for 2 min to obtain common Ganoderma lucidum coarse powder.

[0163] (2) Dry the ordinary Ganoderma lucidum powder in a temperature-controlled forced air drying oven at 65°C to a constant temperature of 7%; and further grind 600 g of the ordinary Ganoderma lucidum powder using a BW low-temperature ultrafine grinder at -10°C for 50 minutes to obtain ordinary Ganoderma lucidum nanopowder.

[0164] Preparation of common Ganoderma lucidum polysaccharide extract (CKCF-GLP): Weigh 4 g of the common Ganoderma lucidum crude powder prepared in step (1) into a round-bottom flask, add 120 mL of water, shake the flask to mix, let stand for 30 minutes, heat to reflux and keep it at a slight boiling point for 2 hours, filter, collect the first filtrate and the first filter residue, put the first filter residue back into the flask, add 112 mL of water, shake the flask to mix, heat to reflux and keep it at a slight boiling point for 2 hours, filter, collect the second filtrate, combine the first filtrate and the second filtrate to obtain a total filtrate, concentrate the total filtrate under reduced pressure, and finally freeze-dry to obtain a common Ganoderma lucidum polysaccharide extract (CKCF-GLP).

[0165] Preparation of common Ganoderma lucidum triterpene extract (CKCF-GLT): Take 5g of the common Ganoderma lucidum crude powder prepared in step (1) and put it into a conical flask, add 125mL of 70% ethanol solution, seal and let it stand for 30min, then perform ultrasound, after ultrasound at room temperature with a power of 300W for 60min, filter, collect filtrate 1 and residue 1, put the residue 1 back into the conical flask, add 115mL of 70% ethanol solution, seal and perform ultrasound, after ultrasound at room temperature with a power of 300W for 60min, filter, collect filtrate 2, combine filtrate 1 and filtrate 2 to obtain total filtrate a, concentrate the total filtrate a under reduced pressure, and finally freeze-dry to obtain common Ganoderma lucidum triterpene extract (CKCF-GLT).

[0166] Preparation of common Ganoderma lucidum polysaccharide extract (CKNM-GLP): Weigh 4 g of the common Ganoderma lucidum nanopowder prepared in step (2) into a round-bottom flask, add 120 mL of water, shake the flask to mix, let stand for 30 minutes, heat to reflux and keep it at a slight boiling point for 2 hours, filter, collect the first filtrate and the first filter residue, put the first filter residue back into the flask, add 112 mL of water, shake the flask to mix, heat to reflux and keep it at a slight boiling point for 2 hours, filter, collect the second filtrate, combine the first filtrate and the second filtrate to obtain a total filtrate, concentrate the total filtrate under reduced pressure, and finally freeze-dry to obtain a common Ganoderma lucidum polysaccharide extract (CKNM-GLP).

[0167] Preparation of common Ganoderma lucidum triterpene extract (CKNM-GLT): Take 5g of common Ganoderma lucidum nanopowder prepared in step (2) and put it into a conical flask, add 125mL of 70% ethanol solution, seal and let it stand for 30min, then perform ultrasound, after ultrasound at room temperature with a power of 300W for 60min, filter, collect filtrate 1 and residue 1, put the residue 1 back into the conical flask, add 115mL of 70% ethanol solution, seal and perform ultrasound, after ultrasound at room temperature with a power of 300W for 60min, filter, collect filtrate 2, combine filtrate 1 and filtrate 2 to obtain total filtrate a, concentrate the total filtrate a under reduced pressure, and finally freeze-dry to obtain common Ganoderma lucidum triterpene extract (CKNM-GLT).

[0168] The selenium-enriched Ganoderma lucidum polysaccharide extract (FXNM-GLP) is the selenium-enriched Ganoderma lucidum polysaccharide extract prepared by step S1 of Example 2.

[0169] The selenium-enriched Ganoderma triterpene extract (FXNM-GLT) is the selenium-enriched Ganoderma triterpene extract prepared by step S2 of Example 2.

[0170] The selenium-enriched Ganoderma lucidum crude powder polysaccharide extract (FXCF-GLP) is the selenium-enriched Ganoderma lucidum polysaccharide extract a prepared in step S1 of Example 2.

[0171] The selenium-enriched Ganoderma lucidum crude powder triterpene extract (FXCF-GLT) is the selenium-enriched Ganoderma lucidum triterpene extract a prepared in step S2 of Example 2.

[0172] 2. Test methods

[0173] The CCK8 method was used for evaluation.

[0174] Plating: U251 cells were seeded into 96-well plates at a cell density of 3000 cells / well and a total culture medium of 100 μL / well.

[0175] Administration: CKCF-GLP at a concentration of 0, 0.4 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, FXCF-GLP at a concentration of 0, 0.4 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, CKNM-GLP at a concentration of 0, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, and FXNM-GLP at a concentration of 0, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL were added to a 96-well plate at 100 μL / well. After 24 hours, the old solution was removed by aspiration. The solution was added again, and 0, 0.4 mg / mL, 0.8 mg / mL, 1 mg / mL, and 1.2 mg / mL CKCF-GLP, 0, 0.4 mg / mL, 0.8 mg / mL, 1 mg / mL, and 1.2 mg / mL FXCF-GLP, 0, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1 mg / mL, and 1.2 mg / mL CKNM-GLP, and 0, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1 mg / mL, and 1.2 mg / mL FXNM-GLP were added to the 96-well plate at 100 μL / well and cultured for 24 h.

[0176] CKCF-GLT at a concentration of 0, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, and 1 mg / mL, FXCF-GLT at a concentration of 0, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, and 1 mg / mL, CKNM-GLT at a concentration of 0, 0.2 mg / mL, 0.8 mg / mL, 1 mg / mL, and 1.2 mg / mL, and FXNM-GLT at a concentration of 0, 0.2 mg / mL, 0.4 mg / mL, and 0.8 mg / mL were added to a 96-well plate at 100 μL / well. After 24 hours, the old solution was removed by aspiration. The plate was then added with 100 μL / well of 0, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, and 1 mg / mL CKCF-GLT, 0, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, and 1 mg / mL FXCF-GLT, 0, 0.2 mg / mL, 0.8 mg / mL, 1 mg / mL, and 1.2 mg / mL CKNM-GLT, and 0, 0.2 mg / mL, 0.4 mg / mL, and 0.8 mg / mL FXNM-GLT, and cultured for 24 h.

[0177] Test: After 48 hours of co-culture, add 10% CCK8 and mix well in the dark, culture for 30 minutes, and measure the OD value with a microplate reader. Data processing uses Prism software to draw graphs and perform statistical analysis. Calculate cell viability (%) = [A (drug added) - A (blank)] / [A (0 drug added) - A (blank)] × 100, where A (drug added): OD value of wells with cells, CCK-8 solution and drug solution; A (0 drug added): OD value of wells with cells, CCK-8 solution but no drug solution; A (blank): OD value of wells without cells.

[0178] 3. Study on the effects of different Ganoderma lucidum polysaccharide extracts on the proliferation activity of U251 cells

[0179] The results showed that CKCF-GLP prepared from common Ganoderma lucidum crude powder had no in vitro anti-U251 cell proliferation activity ( Figure 6 a). After ultrafine grinding, CKNM-GLP prepared from ordinary Ganoderma lucidum nanopowder promoted cancer cell proliferation as the concentration increased ( Figure 6 b) in the above.

[0180] FXCF-GLP prepared from selenium-rich Ganoderma lucidum powder showed a certain inhibitory activity on the proliferation of U251 cancer cells as the concentration increased. Figure 6 c), which should be due to the selenium-containing substances in FXCF-GLP, such as selenium polysaccharides, which play an inhibitory role in the proliferation of U251 cancer cells. The use of FXNM-GLP extracted from selenium-rich Ganoderma lucidum nanopowder did not find the effect of FXCF-GLP on promoting cancer cell proliferation ( Figure 6 d), which may be because the richer selenium polysaccharides in FXNM-GLP extracted from selenium-enriched Ganoderma lucidum nanopowder inhibit the proliferation activity of U251 cancer cells, offsetting the proliferation effect of cancer cells due to the increase in sugar components after nano-sizing.

[0181] 4. Effects of different Ganoderma lucidum triterpenoid extracts on the proliferation activity of U251 cells

[0182] Different Ganoderma triterpenoid extracts have different degrees of inhibitory effects on the proliferation of U251 cancer cells cultured in vitro (see Figure 7 CKNM-GLT ( Figure 7 b) The anti-proliferation effect of U251 cancer cells was significantly better than that of the triterpene extract of Ganoderma lucidum powder CKCF-GLT ( Figure 7 a) in the figure.

[0183] Selenium-enriched Ganoderma lucidum powder triterpene extract FXCF-GLT( Figure 7 The anti-proliferation effect of U251 cancer cells in c) was significantly better than that of the triterpene extract of common Ganoderma lucidum powder CKCF-GLT ( Figure 7(a). The selenium-enriched Ganoderma lucidum triterpenoid extract FXNM-GLT ( Figure 7 d) than the selenium-enriched Ganoderma lucidum powder triterpene extract FXCF-GLT ( Figure 7 c) has better anti-proliferation effect on U251 cancer cells, and its IC 50 Can reach 80μg / mL (see Table 10).

[0184] The results in Table 10 show that the selenium-enriched Ganoderma lucidum triterpene extract FXNM-GLT has the highest anti-proliferation effect on U251 cancer cells.

[0185] Table 10 IC of different Ganoderma triterpenoid extracts in inhibiting U251 cell proliferation 50 value

[0186] Ganoderma lucidum triterpenoid extract CKCF-GLT CKNM-GLT FXCF-GLT FXNM-GLT <![CDATA[IC 50 (mg / mL)]]> 0.50 0.18 0.32 0.08

[0187] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing Ganoderma lucidum nanopowder, characterized in that: The following steps are involved: Dry the ganoderma lucidum fruiting bodies for 1 to 2 days, cut them into ganoderma lucidum particles, and then dry them at a constant temperature of 50 to 65° C. until the water content is 10 to 15% to obtain dried ganoderma lucidum particles, and grind them at 20 to 30° C. for 1 to 3 minutes to obtain ganoderma lucidum coarse powder; The ganoderma lucidum powder is dried at a constant temperature of 50-65° C. until the water content is below 7% to obtain dry ganoderma lucidum powder; 250-650 g of the dry ganoderma lucidum powder is ultrafinely ground at -20-0° C. for 15-55 minutes to obtain ganoderma lucidum nanopowder.

2. The preparation method according to claim 1, characterized in that: The ganoderma lucidum is selenium-enriched ganoderma lucidum.

3. The preparation method according to claim 1, characterized in that: The ultrafine grinding method is to ultrafine grind 500-600g of ganoderma lucidum coarse powder at -20--10°C for 40-50min.

4. The preparation method according to claim 1, characterized in that: The ultrafine grinding method is to ultrafine grind 600g of coarse powder of Ganoderma lucidum for 50 minutes at -20 to -10°C.

5. A method for preparing a ganoderma lucidum polysaccharide extract, characterized in that: The following steps are involved: The ganoderma nano powder according to any one of claims 1 to 4 is mixed with water, subjected to heating and reflux extraction, filtered to collect the filtrate, concentrated under reduced pressure and dried to obtain the powder.

6. The preparation method according to claim 5, characterized in that: The mass volume ratio of the ganoderma nano powder to water is 1 g: 25-32 mL; the number of reflux extractions is 1-3 times, and the time for each extraction is 1.5-2.5 hours.

7. A method for preparing a triterpene extract of Ganoderma lucidum, characterized in that: The following steps are involved: The nano-powder of Ganoderma lucidum described in any one of claims 1 to 4 is mixed with 65% to 75% ethanol solution, subjected to ultrasonic extraction, filtered and collected the filtrate, concentrated under reduced pressure and dried to obtain the powder.

8. The preparation method according to claim 7, characterized in that: The mass volume ratio of the ganoderma nano powder to the 65%-75% ethanol solution is 1g:20-27mL; the ultrasonic extraction is performed 1-3 times at room temperature and a power of 250-350W, and the time of each ultrasonic extraction is 55-65min.

9. Use of the Ganoderma lucidum polysaccharide extract according to claim 5 or 6 or the Ganoderma lucidum triterpene extract according to claim 7 or 8 in the preparation of antioxidant products.

10. Use of the Ganoderma triterpene extract according to claim 7 or 8 in the preparation of an anti-glioma product.

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