A process for freeze-drying a Cordyceps and Polyporus umbellatus antitumor complex

Abstract

This invention discloses a freeze-dried preparation process of a Cordyceps and Polyporus umbellatus antitumor complex, specifically relating to the field of biomedical technology. Specifically, it uses Cordyceps powder and Polyporus umbellatus powder as raw materials, employing a simulated intestinal solution for repeated low-temperature pulsed ultrasound extraction under specific pH conditions. Following low-temperature clarification, filtration, reduced-pressure concentration, membrane fractionation, and freeze-drying protection, a freeze-dried antitumor complex powder is obtained, exhibiting both active ingredient retention, reconstitution stability, and the ability to promote macrophage proliferation and inhibit tumor cell proliferation. This invention, through the synergistic effect of intestinal simulated solution extraction, low-temperature pulsed ultrasound, membrane fractionation, and freeze-drying protection, fully releases and stably retains the active ingredients of Cordyceps and Polyporus umbellatus, improving extraction efficiency, reconstitution, and storage stability, enabling the freeze-dried powder to simultaneously promote macrophage proliferation and inhibit tumor cell proliferation.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a process for preparing a freeze-dried Cordyceps and Polyporus umbellatus antitumor complex. Background Technology

[0002] Medicinal fungi possess both nutritional and medicinal value. Their active components, such as polysaccharides, glycoproteins, nucleosides, alkaloids, and glycosides, have potential applications in immunomodulation, antioxidation, anti-inflammation, and anti-tumor activity. Cordyceps contains cordycepin and cordyceps polysaccharides, which can exert their effects by inhibiting tumor cell proliferation, inducing apoptosis, and regulating the body's immunity. Polyporus umbellatus contains active substances such as polyporus polysaccharides, which can regulate macrophage function and participate in anti-tumor immune responses.

[0003] In existing technologies, research on Cordyceps or Polyporus umbellatus mostly focuses on single extracts, such as extracting polysaccharides or nucleosides alone. While this approach facilitates component analysis, it easily overlooks the synergistic effects between multiple active ingredients in the whole fungus. Furthermore, conventional hot water extraction, high-temperature reflux extraction, or simple water extraction methods suffer from insufficient selectivity, loss of heat-sensitive components, incomplete release of active ingredients, and poor stability during subsequent storage.

[0004] Furthermore, Cordyceps and Polyporus umbellatus products are mostly ingested orally, and the release and absorption of their active ingredients are closely related to the gastrointestinal environment. If only ordinary water extraction or high-temperature extraction is used, it is difficult to simulate the release process of polysaccharides, glycoproteins, and small molecule active substances in the intestinal environment, and it is also difficult to obtain a complex active component that has both the effect of promoting immune cell proliferation and the direct inhibitory effect of tumor cells.

[0005] Therefore, there is an urgent need to provide a freeze-drying preparation process for the Cordyceps and Polyporus umbellatus antitumor complex. This process should combine intestinal mimicry solution, low-temperature pulsed ultrasound, graded clarification filtration, active component compounding, and freeze-drying protection to maintain the synergistic activity of multiple components while improving extraction efficiency, formulation stability, and reconstitution. Summary of the Invention

[0006] The main objective of this invention is to provide a freeze-drying process for a Cordyceps militaris and Polyporus umbellatus antitumor complex, which can effectively solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A process for preparing a lyophilized Cordyceps militaris and Polyporus umbellatus antitumor complex includes the following steps: S1. Mix Cordyceps powder and Polyporus umbellatus powder in a certain proportion to obtain Cordyceps-Polyporus umbellatus compound powder; S2. Add artificial intestinal simulation solution to the Cordyceps and Polyporus umbellatus compound powder, adjust the pH of the system to 6.5-7.8, and obtain the suspension to be extracted; S3. The suspension to be extracted is subjected to low-temperature pulsed ultrasonic extraction, and the temperature of the extraction system is controlled not to exceed 45°C during the ultrasonic process. After the extraction is completed, the supernatant is collected by centrifugation to obtain the first extract. S4. Add artificial intestinal simulation solution to the solid residue after centrifugation, repeat low-temperature pulsed ultrasonic extraction and centrifuge to obtain the supernatant, and obtain the second extract. S5. Combine the first extract and the second extract, and then clarify, filter and concentrate under reduced pressure to obtain a concentrated extract; S6. Add a freeze-drying protectant to the concentrated extract, and after pre-freezing, vacuum freeze-drying, pulverizing and sieving, obtain the freeze-dried powder of Cordyceps and Polyporus umbellatus antitumor complex.

[0008] Preferably, the mass ratio of cordyceps powder to poria cocos powder is 1:0.2 to 1:5, more preferably 1:0.5 to 1:2; the cordyceps powder and poria cocos powder are passed through a 60-200 mesh sieve before mixing, and the moisture content is controlled to be no higher than 10%.

[0009] Preferably, in step S2, the artificial intestinal simulation solution includes a phosphate buffer system, pancreatic enzymes, and bile salts, wherein the concentration of pancreatic enzymes is 0.1–20 mg / ml, and the concentration of bile salts is 0.05–10 mg / ml; the ratio of the Cordyceps militaris compound powder to the artificial intestinal simulation solution is 1 g:8 ml to 1 g:30 ml.

[0010] Preferably, in steps S3 and S4, the ultrasonic frequency of the low-temperature pulsed ultrasound extraction is 20–45 kHz, the ultrasonic power is 100–600 W, the single ultrasound time is 20–60 min, and the extraction is performed using a pulse mode with 3–10 min ultrasound and 1–5 min intervals.

[0011] Preferably, in step S5, the low-temperature clarification includes centrifuging the combined extract after standing at 2–10°C for 2–12 h; the filtration includes passing the extract through filter membranes of 0.8 μm–1.2 μm and 0.22 μm–0.45 μm in sequence to remove insoluble particles and retain water-soluble polysaccharides, glycoproteins, nucleosides and small molecule active ingredients.

[0012] Preferably, in step S5, after filtration, a membrane fractionation process is further performed. The membrane fractionation process includes using an ultrafiltration membrane with a molecular weight cutoff of 3 to 10 kDa to separate the filtrate into high molecular weight active components and low molecular weight active components, and then combining the high molecular weight active components and low molecular weight active components at a solid mass ratio of 1:0.1 to 1:3 to obtain a concentrated extract.

[0013] Preferably, in step S6, the freeze-drying protectant includes one or more of trehalose, mannitol, glycine, dextran, and β-cyclodextrin, and the amount of the freeze-drying protectant added is 1% to 20% of the total solids in the concentrated extract; the pre-freezing temperature is -20℃ to -80℃, the pre-freezing time is 4 to 24 h, the vacuum degree of the vacuum freeze-drying is 5 to 30 Pa, and the freeze-drying time is 24 to 72 h.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes artificial intestinal simulated fluid extraction, low-temperature pulsed ultrasound, repeated extraction, and low-temperature clarification filtration to fully release and retain the active components such as polysaccharides, glycoproteins, and nucleosides in Cordyceps militaris and Polyporus umbellatus, thereby improving extraction efficiency and component stability. Through membrane-based fractionation and compounding, high-molecular-weight immunomodulatory components and low-molecular-weight tumor-suppressing components work synergistically. By adding a lyophilization protectant and performing vacuum freeze-drying, the freeze-dried powder's shape retention, resolubility, and storage stability are improved, thus obtaining an anti-tumor complex lyophilized powder that promotes macrophage proliferation and inhibits tumor cell proliferation. Attached Figure Description

[0015] Figure 1 This is a flowchart of the preparation method of the present invention. Detailed Implementation

[0016] The technical solution of the present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. Conventional adjustments or equivalent substitutions made by those skilled in the art to various process parameters, raw material ratios and processing conditions without departing from the concept of the present invention should all fall within the scope of protection of the present invention.

[0017] This invention provides a freeze-dried preparation process for a Cordyceps and Polyporus umbellatus antitumor complex, mainly including steps such as compounding Cordyceps powder and Polyporus umbellatus powder, extraction with artificial intestinal simulated fluid, low-temperature pulsed ultrasound, repeated extraction, low-temperature clarification and filtration, vacuum concentration, and freeze-drying. Through the combination of these steps, the polysaccharides, glycoproteins, nucleosides, glycosides, and small molecule active ingredients in Cordyceps and Polyporus umbellatus can be fully released and stably retained, thereby obtaining a freeze-dried complex powder with the basic effects of promoting immune cell proliferation and inhibiting tumor cell proliferation.

[0018] Example 1, as Figure 1 This embodiment provides a process for preparing freeze-dried Cordyceps and Polyporus umbellatus antitumor complex, including the following steps: S1. Take Cordyceps powder and Polyporus umbellatus powder and place them in a dry environment for later use. Pass the Cordyceps powder and Polyporus umbellatus powder through a 100-mesh sieve, and control the moisture content of both to be no higher than 10%. Weigh the raw materials according to the mass ratio of Cordyceps powder to Polyporus umbellatus powder of 1:1, mix them thoroughly, and obtain Cordyceps-Polyporus umbellatus compound powder.

[0019] S2. Add an artificial intestinal simulation solution to the Cordyceps and Polyporus umbellatus compound powder. The artificial intestinal simulation solution includes a phosphate buffer system, pancreatic enzymes, and bile salts. The pancreatic enzyme concentration is 5 mg / ml, and the bile salt concentration is 1 mg / ml. Control the ratio of Cordyceps and Polyporus umbellatus compound powder to the artificial intestinal simulation solution at 1 g:10 ml. After stirring and dispersing, adjust the pH of the system to 7.2 to obtain the suspension to be extracted.

[0020] S3. The suspension to be extracted is placed in an ultrasonic extraction device for low-temperature pulsed ultrasonic extraction. The ultrasonic frequency is 40kHz, the ultrasonic power is 300W, and a pulse mode of 5 minutes of ultrasonication followed by 2 minutes of intermittent sonication is adopted, with a total ultrasonic time of 30 minutes. During the ultrasonication process, the temperature of the extraction system is controlled not to exceed 45℃ by using an ice bath or circulating cooling water. After extraction, the mixture is centrifuged at 2400 rpm for 15 minutes, and the supernatant is collected to obtain the first extract.

[0021] S4. Add an equal volume of artificial intestine simulation solution to the solid residue after centrifugation in step S3, and perform a second extraction under the same low-temperature pulsed ultrasound conditions as in step S3. After extraction, centrifuge and collect the supernatant to obtain the second extract.

[0022] S5. Combine the first and second extracts, and let the combined extract stand at 4°C for 6 hours for low-temperature clarification, then centrifuge to remove the precipitate. Filter the clarified supernatant sequentially through a 1.0 μm filter membrane and a 0.45 μm filter membrane to remove insoluble particles and coarse impurities. Concentrate the filtered extract under reduced pressure at low temperature to obtain a concentrated extract.

[0023] S6. Add a freeze-drying protectant to the concentrated extract. The freeze-drying protectant is a combination of trehalose and mannitol, wherein the mass ratio of trehalose to mannitol is 1:1, and the amount of freeze-drying protectant added is 10% of the total solids in the concentrated extract. After dispensing the mixture, pre-freeze it at -80℃ for 12 hours, and then freeze-dry it in a vacuum freeze dryer at a vacuum degree controlled at 10 Pa for 48 hours. After freeze-drying, pulverize the obtained freeze-dried material and pass it through an 80-mesh sieve to obtain the freeze-dried powder of the Cordyceps militaris and Polyporus umbellatus antitumor complex.

[0024] This embodiment utilizes the synergistic effect of artificial intestinal simulation fluid and low-temperature pulsed ultrasound to simulate the release process of Cordyceps militaris and Polyporus umbellatus complex in the intestinal environment, and reduces the damage to active ingredients caused by high temperature; the two extractions can improve the sufficiency of the release of effective ingredients; and the addition of a freeze-drying protectant and vacuum freeze-drying can improve the shapeability, resolubility and storage stability of the freeze-dried powder.

[0025] Example 2 provides another preparation process for freeze-drying of Cordyceps and Polyporus umbellatus antitumor complex. The main difference between this example and Example 1 is the difference in the raw material ratio, the material-liquid ratio, and the membrane grading treatment conditions.

[0026] S1. Pass the cordyceps powder and poria powder through an 80-mesh sieve separately, control the moisture content to be no more than 10%, and then mix them according to the mass ratio of cordyceps powder to poria powder of 1:0.5 to obtain cordyceps and poria compound powder.

[0027] S2. Add artificial intestinal simulation solution to the Cordyceps and Polyporus umbellatus compound powder. The artificial intestinal simulation solution has a pancreatic enzyme concentration of 2 mg / ml and a bile salt concentration of 0.5 mg / ml. The material-liquid ratio of Cordyceps and Polyporus umbellatus compound powder to artificial intestinal simulation solution is 1g:15ml. Adjust the pH of the system to 6.8 to obtain the suspension to be extracted.

[0028] S3. The suspension to be extracted is subjected to low-temperature pulsed ultrasonic extraction. The ultrasonic frequency is 25kHz, the ultrasonic power is 250W, and a pulse mode of 3 minutes of ultrasonication followed by 1 minute of intermittent sonication is adopted, with a total ultrasonic time of 40 minutes. The temperature of the extraction system is controlled below 40℃. After extraction, the supernatant is collected by centrifugation to obtain the first extract.

[0029] S4. Add artificial intestinal simulation solution to the solid residue after centrifugation, repeat low-temperature pulsed ultrasonic extraction and centrifuge to obtain the supernatant, and obtain the second extract.

[0030] S5. Combine the first and second extracts, let stand at 2°C for 8 hours, then centrifuge to clarify. Filter sequentially through 0.8 μm and 0.22 μm membranes. After filtration, perform membrane fractionation using a 10 kDa ultrafiltration membrane to obtain high molecular weight active components and low molecular weight active components. Combine the high molecular weight active components and low molecular weight active components at a solids-to-liquid mass ratio of 1:0.5, then concentrate under reduced pressure to obtain a concentrated extract.

[0031] S6. Mannitol and dextran were added to the concentrated extract as freeze-drying protectants, with the amount of freeze-drying protectants added being 8% of the total solids in the concentrated extract. The extract was pre-frozen at -60℃ for 10 hours, followed by freeze-drying under a vacuum of 15 Pa for 60 hours. After freeze-drying, the extract was pulverized and passed through a 100-mesh sieve to obtain the freeze-dried powder of the Cordyceps militaris and Polyporus umbellatus antitumor complex.

[0032] In this embodiment, membrane fractionation is used to enrich high-molecular-weight polysaccharides, glycoproteins and low-molecular-weight nucleosides and glycosides separately before compounding, which is conducive to forming a complex active system with both immunomodulatory and direct tumor-suppressive properties.

[0033] Example 3 provides another preparation process for freeze-drying of Cordyceps and Polyporus umbellatus antitumor complex. The main difference between this example and Example 1 is the ratio of Cordyceps powder to Polyporus umbellatus powder and the composition of the freeze-drying protectant.

[0034] S1. Pass the cordyceps powder and poria cocos powder through a 150-mesh sieve, and control the moisture content to be no higher than 8%. Weigh and mix the cordyceps powder and poria cocos powder at a mass ratio of 1:2 to obtain cordyceps and poria cocos compound powder.

[0035] S2. Add artificial intestinal simulation solution to the Cordyceps and Polyporus umbellatus compound powder. The artificial intestinal simulation solution has a pancreatic enzyme concentration of 10 mg / ml, a bile salt concentration of 2 mg / ml, a material-to-liquid ratio of 1g:20ml, and adjust the pH of the system to 7.5 to obtain the suspension to be extracted.

[0036] S3. The suspension to be extracted was subjected to low-temperature pulsed ultrasonic extraction at a frequency of 35 kHz and a power of 400 W. A pulsed pattern of 8 minutes of sonication followed by 3 minutes of intermittent sonication was used, for a total sonication time of 45 minutes. The system temperature was controlled to not exceed 45℃ during the sonication process. After extraction, the supernatant was collected by centrifugation to obtain the first extract.

[0037] S4. Add artificial intestinal simulation solution to the solid residue again, repeat low-temperature pulsed ultrasonic extraction, and obtain the second extract after centrifugation.

[0038] S5. Combine the first and second extracts, and let them stand at 10℃ for 4 hours for low-temperature clarification. After centrifugation, filter them sequentially through a 1.2μm filter membrane and a 0.45μm filter membrane. After filtration, perform membrane fractionation using a 3kDa ultrafiltration membrane to collect the high molecular weight active components and low molecular weight active components separately. These components are then compounded at a solids-to-liquid mass ratio of 1:1.5 and concentrated under reduced pressure to obtain the concentrated extract.

[0039] S6. Trehalose, glycine, and β-cyclodextrin were added to the concentrated extract as freeze-drying protectants in a mass ratio of 2:1:1, and the amount of freeze-drying protectant added was 15% of the total solids in the concentrated extract. The extract was pre-frozen at -40℃ for 24 hours, and then freeze-dried under a vacuum of 8 Pa for 72 hours. After freeze-drying, the extract was pulverized and passed through a 60-mesh sieve to obtain the freeze-dried powder of the Cordyceps militaris and Polyporus umbellatus antitumor complex.

[0040] This embodiment increases the proportion of poria cocos powder and combines it with membrane grading to enhance the proportion of immunomodulatory components such as poria cocos polysaccharide in the complex; by using trehalose, glycine and β-cyclodextrin as freeze-drying protectants, it helps to reduce the structural changes of active components and the risk of powder collapse during freeze-drying.

[0041] Comparative Example 1 differs from Example 1 in that, in step S2, artificial intestinal simulation fluid is not used, but double-distilled water is used as the extraction medium. The remaining steps are the same as in Example 1.

[0042] In comparison, although the double-distilled water extraction method can obtain a certain amount of water-soluble components, its ability to release polysaccharides, glycoproteins and small molecule active ingredients related to intestinal release in the Cordyceps and Polyporus umbellatus complex is relatively insufficient. The resulting freeze-dried powder has a weaker overall effect on promoting macrophage proliferation and inhibiting tumor cell proliferation than in Example 1.

[0043] Comparative Example 2 differs from Example 1 in that, in steps S3 and S4, low-temperature pulsed ultrasonic extraction is not used, but 80°C water bath heating extraction is used, with each extraction time being 30 minutes. The remaining steps are the same as in Example 1.

[0044] In comparison, high-temperature heating extraction can promote the dissolution of some water-soluble components, but the high-temperature environment may cause some heat-sensitive active components to degrade or change in structure, thereby affecting the overall activity of the resulting lyophilized compound powder.

[0045] Comparative Example 3 differs from Example 1 in that no freeze-drying protectant is added in step S6, and the concentrated extract is directly pre-frozen and vacuum freeze-dried. The remaining steps are the same as in Example 1.

[0046] In comparison, without the addition of a freeze-drying protectant, the freeze-dried product is prone to problems such as loose and uneven powder structure, local collapse, or decreased reconstitution rate. However, in Example 1, the addition of trehalose and mannitol as freeze-drying protectants can improve the shapeability and reconstitution of the freeze-dried powder, and is beneficial to the stable maintenance of active ingredients during freeze-drying and storage.

[0047] In vitro activity verification: To verify the application effect of the lyophilized Cordyceps and Polyporus umbellatus antitumor complex obtained in this invention, in vitro evaluation can be carried out using CCK-8 assay, fluorescence staining assay, and microscopic observation.

[0048] Specifically, RAW264.7 macrophages and HepG2 tumor cells were seeded into 96-well plates, with 5000–10000 cells per well, and cultured at 37°C and 5% CO2 for 24 h. Blank, control, and experimental groups were set up. The experimental groups were treated with different concentrations of the drug solution prepared by reconstitution of the lyophilized powder of this invention, with concentrations of 1 mg / ml, 2 mg / ml, 4 mg / ml, 8 mg / ml, 16 mg / ml, and 32 mg / ml. After 24 h or 48 h of treatment, CCK-8 reagent was added, and the absorbance was measured at 450 nm. Cell proliferation was calculated for each group according to the cell proliferation rate formula.

[0049] The cell proliferation rate is calculated using the following formula: Cell proliferation rate (%) = Corrected OD value of experimental group / Corrected OD value of control group × 100%.

[0050] Experimental results show that the lyophilized powder of the Cordyceps and Polyporus umbellatus antitumor complex prepared in this invention has a proliferation-promoting effect on RAW264.7 macrophages in the concentration range of 1 mg / ml to 4 mg / ml, indicating that it has a certain basis for immune cell regulation; at a concentration of 4 mg / ml and above, it has a proliferation-inhibiting effect on HepG2 tumor cells, indicating that it has a certain basis for direct tumor inhibition.

[0051] Furthermore, the viability and mortality of RAW264.7 cells were observed using the Calcein-AM / PI double staining method. RAW264.7 cells were treated with the lyophilized powder of this invention for 24 hours, then incubated with Calcein-AM and PI dye working solutions. Subsequently, green fluorescent live cells and red fluorescent dead cells were observed under an inverted fluorescence microscope. The results showed that within a suitable concentration range, RAW264.7 cells were predominantly green fluorescent live cells with a low proportion of dead cells, indicating that the lyophilized powder of this invention has no significant toxicity to macrophages and can promote macrophage proliferation within a certain concentration range.

[0052] As can be seen from the above embodiments and verification results, the present invention, through the synergistic combination of process steps such as artificial intestinal simulated fluid extraction, low-temperature pulsed ultrasound, repeated extraction, low-temperature clarification filtration, membrane graded compounding and freeze-drying protection, can obtain a freeze-dried powder of Cordyceps and Polyporus umbellatus antitumor complex with sufficient component release, good resolubility, high stability, and both immune cell proliferation promotion and tumor cell proliferation inhibition effects.

[0053] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A preparation process of a freeze-dried antitumor compound of Cordyceps militaris and Polyporus umbellatus, characterized in that, Includes the following steps: S1. Mix Cordyceps powder and Polyporus umbellatus powder in a certain proportion to obtain Cordyceps-Polyporus umbellatus compound powder; S2. Add artificial intestinal simulation solution to the Cordyceps and Polyporus umbellatus compound powder, adjust the pH of the system to 6.5-7.8, and obtain the suspension to be extracted; S3. The suspension to be extracted is subjected to low-temperature pulsed ultrasonic extraction, and the temperature of the extraction system is controlled not to exceed 45°C during the ultrasonic process. After the extraction is completed, the supernatant is collected by centrifugation to obtain the first extract. S4. Add artificial intestinal simulation solution to the solid residue after centrifugation, repeat low-temperature pulsed ultrasonic extraction and centrifuge to obtain the supernatant, and obtain the second extract. S5. Combine the first extract and the second extract, and then clarify, filter and concentrate under reduced pressure to obtain a concentrated extract; S6. Add a freeze-drying protectant to the concentrated extract, and after pre-freezing, vacuum freeze-drying, pulverizing and sieving, obtain the freeze-dried powder of Cordyceps and Polyporus umbellatus antitumor complex.

2. The preparation process of the freeze-dried Cordyceps and Polyporus umbellatus antitumor complex according to claim 1, characterized in that: The mass ratio of cordyceps powder to poria cocos powder is 1:0.2 to 1:5, preferably 1:0.5 to 1:2; the cordyceps powder and poria cocos powder are passed through a 60-200 mesh sieve before mixing, and the moisture content is controlled to be no higher than 10%.

3. The preparation process of the freeze-dried antitumor compound of Cordyceps militaris and Polyporus umbellatus according to claim 1, characterized in that: In step S2, the artificial intestinal simulation solution includes a phosphate buffer system, pancreatic enzymes, and bile salts. The concentration of pancreatic enzymes is 0.1–20 mg / ml, and the concentration of bile salts is 0.05–10 mg / ml. The ratio of the Cordyceps militaris compound powder to the artificial intestinal simulation solution is 1 g:8 ml to 1 g:30 ml.

4. The preparation process of the freeze-dried Cordyceps-Polyporus antitumor complex according to claim 1, characterized in that: In steps S3 and S4, the ultrasonic frequency of the low-temperature pulsed ultrasound extraction is 20–45 kHz, the ultrasonic power is 100–600 W, the single ultrasound time is 20–60 min, and the extraction is performed using a pulse mode with 3–10 min ultrasound and 1–5 min intervals.

5. The preparation process of the freeze-dried antitumor compound of Cordyceps militaris and Polyporus umbellatus according to claim 1, characterized in that: In step S5, the low-temperature clarification includes centrifuging the combined extract after standing at 2–10°C for 2–12 h; the filtration includes passing the extract through filter membranes of 0.8 μm–1.2 μm and 0.22 μm–0.45 μm in sequence to remove insoluble particles and retain water-soluble polysaccharides, glycoproteins, nucleosides and small molecule active ingredients.

6. The preparation process of the freeze-dried antitumor compound of Cordyceps militaris and Polyporus umbellatus according to claim 1, characterized in that: In step S5, after filtration, a membrane fractionation process is further performed. The membrane fractionation process includes using an ultrafiltration membrane with a molecular weight cutoff of 3 to 10 kDa to separate the filtrate into high molecular weight active components and low molecular weight active components. The high molecular weight active components and low molecular weight active components are then compounded at a solid mass ratio of 1:0.1 to 1:3 to obtain a concentrated extract.

7. The preparation process of the freeze-dried Cordyceps and Polyporus umbellatus antitumor complex according to claim 1, characterized in that: In step S6, the freeze-drying protectant includes one or more of trehalose, mannitol, glycine, dextran, and β-cyclodextrin. The amount of freeze-drying protectant added is 1% to 20% of the total solids in the concentrated extract. The pre-freezing temperature is -20℃ to -80℃, and the pre-freezing time is 4 to 24 h. The vacuum degree of the vacuum freeze-drying is 5 to 30 Pa, and the freeze-drying time is 24 to 72 h.

Images