Method for preparing high-activity mulberry and silkworm chrysalis cordyceps polysaccharide through ultrasonic combined microjet

Abstract

The invention belongs to the technical field of preparation of natural active products, and particularly relates to an ultrasonic combined microjet preparation method and application of high-activity mulberry and silkworm chrysalis cordyceps polysaccharide. The purified mulberry and silkworm chrysalis cordyceps polysaccharide prepared by using the ultrasonic extraction technology provided by the invention has the characteristics of high total sugar content and strong antioxidant activity; after further microjet treatment, the total sugar content is further increased, the protein impurity content is reduced, and the antioxidant activity is enhanced. Besides, the mulberry and silkworm chrysalis cordyceps polysaccharide prepared by the ultrasonic combined microjet technology provided by the invention can remarkably inhibit proliferation of human non-small cell lung cancer A549 cells, and the cell survival rate is 10.67% + / -1.87% under the conditions that the concentration is 0.5 mg / mL and the action is performed for 72 hours, and is remarkably lower than that of polysaccharide which is not subjected to microjet treatment and polysaccharide (Plt; 0.05 or Plt; 0.01) of the substrate.

Description

Technical Field

[0001] This invention relates to the field of natural active product preparation technology. Specifically, it provides a method for preparing highly active silkworm pupa cordyceps polysaccharide using ultrasound combined with dynamic ultra-high pressure microfluidic technology, and its use in anti-oxidation and anti-non-small cell lung cancer. Background Technology

[0002] In my country, lung cancer ranks first in incidence among all malignant tumors, with approximately 820,000 new cases annually. Lung cancer is mainly divided into two types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with NSCLC accounting for approximately 85% (Nature Reviews Disease Primers, 2024, 10:71), making it the most common type of lung cancer in clinical practice. Although significant progress has been made in the intervention and treatment of NSCLC in recent years, the overall prognosis for patients remains unsatisfactory, with a five-year survival rate of less than 15% (Journal of Oncology, 2021, 2021:836264). The occurrence and development of NSCLC are closely related to oxidative stress. In the early stages of the disease, environmental factors such as smoking and air pollution can induce excessive accumulation of reactive oxygen species (ROS), disrupting the redox balance in lung tissue. This leads to DNA damage, mitochondrial dysfunction, and abnormal activation of key signaling pathways (such as PI3K / Akt, MAPK, and NF-κB), promoting tumor cell proliferation, angiogenesis, and malignant transformation (American Journal of Cancer Research, 2023, 13:2598-2616). As the disease progresses, NSCLC cells can enhance their ability to adapt to oxidative stress by upregulating endogenous antioxidant systems (such as HO-1, SOD, and GSH-Px) and related signaling axes (such as the Nrf2 pathway). They can even use moderately elevated ROS levels to drive tumor growth, invasion, and drug resistance (Phytotherapy Research, 2025, 39:3353-3385). In recent years, restoring cellular redox homeostasis through exogenous supplementation of antioxidants or targeted regulation of oxidative stress-related pathways has gradually become a new direction for adjuvant therapy of NSCLC. Therefore, developing natural functional ingredients or novel drugs with antioxidant activity that can effectively inhibit the development of NSCLC has significant scientific value and application prospects.

[0003] Polysaccharides, as naturally derived macromolecules, have shown promising applications in the fields of anti-oxidation and anti-NSCLC. Their antioxidant effects are mainly achieved through direct scavenging of free radicals and regulation of endogenous antioxidant enzyme systems (International Journal of Biological Macromolecules, 2024, 270:132391). For example, *Zizania latifolia* polysaccharides not only have significant scavenging effects on DPPH and hydroxyl radicals but also significantly inhibit the proliferation of human non-small cell lung cancer A549 cells (Ultrasonics Sonochemistry, 2024, 103:106803). In the anti-NSCLC field, polysaccharides exert their anti-tumor effects through multiple mechanisms, including inhibiting tumor cell proliferation, inducing apoptosis, and regulating the tumor microenvironment (International Journal of Biological Macromolecules, 2025, 319:145142). However, natural polysaccharides have complex structures and large molecular weights, and traditional extraction methods suffer from low extraction efficiency, long processing times, and high energy consumption, making it difficult to meet the industrial production needs of bioactive polysaccharides and limiting their further development and application.

[0004] Ultrasonic-assisted extraction (UAE) is a non-thermal processing technique whose unique cavitation effect effectively disrupts plant cells, promotes the dissolution of intracellular polysaccharides, shortens extraction time, and increases yield (Ultrasonics Sonochemistry, 2023, 101:106646). Power is a key parameter affecting UAE performance; appropriately increasing ultrasonic power enhances shearing and increases polysaccharide yield, but excessive power can lead to polysaccharide chain breakage, affecting structural integrity and bioactivity (Ultrasonics Sonochemistry, 2023, 98:106487). Therefore, power optimization is necessary to achieve an efficient yet gentle extraction process. Dynamic ultra-high pressure microfluidization (DHPM), through the synergistic effect of ultra-high pressure and high-speed shearing, achieves efficient release and moderate degradation of polysaccharides without the need for high temperatures or chemical reagents, significantly improving their physicochemical properties and bioavailability.

[0005] The silkworm (Bombyx mori L.), also known as the domestic silkworm, belongs to the family Bombycidae in the order Lepidoptera and is an important silk-spinning insect. After spinning its cocoon, the silkworm develops into a spindle-shaped pupa, the silkworm chrysalis, in about four days. Silkworm pupae are one of the main by-products of my country's sericulture industry, currently mostly discarded as a by-product of silk reeling or used as low-value feed, resulting in low resource utilization. Cultivating silkworm cordyceps using silkworm pupae as a substrate is an effective way to obtain cordyceps fruiting bodies through substrate cultivation. Because silkworm pupae themselves have high nutritional value, the cultivated silkworm cordyceps has superior nutritional value compared to other substrate-cultivated fruiting bodies, and the cultivation technology is easy to master, making it an effective way to achieve high-value comprehensive utilization of silkworm pupae (Edible Fungi, 2019, 41:46-48). Polysaccharides are key functional components in silkworm pupae and their cordyceps fruiting bodies. Tan Lihe et al. optimized the extraction process of silkworm pupa polysaccharides using ultrasound-assisted extraction (UAE). Under conditions of 760W ultrasound power, the polysaccharide yield reached 15.28%, and it has been developed into a capsule formulation (Traditional Chinese Medicine, 2016, 38:1254-1259; Traditional Chinese Medicine, 2017, 39:76-79). Shi et al. prepared crude polysaccharides from silkworm pupae and Cordyceps using hot water extraction, with a yield of 7.15%. After separation, polysaccharide components SCP II-1 and SCP II-2 were obtained, both of which showed strong antioxidant activity and inhibitory effects on human liver cancer HepG2 cells, with SCP II-1 showing more significant activity (Journal of Food Biochemistry, 2020, 44:e13482).

[0006] However, existing research still has limitations: on the one hand, the optimization of extraction processes and the evaluation of the activity of Cordyceps militaris polysaccharides from silkworm pupae are insufficient; on the other hand, although UAE showed a high yield in the extraction of silkworm pupae polysaccharides, its application in the extraction of Cordyceps militaris polysaccharides from silkworm pupae has not been reported. Furthermore, the balance between yield and bioactivity in existing extraction methods still needs improvement, especially regarding the antioxidant and lung cancer therapeutic activities of the extracts, which need to be elucidated or enhanced. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention employs ultrasound-assisted extraction (UAE) to prepare Cordyceps militaris polysaccharides from silkworm pupae, and introduces a dual-indicator-guided strategy focusing on yield and antioxidant activity to optimize ultrasonic extraction power. Furthermore, dynamic ultra-high pressure microfluidic (DHPM) is used to modify the extracted polysaccharides, surprisingly revealing a significant enhancement in their bioactivity. This combined process is simple to operate, has low energy consumption, and shows promising prospects for industrial application. The Cordyceps militaris polysaccharides obtained through UAE extraction and DHPM treatment exhibit significant antioxidant and anti-non-small cell lung cancer activities, making them potential candidate lead compounds for novel functional foods or pharmaceuticals.

[0008] The purpose of this invention is to provide a method for preparing highly active silkworm pupa cordyceps polysaccharide using ultrasound combined with microfluidics. The silkworm pupa cordyceps polysaccharide prepared using this method exhibits significant antioxidant and antitumor (especially non-small cell lung cancer) activities, and can be applied to the preparation of related functional products or pharmaceuticals.

[0009] According to a first aspect of the present invention, the present invention provides a method for preparing Cordyceps militaris polysaccharide: using defatted Cordyceps militaris fruiting body powder as raw material, crude Cordyceps militaris polysaccharide is prepared in water by ultrasonic-assisted extraction; after deproteinization and chromatography column purification, purified polysaccharide is obtained, and further purified polysaccharide is obtained by microfluidic homogenization to obtain highly active Cordyceps militaris polysaccharide.

[0010] In some embodiments of the present invention, the method for preparing the crude polysaccharide from silkworm pupae and Cordyceps militaris specifically includes the following steps:

[0011] The *Cordyceps militaris* was pulverized and passed through a 60-mesh sieve. The sample was then immersed in petroleum ether and defatted for 12 hours in the dark. The mixture was then filtered under reduced pressure, and the residue was dried at 60℃ to constant weight to obtain defatted *Cordyceps militaris* powder for later use. 10g of this defatted powder was weighed and added to water at a ratio of 1:30 (g / mL). After soaking for 4 hours, the mixture was transferred to an ultrasonic extractor and ultrasonically extracted for 20 minutes at a specific ultrasonic power and temperature of 60℃. The extract was cooled and filtered. The filtrate was concentrated under reduced pressure to 1 / 4 of its original volume. Anhydrous ethanol was added to adjust the final ethanol concentration to 80% (v / v). The mixture was allowed to stand at 4℃ for 12 hours to precipitate. The precipitate was collected by centrifugation and freeze-dried to obtain crude *Cordyceps militaris* polysaccharide.

[0012] Preferably, the ultrasonic extraction power is 100W to 500W, and more preferably 200W;

[0013] Preferably, the ultrasonic extraction power is 200W. Under these conditions, the yield of crude polysaccharide from *Cordyceps militaris* is 14.68±1.57%, and the DPPH free radical scavenging rate at 0.5 mg / mL is 62.82±1.42%. This method achieves both a high crude polysaccharide yield and excellent antioxidant activity, significantly outperforming other ultrasonic power and hot water extraction methods.

[0014] In some embodiments of the present invention, the preparation method of Cordyceps militaris polysaccharide described herein involves deproteinization and column chromatography purification, which refers to the deproteinization of crude Cordyceps militaris polysaccharide using Sevag reagent followed by purification using a diethylaminoethyl cellulose column. Specifically, the crude Cordyceps militaris polysaccharide is dissolved in an appropriate amount of distilled water, and 1 / 4 of its volume of Sevag reagent (chloroform: n-butanol = 4:1, v / v) is added. After mixing and shaking for 15 min, the lower organic phase and the middle denatured protein precipitate are discarded after centrifugation, while the upper polysaccharide aqueous phase is retained. This process is repeated until no significant protein is produced. The deproteinized polysaccharide solution is collected and freeze-dried to obtain deproteinized Cordyceps militaris polysaccharide.

[0015] Further, the above-mentioned deproteinized polysaccharide was prepared into a deionized water solution with a concentration of 5 mg / mL and loaded onto a pre-equilibrated diethylaminoethyl cellulose chromatography column (30 mm × 400 mm). After loading, elution was performed with 0.5 mol / L NaCl solution at a flow rate of approximately 1 mL / min. The eluent was collected, concentrated to 1 / 5 of its original volume, and dialyzed three times (with deionized water replaced every 12 hours). The dialysate was freeze-dried to obtain purified polysaccharide from *Cordyceps militaris*. Under these conditions, the total sugar content of the purified polysaccharide was 82.14 ± 1.46%, the protein content was 6.75 ± 0.25%, and the uronic acid content was 3.21 ± 0.12%. At 0.5 mg / mL, the DPPH free radical scavenging rate reached 93.96 ± 2.87%, significantly higher than that of the crude polysaccharide (P < 0.01).

[0016] In some embodiments of the present invention, the preparation method of Cordyceps militaris polysaccharide further includes microfluidic treatment of the prepared purified Cordyceps militaris polysaccharide. Specifically, 1.0 g of purified Cordyceps militaris polysaccharide is dissolved in 50 mL of deionized water to prepare a polysaccharide solution with a concentration of 20 mg / mL. The resulting solution is injected into a dynamic ultra-high pressure microfluidic homogenizer and circulated three times under a pressure of 100 MPa, with a residence time of 2 min in the high-pressure zone each time. After treatment, to avoid the influence of pressure instability during equipment startup and shutdown on sample uniformity, only 40 mL of uniformly flowing liquid from the middle section of the microfluidic outlet is collected. The collected polysaccharide treatment solution is freeze-dried to obtain Cordyceps militaris polysaccharide. Under these conditions, the total sugar content of Cordyceps militaris polysaccharide increased to 89.86±1.36%, the protein impurity content decreased to 2.18±0.47%, and the uronic acid content increased to 3.88±0.15%. At 0.5 mg / mL, the scavenging rate of DPPH free radicals increased to 96.66%±2.97%, which was significantly higher than that of polysaccharide without microfluidic treatment and polysaccharide prepared by hot water extraction (P<0.05 or P<0.01).

[0017] The applicant surprisingly discovered that the silkworm pupa cordyceps polysaccharide prepared under these conditions had a significant inhibitory effect on human non-small cell lung cancer (NSCLC) A549 cells, demonstrating significant anti-NSCLC activity. At 0.5 mg / mL, after 48 h of treatment with human NSCLC A549 cells, the cell survival rate was 40.45 ± 5.52%; after 72 h of treatment, the cell survival rate was 10.67% ± 1.87%, significantly lower than that of polysaccharides prepared without microfluidic treatment and polysaccharides prepared by hot water extraction (P < 0.05 or P < 0.01).

[0018] According to a second aspect of the present invention, a silkworm pupa cordyceps polysaccharide is provided, wherein the total sugar content of the silkworm pupa cordyceps polysaccharide is 89.86±1.36%, the protein content is 2.18±0.47%, and the uronic acid content is 3.88±0.15%; the silkworm pupa cordyceps polysaccharide is a heterogeneous polysaccharide with an average molecular weight of 71.97 kDa; the silkworm pupa cordyceps polysaccharide is composed of fucose (1.36%), arabinose (3.9%), galactose (15.4%), glucose (52.48%), and xylose (26.86%); the silkworm pupa cordyceps polysaccharide has a high specific surface area and porosity; the silkworm pupa cordyceps polysaccharide not only has a high yield but also has significant antioxidant and anti-non-small cell lung cancer activity. The infrared spectrum of the silkworm pupa cordyceps polysaccharide is basically as follows. Figure 6 As shown.

[0019] According to a third aspect of the present invention, the present invention provides the use of the aforementioned silkworm pupa cordyceps polysaccharide in the preparation of antioxidant and anti-non-small cell lung cancer products; it can be used to prepare tablets, capsules, oral liquids and other products.

[0020] The method described in the first aspect of the present invention can significantly improve the antioxidant and antitumor effects of Cordyceps militaris polysaccharide extract, that is, as an extraction method to improve the antioxidant and / or antitumor activity of Cordyceps militaris polysaccharide extract, and the product obtained by the method can be used to prepare related drugs and functional products.

[0021] The present invention has the following beneficial effects:

[0022] ① The ultrasound-assisted extraction technology provided by this invention yields purified Cordyceps militaris polysaccharides with high yield, high total sugar content, and high antioxidant activity. Further microfluidic treatment further increases the total sugar content, reduces protein impurities, and enhances antioxidant activity. Therefore, the ultrasound-assisted microfluidic technology provided by this invention is a low-energy-consumption method for efficiently preparing highly active Cordyceps militaris polysaccharides.

[0023] ②In addition, the silkworm pupa cordyceps polysaccharide prepared using the ultrasound-assisted microfluidic technology provided by this invention can significantly inhibit the proliferation of human non-small cell lung cancer A549 cells, showing strong anti-non-small cell lung cancer activity.

[0024] ③ The ultrasonic-assisted microfluidic technology and highly active silkworm pupa cordyceps polysaccharide provided by this invention offer a practical and feasible technical solution for the high-value utilization of silkworm pupa cordyceps, effectively improving the comprehensive utilization efficiency of silkworm pupa resources and helping to solve the problem of low utilization rate. Attached Figure Description

[0025] Figure 1 Effects of different ultrasonic powers and hot water extraction on the yield of crude polysaccharides from silkworm pupae and Cordyceps militaris (different letters above the error line indicate statistically significant differences);

[0026] Figure 2 Effects of different ultrasonic powers and hot water extraction on the antioxidant activity of crude polysaccharides from silkworm pupae and Cordyceps militaris (different letters above the error lines indicate statistically significant differences);

[0027] Figure 3 Antioxidant activity of different Cordyceps militaris polysaccharides (different letters above the error line indicate statistically significant differences); UE: purified Cordyceps militaris polysaccharide-200; UEMP: purified Cordyceps militaris polysaccharide-200 treated with microfluidic jet; HW: purified Cordyceps militaris polysaccharide-hot water; HWMP: purified Cordyceps militaris polysaccharide-hot water treated with microfluidic jet;

[0028] Figure 4 Effects of different Cordyceps militaris polysaccharides on A549 cell viability at 48 h (different letters above the error line indicate statistically significant differences); UE: Cordyceps militaris purified polysaccharide-200; UEMP: Cordyceps militaris purified polysaccharide-200 treated with microfluidic jet; HW: Cordyceps militaris purified polysaccharide-hot water; HWMP: Cordyceps militaris purified polysaccharide-hot water treated with microfluidic jet;

[0029] Figure 5 Effects of different Cordyceps militaris polysaccharides on A549 cell viability at 72 h (different letters above the error line indicate statistically significant differences); UE: Cordyceps militaris purified polysaccharide-200; UEMP: Cordyceps militaris purified polysaccharide-200 treated with microfluidic jet; HW: Cordyceps militaris purified polysaccharide-hot water; HWMP: Cordyceps militaris purified polysaccharide-hot water treated with microfluidic jet;

[0030] Figure 6 .UEMP infrared spectrum;

[0031] Figure 7 .Molecular weight distribution of UEMP;

[0032] Figure 8. UEMP monosaccharide composition chromatogram (peak 1-fucose; peak 2-rhamnose; peak 3-arabinose; peak 4-galactose; peak 5-glucose; peak 6-xylose; peak 7-mannose; peak 8-fructose; peak 9-galacturonic acid; peak 10-glucuronic acid);

[0033] Figure 9 Scanning electron microscope image of UEMP. Detailed Implementation

[0034] The dried fruiting bodies of silkworm pupae and Cordyceps were provided by Suzhou Jiahe Sericulture Biotechnology Co., Ltd.; the ultrasonic extractor was provided by Beijing Xianghu Technology Development Co., Ltd., model XH-2008D; the dynamic ultra-high pressure microfluidic homogenizer was provided by Genizer Inc., USA, model Nano GenizerⅡ; infrared spectroscopy was determined using a Fourier transform infrared spectrometer (FTIR-650) from Tianjin Gangdong Technology Co., Ltd.; monosaccharide composition was determined using an ion chromatograph (ICS-5000) from Dionex; molecular weight was determined using a high performance liquid chromatograph (LC-16) from Shimadzu; scanning electron microscopy images were obtained using a Hitachi ultra-high resolution field emission scanning electron microscope (Regulus 8100); and human non-small cell lung cancer A549 cells were provided by Nanjing Kebai Biotechnology Co., Ltd.

[0035] In this embodiment, experimental data are expressed as mean ± standard deviation (n=3); one-way ANOVA was used for statistical analysis, and a difference was considered statistically significant when P < 0.05.

[0036] Example 1: Ultrasonic-assisted extraction of crude polysaccharides from silkworm pupae and Cordyceps sinensis

[0037] The *Cordyceps militaris* was pulverized and passed through a 60-mesh sieve. The sample was then immersed in petroleum ether and defatted for 12 hours in the dark. The mixture was then filtered under reduced pressure, and the residue was dried at 60℃ to constant weight to obtain defatted *Cordyceps militaris* powder for later use. 10g of this defatted powder was weighed and added to water at a ratio of 1:30 (g / mL). After soaking for 4 hours, the mixture was transferred to an ultrasonic extractor and ultrasonically extracted for 20 minutes at 200W and 60℃. The extract was cooled and filtered. The filtrate was concentrated under reduced pressure to 1 / 4 of its original volume. Anhydrous ethanol was added to adjust the final ethanol concentration to 80% (v / v). The mixture was allowed to stand at 4℃ for 12 hours to precipitate. The precipitate was collected by centrifugation and freeze-dried to obtain crude *Cordyceps militaris* polysaccharide-200. The crude polysaccharide yield (%) = (m / M) × 100, where m is the mass (g) of the freeze-dried crude *Cordyceps militaris* polysaccharide obtained, and M is the mass (g) of the defatted *Cordyceps militaris* powder used. The calculated yield of crude polysaccharide-200 was 14.68 ± 1.57%.

[0038] The antioxidant activity of crude polysaccharide-200 from *Cordyceps militaris* was determined using DPPH free radical scavenging capacity as the evaluation index. A 0.5 mg / mL distilled aqueous solution of crude polysaccharide-200 was prepared. 2 mL of this solution was mixed with 2 mL of DPPH ethanol solution (0.1 mmol / L), shaken thoroughly, and reacted at room temperature in the dark for 30 min. The absorbance was measured at 517 nm. DPPH scavenging rate (%) = [1 - (A1 - A2) / A3] × 100, where A1 is the absorbance of crude polysaccharide-200, A2 is the absorbance of the control group (using anhydrous ethanol instead of DPPH), and A3 is the absorbance of the blank group (using distilled water instead of the sample). The calculated DPPH free radical scavenging rate of crude polysaccharide-200 was 62.82 ± 1.42%.

[0039] Comparative Example

[0040] (1) Based on Example 1, the ultrasonic extraction power was adjusted to 100W, and other experimental steps were the same as in Example 1, to obtain crude polysaccharide-100 from Cordyceps militaris. The calculated yield of crude polysaccharide-100 was 12.46±0.52%, and the scavenging rate of DPPH free radicals was 58.27±1.57%.

[0041] (2) Based on Example 1, the ultrasonic extraction power was adjusted to 300W, and other experimental steps were the same as in Example 1, to obtain crude polysaccharide-300 from silkworm pupa and Cordyceps militaris. The calculated yield of crude polysaccharide-300 was 13.08±0.67%, and the scavenging rate of DPPH free radicals was 63.32±1.61%.

[0042] (3) Based on Example 1, the ultrasonic extraction power was adjusted to 400W, and other experimental steps were the same as in Example 1, to obtain crude polysaccharide-400 from silkworm pupa and Cordyceps militaris. The calculated yield of crude polysaccharide-400 was 12.81±0.73%, and the scavenging rate of DPPH free radicals was 59.73±2.19%.

[0043] (4) Based on Example 1, the ultrasonic extraction power was adjusted to 500W, and other experimental steps were the same as in Example 1, to obtain crude polysaccharide-500 from silkworm pupa and Cordyceps. The calculated yield of crude polysaccharide-500 was 12.0±1.34%, and the scavenging rate of DPPH free radicals was 57.11±1.00%.

[0044] (5) Based on Example 1, the ultrasound-assisted extraction step was replaced with hot water extraction: the extraction temperature was 80℃, the extraction time was 2h, and the remaining extraction conditions and operation steps were the same as in Example 1, yielding crude polysaccharide-hot water from silkworm pupae and Cordyceps militaris. The calculated yield of crude polysaccharide-HW was 10.66±1.06%, and the scavenging rate of DPPH free radicals was 55.25±2.06%.

[0045] By comparing bar charts and conducting statistical analysis, the effects of different ultrasonic powers and traditional hot water extraction methods on the yield and antioxidant activity of crude polysaccharides from silkworm pupae and Cordyceps militaris can be more intuitively reflected.

[0046] like Figure 1 As shown, the crude polysaccharide yield reached its maximum value of 14.68 ± 1.57% when the ultrasonic power was 200 W. This value was significantly higher than the yield under the 100 W condition (12.46 ± 0.52%, P < 0.05) and also significantly higher than the yield of conventional hot water extraction (10.66 ± 1.06%, P < 0.01). When the ultrasonic power exceeded 200 W, the polysaccharide yield showed a decreasing trend with further increases in power. The above results indicate that, compared with the traditional hot water extraction method, ultrasonic-assisted extraction technology can significantly improve the extraction efficiency of crude polysaccharides from Cordyceps militaris. This effect is mainly attributed to the cavitation effect generated during the ultrasonic process and the enhancement of the mass transfer process. These physical effects help to disrupt the cell wall structure and promote the dissolution of intracellular polysaccharides. However, excessively high ultrasonic power may induce mechanical degradation or structural damage to polysaccharide molecules, leading to a decrease in polysaccharide yield (UltrasonicsSonochemistry, 2023, 100:106626). Therefore, in this embodiment, 200W is the optimal ultrasonic power condition for achieving efficient extraction of crude polysaccharides from silkworm pupae and Cordyceps sinensis.

[0047] like Figure 2 As shown, at an ultrasonic power of 200W, the DPPH free radical scavenging rate of crude polysaccharide from *Cordyceps militaris* reached 62.82±1.42%, significantly higher than that at 100W (58.27±1.57%, P<0.05), and also significantly better than that of conventional hot water extraction (55.25±2.06%, P<0.01). Although the scavenging rate at 300W was 63.32±1.61%, slightly higher than that at 200W, there was no statistically significant difference between the two (P>0.05). Similar to the trend of polysaccharide extraction yield, when the ultrasonic power exceeded 300W, the DPPH free radical scavenging rate began to decrease with further increases in power. Combined with the polysaccharide yield ( Figure 1 Based on comprehensive analysis of extraction efficiency and antioxidant activity, both 200W and 300W achieved high free radical scavenging effects. However, since 300W did not bring a statistically significant increase in activity compared to 200W, and its energy consumption was higher, considering energy saving, consumption reduction, and process economy, 200W is more advantageous as the extraction power, provided that extraction efficiency and bioactivity are maintained.

[0048] In summary, in this embodiment, 200W ultrasonic power not only achieves a high yield of crude polysaccharides but also imparts excellent antioxidant activity to the extract and helps reduce energy consumption, making it a preferred process parameter for extracting crude polysaccharides from silkworm pupae and Cordyceps.

[0049] Example 2: Purification of Cordyceps militaris crude polysaccharide-200

[0050] Further, the crude polysaccharide (Cordyceps militaris crude polysaccharide-200) extracted under 200W ultrasonic power was purified. The specific steps are as follows: Cordyceps militaris crude polysaccharide-200 was dissolved in an appropriate amount of distilled water, and 1 / 4 of its volume of Sevag reagent (chloroform: n-butanol = 4:1, v / v) was added. After mixing, the mixture was shaken for 15 min, centrifuged, and the lower organic phase and the middle denatured protein precipitate were discarded. The upper polysaccharide aqueous phase was retained. The above operation was repeated until no obvious protein was produced. The deproteinized polysaccharide solution was collected and freeze-dried to obtain deproteinized Cordyceps militaris polysaccharide-200.

[0051] Further, the above-mentioned deproteinized Cordyceps militaris polysaccharide was prepared into a deionized aqueous solution with a concentration of 5 mg / mL and loaded onto a pre-equilibrated diethylaminoethyl cellulose column (30 mm × 400 mm). After loading, elution was performed with 0.5 mol / L NaCl solution at a flow rate of approximately 1 mL / min. The eluent was collected, concentrated to 1 / 5 of its original volume, and dialyzed three times (with deionized water replaced every 12 hours). The dialysate was freeze-dried to obtain purified Cordyceps militaris polysaccharide-200, named UE. The phenol-sulfuric acid method was used, and the glucose standard curve (Y = 10.893X - 0.0098, R0) was used for analysis. 2 The total sugar content was calculated to be 82.14 ± 1.46% based on the formula (=0.9997).

[0052] Comparative Example

[0053] Based on Example 2, the crude polysaccharide-200 from *Cordyceps militaris* was replaced with crude polysaccharide extracted under hot water conditions (*Cordyceps militaris* crude polysaccharide-hot water). The remaining purification conditions and operating steps were the same as in Example 2, yielding purified *Cordyceps militaris* polysaccharide-hot water, named HW. The total sugar content was determined to be 79.3 ± 0.49% using the phenol-sulfuric acid method.

[0054] Example 3: Microfluidic treatment of purified polysaccharide-200 (UE) from silkworm pupae and Cordyceps sinensis

[0055] 1.0 g of UE was dissolved in 50 mL of deionized water to prepare a polysaccharide solution with a concentration of 20 mg / mL. The resulting solution was injected into a dynamic ultra-high pressure microfluidic homogenizer and circulated three times under a pressure of 100 MPa, with a residence time of 2 min in the high-pressure zone each time. After treatment, to avoid the influence of pressure instability during equipment start-up and termination on sample homogeneity, only 40 mL of homogeneous effluent from the middle section of the microfluidic outlet was collected. The collected polysaccharide solution was freeze-dried to obtain the microfluidic-treated UE, named UEMP.

[0056] Comparative Example

[0057] Based on Example 3, the UE was replaced with purified polysaccharide-hot water (HW) from silkworm pupae and cordyceps. Other microfluidic treatment conditions and operating steps were the same as in Example 3, and the microfluidic treated HW was obtained and named HWMP.

[0058] Example 4: Component Analysis of Cordyceps Polysaccharides from Different Silkworm Pupae

[0059] Total sugar content was determined using the phenol-sulfuric acid method described in Example 2; protein content was determined using the Coomassie brilliant blue method, with bovine serum albumin as the standard. The standard curve was: Y = 4.865X + 0.0252, R0 2 =0.9991; The uronic acid content was determined using the m-hydroxybiphenyl method, with galacturonic acid as the standard. The standard curve was: Y = 9.84X + 0.0195, R 2 =0.9995.

[0060] As shown in Table 1, the total sugar content of UEMP was significantly higher than that of UE (P<0.01), and the total sugar content of HWMP was significantly higher than that of HW (P<0.01); the protein content of UEMP was significantly lower than that of UE (P<0.01), and the protein content of HWMP was significantly lower than that of HW (P<0.01); the uronic acid content of UEMP was significantly higher than that of UE (P<0.01), and the uronic acid content of HWMP was significantly higher than that of HW (P<0.01). The above results indicate that microfluidic treatment can significantly increase the total sugar and uronic acid content of Cordyceps militaris polysaccharides; it can significantly reduce the content of protein impurities and can be used as an auxiliary deproteinization purification method with the potential to be applied to polysaccharide refining processes; at the same time, dynamic ultra-high pressure microfluidic homogenizer microfluidic treatment and ultrasonic extraction have a certain synergistic effect. Simple dynamic ultra-high pressure microfluidic homogenizer microfluidic treatment cannot adjust the purified Cordyceps militaris polysaccharides obtained by different extraction methods to the same product quality, especially the protein and uronic acid content; for uronic acid, the enrichment effect is significantly enhanced after HW microfluidic treatment.

[0061] Table 1. Component analysis results of different silkworm pupa cordyceps polysaccharides

[0062]

[0063] In the same column, different superscript letters indicate statistically significant differences; UE: Purified polysaccharide from Cordyceps militaris-200; UEMP: Purified polysaccharide from Cordyceps militaris treated with microfluidic jets-200; HW: Purified polysaccharide from Cordyceps militaris-hot water; HWMP: Purified polysaccharide from Cordyceps militaris-hot water; ND: Not detected.

[0064] Example 5: Comparison of antioxidant activities of different silkworm pupa cordyceps polysaccharides

[0065] The in vitro antioxidant activity of different silkworm pupa cordyceps polysaccharides (HW, HWMP, UE, UEMP) was investigated using DPPH free radical scavenging capacity as the evaluation index. The specific method is as follows: different silkworm pupa cordyceps polysaccharide samples were prepared into distilled aqueous solutions with concentrations ranging from 0.05 mg / mL to 0.5 mg / mL. Subsequent procedures were performed according to the DPPH free radical scavenging experimental method described in Example 1.

[0066] like Figure 3 As shown, different silkworm pupa cordyceps polysaccharide samples all exhibited certain scavenging effects on DPPH free radicals, and their antioxidant activity increased with increasing sample concentration. At a concentration of 0.5 mg / mL, the scavenging rates of HW and UE on DPPH free radicals were higher than those of the corresponding crude polysaccharides (see...). Figure 2 For example, the DPPH radical scavenging rate of UE was 93.96±2.87%, significantly higher than that of crude polysaccharide (P<0.01), indicating that the purification process helps to enhance the antioxidant activity of Cordyceps militaris polysaccharide. At 0.5 mg / mL, UEMP had the highest DPPH radical scavenging rate, reaching 96.66%±2.97%, significantly higher than other polysaccharide samples (P<0.05 or P<0.01). In addition, the scavenging rate of UE was significantly higher than that of HW (P<0.01), and the scavenging rate of HWMP was significantly higher than that of HW (P<0.01).

[0067] The above results indicate that, compared with the traditional hot water extraction method, ultrasound-assisted extraction not only has high extraction efficiency and low energy consumption, but also significantly improves the antioxidant activity of the obtained polysaccharides; after further microfluidic treatment, the antioxidant activity of the polysaccharides is further enhanced, which plays a synergistic role in antioxidant performance.

[0068] The above effects are related to the significant increase in total sugar and uronic acid content in polysaccharides by microfluidic treatment (see Table 1) (Journal of the Science of Food and Agriculture, 2025, 105: 3170-3183), suggesting that this combined treatment method has a positive effect on improving the bioactivity of polysaccharides. It should be noted that the antioxidant activity of polysaccharides is not simply positively correlated with their total sugar content. Total sugar content reflects the total amount of glycosyl groups in the polysaccharide, but neutral monosaccharides (such as glucose) themselves have a weak ability to scavenge free radicals. Therefore, an increase in total sugar does not necessarily lead to an increase in antioxidant activity (International Journal of Biological Macromolecules, 2024, 254: 127955). In contrast, uronic acid, as an acidic monosaccharide in polysaccharides, is rich in active functional groups such as carboxyl groups. It can effectively scavenge free radicals by providing hydrogen atoms or electrons, and has been confirmed by multiple studies to be significantly positively correlated with the antioxidant capacity of polysaccharides (Antioxidants, 2022, 11: 2491).

[0069] The ultrasound-assisted extraction-microfluidic treatment of silkworm pupa cordyceps polysaccharide (UEMP) prepared in this invention exhibits strong scavenging ability against DPPH free radicals, with an IC50 value of [missing information]. 50 The value was 0.15 ± 0.008 mg / mL. Compared with the Cordyceps militaris polysaccharide prepared by hot water extraction as reported in the literature (Journal of Food Biochemistry, 2020, 44:e13482), UEMP had a higher total sugar content (89.86% vs. 69.63%) and a significantly higher uronic acid content (3.88% vs. not detected), while its DPPH free radical scavenging activity (IC50) was also higher. 50 =0.15mg / mL) is significantly better than the sample reported in the literature (IC50). 50 (≈0.5 mg / mL). The above results indicate that the extraction and purification process provided by this invention helps to obtain silkworm pupa cordyceps polysaccharides with better structure and stronger antioxidant activity, confirming the significant advantages of this technical solution in improving polysaccharide quality and bioactivity.

[0070] In addition, the antioxidant performance value IC of this application 50 The levels were significantly lower than those of various previously reported natural polysaccharides, indicating that UEMP possesses superior antioxidant capacity. For example, compared to the polysaccharide *Hippophae rhamnoides* (IC50) mentioned in the literature... 50 =1.22mg / mL)(Food Industry Technology, 2023, 44:236-243), Tremella polysaccharide (IC50) 50=4.49 mg / mL (Food and Nutrition Science, 2025, 14:129-142) and burdock seed polysaccharide (IC50) 50 =1.05 mg / mL (Food and Fermentation Industries, 2015, 6:207-212), indicating that UEMP has a significantly higher free radical scavenging efficiency. It should be noted that experimental conditions may differ in different studies, but a comprehensive comparison still reflects the significant advantage of the UEMP obtained in this invention in terms of antioxidant activity. This advantage is closely related to the preparation process using ultrasound-assisted extraction combined with microfluidic treatment, indicating that this combined technology not only improves the purity and content of active ingredients in the polysaccharide (see Table 1), but also significantly enhances its biological activity.

[0071] Therefore, the silkworm pupa cordyceps polysaccharide, especially UEMP, prepared by this method has excellent antioxidant activity and has the potential to be developed as a functional ingredient for antioxidant-related health products or drugs. It can be used to prepare food, health food or pharmaceutical compositions with antioxidant functions.

[0072] Example 6: Anti-non-small cell lung cancer activity of different silkworm pupa cordyceps polysaccharides

[0073] Using human non-small cell lung cancer (NSCLC) A549 cells as a model, the anti-NSCLC activity of different silkworm pupa and Cordyceps polysaccharides was evaluated. The MTT assay was used to determine the viability of A549 cells. Specifically, different silkworm pupa and Cordyceps polysaccharide samples were prepared into distilled aqueous solutions with a concentration of 0.5 mg / mL for later use. Human NSCLC A549 cell suspension (5 × 10⁻⁶ cells / mL) was then used... 4 Cells (cells / mL) were seeded into 96-well plates, and then different polysaccharide solutions were added. After 48 h and 72 h of treatment, 10 μL of MTT solution (5 mg / mL, dissolved in PBS buffer) was added, and the plates were incubated at 37 °C for 4 h to precipitate formaldehyde. The precipitate was then dissolved in DMSO, and the absorbance (As) at 490 nm was measured. The culture medium without samples was the control (A1). The culture medium without cells and samples was the blank (A2). Cell viability (%) = [(As-A2) / (A1-A2)] × 100.

[0074] like Figure 4As shown, after treatment at a concentration of 0.5 mg / mL for 48 h, the cell viability of the UE group was significantly lower than that of the HW group (P<0.05), and the cell viability of the UEMP group was significantly lower than that of the UE group (P<0.05). These results indicate that, compared to the traditional hot water extraction method (HW), the polysaccharide (UE) obtained by ultrasound-assisted extraction has superior anti-non-small cell lung cancer activity; and the polysaccharide (UEMP) obtained by further combining it with microfluidic homogenization treatment has significantly enhanced activity. The cell viability of the UEMP group was significantly lower than that of the HWMP and UE groups; this indicates that simple ultrasound and microfluidic treatment cannot achieve the expected results, and the combination of ultrasound and microfluidic treatment has a certain synergistic effect on anti-tumor activity (especially non-small cell lung cancer).

[0075] like Figure 5 As shown, after 72 hours of treatment at a concentration of 0.5 mg / mL, the inhibitory effect of each Cordyceps militaris polysaccharide treatment group on A549 cells was enhanced compared to 48 hours, and the cell survival rate further decreased, indicating that its antitumor activity is time-dependent. Among them, the UEMP group had the lowest cell survival rate, at 10.67% ± 1.87%, significantly lower than the UE group (P<0.05), HWMP group (P<0.01), and HW group (P<0.01), showing the strongest anti-non-small cell lung cancer activity. The above results further confirm that the polysaccharide (UEMP) prepared by ultrasound-assisted extraction combined with microfluidic treatment maintains and continuously enhances its inhibitory ability on tumor cell proliferation even after prolonged treatment time.

[0076] Example 7: Structural Characterization of UEMP

[0077] Given that UEMP possesses strong antioxidant and anti-non-small cell lung cancer activities and has significant application and development potential, its structure was further characterized. As shown in Table 1, the total sugar content of UEMP was 89.86±1.36%; the protein content was 2.18±0.47%; and the uronic acid content was 3.88±0.15%.

[0078] Infrared spectroscopy analysis: Weigh 2 mg of UEMP and 100 mg of potassium bromide, mix thoroughly, compress into a tablet, and place in an infrared spectrometer to scan the infrared spectrum. The scanning range is 4000 cm⁻¹. -1 ~400cm -1 .

[0079] like Figure 6 As shown, 3411cm -1 The nearby absorption peak represents the stretching vibration of the hydroxyl group (-OH); 2932 cm⁻¹ -1 The absorption peak at 1647 cm⁻¹ represents the stretching or bending vibration of hydrocarbons (CH₂); -1 and 1400cm -1The absorption peaks at 1079 cm⁻¹ represent the asymmetric and symmetric stretching vibrations of the carbonyl group (-C=O), respectively; -1 and 1040cm -1 The absorption peak at that location is a characteristic absorption peak of the pyran ring. Infrared spectroscopy further confirms that UEMP conforms to the structural characteristics of polysaccharides.

[0080] Molecular weight determination: UEMP was prepared into a 2 mg / mL deionized water solution, filtered through a microporous membrane, and then injected into a high-performance liquid chromatograph. The chromatographic column was an Ultrahydrogel. TM Linear (2μm, 7.8mm×300mm); mobile phase: 0.1M NaNO3 deionized water; flow rate: 0.8mL / min; detector: differential refractive index detector; molecular weight was calculated by establishing a third-order polynomial calibration curve using a series of dextrans with known molecular weights as standard sugars, with the logarithm of the molecular weight of the standard as the ordinate and the retention time as the abscissa.

[0081] like Figure 7 As shown, UEMP is a heterogeneous polysaccharide, with a broad distribution in region II accounting for 92.17% and a peak in region I accounting for 7.83%. Calculations show that the average molecular weight of UEMP is: 6.15kDa × 92.17% + 846.74kDa × 7.83% = 71.97kDa.

[0082] Monosaccharide composition determination: Weigh 5 mg UEMP and place it in a sealed hydrolysis tube. Add 1 mL of 2 M trifluoroacetic acid and hydrolyze at 120 °C for 2 h. After hydrolysis, cool to room temperature, transfer the solution to a volumetric flask, wash the residue several times with distilled water, combine the washings, and dilute to 50 mL. Filter through a microporous membrane and inject into the ion chromatograph. The chromatographic column was a CarboPac PA20 (6.5 μm, 3 mm × 150 mm); gradient elution was used: 0-21 min mobile phase was 97% water / 3% sodium hydroxide solution (250 mM), 21.1-30 min mobile phase was 94% water / 2% sodium hydroxide solution (250 mM) / 4% sodium acetate solution (1 M), 30.1-50 min mobile phase was 20% water / 80% sodium hydroxide solution (250 mM); flow rate was 0.6 mL / min; detector was a pulsed amperometric detector.

[0083] like Figure 8 As shown, by comparison with standard sugars, UEMP was identified as mainly composed of fucose (1.36%), arabinose (3.9%), galactose (15.4%), glucose (52.48%) and xylose (26.86%).

[0084] Scanning electron microscopy analysis: UEMP was uniformly dispersed on a conductive adhesive, and the sample surface was sputtered with gold under high vacuum conditions using an ion sputtering system. The morphology of UEMP was observed using a Hitachi Regulus 8100 field emission scanning electron microscope at 5 kV and 1,000x magnification.

[0085] like Figure 9 As shown, at 1000x magnification, the surface of UEMP exhibits a loose and irregular network or lamellar structure. Furthermore, particles of varying sizes and shapes, including rhombic crystalline structures and nearly spherical particles, are dispersed on its surface. This microstructural feature indicates that UEMP possesses a high specific surface area and porosity, which may contribute to improved solubility and exposure of active sites, thereby enhancing its biological activity.

[0086] Example 8: Preparation of UEMP tablets

[0087] Take 5.0g of UEMP, add 300mg of polyvinylpyrrolidone (PVP) and 4.0mg of dicalcium phosphate, mix evenly, grind finely and pass through a 100-mesh sieve; under stirring conditions, slowly add 5% of 95% ethanol solution of povidone K30 to obtain a soft mass, granulate by wet method and pass through a 16-mesh sieve; dry the wet granules at 60℃, granulate and add 1% magnesium stearate and 2% micronized silica gel as lubricant and flow aid, mix evenly and compress under appropriate pressure to obtain UEMP tablets.

[0088] Example 9: Preparation of UEMP Capsules

[0089] Take 5.0g of UEMP that has passed through a 40-mesh sieve, spray it with 90% ethanol at a 1:1 ratio and mix thoroughly. Add 10% starch as a binder to prepare a soft material. Perform wet granulation through a 20-mesh sieve. Dry the resulting wet granules in an oven at 60°C for 1 hour. Then granulate them through a 20-mesh sieve. Quickly fill the resulting granules into No. 3 empty capsules in an environment with a relative humidity of less than 65% to obtain UEMP capsules.

[0090] Example 10: Preparation of UEMP Oral Solution

[0091] Add 2.0g of UEMP to 100mL of purified water and stir at room temperature until completely dissolved. Then add 0.5g of erythritol as a sweetener and 0.2g of potassium sorbate as a preservative and continue stirring until uniformly dissolved. After the resulting solution is bottled, it is subjected to instant sterilization and then immediately bottled and sealed to obtain UEMP oral solution.

[0092] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing Cordyceps polysaccharide from silkworm pupae: Crude polysaccharide of Cordyceps militaris was prepared by ultrasonic-assisted extraction in water using defatted powder of Cordyceps militaris fruiting bodies as raw material. After deproteinization and chromatography, purified polysaccharide was obtained. The purified polysaccharide was further purified by microfluidic homogenizer to obtain highly active Cordyceps militaris polysaccharide.

2. The method according to claim 1, wherein the ultrasonic extraction power is 100W to 500W.

3. The method according to claim 1, wherein the deproteinization and column chromatography purification refer to the process of deproteinizing the crude polysaccharide of silkworm pupa and Cordyceps sinensis using Sevag reagent, followed by purification using a diethylaminoethyl cellulose column chromatography.

4. The method according to any one of claims 1-3, (1) The crude polysaccharide from silkworm pupae and Cordyceps sinensis was prepared by the following method: The *Cordyceps militaris* was pulverized and sieved. The sample was then immersed in petroleum ether, defatted in the dark, filtered under reduced pressure, and the residue was dried to constant weight to obtain defatted *Cordyceps militaris* powder. The defatted powder was weighed, added to water, soaked, and then transferred to an ultrasonic extractor for ultrasonic extraction under specific ultrasonic power and temperature conditions. The extract was cooled and filtered. The filtrate was concentrated under reduced pressure, and anhydrous ethanol was added to adjust the final ethanol concentration. The mixture was allowed to stand and precipitate, then centrifuged to collect the precipitate. The precipitate was then freeze-dried to obtain crude *Cordyceps militaris* polysaccharide; and / or (2) The ultrasonic extraction power is 200W; and / or; (3) The specific steps of the deproteinization and column chromatography purification are as follows: The crude polysaccharide of Cordyceps militaris was dissolved in an appropriate amount of distilled water, Sevag reagent was added, the mixture was shaken, centrifuged and the lower organic phase and the middle denatured protein precipitate were discarded, and the upper polysaccharide aqueous phase was retained. The above operation was repeated until no obvious protein was produced. The deproteinized polysaccharide solution was collected and freeze-dried to obtain deproteinized Cordyceps militaris polysaccharide. Deproteinized Cordyceps militaris polysaccharides were prepared into a deionized aqueous solution and loaded onto a pre-equilibrated diethylaminoethyl cellulose chromatography column. After loading, elution was performed with NaCl solution, the flow rate was controlled, the eluent was collected, concentrated, dialyzed, and the dialysate was freeze-dried to obtain purified Cordyceps militaris polysaccharides; and / or (4) The specific steps for processing and purifying polysaccharides using a microfluidic homogenizer are as follows: take the purified polysaccharide of silkworm pupa and cordyceps, dissolve it in deionized water, and prepare a polysaccharide solution; inject the obtained solution into a dynamic ultra-high pressure microfluidic homogenizer, and process it in a cycle under a certain pressure condition, and stay in the high pressure zone for a certain time each time; after the processing is completed, in order to avoid the influence of unstable pressure during the start-up and termination of the equipment on the uniformity of the sample, only the uniform effluent from the middle section of the microfluidic outlet is collected; freeze-dry the collected polysaccharide treatment solution to obtain silkworm pupa and cordyceps polysaccharide.

5. A highly active silkworm pupa cordyceps polysaccharide prepared by the method of claim 1, wherein the total sugar content is 89.86±1.36%, the protein content is 2.18±0.47%, and the uronic acid content is 3.88±0.15%; And / or, the highly active silkworm pupa cordyceps polysaccharide is a heterogeneous polysaccharide; And / or, the average molecular weight of the highly active silkworm pupa cordyceps polysaccharide is 71.97 kDa; And / or, the highly active silkworm pupa cordyceps polysaccharide is composed of fucose (1.36%), arabinose (3.9%), galactose (15.4%), glucose (52.48%) and xylose (26.86%); And / or, the highly active silkworm pupa cordyceps polysaccharide has a high specific surface area and porosity; And / or, the infrared spectrum of the highly active silkworm pupa cordyceps polysaccharide is basically as shown in Figure 6.

6. A composition containing highly active silkworm pupa cordyceps polysaccharide prepared by the method of claim 1, wherein the composition can be prepared as tablets, capsules or oral liquid.

7. Use of a highly active silkworm pupa cordyceps polysaccharide prepared by the method of claim 1 or the composition of claim 6 in the preparation of an antitumor drug.

8. The use according to claim 6, wherein the tumor is lung cancer, more preferably non-small cell lung cancer.

9. An extraction method for improving the antioxidant properties of Cordyceps militaris polysaccharide extract, comprising: using defatted Cordyceps militaris fruiting body powder as raw material, preparing crude Cordyceps militaris polysaccharide in water by ultrasonic-assisted extraction; obtaining purified polysaccharide after deproteinization and chromatography column purification, and further purifying the polysaccharide using a microfluidic homogenizer to obtain highly active Cordyceps militaris polysaccharide.

10. An extraction method for improving the antitumor activity of Cordyceps militaris polysaccharide extract, comprising: using defatted Cordyceps militaris fruiting body powder as raw material, preparing crude Cordyceps militaris polysaccharide in water by ultrasonic-assisted extraction; obtaining purified polysaccharide after deproteinization and chromatography column purification, and further purifying the polysaccharide by microfluidic homogenizer to obtain highly active Cordyceps militaris polysaccharide.

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