A method for cultivating artificial chen flower, furan-type galactomannan and preparation method and application thereof

Abstract

This invention relates to the field of directed biotransformation technology of fungal intracellular polysaccharides, disclosing a method for cultivating artificial Cordyceps militaris, a furan-type galactomannan, its preparation method, and its applications. The invention involves deep solid-state fermentation of Cordyceps militaris strains in a culture medium supplemented with an aqueous extract of a traditional Chinese medicine composed of Prunella vulgaris. During the fermentation process, alternating red, blue, and white light illumination is used to perform directed biotransformation of the component, ultimately obtaining artificially cultivated Cordyceps militaris fruiting bodies. Furan-type galactomannan is then purified to obtain the main chain. This furan-type galactomannan contains three residues: α-1,2-Manp-mannopyranose, 6-O-Me-α-1,3-Manf-mannofuranose, and α-1,2-Galf-galactofuranose. The O5 position of α-Manf and the O6 position of α-Galf on the side chains are replaced by T-β-D-Manf terminal mannofuranose, exhibiting significant immunomodulatory effects and can be used as an immunomodulator or for the preparation of immunomodulators.

Description

Technical Field

[0001] This invention relates to the field of directed biotransformation technology of fungal intracellular polysaccharides, specifically to a method for cultivating artificial cicada flowers, furan-type galactomannan, its preparation method, and its application. Background Technology

[0002] Cordyceps cicadae (also known as cicada nymph) has been used in traditional Chinese medicine for 1500 years, with its medicinal record predating that of Cordyceps sinensis by about 800 years. In recent years, its artificially cultivated fruiting body (Artificially cultivated Cordyceps cicadae) has been approved by the National Health Commission as a new food ingredient. It contains various bioactive components such as adenosine, polysaccharides, cordycepin, proteins, and amino acids, and possesses important functions including immune regulation, anti-tumor activity, antioxidant activity, anti-fatigue, anti-aging, sedation, and improvement of kidney function, attracting considerable attention from scholars. Active polysaccharides, as one of the main secondary metabolites of Cordyceps cicadae, vary significantly in monosaccharide composition and molecular chain conformation due to the influence of different culture medium formulations, temperatures, and humidity conditions.

[0003] Besides the factors mentioned above, different light qualities, durations, and intensities have significantly different effects on the growth and development stages of edible fungi mycelia and fruiting bodies. These effects not only significantly influence growth and developmental traits but also stimulate the synthesis of different secondary metabolites, thus affecting the structure and content of terminal secondary metabolites. In particular, red light (640nm-660nm) and blue light (430-450nm) can affect key carbohydrate synthases in the growth process of edible fungi, ultimately influencing the composition and structure of functional polysaccharide macromolecules. For example, Wang Lihua et al. found that SOD enzyme activity was higher in Ganoderma lucidum after different light quality treatments, with POD enzyme activity being highest after yellow light treatment. Mei Xiling et al. showed that blue light treatment significantly increased the synthesis of α-PCM and PGI in Ganoderma lucidum mycelia, leading to an increase in the content of active polysaccharides. Jang et al. found that different light qualities significantly affected sugar accumulation during the fermentation process of Pleurotus eryngii, with shorter wavelengths of light being more beneficial to polysaccharide content. Chinese invention patents, namely a bidirectional artificial culture method for Cordyceps sinensis (CN201510341818.4) and a culture method for Cordyceps sinensis spore powder (CN202110024079.1), both employ staged white light irradiation of Cordyceps sinensis. Although the biomass of the fruiting bodies is improved to some extent, the directional biosynthesis of certain active polysaccharides cannot be achieved. Summary of the Invention

[0004] One objective of this invention is to provide a method for cultivating artificial cicada flowers, thereby achieving the directional conversion of furan-type galactomannan in artificial cicada flowers.

[0005] Another object of the present invention is to provide a cicada fruiting body rich in furan-type galactomannan.

[0006] Another object of the present invention is to provide a characteristic artificial cicada flower furan-type galactomannan.

[0007] Another objective of this invention is to provide a method for preparing and using the artificial cicada flower furan-type galactomannan, which has the advantages of simple operation and easy control, and significantly improves immunomodulatory activity.

[0008] The technical solution adopted in this invention is:

[0009] A method for cultivating artificial *Cicada nymph*, comprising the following steps: inoculating *Cicada nymph* strains into a solid culture medium and cultivating them by alternating irradiation with red, blue, and white light; the solid culture medium comprises: 10-20g glucose, 2-10g peptone, 0.02-0.05g vitamin B1, 1-3g KH2PO4 and 1.5-3.5g MgSO4·7H2O, 10-20mL *Prunella vulgaris* extract, and 20-30g wheat.

[0010] In one implementation, the red light irradiation time is 10-12 hours, the blue light irradiation time is 6-9 hours, and the white light irradiation time is 3-8 hours.

[0011] In one embodiment, the illuminance of the red light is 120-160 Lux, the illuminance of the blue light is 100-130 Lux, and the illuminance of the white light is 100-150 Lux.

[0012] In one implementation, the cultivation temperature is 20°C-28°C and the humidity is 65-75%.

[0013] The present invention also provides the fruiting body of Cicada Flower obtained by the above method, wherein the fruiting body contains furan-type galactomannan.

[0014] The present invention also provides a furan-type galactomannan, which is composed of polysaccharides with a weight percentage of more than 99%; the main chain of the polysaccharide includes three residues: α-1,2-Manp mannopyranose, 6-O-Me-α-1,3-Manf mannofuranose, and α-1,2-Galf galactofuranose, and the O5 position of α-Manf and the O6 position of α-Galf on the side chain are replaced by T-β-D-Manf terminal mannofuranose, respectively; every 77 (1→2) linked α-mannose residues form a repeating unit.

[0015] The weight-average molecular weight of the furan-type galactomannan is 50 kDa-60 kDa.

[0016] The present invention also provides a method for preparing the above-mentioned furan-type galactomannan, wherein the fruiting body of the cicada flower described in the present invention is degreased, dried and sieved, and extracted by water reflux. The obtained filtrate is enzymatically hydrolyzed with papain to obtain an enzymatic hydrolysate. The enzymatic hydrolysate is precipitated with alcohol and freeze-dried to obtain crude furan-type galactomannan polysaccharide.

[0017] Furthermore, the crude furan-type galactomannan polysaccharide was deproteinized and dialyzed, followed by DEAE-agarose fast flow ion exchange chromatography and gel filtration affinity chromatography to obtain purified furan-type galactomannan.

[0018] The application of the furan-type galactomannan described above, or the furan-type galactomannan prepared by the above method, in enhancing immunity or preparing immunomodulatory products.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] This invention is the first to use different light formulations to conduct targeted synthesis and transformation of artificial Cordyceps militaris functional polysaccharides. By alternating combinations of red, blue, and white light, the permeability of Cordyceps militaris cell membranes can be effectively enhanced. In addition, the stimulation of Prunella vulgaris extract affects sugar metabolism and induces the synthesis of phosphoganoyl isomerase, phosphoganoyl mutase, glycosyltransferase, carbohydrate esterase, and transcriptome expression. Through the dual excitation of light signals and extracts, the accumulation of mannan and the targeted transformation of furan-type mannan are significantly promoted.

[0021] This invention yields a cicada flower furan-type galactomannan, whose polysaccharide weight percentage is over 99%. Monosaccharide composition identification revealed that the polysaccharide is composed of mannose and is a polysaccharide. Molecular weight analysis showed that its weight average molecular weight is 50 kDa-60 kDa. Nuclear magnetic resonance spectroscopy showed that it has an α configuration, and its glycosidic bond linkage mode was determined. The main chain includes three residues: α-1,2-Manp mannopyranose, 6-O-Me-α-1,3-Manf mannofuranose, and α-1,2-Galf galactofuranose. The O5 position of α-Manf and the O6 position of α-Galf on the side chain are replaced by T-β-D-Manf terminal mannofuranose, respectively.

[0022] In the method for preparing the above-mentioned cicada flower furan-type galactomannan of the present invention, papain is used for enzymatic hydrolysis. Papain can destroy the cell wall structure of the cicada flower fruiting body, effectively remove complex components such as proteins that interfere, and accelerate the dissolution of the furan-type galactomannan target.

[0023] This invention discloses a furan-type galactomannan from Cordyceps militaris that exhibits significant immunomodulatory effects. It promotes the release of NO and cytokines TNF-α and IL-6 from mouse macrophage line RAW264.7, thus promoting the proliferation and release of TNF-α and IL-6. It also demonstrates immunomodulatory activity in mouse macrophage RAW264.7 cells and can be used directly as an immunomodulator. Furthermore, it can be used to prepare immunomodulatory products, such as functional foods, health supplements, animal feed additives, and pharmaceuticals. The artificially cultured furan-type galactomannan from Cordyceps militaris achieves its immunomodulatory effect by activating the MYD88 / IRAK-1 / TRAF6 / MAPKs / NF-κB signaling pathway. Attached Figure Description

[0024] Figure 1 The absorbance curve of the crude polysaccharide from the artificial Cordyceps militaris of this invention, purified by Sephacryl S-200HR gel, at 490 nm; where the ordinate is the absorbance at 490 nm and the abscissa is the number of tubes.

[0025] Figure 2 This is a laser light scattering diagram of the CXP-Ⅰ of the present invention; where the vertical axis represents the relative scale and the horizontal axis represents time (minutes).

[0026] Figure 3 The first image shows the HPLC chromatograms of various monosaccharide standard solutions, and the second image shows the reference chromatogram. Figure 4 The image shows the HPLC chromatogram of CXP-I after acetylation, and the sample chromatogram. The vertical axis represents the response value, and the horizontal axis represents the retention time (minutes). Rham represents rhamnose, Rib represents ribose, Fuc represents fucose, Ara represents arabinose, Xyl represents xylose, Man represents mannose, Glc represents glucose, Gal represents galactose, GlcUA represents glucuronic acid, and GalUA represents galacturonic acid.

[0027] Figure 5 For the present invention CXP-Ⅰ 1 H-NMR spectrum (a) and 13 C-NMR spectrum (b);

[0028] Figure 6 For the present invention CXP-Ⅰ 1 H / 13 C HMBC map (a) and 1 H / 13 C HMQC map (b);

[0029] Figure 7This is a graph showing the effect of CXP-Ⅰ of the present invention on NO release in mouse macrophages RAW264.7; where Ctrl is the negative control, LPS is the positive control, and the horizontal axis is the sample dose (μg / mL).

[0030] Figures 8 to 9 The following figures show the effects of CXP-Ⅰ of the present invention on the release of TNF-α and IL-6 cytokines from mouse macrophages RAW264.7; where Ctrl is the negative control, LPS is the positive control, and the horizontal axis represents the sample dose (μg / mL).

[0031] Figures 10 to 11 The effects of CXP-I on the MyD88, IRAK-1, and TRAF-6 pathways in RAW264.7 cells are shown in the figure below. The effects of CXP-I on the MAPKs and NF-κB pathways in RAW264.7 cells are shown in the figure below. In the figure below, control is the negative control and LPS is the positive control. The horizontal axis represents the sample dose (μg / mL) and the vertical axis represents the ratio of the target protein to the total protein. Detailed Implementation

[0032] This invention provides a novel method for cultivating artificial cicada flowers. Based on solid culture, by adding different light quality formulations to induce the transport and metabolism of furan-type carbohydrates, the activity of key furan-type galactomannan synthase is significantly enhanced, thereby stimulating the efficient and directional conversion of furan-type galactomannan.

[0033] The method for cultivating artificial cicada flower of the present invention includes the following steps: inoculating the cicada flower strain into a solid culture medium and cultivating it by alternating red light, blue light, and white light; the formula of the solid culture medium includes: 10-20g glucose, 2-10g peptone, 0.02-0.05g vitamin B1, 1-3g KH2PO4 and 1.5-3.5g MgSO4·7H2O, 10-20mL of Prunella vulgaris extract, and 20-30g wheat.

[0034] The *Paecilomyces cicadae* strain described in this invention can be any known *Paecilomyces cicadae* strain, and commercially available products can be used. For example, the *Paecilomyces cicadae* strain *Paecilomyces cicadae* (Miq.) Samson CGMCC No. 3453 was registered and deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 18, 2009.

[0035] In this invention, *Cicada nymph* is inoculated into a solid culture medium at an inoculation rate of 5%-10%. The inoculation rate refers to the percentage ratio of the volume of the inoculated strain to the volume of the culture medium.

[0036] The solid culture medium described in this invention contains Prunella vulgaris extract. Prunella vulgaris is rich in active substances and vitamins, which significantly promote the synthesis of polysaccharide synthase and mycelial growth in Cordyceps militaris. This invention does not specifically limit the source of Prunella vulgaris, and the Prunella vulgaris medicinal material used meets the requirements of the Pharmacopoeia of the People's Republic of China.

[0037] The *Prunella vulgaris* extract of this invention is an aqueous extract of *Prunella vulgaris*, prepared after water extraction and sterilization. As one embodiment, the *Prunella vulgaris* extract is prepared by the following method: *Prunella vulgaris* is pulverized to a powder of 60-100 mesh, soaked in 2-10 times its volume of water for 0.5-1 hour, then heated and simmered at a gentle boiling state for 20-60 minutes, and the extract is collected by filtration. As a preferred method, *Prunella vulgaris* is extracted 1-4 times, and the filtrates are combined to obtain the *Prunella vulgaris* extract.

[0038] The basic components of the solid culture medium described in this invention are all selected from Shanghai Maclean Biochemical Technology Co., Ltd., and the wheat raw material is selected from Henan Jinli Wheat Industry Co., Ltd., but this is not a limitation of the invention.

[0039] In this invention, the method for cultivating artificial cicada nymphs utilizes red, blue, and white light to efficiently and directionally transform the cicada nymphs. As one embodiment, the red light irradiation time is 10-12 hours, the blue light irradiation time is 6-9 hours, and the white light irradiation time is 3-8 hours, with the three lights irradiated alternately. As another embodiment, the red light intensity is 120-160 Lux, the blue light intensity is 100-130 Lux, and the white light intensity is 100-150 Lux. Under these light intensities, the conversion efficiency of polysaccharides in the cicada nymphs is high. This invention does not particularly limit the source of the red, blue, and white light. In a specific embodiment, red, blue, and white light tubes are used for illumination, purchased from Shenzhen Yanxiang Intelligent Optoelectronics Co., Ltd. The tube size is 10×120cm, power is 24W, luminous flux is 2400lm, and beam angle is 120 degrees. Those skilled in the art can select the appropriate light source based on actual conditions.

[0040] In the method for cultivating artificial cicada nymphs of the present invention, the cultivation temperature is 20℃-28℃, preferably 23-26℃; the humidity is 65-75%, preferably 68-72%. The cultivation period is preferably 30-40 days to obtain artificially cultivated cicada nymph fruiting bodies.

[0041] This invention extracts a furan-type galactomannan from the fruiting bodies of cultivated cicada flowers, composed of polysaccharides with a weight percentage of over 99%. The main chain of the polysaccharide includes three residues: α-1,2-Manp mannopyranose, 6-O-Me-α-1,3-Manf mannofuranose, and α-1,2-Galf galactofuranose. The O5 position of α-Manf and the O6 position of α-Galf on the side chains are replaced by T-β-D-Manf terminal mannofuranose, respectively. Every 77 (1→2) linked α-mannose residues form a repeating unit. Wherein, Manp is mannopyranose; Manf is mannofuranose; and Galf is galactofuranose.

[0042] Optionally, the polysaccharide is α-D-mannose, and the α-mannose residue is an α-D-mannose residue.

[0043] The repeating unit of the polysaccharide described in this invention has various structural variations, with the O5 position of α-Manf and the O6 position of α-Galf on the side chain being replaced by T-β-D-Manf terminal mannofuranose, respectively. These various variations all possess immunomodulatory activity; for example, repeating units with the structure shown in Formula I can be used.

[0044]

[0045] The number of repeating units can be determined by the weight-average molecular weight.

[0046] Optionally, the weight-average molecular weight of the artificial Cordyceps militaris galactomannan is 50 kDa-60 kDa, where kDa is kilodaltons.

[0047] The present invention also provides a method for preparing the above-mentioned furan-type galactomannan, comprising the following steps: after defatting the fruiting body of the cicada flower mentioned above, drying and sieving, extracting by water reflux, and hydrolyzing the obtained filtrate with papain to obtain an enzymatic hydrolysate; precipitating the enzymatic hydrolysate with alcohol to obtain crude furan-type galactomannan polysaccharide.

[0048] In one embodiment, the present invention removes grease from the fruiting bodies of *Cicada nymph* by reflux in an ethanol water bath. Preferably, the ethanol is 75%. The ratio of the fruiting bodies to the ethanol is 1:8-15, preferably 1:10-12. The reflux temperature is preferably 90-95°C, and the reflux is performed 2-3 times.

[0049] This invention involves drying the defatted fruiting bodies of *Cicada nymph* at a temperature preferably of 50-55℃. After drying, the dried fruiting bodies are passed through a 40-50 mesh sieve. The sieved *Cicada nymph* fruiting body powder is then extracted by reflux with water, preferably at a temperature of 80-100℃, more preferably 85-95℃. The water used is preferably distilled water with a pH of 7.0. This invention preferably involves two reflux extractions, each lasting 2-3 hours, and the filtrates are combined. Papain is added at 0.5-3% of the filtrate volume, and the mixture is incubated in a water bath at 50℃-80℃ for 2-2.5 hours. The mixture is stirred multiple times during hydrolysis. After hydrolysis, the mixture is centrifuged, and the supernatant is collected as the hydrolysate. This invention utilizes papain to disrupt the cell wall structure of *Cicada nymph* fruiting bodies, effectively remove complex components such as proteins, and accelerate the dissolution of polysaccharide targets.

[0050] Preferably, the enzymatic hydrolysate is concentrated under reduced pressure and then precipitated with alcohol. The method of alcohol precipitation is not particularly limited in this invention; conventional alcohol precipitation methods in the art can be used, such as adding an aqueous ethanol solution to the supernatant, mixing well, allowing precipitation overnight, centrifuging, and collecting the precipitate. As one embodiment, this invention precipitates the concentrated solution with 95% ethanol at a volume ratio of 1:3-5, centrifuges at 5000 r / min for 8 min, and collects the precipitate. The overnight incubation temperature is preferably 2℃-5℃. After freeze-drying, the artificial cicada flower crude polysaccharide is obtained.

[0051] This invention involves deproteinizing and dialyzing crude polysaccharides. As one embodiment, the crude polysaccharide is prepared into an aqueous solution. The volume ratio of Sevag reagent (chloroform: n-butanol = 4:1) to the sample is 1:3. After thorough shaking, the mixture is centrifuged, and layering is observed. The upper aqueous phase is collected, and the deproteinization process is repeated 4-6 times until no obvious white precipitate is produced. Finally, the aqueous phase is collected and dialyzed in a dialysis bag (4000-6000 Da). After freeze-drying, the deproteinized crude cicada flower polysaccharide is obtained. Preferably, the dialysis time in deionized water is 60-120 hours.

[0052] This invention prepares a solution of crude cicada flower polysaccharide after protein removal, centrifuges the solution, and then performs DEAE-agarose fast flow ion exchange chromatography and gel filtration affinity chromatography on the supernatant to obtain purified furan-type galactomannan. As one embodiment, 20-50 mL of the supernatant is loaded onto a DEAE-Sepharose Fast Flow column, concentrated, dialyzed, and lyophilized to obtain the separated polysaccharide. The above components are further purified using a Sephacryl S-200HR gel column, concentrated, dialyzed, and lyophilized to obtain the purified, white, cotton-like artificial cicada flower furan-type galactomannan, named CXP-Ⅰ.

[0053] The cicada flower furan-type galactomannan described in this invention has a significant immune-enhancing effect, promoting the release of nitric oxide (NO) and cytokines TNF-α and IL-6 from mouse macrophage RAW264.7 cells. It can be used to promote the proliferation of mouse macrophage line RAW264.7 and has immunomodulatory activity on mouse macrophage RAW264.7 cells. It can also achieve immunomodulatory effects by activating the MYD88 / IRAK-1 / TRAF6 / MAPKs / NF-κB signaling pathway. It can be used directly as an immunomodulator / product, or it can be used to prepare immunomodulators for use in functional foods, health products, animal feed additives, and pharmaceuticals.

[0054] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0055] Penicillium cicadae strain was purchased from Jinzhai Jiade Traditional Chinese Medicine Co., Ltd. Prunella vulgaris was purchased from Nanjing Chunqiu Bioengineering Co., Ltd.

[0056] Example 1

[0057] (1) Solid culture: Inoculate the Cordyceps militaris strain into the culture medium at an inoculation amount of 5% (volume percentage). The culture medium used contains: 10g glucose, 10g peptone, 0.02g vitamin B1, 1g KH2PO4 and 1.5g MgSO4·7H2O, 10mL of Prunella vulgaris extract and 20g wheat.

[0058] (2) Directed transformation: After successful inoculation, the temperature was controlled at 20℃ and the humidity at 65%. Stimulation was carried out under different light conditions. Red (light intensity of 120 Lux), blue (light intensity of 100 Lux), and white (light intensity of 100 Lux) light were alternately irradiated. The stages were red light for 10 hours, blue light for 6 hours, and white light for 8 hours. After 30 days of cultivation, artificially cultivated cicada flower fruiting bodies were obtained.

[0059] (3) Extraction: The fruiting bodies of *Cicada spores* were defatted with 95% ethanol at a ratio of 1:10 (g / L), refluxed in a water bath at 90℃ for 3 hours each time, dried in an oven at 55℃, and passed through a 40-mesh sieve. A certain amount of *Cicada spores* fruiting body powder was taken, and distilled water was used as the solvent to adjust the pH to 7.0. Extraction was carried out by reflux at 80℃ for 2 hours each time, filtered, and the residue was extracted again. The filtrates obtained from the two extractions were mixed, and papain was added at 0.5% of the solution volume. The mixture was placed in an oven at 70℃ for 2 hours for enzymatic hydrolysis, with multiple stirrings during the process. After enzymatic hydrolysis, the mixture was centrifuged at 5000 r / min for 5 minutes, and the supernatant was collected. The mixture was concentrated under reduced pressure at 60℃, and the concentrate was precipitated with 95% ethanol at a volume ratio of 1:4 (4℃, 12 hours). The mixture was centrifuged at 5000 r / min for 8 minutes, the precipitate was collected, and freeze-dried to obtain the artificial *Cicada spores* crude polysaccharide.

[0060] (4) Dialysis: The crude polysaccharide was prepared into an aqueous solution. The volume ratio of Sevag reagent (chloroform: n-butanol = 4:1) to the sample was 1:3. After the mixed solution was shaken thoroughly for 30 minutes, it was centrifuged at 4000 r / min for 10 min. When the layers were observed, the upper aqueous phase was carefully removed. The above deproteinization operation was repeated 4 times until no obvious white precipitate was produced. Finally, the aqueous phase was collected in a dialysis bag (4000-6000 Da) and dialyzed for two days. After freeze-drying, the crude artificially cultivated Cordyceps sinensis polysaccharide was obtained after protein removal.

[0061] (5) Purification: The crude cicada polysaccharide after deproteinization was prepared into a solution, centrifuged at 5000 r / min, and 30 mL of the supernatant was loaded onto a DEAE-Sepharose Fast Flow chromatography column (5.0 cm × 60 cm). After concentration, dialyzing for 2 days, and lyophilization, the separated polysaccharide was obtained. The above components were further purified by Sephacryl S-200HR gel column, concentrated, dialyzed for 2 days, and lyophilized to obtain the purified white cotton-like artificial cicada furan-type galactomannan, named CXP-Ⅰ.

[0062] Example 2

[0063] (1) Solid culture: Inoculate the Cordyceps militaris strain into the culture medium at an inoculation amount of 5% (volume percentage). The culture medium used contains: 20g glucose, 2g peptone, 0.05g vitamin B1, 3g KH2PO4 and 3.5g MgSO4·7H2O, 20mL of Prunella vulgaris extract and 30g wheat.

[0064] (2) Directed transformation: After successful inoculation, the temperature was controlled at 28℃ and the humidity at 75%. Stimulation was carried out under different light conditions. Red (light intensity of 160 Lux), blue (light intensity of 130 Lux), and white (light intensity of 150 Lux) light were alternately irradiated. The stages were red light for 12 hours, blue light for 9 hours, and white light for 3 hours. After 40 days of cultivation, artificially cultivated cicada flower fruiting bodies were obtained.

[0065] (3) Extraction: The fruiting bodies of *Cicada spores* were defatted with 95% ethanol at a ratio of 1:10 (g / L), refluxed in a water bath at 90℃ for 3 hours each time, dried in an oven at 55℃, and passed through a 40-mesh sieve. A certain amount of *Cicada spores* fruiting body powder was taken, and distilled water was used as the solvent to adjust the pH to 7.0. Extraction was carried out by reflux at 100℃ for 2 hours each time, filtered, and the residue was extracted again. The filtrates obtained from the two extractions were mixed, and papain was added at 3% of the solution volume. The mixture was placed in an oven at 70℃ for 2 hours for enzymatic hydrolysis, with multiple stirrings during the process. After enzymatic hydrolysis, the mixture was centrifuged at 5000 r / min for 5 minutes, and the supernatant was collected. The mixture was concentrated under reduced pressure at 60℃, with the volume ratio of the concentrate to 95% ethanol at 1:4. Alcohol precipitation was performed (4℃, 12 hours), centrifuged at 5000 r / min for 8 minutes, the precipitate was collected, and freeze-dried to obtain the artificial *Cicada spores* crude polysaccharide.

[0066] (4) Dialysis: The crude polysaccharide was prepared into an aqueous solution. The volume ratio of Sevag reagent (chloroform: n-butanol = 4:1) to the sample was 1:3. After the mixed solution was shaken thoroughly for 30 minutes, it was centrifuged at 4000 r / min for 10 min. When the layers were observed, the upper aqueous phase was carefully removed. The above deproteinization operation was repeated 5 times until no obvious white precipitate was produced. Finally, the aqueous phase was collected in a dialysis bag (4000-6000 Da) and dialyzed for two days. After freeze-drying, the crude artificially cultivated Cordyceps militaris polysaccharide was obtained after protein removal.

[0067] (5) Purification: The crude cicada polysaccharide after deproteinization was prepared into a solution, centrifuged at 5000 r / min, and 40 mL of the supernatant was loaded onto a DEAE-Sepharose Fast Flow column (5.0 cm × 60 cm). After concentration, dialyzing for 2 days, and lyophilization, the separated polysaccharide was obtained. The above components were further purified by Sephacryl S-200HR gel column, concentrated, dialyzed for 2 days, and lyophilized to obtain the purified white cotton-like artificial cicada furan-type galactomannan, named CXP-Ⅰ.

[0068] Example 3

[0069] (1) Solid culture: Inoculate the Cordyceps militaris strain into the culture medium at an inoculation amount of 7% (volume percentage). The culture medium used contains: 15g glucose, 8g peptone, 0.03g vitamin B1, 2g KH2PO4 and 2.5g MgSO4·7H2O, 15mL of Prunella vulgaris extract and 25g wheat.

[0070] (2) Directed transformation: After successful inoculation, the temperature was controlled at 25℃ and the humidity at 70%. Stimulation was carried out under different light conditions. Red (light intensity of 140 Lux), blue (light intensity of 120 Lux), and white (light intensity of 130 Lux) light were alternately irradiated. The stages were red light for 11 hours, blue light for 8 hours, and white light for 5 hours. After 35 days of cultivation, artificially cultivated cicada flower fruiting bodies were obtained.

[0071] (3) Extraction: The fruiting bodies of *Cicada spores* were defatted with 95% ethanol at a ratio of 1:10 (g / L), refluxed in a water bath at 90℃ for 3 hours each time, dried in an oven at 55℃, and passed through a 40-mesh sieve. A certain amount of *Cicada spores* fruiting body powder was taken, and the pH was adjusted to 7.0 using distilled water as a solvent. It was extracted by reflux at 80-100℃ for 2 hours each time, filtered, and the residue was extracted again. The filtrates obtained from the two extractions were mixed, and papain was added at 2% of the solution volume. The mixture was placed in an oven at 70℃ for 2 hours for enzymatic hydrolysis, with multiple stirrings during the process. After enzymatic hydrolysis, the mixture was centrifuged at 5000 r / min for 5 minutes, and the supernatant was collected. The mixture was concentrated under reduced pressure at 60℃, with the volume ratio of the concentrate to 95% ethanol at 1:4. Alcohol precipitation was performed (4℃, 12 hours), centrifuged at 5000 r / min for 8 minutes, the precipitate was collected, and freeze-dried to obtain the artificial *Cicada spores* crude polysaccharide.

[0072] (4) Dialysis: Prepare an aqueous solution of crude polysaccharide. The volume ratio of Sevag reagent (chloroform: n-butanol = 4:1) to the sample is 1:3. After shaking the mixed solution thoroughly for 30 minutes, centrifuge at 4000 r / min for 10 min. Observe the appearance of layering. Carefully remove the upper aqueous phase. Repeat the above deproteinization operation 4-6 times until no obvious white precipitate is produced. Finally, collect the aqueous phase in a dialysis bag (4000-6000 Da) and dialyze for two days. After freeze-drying, the crude artificially cultivated Cordyceps militaris polysaccharide after protein removal is obtained.

[0073] (5) Purification: The crude cicada flower polysaccharide after deproteinization was prepared into a solution, centrifuged at 5000 r / min, and 20-50 mL of the supernatant was loaded onto a DEAE-Sepharose Fast Flow chromatography column (5.0 cm × 60 cm). After concentration, dialyzing for 2 days, and lyophilization, the separated polysaccharide was obtained. The above components were further purified by Sephacryl S-200HR gel column, concentrated, dialyzed for 2 days, and lyophilized to obtain the purified white cotton-like artificial cicada flower cordyceps furan-type galactomannan, named CXP-Ⅰ.

[0074] Example 4

[0075] (1) Solid culture: Inoculate the Cordyceps militaris strain into the culture medium at an inoculation amount of 8% (volume percentage). The culture medium used contains: 18g glucose, 8g peptone, 0.35g vitamin B1, 2.5g KH2PO4 and 3g MgSO4·7H2O, 18mL of Prunella vulgaris extract and 28g wheat.

[0076] (2) Directed transformation: After successful inoculation, the temperature was controlled at 26℃ and the humidity at 70%. Stimulation was carried out under different light conditions. Red (light intensity of 150 Lux), blue (light intensity of 120 Lux), and white (light intensity of 140 Lux) light were alternately irradiated. The stages were red light for 11 hours, blue light for 7 hours, and white light for 6 hours. After 36 days of cultivation, artificially cultivated cicada flower fruiting bodies were obtained.

[0077] (3) Extraction: The fruiting bodies of *Cicada spores* were defatted with 95% ethanol at a ratio of 1:10 (g / L), refluxed in a water bath at 90℃ for 3 hours each time, dried in an oven at 55℃, and passed through a 40-mesh sieve. A certain amount of *Cicada spores* fruiting body powder was taken, and distilled water was used as the solvent to adjust the pH to 7.0. Extraction was carried out by reflux at 95℃ for 2 hours each time, filtered, and the residue was extracted again. The filtrates obtained from the two extractions were mixed, and papain was added at 2.5% of the solution volume. The mixture was placed in an oven at 70℃ for 2 hours for enzymatic hydrolysis, with multiple stirrings during the process. After enzymatic hydrolysis, the mixture was centrifuged at 5000 r / min for 5 minutes, and the supernatant was collected. The mixture was concentrated under reduced pressure at 60℃, with the volume ratio of the concentrate to 95% ethanol at 1:4. Alcohol precipitation was carried out (4℃, 12 hours), centrifuged at 5000 r / min for 8 minutes, the precipitate was collected, and freeze-dried to obtain the artificial *Cicada spores* crude polysaccharide.

[0078] (4) Dialysis: The crude polysaccharide was prepared into an aqueous solution. The volume ratio of Sevag reagent (chloroform: n-butanol = 4:1) to the sample was 1:3. After the mixed solution was shaken thoroughly for 30 minutes, it was centrifuged at 4000 r / min for 10 min. When the layers were observed, the upper aqueous phase was carefully removed. The above deproteinization operation was repeated 5 times until no obvious white precipitate was produced. Finally, the aqueous phase was collected in a dialysis bag (4000-6000 Da) and dialyzed for two days. After freeze-drying, the crude artificially cultivated Cordyceps militaris polysaccharide was obtained after protein removal.

[0079] (5) Purification: The crude cicada flower polysaccharide after deproteinization was prepared into a solution, centrifuged at 5000 r / min, and 38 mL of the supernatant was loaded onto a DEAE-Sepharose Fast Flow chromatography column (5.0 cm × 60 cm). After concentration, dialyzing for 2 days, and lyophilization, the separated polysaccharide was obtained. The above components were further purified by Sephacryl S-200HR gel column, concentrated, dialyzed for 2 days, and lyophilized to obtain the purified white cotton-like artificial cicada flower cordyceps furan-type galactomannan, named CXP-Ⅰ.

[0080] Comparative Example 1

[0081] Except for adjusting steps (1) and (2) to “not adding Prunella vulgaris water extract to the solid fermentation medium of Cordyceps militaris, and directly using the basic culture medium components; using white light for 24 hours for artificial lighting of Cordyceps militaris, and not using alternating red, blue and white light”, the rest of the operation is the same as in Example 1.

[0082] The eluent collected by gel filtration chromatography was analyzed for polysaccharides using 1H NMR spectroscopy. The results showed no furanoid polysaccharide components. This indicates that the addition of Prunella vulgaris extract and alternating red, blue, and white light, along with the external aid of Prunella vulgaris aqueous extract and a specially formulated Penicillium cicadae fermentation medium, ensures the directional generation of new secondary metabolites, furanoid galactomannan, during the artificial cultivation of Cordyceps militaris.

[0083] Comparative Example 2

[0084] Except for adjusting steps (1) and (2) to “not adding Prunella vulgaris water extract to the solid fermentation medium of Cordyceps militaris, and directly using the basic culture medium components; using red light alone for 24 hours for artificial lighting of Cordyceps militaris, without alternating red, blue and white light”, the rest of the operation is the same as in Example 1.

[0085] The eluent collected by gel filtration chromatography was analyzed for polysaccharides using 1H NMR spectroscopy, and the results showed that no furanoid polysaccharide components were detected.

[0086] Comparative Example 3

[0087] Except for adjusting steps (1) and (2) to “not adding Prunella vulgaris water extract to the solid fermentation medium of Cordyceps militaris, and directly using the basic culture medium components; using only blue light for 24 hours for artificial lighting of Cordyceps militaris, without alternating red, blue and white light”, the rest of the operation is the same as in Example 1.

[0088] The eluent collected by gel filtration chromatography was analyzed for polysaccharides using 1H NMR spectroscopy, and the results showed that no furanoid polysaccharide components were detected.

[0089] Comparative Example 4

[0090] Except for adjusting step (1) to “not adding Prunella vulgaris water extract to the solid fermentation medium of Cicada Flower and directly using the basic culture medium components”, the rest of the operation is the same as in Example 1.

[0091] The eluent collected by gel filtration chromatography was analyzed for polysaccharides using 1H NMR spectroscopy, and the results showed that no furanoid polysaccharide components were detected.

[0092] Comparative Example 5

[0093] Except for adjusting step (2) to “using only white light for 24 hours for artificial lighting of cicada flowers, without alternating red, blue and white light”, the rest of the operation is the same as in Example 1.

[0094] The eluent collected by gel filtration chromatography was analyzed for polysaccharides using 1H NMR spectroscopy, and the results showed that no furanoid polysaccharide components were detected.

[0095] Examples of CXP-Ⅰ structural qualification or performance analysis:

[0096] Example 5: Detection of Physicochemical Properties, Components, and Molecular Weight

[0097] The artificial Cordyceps militaris mannan CXP-Ⅰ prepared in Example 1 was found to have a total polysaccharide weight percentage of 99.4% by the phenol-sulfuric acid method. Figure 2 It can be seen that the peaks of the 90° light scattering (LS), differential detection (RI), and viscosity detector (VIS) signals have similar shapes and almost completely overlap, indicating that the delay between the two detectors has been accurately corrected. Clearly, the retention time of sample CXP-Ⅰ is mainly distributed between 20 min and 28 min. The RI signal shows that the polysaccharide exhibits a single symmetrical peak shape, indicating that CXP-Ⅰ is a homogeneous polysaccharide. Furthermore, the molecular weight distribution is represented by Mw / Mn, i.e., the dispersion of the sample; the wider the molecular weight distribution, the greater the dispersion. The Mw / Mn ratio of this artificial cod liver oil mannan CXP-Ⅰ is 1.035, which is close to 1, indicating that CXP-Ⅰ is a low-dispersion, relatively homogeneous polysaccharide component with a molecular weight of Mw = 5.9 × 10⁻⁶. 4 Da.

[0098] The artificial cicada nymph mannan CXP-Ⅰ prepared in Example 2 was analyzed using the phenol-sulfuric acid method, and the total polysaccharide weight percentage was found to be 99.3%. Its laser light scattering pattern is similar to... Figure 2 The same. The Mw / Mn ratio of this artificial cicada nymph mannan CXP-Ⅰ is 1.028, which is close to 1, and its molecular weight Mw = 8.3 × 10⁻⁶. 4 Da.

[0099] The artificial Cordyceps militaris mannan CXP-Ⅰ prepared in Example 3 was tested using the phenol-sulfuric acid method, and the total polysaccharide weight percentage was found to be 99.2%. Its laser light scattering pattern is similar to... Figure 2 The same. The Mw / Mn ratio of this artificial cicada nymph mannan CXP-Ⅰ is 1.016, which is close to 1, and its molecular weight Mw = 6.8 × 10⁻⁶. 4 Da.

[0100] The artificial Cordyceps militaris mannan CXP-Ⅰ prepared in Example 4 was tested using the phenol-sulfuric acid method, and the total polysaccharide weight percentage was found to be 99.5%. Its laser light scattering pattern is similar to... Figure 2The same. The Mw / Mn ratio of this artificial cicada nymph mannan CXP-Ⅰ is 1.019, which is close to 1, and its molecular weight Mw = 6.2 × 10⁻⁶. 4 Da.

[0101] Example 6: Monosaccharide Composition

[0102] 3 mg of artificial Cordyceps militaris galactomannan CXP-Ⅰ prepared in Examples 1, 2, 3, or 4 was added to 1 mL of 4 mol·L⁻¹ -1 Hydrolyze the sample in an aqueous solution of trifluoroacetic acid (TFA) in a pear-shaped flask at 120°C for 6 hours. After cooling to room temperature, add 1 mL of methanol and concentrate to dryness by vacuum centrifugation at 60°C. Repeat the process of "cooling to room temperature, adding 1 mL of methanol, and concentrating to dryness by vacuum centrifugation at 60°C" three times to remove residual TFA and proceed with derivatization. Place the open ends of the rotary-dried standards and samples in a desiccator, with a petri dish containing phosphorus pentoxide added to the bottom of the desiccator. Seal the desiccator, evacuate, and dry overnight. Add 1 mL of hydroxylamine hydrochloride solution to each dried sample, seal, shake thoroughly, and oxidize in a vacuum oven at 90°C for 30 minutes. After cooling to room temperature, add 0.2 mL of 1-methylimidazole and 1 mL of acetic anhydride and shake thoroughly to mix. Seal and place in a vacuum oven at 90°C for 30 minutes. Cool to room temperature to form acetylated derivatives. Add 1 ml of chloroform and 1 ml of water to the acetylated derivative and shake thoroughly; let stand for a few minutes to separate the layers and remove the upper aqueous phase; add 1 ml of water to the chloroform layer, shake thoroughly, let stand, remove the aqueous phase, and repeat 5 times. Add an appropriate amount of anhydrous sodium sulfate to the obtained chloroform layer to remove water. Aspirate the chloroform layer using a syringe, pass it through a 0.45 μm organic filter membrane, and place it in a sample vial for HPLC analysis.

[0103] HPLC chromatographic conditions: wavelength 245 nm, column temperature: 30 ℃, column: Waters XBridge C18 (4.6 × 250 mm, 5 μm), flow rate: 1.0 mL / min, injection volume: 10 μL, mobile phase A (acetonitrile): mobile phase B (0.05 mol / L phosphate buffer, pH 6.8) = 17:83.

[0104] like Figure 3-4 Corresponding to various monosaccharide standards, CXP-I in Examples 1-4 includes polysaccharides with a weight percentage of over 99%, and the monosaccharides in the polysaccharide portion are composed of mannose; indicating that CXP-I is a polysaccharide with mannose as the main chain and containing branches.

[0105] Example 7: Methylation Analysis

[0106] Take 2 mg of the artificial Cordyceps sinensis mannan CXP-Ⅰ sample prepared in Example 1, Example 2, Example 3 or Example 4, dissolve it in 1 ml of dimethyl sulfoxide (DMSO), seal with nitrogen gas, sonicate for a while to aid dissolution, and then prepare by methylation according to the method of Ciucanu, et al. (Ciucanu, L., & Kerek, F.. A simple and repid method for permethylation of carbohydrates. Carbohydrate Research, 131, 209-217).

[0107] After three methylation processes, CXP-I was acid-hydrolyzed, reduced, and acetylated to prepare a partially methylated ardiol acetate derivative, which was then analyzed by GC-MS (see Table 1). Table 1 shows that the mannose residues were linked in five ways: (1→) linked mannose residues (1-linked Manf), (1→2) linked mannose residues (1,2-linked Manp), (1→3,5) linked mannose residues (1,3,5-linked Manf), (1→4) linked glucose residues (1,4-linked Glcp), and (1→2,6) linked galactose residues (1,2,6-linked Galf). Their molar ratio was 32.65:27.85:11.39:4.74:23.07. Comparison of the methylation results of CXP-I revealed that it is mainly composed of galactose and mannose, with a very small amount of glucose. Mannose constitutes the majority of the composition, primarily in the form of a furan ring, followed by galactose, with 1, 2, and 6 rings being the most common linkages. Based on the relative molar ratios, mannose is the most abundant component in CXP-I, followed by galactose, and then glucose, which is largely consistent with the monosaccharide composition results mentioned above.

[0108] Table 1. CXP-I Methylation Analysis

[0109]

[0110]

[0111] Example 8: Nuclear Magnetic Resonance

[0112] 60 mg of the artificial Cordyceps sinensis mannan CXP-Ⅰ prepared in Example 1, Example 2, Example 3 or Example 4 was dissolved in 1 ml of deuterium water and scanned at 600 MHz using a Bruker-AVIII500M (Switzerland).

[0113] According to CXP-Ⅰ 1 H-NMR spectrum (see) Figure 5 a)13 C-NMR spectra (see) Figure 5 b) Combining the HMQC map (see...) Figure 6 (b) Five peaks were detected and can be used for analysis. 1 In the H-NMR spectrum, the anomeric proton region (δ 5.10-5.25 ppm) of CXP-Ⅰ mainly exhibits four anomeric hydrogen signals (see...). Figure 5 a) There are four significant vibrational peaks, with hydrogen proton signals of δ5.23, δ5.21, δ5.16, and δ5.13 ppm, which are named sugar residues A, B, C, and D, respectively; according to Figure 6 Analysis revealed that the anomeric proton region of CXP I ranged from δ 103 to 110 ppm, with four distinct anomeric carbon signals corresponding to δ 103.03, δ 106.86, δ 103.46, and δ 108.4 ppm, where A, B, and C are mannose residues, and D is a galactose residue. A distinct vibrational absorption peak at δ 3.23 / 56.77 ppm was observed in the HSQC spectrum, indicating the presence of 6-O-Me. The AE sugar residue... 1 H-NMR, 13 C-NMR chemical shifts were combined with 1 H- 1 The spectral assignments for H COSY, TCOSY, HMQC, and HMBC have been completed (see Table 2).

[0114] Typically, the chemical shift δ of α-configuration sugar residue H-1 is >4.95 ppm, while the chemical shift δ of β-configuration sugar residue H-1 is <4.95 ppm. Based on the chemical shifts of each residue in Table 2, and the hydrogen and carbon chemical shifts of all residues, the coupling constants are compared with standard residues. A and B are α-Man sugar residues, C is a β-Man sugar residue, and D is an α-Gal sugar residue. Based on the chemical shift values ​​of standard carbon atoms, C2 or C4 with δ values ​​greater than δ82-84 are furanose, while C3 or C5 with δ values ​​less than 80 ppm are pyranose. According to Table 2, CXP-I contains both pyranose and furanose rings.

[0115] NMR data comparison (see Table 2) revealed that the H1 / C1 (5.23 / 103.03ppm), H2 / C2 (4.18 / 78.14ppm), H3 / C3 (3.86 / 73.17ppm), and H4 / C4 (3.69 / 68.82ppm) values ​​of residue A gradually decreased, shifting towards a lower field, suggesting that residue A is 1,2-α-Manp. Similarly, residue B can be deduced to be 1,3,5-α-Manp. -α-Manf (H1 / C1: 5.21 / 106.86ppm, H2 / C2: 4.19 / 78.91ppm, H3 / C3: 4.14 / 83.61ppm, H4 / C4: 4.09 / 79.7ppm); D residue is 1,2,6-α-Galf; since only C1 is substituted, C residue is inferred to be 1-β-Manf (where C1: 103.46, C4: 83.6).

[0116] according to Figure 6 Based on HMQC, HMBC, and the above analysis, the connection sequence between CXP I sugar residues can be deduced. Residues A and B show cross-signal, with a cross-signal peak at δ 5.23 / 75.89 ppm indicating that AH1 is connected to B C5: →2)-α-Manp-(1→3,5)-α-Manf-(1→); Residues B and C show cross-signal peaks, with a signal cross between B and C: δ 4.27 / 103.46 ppm indicating that BH5 is connected to C C1: T-β-Manf-(1,3,5)-α-Manf-(1→); Residues B and D show cross-signal, with a signal cross at δ 5.21 / 89.73 ppm indicating that B C1 is connected to DH H2, with the connection mode: →3,5)-α-Manf-(1→2,6)-α-Galf-(1→); Residues C and D show signal cross-signal, indicating that C C1 is connected to D... The H6 phase is linked in the following manner: β-Manf-(1→2,6)-α-Galf-(1→). Due to the low glucose content, no signal was observed in the NMR spectrum. Therefore, it is speculated that the CXP-I sugar residue linkage is as shown in Figure I above. The main chain includes three residues: α-1,2-Manp mannopyranose, 6-O-Me-α-1,3-Manf mannofuranose, and α-1,2-Galf galactofuranose. The O5 position of α-Manf and the O6 position of α-Galf on the side chain are replaced by T-β-D-Manf terminal mannofuranose, respectively. The above results are consistent with the results of methylation analysis.

[0117] Table 2. Assignment of chemical shifts of CXP-I sugar residues

[0118]

[0119] Based on the analysis results of Examples 1-4, it was confirmed that CXP-Ⅰ is composed of a polysaccharide with a weight percentage of more than 99%; the polysaccharide is composed of furan-type galactomannose; the main chain includes three residues: α-1,2-Manp mannopyranose, 6-O-Me-α-1,3-Manf mannofuranose, and α-1,2-Galf galactofuranose, and the O5 position of α-Manf and the O6 position of α-Galf on the side chain are replaced by T-β-D-Manf terminal mannofuranose, respectively.

[0120] Example 9: Evaluation of Immunoreactivity against CXP-I

[0121] The artificial Cordyceps sinensis mannan CXP-Ⅰ prepared in Examples 1, 2, 3 or 4 was used to treat mouse monocyte macrophages RAW264.7 cells, and the effect of CXP-Ⅰ on the release of NO and cytokines (TNF-α, IL-6) from RAW264.7 cells was detected.

[0122] Mouse mononuclear macrophage RAW264.7 cells were cultured in DMEM complete medium (containing 20 μg / mL polymyxin B (PMB), 10% fetal bovine serum, and 1% penicillin-dextrin antibodies, % being weight percentage) at 37°C in a 5% (volume percentage) CO2 cell culture incubator. Cells in the exponential growth phase from passage 4 to 6 were used for experiments.

[0123] Add 3 × 10⁶ RAW264.7 cell suspension to each well of a 96-well plate. 5 Cells were counted at 95% viability using a cell counter (cells / ml). 100 μl of the culture medium was added and incubated at 37°C in a 5% CO2 incubator for 2 hours. The culture medium in each well was discarded, and 100 μl of fresh DMEM complete culture medium was added per well (for positive control lipopolysaccharide (LPS) stimulation wells, DMEM complete culture medium without PMB was added). Culture was continued for 24 hours. The supernatant was removed, and then 200 μl each of DMEM complete culture medium, LPS dilution (1 μg / mL), or different concentrations of CXP-I dilution (12.5, 25, 50, 100, 200 μg / mL) were added, repeating for 5 wells. Cells were cultured at 37℃ and 5% CO2 for 24 h. 100 μL of cell supernatant was transferred to a new 96-well cell culture plate. 100 μL of a 1:1 mixture of Griess A and Griess B solutions (volume ratio) was added to each well. The plate was incubated in the dark for 20 min. The OD value was measured at 540 nm. The average value of three parallel experiments was taken. Results are shown in the table below. Figure 7 .

[0124] Add 2.0 × 10⁻⁶ RAW264.7 cell suspension to each well of a 24-well cell culture plate. 5Cells were counted at 95% viability using a cell counter to ensure cell viability was above 95%. 500 μL of the culture medium was added to each well (for positive control LPS stimulation wells, DMEM without PMB was added), and the cells were incubated for 2 hours at 37°C in a 5% CO2 incubator. The culture medium was discarded, and 500 μL of fresh DMEM complete culture medium was added to each well (for positive control LPS stimulation wells, DMEM complete culture medium without PMB was added). The cells were then incubated for another 24 hours. The supernatant was removed, and then 500 μL each of DMEM complete culture medium, LPS dilution (1 μg / mL), or different concentrations of CXP-I dilution (12.5, 25, 50, 100, 200 μg / mL) were added. The cells were incubated for 24 hours at 37°C in a 5% CO2 incubator. The cell supernatant was collected, and the levels of each cytokine were measured and calculated according to the instructions of the ELISA kit for detecting NO release from mouse cells and cytokines TNF-α and IL-6. The results are shown in the table below. Figure 7 , Figure 8 , Figure 9 .

[0125] The amount of NO released in RAW264.7 cells is directly proportional to cell proliferation, such as... Figure 7 As shown, compared with the negative control group, CXP-I concentrations of 12.5, 25, 50, 100, and 200 μg / mL significantly promoted NO release from RAW264.7 cells (P < 0.05) and significantly promoted RAW264.7 cell proliferation (P < 0.05). The promoting effect reached its peak at 100 μg / mL, and the promoting effect at 200 μg / mL was not significantly different from that at 100 μg / mL. When the CXP-I concentration was increased to 200 μg / mL, it significantly promoted the proliferation of RAW264.7 cells without cytotoxicity. Therefore, 100 μg / mL was determined to be the optimal concentration of CXP-I, providing a basis for subsequent experiments.

[0126] The results of the study on whether CXP-I could promote the secretion of cytokines TNF-α and IL-6 by RAW264.7 cells were as follows: Figure 8 , Figure 9As shown, compared with the negative control group, CXP-I significantly stimulated RAW264.7 cells to secrete cytokines TNF-α and IL-6 within the experimental concentration range (P < 0.05), exhibiting a dose-dependent effect. All experimental concentrations of CXP-I stimulated RAW264.7 cells to secrete tumor necrosis factor TNF-α, showing a highly significant difference compared to the control group. Unlike the results for interleukin-6 (IL-6), the secretion of TNF-α showed a trend of first increasing and then slightly decreasing, reaching its highest value of 692 pg / mL at CXP-I concentration of 100 μg / mL, only slightly lower than the positive control. The secretion of IL-6 showed a trend of continuously increasing with increasing CXP-I concentration. In conclusion, CXP-I possesses immunomodulatory activity against mouse macrophage RAW264.7 cells and can be used directly as an immunomodulator or for the preparation of immunomodulators, such as by combining CXP-I with appropriate excipients to prepare immunomodulators.

[0127] This invention characterized the higher-order structure of artificial cod celestial furan-type galactomannan CXP-Ⅰ and found that artificial cod celestial furan-type galactomannan possessing this structural feature has immunomodulatory activity, and its immunomodulatory activity is closely related to its specific structural features.

[0128] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing furan-type galactomannan, characterized in that, The preparation method includes the following steps: after removing the fat from the fruiting bodies of Cordyceps sinensis, drying and sieving, extracting by water reflux, and hydrolyzing the obtained filtrate with papain to obtain an enzymatic hydrolysate; precipitating the enzymatic hydrolysate with alcohol to obtain furan-type galactomannan crude polysaccharide; The cicada flower seeds are obtained by cultivating artificial cicada flowers. The cultivation method of artificial cicada flowers includes the following steps: inoculating the cicada flower strain into a solid culture medium and cultivating it by alternating red light, blue light, and white light; the formula of the solid culture medium includes: 10-20g glucose, 2-10g peptone, 0.02-0.05g vitamin B1, 1-3g KH2PO4 and 1.5-3.5g MgSO4•7H2O, 10-20mL of Prunella vulgaris extract, and 20-30g wheat. The red light irradiation time is 10-12 hours, the blue light irradiation time is 6-9 hours, and the white light irradiation time is 3-8 hours.

2. The preparation method according to claim 1, characterized in that, The intensity of the red light is 120-160 Lux, the intensity of the blue light is 100-130 Lux, and the intensity of the white light is 100-150 Lux.

3. The preparation method according to claim 1, characterized in that, The cultivation temperature is 20℃-28℃, and the humidity is 65-75%.

4. The preparation method according to claim 1, characterized in that, The crude furan-type galactomannan polysaccharide was deproteinized and dialyzed, followed by DEAE-agarose fast flow ion exchange chromatography and gel filtration affinity chromatography to obtain purified furan-type galactomannan.

5. The furan-type galactomannan prepared by the preparation method according to claim 1, characterized in that, It is composed of polysaccharides with a weight percentage of more than 99%; the main chain of the polysaccharide includes three residues: α-1,2-Manp mannopyranose, 6-O-Me-α-1,3-Manf mannofuranose and α-1,2-Galf galactofuranose, and the 05 position of α-Manf and the 06 position of α-Galf on the side chain are replaced by T-β-D-Manf terminal mannofuranose, respectively; every 77 (1→2) linked α-mannose residues form a repeating unit.

6. The furan-type galactomannan according to claim 5, characterized in that, The weight-average molecular weight of the furan-type galactomannan is 50 kDa-60 kDa.

7. The use of the furan-type galactomannan according to claim 5 or 6, or the furan-type galactomannan prepared by the method according to claim 1, in enhancing immunity or preparing immunomodulatory products.

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