CN117298139A

CN117298139A - Application of paecilomyces hepiali polysaccharide fermentation product in preparation of immunoregulation product

Info

Publication number: CN117298139A
Application number: CN202311224894.8A
Authority: CN
Inventors: 杨开; 刘仁剑; 田宝明; 蔡铭; 李肖娟; 孙培龙
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2023-09-21
Filing date: 2023-09-21
Publication date: 2023-12-29

Abstract

The invention relates to the field of biological products, and discloses application of paecilomyces hepiali polysaccharide fermentation products in preparation of immunoregulatory products. The invention uses paecilomyces hepialid fermentation liquor powder as a raw material, obtains a purified polysaccharide through an improved hot water extraction method and degreasing alcohol precipitation, then extracts a uniform polysaccharide fermentation product PHP40-2 through chromatography and dialysis of the purified polysaccharide, and carries out a series of pharmacological effect researches, so that the uniform polysaccharide has bidirectional immunocompetence. The invention fills the application gap of the paecilomyces hepialid fermentation product and provides another safe and effective substance for bidirectional immunoregulation.

Description

Application of paecilomyces hepiali polysaccharide fermentation product in preparation of immunoregulation product

Technical Field

The invention relates to the field of biological products, in particular to application of paecilomyces hepiali polysaccharide fermentation products in preparation of immunoregulatory products.

Background

Bi-directional immunomodulation is an immunomodulating model of the immune system that can exert both suppression and activation of the immune system. In the immunomodulation process, immune cells and molecules may have the ability to bi-directionally modulate, both to suppress immune responses and to activate or enhance immune responses. Bi-directional immunomodulation can be achieved by a variety of mechanisms. One of these mechanisms is through the modulation of immune cell activity and function. For example, certain cytokines may simultaneously promote and inhibit the activity of immune cells, thereby playing a balancing role in the immune response. Some immune cells may have the ability to both inhibit and activate other immune cells, another mechanism being achieved by modulating the number and distribution of immune cells, thereby maintaining the balance of the overall immune system. Bi-directional immunomodulation plays an important role in the normal function and immune response of the immune system, and it can help maintain the stability of the immune system, preventing the body from being adversely affected by excessive activation or suppression of the immune response, resulting in immune dysfunction and disease.

Therefore, searching more practical and effective bidirectional immunoregulation products has important significance for protecting human health.

Paecilomyces hepiali (Paecilomyces hepialid, PH) is a asexual strain obtained by separating Cordyceps sinensis, belongs to Paecilomyces (Paecilomyces Bain), is widely existing in Cordyceps sinensis, and mycelium products can be obtained by fermentation culture and drying treatment. In the fungus health food review rule issued in 2001, paecilomyces hepiali and Cordyceps militaris are listed as two strains produced by artificial Cordyceps, and are widely used in industrial fermentation production.

At present, the production of the paecilomyces hepialid mainly utilizes mycelia thereof, and related researches on the pharmacological actions of the mycelia of the paecilomyces hepialid are mature, for example, a plurality of reports reveal the immunocompetence of the mycelia of the paecilomyces hepialid, the content of inflammatory factors such as IL-10, IL-12, IFN-alpha and the like can be improved, and a plurality of reports indicate that mycelia polysaccharide can inhibit the generation and expression of NO, iNOS and pro-inflammatory cytokines in macrophages activated by LPS through inhibiting NF-kappa B and AP-1, so that the mycelia polysaccharide can play an important role in inflammation.

In the prior art, the paecilomyces hepialid fermentation liquid is not fully utilized, and is generally directly discarded or used as fertilizer, so that resource waste and environmental pressure are caused. Zhang Yu et al, on the journal of the bacteriology, on the article "composition of the fermented filtrate of Paecilomyces hepiali and analysis of acute toxicity", show that the fermented filtrate of Paecilomyces hepiali after drying contains 21.33% of crude fat, 27.34% of total protein, 16.79% of hydrolyzed amino acid, 11.16% of crude polysaccharide, and also contains various nucleoside substances and various mineral elements necessary for human body; meanwhile, the mice are free from death phenomenon and obvious poisoning reaction in the experimental period. The acute toxicity results of the above articles indicate the applicable prospect of the Paecilomyces hepiali fermentation product.

In the related patent aspect of the paecilomyces hepialid fermentation product, chinese patent with application publication number of CN104762341A discloses a preparation method of low molecular weight paecilomyces hepialid extracellular polysaccharide, which comprises the steps of separating paecilomyces hepialid fermentation liquor by an ultrafiltration membrane, concentrating under reduced pressure, precipitating by ethanol, redissolving, freeze-drying and the like to obtain a polysaccharide component with molecular weight of 3000-10000 daltons, and discloses application of the polysaccharide component as a raw material of a prebiotic product.

Thus, more applications of the Paecilomyces hepiali fermentation product are still to be further mined.

Disclosure of Invention

In order to solve one or more of the technical problems, the invention provides a new application of paecilomyces hepiali polysaccharide fermentation products: its use in the preparation of immunomodulating products. The invention uses paecilomyces hepialid fermentation liquor powder as a raw material, obtains a purified polysaccharide through an improved hot water extraction method and degreasing alcohol precipitation, then extracts a uniform polysaccharide fermentation product through chromatography and dialysis of the purified polysaccharide, and carries out a series of pharmacological effect researches, so that the uniform polysaccharide has bidirectional immunocompetence.

The specific technical scheme of the invention is as follows:

in one aspect, the invention provides an application of paecilomyces hepiali polysaccharide fermentation product in preparing an immunomodulatory product, wherein the polysaccharide fermentation product comprises the following structures:

。

at present, the production of the paecilomyces hepialid mainly utilizes mycelia thereof, and the related research of the pharmacological action of the mycelia of the paecilomyces hepialid is mature, however, the utilization of the fermentation liquid of the paecilomyces hepialid is insufficient, and the fermentation liquid is directly discarded, so that the environment is polluted and the data is wasted. According to the invention, paecilomyces hepialid polysaccharide substances are extracted from the paecilomyces hepialid fermentation liquid in a powdery state sequentially through an improved hot water extraction method and degreasing alcohol precipitation, and then uniform paecilomyces hepialid fermentation product PHP40-2 is obtained through chromatography, dialysis and purification. The backbone structure of PHP40-2 includes the following:

。

by adopting in-vitro cultured mouse mononuclear macrophage RAW264.7 as a cell model, the invention carries out a series of pharmacological effect researches on paecilomyces hepiali fermentation product polysaccharide PHP40-2, such as NO secretion amount, phagocytic activity, IL-1 beta, IL-6, TNF-alpha and other cytokines of normal RAW264.7 macrophages, surprisingly discovers that PHP40-2 has high immune activity and the effect of reducing the immune activity, and determines the novel application of the paecilomyces hepiali polysaccharide fermentation product in preparing an immune regulation product.

Preferably, according to the above aspect of the present invention, the immunomodulation is bidirectional immunomodulation.

According to the invention, experiments prove that the PHP40-2 can improve the phagocytic activity of the macrophage RAW264.7 in a dose-dependent manner, has obvious difference (P is less than 0.01), and has the effect of regulating the immunity in the forward direction when the concentration of the PHP40-2 is 50-400 mug/mL and the higher the dosage is, the higher the phagocytic activity is close to that of a positive control group; meanwhile, the invention also incubates RAW264.7 macrophages with PHP40-2 with different concentrations, and discovers that when the PHP40-2 concentration reaches 200 and 400 mug/mL, compared with a positive control group, the phagocytic activity of the RAW264.7 macrophages is obviously reduced (P < 0.01), and the RAW264.7 macrophages have the function of negative immune regulation. It is proved that the PHP40-2 can be used for bidirectional immunoregulation of organisms.

Bidirectional immunomodulation includes increasing immune activity and decreasing immune activity. Maintaining balance of immune function is critical to the body to avoid adverse effects of immunodeficiency or hyperimmunity on the body. The bidirectional immunoregulation can regulate the immune state of the organism, improve the immunity, and relieve the excessive immune response at the same time, so that the organism returns to the normal state from the state of immunity. Therefore, the proposal of the new application of the paecilomyces hepialid polysaccharide fermentation product in preparing the immunoregulation product has important significance for maintaining the health of human bodies.

As the optimization of the technical scheme, the product is a medicine, a health product, a food additive or a daily chemical product. Based on the above, the paecilomyces hepialid polysaccharide fermentation product PHP40-2 can be used as a medicine, a health product, a food additive or a daily chemical product in different product forms, so that the effect of the paecilomyces hepialid polysaccharide fermentation product on immunoregulation is realized.

In another aspect, the invention provides an article of manufacture for immunomodulation, the active ingredient of the article of manufacture comprising paecilomyces hepiali polysaccharide fermentation product.

According to the invention, paecilomyces hepialid polysaccharide substances are extracted from powdery paecilomyces hepialid fermentation liquid, a series of pharmacological effect researches are carried out by taking mouse mononuclear macrophage RAW264.7 cultured in vitro as a cell model, wherein the pharmacological effect researches comprise NO secretion amount, phagocytic activity, IL-1 beta, IL-6, TNF-alpha and other cytokines of normal RAW264.7 macrophages, and surprisingly, PHP40-2 has the effects of high immune activity and reduced immune activity, so that the novel application of paecilomyces hepialid polysaccharide fermentation products in preparing immune regulation products is determined.

As the preferable choice of the technical scheme, the active ingredient of the product is paecilomyces hepiali polysaccharide fermentation product.

As a preferred aspect of the above technical solution of the present invention, the active ingredient of the product comprises the following structure:

。

preferably, the product is a medicine, a health product, a food additive or a daily chemical product. Based on the above, the paecilomyces hepialid polysaccharide fermentation product PHP40-2 can be used as a medicine, a health product, a food additive or a daily chemical product in different product forms, so that the effect of the paecilomyces hepialid polysaccharide fermentation product on immunoregulation is realized.

Further preferably, the product is a pharmaceutical product, a health product, a food additive or a daily chemical product which can perform bidirectional regulation of enhancing the immune activity and reducing the immune activity.

According to the invention, through multiple experiments, the paecilomyces hepialid polysaccharide fermentation product can improve the phagocytic activity of the macrophage RAW264.7 in a dose-dependent manner, has obvious difference (P is less than 0.01), has the concentration of the polysaccharide PHP40-2 of 50-400 mug/mL, and has the effect of regulating the immunity in the forward direction as the phagocytic activity is closer to a positive control group when the dosage is higher; meanwhile, the invention also uses the paecilomyces hepiali polysaccharide fermentation products PHP40-2 with different concentrations to incubate RAW264.7 macrophages, and discovers that when the PHP40-2 concentration reaches 200 and 400 mug/mL, compared with a positive control group, the phagocytic activity of the RAW264.7 macrophages is obviously reduced (P < 0.01), and the paecilomyces hepiali polysaccharide fermentation products PHP40-2 have the function of negative immune regulation. It was thus demonstrated that paecilomyces hepiali polysaccharide fermentation product can perform bidirectional immunomodulation including enhancement of immune activity and reduction of immune activity on the body.

Compared with the prior art, the invention has the following technical effects:

the invention uses paecilomyces hepialid fermentation liquor powder as a raw material, obtains a purified polysaccharide through an improved hot water extraction method, degreasing and alcohol precipitation, chromatography, dialysis, concentration and freeze-drying, then extracts a uniform polysaccharide fermentation product from the purified polysaccharide, and carries out a series of pharmacological effect researches, so that the uniform polysaccharide has bidirectional immunocompetence. The application provides the proposal to make up the application gap of the paecilomyces hepialid fermentation product and also provides another safe and effective substance for bidirectional immunoregulation.

Drawings

FIG. 1 is a graph showing the elution profile of a DEAE Sepharose Fast Flow ion exchange column chromatography of PHP40 obtained by the measurement in example 1 of the present invention;

FIG. 2 is a high performance liquid chromatogram of PHP40-2 molecular weight determined in example 1 of the present invention;

FIG. 3 is a high performance liquid chromatogram of the PHP40-2 monosaccharide composition determined in example 1 of the present invention;

FIG. 4 is a Fourier infrared spectrum of PHP40-2 measured in example 1 of the present invention;

FIG. 5 is a chart showing the Congo red absorption spectrum of PHP40-2 measured in example 1 of the present invention;

FIG. 6 is a graph showing the effect of PHP40-2 at different concentrations on phagocytic activity of macrophage RAW264.7 in example 2 of the present invention;

FIG. 7 is a graph showing the effect of PHP40-2 at various concentrations on the NO production amount of macrophage RAW264.7 and the expression amounts of IL-1β, IL-6, IL-10 and TNF- α in example 2 of the present invention;

FIG. 8 is a graph showing the effect of PHP40-2 at various concentrations on mRNA expression of iNOS and (B) IL-1β, IL-6 and TNF- α in macrophage RAW264.7 according to example 3 of the present invention;

FIG. 9 is a graph showing the effect of PHP40-2 at various concentrations on phagocytic activity of macrophage RAW264.7 in example 3 of the present invention;

FIG. 10 is a graph showing the effect of PHP40-2 at various concentrations on NO production and IL-1β, IL-6, IL-10, TNF- α expression levels of macrophage RAW264.7 in example 3 of the present invention;

FIG. 11 is a graph showing the results of expression of key target proteins I.kappa.B-alpha and NF-kappa B p65 and TLR4 in the NF-kappa B signaling pathway of cells after PHP40-2 effects at different concentrations in example 4 of the present invention;

FIG. 12 is a graph showing the results of expression of the key target proteins IκB- α and NF- κ B p65 and TLR4 in the NF- κB signaling pathway of cells after PHP40-2 and LPS at different concentrations according to example 4 of the present invention.

Detailed Description

The invention is further described below with reference to examples and figures. Those of ordinary skill in the art will be able to implement the invention based on these descriptions. In addition, the embodiments of the present invention referred to in the following description are typically only some, but not all, embodiments of the present invention. Therefore, all other embodiments, which can be made by one of ordinary skill in the art without undue burden, are intended to be within the scope of the present invention, based on the embodiments of the present invention.

In the polysaccharide component measurement, glucose is used as a standard substance, and the phenol-sulfuric acid method is adopted to measure the total sugar content; glucose is used as a standard substance, and the content of reducing sugar is measured by a dinitrosalicylic acid method; d-galacturonic acid is used as a standard substance, and the content of uronic acid is measured by a m-hydroxybiphenyl method.

EXAMPLE 1 extraction, purification and identification of Paecilomyces hepiali polysaccharide fermentation product

The extraction, purification and identification of the paecilomyces hepiali polysaccharide fermentation product are carried out according to the following steps:

s1: taking paecilomyces hepialid fermentation liquor powder, drying in a 60 ℃ oven for 8 h, crushing, sieving with a 40-mesh sieve, adding 90% ethanol, degreasing at 25 ℃ for 24 h, centrifuging at 4500 r/min for 20 min to remove ethanol, and air-drying.

S2: adding deionized water into the air-dried powder according to a feed liquid ratio of 1:20 (w/v), leaching 5 h, centrifuging at a rotating speed of 4500 r/min for 25 min, and concentrating the supernatant by a rotary evaporation method to obtain the paecilomyces hepialid fermentation broth water extract.

S3: adding absolute food grade ethanol into the paecilomyces hepialid fermentation broth water extract to ensure that the final ethanol concentration is 40%, precipitating at 25 ℃ overnight, centrifuging at 4500 r/min for 25 min, taking the lower precipitate to be redissolved in a proper amount of water, adding a proper amount of trichloroacetic acid, wherein the mass ratio of the precipitate to the water to the trichloroacetic acid is 1:100:30, regulating the final concentration to 50 g/L, and standing at 4 ℃ overnight; centrifuging at 3000 r/min for 5 min overnight, discarding precipitate, collecting liquid, dialyzing with 3500 Daltons dialysis bag to remove small molecule substances, rotary evaporating, concentrating, and lyophilizing to obtain crude polysaccharide named PHP40.

S4: and (3) desalting and purifying the obtained crude polysaccharide PHP40 sequentially by ion exchange column chromatography and dialysis bags, collecting components, concentrating to 1/5 of the original volume, and freeze-drying to obtain purified polysaccharide. And is designated PHP40-2.

Wherein the ion exchange column chromatography step of polysaccharide purification is as follows: an aqueous PHP40 solution (5. 5 mg/mL) was prepared and filtered through a 0.45 μm aqueous filter. Each time 50 mL of the membrane polysaccharide was taken for chromatography through DEAE Sepharose Fast Flow chromatography columns, followed by gradient elution with deionized water 0.1 mol/L, 0.2 mol/L and 0.4 mol/L sodium chloride (NaCl) solution, followed by washing with 2 mol/L NaCl to remove residual polysaccharide impurities in the column packing, and identification washing with silver nitrate was complete. Elution rate was 1 mL/min, 10 min per tube, 50 tubes per gradient. The elution gradient with the highest polysaccharide content was collected for desalting in dialysis bags. The elution profile of the DEAE Sepharose Fast Flow ion exchange column chromatography of PHP40 is shown in FIG. 1.

The desalination steps of the dialysis bag are as follows: the samples were filled into dialysis bags with a water change time of 4 h for a total duration of 24 h. The desalting dialysis was ensured to have been completely completed by silver nitrate.

S5: the purified polysaccharide PHP40-2 is taken to measure the molecular weight and purity, monosaccharide composition and Fourier infrared spectrum, and is observed by a scanning electron microscope, and Congo red experiments, methylation analysis and nuclear magnetic resonance analysis are carried out.

In the molecular weight measurement, high Performance Gel Permeation Chromatography (HPGPC) was used. Wherein the differential detector: RID-20 (Shimazu, japan); gel column: 7.8 TSKgel G3000PWXL, TSKgel G4000PWXL, TSKgel G5000PWXL of mm. Times.300 mm; preparing a sample with a concentration of 2 mg/mL; the columns were calibrated using dextran standards with molecular weights of 3620, 7000, 9000, 12600, 20000, 50000, 126000, 250000, 490000 and 1000000 Daltons and standard curves were established.

In the determination of the molecular weight, the liquid phase conditions of the high performance gel permeation chromatography are: the sample injection amount is 20 mu L, the column temperature is set at 35 ℃, and the flow rate is 0.4 mL/min.

In the monosaccharide composition analysis, the monosaccharide composition is determined by combining a PMP pre-column derivatization high performance liquid chromatography method and a chemical method. The method comprises the following specific steps: preparing 5 mg/mL polysaccharide solution, adding 100 mu L standard solution into an EP tube, adding 3 mL 2.0 mol/L trifluoroacetic acid solution, reacting at 110 ℃ for 5 h, cooling, adding 3 mL methanol, rotary evaporating, adding methanol for multiple times to remove trifluoroacetic acid, adding 1 mL distilled water into a flask, and dissolving by ultrasonic. Using high performance liquid chromatography, 5 mg/mL of different monosaccharide standards (mannose (Man), rhamnose (Rha), galacturonic acid (GlaA), glucose (Glc), galactose (Gal), arabinose (Ara) 6 monosaccharide standards), 200. Mu.L of each were mixed, 100. Mu.L of the standard solution was added to the EP tube, 100. Mu.L of 0.5 mol/L PMP methanol solution, 100. Mu.L of 0.3 mol/L sodium hydroxide solution were added thereto, 1 h was hydrolyzed in a 70℃water bath, cooled to room temperature, 100. Mu.L of 0.3 mol/L hydrochloric acid was added thereto, 1.0 mL chloroform extraction was performed, 12000 r/min centrifugation was performed for 2min.

In the monosaccharide composition analysis, the liquid phase conditions of the high performance liquid chromatography are as follows: the mobile phase was acetonitrile: phosphate buffer = 82:18; chromatographic column: c (C) ₁₈ A chromatographic column; mobile phase flow rate: 0.5 mL/min; detection wavelength: 245 nm; sample injection amount: 20. mu L (mu L)。

The method for observing the Fourier infrared spectrum (FT-IR) comprises the following steps: dry sample powder 2 mg was weighed, and after 300 mg potassium bromide powder was mixed with the sample, it was pressed into a transparent cake for scanning. The scanning wave number range is 4000-400 cm ^-1 Repeated 16 times. The resulting data is analyzed and processed by using OMNIC software.

The method for analyzing the microstructure of the polysaccharide by using a scanning electron microscope comprises the following steps: a quantity of dry sample powder is placed on a sample holder and metallized using a vacuum plating apparatus. Subsequently, observation was performed under a vacuum atmosphere.

The congo red experiment steps are as follows: PHP40-2 at a concentration of 1 mL of 1 mg/mL was taken and mixed with the same volume of 80. Mu. Mol/L Congo red solution. 1 mol/L NaOH was added so that the final concentrations became 0.1, 0.2, 0.3, 0.4 and 0.5 mol/L, respectively, and the reaction was carried out at room temperature for 15 minutes. And measuring the maximum absorption wavelength by using an ultraviolet full-scanning spectrophotometer, wherein the wavelength is 400-600 nm.

The methylation analysis steps are as follows: taking polysaccharide 2 mg, drying 48 h in a vacuum drying oven, adding anhydrous DMSO 1 mL, magnetically stirring at room temperature for 1 h, adding dried NaH 20 mg, stirring at 50deg.C for 1 h, adding methyl iodide to react and turn bright yellow, adding deionized water to terminate the reaction, extracting with CHCl3, and washing the CHCl ₃ The layers were dried under reduced pressure. After methylation, the mixture is hydrolyzed by TFA, methanol is added, naOH solution is dissolved, naBD4 is added for reaction at room temperature for 4 h, glacial acetic acid is added for acid removal, and the mixture is dried under reduced pressure. The reduction reaction was completed by sealing with pyridine 0.5 h, adding acetic anhydride 1 h, and deionized water, and terminating the reaction with CH ₂ Cl ₂ Extracting, and drying under reduced pressure for use.

The chromatographic parameters for methylation analysis were: agilent gas chromatography system (Agilent 7890A;Agilent Technologies,USA), column: BPX70 (30 m ×0.25×0.25 μm, SGE, australia). The sample injection amount is 1 mu L, the split ratio is 10:1, and the carrier gas is high-purity helium; the initial temperature of the column oven was kept at 140℃for 2.0 min, and the temperature was programmed to 230℃at 3℃per minute for 3 min. Mass spectrometry system: quadrupole mass spectrometry detection system (Agilent 5977B, agilent Technologies, USA), mass scan range (m/z): 50-350.

The nuclear magnetic resonance analysis comprises the following steps: weighing 30 mg solid polysaccharide, placing in 25 mL rotary steaming bottle, adding 1 mL weight of water (D ₂ O) dissolving, freeze drying, and adding D again ₂ O is dissolved, freeze-dried repeatedly for 3 times, and finally added with 500 mu L D ₂ O is dissolved, centrifuged and transferred to a nuclear magnetic tube. Recording spectrogram by nuclear magneto-optical spectrometer, one-dimensional nuclear magnetism comprises ¹ H and ¹³ c NMR; the two-dimensional nuclear magnetism comprises ¹ H– ¹ H COSY、 ¹ H– ¹³ CHMQC ¹ H– ¹³ C HMBC。

In this example, the purity and molecular weight of purified polysaccharide PHP40-2 were measured by HPGPC, and the results are shown in FIG. 2, which shows a PHP40-2 molecular weight high performance liquid chromatogram. As can be seen from FIG. 2, the single peak ratio is high and shows uniform symmetry, which means that the purity of the polysaccharide component after separation is over 90%, the purity is high, and the weight average molecular weight of the purified polysaccharide PHP40-2 is 6.2X10 according to the peak time (39.90833 min) ⁵ Daltons。

The monosaccharide composition analysis result is shown in figure 3, and is a PHP40-2 monosaccharide composition high performance liquid chromatogram. The monosaccharide composition of purified PHP40-2 was analyzed according to the retention time of the standard, and as can be seen from FIG. 3, PHP40-2 consisted of three monosaccharides of mannose, glucose and galactose in a molar ratio of 0.3:1.0:3.0.

The result of the Fourier IR spectrum of PHP40-2 is shown in FIG. 4. As can be seen from FIG. 4, PHP40-2 is shown in 3312.9 cm ^-1 The strong absorption at this point is caused by the stretching vibration of O-H, at 2927.5 cm ^-1 The nearby signal is related to C-H single bond stretching vibration; 1643.3 cm ^-1 The nearby absorption peaks may be due to C-O or N-H stretching vibrations, possibly the presence of glycoproteins; 1149 cm ^-1 Sum 1013 cm ^-1 Nearby peaks are attributed to the C-O pendant groups and the C-O-C glycoside ring characteristics. FIG. 4 can demonstrate the characteristic absorbance peaks of PHP40-2 with polysaccharide chemical bonds and functional groups.

The absorption spectrum of Congo red of PHP40-2 is shown in FIG. 5. As can be seen from FIG. 5, the results of Congo red experiments show that PHP40-2 has a gradually decreasing maximum absorption wavelength compared to Congo red solution without the occurrence of red shift, indicating that PHP40-2 may exist in a random coil form.

The methylation analysis results of PHP40-2 are shown in Table 1. As shown in Table 1, PHP40-2 has 4 methylation products, and has GlcP- (1. Fwdarw.,. Fwdarw.6) -Manp- (1. Fwdarw.4) -Galp- (1. Fwdarw.and. Fwdarw.4, 6) -GlcP- (1. Fwdarw.four connection modes, and the molar ratio is 1.00:0.42:4.69:0.55.

TABLE 1 PHP40-2 methylation analysis results

In the nuclear magnetic resonance results, chemical shifts of the respective residues are shown in table 2.

TABLE 2 chemical shifts of residues in PHP40-2

From this, the backbone structure of PHP40-2 includes residues A-D, specifically the following structure:

。

EXAMPLE 2 Positive immunomodulation study of PHP40-2

The cytotoxicity and the forward immunoregulatory effect of PHP40-2 were evaluated using in vitro cultured mouse mononuclear macrophage RAW264.7 as a cell model. The method comprises the following specific steps:

(1) Cytotoxicity evaluation

Cell activity was measured using the MTT method.

The polysaccharide PHP40-2 and LPS were formulated with RPMI-1640 complete medium to give solutions of specific concentration, and were diluted in gradient to give polysaccharide PHP40-2 at concentrations of 5, 10, 50, 100, 200 and 400. Mu.g/mL and LPS at concentrations of 0.1. Mu.g/mL. Cell number was adjusted to 1X 10 ⁶ And each mL. The cell suspension was inoculated into 96-well plates with 5 multiplex wells per set, 100. Mu.L per well, containing 5% CO ₂ 24 h in a 37℃incubator. After 24-h incubation, the complete medium was aspirated, 100. Mu.L each of the polysaccharide PHP40-2 and LPS was added at different concentrations per well, and 100. Mu.L of RPMI-1640 complete medium was incubated in the blank wells and incubation was continued for 24 h.

After completion, 20. Mu.L of 5.0 mg/mL MTT solution was added, and the subsequent incubation continued for a period of 4 h. The supernatant was aspirated, 150. Mu.L of dimethyl sulfoxide (DMSO) was added thereto, and the mixture was shaken well to measure the absorbance at a wavelength of 490 and nm. The cell viability was calculated as follows: (absorbance experimental group-absorbance blank wells)/(absorbance blank wells-absorbance blank medium) ×100%.

The survival results are shown in figure 6, panel a.

As can be seen from FIG. 6 (A), the fermentation product PHP40-2 of Paecilomyces hepiali polysaccharide showed no cytotoxicity to RAW264.7 macrophages in the experimental concentration range, thus demonstrating the safety of PHP40-2.

(2) Evaluation of Forward immunoregulatory Effect

The Paecilomyces hepialid polysaccharide fermentation product PHP40-2 was diluted with RPMI-1640 complete medium to prepare culture solutions containing 5, 10, 50, 100, 200 and 400. Mu.g/mL polysaccharide PHP40-2 concentration levels and 0.1. Mu.g/mL Lipopolysaccharide (LPS) concentration levels, respectively. Inoculating macrophage RAW264.7 into the culture solution, culturing, and adjusting cell number to 1×10 ⁶ And each mL. The cell suspension was inoculated into 96-well plates with 5 multiplex wells per set, 100. Mu.L per well, containing 5% CO ² 24 h in a 37℃incubator. After 24 h incubation, 100 mu L of Paecilomyces hepialid polysaccharide component with corresponding concentration is added to each hole of the experiment groups with different concentrations, 100 mu L of LPS is added to the LPS positive control group, 100 mu L of RPMI-1640 complete culture medium is added to the blank control group for culture, and incubation is continued for 24 h.

Phagocytic activity was measured after incubation was completed as follows: the supernatant was discarded, 100. Mu.L of neutral red solution (0.1%, w/w) was added to each well, incubation was continued at 37℃for 1 h, the supernatant was discarded, and the wells were washed 3 times with PBS buffer. mu.L of cell lysate (ethanol/acetic acid, 1:1, v/v) was added to each well and after standing at room temperature for 2 h, the absorbance was measured using an enzyme-labeled instrument at 540: 540 nm wavelength.

After the incubation, the amount of NO produced by the cells was measured as follows: the amount of NO in the cell supernatant was calculated from the standard curve of the assay, as measured by the ELISA kit (purchased from Nanjinouzan Biotechnology Co., ltd.). Meanwhile, the secretion levels of cytokines IL-1. Beta., IL-6, IL-10 and TNF-. Alpha.in the supernatant were determined according to ELISA kit instructions.

In this example, the effect of PHP40-2 at different concentrations on phagocytic activity of macrophage RAW264.7 is shown in FIG. 6, panel B.

As can be seen from fig. 6 (B), the phagocytic activity of the positive control group was significantly increased (P < 0.01) compared with the blank control group, and the phagocytic activity of the macrophage RAW264.7 was increased in a dose-dependent manner under the influence of PHP40-2, and the phagocytic activity was significantly different (P < 0.01), and the higher the PHP40-2 concentration was 50 to 400 μg/mL, the closer the phagocytic activity was to that of the positive control group. Thus, PHP40-2 has an immunopositive regulatory effect.

The results of the effect of PHP40-2 at various concentrations on the NO production of macrophage RAW264.7 and the expression levels of IL-1β, IL-6, IL-10 and TNF- α are shown in FIG. 7. Wherein, the A, B, C, D and E correspond to the effects of NO, IL-1 beta, IL-6, IL-10 and TNF-alpha, respectively, and P <0.01 compared with the blank group.

As can be seen from FIG. 7, the secretion amount of NO, IL-1. Beta., IL-6, IL-10, TNF-. Alpha.was significantly higher than that of the blank group under the intervention of PHP40-2.

The effect of PHP40-2 at various concentrations on mRNA expression of (A) iNOS and (B) IL-1β, (C) IL-6 and (D) TNF- α in RAW264.7 macrophages is shown in FIG. 8.

As can be seen from FIG. 8, the mRNA expression levels of cytokines including iNOS, IL-6, IL-1β and TNF-. Alpha.were significantly increased (P < 0.01) in the LPS group as compared with the blank group. Under the action of PHP40-2, the expression levels of iNOS, IL-6, IL-1β and TNF- α in RAW264.7 were increased in a dose-dependent manner, and were significantly different from those in the blank group at concentrations of 200 and 400. Mu.g/mL (P < 0.01).

EXAMPLE 3 negative immune regulation study of PHP40-2

The negative immune regulation effect of PHP40-2 was evaluated using in vitro cultured mouse mononuclear macrophage RAW264.7 as a cell model. The method comprises the following specific steps:

diluting the Paecilomyces hepiali polysaccharide fermentation product PHP40-2 with RPMI-1640 complete culture medium, preparing culture solutions containing 5, 10, 50, 100, 200 and 400 mug/mL polysaccharide PHP40-2 concentration respectively, and adding Lipopolysaccharide (LPS) respectively to make the concentration of LPS in the culture solution be 2.5 mug/mL; and preparing a positive control group culture solution which is RPMI-1640 complete culture medium culture solution containing LPS with the concentration of 2.5 mug/mL. Inoculating macrophage RAW264.7 into the culture solution, culturing, and adjusting cell number to 1×10 ⁶ And each mL. The cell suspension was inoculated into 96-well plates with 5 multiplex wells per set, 100. Mu.L per well, containing 5% CO ² 24 h in a 37℃incubator. After 24 h incubation, 100 mu L of Paecilomyces hepialid polysaccharide component with corresponding concentration is added to each hole of the experiment groups with different concentrations, 100 mu L of LPS is added to the LPS positive control group, 100 mu L of RPMI-1640 complete culture medium is added to the blank control group for culture, and incubation is continued for 24 h.

Cell viability was determined after incubation was completed as follows: mu.L of 5.0 mg/mL MTT solution was added, followed by continuous cultivation of 4 h. The supernatant was then aspirated, 150. Mu.L of dimethyl sulfoxide (DMSO) was added thereto, and the mixture was shaken well to measure the absorbance at a wavelength of 490 and nm. The cell viability was calculated as follows: (absorbance experimental group-absorbance blank wells)/(absorbance blank wells-absorbance blank medium) ×100%.

After the incubation, the amount of NO produced by the cells was measured as follows: detecting according to the specification method of ELISA kit, and calculating the NO content in the cell supernatant according to the standard curve. Meanwhile, the secretion levels of cytokines IL-1. Beta., IL-6, IL-10 and TNF-. Alpha.in the supernatant were determined according to ELISA kit instructions.

The RT-PCR method comprises the following steps: RAW264.7 cells in log phase were taken and washed 2 times with PBS buffer, then 1 mL of RNA Isolater reagent was added, and the pipette was repeatedly used. Transferring the suspension after cell lysis into a centrifuge tube without RNase, adding chloroform and mixing, centrifuging after precipitation, and taking out the upper colorless aqueous phase liquid containing total RNA. RNA was precipitated by adding isopropanol solution, centrifuged and the supernatant discarded. Adding ethanol solution to precipitate RNA, centrifuging and removing supernatant, and finally dissolving RNA with DEPC water and detecting concentration and purity with Nanodrop. After reverse transcription of total RNA into cDNA, the cDNA was used as a template, and the corresponding reagent was added to a PCR 8-well tube in a reaction system (20. Mu.L overall) prepared in advance, and 2. Mu.L of DEPC water was used as a blank group instead of cDNA. The gene primer sequences were queried by using NCBI website in advance. Wherein, the RNA amplification procedure of RT-PCR is as follows: 95. pre-denaturation at 10 min; 95. denaturation at 15℃and s, annealing at 59℃for 1 min, extension, 40 cycles; 25. 4 min at the temperature. Beta-actin is used as an internal reference, and a 2-delta Ct method is adopted to quantitatively analyze the target gene of the sample.

In this example, the effect of PHP40-2 at various concentrations on phagocytic activity of macrophage RAW264.7 is shown in FIG. 9.

As can be seen from fig. 8, after induction of LPS at 2 h, RAW264.7 macrophages were incubated with different concentrations of PHP40-2, and phagocytic activity was significantly reduced (P < 0.01) when PHP40-2 concentrations reached 200 and 400 μg/mL relative to the positive control group. Therefore, PHP40-2 has the function of negative immune regulation.

The results of the effect of PHP40-2 at various concentrations on the NO production of macrophage RAW264.7 and the expression levels of IL-1β, IL-6, IL-10 and TNF- α are shown in FIG. 10. Wherein, the A, B, C, D and E graphs respectively correspond to the influence results of NO, IL-1 beta, IL-6, IL-10 and TNF-alpha, and compared with a blank control group, P is less than 0.01, and compared with a positive control group, the # P is less than 0.05, and the # P is less than 0.01.

As can be seen from FIG. 9, under the induction of LPS, the secretion amounts of NO, IL-1 beta, IL-6, IL-10 and TNF-alpha were significantly lower than those of the positive control group under the intervention of PHP40-2 at different concentrations.

Comprehensive examples 2-3 show that PHP40-2 can improve phagocytic activity of macrophage RAW264.7 in a dose-dependent manner, has significant difference (P < 0.01), and has an effect of regulating immune forward when the concentration of PHP40-2 is 50-400 mug/mL and the higher the dosage is, the more the phagocytic activity is close to that of a positive control group; meanwhile, when the PHP40-2 concentration reaches 200 and 400 mug/mL, compared with a positive control group, the phagocytic activity of RAW264.7 macrophages is obviously reduced (P is less than 0.01), and the PHP has the function of negative immune regulation. From this, PHP40-2 was demonstrated to have a bi-directional immunomodulating effect of enhancing and reducing immune activity. Thus, the new application of the paecilomyces hepiali polysaccharide fermentation product in preparing the immunoregulation product is provided. The application provides the proposal to make up the application gap of the paecilomyces hepialid fermentation product and also provides another safe and effective substance for bidirectional immunoregulation.

Example 4 study of bidirectional immunoregulatory mechanism of PHP40-2

The two-way immunoregulation mechanism of PHP40-2 is studied by taking in vitro cultured mouse mononuclear macrophage RAW264.7 as a cell model. The method comprises the following specific steps:

western Blot detects expression of TLR4/NF- κB pathway-associated proteins.

(1) Sample preparation

Taking RAW264.7 cells in the logarithmic phase, adding 500 mu L of prepared RIPA lysate, repeatedly blowing after the lysate contacts the cells for 1-2 seconds, carefully transferring the cell suspension into a newly pre-cooled 1.5 mL centrifuge tube, centrifuging at 11000 r/min for 20 min at 4 ℃, and taking the supernatant in the newly pre-cooled centrifuge tube. The dilution ratios of each primary antibody are as follows: TLR4 (Rabbit, 1:2500), NF- κ B p65 (Rabbit, 1:2500), p-NF- κ B p65 (Rabbit, 1:2500), IκB (Rabbit, 1:2500), p-IκB (Rabbit, 1:2500) and β -actin (Rabbit, 1:2500).

(2) Determination of protein content and denaturation

Transferring the supernatant to a new centrifuge tube, measuring the total protein concentration of each group by using a BCA kit, adjusting the protein concentration to be consistent by using RIPA lysate, adding 40 mu L of 5 Xloading buffer solution, heating in a boiling water bath for 5 min, sub-packaging, and storing in a refrigerator at-80 ℃.

(3) SDS-PAGE gel electrophoresis

Preparing 12% of separation gel and 5% of concentrated gel to prepare polyacrylamide gel, inserting a comb into the concentrated gel, transferring a gel tank into an electrophoresis tank after the gel is solidified, adding electrophoresis liquid, extracting the comb, and adding the same equivalent of denatured protein solution (20 mug) and 3 mug of pre-dyed protein Marker into a sample loading hole. After the sample addition is finished, the constant pressure is 110V 2 h.

(4) Transfer film

Preparing a membrane transferring buffer solution, uncovering the glass plate, taking out the gel, and according to a sandwich membrane transferring structure. And placing the transfer film in a groove, adding transfer film buffer solution, connecting an electrophoresis apparatus, and keeping the constant pressure for 150V 2 h.

(5) Closure

5% of skim milk sealing liquid is prepared in advance by using 1 XTBST, the PVDF film after the film transfer is taken out is placed in the sealing liquid, and the sealing liquid is oscillated at room temperature for sealing 1 h. After the end of the blocking, the PVDF membrane was washed 3 times for 5 min each.

(6) Antibody incubation

The washed PVDF membrane was removed, the residue was sucked with filter paper, placed in a primary antibody diluted with 5% bovine serum albumin solution, and incubated overnight at 4 ℃. After the incubation, the PVDF membrane was washed 3 times for 5 min each. The washed PVDF membrane was incubated in secondary antibodies for 1 h with shaking at room temperature, and after the completion, the secondary antibodies were recovered and washed 3 times with 1 XTBST buffer for 5 min each.

(7) Chemiluminescent development

The ECL developer was taken out, and A, B solutions were mixed uniformly in a ratio of 1:1, photographed by observation in a gel imager, and the band gray scale was quantified using Image jv.1.52a.

The cell (experimental concentration group of examples 2-3 is followed) after PHP40-2 is acted by Western blot to detect the expression of key target proteins I kappa B-alpha and NF-kappa B p65 and TLR4 in NF-kappa B signal channel, and the results are shown in FIG. 11 and FIG. 12.

Wherein, in fig. 11: (A) Protein expression for TLR4, ikb and p65 and their phosphorylation; (B) quantitative analysis of TLR4 protein; (C) Quantitative analysis of p-NF- κ B p65/p65 protein phosphorylation; (D) Is the quantitative analysis of the phosphorylation level of the p-IκB/IκB protein. Significant differences were recorded as P <0.05, < P <0.01 compared to normal group.

Wherein, in fig. 12: (a) TLR4, ikb and p65 and phosphorylated protein expression; (B) TLR4 protein quantification; (C) Quantitative analysis of p-NF- κ B p65/p65 protein phosphorylation; (D) Quantitative analysis of P-ikb/ikb protein phosphorylation levels, compared to the blank, significant differences were recorded as P <0.05, < P <0.01. Compared to the positive control group, the significant differences were recorded as #p <0.05, #p <0.01.

As can be seen from fig. 11, PHP40-2 significantly up-regulates nuclear TLR4, NF- κ B p65 and ikb- α expression in RAW264.7 without LPS induction, thus demonstrating that PHP40-2 can activate TLR4/NF- κb pathway.

As can be seen from fig. 12, PHP40-2 can significantly reduce nuclear TLR4, NF- κ B p65 and ikb- α expression under LPS induction, inhibiting TLR4/NF- κb pathway and reducing inflammation. Further described is the mechanism by which PHP40-2 can exhibit bi-directional immunological activity by modulating signaling pathways.

The paecilomyces hepiali fermentation broth powder used in the invention is provided by Hangzhou snow area biotechnology Co.

The raw materials and equipment used in the invention are common raw materials and equipment in the field unless specified otherwise; the methods used in the present invention are conventional in the art unless otherwise specified.

The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification, variation and equivalent transformation of the above embodiment according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. The application of paecilomyces hepiali polysaccharide fermentation product in preparing an immunoregulatory product is characterized in that: the polysaccharide fermentation product comprises the following structures:

。

2. the use according to claim 1, wherein: the immunomodulation is bi-directional immunomodulation.

3. The use according to claim 1, wherein: the product is a medicine, a health product, a food additive or a daily chemical product.

4. An article of manufacture for immunomodulation, characterized in that: the active ingredient of the product comprises paecilomyces hepiali polysaccharide fermentation products.

5. An article of manufacture for immunomodulation according to claim 4, wherein: the active ingredient of the product is paecilomyces hepiali polysaccharide fermentation product.

6. An article of manufacture for immunomodulation according to claim 4 or 5, wherein: the active ingredients of the article comprise the following structure:

。

7. an article of manufacture for immunomodulation according to claim 4, wherein: the product is a medicine, a health product, a food additive or a daily chemical product.

8. An article of manufacture for immunomodulation according to claim 7, wherein: the product is a medicine, a health product, a food additive or a daily chemical product which can perform bidirectional regulation of improving the immune activity and reducing the immune activity.