Periplaneta americana compound extract as well as preparation method and application thereof

Abstract

The invention discloses a periplaneta americana composite extract as well as a preparation method and application thereof, the periplaneta americana composite extract comprises periplaneta americana mucoglycoprotein and protocatechuic acid-4-O-glucoside, and can be used for preparing medicines or health care products with the effect of regulating and controlling polarization of liver macrophages. The compound can also be used for preparing anti-hepatic fibrosis drugs or health care products, especially anti-immune hepatic fibrosis drugs or health care products.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceuticals, and particularly relates to a composite extract of Periplaneta americana, a preparation method thereof, and an application thereof. Background Art

[0002] Mucoprotein is a class of glycosylated conjugate proteins formed by covalent connection of oligosaccharides with polypeptides or proteins. Its structure mainly includes two parts: a polypeptide chain and a sugar, which are connected by a covalent bond. As a class of conjugate proteins, mucoproteins exist widely in animals, plants, and microorganisms in various forms and play roles such as cell signal recognition, growth regulation, intercellular information transmission, and immune activity regulation in organisms. Research shows that mucoproteins have effects such as enhancing immune regulation, inhibiting tumors, reducing blood sugar, reducing blood lipids, antioxidation, and anti-aging.

[0003] Periplaneta americana ( Periplaneta americana L.) is one of the insects in the genus Periplaneta of the family Blattidae. It is commonly called cockroach, oil thief, kitchen cricket, etc. in folk and is considered one of the four pests. However, as a traditional Chinese medicine, Periplaneta americana plays an important role in treating diseases. It was recorded as early as more than 2000 years ago. In "Shennong Ben Cao Jing", it was recorded that "Feilian (Periplaneta americana) is mainly used for blood stasis, firm masses, cold and heat, breaking up accumulations, and sore throat obstruction". In "Compendium of Materia Medica - Insect Part - Blattidae", it was recorded that its main treatment is "blood stasis, firm masses, cold and heat, lowering qi, and promoting blood circulation". Modern Chinese medicine research has found that the components of Periplaneta americana contain polysaccharides, proteins, polypeptides, amino acids, and some small molecule substances, etc. The extract of Periplaneta americana has effects such as promoting wound healing, antioxidation, anti-tumor, and anti-inflammatory.

[0004] The liver immune microenvironment is a dynamic network system composed of various immune cells, cytokines, and related ECMs, and is closely related to the occurrence and development of liver fibrosis. Under liver injury conditions, multiple hepatocytes participate in complex inflammatory responses. Damaged hepatocytes release damage-associated and pathogen-associated molecular patterns (MAMS) signals, stimulating the recruitment and activation of immune cells in diseased liver tissue. Hepatic Kuffer cells (KCs) differentiate into classically activated macrophages (M1 type) when the liver is injured. These macrophages interact with Toll-like receptors (TLRs) or recombinant purinergic receptor P2X and ligand-gated ion channels, leading to the activation of inflammasomes and the release of interleukin-1β (IL-1β), IL-18, and various other pro-inflammatory cytokines and chemokines. This further promotes the recruitment of circulating leukocytes (monocytes and neutrophils) and activates adaptive immune T cell function. When the damage is controlled, KCs transform into an anti-inflammatory and tissue-repair phenotype (M2 type) to control the excessive inflammatory response. However, persistent liver damage stimulates M1 macrophages to continuously release inflammatory cytokines, promoting excessive activation of hepatic stellate cells (HSCs), leading to the development of liver fibrosis and further aggravating cirrhosis and even liver cancer. Studies have confirmed that after drug treatment, macrophage phenotypes change, with pro-inflammatory M1 macrophages transforming into anti-inflammatory M2 macrophages. The massive release of inflammatory factors is inhibited, thereby reducing the stimulation of hepatic stellate cell activation by inflammatory factors and exerting anti-inflammatory and anti-hepatic fibrosis effects. Therefore, macrophages play an important regulatory role in the development of liver fibrosis.

[0005] To identify the immunomodulatory extracts from the American cockroach medicinal material, this invention studies the pharmaceutical process of the American cockroach compound extract. Based on the in vitro co-incubation experiment of liver fibroblasts (LX-2) and M1 cells, the immunomodulatory effects and mechanisms of the American cockroach compound extract are further studied using immune-induced liver fibrosis mice as the research subjects. Summary of the Invention

[0006] The purpose of this invention is to provide a compound extract of American cockroaches and its preparation method, wherein the American cockroach compound extract can regulate the polarization of liver macrophages and exert an anti-liver fibrosis effect.

[0007] The objective of this invention is achieved through the following technical solution: A complex extract of American cockroaches, composed of American cockroach mucoglycoprotein and protocatechuic acid-4-O-glucoside.

[0008] Preferably, the weight ratio of American cockroach mucoprotein to protocatechuic acid-4-O-glucoside is 4~6:0.6~1.

[0009] The preparation method of the American cockroach complex extract includes the following steps: (1) After crushing the American cockroach medicinal material, extract and defatting it, and then process it into two parts separately; (2) A portion of the extract was glycoprotein extracted by reflux with water, filtered, and the filtrate was concentrated to obtain a defatted extract. Ethanol was added to the defatted extract to a concentration of 80%–85%, and the mixture was allowed to stand overnight at 10–4°C. The precipitate was then collected by centrifugation. The precipitate was dissolved in water, and a chloroform-n-butanol mixture was added. The mixture was stirred, centrifuged, and the precipitate was collected. This process was repeated several times. The precipitate was then dissolved in water and subjected to gel column chromatography. The precipitate was eluted with pure water, the eluent was collected, concentrated, and freeze-dried to obtain American cockroach glycoprotein. (3) Another part of the extraction of protocatechuic acid-4-O-glucoside: the defatted American cockroach medicinal material in step (1) was extracted by ultrasonic extraction with 70%~80% ethanol solution, centrifuged, filtered, and the filtrate was concentrated to dryness to obtain the extract. It was fully dissolved in 1% acetic acid aqueous solution, filtered, and the filtrate was subjected to column chromatography with C18 material. The eluent was collected, concentrated, and dried to obtain protocatechuic acid-4-O-glucoside (3,4-dihydroxybenzoic acid glucoside).

[0010] (4) Mix the products from steps (2) and (3) to obtain the American cockroach complex extract (named PAGP).

[0011] Preferably, in step (2), water is added at a mass-to-volume ratio of 1:6~10g / mL, and the mixture is heated and refluxed at 80~90℃ for 2~4h.

[0012] Preferably, in step (2), the volume ratio of chloroform to n-butanol in the chloroform-n-butanol mixed solution is 4:1.

[0013] Preferably, in step (2), the volume ratio of the aqueous solution obtained by adding water to the precipitate until it is completely dissolved to the chloroform-n-butanol mixed solution is 4:1.

[0014] Preferably, in step (3), the mass-to-volume ratio of the extract to the 1% acetic acid aqueous solution is 1:20~30 g / mL.

[0015] Preferably, in step (3), the elution step is as follows: elute with 3 to 5 column volumes of 5% methanol solution or ethanol solution, and discard the eluent; then perform isocratic elution with 0.1% acetic acid aqueous solution-ethanol solution, the volume ratio of 0.1% acetic acid aqueous solution to ethanol solution is 92% to 85%: 8% to 15%, and collect this part of the eluent.

[0016] The American cockroach compound extract PAGP can inhibit the polarization of M1 macrophages in the liver and reduce the expression of inflammatory factors, while also promoting the polarization of M2 macrophages, thus exhibiting a regulatory effect on hepatic macrophage polarization. Simultaneously, the American cockroach compound extract can also regulate the balance of the M1 / M2 ratio of mouse hepatic macrophages and the Th1 / Th2 ratio of spleen cells, and regulate the PPAR signaling pathway, demonstrating a strong anti-hepatic fibrosis effect, especially against immune-mediated hepatic fibrosis. Therefore, the American cockroach compound extract can be used to prepare drugs or health products that regulate hepatic macrophage polarization, and also to prepare drugs or health products that combat hepatic fibrosis, especially those against immune-mediated hepatic fibrosis.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The American cockroach compound extract PAGP described in this invention can downregulate the polarization ratio of M1 macrophages and upregulate the ratio of M2 macrophages, significantly inhibit the expression of inflammatory factors in M1 macrophages, improve the inflammatory state of the cell model, thereby inhibiting the expression of activation markers in LX-2 cells, promoting apoptosis of activated LX-2 cells, and exerting an anti-liver fibrosis effect.

[0018] 2. The American cockroach compound extract PAGP described in this invention also has a significant anti-immune liver fibrosis effect. PAGP alleviated Con A-induced liver pathological damage in mice, restored liver function, reduced the degree of macrophage infiltration in the liver, and alleviated immune liver fibrosis. Furthermore, PAGP also regulated the balance of the M1 / M2 ratio of liver macrophages and the Th1 / Th2 ratio of spleen macrophages in mice, and reduced the expression of inflammatory factors in the liver. The differential gene expression of PAGP in the livers of mice with immune liver fibrosis was mainly enriched in the PPAR signaling pathway, indicating that PAGP exerts its anti-immune liver fibrosis effect by regulating the PPAR signaling pathway.

[0019] 3. The inventors of this invention have discovered that the extract of American cockroach mucoprotein has good immunomodulatory activity.

[0020] 4. This invention provides a scientific basis for the development of active substances from American cockroaches for the treatment of immune liver fibrosis (ILF), and also provides a new approach for the development of therapeutic drugs for diseases related to macrophage polarization. Attached Figure Description

[0021] Figure 1 The image shows an HPLC chromatogram of the amino acid composition of the American cockroach mucoprotein obtained in Example 1.

[0022] Figure 2 The image shows an HPLC chromatogram of the monosaccharide composition of the American cockroach mucoprotein obtained in Example 1.

[0023] Figure 3 The image shows an SDS-PAGE gel image of the American cockroach mucoprotein prepared in Example 1. In the image, A is a mucoprotein with a molecular weight of 10-200 kDa, and B is a mucoprotein with a molecular weight of 2-40 kDa. a is Coomassie brilliant blue staining, and b is periodic acid-Schiff staining.

[0024] Figure 4 This is a SEM scan image of the American cockroach mucoprotein prepared in Example 1.

[0025] Figure 5 The image shows the FT-IR spectrum of the American cockroach mucoprotein obtained in Example 1.

[0026] Figure 6 The UV spectra of the β-elimination reaction of the American cockroach mucoprotein prepared in Example 1 before and after NaOH treatment are shown.

[0027] Figure 7 The image shows the HPLC chromatogram of the American cockroach medicinal material in Example 2, where peak 1 is protocatechuic acid-4-O-glucoside.

[0028] Figure 8 The image shows the HPLC chromatogram of the protocatechuic acid-4-O-glucoside reference standard prepared in Example 2, with peak 1 representing protocatechuic acid-4-O-glucoside.

[0029] Figure 9 The structure of protocatechuic acid-4-O-glucoside obtained in Example 2 is shown below.

[0030] Figure 10 The effect of PAGP on the polarization ratio of M1 macrophages in Example 1 was applied (n=3).

[0031] Figure 11 The effect of PAGP on the polarization ratio of M2 macrophages in Example 1 was applied (n=3).

[0032] Figure 12 To illustrate the effect of PAGP on the ratio of apoptotic cells to total cells in LX-2 cells (n=3) as described in Example 1.

[0033] Figure 13 To illustrate the effect of PAGP on the expression of inflammatory factors in macrophages in Example 1 (n=3), where A is TNF-α, B is IL-1β, C is IL-6, and D is IL-8.

[0034] Figure 14 The effect of PAGP on LX-2 cells in Example 1 (n=3) is shown, where A represents COL1A1, B represents COL3A1, and C represents the expression of α-SMA.

[0035] Figure 15 The effect of PAGP on the fluorescence intensity of α-SMA in LX-2 cells was investigated in Example 1 (n=3).

[0036] Figure 16 The effect of PAGP on the morphology of mouse liver and spleen was applied in Example 2.

[0037] Figure 17 To illustrate the effect of PAGP on the liver and spleen indices of mice in Example 2, where A represents the liver index and B represents the spleen index.

[0038] Figure 18 To illustrate the effect of PAGP on liver function and LF index in mouse serum as described in Example 2, where A represents ALT, B represents AST, C represents HA, and D represents LN.

[0039] Figure 19 The effect of PAGP on the pathological changes of liver tissue in mice in each group was shown in Example 2 (200×), where a represents inflammatory cell infiltration, b represents increased nuclear volume, and c represents cytoplasmic cavitation.

[0040] Figure 20 The effect of PAGP on the histopathology of spleen tissue in each group of mice in Example 2 (200×) is shown, where a represents white pulp, b represents red pulp, and c represents disordered spleen tissue structure.

[0041] Figure 21 Masson staining (200×) was performed on the livers of mice in each group in Example 2, where a represents collagen fibers and b represents pseudolobules.

[0042] Figure 22 Sirius red staining (200×) was performed on the livers of mice in each group in Example 2, where a represents pseudolobules and b represents collagen fibers.

[0043] Figure 23 Immunohistochemical staining images (200×) of macrophage infiltration in the livers of mice in each group in Example 2, where a is F4 / 80 positive.

[0044] Figure 24 This study investigated the effect of PAGP on the polarization of M1 type hepatic macrophages in mouse liver tissue, as described in Example 2.

[0045] Figure 25 This study investigated the effect of PAGP on the polarization of M2 hepatic macrophages in mouse liver tissue, as described in Example 2.

[0046] Figure 26 This study investigated the effect of PAGP on the polarization of Th1 T lymphocytes in mouse spleen tissue, as described in Example 2.

[0047] Figure 27 This study investigated the effect of PAGP on the polarization of Th2-type T lymphocytes in mouse spleen tissue, as described in Example 2.

[0048] Figure 28 To illustrate the effect of PAGP on the expression of Pparα, Foxa1, Fapp1, and Hnf4α mRNA in mouse liver in Example 2, A represents Pparα, B represents Foxa1, C represents Fapp1, and D represents Hnf4α.

[0049] Figure 29 This is a protein band diagram showing the effect of PAGP on the expression of Pparα, Foxa1, Fapp1, and Hnf4α proteins in mouse liver, as described in Example 2.

[0050] Figure 30 This is a statistical graph showing the effect of PAGP on the expression of Pparα, Foxa1, Fapp1, and Hnf4α proteins in mouse liver in Example 2. A represents Pparα, B represents Foxa1, C represents Fapp1, and D represents Hnf4α.

[0051] In the above figure, compared with the normal group, # P <0.05, ## P <0.01; compared with the model group, * P <0.05, ** P <0.01. Detailed Implementation

[0052] The present invention will be further described below with reference to the embodiments, but the present invention is not limited to the embodiments. Example 1 – Extraction, purification and identification of American cockroach mucoprotein

[0053] 1. Extraction and purification of American cockroach mucoprotein The American cockroach mucoprotein is a glycocomplex mainly formed by O-glycosidic bonds, with a weight-average molecular weight of approximately 75-85% in the 10-18 kDa range and approximately 15-25% in the 70-75 kDa range. The preparation method is as follows: American cockroach material is pulverized and passed through an 8-20 mesh sieve, then extracted and defatted. Water is added at a material-to-liquid ratio of 1:6-10 g / mL, and the mixture is heated to reflux at 80-90℃ for 2-4 hours. After filtration, the filtrate is collected, filtered through a 2000-4000 mesh sieve, and concentrated under reduced pressure to a specific gravity of approximately 1.15-1.35 to obtain the defatted extract of American cockroach. 95% ethanol is added to the defatted extract, stirred, and the ethanol concentration is adjusted to 80-85%. The mixture is allowed to stand overnight at 10-4℃, centrifuged at 4000 rpm for 10-15 minutes, and the precipitate is collected. Dissolve the precipitate in water, add a chloroform-n-butanol mixed solution (chloroform to n-butanol volume ratio 4:1), and add water to the precipitate until completely dissolved. The volume ratio of the aqueous solution obtained to the chloroform-n-butanol mixed solution is 4:1. Stir thoroughly at 4000-8000 rpm for 4-8 min, centrifuge at 4000 rpm for 10 min, collect the precipitate, and repeat 7-10 times to remove free proteins from the American cockroach extract. Add water to the precipitate and stir until the precipitate is completely dissolved to prepare an extract concentration (w / v) of 3%-5% g / mL. Add the solution to an activated Sephadex G gel column and allow static adsorption for 8-16 h. Elute with 3-5 column volumes of pure water at a rate of 0.01-0.02 column volumes / min, collect the eluent, concentrate under reduced pressure at 60℃, and freeze-dry to prepare American cockroach mucoglycoprotein with an extraction rate of 4%-7% (depending on the quality of the medicinal material). The extracted American cockroach mucoprotein contained 65±5% total protein, with the main amino acids being arginine, glutamic acid, aspartic acid, tyrosine, threonine, serine, glycine, histidine, and alanine. The total sugar content of the extracted American cockroach mucoprotein was 18±1% (phenol-sulfuric acid method). The main monosaccharides constituting the polysaccharide were glucose, galactose, mannose, xylose, arabinose, fucose, and rhamnose, with glucose, galactose, and mannose accounting for ≥70% of the total.

[0054] 2. Identification of mucoproteins from the American cockroach 2.1 Amino acid composition analysis The protein content of the mucoglycoprotein in the American cockroach is 65±5%, and its amino acid composition and content are shown in Table 1. Figure 1 As shown, a total of 9 amino acids were detected, including arginine, glutamic acid, aspartic acid, tyrosine, threonine, serine, glycine, histidine, and alanine, with a molar ratio of 6.7:9.3:3.6:1.6:3.8:18.2:1.0:5.8:3.0.

[0055]

[0056] 2.2 Monosaccharide Composition Analysis The total sugar content of the American cockroach mucoprotein was determined to be 18±1% using the phenol-sulfuric acid method. HPLC analysis revealed seven monosaccharides in the American cockroach mucoprotein: mannose, rhamnose, glucose, galactose, xylose, arabinose, and fucose, with a molar ratio of 2.1:4.4:4.4:32.8:3.3:4.3:1.0. The molar percentages are shown in Table 2. HPLC analysis is described in [Table 2]. Figure 2 .

[0057]

[0058] 3. Molecular weight characterization of American cockroach mucoprotein 3.1 Molecular weight determination of American cockroach mucoprotein using SDS-PAGE Coomassie Brilliant Blue and PAS reactions were used to identify the mucoproteins of the American cockroach. After staining the gel containing the isolated American cockroach mucoproteins with Coomassie Brilliant Blue and PAS, two protein bands overlapped at the 10-18 kDa and 73 kDa molecular weight ranges in both staining methods. Figure 3 This indicates that the American cockroach mucoproteins at these two molecular weight positions can be stained by both Coomassie Brilliant Blue and PAS, suggesting that they are glycoproteins.

[0059] 3.2 SEM analysis of the microstructure of American cockroach mucoprotein SEM scan results of American cockroach mucoproteins are as follows: Figure 4 As shown, the surface of the American cockroach mucoprotein is rough, with a relatively obvious porous structure and a layered structure, which is consistent with the structure of the protein and polysaccharide after binding.

[0060] 3.3 FT-IR analysis of functional groups of American cockroach mucoprotein FT-IR is a method for detecting polymer functional groups and chemical structures. The FT-IR spectrum of the mucoprotein from the American cockroach is shown below. Figure 5 As shown, the absorption peak of the American cockroach mucoprotein is located at 3500-3200 cm⁻¹ due to the superposition of OH bonds and the stretching vibration of NH bonds. -1 A distinctly broad peak appears within the range, which is characteristic of polysaccharide and protein molecules. 2928 cm⁻¹ -1 The absorption peak at 1655 cm⁻¹ indicates the stretching vibration of the CH bond in the alkyl group. -1 The absorption peak at 1400 cm⁻¹ corresponds to the stretching vibration of the CO bond in the acetylamino group. -1 Variable-angle vibrational absorption is a major characteristic of the CH molecules in peptide bonds of proteins. (1000-1200 cm⁻¹)-1 The strong absorption peaks within this range are related to the stretching vibrations of COH and COC. (1081, 1050 cm⁻¹) -1 The absorption peak at 877 cm⁻¹ indicates the presence of a pyran ring, suggesting that the glycoside of the American cockroach mucin is pyran-type. -1 The absorption peak at this point indicates the presence of β-glycosidic bonds, suggesting that the polysaccharides in the American cockroach mucoprotein are linked by β-glycosidic bonds.

[0061] 3.4 Analysis of the types of glycosidic bonds in the mucoproteins of the American cockroach β-elimination reactions can be used to determine the type of glycosidic bond. O-type glycosidic bonds are unstable in bases and readily undergo β-elimination reactions in NaOH solution. Serine and threonine residues on the glycosidic bond are converted to α-aminoacrylic acid and α-butylacrylic acid under the action of a base, respectively. These two compounds produce significant ultraviolet absorption at 240 nm. Experimental results are as follows... Figure 6 As shown, compared with aqueous solution, the American cockroach mucoprotein exhibits enhanced ultraviolet absorption at 240 nm in alkaline solution (0.2 M NaOH), indicating that the glycopeptide bonds in the American cockroach mucoprotein are of the O-type glycosidic bond type.

[0062] Example 2 – Extraction, purification and identification of protocatechuic acid-4-O-glucoside from American cockroaches 1. Extraction and purification of protocatechuic acid-4-O-glucoside (3,4-dihydroxybenzoic acid glucoside) from American cockroach. After extraction and defatting, the American cockroach medicinal material was added to an ethanol (80%~70%) solution at a material-solvent mass-to-volume ratio of 1:8~15 g / mL. The mixture was ultrasonically extracted for 30~60 min, centrifuged at 3000~4000 r / min for 5~10 min, filtered, and the filtrate was concentrated to dryness to obtain the extract. The extract was then dissolved in a 1% acetic acid aqueous solution at a extract:solvent (mass-to-volume ratio) of 1:20~30 g / mL and filtered through a 3000~5000 mesh filter. The filtrate was adsorbed onto a C18 material for 8–12 h, and then eluted with 3–5 column volumes of 5% methanol solution (or ethanol solution), discarding the eluent. Next, isocratic elution was performed with a 0.1% acetic acid aqueous solution-ethanol solution, with a volume ratio of 92%–85%:8%–15%. This eluent was collected, concentrated, and dried to obtain protocatechuic acid-4-O-glucoside with a purity ≥80% and an extraction rate of 0.5–1% (dependent on the quality of the medicinal material).

[0063] 2. Identification of protocatechuic acid-4-O-glucoside (3,4-dihydroxybenzoic acid glucoside) in American cockroaches Figure 7 and Figure 8 Peak 1 shown is protocatechuic acid-4-O-glucoside, and its structural formula is as follows: Figure 9 As shown.

[0064] Example 3 – American cockroach complex extract PAGP The American cockroach mucoprotein obtained in Example 1 was mixed with the protocatechuic acid-4-O-glucoside obtained in Example 2 to obtain the American cockroach complex extract PAGP.

[0065] Application Example 1 – Effect of PAGP, the American cockroach compound extract of the present invention, on the in vitro regulation of macrophage (THP-1 cell) polarization on LX-2 cells. Macrophages are the main immune cells in the liver, primarily classified into M1 type (pro-inflammatory, classically activated) and M2 type (anti-inflammatory, alternatively activated). Studies have shown that regulating macrophage M1 polarization, altering the macrophage M1 / M2 polarization ratio, and inhibiting the excessive secretion of inflammatory cytokines can effectively reduce the activation of hepatic stellate cells (HSCs) and promote their apoptosis, thereby improving liver fibrosis.

[0066] 1 Experimental Methods THP-1 and LX-2 cells were induced separately. THP-1 cells were induced with PMA 100 ng / mL for 48 h to become M0 macrophages, and then induced with LPS 1000 ng / mL for 24 h to become M1 macrophages, or induced with IL-4 20 ng / mL + IL-10 20 ng / mL for 24 h to become M2 macrophages. LX-2 cells were induced with TGF-β1 for 48 h. Then, using Transwell chambers, the two cell lines were adaptively co-cultured for 12 hours. The cells were then grouped and administered the following drugs: normal group (M0+LX-2), model group (M1 or M2+LX-2), low-dose group (M1 or M2+LX-2+PAGP 80μg / mL), medium-dose group (M1 or M2+LX-2+PAGP 120μg / mL), and high-dose group (M1 or M2+LX-2+PAGP 160μg / mL). PAGP was prepared in Example 3. In this application example, the mass ratio of American cockroach mucoprotein to protocatechuic acid-4-O-glucoside was any ratio within the range of 4~6:0.6~1.

[0067] The study used flow cytometry to detect changes in the M1 / M2 polarization ratio of macrophages and the apoptosis of LX-2 cells, RT-qPCR to detect the expression of inflammatory factors in M1 macrophages and the expression of activating factors in LX-2 cells, and immunofluorescence to observe the activation level and apoptosis of LX-2 cells. This study elucidated how PAGP exerts its anti-liver fibrosis effect by regulating macrophages and inhibiting LX-2 cells.

[0068] 2. Experimental Results 2.1 Effect of PAGP on the M1 / M2 polarization ratio of macrophages Under co-culture conditions, flow cytometry was used to detect the effects of PAGP on M1 and M2 macrophage polarization: compared with the normal group, the number of M1 macrophages in the model group was significantly increased, while compared with the model group, the proportion of M1 macrophages in the PAGP intervention group was significantly decreased (P<0.01), showing a dose-dependent effect (e.g., ...). Figure 10 As shown); the proportion of M2 macrophages in the PAGP intervention group increased to varying degrees compared with the model group, with the high-dose and medium-dose groups showing the most significant effects (e.g. Figure 11 As shown in the figure, the results indicate that PAGP can downregulate the polarization ratio of M1 macrophages and upregulate the ratio of M2 macrophages.

[0069] 2.2 Apoptosis in LX-2 cells The above experiments confirmed that PAGP can reduce the polarization ratio of M1 macrophages. To confirm the effect of PAGP-regulated macrophages on LX-2 cell apoptosis, flow cytometry was used to detect changes in the apoptosis ratio of LX-2 cells. The results are as follows: Figure 12 As shown, compared with the model group, the proportion of apoptotic cells in both the medium-dose and high-dose PAGP groups increased to varying degrees, with the high-dose group showing the most significant increase (P<0.05), indicating that PAGP can promote apoptosis of LX-2 cells and exert an anti-liver fibrosis effect.

[0070] 2.3 Effects of PAGP on the expression of inflammatory factors in M1 macrophages RT-qPCR was used to further detect the expression of inflammatory factors in M1 macrophages under co-culture conditions. Compared with the normal group, the expression of inflammatory factors (TNF-α, IL-1β, IL-6, IL-8) in M1 macrophages in the model group was significantly increased (P<0.01). After PAGP intervention, the expression of inflammatory factors TNF-α, IL-1β, IL-6, and IL-8 in M1 macrophages was regulated to varying degrees. Among them, the high-dose PAGP group had the most significant inhibitory effect on inflammation in M1 macrophages (P<0.01). The results indicate that PAGP can effectively regulate the expression of inflammatory factors in M1 macrophages (see...). Figure 13 ).

[0071] 2.4 Expression of LX-2 cell activating factors For LX-2 cells, after PAGP was administered under co-culture conditions to inhibit the expression of inflammatory factors in M1 macrophages, compared with the model group, the levels of COL1A1, COL3A1, and α-SMA in LX-2 cells treated with PAGP decreased to varying degrees, with the highest decrease observed in the high-dose group (P<0.01) (see [link to article]). Figure 14In the PAGP-treated group, the degree of fibrosis in LX-2 cells was inhibited, and the deposition of extracellular collagen was improved. This indicates that PAGP has a certain inhibitory effect on LX-2 cell activation by regulating the expression of inflammatory factors in M1 macrophages.

[0072] 2.5 Expression of α-SMA agonist in LX-2 cells The expression of α-SMA was detected by immunofluorescence to further evaluate the activation level of LX-2 cells. Results are shown below. Figure 15 Compared with the model group, the expression of α-SMA in LX-2 cells in the PAGP-treated group was reduced to varying degrees, indicating that the activation of LX-2 cells was inhibited, suggesting that PAGP has a positive effect on improving liver fibrosis.

[0073] 3. Conclusion Under co-culture conditions, PAGP downregulated the polarization ratio of M1 macrophages and upregulated the ratio of M2 macrophages, significantly inhibited the expression of inflammatory factors in M1 macrophages, and improved the inflammatory state of the cell model, thereby affecting the activation and apoptosis of LX-2 cells. PAGP intervention inhibited the expression of type I and III collagen in LX-2 cells, reduced α-SMA secretion, and increased apoptosis in activated LX-2 cells, ultimately exerting an anti-liver fibrosis effect.

[0074] Application Example 2 – Effects of PAGP on Immune Liver Fibrosis (ILF) in Mice 1 Experimental Methods SPF female Balb / c mice at 6 - 8 weeks of age (16 - 18 g) (Animal License: SCXK (Beijing) 2019 - 0010). After one - week adaptive feeding, the mice were randomly divided into 5 groups according to body weight: normal group, model group, positive group, low - dose PAGP group, and high - dose PAGP group (8 mice in each group). Mice in the normal group were injected with 0.9% normal saline via the tail vein. Mice in the model group, positive group, and each PAGP group were injected with Con A twice a week. Among them, Con A was injected at doses of 6, 8, 10, 11, 12, 13, 14, 14 mg / kg (10 μL / g) from week 1 to week 8 respectively. Since the first injection of Con A, the normal group and the model group were given normal saline, the positive group was given a solution made from Fuzheng Huayu Capsules (FZHY, 680 mg / kg, 10 μL / g), and the low - dose PAGP group and the high - dose PAGP group were given 50 and 100 mg / kg (10 μL / g) of PAGP (PAGP was prepared according to Example 3) respectively, and administered by gavage once a day for 8 consecutive weeks. At the end of the 8th week of drug administration in mice, the mice were fasted for 12 hours without water restriction. After anesthetizing the mice, blood was collected. The blood was centrifuged at 4000 r / min for 10 min, and the upper - layer serum was collected and stored at - 80 °C. After sacrificing the mice, the liver and spleen were carefully removed intact, excess tissue was removed, washed with normal saline, weighed, used to calculate the organ index, and photographed to record the morphological changes of the liver and spleen of the mice. The formula for calculating the organ index: organ index = organ weight / mouse body weight.

[0075] The contents of ALT, AST, LN, and HA in the serum of mice were detected by kits; the histopathological changes of the liver and spleen tissues of mice in each group were observed by H&E staining, and the deposition of collagen fibers and macrophage infiltration in the liver tissues were detected by Masson, Sirius red staining, and immunohistochemistry, and the changes in the polarization ratio of M1 / M2 macrophages in the liver tissues of mice were detected by immunofluorescence staining; the changes in the polarization ratio of Th1 / Th2 T lymphocytes in the spleen of mice were detected by flow cytometry; differential genes in the livers of mice in each group were screened by RNA - seq, the molecular mechanism of PAGP in treating ILF was screened by KEGG analysis, and the accuracy of RNA - seq was further verified by qPCR and Western blot. The mechanism of action of PAGP in treating ILF in mice was clarified by the above - mentioned methods.

[0076] 2 Experimental results 2.1 Effects of PAGP on the morphology and organ index of the liver and spleen of mice Clinically, chronic liver disease is often accompanied by portal hypertension, leading to splenomegaly and hypersplenism. In this application example, the livers and spleens of mice in the model group were significantly enlarged, with a dark red surface and uneven granular texture in the liver. The spleens were irregular in shape and showed some fat accumulation. Furthermore, the liver and spleen indices in the model group were higher than those in the normal group (P<0.01). After PAGP intervention, the liver and spleen volumes of mice decreased, the liver surface became smooth without uneven granular texture, and the spleen shape became more regular. In addition, the liver and spleen indices in the positive control group and the high-dose PAGP group were significantly lower than those in the model group (P<0.01). Experimental results are shown below. Figure 16 , 17 The results showed that FZHY and PAGP could effectively prevent morphological changes in the liver and spleen and changes in organ indices in ILF mice.

[0077] 2.2 Effects of PAGP on serum ALT, AST, LN, and HA levels Immunohistochemical detection results as follows Figure 18 As shown, the ALT, AST, HA, and LN values ​​in the model group were all higher than those in the normal group, while the above indicators in each PAGP group were significantly lower than those in the model group (P<0.05), with the high-dose group showing the most significant effect (P<0.01). Furthermore, the LN and HA indicators in the positive group were significantly lower (P<0.01), but the ALT and AST values ​​were not statistically significantly lower than those in the model group. This suggests that both FZHY and PAGP effectively improve liver injury and ILF in ILF mice.

[0078] 2.3 Effects of PAGP on liver and spleen histopathology Observe the results of H&E staining pathological tissue analysis of mouse liver (see) Figure 19 The study found that in the normal group of mice, hepatocytes were evenly distributed, orderly arranged, and clearly structured, with obvious hepatic cord structures. In the model group of mice, hepatocytes were disordered, hepatic cord structures were destroyed, cell nuclei were enlarged, cell numbers were reduced, and extensive cytoplasmic vacuolation was observed, accompanied by inflammatory cell infiltration. Both the positive control group and all PAGP dose groups improved the above pathological conditions, with the high-dose PAGP group showing the most significant effect. This suggests that FZHY and PAGP can effectively prevent liver damage in ILF mice.

[0079] H&E staining of spleen tissue (see) Figure 20 The dark blue color is white pith ( Figure 20 (a) The red area is red marrow ( Figure 20 (b) Staining results showed that the spleen tissue of normal mice had clear structures in each layer, with a clear boundary between the red and white pulp. However, the spleen tissue structure of the model group was disordered. Figure 20(c) The boundary between the red and white pulp was blurred. Compared with the model group, the spleen tissue structure of mice in the high-dose PAGP group and the positive group was significantly clearer, and the boundary between the red and white pulp was also significantly clearer, indicating that PAGP and FZHY can effectively improve spleen damage induced by Con A in ILF mice.

[0080] Masson and Sirius red staining results are as follows: Figure 21 , 22 As shown, in the normal group, a small amount of collagen fibers were visible around the blood vessel walls in the liver tissue, which is within the normal range, and no pseudolobules were formed. In the model group, a large amount of collagen fiber deposition was visible around the portal areas in the liver tissue. Figure 21 a, Figure 22 (b) extends outward from the periphery of the manifold area, forming pseudolobes ( Figure 21 b, Figure 22 In the model group (a), the collagen fibers were thicker and darker in color. Quantitative analysis showed that the collagen fiber area in the liver of the model group mice was significantly higher than that in the normal group (P<0.01). After PAGP treatment, the blue staining of the collagen fibers in the low-dose group was reduced (P<0.01), but pseudolobules still formed. In the high-dose group and the positive group, the blue staining of the collagen fibers was significantly reduced and the color was significantly lighter (P<0.01), and no pseudolobules were observed. The high-dose group showed the best effect. This suggests that PAGP and FZHY can effectively inhibit the deposition of collagen fibers in liver tissue and improve ILF.

[0081] 2.4 Effects of PAGP on hepatic macrophages Macrophage infiltration is an inflammatory response, and the F4 / 80 molecule has been identified as a unique marker of macrophages; therefore, detecting the positive expression level of F4 / 80 in the liver ( Figure 23 (a) can determine the degree of liver inflammation in mice. Immunohistochemical results are as follows: Figure 23 As shown, compared with the normal group, the F4 / 80 positive expression level in the model group was significantly increased (P<0.01). Compared with the model group, the F4 / 80 positive expression levels in the positive group and each PAGP group were decreased to varying degrees, and the differences were statistically significant (P<0.01). The results suggest that PAGP can alleviate the inflammatory response in liver tissue and reduce the degree of liver damage in ILF mice. Moreover, based on the animal experiment results, the high-dose PAGP group (100 mg / kg) showed better therapeutic effects than the positive group (680 mg / kg).

[0082] 2.5 Effects of PAGP on M1 / M2 type hepatic macrophages The above results confirm that PAGP can reduce the positive expression of F4 / 80 in liver macrophages of ILF mice. To further confirm the regulation of liver macrophages by PAGP, immunofluorescence was used to detect changes in M1 (DAPI, F4 / 80, iNOS) and M2 (DAPI, F4 / 80, Arg-1) macrophages in mouse liver tissue. iNOS is typically a surface marker for M1 macrophages, and Arg-1 is a surface marker for M2 macrophages. Figure 24 It was found that, compared with the normal group, the fluorescence intensity of F4 / 80 and iNOS in the model group was significantly increased (P<0.01). Compared with the model group, the fluorescence intensity of F4 / 80 and iNOS in each PAGP group and the positive group was significantly decreased (P<0.01), with the high-dose group and the positive group showing the most significant effects, their fluorescence intensity being close to that of the normal group, followed by the low-dose group. This indicates that both PAGP and FZHY can inhibit the increase in the proportion of M1 macrophages induced by Con A-induced ILF. Conversely, the increase in the proportion of M1 macrophages induced by Con A-induced ILF was significantly reduced. Figure 25 It was found that, compared with the normal group, the F4 / 80 and Arg-1 intensities were significantly reduced in the model group (P<0.01). After intervention with PAGP and FZHY, the fluorescence intensity of F4 / 80 and Arg-1 increased to varying degrees in both the PAGP group and the positive group (P<0.01), with the fluorescence intensity in the high-dose group being higher than that in the positive and low-dose groups. This indicates that PAGP and FZHY can effectively enhance the polarization ratio of M2 macrophages in the liver tissue of ILF mice. This suggests that PAGP and FZHY can exert a therapeutic effect on mouse ILF by regulating the polarization ratio of hepatic macrophages.

[0083] 2.6 Effects of PAGP on Th1 / Th2 lymphocytes in the spleen This application example found that PAGP significantly improved splenomegaly and increased spleen index in mice. Therefore, it is hypothesized that PAGP exerts its anti-ILF effect by regulating splenic immune cells. To investigate the effect of PAGP on splenic immune cells in ILF mice, this example examined changes in the proportions of Th1 (CD3+, CD4+, and IFN-γ) and Th2 (CD3+, CD4+, and IL-4) cells in the mouse spleen. IFN-γ is typically a marker of the Th1 cell subset, while IL-4 is a marker of the Th2 cell subset. The results are as follows... Figure 26 , 27 As shown in the figure. The results indicated that the proportion of Th1 cells in the model group mice was significantly increased (P<0.01), while the proportion of Th2 cells was significantly decreased (P<0.05). Compared with the model group, both the positive control group and all PAGP dose groups decreased the proportion of Th1 cells and increased the proportion of Th2 cells, with a significant difference in the high-dose group (P<0.01), but no statistically significant difference between the positive control group and the low-dose group. This suggests that PAGP improves the change in the Th1 / Th2 cell ratio in the spleen of ILF.

[0084] 2.7 RNA-seq analysis To gain a deeper understanding of the molecular mechanism of PAGP treatment for ILF in mice, we performed RNA-seq analysis on mouse livers. The OD 260 / 280 values ​​of total RNA in the samples were all within the range of 1.8-2.2, indicating good purity of total RNA. Furthermore, the samples were free of DNA and impurities, and showed no or only slight degradation, meeting the requirements for library construction. In this example, a total of 21,899 genes were obtained from mouse livers, and differentially expressed genes were screened according to the criteria of FDR < 0.05 and P < 0.05. In the normal group vs. the model group, 1,928 genes were upregulated and 1,917 genes were downregulated. In the model group vs. the high-dose PAGP group, 528 genes were upregulated and 614 genes were downregulated. Notably, there was gene dispersion between the normal group and the model group, and between the model group and the high-dose PAGP group, indicating significant differences in genes among them.

[0085] To further clarify the biological processes and signaling pathways involved in the development of ILF in mice and PAGP treatment, GO and KEGG analyses were performed on the differentially expressed genes. GO analysis showed that the differentially expressed genes were enriched in biological processes, molecular functions, and cellular components, including binding, cellular anatomical entities, and cellular processes. KEGG results showed that the differentially expressed genes in mouse liver were mainly enriched in the PPAR, p53, TNF, and TGF-beta signaling pathways, with the PPAR signaling pathway showing the strongest correlation with the development of ILF and PAGP treatment in mice.

[0086] Analysis of the expression of common differentially expressed genes revealed that, in the model group versus the normal group, the main upregulated genes were Cyp4a12a, Scd2, Slc27a2, and Cyp7a1, while the main downregulated genes were Pparα, Fapp1, Hmgcs2, Cyp4a14, Cyp4a10, Apoc3, Apoc5, Foxa1, and Hnf4α. However, the upregulation and downregulation of genes in the high-dose PAGP group versus the model group were the opposite of those in the model group versus the normal group, suggesting that PAGP may improve mouse ILF by regulating the expression of related genes in the aforementioned PPAR signaling pathway.

[0087] 2.8 Effects of PAGP on the expression of Pparα, Foxa1, Fabp1, and Hnf4α mRNA and protein in mouse liver tissue To further verify the intervention mechanism of PAGP in ILF mice, based on relevant research reports, we screened the genes Pparα, Fabp1, and the upstream and downstream genes of Fabp1, Foxa1 and Hnf4α, which are highly associated with ILF. We then used qPCR and Western Blot to verify the expression of Pparα, Foxa1, Fabp1, and Hnf4α mRNA and protein in mouse liver tissue. Figures 28-30 As shown, the experimental results indicated that the expression of Pparα, Foxa1, Fapp1, and Hnf4α mRNA and protein was significantly reduced in the model group (P<0.01), while PAGP could restore the expression of the above mRNA and protein. This suggests that related genes in the Ppar signaling pathway (Pparα, Foxa1, Fapp1, Hnf4α) may be involved in the treatment of ILF by PAGP.

[0088] 3. Conclusion This invention successfully isolated and purified PAGP, a complex extract that regulates the polarization of M1 macrophages in the liver, from the American cockroach (Periplaneta americana), and identified and characterized it. Furthermore, this invention preliminarily explored the mechanism of action of PAGP in improving intrahepatic fibrosis (ILF) in mice. Studies showed that PAGP can alleviate Con A-induced liver pathological damage in mice, restore liver function, reduce the degree of macrophage infiltration in the liver, and alleviate ILF. This invention also found that PAGP can regulate the balance of the M1 / M2 ratio of liver macrophages and the Th1 / Th2 cell ratio in the spleen of mice. RNA-seq experiments showed that the PPAR signaling pathway is involved in the occurrence and treatment of ILF in mice, elucidating the molecular mechanism of PAGP in treating ILF.

[0089] This invention provides the first evidence that the pharmaceutical composition PAGP can inhibit the polarization of M1 macrophages in the liver, reduce the expression of inflammatory factors, and simultaneously promote the polarization of M2 macrophages. It also elucidates the mechanism by which PAGP regulates M1 macrophage polarization. This provides a scientific basis for the development of anti-ILF active substances from the American cockroach and offers a new avenue for the development of drugs to treat macrophage polarization-related diseases.

Claims

1. A compound extract of American cockroaches, characterized in that, The American cockroach complex extract includes American cockroach mucoprotein and protocatechuic acid-4-O-glucoside.

2. The method for preparing the American cockroach complex extract according to claim 1, characterized in that, The preparation method of American cockroach mucoprotein includes the following steps: American cockroach medicinal material is pulverized, extracted and defatted, then extracted by heating and reflux with water, filtered, and the filtrate is concentrated to obtain defatted extract; ethanol is added to the defatted extract to an ethanol concentration of 80%~85%, and the mixture is allowed to stand overnight at 10~4℃, centrifuged, and the precipitate is collected; the precipitate is dissolved in water, a chloroform-n-butanol mixed solution is added, stirred, centrifuged, and the precipitate is collected, and this process is repeated several times; the precipitate is then dissolved in water, subjected to gel column chromatography, eluted with pure water, the eluent is collected, concentrated, and freeze-dried to obtain American cockroach mucoprotein.

3. The method for preparing the American cockroach complex extract according to claim 2, characterized in that, In the chloroform-n-butanol mixed solution, the volume ratio of chloroform to n-butanol is 4:

1.

4. The method for preparing the American cockroach complex extract according to claim 2, characterized in that, The volume ratio of the aqueous solution obtained by adding water to the precipitate until it is completely dissolved to the chloroform-n-butanol mixed solution is 4:

1.

5. The method for preparing the American cockroach complex extract according to claim 1, characterized in that, The preparation method of protocatechuic acid-4-O-glucoside includes the following steps: American cockroach medicinal material is pulverized, extracted and defatted, then ultrasonically extracted with 70%~80% ethanol solution, centrifuged, filtered, the filtrate is concentrated to dryness to obtain the extract, 1% acetic acid aqueous solution is added to fully dissolve, filtered, the filtrate is subjected to column chromatography with C18 material, the eluent is collected, concentrated, and dried to obtain protocatechuic acid-4-O-glucoside.

6. The method for preparing the American cockroach complex extract according to claim 5, characterized in that, The mass-to-volume ratio of the extract to 1% acetic acid aqueous solution is 1:20~30 g / mL.

7. The method for preparing the American cockroach complex extract according to claim 5, characterized in that, The elution steps are as follows: elute with 3 to 5 column volumes of 5% methanol or ethanol solution, and discard the eluent; then elute isocratically with 0.1% acetic acid aqueous solution-ethanol solution, with the ratio of 0.1% acetic acid aqueous solution to ethanol solution being 92% to 85%: 8% to 15%, and collect this part of the eluent.

8. The application of the American cockroach complex extract according to claim 1, characterized in that, The American cockroach compound extract is used to prepare drugs or health products that regulate the polarization of liver macrophages.

9. The application of the American cockroach complex extract according to claim 1, characterized in that, The American cockroach compound extract is used to prepare drugs or health products for treating liver fibrosis.

10. The application of the American cockroach complex extract according to claim 9, characterized in that, The American cockroach compound extract is used to prepare drugs or health products for the treatment of immune-mediated liver fibrosis.

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