Preparation and application of medicinal dregs polysaccharide of periplaneta americana for treating ulcerative colitis

Abstract

The present application relates to the field of traditional Chinese medicine extraction, in particular to a preparation and application of a Periplaneta americana residue polysaccharide for treating ulcerative colitis. The polysaccharide is extracted from Periplaneta americana residue by ultrasonic-assisted enzyme method, and the extraction process is optimized by combining single factor experiment and response surface method, thereby improving the extraction yield and reducing the use of organic reagents. Then, the polysaccharide is obtained by decolorization with macroporous resin, alcohol precipitation and Sevage method for removing protein. In a DSS-induced ulcerative colitis mouse model, the polysaccharide can significantly reduce the DAI, CMDI and HS scores of mice, improve the histopathological damage of colon tissue, inhibit the expression of pro-inflammatory factors and regulate the level of anti-inflammatory factors, and has a significant treatment effect. The Periplaneta americana residue polysaccharide product prepared by the present application has high purity and strong biological activity, and the process is simple, environmentally friendly, suitable for industrial large-scale production, and has a significant therapeutic effect on ulcerative colitis.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extraction, specifically to the preparation and application of polysaccharides from the residue of American cockroaches used to treat ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease caused by the interaction of multiple factors, including genetics, immunity, environment, and gut microbiota. Its pathological features include persistent inflammation and ulceration of the colonic mucosa. Clinically, it is characterized by alternating episodes of exacerbation, remission, and relapse. Common symptoms include abdominal pain, diarrhea, bloody and mucous stools, and tenesmus. The incidence of this disease in my country is increasing year by year, and it carries a risk of cancer development. It has been listed as a modern intractable disease by the World Health Organization. According to the "Expert Consensus on the Integrated Traditional Chinese and Western Medicine Diagnosis and Treatment of Ulcerative Colitis," Traditional Chinese Medicine classifies it into eight syndrome types, with the damp-heat type of the large intestine being the most common clinically. Currently, Western medicine treatment mainly relies on aminosalicylic acid preparations, corticosteroids, and immunosuppressants. While these can control inflammation, they have significant side effects and a high risk of relapse upon discontinuation. For example, long-term use of aminosalicylic acid may cause adverse reactions such as headache, diarrhea, and nausea, while corticosteroids and immunosuppressants may cause immunosuppression and increase the risk of infection. In contrast, traditional Chinese medicine (TCM) has the advantages of multi-component, multi-target, and holistic regulation, enabling it to regulate immunity, improve intestinal barrier function, and reduce recurrence through syndrome differentiation and treatment. The main challenges of current treatment strategies include how to effectively repair damaged intestinal barriers, regulate gut microbiota imbalances, and optimize mucosal immune responses, while avoiding systemic side effects.

[0003] The American cockroach, a traditional Chinese medicine, derives its medicinal value from the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), which records its ability to "break up accumulations and relieve sore throat." Modern research reveals that it is rich in amino acids, peptides, polysaccharides, and other active ingredients, possessing multiple pharmacological effects including antibacterial, antiviral, anti-inflammatory, antioxidant, immunomodulatory, and tissue-repair-promoting properties, reflecting a deep integration of traditional medicinal properties and modern science. In the treatment of ulcerative colitis (UC), its ethanol extract, Kangfuxin Liquid, plays a key role by "promoting blood circulation and nourishing yin and tissue regeneration." It not only inhibits pro-inflammatory factors and increases anti-inflammatory factor levels to regulate immune balance but also directly promotes intestinal mucosal repair. Clinical practice shows that the combined use of Kangfuxin Liquid and drugs such as mesalazine can significantly improve symptoms of abdominal pain, diarrhea, and bloody mucus in patients with mild to moderate UC, while reducing the toxic side effects of traditional drugs. This demonstrates the therapeutic advantages of multi-target and holistic regulation, providing a treatment option that combines repair and regulation for UC, a chronic and refractory disease. Of particular note is the polysaccharide component in the American cockroach, which is considered one of its important active substances. Studies have shown that polysaccharides have unique advantages in the treatment of colitis. They can act as prebiotics to regulate the balance of intestinal flora; they also have mucosal protective and immunomodulatory functions, reducing intestinal inflammation and promoting tissue repair. Furthermore, polysaccharide materials have good biocompatibility and biodegradability, making them suitable as carrier materials for drug delivery systems. Currently, the extraction of American cockroach polysaccharides mostly uses hot water extraction, which is inefficient and produces many impurities. Ultrasound-assisted enzymatic methods can efficiently disrupt cell walls and improve polysaccharide yield, but systematic research on optimizing process parameters is lacking.

[0004] The strategy of using American cockroach polysaccharide as a natural polysaccharide to treat ulcerative colitis in this invention has the advantage of utilizing its natural and biocompatible properties to achieve multiple effects such as anti-inflammation, anti-oxidation, intestinal barrier repair, and regulation of intestinal flora. This multi-target mode of action is highly consistent with the traditional Chinese medicine principle of treating UC by clearing heat and dampness, promoting blood circulation and removing blood stasis to treat the symptoms, and strengthening the spleen and replenishing qi to treat the root cause. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an extraction, preparation, and application of polysaccharides from the residue of the American cockroach used to treat ulcerative colitis by optimizing the extraction method and process parameters.

[0006] One object of the present invention is to provide a *Periplaneta americana* polysaccharide for treating ulcerative colitis, characterized in that the polysaccharide is derived from *Periplaneta americana* medicinal residue and is obtained by ultrasound-assisted enzymatic extraction, separation and purification.

[0007] Furthermore, the American cockroach residue polysaccharide is characterized in that the enzymes extracted by the ultrasound-assisted enzymatic method are cellulase, pectinase, and papain, the ultrasound time is 20-60 min, the ultrasound power is 160-380 W, the enzymatic hydrolysis time is 30-70 min, and the enzymatic hydrolysis temperature is 25-65℃; furthermore, the enzyme is a complex enzyme (cellulase: pectinase: papain).

[0008] Furthermore, the extraction process parameters were optimized through single-factor experiments and response surface methodology; preferably, the enzyme activities were 400 U / mg cellulase, 500 U / mg pectinase, and 100 U / mg papain.

[0009] Optionally, the amount of compound enzyme added is 0.5% to 2.5%; more preferably, it is 2%.

[0010] Furthermore, the process parameters are: ultrasonic time 40 min, ultrasonic power 300 W, enzymatic hydrolysis time 60 min, and enzymatic hydrolysis temperature 25°C.

[0011] Furthermore, the polysaccharide from the American cockroach residue is extracted as follows: ① The dried residue of the American cockroach, obtained by a pharmaceutical factory producing Kangfuxin liquid, is extracted with ethanol under high temperature and pressure. Small molecules and most pigments have been removed, therefore a defatting step is unnecessary. It is dried at 60°C, pulverized, sieved through an 80-mesh screen, sealed, and retained. ② The defatted American cockroach powder from step ① is added to a buffer solution (pH=6) and ultrasonically extracted. After ultrasonic treatment, different proportions of a compound enzyme are added, and enzymatic hydrolysis is performed at different temperatures. After hydrolysis, the mixture is rapidly cooled to room temperature to terminate enzyme activity. Then, it is centrifuged at 5000 r / min for 15 min, and the supernatant is collected. The residue is extracted again under the same conditions, and the supernatants are combined to improve the yield. The combined supernatant is concentrated under reduced pressure at 56°C to one-fifth of its original volume. ③ The concentrated solution is decolorized using D101 macroporous resin, and the resulting eluent is mixed with ethanol and stored in a refrigerator at 4°C to precipitate the polysaccharide. The precipitate was then centrifuged, collected, washed, and freeze-dried to obtain crude polysaccharide. ④ The freeze-dried precipitate obtained in step ③ was prepared into a 5% aqueous solution, Sevage reagent (chloroform: n-butanol = 4:1) was added, the mixture was vigorously shaken for 20 min, allowed to stand for 15 min, and then centrifuged for 10 min. The precipitate and organic layer were discarded, and the supernatant was collected. This process was repeated until the protein layer disappeared. Dialysis was performed with distilled water for 36 h (using a 3500 Da dialysis bag), changing the water every 6 h. The dialyzed solution was then freeze-dried in a vacuum freeze dryer to obtain purified American cockroach polysaccharide.

[0012] Furthermore, the American cockroach mentioned is the dried whole insect residue.

[0013] Optionally, in step ②, the solid-liquid ratio of defatted American cockroach powder to buffer solution is 1:(10-25); more preferably, it is 1:25; preferably, in step ③, the eluent is adjusted to 70% alcohol content by adding 4 times the amount of ethanol and allowed to stand at 4°C for 24 h. In this invention, anhydrous ethanol is preferably used.

[0014] Preferably, in step ④, the volume ratio of the 5% aqueous solution to the Sevage reagent is 4:1.

[0015] This invention does not impose any particular limitation on the concentration and centrifugation methods; conventional methods in the field can be used.

[0016] Another object of the present invention is to provide the above-mentioned American cockroach polysaccharide.

[0017] Another object of the present invention is to provide the use of the above-mentioned American cockroach polysaccharide in the preparation of a medicament for treating ulcerative colitis.

[0018] Compared with the prior art, the present invention has the following advantages: The *Periplaneta americana* residue polysaccharide of this invention is prepared by ultrasonic treatment, enzymatic hydrolysis with a complex enzyme, centrifugation to collect the supernatant, followed by vacuum concentration and decolorization with D101 macroporous resin. The eluent is then precipitated with ethanol, centrifuged to collect the precipitate, washed, and dried to obtain crude polysaccharide. The crude polysaccharide is then deproteinized with Sevage reagent, dialyzed, and freeze-dried to obtain purified *Periplaneta americana* polysaccharide. The *Periplaneta americana* residue polysaccharide product prepared by this invention has high purity and strong biological activity. The process is simple, environmentally friendly, and suitable for large-scale industrial production.

[0019] This invention, through research on American cockroach polysaccharides, has discovered that American cockroach polysaccharides have a significant therapeutic effect on ulcerative colitis, and has been experimentally proven to be applicable to the preparation of colon-targeted formulations. Attached Figure Description

[0020] Figure 1 The effect of American cockroach polysaccharide on colon tissue of UC mice (HE, ×20).

[0021] Figure 2 This is a representative photograph of the colon of UC mice affected by American cockroach polysaccharide.

[0022] Figure 3 This is a representative photograph of the spleen of UC mice affected by American cockroach polysaccharide. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments. It should be emphasized that the following description is merely exemplary and is not intended to limit the scope and application of the present invention.

[0024] In this invention, the American cockroach medicinal material used is the dried residue of the whole cockroach. This invention does not specify the source of the American cockroach medicinal residue, but can use the residue that has been preliminarily extracted by pharmaceutical companies or commercially available residue.

[0025] In a specific embodiment of the present invention, the American cockroach polysaccharide is referred to as PAPX.

[0026] Example 1

[0027] 10 g of American cockroach residue powder was added to 250 ml of buffer to adjust the pH to 6. After ultrasonic treatment at 300 W (20 min, 30 min, 40 min, 50 min, 60 min), 2% of a compound enzyme was added and enzymatically hydrolyzed at 25℃ for 60 min. After hydrolysis, the mixture was rapidly cooled to room temperature to terminate enzyme activity. The mixture was then centrifuged for 15 min, and the supernatant was collected. The residue was extracted once more under the same conditions, and the supernatants were combined to improve the yield. The combined supernatant was concentrated to one-fifth of its original volume under reduced pressure at 56°C. The concentrate was decolorized using D101 macroporous resin, and the eluent was mixed with 4 times its volume of ethanol to precipitate the polysaccharide. The precipitate was then centrifuged, washed, and dried to obtain crude polysaccharide. The obtained lyophilized precipitate was prepared into a 5% aqueous solution, and one-quarter volume of Sevage reagent (chloroform:n-butanol = 4:1) was added. The mixture was vigorously shaken for 20 min, allowed to stand for 15 min, and then centrifuged for 10 min. The precipitate and organic layer were discarded, and the supernatant was collected. This process was repeated until the protein layer disappeared. The solution was dialyzed with distilled water for 36 h (using a 3500 Da dialysis bag), with the water changed every 6 h. The dialyzed solution was then freeze-dried in a vacuum freeze dryer to obtain purified American cockroach polysaccharide.

[0028] Table 1. Yield and activity evaluation of American cockroach polysaccharides under different ultrasonic times

[0029] According to the results in Table 1, under the same conditions, the polysaccharide yield gradually increased with the increase of ultrasound time, and the anti-inflammatory activity of CCD841 cells was enhanced. When the ultrasound time reached 40 min, the polysaccharide yield reached the maximum. However, when the ultrasound time reached 50 min, the polysaccharide yield decreased and the enhancement of anti-inflammatory activity was not obvious. Considering the cost control of large-scale industrialization, the ultrasound time of 40 min was selected as the optimal time for the next step of the extraction process.

[0030] Example 2 10 g of American cockroach residue powder was added to 250 ml of buffer to adjust the pH to 6. After sonication at various power levels (160 W, 200 W, 260 W, 300 W, 380 W) for 40 min, 2% of a compound enzyme was added and the mixture was enzymatically hydrolyzed at 25°C for 60 min. Following hydrolysis, the mixture was rapidly cooled to room temperature to terminate enzyme activity. The mixture was then centrifuged for 15 min, and the supernatant was collected. The residue was extracted once more under the same conditions, and the supernatants were combined to improve the yield. The combined supernatant was concentrated under reduced pressure at 56°C to one-fifth of its original volume. The concentrate was mixed with four times its volume of ethanol to precipitate the polysaccharide. The precipitate was then centrifuged, washed, and dried to obtain crude polysaccharide. The obtained lyophilized precipitate was prepared into a 5% aqueous solution, and one-quarter volume of Sevage reagent (chloroform:n-butanol = 4:1) was added. The mixture was vigorously shaken for 20 min, allowed to stand for 15 min, and then centrifuged for 10 min. The precipitate and organic layer were discarded, and the supernatant was collected. This process was repeated until the protein layer disappeared. The solution was dialyzed with distilled water for 36 h (using a 3500 Da dialysis bag), with the water changed every 6 h. The dialyzed solution was then freeze-dried in a vacuum freeze dryer to obtain purified American cockroach polysaccharide.

[0031] Table 2. Yield and activity evaluation of American cockroach polysaccharides under different ultrasonic powers

[0032] According to the results in Table 2, under the same conditions, the polysaccharide yield gradually increased with the increase of ultrasonic power, and the anti-inflammatory activity of CCD841 cells was enhanced. The polysaccharide yield was the highest when the ultrasonic power reached 300W, but the polysaccharide yield decreased when the ultrasonic power reached 380W, and the enhancement of anti-inflammatory activity was not obvious. Considering the cost control of large-scale industrialization, the ultrasonic power of 300W is preferred for the next step of the extraction process optimization.

[0033] Example 3

[0034] 10 g of American cockroach residue powder was added to 250 ml of buffer to adjust the pH to 6. After sonication at 300 W for 40 min, a complex enzyme (0.5%, 1%, 1.5%, 2%, 2.5%) was added and enzymatically hydrolyzed at 25°C for 60 min. After hydrolysis, the mixture was rapidly cooled to room temperature to terminate enzyme activity. The mixture was then centrifuged for 15 min, and the supernatant was collected. The residue was extracted again under the same conditions, and the supernatants were combined to improve the yield. The combined supernatant was concentrated to one-fifth of its original volume under reduced pressure at 56°C. The concentrate was mixed with four volumes of ethanol to precipitate the polysaccharide. The precipitate was then centrifuged, washed, and dried to obtain crude polysaccharide. The lyophilized precipitate was prepared into a 5% aqueous solution, and one-quarter volume of Sevage reagent (chloroform: n-butanol = 4:1) was added. The mixture was shaken vigorously for 20 min, allowed to stand for 15 min, and then centrifuged for 10 min. The precipitate and organic layer were discarded, and the supernatant was collected. This process was repeated until the protein layer disappeared. Dialyze the solution with distilled water for 36 hours (using a 3500 Da dialysis bag), changing the bag every 6 hours. Freeze-dry the dialyzed solution in a vacuum freeze dryer to obtain purified American cockroach polysaccharide.

[0035] Table 3. Yield and activity evaluation of American cockroach polysaccharides under different amounts of compound enzymes.

[0036] According to the results in Table 3, under the same conditions, the polysaccharide yield gradually increased with the increase of the amount of compound enzyme added, and the anti-inflammatory activity of CCD841 cells was enhanced. The polysaccharide yield was the highest when the amount of compound enzyme added reached 2%, but the polysaccharide yield decreased when the amount of compound enzyme added reached 2.5%, and the anti-inflammatory activity also decreased. Considering the cost control of large-scale industrialization, the optimal amount of compound enzyme added is 2% for the next step of the extraction process optimization.

[0037] Example 4

[0038] 10 g of American cockroach residue powder was added to 250 ml of buffer to adjust the pH to 6. After sonication at 300 W for 40 min, 2% of a compound enzyme was added, and enzymatic hydrolysis was performed at 25°C for 30 min, 40 min, 50 min, 60 min, and 70 min. After hydrolysis, the mixture was rapidly cooled to room temperature to terminate enzyme activity. The mixture was then centrifuged for 15 min, and the supernatant was collected. The residue was extracted again under the same conditions, and the supernatants were combined to improve the yield. The combined supernatant was concentrated to one-fifth of its original volume under reduced pressure at 56°C. The concentrate was mixed with four volumes of ethanol to precipitate the polysaccharide. The precipitate was then centrifuged, washed, and dried to obtain crude polysaccharide. The lyophilized precipitate was prepared into a 5% aqueous solution, and one-quarter volume of Sevage reagent (chloroform: n-butanol = 4:1) was added. The mixture was shaken vigorously for 20 min, allowed to stand for 15 min, and then centrifuged for 10 min. The precipitate and organic layer were discarded, and the supernatant was collected. This process was repeated until the protein layer disappeared. Dialyze the solution with distilled water for 36 hours (using a 3500 Da dialysis bag), changing the bag every 6 hours. Freeze-dry the dialyzed solution in a vacuum freeze dryer to obtain purified American cockroach polysaccharide.

[0039] Table 4. Yield and activity evaluation of American cockroach polysaccharides at different enzymatic hydrolysis times

[0040] According to the results in Table 4, under the same conditions, the polysaccharide yield gradually increased with the increase of enzymatic hydrolysis time, and the anti-inflammatory activity of CCD841 cells was enhanced. The polysaccharide yield was the highest when the enzymatic hydrolysis time was 60 min, but the polysaccharide yield and anti-inflammatory activity decreased when the enzymatic hydrolysis time reached 70 min. Considering the cost control of large-scale industrialization, the optimal enzymatic hydrolysis time is 60 min for the next step of extraction process optimization.

[0041] Example 5

[0042] 10 g of American cockroach residue powder was added to 250 ml of buffer to adjust the pH to 6. After sonication at 300 W for 40 min, 2% of a compound enzyme was added, and the mixture was enzymatically hydrolyzed at (25℃, 35℃, 45℃, 55℃, 65℃) for 60 min. After hydrolysis, the mixture was rapidly cooled to room temperature to terminate enzyme activity. The mixture was then centrifuged for 15 min, and the supernatant was collected. The residue was extracted again under the same conditions, and the supernatants were combined to improve the yield. The combined supernatant was concentrated to one-fifth of its original volume under reduced pressure at 56°C. The concentrate was mixed with four volumes of ethanol to precipitate the polysaccharide. The precipitate was then centrifuged, washed, and dried to obtain crude polysaccharide. The lyophilized precipitate was prepared into a 5% aqueous solution, and one-quarter volume of Sevage reagent (chloroform: n-butanol = 4:1) was added. The mixture was shaken vigorously for 20 min, allowed to stand for 15 min, and then centrifuged for 10 min. The precipitate and organic layer were discarded, and the supernatant was collected. This process was repeated until the protein layer disappeared. Dialyze the solution with distilled water for 36 hours (using a 3500 Da dialysis bag), changing the bag every 6 hours. Freeze-dry the dialyzed solution in a vacuum freeze dryer to obtain purified American cockroach polysaccharide.

[0043] Table 5. Yield and activity evaluation of American cockroach polysaccharides at different enzymatic hydrolysis temperatures.

[0044] According to the results in Table 5, under the same conditions, the polysaccharide yield gradually decreased with the increase of enzymatic hydrolysis temperature, and the anti-inflammatory activity of CCD841 cells gradually weakened. The polysaccharide yield was highest when the enzymatic hydrolysis temperature was 25℃. Therefore, the optimal enzymatic hydrolysis temperature is 25℃ for further optimization of the extraction process.

[0045] Example 6

[0046] The therapeutic effect of American cockroach residue polysaccharide on a mouse model of DSS-induced ulcerative colitis. 1. Experimental Materials 1.1 Laboratory Animals Thirty-six male BALB / c mice, 6 weeks old, 18-20 g, were obtained from SLAC Laboratory Animal Co., Ltd., Shanghai, China. License No.: [License Number Missing], Batch No.: [Batch Number Missing] 1.2 Test Drug This invention relates to American cockroach polysaccharides; Mesalazine (5-aminosalicylic acid, 5-ASA), (Batch No.: LOT2512, Zhejiang Sanmen Hengkang Pharmaceutical Co., Ltd., Specification: 50 g).

[0047] 1.3 Experimental Reagents Dextran sulfate sodium (DSS), (batch number: J0715F, Dalian Meilun Biotechnology Co., Ltd.); Anhydrous ethanol, AR (batch number: 104021-5L, Xilong Scientific); Phosphate buffer solution with pH=6 (batch number: CD440333, Shanghai Shangbao Biotechnology Co., Ltd.); Cellulase, 400 U / mg (batch number: 20251212, Xiasheng Industrial Group Co., Ltd.); Pectinase, 500 U / mg (batch number: 20251209, Xiasheng Industrial Group Co., Ltd.); Papain, 100 U / mg (batch number: 20251219, Xiasheng Industrial Group Co., Ltd.); Chloroform, AR (batch number: 20251225, Chongqing Chuandong Chemical (Group) Co., Ltd.); n-Butanol, AR (batch number: 20251216, Sinopharm Chemical Reagent Co., Ltd.); Physiological saline, NS (batch number: A15031602, Guizhou Tiandi Pharmaceutical Co., Ltd.); Occult blood test kit (batch number: 20251225, Nanjing Jiancheng Biotechnology Institute); H&E kit; mouse colon IL-17A kit; mouse colon TNF-α kit; mouse colon IL-10 kit; mouse colon GM-CSF kit; mouse colon TGF-β kit; mouse colon MPO kit; all were purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.

[0048] 1.4 Main Instruments Electronic balance (Mettler-Toledo Instruments Ltd.), model: AL204-IC; Precision electronic analytical balance (Mettler-Toledo Instruments Ltd.), Model: METTLERAE240; Rotary evaporator (Shanghai Yarong Biochemical Instrument Factory), Model: RE-52AA; High-speed centrifuge (Anhui Zhongke Zhongjia Scientific Instruments Co., Ltd.), Model: HC-30118R; Freeze dryer (Cold-Sim, USA), model: FD8-4a; Mouse metabolic cage (Suzhou Feng's Laboratory Animal Equipment Co., Ltd.), Model: DXL-D; Pathological tissue drying apparatus (Changzhou Zhongwei Electronic Instruments Co., Ltd.), Model: PHY-Ⅲ; Paraffin slicer (Leica), model: RM2245; The macro camera, model: DIGITAI CAMERA D7100, and the macro lens, model: AF-S Micro 60 / 2 .8GED, were both purchased from Nikon.

[0049] Ultrasonic equipment (Anhui Zhongke Zhongjia Scientific Instruments Co., Ltd.), model: HC-30118R; 2. Experimental Methods 2.1 Establishment of experimental animal models Male BALB / c mice were selected and acclimatized for one week, then randomly divided into 6 groups (n=6 per group): normal control group, model group, mesalazine group, low-dose American cockroach polysaccharide group, medium-dose American cockroach polysaccharide group, and high-dose American cockroach polysaccharide group. Mice in the normal control group were given normal saline for drinking. The experimental animals in the model group, mesalazine group, low-dose American cockroach polysaccharide group, medium-dose American cockroach polysaccharide group, and high-dose American cockroach polysaccharide group were given free access to DSS aqueous solution (2.5%, w / v) for 10 consecutive days to establish an experimental acute colitis model. The Disease Activity Index (DAI) was scored daily on the model mice from day 0 to day 14.

[0050] 2.2 Grouping and administration of experimental animals On day 8 of the experiment, animals were grouped according to their Disease Activity Index (DAI) scores. After grouping, the normal group, model group, mesalazine group, low-dose American cockroach polysaccharide group, medium-dose American cockroach polysaccharide group, and high-dose American cockroach polysaccharide group were administered 0.4 mL of normal saline (NS, 0.4 mL / animal), normal saline (NS, 0.4 mL / animal), mesalazine (200 mg / kg), American cockroach polysaccharide (50 mg / kg), American cockroach polysaccharide (100 mg / kg), and American cockroach polysaccharide (200 mg / kg) by gavage daily.

[0051] 2.3 Indicator Observation and Detection Methods 2.3.1 Disease Activity Index The disease activity index (DAI) of each group of experimental individuals was recorded daily. The DAI assessment included weight loss (0-4 points), fecal characteristics (0-4 points), and fecal bleeding (0-4 points). The DAI score of each mouse was calculated to assess the disease activity.

[0052] 2.3.2 Organ Index At the end of the experiment, mice were euthanized by cervical dislocation. The colon, spleen, and thymus were dissected and collected. After rinsing with physiological saline, the surface moisture was blotted dry with filter paper. The organs were weighed, and the organ index was calculated: Organ Index (mg / g) = Organ wet mass (mg) / Body mass (g).

[0053] 2.3.3 Colonic mucosal injury index After dissecting the mice, the entire colon, from about 1 cm from the anus to the end of the cecum, was removed. The colon was cut along the mesentery, and the contents of the intestinal lumen were washed with ice-cold saline. The colon was laid flat on a white porcelain plate, and the length and width of the colon were measured with a steel ruler. The colonic mucosal damage index (CMDI) was scored according to the literature standards. Then, the surface moisture of the colon was dried with filter paper, weighed, and the colonic index was calculated.

[0054] 2.3.4 Histopathological scoring Tissue samples were fixed in 4% paraformaldehyde solution, embedded in paraffin, and cut into 4 μm sections for hematoxylin and eosin (H&E) staining. The stained sections were observed under a fluorescence microscope to assess the extent of damage within the colon. Histopathological analysis included assessing the severity of inflammation, depth of tissue damage, extent of crypt damage, and percentage of affected tissue. Colonic histopathological score (HS score) was calculated according to literature standards.

[0055] 2.3.5 Colonic tissue cytokine assay The other half of the longitudinally cut colon tissue was stored at -80°C for later use. During the experiment, the stored colon tissue was removed and subjected to freeze-thaw cycles at -20°C and 4°C, respectively. A 10% (w / v) tissue homogenate was prepared using pre-cooled physiological saline. The homogenate was centrifuged at 3000 rpm for 10 min at 4°C, and the supernatant was collected. The levels of IL-17A, TNF-α, IL-10, GM-CSF, TGF-β, and MPO in the colon tissue were measured strictly according to the relevant kit instructions.

[0056] 3 Results 3.1 Effects of American cockroach polysaccharide on the general condition of UC mice Mice in the normal group had normal diet and water intake, smooth and clean fur, good mental state, black, hard, oval-shaped feces, and pale yellow urine of normal color. Mice in the model group showed some improvement in mental state over time, with a slight increase in food intake, but loose stools remained attached to their tails, or blood was present in their feces. Mice in the mesalazine group showed significant improvement in mental state, increased activity, and no yellow, sticky, loose, or bloody stools were observed; their feces were basically formed and turned black; and their weight gradually increased. Mice in the low-dose American cockroach polysaccharide group showed improved mental state, reduced huddling and curling up, less anal soiling, and their feces were basically formed and turned black; their weight gradually recovered. Mice in the medium-dose American cockroach polysaccharide group showed improved mental state, increased activity, no feces adhering to the anus, formed feces that turned black, and their weight gradually increased. Mice in the high-dose American cockroach polysaccharide group showed good mental state, increased activity, no feces adhering to the anus, formed feces that turned black, and their weight gradually increased.

[0057] 3.2 Effects of American cockroach polysaccharide on DAI score in UC mice (see Tables 6 and 7) Repeated measures ANOVA was performed on the DAI scores of mice in each group from day 0 to day 14. The Mauchly test of sphericity showed that the data did not meet the football-shaped hypothesis (P=0.011<0.05), so the results after Greenhouse-Geisser correction were used. The results of repeated measures ANOVA showed (Table 7): (1) The main effect of grouping was significant: the inter-group effect of mouse DAI scores was statistically significant (F=9.683, P<0.01), indicating that there was an overall difference in DAI scores between different groups and that the DAI scores of mice in each group were different. (2) The main effect of time was significant: the time effect of mouse DAI scores was statistically significant (F=27.148, P<0.01), indicating that DAI scores changed over time and that there were differences in mouse DAI scores at different time points. (3) The interaction between time and grouping was significant: the interaction between mouse DAI scores was statistically significant (F=3.093, P<0.01), indicating that the trend of DAI scores in different groups changed over time differently. To evaluate the differences between groups at the same time point, one-way ANOVA showed that on day 0, the DAI scores of all groups were low and there was no significant difference (P > 0.05), indicating that all mice were normal and at the same level. On day 7, the UC model was established. The DAI score of the normal group was low, and the DAI scores of all model mice were significantly higher than those of the normal group (P < 0.05 or P < 0.01), with no significant difference between the model groups, indicating that the UC model was successfully replicated. Compared with the model group, all drug-treated groups showed a significant decrease from day 12 (P < 0.05 or P < 0.01). Among them, the DAI scores of the positive control group and the low-dose and medium-dose groups of American cockroach polysaccharide had dropped to near the level of the normal group by day 14 (no significant difference from the normal group). The high-dose group of American cockroach polysaccharide also showed a significant improvement, but it was still higher than the normal group on day 14 (P < 0.05). Overall, all treatment groups effectively reduced the DAI score of UC mice, and the reduction was dose-dependent.

[0058] Table 6. Effects of American cockroach polysaccharide on DAI score in UC mice (n=6)

[0059] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, # P < 0.05 ## P < 0.01.

[0060] Table 7. Analysis of variance plot showing the effect of American cockroach polysaccharide on the DAI score of UC mice. Grouping 323.244 5 64.649 9.683 ＜0.01 time 332.156 8.177 40.622 27.148 ＜0.01 Time * Grouping 189.200 40.884 4.628 3.093 ＜0.01 Note:¹ Degrees of freedom after Greenhouse-Geisser correction (Mauchly sphericity test P=0.011<0.05, Greenhouse-Geisser correction coefficient=0.584).

[0061] 3.3 Effects of American cockroach polysaccharide on colon-related parameters in UC mice (see Table 8) Macroscopic observation of the colon tissue of mice in each group revealed the following: In the normal group, the colonic mucosa of mice showed no congestion, edema, erosion, or ulceration; in the model group, the intestinal wall showed severe congestion and edema, with scattered ulcers and erosions on the intestinal mucosa; the colonic mucosal lesions of mice in each drug treatment group were improved. Among them, the positive control group showed mild congestion and edema, without erosion or ulceration; the low-dose group of American cockroach polysaccharide showed congestion and edema, thickening of the intestinal wall, and formation of inflammatory polyps; the medium-dose group of American cockroach polysaccharide showed congestion and edema, with a rough, granular mucosa; and the high-dose group of American cockroach polysaccharide showed mild congestion and edema, with a smooth surface and no erosion or ulceration. Compared with the normal group, the model group mice showed significantly shorter colon length (P<0.01), significantly increased colonic index (P<0.01), significantly increased colon width (P<0.01), and significantly increased CMDI score (P<0.01), indicating that the model group had colonic tissue edema, inflammatory damage, and mucosal ulceration. Compared with the model group: the positive control group showed significantly decreased colon width and CMDI score (P<0.01), but no significant improvement in colon length and colonic index; the low-dose group showed significantly decreased colonic index (P<0.01), colon width, and CMDI score (P<0.01); the medium-dose group showed significantly decreased CMDI score (P<0.05), colon width, and colonic index (P<0.01); and the high-dose group showed significantly increased colon length (P<0.05), significantly decreased CMDI score (P<0.05), and significantly decreased colon width and colonic index (P<0.01).

[0062] Table 8. Effects of American cockroach polysaccharide on colon-related parameters in UC mice (n=6) normal group - 8.24±1.26 10.52±1.26 0.34±0.01 0.67±0.52 Model group - <![CDATA[10.09±0.57 ** ]]> <![CDATA[8.96±0.62 ** ]]> <![CDATA[0.46±0.02 ** ]]> <![CDATA[2.33±0.52 ** ]]> Mesalazine group 200 <![CDATA[8.74±0.77 ## ]]> 9.73±0.46 <![CDATA[0.35±0.02 ## ]]> <![CDATA[1.33±0.52 *## ]]> American cockroach polysaccharide low-dose group 50 <![CDATA[8.79±0.52 ## ]]> 9.73±0.51 <![CDATA[0.39±0.02 **## ]]> <![CDATA[1.50±0.55 *# ]]> American cockroach polysaccharide medium dose group 100 <![CDATA[8.74±0.87 ## ]]> <![CDATA[9.60±0.58 * ]]> <![CDATA[0.39±0.02 **## ]]> <![CDATA[1.50±0.55 *# ]]> High-dose group of American cockroach polysaccharide 200 <![CDATA[8.65±0.40 ## ]]> <![CDATA[9.83±0.71 # ]]> <![CDATA[0.38±0.02 **## ]]> <![CDATA[1.50±0.55 *# ]]> Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, # P < 0.05 ## P < 0.01.

[0063] 3.4 Effects of American cockroach polysaccharide on colonic HS score in UC mice (see Table 9) Microscopic observation of colonic pathological sections revealed that the normal group had an intact and clear colonic structure, with neatly arranged glands and virtually no congestion, edema, or inflammatory cell infiltration. The model group showed significant damage to the colonic mucosa, disordered crypt structure, and extensive inflammatory cell infiltration. The mesalazine group and various doses of American cockroach polysaccharide showed varying degrees of reduction in colonic mucosal damage, partial repair of goblet cells and crypt structures, and decreased inflammatory cell infiltration. Compared with the normal group, the model group showed significantly increased epithelial cell scores, inflammatory cell infiltration scores, and total HS scores (P<0.01). Compared with the model group, the mesalazine group and various doses of American cockroach polysaccharide showed significantly decreased epithelial cell scores, inflammatory cell infiltration scores, and total HS scores (P<0.01 or P<0.05). The mesalazine group showed a more significant decrease in inflammatory cell infiltration scores and total HS scores (P<0.01); most other comparisons showed P<0.05 or P<0.01, as detailed in Table 9. Furthermore, the scores of the medium and high dose groups of American cockroach polysaccharide were similar across all categories.

[0064] Table 9. Effects of American cockroach polysaccharide on colonic HS score in UC mice (n=6)

[0065] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, # P < 0.05 ## P < 0.01.

[0066] 3.5 Effects of American cockroach polysaccharide on pro-inflammatory cytokines in colon tissue of UC mice (see Table 10) Compared with the normal group, the levels of four pro-inflammatory cytokines (TNF-α, IL-17A, MPO, and GM-CSF) in the colon tissue of the model group mice were significantly increased (P<0.01). After intervention with American cockroach polysaccharide, the levels of each pro-inflammatory cytokine decreased in a dose-dependent manner. The high-dose group of American cockroach polysaccharide showed the most significant effect, restoring the four pro-inflammatory cytokines to levels not statistically different from those in the normal group (P>0.05), an effect comparable to the positive control drug mesalazine. The results indicate that American cockroach polysaccharide, especially at high doses, can effectively inhibit the colonic inflammatory response in mice with ulcerative colitis.

[0067] Table 10 Effects of American cockroach polysaccharide on pro-inflammatory cytokines in colon tissue of UC mice (n=6) normal group - 48.13±4.52 15.80±7.66 556.00±30.99 47.21±4.89 Model group - <![CDATA[210.36±33.29 ** ]]> <![CDATA[68.09±18.38 ** ]]> <![CDATA[1321.54±195.77 ** ]]> <![CDATA[137.71±21.39 ** ]]> Mesalazine group 200 <![CDATA[61.93±7.26 ## ]]> <![CDATA[22.49±1.89 ## ]]> <![CDATA[550.95±47.81 ## ]]> <![CDATA[48.23±4.66 ## ]]> American cockroach polysaccharide low-dose group 50 <![CDATA[147.61±24.78 **## ]]> <![CDATA[49.62±9.65 **## ]]> <![CDATA[858.54±62.31 **## ]]> <![CDATA[73.49±7.46 **## ]]> American cockroach polysaccharide medium dose group 100 <![CDATA[124.15±14.94 **## ]]> <![CDATA[43.65±2.93 **## ]]> <![CDATA[798.35±52.97 **## ]]> <![CDATA[58.83±8.93 ## ]]> High-dose group of American cockroach polysaccharide 200 <![CDATA[60.64±7.21 ## ]]> <![CDATA[25.60±4.42 ## ]]> <![CDATA[533.80±68.11 ## ]]> <![CDATA[42.03±3.32 ## ]]> Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, # P < 0.05 ## P < 0.01.

[0068] 3.6 Effects of American cockroach polysaccharide on anti-inflammatory cytokines in colon tissue of UC mice (see Table 11) Compared with the normal group, the model group mice showed a significant decrease in both anti-inflammatory factor indices (P<0.01), confirming the successful establishment of the UC model. After drug intervention, all treatment groups showed significant therapeutic effects, but the recovery patterns of the two indices differed slightly from those in the model group. For IL-10, both mesalazine and American cockroach polysaccharide significantly inhibited its abnormal decline (P<0.05), with the medium-dose group showing the largest recovery (P<0.01), and the values ​​in each treatment group did not show statistical differences compared with the normal group (P>0.05). For TGF-β, all treatment groups significantly increased its level (P<0.01), with the medium-dose group showing the best efficacy, slightly better than the positive control group. The results indicate that American cockroach polysaccharide, especially at a medium dose, can effectively regulate the pathological process of UC mice, demonstrating good application potential.

[0069] Table 11 Effects of American cockroach polysaccharide on anti-inflammatory cytokines in colon tissue of UC mice (n=6) normal group - 59.96±7.09 1746.96±137.57 Model group - <![CDATA[33.13±6.11 ** ]]> <![CDATA[1070.68±156.60 ** ]]> Mesalazine group 200 <![CDATA[51.42±4.94 # ]]> <![CDATA[1498.93±115.69 ## ]]> American cockroach polysaccharide low-dose group 50 <![CDATA[50.87±5.83 # ]]> <![CDATA[1691.46±158.48 ## ]]> American cockroach polysaccharide medium dose group 100 <![CDATA[54.31±3.68 ## ]]> <![CDATA[1789.42±150.55 ## ]]> High-dose group of American cockroach polysaccharide 200 <![CDATA[51.33±7.37 # ]]> <![CDATA[1453.38±91.39 ## ]]> Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, # P < 0.05 ## P < 0.01.

Claims

1. A polysaccharide derived from the residue of the American cockroach (Periplaneta americana) for treating ulcerative colitis, characterized in that, The polysaccharide was obtained from the residue of American cockroaches through ultrasonic-assisted enzymatic extraction, separation, and purification.

2. The American cockroach residue polysaccharide according to claim 1, characterized in that, The enzyme extracted by the ultrasound-assisted enzymatic method is a complex enzyme. The ultrasound time is 20-60 min, the ultrasound power is 160-380 W, the enzymatic hydrolysis time is 30-70 min, and the enzymatic hydrolysis temperature is 25-65℃. Further, the complex enzyme is (cellulase: pectinase: papain).

3. The American cockroach residue polysaccharide according to claim 1, characterized in that, The extraction process was optimized through single-factor experiments and response surface methodology. Preferably, the enzyme activities are 400 U / mg cellulase, 500 U / mg pectinase, and 100 U / mg papain. Optionally, the amount of the compound enzyme added is 0.5%~2.5%; more preferably, it is 2%. Preferably, the process parameters are: ultrasonic time 40 min, ultrasonic power 300 W, enzymatic hydrolysis time 60 min, and enzymatic hydrolysis temperature 25°C. More preferably, the polysaccharide from the American cockroach residue is extracted as follows: ① The dried residue of the American cockroach (Periplaneta americana) is obtained by high-temperature and high-pressure extraction with ethanol by a pharmaceutical factory producing Kangfuxin liquid. Small molecules and most pigments have been removed, therefore a defatting step is unnecessary. It is dried at 60°C, pulverized, sieved through an 80-mesh sieve, sealed, and stored. ② The defatted American cockroach powder from step ① was extracted with a buffer solution (pH=6) and sonicated. After sonication, a proportionate amount of compound enzyme was added, and the mixture was enzymatically hydrolyzed at different temperatures. After hydrolysis, the mixture was rapidly cooled to room temperature to terminate enzyme activity. Then, it was centrifuged at 5000 r / min for 15 min, and the supernatant was collected. The residue was extracted again under the same conditions, and the supernatants were combined to improve the yield. The combined supernatant was concentrated under reduced pressure at 56°C to one-fifth of its original volume. ③ The concentrate was decolorized with D101 macroporous resin, and the eluent was mixed with ethanol and stored in a refrigerator at 4°C to precipitate the polysaccharide. The precipitate was then centrifuged, collected, washed, and freeze-dried to obtain crude polysaccharide. ④ The freeze-dried precipitate obtained in step ③ was prepared into a 5% aqueous solution, and Sevage reagent (chloroform: n-butanol = 4:1) was added. The mixture was shaken vigorously for 20 min, allowed to stand for 15 min, and then centrifuged for 10 min. The precipitate and organic layer were discarded, and the supernatant was collected. The operation was repeated until the protein layer disappeared. Dialyze the solution with distilled water for 36 hours (using a 3500 Da dialysis bag), changing the bag every 6 hours. Freeze-dry the dialyzed solution in a vacuum freeze dryer to obtain purified American cockroach polysaccharide.

4. The American cockroach polysaccharide according to claim 3, characterized in that, The American cockroaches are dried whole insect residues; optionally, in step ②, the solid-liquid ratio of defatted American cockroach powder to buffer solution is 1:(10-25); more preferably, it is 1:25; preferably, in step ③, the eluent is adjusted to 80% alcohol content with 4 times the volume of anhydrous ethanol and allowed to stand at 4°C for 24 hours. Preferably, in step ④, the volume ratio of 5% aqueous solution to Sevage reagent is 4:

1.

5. A polysaccharide from the American cockroach species, characterized in that, Extraction was performed as follows: ① The dried residue of American cockroaches, obtained by a pharmaceutical factory producing Kangfuxin liquid, was extracted with ethanol under high temperature and pressure. Small molecules and most pigments had been removed, thus eliminating the need for defatting. It was dried at 60°C, pulverized, sieved through an 80-mesh sieve, sealed, and stored. ② The defatted American cockroach powder from step ① was added to a buffer solution (pH=6) and ultrasonically extracted. After ultrasonic treatment, a proportionate amount of compound enzyme was added, and enzymatic hydrolysis was performed at different temperatures. After hydrolysis, the mixture was rapidly cooled to room temperature to terminate enzyme activity. Then, it was centrifuged at 5000 r / min for 15 min, and the supernatant was collected. The residue was extracted again under the same conditions, and the supernatants were combined to improve the yield. The combined supernatant was concentrated under reduced pressure at 56°C to one-fifth of its original volume. ③ The concentrated solution was decolorized using D101 macroporous resin, and the resulting eluent was mixed with ethanol and stored in a refrigerator at 4°C to precipitate the polysaccharides. The precipitate was then centrifuged, collected, washed, and freeze-dried to obtain crude polysaccharide. ④ The freeze-dried precipitate obtained in step ③ was prepared into a 5% aqueous solution, Sevage reagent (chloroform:n-butanol = 4:1) was added, the mixture was vigorously shaken for 20 min, allowed to stand for 15 min, and then centrifuged for 10 min. The precipitate and organic layer were discarded, and the supernatant was collected. This process was repeated until the protein layer disappeared. Dialysis was performed with distilled water for 36 h (using a 3500 Da dialysis bag), changing the solution every 6 h. The dialyzed solution was then freeze-dried in a vacuum freeze dryer to obtain purified American cockroach polysaccharide.

6. The use of the compound traditional Chinese medicine according to any one of claims 1-4 or the American cockroach residue polysaccharide according to claim 5 in the treatment of ulcerative colitis.

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