Application of fructus cannabis protein hydrolysate in prevention and treatment of ulcerative colitis

Abstract

The invention relates to a fructus cannabis protein hydrolysate and application thereof in preventing and treating ulcerative colitis and intestinal barrier dysfunction.

Description

Technical Field

[0001] This invention relates to the field of biology, and more particularly to a hemp seed protein hydrolysate and its use in the prevention and treatment of colitis. Background Technology

[0002] Hemp seeds are the dried, mature seeds of the cannabis plant (Cannabis sativa L.), a member of the Moraceae family. They are both medicinal and edible, cultivated throughout my country, and also found in semi-wild areas, widely distributed in Northeast, North, East, and Central South my country. Hemp seeds are neutral in nature and sweet in taste, possessing laxative properties. They are also rich in protein, vitamins, lecithin, volatile oils, and trace elements such as calcium and magnesium. While hemp seeds are a traditional Chinese medicine and food ingredient with various physiological functions, research on them is not in-depth, and their potential applications in medicine, health products, and food have not yet been fully explored.

[0003] In recent years, naturally derived bioactive peptides have become a research hotspot in the development of pharmaceuticals and functional foods due to their advantages such as mild effects, well-defined functions, high safety, and few side effects. The preparation of hemp seed bioactive peptides from hemp seeds to develop bioactive peptide products with medicinal, health, and nutritional applications has broad application prospects.

[0004] Inflammatory bowel disease (IBD), a chronic, relapsing inflammatory condition of the intestines, not only harms patients' physical health but also has long-term effects on their daily life patterns and mental health, gradually becoming a focus of public attention. Its main form, ulcerative colitis (UC), has an unclear etiology. This disease is characterized by recurrent flare-ups and alternating remissions, severely impacting patients' quality of life, and current research continues to search for more effective treatments. Summary of the Invention

[0005] Therefore, the purpose of this application is to provide a protease hydrolysate derived from hemp seeds and its therapeutic uses.

[0006] Specifically, this application provides the use of hemp seed protein hydrolysate in the preparation of anti-inflammatory drugs.

[0007] Furthermore, the use of hemp seed protein hydrolysate in the preparation of medicines for the prevention, treatment or improvement of intestinal diseases is also provided.

[0008] Furthermore, the intestinal disease is inflammatory bowel disease. More preferably, it is ulcerative colitis.

[0009] Furthermore, the intestinal disease is a disease of intestinal barrier damage or intestinal barrier dysfunction.

[0010] Furthermore, the intestinal barrier damage or intestinal barrier dysfunction diseases include colorectal cancer, colon cancer, constipation, irritable bowel syndrome, or enterogenic infections. Furthermore, the hemp seed protein hydrolysate provided in this application is prepared by the following method:

[0011] The hemp seed protein was enzymatically hydrolyzed with a protease, and the hydrolysate was recovered to obtain the hemp seed protease hydrolysate.

[0012] Furthermore, the hemp seed protein is obtained by extracting hemp seed powder.

[0013] Furthermore, the extraction method includes ultrasound-assisted organic extraction, ultrasound-assisted alkaline extraction and acid precipitation extraction, alkaline extraction and acid precipitation extraction, or salt extraction.

[0014] Furthermore, the protease is selected from single enzymes or complex enzymes. The single enzyme is selected from pepsin, trypsin, α-chymotrypsin, papain, flavor protease, proteinase K, neutral protease, alkaline protease, or thermophilic protease. The complex enzyme is selected from gastric / pancreatic complex enzymes or chymotrypsin / pancreatic complex enzymes.

[0015] More preferably, the protease is papain.

[0016] Furthermore, the hemp seed protein hydrolysate is prepared by the following method:

[0017] Papain was added to hemp seed protein at a concentration of 8000–10000 U / g, and enzymatic hydrolysis was carried out at pH 6.0–8.5 to obtain the hemp seed protein hydrolysate.

[0018] Furthermore, the enzymatic hydrolysate is filtered, centrifuged, and concentrated to obtain the hemp seed enzymatic hydrolysate.

[0019] Invention Effects

[0020] 1. The hemp seed protein hydrolysate provided in this application has anti-inflammatory effects and significantly inhibits the secretion of inflammatory factors IL-6, TNF-α and NO.

[0021] 2. The hemp seed protein hydrolysate has a significant ameliorative effect on ulcerative colitis.

[0022] 3. The hemp seed protein hydrolysate also has the ability to maintain the intestinal epithelial barrier and can be used for the prevention and treatment of diseases related to intestinal barrier damage or impairment.

[0023] The hemp seed enzymatic extract provided in this application is derived from the shelled kernel of hemp seeds (hemp kernel), a crop that is both medicinal and edible. It has virtually no side effects when consumed and is therefore safer for human use. Furthermore, it can achieve therapeutic effects against colitis through oral administration, making it safer and more convenient to take. Attached Figure Description

[0024] Figure 1 The diagram shows the in vitro anti-inflammatory effects of different hemp seed protein hydrolysates, where a: IL-6; b: TNF-α; c: NO.

[0025] Figure 2 The table shows a comparison of basic indicators in UC mice treated with different concentrations of hemp seed protein hydrolysate, pilot-scale product, and 5ASA (n=8). a: Schematic diagram of the dosing regimen and experimental procedure; b: Mouse weight change graph; c: Mouse water intake change graph; d: Statistical results of mouse colon length; e: Representative colon image; f: Statistical results of mouse liver weight index; g: Statistical results of mouse spleen weight index; h: Representative spleen and fecal images; i: Statistical results of DAI score; j: Representative colon HE-stained pathological section image.

[0026] Figure 3 The image shows the effect of hemp seed protein hydrolysate on tight junction proteins in DSS-treated colonic epithelial cells NCM460, where: a: ZO-1; b: Claudin-1; c: Occludin. Detailed Implementation

[0027] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are intended to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0028] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0029] This application provides a hemp seed protein hydrolysate extracted from hemp seed protein and its use in anti-inflammatory applications.

[0030] The hemp seed protein hydrolysate in this application is derived from natural hemp seeds and is prepared by the following method:

[0031] The hemp seed protein was enzymatically hydrolyzed with a protease, and the hydrolysate was filtered, centrifuged, and concentrated to obtain the hemp seed protease hydrolysate.

[0032] In one specific embodiment, the hemp seed protein is obtained by defatting and removing oil from hemp seed raw material powder with an organic solvent to obtain defatted hemp seed powder, and then extracting hemp seed protein from the defatted hemp seed powder.

[0033] In this application, no restrictions are placed on the preparation method of hemp seed raw material powder. It can be prepared in accordance with conventional methods in the field, including but not limited to grinding.

[0034] In a specific embodiment, the method for degreasing and removing oil using an organic solvent includes: adding hemp seed powder to an extraction reagent, stirring, and centrifuging to remove the oil from the hemp seed powder. The extraction reagent is an organic solvent, which in specific embodiments can be selected from one or more of pure petroleum ether, n-hexane, n-butanol, and anhydrous ethanol. In some specific embodiments, the organic solvent is a mixture of petroleum ether / n-butanol, n-hexane / n-butanol, petroleum ether / ethanol, or n-hexane / ethanol in a certain proportion. In some specific embodiments, the organic solvent is a 5:2 ratio of petroleum ether / n-butanol, 5:2 ratio of n-hexane / n-butanol, 5:2 ratio of petroleum ether / ethanol, or 5:2 ratio of n-hexane / ethanol. In a preferred embodiment, the organic solvent is anhydrous ethanol.

[0035] In some specific embodiments, the ratio of hemp seed powder to the above-mentioned organic solvent is 1:10 to 1:20 (g / mL).

[0036] In this application, there are no restrictions on the method for extracting oil from hemp seed powder. It can be carried out in accordance with conventional methods in the art. In some specific embodiments, static stirring or ultrasonic extraction methods can be used.

[0037] One specific implementation of the static stirring method is as follows: the mixture of hemp seed powder and extraction reagent is stirred at room temperature of 20-25℃ for 12 hours, the supernatant is removed by centrifugation, the extraction reagent is added to the precipitate again and stirred at room temperature of 20-25℃ for 6 hours, the supernatant is removed by centrifugation, the precipitate is first evaporated in a water bath at about 60℃ until no organic matter remains, and then dried and cooled to obtain defatted hemp seed powder.

[0038] One specific implementation of the ultrasonic extraction method is as follows: the mixture of hemp seed powder and extraction reagent is stirred at room temperature of 20-25℃ for 10 min, ultrasonicated (100 kHz) for 10 min, centrifuged to remove the supernatant, the precipitate is added to the extraction reagent again and stirred and ultrasonicated repeatedly, centrifuged to remove the supernatant, the precipitate is first evaporated in a water bath at about 60℃ until no organic matter remains, and then dried and cooled to obtain defatted hemp seed powder.

[0039] In this application, no restrictions are placed on the extraction method of hemp seed protein. It can be carried out in accordance with conventional methods in the art. In some specific embodiments, hemp seed protein can be extracted from defatted hemp seed powder by ultrasound-assisted organic extraction, ultrasound-assisted alkaline extraction and acid precipitation extraction, alkaline extraction and acid precipitation extraction, or salt extraction.

[0040] One specific implementation of the ultrasound-assisted organic extraction method is as follows: defatted hemp seed powder is mixed with n-hexane, centrifuged under ultrasound, and the supernatant is collected. The remaining substances can be repeated several times. The supernatants obtained are combined and evaporated until no organic matter remains, and then dried to obtain the final product.

[0041] In a preferred embodiment, defatted hemp seed powder and n-hexane are mixed at a ratio of 1:30 to 1:50 (g / mL); the ultrasonic frequency is 2 kHz; the ultrasonic time is 20 to 40 min each time; the mixture is evaporated in a water bath at 55 to 65 °C; and then dried in an oven at 55 to 65 °C.

[0042] One specific implementation of the ultrasound-assisted alkaline extraction and acid precipitation method is as follows: defatted hemp seed powder is mixed with ultrapure water (pH 8.5), ultrasonicated (200W), and centrifuged to obtain the supernatant. The remaining substances can be repeated several times. The supernatants obtained are combined and the pH is adjusted to 4.5 to precipitate the protein. After washing with water, the pH is adjusted to 7.0 and then dried to obtain the final product.

[0043] In a preferred embodiment, defatted hemp seed powder is mixed with ultrapure water at a ratio of 1:10 to 1:30 (g / mL); the ultrasonic frequency is 100 to 300 W; the ultrasonic treatment temperature is 20 to 25°C; the ultrasonic treatment time is 20 to 40 min each time; and the product is dried in an oven at 55 to 65°C.

[0044] One specific implementation method of the alkaline extraction and acid precipitation extraction method is as follows: Defatted hemp seed powder is mixed with ultrapure water (pH 10.0), stirred, centrifuged and the supernatant is collected. The remaining substances can be repeated several times. The supernatants obtained are combined and the pH is adjusted to 5.0 to precipitate the protein. After washing with water, the pH is adjusted to 7.0 and then dried or freeze-dried to obtain the final product.

[0045] In a preferred embodiment, defatted hemp seed powder is mixed with ultrapure water at a ratio of 1:5 to 1:20 (g / mL); the stirring temperature is 30 to 40°C; the stirring time is 1 to 3 hours each time; and the mixture is dried in an oven at 55 to 65°C.

[0046] One specific implementation of the salt extraction method is as follows: Mix defatted hemp seed powder with NaCl solution (pH 7.0), stir, centrifuge and collect the supernatant. The process can be repeated several times for the remaining material. Add ultrapure water (pH 10.0) to the remaining material, stir, centrifuge and collect the supernatant. Combine the supernatants and adjust the pH to 4.5 to precipitate the protein. After washing with water, adjust the pH to 7.0 and dry or freeze-dry to obtain the final product.

[0047] In a preferred embodiment, the NaCl solution concentration is 0.5–1.0 M; defatted hemp seed powder is mixed with NaCl solution at a ratio of 1:5 to 1:20 (g / mL); the stirring temperature is 30–40 °C; the stirring time is 1–3 h each time; and drying is performed in an oven at 55–65 °C.

[0048] In a preferred embodiment of this application, hemp seed protein is extracted using a salt extraction method.

[0049] In this application, when hemp seed protein is enzymatically hydrolyzed with a protease, the protease is selected from single enzymes or complex enzymes.

[0050] In a specific embodiment, the single enzyme is selected from pepsin, trypsin, α-chymotrypsin, papain, flavor protease, proteinase K, neutral protease, alkaline protease, or thermophilic protease, and the complex enzyme is selected from gastric / pancreatic complex enzyme or chymotrypsin / pancreatic complex enzyme.

[0051] In some preferred embodiments, the protease is selected from papain.

[0052] In some embodiments, hemp seed protein is prepared into a protein solution of a certain mass concentration, and protease is added at a concentration of 8000-10000 U / g for enzymatic hydrolysis.

[0053] In specific embodiments, the enzymatic hydrolysis temperature is 35-60℃; the pH value is 7-11; the enzymatic hydrolysis time is 3-6h; in some preferred embodiments, the pH value is 6.0-8.5.

[0054] This application provides the anti-inflammatory effects of hemp seed protein hydrolysate prepared by the above method.

[0055] In some specific embodiments, the hemp seed protein hydrolysate has a significant inhibitory effect on the secretion of inflammatory factors IL-6, TNF-α and NO, and can be used in the preparation of anti-inflammatory drugs.

[0056] In a specific embodiment, this application provides the use of the hemp seed protein hydrolysate in the preparation of a drug with anti-inflammatory effects.

[0057] This application also provides the efficacy of the hemp seed protein hydrolysate in the prevention, treatment and improvement of intestinal diseases.

[0058] In a specific embodiment, this application provides the use of the hemp seed protein hydrolysate in the preparation of a medicament for the prevention, treatment or improvement of intestinal diseases.

[0059] In specific implementations, the intestinal diseases include inflammatory bowel disease, intestinal barrier damage, or intestinal barrier dysfunction.

[0060] In a specific implementation, the inflammatory bowel disease is ulcerative colitis (UC) or Crohn's disease (CD).

[0061] In some specific embodiments, the hemp seed protein hydrolysate of this application can effectively relieve or inhibit the symptoms of ulcerative colitis.

[0062] In some specific implementations, the intestinal barrier damage or intestinal barrier dysfunction includes rectal cancer, colorectal cancer, constipation, irritable bowel syndrome, or enterogenic infections.

[0063] The intestinal barrier refers to the sum of structures and functions within the intestine that prevent harmful substances such as bacteria and toxins from crossing the intestinal mucosa and entering other tissues, organs, and the bloodstream. It is one of the largest and most important internal barriers in the body, composed of a mucus layer, symbiotic bacteria, epithelial cells, and immune cells in the lamina propria. The mucus layer is the first physical barrier encountered by external molecules reaching the intestinal lumen, preventing bacteria from directly contacting epithelial cells. Its main component is highly glycosylated mucin, which forms a gel-like sieve structure covering the intestinal epithelium. Beneath the mucus layer, epithelial cells are the decisive factor in the formation of the physical intestinal barrier. The intestinal epithelium is a single layer of cells lining the intestinal lumen and has two key functions: first, it acts as a barrier, preventing the passage of harmful substances (including foreign antigens, microorganisms, and their toxins) into the lumen; second, it acts as a selective filter, allowing essential dietary nutrients, electrolytes, and water to be transferred from the intestinal lumen into the circulation. The epithelium maintains its selective barrier function by forming complex protein-protein networks that mechanically connect adjacent cells and seal intercellular spaces. The protein network connecting epithelial cells forms three adhesion complexes: desmosomes, adhesion junctions, and tight junctions.

[0064] Tight junctions are the most adhesive junctional complexes at the apex of mammalian epithelial cells, forming a continuous band-like loop around the epithelial cell at the boundary between the apical and lateral membrane regions. They facilitate the passage of ions and solutes through the intercellular space while preventing the translocation of luminal antigens, microorganisms, and their toxins. Tight junctions are composed of four distinct families of transmembrane proteins (Occludin, Claudins, junctional adhesion molecules (JAMs), and three-cell tight junction proteins). The extracellular regions of transmembrane tight junction proteins in adjacent cells anastomose to form the tight junction seal region. Their intracellular domains interact with various scaffold proteins, adaptor proteins, and signaling complexes, thereby regulating cytoskeleton attachment, cell polarity, cell signaling, and vesicle transport. The closure protein Occludin (60-82 kDa) is a tetraspanic membrane integrated protein with two extracellular loops, a short cytoplasmic N-terminus, and a long cytoplasmic C-terminus. Occludin is primarily expressed at tight junctions in epithelial and endothelial cells, and also in astrocytes, neurons, and dendritic cells. Functional analysis indicates that the extracellular loop and transmembrane domain of ocludin regulate selective paracellular permeability. Intracellularly, the C-terminus of ocludin interacts with closure band 1 (ZO-1), which contains a PDZ domain, allowing it to attach to the actin cytoskeleton. Claudin, a tight junction protein, is a 20-27 kDa intact membrane protein with four hydrophobic transmembrane domains, two extracellular loops, and N- and C-terminal cytoplasmic domains. The extracellular loops are crucial for homologous and heterologous tight junction protein-protein interactions and the formation of ion-selective channels. Intracellularly, the C-terminal domain anchors Claudin to the cytoskeleton through interactions with PDZ-binding domain proteins, including ZO-1, ZO-2, and ZO-3. Claudin-Claudin interactions between adjacent cells can be homologous or heterologous. Selective interactions contribute to the diversity of tight junction formation and provide the molecular basis for the tissue heterogeneity of barrier function. Damage to or disruption of the intestinal barrier due to various reasons can prevent it from functioning properly, allowing harmful substances to enter the body and causing systemic inflammation. For example, bacteria can escape from the digestive tract, causing fatal systemic inflammatory response syndrome; bacterial endotoxins can damage the intestinal mucosa barrier, leading to systemic circulatory failure.

[0065] In some specific embodiments, the hemp seed protein hydrolysate of this application can increase the mRNA expression levels of tight junction proteins ZO-1 and Occludin, suggesting that hemp seed polypeptides may have the ability to maintain the intestinal epithelial barrier.

[0066] The medicament provided in this application may further include pharmaceutically acceptable carriers or excipients.

[0067] Specifically, examples of pharmaceutically acceptable carriers include excipients, binders, buffers, antioxidants, solubilizers, thickeners, lubricants, disintegrants, diluents, stabilizers, preservatives, colorants, flavorings, solubilizers, emulsifiers, isotonic agents, and the like.

[0068] The excipients may be selected from, but are not limited to, starch, lactose, sucrose, calcium carbonate, and calcium phosphate; the binders may be selected from, but are not limited to, starch, gum arabic, carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose, alginic acid, gels, and polyvinylpyrrolidone; the buffers may be selected from, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, borate buffer solutions, Tris salt buffer solutions, and the like; the antioxidants may be selected from, but are not limited to, butylated hydroxytoluene, butylated hydroxyanisole, sodium sulfite, sodium bisulfite, sodium metabisulfite, ascorbic acid, cysteine ​​hydrochloride, cystine, lipoic acid, thioglycerol, and the like; the lubricants may be selected from... However, the following are not limited to magnesium stearate, calcium stearate, and talc; the thickener may be selected from, but is not limited to, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, amorphous cellulose, polysaccharides (including starch derivatives), polyvinyl alcohol, and polyvinylpyrrolidone or mixtures thereof; the disintegrant may be selected from, but is not limited to, calcium carboxymethylcellulose and talc; the diluent may be selected from, but is not limited to, water for injection and saline; the preservative may be selected from, but is not limited to, sodium bisulfite, sodium bisulfite, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, methylparaben, propylparaben, and phenylethanol; the isotonic agent may be selected from, but is not limited to, chlorides and sugars.

[0069] This application also provides that a pharmaceutical preparation containing the hemp seed protein hydrolysate can be prepared by mixing the polypeptide with a pharmaceutically acceptable carrier, for example, to obtain oral formulations such as tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets), capsules (including soft capsules, microcapsules), granules, powders, lozenges, syrups, emulsions, suspensions, films (e.g., orally disintegrating films), parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, drops), topical preparations (e.g., skin preparations, ointments), suppositories (e.g., rectal suppositories, vaginal suppositories), pills, nasal drops, respiratory preparations (inhalers), eye drops, etc. In addition, these preparations can be used as controlled-release preparations (e.g., sustained-release microcapsules), such as immediate-release preparations, sustained-release preparations, etc. Such preparations can be obtained by preparation methods conventionally used in this art.

[0070] Drugs containing the hemp seed protein hydrolysate described in this application can be administered to mammals (e.g., humans, mice, rats, rabbits, dogs, cats, cattle, horses, pigs, and monkeys). The administration route can be oral or parenteral (e.g., intravenous, intramuscular, subcutaneous, intra-organ, intranasal, intradermal, intravenous drip, intracerebral, rectal, vaginal, intraperitoneal, etc.).

[0071] The dosage of hemp seed protein hydrolysate administered to subjects varies depending on the route of administration, symptoms, patient age, etc., and can be determined by clinicians in practice.

[0072] The drugs described in this application can also be used in conjunction with other existing known drugs for treating inflammatory diseases and intestinal diseases. When used together, there are no restrictions on the timing of administration of each drug; two or more different drugs can be administered simultaneously, or at different times. The dosage of the known drugs can be determined according to clinically used dosages and appropriately selected based on the patient, route of administration, etc.

[0073] Example

[0074] The hemp seeds used in the following examples of this application were obtained from Bama, Guangxi, and the papain was purchased from Nanning Pangbo Biotechnology Co., Ltd. Other materials and reagents, unless otherwise specified, are commercially available.

[0075] Example 1: Preparation of hemp seed protein hydrolysate

[0076] The hemp seed protein hydrolysate was prepared using the following method:

[0077] (1) Enzymatic hydrolysis of hemp seed protein

[0078] Hemp seed protein was enzymatically hydrolyzed using pepsin, trypsin, α-chymotrypsin, papain, flavorzyme, proteinase K, neutral protease, alkaline protease, thermolysin, and gastric / pancreatic (trypsin / pepsin) and chymotrypsin / pancreatic (trypsin / chymotrypsin) complex enzymes, respectively.

[0079] The specific method is as follows: Weigh 2g of hemp seed powder into 40mL of water (1:20 material-to-liquid ratio, w / v, g / mL) to prepare a protein solution. Add protease at a protein sample concentration of 10000U / g (8000U / g thermophilic protease). Perform enzymatic hydrolysis for 5h according to the action conditions of each enzyme in Table 1. After enzymatic hydrolysis, centrifuge at 5000rpm for 20min and collect the supernatant.

[0080] Table 1. Types of enzymes and their enzymatic hydrolysis conditions

[0081]

[0082] (2) Freeze-drying

[0083] Freeze-dry the above enzymatic hydrolysate to obtain the various enzymatic hydrolysates of hemp seed protein.

[0084] Example 2: Anti-inflammatory ability test of hemp seed protein hydrolysate

[0085] The anti-inflammatory effects of various enzymatic hydrolysates of hemp seed protein were investigated using a cell model. Specifically, a mouse macrophage cell line (RAW264.7) stimulated with LPS (purchased from Sigma) was used, and the in vitro anti-inflammatory activity of the hemp seed protein hydrolysates was quantitatively assessed by ELISA. The experimental protocol is as follows:

[0086] Resuscitated RAW 264.7 cells were cultured in DMEM (containing 10% FBS + 1% penicillin antibody) at a concentration of 3 × 10⁻⁶ cells / mL. 5 Cells were seeded at a concentration of 100 μL / mL in 96-well plates, with 100 μL of culture medium per well. After about 16 hours, when cell conjugation reached 70%–80%, the old culture medium was discarded. The control group (Blank) was treated with 180 μL of Opti-MEM medium, while the other experimental groups were treated with 180 μL of Opti-MEM medium containing 100 ng / mL LPS. The plates were then stimulated at 37°C for 1 hour.

[0087] During the process, the enzymatic hydrolysates of hemp seed protein obtained in Example 1 were dissolved in DMEM (containing 10% FBS + 1% penicillin antibody) at a concentration of 500 μg / mL to prepare stock solutions, which were then filtered and stored for later use. Subsequently, 20 μL of the stock solution was added to the wells of the drug-treated group to bring the final concentration of the enzymatic hydrolysates in the cell culture medium to 500 μg / mL, and the plates were incubated at 37°C for 24 h. After peptide treatment, the plates were centrifuged at 300g for 5 min at 4°C, and 50 μL of cell-free culture medium supernatant was collected, diluted 10-fold with PBS, and stored at 4°C for later use. The levels of IL-6 and TNF-α in the culture medium supernatant were then detected using an ELISA kit (Invitrogen, USA), and the NO content was measured using a Gres (Beyotime, China) assay. The results are as follows: Figure 1 As shown.

[0088] Cellular experiments showed that the enzymatic hydrolysates of hemp seed possess certain anti-inflammatory activities. Among them, the anti-inflammatory effect of the papain hydrolysate mixture was more significant than that of the other 11 enzymatic hydrolysates mixture, and it significantly inhibited the secretion of inflammatory factors IL-6, TNF-α, and NO. This mixture was selected as the active peptide of hemp seed for further efficacy verification.

[0089] Example 3: Pilot production of hemp seed protein hydrolysate

[0090] Papain was selected for pilot-scale production of hemp seed enzymatic hydrolysis. The pilot-scale production conditions were: a material-to-liquid ratio of 1:20, a temperature of 55℃, a hydrolysis time of 4 hours, and an enzyme activity of 10000 U / g. Specific product data under these conditions are as follows:

[0091] 8000g of hemp seed powder and 80g of papain were added. The mixture was hydrolyzed at 55℃ with 160L of water at 30rpm for 4 hours. After hydrolysis, the solution was filtered and centrifuged. The filtrate was collected and concentrated at 55℃ under a vacuum of 0.8–1.0 until it reached one-third of its initial volume. Spray drying was then initiated with an inlet air temperature of 180℃, an nebulizer frequency of 350Hz, an outlet air temperature of 90℃, and a sample pump speed of 35rpm. A total of 3957g of dried powder was obtained, with a yield of 49.46%.

[0092] Example 4: Hemp seed active peptides alleviate DSS-induced acute ulcerative colitis in mice

[0093] 1. Experimental System:

[0094] a. Laboratory animals

[0095] C57BL / 6J mouse species

[0096] SPF level

[0097] Supplier Viton Lihua

[0098] Weight and sex: 18-20g, male, 6-8 weeks old

[0099] The expected starting weight for administration is 22 ± 2 g.

[0100] b. Rearing environment

[0101] Mice were housed in an SPF barrier environment with a room temperature of 20-25℃, a daily temperature difference of ≤3℃, an air exchange rate of 10-20 times / h, a humidity of 40-70%, a pressure gradient of 20-50Pa, and a day / night light / dark cycle of 12h / 12h. The cages and bedding were changed weekly.

[0102] c. Dosage design and basis

[0103] Rationale for Dosage Design: The test sample dosage was designed based on the pharmacopoeia and earlier preliminary experiments. The pharmacopoeia recommends an intake of 10–15 g / day of hemp seed, and three concentrations of 80, 160, and 320 mg / kg / day were tested in the preliminary experiments. The recommended intake of 5-ASA for adults is 1200–2400 mg / day, which translates to 156–312 mg / kg / day for mice.

[0104] Dosage design: The positive control dose was 160 mg / kg / day / animal, the drug storage concentration was 16 mg / mL, and the total volume of solution administered by gavage was determined according to body weight (10 μL / g). The dosage design for this experiment is shown in Table 2.

[0105] Table 2 Dosage Design Table

[0106]

[0107] 2. Experimental Methods

[0108] (1) SPF-grade healthy male C57BL / 6J mice were acclimatized to the new experimental environment for 7 days (average weight reached 22±2g), and then randomly divided according to weight. In accordance with the experimental requirements, the number of mice in each group was kept consistent to ensure that there was no significant difference in weight between the initial groups.

[0109] (2) The mice in the treatment group were given the test product by gavage daily. The dosage was as shown in Table 2. The mice in the WT group and DSS group were given an equal amount of water by gavage according to their body weight.

[0110] (3) Starting from the 7th day after gavage, except for the WT group, other mice were given free access to a solution containing 3% DSS (prepared fresh and changed every other day; the high-concentration stock solution was filtered through a 0.22μM filter membrane).

[0111] (4) No fasting or water restriction is required throughout the entire experiment.

[0112] (5) On the 7th day after DSS induction (the 14th day of the entire experimental process, no drug administration is required on Day 14), the mice were euthanized and immediately dissected. Blood was collected from the orbital rim to obtain whole blood from the mice. The mice were then dissected and the colon was removed (placed in ice-cold PBS) for subsequent experiments.

[0113] Dosing regimen and experimental procedure as follows Figure 2 As shown in a.

[0114] 3. Indicator Inspection

[0115] Evaluation criteria for successful model establishment: simultaneously meeting the following conditions: weight loss of more than 12%, shortening of colon length, and obvious bloody and loose stools in the intestines.

[0116] a. General observation

[0117] During the mouse modeling period, the Disease Activity Index (DAI) was used to score the animals based on their body weight, stool characteristics, and fecal occult blood test results, and their daily water intake was recorded. The DAI scoring table is shown in Table 3.

[0118] Table 3DAI Scoring Table

[0119]

[0120] b. Post-dissection observation indicators

[0121] After dissection, the colon length of mice was measured, and the spleen and liver were weighed. H&E staining of the colonic epithelium was performed to observe the extent of colonic tissue damage in each group of mice. Results are as follows: Figure 2 As shown.

[0122] like Figure 2 As shown in the figure (bi), compared with the WT group, mice in the DSS group exhibited significant weight loss, shortened colon, enlarged spleen, and loose or bloody stools. Oral administration of hemp seed active peptides alleviated these symptoms in a dose-dependent manner. Simultaneously, the pilot-scale product also demonstrated certain biological activity in alleviating UC. Based on pathological section results (… Figure 2 According to the results, hemp seed active peptides can significantly protect the colonic epithelial tissue of mice, and this effect is dose-dependent. In the high-dose group, the colonic epithelial tissue structure was intact, the crypt shape was normal, the crypts were regularly arranged, and there were normally distributed goblet cells and a small number of neutrophil infiltrations. The swelling of the submucosal tissue was also significantly improved.

[0123] All of the above results indicate that gavage administration of hemp seed polypeptide has a certain preventive effect against DSS-induced colitis in mice.

[0124] Example 5: Protective effect of hemp seed enzymatic hydrolysate on the intestinal epithelial barrier

[0125] The anti-colitis activity of hemp seed enzymatic hydrolysate was verified using the above animal models, and the ability of hemp seed active peptides to protect the intestinal epithelial barrier was further explored using cell models.

[0126] A human colonic epithelial cell (NCM460) model stimulated by DSS (purchased from MP) was used, and the ability of hemp seed enzymatic hydrolysates to maintain the colonic barrier was investigated by qPCR. The specific experimental protocol is as follows.

[0127] Table 4 NCM460 Dosing Regimen

[0128]

[0129] The enzymatic hydrolysates of hemp seeds obtained in Example 1 and 5ASA were dissolved in Opti-MEM at a concentration of 5 mg / mL to prepare a stock solution, which was then filtered for later use. Resuscitated NCM460 cells were cultured in RPMI 1640 (containing 10% FBS + 1% penicillin antibiotics) at a concentration of 2 × 10⁻⁶ cells / mL. 5 Cells were seeded at a concentration of 10 cells / mL in 6-well plates, with 2 mL of culture medium per well. Once cell conjugation reached 70%–80%, culture medium was added to each well according to the administration protocol shown in Table 4, with 3 replicates per group.

[0130] After treatment, cellular RNA was collected using TRIzol reagent and reverse transcribed to obtain cDNA samples according to the TIANGEN (KR116) manufacturer's instructions. The mRNA expression levels of ZO-1, Claudin-1, and Occludin in the samples were determined using the Genstar kit (A304). The experimental results are as follows: Figure 3 As shown.

[0131] The results showed that pretreatment with the mixture of peptides by enzymatic hydrolysis of the papain at a concentration of 0.5 mg / mL significantly increased the mRNA expression levels of tight junction proteins ZO-1 and Occludin in DSS-induced NCM460 cells, suggesting that hemp seed peptides may have the ability to maintain the intestinal epithelial barrier.

[0132] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application, without departing from the scope of the technical solution of this application, shall still fall within the protection scope of this application.

Claims

1. Use of hemp seed protein enzymatic hydrolysate in the preparation of an anti-inflammatory drug.

2. Use of hemp seed protein enzymatic hydrolysate in the preparation of a drug for preventing, treating or improving intestinal diseases.

3. Use according to claim 2, wherein, The intestinal diseases are inflammatory bowel diseases, preferably ulcerative colitis.

4. The use according to claim 2, wherein, The intestinal diseases are intestinal barrier injury or intestinal barrier dysfunction diseases.

5. Use according to claim 4, wherein, The intestinal barrier injury or intestinal barrier dysfunction diseases include rectal cancer, colon cancer, constipation, irritable bowel syndrome or enterogenous infection.

6. Use according to any one of claims 1 to 5, wherein, The hemp seed protein enzymatic hydrolysate is prepared by the following method: The hemp seed protein is extracted from hemp seed powder, and the extraction method includes ultrasonic-assisted organic extraction, ultrasonic-assisted alkali extraction and acid precipitation extraction, alkali extraction and acid precipitation extraction, or salt extraction.

7. Use according to claim 6, wherein, The protease is selected from a single enzyme or a complex enzyme, the single enzyme is selected from pepsin, trypsin, α-chymotrypsin, papain, flavor protease, protease K, neutral protease, alkaline protease or thermolysin, and the complex enzyme is selected from stomach / pancreas complex enzyme or chymotrypsin / pancreas complex enzyme, preferably papain.

8. The use according to claim 6, wherein, The hemp seed protein enzymatic hydrolysate is prepared by the following method: papain is added to hemp seed protein at a concentration of 8000-10000 U / g, and enzymatic hydrolysis is carried out at a pH value of 6.0-8.5 to obtain the hemp seed protein enzymatic hydrolysate.

9. Use according to claim 8, wherein, The enzymatic hydrolysate is filtered, centrifuged and concentrated to obtain the hemp seed protein enzymatic hydrolysate.

10. The use according to claim 6, wherein, ​

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