Highland barley fried malt polysaccharide and application thereof in preparation of anti-tumor preparation

Abstract

The invention discloses an application of fried highland barley malt polysaccharide in preparation of an anti-tumor preparation. The highland barley roasted malt polysaccharide is heteropolysaccharide of which monosaccharide composition comprises arabinose, glucose and xylose in a molar mass ratio of 1.00: 3.75: 1.54. Research finds that the barley fried malt polysaccharide can significantly inhibit proliferation of liver cancer cells, lung cancer cells, breast cancer cells and colorectal cancer cells, especially has significant anti-tumor activity on the colorectal cancer cells, and is expected to be applied to food, health care products or drugs.

Description

Technical Field

[0001] This invention belongs to the fields of food, health products, and medicine, and specifically relates to the use of roasted barley malt polysaccharide in the preparation of anti-tumor agents. Background Technology

[0002] Highland barley ( Hordeum vulgare L. var. nudum is a cold-resistant grass unique to the Qinghai-Tibet Plateau, with a long history of application in Tibetan medicine, traditionally used to regulate the spleen and stomach, and harmonize qi and blood. Modern scientific research shows that roasted barley malt, made by germinating barley grains using a specific process, not only retains many of the original nutrients and bioactive components of barley (such as β-glucan and phenolic compounds), but also transforms into new physiologically active substances during processing due to Maillard reactions and endogenous enzymatic hydrolysis. Specifically, barley malt and barley grains are not the same. Barley grains are dormant seeds, while barley malt is an activated seed that begins to grow. Germination (or sprouting) activates the seed from its dormant state, causing a series of dramatic biochemical changes: such as the degradation of starch and protein, a decrease in the content of phytic acid, tannins, saponins, etc., an increase in the content of antioxidants such as vitamins, phenolic compounds, and flavonoids, and structural changes in dietary fiber, with some insoluble fiber (such as cellulose) being enzymatically broken down, and some new soluble dietary fiber may be produced at the same time. Wang Xiaoyan et al.'s paper points out that the physiological characteristics and main components of peanut seeds undergo significant changes during germination. With the extension of germination time, germination initially increases and then tends to stabilize. Moisture content and respiration intensity gradually increase, fat content gradually decreases, starch content gradually increases, and the content of soluble total sugar and non-starch polysaccharides shows a trend of first decreasing and then increasing, while the crude protein content does not change significantly (DOI:10.13684 / j.cnki.spkj.2018.05.032). Pei Yingying et al.'s research shows that the galactomannan of saponins changes at different germination stages: with the extension of germination time, the mass ratio of mannose to galactose (M / G value) gradually increases, and the molecular weight gradually decreases (Forest Products Chemistry and Industry, 2023, 43(01):133-139). In addition, roasting changes the physical and chemical properties of raw malt, thereby expanding its uses. Stir-frying denatures proteins, causes the loss of polyphenols and vitamins, and breaks some glycosidic bonds in polysaccharides, thereby altering the structure of polysaccharides.

[0003] Roasted malt, a typical food and medicine resource, has its medicinal value clearly recorded in the *Compendium of Materia Medica*, describing its effects as "aiding digestion, dispelling cold qi, and relieving abdominal distension." It is noteworthy that compared to ordinary barley malt, barley grown in the unique ecological environment of the Qinghai-Tibet Plateau (strong ultraviolet radiation, significant diurnal temperature variation) exhibits different molecular structures in its malt polysaccharides, primarily characterized by a higher relative abundance of arabinose and xylose residues. Existing research suggests that the composition and linkage of polysaccharides may be closely related to their potential for superior biological activity. Liu Wenwen et al., in their research progress on the chemical composition and functional activities of malt, pointed out that the functional activities of malt include promoting digestion, strengthening the spleen and stomach, reducing lactation and relieving bloating, and possessing pharmacological effects such as antioxidation, antitumor, anti-inflammation, digestion aid, hypoglycemia, and hepatoprotective effects, making it widely applicable in clinical practice. Specifically, barley malt contains abundant hydrolytic enzymes such as α-amylase and β-amylase; starch is hydrolyzed by α-amylase to form dextrin, while β-amylase breaks down dextrin into maltose, thereby promoting the body's absorption of nutrients. Nutrients in barley malt, such as glutenin and hemicellulose, have good therapeutic effects on ulcerative colitis. The antitumor activity of malt protein is related to phenolic compounds; and barley free phenol extract can reduce CCl4-induced hepatocyte apoptosis and damage, thus protecting the liver. Crude barley polysaccharide can lower fasting blood glucose in mice; in vitro experiments have shown that malt oligosaccharides have hypoglycemic activity. Furthermore, Zhu Muyuan et al. studied the interaction between phenolic substances in several types of hulled highland barley and HepG2 cells, finding that both free and bound polyphenols could inhibit HepG2 cell proliferation, with free polyphenols exhibiting stronger anti-proliferative capacity than bound polyphenols. The material basis for this anti-proliferative effect is chlorogenic acid and (+)-catechin. Studies by Rao et al. have found that high concentrations of phenolic compounds in barley can induce apoptosis in SW480 rectal cancer cells. The mechanism may be that phenolic substances stimulate the expression of p53 protein, triggering a cascade effect that activates caspase-3 and caspase-7, ultimately leading to cancer cell apoptosis. Furthermore, barley contains lunasin, an active peptide with anti-cancer activity. The essential amino acid lysine, abundant in barley, has the effect of indirectly fighting tumors by enhancing immunity. The study also points out that current research on malt still faces some challenges. Regarding chemical composition, there is limited research on malt enzymes, proteins, and polysaccharides. In terms of functional activity, current research on malt focuses primarily on verifying and elucidating traditional efficacy and mechanisms, while research and development on modern pharmacological effects are relatively limited.

[0004] Currently, conventional chemotherapy and radiotherapy, widely used in cancer treatment, face numerous challenges, including acquired drug resistance in tumor cells and the resulting systemic toxicity. Against this backdrop, plant-derived bioactive polysaccharides, due to their multi-target, low toxicity, and potential immunomodulatory mechanisms of action, have become an important direction in cancer prevention and treatment research. Plant polysaccharides typically possess advantages such as diverse pathways of action, multiple targets, relatively mild adverse reactions, and the ability to generate synergistic effects with other treatments, demonstrating their potential for development into novel anticancer adjuvants or drugs. However, systematic research on polysaccharides from roasted barley malt, particularly exploring their antitumor activity and mechanisms of action, is relatively scarce both domestically and internationally. Based on the reference and optimization of research methods for cereal polysaccharides, our research team has successfully developed and optimized an extraction process for efficiently enriching polysaccharide components with potential anticancer activity from roasted barley malt.

[0005] In conclusion, in-depth research and development of the antitumor activity of roasted barley malt polysaccharides will not only help expand the application areas of barley resources and increase the added value of its deep-processed products, but may also provide new solutions to overcome the limitations of traditional anticancer drugs, such as significant toxic side effects and high treatment costs. Summary of the Invention

[0006] This invention provides the use of roasted barley malt polysaccharide in the preparation of antitumor agents; it provides a new candidate material basis and process technology support for the development of anticancer drugs based on natural products, and has important application potential.

[0007] The objective of this invention is achieved through the following technical solution: This invention provides the use of roasted barley malt polysaccharide in the preparation of antitumor agents; the roasted barley malt polysaccharide is a (neutral) heteropolysaccharide with a monosaccharide composition of arabinose, glucose and xylose.

[0008] As one implementation, the tumor includes liver cancer, lung cancer, breast cancer, and colorectal cancer.

[0009] As one implementation, the antitumor agent is an anti-colorectal cancer agent.

[0010] As one embodiment, the monosaccharide composition of the roasted barley malt polysaccharide is arabinose, glucose, and xylose, with a molar mass ratio of 1.00:3.75:1.54. Different monosaccharide compositions determine the function of the polysaccharide, and different monosaccharide ratios may result in completely different functions. The roasted barley malt polysaccharide of this application is a non-β-glucan, a heteropolysaccharide, with glucose being the most abundant component.

[0011] As one implementation method, the roasted barley malt polysaccharide is prepared by a method comprising the following steps: S1. Barley roasted malt powder is defatted, enzymatically hydrolyzed, alcohol-precipitated, and freeze-dried to obtain crude barley roasted malt polysaccharide; S2. The crude polysaccharide of roasted barley malt was purified by dynamic axial compression chromatography column to obtain roasted barley malt polysaccharide.

[0012] As one implementation scheme, in step S1, the roasted barley malt powder is obtained by soaking and culturing barley seeds to germinate, then drying, roasting, grinding, and sieving the malt before collection.

[0013] The preferred soaking time for the seeds is 3-5 hours. The optimal seed culture temperature is 23~27℃, the culture time is 42~48 h, the humidity is 95%~100%, and 4~5 mL of water is added every 12 h.

[0014] The preferred drying conditions are drying at 50-60°C for 2-3 hours, followed by heating to 70-80°C for 5-10 minutes.

[0015] Preferably, the frying temperature is 220~230℃ and the time is 10~15 min.

[0016] As one implementation, the roasted barley malt powder is passed through a 60-mesh sieve.

[0017] As one implementation, in step S1, defatting involves mixing roasted barley malt powder and petroleum ether at a ratio of 1 g: 40-50 mL.

[0018] As one implementation scheme, in step S1, the enzymatic hydrolysis involves mixing roasted barley malt powder and water at a ratio of 1 g: 30-40 mL, adding 5-10 μL / g of the roasted barley malt powder containing thermostable α-amylase, incubating at 75-80℃ for 20-30 min, and then inactivating at 100℃ for 10-15 min; after the solution cools, adding 5-10 μL / g of the roasted barley malt powder containing papain, incubating at 50-60℃ for 1-2 h, and then inactivating at 100℃ for 5-15 min; after the solution cools, adding 30-40 μL / g of the roasted barley malt powder containing saccharifying enzyme, incubating at 50-60℃ for 1-2 h, and then inactivating at 100℃ for 10-15 min, followed by incubation at 80-90℃ for 3-4 h, and collecting the supernatant after extraction.

[0019] As one implementation scheme, in step S1, the alcohol precipitation involves filtering the obtained supernatant twice, concentrating the collected supernatant in a water bath at 70-80°C to 1 / 20-1 / 10 of its original volume, cooling it, adding 3-4 times the volume of anhydrous ethanol, letting the solution stand overnight, centrifuging it, collecting the precipitate, redissolving the precipitate with an appropriate amount of deionized water in a water bath at 70-80°C and concentrating it to 20-30 ml, adding 3-4 times the volume of anhydrous ethanol again, letting it stand, centrifuging it, and redissolving it with water.

[0020] As one implementation scheme, the vacuum freeze-drying involves freezing the precipitate, which has been reconstituted with deionized water, at -80°C and then performing vacuum freeze-drying under the following conditions: -35°C for 4 hours; -20°C for 20 hours; and 10°C for 12 hours, in sequence.

[0021] As one implementation, in step S2, the dynamic axial compression column is packed with DEAE. 32 or Sephacryl S-400 packing.

[0022] As an implementation plan, first use DEAE Separation was performed using a 32 cellulose ion exchange column with distilled water as the eluent, at a rate of 2–5 mL / min and a time of 750–1500 min. The resulting gel was then purified using a Sephacryl S-400 dextran gel column with distilled water as the eluent, at a rate of 0.2–0.4 mL / min and a time of 2000–3000 min.

[0023] As one implementation, in the structure of the roasted barley malt polysaccharide, the main chain consists of 1,4-linked β-D-Glc p 1.3-connected β-d-Glc p 1,5-connected α-l-Ara f β-d-Xyl linked by 1,4 p β-D-Xyl and 1,4-linked p Composed of α-l-Ara with side chains connected by 1, 2 or 1, 3. f α-d-Glc connected to 1, 4 p constitute.

[0024] Compared with the prior art, the present invention has the following beneficial effects: This invention demonstrates through cytotoxicity experiments that roasted barley malt polysaccharide has a good inhibitory effect on liver cancer, lung cancer, breast cancer, and colorectal cancer, especially on HCT116 colorectal cancer cells, which promotes the application of polysaccharide as an anti-cancer drug and provides a new direction for the utilization of barley resources. Attached Figure Description

[0025] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The DE-32 cellulose column elution curve for barley roasted malt polysaccharide; Figure 2 Elution curve of dextran gel column for highland barley roasted malt polysaccharide; Figure 3 HPSEC spectrum of roasted barley malt polysaccharide; Figure 4 The ion chromatogram is for a standard monosaccharide. Figure 5 The ion chromatogram of roasted barley malt polysaccharide; Figure 6 Infrared spectrum of roasted barley malt polysaccharide; Figure 7 For barley roasted malt polysaccharide 1 H NMR spectrum; Figure 8 For barley roasted malt polysaccharide 13 C NMR spectrum; Figure 9 DEPT spectrum of roasted barley malt polysaccharide; Figure 10 The HSQC spectrum of roasted barley malt polysaccharide; Figure 11 For barley roasted malt polysaccharide 1 H 1 H COSY spectrum; Figure 12 The HMBC spectrum of roasted barley malt polysaccharide; Figure 13 The diagram shows the repeating unit structure of roasted barley malt polysaccharide; Figure 14 The inhibition rate of highland barley roasted malt polysaccharide on HepG2; Figure 15 The inhibition rate of 4T1 by roasted barley malt polysaccharide; Figure 16 The inhibition rate of highland barley roasted malt polysaccharide on A549; Figure 17 The inhibition rate of roasted barley malt polysaccharide on HCT116 is shown. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0027] Example 1 This embodiment provides a method for preparing roasted barley malt polysaccharide, including the following steps: (1) Soak barley seeds at room temperature for 5 h, then incubate them in a 25℃ incubator for 48 h, maintaining humidity above 95% during germination, adding 4 mL of water every 12 h. After germination, place them in an oven at 60℃ for 2 h, then raise the temperature to 80℃ for 5 min. Then roast them at 220℃ for 10 min, collect the powder, grind it, and sieve it to obtain roasted barley malt powder.

[0028] (2) Barley roasted malt powder was defatted, enzymatically hydrolyzed, precipitated with alcohol, and freeze-dried to obtain barley roasted malt polysaccharide. The defatting step involved mixing barley roasted malt powder and petroleum ether at a ratio of 1 g: 40 mL, and removing the petroleum ether after defatting. The enzymatic hydrolysis involved mixing barley roasted malt powder and water at a ratio of 1 g: 30 mL, adding 10 μL / g of the thermostable α-amylase from the barley roasted malt powder, incubating in a water bath at 80°C for 30 min, and then inactivating the enzyme by incubating in a water bath at 100°C for 10 min. After the solution cooled, 10 μL / g of the papain from the barley roasted malt powder was added, incubating in a water bath at 60°C for 1 h, and then inactivating the enzyme by incubating in a water bath at 100°C for 10 min. After the solution cooled, 40 μL / g of the saccharifying enzyme from the barley roasted malt powder was added, incubating in a water bath at 60°C for 1 h, and then inactivating the enzyme by incubating in a water bath at 100°C for 10 min, and then incubating in a water bath at 90°C for 4 hours. After extraction, the supernatant was collected. The supernatant was filtered twice, and the collected supernatant was concentrated to 1 / 10 of its original volume in a water bath at 80℃. After cooling, 4 times the volume of anhydrous ethanol was added, the solution was allowed to stand overnight, centrifuged, and the precipitate was collected. The precipitate was reconstituted with an appropriate amount of deionized water in a water bath at 80℃ and concentrated to 1 / 10 of its original volume. 4 times the volume of anhydrous ethanol was added again, the solution was allowed to stand overnight, centrifuged, and reconstituted with 20 mL of water. The reconstituted precipitate was frozen at -80℃ and then freeze-dried under vacuum for 36 h (-35℃, 4 h; -20℃, 20 h; 10℃, 12 h) to obtain powdered water-soluble crude polysaccharide from roasted barley malt.

[0029] (3) Weigh 50 g of crude polysaccharide from roasted barley malt and dissolve it in 500 mL of distilled water. After filtering through a 0.45 μm filter membrane, add the solution dropwise to a dynamic axial compression chromatographic column (packing material: DEAE-32). Elute with distilled water, 0.1 M, 0.2 M, and 0.3 M NaCl solutions sequentially at a rate of 2 mL / min for 750 min. Take samples from every 4 test tubes, collect 0.1 mL of the solution, add 0.9 mL of deionized water, add 1 mL of 5% phenol solution, vortex, add 5 mL of concentrated sulfuric acid, and measure the absorbance at 490 nm. Plot the corresponding elution curve as shown below. Figure 1 The fractions eluted with distilled water were combined and concentrated to 1 / 10 of their original volume, then dialyzed in a 3000 Da dialysis bag for 36 h, with water changed every 12 h. The dialyzed sample was then freeze-dried under vacuum for 36 h (-35℃, 4 h; -20℃, 20 h; 10℃, 12 h). 5 g of the solution was dissolved in 50 mL of distilled water, filtered through a 0.45 μm filter membrane, and then added dropwise to a dynamic axial compression column (Sephacryl S-400 packing material). The eluent was distilled water, the elution rate was 0.2 mL / min, and the elution time was 2250 min. The total sugar content was determined using the phenol-sulfuric acid method, and the corresponding elution curves were plotted as follows: Figure 2 The eluent fraction corresponding to the peak of the maximum total sugar content in the elution curve was used to prepare roasted barley malt polysaccharide.

[0030] Comparative Example 1 This comparative example relates to an extract of roasted barley malt, and the preparation steps are as follows: Roasted barley malt powder (same as in Example 1) and water were mixed at a ratio of 1 g: 30 mL. 10 μL / g of the thermostable α-amylase from roasted barley malt powder was added, and the mixture was inactivated by water bath at 80°C for 30 min and then at 100°C for 10 min. After the solution cooled, 10 μL / g of the papain from roasted barley malt powder was added, and the mixture was inactivated by water bath at 60°C for 1 h and then at 100°C for 10 min. After the solution cooled, 40 μL / g of the saccharifying enzyme from roasted barley malt powder was added, and the mixture was inactivated by water bath at 60°C for 1 h and then at 100°C for 10 min. After extraction, the mixture was incubated at 90°C for 4 h. After extraction, the supernatant was collected. The supernatant was filtered twice, and the collected supernatant was concentrated to 1 / 10 of its original volume in a water bath at 80℃. It was then freeze-dried under vacuum for 36 h (-35℃, 4 h; -20℃, 20 h; 10℃, 12 h) to obtain powdered water-soluble barley roasted malt extract.

[0031] Comparative Example 2 This comparative example relates to a crude polysaccharide made from roasted highland barley malt, and the preparation steps are as follows: Roasted barley malt powder and water were mixed at a ratio of 1 g: 30 mL. 10 μL / g of the roasted barley malt powder was added to the thermostable α-amylase, and the mixture was inactivated by water bath at 80℃ for 30 min and then at 100℃ for 10 min. After the solution cooled, 10 μL / g of the roasted barley malt powder was added to the papain, and the mixture was inactivated by water bath at 60℃ for 1 h and then at 100℃ for 10 min. After the solution cooled, 40 μL / g of the roasted barley malt powder was added to the saccharifying enzyme, and the mixture was inactivated by water bath at 60℃ for 1 h and then at 100℃ for 10 min. The mixture was then incubated at 90℃ for 4 h. After extraction, the supernatant was collected. The supernatant was filtered twice, and the collected supernatant was concentrated to 1 / 10 of its original volume in a water bath at 80℃. After cooling, 4 times the volume of anhydrous ethanol was added, the solution was allowed to stand overnight, centrifuged, and the precipitate was collected. The precipitate was reconstituted with an appropriate amount of deionized water in a water bath at 80℃ and concentrated to 1 / 10 of its original volume. 4 times the volume of anhydrous ethanol was added again, the solution was allowed to stand overnight, centrifuged, and reconstituted with 20 ml of water. The reconstituted precipitate was frozen at -80℃ and then freeze-dried under vacuum for 36 h (-35℃, 4 h; -20℃, 20 h; 10℃, 12 h) to obtain powdered water-soluble crude polysaccharide from roasted barley malt.

[0032] Comparative Example 3 This comparative example relates to a barley grain polysaccharide, and the preparation steps are as follows: (1) Grind the barley grains into powder and sieve them to obtain barley grain powder. Barley grain powder was defatted, enzymatically hydrolyzed, precipitated with alcohol, and freeze-dried to obtain barley grain polysaccharide. The defatting step involved mixing barley grain powder and petroleum ether at a ratio of 1 g: 40 mL, and removing the petroleum ether after defatting. The enzymatic hydrolysis involved mixing barley grain powder and water at a ratio of 1 g: 30 mL, adding 10 μL / g of thermostable α-amylase from barley grain powder, incubating at 80°C for 30 min, and then inactivating the enzyme at 100°C for 10 min. After the solution cooled, 10 μL / g of papain from barley grain powder was added, incubating at 60°C for 1 h, and then inactivating the enzyme at 100°C for 10 min. After the solution cooled, 40 μL / g of saccharifying enzyme from barley grain powder was added, incubating at 60°C for 1 h, and then inactivating the enzyme at 100°C for 10 min. Finally, the solution was incubated at 90°C for 4 h. After extraction, the supernatant was collected. The supernatant was filtered twice, and the collected supernatant was concentrated to 1 / 10 of its original volume in a water bath at 80℃. After cooling, 4 times the volume of anhydrous ethanol was added, the solution was allowed to stand overnight, centrifuged, and the precipitate was collected. The precipitate was reconstituted with an appropriate amount of deionized water in a water bath at 80℃ and concentrated to 1 / 10 of its original volume. 4 times the volume of anhydrous ethanol was added again, the solution was allowed to stand overnight, centrifuged, and reconstituted with 20 mL of water. The reconstituted precipitate was frozen at -80℃ and then freeze-dried under vacuum for 36 h (-35℃, 4 h; -20℃, 20 h; 10℃, 12 h) to obtain powdered water-soluble barley grain polysaccharide.

[0033] (2) Weigh 50 g of barley grain polysaccharide and dissolve it in 500 mL of distilled water. After passing through a 0.45 μm filter membrane, add it dropwise to a dynamic axial compression chromatographic column (packing material: DEAE-32). Elute with distilled water, 0.1 M, 0.2 M and 0.3 M NaCl solutions sequentially. The elution rate is 2 mL / min and the elution time is 750 min. Combine the fractions eluted with distilled water and concentrate them to 1 / 10 of the original volume. Dialyze them in a dialysis bag for 36 h, changing the water every 12 h. After dialysis, freeze-dry the sample under vacuum. Take 5 g of the solution and dissolve it in 50 mL of distilled water. After passing through a 0.45 μm filter membrane, add it dropwise to a dynamic axial compression chromatographic column (packing material: Sephacryl S-400). The eluent is distilled water. The elution rate is 0.2 mL / min and the elution time is 2250 min to obtain barley grain polysaccharide.

[0034] Example 2 This embodiment is used to illustrate the physicochemical properties of the roasted barley malt polysaccharide obtained in Example 1. The specific characterization methods are as follows: Molecular weight analysis: Molecular weight distribution was determined using high-performance size exclusion chromatography (USG) equipped with multi-angle laser scattering detection. A barley roasted malt polysaccharide solution (2 mg / mL) was prepared using ultrapure water. NaNO3 was injected as the mobile phase using a TSK-GEL G6000 PWXL column at 45°C and a flow rate of 0.5 mL / min. Figure 3 As shown, roasted barley malt polysaccharide has high homogeneity, with a polydispersity index (Mw / Mn) of 1.28 and a weight-average molecular weight (Mw) of 37.71 kDa.

[0035] Monosaccharide composition: Trifluoroacetic acid (2 M) was added to 5 mg of roasted barley malt polysaccharide and hydrolyzed at 121°C for 2 h. The mixture was then washed three times with methanol to remove trifluoroacetic acid. Fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, guluronic acid, glucuronic acid, and mannuronic acid were prepared as monosaccharide standards. The residue of roasted barley malt polysaccharide was redissolved in deionized water and filtered through a 0.22 μm microporous membrane. The monosaccharide composition of roasted barley malt polysaccharide was analyzed by high performance anion exchange chromatography (HPLC) on a CarboPac PA-20 anion exchange column (3 × 150 mm; Dionez) using a pulsed amperometric detector (PAD; Dionex ICS 5000+ system). Solvent system A is ddH2O, solvent system B is 0.1 M NaOH, and solvent system C is 0.1 M NaOH and 0.2 M NaAc.

[0036] The volume ratio of solutions A, B, and C was 95:5:0 at 0 min, 85:5:10 at 26 min, 85:5:10 at 42 min, 60:0:40 at 42.1 min, 60:40:0 at 52 min, 95:5:0 at 52.1 min, and 95:5:0 at 60 min. Flow rate: 0.5 mL / min; injection volume: 5 μL.

[0037] Figure 4 The ion chromatograms of the monosaccharide standards are shown below. The elution order of the monosaccharide standards is as follows: fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, guluronic acid, glucuronic acid, and mannuronic acid. The ion chromatogram of roasted barley malt polysaccharide is shown below. Figure 5 As shown, it is composed of arabinose, glucose and xylose in a molar ratio of 1.00:3.75:1.54.

[0038] Infrared spectroscopy: 5 mg of roasted barley malt polysaccharide sample was mixed evenly with 750 mg of KBr solid, ground into powder, and then pressed into a 1 mm thick disc for pelleting. A Nicolet 6700 Fourier transform infrared spectrometer was used to scan the region from 4000 to 400 cm⁻¹. -1 Perform infrared spectroscopy analysis. Infrared spectra, such as... Figure 6 As shown, at 3313.07 cm -1 The absorption peak intensity is the highest and the peak shape is the widest at point O, which may be due to O. This is caused by the stretching vibration of the H bond, indicating that the polysaccharide molecule may contain a carboxyl group (O). H; at 1648 cm -1 The absorption peak at 1154.87 cm⁻¹ is located in the double bond stretching vibration region, which is caused by the stretching vibration of the C=O bond; -1 The absorption peak at C is O This is due to the absorption vibration of the C bond, 1070.69 cm⁻¹ -1 The absorption peak at C is O This is due to the absorption vibration of the H bond, indicating the presence of pyranose residues in the sample, 897.27 cm⁻¹. -1 The absorption peak at that location indicates the presence of the β configuration.

[0039] Methylation analysis: 3 mg of roasted barley malt polysaccharide was dissolved in DMSO, 1 mg of NaOH was added, and the mixture was incubated for 30 min to ensure complete dissolution. Then, 50 μl of CH3I was added and the reaction was carried out for 1 h. The reaction was terminated by adding ddH2O (1 mL), and the sample was extracted by adding CH2Cl2. The lower CH2Cl2 phase was then collected, dried under nitrogen, hydrolyzed with trifluoroacetic acid, reduced with NaBD4, and terminated with acetic acid. Acetic anhydride was added, and the reaction was carried out at 100 °C for 2.5 h. After terminating the reaction with water, CH2Cl2 was added to obtain partially methylated sugar alcohol acetates. Analysis was performed on an Agilent 6890A-5977B instrument equipped with an Agilent BPX70 column.

[0040] As shown in Table 1, the main repeating structural units of roasted barley malt polysaccharide are composed of the following linear residues: →4)-β-D-Glc p -(1→(50.53%),→3)-β-D-Glc p -(1→(15.24%),→4)-β-D-Xyl p -(1→(13.96%),α-L-Ara f - (1→(9.9%), and a small amount of α-D-Glc p -(1→(4.87%),→3, 4)-β-D-Xyl p -(1→(3.49%) and →2, 3, 5)-α-L-Ara f - (1 → (2.01%).

[0041] Table 1. Analysis of methylation results of roasted barley malt polysaccharides

[0042] Nuclear magnetic resonance (NMR) analysis: Roasted barley malt polysaccharide was dissolved in D2O and stirred overnight at room temperature. One-dimensional NMR spectra were obtained using a Bruker AV600 spectrometer. 1 H NMR, 13 C NMR and two-dimensional NMR (DEPT-135, COSY, HSQC, HMBC).

[0043] 1 H NMR spectroscopy ( Figure 7 The characteristic signal of the polysaccharide was mainly between 3.0 and 5.5 ppm, with multiple angiopon signals identified between 4.3 and 5.4 ppm, indicating heterogeneous glycosylation. Seven distinct angiopons were observed at δ 4.49, 4.54, 4.64, 4.79, 5.23, 5.28, and 5.40 ppm. Correspondingly, 13 C NMR spectroscopy ( Figure 8Seven distinct anodic carbon signals were observed at δ 108.7, 108.1, 102.5, 102.3, 101.6, 95.9, and 92.2 ppm. DEPT-135 ( Figure 9 The spectrum provided additional structural information through the methylene signal in the 60.0-70.0 ppm range, corresponding to the CH2 group of the sugar ring. This was further supported by... 1 H and 13 Comprehensive analysis of C chemical shift, HSQC spectroscopy ( Figure 10 The anomaly correlations (cross peaks) can be clearly assigned as follows: δH / C 5.28 / 108.7 (residue A), 5.40 / 108.1 (B), 5.23 / 92.2 (D), 4.49 / 101.6 (E), 4.54 / 102.3 (F), 4.79 / 102.5 (G) and 4.64 / 95.9 (I). The cross peaks at δH / C 5.28 / 108.7 and 5.40 / 108.1 are respectively attributed to H-1 / C-1 of α-L-Raf-(1→,→2,3,5)-α-L-Rap-(1→). Other characteristic anomaly signals were detected at 5.23 / 92.2, 4.49 / 101.6, 4.54 / 102.3, 4.79 / 102.5, and 4.64 / 95.9 ppm and were assigned to α-D-Glcp-(1→,→3)-β-D-Glc p -(1→、→4)-β-D-Glc p -H-1 / C-1. The anomeric configuration of each residue was determined based on the characteristic chemical shift value of the anomeric proton (H-1). For pyran residues, the chemical shift of the hydrogen proton at the α-configuration anomeric proton is typically greater than 4.9 ppm, while the anomeric hydrogen of the β-configuration glycoside is less than 4.9 ppm. Therefore, residues A, B, and D were identified as α-configuration, while residues E, F, G, and I were identified as β-configuration. Furthermore, based on the chemical shift of the anomeric proton, by 1 H- 1 H COSY spectrum ( Figure 11 The H-2 signal was obtained through the cross-peaks of H-1 and H-2. The same analogy was used for the assignment of chemical shifts for the remaining proton signals (H-3 to H-6). Supplementary HSQC analysis allowed for the assignment of other carbon chemical shifts (C-2 to C-6) for the remaining glycosyl residues. Table 2 systematically summarizes the complete glycosyl residue profile of roasted barley malt polysaccharide. 1 H and 13 C chemical shift partitioning.

[0044] Table 2. Highland barley roasted malt polysaccharides 1 H and 13 Chemical shift of C

[0045] Through HMBC ( Figure 12 Spectroscopic analysis of the glycosyl residue sequence and linkage sites revealed a cross-peak at δH / C 5.28 / 83.8 ppm (A H-1 / B C-2), indicating that the O-1 of residue A is linked to the C-2 of residue B. Similarly, at δH / C 5.2.8 / 83.8 ppm (A H-1 / B / C-3), δH / C 5.40 / 77.6 ppm (B H-1 / G C-4), δH / C 5.23 / 78.2 ppm (D H-1 / I C-3), and δH / C 4.79 / 83.8 ppm, the O-1 of residue B is associated with the C-4 of residue G; the O-1 of residue D is associated with the C-3 of residue I; and the O-1 of residue G is associated with the C-4 of residue C. Other HMBC correlations at δH / C 4.54 / 77.2 ppm (F H-1 / E C-3), 3.53 / 101.6 ppm (BH-5 / E C-1), and 3.53 / 102.5 ppm (I H-4 / G C-1) indicate that the O-1 of residue F is correlated with the C-3 of residue E; the O-5 of residue B is correlated with the C-1 of residue E; and the O-4 of residue I is correlated with the C-1 of residue G. Notably, the cross-peaks at δH / C 4.54 / 78.4 ppm (F H-1 / F C-4) and 4.49 / 77.2 ppm (E H-1 / E C-3) indicate that the O-1 of residue F is linked to the C-4 of residue F, and the O-1 of residue E is linked to the C-3 of residue E, suggesting the presence of repeating units in residues E and F. In summary, the repeating unit inference model for highland barley roasted malt polysaccharide is as follows: Figure 13 As shown.

[0046] Verification Example 3 This validation example demonstrates the application of roasted barley malt polysaccharide in anti-cancer activity. The specific implementation process is as follows: I. Inhibitory effect of roasted barley malt polysaccharide on cancer cell proliferation HepG2 liver cancer cells, A549 lung cancer cells, 4T1 breast cancer cells, and HCT116 colorectal cancer cells were resuscitated and cultured in DMEM medium containing 10% FBS in an incubator at 37°C and 5% CO2. When the cell confluence reached 80%, the cells were digested with trypsin, centrifuged, and the supernatant was discarded. 2 mL of culture medium was added to the pellet, and the cells were pipetted and counted using a hemocytometer. The cells were then diluted proportionally and seeded into 96-well plates (100 μL of cell suspension per well, i.e., 5000 cells) and cultured for 24 h. Barley roasted malt polysaccharide (Example 1), barley roasted malt extract (Comparative Example 1), barley roasted malt crude polysaccharide (Comparative Example 2), or barley grain polysaccharide (Comparative Example 3) were added to 96-well plates to achieve final concentrations of 0, 0.625, 1.25, 2.5, 5, and 10 mg / mL, respectively, and incubated at 37°C for 48 h. 5 μL of CCK-8 was added to each well, and the plates were incubated at 37°C for another 4 h. The OD value of each well was measured at 450 nm. The cell inhibition rate was calculated using the following formula: Inhibition rate = (1 - OD value of experimental group / OD value of control group) × 100%. Results are as follows: Figure 14-17 As shown, after initial screening of four types of tumor cells, the polysaccharides from roasted barley malt, roasted barley malt extract, crude roasted barley malt polysaccharide, and barley grain polysaccharide all inhibited the proliferation of HepG2, 4T1, A549, and HCT116 cells. Among them, roasted barley malt polysaccharide showed the best inhibitory effect and the most significant inhibitory effect on the proliferation of colorectal cancer cells HCT116. The effects of different concentrations of roasted barley malt polysaccharide solutions (0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL, and 10 mg / mL) on the growth of colorectal cancer cells HCT116 after 48 h of treatment were compared with the control group, and the inhibition rates were 5.24%, 13.13%, 20.62%, 26.94%, and 39.04%, respectively.

[0047] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. The use of roasted barley malt polysaccharide in the preparation of antitumor agents; wherein the roasted barley malt polysaccharide is a heteropolysaccharide with monosaccharide composition of arabinose, glucose and xylose.

2. The use according to claim 1, characterized in that, The tumors include liver cancer, lung cancer, breast cancer, and colorectal cancer.

3. The use according to claim 1, characterized in that, The antitumor agent is an anti-colorectal cancer agent.

4. The use according to claim 1, characterized in that, The molar mass ratio of arabinose, glucose and xylose is 1.00:3.75:1.

54.

5. The use according to claim 1, characterized in that, The roasted barley malt polysaccharide is prepared by a method comprising the following steps: S1. Barley roasted malt powder is defatted, enzymatically hydrolyzed, alcohol-precipitated, and freeze-dried to obtain crude barley roasted malt polysaccharide; S2. The crude polysaccharide of roasted barley malt was purified by dynamic axial compression chromatography column to obtain roasted barley malt polysaccharide.

6. The use according to claim 5, characterized in that, In step S1, the roasted barley malt powder is obtained by soaking and culturing barley seeds to germinate, then drying, roasting, grinding, and sieving the malt before collection.

7. The use according to claim 6, characterized in that, The seeds are soaked for 3-5 hours, cultured at 23-27°C for 42-48 hours, with a humidity of 95%-100%, and 4-5 mL of water is added every 12 hours. The drying conditions are: drying at 50-60°C for 2-3 hours, followed by drying at 70-80°C for 5-10 minutes. The roasting temperature is 220-230°C for 10-15 minutes.

8. The use according to claim 5, characterized in that, In step S1, the defatting is achieved by mixing roasted barley malt powder and petroleum ether at a ratio of 1 g: 40~50 mL; And / or, the enzymatic hydrolysis is performed by mixing roasted barley malt powder and water at a ratio of 1 g: 30-40 mL, adding 5-10 μL / g of the thermostable α-amylase from the roasted barley malt powder, incubating in a water bath at 75-80℃ for 20-30 min, and then inactivating in a water bath at 100℃ for 10-15 min; after the solution has cooled, adding 5-10 μL / g of the papain from the roasted barley malt powder, incubating in a water bath at 50-60℃ for 1-2 h, and then inactivating in a water bath at 100℃ for 5-15 min; after the solution has cooled, adding 30-40 μL / g of the saccharifying enzyme from the roasted barley malt powder, incubating in a water bath at 50-60℃ for 1-2 h, and then inactivating in a water bath at 100℃ for 10-15 min, followed by incubation in a water bath at 80-90℃ for 3-4 h, and collecting the supernatant after extraction; And / or, the alcohol precipitation is performed by filtering the obtained supernatant twice, concentrating the collected supernatant in a water bath at 70-80°C to 1 / 20-1 / 10 of its original volume, cooling, adding 3-4 times the volume of anhydrous ethanol, letting the solution stand overnight, centrifuging, collecting the precipitate, redissolving the precipitate with an appropriate amount of deionized water in a water bath at 70-80°C and concentrating it to 20-30 ml, adding 3-4 times the volume of anhydrous ethanol again, letting it stand, centrifuging, and redissolving it with water.

9. The use according to claim 5, characterized in that, In step S2, the dynamic axial compression column is packed with DEAE. 32 or Sephacryl S-400 packing; first use DEAE Separation was performed using a 32 cellulose ion exchange column with distilled water as the eluent, at a rate of 2–5 mL / min and a time of 750–1500 min. The resulting gel was then purified using a Sephacryl S-400 dextran gel column with distilled water as the eluent, at a rate of 0.2–0.4 mL / min and a time of 2000–3000 min.

10. The use according to claim 1 or 5, characterized in that, In the structure of the roasted barley malt polysaccharide, the main chain consists of 1,4-linked β-D-Glc p 1.3-connected β-d-Glc p 1,5-connected α-l-Ara f β-d-Xyl linked by 1,4 p β-D-Xyl and 1,4-linked p Composed of α-l-Ara with side chains connected by 1, 2 or 1, 3. f α-d-Glc connected to 1, 4 p constitute.

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