Extraction method of fructus alpiniae oxyphyllae

Abstract

The invention discloses an extraction method of fructus alpiniae oxyphyllae polysaccharide, which comprises the following steps: (1) selecting high-quality fructus alpiniae oxyphyllae fruits, cleaning, removing impurities, and carrying out microwave vacuum drying treatment; (2) crushing the dried fructus alpiniae oxyphyllae, and sieving to obtain powder; (3) mixing the crushed fructus alpiniae oxyphyllae powder with deionized water, and adding lactobacillus plantarum ATCC14917 for fermentation; (4) adding a compound enzyme preparation, carrying out enzymolysis, and after enzymolysis is completed, putting the mixed solution into an ultrasonic-microwave synergistic extraction device, and carrying out ultrasonic treatment; (5) cooling the extracting solution to room temperature, adding an ethanol-water mixed solvent, standing and extracting; (6) primarily purifying the supernate through an ultrafiltration membrane, removing macromolecular impurities and part of protein, further decoloring and purifying the filtrate after ultrafiltration through cation exchange resin, eluting with deionized water, and collecting the eluent; and (7) concentrating and drying to obtain the fructus alpiniae oxyphyllae polysaccharide extract. The fructus alpiniae oxyphyllae polysaccharide extract extracted by the method disclosed by the invention has an obvious effect of improving the diabetic liver injury.

Description

Technical Field

[0001] This invention relates to the field of extraction technology of traditional Chinese medicine, and in particular to a method for extracting Alpinia oxyphylla. Background Technology

[0002] Alpinia oxyphylla Miq., the dried, ripe fruit of the ginger family plant, is first recorded in the *Compendium of Materia Medica*. Traditionally used to warm the spleen and stop diarrhea, control salivation, warm the kidneys, and strengthen the kidneys to reduce urination. Modern pharmacological research indicates that Alpinia oxyphylla is a commonly used herb in Traditional Chinese Medicine, traditionally believed to have effects such as warming the spleen and stopping diarrhea, and strengthening the kidneys to reduce urination. Modern research suggests that the volatile oils, flavonoids, and polysaccharides in Alpinia oxyphylla may have potential for auxiliary regulation of diabetes-related symptoms, but there is no clear evidence that it can treat diabetes alone, nor is there evidence that it has a protective effect against liver damage caused by diabetes.

[0003] The current extraction technology for Alpinia oxyphylla has the following shortcomings, which seriously restrict its industrial application and efficacy:

[0004] Solvent extraction is inefficient: traditional ethanol reflux or water extraction and alcohol precipitation processes have long extraction times (2-4 hours) and large solvent consumption (solid-liquid ratio 1:10-1:20). The transfer rate of active ingredients such as oxytocin A and polysaccharides is less than 60%, and high temperatures can easily lead to the degradation of heat-sensitive components.

[0005] The current limitations of Alpinia oxyphylla extraction and separation technology are severely restricting its industrial application and the full release of its medicinal potential. Solvent extraction efficiency is low, and the loss of active ingredients is significant. Traditional extraction processes mostly use ethanol reflux or water extraction with alcohol precipitation, which have long extraction cycles and low equipment utilization. The large consumption of solvents not only increases production costs but also brings environmental pressure. The transfer rate of key active ingredients is low, and the yield of polysaccharides is insufficient due to molecular weight fractionation during water extraction. The overall transfer rate of active ingredients is generally below 60%, which is difficult to meet the requirements of industrial production for raw material utilization. The purification process is crude and cannot meet the standardization requirements. Existing purification techniques largely rely on macroporous adsorption resins or silica gel column chromatography. While these can achieve preliminary enrichment, they have significant drawbacks: cumbersome operation steps, long processing times, and high labor costs; enormous consumption of organic solvents, easily causing environmental pollution and safety hazards; complex resin regeneration processes; and declining adsorption performance after long-term use, making it difficult to stably control product quality. Furthermore, existing technologies lack the ability to accurately identify and target components, resulting in high impurity content and large batch-to-batch variations in extracts, failing to meet the stringent requirements of Good Manufacturing Practices (GMP) for raw material homogeneity. Efficacy correlation studies are weak, and clinical positioning is unclear. Currently, commercially available Alpinia oxyphylla extracts mostly use traditional indicators such as "total flavonoid content" and "volatile oil yield" as quality control standards, but these macroscopic indicators lack direct correlation with the therapeutic targets of specific diseases (such as diabetic liver damage). Diabetic liver injury, a common complication of diabetes, involves a multi-dimensional pathological process involving oxidative stress imbalance (excessive ROS production), neuroinflammatory response (excessive activation of microglia), and cholinergic nervous system damage (decreased acetylcholine levels). However, current research has not yet clarified the regulatory effects of various active ingredients in Alpinia oxyphylla extract on the above-mentioned key targets and their synergistic effects, resulting in unstable efficacy in clinical applications and difficulty in forming precise indications.

[0006] The aforementioned deficiencies indicate that the shortcomings of existing technologies have severely hindered the transformation of Alpinia oxyphylla from a traditional medicinal herb into a high-value-added functional product. Developing a novel Alpinia oxyphylla extraction method that is highly efficient and energy-saving, has controllable processes, is suitable for large-scale production, and can enhance the efficacy of improving diabetic liver damage through precise enrichment of active ingredients is not only crucial for overcoming the bottlenecks in industrial development but also an inevitable choice for promoting the modernization of traditional Chinese medicine and meeting clinical needs. This technological innovation will provide scientific support for the in-depth development of Alpinia oxyphylla and open up new pathways for the application of natural products in the protection against liver damage in diabetes. Summary of the Invention

[0007] The purpose of this invention is to provide a highly efficient extraction method for Alpinia oxyphylla polysaccharides suitable for industrial production. The Alpinia oxyphylla polysaccharide extract obtained by this method has significant advantages in improving the liver damage caused by diabetes.

[0008] The technical solution adopted in this invention is:

[0009] A method for extracting polysaccharides from Alpinia oxyphylla includes the following steps:

[0010] (1) Select high-quality Alpinia oxyphylla fruits, wash and remove impurities, and then use microwave vacuum drying treatment;

[0011] (2) The dried Alpinia oxyphylla kernels are crushed and sieved to obtain powder;

[0012] (3) Mix the crushed Alpinia oxyphylla powder with deionized water and add Lactobacillus plantarum ATCC14917 for fermentation;

[0013] (4) Add the compound enzyme preparation and perform enzymatic hydrolysis. After the enzymatic hydrolysis is completed, place the mixture in an ultrasonic-microwave synergistic extraction device for ultrasonic treatment.

[0014] (5) After cooling the extract to room temperature, add an ethanol-water mixture and allow it to stand for extraction;

[0015] (6) The supernatant was initially purified by ultrafiltration membrane to remove macromolecular impurities and some proteins. The filtrate after ultrafiltration was further decolorized and purified by cation exchange resin, eluted with deionized water, and the eluent was collected.

[0016] (7) Alpinia oxyphylla polysaccharide extract obtained by concentration and drying.

[0017] Specifically, step (2) involves pulverizing the dried Alpinia oxyphylla kernels, sieving them through 200 mesh and 400 mesh sieves respectively, and selecting particles between 200 mesh and 400 mesh.

[0018] The compound enzyme preparation in step (4) is a mixture of cellulase, pectinase and protease, with the enzyme activity ratio of cellulase, pectinase and protease being (2-3):2:(1-2).

[0019] In step (3), the mass ratio of Alpinia oxyphylla powder to deionized water is 1:(15-20).

[0020] In step (3), the fermentation time of Lactobacillus plantarum ATCC14917 is 18-24 hours.

[0021] In step (4), the amount of compound enzyme preparation added is 0.8-1.2 wt%.

[0022] In step (4), the enzymatic hydrolysis conditions are 40°C for 1.5 hours.

[0023] In step (4), the ultrasonic conditions are 200 watts ultrasonic power, 35 kHz frequency, 500 watts microwave power, and 20 minutes processing time.

[0024] In step (5), the volume ratio of ethanol to water in the ethanol-water mixed solvent is (1-2):(1-2).

[0025] The extraction conditions for step (5) are: extraction at 4°C for 12 hours.

[0026] In step (6), the ultrafiltration membrane has a molecular weight cutoff of 3000 Daltons, and the filtrate is further decolorized and purified by cation exchange resin AB-8, with the flow rate controlled at 1.0 times column volume / hour.

[0027] A method for extracting polysaccharides from Alpinia oxyphylla includes the following steps:

[0028] (1) Select high-quality Alpinia oxyphylla fruits, wash and remove impurities, and then microwave vacuum dry for 8 hours;

[0029] (2) After drying, the Alpinia oxyphylla kernels are crushed and sieved through 200 mesh and 400 mesh respectively, and particles between 200 mesh and 400 mesh are selected;

[0030] (3) Mix the crushed Alpinia oxyphylla powder with deionized water at a mass ratio of 1:(15-20), add Lactobacillus plantarum ATCC14917 and ferment for 18-24 hours;

[0031] (4) Add a compound enzyme preparation (cellulase, pectinase and protease, with an enzyme activity ratio of (2-3):2:(1-2)). The amount of compound enzyme preparation added is 0.8-1.2wt%. Enzymatic hydrolysis is carried out at 40℃ for 1.5 hours. The enzymatic hydrolysis process can effectively destroy the cell wall and release polysaccharide components. After the enzymatic hydrolysis is completed, the mixture is placed in an ultrasonic-microwave synergistic extraction device with an ultrasonic power of 200 watts and a frequency of 35 kHz, a microwave power of 500 watts, and a processing time of 20 minutes.

[0032] (5) After cooling the extract to room temperature, add an ethanol-water mixed solvent at a volume ratio of 4:1 and let it stand at 4°C for 12 hours for extraction.

[0033] (6) The supernatant was initially purified by passing it through an ultrafiltration membrane (with a molecular weight cutoff of 3000 Daltons) to remove macromolecular impurities and some proteins. The filtrate after ultrafiltration was further decolorized and purified by passing it through a cation exchange resin (AB-8) with a flow rate of 1.0 column volume / hour. The filtrate was eluted with deionized water and the eluent was collected.

[0034] (7) Alpinia oxyphylla polysaccharide extract obtained by concentration and drying.

[0035] Compared with the prior art, the present invention provides a method for extracting polysaccharides from Alpinia oxyphylla, which includes the following steps: (1) Selecting high-quality Alpinia oxyphylla fruits, washing and removing impurities, and then drying them using microwave vacuum drying; (2) Crushing the dried Alpinia oxyphylla and sieving it to obtain powder; (3) Mixing the crushed Alpinia oxyphylla powder with deionized water and adding Lactobacillus plantarum ATCC14917 for fermentation; (4) Adding a compound enzyme preparation for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the mixture is placed in an ultrasonic-microwave synergistic extraction device for ultrasonic treatment; (5) Cooling the extract to room temperature and adding an ethanol-water mixed solvent for static extraction; (6) Preliminarily purifying the supernatant through an ultrafiltration membrane to remove macromolecular impurities and some proteins, further decolorizing and purifying the ultrafiltration filtrate through a cation exchange resin, eluting with deionized water, and collecting the eluent; (7) Concentrating and drying the obtained Alpinia oxyphylla polysaccharide extract. The Alpinia oxyphylla extraction method of the present invention, through the addition of specific bacterial strains for fermentation and control of the particle size of the powder, yields an Alpinia oxyphylla polysaccharide extract with high polysaccharide content and high content of Alpinia oxyphylla methyl ester, which has significant advantages in improving liver function damage caused by diabetes. Attached Figure Description

[0036] Figure 1 A standard curve for polysaccharide content determination;

[0037] Figure 2 A graph showing the comparison of GSH activity in mice;

[0038] Figure 3 A graph showing the comparison of MDA activity in mice;

[0039] Figure 4 A graph showing the comparison of SOD activity in mice;

[0040] Figure 5 This is a comparison chart of CAT levels in mice. Detailed Implementation

[0041] To better demonstrate the technical solution and beneficial effects of the present invention, the technical solution of the present invention will be further described below in conjunction with embodiments, but this is not intended to limit the scope of protection of the present invention.

[0042] Raw material description

[0043] Alpinia oxyphylla: Bozhou Wufang Jiuru Pharmaceutical Co., Ltd. (Batch No.: 20250306);

[0044] Cellulase: Guangdong Mingcheng Biotechnology Co., Ltd.;

[0045] Pectinase: Guangdong Mingcheng Biotechnology Co., Ltd.;

[0046] Protease: Guangdong Mingcheng Biotechnology Co., Ltd.;

[0047] Resin: Macroporous resin AB-8, Ruida Henghui;

[0048] Lactobacillus plantarum ATCC14917: Shanghai Fuxiang Biotechnology Co., Ltd.;

[0049] Lactobacillus plantarum CICC 6009: Shanghai Beinuo Biotechnology Co., Ltd.;

[0050] Lactobacillus plantarum CICC 6076 ATCC 8014: Shanghai Beinuo Biotechnology Co., Ltd.

[0051] The remaining materials are sourced from commercially available sources.

[0052] Example 1

[0053] A method for extracting polysaccharides from Alpinia oxyphylla includes the following steps:

[0054] (1) Select high-quality Alpinia oxyphylla fruits, wash and remove impurities, and then microwave vacuum dry for 8 hours;

[0055] (2) After drying, the Alpinia oxyphylla kernels are crushed and sieved through 200 mesh and 400 mesh respectively, and particles between 200 mesh and 400 mesh are selected;

[0056] (3) Mix the crushed Alpinia oxyphylla powder with deionized water at a mass ratio of 1:18, add Lactobacillus plantarum ATCC14917 and ferment for 20 hours;

[0057] (4) Add a compound enzyme preparation (cellulase, pectinase and protease, with an enzyme activity ratio of 3:2:1). The amount of compound enzyme preparation added is 1 wt%. Enzymatic hydrolysis is carried out at 40°C for 1.5 hours. The enzymatic hydrolysis process can effectively destroy the cell wall and release polysaccharide components. After the enzymatic hydrolysis is completed, the mixture is placed in an ultrasonic-microwave synergistic extraction device with an ultrasonic power of 200 watts and a frequency of 35 kHz, a microwave power of 500 watts, and a processing time of 20 minutes.

[0058] (5) After cooling the extract to room temperature, add an ethanol-water mixed solvent (ethanol-water volume ratio of 4:1) and let it stand for 12 hours at 4°C for extraction.

[0059] (6) The supernatant was initially purified by passing it through an ultrafiltration membrane (with a molecular weight cutoff of 3000 Daltons) to remove macromolecular impurities and some proteins. The filtrate after ultrafiltration was further decolorized and purified by passing it through a cation exchange resin (AB-8) with a flow rate of 1.0 column volume / hour. The filtrate was eluted with deionized water and the eluent was collected.

[0060] (7) Alpinia oxyphylla polysaccharide extract obtained by concentration and drying.

[0061] Example 2

[0062] A method for extracting polysaccharides from Alpinia oxyphylla includes the following steps:

[0063] (1) Select high-quality Alpinia oxyphylla fruits, wash and remove impurities, and then microwave vacuum dry for 8 hours;

[0064] (2) After drying, the Alpinia oxyphylla kernels are crushed and sieved through 200 mesh and 400 mesh respectively, and particles between 200 mesh and 400 mesh are selected;

[0065] (3) Mix the crushed Alpinia oxyphylla powder with deionized water at a mass ratio of 1:15, add Lactobacillus plantarum ATCC14917 and ferment for 24 hours;

[0066] (4) Add a compound enzyme preparation (cellulase, pectinase and protease, with an enzyme activity ratio of 3:2:2). The amount of compound enzyme preparation added is 0.8wt%. Enzymatic hydrolysis is carried out at 40℃ for 1.5 hours. The enzymatic hydrolysis process can effectively destroy the cell wall and release polysaccharide components. After the enzymatic hydrolysis is completed, the mixture is placed in an ultrasonic-microwave synergistic extraction device with an ultrasonic power of 200 watts and a frequency of 35 kHz, a microwave power of 500 watts, and a processing time of 20 minutes.

[0067] (5) After cooling the extract to room temperature, add an ethanol-water mixed solvent (ethanol-water volume ratio of 4:1) and let it stand at 4°C for 12 hours for extraction.

[0068] (6) The supernatant was initially purified by passing it through an ultrafiltration membrane (with a molecular weight cutoff of 3000 Daltons) to remove macromolecular impurities and some proteins. The filtrate after ultrafiltration was further decolorized and purified by passing it through a cation exchange resin (AB-8) with a flow rate of 1.0 column volume / hour. The filtrate was eluted with deionized water and the eluent was collected.

[0069] (7) Alpinia oxyphylla polysaccharide extract obtained by concentration and drying.

[0070] Example 3

[0071] A method for extracting polysaccharides from Alpinia oxyphylla includes the following steps:

[0072] (1) Select high-quality Alpinia oxyphylla fruits, wash and remove impurities, and then microwave vacuum dry for 8 hours;

[0073] (2) After drying, the Alpinia oxyphylla kernels are crushed and sieved through 200 mesh and 400 mesh respectively, and particles between 200 mesh and 400 mesh are selected;

[0074] (3) Mix the crushed Alpinia oxyphylla powder with deionized water at a mass ratio of 1:20, add Lactobacillus plantarum ATCC14917 and ferment for 18 hours;

[0075] (4) Add a compound enzyme preparation (cellulase, pectinase and protease, with an enzyme activity ratio of 2:2:1). The amount of compound enzyme preparation added is 1.2 wt%. Enzymatic hydrolysis is carried out at 40°C for 1.5 hours. The enzymatic hydrolysis process can effectively destroy the cell wall and release polysaccharide components. After the enzymatic hydrolysis is completed, the mixture is placed in an ultrasonic-microwave synergistic extraction device with an ultrasonic power of 200 watts and a frequency of 35 kHz, a microwave power of 500 watts, and a processing time of 20 minutes.

[0076] (5) After cooling the extract to room temperature, add an ethanol-water mixed solvent (ethanol-water volume ratio of 4:1) and let it stand for 12 hours at 4°C for extraction.

[0077] (6) The supernatant was initially purified by passing it through an ultrafiltration membrane (with a molecular weight cutoff of 3000 Daltons) to remove macromolecular impurities and some proteins. The filtrate after ultrafiltration was further decolorized and purified by passing it through a cation exchange resin (AB-8) with a flow rate of 1.0 column volume / hour. The filtrate was eluted with deionized water and the eluent was collected.

[0078] (7) Alpinia oxyphylla polysaccharide extract obtained by concentration and drying.

[0079] Comparative Example 1

[0080] A method for extracting polysaccharides from Alpinia oxyphylla includes the following steps:

[0081] (1) Select high-quality Alpinia oxyphylla fruits, wash and remove impurities, and then microwave vacuum dry for 8 hours;

[0082] (2) After drying, crush the Alpinia oxyphylla seeds and pass them through a 400-mesh sieve. Select powder with a mesh size greater than 400 mesh (particle size less than 400 mesh).

[0083] (3) Mix the crushed Alpinia oxyphylla powder with deionized water at a mass ratio of 1:18, add Lactobacillus plantarum ATCC14917 and ferment for 20 hours;

[0084] (4) Add a compound enzyme preparation (cellulase, pectinase and protease, with an enzyme activity ratio of 3:2:1). The amount of compound enzyme preparation added is 1 wt%. Enzymatic hydrolysis is carried out at 40°C for 1.5 hours. The enzymatic hydrolysis process can effectively destroy the cell wall and release polysaccharide components. After the enzymatic hydrolysis is completed, the mixture is placed in an ultrasonic-microwave synergistic extraction device with an ultrasonic power of 200 watts and a frequency of 35 kHz, a microwave power of 500 watts, and a processing time of 20 minutes.

[0085] (5) After cooling the extract to room temperature, add an ethanol-water mixed solvent (ethanol-water volume ratio of 4:1) and let it stand for 12 hours at 4°C for extraction.

[0086] (6) The supernatant was initially purified by passing it through an ultrafiltration membrane (with a molecular weight cutoff of 3000 Daltons) to remove macromolecular impurities and some proteins. The filtrate after ultrafiltration was further decolorized and purified by passing it through a cation exchange resin (AB-8) with a flow rate of 1.0 column volume / hour. The filtrate was eluted with deionized water and the eluent was collected.

[0087] (7) Alpinia oxyphylla polysaccharide extract obtained by concentration and drying.

[0088] Comparative Example 2

[0089] A method for extracting polysaccharides from Alpinia oxyphylla includes the following steps:

[0090] (1) Select high-quality Alpinia oxyphylla fruits, wash and remove impurities, and then microwave vacuum dry for 8 hours;

[0091] (2) After drying, crush the Alpinia oxyphylla kernels and pass them through a 200-mesh sieve. Select particles with a mesh size of less than 200 mesh (particle size greater than the corresponding particle size of 200 mesh).

[0092] (3) Mix the crushed Alpinia oxyphylla powder with deionized water at a mass ratio of 1:18, add Lactobacillus plantarum ATCC14917 and ferment for 20 hours;

[0093] (4) Add a compound enzyme preparation (cellulase, pectinase and protease, with an enzyme activity ratio of 3:2:1). The amount of compound enzyme preparation added is 1 wt%. Enzymatic hydrolysis is carried out at 40°C for 1.5 hours. The enzymatic hydrolysis process can effectively destroy the cell wall and release polysaccharide components. After the enzymatic hydrolysis is completed, the mixture is placed in an ultrasonic-microwave synergistic extraction device with an ultrasonic power of 200 watts and a frequency of 35 kHz, a microwave power of 500 watts, and a processing time of 20 minutes.

[0094] (5) After cooling the extract to room temperature, add an ethanol-water mixed solvent (ethanol-water volume ratio of 4:1) and let it stand for 12 hours at 4°C for extraction.

[0095] (6) The supernatant was initially purified by passing it through an ultrafiltration membrane (with a molecular weight cutoff of 3000 Daltons) to remove macromolecular impurities and some proteins. The filtrate after ultrafiltration was further decolorized and purified by passing it through a cation exchange resin (AB-8) with a flow rate of 1.0 column volume / hour. The filtrate was eluted with deionized water and the eluent was collected.

[0096] (7) Alpinia oxyphylla polysaccharide extract obtained by concentration and drying.

[0097] Comparative Example 3

[0098] A method for extracting polysaccharides from Alpinia oxyphylla includes the following steps:

[0099] (1) Select high-quality Alpinia oxyphylla fruits, wash and remove impurities, and then microwave vacuum dry for 8 hours;

[0100] (2) After drying, the Alpinia oxyphylla kernels are crushed and sieved through 200 mesh and 400 mesh respectively, and particles between 200 mesh and 400 mesh are selected;

[0101] (3) Mix the crushed Alpinia oxyphylla powder with deionized water at a mass ratio of 1:18;

[0102] (4) Add a compound enzyme preparation (cellulase, pectinase and protease, with an enzyme activity ratio of 3:2:1). The amount of compound enzyme preparation added is 1 wt%. Enzymatic hydrolysis is carried out at 40°C for 1.5 hours. The enzymatic hydrolysis process can effectively destroy the cell wall and release polysaccharide components. After the enzymatic hydrolysis is completed, the mixture is placed in an ultrasonic-microwave synergistic extraction device with an ultrasonic power of 200 watts and a frequency of 35 kHz, a microwave power of 500 watts, and a processing time of 20 minutes.

[0103] (5) After cooling the extract to room temperature, add an ethanol-water mixed solvent (ethanol-water volume ratio of 4:1) and let it stand for 12 hours at 4°C for extraction.

[0104] (6) The supernatant was initially purified by passing it through an ultrafiltration membrane (with a molecular weight cutoff of 3000 Daltons) to remove macromolecular impurities and some proteins. The filtrate after ultrafiltration was further decolorized and purified by passing it through a cation exchange resin (AB-8) with a flow rate of 1.0 column volume / hour. The filtrate was eluted with deionized water and the eluent was collected.

[0105] (7) Alpinia oxyphylla polysaccharide extract obtained by concentration and drying.

[0106] Comparative Example 4

[0107] A method for extracting polysaccharides from Alpinia oxyphylla includes the following steps:

[0108] (1) Select high-quality Alpinia oxyphylla fruits, wash and remove impurities, and then microwave vacuum dry for 8 hours;

[0109] (2) After drying, the Alpinia oxyphylla kernels are crushed and sieved through 200 mesh and 400 mesh respectively, and particles between 200 mesh and 400 mesh are selected;

[0110] (3) Mix the crushed Alpinia oxyphylla powder with deionized water at a mass ratio of 1:18, add Lactobacillus plantarum ATCC14917 and ferment for 20 hours;

[0111] (4) Add pectinase at a rate of 1 wt% and hydrolyze at 40°C for 1.5 hours. The hydrolysis process can effectively destroy the cell wall and release polysaccharide components. After the hydrolysis is completed, place the mixture in an ultrasonic-microwave synergistic extraction device with an ultrasonic power of 200 watts and a frequency of 35 kHz, a microwave power of 500 watts, and a processing time of 20 minutes.

[0112] (5) After cooling the extract to room temperature, add an ethanol-water mixed solvent (ethanol-water volume ratio of 4:1) and let it stand for 12 hours at 4°C for extraction.

[0113] (6) The supernatant was initially purified by passing it through an ultrafiltration membrane (with a molecular weight cutoff of 3000 Daltons) to remove macromolecular impurities and some proteins. The filtrate after ultrafiltration was further decolorized and purified by passing it through a cation exchange resin (AB-8) with a flow rate of 1.0 column volume / hour. The filtrate was eluted with deionized water and the eluent was collected.

[0114] (7) Alpinia oxyphylla polysaccharide extract obtained by concentration and drying.

[0115] Comparative Example 5

[0116] A method for extracting polysaccharides from Alpinia oxyphylla includes the following steps:

[0117] (1) Select high-quality Alpinia oxyphylla fruits, wash and remove impurities, and then microwave vacuum dry for 8 hours;

[0118] (2) After drying, the Alpinia oxyphylla kernels are crushed and sieved through 200 mesh and 400 mesh respectively, and particles between 200 mesh and 400 mesh are selected;

[0119] (3) Mix the crushed Alpinia oxyphylla powder with deionized water at a mass ratio of 1:18, add Lactobacillus plantarum CICC 6009 and ferment for 20 hours;

[0120] (4) Add a compound enzyme preparation (cellulase, pectinase and protease, with an enzyme activity ratio of 3:2:1). The amount of compound enzyme preparation added is 1 wt%. Enzymatic hydrolysis is carried out at 40°C for 1.5 hours. The enzymatic hydrolysis process can effectively destroy the cell wall and release polysaccharide components. After the enzymatic hydrolysis is completed, the mixture is placed in an ultrasonic-microwave synergistic extraction device with an ultrasonic power of 200 watts and a frequency of 35 kHz, a microwave power of 500 watts, and a processing time of 20 minutes.

[0121] (5) After cooling the extract to room temperature, add an ethanol-water mixed solvent (ethanol-water volume ratio of 4:1) and let it stand for 12 hours at 4°C for extraction.

[0122] (6) The supernatant was initially purified by passing it through an ultrafiltration membrane (with a molecular weight cutoff of 3000 Daltons) to remove macromolecular impurities and some proteins. The filtrate after ultrafiltration was further decolorized and purified by passing it through a cation exchange resin (AB-8) with a flow rate of 1.0 column volume / hour. The filtrate was eluted with deionized water and the eluent was collected.

[0123] (7) Alpinia oxyphylla polysaccharide extract obtained by concentration and drying.

[0124] Comparative Example 6

[0125] A method for extracting polysaccharides from Alpinia oxyphylla includes the following steps:

[0126] (1) Select high-quality Alpinia oxyphylla fruits, wash and remove impurities, and then microwave vacuum dry for 8 hours;

[0127] (2) After drying, the Alpinia oxyphylla kernels are crushed and sieved through 200 mesh and 400 mesh respectively, and particles between 200 mesh and 400 mesh are selected;

[0128] (3) Mix the crushed Alpinia oxyphylla powder with deionized water at a mass ratio of 1:18, add Lactobacillus plantarum CICC 6076 ATCC 8014 and ferment for 20 hours;

[0129] (4) Add a compound enzyme preparation (cellulase, pectinase and protease, with an enzyme activity ratio of 3:2:1). The amount of compound enzyme preparation added is 1 wt%. Enzymatic hydrolysis is carried out at 40°C for 1.5 hours. The enzymatic hydrolysis process can effectively destroy the cell wall and release polysaccharide components. After the enzymatic hydrolysis is completed, the mixture is placed in an ultrasonic-microwave synergistic extraction device with an ultrasonic power of 200 watts and a frequency of 35 kHz, a microwave power of 500 watts, and a processing time of 20 minutes.

[0130] (5) After cooling the extract to room temperature, add an ethanol-water mixed solvent (ethanol-water volume ratio of 4:1) and let it stand for 12 hours at 4°C for extraction.

[0131] (6) The supernatant was initially purified by passing it through an ultrafiltration membrane (with a molecular weight cutoff of 3000 Daltons) to remove macromolecular impurities and some proteins. The filtrate after ultrafiltration was further decolorized and purified by passing it through a cation exchange resin (AB-8) with a flow rate of 1.0 column volume / hour. The filtrate was eluted with deionized water and the eluent was collected.

[0132] (7) Alpinia oxyphylla polysaccharide extract obtained by concentration and drying.

[0133] Verification Experiment

[0134] I. Extraction rate of Alpinia oxyphylla polysaccharides

[0135] 1. The detection method for polysaccharides from Alpinia oxyphylla is as follows:

[0136] (1) Preparation of Glucose Standard Curve

[0137] Pipette 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL of 100 mg / mL glucose standard solution into colorimetric tubes, and make up the volume to 1 mL with water respectively. Then add 1.0 mL of 5% phenol solution to each colorimetric tube, and then add 5.0 mL of concentrated sulfuric acid. Shake well, let stand for 10 min, then place in a water bath at 30 °C for 20 min. Using the blank as a control, measure the absorbance at a wavelength of 490 nm. Plot the standard curve with absorbance as the ordinate and glucose content as the abscissa to obtain the regression equation. The results are shown in Figure 1 .

[0138] (2) Determination of Polysaccharide Content in Samples

[0139] Take 1 mL of the prepared aqueous sample solution and place it in a 10 mL dry colorimetric tube respectively. The remaining operations are the same as (1), and measure the absorbance at a wavelength of 490 nm.

[0140] (3) Calculation of the Content of Polysaccharides from Alpinia oxyphylla

[0141] Polysaccharide content (%) = m / W × dilution factor × 100%

[0142] m: Glucose content (mg) calculated according to the regression equation

[0143] w: Dry weight of the sample (mg)

[0144] List the extraction rates of polysaccharides from Alpinia oxyphylla in the samples of the examples and comparative examples in Table 1.

[0145] Table 1 Comparison of Extraction Rates of Polysaccharides from Alpinia oxyphylla

[0146] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Extraction rate of polysaccharides from Alpinia oxyphylla (%) 17.8±0.21 16.5±0.33 15.8±0.47 8.9±0.26 11.2±0.51 10.5±0.35 6.9±0.42 13.2±0.3 12.9±0.4

[0147] II. Detection of the Effect of Polysaccharides from Alpinia oxyphylla on Improving (Liver) Injury

[0148] 1. Animals, Materials and Reagents

[0149] Purchase 120 healthy Kunming mice (SPF grade, female) from Zhuhai Bestong Biotechnology Co., Ltd. and raise them in the animal house of Guangdong Ocean University (environmental conditions: 12 h light / 12 h dark cycle using a bull automatic timer, temperature 22 ± 2 °C, humidity 50 ± 10%). They are allowed to freely ingest standard rodent feed and sterile drinking water, and are adaptively raised for 10 days. During the whole experiment, the bedding is changed every two days, and all operations strictly follow the animal ethics norms (license number: SCXK(Guangdong)2025 - 0051; NO.20250517).

[0150] 2. Establishment and grouping of T2DM mouse model

[0151] One hundred and twenty mice were acclimatized for one week and then randomly divided into two groups according to body weight. Ten mice served as the blank control group (NC) and were fed a standard diet. The other 110 mice served as the model group and were fed a high-fat, high-sugar diet with 60% fat as the energy source for four weeks to induce insulin resistance. After four weeks of high-fat feeding, the mice in the model group were fasted for 12 hours but allowed free access to water. Streptozotocin (STZ, Sigma) was administered intraperitoneally to induce insulin resistance. A 40 mg / mL STZ solution was prepared fresh (0.1 M, pH 4.5) and injected at a dose of 100 mg / kg. Simultaneously, the NC group received an equal volume of physiological saline. Seventy-two hours after model induction, fasting blood glucose (FBG) was measured by tail vein sampling. Two consecutive FBG values ​​≥11.1 mg / kg were required. Mice with a blood glucose level of mmol / L were considered to have successfully developed type 2 diabetes mellitus (T2DM). Subsequently, the 110 successfully modeled mice were randomly divided into 11 groups (n=10 per group): Model Group (M, continued high-fat diet), Positive Control Group (P, high-fat diet + metformin 200 mg / kg), Example Experimental Group 1 (H1, high-fat diet + Alpinia oxyphylla polysaccharide extract 200 mg / kg), Example Experimental Group 2 (H2, high-fat diet + Alpinia oxyphylla polysaccharide extract 200 mg / kg), Example Experimental Group 3 (H3, high-fat diet + Alpinia oxyphylla polysaccharide extract 200 mg / kg), and Comparative Experimental Group 1 (L1, high-fat diet + comparative Alpinia oxyphylla polysaccharide extract 200 mg / kg), Comparative Experimental Group 2 (L2, high-fat diet + comparative Alpinia oxyphylla polysaccharide extract 200 mg / kg), and Comparative Experimental Group 3 (L3, high-fat diet + comparative Alpinia oxyphylla polysaccharide extract 200 mg / kg). The experimental groups were divided into three groups: control group (L4, high-fat diet + control group 4 Alpinia oxyphylla polysaccharide extract 200 mg / kg), control group 5 (L5, high-fat diet + control group 5 Alpinia oxyphylla polysaccharide extract 200 mg / kg), and control group 6 (L6, high-fat diet + control group 6 Alpinia oxyphylla polysaccharide extract 200 mg / kg). The control group (NC) maintained a normal diet, and the control group (M) maintained a high-fat diet and was given an equal volume of physiological saline by gavage. All interventions were performed once daily throughout the experiment, and body weight and fasting blood glucose were monitored weekly (fasting was the same as before). Data were recorded for dynamic analysis.

[0152] 4. Measurement of antioxidant enzyme levels in mouse liver tissue

[0153] Mice in each group were euthanized by cervical dislocation. Liver tissue was rapidly removed, accurately weighed, and then added to pre-cooled phosphate buffer / physiological saline at a mass-to-volume ratio of 1:9 (g / mL). The mixture was homogenized in a cryo-homogeneous grinder to prepare a 10% tissue solution. After centrifugation at 4 °C, the supernatant was collected, and the activities of GSH, MDA, SOD, and CAT were determined according to the kit instructions. Results are shown in the table below. Figures 2-5 .

[0154] Table 2. Efficacy of Alpinia oxyphylla polysaccharide extract in improving liver damage

[0155] Blank group Model group Example 1 Experimental Group 1 Example 2 Experimental Group Example 3 Experimental Group 3 Comparative Experimental Group 1 Comparative Experimental Group 2 Comparative experimental group 3 Comparative experimental group 4 Comparative experimental group 5 Comparative experimental group 6 Positive control group GSH activity (mol / L) 53.16 ± 0.92 * ]] 32.31 ± 1.07 * ]] 45.76±0.64 44.58 ± 0.16 * ]] 45.24±0.72 40.63 ± 0.74 * ]] 38.55 ± 0.64 * ]] 39.29 ± 0.62 * ]] 36.53 ± 0.67 * ]] 40.68 ± 0.32 * ]] 40.97 ± 0.81 * ]] 48.15 ± 0.23 * ]] MDA activity (nmol / g) 5.97 ± 0.29 * ]] 13.03 ± 0.94 * ]] 7.56±0.16 7.92±0.26 7.72±0.73 11.09 ± 0.56 * ]] 10.95 ± 0.40 * ]] 10.53 ± 0.37 * ]] 10.19 ± 0.33 * ]] 9.88 ± 0.23 * ]] 10.37 ± 0.29 * ]] 8.83 ± 0.17 * <!-- 8 -->]] SOD activity (U / mgprot) 166.29 ± 0.70 * ]] 119.63 ± 1.92 * ]] 148.18±0.69 146.06 ± 0.72 * ]] 145.25 ± 1.03 * ]] 129.89 ± 1.14 * ]] 131.97 ± 0.57 * ]] 128.79 ± 1.62 * ]] 134.74 ± 0.69 * ]] 136.95 ± 0.62 * ]] <![CDATA[133.71±0.91 * ]]> 151.20±1.25 CAT(U / mgprot) <![CDATA[9.67±0.18 * ]]> <![CDATA[7.07±0.97 * ]]> 8.69±0.32 8.53±0.17 8.56±0.45 <![CDATA[7.70±0.29 * ]]> <![CDATA[7.57±0.32 * ]]> <![CDATA[7.88±0.42 * ]]> <![CDATA[7.73±0.30 * ]]> <![CDATA[7.56±0.14 * ]]> <![CDATA[7.46±0.29 * ]]> 8.83±0.17

[0156] Statistical analysis was performed using SPSS 27.0 software. Compared with Example 1, (*) indicates p<0.05, which means the difference is statistically significant.

[0157] The results above show that the Alpinia oxyphylla polysaccharide extraction method of the present invention, through fermentation with specific strains and control of particle size, yields Alpinia oxyphylla polysaccharide extract with high polysaccharide content. In vivo experiments show that after 4 weeks of intervention in the high-dose group, fasting blood glucose continued to decrease, and OGTT decreased more sharply; serum insulin levels decreased, liver SOD / CAT / GSH-Px activity increased, MDA decreased, and oxidative stress was reduced; it also showed multi-target regulation of glucose and lipid metabolism, and has significant advantages in improving diabetic liver damage.

[0158] The method for extracting polysaccharides from Alpinia oxyphylla of this invention involves fermentation with Lactobacillus plantarum ATCC14917, followed by extraction using a complex enzyme system of cellulase, pectinase, and protease, along with ultrasound-microwave synergistic extraction. Lactobacillus plantarum ATCC14917, as a probiotic, produces various extracellular enzymes during fermentation. These enzymes pre-decompose the polysaccharide and protein structures in the Alpinia oxyphylla cell wall, making them easier for subsequent enzymatic hydrolysis and extraction. Simultaneously, short-chain fatty acids, peptides, and small-molecule metabolites may be produced during fermentation. These substances possess anti-inflammatory and antioxidant activities, which can synergistically enhance the bioactivity of Alpinia oxyphylla polysaccharides. The complex enzyme system of cellulase, pectinase, and protease specifically disrupts the cellulose, pectin, and protein networks in the Alpinia oxyphylla cell wall, allowing for a more complete release of intracellular polysaccharides, flavonoids, sesquiterpenes, and other active components. During enzymatic hydrolysis, some large-molecule polysaccharides may be degraded into small-molecule polysaccharides or oligosaccharides, which are more easily absorbed and possess stronger immunomodulatory and antioxidant activities. Furthermore, the cavitation effect of ultrasound and the rapid heating of microwaves combined with molecular vibrations can further disrupt cell structure, increase polysaccharide dissolution rate, and avoid degradation of heat-sensitive components caused by prolonged high-temperature treatment. The Alpinia oxyphylla polysaccharide extract obtained through this process shows a significantly increased content of polysaccharide components (especially small-molecule polysaccharides and heteropolysaccharides), while retaining active substances such as Alpinia oxyphylla methyl ester and flavonoids. These components synergistically improve diabetic liver damage, demonstrating significant advantages.

[0159] The results above show that the Alpinia oxyphylla extraction method of the present invention, through fermentation with specific strains and control of particle size, yields Alpinia oxyphylla polysaccharide extract with high polysaccharide content, which has significant advantages in improving diabetic liver damage.

[0160] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting polysaccharides from Alpinia oxyphylla, comprising the following steps: (1) Select high-quality Alpinia oxyphylla fruits, wash and remove impurities, and then use microwave vacuum drying treatment; (2) The dried Alpinia oxyphylla kernels are crushed and sieved to obtain powder; (3) Mix the crushed Alpinia oxyphylla powder with deionized water and add Lactobacillus plantarum ATCC14917 for fermentation; (4) Add the compound enzyme preparation and perform enzymatic hydrolysis. After the enzymatic hydrolysis is completed, place the mixture in an ultrasonic-microwave synergistic extraction device for ultrasonic treatment. (5) After cooling the extract to room temperature, add an ethanol-water mixture and allow it to stand for extraction; (6) The supernatant was initially purified by ultrafiltration membrane to remove macromolecular impurities and some proteins. The filtrate after ultrafiltration was further decolorized and purified by cation exchange resin, eluted with deionized water, and the eluent was collected. (7) Alpinia oxyphylla polysaccharide extract obtained by concentration and drying.

2. The method for extracting Alpinia oxyphylla polysaccharide as described in claim 1, comprising the following steps: The specific steps (2) are as follows: the dried Alpinia oxyphylla kernels are crushed and sieved through 200 mesh and 400 mesh respectively, and particles between 200 mesh and 400 mesh are selected.

3. The method for extracting Alpinia oxyphylla polysaccharide as described in claim 1, comprising the following steps: The compound enzyme preparation in step (4) is a mixture of cellulase, pectinase and protease, with the enzyme activity ratio of cellulase, pectinase and protease being (2-3):2:(1-2).

4. The method for extracting Alpinia oxyphylla polysaccharide as described in claim 1, comprising the following steps: In step (3), the mass ratio of Alpinia oxyphylla powder to deionized water is 1:(15-20).

5. The method for extracting Alpinia oxyphylla polysaccharide as described in claim 1, comprising the following steps: The fermentation time of Lactobacillus plantarum ATCC14917 in step (3) is 18-24 hours.

6. The method for extracting Alpinia oxyphylla polysaccharide as described in claim 1, comprising the following steps: The amount of compound enzyme preparation added in step (4) is 0.8-1.2 wt%.

7. The method for extracting Alpinia oxyphylla polysaccharide as described in claim 1, comprising the following steps: In step (4), the enzymatic hydrolysis conditions are 40°C for 1.5 hours.

8. The method for extracting Alpinia oxyphylla polysaccharide as described in claim 1, comprising the following steps: In step (4), the ultrasonic conditions are 200 watts ultrasonic power, 35 kHz frequency, 500 watts microwave power, and 20 minutes processing time.

9. The method for extracting Alpinia oxyphylla polysaccharide as described in claim 1, comprising the following steps: In step (5), the volume ratio of ethanol to water in the ethanol-water mixed solvent is 4:

1.

10. The method for extracting Alpinia oxyphylla polysaccharide as described in claim 1, comprising the following steps: In step (6), the ultrafiltration membrane has a molecular weight cutoff of 3000 Daltons, and the filtrate is further decolorized and purified by cation exchange resin AB-8, with the flow rate controlled at 1.0 column volume / hour.

Images