Polygonatum cyrtonema leaf enzyme extraction polysaccharide as well as preparation method and application thereof

Abstract

The preparation method comprises the following steps: degreasing polygonatum cyrtonema leaf superfine powder, sequentially carrying out ultrasonic-assisted enzymolysis treatment on cellulase, xylanase, high-temperature-resistant alpha-amylase, papain and saccharifying enzyme, and carrying out alcohol precipitation, dialysis and vacuum freeze drying treatment to obtain the polygonatum cyrtonema leaf polysaccharide. The polygonatum cyrtonema leaf enzyme extraction crude polysaccharide is obtained; and separating the enzyme-extracted crude polysaccharide through a cellulose column, and purifying through a sephadex column to obtain the polygonatum cyrtonema leaf enzyme-extracted polysaccharide. Polysaccharide in polygonatum cyrtonema leaves is extracted through ultrasonic-assisted compound enzyme, single pure polysaccharide is obtained through alcohol precipitation, dialysis, cellulose column chromatography and gel column chromatography, and the purity can reach 99.86%; the obtained enzyme extraction polysaccharide has good anti-cancer activity, can significantly inhibit proliferation of breast cancer cells and colorectal cancer cells, has the characteristics of good biocompatibility, strong specificity, low toxic and side effects and the like, and has wide application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of polysaccharide extraction, and in particular to an enzyme-extracted polysaccharide from polygonatum cyrtonema leaves and a preparation method and application thereof. Background Art

[0002] my country's "medicine and food have the same origin" culture has a long history. There are currently 101 types of medicine and food on the list. These resources have not only been continuously verified efficacy, but also food safety, and have played an important role in protecting people's health. Polygonatum sibiricum Red is one of the representatives. Polygonatum sibiricum was first recorded in the "Famous Doctors' Records" of the Jin Dynasty, which states that "it tastes sweet, flat, and non-toxic. It mainly replenishes the middle and replenishes qi, removes dampness, and calms the five internal organs. Long-term use can make the body light, prolong life, and prevent hunger." It has the effects of nourishing the kidney and essence, nourishing yin and moistening dryness, and is widely used as a tonic. There are three types of Polygonatum sibiricum: Polygonatum sibiricum, Yunnan Polygonatum sibiricum and Polygonatum sibiricum. Compared with the first two, Polygonatum sibiricum has been less studied. Polygonatum sibiricum is a local Chinese medicinal material for both medicine and food in the southern region. Modern pharmacological effects have found that Polygonatum sibiricum has the effects of lowering blood sugar, improving immunity, anti-fatigue, anti-tumor, antibacterial and antiviral. In recent years, as people pay more and more attention to their health, the market demand for Polygonatum cyrtonema has been increasing. Wild Polygonatum cyrtonema resources are far from meeting people's needs. Artificially cultivated Polygonatum cyrtonema is the main source of Polygonatum cyrtonema on the market, and the cultivation area and scale are constantly expanding. However, the medicinal and edible parts of Polygonatum cyrtonema are mainly its rhizomes, and the research on the extraction, processing and pharmacological effects of bioactive ingredients is also mainly focused on the roots and stems. The non-medicinal parts of Polygonatum cyrtonema: its buds, fibrous roots, flowers and leaves are discarded as by-products, resulting in a large amount of resource waste due to the ineffective utilization.

[0003] At present, the research on the molecular cell activity of plant polysaccharides is mainly based on anti-inflammatory, antioxidant and anti-tumor effects. There are many studies on plant polysaccharides in anti-cancer. Due to the high harm of cancer to human health and the difficulty of treatment, many scholars have turned their attention to natural medicines, hoping to use natural substances such as plant polysaccharides for the prevention and treatment of tumors. Compared with chemotherapy and radiotherapy, naturally extracted polysaccharides, as anti-tumor drugs, have the advantages of good biocompatibility, gentle action, low toxicity and side effects, and not easy to produce drug resistance. There is no relevant research on enzyme-extracted polysaccharides in Polygonatum cyrtonema leaves at home and abroad. Patent CN 114349878A and Patent CN 117801132 A disclose a method for extracting polysaccharides from Polygonatum cyrtonema leaves, but the extraction processes of the two patents are water extraction and alcohol precipitation, which are different from the composition of enzyme-extracted polysaccharides, and thus the effects are different. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides an enzyme-extracted polysaccharide from the leaves of Polygonatum cyrtonema, and a preparation method and application thereof. The polysaccharide in the leaves of Polygonatum cyrtonema is extracted by ultrasound-assisted composite enzyme, and a single pure polysaccharide is obtained through alcohol precipitation, dialysis, cellulose column chromatography and gel column chromatography, with a purity of up to 99.86%. The obtained enzyme-extracted polysaccharide has good anti-cancer activity, can significantly inhibit the proliferation of breast cancer cells and colorectal cancer cells, has the characteristics of good biocompatibility, strong specificity, low toxic and side effects, etc., can make up for the deficiencies of traditional cancer treatment methods, and has broad application prospects.

[0005] In order to solve the above technical problems, the first aspect of the present invention provides a method for preparing polysaccharides extracted from leaves of Polygonatum cyrtonema by enzyme, comprising the following steps:

[0006] S1. After defatting, the ultrafine powder of Polygonatum cyrtonema leaf is mixed with pure water and placed in an ultrasonic water bath, ultrasonically treated at 40-60° C. for 30-60 min, and then alkali solution is added to adjust the pH to 4-6;

[0007] S2, enzymatic hydrolysis: add cellulase and xylanase, and water bath at 50-70°C; then add high temperature resistant α-amylase, and water bath at 70-90°C; cool the water bath to 50-70°C, add papain and water bath; heat to 90-100°C to inactivate enzymes, cool to 50-70°C and add saccharifying enzymes and water bath; heat to 90-100°C to inactivate enzymes, centrifuge and collect supernatant;

[0008] S3, subjecting the supernatant to alcohol precipitation, dialysis, and vacuum freeze-drying treatment to obtain crude polysaccharide extracted from Polygonatum cyrtonema leaves;

[0009] S4, separating the crude polysaccharide extracted by enzyme through a cellulose column, and then purifying it through a dextran gel column to obtain the polysaccharide extracted by enzyme from the leaves of Polygonatum cyrtonema.

[0010] Further, in S1, the degreasing treatment is specifically: using petroleum ether as a solvent, soaking treatment at 55-65°C for 90-120 minutes, heating to 65-70°C for extraction treatment for 120-150 minutes, then heating to 75-80°C for recovery treatment for 15-20 minutes, and after solid-liquid separation, obtaining the defatted product for subsequent treatment steps. The ratio of the Polygonatum cyrtonema leaf ultrafine powder to petroleum ether is 1g:40-50mL.

[0011] Furthermore, in S1, the mass ratio of the defatted Polygonatum cyrtonema leaf ultrafine powder to pure water is 1:(20-40); and the alkali solution is NaOH solution.

[0012] Furthermore, in S2, the added amounts of cellulase and xylanase are 800-1000U / g of the defatted Polygonatum cyrtonema leaf superfine powder, respectively; the added amount of α-amylase is 4-6μL / g of the defatted Polygonatum cyrtonema leaf superfine powder; the added amount of papain is 4-6μL / g of the defatted Polygonatum cyrtonema leaf superfine powder; and the added amount of saccharifying enzyme is 30-50μL / g of the defatted Polygonatum cyrtonema leaf superfine powder.

[0013] Furthermore, in S3, the alcohol precipitation is specifically as follows: adding anhydrous ethanol to the supernatant until the ethanol mass concentration is 10%-20%, standing overnight, centrifuging to remove the precipitate, adding anhydrous ethanol until the ethanol mass concentration is 60-80%, standing overnight, and centrifuging to collect the precipitate.

[0014] Furthermore, the supernatant is concentrated to 1 / 4-1 / 6 of the original volume by a rotary evaporator before alcohol precipitation.

[0015] Furthermore, in S3, the dialysis is specifically as follows: dissolving the precipitate collected by alcohol precipitation in pure water, dialyzing it with flowing water in a 3000-5000Da dialysis bag for 2-3 days, then dialyzing it with ultrapure water for 1-2 days, and then performing a freeze-drying step after concentrating under reduced pressure.

[0016] Furthermore, in S3, the vacuum freeze-drying is specifically as follows: after being placed at -50°C to -60°C for 4-6 hours, placed at -35 to -40°C for 10-15 hours; then placed at -10 to -20°C for 10-15 hours, and finally placed at 20-25°C for 24-48 hours.

[0017] Furthermore, in S1, the preparation method of the Polygonatum cyrtonema leaf ultrafine powder is: drying the Polygonatum cyrtonema leaf, crushing it to 200 mesh sieve, and grinding it with an ultrafine pulverizer to obtain micron-grade ultrafine powder.

[0018] Furthermore, in S4, the eluent for separation and purification is distilled water, and the elution rate is 0.5-0.7 mL / min.

[0019] The second aspect of the present invention provides the preparation method described in the first aspect for preparing polysaccharides extracted from leaves of Polygonatum cyrtonema, wherein the molecular weight of the polysaccharides extracted from the leaves of Polygonatum cyrtonema is 20kD-30kD, and the polydispersity coefficient is 1.04.

[0020] The third aspect of the present invention provides the use of the polysaccharide extracted from the leaves of Polygonatum cyrtonema as described in the second aspect in the preparation of anticancer drugs.

[0021] Furthermore, the anticancer drug is an anti-breast cancer drug or an anti-colorectal cancer drug.

[0022] Furthermore, the polysaccharide extracted from the leaves of Polygonatum cyrtonema can achieve an inhibition rate of 60%-70% on breast cancer cells 4T1, and can achieve an inhibition rate of 70%-80%, 80-90% and 50%-60% on colorectal cancer cells HT29, Caco-2 and CT26, respectively, and still has good cell safety for the normal colon mucosal epithelial cell line NCM460 when the dosage concentration reaches 10 mg / mL.

[0023] The fourth aspect of the present invention provides an anticancer drug comprising the polysaccharide extracted from the leaves of Polygonatum cyrtonema as described in the second aspect.

[0024] Beneficial effects of the present invention:

[0025] The invention adopts ultrasound to assist the action of cellulase and xylanase to destroy the cell wall of polygonatum sibiricum leaf and promote the dissolution of polysaccharide, thereby increasing the yield of polysaccharide extracted by enzyme from polygonatum sibiricum leaf; by adding papain and high temperature resistant alpha-amylase, the removal rate of protein and starch is greatly improved, and the purity of polysaccharide extracted by enzyme from polygonatum sibiricum leaf is improved; through alcohol precipitation, dialysis, cellulose column chromatography and gel column chromatography, a new single pure polysaccharide is obtained, and the purity can reach 99.86%.

[0026] The enzyme-extracted polysaccharide obtained by the present invention has good anti-cancer activity, can significantly inhibit the proliferation of breast cancer cells and colorectal cancer cells, especially colorectal cancer HT-29 and Caco-2 cells, and promotes the application of polysaccharides as anti-cancer drugs; at the same time, it can also avoid damage to the proliferation ability and cell state of normal cells, making up for the shortcomings of existing chemical anti-cancer drugs with low specificity and large side effects on the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the embodiments are briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 This is the fiber column elution curve of crude polysaccharide extracted by enzyme from Polygonatum cyrtonema leaves in Example 1 of the present invention;

[0029] Figure 2 This is a high performance liquid chromatogram (HPLC) of crude polysaccharide extracted by enzyme from leaves of Polygonatum cyrtonema in Example 1 of the present invention;

[0030] Figure 3 This is the dextran column elution curve of the polysaccharide extracted from the leaves of Polygonatum cyrtonema in Example 1 of the present invention;

[0031] Figure 4 This is a high performance liquid chromatogram (HPLC) of polysaccharides extracted from leaves of Polygonatum cyrtonema in Example 1 of the present invention;

[0032] Figure 5 This is a relative molecular mass standard curve of the polysaccharide extracted from the leaves of Polygonatum cyrtonema in Example 1 of the present invention;

[0033] Figure 6 is the inhibition rate of the polysaccharide extracted from the leaves of Polygonatum cyrtonema on HT29 in Application Example 1 of the present invention;

[0034] Figure 7 is the inhibition rate of polysaccharide extracted from Polygonatum cyrtonema leaves on Caco-2 in Application Example 1 of the present invention;

[0035] Figure 8 is the inhibition rate of the polysaccharide extracted from the leaves of Polygonatum cyrtonema on CT26 in Application Example 1 of the present invention;

[0036] Fig. 9 is the inhibition rate of the polysaccharide extracted from the leaves of Polygonatum cyrtonema against 4T1 in Application Example 1 of the present invention;

[0037] Fig.10 It is the survival rate of NCM460 after being treated with enzyme-extracted polysaccharides from the leaves of Polygonatum cyrtonema in Application Example 1 of the present invention.

[0038] Fig.11 The inhibition rate of the water-extracted polysaccharide from Polygonatum cyrtonema leaves on HT29 in Comparative Example 1;

[0039] Fig.12 The inhibition rate of the polysaccharide extracted from the leaves of Polygonatum cyrtonema on Caco-2 in Comparative Example 1;

[0040] Fig.13 The inhibition rate of the water-extracted polysaccharide from Polygonatum cyrtonema leaves on CT26 in Comparative Example 1;

[0041] Fig.14 This is the inhibition rate of the water-extracted polysaccharides from Polygonatum cyrtonema leaves on 4T1 in Comparative Example 1. DETAILED DESCRIPTION

[0042] The technical solution of the present invention will be described clearly and completely below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Example 1

[0044] This embodiment relates to a method for preparing polysaccharides extracted by enzyme from Polygonatum cyrtonema leaves, comprising the following steps:

[0045] (1) Preparation of Polygonatum cyrtonema leaf ultrafine powder

[0046] The leaves of Polygonatum cyrtonema were placed in a 40°C oven for overnight drying, ground and passed through a 200-mesh sieve, and the powder was then ground with an ultra-high-speed ultra-fine grinder to obtain ultra-fine powder of the leaves of Polygonatum cyrtonema.

[0047] (2) Preparation of crude polysaccharides extracted from Polygonatum cyrtonema leaves

[0048] Degreasing: Weigh about 1g of Polygonatum cyrtonema leaf ultrafine powder, wrap it in qualitative filter paper (diameter: 18cm), tie the filter paper with cotton rope and place it in a Soxhlet fat extractor, and use 50mL petroleum ether as solvent for degreasing. The degreasing conditions are immersion at 65℃ for 90min, extraction at 70℃ for 120min, recovery at 80℃ for 15min, and then drying at 50℃ to obtain the defatted powder sample.

[0049] Enzyme extraction: weigh about 10g of defatted Polygonatum cyrtonema leaf powder and place it in a beaker, add 300mL of pure water, stir and mix thoroughly, then place in an ultrasonic water bath, ultrasonicate at 40℃ for 30min. Use NaOH solution to adjust the pH of the Polygonatum cyrtonema pollen solution to 5. Add 1000U / g of cellulase and xylanase from Polygonatum cyrtonema leaf superfine powder, bathe at 60℃ for 3h, then add 5μL / g of thermostable α-amylase from Polygonatum cyrtonema leaf superfine powder, bathe at 80℃ for 1h; adjust the water bath temperature to 60℃, add 5μL / g of papain from Polygonatum cyrtonema leaf superfine powder, bathe at 100℃ for 10min, inactivate the enzyme at 100℃ for 10min, add 50μL / g of saccharifying enzyme from Polygonatum cyrtonema leaf superfine powder after cooling, bathe at 60℃ for 3h; inactivate the enzyme at 100℃ for 10min. Centrifuge after extraction and collect the supernatant.

[0050] Alcohol precipitation: After the supernatant obtained by enzymatic hydrolysis is concentrated to 1 / 5 of the original volume in a rotary evaporator, anhydrous ethanol is added dropwise while stirring to make the ethanol concentration in the solution reach 10%, let it stand overnight, and centrifuge to remove the precipitate; anhydrous ethanol is added to the supernatant to ensure that the final ethanol concentration of the system is 70%, let it stand overnight, and centrifuge to collect the precipitate.

[0051] Dialysis: The precipitate obtained after the above centrifugation was re-dissolved with pure water, added to a 3500Da dialysis bag and dialyzed with flowing water for 2 days, and dialyzed with ultrapure water for 1 day. The dialyzed polysaccharide solution was concentrated under reduced pressure, placed at -50℃~-60℃ for 4-6 hours, then placed at -35~-40℃ for 10-15 hours; then placed at -10~-20℃ for 10-15 hours, and finally placed at 20-25℃ for 24-48 hours for freeze-drying, thereby obtaining the polysaccharide extracted from the leaves of Polygonatum cyrtonema.

[0052] (3) Preparation of Polysaccharides Extracted from Polygonatum cyrtonema Leaves

[0053] Weigh 0.5g of crude polysaccharide sample extracted from Polygonatum cyrtonema leaf enzyme, dissolve in 10mL pure water, place in a metal bath at 55℃ for 1h, then stir overnight at 4℃, centrifuge and draw the supernatant and add it dropwise to the top of the chromatography column filled with DEAE-52, and elute with distilled water and 0.25M, 0.5M, and 1M sodium chloride solutions in sequence. The flow rate is: 0.7mL / min, 5min / tube collection, and 80 tubes are collected each time. Samples are taken from 4 test tubes at intervals, 0.1mL of the collected liquid is taken, 0.9mL of pure water is added, 1mL of 5% phenol solution is added and vortexed, 5mL of concentrated sulfuric acid is added, and the colorimetry is compared with the blank sample after vortexing and the absorbance is recorded, and the corresponding elution curve is drawn as shown in the figure. Figure 1 .Depend on Figure 1 It can be seen that different concentrations of eluent can effectively separate these polysaccharide components with different molecular weights, among which the highest sugar content is in tubes 20 to 50 eluted with water as the eluent. Since the sugar content in the sugar-containing test tubes eluted with 0.25M NaCl solution and 0.5M NaCl solution was too low, the subsequent experiments did not continue to collect the sugar-containing test tubes eluted with 0.25M NaCl solution and 0.5M NaCl solution. The polysaccharide components eluted with pure water were combined and concentrated to about 5mL, frozen at -80℃ for one day, and vacuum freeze-dried to obtain the polysaccharide extracted from the leaves of Polygonatum multiflorum by DEAE-52 column.

[0054] The polysaccharide sample eluted with pure water was prepared into a 10 mg / mL solution with pure water. After passing through a 0.45 μm filter membrane, 20 μL was sampled and injected into a high performance liquid chromatograph for detection. The polysaccharide sample with the highest polysaccharide purity was retained. Its HPLC chromatogram is shown in FIG. Figure 2 It can be seen that the polysaccharide component after elution from the DEAE-52 column is not single, but contains two main peaks, which needs to be further purified with a Sephacryl-400 column. Pipette the DEAE-52 eluate and add it dropwise to the upper end of the column filled with Sephacryl-100, and elute with distilled water. The flow rate is: 0.7mL / min, 5min / tube collection, and 100 tubes are collected. Take samples every 4 test tubes, take 0.1mL of the collection liquid, add 0.9mL of pure water, add 1mL of 5% phenol solution and vortex, add 5mL of concentrated sulfuric acid, vortex and compare colorimetry with the blank sample and record the absorbance, and draw the elution curve as shown in the figure. Figure 3 It can be seen that after elution and purification by Sephacryl-400 column, only sugar components were separated from tubes 25 to 60, and the sugar components were collected and concentrated to about 5 mL. The sugar solution was frozen at -80°C for one day and vacuum freeze-dried to obtain the polysaccharide extracted from the leaves of Polygonatum cyrtonema by Sephacryl-100 column.

[0055] The eluted polysaccharide extracted from the leaves of Polygonatum cyrtonema was prepared into a 10 mg / mL solution with pure water, filtered through a 0.45 μm filter membrane, and then injected into a high performance liquid chromatograph for detection. The detection instrument was an e2695 high performance liquid chromatograph equipped with a Waters 2414 differential detector and a SUGAR KS-805 (8.0mm ID×300mm L) sugar column; mobile phase: ultrapure water; flow rate: 1.0mL / min; column temperature: 35℃; detector temperature: 30℃; injection volume: 20μL. The results are as follows Figure 4 It can be seen that after elution with Sephacryl-100 column, there was no obvious impurity peak in the HPLC chromatogram of the enzyme-extracted polysaccharide from the leaves of polygonatum sibiricum. The polysaccharide of a single component was successfully separated. The purity was 99.86% calculated by peak area. The refined enzyme-extracted polysaccharide from the leaves of polygonatum sibiricum was obtained, and the total extraction rate was 8.92%.

[0056] Test Example 1

[0057] The polysaccharide extracted from the leaves of Polygonatum cyrtonema purified in Example 1 was prepared into a 5 mg / mL aqueous solution with pure water, and the sugar anhydride standards with relative molecular weights of 4400, 9900, 21400, 43500, 124000, and 805000 were prepared into a 5 mg / mL aqueous solution with pure water. Each solution was filtered through a 0.45 μm filter membrane and then sampled and tested using an EcoSEC HLC-8320GPC detection instrument. The corresponding standard curve was drawn according to the peak time of each sugar anhydride standard, as shown in FIG. Figure 5 As shown. The relative molecular mass of the sample can be calculated from the standard curve of relative molecular mass, as shown in Table 1. Its weight average molecular weight Mw and number average molecular weight Mn are 26796Da and 25765Da respectively, and the polydispersity index (PDI=Mw / Mn) of the corresponding polysaccharide molecule is 1.04. Since the closer the PDI value is to 1, the better the uniformity of the corresponding substance, it can be considered that the prepared polysaccharide sample extracted from the leaves of Polygonatum cyrtonema is a relatively uniform polysaccharide.

[0058] Table 1 Determination results of relative molecular weight of polysaccharides extracted from Polygonatum cyrtonema leaves

[0059] Retention time / min Weight average molecular weight Mw / Da Number average molecular weight Mn / Da Polydispersity coefficient 26.80 26796 25765 1.04

[0060] Application Example 1

[0061] This application example involves the application of polysaccharide extracted from leaves of Polygonatum cyrtonema (obtained in Example 1) in anticancer treatment, and the specific implementation process is as follows:

[0062] 1. The polysaccharides extracted from the leaves of Polygonatum cyrtonema have an inhibitory effect on the proliferation of colorectal cancer (HT29, Caco-2, CT26) cells and breast cancer 4T1 cells.

[0063] After the colorectal cancer (HT29, Caco-2, CT26) cells and breast cancer 4T1 cells were revived, they were cultured in DMEM medium containing 10% FBS and placed in an incubator at 37°C and 5% CO2. When the cells covered the bottom of the cell bottle, they were digested with trypsin, centrifuged, the supernatant was discarded, 2 mL of medium was added to the precipitate, blown, counted using a hemocytometer, and the cells were diluted proportionally. The cells were inoculated into a 96-well plate (100 μL of cell suspension per well, i.e., 5,000 cells), and cultured in an incubator for 24 hours. DMEM medium containing polysaccharides extracted from Polygonatum cyrtonema leaves at concentrations of 0, 0.625, 1.25, 2.5, 5, and 10 mg / mL was added to the 96-well plate and incubated at 37°C for 48 hours. Carefully discard the supernatant, add 100 μL of 0.5 mg / mL MTT to each well, continue incubating in a 37°C incubator for 4 hours, carefully remove the MTT solution, add 100 μL of DMSO to each well, continue incubating in a 37°C incubator for 10 minutes, fully oscillate on an oscillator until the blue-purple crystals are completely dissolved, and detect the OD value of each well at 570 nm using an automatic microplate reader. Calculate cell viability according to the following formula. Cell viability (%) = OD value of experimental group / OD value of control group × 100%. The results are as follows Figure 6-9 As shown, after preliminary screening of four types of tumor cells, it was found that the polysaccharide extracted from the leaves of Polygonatum cyrtonema of the present invention had a significant inhibitory effect on the proliferation of breast cancer and colorectal cancer cells, among which the inhibitory effect on HT29 and Caco-2 cells was particularly significant. After 48 hours of treatment with 0.625 mg / mL of polysaccharide extracted from the leaves of Polygonatum cyrtonema, the proliferation and growth of HT29 and Caco-2 colorectal cancer cells were significantly affected, with inhibition rates of 30.96% and 36.52%, respectively; and after 48 hours of treatment with 10 mg / mL of polysaccharide extracted from the leaves of Polygonatum cyrtonema, the proliferation of HT29 and Caco-2 colorectal cancer cells was almost completely inhibited, with inhibition rates of 78.37% and 86.25%, respectively.

[0064] 2. The inhibitory effect of polysaccharides extracted from Polygonatum cyrtonema leaves on the proliferation of human normal colon mucosal epithelial cells NCM460.

[0065] After the human normal colon mucosal epithelial cells NCM460 were revived, they were cultured in 1640 medium containing 10% FBS and placed in an incubator at 37°C and 5% CO2. When the cells covered the bottom of the cell bottle, they were digested with trypsin, centrifuged, the supernatant was discarded, 2 mL of medium was added to the precipitate, blown, counted using a hemocytometer, and the cells were diluted proportionally. The cells were inoculated into a 96-well plate (100 μL of cell suspension per well, i.e., 5,000 cells), and cultured in an incubator for 48 hours. 1640 medium containing polysaccharides extracted from Polygonatum cyrtonema leaves at concentrations of 0, 0.625, 1.25, 2.5, 5, and 10 mg / mL was added to the 96-well plate and incubated at 37°C for 24 hours. Carefully discard the supernatant, add 100 μL of 0.5 mg / mL MTT to each well, continue incubating in a 37°C incubator for 4 hours, carefully remove the MTT solution, add 100 μL of DMSO to each well, continue incubating in a 37°C incubator for 10 minutes, fully oscillate on an oscillator until the blue-purple crystals are completely dissolved, and detect the OD value of each well at 570 nm using an automatic microplate reader. Calculate cell viability according to the following formula. Cell viability (%) = OD value of experimental group / OD value of control group × 100%. The cell survival rate results are as follows: Fig.10 As shown, it can be seen that after incubating NCM460 cells with polysaccharides extracted from the leaves of Polygonatum cyrtonema, the cell survival rate has no obvious downward trend, which proves that the polysaccharides extracted from the leaves of Polygonatum cyrtonema have no inhibitory effect on the proliferation of normal cells.

[0066] Comparative Example 1

[0067] This comparative example involves the preparation and application of a polysaccharide from Polygonatum cyrtonema leaf. The preparation method is the same as that in the embodiment of patent CN 114349878A. The obtained polysaccharide has a molecular weight of 44.7 kD and a yield of about 4.63%.

[0068] After the colorectal cancer (HT29, Caco-2, CT26) cells and breast cancer 4T1 cells were revived, they were cultured in DMEM medium containing 10% FBS and placed in an incubator at 37°C and 5% CO2. When the cells covered the bottom of the cell bottle, the cells were digested with trypsin, centrifuged, the supernatant was discarded, 2 mL of culture medium was added to the precipitate, blown, counted using a hemocytometer, and the cells were diluted proportionally. The cells were inoculated into a 96-well plate (100 μL of cell suspension per well, i.e., 5000 cells), and cultured in an incubator for 24 hours. The Polygonatum sibiricum polysaccharide culture medium prepared in Comparative Example 1 at the same concentration as in Application Example 1 was added to the 96-well plate and incubated at 37°C for 48 hours. Carefully discard the supernatant, add 100 μL of 0.5 mg / mL MTT to each well, continue incubating in a 37°C incubator for 4 hours, carefully remove the MTT solution, add 100 μL of DMSO to each well, continue incubating in a 37°C incubator for 10 minutes, fully oscillate on an oscillator until the blue-purple crystals are completely dissolved, and detect the OD value of each well at 570 nm using an automatic microplate reader. Calculate cell viability according to the following formula. Cell viability (%) = OD value of experimental group / OD value of control group × 100%. The results are as follows Figure 11-14 As shown, after preliminary screening of four tumor cells, it was found that the polysaccharide sample prepared in comparative example 1 only had a significant inhibitory effect on the proliferation of mouse colorectal cancer cell HT29, which was equivalent to the effect of enzyme-extracted polysaccharide, while the inhibitory activity on the other three tumor cells was significantly reduced compared with enzyme-extracted polysaccharide. The enzyme-extracted polysaccharide prepared in Example 1 significantly improved the inhibitory effect on Caco-2 cells. At a concentration of 10 mg / mL, the inhibition rate can reach more than 80%, while the water-extracted polysaccharide prepared in comparative example 1 had an inhibition rate of only about 50% on Caco-2 cells at the same concentration.

[0069] The present invention can further hydrolyze xylan and cellulose in the leaves of Polygonatum cyrtonema by adding xylanase and cellulase, so the enzyme extraction can significantly increase the polysaccharide yield from 4.63% to 8.92%. The molecular weight of the water-extracted polysaccharide in Comparative Example 1 is 44.7 kD, while the molecular weight of the enzyme-extracted polysaccharide is 26.7 kD, which proves that the polysaccharide structures obtained by the two extraction methods are not the same, which is also the reason why the enzyme-extracted polysaccharide has a significantly better inhibitory effect on four tumor cells than the water-extracted polysaccharide.

[0070] In summary, the present invention uses ultrasound-assisted composite enzyme to extract polysaccharides from the leaves of Polygonatum cyrtonema, and obtains a single pure polysaccharide with a purity of up to 99.86% through alcohol precipitation, dialysis, cellulose column chromatography and gel column chromatography; the obtained enzyme-extracted polysaccharide has good anti-cancer activity, can significantly inhibit the proliferation of breast cancer cells and colorectal cancer cells, has the characteristics of good biocompatibility, strong specificity, low toxic and side effects, can make up for the shortcomings of traditional cancer treatment methods, and has broad application prospects.

[0071] The present invention has been described in detail above in conjunction with specific implementations and exemplary examples, but these descriptions cannot be understood as limiting the present invention. Those skilled in the art understand that, without departing from the spirit and scope of the present invention, a variety of equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its implementation methods, all of which fall within the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing polysaccharides extracted from Polygonatum cyrtonema leaves by enzyme extraction, characterized in that: The steps include: S1. After defatting, the ultrafine powder of Polygonatum cyrtonema leaf is mixed with pure water and placed in an ultrasonic water bath, ultrasonically treated at 40-60° C., and then alkali solution is added to adjust the pH to 4-6; S2, enzymatic hydrolysis: add cellulase and xylanase, and water bath at 50-70°C; then add high temperature resistant α-amylase, and water bath at 70-90°C; cool the water bath to 50-70°C, add papain and water bath; heat to 90-100°C to inactivate enzymes, cool to 50-70°C and add saccharifying enzymes and water bath; heat to 90-100°C to inactivate enzymes, centrifuge and collect supernatant; S3, subjecting the supernatant to alcohol precipitation, dialysis, and vacuum freeze-drying treatment to obtain crude polysaccharide extracted from Polygonatum cyrtonema leaves; S4, separating the crude polysaccharide extracted by enzyme through a cellulose column, and then purifying it through a dextran gel column to obtain the polysaccharide extracted by enzyme from the leaves of Polygonatum cyrtonema.

2. The method for preparing polysaccharides extracted from leaves of Polygonatum cyrtonema as claimed in claim 1, characterized in that: In S1, the degreasing treatment is specifically: using petroleum ether as a solvent, after soaking treatment at 55-65°C, heating to 65-70°C for extraction treatment, and then heating to 75-80°C for recovery treatment.

3. The method for preparing polysaccharides extracted from leaves of Polygonatum cyrtonema as claimed in claim 1, characterized in that: In S3, the alcohol precipitation is specifically as follows: adding anhydrous ethanol to the supernatant until the ethanol mass concentration is 10%-20%, standing and centrifuging to remove the precipitate, adding anhydrous ethanol until the ethanol mass concentration is 60-80%, standing and centrifuging to collect the precipitate.

4. The method for preparing polysaccharides extracted from leaves of Polygonatum cyrtonema as claimed in claim 1, characterized in that: In S3, the dialysis specifically comprises: dissolving the precipitate collected by alcohol precipitation in pure water, dialyzing it with flowing water in a 3000-5000Da dialysis bag, and then dialyzing it with ultrapure water.

5. The method for preparing polysaccharides extracted from leaves of Polygonatum cyrtonema as claimed in claim 1, characterized in that: In S3, the vacuum freeze drying is specifically: placed at -50℃~-60℃ for 4-6h, then placed at -35~-40℃ for 10-15h; then placed at -10~-20℃ for 10-15h, and finally placed at 20-25℃ for 24-48h.

6. The method for preparing polysaccharides extracted from leaves of Polygonatum cyrtonema as claimed in claim 1, characterized in that: In S1, the preparation method of the Polygonatum cyrtonema leaf ultrafine powder is: drying the Polygonatum cyrtonema leaf, crushing and sieving it, and then grinding it with an ultrafine grinder.

7. The method for preparing polysaccharides extracted from leaves of Polygonatum cyrtonema as claimed in claim 1, characterized in that: In S4, the separation and purification eluent is distilled water, and the elution rate is 0.5-0.7 mL / min.

8. A method for preparing polysaccharide extracted from leaves of Polygonatum cyrtonema by enzyme according to any one of claims 1 to 7, characterized in that: The molecular weight of the enzyme-extracted polysaccharide is 20kD-30kD.

9. A use of the polysaccharide extracted from the leaves of Polygonatum cyrtonema according to claim 8 in the preparation of anticancer drugs, characterized in that: The anticancer drug is an anti-breast cancer drug or an anti-colorectal cancer drug.

10. An anticancer drug, characterized in that: The invention comprises the polysaccharide extracted from the leaves of Polygonatum cyrtonema as claimed in claim 8.

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