CN115725004B - Preparation method and application of highland barley tender leaf enzyme extracted polysaccharide - Google Patents
Preparation method and application of highland barley tender leaf enzyme extracted polysaccharide Download PDFInfo
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Abstract
The invention discloses a preparation method and application of highland barley tender leaf enzyme extracted polysaccharide; the preparation method of the highland barley tender leaf enzyme-extracted polysaccharide can obtain high-purity enzyme-extracted polysaccharide, wherein the extraction rate of the enzyme-extracted polysaccharide can reach 7.68%, and the purity can reach 99.75%. The research of the invention discovers that the highland barley tender leaf enzyme extracted polysaccharide has good anticancer activity and can obviously inhibit proliferation of breast cancer cells and colorectal cancer cells. Especially can inhibit proliferation of colorectal cancer cells specifically, does not influence proliferation of normal colon mucosa epithelial cells, has the characteristics of good biocompatibility, strong specificity, low toxic and side effects and the like, can make up for the defects of the traditional cancer treatment means, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of anticancer active ingredients, relates to a preparation method of highland barley tender leaf enzyme-extracted polysaccharide and application thereof, in particular to a preparation method of highland barley tender leaf enzyme-extracted polysaccharide and application thereof in anticancer, and especially relates to a preparation method of highland barley tender leaf enzyme-extracted polysaccharide and application thereof in specifically inhibiting colorectal cancer cell proliferation.
Background
Highland barley grows in the severe cold and anoxic highland area, is the most important carbohydrate source in daily diet of Tibetan people, has outstanding medical health care effect, and is considered as one of the important factors of the Tibetan people for longevity. The highland barley obtains extremely strong stress resistance under the special environments of severe cold, hypoxia, dryness, strong light irradiation and the like, and generates extremely rich secondary metabolites. Highland barley is popular with people because of its comprehensive and unique nutritional composition and high dietary therapy value. The scholars indicate that the content of enzyme-extracted polysaccharide in highland barley is generally higher than that of barley varieties planted in other parts of the world. Therefore, the enzyme-extracted polysaccharide is also considered as a nutrient substance which is most closely related to the health and longevity of human bodies in highland barley. According to the report, the enzyme-extracted polysaccharide has: 1) Reducing serum cholesterol, and preventing cardiovascular diseases; 2) Regulating blood sugar level, and preventing diabetes; 3) Promoting intestinal probiotics proliferation and preventing intestinal cancer; 4) Regulating the immune function of the organism and the like, and has wide application prospect. At present, research on plant polysaccharide is mainly focused on cereal crops such as wheat, barley, corn and the like, and extraction methods thereof are mainly divided into a water method, an alkaline method, an enzyme method and a combination of the methods. Wherein, the enzymatic modification mainly comprises removing starch and protein by adding enzyme such as amylase and protease, and adding cellulase to cut off beta-1, 4-glycosidic bond of insoluble dietary fiber, so as to enhance physical and chemical properties such as solubility and water-holding capacity of polysaccharide. The method has the advantages of simple reaction conditions, low requirements on equipment and convenient operation; in addition, the enzymatic modification has the characteristic of specificity. The method of the invention adopts a complex enzyme method to extract, which is the best method after laboratory optimization, increases the yield of polysaccharide to the maximum extent and ensures the bioactivity of the polysaccharide.
At present, the research on the cell activity of plant polysaccharide molecules mainly comprises anti-inflammatory effect, antioxidant effect and anti-tumor effect, the research on the plant polysaccharide in anti-cancer aspect is numerous, and due to the high harm of cancer to human health and high treatment difficulty, many scholars aim at natural medicines, and hope to use natural substances such as plant polysaccharide for preventing and treating tumors. There are relatively few studies on enzyme-extracted polysaccharides from naked barley (highland barley is one of naked barley) at home and abroad. By referring to the research method of expert scholars on enzyme-extracted polysaccharide in grains, the extraction method and the process thereof are optimized, and the enzyme-extracted polysaccharide with high purity is successfully obtained by separating and purifying highland barley tender leaves. In addition, compared with chemotherapy and radiotherapy, the polysaccharide extracted naturally has the advantages of good biocompatibility, mild effect, low toxic and side effect, difficult generation of drug resistance and the like as an anti-tumor drug. Therefore, the development of the anticancer activity of the highland barley tender leaf enzyme-extracted polysaccharide not only can expand the application range of highland barley tender leaves and improve the additional value of related products, but also can overcome the defects of large toxic and side effects, high price and the like of the traditional anticancer drugs. In conclusion, the invention has wide application prospect in developing natural and safe anticancer drugs.
Disclosure of Invention
The invention provides a preparation method and application of highland barley tender leaf enzyme extracted polysaccharide. In particular, the method comprises the steps of,
the first aim is to provide a novel enzyme extracted polysaccharide active ingredient extracted and purified from highland barley tender leaves.
The second aim of the invention is to provide a preparation method of the highland barley tender leaf enzyme extracted polysaccharide.
The third object of the invention is to provide the application of the highland barley tender leaf enzyme extracted polysaccharide active ingredient in preparing anticancer drugs.
The aim of the invention is realized by the following technical scheme:
in a first aspect, the invention relates to a preparation method for extracting polysaccharide active ingredients from highland barley tender leaf enzymes, which comprises the following steps:
s1, vacuum freeze-drying highland barley leaves, powdering and collecting highland barley tender leaf powder;
s2, degreasing, enzyme extracting and alcohol precipitating highland barley tender leaf powder to obtain crude highland barley enzyme extracted polysaccharide;
s3, separating crude polysaccharide extracted from highland barley enzyme by using a cellulose column, and purifying by using a sephadex column to obtain active components of polysaccharide extracted from highland barley tender leaf enzyme.
As one embodiment of the invention, highland barley is Qinghai yellow, and powder is obtained by sieving the highland barley with a 200-mesh sieve after powdering.
As one embodiment of the invention, in the step S1, the condition of vacuum freeze drying is-50 to-60 ℃ for 4 to 6 hours; -35 to-40 ℃ for 10-15 hours; -10 to-20 ℃ for 10 to 15 hours; 20-25 ℃ for 24-48 hours. Preferably, the conditions of vacuum freeze drying are-60 ℃ for 6 hours; -35 ℃,15 hours; -20 ℃,15 hours; at 25℃for 48 hours.
In some embodiments, step S1, planting highland barley seeds in a planting tray, cutting highland barley leaves from roots when the length of the highland barley leaves is about 15cm, cleaning, refrigerating, vacuum freeze-drying, and collecting highland barley tender leaf powder after powdering.
Specifically, about 100g of Qinghai yellow highland barley seeds are uniformly planted in each planting tray, the Qinghai yellow highland barley seeds are soaked in clear water for overnight, then a small amount of nutrient soil is used for covering the highland barley seeds, the highland barley seeds are fully watered until the nutrient soil is thoroughly wet, and then a small amount of highland barley seeds are watered every day, wherein the planting period is 14-21 days.
In the step S2, petroleum ether is used as a solvent for degreasing, and the ratio of highland barley tender leaf powder to petroleum ether is 1g:40-50mL. Soaking at 55-60deg.C for 90-120 min, extracting at 65-70deg.C for 120-150min, and recovering at 75-80deg.C for 15-20min; after degreasing treatment, solid-liquid separation is carried out, and the obtained degreased matter is subjected to subsequent extraction.
In the step S2, water is added according to the weight ratio of highland barley powder to water of 1:20-1:30.
In step S2, the enzyme hydrolysis is performed by adding high temperature resistant α -amylase, papain, saccharifying enzyme, xylanase and cellulase to the above solution.
In the step S2, 5-10 mu L/g of high temperature resistant alpha-amylase of highland barley powder is added into the highland barley powder water solution, and water bath is carried out for 1-2 hours at 80-90 ℃; regulating the temperature of the water bath to 60-80 ℃, adding 5-10 mu L/g of papain of highland barley powder, and carrying out water bath for 1-2h; inactivating enzyme in 100deg.C water bath for 10-20min, cooling, adding saccharifying enzyme of 40-50 μl/g highland barley powder, and 60-80deg.C water bath for 1-2 hr; inactivating enzyme in water bath at 100deg.C for 10-20min, cooling, and water-bathing at 60-90deg.C for 2-4 hr to obtain enzymolysis liquid.
As one embodiment of the invention, the pH of the obtained enzymolysis liquid is regulated to 5-6 by NaOH solution, 600-1000U/g xylanase of highland barley tender leaf powder and 200-600U/g cellulase of highland barley tender leaf powder are added, and after the temperature is kept at 40-60 ℃ for 2-3h, the enzyme is inactivated in a water bath at 100 ℃ for 10-20min. Concentrating the supernatant obtained by enzymolysis in water bath at 60-80deg.C to 50-100 mL, cooling, adding 3-5 times of ethanol, standing, centrifuging, and collecting precipitate; re-dissolving with water, and vacuum freeze drying to obtain crude polysaccharide extracted from tender highland barley leaf.
In some embodiments, the high temperature resistant alpha-amylase is added to the supernatant, water-bath at 80 ℃ for 30min, water-bath at 100 ℃ for 10min for inactivation; cooling the solution, adding papain, water-bathing at 60deg.C for 2 hr, and inactivating at 100deg.C for 10 min; and cooling the solution, adding saccharifying enzyme, performing water bath at 60 ℃ for 1h, and performing water bath at 100 ℃ for 10min for inactivation. Then NaOH solution is used for adjusting the pH value to 5, 800U/g xylanase of highland barley tender leaf powder and 400U/g cellulase of highland barley tender leaf powder are added, and after the temperature is kept for 2 hours at 50 ℃, enzyme is inactivated in water bath at 100 ℃ for 10 minutes.
In the step S2, the supernatant obtained by enzymolysis is concentrated to 50mL-100mL in a water bath at 60-80 ℃, cooled, added with 3-5 times of ethanol, and then left stand, centrifuged and the precipitate is collected; re-dissolving with water, and vacuum freeze drying to obtain crude polysaccharide extracted from tender highland barley leaf.
In some embodiments, the supernatant from the enzymatic hydrolysis is concentrated to 50mL-100mL in a water bath at 80℃and cooled and then added dropwise with 3-5 volumes of ethanol under stirring. The solution was allowed to stand for one day, centrifuged, the precipitate collected, redissolved with 100mL deionized water and 3-5 volumes of ethanol were added. Repeating the process for 3 times, centrifuging, re-dissolving the precipitate with 50mL deionized water, freezing at-80deg.C for one day, and vacuum freeze drying to obtain crude polysaccharide extracted from semen Avenae Nudae tender leaf enzyme.
As one embodiment of the invention, the conditions of vacuum freeze drying are-50 to-60 ℃ for 4 to 6 hours; -35 to-40 ℃ for 10-15 hours; -10 to-20 ℃ for 10 to 15 hours; 20-25 ℃ for 24-48 hours. Preferably, the conditions of vacuum freeze drying are-60 ℃ for 6 hours; -35 ℃,15 hours; -20 ℃,15 hours; at 25℃for 48 hours.
As an embodiment of the present invention, in step S3, the cellulose column is a DEAE-32 cellulose ion exchange column; the sephadex column is a Sephacryl-400 sephadex column.
As one embodiment of the present invention, in the step S3, the eluent for separation is distilled water, the elution rate is 0.5-0.7mL/min, and the elution time is 500-1000min.
As one embodiment of the present invention, in the step S3, the eluent for purification is distilled water, the elution rate is 0.5-0.7mL/min, and the elution time is 500-1000min.
In some embodiments, the invention provides a preparation method for extracting polysaccharide active ingredients from highland barley tender leaf enzymes, which comprises the following steps:
step (1): uniformly planting about 100g of Qinghai yellow highland barley seeds in each planting tray, soaking the Qinghai yellow highland barley seeds overnight with clear water, covering the highland barley seeds with a small amount of nutrient soil, fully watering until the nutrient soil is wet, and watering a small amount every day, wherein the planting period is 14-21 days; cutting off highland barley leaves from roots when the length of the highland barley leaves is about 15cm, cleaning, refrigerating, freeze-drying in vacuum, powdering, and sieving and collecting the obtained highland barley tender leaf powder;
step (2): degreasing highland barley tender leaf powder by using petroleum ether as a solvent, wherein the ratio of the highland barley tender leaf powder to the petroleum ether is 1g:50ml, soaking at 55-65deg.C for 60-90min, extracting at 65-85deg.C for 90-150min, and recovering at 75-95deg.C for 10-30min. Solid-liquid separation is carried out after degreasing treatment, and the obtained degreased material is subjected to the subsequent enzyme extraction step;
step (3): highland barley young leaf powder and water are mixed according to the proportion of 1:20 to 1:30 weight percent, adding 5-10 mu L/g highland barley powder high temperature resistant alpha-amylase into the solution, and carrying out water bath at 80-90 ℃ for 1-2h; regulating the temperature of the water bath to 60-80 ℃, adding 5-10 mu L/g of papain of highland barley powder, and carrying out water bath for 1-2h; inactivating enzyme in 100deg.C water bath for 10-20min, cooling, adding saccharifying enzyme of 40-50 μl/g highland barley powder, and 60-80deg.C water bath for 1-2 hr; inactivating enzyme in water bath at 100deg.C for 10-20min, cooling, and water-bathing at 60-90deg.C for 2-4 hr. Then NaOH solution is used for regulating pH to 5-6, xylanase of 600-1000U/g highland barley tender leaf powder and cellulase of 200-600U/g highland barley tender leaf powder are added, and after heat preservation is carried out for 2-3h at 40-60 ℃, enzyme is inactivated in water bath at 100 ℃ for 10-20min;
step (4): concentrating the collected supernatant in water bath at 80 ℃ to 50-100 mL, cooling, and dropwise adding 3-5 times of ethanol under stirring. The solution was allowed to stand for one day, centrifuged, the precipitate collected, redissolved with 100mL deionized water and 3-5 volumes of ethanol were added. Repeating the steps for 3 times, centrifuging, re-dissolving the precipitate with 50mL of deionized water, freezing at-80 ℃ for one day, and vacuum freeze-drying to obtain crude polysaccharide extracted from highland barley tender leaf enzyme;
step (5): separating crude polysaccharide extracted from semen Avenae Nudae tender leaf enzyme obtained in step 4 by cellulose column chromatography (preferably DEAE-32 cellulose ion exchange column), eluting with pure water;
step (6): concentrating the obtained eluate, purifying with Sephacryl-400 Sephadex column, eluting with distilled water, collecting eluate, concentrating, and lyophilizing to obtain polysaccharide active ingredient extracted from semen Avenae Nudae tender leaf enzyme.
In a second aspect, the invention relates to a highland barley tender leaf enzyme extracted polysaccharide active ingredient prepared by the method, the molecular weight of the highland barley tender leaf enzyme extracted polysaccharide active ingredient is 20000-30000Da, and the polydispersity coefficient of the highland barley tender leaf enzyme extracted polysaccharide active ingredient is 1.14. Proved that the enzyme extracted polysaccharide is homogeneous polysaccharide. According to the research of the invention, the novel highland barley tender leaf enzyme polysaccharide active ingredient can be obtained by the method and the elution process. The novel polysaccharide compound can have good specific anticancer activity, for example, has excellent inhibiting and killing effects on cancer cells, and in addition, the polysaccharide with the structure has no inhibiting activity on normal cells.
As one embodiment of the invention, the highland barley tender leaf enzyme extracted polysaccharide consists of arabinose, galactose, xylose and glucose, wherein the content ratio of the arabinose, the xylose, the glucose and the galactose is about 10:11:1:4.
as one embodiment of the invention, the active component of the highland barley tender leaf enzyme extracted polysaccharide is odorless and tasteless light yellow floccule which is easy to dissolve in water.
As one embodiment of the invention, the active ingredient of the highland barley tender leaf enzyme polysaccharide contains galactose residues.
In a third aspect, the invention relates to an application of highland barley tender leaf enzyme extracted polysaccharide active ingredient in preparing anticancer drugs.
As an embodiment of the present invention, the anticancer drug comprises an anti-breast cancer drug or an anti-colorectal cancer drug. The inhibition rate of the highland barley tender leaf enzyme extracted polysaccharide active ingredient on the breast cancer cells 4T1 can reach 40% -50%, and the inhibition rate on colorectal cancer cells HT29, caco-2 and CT26 can reach 80% -90%, 60% -70% and 30% -40%; it has good cell safety to normal colon mucosa epithelial cell line NCM460 at the administration concentration reaching 10 mg/mL.
In a fourth aspect, the invention relates to an anticancer drug, which comprises a pharmaceutically effective amount of polysaccharide active ingredient extracted from highland barley tender leaf enzymes. The highland barley tender leaf enzyme polysaccharide prepared by the invention can effectively inhibit proliferation of breast cancer 4T1 cells, colorectal cancer HT29, caco-2 and CT26 cells, and particularly can specifically inhibit proliferation of colorectal cancer HT29 cells without obviously affecting normal physiological activities of normal cells NCM 460. The specific inhibition of the physiological activities of colorectal cancer and breast cancer cells by the polysaccharide extracted from highland barley tender leaf enzyme can be proved by a cell proliferation toxicity experiment. Experimental results show that the polysaccharide extracted from highland barley tender leaf enzyme can greatly reduce the survival rate of two cancer cells in liver, and especially can specifically inhibit and kill colorectal cancer HT-29 cells without obvious influence on proliferation of normal cells.
The research of the invention discovers that the novel highland barley tender leaf enzyme polysaccharide active ingredient has good inhibition effect on various breast cancer and colorectal cancer cells, can specifically inhibit and kill colorectal cancer cells, and basically has no inhibition effect on normal cells. The invention demonstrates the anticancer selectivity of the polysaccharide active ingredient by breast cancer and colorectal cancer cell models (e.g., HT29, caco-2 and CT26 colorectal cancer cells, and 4T1 breast cancer cells) and normal cell models (human normal colon mucosal epithelial cells NCM 460).
Compared with the prior art, the invention has the following beneficial effects:
1) The invention provides a brand-new method for extracting polysaccharide active ingredients from highland barley tender leaf enzyme, which proves that the polysaccharide extracted from highland barley tender leaf enzyme is a brand-new homogeneous polysaccharide through means of purity analysis, molecular weight measurement, monosaccharide composition analysis and the like;
2) The invention proves that the highland barley tender leaf enzyme extracted polysaccharide has good inhibition effect on breast cancer and colorectal cancer, especially colorectal cancer HT-29 cells, and plays a promotion role in the application of polysaccharide as an anticancer drug; in addition, the novel polysaccharide substance can not only effectively inhibit proliferation of cancer cells such as breast cancer and colorectal cancer cells, but also avoid damage to normal cell proliferation capacity and cell state, and make up for the defects of weak specificity and large side effect on human bodies of the existing chemical anticancer drugs;
3) According to the preparation method for extracting the polysaccharide active ingredient from the highland barley tender leaf enzyme, the removal rate of protein and starch is greatly improved and the yield of the polysaccharide extracted from the highland barley tender leaf enzyme is improved by adding papain and high-temperature resistant alpha-amylase; through the elution method, the highland barley tender leaf enzyme polysaccharide can be obtained, wherein the apoptosis of colorectal cancer cells can be specifically regulated and controlled, and normal cell metabolic proliferation is not influenced.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:
FIG. 1 is a fiber column elution curve of crude polysaccharide extracted from highland barley tender leaf enzyme;
FIG. 2 is a High Performance Liquid Chromatography (HPLC) of crude polysaccharide extracted from highland barley tender leaf enzyme;
FIG. 3 is a graph showing elution of glucan column of enzyme-extracted polysaccharide from tender highland barley leaves;
FIG. 4 is a High Performance Liquid Chromatography (HPLC) of the enzyme extracted from the tender highland barley leaves;
FIG. 5 is a graph showing the relative molecular mass standard of the enzyme extracted from the tender highland barley leaves;
FIG. 6 is a Gas Chromatogram (GC) of a standard monosaccharide;
FIG. 7 is a Gas Chromatogram (GC) of the enzyme extracted from the tender highland barley leaves;
FIG. 8 is an infrared spectrogram (FT-IR) of the enzyme extracted polysaccharide from the tender leaves of highland barley;
FIG. 9 shows the HT29 inhibition rate of highland barley tender leaf enzyme extracted polysaccharide;
FIG. 10 shows the inhibition ratio of enzyme extracted polysaccharide of highland barley tender leaves to Caco-2;
FIG. 11 shows the inhibition ratio of enzyme extracted polysaccharide from tender highland barley leaves to CT 26;
FIG. 12 shows the inhibition ratio of highland barley tender leaf enzyme extracted polysaccharide to 4T 1;
FIG. 13 shows the survival rate of enzyme extracted polysaccharides from highland barley young leaves to NCM460 after treatment.
FIG. 14 shows the HT29 inhibition ratio of highland barley tender leaf water extract polysaccharide;
FIG. 15 shows the inhibition ratio of water-extracted polysaccharides of highland barley young leaves to Caco-2;
FIG. 16 shows the inhibition ratio of water-extracted polysaccharides of highland barley young leaves to CT 26;
FIG. 17 shows the inhibition ratio of water-extracted polysaccharides from tender highland barley leaves to 4T 1.
Detailed Description
The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that several modifications and improvements can be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.
Example 1
The embodiment provides a preparation method for extracting polysaccharide from highland barley tender leaf enzyme, which comprises the following specific implementation steps:
1. preparation of highland barley tender leaf powder
The planting trays are fully paved with nutrient soil with a thickness of about one finger, each planting tray is uniformly planted with about 100g of Qinghai yellow highland barley seeds soaked by clear water for overnight, then the highland barley seeds are covered by a small amount of nutrient soil, the highland barley seeds are fully watered until the nutrient soil is thoroughly wet, and then a small amount of highland barley seeds are watered every day. And when the length of the highland barley tender leaves is about 15cm after 14 days to 21 days, shearing the highland barley tender leaves from the roots, cleaning, refrigerating, vacuum freeze-drying, and collecting the obtained highland barley tender leaf powder after powdering. Vacuum freeze drying at-50deg.C for 4 hr; -35 ℃,10 hours; -10 ℃,10 hours; 25℃for 24 hours.
2. Preparation of crude polysaccharide extracted from highland barley tender leaf by enzyme
Degreasing: about 1g of highland barley tender leaf powder is weighed, wrapped in qualitative filter paper (diameter: 18 cm), and the filter paper is packed by cotton ropes and placed in a Soxhlet fat extractor, and 50mL of petroleum ether is used as a solvent for degreasing. Soaking at 65deg.C for 90min, extracting at 70deg.C for 120min, recovering at 80deg.C for 15min, and oven drying at 50deg.C to obtain defatted powder sample.
Enzyme extraction: weighing about 10g of defatted highland barley tender leaf powder, adding 300mL of deionized water into a beaker, adding 5 mu L/g of high temperature resistant alpha-amylase of highland barley powder, carrying out water bath at 80 ℃ for 30min, and carrying out water bath at 100 ℃ for 10min for inactivation; after the solution is cooled, adding 5 mu L/g of papain of highland barley powder, carrying out water bath at 60 ℃ for 2 hours, and carrying out water bath at 100 ℃ for 10 minutes for inactivation; after the solution is cooled, adding 40 mu L/g saccharifying enzyme of highland barley powder, water-bathing at 60 ℃ for 1h, and water-bathing at 100 ℃ for 10min for inactivation. Then NaOH solution is used for regulating pH to 5, 800U/g xylanase of highland barley tender leaf powder and 400U/g cellulase of highland barley tender leaf powder are added, the temperature is kept at 50 ℃ for 2 hours, enzyme is inactivated in a water bath at 100 ℃ for 10 minutes, and the supernatant is collected after centrifugation.
Alcohol precipitation: concentrating the collected supernatant in water bath at 80 ℃ to 50-100 mL, cooling, and dropwise adding 3-4 times of ethanol under stirring. The solution was allowed to stand for one day, centrifuged, the precipitate collected, redissolved with 100mL deionized water and 3-4 volumes of ethanol were added. Repeating for 3 times, centrifuging, re-dissolving the precipitate with 50mL deionized water, freezing at-80deg.C for one day, and vacuum freeze drying to obtain crude polysaccharide extracted from semen Avenae Nudae tender leaf enzyme.
3. Preparation of highland barley tender leaf enzyme extracted polysaccharide
0.5g of crude polysaccharide sample was weighed and dissolved in 10mL of deionized water, metal-bathed at 55℃for 1 hour, then stirred overnight at 4℃and centrifuged and the supernatant was pipetted dropwise onto the upper end of a column packed with DEAE-32, eluting with distilled water followed by 0.25M, 0.5M, 1M sodium chloride solution. The flow rate is: 0.7mL/min,5 min/tube collection, 80 tubes each. Every 4 test tubes are separated for sampling, 0.1mL of collecting liquid is taken, 0.9mL of deionized water is added, 1mL of 5% phenol solution is added, then the mixture is vortexed, 5mL of concentrated sulfuric acid is added, after vortexing, color comparison with a blank sample is carried out, absorbance is recorded, and a corresponding elution curve is drawn as shown in figure 1. The sugar components of different eluents are separated, combined and collected, and the combined liquid eluted by sodium chloride is placed in a dialysis bag for dialysis for 36 hours, and deionized water is changed every 12 hours. Concentrating the dialyzed sample solution to about 5mL, freezing at-80 ℃ for one day, and vacuum freeze-drying to obtain highland barley enzyme extracted crude polysaccharide eluted by a DEAE-32 chromatographic column.
Polysaccharide samples eluted by different eluents are prepared into 10mg/mL solution by deionized water, 20 mu L of solution is sampled to be detected by a high performance liquid chromatograph after passing through a 0.45 mu m filter membrane, and the polysaccharide sample with the highest polysaccharide purity is reserved as shown in figure 2. The adsorbed DEAE-32 eluate was applied dropwise to the upper end of the column packed with Sephacryl-400, and eluted with distilled water. The flow rate is: 0.7mL/min,5 min/tube collection, 100 tubes. Sampling with 4 test tubes at intervals, collecting 0.1mL of collecting liquid, adding 0.9mL of deionized water, adding 1mL of 5% phenol solution, adding 5mL of concentrated sulfuric acid, performing color comparison with a blank sample after vortex, recording absorbance, drawing an elution curve as shown in figure 3, and combining and collecting sugar components. Freezing sugar-containing solution at-80deg.C for one day, and vacuum freeze drying to obtain semen Avenae Nudae tender leaf enzyme-extracted polysaccharide eluted by Sephacryl-400 chromatographic column.
The eluted highland barley tender leaf enzyme polysaccharide is prepared into 10mg/mL solution by deionized water, and the solution is filtered by a 0.45 mu m filter membrane and then is injected into a high performance liquid chromatograph for detection, and the purity is 99.75% as shown in figure 4. The refined highland barley tender leaf enzyme extracted polysaccharide is obtained, and the total extraction rate is 7.68 percent. The detection instrument is e2695 high performance liquid chromatograph equipped with Waters2414 differential detector and SUGAR KS-805(8.0 mm ID. Times.300 mm L) sugar column; mobile phase: ultrapure water; flow rate: 1.0mL/min; column temperature: 35 ℃; detector temperature: 30 ℃; sample injection amount: 20. Mu.L.
Example 2
Example 2 illustrates the physicochemical properties of the enzyme extracted polysaccharide from highland barley young leaves (obtained in example 1), and the specific characterization method is as follows:
molecular weight analysis: the purified highland barley tender leaf enzyme polysaccharide is prepared into 5mg/mL aqueous solution by deionized water, and the dextran standard products with relative molecular weights of 4400, 9900, 21400, 43500, 124000 and 805000 are respectively prepared into 5mg/mL aqueous solution by deionized water, and the aqueous solution is subjected to sample injection detection after passing through a 0.45 mu m filter membrane, and the detection instrument is EcoSEC HLC-8320GPC. Corresponding standard curves were plotted according to the peak time of each dextran standard, as shown in fig. 5. The relative molecular masses of the samples were calculated from the standard curves of the relative molecular masses, as shown in table 1, with weight average molecular weights Mw and number average molecular weights Mn of 22575Da and 25996Da, respectively, and the polydispersity coefficients (polydispersity index, pdi=mw/Mn) of the corresponding polysaccharide molecules were 1.14. Since the PDI value is closer to 1, the uniformity of the corresponding substances is better, the prepared highland barley tender leaf enzyme extracted polysaccharide sample can be considered to be relatively uniform polysaccharide.
TABLE 1 determination of relative molecular Mass of polysaccharide extracted from highland barley tender leaf enzyme
| RT | Area | ％Area | Height | Mn/Daltons | Mw/Daltons | Mw/Mn | Poly dispersity |
| 21.30 | 3132379 | 85.12 | 50103 | 22575 | 25996 | 1.14 | 1.14 |
Monosaccharide composition: taking a purified highland barley tender leaf enzyme polysaccharide extraction sample, and preparing a highland barley polysaccharide solution with the concentration of 500 mug/mL. 1mL of the solution was taken, 1mL of a 20. Mu.g/mL inositol solution was added, and vortexed. Then 0.5mL of trifluoroacetic acid solution was added and vortexed. And (3) placing the mixed solution in a metal bath at 120 ℃ for 2 hours, and drying under the protection of nitrogen. After that, 250. Mu.L of isopropyl alcohol was added, vortexed, and nitrogen blown to complete dryness, and repeated 2 times.
Six monosaccharide standards (rhamnose, glucose, xylose, mannose, arabinose, galactose) and mixtures thereof were prepared as 20. Mu.g/mL solutions, respectively. 1mL of each solution was taken, 1mL of a 20. Mu.g/mL inositol solution was added, vortexed, and blow-dried under nitrogen.
And sequentially adding 100 mu L of ammonia water and 0.5mL of 20mg/mL sodium borohydride solution (dimethyl sulfoxide is taken as a solvent) into the treated highland barley tender leaf enzyme polysaccharide extraction sample, each monosaccharide standard substance and the mixed standard substance, and fully swirling. The mixed solution is placed in a metal bath at 40 ℃ for 90min, and then 6-9 glacial acetic acid is added dropwise into the mixed solution until no bubbles emerge. To each sample was added 100. Mu.L of 1-methylimidazole and 0.5mL of acetic anhydride in this order, followed by vortexing sufficiently and reacting at room temperature for 10 minutes. 4mL of deionized water and 1mL of dichloromethane were added and vortexed well, leaving a layer of organic phase. The upper aqueous phase was transferred to another tube, 1mL of dichloromethane was added, vortexing well, leaving the organic phase of the layer. The organic phases obtained in the two steps are combined and washed twice with 4mL of deionized water, dried under the protection of nitrogen, and placed in a refrigerator for refrigeration to be tested.
The sample to be tested was dissolved with 0.6mL of acetone, vortexed well, filtered through a 0.45 μm filter and detected by gas chromatograph. The detection instrument is a GC-2010plus gas chromatograph equipped with a (30meter,0.25mm ID,0.25 μm df) chromatographic column; sample inlet temperature: 240 ℃; column temperature: 100 ℃ for 2min;10 ℃/min, and raising the temperature to 180 ℃;180 ℃ for 2min;4 ℃/min, and raising the temperature to 240 ℃;240 ℃ for 5min;10 ℃/min, and raising the temperature to 255 ℃;255 ℃ for 5min; the carrier gas is nitrogen, and the flow rate is 1.9mL/min; sample injection amount: 1 mul.
The monosaccharide samples represented by each peak were determined in the mix from the peak time of each monosaccharide standard and inositol, as shown in fig. 6. The peak-out sequence of each monosaccharide standard is as follows: rhamnose, arabinose, xylose, mannose, glucose, galactose.
TABLE 2 gas chromatography data for monosaccharide standards
| Peak number | Retention time | Area of | Peak height | Component (A) | Area percent |
| 1 | 17.955 | 3222.9 | 805.9 | Rha | 5.8450 |
| 2 | 18.260 | 10342.4 | 2682.3 | Ara | 18.7568 |
| 3 | 18.665 | 7637.2 | 1921.1 | Xyl | 13.7748 |
| 4 | 23.174 | 8698.2 | 1938.7 | Inositol (inositol) | 15.7748 |
| 5 | 23.515 | 8519.0 | 2126.1 | Man | 15.4499 |
| 6 | 23.720 | 7111.7 | 1495.9 | Glu | 12.8975 |
| 7 | 23.893 | 9608.3 | 2345.8 | Gla | 17.4254 |
The gas chromatogram of the highland barley tender leaf enzyme polysaccharide is shown in figure 7, the peak time of each monosaccharide standard product of mixed label determines the monosaccharides represented by each peak in the sample, and the sequence of the peak time is as follows: arabinose, xylose, glucose and galactose, wherein the main components of the xylose and arabinose contained a small amount of galactose and glucose, and the specific peak information is shown in Table 3. The relative correction factor of the monosaccharide standard calculated from the mixed standard gas chromatogram can be about 10 for the content ratio of arabinose, xylose, glucose and galactose in the polysaccharide sample: 11:1:4. the main chain of the highland barley tender leaf enzyme polysaccharide is presumed to be composed of arabinose and xylose residues from the content of each monosaccharide.
TABLE 3 gas chromatography data for enzyme-extracted polysaccharide from highland barley young leaves
| Peak number | Retention time | Area of | Peak height | Component (A) | Area percent |
| 1 | 18.255 | 10474.3 | 2691.1 | Ara | 24.8778 |
| 2 | 18.659 | 11445.9 | 2948.2 | Xyl | 27.1856 |
| 3 | 23.171 | 14383.0 | 3827.9 | Inositol (inositol) | 34.1615 |
| 4 | 23.718 | 1278.4 | 277.4 | Glu | 3.0364 |
| 5 | 23.887 | 4521.3 | 1029.0 | Gla | 10.7387 |
Infrared spectroscopy: taking 5mg of highland barley tender leaf enzyme extracted polysaccharide freeze-dried sample, uniformly mixing with 0.75g KBr solid, grinding into powder, pressing the powder into a disc with the thickness of 1mm for tabletting, using a Nicolet 6700 type Fourier infrared transformation spectrometer, and providing an IR device, wherein the scanning interval is 4000-400cm -1 Infrared spectroscopic analysis was performed. The IR spectrum is shown in FIG. 8, in whichAt 3438.15cm -1 The absorption peak intensity is the largest, the peak shape is the widest, and the absorption peak is probably caused by the stretching vibration of O-H bonds, which indicates that the molecular structure of the polysaccharide possibly contains carboxyl O-H; at 1640.96cm -1 The absorption peak at the position is in a double bond stretching vibration area, which is possibly caused by stretching vibration of a C=C bond or a C=O bond, and the absorption peak is considered to have higher intensity, and is more possibly a stretching vibration peak of the C=O bond; at 1415.91cm -1 The absorption peak at this point may be-CH 3 Is caused by the telescopic vibration of the device; at 1043.73cm -1 The absorption peak at this point may be caused by absorption vibration of C-O-C bond or C-O-H bond, indicating that there is a pyranose residue in the sample, and the combined monosaccharide composition analysis result may be a characteristic peak of galactose residue.
Example 3
Example 3 provides the application of highland barley tender leaf enzyme extracted polysaccharide (obtained from example 1) in anticancer, and the specific implementation process is as follows:
1. proliferation inhibition of colorectal cancer (HT 29, caco-2, CT 26) cells and breast cancer 4T1 cells by extracting polysaccharide from highland barley tender leaf enzyme
Resuscitates colorectal cancer (HT 29, caco-2, CT 26) cells and breast cancer 4T1 cells, cultures in DMEM medium containing 10% FBS, and places at 37deg.C in 5% CO 2 The conditions were in an incubator. When the cells were grown to the bottom of the cell flask, the cells were digested with pancreatin, centrifuged, the supernatant was discarded, 2mL of medium was added to the pellet, and after blowing, counted with a hemocytometer, and the cells were diluted in proportion, inoculated into 96-well plates (100 μl of cell suspension per well, i.e. 5000 cells), and cultured in an incubator for 24 hours. DMEM culture medium containing highland barley tender leaf enzyme extract polysaccharide concentration of 0, 0.625, 1.25, 2.5, 5 and 10mg/mL is added into a 96-well plate respectively, and incubated for 48h at 37 ℃. Carefully pipetting the supernatant, adding 0.5mg/mL MTT 100. Mu.L per well, continuing to incubate for 4 hours in a 37 ℃ incubator, carefully blotting the MTT solution, adding DMSO 100. Mu.L per well, continuing to incubate for 10 minutes in a 37 ℃ incubator, sufficiently oscillating on an oscillator until the bluish-violet crystals are completely dissolved, and detecting the OD value of each well at 570nm by a full-automatic microplate reader. Cell viability was calculated as follows. Cell viability (%) = experimental OD value/control OD value x 100%. The results are shown in FIGS. 9-12After four tumor cell primary screening, the polysaccharide sample extracted from highland barley tender leaf enzyme has no obvious effect of inhibiting the growth of 4T1 of mouse breast cancer cells, and has obvious effect of inhibiting proliferation of colorectal cancer cells, wherein the effect of inhibiting HT29 cells is particularly obvious. After 0.625mg/mL highland barley tender leaf enzyme polysaccharide extracting solution is used for treating colorectal cancer cells HT29 h, the proliferation and growth of the colorectal cancer cells HT can be obviously influenced, and the inhibition rate can reach 37.82%; and after 10mg/mL highland barley tender leaf enzyme polysaccharide extracting solution is treated for 29 hours with HT, the proliferation of cells can be almost completely inhibited, and the inhibition rate reaches 86.37 percent.
2. Proliferation inhibition effect of highland barley tender leaf enzyme extracted polysaccharide on human normal colon mucosa epithelial cell NCM460
Resuscitates human normal colon mucosa epithelial cells NCM460, cultures in 1640 medium containing 10% FBS, and places in 37deg.C and 5% CO 2 The conditions were in an incubator. When the cells were grown to the bottom of the cell flask, the cells were digested with pancreatin, centrifuged, the supernatant was discarded, 2mL of medium was added to the pellet, and after blowing, counted with a hemocytometer, and the cells were diluted in proportion, inoculated into 96-well plates (100 μl of cell suspension per well, i.e. 5000 cells), and cultured in an incubator for 48 hours. 1640 culture medium with the concentration of 0, 0.625, 1.25, 2.5, 5 and 10mg/mL of highland barley tender leaf enzyme extract polysaccharide is respectively added into a 96-well plate, and incubated for 24 hours at 37 ℃. Carefully pipetting the supernatant, adding 0.5mg/mL MTT 100. Mu.L per well, continuing to incubate for 4 hours in a 37 ℃ incubator, carefully blotting the MTT solution, adding DMSO 100. Mu.L per well, continuing to incubate for 10 minutes in a 37 ℃ incubator, sufficiently oscillating on an oscillator until the bluish-violet crystals are completely dissolved, and detecting the OD value of each well at 570nm by a full-automatic microplate reader. Cell viability was calculated as follows. Cell viability (%) = experimental OD value/control OD value x 100%. The cell viability results are shown in FIG. 13, where NCM460 cells showed no significant decrease in cell viability after incubation with highland barley tender leaf enzyme polysaccharide extraction. Proved that the highland barley tender leaf enzyme extract has no inhibition effect on the proliferation of normal cells.
Comparative example 1
The comparative example provides the proliferation inhibition effect of highland barley young leaf water extract polysaccharide on colorectal cancer (HT 29, caco-2, CT 26) cells and breast cancer 4T1 cells. The highland barley young leaf water extraction polysaccharide is prepared by the team of the present inventors, and the preparation method is described in detail in an article (Chemical Characterization and In Vitro Anti-Cancer Activities of a Hot Water Soluble Polysaccharide from Hulless Barley Grass "(https:// doi.org/10.3390/foos 11050677), and the monosaccharide composition is 35.1% galactose, 25.6% arabinose, 5.5% glucose and 5.3% xylose.
Colorectal cancer (HT 29, caco-2, CT 26) cells and breast cancer 4T1 cells were resuscitated and cultured in DMEM medium containing 10% FBS and placed in an incubator under conditions of 5% CO2 at 37 ℃. When the cells were grown to the bottom of the cell flask, the cells were digested with pancreatin, centrifuged, the supernatant was discarded, 2mL of medium was added to the pellet, and after blowing, counted with a hemocytometer, and the cells were diluted in proportion, inoculated into 96-well plates (100 μl of cell suspension per well, i.e. 5000 cells), and cultured in an incubator for 24 hours. And respectively adding highland barley tender leaf water-extracted polysaccharide with the same concentration into a 96-well plate, and incubating for 48 hours at 37 ℃. Carefully pipetting the supernatant, adding 0.5mg/mL MTT 100. Mu.L per well, continuing to incubate for 4 hours in a 37 ℃ incubator, carefully blotting the MTT solution, adding DMSO 100. Mu.L per well, continuing to incubate for 10 minutes in a 37 ℃ incubator, sufficiently oscillating on an oscillator until the bluish-violet crystals are completely dissolved, and detecting the OD value of each well at 570nm by a full-automatic microplate reader. Cell viability was calculated as follows. Cell viability (%) = experimental OD value/control OD value x 100%. As shown in fig. 14-17, after primary screening of four tumor cells, the water-extracted polysaccharide sample of highland barley tender leaves has a remarkable inhibition effect on proliferation of only mouse colorectal cancer cells HT29, and has an effect equivalent to that of enzyme-extracted polysaccharide, but has remarkably reduced inhibition activity on other three tumor cells compared with that of enzyme-extracted polysaccharide. The inhibition effect of the enzyme-extracted polysaccharide on Caco-2 cells is obviously improved, the inhibition rate can reach more than 70% at the concentration of 10mg/mL, and the inhibition rate of the water-extracted polysaccharide on the Caco-2 cells is only about 40% at the same concentration.
In the process of preparing the enzyme-extracted polysaccharide, high temperature resistant alpha-amylase, papain and saccharifying enzyme are used for enzymolysis, which inevitably leads to that a part of water-extracted polysaccharide is dissolved in enzymolysis liquid, and xylan (composed of xylose) and cellulose (composed of glucose) in highland barley tender leaves are continuously hydrolyzed by adding xylanase and cellulase, so that the finally obtained enzyme-extracted polysaccharide has the same monosaccharide composition type as the water-extracted polysaccharide, but the ratio of xylose to glucose is increased. Compared with the water extraction method, the enzyme extraction can obviously increase the polysaccharide yield from 2.3% to 7.68%. The monosaccharide composition result of the water-extracted polysaccharide shows that the water-extracted polysaccharide is a polysaccharide structure with galactose forming a main chain skeleton and other monosaccharide combinations on a branched chain; the monosaccharide composition structure of the enzyme-extracted polysaccharide shows that the enzyme-extracted polysaccharide is a polysaccharide structure with arabinose and xylose forming a main chain skeleton and other monosaccharide combinations on a branched chain. The polysaccharide structures obtained by the two extraction methods are not identical. And the inhibition effect of the enzyme-extracted polysaccharide on four tumor cells is obviously better than that of the water-extracted polysaccharide. The invention provides a polysaccharide extracted from highland barley tender leaf enzyme, which has excellent biological activity of inhibiting tumor cell proliferation, and has wide application prospect in the aspect of developing safe anticancer drugs without toxic or side effects.
The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.
Claims (5)
1. The application of the highland barley tender leaf enzyme polysaccharide extraction active ingredient in preparing a medicament for inhibiting proliferation activity of colorectal cancer cells Caco-2 or CT26 is characterized in that the preparation method of the highland barley tender leaf enzyme polysaccharide extraction active ingredient comprises the following steps:
s1, vacuum freeze-drying highland barley leaves, powdering and collecting highland barley tender leaf powder;
s2, degreasing, enzyme extracting, alcohol precipitating and drying highland barley tender leaf powder to obtain highland barley enzyme extracted crude polysaccharide;
the enzyme extraction process comprises the steps of adding water into the highland barley tender leaf powder and water in a weight ratio of 1:20-1:30, and adding 5-10 mu L/g high temperature resistant alpha-amylase of the highland barley powder into the solution, wherein the water bath is carried out at 80-90 ℃ for 1-2h; regulating the temperature of the water bath to 60-80 ℃, adding 5-10 mu L/g of papain of highland barley powder, and carrying out water bath 1-2h; inactivating enzyme in 100deg.C water bath for 10-20min, cooling, adding saccharifying enzyme of 40-50 μl/g highland barley powder, and in 60-80deg.C water bath for 1-2h; inactivating enzyme in 100deg.C water bath for 10-20min, cooling, and cooling in 60-90deg.C water bath for 2-4h to obtain enzymolysis solution; regulating pH of the obtained enzymolysis solution to 5-6 with NaOH solution, adding 600-1000U/g xylanase of highland barley tender leaf powder and 200-600U/g cellulase of highland barley tender leaf powder, maintaining the temperature at 40-60deg.C for 2-3h, inactivating enzyme in 100deg.C water bath for 10-20min, concentrating supernatant obtained by enzymolysis in 60deg.C water bath to 50mL-100mL, cooling, adding 3-5 times volume of ethanol, standing, centrifuging, and collecting precipitate; re-dissolving with water, and vacuum freeze drying to obtain crude polysaccharide extracted from tender highland barley leaf;
s3, separating crude polysaccharide extracted from highland barley enzyme by using a cellulose column, and purifying by using a sephadex column to obtain an active ingredient of polysaccharide extracted from highland barley tender leaf enzyme; the cellulose column is a DEAE-32 cellulose ion exchange column; the sephadex column is a Sephacryl-400 sephadex column; the eluent for separation is distilled water, the elution rate is 0.5-0.7mL/min, and the elution time is 500-1000 min; the eluent for purification is distilled water, the elution rate is 0.5-0.7mL/min, and the elution time is 500-1000min.
2. The use according to claim 1, wherein in step S2, petroleum ether is used as solvent for degreasing, and the ratio of highland barley young leaf powder to petroleum ether is 1g:40-50 mL; soaking at 55-60deg.C for 90-120 min, extracting at 65-70deg.C for 120-150min, and recovering at 75-80deg.C for 15-20min; after degreasing treatment, solid-liquid separation is carried out, and the obtained degreased matter is subjected to a subsequent extraction process.
3. The use according to claim 1, wherein the conditions of vacuum freeze-drying are-50 to-60 ℃ for 4-6 hours; -35 to-40 ℃ for 10-15 hours; -10 to-20 ℃ for 10 to 15 hours; 20-25 ℃ for 24-48 hours.
4. The use according to claim 1, wherein the molecular weight of the polysaccharide active ingredient extracted from highland barley young leaf enzyme is 20000-30000Da and the polydispersity coefficient is 1.14.
5. The use according to claim 4, wherein the highland barley young leaf enzyme extract polysaccharide consists of arabinose, galactose, xylose and glucose, and the content ratio of arabinose, xylose, glucose and galactose is 10:11:1:4.
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| US5614242A (en) * | 1995-09-27 | 1997-03-25 | Barkley Seed, Inc. | Food ingredients derived from viscous barley grain and the process of making |
| CN106117389A (en) * | 2016-08-23 | 2016-11-16 | 上海交通大学 | Extract from Semen avenae nudae grain and the method for purification beta glucan |
| CN111378055A (en) * | 2020-03-26 | 2020-07-07 | 南昌大学 | Method for continuously extracting and preparing non-starch polysaccharide from highland barley |
| CN114874344A (en) * | 2022-04-01 | 2022-08-09 | 上海交通大学 | Preparation method and application of alkali-extracted polysaccharide of highland barley tender leaves |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5614242A (en) * | 1995-09-27 | 1997-03-25 | Barkley Seed, Inc. | Food ingredients derived from viscous barley grain and the process of making |
| CN106117389A (en) * | 2016-08-23 | 2016-11-16 | 上海交通大学 | Extract from Semen avenae nudae grain and the method for purification beta glucan |
| CN111378055A (en) * | 2020-03-26 | 2020-07-07 | 南昌大学 | Method for continuously extracting and preparing non-starch polysaccharide from highland barley |
| CN114874344A (en) * | 2022-04-01 | 2022-08-09 | 上海交通大学 | Preparation method and application of alkali-extracted polysaccharide of highland barley tender leaves |
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| Title |
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| "Chemical Characterization and In Vitro Anti-Cancer Activities of a Hot Water Soluble Polysaccharide from Hulless Barley Grass";Yijuan Xu等;《Foods》;第11卷;第1-11页 * |
| "Preparation, Physicochemical Properties, and Antioxidant Capacity of Edible Agar Films Containing Hull-Less Barley Grass Polysaccharide Extracts";Shan Gao等;《Starch - Stärke》;第75卷;第1-8页 * |
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