CN106692255B - Brassica oleracea polysaccharide hydrolysate and medical application thereof - Google Patents
Brassica oleracea polysaccharide hydrolysate and medical application thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/36—Caryophyllaceae (Pink family), e.g. babysbreath or soapwort
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2866—Grinding or homogeneising
Abstract
The invention discloses a broccoli polysaccharide hydrolysate and medical application thereof, wherein the broccoli polysaccharide hydrolysate is obtained by hydrolyzing broccoli seed polysaccharide, and has a molecular weight of 1.7-50 kDa. The preparation method of the ceruleus went seed polysaccharide comprises the following steps: extracting the seed of the Brassica oleracea with distilled water, filtering the extractive solution, collecting filtrate, and concentrating the filtrate under heating; adding ethanol into the concentrated extractive solution under stirring to ethanol concentration of 50-95%, standing or centrifuging, and collecting precipitate. The hydrolysis method of the ceruleus went seed polysaccharide comprises the following steps: dissolving the seed polysaccharide of Brassica oleracea L.var.latifolia in hydrochloric acid solution, heating for hydrolysis, adjusting pH to neutral with alkali, and desalting. The biological activity of the polysaccharide depends on the chemical structure, wherein the molecular weight range of the polysaccharide is an important factor influencing the activity of the polysaccharide, and compared with the existing cerrena unicolor polysaccharide, the cerrena unicolor polysaccharide hydrolysate provided by the invention has the advantages of clear and narrower molecular weight range, stronger pharmacological activity, high water solubility, small administration dosage, quick response and excellent drug forming performance.
Description
Technical Field
The invention belongs to the field of medicines, relates to preparation and activity research of polysaccharide, and particularly relates to a broccoli polysaccharide hydrolysate and medical application thereof, which are further development and development of broccoli polysaccharide.
Background
Polysaccharides, also known as polysaccharides (polysaccharides), are chain polymers formed by dehydration of aldoses or ketoses to form glycosidic bonds and linear or branched glycosidic bonds. Polysaccharides are one of four basic substances constituting life activities, are widely present in organisms, and are closely related to various physiological functions of life. From the biochemical and structural chemical points of view, the physiological efficacy and biological activity of polysaccharide are related to the molecular weight, polymerization degree, branching degree, high-grade conformation in solution and the like, and the reduction of activity is caused by the fact that the molecular weight of polysaccharide is too large or too small. The structure of polysaccharides is extremely complex, and the structure and composition of polysaccharides from different sources vary, and the current research on the structure of polysaccharides mainly focuses on the research on the primary structure of polysaccharides, while little is known about their secondary structure and spatial structure.
Vaccinium macrocarpum segetalis (Neck.) Garcke is a plant of the genus Vaccinium of the family Caryophyllaceae, also known as seed of cowherb, milk rice, barley, cowherb, cow of cowherb, and semen Vaccariae. The Shen nong Ben Cao Jing listed as the superior one can be taken frequently without toxic and side effects. The lettuce is mainly used as a medicine by using seeds. The traditional Chinese medicine of the kalanchoe odorata seeds is named as cowherb seed, forbidden flower, golden scissors grass, calendula, kalanchoe and the like. The traditional clinical application is mainly used for treating dysmenorrhea and amenorrhea of women; the milk does not fall; acute mastitis; diuresis inducing and stranguria treating. According to the records of Ben Cao Jing Shu Lun, the flavor of Wang cowherb seed due to native, gold and fire is bitter, sweet and mild. Even if it is pungent, its qi should be warm and nontoxic; bitter can purge, pungent can disperse, sweet can enter blood, warm can move, enter liver meridian of foot jueyin. The main components of the cowherb seed include flavonoids, alkaloids, triterpenoid saponins, cyclic peptides, lipids and the like, but the active components of the cowherb seed are not clear yet. The prior art has less research on polysaccharide components in the broccoli.
The inventor of the Yanhaijiang has been engaged in the research of the broccoli polysaccharide for a long time, submits two applications (application numbers are 201110072211.2 and 201110158539.6 respectively) respectively in 2011, 3 and 16 months and 2011, 6 and 3 months, and discloses a preparation method of the broccoli polysaccharide and application of the broccoli polysaccharide in preparing a medicine for treating prostatic hyperplasia or urinary tract infection, and the application is authorized in 2013.
The inventor finds that the molecular weight of the previously claimed lettuce polysaccharide is large, the water solubility is poor, and the dosage is large, so that the pharmaceutical performance of the lettuce polysaccharide is influenced. Therefore, the inventor further improves the macleaya cordata polysaccharide to improve the pharmacological activity and the pharmaceutical performance of the macleaya cordata polysaccharide.
Disclosure of Invention
The invention aims to improve the prior art, further develop a lettuce polysaccharide product, provide a lettuce polysaccharide product with high water solubility, small taking dose, quick response and high curative effect, and improve the performance of finished medicines.
The above purpose is realized by the following technical scheme:
a herba Brassicae campestris polysaccharide hydrolysate is prepared by hydrolyzing herba Brassicae campestris seed polysaccharide with molecular weight of 1.7-50 kDa.
Furthermore, the polysaccharide content in the hydrolysate is more than or equal to 45 percent.
Furthermore, the broccoli seed polysaccharide is extracted from the broccoli seeds, and the polysaccharide content is more than or equal to 40 percent.
Further, the ceruleus went seed polysaccharide is obtained by water extraction and alcohol precipitation, and the steps are as follows: extracting the seed of the Brassica oleracea with distilled water, filtering the extractive solution, collecting filtrate, and concentrating the filtrate under heating; adding ethanol into the concentrated extractive solution under stirring to ethanol concentration of 50-95%, standing or centrifuging, and collecting precipitate.
Further, the water-material ratio of the distilled water to the cabbage seeds is 5-15m L/g, the filtrate is heated and concentrated to the extract density of 1.05-1.15g/m L, and the hot water extraction method is adopted for extraction, the extraction temperature is 60-100 ℃, the extraction is carried out for 1-5 times, and the extraction time is 0.5-5 hours each time.
Further, the hydrolysis method comprises the following steps: dissolving the seed polysaccharide of Brassica oleracea L.var.sativa L.in diluted acid with pH of 0.5-3, heating for hydrolysis, adjusting pH to neutral with alkali, and desalting.
Further, the acid is hydrochloric acid or sulfuric acid; the temperature of heating hydrolysis is 30-45 ℃, and the hydrolysis time is 10-60 minutes.
Further, the alkali is selected from one or more of sodium hydroxide, sodium carbonate or sodium bicarbonate.
Further, the desalting method is selected from method a or B or a combination of both; the method A comprises the following steps: concentrating the neutral hydrolysate to be dry, adding a desalting solvent, shaking and washing, centrifuging, collecting precipitate, and repeating for one to more times; the method B comprises the following steps: concentrating the neutral hydrolysate, filtering with nanofiltration machine to remove salt, and concentrating under reduced pressure to dry; the desalting solvent is selected from one or more of methanol, ethanol or acetone.
The application of the above herba Brassicae campestris polysaccharide hydrolysate in preparing medicine for treating urinary tract infection and prostatic hyperplasia comprises herba Brassicae campestris polysaccharide hydrolysate and pharmaceutically acceptable carrier or excipient, and making into pharmaceutically acceptable dosage forms.
The invention has the beneficial effects that:
the biological activity of the polysaccharide is known to be related to the molecular weight, the polymerization degree, the high-level conformation and the like of the polysaccharide, and the reduction of the activity is caused by the overlarge molecular weight or the overlarge molecular weight of the polysaccharide, but compared with the existing ceruleus went polysaccharide, the ceruleus went polysaccharide hydrolysate provided by the invention has stronger pharmacological activity, high water solubility, small dosage, quick response and excellent drug forming performance. Compared with the prior art, the invention has outstanding substantive characteristics and remarkable progress.
Detailed Description
The technical solution of the present invention will be specifically described below with reference to examples.
Example 1: preparation of cabbage polysaccharide hydrolysate
1. Experimental methods
1.1 extraction of polysaccharides
Extracting the cerrena unicolor polysaccharide by a hot water extraction method under the conditions of extraction temperature of 100 ℃, extraction time of 2 hours, water-material ratio of 10m L/g, extraction for 3 times, filtering by 100-mesh filter cloth, merging extracting solutions, concentrating until the density of an extract is 1.05-1.15g/m L, adding absolute ethyl alcohol to reach the concentration of about 75%, carrying out alcohol precipitation for 12 hours, pouring out supernatant, adding a proper amount of 80% ethanol into a precipitate, carrying out alcohol precipitation for 8 hours, centrifuging at 5000r/min, drying the precipitate under reduced pressure at 60 ℃, and grinding to obtain the cerrena unicolor polysaccharide.
1.2 polysaccharide hydrolysis
Dissolving the above herba Brassicae Capitatae polysaccharide in appropriate amount of hydrochloric acid solution with pH of 1, stirring at 37 deg.C for hydrolysis for 30min, adding sodium hydroxide solution for neutralization to neutrality, centrifuging, and collecting supernatant. Concentrating the supernatant under reduced pressure, filtering with nanofiltration machine to remove salt, and concentrating under reduced pressure to dry to obtain light gray solid powder; or concentrating the supernatant to dry, adding methanol to dissolve, centrifuging, dissolving the precipitate with methanol, centrifuging, and drying the precipitate under reduced pressure to obtain light gray solid powder. The above hydrolysis method is adopted to obtain the cerulenin hydrolysate.
1.3 measurement of polysaccharide content
Accurately weighing 100mg of the cerulenin or cerulenin hydrolysate in an ampoule bottle, adding 15m L2 mol/L TFA, sealing by filling nitrogen, hydrolyzing at 120 ℃ for 4h, taking out and cooling, then rotationally evaporating the hydrolysate to dryness, adding about 2m L methanol, evaporating to dryness, repeating the steps for multiple times to remove TFA, adding a small amount of deionized water, ultrasonically dissolving, transferring to a 10m L volumetric flask, washing and transferring for multiple times, fixing the volume, shaking up, filtering through a 0.45 mu m microporous membrane to obtain a to-be-tested liquid of acid hydrolysis, measuring 1m L to-be-tested liquid of acid hydrolysis in a 25m L volumetric flask by using a pipette, fixing the volume by using the deionized water, and shaking up to obtain the to-be-tested solution A.
Accurately weighing 100mg of cerulenin or cerulenin hydrolysate, adding deionized water, performing ultrasonic dissolution, transferring to a 10m L volumetric flask, cleaning, transferring, fixing the volume, shaking uniformly, filtering by a 0.45 mu m microporous filter membrane to obtain a sample aqueous solution, measuring 1m L sample aqueous solution by a pipette into a 25m L volumetric flask, fixing the volume by deionized water, shaking uniformly to obtain a sample solution B, referring to a Chinese pharmacopoeia 2015 edition and a DNS method reported in literature, slightly improving, preparing a standard curve by taking glucose as a standard substance, accurately weighing 500mg of anhydrous glucose dried at 105 ℃ to constant weight in a 50m L volumetric flask, adding deionized water to the scales, performing ultrasonic dissolution to obtain a standard glucose mother solution of 10mg/m L, preparing a 0.2, 0.3, 0.4, 0.5 and 0.6mg/m L series of glucose standard solutions based on the mother solution, respectively absorbing the glucose standard solution of 2.0m L of the above concentration into a L m volumetric solution, adding a constant volume plug of L.8 mg/m of deionized water, heating the solution to obtain a water bath contrast water solution, and measuring absorbance of the water at room temperature by using a water bath, heating to obtain a water bath, and measuring the same coordinate of the absorbance of the.
Respectively taking 2m L sample solution A and B with a pipette from a 25m L color comparison tube with a plug, fixing the volume, shaking up, adding DNS reagent 4m L, shaking up, heating in a boiling water bath for 7min, cooling with running water to room temperature, fixing the volume with deionized water, taking 2.0m L deionized water for the same operation as a blank control, measuring absorbance at 540nm, respectively calculating the sample solution A and the sample solution B according to a standard curveB has a glucose content of CAAnd CB. The content of polysaccharide in the Brassica oleracea polysaccharide or Brassica oleracea polysaccharide hydrolysate is CA-CB。
1.4 molecular weight Range determination
Molecular weight of the Brassica oleracea polysaccharide and Brassica oleracea polysaccharide hydrolysate was determined by size exclusion high performance liquid chromatography (HPGPC).
2. Results of the experiment
The results of the content measurement and the results of the molecular weight range measurement are shown in the following table.
Example 2: preparation of cabbage polysaccharide hydrolysate
1. Experimental methods
1.1 extraction of polysaccharides
Extracting the cerrena unicolor polysaccharide by a hot water extraction method under the conditions of extraction temperature of 100 ℃, extraction time of 2 hours, water-material ratio of 10m L/g, extraction for 3 times, filtering by 100-mesh filter cloth, merging extracting solutions, concentrating until the density of an extract is 1.05-1.15g/m L, adding absolute ethyl alcohol to reach the concentration of about 65%, carrying out alcohol precipitation for 12 hours, pouring out supernatant, adding a proper amount of 70% ethanol into a precipitate, carrying out alcohol precipitation for 8 hours, centrifuging at 5000r/min, drying the precipitate under reduced pressure at 60 ℃, and grinding to obtain the cerrena unicolor polysaccharide.
1.2 polysaccharide hydrolysis
Dissolving the above herba Brassicae Capitatae polysaccharide in appropriate amount of hydrochloric acid solution with pH of 1, stirring at 37 deg.C for hydrolysis for 30min, adding sodium hydroxide solution for neutralization to neutrality, centrifuging, and collecting supernatant. Concentrating the supernatant under reduced pressure, filtering with nanofiltration machine to remove salt, and concentrating under reduced pressure to dry to obtain light gray solid powder; or concentrating the supernatant to dry, adding methanol to dissolve, centrifuging, dissolving the precipitate with methanol, centrifuging, and drying the precipitate under reduced pressure to obtain light gray solid powder. The above hydrolysis method is adopted to obtain the cerulenin hydrolysate.
1.3 measurement of polysaccharide content
Accurately weighing 100mg of the cerulenin or cerulenin hydrolysate in an ampoule bottle, adding 15m L2 mol/L TFA, sealing by filling nitrogen, hydrolyzing at 120 ℃ for 4h, taking out and cooling, then rotationally evaporating the hydrolysate to dryness, adding about 2m L methanol, evaporating to dryness, repeating the steps for multiple times to remove TFA, adding a small amount of deionized water, ultrasonically dissolving, transferring to a 10m L volumetric flask, washing and transferring for multiple times, fixing the volume, shaking up, filtering through a 0.45 mu m microporous membrane to obtain a to-be-tested liquid of acid hydrolysis, measuring 1m L to-be-tested liquid of acid hydrolysis in a 25m L volumetric flask by using a pipette, fixing the volume by using the deionized water, and shaking up to obtain the to-be-tested solution A.
Accurately weighing 100mg of the ceruleus polysaccharide or the ceruleus polysaccharide hydrolysate, adding deionized water, ultrasonically dissolving, transferring into a 10m L volumetric flask, cleaning, transferring, fixing the volume, shaking up, filtering through a 0.45 mu m microporous membrane to obtain a sample aqueous solution, measuring 1m L of the sample aqueous solution into a 25m L volumetric flask by using a pipette, fixing the volume by using the deionized water, and shaking up to obtain a test solution B.
The method comprises the following steps of referring to a DNS method reported by Chinese pharmacopoeia 2015 edition and literature, slightly improving, preparing a standard curve by taking glucose as a standard substance, accurately weighing 500mg of anhydrous glucose dried to constant weight at 105 ℃, putting the anhydrous glucose into a 50m L volumetric flask, adding deionized water to scale, and carrying out ultrasonic dissolution to obtain a standard glucose mother solution of 10mg/m L, preparing a series of glucose standard solutions of 0.2, 0.3, 0.4, 0.5 and 0.6mg/m L based on the mother solution, respectively sucking 2.0m L of the glucose standard solution with the concentration into a 25m L colorimetric tube with a plug by using a pipette gun, then adding a DNS reagent of 4m L, shaking uniformly, heating in a boiling water bath for 7min, cooling running water to room temperature, fixing the volume by using deionized water, carrying out the same operation by using 2.0m L of deionized water as a blank control, measuring absorbance at 540nm, taking the glucose concentration as a horizontal coordinate and the absorbance as a vertical coordinate, and drawing the standard curve.
Respectively taking 2m L sample solution A and B in 25m L color comparison tube with plug by pipette, fixing volume, shaking up, adding DNS reagent 4m L, shaking up, heating in boiling water bath for 7min, cooling with running water to room temperature, fixing volume with deionized water, taking 2.0m L deionized water for the same operation as blank control, measuring absorbance at 540nm, and calculating glucose content C of sample solution A and sample solution B according to standard curveAAnd CB. The content of polysaccharide in the Brassica oleracea polysaccharide or Brassica oleracea polysaccharide hydrolysate is CA-CB。
1.4 molecular weight Range determination
Molecular weight of the Brassica oleracea polysaccharide and Brassica oleracea polysaccharide hydrolysate was determined by size exclusion high performance liquid chromatography (HPGPC).
2. Results of the experiment
The results of the content measurement and the results of the molecular weight range measurement are shown in the following table.
Example 3: preparation of cabbage polysaccharide hydrolysate
1. Experimental methods
1.1 extraction of polysaccharides
Extracting the cerrena unicolor polysaccharide by a hot water extraction method under the conditions of extraction temperature of 100 ℃, extraction time of 2 hours, water-material ratio of 10m L/g, extraction for 3 times, filtering by 100-mesh filter cloth, merging extracting solutions, concentrating until the density of an extract is 1.05-1.15g/m L, adding absolute ethyl alcohol to reach the concentration of about 80%, carrying out alcohol precipitation for 12 hours, pouring out supernatant, adding a proper amount of 85% ethyl alcohol into a precipitate, carrying out alcohol precipitation for 8 hours, centrifuging at 5000r/min, drying the precipitate under reduced pressure at 60 ℃, and grinding to obtain the cerrena unicolor polysaccharide.
1.2 polysaccharide hydrolysis
Dissolving the above herba Brassicae Capitatae polysaccharide in appropriate amount of hydrochloric acid solution with pH of 1, stirring at 37 deg.C for hydrolysis for 30min, adding sodium hydroxide solution for neutralization to neutrality, centrifuging, and collecting supernatant. Concentrating the supernatant under reduced pressure, filtering with nanofiltration machine to remove salt, and concentrating under reduced pressure to dry to obtain light gray solid powder; or concentrating the supernatant to dry, adding methanol to dissolve, centrifuging, dissolving the precipitate with methanol, centrifuging, and drying the precipitate under reduced pressure to obtain light gray solid powder. The above hydrolysis method is adopted to obtain the cerulenin hydrolysate.
1.3 measurement of polysaccharide content
Accurately weighing 100mg of the cerulenin or cerulenin hydrolysate in an ampoule bottle, adding 15m L2 mol/L TFA, sealing by filling nitrogen, hydrolyzing at 120 ℃ for 4h, taking out and cooling, then rotationally evaporating the hydrolysate to dryness, adding about 2m L methanol, evaporating to dryness, repeating the steps for multiple times to remove TFA, adding a small amount of deionized water, ultrasonically dissolving, transferring to a 10m L volumetric flask, washing and transferring for multiple times, fixing the volume, shaking up, filtering through a 0.45 mu m microporous membrane to obtain a to-be-tested liquid of acid hydrolysis, measuring 1m L to-be-tested liquid of acid hydrolysis in a 25m L volumetric flask by using a pipette, fixing the volume by using the deionized water, and shaking up to obtain the to-be-tested solution A.
Accurately weighing 100mg of the ceruleus polysaccharide or the ceruleus polysaccharide hydrolysate, adding deionized water, ultrasonically dissolving, transferring into a 10m L volumetric flask, cleaning, transferring, fixing the volume, shaking up, filtering through a 0.45 mu m microporous membrane to obtain a sample aqueous solution, measuring 1m L of the sample aqueous solution into a 25m L volumetric flask by using a pipette, fixing the volume by using the deionized water, and shaking up to obtain a test solution B.
The method comprises the following steps of referring to a DNS method reported by Chinese pharmacopoeia 2015 edition and literature, slightly improving, preparing a standard curve by taking glucose as a standard substance, accurately weighing 500mg of anhydrous glucose dried to constant weight at 105 ℃, putting the anhydrous glucose into a 50m L volumetric flask, adding deionized water to scale, and carrying out ultrasonic dissolution to obtain a standard glucose mother solution of 10mg/m L, preparing a series of glucose standard solutions of 0.2, 0.3, 0.4, 0.5 and 0.6mg/m L based on the mother solution, respectively sucking 2.0m L of the glucose standard solution with the concentration into a 25m L colorimetric tube with a plug by using a pipette gun, then adding a DNS reagent of 4m L, shaking uniformly, heating in a boiling water bath for 7min, cooling running water to room temperature, fixing the volume by using deionized water, carrying out the same operation by using 2.0m L of deionized water as a blank control, measuring absorbance at 540nm, taking the glucose concentration as a horizontal coordinate and the absorbance as a vertical coordinate, and drawing the standard curve.
Respectively taking 2m L sample solution A and B in 25m L color comparison tube with plug by pipette, fixing volume, shaking up, adding DNS reagent 4m L, shaking up, heating in boiling water bath for 7min, cooling with running water to room temperature, fixing volume with deionized water, taking 2.0m L deionized water for the same operation as blank control, measuring absorbance at 540nm, and calculating glucose content C of sample solution A and sample solution B according to standard curveAAnd CB. The content of polysaccharide in the Brassica oleracea polysaccharide or Brassica oleracea polysaccharide hydrolysate is CA-CB。
1.4 molecular weight Range determination
Molecular weight of the Brassica oleracea polysaccharide and Brassica oleracea polysaccharide hydrolysate was determined by size exclusion high performance liquid chromatography (HPGPC).
2. Results of the experiment
The results of the content measurement and the results of the molecular weight range measurement are shown in the following table.
Example 4: preparation of cabbage polysaccharide hydrolysate
1. Experimental methods
1.1 extraction of polysaccharides
Extracting the cerrena unicolor polysaccharide by a hot water extraction method under the conditions of 60 ℃ of extraction temperature, 5 hours of extraction time and 5m L/g of water-material ratio, extracting for 5 times, filtering by 250-mesh filter cloth, merging extracting solutions, concentrating until the density of an extract is 1.05-1.15g/m L, adding absolute ethyl alcohol to reach the concentration of about 50%, carrying out alcohol precipitation for 12 hours, pouring out supernatant, adding a proper amount of 95% ethanol into a precipitate, carrying out alcohol precipitation for 8 hours, centrifuging at 5000r/min, drying the precipitate under reduced pressure at 60 ℃, and grinding to obtain the cerrena unicolor polysaccharide.
1.2 polysaccharide hydrolysis
Dissolving the above herba Brassicae Capitatae polysaccharide in appropriate amount of hydrochloric acid solution with pH of 0.5, stirring at 30 deg.C for hydrolysis for 10min, adding sodium hydroxide solution for neutralization to neutrality, centrifuging, and collecting supernatant. Concentrating the supernatant under reduced pressure, filtering with nanofiltration machine to remove salt, and concentrating under reduced pressure to dry to obtain light gray solid powder; or concentrating the supernatant to dry, adding methanol to dissolve, centrifuging, dissolving the precipitate with methanol, centrifuging, and drying the precipitate under reduced pressure to obtain light gray solid powder. The above hydrolysis method is adopted to obtain the cerulenin hydrolysate.
1.3 measurement of polysaccharide content
Accurately weighing 100mg of the cerulenin or cerulenin hydrolysate in an ampoule bottle, adding 15m L2 mol/L TFA, sealing by filling nitrogen, hydrolyzing at 120 ℃ for 4h, taking out and cooling, then rotationally evaporating the hydrolysate to dryness, adding about 2m L methanol, evaporating to dryness, repeating the steps for multiple times to remove TFA, adding a small amount of deionized water, ultrasonically dissolving, transferring to a 10m L volumetric flask, washing and transferring for multiple times, fixing the volume, shaking up, filtering through a 0.45 mu m microporous membrane to obtain a to-be-tested liquid of acid hydrolysis, measuring 1m L to-be-tested liquid of acid hydrolysis in a 25m L volumetric flask by using a pipette, fixing the volume by using the deionized water, and shaking up to obtain the to-be-tested solution A.
Accurately weighing 100mg of the cerulenin or cerulenin hydrolysate, adding deionized water, ultrasonically dissolving, transferring into a 10m L volumetric flask, washing, transferring, fixing the volume, shaking up, filtering with a 0.45 μm microporous membrane to obtain a sample aqueous solution, measuring 1m L sample aqueous solution into a 25m L volumetric flask by using a pipette, fixing the volume with deionized water, and shaking up to obtain a test solution B.
The method comprises the following steps of referring to a DNS method reported by Chinese pharmacopoeia 2015 edition and literature, slightly improving, preparing a standard curve by taking glucose as a standard substance, accurately weighing 500mg of anhydrous glucose dried to constant weight at 105 ℃, putting the anhydrous glucose into a 50m L volumetric flask, adding deionized water to scale, and carrying out ultrasonic dissolution to obtain a standard glucose mother solution of 10mg/m L, preparing a series of glucose standard solutions of 0.2, 0.3, 0.4, 0.5 and 0.6mg/m L based on the mother solution, respectively sucking 2.0m L of the glucose standard solution with the concentration into a 25m L colorimetric tube with a plug by using a pipette gun, then adding a DNS reagent of 4m L, shaking uniformly, heating in a boiling water bath for 7min, cooling running water to room temperature, fixing the volume by using deionized water, carrying out the same operation by using 2.0m L of deionized water as a blank control, measuring absorbance at 540nm, taking the glucose concentration as a horizontal coordinate and the absorbance as a vertical coordinate, and drawing the standard curve.
Respectively taking 2m L sample solution A and B in 25m L color comparison tube with plug by pipette, fixing volume, shaking up, adding DNS reagent 4m L, shaking up, heating in boiling water bath for 7min, cooling with running water to room temperature, fixing volume with deionized water, taking 2.0m L deionized water for the same operation as blank control, measuring absorbance at 540nm, and calculating glucose content C of sample solution A and sample solution B according to standard curveAAnd CB. The content of polysaccharide in the Brassica oleracea polysaccharide or Brassica oleracea polysaccharide hydrolysate is CA-CB。
1.4 molecular weight Range determination
Molecular weight of the Brassica oleracea polysaccharide and Brassica oleracea polysaccharide hydrolysate was determined by size exclusion high performance liquid chromatography (HPGPC).
2. Results of the experiment
The results of the content measurement and the results of the molecular weight range measurement are shown in the following table.
Example 5: preparation of cabbage polysaccharide hydrolysate
1. Experimental methods
1.1 extraction of polysaccharides
Extracting the cerulenin by a hot water extraction method under the conditions of extraction temperature of 100 ℃, extraction time of 0.5h and water-material ratio of 15m L/g, extracting for 1 time, filtering by 250-mesh filter cloth, combining extracting solutions, concentrating until the density of an extract is 1.05-1.15g/m L, adding absolute ethyl alcohol to the concentration of about 75%, precipitating with ethanol for 12h, pouring out supernatant, adding an appropriate amount of 80% ethanol into the precipitate, precipitating with ethanol for 8h, centrifuging at 5000r/min, adding distilled water to fully redissolve the precipitate, adding trichloroacetic acid to the concentration of 8%, stirring, standing for 3h, heating and concentrating the solution to 1/3 of the original volume, adding 4 times of ethanol into the concentrated extracting solution under stirring, standing for 8h, centrifuging at 5000r/min, collecting precipitate, drying the precipitate under reduced pressure at 60 ℃, and grinding to obtain the cerulenin.
1.2 polysaccharide hydrolysis
Dissolving the above herba Brassicae Capitatae polysaccharide in appropriate amount of hydrochloric acid solution with pH of 3, stirring at 45 deg.C for hydrolysis for 60min, adding sodium hydroxide solution for neutralization to neutrality, centrifuging, and collecting supernatant. Concentrating the supernatant under reduced pressure, filtering with nanofiltration machine to remove salt, and concentrating under reduced pressure to dry to obtain light gray solid powder; or concentrating the supernatant to dry, adding methanol to dissolve, centrifuging, dissolving the precipitate with methanol, centrifuging, and drying the precipitate under reduced pressure to obtain light gray solid powder. The above hydrolysis method is adopted to obtain the cerulenin hydrolysate.
1.3 measurement of polysaccharide content
Accurately weighing 100mg of the cerulenin or cerulenin hydrolysate in an ampoule bottle, adding 15m L2 mol/L TFA, sealing by filling nitrogen, hydrolyzing at 120 ℃ for 4h, taking out and cooling, then rotationally evaporating the hydrolysate to dryness, adding about 2m L methanol, evaporating to dryness, repeating the steps for multiple times to remove TFA, adding a small amount of deionized water, ultrasonically dissolving, transferring to a 10m L volumetric flask, washing and transferring for multiple times, fixing the volume, shaking up, filtering through a 0.45 mu m microporous membrane to obtain a to-be-tested liquid of acid hydrolysis, measuring 1m L to-be-tested liquid of acid hydrolysis in a 25m L volumetric flask by using a pipette, fixing the volume by using the deionized water, and shaking up to obtain the to-be-tested solution A.
Accurately weighing 100mg of the ceruleus polysaccharide or the ceruleus polysaccharide hydrolysate, adding deionized water, ultrasonically dissolving, transferring into a 10m L volumetric flask, cleaning, transferring, fixing the volume, shaking up, filtering through a 0.45 mu m microporous membrane to obtain a sample aqueous solution, measuring 1m L of the sample aqueous solution into a 25m L volumetric flask by using a pipette, fixing the volume by using the deionized water, and shaking up to obtain a test solution B.
The method comprises the following steps of referring to a DNS method reported by Chinese pharmacopoeia 2015 edition and literature, slightly improving, preparing a standard curve by taking glucose as a standard substance, accurately weighing 500mg of anhydrous glucose dried to constant weight at 105 ℃, putting the anhydrous glucose into a 50m L volumetric flask, adding deionized water to scale, and carrying out ultrasonic dissolution to obtain a standard glucose mother solution of 10mg/m L, preparing a series of glucose standard solutions of 0.2, 0.3, 0.4, 0.5 and 0.6mg/m L based on the mother solution, respectively sucking 2.0m L of the glucose standard solution with the concentration into a 25m L colorimetric tube with a plug by using a pipette gun, then adding a DNS reagent of 4m L, shaking uniformly, heating in a boiling water bath for 7min, cooling running water to room temperature, fixing the volume by using deionized water, carrying out the same operation by using 2.0m L of deionized water as a blank control, measuring absorbance at 540nm, taking the glucose concentration as a horizontal coordinate and the absorbance as a vertical coordinate, and drawing the standard curve.
Respectively taking 2m L sample solution A and B in 25m L color comparison tube with plug by pipette, fixing volume, shaking up, adding DNS reagent 4m L, shaking up, heating in boiling water bath for 7min, cooling with running water to room temperature, fixing volume with deionized water, taking 2.0m L deionized water for the same operation as blank control, measuring absorbance at 540nm, and calculating glucose content C of sample solution A and sample solution B according to standard curveAAnd CB. The content of polysaccharide in the Brassica oleracea polysaccharide or Brassica oleracea polysaccharide hydrolysate is CA-CB。
1.4 molecular weight Range determination
Molecular weight of the Brassica oleracea polysaccharide and Brassica oleracea polysaccharide hydrolysate was determined by size exclusion high performance liquid chromatography (HPGPC).
2. Results of the experiment
The results of the content measurement and the results of the molecular weight range measurement are shown in the following table.
In examples 1 to 5, sodium carbonate or sodium bicarbonate may be used instead of sodium hydroxide, and sulfuric acid may be used instead of hydrochloric acid in the hydrolysis.
Example 6: safety experiment of cabbage polysaccharide hydrolysate
Toxicity experiments were performed using the ceriphyllum micranthum polysaccharide hydrolysate prepared in example 1 of the present invention. 64 Kunming mice are taken and randomly grouped, and toxicity comparison experiment research is carried out by taking and not taking the ceruleus wenyujin polysaccharide hydrolysate. The result shows that the ceruleus polysaccharide hydrolysate group has no toxic or side effect under the dosage of 500mg/kg, has no abnormality through anatomical observation on heart, liver, kidney and lung, and has increased weight average. The results are shown in Table 1.
TABLE 1 toxicity test of Brassica oleracea polysaccharide hydrolysate
The ceruleus polysaccharide hydrolysates prepared in examples 2-5 have similar safety.
Example 7: therapeutic effect of ceruleus went polysaccharide hydrolysate on urinary tract infection model rat
70 SD mice, females, were randomly divided into 7 groups by body weight, namely blank control group (distilled water 10m L/kg), model group (distilled water 10m L/kg), three gold tablet group (2.5g/kg), low, medium and high dose groups of Brassica oleracea polysaccharide hydrolysate (corresponding to 0.1g/kg, 0.2g/kg, 0.4g/kg, respectively) and Brassica oleracea polysaccharide group (1.0 g/kg). preparation methods of Brassica oleracea polysaccharide and Brassica oleracea polysaccharide hydrolysate are shown in example 1.
Except for the blank control group, the animals were kept from water for 15h before the experiment, anesthetized with ether, fixed, unhaired at lower abdominal wall, and incised at the middle part to approximate the abdominal wallExposing abdominal cavity, threading 4-gauge suture angle needle from two sides of middle section of left nephroureter to posterior abdominal wall, respectively, guiding two ends of the thread out of abdominal wall, and injecting 0.6m L Escherichia coli solution (concentration 6 × 10) into bladder7g/m L), stopping injection when bacterial liquid flows out of the urethral orifice, continuously measuring the following indexes for improving the success rate of the model, ligating the ureter in a short time, suturing the incision, recovering drinking water and feeding feed, removing the ligature of the ureter after 24 hours of operation, intragastrically irrigating corresponding medicaments according to the dose for 1 time every day, and continuously measuring 7 days, (1) killing the animals, extracting 0.3m L of urine from the bladder, and detecting the positive rate of escherichia coli in the urine of the bladder, (2) taking the right kidney of each group of rats, longitudinally cutting the kidney into two halves by using a scalpel, weighing one half of the right kidney, adding 5m L of sterilized physiological saline for homogenizing, and detecting the positive rate of bacteria, wherein all data are expressed by mean +/-standard deviation, are processed by SPSS13.0 statistical software, and comparison is carried out by X2 test among groups, and p is less than 0.05, thus having statistical significance.
The measurement results of each group of indexes are shown in tables 2-3.
TABLE 2 influence of the polysaccharide hydrolysate of Brassica oleracea on the positivity of Escherichia coli in rat urine
Note: comparison with model groups:##p＜0.01，#p＜0.05。
the results show that the detection rate of escherichia coli in the urine of rats in the three-golden tablet group, the high-dose group and the medium-dose group of the ceruleus polysaccharide hydrolysate is obviously reduced, and the difference has statistical significance (p is less than 0.01 or p is less than 0.05) compared with the model group.
TABLE 3 influence of Brassica oleracea polysaccharide hydrolysate on the positivity of rat nephrohistobacter
Note: comparison with model groups:##p＜0.01，#p＜0.05。
the results show that the detection rate of escherichia coli in the homogenates of the kidney tissues of rats in the three-gold-tablet group, the large, medium and low-dosage groups of the cerulenin hydrolysate and the cerulenin hydrolysate is obviously reduced, and compared with the model group, the differences have statistical significance (p is less than 0.01 or p is less than 0.05), and the cerulenin hydrolysate prepared in the examples 2 to 5 has similar therapeutic activity on the rats with urinary tract infection models.
Example 8: therapeutic effect of ceruleus went polysaccharide hydrolysate on prostatic hyperplasia model mice
84 KM mice are divided into 7 groups according to body weight at random, namely a normal group, a model group, a plerian group, a cabbage polysaccharide group and a cabbage polysaccharide hydrolysate high, medium and low dose group, wherein each group comprises 12 mice after being adapted for 3d according to conventional feed, a blank control group is subcutaneously injected with sterile NS 10m L/kg every day, the other mice are subcutaneously injected with testosterone propionate 5 mg/(kg. d) every day, the mice are continuously injected for 12 days, meanwhile, the plerian group mice are injected with 1.20g/kg of plerian suspension every day, the cabbage polysaccharide group mice are injected with stomach gravy polysaccharide extract 1.0g/kg every day, the cabbage polysaccharide hydrolysate is respectively injected with stomach gravy 0.1g/kg, 0.2g/kg and 0.4g/kg, the normal group mice and the model group mice are injected with physiological saline with the same volume, the mice on the 13 th day are weighed, the mice are killed by dislocation, the prostate tissue and testis are weighed, the prostate tissue and the testis weight index (the prostate weight index) is calculated, the testis weight index (the prostate weight) is less than the prostate weight index (0.05. t), and the statistical result is treated by using bilateral wet test (SPwet test).
TABLE 4 Effect on testicular index and prostate index of mouse Experimental prostatic hyperplasia model
Note: comparison with the normal group: p < 0.01, p < 0.05; comparison with model groups:##p＜0.01，#p＜0.05
the results show that the prostate wet weight of the mice in the model group is increased, the difference has statistical significance (p is less than 0.01) compared with the normal group, the pulean, the lettuce polysaccharide and the lettuce polysaccharide hydrolysate can reduce the prostate wet weight, and the difference has statistical significance (p is less than 0.01 or p is less than 0.05) compared with the model group, but the difference between the administration groups has no statistical significance; the wet weight of the mouse testis caused by testosterone propionate is increased, and compared with a normal group, the difference is not statistically significant, the wet weight of the mouse testis can be reduced by the medium and high dose groups of the pulean and the kalanchoe polysaccharide hydrolysate, and compared with a model group, the difference is statistically significant (p is less than 0.01 or p is less than 0.05).
The ceruleus went polysaccharide hydrolysates prepared in examples 2-5 had similar therapeutic activity against prostate hyperplasia model mice.
The results of examples 6 and 7 show that the ceruleus went polysaccharide hydrolysate has stronger pharmacological activity than ceruleus went polysaccharide, and has the advantages of high water solubility, small dosage, quick response and excellent drug performance.
The above embodiments are only for illustrating the essential contents of the present invention, and it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Claims (7)
1. A ceruleus went polysaccharide hydrolysate is characterized in that: is obtained by hydrolyzing the seed polysaccharide of the kalanchoe indica, and has the molecular weight of 1.7-50 kDa; the hydrolysis method comprises the following steps: dissolving the seed polysaccharide of Brassica oleracea L.var.latifolia in hydrochloric acid or sulfuric acid with pH of 0.5-3, heating for hydrolysis at 30-45 deg.C for 10-60 min, adjusting pH to neutral with alkali, and desalting;
the ceruleus went seed polysaccharide is obtained by water extraction and alcohol precipitation, and the steps are as follows: extracting the seed of the Brassica oleracea with distilled water, filtering the extractive solution, collecting filtrate, and concentrating the filtrate under heating; adding ethanol into the concentrated extractive solution under stirring to ethanol concentration of 50-95%, standing or centrifuging, and collecting precipitate.
2. The cerulenin hydrolysate according to claim 1, wherein: the content of polysaccharide in the hydrolysate is more than or equal to 45 percent.
3. The cerulenin hydrolysate according to claim 1, wherein: the above-mentioned cabbage seed polysaccharide is extracted from cabbage seed, and its polysaccharide content is greater than or equal to 40%.
4. The ceruleus polysaccharide hydrolysate of claim 1, wherein the water-to-material ratio of the distilled water to the ceruleus seeds is 5-15m L/g, the filtrate is heated and concentrated to an extract density of 1.05-1.15g/m L, and the extract is extracted by a hot water extraction method at 60-100 ℃ for 1-5 times, wherein the extraction time is 0.5-5 hours each time.
5. The cerulenin hydrolysate according to claim 1, wherein: the alkali is selected from one or more of sodium hydroxide, sodium carbonate or sodium bicarbonate.
6. The cerulenin hydrolysate according to claim 1, wherein the desalting method is selected from method a or B or a combination of both; the method A comprises the following steps: concentrating the neutral hydrolysate to be dry, adding a desalting solvent, shaking and washing, centrifuging, collecting precipitate, and repeating for one to more times; the method B comprises the following steps: concentrating the neutral hydrolysate, filtering with nanofiltration machine to remove salt, and concentrating under reduced pressure to dry; the desalting solvent is selected from one or more of methanol, ethanol or acetone.
7. Use of the Brassica campestris polysaccharide hydrolysate of any one of claims 1-6 in the preparation of a medicament for treating urinary tract infection and prostatic hyperplasia, wherein the medicament comprises the Brassica campestris polysaccharide hydrolysate and a pharmaceutically acceptable carrier or excipient, and is prepared into a pharmaceutically acceptable dosage form.
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