CN118078867A - Inonotus obliquus extraction composition for eliminating nodules and improving immunity and preparation method thereof - Google Patents
Inonotus obliquus extraction composition for eliminating nodules and improving immunity and preparation method thereof Download PDFInfo
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- CN118078867A CN118078867A CN202310329530.XA CN202310329530A CN118078867A CN 118078867 A CN118078867 A CN 118078867A CN 202310329530 A CN202310329530 A CN 202310329530A CN 118078867 A CN118078867 A CN 118078867A
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- inonotus obliquus
- parts
- extract
- nodules
- eliminating
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Abstract
The invention discloses an inonotus obliquus extraction composition for eliminating nodules and improving immunity and a preparation method thereof, belonging to the technical field of traditional Chinese medicine compositions, and comprising the following components in parts by weight: 54 parts of inonotus obliquus polysaccharide, 18 parts of grifola frondosa extract, 10 parts of hericium erinaceus extract, 7 parts of dandelion extract, 5 parts of cinnamon extract, 14 parts of mulberry leaf extract and 15 parts of xylooligosaccharide, and a shell, wherein the shell comprises carboxymethyl cellulose, sodium alginate and CaCl 2; meanwhile, the traditional Chinese medicine composition is reasonably compatible, and plays a role in synergy promotion by adopting a monarch, minister, assistant and guide principle; in addition, the complex enzyme is added to rapidly degrade the cell wall to generate soluble glycopeptides and polysaccharide, so that purification with overlarge loss rate such as column chromatography is avoided.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine compositions, and particularly relates to an inonotus obliquus extraction composition for eliminating nodules and improving immunity and a preparation method thereof.
Background
Inonotus obliquus, commonly called as Inonotus obliquus or Inonotus obliquus, is a rare medical and edible dual-purpose fungus, has complex chemical components, and polysaccharide of the Inonotus obliquus has proved to be one of the main active components, and modern pharmacology shows that the Inonotus obliquus polysaccharide has pharmacological activities of resisting tumor, reducing blood sugar, regulating immunity, resisting inflammation, eliminating nodules, resisting oxidization and the like.
In the prior report, mostly the crude polysaccharide component is taken as a research object, and the crude polysaccharide contains non-polysaccharide substances such as protein, pigment, inorganic salt and the like, and the crude polysaccharide is purified by means of deproteinization, depigmentation, deionization, fractionation and the like, and at present, the TCA method, the Sevage method and the hydrochloric acid method mainly exist, namely, the protein is removed by utilizing the denaturation principle of the protein in an organic solvent, but the operation is complicated, the solvent consumption is large and not thorough enough, the efficiency is low, and the repeated times are needed; the cost is high by an active carbon method, a polyamide method, a hydrogen peroxide method, a macroporous resin method and the like, and the patent with the publication number of CN202011478246.1 discloses an extraction and purification method of inonotus obliquus polysaccharide, which sequentially comprises ethyl acetate and alcohol extraction, ultrasonic and cellulase enzymatic hydrolysis and cellulose removal, papain deproteinization, polyamide resin column decolorization and AB-8 resin decolorization, so that the yield of inonotus obliquus polysaccharide is improved, but the loss rate of organic matters is very high by adopting a polyamide resin column decolorization and AB-8 resin decolorization method.
Disclosure of Invention
Aiming at the situation, in order to overcome the defects of the prior art, the invention provides the inonotus obliquus extraction composition for eliminating nodules and improving immunity and a preparation method thereof, and in order to relieve psychological stress of patients when taking traditional Chinese medicines, the invention provides a mode of coating pellets by shells, so that the inonotus obliquus extraction composition is continuously released in the stomach and intestine; meanwhile, the traditional Chinese medicine composition is reasonably compatible, and plays a role in synergy promotion by adopting a monarch, minister, assistant and guide principle; in addition, the complex enzyme is added to rapidly degrade the cell wall to generate soluble glycopeptides and polysaccharide, so that purification with overlarge loss rate such as column chromatography is avoided.
The invention provides inonotus obliquus polysaccharide and an extraction method thereof, which can effectively improve the yield of inonotus obliquus polysaccharide, in particular to the extraction rate of water-soluble inonotus obliquus polysaccharide by combining an ionic liquid with a complex enzyme enzymolysis method.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: the invention provides an inonotus obliquus extraction composition for eliminating nodules and improving immunity and a preparation method thereof, wherein the inonotus obliquus extraction composition comprises the following components in parts by weight:
Inonotus obliquus polysaccharide: 30-70 parts of a lubricant;
grifola frondosa extract: 7-30 parts;
hericium erinaceus extract: 6-25 parts of a lubricant;
Dandelion extract: 5-15 parts;
cinnamon extract: 3-10 parts;
Mulberry leaf extract: 10-20 parts of a lubricant;
xylo-oligosaccharide: 10-20 parts.
Preferably, the inonotus obliquus extraction composition comprises the following components in parts by weight:
Inonotus obliquus polysaccharide: 50-57 parts;
grifola frondosa extract: 14-21 parts;
Hericium erinaceus extract: 8-15 parts;
Dandelion extract: 5-12 parts;
cinnamon extract: 4-10 parts of a lubricant;
mulberry leaf extract: 11-18 parts;
Xylo-oligosaccharide: 12-20 parts.
Further, the inonotus obliquus extraction composition further comprises a shell, wherein the shell comprises carboxymethyl cellulose, sodium alginate and CaCl 2.
The invention also provides a preparation method of the inonotus obliquus extract composition for eliminating nodules and improving immunity, which comprises the following steps:
(1) Weighing the prepared inonotus obliquus extract composition, adding 10 times of water, and soaking for 2 hours;
(2) Decocting at 100deg.C for 30min, filtering, concentrating under reduced pressure, vacuum lyophilizing, and sieving with 40 mesh sieve to obtain lyophilized powder of Inonotus obliquus extract composition;
(3) Adding sterile water into the freeze-dried powder of the inonotus obliquus extract composition, kneading to prepare a soft material until the color is uniform and the humidity is proper;
(4) Placing the soft material in an extrusion spheronizer, placing the spheronized pellets in a 40 ℃ oven for drying for 12 hours, and sieving to obtain inonotus obliquus extract composition pellets;
(5) Uniformly dispersing the inonotus obliquus extract composition pellets into a solution composed of carboxymethyl cellulose and sodium alginate, and dripping the mixture into a CaCl 2 solution by using a 5mL syringe;
(6) Washing with deionized water to obtain shell type micro-pill Inonotus obliquus extract composition.
Preferably, in the step (4), the extrusion and rounding machine needs to set the diameter of an orifice plate to be 1mm, the extrusion speed to be 30rpm/min, the rounding speed to be 1400rpm/min and the rounding time to be 5min.
Preferably, the concentration of carboxymethyl cellulose in step (5) is 8% (v/v) and the concentration of sodium alginate is 5% (v/v).
Preferably, the CaCl 2 solution in step (5) has a concentration of 0.03mol/L and a volume of 80mL.
Further, the preparation method of the inonotus obliquus polysaccharide specifically comprises the following steps:
(1) Pulverizing Inonotus obliquus fruiting body, sieving with 120 mesh sieve, hot air drying at 60deg.C to constant weight, adding sterile water at feed-liquid ratio of 1:40, extracting at 100deg.C for 30min, repeating for 2 times, mixing extractive solutions, concentrating to 1/8 of original volume, cooling, centrifuging at 7200r/min for 10min, and collecting supernatant;
(2) Adjusting the pH value of the supernatant, adding cellulase and lysozyme, stirring for enzymolysis, and filtering to obtain an enzymolysis extract;
(3) Adding 95% (v/v) ethanol with volume of 4 times of that of the enzymolysis extract, precipitating with ethanol at 4deg.C for 12 hr, centrifuging 7200r/min for 10min, collecting precipitate, soaking in ethanol for 3 times, and concentrating under reduced pressure to obtain ethanol extract;
(4) Dispersing the ethanol extract into suspension with water, extracting with 98% (v/v) ethyl acetate, concentrating under reduced pressure, and vacuum freeze drying to obtain Inonotus obliquus polysaccharide.
Preferably, the supernatant in step (2) has a pH of 6.
Preferably, the stirring conditions in step (2) are 40℃for 1.5h.
Preferably, in the step (2), the cellulase activity is controlled to be 58.8U/mL and the lysozyme activity is controlled to be 70.5U/mL.
The beneficial effects obtained by the invention are as follows:
(1) The traditional Chinese medicine composition provided by the invention has the following effects: inonotus obliquus: promoting blood circulation, resolving hard mass, soothing liver, relieving depression, invigorating qi, nourishing blood, strengthening spleen, regulating stomach, resisting tumor, improving body defense and disease resistance, and regulating intestinal flora; grifola frondosa: neutral nature, sweet taste, efficacy: replenishing qi to invigorate the spleen, tonifying deficiency, strengthening the body resistance, clearing heat and detoxicating, treating spleen deficiency and weak qi, arteriosclerosis, reducing blood sugar, improving immunity and relieving pain; hericium erinaceus: neutral nature, sweet taste, efficacy: invigorating spleen and stomach, invigorating qi and promoting fluid production, and treating asthenia and chronic gastritis; dandelion: cold nature, bitter and sweet taste, efficacy: clearing away heat and toxic materials, protecting liver, promoting urination, resolving carbuncle, resolving hard mass, and eliminating damp-heat, and can be used for treating acute mastitis, lymphadenitis, acute conjunctivitis, acute tonsillitis, and urinary tract infection; cinnamon: sex heat, pungent and sweet taste, efficacy: tonifying fire and yang, warming spleen and stomach, removing accumulated cold, tonifying kidney deficiency, promoting blood circulation and dredging channels, treating limb cold pulse weakness, and treating yang deficiency and deficiency yang floating; mulberry leaf: cold nature, bitter and sweet taste, efficacy: dispelling wind and clearing heat, cooling blood and improving eyesight, anti-inflammatory and diuretic, and treating wind-warm fever, headache, cough due to lung heat and wind arthralgia; xylo-oligosaccharide: inhibiting pathogenic bacteria and diarrhea, protecting liver, reducing blood lipid, enhancing immunity, and resisting cancer;
(2) The invention creatively adopts shell type pellets, improves the taste of the traditional Chinese medicine, improves the medicine carrying quantity and reduces the medicine using times;
(3) The calcium alginate gel is commonly used for constructing a shell structure, but is very unstable in intestinal tracts, and the calcium alginate gel is prepared by adding carboxymethyl cellulose and combining with calcium alginate, and the polymer chains are more compact due to the hydrogen bonding action between the carboxymethyl cellulose and the calcium alginate, so that the pore diameter of the gel is reduced, the microstructure of the shell is regulated, and the calcium alginate gel has higher stability and pH sensitivity;
(4) The inonotus obliquus extraction composition shell type pellets prepared by the invention have better health care effects of eliminating nodules and enhancing immunity by adopting a relatively low-temperature extraction mode and retaining more flavonoids and polysaccharides;
(5) The invention adopts cellulase and lysozyme as compound enzyme for enzymolysis, and the lysozyme breaks down beta-1, 4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in cell wall to decompose insoluble mucopolysaccharide in cell wall into soluble glycopeptide with biological pharmacological activity; cellulose is degraded into glucose by cellulase; according to the invention, through the combination of the cellulase and the lysozyme, the cell wall can be rapidly degraded, and meanwhile, the soluble glycopeptide and polysaccharide are generated, so that the separation from macromolecular organic matters is facilitated, and the purification with overlarge loss rate such as column chromatography is avoided;
Drawings
FIG. 1 glucose standard curve;
FIG. 2 SEM images of shells of different carboxymethyl cellulose content;
FIG. 3 is a graph comparing the effect of each treatment on the release of NO by giant cells RAW-264.7.
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention; all other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are illustrative only and should not be construed as limiting the application.
The experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials and test strains used in the examples described below, unless otherwise specified, were commercially available.
Example 1
The inonotus obliquus extraction composition for eliminating nodules and improving immunity is a shell type micro-pellet inonotus obliquus extraction composition, and comprises the following components in parts by weight: 54 parts of inonotus obliquus polysaccharide, 18 parts of grifola frondosa extract, 10 parts of hericium erinaceus extract, 7 parts of dandelion extract, 5 parts of cinnamon extract, 14 parts of mulberry leaf extract and 15 parts of xylooligosaccharide.
Wherein, the shell type micro-pill inonotus obliquus extraction composition also comprises a shell, and the shell comprises carboxymethyl cellulose, sodium alginate and CaCl 2.
The invention also provides a preparation method of the inonotus obliquus extract composition for eliminating nodules and improving immunity, which comprises the following steps:
(1) Weighing the prepared inonotus obliquus extract composition, adding 10 times of water, and soaking for 2 hours;
(2) Decocting at 100deg.C for 30min, filtering, concentrating under reduced pressure, vacuum lyophilizing, and sieving with 40 mesh sieve to obtain lyophilized powder of Inonotus obliquus extract composition;
(3) Adding sterile water into the freeze-dried powder of the inonotus obliquus extract composition, kneading to prepare a soft material until the color is uniform and the humidity is proper;
(4) Placing the soft material in an extrusion spheronizer, placing the spheronized pellets in a 40 ℃ oven for drying for 12 hours, and sieving to obtain inonotus obliquus extract composition pellets;
(5) Uniformly dispersing the inonotus obliquus extract composition pellets into a solution consisting of 8% (v/v) carboxymethyl cellulose and 5% (v/v) sodium alginate, and dripping the mixture into CaCl 2 solution by using a 5mL syringe;
(6) Washing with deionized water to obtain shell type micro-pill Inonotus obliquus extract composition.
Wherein, in the step (4), the diameter of the orifice plate is required to be set to be 1mm, the extrusion speed is 30rpm/min, the rounding speed is 1400rpm/min, and the rounding time is 5min.
Wherein, the concentration of CaCl 2 solution in the step (5) is 0.03mol/L and the volume is 80mL.
The preparation method of the inonotus obliquus polysaccharide specifically comprises the following steps:
(1) Pulverizing the blocky inonotus obliquus fruiting body, sieving with 120 mesh sieve, hot air drying at 60deg.C to constant weight, adding distilled water according to feed-liquid ratio of 1:40, extracting at 100deg.C for 30min, repeating for 2 times, mixing extractive solutions, concentrating to 1/8 of original volume, cooling, centrifuging at 7200r/min for 10min, and collecting supernatant;
(2) Adjusting pH of the supernatant to 6, adding cellulase and lysozyme, stirring at 40deg.C for enzymolysis for 1.5 hr, and filtering to obtain supernatant as enzymolysis extractive solution;
(3) Adding 95% (v/v) ethanol with volume of 4 times of that of the enzymolysis extract, precipitating with ethanol at 4deg.C for 12 hr, centrifuging 7200r/min for 10min, collecting precipitate, soaking in ethanol for 3 times, and concentrating under reduced pressure to obtain ethanol extract;
(4) Dispersing the ethanol extract into suspension with water, extracting with 98% (v/v) ethyl acetate, concentrating under reduced pressure, and vacuum freeze drying to obtain Inonotus obliquus polysaccharide.
Wherein, in the step (2), the cellulase activity is controlled to be 58.8U/mL and the lysozyme activity is controlled to be 70.5U/mL.
Carboxymethyl cellulose is purchased from Delta (Delta) organisms; cellulase and lysozyme were purchased from Shanghai leaf Biotech company; the rest chemical reagents are all of domestic analytical purity.
Example 2
The inonotus obliquus extraction composition for eliminating nodules and improving immunity is a shell type micro-pellet inonotus obliquus extraction composition, and comprises the following components in parts by weight: 50 parts of inonotus obliquus polysaccharide, 14 parts of grifola frondosa extract, 8 parts of hericium erinaceus extract, 5 parts of dandelion extract, 4 parts of cinnamon extract, 11 parts of mulberry leaf extract and 12 parts of xylooligosaccharide.
Wherein, the shell type micro-pill inonotus obliquus extraction composition also comprises a shell, and the shell comprises carboxymethyl cellulose, sodium alginate and CaCl 2.
In addition, the invention also provides a preparation method of the inonotus obliquus extract composition for eliminating nodules and improving immunity, and the preparation method is implemented according to the embodiment 1.
Example 3
The inonotus obliquus extraction composition for eliminating nodules and improving immunity is a shell type micro-pellet inonotus obliquus extraction composition, and comprises the following components in parts by weight: 57 parts of inonotus obliquus polysaccharide, 21 parts of grifola frondosa extract, 15 parts of hericium erinaceus extract, 12 parts of dandelion extract, 10 parts of cinnamon extract, 18 parts of mulberry leaf extract and 20 parts of xylooligosaccharide.
Wherein, the shell type micro-pill inonotus obliquus extraction composition also comprises a shell, and the shell comprises carboxymethyl cellulose, sodium alginate and CaCl 2.
In addition, the invention also provides a preparation method of the inonotus obliquus extract composition for eliminating nodules and improving immunity, and the preparation method is implemented according to the embodiment 1.
Comparative example 1
This comparative example provides an inonotus obliquus extract composition for eliminating nodules and improving immunity and a preparation method thereof, which is different from example 1 only in that the concentration of carboxymethyl cellulose in step (5) is 3% (v/v), and the content of the remaining components is the same as that of example 1.
Comparative example 2
This comparative example provides an inonotus obliquus extract composition for eliminating nodules and improving immunity and a preparation method thereof, which is different from example 1 only in that the shell type micro-pellet inonotus obliquus extract composition does not contain carboxymethyl cellulose, and the rest components and the content of the components are the same as those in example 1.
Comparative example 3
This comparative example provides an inonotus obliquus extract composition for eliminating nodules and improving immunity and a preparation method thereof, which is different from example 1 only in that the composition is in the form of inonotus obliquus extract composition pellets.
Specifically, the inonotus obliquus extract composition for eliminating nodules and improving immunity is an inonotus obliquus extract composition pellet, and comprises the following components in parts by weight: 54 parts of inonotus obliquus polysaccharide, 18 parts of grifola frondosa extract, 10 parts of hericium erinaceus extract, 7 parts of dandelion extract, 5 parts of cinnamon extract, 14 parts of mulberry leaf extract and 15 parts of xylooligosaccharide.
The invention also provides a preparation method of the inonotus obliquus extract composition for eliminating nodules and improving immunity, which comprises the following steps:
(1) Weighing the prepared inonotus obliquus extract composition, adding 10 times of water, and soaking for 2 hours;
(2) Decocting at 100deg.C for 30min, filtering, concentrating under reduced pressure, vacuum lyophilizing, and sieving with 40 mesh sieve to obtain lyophilized powder of Inonotus obliquus extract composition;
(3) Adding sterile water into the freeze-dried powder of the inonotus obliquus extract composition, kneading to prepare a soft material until the color is uniform and the humidity is proper;
(4) Placing the soft material in an extrusion spheronizer, placing the spheronized pellets in a 40 ℃ oven for drying for 12 hours, and sieving to obtain inonotus obliquus extract composition pellets;
Wherein, in the step (4), the diameter of the orifice plate is required to be set to be 1mm, the extrusion speed is 30rpm/min, the rounding speed is 1400rpm/min, and the rounding time is 5min.
The preparation method of the inonotus obliquus polysaccharide specifically comprises the following steps:
(1) Pulverizing the blocky inonotus obliquus fruiting body, sieving with 120 mesh sieve, hot air drying at 60deg.C to constant weight, adding distilled water according to feed-liquid ratio of 1:40, extracting at 100deg.C for 30min, repeating for 2 times, mixing extractive solutions, concentrating to 1/8 of original volume, cooling, centrifuging at 7200r/min for 10min, and collecting supernatant;
(2) Adjusting pH of the supernatant to 6, adding cellulase and lysozyme, stirring at 40deg.C for enzymolysis for 1.5 hr, and filtering to obtain supernatant as enzymolysis extractive solution;
(3) Adding 95% (v/v) ethanol with volume of 4 times of that of the enzymolysis extract, precipitating with ethanol at 4deg.C for 12 hr, centrifuging 7200r/min for 10min, collecting precipitate, soaking in ethanol for 3 times, and concentrating under reduced pressure to obtain ethanol extract;
(4) Dispersing the ethanol extract into suspension with water, extracting with 98% (v/v) ethyl acetate, concentrating under reduced pressure, and vacuum freeze drying to obtain Inonotus obliquus polysaccharide.
Wherein, in the step (2), the cellulase activity is controlled to be 58.8U/mL and the lysozyme activity is controlled to be 70.5U/mL.
Comparative example 4
The comparative example provides an inonotus obliquus extraction composition for eliminating nodules and improving immunity and a preparation method thereof, which is different from example 1 only in that the preparation method of inonotus obliquus polysaccharide does not comprise step (2), and the rest components and the content of the components are the same as those in example 1.
Experimental example 1
Determination of polysaccharide content
Experiments were performed with the inonotus obliquus polysaccharide prepared in example 1, comparative example 4:
(1) Experimental principle: the polysaccharide is hydrolyzed into monosaccharide under the action of concentrated sulfuric acid, and is dehydrated rapidly to generate a furfural derivative, and the furfural derivative reacts with phenol to generate orange-yellow solution, has characteristic absorption at 490nm, and is quantified compared with a standard series.
(2) Experimental reagent: concentrated sulfuric acid (H 2SO4), ρ=1.84 g/mL, and the like are all analytically pure.
(3) Glucose standard curve: as shown in fig. 1, the glucose standard and the absorbance show good linear relationship, and a regression equation is obtained, wherein y= 5.2442x-0.0793, and the correlation coefficient is r= 0.9932.
(4) Polysaccharide content calculation formula: p= (m×v 1)/(V2×V3) ×0.9
Wherein: p-polysaccharide content per treatment (mg/mL);
M-obtaining sugar content (mg) in the sample measurement solution from the standard curve;
v 1 -sample constant volume (mL);
V 2 -volume of sample measurement solution (mL) moved during colorimetric measurement;
v 3 -aspirate volume upon alcohol precipitation (mL);
0.9-the coefficient of conversion of glucose to polysaccharide.
(5) And (3) calculating: and (3) carrying the sugar content M in the sample measurement solution obtained by checking each treatment on a standard curve into a formula, and calculating to obtain the polysaccharide content of each treatment.
As a result of analysis, the polysaccharide content of the Inonotus obliquus extract composition prepared in example 1 was 352mg/kg, the polysaccharide content of the Inonotus obliquus extract composition prepared in comparative example 4 was 280mg/kg, and the polysaccharide content of the Inonotus obliquus prepared in comparative example 4 was lower because the polysaccharide was not treated by complex enzyme, and was a crude polysaccharide.
Experimental example 2
SEM image of each processing enclosure
Fig. 2a, b, c are SEM images of the casings prepared in example 1 and comparative examples 1 and 2, respectively.
As a result analysis, as shown in fig. 2, SEM images show the microstructure of the shell, in general, all samples are in a network structure, the average pore size of the shell is continuously increased with the decrease of the content of carboxymethyl cellulose, and the network structure is more sparse, and these results indicate that the content of carboxymethyl cellulose in the shell can regulate the microstructure of the shell, which is due to the hydrogen bonding effect between carboxymethyl cellulose and sodium alginate, so that polymer chains are more compact to each other, thereby reducing the pore size of the gel, and the microstructure of the shell has a significant influence on the release behavior of the inonotus obliquus extract composition, because the more compact shell can not only act as a barrier, reduce the direct exposure of the inonotus extract composition in the stomach and intestine, but also help to slow down the release rate thereof, and therefore, the preparation method provided by the present invention is more advantageous in the continuous release of the inonotus obliquus extract composition in the stomach and intestine.
Experimental example 3
Effect of each treatment on the release of NO by megakaryocytes RAW-264.7
Experiments were performed with the inonotus obliquus extraction compositions prepared in example 1, example 2, example 3, comparative example 1, comparative example 2, comparative example 3, comparative example 4, specifically comprising the following steps:
(1) Diluting the Inonotus obliquus extract composition prepared by each treatment to 200 mug/mL with RPMI-1640 medium, determining the amount of NO released by macro-release cells RAW-264.7, wherein the positive control is bacterial lipopolysaccharide (LPS, the final concentration is 1 mug/mL), and the negative control is Phosphate Buffer (PBS);
(2) Placing in a CO 2 incubator at 37 ℃ for constant temperature culture for 48 hours, taking 100 mu L of supernatant in a 96-well plate, adding 50 mu L of Griess reagent, incubating for 10 minutes at constant temperature, measuring 543nm absorbance value, and calculating the amount of NO released by macrophages according to a standard curve.
As shown in FIG. 3, the results of the in vitro activity test of NO produced by macrophage stimulation can be seen, and the Inonotus obliquus extract composition prepared in example 1 has better immune activity; whereas examples 2 and 3 were relatively weak in immune activity due to the change in the proportions of the components of the inonotus obliquus extract composition; comparative example 1, comparative example 2, comparative example 3 changed the content of carboxymethyl cellulose, resulting in poor sustained release performance of the inonotus obliquus extraction composition; the inonotus obliquus polysaccharide prepared in comparative example 4 is crude polysaccharide, and has low active ingredient content, so that NO generated by macrophages is less.
Experimental example 4
Efficacy assessment test
Clinical data: collecting 80 patients with thyroid nodule 18-60 years old treated by clinic, wherein the average age is 32.6 years, 28 men and 52 women are randomly divided into four groups, 20 people are treated, and one group is control.
Diagnostic criteria for traditional Chinese medicine: the method is mainly formulated by referring to the related content of blood stasis phlegm coagulation type goiter in 2007 edition of Chinese medicine science and 2002 edition of Chinese medicine new drug clinical study guiding principle.
Inclusion criteria: meets the diagnosis standard of the nodular goiter in traditional Chinese medicine, such as blood stasis and phlegm coagulation, has basically normal heart, kidney and liver functions, and has no hereditary, infectious and malignant diseases.
The treatment method comprises the following steps: the inonotus obliquus extract composition shell type pellets prepared in the example 1 and the comparative example 4 and the inonotus obliquus extract composition pellets prepared in the comparative example 3 are respectively taken for the treatment of 2 pellets each time and 3 times a day; the control group was given a commercially available pharmaceutical, namely, a UJiale tablet (manufactured by Merck Co.) 1 tablet (25. Mu.g dose) taken in the morning each day, and the treatment effect was measured after expiration of 3 months as a treatment course.
(1) Maximum diameter (cm) of thyroid nodules was measured using a color Doppler flow imager and the experimental results are shown in Table 1.
TABLE 1 comparison of maximum diameters (cm) of thyroid nodules before and after treatment
From the results of the analysis of the results in Table 1, it is clear that the maximum diameter of thyroid nodule of patients can be effectively reduced after patients in treatment group take the inonotus obliquus extract composition shell-type pellets prepared in example 1 and comparative example 4 and the inonotus obliquus extract composition pellets prepared in comparative example 3 for three months; wherein the therapeutic effect of example 1 is optimal; in contrast, the maximum diameter of thyroid nodules was not significantly changed in the patients of the control group who took the Youjiale tablet.
(2) The thyroid function level was measured, and the experimental results are shown in table 2.
TABLE 2 comparison of thyroid function levels (FT 3, FT4, TSH) before and after treatment
Results analysis As shown in Table 2, after patients in treatment group took the Inonotus obliquus extract composition shell-type pellets prepared in example 1 of the present invention for three months, the thyroid function level of the patients remained essentially unchanged; in contrast, the TSH levels of the control group of patients taking the youjiale tablet were significantly reduced compared to those before treatment, indicating impaired thyroid function levels.
In conclusion, the traditional Chinese medicine formula preparation can effectively improve the nodular symptoms of a patient through reasonable compatibility of the traditional Chinese medicine raw materials and optimization of the preparation process, has high clinical application value, is combined with various medicines, can eliminate stagnation and gall, sooth liver and relieve depression, activate blood and remove stasis, can remarkably inhibit and reduce thyroid nodule of the patient, does not influence the first function level of the patient, and has high safety and no toxic or side effect.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
The invention and its embodiments have been described above with no limitation, and the invention is illustrated in the figures of the accompanying drawings as one of its embodiments, without limitation in practice. In summary, those skilled in the art, having benefit of this disclosure, will appreciate that the invention can be practiced without the specific details disclosed herein.
Claims (10)
1. The preparation method of the inonotus obliquus extraction composition for eliminating nodules and improving immunity is characterized by comprising the following steps of:
(1) Weighing the prepared inonotus obliquus extract composition, adding 10 times of water, and soaking for 2 hours;
(2) Decocting at 100deg.C for 30min, filtering, concentrating under reduced pressure, vacuum lyophilizing, and sieving with 40 mesh sieve to obtain lyophilized powder of Inonotus obliquus extract composition;
(3) Adding sterile water into the freeze-dried powder of the inonotus obliquus extract composition, kneading to prepare a soft material until the color is uniform and the humidity is proper;
(4) Placing the soft material in an extrusion spheronizer, placing the spheronized pellets in a 40 ℃ oven for drying for 12 hours, and sieving to obtain inonotus obliquus extract composition pellets;
(5) Uniformly dispersing the inonotus obliquus extract composition pellets into a solution composed of carboxymethyl cellulose and sodium alginate, and dripping the mixture into a CaCl 2 solution by using a 5mL syringe;
(6) Washing with deionized water to obtain shell type micro-pill Inonotus obliquus extract composition.
2. The method for preparing the inonotus obliquus extract composition for eliminating nodules and improving immunity according to claim 1, which is characterized in that: in the step (4), the extrusion and rounding machine is required to set the diameter of an orifice plate to be 1mm, the extrusion speed to be 30rpm/min, the rounding speed to be 1400rpm/min and the rounding time to be 5min.
3. The method for preparing the inonotus obliquus extract composition for eliminating nodules and improving immunity according to claim 2, which is characterized in that: in the step (5), the concentration of carboxymethyl cellulose is 8% (v/v), and the concentration of sodium alginate is 5% (v/v).
4. The method for preparing an anti-tubercle and immunity-enhancing Inonotus obliquus extract composition as claimed in claim 3, wherein the concentration of CaCl 2 solution in the step (5) is 0.03mol/L and the volume is 80mL.
5. The method for preparing the inonotus obliquus extract composition for eliminating nodules and improving immunity according to claim 4, wherein the method for preparing the inonotus obliquus polysaccharide specifically comprises the following steps:
(1) Pulverizing Inonotus obliquus fruiting body, sieving with 120 mesh sieve, hot air drying at 60deg.C to constant weight, adding sterile water at feed-liquid ratio of 1:40, extracting at 100deg.C for 30min, repeating for 2 times, mixing extractive solutions, concentrating to 1/8 of original volume, cooling, centrifuging at 7200r/min for 10min, and collecting supernatant;
(2) Adjusting the pH value of the supernatant, adding cellulase and lysozyme, stirring for enzymolysis, and filtering to obtain an enzymolysis extract;
(3) Adding 95% (v/v) ethanol with volume of 4 times of that of the enzymolysis extract, precipitating with ethanol at 4deg.C for 12 hr, centrifuging 7200r/min for 10min, collecting precipitate, soaking in ethanol for 3 times, and concentrating under reduced pressure to obtain ethanol extract;
(4) Dispersing the ethanol extract into suspension with water, extracting with 98% (v/v) ethyl acetate, concentrating under reduced pressure, and vacuum freeze drying to obtain Inonotus obliquus polysaccharide.
6. The method for preparing the inonotus obliquus extract composition for eliminating nodules and improving immunity according to claim 5, which is characterized in that: the pH value of the supernatant in the step (2) is 6.
7. The method for preparing the inonotus obliquus extract composition for eliminating nodules and improving immunity according to claim 6, which is characterized in that: the stirring condition in the step (2) is that stirring is carried out for 1.5h at 40 ℃.
8. A method for preparing an inonotus obliquus extract composition for eliminating nodules and improving immunity according to claim 7, which is characterized in that: in the step (2), the cellulase activity is controlled to be 58.8U/mL and the lysozyme activity is controlled to be 70.5U/mL.
9. The method for preparing the inonotus obliquus extract composition for eliminating nodules and improving immunity according to claim 8, wherein the inonotus obliquus extract composition comprises the following components in parts by weight: 50-57 parts of inonotus obliquus polysaccharide, 14-21 parts of grifola frondosa extract, 8-15 parts of hericium erinaceus extract, 5-12 parts of dandelion extract, 4-10 parts of cinnamon extract, 11-18 parts of mulberry leaf extract and 12-20 parts of xylooligosaccharide.
10. The method for preparing the inonotus obliquus extract composition for eliminating nodules and improving immunity according to claim 9, which is characterized in that: the inonotus obliquus extraction composition further comprises a shell, wherein the shell comprises carboxymethyl cellulose, sodium alginate and CaCl 2.
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CN104997883A (en) * | 2015-06-18 | 2015-10-28 | 哈尔滨贝加尔泰生物工程有限公司 | Inonotus obliquus raspberry tree red raspberry composition, inonotus obliquus raspberry tree red raspberry composite oral solution and preparation method and application thereof |
US20180085305A1 (en) * | 2016-09-29 | 2018-03-29 | Gesea Biosciences Inc. | Bioerodible implant for long-term drug delivery and associated methods of manufacture and use |
CN113116862A (en) * | 2019-12-30 | 2021-07-16 | 辽宁上药好护士药业(集团)有限公司 | Blood stasis arthralgia micro-pill and preparation method and application thereof |
CN113402625A (en) * | 2021-06-28 | 2021-09-17 | 安发(福建)生物科技有限公司 | Inonotus obliquus polysaccharide and extraction method thereof |
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Publication number | Priority date | Publication date | Assignee | Title |
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CN104997883A (en) * | 2015-06-18 | 2015-10-28 | 哈尔滨贝加尔泰生物工程有限公司 | Inonotus obliquus raspberry tree red raspberry composition, inonotus obliquus raspberry tree red raspberry composite oral solution and preparation method and application thereof |
US20180085305A1 (en) * | 2016-09-29 | 2018-03-29 | Gesea Biosciences Inc. | Bioerodible implant for long-term drug delivery and associated methods of manufacture and use |
CN113116862A (en) * | 2019-12-30 | 2021-07-16 | 辽宁上药好护士药业(集团)有限公司 | Blood stasis arthralgia micro-pill and preparation method and application thereof |
CN113402625A (en) * | 2021-06-28 | 2021-09-17 | 安发(福建)生物科技有限公司 | Inonotus obliquus polysaccharide and extraction method thereof |
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