CN112759663B - Liuan Guapian polysaccharide with immunoregulatory activity and preparation method and application thereof - Google Patents
Liuan Guapian polysaccharide with immunoregulatory activity and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses Liuan Guapian polysaccharide with immunoregulation activity, and a preparation method and application thereof, wherein the molecular weight of the polysaccharide is 2.35 multiplied by 105Da. The Liuan Guapian polysaccharide can regulate the cellular immune activity, and the characteristic of the Liuan Guapian polysaccharide for enhancing immune cells in vitro enables the Liuan Guapian polysaccharide to be used as an immunomodulator, can be widely applied to the fields of health-care foods and medicines, and has important research, development and utilization prospects.
Description
Technical Field
The invention relates to Liuan Guapian polysaccharide with immunoregulation activity and a preparation method and application thereof, belonging to the technical field of food and medicine.
Background
The immunity is an important physiological function of human body, and the organism can recognize self and non-self components by means of the function, thereby destroying and rejecting heterogeneous substances entering the organism or damaged and variant cells generated by the organism to maintain the health of the human body. Walford proposed the theory of immunosenescence in 1962, and thought that the decline of immune function is an important factor causing body senescence, with the increase of age, the immune organs age, the number of immune cells is reduced, the immune function of the body is gradually weakened, and the incidence of infectious diseases and tumors is gradually increased. The decline of the immune function can further accelerate the aging of the body.
The medicine for improving the human body immune function can bring a plurality of side effects such as drug resistance, drug dependence, teratogenicity and carcinogenicity while regulating the immune activity. Therefore, the search for a natural, highly effective, non-toxic immune modulator is the focus of research by researchers. With the intensive research on natural active substances, more and more researchers pay attention to polysaccharides, and in recent years, polysaccharides of green tea, black tea and the like have been proved to have good immunoregulatory efficacy.
Liuan Guapian, abbreviated as Guapian and piece tea, is a traditional historical famous tea in China, also one of ten famous teas in China, is produced from Dabie mountain area in Liuan City, Anhui province, and is a special tea type of green tea. In all tea leaves in the world, Liuan Guapian is the only tea without buds and stalks and is prepared from single raw leaves. The bud removal not only keeps a single-piece body, but also has no green grass flavor; the stems are lignified in the making process, and after the stems are removed, the tea can be ensured to have thick but not bitter taste and fragrant but not astringent taste. As a local characteristic biomass resource, the immunocompetence of the green tea polysaccharide is not reported at present, so that the green tea polysaccharide has potential research and development values.
Disclosure of Invention
The invention aims to provide Liuan Guapian polysaccharide with immunoregulation activity and a preparation method and application thereof. The Liuan Guapian polysaccharide found by the invention has good immunoregulation activity, has NO obvious cytotoxicity to RAW264.7 macrophages when the Liuan Guapian polysaccharide is 25-400 mu g/mL in vitro immunocompetence detection, can greatly increase NO release amount of RAW264.7 macrophages, activates immunocyte activity, and has important research, development and utilization prospects.
In order to realize the purpose of the invention, the following technical scheme is adopted:
a Liuan Guapian polysaccharide with immunoregulatory activity and molecular weight of 2.35 × 105Da, an infrared spectrum shows that the polysaccharide has a remarkable infrared characteristic absorption peak.
The preparation method of Liuan Guapian polysaccharide with immunoregulation activity, provided by the invention, comprises the following steps:
(1) crushing Liuan Guapian tea leaves by a crusher, and sieving by a 80-mesh sieve to obtain powder;
(2) soaking the obtained powder in 50-80 deg.C anhydrous ethanol for 2-4h, filtering with gauze, and vacuum drying to obtain filter residue;
(3) leaching the obtained filter residue in hot water at 50-100 deg.C for 1-5 hr according to a material-liquid ratio of 1g:10-40mL, repeating for 2-3 times, and mixing extractive solutions;
(4) concentrating the extracting solution obtained in the step (3) to 1/3-1/10, adding ethanol into the obtained concentrated solution until the final volume concentration of the ethanol is 75-90%, standing for 8-12h at 4 ℃, centrifuging for 10-30min at 8000rpm of 5000-;
(5) dissolving the precipitate obtained in the step (4) with water, centrifuging to remove insoluble substances, and freeze-drying to obtain crude Liuan Guapian polysaccharide;
(6) preparing the crude polysaccharide of the Liuan Guapian tablet obtained in the step (5) into a crude polysaccharide solution with the concentration of 20-40mg/mL by using deionized water, centrifuging and filtering by using a 0.22 mu m filter membrane, putting the obtained trapped solution into a DEAE-cellulose 52 ion exchange column for purification, wherein the mobile phase is a 0.3mol/L sodium chloride solution, and the flow rate is 0.5-1.0 mL/min; collecting the product, purifying by using a dialysis bag with the molecular weight cutoff of 3.5-5.5 kDa, and freeze-drying to obtain the Liuan Guapian polysaccharide.
Preferably, in the step (2), the temperature of the vacuum drying is 60-70 ℃ and the time is 2-4 h.
Preferably, in the steps (5) and (6), the rotation speed of the centrifugation is 7000-10000 rpm.
The Liuan Guapian polysaccharide found by the invention has good immunoregulation activity and can be used for preparing an immunomodulator.
The invention has the beneficial effects that:
1. the invention discovers that the Liuan Guapian polysaccharide has obvious immunoregulation effect on macrophage RAW264.7, has no toxicity on normal cells, and has higher survival rate of the macrophage even under the condition of high concentration (400 mu g/mL); the immune activity test shows that the Liuan Guapian polysaccharide also shows obvious stimulation effect at low concentration (25 mu g/mL), which indicates that the Liuan Guapian polysaccharide can regulate the cellular immune activity. At present, no immunoreaction report of the Liuan Guapian polysaccharide is found.
2. The Liuan Guapian polysaccharide prepared by the method has only one absorption peak and the molecular weight is 2.35 multiplied by 10 through High Performance Liquid Chromatography (HPLC) detection5Da, has strong immunoregulation activity, and can be used as a novel immunoregulator to be applied to health-care foods and medicines.
3. The method for preparing the active polysaccharide has high efficiency and stable activity.
Drawings
FIG. 1 is a high performance liquid chromatogram of Liuan Guapian polysaccharide obtained in example 1.
FIG. 2 is a graph showing the toxicity test of Liuan Guapian polysaccharide obtained in example 1 on macrophages. As can be seen from FIG. 2, Liu' an Guapian polysaccharide has no toxic effect on the macrophage RAW264.7, and can promote the growth of RAW264.7 macrophages within a certain concentration range.
FIG. 3 is a graph showing the measurement of the NO release amount of macrophages by the Liuan Guapian polysaccharide obtained in example 1. As can be seen from FIG. 3, compared with the blank group, Liuan Guapian polysaccharide has an obvious promotion effect on NO secretion of macrophages, which indicates that Liuan Guapian polysaccharide has stronger immunoregulatory activity.
FIG. 4 shows the phagocytosis of macrophages by Liuan Guapian obtained in example 1. As can be seen from FIG. 4, phagocytosis of macrophages after 24h of Liuan Guapian polysaccharide action is obviously increased and is concentration-dependent, and compared with the control group, the phagocytosis activity of Liuan Guapian polysaccharide is obviously increased and is dose-dependent when the concentration of Liuan Guapian polysaccharide is 400 mug/mL.
FIG. 5 is the morphological observation of macrophages after phagocytosis in example 1, wherein FIG. 5a corresponds to the blank control group, FIG. 5b corresponds to the 1 μ g/mLLPS group, FIG. 5c corresponds to the 100 μ g/mL Liuan Guapian polysaccharide group, and FIG. 5d corresponds to the 400 μ g/mL Liuan Guapian polysaccharide group. As can be seen from fig. 5, the cells of the blank control group were round and had a normal morphology, whereas the morphology of the cells was significantly changed after the treatment with the polysaccharide of liuan guapian, and it can be clearly seen that many spikes were generated around the cells and had a concentration dependence.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. The following disclosure is merely exemplary and illustrative of the inventive concept, and those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
Example 1
In the embodiment, Liuan Guapian tea is taken as a raw material, and Liuan Guapian polysaccharide is extracted, separated and purified according to the following steps:
(1) 100g of Liuan Guapian tea leaves are ground by a grinder and then sieved by a 80-mesh sieve to obtain powder.
(2) Soaking the obtained powder in 1L of 70 deg.C anhydrous ethanol for 2 hr, filtering with gauze, and vacuum drying at 70 deg.C for 3 hr to obtain filter residue.
(3) Leaching the obtained filter residue in hot water at 90 deg.C for 2h according to a material-liquid ratio of 1g:20mL, repeating for 2 times, and mixing extractive solutions.
(4) Concentrating the extracting solution obtained in the step (3) to 1/10 volume, adding ethanol into the obtained concentrated solution until the final volume concentration of the ethanol is 75 volume%, standing at 4 ℃ for 10h, centrifuging at 10000rpm for 10min, and collecting precipitate.
(5) And (4) dissolving the precipitate obtained in the step (4) with water, centrifuging at 10000rpm for 10min to remove insoluble substances, and freeze-drying to obtain the crude Liuan Guapian polysaccharide.
(6) Preparing the crude polysaccharide of the Liuan Guapian slices obtained in the step (5) into a crude polysaccharide solution with the concentration of 40mg/mL by using deionized water, centrifuging for 1min at 10000rpm, filtering by using a 0.22 mu m filter membrane, and purifying the obtained trapped solution in a DEAE-cellulose 52 ion exchange column at the flow rate of 0.75mL/min and with the mobile phase of 0.3mol/L sodium chloride solution; collecting the product, purifying by using a dialysis bag with the molecular weight cutoff of 3.5kDa, and freeze-drying to obtain the Liuan Guapian polysaccharide.
Fig. 1 is a high performance liquid chromatogram of liuan guapian polysaccharide obtained in this example, and it can be seen from the figure that: the polysaccharide sample has a single absorption peak with retention time of 5.45min, and its molecular weight is 2.35 × 105Da。
1. Cytotoxicity assays
The Liuan Guapian polysaccharide was diluted to different concentrations with growth medium. RAW264.7 cells were cultured in growth medium at 37 ℃ and then seeded into 96-well plates (1X 10)4Cells/well) and in 96-well plates, respectivelyIncreasing concentrations of 100. mu.L Liuan Guapian polysaccharide (25-400. mu.g/mL) were added for treatment. Meanwhile, 1. mu.g/mL of LPS was used as a positive control group.
After 24h incubation, cell viability was determined using the MTT method: the cell supernatant was first removed and 100mL of MTT solution (0.5mg/mL) was added to each well and incubated for an additional 4 h. Next, the supernatant was discarded and 100 μ L DMSO was added to the cell culture, followed by shaking for 10min away from light until no visible particulate matter was detected. The absorbance at 570nm was measured using a microplate reader. The toxic effect result of Liuan Guapian polysaccharide on macrophages is shown in figure 2, and within the concentration range of 25 mug/mL-400 mug/mL, Liuan Guapian polysaccharide has no toxic effect on macrophages and has promotion effect.
2. NO production amount measurement (Griess method)
The Liuan Guapian polysaccharide was diluted to different concentrations with growth medium. RAW264.7 cells were plated in 48-well plates (1X 10)5Individual cells/well) were preincubated at 37 ℃ for 24 h. Then 200. mu.L of Liuan Guapian polysaccharide (25-400. mu.g/ml) with increasing concentration is added into the well plate for 24 h. Meanwhile, cells were treated with LPS (1. mu.g/mL) for 24h under the same conditions as a positive control group. The cell culture supernatant was then collected and mixed with an equal volume of Griess reagent, incubated at room temperature for 30min, and measured with a microplate reader at 540 nm. The results are shown in FIG. 3.
As can be seen from fig. 3, the production of NO in the blank control was much lower than the positive control of LPS and hexaampere papyrhyacinthine polysaccharide treated groups, with significant differences. Meanwhile, after the Liuan Guapian polysaccharide with different concentrations is treated for 24 hours, the generation of NO is concentration-dependent, and the NO release amount of macrophages is gradually increased. The experimental result shows that the Liuan Guapian polysaccharide has a remarkable stimulation effect on RAW264.7 cells, and particularly has a promoting effect on the generation of NO.
3. Phagocytic Activity assay and morphological Observation
Phagocytic capacity of macrophages was measured by a neutral red uptake assay. Mixing cells (1X 10) with Liuan Guapian polysaccharide (25-400 μ g/mL) or LPS (1 μ g/mL)5Individual cells/well) were placed in 48-well plates in an incubator (5% CO) at 37 ℃2) And (4) performing medium incubation for 24 h. Removing the supernatantSolution, and neutral red solution (v/v, 0.05%) was added to the wells and incubation continued for 20 min. Then, the supernatant was discarded and the cells were washed three times with PBS to remove excess neutral red not phagocytosed by RAW264.7 cells, added to the lysate, and lysed at 4 ℃ for 2 h. Cells were incubated overnight at room temperature and assayed at 540 nm. The morphology of RAW264.7 cells phagocytosing neutral red was observed by an inverted fluorescence microscope.
As can be seen from fig. 4, the phagocytosis index of the polysaccharide-treated Liuan Guapian group was significantly higher than the blank after 24h and showed good concentration dependence. Meanwhile, the maximum concentration of polysaccharide in Liuan Guapian (400 mu g/mL) is significantly higher than that in the LPS positive control group. The Liuan Guapian polysaccharide can remarkably promote the phagocytic capacity of RAW264.7 cells.
To further observe the morphological changes of the cells during phagocytosis, the morphology of RAW264.7 cells was examined. As can be seen in fig. 5, the blank control cells were round with normal morphology (fig. 5 a). However, the cell morphology after treatment with LPS (FIG. 5b, LPS concentration 1. mu.g/mL) and Liuan Guapian polysaccharide (FIGS. 5c-d, Liuan Guapian polysaccharide concentrations 100, 400. mu.g/mL, respectively) changed significantly. It is clearly seen that a number of spikes are produced around the cell, and this structure is advantageous in increasing the contact area of the cell with the external environment, contributing to phagocytosis. When the concentration of Liuan Guapian polysaccharide reached 400. mu.g/mL, the spikes were significantly increased (FIG. 5d), exhibiting good concentration dependence.
Example 2
In the embodiment, Liuan Guapian tea is taken as a raw material, and Liuan Guapian polysaccharide is extracted, separated and purified according to the following steps:
(1) 100g of Liuan Guapian tea leaves are ground by a grinder and then sieved by a 80-mesh sieve to obtain powder.
(2) Soaking the obtained powder in 1L of 70 deg.C anhydrous ethanol for 2 hr, filtering with gauze, and vacuum drying at 70 deg.C for 3 hr to obtain filter residue.
(3) Leaching the obtained filter residue in hot water at 80 deg.C for 3h according to a material-liquid ratio of 1g:20mL, repeating for 3 times, and mixing extractive solutions.
(4) Concentrating the extracting solution obtained in the step (3) to 1/10 volume, adding ethanol into the obtained concentrated solution until the final volume concentration of the ethanol is 75 volume%, standing at 4 ℃ for 12h, centrifuging at 8000rpm for 10min, and collecting precipitate.
(5) And (4) dissolving the precipitate obtained in the step (4) with water, centrifuging at 10000rpm for 10min to remove insoluble substances, and freeze-drying to obtain the crude Liuan Guapian polysaccharide.
(6) Preparing the crude polysaccharide of the Liuan Guapian slices obtained in the step (5) into a crude polysaccharide solution with the concentration of 40mg/mL by using deionized water, centrifuging for 1min at 10000rpm, filtering by using a 0.22 mu m filter membrane, and purifying the obtained trapped solution in a DEAE-cellulose 52 ion exchange column at the flow rate of 0.5mL/min and with the mobile phase of 0.3mol/L sodium chloride solution; collecting the product, purifying by using a dialysis bag with the molecular weight cutoff of 3.5kDa, and freeze-drying to obtain the Liuan Guapian polysaccharide.
The molecular weight of the Liuan Guapian polysaccharide obtained in the example is tested to be 2.35 multiplied by 105Da and has good immunoregulation activity.
Example 3
In the embodiment, Liuan Guapian tea is taken as a raw material, and Liuan Guapian polysaccharide is extracted, separated and purified according to the following steps:
(1) 100g of Liuan Guapian tea leaves are ground by a grinder and then sieved by a 80-mesh sieve to obtain powder.
(2) Soaking the obtained powder in 2L of 80 deg.C anhydrous ethanol for 3 hr, filtering with gauze, and vacuum drying at 70 deg.C for 3 hr to obtain filter residue.
(3) Leaching the obtained filter residue in hot water at 80 deg.C for 2h according to a material-liquid ratio of 1g:30mL, repeating for 3 times, and mixing extractive solutions.
(4) Concentrating the extracting solution obtained in the step (3) to 1/10 volume, adding ethanol into the obtained concentrated solution until the final volume concentration of the ethanol is 80%, standing at 4 ℃ for 8h, centrifuging at 8000rpm for 20min, and collecting precipitate.
(5) And (4) dissolving the precipitate obtained in the step (4) with water, centrifuging at 10000rpm for 10min to remove insoluble substances, and freeze-drying to obtain the crude Liuan Guapian polysaccharide.
(6) Preparing the crude polysaccharide of the Liuan Guapian slices obtained in the step (5) into a crude polysaccharide solution with the concentration of 50mg/mL by using deionized water, centrifuging for 1min at 10000rpm, filtering by using a 0.22 mu m filter membrane, and purifying the obtained trapped solution in a DEAE-cellulose 52 ion exchange column at the flow rate of 1mL/min, wherein the mobile phase is a 0.3mol/L sodium chloride solution; collecting the product, purifying by using a dialysis bag with the molecular weight cutoff of 3.5kDa, and freeze-drying to obtain the Liuan Guapian polysaccharide.
The molecular weight of the Liuan Guapian polysaccharide obtained in the example is tested to be 2.35 multiplied by 105Da and has good immunoregulation activity.
The present invention is not limited to the above exemplary embodiments, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. The Liuan Guapian polysaccharide with immunoregulation activity is characterized in that: the molecular weight of the Liuan Guapian polysaccharide is 2.35 × 105Da; the preparation method of the Liuan Guapian polysaccharide comprises the following steps:
(1) crushing Liuan Guapian tea leaves by a crusher, and sieving by a 80-mesh sieve to obtain powder;
(2) soaking the obtained powder in 50-80 deg.C anhydrous ethanol for 2-4h, filtering with gauze, and vacuum drying to obtain filter residue;
(3) leaching the obtained filter residue in hot water at 50-100 deg.C for 1-5 hr according to a material-liquid ratio of 1g:10-40mL, repeating for 2-3 times, and mixing extractive solutions;
(4) concentrating the extracting solution obtained in the step (3) to 1/3-1/10, adding ethanol into the obtained concentrated solution until the final volume concentration of the ethanol is 75-90%, standing for 8-12h at 4 ℃, centrifuging for 10-30min at 8000rpm of 5000-;
(5) dissolving the precipitate obtained in the step (4) with water, centrifuging to remove insoluble substances, and freeze-drying to obtain crude Liuan Guapian polysaccharide;
(6) preparing the crude polysaccharide of the Liuan Guapian tablet obtained in the step (5) into a crude polysaccharide solution with the concentration of 20-50mg/mL by using deionized water, centrifuging and filtering by using a 0.22 mu m filter membrane, putting the obtained trapped solution into a DEAE-cellulose 52 ion exchange column for purification, wherein the mobile phase is a 0.3mol/L sodium chloride solution, and the flow rate is 0.5-1.0 mL/min; collecting the product, purifying by using a dialysis bag, and freeze-drying to obtain the Liuan Guapian polysaccharide.
2. The Liuan Guapian polysaccharide having immunomodulatory activity, according to claim 1, wherein: in the step (2), the temperature of the vacuum drying is 60-70 ℃ and the time is 2-4 h.
3. The Liuan Guapian polysaccharide having immunomodulatory activity, according to claim 1, wherein: in the step (6), the molecular weight cut-off of the dialysis bag is 3.5kDa to 5.5 kDa.
4. The Liuan Guapian polysaccharide having immunomodulatory activity, according to claim 1, wherein: in the steps (5) and (6), the rotation speed of the centrifugation is 7000-10000 rpm.
5. The use of the polysaccharide of liuan guapian with immunomodulatory activity of claim 1, wherein: can be used for preparing immunomodulator.
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| CN101220100A (en) * | 2007-01-10 | 2008-07-16 | 兰州大学 | Separation and purification method for squash polyoses and use of obtained component |
| CN102887962A (en) * | 2012-10-10 | 2013-01-23 | 安徽工程大学 | Method for preparing green tea polysaccharide through separation and purification and application of polysaccharide |
| KR20150100599A (en) * | 2015-08-18 | 2015-09-02 | (주)아모레퍼시픽 | Method for preparing polysaccharide of green tea |
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| CN105367679A (en) * | 2015-12-03 | 2016-03-02 | 镇江市丹徒区南山溪园茶叶专业合作社 | Green tea polysaccharide extraction method |
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| CN101220100A (en) * | 2007-01-10 | 2008-07-16 | 兰州大学 | Separation and purification method for squash polyoses and use of obtained component |
| CN102887962A (en) * | 2012-10-10 | 2013-01-23 | 安徽工程大学 | Method for preparing green tea polysaccharide through separation and purification and application of polysaccharide |
| KR20150100599A (en) * | 2015-08-18 | 2015-09-02 | (주)아모레퍼시픽 | Method for preparing polysaccharide of green tea |
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