CN102276745B - Franchet groundcherry fruit stem polysaccharide with immunological activity and preparation method thereof - Google Patents
Franchet groundcherry fruit stem polysaccharide with immunological activity and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to polysaccharide with immunological activity, in particular to a franchet groundcherry fruit stem polysaccharide with immunological activity and a preparation method thereof. The method comprises the following steps of: extracting through a water immersion method; precipitating water immersion liquor extract; deproteinizing by using an enzyme method in combination with a Sevage method; drying to obtain rough polysaccharide; eluting with physiological saline; freeze-drying to obtain polysaccharide Q-WSP; and further purifying through HPLC (High Performance Liquid Chromatography) to obtain WSP (Water Soluble Polysaccharide). The molecular weight of the WSP obtained by using the method is about 7,000Da through HPLC measurement and the components are uniform. Monosaccharide components obtained through gas chromatographic analysis are Rha, Ara, Gal, Glc and GalA. The franchet groundcherry fruit stem polysaccharide has an immunological enhancement effect and can be used for foods and health-care products for improving the immunologic function of the organism. The preparation method is simple and reliable. The franchet groundcherry fruit stem has a wide source and is changed into things of value through development and utilization of the franchet groundcherry fruit stem polysaccharide.
Description
Technical field
The invention belongs to the immunocompetence polysaccharide, be specifically related to a kind of immunocompetent Franchet Groundcherry Calyx and Fruit stem polysaccharide and its preparation method and application that has.
Background technology
The Franchet Groundcherry Calyx and Fruit formal name used at school is wintercherry, calls red Miss, Huechys sanguinea De Geer., Calyx seu Fructus physalis, bitter Miss, root of Manchurian Currant.Fruit is berry, and sphere is orange-yellow when ripe; The place is deposited calyx and is grown up with fruit and increase, orange, the bulk berry outside that wraps in, likeness in form lantern and gaining the name.
Franchet Groundcherry Calyx and Fruit is Solanaceae, Physalis (Physalis alkekengi L.) per nnial herb, and all there is distribution all parts of the country, are the medicine food dual purpose plants of commonly using.The Franchet Groundcherry Calyx and Fruit herb all can be used as medicine; Its fruit flavor is sweet, sour, cold, edible.Have clearing heat and detoxicating, the effects such as relieve sore throat reduces phlegm, diuresis, be used for pharyngalgia, hoarsen, phlegm heat cough, dysuria, control pemphigus, eczema etc. outward.
In recent years, along with developing rapidly of the medicine take Franchet Groundcherry Calyx and Fruit as development of raw materials and food, the demand that wild Franchet Groundcherry Calyx and Fruit supply far away can not satisfying the market, therefore the Franchet Groundcherry Calyx and Fruit of a large amount of plantations arise at the historic moment.At present, the efficient part of the Franchet Groundcherry Calyx and Fruit that is widely used remains the Franchet Groundcherry Calyx and Fruit fruit, and its stem is discarded because can not get effectively utilizing.Therefore the research of Franchet Groundcherry Calyx and Fruit stem polysaccharide has great importance to comprehensive, effective development and utilization Franchet Groundcherry Calyx and Fruit.
Summary of the invention
The purpose of this invention is to provide a kind of have immunocompetent wintercherry fruit polysaccharide, preparation method and purposes.Concrete steps of the present invention:
(1) the dry stem with Franchet Groundcherry Calyx and Fruit is cut into long the breaking into pieces of 2cm, soaked overnight, and solid-liquid ratio is 1g: 20ml to 1g: 50ml;
(2) water seaoning was extracted 1-4 hour, three lixiviates, temperature 50-100 ℃;
(3) with ethanol the infusion extract is precipitated, the final concentration of ethanol is 65-85%;
(4) multigelation is extremely without after the precipitation, with enzyme process and Sevage method associating deproteinated, the dry Crude polysaccharides that gets;
(5) Crude polysaccharides QAE SephadexA-25 carries out wash-out with physiological saline, gets polysaccharide Q-WSP after the lyophilize;
(6) Q-WSP is further purified through HPLC and obtains WSP.
The WSP that aforesaid method obtains measures its molecular weight through HPLC and is approximately 7000Da, its component homogeneous.Getting monosaccharide component through gas chromatographic analysis is Rha, Ara, Gal, Glc and GalA, and its mol ratio is 2.6: 1.0: 6.5: 3.5: 2.4.
Gained Franchet Groundcherry Calyx and Fruit stem polysaccharide of the present invention has immuno-potentiation, can be used for improving food and the healthcare products of body's immunity.The preparation method is simple, reliable.Franchet Groundcherry Calyx and Fruit stem wide material sources, the development and use of Franchet Groundcherry Calyx and Fruit stem polysaccharide will make the Franchet Groundcherry Calyx and Fruit stem turn waste into wealth.
Embodiment:
Embodiment 1:
One, the preparation of Franchet Groundcherry Calyx and Fruit stem polysaccharide
The 1 dry stem 500g with Franchet Groundcherry Calyx and Fruit is cut into long the breaking into pieces of 2cm with scissors, and the 3000ml water soaking spends the night;
2 water seaonings were extracted 2 hours, three lixiviates, 100 ℃ of temperature;
3 usefulness ethanol precipitate the infusion extract, and the final concentration of ethanol is 85%;
4 multigelations are extremely without after the precipitation, with enzyme process and Sevage method associating deproteinated, the dry Crude polysaccharides that gets;
5 Crude polysaccharides carry out wash-out with QAE Sephadex A-25 column chromatography with physiological saline, get polysaccharide Q-WSP after the lyophilize;
6Q-WSP is further purified through HPLC and obtains WSP;
WSP measures its molecular weight through HPLC and is approximately 7000Da, is polysaccharide homogeneous component.Getting monosaccharide component through gas chromatographic analysis is Rha, Ara, Gal, Glc and GalA, and its mol ratio is 2.6: 1.0: 6.5: 3.5: 2.4.
Two, Franchet Groundcherry Calyx and Fruit stem polysaccharide immune-enhancing activity experiment
(1) animal grouping and immunization method thereof
Five age in week male ICR mouse be used for experimentation on animals, 5 every group; Take chicken egg white (OVA) as antigen, subcutaneous injection.
Experiment is grouped as follows:
First group (Saline): injecting normal saline, as blank
Second group (OVA): g/ of injection OVA100 μ is as negative control
The 3rd group (QuilA): only inject g/+QuilA10 μ g/ of OVA100 μ, the positive contrast of QuilA (South America quillaja-saponin)
The 4th group (WSP): g/+Franchet Groundcherry Calyx and Fruit stem polysaccharide 200 μ g/ of injection OVA100 μ only
More than all immunity twice of each group, interval 14 days.Rear 14 days of for the second time immunity, mtt assay detect animal splenic lymphocyte increment reaction; Detect the variation of anti-OVA specific antibody IgG in the animal antiserum with the ELISA method.
(2) mouse spleen lymphocyte transformation experiment
1 experimental technique
(1) the aseptic mouse spleen of getting, and make single cell suspension, with the RPMI1640 nutrient solution cell dilution is become 2x10
6/ ml.
(2) in 96 well culture plates, every hole adds 100 μ l splenocyte suspensions, and each hole adds respectively isopyknic ConA solution (final concentration is 5 μ g/ml) or LPS solution (final concentration is 10 μ g/ml), and establishes the blank group.
(3) 37 ℃, 5%CO
2Cultivate in the incubator after 68 hours, each hole adds respectively 10 μ l MTT solution (5mg/ml), continues to cultivate 4 hours, centrifugal after, every hole sucks respectively supernatant, adds acid dimethyl sulfoxide (DMSO) (DMSO) solution of 200 μ l, vibration is placed 15min in room temperature.
(4) measure the OD value in the 570/630nm place with microplate reader.Be calculated as follows stimulation index: SI=is added with the OD value of a mitogen culture/the be not added with OD value of a mitogen culture.
2 experimental results
(1) impact of Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP mice spleen lymphocytes proliferation reaction that ConA is induced sees Table 1.
The impact that the mice spleen lymphocytes proliferation that table 1 Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP induces ConA reacts
| Group | The SI value |
| Saline | 1.37±0.12 |
| OVA | 1.420±0.11 |
| QiulA | 1.69±0.10* |
| WSP | 1.72±0.12* |
Annotate: experimental result represents (n=5) with the SI mean+SD, and * represents relatively P＜0.01 of OVA control group;
WSP is Franchet Groundcherry Calyx and Fruit stem polysaccharide.
Experimental result (such as table 1) shows: compare with the OVA group, Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP group has obvious enhancement (P＜0.01) to the mice spleen lymphocytes proliferation reaction that ConA induces, and its effect is suitable with positive control QuilA.
(2) impact of Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP mice spleen lymphocytes proliferation reaction that LPS is induced sees Table 2.
The impact that the mice spleen lymphocytes proliferation that table 2 Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP induces LPS reacts
Annotate: experimental result represents (n=5) with the SI mean+SD, and * represents relatively P＜0.01 of OVA control group
WSP is Franchet Groundcherry Calyx and Fruit stem polysaccharide.
Experimental result (such as table 2) shows: compare with the OVA group, Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP has obvious enhancement (P＜0.01) to the mice spleen lymphocytes proliferation reaction that LPS induces, and its action effect is suitable with positive controls QuilA.
(3) ELISA detects Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP to the impact of anti-OVA specific antibody IgG in the mouse resisting anteserum
1 experimental technique
(1) on 96 hole enzyme plates, every hole adds 100 μ l coating buffers (0.05M that contains 50 μ g/ml OVA, the carbonate buffer solution of PH9.6), puts 4 ℃ and hatches 24 hours.
(2) with lavation buffer solution liquid washing 3 times, each 3 minutes.
(3) every hole adds confining liquid 100 μ l, in 37 ℃ hatch 1 hour after the washing.
(4) every hole adds respectively 100 μ l mouse resisting anteserum diluent to be measured, hatches 1 hour, and washs with lavation buffer solution for 37 ℃.
(5) add respectively the goat anti-mouse IgG antibody diluent 100 μ l of horseradish peroxidase-labeled, hatched 0.5 hour for 37 ℃, washing.
(6) every hole adds respectively 50 μ l substrate nitrite ion A and substrate nitrite ion B, and room temperature was placed 15 minutes, and every hole adds stop buffer (2MH
2S0
4) 50 μ l termination reactions, measure the OD value in wavelength 450/630nm place with microplate reader.
2 experimental results
Experimental result (seeing Table 3) shows: compares with the OVA group, and the anti-OVA specific antibody of Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP group antiserum(antisera) IgG significant difference, its action effect and QuilA group are quite.
Table 3: Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP is on the impact of anti-OVA IgG antibody content in the mouse resisting anteserum
Annotate: measured value of experiment represents (n=5) with mean+SD, and * represents relatively P＜0.01 of OVA control group
Immune-enhancing activity experiment shows: Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP had not only strengthened the cellular immunization of body but also had strengthened the humoral immunization of body, and its action effect and QuilA are suitable, can be used for improving food and the healthcare products of body immunity.
Embodiment 2:
The preparation method of Franchet Groundcherry Calyx and Fruit stem polysaccharide:
The 1 dry stem 500g with Franchet Groundcherry Calyx and Fruit is cut into long the breaking into pieces of 2cm with scissors, and the 3000ml water soaking spends the night;
2 water seaonings were extracted 1.5 hours, three lixiviates, 90 ℃ of temperature;
3 usefulness ethanol precipitate the infusion extract, and the final concentration of ethanol is 70%;
4 multigelations are extremely without after the precipitation, with enzyme process and Sevage method associating deproteinated, the dry Crude polysaccharides that gets;
5 Crude polysaccharides carry out wash-out with QAE Sephadex A-25 column chromatography with physiological saline, get polysaccharide Q-WSP after the lyophilize;
6Q-WSP is further purified through HPLC and obtains WSP;
WSP measures its molecular weight through HPLC and is approximately 7000Da, is polysaccharide homogeneous component.Getting monosaccharide component through gas chromatographic analysis is Rha, Ara, Gal, Glc and GalA, and its mol ratio is 2.6: 1.0: 6.5: 3.5: 2.4.
Embodiment 3:
The preparation method of Franchet Groundcherry Calyx and Fruit stem polysaccharide:
The 1 dry stem 500g with Franchet Groundcherry Calyx and Fruit is cut into long the breaking into pieces of 2cm with scissors, and the 2500ml water soaking spends the night;
2 water seaonings were extracted 4 hours, three lixiviates, 80 ℃ of temperature;
3 usefulness ethanol precipitate the infusion extract, and the final concentration of ethanol is 80%;
4 multigelations are extremely without after the precipitation, with enzyme process and Sevage method associating deproteinated, the dry Crude polysaccharides that gets;
5 Crude polysaccharides carry out wash-out with QAE Sephadex A-25 column chromatography with physiological saline, get polysaccharide Q-WSP after the lyophilize;
6Q-WSP is further purified through HPLC and obtains WSP.
WSP measures its molecular weight through HPLC and is approximately 7000Da, is polysaccharide homogeneous component.Getting monosaccharide component through gas chromatographic analysis is Rha, Ara, Gal, Glc and GalA, and its mol ratio is 2.6: 1.0: 6.5: 3.5: 2.4.
Claims (3)
1. preparation method with immunocompetent Franchet Groundcherry Calyx and Fruit stem polysaccharide is characterized in that concrete steps are as follows:
(1) the dry stem with Franchet Groundcherry Calyx and Fruit is cut into the long cun section of 2cm, soaked overnight, and solid-liquid ratio is 1g: 20mL to 1g: 50mL;
(2) water seaoning was extracted 1-4 hour, three lixiviates, temperature 50-100 ℃;
(3) with ethanol the infusion extract is precipitated, the final concentration of ethanol is 65-85%;
(4) multigelation is extremely without after the precipitation, with enzyme process and Sevage method associating deproteinated, the dry Crude polysaccharides that gets;
(5) Crude polysaccharides carries out wash-out with QAE Sephadex A-25 with physiological saline, gets polysaccharide Q-WSP after the lyophilize;
(6) Q-WSP is further purified through HPLC and obtains WSP.
2. according to the immunocompetent Franchet Groundcherry Calyx and Fruit stem polysaccharide that has of preparation method claimed in claim 1 preparation.
3. has immunocompetent Franchet Groundcherry Calyx and Fruit stem polysaccharide in the food of preparation raising body's immunity and the application aspect the healthcare products according to claimed in claim 2.
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| CN103819576B (en) * | 2014-03-19 | 2016-11-23 | 上海市农业科学院 | A kind of Hericium erinaceus (Bull. Ex Fr.) Pers. large molecular weight polysaccharides and preparation method thereof |
| CN106266217A (en) * | 2016-09-28 | 2017-01-04 | 广州凯耀资产管理有限公司 | A kind of pharmaceutical composition with anti-infectious function and preparation method thereof |
| CN106977619A (en) * | 2017-05-10 | 2017-07-25 | 佛山职业技术学院 | Straw berry tomato food, straw berry tomato polysaccharide and preparation method thereof |
| CN109705229B (en) * | 2018-12-14 | 2021-06-04 | 浙江中医药大学 | Radix tetrastigme polysaccharide and application thereof in preparing immunomodulator |
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| CN101474235B (en) * | 2009-01-19 | 2012-09-05 | 湛江师范学院 | Preparation of wild cactus polysaccharide extract and high-efficient serum cholesterol-reducing function |
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