CN102276745A - 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 production and application that has.
Background technology
The Franchet Groundcherry Calyx and Fruit formal name used at school is a wintercherry, calls red Miss, Huechys sanguinea De Geer., Calyx seu Fructus physalis, bitter Miss, root of Manchurian Currant.Fruit is a 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 using always.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, 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 the Franchet Groundcherry Calyx and Fruit being the medicine of development of raw materials and developing rapidly of food, wild Franchet Groundcherry Calyx and Fruit supply can not have been satisfied the demand in market far away, and therefore the Franchet Groundcherry Calyx and Fruit of a large amount of plantations arises 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 of can not get effectively utilizing.Therefore Franchet Groundcherry Calyx and Fruit stem STUDY ON POLYSACHAROSE 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 alcoholic acid final concentration is 65-85%;
(4) multigelation is not to there being post 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 the food and the healthcare products of enhance immunity function.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 3000ml water logging bubble spends the night;
2 water seaonings were extracted 2 hours, three lixiviates, 100 ℃ of temperature;
3 usefulness ethanol precipitate the infusion extract, and the alcoholic acid final concentration is 85%;
4 multigelations are not to there being post 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 the week age male ICR mouse be used for experimentation on animals, 5 every group; With chicken egg white (OVA) is 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, 14 days at interval.Back 14 days of immunity for the second time, 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 isopyknic ConA solution (final concentration is 5 μ g/ml) or LPS solution (final concentration is 10 μ g/ml) respectively, and establishes the blank group.
(3) 37 ℃, 5%CO
2Cultivate in the incubator after 68 hours, each hole adds 10 μ l MTT solution (5mg/ml) respectively, continues to cultivate 4 hours, centrifugal after, supernatant is inhaled in every hole respectively, 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) Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP sees Table 1. to the influence of ConA inductive mice spleen lymphocytes proliferation reaction
Table 1 Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP is to the influence of ConA inductive mice spleen lymphocytes proliferation reaction
| 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 is represented (n=5) with the SI mean+SD, and * represents relatively P＜0.01 of OVA control group;
WSP is a Franchet Groundcherry Calyx and Fruit stem polysaccharide.
Experimental result (as table 1) shows: compare with the OVA group, reaction has tangible enhancement (P＜0.01) to Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP group to ConA inductive mice spleen lymphocytes proliferation, and its effect is suitable with positive control QuilA.
(2) Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP sees Table 2. to the influence of LPS inductive mice spleen lymphocytes proliferation reaction
Table 2 Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP is to the influence of LPS inductive mice spleen lymphocytes proliferation reaction
Annotate: experimental result is represented (n=5) with the SI mean+SD, and * represents relatively P＜0.01 of OVA control group
WSP is a Franchet Groundcherry Calyx and Fruit stem polysaccharide.
Experimental result (as table 2) shows: compare with the OVA group, reaction has tangible enhancement (P＜0.01) to Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP to LPS inductive mice spleen lymphocytes proliferation, and its action effect is suitable with positive controls QuilA.
(3) ELISA detects the influence of Franchet Groundcherry Calyx and Fruit stem polysaccharide WSP to 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, hatches 1 hour after scouring in 37 ℃.
(4) every hole adds 100 μ l mouse resisting anteserum diluent to be measured respectively, hatches 1 hour, and washs with lavation buffer solution for 37 ℃.
(5) add the goat anti-mouse IgG antibody diluent 100 μ l of horseradish peroxidase-labeled respectively, hatched 0.5 hour for 37 ℃, washing.
(6) every hole adds 50 μ l substrates colour developing liquid A and substrate colour developing liquid B respectively, 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 to the influence of anti-OVA IgG antibody content in the mouse resisting anteserum
Annotate: measured value of experiment is represented (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 the food and the healthcare products of enhance immunity ability.
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 3000ml water logging bubble 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 alcoholic acid final concentration is 70%;
4 multigelations are not to there being post 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 2500ml water logging bubble spends the night;
2 water seaonings were extracted 4 hours, three lixiviates, 80 ℃ of temperature;
3 usefulness ethanol precipitate the infusion extract, and the alcoholic acid final concentration is 80%;
4 multigelations are not to there being post 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 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 alcoholic acid final concentration is 65-85%;
(4) multigelation is not to there being post 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.
2. according to the immunocompetent Franchet Groundcherry Calyx and Fruit stem polysaccharide that has of the described preparation method of claim 1 preparation.
3. has immunocompetent Franchet Groundcherry Calyx and Fruit stem polysaccharide in the food of preparation enhance immunity function and the application aspect the healthcare products according to claim 2 is described.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103819576A (en) * | 2014-03-19 | 2014-05-28 | 上海市农业科学院 | Hericium erinaceus large-molecular polysaccharide 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 |
| CN109705229A (en) * | 2018-12-14 | 2019-05-03 | 浙江中医药大学 | A kind of radix tetrastigme polysaccharide and its preparing the application in immunomodulator |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101371851A (en) * | 2008-09-24 | 2009-02-25 | 东北师范大学 | Immunity enhancement use of wintercherry fruit polysaccharide |
| CN101474235A (en) * | 2009-01-19 | 2009-07-08 | 湛江师范学院 | Preparation of wild cactus polysaccharide extract and high-efficient serum cholesterol-reducing function |
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2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101371851A (en) * | 2008-09-24 | 2009-02-25 | 东北师范大学 | Immunity enhancement use of wintercherry fruit polysaccharide |
| CN101474235A (en) * | 2009-01-19 | 2009-07-08 | 湛江师范学院 | Preparation of wild cactus polysaccharide extract and high-efficient serum cholesterol-reducing function |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103819576A (en) * | 2014-03-19 | 2014-05-28 | 上海市农业科学院 | Hericium erinaceus large-molecular polysaccharide and preparation method thereof |
| 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 |
| CN109705229A (en) * | 2018-12-14 | 2019-05-03 | 浙江中医药大学 | A kind of radix tetrastigme polysaccharide and its preparing the application in immunomodulator |
| 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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