CN117209620A - Purslane galactoglucan and composition thereof, and preparation method and application thereof - Google Patents
Purslane galactoglucan and composition thereof, and preparation method and application thereof Download PDFInfo
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- CN117209620A CN117209620A CN202311234681.3A CN202311234681A CN117209620A CN 117209620 A CN117209620 A CN 117209620A CN 202311234681 A CN202311234681 A CN 202311234681A CN 117209620 A CN117209620 A CN 117209620A
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- Prior art keywords
- purslane
- galactoglucan
- application
- ethanol
- composition
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Landscapes
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The application provides purslane galactoglucan, a composition thereof and a preparation method and application thereof, belonging to the technical field of medicines, foods and cosmetics. The purslane galactoglucan has a structure represented by a general formula shown in fig. 7, wherein n represents the number of sugar repeating units, and n is a natural number. The weight average molecular weight of the purslane galactoglucan is 10 kDa-30 kDa, and the polydispersity coefficient is 1-3. Galactose in monosaccharide composition, glucose, molar ratio about 3.3:1. both the purslane galactoglucan and the purslane galactoglucan-containing composition have obvious activity of activating macrophages to release immune medium, and the activity is related to the activation of NF- κB signal channels. The application also discloses the purslane galactoglucan and the composition containing the purslane galactoglucan, and the application of the purslane galactoglucan and the composition in preparing the medicaments or foods for enhancing the immune activity.
Description
Technical Field
The application relates to the technical fields of foods, medicines and cosmetics, in particular to purslane galactoglucan with a novel structure, a composition thereof, a preparation method and application thereof.
Background
Purslane (Portulaca oleracea l.) is a annual fleshy herb of the portulaca genus of the Portulacaceae family, and is received in the national catalogue of homology of medicine and food. The natural green food is widely distributed worldwide, commonly called as long-life dish (longevity vegetable) and universal medicament (global panacea), and is listed by the world health organization as one of the most widely used medicinal plants in the world. The medical and edible history of the purslane is long, the research on the effective chemical components of the purslane is mainly focused on alkaloids, polysaccharide, flavonoid and organic acid substances, and the purslane has obvious curative effects on anti-inflammatory, anti-tumor, antibacterial, blood sugar reducing, skin disease treating and other aspects. However, the chemical structure of the purslane plant polysaccharide is extremely complex, and the same plant often contains a plurality of different types of polysaccharides, and the research on the biological efficacy of the polysaccharide is extremely difficult and needs to be intensively studied. The inventor conducts systematic chemical and pharmacological research on purslane polysaccharide, reports a novel purslane galactoglucan with a novel structure for the first time, and surprisingly discovers that the purslane galactoglucan has good immunoregulatory activity.
Disclosure of Invention
The application aims to provide a novel purslane galactoglucan, a pharmaceutical composition taking the purslane galactoglucan as an active ingredient, cosmetics comprising the purslane galactoglucan, foods containing the purslane galactoglucan, a preparation method of the purslane galactoglucan, application of the purslane galactoglucan in preparing a medicament for regulating the immune activity, and application of the purslane galactoglucan in preparing a product for improving the immunity of organisms.
In order to achieve the above object of the present application, the present application provides the following technical solutions:
the application firstly provides purslane galactoglucan with a novel structure, which has a chemical structure represented by the following general formula:
wherein: n represents the number of sugar repeating units, and n is a natural number.
The weight average molecular weight of the purslane galactoglucan is 10 kDa-30 kDa, and the polydispersity coefficient is 1-3. The galactose/glucose molar ratio in the monosaccharide composition is about 3.3:1.
The purslane galactoglucan is obtained by extracting and purifying dry aerial parts of purslane Portulaca oleracea L of Portulaca oleracea of Portulaca of Portulacaceae.
The activity research of the inventor shows that the purslane galactoglucan with novel structure can remarkably promote RAW264.7 cells to secrete cytokines such as IL-6, TNF-alpha, NO and the like, thereby having the capability of regulating organism immunity.
The inventor has found through intensive pharmacological research that the purslane galactoglucan can activate NF- κB signaling pathway by promoting phosphorylation of P65 and IκB-alpha in RAW264.7 cells, thereby exhibiting potential immunopotentiating properties.
In order to obtain the purslane galactoglucan with novel structure and outstanding activity, the application also provides a method for preparing the purslane galactoglucan by separating and extracting the purslane Portulaca oleracea L.
The preparation method of the purslane galactoglucan comprises the following steps:
step one: extracting the dried upper part of the purslane with 95% ethanol, discarding the extracting solution, and retaining the residue.
Step two: extracting the residues with warm water or hot water to obtain herba Portulacae crude polysaccharide solution;
step three: grading and alcohol precipitation to obtain coarse purslane polysaccharide;
step four: refining the components with the weight average molecular weight of 10 kDa-30 kDa in the purslane crude polysaccharide to obtain the purslane galactoglucan.
Further, the preparation method of the purslane galactoglucan disclosed by the application specifically comprises the following steps of:
the first to third steps include the following steps: pulverizing dry aerial parts of herba Portulacae, and defatting with 95% ethanol. Extracting with warm water or hot water, centrifuging to remove residue, sequentially precipitating the supernatant with ethanol to reach alcohol concentration of 40% and 60%, respectively, centrifuging, and collecting precipitate.
In the application, the step of extracting purslane galactoglucan further comprises the following steps of:
degreasing the powder of the dry aerial parts of the purslane twice with 95% ethanol in a feed liquid ratio of 1:4-1:6. The preferred feed to liquid ratio is 1:5. Filtering, discarding the extracting solution, and extracting the residues twice with hot water or warm water in a feed liquid ratio of 1:4-1:6. The preferred feed to liquid ratio is 1:5.
Further, extracting the obtained solution with hot water or warm water, centrifuging, adding ethanol with the final concentration of 35% -45%, 55% -65% and 75% -85% into the supernatant, carrying out fractional precipitation, centrifuging, and collecting 55% -65% ethanol precipitation precipitate to obtain the purslane galactoglucan. Preferably, the ethanol concentration of the fractionated alcohol precipitation is 40% and 60%, respectively.
In the present application, the method for refining the dry aerial part of the purslane as described above is one or more selected from the group consisting of quaternary ammonium salt precipitation, fractional alcohol precipitation, anion exchange chromatography and gel exclusion chromatography. Preferably, one or more of quaternary ammonium salt precipitation, fractional alcohol precipitation, anion exchange chromatography.
According to the preparation method provided by the application, the weight average molecular weight of the purslane galactoglucan is 10 kDa-30 kDa, beta (1-4) galactoglucan and alpha (1-4) glucan are taken as main structures, and the monosaccharide molar ratio of galactose to glucose is 3.3:1.
The purslane galactoglucan with the novel structure remarkably promotes RAW264.7 cells to secrete NO, cytokines IL-6, TNF-alpha and the like, so that the purslane galactoglucan has the capability of regulating organism immunity. The purslane galactoglucan can activate NF- κB signaling pathway by promoting P65 and IκB-alpha phosphorylation in RAW264.7 cells, thereby exhibiting potential immunopotentiating properties.
The application further provides a pharmaceutical composition comprising the effective amount of purslane galactoglucan and a pharmaceutically acceptable carrier.
A cosmetic comprising said effective amount of purslane galactoglucan and cosmetic usual adjuvants.
A food product comprising said purslane galactoglucan.
The purslane galactoglucan or the pharmaceutical composition thereof is applied to the preparation of the medicine for regulating the immunocompetence.
The purslane galactoglucan is applied to the preparation of cosmetics or foods.
Further, the purslane galactoglucan and the pharmaceutical composition thereof can be in a dosage form for oral or parenteral administration. Specifically, the dosage form optionally comprises a tablet, a capsule, a freeze-dried powder, an oral liquid or a pellet of purslane galactoglucan as an active ingredient.
Further, the purslane galactoglucan and the cosmetics thereof can be used locally and can be prepared into the following dosage forms: masks, creams, ointments, gels, and the like.
The application of the purslane galactoglucan or the pharmaceutical composition in the product for improving the immunity of organisms is provided.
Use of purslane galactoglucan or a combination thereof in the preparation of a medicament or food or cosmetic with modulated immune activity.
In the application, the dosage forms of the purslane galactoglucan and the purslane galactoglucan composition comprise, but are not limited to, freeze-dried powder, oral liquid, capsules, tablets and the like.
In summary, due to the adoption of the technical scheme, the beneficial effects of the application are as follows:
the purslane galactoglucan prepared by the application is reported for the first time, and the inventor cell experimental study proves that the purslane galactoglucan has the effects of promoting the secretion of macrophage factors and improving the immunity, and has important medicine application potential.
The innovative method provided by the application is used for extracting and purifying the dry aerial parts of the purslane, so that the purslane galactoglucan with a novel structure is obtained. Further pharmacological activity research shows that the newly discovered purslane galactoglucan has the function of regulating immunity. Therefore, the purslane galactoglucan with the novel structure, the preparation method and the composition thereof and the application thereof in immunoregulation medicines and/or foods and/or cosmetics are all reported for the first time.
Drawings
FIG. 1 is a graph showing the molecular weight distribution of purslane galactoglucan POL-2;
FIG. 2 is a HPLC diagram of the composition of purslane galactoglucan POL-2 monosaccharide;
FIG. 3 is a total ion flow diagram of purslane galactoglucan POL-2 methylation;
FIG. 4 shows the nuclear magnetic spectrum of the purslane galactoglucan POL-2, (A) one-dimensional nuclear magnetic resonance 1 H spectrogram; (B) One-dimensional nuclear magnetism 13 C, spectrogram; (C) 1 H- 1 H COSY profile; (D) 1 H- 1 H TOCSY profile; (E) 1 H- 13 C HSQC spectrum; (F) 1 H- 13 C HMBC spectra; (G) 1 H- 1 H ROESY profile; (H) HSQC-TOCSY spectra;
FIG. 5 is the effect of purslane galactoglucan (POL-2) on the secretion of NO, TNF- α and IL-6 by RAW264.7 cells: (a) effect on RAW264.7 cell viability; (B) effect on RAW264.7 cells to secrete NO; (C) effect on TNF- α secretion by RAW264.7 cells; (D) effect on RAW264.7 cells to secrete IL-6; in the figure "" represents significance compared to the blank group, representing p <0.05, "" represents p <0.01, "" represents p <0.001, n=3;
FIG. 6 is an effect of purslane galactoglucan (POL-2) on expression of related proteins in the NF- κB pathway of RAW264.7 cells; (A) Protein band diagrams of P65, IκB- α, P-P65 and P-IκB- α; (B) protein quantification map of P-P65/P65; (C) protein quantification map of p-IκB- α/IκB- α; in the figure "" represents significance compared to the blank group, representing p <0.05, "" represents p <0.01, "" represents p <0.001, n=3;
FIG. 7 is a schematic structural diagram of purslane galactoglucan POL-2 of the present application, wherein: n represents the number of sugar repeating units, and n is a natural number.
Detailed Description
The present application will be described more fully hereinafter in order to facilitate an understanding of the present application, and preferred embodiments of the present application are set forth. This application may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
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 to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The purslane galactoglucan of the embodiment of the application has a structure represented by the following general formula:
wherein: n represents the number of sugar repeating units, and n is a natural number.
The purslane galactoglucan takes beta (1-4) galactose glycosidic bond as a main chain, and the side chain is replaced by alpha (1-4) glucose and alpha (1-6) glucose.
The preparation method of the purslane galactoglucan provided by the embodiment of the application comprises the following steps S1-S4:
s1: extracting the dried upper part of the purslane with 95% ethanol, discarding the extracting solution, and retaining the residue.
S2: extracting the residues with warm water or hot water to obtain herba Portulacae crude polysaccharide solution;
s3: grading and alcohol precipitation to obtain coarse purslane polysaccharide;
s4: refining the components with the weight average molecular weight of 10 kDa-30 kDa in the purslane crude polysaccharide to obtain the purslane galactoglucan.
In one specific example, S1 includes the steps of: pulverizing dry aerial parts of herba Portulacae, extracting with 95% ethanol, filtering, and retaining residue.
S2, mainly extracting with warm water or hot water twice, centrifuging to remove slag, and combining the supernatant.
Then adding 95% ethanol into the supernatant to a final concentration of 35% -45%, and centrifuging (4000 rpm is multiplied by 10 min) to obtain 35% -45% alcohol sediment and 35% -45% alcohol sediment supernatant; adding 95% ethanol into the 35% -45% ethanol precipitation supernatant under stirring until the final concentration of the ethanol is 55% -65%, centrifuging to obtain 55% -65% ethanol precipitation and ethanol precipitation supernatant, washing 55% -65% ethanol precipitation 3 times with 95% ethanol, adding water for redissolution, and freeze-drying; the crude purslane polysaccharide obtained by two times of centrifugation is named POL40 and POL60 respectively.
The method for refining herba Portulacae galactoglucan is selected from one or more of anion exchange column chromatography, gel exclusion column chromatography, dialysis and ultrafiltration. Specifically, the crude purslane galactoglucan component can be added with deionized water for re-dissolution, a small amount of insoluble matters are removed by centrifugation, and the purification is carried out by optional methods such as fractional alcohol precipitation, gel exclusion chromatography, dialysis or ultrafiltration, etc., and fractions containing the galactoglucan or trapped fluid or permeate liquid is collected, and the refined purslane galactoglucan is obtained after direct vacuum freeze drying or reduced pressure concentration and alcohol precipitation and reduced pressure drying.
As will be readily understood by those skilled in the art, for anion exchange column chromatography, column materials such as DEAE-Sepharose Fast Flow series, DEAE-52 series, DEAE-32 series, etc. are reasonably selected according to how much charge is carried out in the purslane polysaccharide component, and then column packing, loading and sequential elution with saline or non-saline elution solutions are performed according to the actual properties of each of the packing materials and fractions are collected. Specifically, the fraction eluted by deionized water is generally uncharged, i.e., neutral sugar, and polysaccharide fractions with different charge amounts can be fractionated by subsequent elution with a gradient of salt solution. Concentrating the effluent liquid or not, loading into dialysis bag for dialysis or ultrafiltration membrane for ultrafiltration and desalination, collecting desalted retentate, and vacuum freeze drying or vacuum reduced pressure drying to obtain purified herba Portulacae galactodextran.
The application also provides a structural analysis method of the purslane galactoglucan, which comprises the following steps:
(1) Molecular weight measurement: taking a purslane galactoglucan sample, and adopting a high-efficiency gel exclusion chromatography-differential detector detection method.
(2) Monosaccharide composition analysis: taking a purslane galactoglucan sample, carrying out acid hydrolysis to obtain monosaccharide, carrying out derivatization on the monosaccharide by 1-phenyl-3-methyl-5-pyrazolone (PMP), and analyzing the monosaccharide composition of the purslane galactoglucan by using a chromatographic column with octadecylsilane chemically bonded silica gel as a filler through a high performance liquid chromatograph.
(3) Methylation analysis: taking a purslane galactoglucan sample, carrying out polysaccharide methylation by using a halomethane reagent such as methyl iodide and the like under alkaline conditions, reducing by using sodium borohydride after hydrolysis under alkaline conditions, acetylating methylated sugar alcohol by using anhydride, and carrying out gas chromatograph-mass spectrometer (GC-MS) analysis after extraction to judge the glycosidic bond connection mode of the purslane galactoglucan.
(4) Nuclear magnetic resonance analysis: dissolving herba Portulacae galactoglucan sample in heavy water, freeze vacuum drying, repeating three times of heavy water exchange, dissolving in heavy water, and detecting nuclear magnetic resonance spectrum including one dimension 1 H、 13 C-spectrum and two dimensions 1 H- 1 H COSY、 1 H- 1 H TOCSY、 1 H- 1 H ROESY、 1 H- 13 C HSQC、 1 H- 13 CHMBC, HSQC-TOCSY correlation spectra.
(5) And (3) comprehensive data analysis: analyzing the analysis data of the steps comprehensively, and analyzing the chemical structure of the purslane galactoglucan. According to the structure analysis methodThe preparation method provided by the application is used for extracting and purifying purslane galactoglucan from purslane, and carrying out structural analysis. The result shows that the purslane galactoglucan only shows a chromatographic peak with good symmetry on HPGPC gel chromatography, the weight average molecular weight of the refined polysaccharide POL-2 after the purslane is subjected to 60% ethanol fractionation and ethanol precipitation is about 21000Da, and the polydispersity index is 1.9; monosaccharide composition showed that the purslane galactoglucan consisted of galactose and glucose at 3.3:1, the composition is as follows; methylation analysis showed that the purslane galactoglucan has 6 different glycosidic linkages: glcp- (1. Fwdarw., galp- (1. Fwdarw.,. Fwdarw.4) -GlcP- (1. Fwdarw., 4) -Galp- (1. Fwdarw.,. Fwdarw.6) -GlcP- (1. Fwdarw., 4, 6) -Galp- (1. Fwdarw., according to NMR spectrum data, purslane galactoglucan can be attributed to 1 H and 13 and C.
In summary of the above data, the purslane galactoglucan has the structural features shown in fig. 7: the molar ratio of galactose to glucose was 3.3:1, a step of; beta (1-4) galactose glycosidic bond is taken as main chain, and side chains are replaced by alpha (1-4) glucose glycosidic bond and alpha (1-6) glucose glycosidic bond.
According to the application, pharmacological studies have surprisingly found that the purslane galactoglucan can activate macrophages to generate NO and release immune related active substances such as cytokines such as IL-6, TNF-alpha and the like, and has remarkable immunoregulatory activity. In addition, the immunomodulatory activity of the polysaccharide was found to be related to activation of NF- κB signaling pathways.
Therefore, the application also provides application of the purslane galactoglucan in preparing an immunoregulatory medicament and/or food.
The pharmaceutical composition provided by the embodiment of the application comprises the purslane galactoglucan or pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof and pharmaceutically and/or food and/or cosmetic acceptable auxiliary materials. Optionally, the auxiliary materials comprise pharmaceutically acceptable excipients, carriers and/or diluents and/or film forming agents and the like.
In a specific example, the dosage form of the pharmaceutical composition is injection or solid preparation or oral liquid, such as water injection, freeze-dried powder injection for injection, capsules, pellets, oral liquid and the like, and the auxiliary materials comprise excipient, corrective and the like.
The cosmetic composition provided by the embodiment of the application comprises the purslane galactoglucan, a cosmetically acceptable salt or a cosmetically acceptable solvate thereof and a cosmetically acceptable auxiliary material. Optionally, the auxiliary materials comprise cosmetic excipient, carrier and/or diluent and/or film forming agent, etc.
In a specific example, the formulation of the cosmetic is cream or gel, and the auxiliary materials comprise excipient and/or diluent and the like.
The present application will be described in detail below with reference to specific embodiments in conjunction with the attached drawings, but these embodiments do not limit the scope of the claims of the present application in any way.
Example 1
Extracting and refining purslane galactoglucan.
1. Extraction of purslane crude polysaccharide
500g of crushed dry aerial parts of purslane are weighed, added with 2.5L of 95% ethanol and extracted for 2 times at 65 ℃ for 3 hours each time. The residue obtained after filtration was extracted with deionized water at 80℃for 2 times, each for 2 hours. Centrifuging (4000 rpm. Times.20 min) the obtained extract to obtain supernatant; adding 95% ethanol into the supernatant until the ethanol concentration is 40%, centrifuging (4000 rpm×20 min) to obtain 40% ethanol precipitate and 40% ethanol supernatant; adding 95% ethanol into the 40% ethanol supernatant under stirring until the ethanol concentration is 60%, and centrifuging (4000 rpm×20 min) to obtain 60% ethanol precipitate and ethanol supernatant; washing 40% ethanol and 60% ethanol respectively for 1 time, centrifuging, adding water into the precipitate, re-dissolving, and lyophilizing to obtain crude polysaccharides POL40 and POL60 29.3g and 7.5g respectively.
2. Refining of purslane galactoglucan
Dissolving crude polysaccharide POL60 and 100mL deionized water, adding equal volume of 10% benzethonium chloride, stirring, standing, centrifuging (4000 rpm,20 deg.C, 15 min), adding 95% ethanol to the supernatant to reach final alcohol content of 80%, stirring, and standing for alcohol precipitation. The precipitate obtained after centrifugation (4000 rpm,20 ℃,15 min) was the crude purslane polysaccharide POL60q. Taking freeze-dried crude polysaccharide POL60q, adding 95% ethanol after redissolving deionized water to make the final concentration of ethanol 40%, and centrifuging (4000 rpm,20 ℃ C., 15 min) to obtain a precipitate POL60q40. 100mg of POL60q40 was lyophilized, dissolved in 4mL of deionized water, centrifuged (4000 rpm,20 ℃ C., 15 min), and the supernatant was filtered through a 0.45 μm filter membrane and slowly and uniformly injected into the top of the DEAE-Sepharose Fast Flow column. Eluting with deionized water, controlling flow rate to 0.1mL/min, collecting 2mL of the solution per tube, and detecting polysaccharide content by phenol-sulfuric acid method. And drawing an elution curve of the polysaccharide component by taking the light absorption value as an ordinate and the tube number as an abscissa. And combining eluents belonging to the same elution peak, collecting a sample with the largest area of an elution curve, concentrating under reduced pressure, freezing, drying in vacuum, and enriching for multiple times to obtain 100mg of refined purslane galactoglucan.
Example 2
Structural analysis of purslane galactoglucan.
1. Experimental method
1.1. Molecular weight and distribution thereof
The molecular weight of purslane galactoglucan prepared in example 1 and its distribution were analyzed using high performance gel exclusion chromatography-differential detector detection (HPGPC-RID).
Chromatographic instrument: agilent technologies 1260series high performance liquid chromatograph;
chromatographic conditions: shodex OHPak SB-804HQ (8 mm. Times.300 mm) column; the column temperature is 35 ℃; a differential detector; the mobile phase is 0.1M NaCl, and the flow rate is 0.5mL/min;
the measuring process comprises the following steps: 5mg of purslane galactoglucan samples or dextran reference substances with known molecular weight are respectively taken, a mobile phase is added to prepare a solution with the concentration of 5mg/mL, the solution is filtered by a microporous membrane with the concentration of 0.2 mu m, and 30 mu L of filtrate is analyzed by a high performance liquid chromatograph and a chromatogram is recorded. The data are processed by GPC software, a standard curve is drawn, the data are brought into an equation, and the molecular weight is calculated.
1.2 analysis of monosaccharide composition
1mL of monosaccharide standard solution and 1mL of sample solution are taken and placed in a hydrolysis tube, 1mL of 4M trifluoroacetic acid (TFA) solution is added, and the mixture is uniformly mixed and hydrolyzed for 4h in an oven at 110 ℃. After the hydrolysis reaction is finished, the hydrolysis solution is dried by rotary evaporation, 200 mu L of deionized water is added for dissolution until TFA is completely removed, 200 mu L of 0.6M NaOH solution and 400 mu L of 0.5M PMP-methanol solution are sequentially added, the mixture is uniformly mixed, the derivatization reaction is carried out for 1h in a 70 ℃ oven, the temperature is cooled to room temperature, and 200 mu L of 0.6M hydrochloric acid is added for neutralization. Then, chloroform was added thereto and the mixture was extracted sufficiently 3 times (2 mL each time), and the aqueous phase was centrifuged (15000 rpm. Times.10 min) and filtered through a 0.2 μm microporous membrane for liquid chromatography.
Chromatographic conditions:
instrument: agilent technologies 1260series high performance liquid chromatograph;
chromatographic column: hadesil C18-Bio (250 mm. Times.4.6 mm,5 μm); column temperature: 25 ℃;
mobile phase: 0.1M phosphate buffer-acetonitrile (82:18);
eluting: the flow rate is 1mL/min; the sample injection amount is 5 mu L; the time is 50min;
and (3) detection: DAD detector, wavelength 250nm.
1.3 methylation analysis
1) Methylation reaction: polysaccharide sample 3-5 mg is taken and placed in a 10mL COD tube. After 2mL of methanol was added to the COD tube, the mixture was dried in a vacuum oven and repeated twice to completely dehydrate the sample. Adding 0.5mL of DMSO into the dehydrated sample, and carrying out ultrasonic treatment for 20-30 min. Adding 500 mu L of 120mg/mL NaOH/DMSO suspension into a sample (injection: naOH/DMSO suspension is prepared in advance: weighing NaOH solid, adding a proper amount of DMSO to reach the required concentration, stirring at 25 ℃ by using a magnetic stirrer, sealing and stirring overnight), directly dripping the uniform solid NaOH suspension onto the sample, and carrying out ultrasonic treatment for 30min, wherein the temperature of the ultrasonic treatment process is kept below 40 ℃.
To the suspension completely dissolved in NaOH/DMSO, 800. Mu.L of CH was added in portions 3 I, adding 200 μL of each of the two times, performing ultrasonic treatment for 10min, and finally adding 400 μL of CH 3 And carrying out ultrasonic treatment for 30min (the temperature of the ultrasonic process cannot exceed 40 ℃) and carrying out light-proof reaction. After the reaction, 1mL of water was added and the reaction was terminated, 3mL of CH was added 2 Cl 2 The sample was extracted, and the aqueous phase was removed and discarded. Back-extracting CH with an equal volume of water 2 Cl 2 The phases were spun dry 3 times.
2) Acid hydrolysis of methylated polysaccharides:
to the spin-dried sample was added 2mL of 2m TFA, hydrolyzed at 120 ℃ for 2h, and after spinning-dried sample was added 2mL of methanol and spun-dried again, repeated 2 times.
3) Reduction of methylated monosaccharides:
the hydrolyzed sample was dissolved in 1mL of 2M NH 4 OH (ready-to-prepare) and 1mL of ready-to-prepare 1M NaBD was added 4 (at 2MNH 4 OH as solvent). After the sample was sonicated to dissolution, incubated at room temperature (25 ℃) for 2.5h, 400. Mu.L CH was carefully added after the reaction was completed 3 COOH. Spin-dry sample and continue with 1mL5% (v/v) CH 3 COOH-CH 3 The OH solution was dried twice with 1mL of methanol to remove boric acid. Finally, the spin-dried sample is placed in a vacuum drying oven to be dried until the sample in the tube does not flow.
4) Acetylation of methylated sugar alcohols:
1mL of acetic anhydride and 1mL of pyridine are added into the sample, the mixture is dissolved by vortex and then is placed at 100 ℃ for reaction for 1.5h, and after the reaction is finished, 1mL of water is added for stopping the reaction. By CHCl 3 Extracting the reaction mixture three times, CHCl 3 The layers were back-extracted three more times with water and CHCl was extracted 3 Spin-drying the layer, and adding appropriate amount of CH 3 The OH was dried twice. Spin-dry sample was added with 1mL of chromatographic grade CHCl 3 Dissolving, filtering with 0.2 μm organic filter membrane, and performing GC-MS analysis.
GC conditions: DB-5MS quartz capillary column (30 m x 0.25mm x 0.25 μm); column temperature: the initial temperature is 80 ℃, the temperature is kept for 1min, the temperature is programmed to be 5 ℃/min to 250 ℃ and the temperature is kept for 40min; the column flow rate is 1.5mL/min; the temperature of the sample inlet is 250 ℃; the split ratio is 10:1; the carrier gas is high-purity helium, and the sample injection amount is 1 mu L.
MS conditions: ionization mode EI; electron energy 70eV; the temperature of the transmission line is 290 ℃; the ion source temperature is 230 ℃; the temperature of the quadrupole rods is 150 ℃; the mass range is 50-600; and comparing the result with a CCRC spectrum database so as to judge the type of the glycosidic bond.
1.4 Nuclear magnetic resonance analysis
Taking 15mg of purslane galactoglucan sample and dissolving in 0.5mL D 2 O, freeze-drying, D 2 After three O exchanges, the lyophilized sample was dissolved in 0.5mL D 2 O (99.9 atom% D, containing 0.05wt.%3- (trimethyllyl) propionic-2, 3-d as internal standard) 4 acid,sodium salt) Is a kind of medium. Determination using Bruker 800MHz Nuclear magnetic resonance spectrometer 1 H/ 13 CNMR spectrogram and two-dimensional spectrogram 1 H- 1 H COSY、 1 H- 1 H TOCSY、 1 H- 1 H ROESY、 1 H- 13 C HMBC、 1 H- 13 CHSQC and HSQC-TOCSY were analyzed using MestReNova software.
2. Experimental results
The characteristics of the purslane galactoglucan disclosed by the application are as follows: white or white-like solid, odorless, easily soluble in water, and moisture-absorbing.
The results of high performance gel exclusion chromatography (FIG. 1) showed that the purified purslane galactoglucan extracted from purslane had only one symmetrical chromatographic peak with a weight average molecular weight (Mw) of 21000Da and a polydispersity of 1.9.
The results of the monosaccharide composition analysis show (fig. 2), the HPLC profile of the purslane galactoglucan after PMP pre-column derivatization shows two peaks, galactose and glucose, with a ratio of about 3.3:1, the purslane galactoglucan monosaccharide composition only contains galactose and glucose.
Methylation analysis showed (fig. 3) that the purslane galactoglucan had 6 glycosidic linkages: glcp- (1→, galp- (1→, →4) -Glcp- (1→, →4) -Galp- (1→, →6) -Glcp- (1→, →4, 6) -Galp- (1→).
The results of the NMR analysis are shown in FIG. 4, and are detailed 1 H and 13 the assignment of the C NMR signals is shown in tables 1 and 2. At the position of 1 In the H NMR spectrum, the low field region delta H The signal at 5.42ppm is attributed to the terminal hydrogen signal at Glc, from which Glc is inferred to be the alpha configuration. Delta H The signal at 4.65ppm was attributed to the terminal hydrogen signal of Gal, and the proton coupling constant (J H1-H2 ) Is 7.8Hz, thereby determining Gal to be in the beta configuration. The protons with weaker signals in the low field region may be the terminal hydrogen signals of the polysaccharide POL-2, which occupy smaller residues, the high field delta H Peaks at 3.40 to 4.30ppm are proton signals at the rest of the sugar ring. Based on the methylation result of polysaccharide POL60ql, the main structural fragment → 4) -Galp- (1 → 4, 6) -Galp- (1 → Glcp- (1 → 4) -Glcp- (1 → 4)And → 6) -Glcp- (1 → designated residues A, B, C, D and E, respectively.
At the position of 13 In the C NMR spectrum, the low field region delta C 104.3ppm and delta C The terminal carbon signals at 99.6ppm are residues A and D, respectively, again validating the configuration of Gal and Glc. In combination with the HSQC signal (E in FIG. 4), the A and corresponding hydrogen signals of the different heads were assigned 4.65/104.3ppm, 5.42/99.6ppm.
The detailed attribution information is provided by 2D NMR maps. At the position of 1 H- 1 In the H COSY spectrum, from the terminal hydrogen of residue A, 3 groups of related hydrogen signals were found, respectively (4.65, 3.69 ppm), (3.69, 3.79 ppm) and (3.79, 4.18 ppm), corresponding to H-2 to H-4 of residue A, respectively. Similarly, H-2 and H-3 of the D residue are also produced by 1 H- 1 The H COSY related signals are attributed. In combination with the HSQC signal, the A residues C-1 to C-4 signal were assigned, wherein C-4 shifted to the low field to 77.6ppm due to glycosylation effects. Similarly, C1-C3 of the D residue is assigned. The A residue and the D residue occupy larger in the polysaccharide POL60ql, so that the signal is stronger, and the residual hydrocarbon signal on the sugar ring can be perfected according to an HMBC spectrogram. The relevant signals at (F in FIG. 4), (4.65, 77.6 ppm) and (4.18, 104.3 ppm) in the HMBC spectra indicate that the A residues are linked by a beta (1.fwdarw.4) glycosidic linkage, which is consistent with the methylation results. In addition, the correlation signals at (5.42, 76.8 ppm) and (3.68, 99.6 ppm) were attributed to the correlation within the D residues, indicating that the D residues are linked by an alpha (1.fwdarw.4) glycosidic linkage. From the methylation results, residue B, C, E was found to be relatively small in polysaccharide POL-2. From the above data, the chemical structural formula of the purslane galactoglucan with novel structure is shown as follows:
wherein n represents the number of sugar repeating units and n is a natural number.
The structure is characterized in that: (1) The purslane galactoglucan is novel-structure galactoglucan extracted from dry aerial parts of purslane, wherein the molar ratio of galactose to glucose is 3.3:1, a step of; (2) The monosaccharide connection mode of the purslane galactoglucan is as follows: beta (1-4) galactose glycosidic bond is taken as a main chain, and side chains are replaced by alpha (1-4) glucose and alpha (1-6) glucose. Through domestic and foreign public literature search, the arrangement mode and chemical structure of the purslane galactoglucan are not disclosed and reported, and are found for the first time by the inventor.
TABLE 1 purslane galactoglucan 1 Assignment of H NMR
TABLE 2 purslane galactoglucan 13 Assignment of C NMR
Example 3
An immunopotentiating activity test of purslane galactoglucan.
1. Test article, reagent and cell
Purslane galactoglucan, abbreviated as POL-2, prepared in accordance with example 1. An improved Eagle medium, a mixture of green streptomycin, solarbio; australian Fetal Bovine Serum (FBS), gibco; lipopolysaccharide (LPS), dimethyl sulfoxide (DMSO), sigma; NO kit, nitric oxide kit, shanghai bi yun biotechnology limited; IL-6, TNF-alpha kit, sizhengbai Biotechnology Co., ltd; TNF-alpha kit Shanghai Biyun biotechnology Co., ltd; antibodies to NF- κ B p65, IκBα, phospho-NF- κ B p65 and phospho-IκBα, wohan Sanying Biotechnology Co. RAW264.7 cells were purchased from the national academy of sciences typical culture collection committee cell bank.
2. Experimental method
2.1 removal of LPS from samples
(1) The detox-GelTM Endotoxin Removing gel column was activated with 5 resin bed volumes of 1% sodium deoxycholate and then washed with 10 resin bed volumes of sterile injection water to regenerate the detoxified gel resin to remove the detergent. The resin was regenerated before each use.
(2) The detoxification gel resin was equilibrated with 5 resin bed volumes of sterilized water for injection.
(3) 20mg of purslane galactoglucan POL-2 sample is weighed and dissolved in 1mL of sterilized water for injection, the sample is added into a column, the collection of the sample is started, the sample enters the column material, an aliquot of pyrogen-free buffer solution or water is added, the column flow is stopped, the mobile phase is collected after incubation for 1h, then 1mL of sterilized water for injection is added, the mobile phase is collected, and 5mL of mobile phase is collected in total.
(4) Freeze-drying to obtain the Portulaca oleracea galactoglucan POL-2.6 mg without LPS.
2.2RAW264.7 cell resuscitation
And taking out the frozen RAW264.7 macrophages from the liquid nitrogen tank, placing the frozen RAW264.7 macrophages in warm water at 37 ℃ which is prepared in advance, melting the cells within 1-2 min, and recovering the cells to room temperature. After that, the cells were transferred to a 15mL centrifuge tube, 3mL DMEM medium was added, centrifuged at 1000r/min for 3min, the supernatant was discarded, 2mL DMEM complete medium was added to blow the cells, and then transferred to 10cm 2 And 10mL of culture medium is added into the culture dish, the culture dish is uniformly shaken, the state and the density of cells are observed under a microscope, and finally the culture dish is placed into a cell culture box.
2.3RAW264.7 cell culture
After RAW264.7 macrophages are resuscitated, they are resuscitated at 37℃with 5% CO 2 Cells were grown in DMEM high glucose medium containing 10% fetal bovine serum), 100U/mL penicillin and 100 μg/mL streptomycin in a constant temperature incubator under conditions. LPS was used as positive control.
2.4 cell viability assay
The effect of POL-2 cell viability was examined using the MTT method. The cells were grown at 1.5X10 4 Density of individual/well was seeded in 96-well plates at 37 ℃ with 5% CO 2 After incubation in an incubator for 24h with complete culture containing 10% fetal bovine serum under the conditions, the upper medium was discarded, 100. Mu.L of complete medium containing different concentrations of POL-2 (400, 200, 100, 50, 25, 12.5. Mu.g/mL) was added, and 6 wells were placed in each group with the same amount of complete medium without polysaccharide as the blank. After 24h of incubation, 10. Mu.L MTT solution was added to each wellIncubation was continued for 4h (5 mg/mL), medium was carefully discarded, 100 μL of LDMSO was added to each well, incubated for 10min and OD was measured at 490 nm.
2.5Griess method for detecting influence of purslane galactoglucan on NO release of macrophages
mu.L of the cell suspension was inoculated into 24-well plates (5X 10) 5 And/or wells) were placed in a cell incubator for incubation for 24h. The supernatant was discarded, the blank was replaced with complete medium, the positive LPS was replaced with complete medium containing 1. Mu.g/mL LPS, and the drug-treated groups were each added with complete medium containing different concentrations of POL-2 (200, 100, 50. Mu.g/mL) and cultured for 24h. 50 μl of each culture was added to a 96-well plate, equal volumes of Griess Reagent I and Griess Reagent II were added to each well, mixed by shaking, OD was measured at 540nm, and the concentration of NO was calculated.
2.6ELISA method for detecting influence of purslane galactoglucan on macrophage cytokine secretion level
Cells were grown in 2X 10 cells 5 The cells/well were inoculated into 24-well plates and cultured for 24 hours. The drug-treated groups were each supplemented with complete medium containing different concentrations of POL-2 (200, 100, 50. Mu.g/mL) and 1. Mu.g/mL LPS as positive control. And (5) continuously culturing for 24 hours, collecting culture supernatant, and preserving at-80 ℃ for later use. And detecting the contents of TNF-alpha and IL-6 in the culture solution according to the instruction of ELISA kit.
2.7Western Blot method for detecting influence of purslane galactoglucan on expression of related protein in NF- κB pathway
The cell suspension density was adjusted to 5X 10 5 2mL of the mixture is sucked into each hole of a 6-hole plate, and the mixture is placed in a cell culture box for culture for 24 hours. The drug-treated groups were each supplemented with complete medium containing different concentrations of POL-2 (200, 100, 50. Mu.g/mL) and 1. Mu.g/mL LPS as positive control. After 24 hours of treatment, adding a proper amount of lysate to extract total protein. The total protein extracted was separated by SDS-PAGE electrophoresis and transferred to PVDF membrane. Subsequently, the primary antibody was incubated at 4℃and overnight, blocked with 5% nonfat milk powder. The membrane was washed with TBST buffer and the secondary antibody was incubated. The protein bands were developed in a Fluorchem Q imaging system. The band gray was quantitatively analyzed using Image J software.
3. Experimental results
3.1 Effect on NO, IL-6 and TNF- α secretion in RAW264.7 cells
As shown in fig. 5, after purslane galactoglucan is treated on RAW264.7 cells for 24 hours, the secretion amount of NO (B in fig. 5) and IL-6 (D in fig. 5) in the cells is significantly increased (p < 0.001) in a dose-dependent manner in a treatment concentration range of 50-200 μg compared with a blank group, and TNF- α secretion amount is significantly increased (p < 0.01) when POL-2 treatment concentration is 50 μg (C in fig. 5), which indicates that purslane galactoglucan POL-2 can promote RAW264.7 cells to secrete cytokines, effectively activate immune response of macrophages, and has significant immunopotentiating activity.
3.2 Effect on expression of related proteins in the NF- κB pathway of RAW264.7 cells
As shown in fig. 6, the expression levels of P-P65 and P-ikb- α in cells were significantly increased after purslane galactoglucan treatment compared to the blank group, demonstrating that POL-2 can wake macrophages to participate in immune response by activating NF- κb signaling pathway, thereby exerting an immunomodulatory effect.
Example 4
Preparation of herba Portulacae galactoglucan tablet.
1. Material
A purslane galactoglucan, food or pharmaceutical grade starch obtained in the same manner as in example 1.
2. Prescription of prescription
| Names of raw and auxiliary materials | Dosage of |
| Purslane galactoglucan | 200g |
| Lactose and lactose | 190g |
| Microcrystalline cellulose 101 | 85g |
| Carboxymethyl starch sodium | 10g |
| 50% ethanol solution of 5% povidone K30 | 200mL |
| Silica dioxide | 5g |
| Magnesium stearate | 5g |
| Is co-manufactured into | 5000 tablets |
3. Preparation process
Pulverizing the raw materials and the auxiliary materials, and sieving for standby; PVPK30 with the prescription amount is weighed according to the prescription, added into 50% ethanol solution, and stirred with strong force to be fully dispersed and dissolved, so as to form transparent and clear solution, and sealed for standby. Weighing purslane galactoglucan, microcrystalline cellulose 101, lactose and sodium carboxymethyl starch according to the prescription amount, dry-mixing for 15 minutes, adding the prepared adhesive, stirring for 3 minutes, and granulating by using an 18-mesh screen. And (3) drying the granules at 50-60 ℃, adding silicon dioxide and magnesium stearate, mixing for 2 minutes, and finishing the granules by using a 16-mesh screen. Measuring the content of the semi-finished product, tabletting, and coating with film coating.
Example 5
Preparation of herba Portulacae galactoglucan capsule.
1. Material
A purslane galactoglucan, food or pharmaceutical grade starch obtained in the same manner as in example 1.
2. Prescription of prescription
| Names of raw and auxiliary materials | Dosage of |
| Purslane galactoglucan | 500g |
| Starch | 300g |
| Is co-manufactured into | 5000 granules |
3. Preparation process
Weighing the prescription amount of purslane galactoglucan and starch, and stirring to completely mix. Adding appropriate amount of pulvis Talci, granulating with ethanol, sieving, drying, and packaging into No. 2 capsule shell, wherein each capsule body is filled with 80mg of herba Portulacae galactoglucan to obtain herba Portulacae galactoglucan capsule.
Example 6
Preparation of herba Portulacae galactoglucan oral liquid.
1. Material
The purslane galactoglucan obtained by the method of the example 1 and the food or pharmaceutical grade flavoring agent are caramel essence.
2. Prescription of prescription
| Original and auxiliary materialsName of the Material | Dosage of |
| Purslane galactoglucan | 100g |
| Sucrose | 0.6g |
| Caramel essence | 0.1g |
| Purified water | 2000mL |
| Is co-manufactured into | 500 counts |
3. Preparation process
Weighing purslane galactoglucan, sucrose and caramel essence with prescription amount, adding purified water for complete dissolution, filtering with a microporous filter membrane of 0.2 mu m, canning the filtrate according to the amount of 4mL per bottle by an oral liquid canning machine, sealing, and sterilizing to obtain the purslane galactoglucan.
Example 7
Preparation of herba Portulacae galactoglucan gel.
1. Material
The purslane galactoglucan obtained in the same way as in example 1 is prepared from cosmetic grade or food grade raw materials.
2. Prescription of prescription
| Names of raw and auxiliary materials | Dosage of |
| Purslane galactoglucan | 50g |
| Carbomer 940 | 30g |
| Glycerol | 250g |
| Hydroxy-phenyl ethyl ester | 2.5g |
| Triethanolamine salt | 25g |
| Polysorbate 80 | 10g |
| Propylene glycol | 250mL |
| Purified water | 5L |
3. Preparation process
Weighing the prescription amount of purslane galactoglucan, and adding 2.5L of water for dissolving for standby; uniformly scattering carbomer 940 powder with a prescription amount on the surface of glycerin, fully wetting the carbomer, adding 2L of water to fully swell the carbomer, adding a polysaccharide solution which is dissolved in advance, and uniformly stirring and mixing to obtain a mixed solution I; dissolving ethylparaben in propylene glycol, and slowly adding into the mixed solution I to obtain a mixed solution II; and then dissolving the polysorbate 80 and the triethanolamine with the prescription amount in 0.5L of water, adding the mixture into the mixed solution II under the stirring condition, uniformly stirring, and canning to obtain the purslane galactoglucan gel.
Example 8
Preparation of purslane galactoglucan facial cleanser.
1. Material
The purslane galactoglucan obtained in the same manner as in example 1 was prepared from cosmetic grade or food grade materials.
2. Formulation of
0.5g of purslane galactoglucan, 3.0g of glycerin, 3.0g of butanediol, 0.6g of propylene glycol, 0.1g of EDTA sodium, 0.3g of guar gum, 2.0g of zinc dioxide, 4.0g of C12-15 alcohol benzoate, 3.0g of C12-20 alkyl glucoside, 0.5g of C14-22 alcohol, 1.2g of cetostearyl alcohol, 0.2g of nipagin ester, 0.3g of sodium stearate, 0.5g of polydimethylsiloxane alcohol, 0.2g of polysorbate and 60g of deionized water.
3. Preparation process
Weighing the formula amount of purslane galactoglucan, and adding purified water for complete dissolution; mixing glycerol, butanediol, propylene glycol, EDTA sodium, guar gum, zinc dioxide, sodium stearate, C12-15 alcohol benzoate, C12-20 alkyl glucoside, C14-22 alcohol, cetostearyl alcohol, and purified water; mixing the nipagin ester, the polydimethylsiloxane alcohol and the polysorbate with purified water. Mixing the above prepared solutions, stirring, and canning.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. The scope of the application should, therefore, not be limited by the description of the application.
Claims (10)
1. The purslane galactoglucan is characterized by having a structure represented by the following general formula:
in the chemical formula: n represents the number of sugar repeating units, and n is a natural number.
2. The purslane galactoglucan of claim 1, wherein the weight average molecular weight of the purslane galactoglucan is 10kDa to 30kDa, the polydispersity is 1 to 3, and the molar ratio of galactose to glucose in the monosaccharide composition is about 3.3:1.
3. The purslane galactoglucan of claim 1, wherein the purslane galactoglucan is obtained by extraction and purification from dry aerial parts of purslane Portulaca oleracea l.
4. A method for preparing purslane galactoglucan as claimed in any one of claims 1 to 3, comprising the steps of:
step one: extracting dry aerial parts of herba Portulacae with 95% ethanol, discarding extractive solution, and retaining residue;
step two: extracting the residues with warm water or hot water to obtain herba Portulacae crude polysaccharide solution;
step three: grading and alcohol precipitation to obtain coarse purslane polysaccharide;
step four: refining the components with weight average molecular weight of 10 kDa-30 kDa in the crude purslane polysaccharide to obtain the purslane galactoglucan.
5. The method for preparing purslane galactoglucan according to claim 4, comprising the following steps: pulverizing dry aerial parts of herba Portulacae, defatting with 95% ethanol, extracting with warm water or hot water, centrifuging to remove residue, sequentially precipitating the supernatant with ethanol to reach alcohol concentration of 40% and 60%, centrifuging to collect precipitate, wherein refining herba Portulacae galactoglucan comprises one or more of fractional alcohol precipitation, anion exchange column chromatography, gel exclusion chromatography, dialysis and ultrafiltration.
6. A pharmaceutical composition comprising an effective amount of purslane galactoglucan as claimed in any one of claims 1 to 3 and a pharmaceutically acceptable carrier.
7. A cosmetic comprising an effective amount of purslane galactoglucan according to any one of claims 1 to 3 and a cosmetically acceptable adjuvant.
8. A food product comprising the purslane galactoglucan of any one of claims 1 to 3.
9. Use of a purslane galactoglucan or a pharmaceutical composition thereof according to any one of claims 1-3 in the manufacture of a medicament for modulating immune activity, in the manufacture of a product for enhancing immunity in an organism.
10. Use of a purslane galactoglucan as claimed in any one of claims 1 to 3 in the preparation of a cosmetic or food product.
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