CN113956370A - Dendrobium officinale polysaccharide purification method based on aminated dendritic nano mesoporous material - Google Patents
Dendrobium officinale polysaccharide purification method based on aminated dendritic nano mesoporous material Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
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- B01D3/14—Fractional distillation or use of a fractionation or rectification column
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- B01J20/26—Synthetic macromolecular compounds
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Abstract
The invention discloses a method for purifying dendrobium officinale polysaccharide based on an aminated dendritic nano mesoporous material, which comprises the following steps: s1: extracting dendrobium officinale polysaccharide; s2: synthesizing aminated dendritic nano mesoporous material DMSN-NH by adopting hot solvent method2(ii) a S3: and (3) purifying the dendrobium officinale polysaccharide: the aminated dendritic nano mesoporous material DMSN-NH is added2Ultrasonically dissolving in the incubation solution, and adding the dendrobium officinale polypeptides extracted in the step S1A sugar sample is subjected to vibration adsorption for at least 3 hours by using a rotary culture mixer, and then the aminated dendritic nano mesoporous material DMSN-NH adsorbed with dendrobium officinale polysaccharide is centrifugally collected2Then washing with 10% SDS for three times, washing the precipitate with distilled water, and adsorbing the redundant liquid with filter paper; adding 70% ethanol solution, oscillating for desorption for 24h, centrifuging at 4000rpm for 15min, and collecting supernatant to obtain purified Dendrobium officinale polysaccharide. The purification method disclosed by the invention is simple in process, and can realize efficient, environment-friendly and economic enrichment and separation of the dendrobium officinale polysaccharide.
Description
Technical Field
The invention belongs to the technical field of dendrobium officinale polysaccharide purification, and particularly relates to a dendrobium officinale polysaccharide purification method based on an aminated dendritic nano mesoporous material.
Background
The dendrobium officinale is an edible plant with high medicinal value as a traditional famous and precious traditional Chinese medicinal material, and the main active ingredients of the dendrobium officinale comprise polysaccharide, alkaloid, flavonoid, polyphenol, trace elements, amino acid and the like, wherein the content of the polysaccharide is highest, and the content of the polysaccharide in the dendrobium officinale recorded in 2020 edition Chinese pharmacopoeia is not less than 25%. The Dendrobium officinale polysaccharide has the effects of reducing blood sugar, protecting liver, inhibiting bacteria, resisting tumors, resisting osteoporosis, protecting intestines and stomach, resisting oxidation, enhancing immunity and the like.
The dendrobium officinale is a natural product, the active ingredients of the dendrobium officinale are complex and low in content, and are difficult to enrich, and how to purify and separate the active ingredients from the complex natural product is an important problem for accelerating the application process of the natural product at present. The traditional separation methods, such as macroporous resin, silica gel column chromatography and the like, are used, although the methods are simple, the method has large solvent consumption, serious pollution, time and energy consumption and high economic cost, and the effective separation of some components with similar structures and properties is difficult to realize. The existing method for separating the dendrobium officinale polysaccharide has the problems of complicated steps, low polysaccharide yield and serious loss, so that the method for purifying the dendrobium officinale polysaccharide with high efficiency, environmental protection and low cost becomes a key. At present, a method for enriching and separating dendrobium officinale polysaccharide and separating and purifying active ingredients of the dendrobium officinale polysaccharide is still lacking in the field.
Disclosure of Invention
An object of the present invention is to provide a method for purifying dendrobium officinale polysaccharide based on an aminated dendritic nano-mesoporous material, which can efficiently, environmentally and economically realize the enrichment and separation of dendrobium officinale polysaccharide, and can provide a technical hint for the research of the method for enriching and separating the active ingredients of traditional Chinese medicines.
In order to realize the aim, the invention provides a dendrobium officinale polysaccharide purification method based on an aminated dendritic nano mesoporous material, which comprises the following steps:
s1: extracting dendrobium officinale polysaccharide;
s2: synthesizing aminated dendritic nano mesoporous material DMSN-NH by adopting hot solvent method2: firstly preparing dendritic mesoporous nano-particle undMPSN, and then carrying out amino modification on the dendritic mesoporous nano-particle undMPSN by a post-grafting method to obtain DMSN-NH2;
S3: and (3) purifying the dendrobium officinale polysaccharide: the aminated dendritic nano mesoporous material DMSN-NH prepared in the step S22Ultrasonically dissolving in an incubation solution, adding the dendrobium officinale polysaccharide sample extracted in the step S1, carrying out vibration adsorption for at least 3 hours by using a rotary culture mixer, and centrifugally collecting the aminated dendritic nano mesoporous material DMSN-NH adsorbed with the dendrobium officinale polysaccharide2Then washing with 10% SDS for three times, washing the precipitate with distilled water, and adsorbing the redundant liquid with filter paper; and then adding 70% ethanol solution, placing in a constant temperature shaking table at 25 ℃, shaking and desorbing at 150rpm for 24h, centrifuging at 4000rpm for 15min, and taking supernatant to obtain purified dendrobium officinale polysaccharide.
Compared with the prior art, the method adopts a hot solvent method to synthesize the dendritic mesoporous material DMSN-NH2Then utilizing dendritic mesoporous material DMSN-NH2The dendrobium candidum polysaccharide purification device has good adsorption and desorption performance on dendrobium candidum polysaccharide compounds, realizes the purification of the dendrobium candidum polysaccharide, and respectively increases the content of the dendrobium candidum polysaccharide from the initial 6.55 percent to 51.14 percent. The method for purifying the dendrobium officinale polysaccharide is simple in process, can realize high-efficiency, environment-friendly and economical enrichment and separation of the dendrobium officinale polysaccharide, and is expected to provide a new idea for enrichment and separation of sugar-containing substances in food industry.
Preferably, step S1 includes the steps of:
s1 a: removing impurities from herba Dendrobii, cutting into small segments, oven drying at 50 deg.C, pulverizing, and sieving;
s1 b: weighing dendrobium officinale medicinal material powder, mixing the powder with distilled water, and extracting dendrobium officinale polysaccharide by adopting a reflux extraction method.
Preferably, in step S1b, the mass ratio of the dendrobium officinale medicinal material powder to the distilled water is 1:100, the extraction times are 2 times, and the extraction time is 1.5 hours.
Preferably, step S2 includes the steps of:
s2 a: preparing dendritic mesoporous nano-particle undmpms: adding TEA into deionized water, stirring in an oil bath kettle at 80 ℃ for 30 minutes, then adding CTAB and NaSal, continuing stirring for 1 hour, dropwise adding TEOS into the mixed solution, and continuing stirring for 4 hours at 80 ℃; after the reaction was completed, the resultant was collected by centrifugation at 9000rpm for 10min, and washed twice with ethanol and water to remove the residual reactant; extracting the obtained precipitate twice at 80 ℃ by using a mixed solution of HCl and ethanol to remove residual organic templates, and finally performing vacuum drying at 50 ℃ for 12 hours to obtain a white solid sample, namely the dendritic mesoporous nanoparticle DMSN;
s2 b: and (3) dispersing the dendritic mesoporous nano particle undmNSN prepared in the step S2a in ethanol, carrying out ultrasonic treatment for 10min, continuing stirring for 15min, then adding ammonia water, deionized water and APTES into the solution under vigorous stirring, continuing to stir vigorously for 12h, finally washing the final product with ethanol, and drying in vacuum at 50 ℃ for more than 8 h.
Preferably, in step S2a, the ratio of the amount of TEA, deionized water, CTAB, NaSal, and TEOS is 0.68g:25mL:0.38g:0.168g:4 mL.
Preferably, in step S2a, the volume ratio of HCl to ethanol in the mixed solution of HCl and ethanol is 1: 9.
Preferably, in step S2a, the precipitate is extracted twice with a mixed solution of HCl and ethanol at 80 ℃ for 24h each time.
Preferably, in step S2b, the usage ratio of the dendritic mesoporous nanoparticle undmdsn, ethanol, ammonia water, deionized water, and APTES is 500mg:100mL:2.5mL: 1 mL.
Preferably, in step S3, the aminated dendritic nano-mesoporous material DMSN-NH2And the amount of the incubation solution was 40mg:30 mL.
Preferably, in step S3, the incubation solution is deionized water.
The invention provides a method for purifying dendrobium officinale polysaccharide based on an aminated dendritic mesoporous nano material, wherein amino dendritic mesoporous nano particles (DMSN-NH)2) Has the advantages of high specific surface area, large pore volume, accessibility of the inner surface and the like, and is a mesoporous material with a dendritic fiber form. DMSN-NH2Is composed of silicon dioxide fiber or nano-corrugation as structural unit, which are arranged along the central radial direction of the particle and undergo the incremental process from 0 generation to 4 generations to form a central radial pore canal structure, the pore diameter of which is gradually increased from the inside of the particle to the surface of the particle, therefore, DMSN-NH2And the dendritic morphology is presented. Furthermore, in an iterative process, uniform small mesopores can be introduced into the fibers or wrinkles, thus DMSN-NH2The silica sphere has a hierarchical pore structure, has central radial pores and uniform small mesopores, and is irregularly distributed on the surface of the silica sphere. The unique layered pore structure can enhance the diffusion of guest molecules with different sizes in the silicon dioxide, so that small molecules are dispersed in small mesopores, and larger molecules such as biomolecules and fluorescent molecules are dispersed in macropores in the central radial direction, therefore, the dendritic mesoporous silicon dioxide with the hierarchical pore structure is expected to be a multifunctional carrier. Compared with the MSN with uniform ordered hexagonal mesopores or disordered large and medium mesopores, the dendritic particles with irregular pore structures have higher loading capacity than the MSN with uniform ordered mesopores and large and medium pore structures. Thus, DMSN-NH2The material is applied to the purification of the dendrobium officinale polysaccharide, provides a high-efficiency, environment-friendly, economic and simple-process way for the enrichment and separation of the dendrobium officinale polysaccharide, and also provides a theoretical basis for the research of the enrichment and separation method of the effective components of traditional Chinese medicines.
Drawings
FIG. 1 shows amino dendritic mesoporous material nanoparticles DMSN-NH2Preparation process diagram
FIG. 2 shows undMPMS and DMSN-NH2In the infrared spectrum
FIG. 3 shows undMPMS and DMSN-NH2N of (A)2Adsorption and desorption isotherm and pore size distribution map
FIG. 4 shows undMPMS and DMSN-NH2Zeta potential distribution diagram
FIG. 5 shows DMSN-NH2Scanning electron microscope image of
FIG. 6 shows DMSN-NH adsorbed with Dendrobium officinale polysaccharide2(A-I) Transmission Electron microscopy
FIG. 7 shows undMPMS and DMSN-NH2Wide angle XRD diffractogram of
FIG. 8 shows DMSN-NH2Ultraviolet and visible absorption spectrum of
FIG. 9 shows a mesoporous material DMSN-NH2Adsorption contrast curve diagram for dendrobium officinale polysaccharide at different times
FIG. 10 is QtAnd t0.5Is shown in
FIG. 11 shows DMSN-NH2Histogram of adsorption capacity for different concentrations of polysaccharide
FIG. 12 is a histogram of polysaccharide elution rates of different eluents on mesoporous materials
Detailed Description
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative of the present invention only, and are not intended to limit the scope of the present invention.
The laboratory instruments and reagents used in the examples are shown in tables 1 and 2:
TABLE 1 Experimental instruments
TABLE 2 Experimental reagents
Example 1: dendrobium officinale polysaccharide purification method based on aminated dendritic nano mesoporous material
The dendrobium officinale polysaccharide is purified according to the following steps:
s1: the method for extracting the dendrobium officinale polysaccharide comprises the following steps:
s1 a: removing impurities from herba Dendrobii original medicinal material, cutting into small segments, oven drying at 50 deg.C, pulverizing, and sieving with a third sieve.
S1 b: weighing 0.5g of medicinal powder, precisely weighing, adding 50mL of distilled water, and extracting Dendrobium officinale polysaccharide by reflux extraction for 2 times with an extraction time of 1.5 h.
S2: synthesizing aminated dendritic nano mesoporous material DMSN-NH by adopting hot solvent method2As shown in fig. 1, the method comprises the following steps:
s2 a: preparation of dendritic mesoporous nanoparticles (undmdsn): 0.68g TEA was added to 25mL deionized water in an 80 ℃ oil bath and stirred for 30 minutes, then 0.38g CTAB and 0.168g NaSal were added and stirring was continued for 1 hour, then 4mL TEOS was added dropwise to the above mixed solution using a constant pressure addition funnel and stirring was continued for 4 hours at 80 ℃. After the reaction was completed, the resultant was collected by centrifugation at 9000rpm for 10 minutes, and washed twice with ethanol and water to remove the residual reactant. Subsequently, the obtained precipitate was extracted twice (24 h each time) with a mixed solution of HCl and ethanol (HCl: ethanol ═ 1: 9) at 80 ℃ to remove the residual organic template, and finally dried under vacuum at 50 ℃ for 12h to give a white solid sample.
S2 b: synthesis of amino-modified dendritic mesoporous nanoparticles (DMSN-NH)2): the modification of the amino group is carried out by the post-grafting method. Firstly, 500mg of dendritic mesoporous nano particles (undmpms) are dispersed in 100mL of ethanol, and after 10 minutes of ultrasonic treatment, the mixture is continuously stirred for 15 minutes. Then 2.5mL of ammonia, 2.5mL of deionized water and 1mL of LAPTES were added to the above solution with vigorous stirring, vigorous stirring was continued for 12h, and finally the final product was washed several times with ethanol and dried in vacuo at 50 ℃ overnight.
S3: and (3) purifying the dendrobium officinale polysaccharide: weighing 40mg of the aminated dendritic nano mesoporous material DMSN-NH prepared in the step S22Ultrasonically dissolving the mixture in 30mL of incubation solution, adding 30mg of the dendrobium officinale polysaccharide sample extracted in the step S1, carrying out vibration adsorption for 3 hours by using a rotary culture mixer, and centrifugally collecting the aminated dendritic nano mesoporous material DMSN-NH adsorbed with the dendrobium officinale polysaccharide2Then washed three times with 10% SDS and then steamed to 100mLThe precipitate was washed with distilled water, and the excess liquid was adsorbed by filter paper. Adding 70% ethanol solution, placing in a constant temperature shaking table at 25 deg.C, oscillating at 150rpm for desorption for 24h, centrifuging at 4000rpm for 15min, and collecting supernatant to obtain purified herba Dendrobii polysaccharide. The incubation solution can be phosphate buffered solution with pH3, phosphate buffered solution with pH8, or deionized water, and preferably, deionized water is used as the incubation solution in the present embodiment.
Test example 1: optimization of extraction process of dendrobium officinale polysaccharide
And (3) polysaccharide content determination: and (3) determining the content of the polysaccharide by using a sulfuric acid-phenol color development method by taking D-anhydrous glucose as a reference, wherein a standard curve equation is that y is 7.66812x +0.00000(r is 0.99926), wherein x is the mass concentration (mg/mL) of the D-anhydrous glucose and y is absorbance, and calculating the content of the polysaccharide according to a regression equation after measuring the absorbance.
The embodiment optimizes the extraction process of the dendrobium officinale polysaccharide: taking dendrobium officinale medicinal material powder, precisely weighing, and observing 3 factors of a material-liquid ratio (A), extraction time (B) and extraction times (C) on the basis of single-factor extraction process observation, wherein each factor is set to be 3 levels. And (3) selecting an L9(33) table to carry out orthogonal experiments, taking the polysaccharide content as an evaluation index, calculating the standard deviation, and comparing t test by using the mean of two independent samples.
(1) Investigation of the extraction method: respectively taking 0.5g of medicinal powder, precisely weighing, adding 80mL of distilled water, extracting the dendrobium officinale polysaccharide by an ultrasonic extraction method and a reflux extraction method, and measuring the absorbance by an ultraviolet spectrophotometer. The result shows that the average content of polysaccharide is 26.25% by adopting the reflux extraction method, the average content of polysaccharide extracted by ultrasonic is 17%, and the extraction rate of the dendrobium officinale and dendrobium officinale polysaccharide by adopting the reflux extraction method is highest. Therefore, in step S1 of example 1, the dendrobium officinale polysaccharide is extracted by a reflux extraction method.
(2) And (3) examining the extraction times: adopting a reflux extraction method, selecting a polysaccharide extraction time of 1.5h and a material-liquid ratio of 0.5: 40(g/mL), and the result of examining the extraction times shows that the extraction rate of the polysaccharide is higher after 3 times of extraction, but the content of the polysaccharide is only different by 0.25 percent between 2 times of extraction and 3 times of extraction, and the extraction times of 2 times is more reasonable from the aspects of reducing energy consumption and time. Thus, in example 1, 2 extractions were performed in step S1 b. The specific examination results are shown in table 3:
TABLE 3 results of frequency of extraction investigation
(4) And (3) extracting time investigation: extracting by a reflux method, wherein the material-liquid ratio is 0.5: 50(g/mL), 2 times of extraction, and the influence of the extraction time on the polysaccharide yield, wherein the extraction time is 1.5h, the polysaccharide yield is the highest, and the result is shown in Table 4.
TABLE 4 extraction time survey
(5) Orthogonal experiment
According to a single-factor experiment, 3 factors of the feed-liquid ratio (A), the extraction time (B) and the extraction times (C) are considered, and an orthogonal experiment is arranged by L9 (33). The results are shown in Table 5. According to the result analysis of the orthogonal test R value: the feed-liquid ratio (A) > the extraction times (C) > the extraction time (B). The factor A has the largest influence on the index K1, the factor C has the largest influence on the index K2, the factor B has the largest influence on the index K3, the orthogonal experiment result shows that the content measured by the factor A1B2C2 is the highest, and the optimal extraction method is determined by integrating all process factors and is a material-liquid ratio of 1:100, the extraction time is 1.5h, and the extraction is performed for 2 times, so that the step S1 of the embodiment 1 adopts the optimal parameters for extraction.
TABLE 5 results of orthogonal experiments
Numbering | A | B | C | Average content (%) |
1 | 1 | 1 | 1 | 20.65 |
2 | 1 | 2 | 2 | 30.12 |
3 | 1 | 3 | 3 | 27.91 |
4 | 2 | 1 | 2 | 15.95 |
5 | 2 | 2 | 3 | 15.69 |
6 | 2 | 3 | 1 | 15.70 |
7 | 3 | 1 | 3 | 29.12 |
8 | 3 | 2 | 1 | 22.04 |
9 | 3 | 3 | 2 | 27.30 |
K1 | 26.23 | 21.1 | 19.46 | |
K2 | 15.78 | 22.62 | 24.46 | |
K3 | 26.15 | 23.64 | 22.24 | |
R | 10.447 | 1.730 | 4.99 |
(6) Determination of polysaccharide content
The optimum extraction process was designed according to the orthogonal test, taking 0.5g of each portion, for a total of 3 portions. Placing in a conical flask, adding 30mL of petroleum ether, heating in a water bath for 30min, taking out, discarding the filtrate, keeping the filter residue to evaporate the dry solvent, adding 80mL of ethanol, shaking uniformly, heating in a water bath for 1h, taking out, discarding the filtrate, keeping the filter residue to evaporate the dry solvent, adding 50mL of distilled water, heating and refluxing for 2 times, 1.5h each time, filtering, combining the two filtrates, cooling in a 200mL volumetric flask, adding pure water to the scale, shaking uniformly, sucking 1mL, placing in a 25mL volumetric flask, fixing the volume, and shaking uniformly to obtain the product. As a result, the average polysaccharide content of the dendrobium officinale extract is 30.65%, and the RSD value is 4.26%. The average polysaccharide content extracted by the orthogonal test is 30.12 percent, and the RSD value is 1.06 percent. The results are shown in Table 6.
TABLE 6 results of content measurement of samples
Therefore, in the single-factor process investigation, the content of polysaccharide in the reflux extraction method is obviously higher than that in the ultrasonic extraction method, the reflux extraction is carried out at a high temperature of direct fire heating, and compared with the ultrasonic extraction, the reflux extraction is only 40 ℃, and is possibly closely related to the extraction temperature. The content measurement in the extraction times shows that the polysaccharide extraction rate is higher when the polysaccharide is extracted for 3 times by refluxing, and the polysaccharide is extracted for 2 times from the aspects of reducing energy consumption and time by considering that the content of the polysaccharide is only different by 0.25 percent between 2 times and 3 times of polysaccharide extraction.
In addition, the optimal polysaccharide extraction process conditions of the experiment are obtained through an orthogonal test method, the content of the polysaccharide in the dendrobium officinale and the dendrobium officinale is measured through an ultraviolet-visible spectrophotometry method, and test results show that the extraction process method is stable and reliable.
In conclusion, the optimal experimental conditions for extracting the dendrobium officinale polysaccharide are as follows: the ratio of material to liquid is 1:100, the extraction time is 1.5h, the extraction times are 2 times, and the average polysaccharide content of the dendrobium officinale polysaccharide extracted under the optimal condition is 30.65 percent through verification experiments.
Test example 2: aminated dendritic nano mesoporous material DMSN-NH2Is characterized by
(1) UnDMSN and DMSN-NH2Infrared spectroscopy (FT-IR) test
Approximately 1mg of the powder sample was weighed out and mixed with 100mg of dry KBr powder and mixed by thorough grinding in a clean dry agate mortar. The mixed powder is pressed into a transparent sheet sample at 4000-400cm by a tablet press-1Scanning in the range to obtain an infrared spectrogram.
As shown in FIG. 2, at 446cm-1And 797cm-1Two obvious characteristic peaks of (A) are caused by vibration of Si-O at 1090cm-1The strong absorption peak is originated from the asymmetric stretching vibration of Si-O-Si and is the characteristic absorption peak of the silicon dioxide material. Amino group-modified DMSN-NH2Compared with infrared data of the undDMSN, the infrared data of the undDMSN are 2850-2950 cm-1There are two weak symmetric stretching vibrations, which are-CH in silane2-a characteristic absorption of. At 1546cm-1The vibration peak is a characteristic absorption peak belonging to-N-H, and the two groups of infrared data indicate that APTES is silanized on the surface of the DMSN and successfully modifies the amino group on the surface of the DMSN.
(2) Nitrogen adsorption and desorption characterization Bamauer-Enunett-Teller (BET)
Mixing a certain amount of undMPMS and DMSN-NH2After the solid sample is dried in a vacuum drying oven for a certain time, about 100mg of the sample is quickly weighed and carefully poured into a special drying tube by means of a paper groove, the difference value is recorded, and then the sample is placed into a nitrogen adsorption instrument for testing.
Nitrogen adsorption-desorptionExperiments are commonly used to analyze the pore volume, pore diameter and specific surface area of mesoporous materials. Shown in FIG. 3 are undMPMS N and DMSN-NH2The BET curve and the BJH pore size distribution diagram show that HI type hysteresis loops of the BET curve and the BJH pore size distribution diagram are clearly visible and belong to a typical type IV curve. Calculating the DMSN and DMSN-NH according to the Bamauer-roundt-teller (bet) method2Compared with pure undmpmsn, the modified DMSN-NH with amino groups2Specific surface area of 414.39m2The/g is reduced to 239.04m2In terms of/g, total pore volume is from 1.17cm3The/g is reduced to 0.80cm3Per g, this is because APTES is in DMSN-NH2Surface hydrolysis leading to DMSN-NH2The specific surface area, pore volume and other parameters are reduced in different degrees. These results indicate that APTES was successful in DMSN-NH2Hydrolysis on the surface. Furthermore, as can be seen from the pore size distribution diagram, DMSN-NH2The material has bimodal pore size distribution, shows that the material has the characteristics of hierarchical pore mesopores and small mesopores which are irregularly distributed, is mainly concentrated in the range of 10-35nm, and is processed according to a Barrett-Joyner-Halenda (BJH) method to obtain the material with the average pore size of 16.3nm, and the result shows that DMSN-NH2Is of mesoporous structure.
(3) Zeta potential characterization of nanoparticles
Taking small amount of undmNSN and DMSN-NH2Dispersing in deionized water, and dispersing for 15min by ultrasonic oscillation. And respectively injecting the dispersed solutions into a potential tank for Zeta potential test.
From the change of the Zeta potential shown in fig. 4 below, it can be seen that the Zeta potential values of the mesoporous materials before and after modification and before and after loading are obviously changed. Pure undmpmsn has negative charges because of abundant hydroxyl groups on the surface of the silicon dioxide, and the potential value is-24.5 +/-0.5 mV. DMSN modified with amino group, surface being largely-NH2Modified and the potential changed to be positive 26.8 +/-0.8 mV. The highly positive potential values indicate that the synthesized nanoparticles have excellent stability. The apparent change in the Zate potential profile indicates successful modification of the amino group and successful in situ generation of noble metal nanoparticles.
(4) Transmission Electron microscopy characterization (TEM) and scanning Electron microscopy characterization (SEM)
Small amount of DMSN-NH is sucked up2The sample is dispersed in ethanol, and the ultrasonic dispersion is uniform. Then a small amount of liquid to be detected is absorbed by a liquid-transferring gun, is dripped on the surface of a copper mesh special for TEM, is dried by an infrared lamp, and is finally observed in a transmission electron microscope instrument. Taking a small amount of DMSN-NH2And uniformly spreading the solid sample on the conductive adhesive, and observing the appearance of the sample in a scanning electron microscope instrument after the gold spraying treatment.
FIGS. 5 and 6 are SEM and TEM images of the prepared aminated dendritic mesoporous material (after adsorbing Dendrobium officinale polysaccharide), respectively. As can be seen from the SEM image of FIG. 5, the synthesized mesoporous material has distinct central radial channels, the pore size of the mesoporous material gradually increases from the inside to the outside of the mesoporous material, the mesoporous material has a wrinkle shape, hierarchical pores formed by wrinkle extrusion are unevenly distributed on the surface of silica spheres, the pore size of the mesoporous material is about 20nm, and the morphology conforms to the N2The results of adsorption-desorption, as well as the results of the uneven distribution of the pore size of BJH, are also confirmed. The unique morphology provides sufficient loading space for the in-situ adsorption of the dendrobium officinale polysaccharide. As can be generally seen from the TEM image of FIG. 6, the prepared nanoparticles have good dispersibility and unique dendritic structures, and each dendritic mesoporous particle has a particle size of about 160 nm. In addition, the A, D, G diagram of fig. 6 is a TEM diagram of the aminated dendritic mesoporous material after adsorbing dendrobium officinale polysaccharides with different qualities, the region of the aminated dendritic mesoporous material is enlarged (B, E, H), and then a region attached with dendrobium officinale polysaccharides is randomly selected (C, F, I) for analysis, so that it can be seen that the dendrobium officinale polysaccharide nanoparticles can be better attached with DMSN-NH2The dendrobium polysaccharide is adsorbed, evenly distributed in the dendritic mesoporous channel and has a clear structure, which proves that the dendrobium polysaccharide is successfully loaded in DMSN-NH2And (4) the following steps.
(5) Characterization by X-ray diffraction (XRD)
Taking appropriate amount of undMPSNN and DMSN-NH2And (3) a sample is ground into fine powder and pressed on a special glass sample table to scan powder diffraction signals.
XRD is a commonly used characterization means to verify nanoparticle crystallinity. As shown in FIG. 7, each XRD pattern had a broad characteristic peak at 22.9 due to the absence of a characteristic peakThe structure of the silica is shaped. UnDMSN and DMSN-NH2Five diffraction peaks at 2 θ of 38.1 °, 44.3 °, 64.5 °, 77.5 ° and 81.8 ° represent the crystal planes of (111), (200), (220), (311), and (222), respectively. These results confirm DMSN-NH2The nanoparticles are successfully generated and all maintain good crystal structure.
(6) Ultraviolet visible absorption Spectroscopy analysis (UV-Vis)
Taking small amount of undmNSN and DMSN-NH2Respectively dispersing in deionized water, and ultrasonically dispersing uniformly. And then, respectively injecting the dispersed solution into cuvettes with two transparent surfaces, and measuring the characteristic absorbance in an ultraviolet-visible spectrophotometer.
Due to the plasma resonance effect, the particles have a distinct uv-visible absorption peak. As can be seen from FIG. 8, DMSN-NH2A characteristic ultraviolet absorption peak at 273nm further illustrates the successful synthesis of amino groups on DMSN.
Test example 3: adsorption kinetics of dendrobium officinale polysaccharide on mesoporous material
Weighing 40mg of mesoporous material DMSN-NH2The incubation solutions were 30ml of phosphate buffered solution at pH3, 8 phosphate buffered solution at pH8 and deionized water, respectively, and sonicated for 5 min. Adding 30mg polysaccharide sample, respectively oscillating and adsorbing with a rotary culture mixer, respectively adsorbing 1mL adsorption solution at 0.5h, 1h, 1.5h, 2h, 3h, 5h, 7h and 9h, centrifuging at 10000r/min for 10min, and collecting supernatant. 0.1mL of the supernatant was aspirated, and the polysaccharide content in the supernatant was measured by the phenol-sulfuric acid method. The adsorption quantity Q of the polysaccharide on the mesoporous material is calculated by using a difference value method, and is shown as a formula (1).
Wherein m represents the mass of the mesoporous material, g; v represents the volume of the adsorption solution, mL; c0And C represents the polysaccharide concentration in the initial solution and the supernatant after adsorption, mg/mL, respectively.
And (3) carrying out adsorption kinetics fitting through a quasi-first order (PFO) model and a quasi-second order adsorption kinetics (PSO) model, wherein the formula is shown as (2) and (3).
ln(Qe-Qt)=lnQe-K1t (2)
Wherein Q istRepresents the adsorption amount at time t (min), mg/g; qeRepresents the equilibrium adsorption capacity, mg/g; k1(min-1)、K2(g·mg-1·min-1) PFO and PSO constants are respectively expressed; v. of0=K2Qe 2The initial velocity of PS adsorption is shown.
When the concentration of the polysaccharide is 1mg/mL, and the incubation solutions are water, phosphoric acid buffer solutions with pH3 and pH8 respectively, the mesoporous material DMSN-NH2The adsorption results of dendrobium officinale polysaccharide at different times are shown in fig. 9. As can be seen from the graph, the adsorption amount increases as the adsorption time increases. Under the incubation solutions with different pH values, adsorption equilibrium is basically achieved within 3 h. Wherein the mesoporous material DMSN-NH is prepared when the incubation solution is water2The maximum adsorption capacity can reach 350 mg/g. The surface of the mesoporous material contains a large number of hydroxyl groups, and the dendrobium officinale polysaccharide mainly comprises glucose, so that the polysaccharide and the mesoporous material are adsorbed by virtue of hydrophilic action, hydrogen bond action and electrostatic action, and under alkaline or acidic conditions, the adsorption amount is reduced due to the inhibition of hydrophilic action, the weakening of hydrogen bond action, electrostatic repulsion and the like. In order to quantitatively research the mesoporous material DMSN-NH2The kinetic adsorption behavior of (1) was obtained by substituting kinetic data into a model, the fitting results are shown in Table 7, and the linear correlation coefficient R 'of the quasi-second order rate equation was found by comparing the fitting coefficient R'>0.99, and Q after fitting. The approximation with the experimental value indicates DMSN-NH2The dynamic adsorption behavior of (a) meets the assumption of a quasi-secondary adsorption dynamic model (PSO). The quasi-second-order reaction dynamic model not only comprises the outer surface diffusion, the surface adsorption and the like for adsorption, but also comprises the process of the inner diffusion of the molecular particles, and compared with the quasi-first-order dynamic model, the model can describe the adsorption reaction process of the mesoporous material to the dendrobium polysaccharide more exactly。
TABLE 7 kinetic adsorption data fitting of polysaccharides on mesopores
In general, the adsorption process consists of (i) external diffusion, where solute molecules diffuse from the bulk of the aqueous phase to the surface of the adsorbent particles, (ii) intra-particle diffusion, where surface adsorbed solute molecules diffuse to the pores inside the particles, and (iii) adsorption, where solute molecules adsorb at internal adsorption sites. Generally, the adsorption of internal adsorption sites is relatively rapid and thus can be neglected. Thus, the adsorption rate control step depends on how fast the out-diffusion or in-particle diffusion is. If intraparticle diffusion is the rate controlling factor for adsorption, the relationship between the amount of adsorption and time follows equation (7).
Qt=Kit0.5+C (7)
In the formula, Qt(mg/g) is the adsorption amount of t (min), C (mg/g) is a coefficient relating to the thickness of the boundary layer, Ki(mg·g-1·min-0.5) Is the intrinsic diffusion rate constant. When Q istAnd t0.5When well linear and passing through the origin, it is shown that the intraparticle diffusion process is the only control step of the adsorption rate.
From Q of FIG. 10tAnd t0.5The graph of (a) shows that the curve consists of a plurality of line segments and, although the origin, illustrates that intraparticle diffusion is not the only control step in which both outdiffusion and outdiffusion play a role. The adsorption process consisted of two steps, the first fast adsorption process consisting of polysaccharide binding to surface active sites, the second slow adsorption process gradually reaching equilibrium due to saturation of adsorption sites, fitting equations in which the C value increased from 98.473mg/g in the first stage to 247.67mg/g in the second stage, indicating that the boundary layer thickness increased as adsorption proceeded.
Test example 4: adsorption thermodynamics of dendrobium officinale polysaccharide on mesoporous material
The incubation solution is deionized water, other conditions are the same as those of the kinetic study, the adsorption isotherm determination is carried out, the adsorption time is 3h, and the initial concentrations of the polysaccharide are respectively 0.1, 0.2, 0.3, 0.5, 0.75, 1 and 105mg/mL
Thermodynamic adsorption data were fitted by a Langmuir model for monolayer adsorption (formula (4)) and a Freundlich model for multilayer adsorption (formula (5)).
Wherein Q iseThe equilibrium adsorption capacity of a material to a target object under a certain concentration is expressed as mg/g; ceRepresents the concentration of the target substance remaining in the solution at the time of equilibrium adsorption, mg/mL; kLRepresents the adsorption binding constant, mL/mg; qmaxRepresents the maximum adsorption capacity of the material to a target object, mg/g; kfAnd n is the Freundlich model constant.
Mesoporous material DMSN-NH2The adsorption thermodynamic behavior for dendrobii polysaccharide is shown in fig. 11. As the sample concentration increases, the amount of adsorption also increases. In order to quantitatively research the mesoporous material DMSN-NH2The fitting results for the thermodynamic adsorption behavior of dendrobii polysaccharide are shown in table 8. The better regression effect of the Langmuir model regression and the Freundlich model regression is found through fitting, wherein the fitting coefficient of the Langmuir model is more than 0.99, and the equation is shown to be more in line with DMSN-NH2The adsorption process of the dendrobium officinale polysaccharide is explained at the same time by DMSN-NH2The adsorption process of (a) is mainly a monolayer and is accompanied by adsorption of a monolayer. The parameter n in the Freundlich model is related to the adsorption strength and the interaction between the adsorbent and the adsorbate, and the value of n is more than 1, which indicates that the dendrobium officinale polysaccharide is easy to adsorb on DMSN-NH2The surface active site of (1).
TABLE 8 Dendrobii officmalis caulis polysaccharide in DMSN-NH2Fitting results of thermodynamic adsorption data of
Test example 5: adsorbing the mesoporous material DMSN-NH2Eluting the dendrobium polysaccharide: eluent optimization
After the mesoporous materials and the polysaccharide samples are oscillated and incubated for 3h, the mesoporous materials adsorbing the polysaccharide are centrifugally collected, washed three times by using 20% ACN, 1mol/L NaCl and 10% SDS respectively, and the elution amount of the polysaccharide in the supernatant is centrifugally determined. The elution rate is the ratio of the elution amount to the adsorption amount. As shown in equation (6). Wherein C (mg/mL) is the concentration of the polysaccharide in the eluent, V (mL) is the volume of the eluent, m (mg) is the mass of the polysaccharide, and Q (mg/g) is the adsorption amount of the polysaccharide on the mesoporous material.
Different eluents (20% ACN solution, Imol/LNaC1, 10% SDS) adsorbed on DMSN-NH2The desorption result of the dendrobium officinale polysaccharide is shown in fig. 12. Dendritic mesoporous material DMSN-NH2And polysaccharide both contain abundant hydroxyl groups, both are hydrophilic substances, 10% SDS has stronger polarity than water, and can destroy DMSN-NH2Hydrogen bonding with polysaccharide to adsorb DMSN-NH2The dendrobium polysaccharide on the surface is desorbed due to DMSN-NH2The electrostatic force between the ionic eluent and the polysaccharide is weak, so the elution capacity of the ionic eluent NaCl is limited; DMSN-NH2And the polysaccharide are hydrophilic substances, and the hydrophobic interaction force between the polysaccharide and the polysaccharide is small, so that the ACN elution capacity is relatively low.
Test example 6: determination of purified dendrobium officinale polysaccharide and other contents
Via DMSN-NH2Polysaccharide eluted by adsorption and 10% SDS is desalted by dialysis and then analyzed by PMP pre-column derivatization and high performance liquid chromatography for polysaccharide composition, and the content of the eluted dendrobium officinale polysaccharide is as follows. (polysaccharide, mannose content and mannose-glucose peak area ratio are measured according to the method of Chinese pharmacopoeia, statistical software SPSS20.0 is adopted for data analysis.)The molar contents of various saccharides in the dendrobium officinale polysaccharide after sugar elution are respectively 31.22 +/-2.36 of polysaccharide, 17.87 +/-1.86 of mannose and 3.26 +/-0.72 of the peak area ratio of mannose to glucose. Through analysis, the purity of the total sugar is improved by 51.14 percent compared with that before enrichment, because the protein substances are DMSN-NH with polarity2DMSN-NH with weak surface adsorption and polar carbohydrate in polarity2The surface is selectively enriched, so that the purity of the enriched polysaccharide is improved.
At present, a method for enriching and separating dendrobium officinale polysaccharide and separating and purifying active ingredients of the dendrobium officinale polysaccharide is still lacking in the field. Aiming at the problems of complicated separation steps of dendrobe polysaccharide, low polysaccharide yield and serious loss, the invention discloses a mesoporous material-based DMSN-NH2The novel process for enriching polysaccharides. The experimental result shows that the mesoporous material DMSN-NH2The adsorption capacity of the dendrobium officinale polysaccharide is 350mg/g, the adsorption kinetics accords with a quasi-second-level adsorption kinetics model, and the adsorption process is controlled by external diffusion and intra-particle diffusion; polysaccharide in DMSN-NH2The adsorption process of (a) is mainly a monolayer and is accompanied by adsorption of a monolayer. The invention is expected to provide guidance for the enrichment and separation of sugar-containing substances in the food industry.
While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
The above description is only a partial example of the present invention, and does not limit the embodiments and the protection scope of the present invention, therefore, it should be recognized that the present invention is covered by the protection scope of the present invention by the equivalent substitution and obvious change made by the description of the present invention for those skilled in the art.
Claims (10)
1. A method for purifying dendrobium officinale polysaccharide based on an aminated dendritic nano mesoporous material is characterized by comprising the following steps: the method comprises the following steps:
s1: extracting dendrobium officinale polysaccharide;
s2: synthesizing aminated dendritic nano mesoporous material DMSN-NH by adopting hot solvent method2: firstly preparing dendritic mesoporous nano-particle undMPSN, and then carrying out amino modification on the dendritic mesoporous nano-particle undMPSN by a post-grafting method to obtain DMSN-NH2;
S3: and (3) purifying the dendrobium officinale polysaccharide: the aminated dendritic nano mesoporous material DMSN-NH prepared in the step S22Ultrasonically dissolving in an incubation solution, adding the dendrobium officinale polysaccharide sample extracted in the step S1, carrying out vibration adsorption for at least 3 hours by using a rotary culture mixer, and centrifugally collecting the aminated dendritic nano mesoporous material DMSN-NH adsorbed with the dendrobium officinale polysaccharide2Then washing with 10% SDS for three times, washing the precipitate with distilled water, and adsorbing the redundant liquid with filter paper; and then adding 70% ethanol solution, placing in a constant temperature shaking table at 25 ℃, shaking and desorbing at 150rpm for 24h, centrifuging at 4000rpm for 15min, and taking supernatant to obtain purified dendrobium officinale polysaccharide.
2. The purification process according to claim 1, characterized in that: step S1 includes the following steps:
s1 a: removing impurities from herba Dendrobii, cutting into small segments, oven drying at 50 deg.C, pulverizing, and sieving;
s1 b: weighing dendrobium officinale medicinal material powder, mixing the powder with distilled water, and extracting dendrobium officinale polysaccharide by adopting a reflux extraction method.
3. The purification method according to claim 2, characterized in that: in the step S1b, the mass ratio of the dendrobium officinale medicinal material powder to the distilled water is 1:100, the extraction times are 2 times, and the extraction time is 1.5 hours.
4. The purification process according to claim 1, characterized in that: step S2 includes the following steps:
s2 a: preparing dendritic mesoporous nano-particle undmpms: adding TEA into deionized water, stirring in an oil bath kettle at 80 ℃ for 30 minutes, then adding CTAB and NaSal, continuing stirring for 1 hour, dropwise adding TEOS into the mixed solution, and continuing stirring for 4 hours at 80 ℃; after the reaction was completed, the resultant was collected by centrifugation at 9000rpm for 10min, and washed twice with ethanol and water to remove the residual reactant; then, extracting the obtained precipitate twice by using a mixed solution of HCl and ethanol at 80 ℃ to remove residual organic templates, and finally, drying in vacuum at 50 ℃ for 12 hours to obtain a white solid sample, namely the dendritic mesoporous nano-particle undMPN;
s2 b: and (3) dispersing the dendritic mesoporous nano particle undmNSN prepared in the step S2a in ethanol, carrying out ultrasonic treatment for 10min, continuing stirring for 15min, then adding ammonia water, deionized water and APTES into the solution under vigorous stirring, continuing to stir vigorously for 12h, finally washing the final product with ethanol, and drying in vacuum at 50 ℃ for more than 8 h.
5. The purification method according to claim 4, characterized in that: in step S2a, the amount ratio of TEA, deionized water, CTAB, NaSal, and TEOS was 0.68g:25mL:0.38g:0.168g:4 mL.
6. The purification method according to claim 4, characterized in that: in step S2a, the volume ratio of HCl to ethanol in the HCl-ethanol mixed solution is 1: 9.
7. The purification method according to claim 4, characterized in that: in step S2a, the precipitate was extracted twice with a mixture of HCl and ethanol at 80 ℃ for 24h each time.
8. The purification method according to claim 4, characterized in that: in step S2b, the dosage ratio of the dendritic mesoporous nano-particles DMSN, the ethanol, the ammonia water, the deionized water and the APTES is 500mg:100mL:2.5mL:2.5mL:1 mL.
9. The purification process according to claim 1, characterized in that: in step S3, amination dendritic nano mesoporous material DMSN-NH2And an incubation solutionThe dosage ratio of the components is 40mg to 30 mL.
10. The purification process according to claim 1, characterized in that: in step S3, the incubation solution is deionized water.
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