CN106008729B - A kind of sunset abelmoschus stem or bark leaf polyose and preparation method and application - Google Patents

Abstract

The invention discloses a hollyhock stem and leaf polysaccharide as well as a preparation method and application thereof. The preparation method comprises: extraction, ethanol precipitation, macroporous adsorption resin column chromatographic purification, hollow cellulose membrane separation, DEAE cellulose resin column chromatographic separation, collecting target fractions, desalting treatment, and freeze-drying to obtain hollyhock stem and leaf polysaccharides. The polysaccharide weight average molecular weight of this marshmallow stem leaf is 13821Da, is made up of rhamnose, mannose, galactose, galacturonic acid, glucose and arabinose, and its mol ratio is arabinose: galactose: glucose: galacturonic acid: Rhamnose:mannose=1:0.53:0.12:0.05:0.16:0.03, has good immune enhancing activity and can be developed as immune enhancing drug and health food. The invention uses the stem and leaf waste resources produced in the production process of the marshmallow medicinal material as raw materials to separate and prepare polysaccharides with immune activity, which lays a foundation for realizing the recycling of traditional Chinese medicine resources.

Description

A kind of hollyhock stem and leaf polysaccharide and its preparation method and application

technical field

The invention relates to a traditional Chinese medicine extract, in particular to a preparation method and application of the stem and leaf polysaccharide of marshmallow with immune activity.

Background technique

Hollyhock (Abelmoschus manihot (L.) Medic.) is a one-year to perennial herb of the Malvaceae genus Abelmoschus. It was first recorded in "Jiayou Materia Medica" written by Zhang Yuxi in the Song Dynasty. All have records, and included in the past editions of "The Pharmacopoeia of the People's Republic of China". In the past dynasties, herbal medicine mostly used its flowers as medicine, followed by seeds, and the roots, stems and leaves were rarely used. Hollyhock flower is currently one of the main raw materials of traditional Chinese medicine for the treatment of kidney disease. The market demand is huge, but the stem and leaf resources after harvesting the flowers are largely discarded due to lack of effective utilization methods, resulting in waste of resources. Therefore, carrying out resource utilization research and industrial development of hollyhock stems and leaves to realize turning waste into treasure can not only promote the healthy development of hollyhock resource industry, but also meet the green development requirements advocated by the state.

Studies have shown that the stems and leaves of hollyhock are rich in polysaccharides, and it has been reported in patents that vegetable gum can be prepared using it as the main raw material. Studies have also shown that natural polysaccharides have good immune activity, so it is very necessary to systematically carry out research on the purification and application of polysaccharides from stems and leaves of hollyhock on the basis of existing research, and to screen them for use in immunoenhancing drugs or drugs. Active components or molecules developed for health food to expand the utilization of hollyhock stem and leaf resources and promote the recycling of hollyhock medicinal biological resources.

Contents of the invention

Purpose of the invention: the purpose of the present invention is to provide a polysaccharide of hollyhock stems and leaves with immune enhancing activity, to realize resource utilization and industrial development of hollyhock stems and leaves, and to turn waste into treasure; another purpose of the present invention is to provide the above hollyhock stems Preparation method and application of leaf polysaccharide.

Technical scheme: in order to realize above object, the technical scheme that the present invention takes is:

A kind of hollyhock stem and leaf polysaccharide is isolated from the hollyhock hollyhock stem and leaf, and its weight-average molecular weight is 13821 Daltons (Da), and the polysaccharide is composed of arabinose, galactose, glucose, galacturonic acid, rhamnose and mannose Sugar composition, its molar ratio is arabinose: galactose: glucose: galacturonic acid: rhamnose: mannose=1:0.53:0.12:0.05:0.16:0.03.

The preparation method of a kind of hollyhock stem and leaf polysaccharide of the present invention comprises extraction, ethanol precipitation, macroporous adsorption resin column chromatographic purification, ultrafiltration, DEAE cellulose resin column chromatographic separation and purification in sequence, and the specific separation steps include:

(1) Take the stems and leaves of hollyhock, after crushing, add water according to the solid-liquid weight ratio of 1:8-1:30, heat and extract at 80-100°C for 2-3 times, each time for 1-3 hours, filter, Get the filtrate.

(2) Take the filtrate obtained in step (1), concentrate it to an appropriate volume, add ethanol until the alcohol content is 50% to 80%, let it settle for 24 hours, and separate the solid and liquid to obtain an alcohol precipitation precipitate.

(3) Take the alcohol precipitation precipitate obtained in step (2), dissolve it in water, separate it through macroporous adsorption resin column chromatography, and elute with pure water and 1%-20% ethanol aqueous solution to obtain an eluent.

(4) Take the eluate obtained in step (3), concentrate it properly, and ultrafilter it through an ultrafiltration membrane, take some samples with a molecular weight ranging from 10,000 to 30,000, and concentrate it properly to obtain a refined polysaccharide sample solution.

(5) Take the purified polysaccharide sample solution obtained in step (4), separate it through DEAE cellulose column chromatography, and elute with water and 0.01-0.05% NaCl solution respectively, then elute with 0.1-0.5% NaCl solution, and collect 0.1-0.5 %NaCl eluting solution, desalted by dialysis, concentrated, and freeze-dried to obtain polysaccharides from stems and leaves of hollyhock.

The stems and leaves of hollyhock described in the present invention refer to the stems and leaves left on the ground after harvesting the flowers of hollyhock.

As a preferred solution, the solid-to-liquid ratio described in step (1) is 1:20, the extraction temperature is 100° C., the extraction is performed twice, and the extraction time is 2 hours.

As a preferred version, in step (2), ethanol is added until the alcohol content is 80%, and the solid-liquid separation method adopts centrifugation.

As a preferred solution, the macroporous adsorption resin column chromatography in step (3) adopts AB-8 or D101 resin, and collects 10% ethanol eluate.

As a preferred version, the DEAE cellulose described in step (5) is DEAE-52.

As a preferred solution, in step (5), the refined polysaccharide sample solution obtained in step (4) is taken, separated by DEAE cellulose column chromatography, eluted with water and 0.05% NaCl solution, and then eluted with 0.1% NaCl solution, and collected The solution was eluted with 0.1% NaCl, desalted by dialysis, concentrated, and freeze-dried to obtain polysaccharides from stems and leaves of hollyhock.

A large number of experimental studies in the present invention show that the polysaccharides from the stems and leaves of the marshmallow provided by the present invention can significantly promote the proliferation of spleen lymphocytes in mice, and there is no significant difference between the high-dose group and the positive drug lipopolysaccharide (LPS) group. It shows that the hollyhock stem and leaf polysaccharide provided by the present invention has immune enhancing activity on mice, and can be applied to prepare medicines or health products with immune enhancing effect.

The present invention uses the stem and leaf resources discarded after harvesting hollyhock flowers in the production process of traditional Chinese medicinal materials as raw materials to carry out the extraction, purification and industrialization development of its active polysaccharides, aiming to turn waste into treasure and effectively improve the utilization efficiency of hollyhock medicinal biological resources. Realize the comprehensive utilization of high added value and high social value of traditional Chinese medicine waste.

In the extraction and preparation process of polysaccharides from hollyhock stems and leaves, the present invention adopts an orthogonal test design, takes material-liquid ratio, extraction time, extraction temperature, and extraction times as investigation factors, and optimizes its extraction process parameters, so that the established extraction process can be effective. Improve the extraction efficiency of polysaccharides, reduce the dissolution rate of impurities, and establish an optimal extraction and purification process.

In the preparation and purification process of hollyhock stem and leaf polysaccharide provided by the present invention, the method of activity tracking is adopted, and the medium and low polarity impurities can be effectively removed through macroporous adsorption resin column chromatography purification, which can effectively prevent membrane module pollution during ultrafiltration treatment and improve production efficiency. efficiency. Through the ultrafiltration treatment, the relative molecular weight range of the target polysaccharides in stems and leaves of hollyhock can be effectively controlled, which facilitates the enrichment of active polysaccharides. Through DEAE cellulose resin column chromatography separation and purification, polysaccharide components with relatively fixed structure and uniform molecular weight can be obtained, which provides favorable conditions for its activity evaluation and product quality control.

Beneficial effects: the invention provides the waste hollyhock stem and leaf resources after harvesting as raw materials, through extraction, ethanol precipitation, macroporous adsorption resin column chromatography coupling ultrafiltration, DEAE cellulose resin column chromatography separation and purification, etc. The hollyhock stem and leaf polysaccharide prepared by the process has the characteristics of relatively fixed chemical structure and uniform molecular weight, etc., and has strong immune enhancement activity, especially the activity of lymphoid B cell proliferation, and can be used to prepare medicines or health products with immune enhancement effect .

In addition, the present invention takes the discarded stems and leaves that seriously restrict the development of the medical biological resource industry of hollyhock and have great potential harm to the environment as the object of utilization. On the one hand, the cost of raw materials is low, and the production cost advantage is obvious; Leaf resources can reduce environmental bearing pressure, realize resource recycling, provide support for the sustainable development of hollyhock medicinal biological resources, and have high social and economic benefits.

Description of drawings

Fig. 1 is the determination chromatogram of the molecular weight of polysaccharides of hollyhock stems and leaves provided by the invention;

Fig. 2 is the determination chromatogram of the molecular weight of polysaccharides of the stem and leaf of hollyhock provided by the present invention;

Fig. 3 is a chromatogram for determining the monosaccharide composition of the stem and leaf polysaccharides of hollyhock provided by the present invention.

Detailed ways

The present invention is further illustrated below in conjunction with specific embodiments, should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention, after having read the present invention, those skilled in the art will understand the various equivalent forms of the present invention All modifications fall within the scope defined by the appended claims of this application.

Example 1 Extraction process of hollyhock stem and leaf polysaccharide is preferred

(1) Optimization of single factor for extracting polysaccharides from stems and leaves of hollyhock

Take the hollyhock stem and leaf samples and divide them into several parts. After proper crushing, weigh them, add water with a weight ratio of (1:10, 1:15, 1:20, 1:30, 1:40) of the raw materials, and set the temperature at (60°C , 70°C, 80°C, 90°C, 100°C) extraction temperature, extraction (0.5h, 1h, 2h, 3h, 4h), extraction (1 time, 2 times, 3 times). Filtrate, take the supernatant, and measure the polysaccharide extraction rate by the phenol-sulfuric acid method, and the optimal factor index results are shown in Table 1.

Table 1 Univariate analysis

(2) Orthogonal optimization test for extraction of polysaccharides from stems and leaves of hollyhock

Taking solid-liquid ratio, extraction time, and extraction temperature as the investigation factors, the optimum level of single factor was selected, and the optimal extraction scheme was determined through orthogonal experiments. The results are shown in Table 2.

Table 2 Orthogonal experiment design and results of polysaccharide extraction process

Table 3 Results of analysis of variance of extraction of polysaccharides from stems and leaves of hollyhock

The extreme difference results show that the degree of extraction process affecting polysaccharides from stems and leaves of hollyhock is as follows: extraction temperature > extraction time > ratio of solid to liquid, and the two factors of extraction temperature and extraction time (P<0.05) have a significant effect on the extraction process of polysaccharides from leaves of hollyhock The influence of solid-liquid ratio has no significant difference. In order to save resources, after comprehensive consideration, the best extraction process is A ₃ B ₃ C ₂ , that is, the solid-liquid ratio is 1:20, the extraction temperature is 100°C, and the extraction time is For 2h, extract twice.

Embodiment 2 A kind of separation and purification process of hollyhock stem and leaf polysaccharide, it comprises the steps:

(1) Get 1.0 kg of hollyhock stems and leaves, after crushing, add 20 kg of water according to the optimized extraction method in Example 1, heat and reflux at 100° C. to extract twice, each time for 2 hours, and filter to obtain the filtrate.

(2) Take the filtrate obtained in step (1), concentrate it to 1 L, add ethanol until the alcohol content is 80%, let it settle for 24 hours, and centrifuge to separate the solid and liquid to obtain the alcohol precipitate.

(3) Take the alcohol precipitation precipitate obtained in step (2), add water to dissolve, separate through AB-8 macroporous adsorption resin column chromatography, and elute with 10% ethanol aqueous solution to obtain the eluent.

(4) Take the eluate obtained in step (3), concentrate it appropriately, and ultrafilter it through a hollow cellulose ultrafiltration membrane, take a sample with a molecular weight ranging from 10,000 to 30,000, and concentrate it appropriately to obtain a refined polysaccharide sample solution.

(5) Take the refined polysaccharide sample solution obtained in step (4), separate it through DEAE-52 cellulose column chromatography, and elute it with water and 0.05% NaCl solution respectively, then elute it with 0.1% NaCl solution, and collect the 0.1% NaCl eluate The solution was desalted by dialysis, concentrated, and freeze-dried to obtain 8.7 g of polysaccharides from stems and leaves of hollyhock.

Embodiment 3 A kind of separation and purification process of hollyhock stem and leaf polysaccharide, it comprises the steps:

(1) Get 2.0 kg of hollyhock stems and leaves, after crushing, add 40 kg of water according to the optimized extraction method in Example 1, heat and reflux at 100° C. to extract 3 times, each time for 1 hour, and filter to obtain the filtrate.

(2) Take the filtrate obtained in step (1), concentrate it to 1 L, add ethanol to reach an alcohol content of 70%, let it settle for 24 hours, and filter to obtain an alcohol precipitate.

(3) Take the alcohol precipitation precipitate obtained in step (2), dissolve it in water, separate it through D101 macroporous adsorption resin column chromatography, and elute with water to obtain an eluent.

(4) Take the eluate obtained in step (3), concentrate it properly, and ultrafilter it through an ultrafiltration membrane, take some samples with a molecular weight ranging from 10,000 to 30,000, and concentrate it properly to obtain a refined polysaccharide sample solution.

(5) Take the refined polysaccharide sample solution obtained in step (4), separate it through DEAE-52 cellulose column chromatography, and elute it with water and 0.05% NaCl solution respectively, then elute it with 0.1% NaCl solution, and collect the 0.1% NaCl eluate The solution was desalted by dialysis, concentrated, and freeze-dried to obtain 13.6 g of polysaccharides from stems and leaves of hollyhock.

Example 4 Research on the chemical structure of polysaccharides from stems and leaves of hollyhock

(1) Determination of weight average molecular weight

Chromatographic conditions: Tsk-gel G4000PWXL0 chromatographic column (7.8×300mm), column temperature 30°C, ultrapure water as mobile phase, isocratic elution, volume flow rate 0.5mL/min; injection volume 20μL; ELSD drift tube temperature: 60 °C; gas pressure: 40psi.

Need testing solution: Accurately weigh 10 mg sample of hollyhock stem and leaf polysaccharide prepared in Example 2, dissolve in 2 ml ultrapure water, fully dissolve, centrifuge, take supernatant, and inject.

Determination results: After comparing with dextran molecular weight standard substance, GPC molecular weight distribution software was used to calculate its number average molecular weight, weight average molecular weight and distribution coefficient. The results are shown in Table 4. It can be seen from Table 4 that its weight average molecular weight is 13821 Da, and its distribution coefficient is 1.9654.

Table 4 Determination results of molecular weight of polysaccharides in hollyhock stems and leaves

(2) Determination of monosaccharide composition and ratio

The stem and leaf polysaccharide of hollyhock obtained in Example 2 was derivatized by PMP, and its monosaccharide composition was determined by HPLC, as shown in FIGS. 1 to 3 . The results show that the polysaccharides of hollyhock stems and leaves are composed of rhamnose, mannose, galactose, galacturonic acid, glucose and arabinose, and its molar ratio is arabinose: galactose: glucose: galacturonic acid: rhamnose: Mannose=1:0.53:0.12:0.05:0.16:0.03.

Example 5 Immunological activity test of hollyhock stem and leaf polysaccharide

Experimental method: According to the preparation method of mouse splenic lymphocyte suspension, take the purified splenic lymphocyte suspension, adjust the cell concentration to 1×10 ⁶ ·mL ^-1 , and place in the control well and the experimental well in a 96-well cell culture plate. Add 100 μL of single cell suspension to all the wells, place in 5% CO ₂ , 37°C incubator for continuous culture for 24 hours, add RPMI 1640 medium, LPS (final concentration 5 μg · mL ^-1 ) and 50 μL of hollyhock stem and leaf polysaccharides obtained in Example 2 at different concentrations (final concentrations were 40, 120, 370, 1110 μg·mL ^-1 ), and 3 replicate wells were set up for each group. After 48 hours of continuous culture, add 10 μL of MTT (5 mg/ml) reagent to each well 4 hours before the end of the culture, continue to culture for 4 hours, centrifuge, discard the culture medium, add 100 μL of dimethyl sulfoxide (DMSO) to each well, and shake to dissolve for 20 minutes Afterwards, the enzyme-linked immunosorbent assay instrument measured the OD ₅₇₀ value (absorbance value) at 570 nm, and calculated the immune stimulation index SI. Wherein, SI=OD _{test group} /OD _{control group} .

Statistical analysis: SPSS 16.0 software was used for statistical analysis, and the results were expressed as mean ± SD. The t test was completed using SPSS 19 statistical software. P<0.05 means significant difference; P<0.01 means extremely significant difference.

Determination results: the research results (Table 5) showed that within the concentration range of 40-120 μg·mL ^-1 , polysaccharides from stems and leaves of hollyhock can significantly promote the proliferation of mouse spleen lymphocytes (P<0.05 or P<0.01). increase, the promotion effect is enhanced. And when the concentration of polysaccharides in stems and leaves of hollyhock was 120 μg·mL ^-1 , its enhancing effect was close to that of B lymphocyte mitogen LPS (lipopolysaccharide), but then the concentration increased again, and the promoting effect decreased, showing a two-way dose-effect relationship.

Table 5 Effects of different concentrations of polysaccharides on mouse spleen cell proliferation

Note: "*" means significant difference compared with blank control group (P<0.05); "**" means extremely significant difference compared with blank control group (P<0.01).

The above experimental results show that the hollyhock stem and leaf polysaccharide provided by the present invention can significantly enhance the proliferative activity of spleen lymphocytes, exhibit strong immune enhancement activity, and can be used to prepare medicines and health food with immune enhancement function. And compared with the existing drugs, the hollyhock stem and leaf polysaccharide is derived from natural traditional medicinal and edible plants, which is safer and has no adverse reactions.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (8)

1. a solution of the hollyhock stem and leaf polysaccharide with immune-enhancing effect is characterized in that: the weight average molecular weight of the hollyhock stem and leaf polysaccharide is 13821 Daltons, composed of arabinose, galactose, glucose, galacturonic acid, rhamnos Sugar and mannose composition, its mol ratio is arabinose: galactose: glucose: galacturonic acid: rhamnose: mannose=1:0.53:0.12:0.05:0.16:0.03; The concentration of the polysaccharide solution of hollyhock stems and leaves is 40-120 μg/mL; The preparation method of the stem and leaf polysaccharide of hollyhock comprises extraction, ethanol precipitation, macroporous adsorption resin column chromatography purification, hollow cellulose membrane separation, DEAE cellulose resin column chromatography separation and purification in sequence, and the specific steps include: (1) Take the stems and leaves of hollyhock, after crushing, add water according to the solid-liquid weight ratio of 1:8-1:30, heat and extract at 80-100°C for 2-3 times, each time for 1-3 hours, filter, get the filtrate; (2) Take the filtrate obtained in step (1), concentrate, add ethanol until the volume concentration of ethanol is 50% to 80%, let stand for precipitation, and separate solid and liquid to obtain alcohol precipitation; (3) Take the alcohol precipitation precipitate obtained in step (2), add water to dissolve, separate through macroporous adsorption resin column chromatography, and elute with pure water and 1% to 20% ethanol aqueous solution in turn to obtain an eluent; (4) Take the eluate obtained in step (3), concentrate it, and ultrafilter it through an ultrafiltration membrane, take a sample with a molecular weight ranging from 10,000 to 30,000, and concentrate it to obtain a refined polysaccharide sample solution; (5) Take the purified polysaccharide sample solution obtained in step (4), separate it through DEAE cellulose column chromatography, and elute with water and 0.01-0.05% NaCl solution respectively, then elute with 0.1-0.5% NaCl solution, and collect 0.1-0.5 %NaCl eluting solution, desalted by dialysis, concentrated, and freeze-dried to obtain polysaccharides from stems and leaves of hollyhock. 2. a kind of preparation method of the solution of the hollyhock stem-leaf polysaccharide with immune enhancement described in claim 1 is characterized in that, the preparation method of hollyhock stem-leaf polysaccharide comprises successively extraction, ethanol precipitation, macroporous adsorption resin column chromatography Purification, hollow cellulose membrane separation, DEAE cellulose resin column chromatography separation and purification, the specific steps include: (1) Take the stems and leaves of hollyhock, after crushing, add water according to the solid-liquid weight ratio of 1:8-1:30, heat and extract at 80-100°C for 2-3 times, each time for 1-3 hours, filter, get the filtrate; (2) Take the filtrate obtained in step (1), concentrate, add ethanol until the volume concentration of ethanol is 50% to 80%, let stand for precipitation, and separate solid and liquid to obtain alcohol precipitation; (3) Take the alcohol precipitation precipitate obtained in step (2), add water to dissolve, separate through macroporous adsorption resin column chromatography, and elute with pure water and 1% to 20% ethanol aqueous solution in turn to obtain an eluent; (4) Take the eluate obtained in step (3), concentrate it, and ultrafilter it through an ultrafiltration membrane, take a sample with a molecular weight ranging from 10,000 to 30,000, and concentrate it to obtain a refined polysaccharide sample solution; (5) Take the purified polysaccharide sample solution obtained in step (4), separate it through DEAE cellulose column chromatography, and elute with water and 0.01-0.05% NaCl solution respectively, then elute with 0.1-0.5% NaCl solution, and collect 0.1-0.5 %NaCl eluting solution, desalted by dialysis, concentrated, freeze-dried to obtain polysaccharides from the stems and leaves of hollyhock. 3. the preparation method of the solution of the hollyhock stem and leaf polysaccharide with immune-enhancing effect according to claim 2, characterized in that, the hollyhock stem and leaf is the remaining ground stem and leaf after harvesting the hollyhock flower. 4. the preparation method of the solution of the hollyhock stem and leaf polysaccharide with immune-enhancing effect according to claim 2, is characterized in that, the solid-liquid ratio described in step (1) is 1:20, and extraction temperature is 100 ℃, extracts The number of times is 2, and the extraction time is 2 hours. 5. the preparation method of the solution of the hollyhock stem and leaf polysaccharide with immune-enhancing effect according to claim 2, is characterized in that, in step (2), adding ethanol to ethanol volume concentration is 80%, and solid-liquid separation method adopts centrifugation Law. 6. the preparation method of the solution of the hollyhock stem and leaf polysaccharide with enhancing immunity according to claim 2 is characterized in that, macroporous adsorption resin column chromatography described in step (3) adopts AB-8 type or D101 type resin , and collect the eluate with a volume concentration of 10% ethanol. 7. The preparation method of the solution of the hollyhock stem and leaf polysaccharide with immune-enhancing effect according to claim 2, characterized in that, the DEAE cellulose described in step (5) is DEAE-52. 8. the preparation method of the solution of the hollyhock stem and leaf polysaccharide with enhancing immunity according to claim 2, is characterized in that, in step (5), get the refined polysaccharide sample solution of step (4) gained, through DEAE cellulose column Chromatographic separation, eluting with water, 0.05% NaCl solution, and 0.1% NaCl solution, collecting the 0.1% NaCl eluting solution, desalting by dialysis, concentrating, and freeze-drying to obtain the stem and leaf polysaccharide of hollyhock.