CN111675770A

CN111675770A - Preparation method and application of orostachys wolfsii polysaccharide

Info

Publication number: CN111675770A
Application number: CN202010681171.0A
Authority: CN
Inventors: 朴炫春; 金美玉; 姜君; 廉美兰
Original assignee: Yanbian University
Current assignee: Yanbian University
Priority date: 2020-07-15
Filing date: 2020-07-15
Publication date: 2020-09-18
Anticipated expiration: 2040-07-15
Also published as: CN111675770B

Abstract

The invention discloses a preparation method and application of orostachys wolfsii polysaccharide, and belongs to the technical field of biology. The invention discloses a preparation method of a radix cynanchi bungei polysaccharide, which takes callus of the radix cynanchi bungei cultured by a bioreactor as a main raw material, and utilizes macroporous adsorption resin to purify polysaccharide of a crude extract of the radix cynanchi bungei callus polysaccharide, so that the preparation method is simple to operate, high in efficiency and low in cost. The prepared artocarpus heterophyllus polysaccharide has antibacterial, anti-inflammatory and anticancer activities, and can be prepared into antibacterial drugs, anti-inflammatory drugs and anticancer drugs.

Description

Preparation method and application of orostachys wolfsii polysaccharide

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method and application of artocarpus heterophyllus polysaccharide.

Background

Radix seu folium Heteropanacis chinensis (Orostachys caralatinosus A. bor), also known as herba Orostachyos chinensis, is a biennial or perennial herb of genus Orostachys of family Crassulaceae. The rhizoma kaempferiae mostly grows in low mountain land and is mainly distributed in inner Mongolia, Liaoning, Jilin, Heilongjiang and other places in China. The marchantia woad has high medicinal value, such as bacteriostasis, anticancer, antivirus, antioxidation, blood sugar reduction, blood fat reduction and the like, and is closely related to various active substances such as polysaccharides, flavonoids, phenols and the like contained in other plants. At present, with the increase of the demand of people on the orostachys wolfsii, the phenomenon of excessive collection is serious, so that wild resources are reduced year by year, and the development and application of the orostachys wolfsii are limited due to unstable components of artificial cultivation and the like. Therefore, the rapid and mass culture of the orostachys tripartita callus by using the bioreactor is one of effective ways for relieving the resource problem of the orostachys tripartita, and has potential application value as a new plant material source.

As a natural active substance extracted from plants, a large number of researches prove that the plant polysaccharide has biological activities of bacteriostasis, cancer resistance, inflammation resistance and the like. The method has been a research hotspot of natural pharmacology and health food science, is an effective way for searching natural medicines without toxic and side effects, and is also one of the research projects of health food functional factors. Because of its wide source, plant polysaccharides play an important role in the medical field. However, in the process of applying the plant polysaccharide, since the structure of the plant body is complex, the bioactive compounds have various kinds in the plant body, and the functions of different compounds are different, it is difficult to implement precise and efficient application when the plant polysaccharide is used, thereby causing waste of resources, it is necessary to separate and purify plant-derived bioactive substances having different functions. The macroporous adsorbent resin is a novel organic polymer adsorbent developed in the 60 th century, and is widely applied to purification and separation of bioactive compounds of natural resources at present, especially flavonoid, polyphenol and polysaccharide compounds, because the macroporous adsorbent resin has stable physicochemical properties, is insoluble in acid, alkali and organic solvents, has good selectivity to organic matters, is not influenced by the existence of inorganic salts, strong ions and low molecular compounds, and can adsorb the solvents in water and organic solvents to swell.

Efficient extraction of natural products is considered to be an important area of green technology in the pharmaceutical and biochemical industries, where the purification of biologically active compounds from crude plant extracts is of particular interest for the utilization of natural products, whereas the purification of desired compounds from extracts containing large amounts of compounds is a very challenging task. At present, various compound purification methods, including supercritical fluid extraction, high-speed counter-current chromatography, two aqueous phase extraction, high performance liquid chromatography, etc., have been developed, but these methods have the disadvantages of complexity, time consumption, high maintenance cost, environmental harm, unsuitability for industrial production, etc.

Therefore, the preparation method of the artocarpus heterophyllus polysaccharide with simple operation, high efficiency and low cost and the application thereof are problems to be solved by the technical personnel in the field.

Disclosure of Invention

In view of this, the invention provides a preparation method and application of orostachys wolfsii polysaccharide.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of the polysaccharide of the radix ranunculi triphylli comprises the following specific steps:

(1) culturing herba Orostachyos callus with bioreactor, harvesting, washing to remove surface culture medium, oven drying at 45 deg.C to constant weight, and collecting dry callus;

(2) preparing crude extract of orostachys tripartita callus polysaccharide

Soaking the callus dry product collected in the step (1) in 70% ethanol with a material-liquid ratio of 1:15, condensing and refluxing for 1h at 60 ℃, performing suction filtration, and collecting filter residue; condensing and refluxing the filter residue again, and repeating for 3 times; mixing the filter residues, volatilizing ethanol on the surface of the filter residues, extracting with 100 deg.C hot water for 1h for three times, and mixing the filtrates after each filtration; concentrating the filtrate at 45 deg.C under reduced pressure, adding ethanol for precipitation, centrifuging, and freeze drying to obtain radix Stellariae Eschatae callus polysaccharide crude extract;

(3) purification of polysaccharide of wakame kadsura root by macroporous adsorption resin method

Taking the pretreated AB-8 resin to be loaded on a column according to the feeding concentration1.6mg·mL^-1The feeding volume is 3BV, the feeding flow rate is 1 BV.h^-1Eluent concentration of 65%, elution volume of 3BV, elution flow rate of 2 BV.h^-1Purifying under the purification conditions of (1), and collecting effluent liquid;

(4) and (4) concentrating the effluent liquid collected in the step (3) under reduced pressure, and drying at 40 ℃ to obtain the purified fimbriate rupestris herb polysaccharide.

Further, the specific steps of culturing the orostachys wolfsii callus by using the bioreactor in the step (1) are as follows: add 4L of medium to a 5L balloon airlift bioreactor: MS + BA 3.5 mg.L^-1+NAA 0.1mg·L^-1+ sucrose 30 g.L^-1pH 5.8; access 15 g.L^-1The callus of the orostachys wolfsii; the aeration rate of the reactor was adjusted to 300 mL/min^-1At a temperature of 25 ℃ and with light irradiation of 30. mu. mol. m per day^-2·s^-1Culturing for 16h under the condition, harvesting callus after 25d, fully washing with tap water to remove surface culture medium, drying in a constant temperature oven at 45 ℃ to constant weight, and collecting dry callus.

Further, the specific steps for pretreating the AB-8 resin in the step (3) are as follows: soaking macroporous adsorbent resin AB-8 in 95% ethanol solution for 24 hr, loading onto column, washing with 95% ethanol, washing with distilled water until no alcohol smell, soaking resin in 4% hydrochloric acid solution for 4 hr, and washing with distilled water until pH of effluent is neutral; adding 4% sodium hydroxide solution to soak the resin for 4h, and washing the column with distilled water until the effluent liquid pH is neutral.

Further, the application of the orostachys wolfsii polysaccharide in preparing antibacterial drugs.

Further, the application of the polysaccharide of the orostachys wolfsii var.

Further, the application of the polysaccharide of the radix ranunculi triphylli in preparing anti-cancer drugs.

According to the technical scheme, compared with the prior art, the preparation method and the application of the orostachys wolfsii polysaccharide are disclosed and provided, the orostachys wolfsii callus cultured by the bioreactor is used as a main raw material, and the macroporous adsorption resin is used for purifying polysaccharide of a crude extract of the orostachys wolfsii callus polysaccharide, so that the operation is simple, the efficiency is high, and the cost is low; the prepared artocarpus heterophyllus polysaccharide has antibacterial, anti-inflammatory and anticancer activities, and can be prepared into antibacterial drugs, anti-inflammatory drugs and anticancer drugs.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a drawing showing the bacteriostatic spectrum of total polysaccharide (OTP) after the callus of the orostachys wolfsii of the invention is purified;

wherein Ec: e.coli; pa: pseudomonas aeruginosa; sa: staphylococcus aureus bacteria; bs: b, bacillus subtilis; OTP: total polysaccharides; control: DMSO;

FIG. 2 is a graph showing the effect of total polysaccharide (OTP) on the activity of RAW 264.7 cells after purification according to the present invention;

wherein ns indicates no significant difference compared to the control;

FIG. 3 is a graph showing the effect of total polysaccharide (OTP) on nitric oxide production after purification according to the present invention;

wherein # is a comparison result between the LPS only group and the control group, and the difference is significant at the 0.05 level; indicates significant differences at 0.05, 0.01, 0.001 levels compared to LPS only group, respectively; adding 3.13 mu g/mL^-1The test data of the OTP group is not different from that of the LPS group only;

FIG. 4 is a graph showing the effect of total polysaccharides (OTP) on AGS cell activity after callus purification according to the present invention;

FIG. 5 is a graph showing the effect of total polysaccharides (OTP) on HCT116 cell activity after callus purification according to the present invention;

FIG. 6 is a graph showing the effect of total polysaccharides (OTP) on the activity of MC38 cells after the purification of callus according to the present invention;

FIG. 7 is a graph showing the effect of total polysaccharide (OTP) on HepG2 cell activity after callus purification according to the present invention;

FIG. 8 is a graph showing the effect of total polysaccharide (OTP) on Hela cell activity after the callus of the present invention was purified.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

A method for preparing radix Stellariae mediae polysaccharide comprises the following steps of taking radix Stellariae mediae callus cultured by a bioreactor as a main raw material, and purifying polysaccharide of a crude extract of the radix Stellariae mediae callus polysaccharide by using macroporous adsorption resin:

(1) culture of orostachys wolfsii callus bioreactor

4L of culture medium (MS + BA 3.5 mg. L) was added to a 5L balloon-type airlift bioreactor^-1+NAA 0.1mg·L^-1+ sucrose 30 g.L^-1pH 5.8), 15 g.L was added^-1The callus of the orostachys wolfsii; the aeration rate of the reactor was adjusted to 300 mL/min^-1At a temperature of 25 ℃ and daily illumination (30. mu. mol. m)^-2·s^-1) Culturing for 16h, harvesting callus after 25d, washing with tap water to remove surface culture medium, drying in a constant temperature oven (45 deg.C) to constant weight, and collecting dry callus.

(2) Preparation of radix Stellariae Evolvuli callus polysaccharide crude extract

Soaking the callus dry product collected in the step (1) in 70% ethanol (material-liquid ratio is 1:15), condensing and refluxing for 1h at 60 ℃, filtering, and collecting filter residue; condensing and refluxing the filter residue again, and repeating for 3 times; mixing the filter residues, volatilizing ethanol on the surface of the filter residues, extracting with 100 deg.C hot water for 1h for three times, and mixing the filtrates after each filtration; concentrating the filtrate at 45 deg.C under reduced pressure, adding ethanol for precipitation, centrifuging, and freeze drying to obtain radix Stellariae Eschatae callus polysaccharide crude extract;

Preprocessing macroporous adsorption resin AB-8: soaking macroporous adsorbent resin AB-8 in 95% ethanol solution for 24 hr, loading onto column, washing with 95% ethanol, washing with distilled water until no alcohol smell, soaking resin in 4% hydrochloric acid solution for 4 hr, and washing with distilled water until pH of effluent is neutral; adding 4% sodium hydroxide solution to soak the resin for 4h, and washing the column with distilled water until the effluent liquid pH is neutral;

② the pretreated AB-8 resin was loaded onto the column at a feed concentration of 1.6 mg. multidot.mL^-1The feeding volume is 3BV, the feeding flow rate is 1 BV.h^-1Eluent concentration of 65%, elution volume of 3BV, elution flow rate of 2 BV.h^-1Purifying under the purification conditions of (1), and collecting effluent liquid;

(4) and (4) concentrating the effluent liquid collected in the step (3) under reduced pressure, and drying at 40 ℃ to obtain purified radix ranunculi triphylli polysaccharide (OTP) with the purity of 96.4%.

Example 2 bacteriostatic Activity of Orostachys jatamansi polysaccharide

The test method comprises the following steps: staphylococcus aureus, pseudomonas aeruginosa, escherichia coli and bacillus subtilis are selected as tested strains, and the antibacterial activity of the purified polysaccharide is proved by measuring the diameter of an inhibition zone and the Minimum Inhibitory Concentration (MIC).

(1) The bacteriostasis zone: and measuring the diameter of the bacteriostatic circle by adopting a filter paper sheet method.

The method specifically comprises the following steps: dissolving purified radix Stephaniae Cepharanthae polysaccharide with dimethyl sulfoxide (DMSO) to obtain final concentration of 128 mg/mL^-1. A6 mm diameter piece of filter paper (CT 0998B; Oxoid, UK) was soaked in the purified polysaccharide solution and DMSO (control), respectively, to make the piece of filter paper imbibed with the solution (about 2 h). Respectively diluting bacterial suspensions of staphylococcus aureus, pseudomonas aeruginosa, escherichia coli and bacillus subtilis to 10⁷CFU·mL^-1Uniformly spreading 300 μ L of the bacterial liquid on beef extract peptone medium (beef extract 3 g.L)^-1+ peptone 10 g.L^-1+ sodium chloride 10 g.L^-1+ agar 10-20 g, pH 7.0-7.2) (the diameter of the flat plate is 9cm), and then placing the soaked filter paper sheet. The plates were incubated at 37 ℃ for 12h, the results are shown in FIG. 1 (two replicates for two OTP); the diameter of the zone of inhibition was measured with a vernier caliper and the results are shown in Table 1.

TABLE 1 bacteriostatic zone diameter of total polysaccharide (OTP) after purification of mace callus

Note: values are mean ± standard deviation (n ═ 3); -means failure to determine.

As can be seen from the bacteriostatic spectrum of the purified polysaccharide, OTP has no bacteriostatic effect on Escherichia coli (figure 1A), but has better bacteriostatic effects on Pseudomonas aeruginosa (figure 1B), Staphylococcus aureus (figure 1C) and Bacillus subtilis (figure 1D), and the bacteriostatic diameters are respectively 8.5mm, 11.2mm and 13mm (Table 1).

(2) The minimum bacteriostatic concentration is as follows: the TTC method is used for determining the minimum inhibitory concentration. 100. mu.L of bacterial suspension (10)⁷CFU·mL^-1) Adding into 96-well plate, adding 100 μ L purified polysaccharide solution, and adjusting the final concentration of sample to 64, 32, 16, 8, 4, 2, 1 mg/mL^-1DMSO is control. Culturing at 37 deg.C for 24 hr, adding 0.2% 2, 3, 5-triphenyltetrazolium chloride (TTC) 20 μ L, culturing at 37 deg.C in dark for 4 hr, and observing the color of the reaction solution to obtain colorless extract with Minimum Inhibitory Concentration (MIC).

As is clear from FIG. 1 and the results in Table 1, the purified polysaccharide had the best effect on the production of Bacillus subtilis; therefore, the Minimum Inhibitory Concentration (MIC) of purified polysaccharide against Bacillus subtilis was determined by TTC method. TTC is a lipid-soluble light-sensitive complex that is colorless in its oxidation state and, following reaction with succinate dehydrogenase in living bacteria, is reduced to form red insoluble formazan. Therefore, the viability of the bacteria can be identified according to the shade of the color. The test adopts a two-fold dilution method, and is determined according to the TTC color development principle, and finds that the red color of the mixed solution gradually becomes lighter along with the increase of the concentration of the sample, and when the concentration of the purified polysaccharide solution is 8 mg/mL^-1In red colorThe precipitate disappeared completely and the results are shown in Table 2. Therefore, 8 mg. mL^-1To purify the MIC of polysaccharide to Bacillus subtilis.

TABLE 2 minimum inhibitory concentration assay results for total polysaccharide (OTP) after purification

Note: +++: deep red; ++: peach-red; +: pink; -: it is colorless.

Example 3 anti-inflammatory Activity of Falsestarwort Stellate polysaccharide

Assay method RAW 264.7 cells were seeded in DMEM, and when the cells grew to log phase, they were trypsinized and collected at 5 × 10 per well³Inoculating into 96-well plate, culturing for 12 hr, adding extract, measuring cell activity by MTT method to evaluate cytotoxicity, and screening suitable concentration to measure Nitric Oxide (NO) release amount, RAW 264.7 cell per well 2.5 × 10⁵Inoculating into 48-well plate, purifying polysaccharide, and treating with lipopolysaccharide (LPS, 0.1. mu.g. L)^-1) And (4) treating, collecting supernatant, and measuring the release amount of NO.

(1) cytotoxicity-Raw 264.7 cells in log phase were grown at 5 × 10 per well³The cells were inoculated into a 96-well plate (containing 0.1mL of DMEM medium) and cultured at 37 ℃ for 12 hours. Adding purified polysaccharide extract to give final concentrations of 0, 25, 50, 100, 200 μ g/mL^-1(ii) a The control group, which was not treated with the extract, was supplemented with an equal amount of DMEM medium. After 24 hours of treatment, the mixture was discarded, and 100. mu.L of MTT (500 ng. multidot.mL) was added to each well^-1) Culturing at 37 deg.C for 3 hr, discarding MTT, adding 150 μ L DMSO, 300 r.min^-1Followed by shaking for 5min, and determining OD at 550nm, the result is shown in FIG. 2. As can be seen from FIG. 2, the concentration of the surfactant is 0 to 200. mu.g/mL^-1In the range, the activity of RAW 264.7 cells is not significantly reduced, which indicates that the purified polysaccharide in the concentration range has no cytotoxicity and can be used as the test concentration for anti-inflammatory research.

(2) NO content in cells of Raw 264.7, 2.5 × 10 per well, in log phase of growth⁵Inoculating to 48-well plate (containing 0.3mL of DMEM medium) at 37 deg.CCulturing for 12 h. Adding purified polysaccharide extract to give final concentrations of 0, 3.13, 6.25, 12.5, and 25 μ g/mL^-1The control group was treated with the same amount of DMEM medium as the control group without the extract, and 1% LPS (0.1. mu.g/mL) was added after 1 hour of treatment^-1) And treating for 24h, and collecting supernatant. The supernatant was mixed with Griess reagent at a ratio of 1:1 by Griess reduction, OD was measured at 550nm, and the NO content was calculated, the result is shown in FIG. 3. As can be seen from FIG. 3, after the treatment of OTP, the amount of NO released from LPS-stimulated cells was reduced, and the lowest concentration at which OTP had a significant inhibitory effect on NO release was 6.25. mu.g/mL^-1It shows that OTP has better anti-inflammatory effect.

Example 4 anticancer Activity of Falsestarwort Stellate Pogostemon polysaccharide

The test method comprises the following steps: the cell lines used in this experiment were provided by the national university of south China university of Central university of Heart fire, West's national center for Chronic disease laboratory. Respectively inoculating human gastric cancer cell line (AGS), human colon cancer cell line (HCT116), human liver cancer cell line (HepG2), human cervical cancer cell line (Hela), and mouse colon cancer cell line (MC38) to a culture medium containing 10% (V.V)^-1) Fetal bovine serum (FBS, Thermoscientific, USA) and 1% (V.V)^-1) Antibiotics (penicillin and streptomycin, 100U. mL)^-1Thermoscientific, USA) in DMEM (Gibco, USA) at 37 deg.C in a cell incubator (95% relative humidity, CO)₂Content 5%), when the cells have grown to log phase, Trypsin (Trypsin-EDTA, Thermo Scientific, USA) digests and collects the cells cancer cells at 5 × 10 per well³The cells were seeded in a 96-well plate (0.1 mL of DMEM per well), cultured for 12 hours, and then treated with OTP, and the cancer cell activity was measured by the MTT method.

The determination method comprises subjecting cancer cells in logarithmic growth phase to 5 × 10 per well³Cells were seeded in 96-well plates (containing 0.1ml of MEM culture medium) and cultured at 37 ℃ (95% relative humidity, CO)₂Content 5%) 12 h. Adding purified polysaccharide extract to final concentrations of 0, 10, 20, 40, 80, 160, and 320 μ g/mL^-1The control group was supplemented with the same amount of DMEM medium, and the control group was blank without the extract. After 24, 48 and 72 hours of treatment, the mixture was discarded, and 100. mu.L of 3- (4, 5-bis) was added to each wellMethylthiazole-2) -2, 5-diphenyltetrazolium bromide salt (MTT, 500 ng. mL)^-1) Culturing at 37 deg.C for 3 hr, discarding MTT, adding 150 μ L DMSO, 300 r.min^-1The cell activity was calculated by measuring the OD at 550nm with shaking for 5 min.

The anticancer activity of the purified polysaccharides against AGS, HCT116, MC38, HepG2 and HeLa cells was evaluated by measurement of cell activity using the MTT method, and the results are shown in FIGS. 4 to 8. As can be seen from FIGS. 4 to 8, the effect of purified polysaccharide on the activity of cancer cell lines was significant, and the concentration was 10. mu.g/mL^-1It has certain inhibiting effect on AGS, HCT116, MC38, HepG2 and Hela cells, and the activity of the cells is reduced along with the increase of the concentration and the treatment time.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A preparation method of the polysaccharide of the marchantia wolvescens is characterized by comprising the following specific steps:

(2) preparing crude extract of orostachys tripartita callus polysaccharide

Loading the pretreated AB-8 resin on a column according to the feeding concentration of 1.6 mg/mL^-1The feeding volume is 3BV, the feeding flow rate is 1 BV.h^-1Eluent concentration of 65%, elution volume of 3BV, elution flow rate of 2 BV.h^-1Purifying under the purification conditions of (1), and collecting effluent liquid;

2. The method for preparing the polysaccharide of the orostachys tripartita according to claim 1, wherein the step (1) of culturing the callus of the orostachys tripartita by using the bioreactor comprises the following steps: add 4L of medium to a 5L balloon airlift bioreactor: MS + BA 3.5 mg.L^-1+NAA 0.1mg·L^-1+ sucrose 30 g.L^-1pH 5.8; access 15 g.L^-1The callus of the orostachys wolfsii; the aeration rate of the reactor was adjusted to 300 mL/min^-1At a temperature of 25 ℃ and with light irradiation of 30. mu. mol. m per day^-2·s^-1Culturing for 16h under the condition, harvesting callus after 25d, fully washing with tap water to remove surface culture medium, drying in a constant temperature oven at 45 ℃ to constant weight, and collecting dry callus.

3. The method for preparing the polysaccharide of the marchantia tripartita according to claim 1, wherein the pre-treating AB-8 resin in step (3) comprises the following steps: soaking macroporous adsorbent resin AB-8 in 95% ethanol solution for 24 hr, loading onto column, washing with 95% ethanol, washing with distilled water until no alcohol smell, soaking resin in 4% hydrochloric acid solution for 4 hr, and washing with distilled water until pH of effluent is neutral; adding 4% sodium hydroxide solution to soak the resin for 4h, and washing the column with distilled water until the effluent liquid pH is neutral.

4. Use of the artocarpus heterophyllus polysaccharide of any one of claims 1 to 3 for the preparation of a bacteriostatic medicament.

5. Use of the marchantia kawachii polysaccharides according to any one of claims 1-3 for the preparation of anti-inflammatory drugs.

6. Use of the marchantia kawakamii polysaccharides of any one of claims 1-3 in the preparation of anti-cancer drugs.