CN113667032A

CN113667032A - Selenized amomum villosum polysaccharide and preparation method and application thereof

Info

Publication number: CN113667032A
Application number: CN202111066126.5A
Authority: CN
Inventors: 赵志敏; 杨得坡; 周洋; 钟镜堂
Original assignee: Guangdong Yayishan Rare Medicinal Materials Planting Co ltd; Sun Yat Sen University
Current assignee: Guangdong Yayishan Rare Medicinal Materials Planting Co ltd; Sun Yat Sen University
Priority date: 2021-09-13
Filing date: 2021-09-13
Publication date: 2021-11-19
Anticipated expiration: 2041-09-13
Also published as: CN113667032B

Abstract

The invention discloses a selenized amomum villosum polysaccharide and a preparation method and application thereof, wherein the selenized amomum villosum polysaccharide is obtained by selenizing and modifying amomum villosum crude polysaccharide AVP. The selenized polysaccharide can effectively supplement organic selenium, AVP has no anti-helicobacter pylori and anti-tumor activity, compared with AVP before selenization, the MIC value of SAVP to helicobacter pylori after selenization modification is 250 mug/mL, and IC of MDA-MB-231 cell₅₀The polysaccharide has 375 +/-32.5 mu g/mL, generates new activity, has unexpected effect, and is expected to further expand the application range of the selenized amomum villosum polysaccharide.

Description

Selenized amomum villosum polysaccharide and preparation method and application thereof

Technical Field

The invention relates to the field of polysaccharides, in particular to a selenized amomum villosum polysaccharide and a preparation method and application thereof.

Background

Polysaccharides are high molecular compounds formed by connecting at least 10 monosaccharides by glycosidic bonds, and play an important role in life activities. The polysaccharide has wide biological activity, such as antioxidant activity, antitumor activity, immunoregulation activity and the like, and becomes a key point of domestic and foreign research due to the characteristics of good activity and low toxicity. The activity of polysaccharides is directly influenced by structure, and there are many factors that make the polysaccharide less active than the application requirements or inactive. At present, chemical modification methods are generally adopted at home and abroad to enhance the biological activity of polysaccharide, and among the methods, selenylation modification is more common.

Selenium (Se) is an essential trace element in the human body, which cannot be synthesized by the human body and can only be obtained by food. Selenium has anti-tumor, antioxidant and immunoregulatory effects in human body. Inorganic selenium has the characteristics of short retention time, low bioavailability, high toxicity and the like in a human body. How to effectively supplement selenium is a problem to be solved.

As a medicinal material with higher economic value and medicinal value, the amomum villosum seleno polysaccharide has deep research, so that the application value of the amomum villosum polysaccharide is improved. The laboratory carries out separation and purification, structure analysis and activity research on the amomum villosum polysaccharide in the early stage, and no report is found on the research on the amomum villosum selenylation polysaccharide. CN112920287A and CN112794925A disclose a preparation method of the crude polysaccharide AVP of the amomum villosum, and simultaneously further separate and purify to obtain the polysaccharide AVPG-1 and AVPG-2 of the amomum villosum. The amomum villosum polysaccharide AVPG-1 can remarkably promote macrophage RAW 264.7 to generate NO and improve the cell activity of the macrophage RAW 264.7 within the concentration range of 50-200 mug/mL. The polysaccharide AVPG-2 of the amomum villosum can obviously promote macrophage RAW 264.7 to generate NO and secrete cell factors IL-6 and TNF-αAnd the cell activity and phagocytic capacity of macrophage RAW 264.7 are improved, and the preparation method is expected to be used for preparing immunoregulation health-care products or medicines. The polysaccharide has a single function.

Disclosure of Invention

The invention aims to overcome at least one defect of the prior art and provides a selenized amomum villosum polysaccharide and a preparation method and application thereof.

The technical scheme adopted by the invention is as follows:

the inorganic selenium and the polysaccharide are combined by a chemical synthesis method, so that the defects can be overcome, the effect of 1+1 > 2 can be exerted, the biological activity of the inorganic selenium and the polysaccharide is improved, and the absorption and utilization of the inorganic selenium and the polysaccharide by the body are facilitated.

In a first aspect of the present invention, there is provided:

selenizing fructus Amomi polysaccharide is prepared from coarse polysaccharide AVP of fructus Amomi by selenizing modification.

In some examples, the fructus amomi crude polysaccharide AVP is obtained by degreasing fructus amomi powder, then carrying out water extraction, and then further removing protein in the fructus amomi powder, and mainly contains fructus amomi polysaccharide AVPG-1 and fructus amomi polysaccharide AVPG-2.

In some examples, the preparation method of the crude polysaccharide AVP of fructus amomi

In some examples, the preparation method of the amomum villosum selenizing polysaccharide comprises the following steps:

s1) adding nitric acid solution with concentration not more than 1% into the crude polysaccharide AVP of the villous amomum fruit, and fully stirring for reaction to obtain reaction liquid A;

s2) adding BaCl into the reaction solution A₂Fully reacting with sodium selenite at 50-90 ℃ to obtain reaction liquid B;

s3) precipitating and removing Ba in the reaction liquid B²⁺Ionizing to obtain clarified liquid;

s4) adjusting the pH of the clarified liquid to neutrality, concentrating, dialyzing and purifying to obtain purified polysaccharide liquid;

s5) separating and drying the polysaccharide in the purified polysaccharide solution to obtain the polysaccharide of the amomum villosum selenide.

In some examples, the mass ratio of the amomum villosum crude polysaccharide AVP to the nitric acid solution is 1: (50-150).

In some examples, the nitric acid solution has a concentration of 0.4 to 1.0%.

In some examples, the nitric acid solution has a concentration of 0.6%.

In some examples, the sodium selenite is added in an amount of 1-3 times of the mass of the AVP.

In some examples, the polysaccharide is precipitated by adding anhydrous ethanol to the purified polysaccharide solution.

In some examples, the concentration of the nitric acid solution is 0.6%, and the mass ratio of the amomum villosum crude polysaccharide AVP to the nitric acid solution is 1: 50.

in some examples, Ba is made by adding a water soluble sulfate salt²⁺And (4) ion precipitation.

In some examples, the water soluble sulfate is selected from at least one of sodium sulfate, potassium sulfate, ammonium sulfate.

In a second aspect of the present invention, there is provided:

a composition comprising amomum villosum selenides polysaccharides according to the first aspect of the invention.

In a third aspect of the present invention, there is provided:

the invention relates to the application of the selenized amomum villosum polysaccharide in preparing the anti-helicobacter pylori composition.

In a fourth aspect of the present invention, there is provided:

the application of the amomum villosum selenylation polysaccharide in the first aspect of the invention in preparing an anti-tumor composition or a tumor adjuvant therapy composition.

The invention has the beneficial effects that:

the amomum villosum selenylation polysaccharide provided by the invention can effectively supplement organic selenium.

The polysaccharide of the amomum villosum selenylation, AVP, of some examples of the invention has no anti-helicobacter pylori and anti-tumor activity, compared with the AVP before selenylation, the MIC value of SAVP to helicobacter pylori after selenylation modification is 250 mug/mL, and the IC of MDA-MB-231 cells₅₀The polysaccharide has 375 +/-32.5 mu g/mL, generates new activity, has unexpected effect, and is expected to further expand the application range of the selenized amomum villosum polysaccharide.

Drawings

FIG. 1 is a relative molecular mass distribution chromatogram of High Performance Gel Permeation Chromatography (HPGPC) measurements of AVP (A) and SAVP (B);

FIG. 2 is a Fourier transform Infrared Spectroscopy (FT-IR) plot of AVP (A) and SAVP (B).

Detailed Description

In a first aspect of the present invention, there is provided:

The concentration of nitric acid can be adjusted accordingly to the extent of the reaction to avoid over-oxidation. In some examples, the nitric acid solution has a concentration of 0.4 to 1.0%.

In some examples, the nitric acid solution has a concentration of 0.6%. The polysaccharide has the required oxidation capacity under the concentration, and is more beneficial to preparing the selenized amomum villosum polysaccharide.

In some examples, the sodium selenite is added in an amount of 1-3 times of the mass of the AVP. The sodium selenite is added excessively, so that all reaction sites can fully react, and the preparation method is more favorable for obtaining the amomum villosum selenides polysaccharide.

The water-soluble sulfate has no special requirements, and does not cause adverse effect on the product quality. In some examples, the water soluble sulfate is selected from at least one of sodium sulfate, potassium sulfate, ammonium sulfate.

In a second aspect of the present invention, there is provided:

In a third aspect of the present invention, there is provided:

When in use, the selenized amomum villosum polysaccharide is taken in an oral mode. Certainly, in order to better ensure the drug effect of the selenized amomum villosum polysaccharide, the corresponding composition can be prepared by the selenized amomum villosum polysaccharide and acceptable pharmaceutical excipients according to a conventional method.

In a fourth aspect of the present invention, there is provided:

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The reagents, equipment and methods employed in the present invention are all reagents, equipment and methods conventionally available in the art and conventionally used methods, unless otherwise specified.

For convenience of comparison, the polysaccharide AVP of fructus Amomi is prepared as follows.

Preparing the amomum villosum polysaccharide AVP:

s1) shelling the amomum villosum seeds, collecting seed groups, crushing, collecting 200 g of crushed amomum villosum powder, adding 4L of 95% ethanol, extracting at 80 ℃ for 3 h, repeating the operation for three times, and finally drying the amomum villosum powder to obtain the degreased powder of the amomum villosum; adding water into the defatted powder of YANGCHUNSHA, adding 4L water into 200 g of defatted powder of YANGCHUNSHA, extracting at 95 deg.C for 3 hr, repeating the operation for 3 times, and concentrating the water extractive solution at 60 deg.C under reduced pressure to obtain concentrated solution;

s2) adding anhydrous ethanol with the volume 4 times that of the concentrated solution, standing for 12 h at 4 ℃, precipitating polysaccharide, then centrifuging and collecting the precipitate, wherein the centrifugal speed is 5000 rpm, and the centrifugal time is 5 min, and dissolving the precipitate in distilled water to obtain a polysaccharide solution;

s3) adding a 4-fold volume of Sevage reagent to the polysaccharide solution, the Sevage reagent being chloroform: the solvent with n-butyl alcohol =4:1 (volume ratio) is centrifuged after violent shaking, the centrifugation speed is 5000 rpm, the centrifugation time is 10 min, and the supernatant is taken and repeated until the protein is completely removed; the deproteinized polysaccharide solution was then concentrated at 50 ℃ until free of organic reagent flavor, followed by lyophilization to yield the crude Amomum villosum polysaccharide AVP.

Example 1:

preparation of Actinolitum villosum selenylation polysaccharide SAVP

Weighing 0.5 g AVP, placing in 50 mL round bottom flask, adding 50 mL dilute nitric acid with concentration of 0.6%, magnetically stirring for 30 min, adding 1.0 g BaCl₂And 1.0 g of sodium selenite, and the mixture is subjected to oil bath reaction at 70 ℃ for 10 hours. After the reaction was complete, a slight excess of Na was added₂SO₄Removal of catalyst BaCl₂Centrifuging to remove precipitate, adding appropriate amount of Na into the clarified solution₂SO₄No precipitate was produced indicating BaCl₂Is completely removed. Adding a proper amount of Na₂CO₃The pH of the solution was adjusted to 7, and the reaction solution was concentrated to about 10 mL by rotary evaporation. Dialyzing the concentrated solution with dialysis bag (3500 Da), changing water every 8 h until the solution outside the dialysis bag does not turn red after dropping Vc, adding 4 times of anhydrous ethanol to precipitate polysaccharide, and freeze drying the precipitate to obtain selenizing derivative of AVP (SAVP) for use.

II) determination of relative molecular mass of fructus amomi selengensis polysaccharide SAVP

The relative molecular masses were determined by High Performance Gel Permeation Chromatography (HPGPC).

AVP and SAVP are prepared into 2.0 mg/mL solution, and the solution is filtered through 0.22 μm filter membrane and injected with 20 μ L sample amount, each sample lasting for 30 min. The chromatographic conditions are as follows: the model of the chromatographic column is PolySep-GFC-P4000, the mobile phase is distilled water, the flow rate is 1.0 mL/min, the column temperature is 35 ℃, the detector is an evaporative light detector (ELSD-LT II) of Shimadzu, the detector temperature is 60 ℃, the Gain value is 10, and the retention time is recorded. The purity of AVP and SAVP was identified from the sample peak shape and the change in relative molecular mass was identified from the change in retention time.

Elution chromatograms for AVP and SAVP are shown in fig. 1A and 1B, respectively. As can be seen from the figure, the AVP contains more components and the retention time is in the range of 3.955-16.240 min. After the AVP is selenized, the selenized derivative SAVP only contains two components, the peak shape is symmetrical and sharp, and the retention time is in the range of 3.715-7.488 min. After the amomum villosum polysaccharide is selenized, the purity of the polysaccharide is increased, the molecular weight is increased, and the phenomenon indicates that the selenization is successful.

III) Infrared spectroscopic analysis of Amomum villosum seleno polysaccharide SAVP

The infrared spectra of AVP and SAVP were recorded using a Fourier transform infrared spectrometer (FT-IR) at a detection range of 4000-400 cm^-1。

FT-IR spectra of AVP and SAVP, respectively, are shown in FIGS. 2A and B. At 3600-^-1The strong absorption peak in the range is the-OH stretching vibration peak. At 2925 cm^-1Nearby absorption peak is-CH₃Middle C-H stretching vibration peak. At 1650-^-1The nearby absorption peak is free-COO^-C = O stretching vibration peak in (a), demonstrating that both AVP and SAVP are acidic polysaccharides. At 1000 cm of the SAVP infrared spectrum^-1New absorption peaks appear in the following region. 918.07 cm^-1And 895.38 cm^-1The absorption peak is the stretching vibration peak of Se-O-C, 769.33 cm^-1The absorption peak at (b) is Se = O stretching vibration peak. The appearance of these characteristic peaks indicates the successful synthesis of selenized derivatives of AVP.

Example 2: biological activity test of amomum villosum selenoglycosan SAVP

Anti-helicobacter pylori activity of amomum villosum seleno polysaccharide SAVP

1. Preparation of culture medium

(1) Blood flat plate preparation: weighing 3.9 g of Columbia agar base into 100 mL of distilled water, shaking and mixing uniformly, sterilizing at 121 ℃ for 20 min under high pressure, cooling to 55 ℃, adding 5% of sterile defibered sheep blood, mixing uniformly, pouring the mixture while the mixture is hot, standing and drying in a biological safety cabinet overnight, and using up within 2 weeks.

(2) Preparing a brain-heart leaching solution: weighing 4.7 g of brain-heart leachate powder into 100 mL of distilled water, shaking, mixing, autoclaving at 121 deg.C for 20 min, and storing in 4 deg.C refrigerator for 2 weeks.

2. Bacterial resuscitation and passage

Strains used in this study included: helicobacter pylori (Hp) standard strain ATCC43504 purchased from American type culture collection (American type culture collection).

Taking out the cryopreserved strain in an ultra-low temperature refrigerator, sucking 100 mu L of the cryopreserved strain to be naturally melted, uniformly coating the cryopreserved strain on a Columbia blood plate, placing the Columbia blood plate in a three-gas incubator at 37 ℃ under a microaerophilic condition (5% O)₂、10% CO₂、85% N₂) Culturing for 48-72 h. After being wetted by a sterile cotton swab, lawn is scraped from the blood plate, suspended in sterile PBS, 100 muL of lawn is sucked and evenly spread on a new Columbia blood plate, and the lawn is cultivated for 48 to 72 hours in an inverted microaerophilic condition as described above.

3. Bacteria cryopreservation

Selecting a blood plate with vigorous growth (the culture time is 48-72 h), wetting the blood plate with a sterile cotton swab, scraping lawn, suspending the lawn in BHI containing 20% FBS, uniformly mixing the bacterial suspension and 50% sterilized glycerol according to a ratio of 1:1 (10% FBS: 25% glycerol), sucking 1 mL of the bacterial suspension into a 2 mL freezing tube, placing the tube in a refrigerator at minus 80 ℃ for storage, wherein the storage life of the glycerol freezing bacteria is 1-2 years, and periodically resuscitating and then storing the strain every year.

4. Strain identification

The Hp strain grows well on a blood plate, the bacterial lawn is connected into sheets to form a ground glass sample, and a single bacterial colony is of a needle point size, is semi-transparent and circular and has a smooth surface. A small amount of the scraped strain is determined to be positive by urease, oxidase and catalase. Gram staining is negative in microscopic examination, and the gram is spiral, sickle-shaped, short rod-shaped and the like.

5. Determination of anti-helicobacter pylori Activity

Dissolving a sample by brain-heart infusion Broth (BHI), centrifuging at 10000 rpm for 3 min, filtering by a 0.22 mu m filter membrane, preparing a plurality of concentration gradient drugs such as 500 mu g/mL, 250 mu g/mL, 125 mu g/mL, 62.5 mu g/mL, 31.2 mu g/mL, 15.6 mu g/mL and the like by a two-fold dilution method, dissolving antibiotics by DMSO to prepare a stock solution, diluting to corresponding concentration gradients (DMSO content is less than or equal to 1%) when in use, sucking 50 mu L of the prepared liquid medicine, adding the liquid medicine into a 96-well plate, setting at least 3 multiple holes for each drug concentration, and setting negative control holes (only containing drugs and not inoculating strains) and growth control holes (not containing any drugs and only inoculating strains). Removing the strain from the incubator, scraping the lawn into PBS with moistened sterilized cotton swab, adjusting turbidity of the suspension to 1 McF with BHI containing 20% FBS, diluting 10 times, and inoculating into the above-mentioned drug-containing 96-well plate (final turbidity of the suspension in 96-well plate is about 1 × 10)⁶CFU/ml), the 96-well plate was immediately placed in an incubator, and the results were taken out after culturing at 37 ℃ under microaerophilic conditions for 72 hours with shaking at 150 rpm. The result can be used when the growth control is button-shaped or obviously turbid, the visual observation from bottom to top shows that the drug concentration when the turbidity of the bacteria is obviously reduced is MIC, each experiment takes clarithromycin as a positive control, the determination of the MIC value is completed within 3 generations after recovery, and the determination of the MIC value is repeated for 3 times.

The MICs for AVP and SAVP are shown in Table 1. The results show that the amomum villosum polysaccharide has almost no anti-helicobacter pylori activity; after selenylation modification, the selenylation derivative has anti-helicobacter pylori activity, and the MIC of SAVP is 250 microgram/mL.

TABLE 1 polysaccharide sample resistanceH. pyloriMinimum Inhibitory Concentration (MIC) of Standard Strain ATCC43504

II) antitumor activity of amomum villosum seleno polysaccharide SAVP

1. Cell recovery and culture

And taking out the pre-cryopreserved cells from the liquid nitrogen tank, quickly placing the cryopreservation tube in a constant-temperature water bath at 37 ℃, enabling the liquid level of the cells to be lower than the water surface, and shaking the cryopreservation tube by a small amplitude, wherein the liquid does not pollute the tube opening. After the cell suspension is completely melted, sucking out the cell suspension in a super clean bench, adding the cell suspension into a 15 mL centrifugal tube filled with 9 mL culture medium, uniformly blowing, and centrifuging at the normal temperature of 1000 rpm for 3 min. The supernatant was removed, the cell pellet resuspended in media containing 10% FBS and plated into a petri dish, and the cells were evenly distributed in the dish by cross-shaking. The cells were incubated at 37 ℃ with 5% CO₂The cells were cultured in an incubator and the state of the cells was observed the next day to determine whether to replace the fresh medium or to passage.

Passages should be performed whenever the degree of cell fusion reaches 80-90%. After the culture medium is discarded, PBS is added to wash the cells for 2-3 times so as to avoid the influence of residual FBS on the digestion of trypsin, then 2-3 mL of trypsin (1 x diluted by PBS) is added to be placed in an incubator, the adherent condition of the cells is observed under a microscope at regular time, when the culture dish is slightly tapped to enable the cells to fall off, a small amount of FBS is added to stop digestion, the cells are collected in a 15 mL centrifuge tube, and the centrifugation is carried out for 3 min at the normal temperature of 1000 rpm. Then removing the supernatant, resuspending the cell sediment by using a culture medium containing 10% FBS, inoculating the cell sediment into a new culture dish according to a certain proportion of the cell growth speed, marking information such as cell generation and the like on a dish cover, and enabling the cells to be uniformly distributed in the dish by cross shaking. The cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator, observing the cell state regularly, and carrying out passage every other day or replacing fresh culture medium. When the cells reach passage 20, they should be disposed of, and newly revived cells are used for subsequent experiments.

The basic culture medium of MDA-MB-231 cells is DMEM high-sugar medium.

2. Determination of antitumor Activity

The cells in logarithmic growth phase were collected by digestion and resuspended in 10% FBS-containing medium, and the concentration was adjusted to 4X 10 after cell counting³Perwell (100. mu.L of liquid per well) into 96-well plates, surroundingReserving a circle of blank holes, only adding a culture medium to serve as a blank group and preventing the cell solution from volatilizing, and culturing in an incubator for 24 hours to allow the cells to adhere to the wall. The AVP and SAVP polysaccharide mother liquors were prepared as solutions with final concentration of 800. mu.g/mL using complete medium and half-diluted with complete medium to 6 concentration gradients (highest concentration of 800. mu.g/mL, lowest concentration of 25. mu.g/mL), respectively. Old medium was aspirated from wells containing cells, 100. mu.L of the above-described concentration gradient was added to the experimental group, 100. mu.L of fresh medium containing the same volume of the highest concentration stock solution DMSO was added to the control group, 3 replicate wells per group were set, and incubation continued in an incubator for 48 h. After the incubation was completed, 20. mu.L of MTT solution was added to each well, and the mixture was returned to the incubator and incubated for 4 hours. Finally, the cell supernatant was carefully aspirated from each well, 100 μ L DMSO was added and placed on a shaker to shake until the purple pellet was completely dissolved, and absorbance was measured at 570 nm using a microplate reader.

Results are expressed as cell viability and median inhibitory concentration IC₅₀And (6) presenting. The cell viability calculation formula is as follows:

data were processed with GraphPad Prism 7 and IC calculated₅₀And presented in mean ± SD.

At the administration concentration, AVP has proliferation effect on MDA-MB-231 cells, and IC of SAVP on MDA-MB-231 cells₅₀375. + -. 32.5. mu.g/mL. After selenylation modification, the selenylation derivative SAVP has anti-breast cancer activity.

The foregoing is a more detailed description of the invention and is not to be taken in a limiting sense. It will be apparent to those skilled in the art that simple deductions or substitutions without departing from the spirit of the invention are within the scope of the invention.

Claims

1. The selenized amomum villosum polysaccharide is characterized in that: is prepared from the crude polysaccharide AVP of amomum villosum through selenylation modification.

2. The amomum villosum selenized polysaccharide of claim 1, wherein: the preparation method comprises the following steps:

3. The amomum villosum selenized polysaccharide of claim 2, wherein: the mass ratio of the coarse polysaccharide AVP of the amomum villosum to the nitric acid solution is 1: (50-150).

4. The amomum villosum selenized polysaccharide of claim 2, wherein: the concentration of the nitric acid solution is 0.4-1.0%.

5. The amomum villosum selenized polysaccharide of claim 2, wherein: the addition amount of the sodium selenite is 1-3 times of the mass of the coarse polysaccharide AVP of the amomum villosum.

6. The amomum villosum selenized polysaccharide of claim 2, wherein: the polysaccharide is precipitated by adding anhydrous ethanol to the purified polysaccharide solution.

7. The amomum villosum selenized polysaccharide of claim 2, wherein: in the step S2), the reaction time is 6-24 h.

8. A composition characterized by: contains the polysaccharide of amomum villosum selenized as described in any one of claims 1 to 7.

9. Use of amomum villosum selenized polysaccharide in the preparation of an anti-helicobacter pylori composition, wherein the amomum villosum selenized polysaccharide is as defined in any one of claims 1 to 7.

10. Use of amomum villosum selenides polysaccharide in the preparation of an anti-tumor composition or a tumor adjuvant therapy composition, wherein the amomum villosum selenides polysaccharide is as defined in any one of claims 1 to 7.