Preparation method of dendrobium officinale polysaccharide fragment

Abstract

The invention relates to the technical field of dendrobium officinale polysaccharide processing and active ingredient function improvement, in particular to a preparation method of a dendrobium officinale polysaccharide fragment. According to the specific technical scheme, the method comprises the following steps: adding mannase with enzyme activity of 0.7-1U into a dendrobium officinale polysaccharide solution, and stirring for 0.5-1.5 hours; and adding absolute ethyl alcohol until the concentration is 65-75%, centrifuging to remove supernate, collecting precipitate, adding water to redissolve, concentrating, and freeze-drying to obtain the dendrobium officinale polysaccharide fragment. The dendrobium officinale polysaccharide with higher immunoregulatory activity can be conveniently and rapidly obtained in an enzymolysis mode.

Description

Technical Field

[0001] The present invention relates to the technical fields of processing technology of polysaccharides from Dendrobium officinale and improvement technology of active ingredient functions, and particularly relates to a preparation method of polysaccharide fragments of Dendrobium officinale. Background Art

[0002] Dendrobium officinale is a perennial epiphytic herb of the genus Dendrobium in the Orchidaceae family. Since ancient times, it has had the reputation of "gold in medicine" and is mainly distributed in Anhui Province and Zhejiang Province in China, Kyushu Island in Japan, and Missouri in the United States. Dendrobium officinale has anti-tumor, hypoglycemic, gastric protection, anti-inflammatory and immunomodulatory effects. It is well known that these beneficial activities are due to their complex bioactive components, including polysaccharides, alkaloids, amino acids, polyphenols, flavonoids and other components, among which polysaccharides are the main active components of Dendrobium officinale. Various biological activities of polysaccharides from Dendrobium officinale have been widely studied and reported, including immunomodulatory, antioxidant, anti-tumor, hypoglycemic and other biological activities, among which the immunomodulatory effect is the most obvious.

[0003] The polysaccharides from Dendrobium officinale directly extracted have a large molecular weight and poor water solubility, which will affect their diffusion and absorption, and are not conducive to passing through the cell membrane and entering the cell to exert their biological activities, which somewhat limits the bioavailability of polysaccharides from Dendrobium officinale. In view of the above problems, there are currently a series of methods for degrading the molecular weight of polysaccharides, such as oxidative degradation, acid degradation and ultrasonic degradation. Although oxidative degradation and ultrasonic degradation are beneficial to reducing the molecular weight, they may damage the structure of polysaccharides; acid degradation will lead to disadvantages such as a wide molecular weight distribution of degradation products and difficult separation and purification. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a preparation method of polysaccharide fragments of Dendrobium officinale, which solves the problems of non-uniform degradation products and complex separation and purification in the prior art.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0006] The present invention discloses a preparation method of polysaccharide fragments of Dendrobium officinale. Mannanase with an enzyme activity of 0.7 - 1 U is added to the Dendrobium officinale polysaccharide solution and stirred for 0.5 - 1.5 h; anhydrous ethanol is added to make its concentration 65 - 75%, the supernatant is removed by centrifugation, the precipitate is collected, redissolved in water, and after concentration and freeze-drying, polysaccharide fragments of Dendrobium officinale are obtained.

[0007] Preferably, the Dendrobium officinale polysaccharide is formulated with water into a Dendrobium officinale polysaccharide solution with a concentration of 2 - 7 mg / mL.

[0008] Preferably, the preparation process of the Dendrobium officinale polysaccharide is as follows:

[0009] (1) Water extraction: Mix the dried powder of Dendrobium officinale with distilled water, stir at 65 - 75 °C for 3 - 5 h, and centrifuge to collect the supernatant; perform water extraction on the precipitate with distilled water multiple times, and mix the centrifuged supernatants.

[0010] (2) Alcohol precipitation: Rotavapor and concentrate the supernatant, then add 90 - 95% ethanol to make its concentration 80%, and let it stand overnight at 4 °C.

[0011] (3) Dialysis and freeze - drying: Centrifuge to remove the supernatant, collect the precipitate, redissolve it in water, and obtain Dendrobium officinale polysaccharide after concentration, dialysis, concentration, and freeze - drying.

[0012] Preferably, the material - liquid ratio of the dried powder of Dendrobium officinale to distilled water is 1:15 - 25, m / v.

[0013] Preferably, the preparation process of the dried powder of Dendrobium officinale is: Cut fresh Dendrobium officinale into slices, dry them at 50 °C, and grind the dried Dendrobium officinale and pass through an 80 - mesh sieve to obtain the dried powder of Dendrobium officinale.

[0014] The present invention has the following beneficial effects:

[0015] The present invention provides a method for improving the immunomodulatory activity of Dendrobium officinale polysaccharide. Through enzymatic hydrolysis, Dendrobium officinale polysaccharide with stronger immunomodulatory activity can be obtained conveniently and quickly. Brief Description of the Drawings

[0016] Figure 1 is the HPGPC chromatogram of Dendrobium officinale polysaccharide;

[0017] Figure 2 is the effect of Dendrobium officinale polysaccharide on the viability of RAW264.7 macrophages; "*" indicates comparison between the example and the control, and "#" indicates comparison between the LPS group and the normal group;

[0018] Figure 3 is the effect of Dendrobium officinale polysaccharide on the phagocytic ability of RAW264.7 macrophages; "*" indicates comparison between the example and the control, and "#" indicates comparison between the LPS group and the normal group;

[0019] Figure 4 is the effect of Dendrobium officinale polysaccharide on the secretion of NO by RAW264.7 macrophages; "*" indicates comparison between the example and the control, and "#" indicates comparison between the LPS group and the normal group;

[0020] Figure 5 is the effect of Dendrobium officinale polysaccharide on the secretion of IL - 6 by RAW264.7 macrophages; "*" indicates comparison between the example and the control, and "#" indicates comparison between the LPS group and the normal group. Detailed Embodiments

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0022] Unless otherwise specified, the technical means used in the implementation examples are conventional means well known to those skilled in the art.

[0023] The present invention discloses a preparation method of a polysaccharide fragment of Dendrobium officinale, comprising the following steps:

[0024] (1) Cut fresh Dendrobium officinale into slices, dry them at 50 °C, and grind the dried Dendrobium officinale and pass through an 80-mesh sieve to obtain Dendrobium officinale dry powder.

[0025] (2) Water extraction: Mix the Dendrobium officinale dry powder with distilled water, with a solid-liquid ratio of 1:15 - 25, m / v, stir at 65 - 75 °C for 3 - 5 h, and centrifuge to collect the supernatant; extract the precipitate with distilled water multiple times and mix the centrifuged and collected supernatants.

[0026] (3) Alcohol precipitation: Rotavapor and concentrate the supernatant to 1 / 3 of the original volume, then add 90 - 95% ethanol to make its concentration 80%, and let it stand overnight at 4 °C.

[0027] (4) Dialysis, freeze-drying: Centrifuge to remove the supernatant, collect the precipitate, dissolve it in water, and after concentration, dialysis, concentration, and freeze-drying, obtain Dendrobium officinale polysaccharide.

[0028] (5) Enzymatic hydrolysis: Add mannanase with an enzyme activity of 0.7 - 1 U to the Dendrobium officinale polysaccharide solution and stir for 0.5 - 1.5 h; the Dendrobium officinale polysaccharide is formulated with water into a Dendrobium officinale polysaccharide solution with a concentration of 2 - 7 mg / mL.

[0029] (6) Alcohol precipitation: Add absolute ethanol to make its concentration 65 - 75%, and let it stand overnight at 4 °C.

[0030] (7) Freeze-drying: Centrifuge to remove the supernatant, collect the precipitate, dissolve it in water, and after concentration and freeze-drying, obtain the polysaccharide fragment of Dendrobium officinale.

[0031] The following further elaborates on the present invention with specific embodiments.

[0032] Example 1

[0033] A processing method of a polysaccharide fragment of Dendrobium officinale, the steps are as follows:

[0034] (1) Raw material treatment: Cut fresh Dendrobium officinale into slices, place them in an oven at 50 °C for drying treatment, and grind the dried Dendrobium officinale into powder and pass it through an 80-mesh sieve to obtain Dendrobium officinale dry powder.

[0035] (2) Water extraction: Add 150 g of Dendrobium officinale dry powder and 3000 mL of distilled water according to a solid-liquid ratio of 1:20, stir at 70 °C for 4 h, centrifuge to collect the supernatant, repeat the extraction step for the precipitate once, and combine the two extraction liquids.

[0036] (3) Ethanol precipitation: Concentrate to 1 / 3 of the original volume using a rotary evaporator, then add 95% ethanol to make the final concentration 80%, and let it stand overnight at 4 °C.

[0037] (4) Dialysis and freeze-drying: Centrifuge to remove the supernatant, collect the precipitate, redissolve it with a small amount of water, and finally obtain Dendrobium officinale polysaccharide through concentration, dialysis, concentration, and freeze-drying.

[0038] (5) Enzymatic hydrolysis: Add 500 mg of Dendrobium officinale polysaccharide to 100 mL of distilled water to prepare a 5 mg / mL polysaccharide solution, and add β-1,4-mannanase with an enzyme activity of 0.9 U and stir magnetically at 37 °C for 1 h.

[0039] (6) Ethanol precipitation: Add absolute ethanol to make the final concentration 70%, and let it stand overnight at 4 °C.

[0040] (7) Freeze-drying: Centrifuge to remove the supernatant, collect the precipitate, redissolve it with a small amount of water, and finally obtain Dendrobium officinale polysaccharide fragments through concentration and freeze-drying.

[0041] Example 2

[0042] A processing method for Dendrobium officinale polysaccharide fragments is as follows:

[0043] (1) Raw material treatment: Cut fresh Dendrobium officinale into slices, place them in an oven at 50 °C for drying treatment, and grind the dried Dendrobium officinale into powder and pass it through an 80-mesh sieve to obtain Dendrobium officinale dry powder.

[0044] (2) Water extraction: Add 150 g of Dendrobium officinale dry powder and 3000 mL of distilled water according to a solid-liquid ratio of 1:20, stir at 70 °C for 4 h, centrifuge to collect the supernatant, repeat the extraction step for the precipitate once, and combine the two extraction liquids.

[0045] (3) Ethanol precipitation: Concentrate to 1 / 3 of the original volume using a rotary evaporator, then add 95% ethanol to make the final concentration 80%, and let it stand overnight at 4 °C.

[0046] (4) Dialysis and freeze-drying: Centrifuge to remove the supernatant, collect the precipitate, redissolve it with a small amount of water, and finally obtain Dendrobium officinale polysaccharide through concentration, dialysis, concentration, and freeze-drying.

[0047] (5) Enzymatic hydrolysis: Add 500 mg of Dendrobium officinale polysaccharide to 100 mL of distilled water to prepare a 5 mg / mL polysaccharide solution. Add mannanase with an enzyme activity of 0.8 U and stir magnetically at 37°C for 1.5 h.

[0048] (6) Alcohol precipitation: Add absolute ethanol to make the final concentration 70%, and let it stand overnight at 4°C.

[0049] (7) Freeze-drying: Centrifuge to remove the supernatant, collect the precipitate, re-dissolve it with a small amount of water, and finally obtain Dendrobium officinale polysaccharide fragments through concentration and freeze-drying.

[0050] Example 3

[0051] A processing method for Dendrobium officinale polysaccharide fragments is as follows:

[0052] (1) Raw material treatment: Cut fresh Dendrobium into slices, place them in an oven at 50°C for drying, and grind the dried Dendrobium officinale into powder and pass through an 80-mesh sieve to obtain Dendrobium officinale dry powder.

[0053] (2) Water extraction: Add 150 g of Dendrobium officinale dry powder and 3000 mL of distilled water according to a solid-liquid ratio of 1:20, stir at 70°C for 4 h, centrifuge to collect the supernatant, repeat the extraction step for the precipitate once, and combine the two extraction solutions.

[0054] (3) Alcohol precipitation: Concentrate to 1 / 3 of the original volume using a rotary evaporator, then add 95% ethanol to make the final concentration 80%, and let it stand overnight at 4°C.

[0055] (4) Dialysis, freeze-drying: Centrifuge to remove the supernatant, collect the precipitate, re-dissolve it with a small amount of water, and finally obtain Dendrobium officinale polysaccharide through concentration, dialysis, concentration, and freeze-drying.

[0056] (5) Enzymatic hydrolysis: Add 500 mg of Dendrobium officinale polysaccharide to 100 mL of distilled water to prepare a 5 mg / mL polysaccharide solution. Add mannanase with an enzyme activity of 0.7 U and stir magnetically at 37°C for 2 h.

[0057] (6) Alcohol precipitation: Add absolute ethanol to make the final concentration 70%, and let it stand overnight at 4°C.

[0058] (7) Freeze-drying: Centrifuge to remove the supernatant, collect the precipitate, re-dissolve it with a small amount of water, and finally obtain Dendrobium officinale polysaccharide fragments through concentration and freeze-drying.

[0059] Comparative Example 1

[0060] The preparation of Dendrobium officinale polysaccharide is as follows:

[0061] (1) Raw material treatment: Fresh Dendrobium officinale was sliced and dried in an oven at 50 °C. After drying, the dried Dendrobium officinale was ground into powder and passed through an 80-mesh sieve to obtain dry Dendrobium officinale powder.

[0062] (2) Water extraction: 150 g of dry Dendrobium officinale powder and 3000 mL of distilled water were added according to a solid-liquid ratio of 1:20. Stirring was carried out at 70 °C for 4 h, and the supernatant was collected by centrifugation. The precipitate was extracted again following the same steps, and the two extraction solutions were combined.

[0063] (3) Alcohol precipitation: It was concentrated to 1 / 3 of the original volume using a rotary evaporator, and then 95% ethanol was added to make its final concentration 80%. It was left to stand overnight at 4 °C.

[0064] (4) Dialysis and freeze-drying: The supernatant was removed by centrifugation, and the precipitate was collected, redissolved with a small amount of water, and finally, through concentration, dialysis, concentration, and freeze-drying, Dendrobium officinale polysaccharide was obtained.

[0065] The molecular weight of the Dendrobium officinale polysaccharide fragment prepared in Example 1 was significantly reduced, from 264 kDa to 28 kDa; in addition, it had a stronger promoting ability for the proliferation, phagocytosis, and production of NO and IL-6 in RAW264.7 macrophages. Taking the Dendrobium officinale polysaccharide fragment prepared in Example 1 and the Dendrobium officinale polysaccharide prepared in Comparative Example 1 as examples, the specific experiments are as follows:

[0066] (1) Determination of molecular weight: The molecular weight of Dendrobium officinale polysaccharide was determined by high-performance liquid gel permeation chromatography. Chromatographic conditions: The mobile phase was 0.1 M NaNO3, and the flow rate was 0.6 mL / min; the chromatographic column was an Ultrahydrogel TM Linear Column with a size of 300 mm × 7.8 mm; the column temperature and differential refractive index detector temperature were both 35 °C; the injection volume was 20 μL.

[0067] (2) Cell viability determination: Cells were evenly added to a 96-well plate at a density of 2×10 5 cells / mL. After adherent culture for 4 h, the supernatant was aspirated, and different concentration gradients of Dendrobium officinale polysaccharide, namely 20, 40, 80, 160, and 320 μg / mL, were added respectively. The solvent was DMEM medium containing 10% FBS; among them, lipopolysaccharide (LPS) was used as the positive control group with a concentration of 1 μg / mL. After culturing for 24 h, serum-free medium containing 1% CCK-8 was added and cultured for another 1 h. Subsequently, the absorbance was measured at a wavelength of 450 nm. The cell viability calculation formula is: Cell viability = (A1 - A0) / (A2 - A0), where A1 represents the absorbance value of the polysaccharide group, A0 represents the absorbance value of the blank group, and A2 represents the absorbance value of the normal group. Among them, the blank group is the group without cell culture, and the normal group is the group without adding polysaccharide but adding medium.

[0068] (2) Determination of cell phagocytosis ability: Cells were evenly added to a 96-well plate at a density of 2×10 5 cells / mL. After adhering for 4 h, the supernatant was aspirated, and different concentration gradients of polysaccharides from Dendrobium officinale, namely 20, 40, 80, 160, and 320 μg / mL, were added respectively. The solvent was DMEM medium containing 10% FBS; among them, LPS was used as the positive control group with a concentration of 1 μg / mL. After culturing for 24 h, complete medium containing 10% neutral red staining solution was added and cultured for another 2 h. Subsequently, it was washed twice with PBS to thoroughly wash away the excess neutral red staining solution. Finally, neutral red detection lysis solution was added and left standing at room temperature for more than 2 h, and then the absorbance value was measured at a wavelength of 540 nm. The calculation formula for cell phagocytosis rate is: Cell phagocytosis rate = (A1 - A0) / (A2 - A0), where A1 represents the absorbance value of the polysaccharide group, A0 represents the absorbance value of the blank group, and A2 represents the absorbance value of the normal group.

[0069] (3) Determination of NO and IL-6 contents: Cells were added to a 12-well plate at a density of 5×10 5 cells / mL. After adhering for 4 h, the supernatant was aspirated, and different concentration gradients of polysaccharides from Dendrobium officinale, namely 40, 80, and 160 μg / mL, were added respectively. The solvent was DMEM medium containing 10% FBS; among them, LPS was used as the positive control group with a concentration of 1 μg / mL. After culturing for 24 h, the cell supernatant was collected by centrifugation. According to the kit protocol, the expression levels of NO and IL-6 were measured.

[0070] It can be seen from Figure 1 that the molecular weight of the polysaccharide fragment of Dendrobium officinale obtained in Example 1 was significantly reduced, from 264 kDa to 28 kDa, and this fragment had only a single symmetric peak, indicating its good homogeneity. It shows that the separation and preparation method provided by the present invention can significantly reduce the molecular weight of polysaccharides from Dendrobium officinale, and the molecular weight distribution is narrower, and the polysaccharides are more homogeneous.

[0071] As Figure 2 shown, the proliferation ability of polysaccharides from Dendrobium officinale and polysaccharide fragments was determined by the CCK-8 method. Both polysaccharides significantly promoted macrophage proliferation in a dose-dependent manner, indicating that the two prepared polysaccharide components are non-toxic and harmless. Compared with the original sugar, the enzymatically hydrolyzed polysaccharides from Dendrobium officinale can more significantly promote macrophage proliferation at the same dose. Macrophages are crucial for innate immunity. Its main functions are to phagocytose and remove pathogens, microorganisms, cell debris, and other foreign substances in the body, as well as to participate in immune regulation and inflammatory processes. Therefore, activating macrophages will enable it to exert its phagocytic function and secrete immune effector factors.

[0072] As Figure 3As shown, compared with the original polysaccharide of Dendrobium officinale, the enzymatically hydrolyzed polysaccharide of Dendrobium officinale can more significantly promote the phagocytic ability of macrophages at the dose of 20-160 μg / mL. Based on the cell viability and cell phagocytosis experiments, the dose of 40-160 μg / mL was selected for the subsequent experiments. As Figure 4 and 5 shown, compared with the original polysaccharide of Dendrobium officinale, the enzymatically hydrolyzed polysaccharide of Dendrobium officinale can more significantly promote the secretion of NO and IL-6 by macrophages at the dose of 40-160 μg / mL. In summary, the polysaccharide fragments prepared by the present invention have good homogeneity and stronger immunomodulatory ability.

[0073] There is no significant difference in the measurement results between the polysaccharide fragments prepared in Example 2 and Example 3 and the polysaccharide fragments prepared in Example 1, which will not be elaborated here.

[0074] Figures 1-5 In [reference], the example is Example 1 and the comparative example is Comparative Example 1.

[0075] The above-described examples are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A preparation method of a polysaccharide fragment of Dendrobium officinale, characterized in that: Add mannanase with an enzyme activity of 0.7 - 1 U to the polysaccharide solution of Dendrobium officinale, and stir for 0.5 - 1.5 h; add absolute ethanol to make its concentration 65 - 75%, centrifuge to remove the supernatant, collect the precipitate, re-dissolve it in water, and after concentration and freeze-drying, obtain the polysaccharide fragment of Dendrobium officinale.

2. The preparation method of a polysaccharide fragment of Dendrobium officinale as claimed in claim 1, wherein: The polysaccharide of Dendrobium officinale is formulated with water into a polysaccharide solution of Dendrobium officinale with a concentration of 2 - 7 mg / mL.

3. The preparation method of a polysaccharide fragment of Dendrobium officinale according to claim 1, characterized in that: The preparation process of the polysaccharide of Dendrobium officinale is as follows: (1) Water extraction: Mix the dry powder of Dendrobium officinale with distilled water, stir at 65 - 75 °C for 3 - 5 h, and centrifuge to collect the supernatant; perform multiple water extractions on the precipitate with distilled water, and mix the centrifugally collected supernatants; (2) Alcohol precipitation: Rotavapor and concentrate the supernatant, then add ethanol with a concentration of 90 - 95% to make its concentration 80%, and let it stand overnight at 4 °C; (3) Dialysis and freeze-drying: Centrifuge to remove the supernatant, collect the precipitate, re-dissolve it in water, and after concentration, dialysis, concentration, and freeze-drying, obtain the polysaccharide of Dendrobium officinale.

4. The preparation method of a polysaccharide fragment of Dendrobium officinale as claimed in claim 3, wherein: The material-liquid ratio of the dry powder of Dendrobium officinale to distilled water is 1:15 - 25, m / v.

5. The preparation method of a polysaccharide fragment of Dendrobium officinale according to claim 1, characterized in that: The preparation process of the dry powder of Dendrobium officinale is as follows: Cut fresh Dendrobium officinale into slices, dry them at 50 °C, and after drying, grind the Dendrobium officinale and pass it through an 80-mesh sieve to obtain the dry powder of Dendrobium officinale.

Images