CN114031693B - Chemical degradation method of dendrobium officinale polysaccharide - Google Patents

Abstract

The invention belongs to the field of polysaccharide preparation, in particular to a chemical degradation method of Dendrobium officinale polysaccharide. Diethylenetriaminepentaacetic acid was used to chelate ferrous ions and hydrogen peroxide to form a Fenton system, and the generated hydroxyl radicals were oxidized to degrade Dendrobium officinale polysaccharides. The degradation method provided by the invention has a simple process, and the method can effectively reduce the molecular weight of the Dendrobium officinale polysaccharide after processing the method, which is beneficial to the subsequent utilization of the Dendrobium officinale polysaccharide.

Description

A kind of chemical degradation method of Dendrobium officinale polysaccharide

technical field

The invention belongs to the field of polysaccharide preparation, in particular to a chemical degradation method of Dendrobium officinale polysaccharide.

Background technique

Dendrobium officinale (scientific name: Dendrobium officinale) is a plant of the orchid family Dendrobium. Dendrobium officinale is often used as medicine with fresh or dried stems. It has the functions of nourishing yin and clearing heat, promoting body fluid and benefiting the stomach, moistening the lungs and relieving cough, improving eyesight and strengthening the body. It is a traditional and precious Chinese medicinal material.

Dendrobium officinale polysaccharide is the main active ingredient in Dendrobium officinale, and its content is mostly in the range of 10-49%. Dendrobium officinale polysaccharide has physiological activities such as enhancing immunity, anti-tumor, lowering blood sugar, and anti-oxidation, and has important edible and medicinal value.

However, the bioabsorption rate of polysaccharides is very low, and the gastrointestinal absorption rate is less than 5% after oral ingestion. Many current studies are based on cell experiments, ignoring the low absorption rate, and the injection method has many safety issues. Therefore, reducing the molecular weight of polysaccharides, improving the absorption rate of the digestive system, and improving or maintaining their physiological activities are the hotspots of polysaccharide research.

At present, the degradation of Dendrobium officinale polysaccharide mainly includes physical degradation, chemical degradation and biodegradation. The physical degradation method mainly includes ultrasonic degradation method and microwave degradation method. It does not need to introduce other reactants. The operation is simple, but the reaction time is long and the degradation efficiency is low. To improve the degradation efficiency, it can be combined with other degradation methods such as acid degradation (patent). No.: CN200510008708.2). The biodegradation method is mainly an enzymatic degradation method with mild reaction conditions and strong specificity. For example, oxidohydrolase can effectively degrade cellulose and chitosan (patent number: CN201710735196.2). The chemical degradation method mainly includes acid degradation method and hydrogen peroxide degradation method. The reaction system is simple and the time is short; however, its application limitation is that some preparations are toxic and harmful, which limits its application in food. The acid degradation method generally uses hydrochloric acid or sulfuric acid to hydrolyze the glycosidic bond of the polysaccharide molecule, which reduces the molecular weight of the polysaccharide, and the reaction is simple and easy. . The hydrogen peroxide degradation method utilizes redox reactions to oxidatively degrade polysaccharides, and the cost is low. The free radical degradation method of polysaccharide invented by Qu Aiqin et al. (patent number: CN1459455A) is to place the polysaccharide in the composite system of organic acid, hydrogen peroxide and vitamin C, which is suitable for most polysaccharides from sources, but the reaction time is limited. For 2-72 hours, the concentration of the solution is in the range of 1-100 mmol/L, and the concentration required for a long reaction time is high.

SUMMARY OF THE INVENTION

In view of the above-mentioned technical problems, the object of the present invention is to provide a kind of chemical degradation method of Dendrobium officinale polysaccharide, adopt diethylenetriaminepentaacetic acid (DTPA) chelated ferrous ion and hydrogen peroxide to react to form Fenton system, the hydroxyl radical that produces For the oxidative degradation of Dendrobium officinale polysaccharide, the degradation method provided by the invention is simple in process, and the method can reduce the molecular weight of the Dendrobium officinale polysaccharide after processing the polysaccharide, improve its antioxidant capacity, and facilitate the subsequent utilization of the Dendrobium officinale polysaccharide.

The present invention is achieved through the following technical solutions:

A method for chemical degradation of Dendrobium officinale polysaccharide, wherein a Fenton system is formed by reacting ferrous ions chelated with diethylenetriaminepentaacetic acid and hydrogen peroxide, and the hydroxyl radicals generated by the Fenton system are used to oxidatively degrade Dendrobium officinale polysaccharide to obtain degradation For the crude product solution, the pH of the degraded crude product solution is adjusted to remove iron ions by precipitation, and after alcohol precipitation and drying, the degraded polysaccharide of Dendrobium officinale is obtained.

Further, the method specifically follows the steps:

(1) dissolve Dendrobium officinale polysaccharide in water to obtain Dendrobium officinale polysaccharide solution;

(2) in described Dendrobium officinale polysaccharide solution, add the ferrous ion (being DTPA-ferrous ion) that diethylenetriaminepentaacetic acid is chelated, then add hydrogen peroxide solution again, form Fenton system, carry out oxidation reaction at room temperature Degrading Dendrobium officinale polysaccharide to obtain a solution of degraded crude product;

(3) adjusting the pH of the degraded crude product solution, discarding the precipitation to obtain the dendrobium officinale polysaccharide degradation solution;

(4) alcohol precipitation is carried out in the dendrobium officinale polysaccharide degradation solution, the liquid phase is discarded, and the precipitation is dried to obtain the dendrobium officinale degraded polysaccharide.

Further, the molecular weight of the Dendrobium officinale polysaccharide is in the range of 2×10 ⁷ -1.5×10 ⁵ Da; the molecular weight of the degraded polysaccharide of Dendrobium officinale is in the range of 5×10 ⁴ -1.5×10 ⁴ Da.

Further, in step (1), the concentration of the Dendrobium officinale polysaccharide solution is 1-10 mg/mL.

Further, step (2) is specifically:

Add diethylenetriaminepentaacetic acid chelated ferrous ion to Dendrobium officinale polysaccharide solution until the concentration of DTPA-ferrous ion in Dendrobium officinale polysaccharide solution is 5-20 μm/L; then add hydrogen peroxide solution to Dendrobium officinale crude polysaccharide The volume ratio concentration of hydrogen peroxide in the aqueous solution is 0.05-0.5%; the oxidation reaction is carried out at room temperature to degrade Dendrobium officinale polysaccharide, and the degraded crude product solution is obtained after a certain time of reaction.

In this step, it is necessary to strictly control the concentration of DTPA-ferrous ions to be 5-20 μm/L and the volume ratio of hydrogen peroxide to be 0.05-0.5%. It is precisely by controlling the concentration of DTPA-ferrous ions in the Fenton system and peroxide The volume ratio of hydrogen concentration can obtain the degradable polysaccharides of Dendrobium officinale with molecular weight between 5×10 ⁴ -1.5×10 ⁴ Da, and at the same time, it can ensure that the polysaccharides of Dendrobium officinale are not excessively degraded into monosaccharides.

Preferably, the selected Dendrobium officinale polysaccharide concentration is 2 mg/mL, the DTPA-ferrous ion concentration in the Dendrobium officinale polysaccharide solution is 5 μm/L, and the volume ratio concentration of the hydrogen peroxide solution is 0.1%.

The method of the invention adopts diethylenetriaminepentaacetic acid (DTPA) as an important ammonia carboxyl chelating agent, which has extremely strong coordination ability to metal ions, especially high-valence chromogenic metal ions, and is widely used in medical pharmaceuticals. After chelating essential trace elements, it can promote the excretion of heavy metal poisons from the body. The stability and antioxidant effect of DTPA on the chelates formed by transition metals are better than those of ethylenediaminetetraacetic acid (EDTA). Possibility of oxidation to ferric ions.

Further, in step (2), in step (2), the pH of the Dendrobium officinale polysaccharide solution is adjusted to 3.0-4.0, and then the ferrous ions chelated by diethylenetriaminepentaacetic acid are added.

Further, in step (2), the degradation time is 10-20min.

Further, in step (3), the pH of the crude degradation product solution is adjusted to be 9.0-10.0.

Further, in step (4), in step (4), the temperature of the alcohol precipitation is 3-5°C, and the time of alcohol precipitation is 6-18h.

Beneficial technical effects of the present invention:

1) The degradation method provided by the present invention adopts the Fenton system to degrade Dendrobium officinale polysaccharide, which has the advantages of short degradation time and low cost, and can significantly reduce the molecular weight of Dendrobium officinale polysaccharide. The Fenton system adopted in the present invention includes ferrous ions chelated by diethylenetriaminepentaacetic acid and hydrogen peroxide, wherein, the hydrogen peroxide is decomposed as a reaction substrate, and the generated hydroxyl radicals attack the Dendrobium officinale polysaccharide molecules to reduce polysaccharide The effect of molecular weight; ferrous ion is used as a catalyst to participate in the decomposition of hydrogen peroxide to achieve the effect of rapid degradation; DTPA is used as a metal ion chelator to chelate ferrous ions to enhance its stability and reduce the possibility of being oxidized. ferric ions to avoid interfering with the reaction.

2) The antioxidant activity of the degraded polysaccharide of Dendrobium officinale prepared by the degradation method provided by the present invention is obviously improved compared with that of the polysaccharide by the traditional water extraction and alcohol precipitation method.

Description of drawings

Fig. 1 is the high performance liquid chromatogram of Dendrobium officinale polysaccharide DP and dendrobium officinale degraded polysaccharides DP-1, DP-2, DP-3 in the embodiment of the present invention.

Fig. 2a is a diagram showing the reducing ability of Dendrobium officinale polysaccharide DP and Dendrobium officinale degraded polysaccharide DP-1 to ferric ions in the embodiment of the present invention; Fig. 2b is a graph of the reduction ability of Dendrobium officinale polysaccharide DP and Dendrobium officinale degraded polysaccharide DP-1 in the embodiment of the present invention. DPPH free radical scavenging ability diagram; Figure 2c is the hydroxyl radical scavenging ability diagram of Dendrobium officinale polysaccharide DP and Dendrobium officinale polysaccharide DP-1 in the embodiment of the present invention; Figure 2d is the Dendrobium officinale polysaccharide DP and Total antioxidant capacity of Dendrobium officinale degrading polysaccharide DP-1.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.

On the contrary, the present invention covers any alternatives, modifications, equivalents and arrangements within the spirit and scope of the present invention as defined by the appended claims. Further, in order to give the public a better understanding of the present invention, some specific details are described in detail in the following detailed description of the present invention. The present invention can be fully understood by those skilled in the art without the description of these detailed parts.

The invention provides a chemical degradation method of Dendrobium officinale polysaccharide, which comprises the reaction of ferrous ions chelated by diethylenetriaminepentaacetic acid and hydrogen peroxide to form a Fenton system, and the hydroxyl radicals generated by the Fenton system are utilized to oxidize and degrade Dendrobium officinale polysaccharides. , obtain a degraded crude product solution, adjust the pH of the degraded crude product solution, remove iron ions by precipitation, and obtain dendrobium officinale degraded polysaccharide after alcohol precipitation and drying. The method specifically includes the following steps:

(1) Dissolving Dendrobium officinale polysaccharide in water to obtain a Dendrobium officinale polysaccharide solution; the concentration of the Dendrobium officinale polysaccharide solution is 1-10 mg/mL, preferably 2 mg/mL.

(2) Add ferrous ion chelated by diethylenetriaminepentaacetic acid to Dendrobium officinale polysaccharide solution until the concentration of DTPA-ferrous ion in Dendrobium officinale polysaccharide solution is 5-20 μm/L; then add hydrogen peroxide solution to The volume concentration ratio of hydrogen peroxide in the aqueous solution of Dendrobium crude polysaccharide is 0.05-0.5%; the oxidation reaction is carried out at room temperature to degrade Dendrobium officinale polysaccharide, and the degraded crude product solution is obtained after a certain time of reaction; the degradation time is 10-20min;

Preferably, the selected Dendrobium officinale polysaccharide concentration is 2 mg/mL, the DTPA-ferrous ion concentration is 5 μm/L, and the volume ratio concentration of the hydrogen peroxide solution is 0.1%.

(3) adjusting the pH of the degraded crude product solution to be 9-10, discard the precipitation, and obtain the dendrobium officinale polysaccharide degradation solution;

(4) alcohol precipitation is carried out in the dendrobium officinale polysaccharide degradation solution, the liquid phase is discarded, and the precipitation is dried to obtain the dendrobium officinale degraded polysaccharide. In this embodiment, the temperature of the alcohol precipitation is 3-5° C., and the time of the alcohol precipitation is 6-18 h.

Described below with specific embodiments:

Comparative Example

The present comparative example provides a method for extracting Dendrobium officinale polysaccharide by hot water extraction method:

Weigh 0.500 g of Dendrobium officinale powder that was dried, crushed and passed through a 60-mesh sieve, add 25 mL of 80% ethanol, vortex and mix, extract by 150 W ultrasonic for 30 min, centrifuge at 6000 r/min, take the precipitate, wash with 10 mL of 80% ethanol and centrifuge for 2- 3 times until the supernatant is colorless; the precipitate is extracted with boiling water for 2 hours at a material-to-liquid ratio of 1:100, suction filtered, concentrated under reduced pressure, added with 4 times the volume of ethanol for overnight alcohol precipitation, centrifuged, and dried to obtain Dendrobium officinale polysaccharide DP.

Example 1

The present embodiment provides a chemical degradation method of Dendrobium officinale polysaccharide, the method comprising:

a) prepare Dendrobium officinale polysaccharide DP according to the method of Comparative Example;

b) adopting diethylenetriaminepentaacetic acid (DTPA) solution to chelate ferrous ion to obtain ferrous ion chelated by diethylenetriaminepentaacetic acid, namely DTPA-ferrous ion;

c) The crude polysaccharide DP of Dendrobium officinale prepared in step a) is prepared into an aqueous solution of Dendrobium officinale polysaccharide with a concentration of 2 mg/mL, and the pH is adjusted to 3.0; The concentration is 5 μm/L; 1% hydrogen peroxide solution is added to the Dendrobium officinale polysaccharide aqueous solution to the hydrogen peroxide concentration of 0.1%; the oxidation reaction is carried out at room temperature;

d) after 20 minutes of reaction, adjust the pH value of the reaction solution to 9.0, and remove the precipitate by centrifugation;

e) adjusting the pH of the supernatant to neutral, concentrating under reduced pressure, and drying to obtain DP-1, a free radical degradation product of Dendrobium officinale polysaccharide.

It was reconstituted in ultrapure water and filtered through a 0.22 μm Nylon membrane for high performance liquid chromatography analysis.

In this example, the chromatographic column used was a C18 column (4.6×250 mm, 5 μm), the mobile phase was 100% ultrapure water, the flow rate was 1 mL/min, the column temperature was 30 °C, the injection volume was 20 μL, and the running time was 15 min. After the run, compare with the standard according to the retention time.

Example 2

This embodiment provides a chemical degradation method of Dendrobium officinale polysaccharide, which is basically the same as Embodiment 1, except that: in step d), the reaction time is 10 min; the degradation product DP-2 of Dendrobium officinale polysaccharide is obtained.

Example 3

This embodiment provides a chemical degradation method of Dendrobium officinale polysaccharide, which is basically the same as Embodiment 1, with the only difference being that: in step c), the DTPA-ferrous ion is replaced with ferrous ion: adding ferrous ion, The ferrous ion concentration in the Dendrobium officinale polysaccharide aqueous solution was 5 μm/L. Finally, the degradation product DP-3 of Dendrobium officinale polysaccharide was obtained.

Fig. 1 is the high performance liquid chromatogram of Dendrobium officinale polysaccharide DP and the degraded polysaccharides DP-1, DP-2 and DP-3 of Dendrobium officinale. It can be seen from Fig. 1 that: DP, DP-1, DP-2 were analyzed by high performance liquid chromatography. , DP-3 molecular weight, it is determined that DP is mainly composed of four molecular weight polysaccharides, which are 2.4 × 10 ⁷ Da, 1.9 × 10 ⁶ Da, 1.3 × 10 ⁶ Da, 1.5 × 10 ⁵ Da; DP-1 is mainly composed of two ^The molecular weight of ^{polysaccharides} after degradation was significantly lower than that before degradation, and the molecular weight distribution was more concentrated. The molecular weights of DP-2 and DP-1 are basically the same, but compared with DP-1, the proportion of low molecular weight polysaccharides is lower. DP-3 is mainly composed of two molecular weight polysaccharides, 8.5×10 ⁵ Da and 2.0×10 ⁴ Da, respectively. The molecular weight of Dendrobium officinale polysaccharide after degradation was significantly lower than that of polysaccharide before degradation, but compared with the degradation of Dendrobium officinale polysaccharide by chelating ferrous ions and hydrogen peroxide, the molecular weight after degradation was higher, and the proportion of low molecular weight polysaccharides was less.

Figure 2 is a graph showing the antioxidant activity of Dendrobium officinale polysaccharide DP and Dendrobium officinale degraded polysaccharide DP-1, Figure 2a is a graph of the reducing ability to ferric ions, Figure 2b is a graph of DPPH free radical scavenging ability, and Figure 2c is a graph of hydroxyl radicals The scavenging capacity map, Figure 2d is the total antioxidant capacity map. It can be seen from Figure 2a-d that the antioxidant capacity of the degraded Dendrobium officinale polysaccharide DP-1 is higher than that of the undegraded Dendrobium officinale polysaccharide DP. At the concentration of 3.0 mg/mL, the total antioxidant capacity of DP-1 is about DP. 2.6 times that of DP-1; the most obvious improvement in antioxidant capacity is the scavenging capacity of DPPH radicals. At the concentration of 0.5 mg/mL, the total antioxidant capacity of DP-1 is about 5.7 times that of DP.

Claims (7)

1. a chemical degradation method of Dendrobium officinale polysaccharide, is characterized in that, forms Fenton system with the ferrous ion chelated by diethylenetriaminepentaacetic acid and hydrogen peroxide reaction, utilizes the hydroxyl radical oxidative degradation that described Fenton system produces Dendrobium officinale polysaccharide is obtained to obtain a degraded crude product solution, the pH of the degraded crude product solution is adjusted to remove iron ions by precipitation, and after alcohol precipitation and drying, the degraded Dendrobium officinale polysaccharide is obtained; The method specifically includes the following steps: (1) Dissolving Dendrobium officinale polysaccharide in water to obtain Dendrobium officinale polysaccharide solution; (2) adding ferrous ions chelated by diethylenetriaminepentaacetic acid to the Dendrobium officinale polysaccharide solution, and then adding a hydrogen peroxide solution to form a Fenton system, and performing an oxidation reaction at room temperature to degrade the Dendrobium officinale polysaccharide to obtain a degraded crude oil. product solution; (3) adjusting the pH of the degraded crude product solution, discarding the precipitation, and obtaining a Dendrobium officinale polysaccharide degradation solution; (4) alcohol precipitation of the Dendrobium officinale polysaccharide degradation solution, discarding the liquid phase, drying and precipitation to obtain dendrobium officinale degraded polysaccharide; Step (2) is as follows: To the Dendrobium officinale polysaccharide solution, add diethylenetriaminepentaacetic acid chelated ferrous ion until the concentration of DTPA-ferrous ion in the Dendrobium officinale polysaccharide solution is 5-20 μM/L; then add hydrogen peroxide solution to the Dendrobium officinale polysaccharide The volume ratio concentration of the hydrogen peroxide in the aqueous solution is 0.05-0.5%; the oxidation reaction is carried out at room temperature to degrade the Dendrobium officinale polysaccharide, and the degraded crude product solution is obtained after a certain time of reaction. 2. the chemical degradation method of a kind of Dendrobium officinale polysaccharide according to claim 1, is characterized in that, the molecular weight of described Dendrobium officinale polysaccharide is in the scope of 1.5×10 ⁵ -2×10 ⁷ Da; Between 1.5×10 ⁴ -5×10 ⁴ Da. 3 . The chemical degradation method of Dendrobium officinale polysaccharide according to claim 1 , wherein in step (1), the concentration of the Dendrobium officinale polysaccharide solution is 1-10 mg/mL. 4 . 4. the chemical degradation method of a kind of Dendrobium officinale polysaccharide according to claim 1, is characterized in that, in step (2), adjust the pH of Dendrobium officinale polysaccharide solution to 3.0-4.0 and then add diethylenetriaminepentaacetic acid chelated Ferrous ions. 5 . The chemical degradation method of Dendrobium officinale polysaccharide according to claim 1 , wherein, in step (2), the degradation time is 10-20 min. 6 . 6 . The chemical degradation method of Dendrobium officinale polysaccharide according to claim 1 , wherein, in step (3), the pH of the degraded crude product solution is adjusted to be 9.0-10.0. 7 . 7 . The chemical degradation method of Dendrobium officinale polysaccharide according to claim 1 , wherein, in step (4), the temperature of the alcohol precipitation is 3-5° C., and the alcohol precipitation time is 6-18 h. 8 .

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