Dendrobium officinale oligosaccharide degradation method, dendrobium officinale degraded polysaccharide and application of dendrobium officinale degraded polysaccharide

Abstract

The invention provides a degradation method of dendrobium officinale oligosaccharide, dendrobium officinale degraded polysaccharide and application of the dendrobium officinale degraded polysaccharide, and the method comprises the following steps: weighing a plurality of dendrobium officinale oligosaccharide samples, and adding deionized water to dissolve the dendrobium officinale oligosaccharide samples to obtain a dendrobium officinale crude polysaccharide solution; preparing an ascorbic acid solution; adding the ascorbic acid solution into the dendrobium officinale crude polysaccharide solution to carry out oxidation reaction, and obtaining a degraded crude product solution after the reaction is finished; adjusting the pH value of the degraded crude product solution to 6-8 to obtain a degraded fine product solution, and performing dialysis, rotary evaporation and freeze-drying on the degraded fine product solution to obtain the dendrobium officinale degraded polysaccharide. According to the preparation method, the problems of clarity and transparency of the dendrobium officinale polysaccharide can be solved, the problem of skin permeability of the dendrobium officinale water extract is improved, the extraction rate and the full utilization rate of the dendrobium officinale are increased, and in addition, the dendrobium officinale degraded polysaccharide obtained through degradation has an anti-aging effect and can be used for preparing the dendrobium officinale polysaccharide. The composition is suitable for skin cosmetics, health-care foods and medicines with anti-aging effects.

Description

Technical Field

[0001] The invention relates to the field of polysaccharide degradation, and in particular to a method for degrading oligosaccharides from Dendrobium officinale, degraded polysaccharides from Dendrobium officinale and uses thereof. Background Art

[0002] Dendrobium officinale Kimura et Migo is a perennial herbaceous plant of the genus Dendrobium in the family Orchidaceae. It is also known as black knot grass and is listed in various editions of the pharmacopoeia. It is an important source of medicinal materials for Dendrobium officinale. It is mainly distributed in Yunnan, Zhejiang, Anhui and Guangdong provinces in China, and also in Myanmar and Vietnam. Dendrobium officinale mainly contains chemical components such as schizonepeta, phenanthrene, polyphenols, polysaccharides, etc., which have antioxidant, hypoglycemic, immune regulation and anti-inflammatory activities. It has the effects of promoting fluid production and nourishing the stomach; nourishing yin and clearing heat; moistening the lungs and kidneys; improving eyesight and strengthening the waist. The stems of Dendrobium officinale can be used as traditional Chinese medicine to moisturize the skin and treat diabetes.

[0003] Polysaccharides are a series of natural macromolecular active substances composed of various monosaccharides and their derivatives, which have attracted increasing attention due to their various active functions. It is reported that high molecular weight polysaccharides are not easy to cross the cell membrane barrier, which limits the absorption and application of polysaccharides in the body. Generally, polysaccharide solutions show viscosity and form gels, and this property is affected by polymer concentration, molecular weight, degree of branching, functional groups and other non-polysaccharide chemicals. At the same time, the viscosity of polysaccharides has a great influence on their physicochemical properties and functions. In the food industry, the texture and processing of products are closely related to the viscosity of polymers. Polysaccharides can also interact with small molecular compounds such as vitamins, polyphenols and other compounds, thereby affecting the quality and function of polysaccharides. L-Ascorbic acid (AA), also known as vitamin C (VC), is a naturally occurring organic compound and an important food additive. It has antioxidant, anti-aging, anti-cancer, and immunomodulatory activities. When low concentrations of ascorbic acid are added as an antioxidant to polysaccharide-rich foods, such as juices, beverages and pasta, they may exhibit a pro-oxidative effect, thereby affecting the functional properties of polysaccharides, such as viscosity. Li et al. used the ESR-electron spin capture technique to find that the addition of ascorbic acid significantly increased the number of free radicals in the reaction system. The systems used to study the oxidation of polysaccharides by ascorbic acid are divided into three categories: the first is ascorbic acid alone (including hydrogen peroxide assistance), the second is metal ion assistance, and the third is ultrasound assistance. When ascorbic acid is added alone to the polysaccharide solution, the viscosity of the polysaccharide solution can be quickly reduced, achieving a significant degradation effect. In addition, since ascorbic acid decomposes during the reaction, the degradation products of this oxidation system are of high purity, thus achieving an efficient and eco-friendly degradation process.

[0004] At present, many researchers at home and abroad have conducted research on the extraction and separation of chemical components from various parts of Dendrobium officinale. Most researchers have obtained polysaccharides with a molecular weight greater than 100,000, which are mainly composed of monosaccharides such as glucose, galactose, xylose, and a small amount of arabinose and mannose. For example, in the study of the effects of Dendrobium officinale polysaccharides of different relative molecular masses on cellular immune activity, the researchers used ultrasound combined with hydrogen peroxide to degrade Dendrobium officinale polysaccharides to obtain four Dendrobium officinale polysaccharides (DOP) with different relative molecular masses, namely DOP, DOP-30, DOP-60 and DOP-150. It was found that the immunomodulatory activity of Dendrobium officinale polysaccharides was greatly affected by the relative molecular mass. For example, researchers extracted two polysaccharides from Dendrobium officinale, with molecular weights of 7.4×10 5 ,5.4×10 5 . For another example, researchers used water extraction, alcohol precipitation, and sulfuric acid hydrolysis methods to identify that the polysaccharides in Dendrobium officinale are mainly composed of three monosaccharides: glucose, xylose, and arabinose. Through activity screening, they found that the polysaccharide components of Dendrobium officinale have an effect on the peripheral leukocyte count of mice, indicating that it has an immunomodulatory effect. For another example, researchers obtained Dendrobium officinale polysaccharides through ultrasonic extraction and found through activity screening that the polysaccharide components of Dendrobium officinale have an effect on the SOD, MDA, and CAT of fruit flies, indicating that it has an antioxidant effect. The present invention has conducted a comprehensive study on the molecular weight, composition, preparation, and anti-aging activity of oligosaccharides, and for the first time proposed that Dendrobium officinale oligosaccharides have anti-aging effects, and are suitable for skin cosmetics, health foods, and medicines with anti-aging effects. Summary of the invention

[0005] The purpose of the present invention is to provide a degradation method of Dendrobium officinale oligosaccharides, degraded Dendrobium officinale polysaccharides and uses thereof. The degradation method has the advantages of high efficiency, low pollution, low energy consumption and high yield, and is suitable for industrial production. In addition, the degraded Dendrobium officinale polysaccharides obtained by degradation have anti-aging effects and can be used as functional additives in skin care products, health care products and medicines.

[0006] In a first aspect, the present invention provides a method for degrading oligosaccharides from Dendrobium officinale, the method comprising:

[0007] Weigh a number of Dendrobium officinale oligosaccharide samples, add deionized water to dissolve, and obtain a crude Dendrobium officinale polysaccharide solution;

[0008] Prepare ascorbic acid solution;

[0009] adding the ascorbic acid solution to the crude polysaccharide solution of Dendrobium officinale to carry out an oxidation reaction, and after the reaction is completed, obtaining a degradation crude product solution;

[0010] The pH of the crude degradation product solution is adjusted to 6-8 to obtain a degradation fine product solution, and the degradation fine product solution is dialyzed, rotary evaporated, and freeze-dried to obtain the degraded polysaccharide of Dendrobium officinale.

[0011] Furthermore, the molecular weight of the Dendrobium officinale polysaccharide in the crude Dendrobium officinale polysaccharide solution is 1×10 4 -1.168×10 7 Da, the molecular weight of the degradable polysaccharide of Dendrobium officinale is 1.49×10 4 Da-9.24×10 5 Da.

[0012] Furthermore, the concentration of the crude polysaccharide solution of Dendrobium officinale is 2-15 mg / mL.

[0013] Furthermore, the concentration of the ascorbic acid solution is 0.01-5 mol / L.

[0014] Furthermore, the volume ratio of the crude polysaccharide solution of Dendrobium officinale to the ascorbic acid solution is 20-60:1-5.

[0015] Furthermore, the reaction temperature of the oxidation reaction is 40-80° C., and the reaction time of the oxidation reaction is 0.5-5 h.

[0016] Furthermore, the Dendrobium officinale polysaccharide comprises component I and component II, and the degradable Dendrobium officinale polysaccharide comprises component II and component III, wherein:

[0017] Component I: tR = 4.50min, relative molecular weight is 1.168×10 7 Da, component II: tR = 5.92min, relative molecular weight 9.24×10 5 Da, component III: tR = 8.24min, relative molecular weight 1.49×10 4 Da.

[0018] In a second aspect, the present invention further provides a degradable polysaccharide of Dendrobium officinale, which is prepared according to the above-mentioned method for degrading oligosaccharides of Dendrobium officinale.

[0019] In a third aspect, the present invention further proposes a use of degraded polysaccharides of Dendrobium officinale in preparing a pharmaceutical composition or functional food having an anti-aging effect, wherein the pharmaceutical composition or functional food comprises:

[0020] Physiologically or pharmaceutically acceptable excipients;

[0021] As mentioned above, Dendrobium officinale degrades polysaccharides.

[0022] In a fourth aspect, the present invention further proposes a use of degraded polysaccharides of Dendrobium officinale in preparing a skin care product with anti-aging effects, wherein the skin care product comprises:

[0023] Cosmetic base solutions;

[0024] As mentioned above, Dendrobium officinale degrades polysaccharides.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] 1. Due to the large molecular weight (≥100,000) of Dendrobium officinale polysaccharides, it is difficult to be absorbed through skin cells and mainly acts on the outside of the skin to moisturize. The degraded Dendrobium officinale polysaccharides obtained after enzymatic hydrolysis have a small molecular weight and are easily absorbed through skin cells, playing an anti-aging role.

[0027] 2. The degradation method of Dendrobium officinale oligosaccharides proposed in the present invention can directly obtain degraded Dendrobium officinale polysaccharides, has the advantages of simple operation and low production cost, and the production process does not use any toxic organic solvents, and the technical process involved has no sewage or waste gas emissions, which is safe and environmentally friendly.

[0028] 3. The present invention can degrade Dendrobium officinale polysaccharide by adding an appropriate amount of VC (ascorbic acid), thereby greatly improving the utilization rate and product yield of Dendrobium officinale and having good economic benefits.

[0029] 4. The degraded polysaccharide of Dendrobium officinale prepared by the present invention has a wide range of uses and can be used as an intermediate for the development of a series of Dendrobium officinale products, such as in the fields of medicine, food, and daily necessities development. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0031] Figure 1 This is a flow chart of a method for degrading oligosaccharides of Dendrobium officinale in one embodiment of the present invention;

[0032] Figure 2 HPLC chart of Dendrobium officinale polysaccharide (DOP) and VC-degraded oligosaccharide (VC-DOP);

[0033] Figure 3 HPLC diagram of monosaccharide composition of DOP and VC-DOP;

[0034] Figure 4 Particle distribution (A) and potential (B) diagram of DOP and VC-DOP;

[0035] Figure 5It is the infrared spectra of DOP and VC-DOP;

[0036] Figure 6 SEM images of DOP and VC-DOP;

[0037] Figure 7 It is the apparent viscosity analysis spectrum of DOP and VC-DOP;

[0038] Figure 8 Schematic diagram of the results of DOP (left) and VC-DOP (right) collagen secretion promotion experiments;

[0039] Fig. 9 Schematic diagram of the results of the elastase inhibition experiment of DOP (left) and VC-DOP (right);

[0040] Fig.10 Schematic diagram of the results of collagenase inhibition experiments with DOP (left) and VC-DOP (right). DETAILED DESCRIPTION

[0041] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0042] The following detailed description is an exemplary description, which is intended to provide further detailed description of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. The terms used in the present invention are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present invention.

[0043] See also Figure 1 , which is a flow chart of a method for degrading oligosaccharides of Dendrobium officinale proposed by the present invention, the method comprises steps S101 to S104, wherein:

[0044] Step S101: weighing a number of Dendrobium officinale oligosaccharide samples, adding deionized water to dissolve, and obtaining a Dendrobium officinale crude polysaccharide solution;

[0045] In some embodiments, the process of preparing the Dendrobium officinale oligosaccharide sample is as follows:

[0046] Remove excess leaves from the stems of Dendrobium and dry them. After crushing the dry stems with a high-speed grinder, add petroleum ether with a solid-liquid ratio of 1:10 for degreasing twice, discard the supernatant, and spin-dry the residue. Add pure water with a solid-liquid ratio of 1:15, extract at 100℃ for 2h, and filter with gauze. Repeat the extraction of the residue once. Combine the filtrate, centrifuge, take the supernatant, and concentrate to an appropriate concentration on a rotary evaporator. Alcohol precipitation: Stir and add 95% ethanol to adjust the final alcohol concentration in the polysaccharide extract to 75%, seal with plastic wrap, and place in a 4℃ refrigerator overnight. Centrifuge, take the polysaccharide precipitate and dissolve it in an appropriate amount of water, concentrate it on a 50℃ rotary evaporator until there is no alcohol taste, and adjust the concentration of the polysaccharide extract to about 15-20mg / mL. Sevage method for protein removal: add 1 / 5 volume of Sevage reagent (chloroform: n-butanol = 5:1) to the polysaccharide extract after alcohol precipitation and mix thoroughly in a separatory funnel, let it stand, discard the upper two layers (organic solvent and protein layer), and use a 50°C rotary evaporator to remove the residual organic solvent from the lower polysaccharide solution and concentrate it to an appropriate volume, and freeze-dry to obtain the protein-free crude polysaccharide (denoted as DOP).

[0047] In some embodiments, the concentration of the prepared Dendrobium officinale crude polysaccharide solution is 2-15 mg / mL.

[0048] Step S102: preparing an ascorbic acid solution;

[0049] In this step, the ascorbic acid solution is configured to be added to the DOP solution. It should be noted that the addition of ascorbic acid can significantly increase the number of free radicals in the reaction system. The system for studying ascorbic acid oxidation polysaccharides is divided into three categories: the first category is ascorbic acid alone (including hydrogen peroxide assistance), the second category is metal ion assistance, and the third category is ultrasound assistance. When ascorbic acid is added separately in the Dendrobium officinale polysaccharide solution, the viscosity of the Dendrobium officinale polysaccharide solution can be rapidly reduced to achieve an obvious degradation effect. In addition, since ascorbic acid decomposes during the reaction, the degradation product purity of this oxidation system is high, thereby achieving an efficient eco-friendly degradation process. Ascorbic acid (VC) can form a Fenton system, and the hydroxyl radicals produced by the Fenton system are used to oxidize and degrade the Dendrobium officinale polysaccharide to obtain degraded Dendrobium officinale degradable polysaccharides (VC-DOP).

[0050] In some embodiments, the concentration of the prepared ascorbic acid solution is 0.01-5 mol / L, preferably 0.05 mol / L.

[0051] Step S103: adding the ascorbic acid solution to the crude polysaccharide solution of Dendrobium officinale to carry out an oxidation reaction, and after the reaction is completed, obtaining a degradation crude product solution;

[0052] In this step, the volume ratio of the crude polysaccharide solution of Dendrobium officinale to the ascorbic acid solution is 20-60:1-5, preferably 40:3.

[0053] In some embodiments, the reaction temperature of the oxidation reaction is 40-80°C, preferably a constant temperature of 40°C, and the reaction time of the oxidation reaction is 0.5-5h, preferably 2h.

[0054] Step S104: adjusting the pH of the crude degradation product solution to 6-8 to obtain a degradation fine product solution, dialyzing, rotary evaporating, and freeze-drying the degradation fine product solution to obtain Dendrobium officinale degraded polysaccharides.

[0055] In addition, the study found that Dendrobium officinale polysaccharide (DOP) is composed of components Ⅰ and Ⅱ, and the chemically degraded Dendrobium officinale polysaccharide (VC-DOP) is composed of components Ⅱ and Ⅲ. Component Ⅰ: tR = 4.50min, relative molecular weight of 1.168×107Da, component Ⅱ: tR = 5.92min, relative molecular weight of 9.24×105Da, component Ⅲ: tR = 8.24min, relative molecular weight of 1.49×104Da. By comparison, it can be found that the molecular weight of DOP is greater than that of VC-DOP, proving that VC is an effective method for degrading Dendrobium officinale polysaccharides into oligosaccharides.

[0056] Example 1

[0057] Remove excess leaves from the stems of Dendrobium and dry them. After crushing the dry stems with a high-speed grinder, add petroleum ether with a solid-liquid ratio of 1:10 for degreasing twice, discard the supernatant, and spin-dry the residue. Add pure water with a solid-liquid ratio of 1:15, extract at 100℃ for 2h, and filter with gauze. Repeat the extraction of the residue once. Combine the filtrate, centrifuge, take the supernatant, and concentrate to an appropriate concentration on a rotary evaporator. Alcohol precipitation: Stir and add 95% ethanol to adjust the final alcohol concentration in the polysaccharide extract to 75%, seal with plastic wrap, and place in a 4℃ refrigerator overnight. Centrifuge, take the polysaccharide precipitate and dissolve it in an appropriate amount of water, concentrate it on a 50℃ rotary evaporator until there is no alcohol taste, and adjust the concentration of the polysaccharide extract to about 15-20mg / mL. Sevage method for protein removal: add 1 / 5 volume of Sevage reagent (chloroform: n-butanol = 5:1) to the polysaccharide extract after alcohol precipitation and mix thoroughly in a separatory funnel, let it stand, discard the upper two layers (organic solvent and protein layer), remove the residual organic solvent from the lower polysaccharide solution by a 50°C rotary evaporator and concentrate to an appropriate volume, and freeze-dry to obtain the protein-free crude polysaccharide (denoted as DOP);

[0058] Accurately weigh a certain amount of DOP sample into a 50 mL centrifuge tube, dissolve it in deionized water, and the concentration of the polysaccharide solution is 5 mg / mL to obtain a crude polysaccharide solution of Dendrobium officinale;

[0059] Prepare VC solution (0.05 mol / L);

[0060] To 200 mL of crude polysaccharide solution of Dendrobium officinale (5 mg / mL), 15 mL of 0.05 mol / L VC solution was added, the reaction temperature was 60°C, the reaction time was 2 h, the reaction was sealed during the reaction, and after the reaction, it was dialyzed with a 3500Da dialysis bag for 48 h, concentrated by rotary evaporation to an appropriate concentration, and freeze-dried to obtain degraded polysaccharides of Dendrobium officinale.

[0061] It should be noted that the DOP samples (crude polysaccharides from Dendrobium officinale) and VC-DOP (degraded polysaccharides from Dendrobium officinale) used in subsequent Examples 2-7 were all prepared in Example 1 unless otherwise specified.

[0062] Example 2

[0063] The DOP samples (crude polysaccharides from Dendrobium officinale) and VC-DOP (degraded polysaccharides from Dendrobium officinale) were analyzed by HPLC. Figure 2 As shown in the HPLC graph, the VC method has a certain degradation effect on the polysaccharides of Dendrobium officinale, and VC will decompose during the reaction. The degradation products of this oxidation system are of high purity and can achieve efficient and eco-friendly degradation. The monosaccharide composition of the crude polysaccharides of Dendrobium officinale and the polysaccharide samples treated by the VC method were analyzed. Figure 3 As shown, the results show that there is no difference in the types of monosaccharides before and after degradation, all of which are neutral polysaccharides, mainly composed of glucose and mannose, indicating that chemical degradation does not change the monosaccharide composition of polysaccharides.

[0064] Example 3

[0065] In order to comprehensively analyze the degradation effect of VC on Dendrobium officinale polysaccharides, cryo-electron microscopy observations were performed on DOP and VC-DOP obtained in Example 1. Figure 6 ( Figure 6 As shown in the SEM images of DOP and VC-DOP, the surface of DOP has more lamellar structures, a small amount of pores, and a rough surface; the characteristics of the polysaccharide VC-DOP after VC treatment are that the surface becomes smoother, a large number of pores can be observed, and the pore structure is more complex, the pores are larger, the number of lamellar structures is reduced, and there are more reticular, filamentous and a small amount of spherical structures; indicating that the structure of the polysaccharide becomes loose after degradation.

[0066] Example 4

[0067] The degree of polymerization and stability of DOP can be preliminarily determined by measuring particle size and potential. The potential and particle distribution of the sample can be measured using a dynamic light scattering particle size analyzer. Figure 4 ( Figure 4The particle distribution (A) and potential (B) of DOP and VC-DOP are shown in the figure. Generally speaking, when the potential of the particle is greater than the electronegativity and close to ±30mV, its stability is relatively strong; when the potential of the particle is low, the stability will be relatively reduced. The potential value of VC-DOP is relatively lower than that of DOP. The particle size was measured and it was found that the particle distribution of VC-DOP was significantly lower than that of DOP. From the changes in particle size and potential, the molecular weight and degree of polymerization of Dendrobium officinale polysaccharide decreased after VC treatment. This result is consistent with the HPLC test results. This may be because after VC treatment, the degree of polymerization of DOP decreased, the low molecular weight components increased, and the stability was relatively reduced.

[0068] Example 5

[0069] Weigh different polysaccharide samples quantitatively, mix them with a certain amount of potassium bromide powder, grind them evenly, and use a tablet press to press the tablets. Put the pressed tablets into the instrument for scanning, and the scanning range is 4000-400cm -1 .

[0070] The FTIR spectra of DOP and VC-DOP are as follows: Figure 5 As shown, 3350cm -1 The absorption peak at 2929cm-1 is attributed to the stretching vibration of CH. -1 The absorption peak at 1540.3cm is attributed to the C=O contraction vibration. -1 The absorption peak at 3433 cm -1 The broad and intense absorption peaks are at 1000-1200 cm -1 The absorption peaks in the region are caused by the oxygen bridges COC between sugar rings, while 872cm -1 and 807cm -1 The absorption peak belongs to the typical absorption peak of β-configuration polysaccharide. The characteristic absorption peaks of DOP and degraded polysaccharide are similar, and the results show that the degradation treatment does not change the main functional group structure of polysaccharide.

[0071] Example 6

[0072] Analysis of DOP apparent viscosity: In order to comprehensively analyze the degradation effect of VC on Dendrobium officinale polysaccharides, the apparent viscosity of DOP and VC-DOP was measured, and three different influencing factors were set up for investigation, such as Figure 7 As shown, the results are as follows:

[0073] A. Effect of different solution concentrations on apparent viscosity

[0074] The apparent viscosity of DOP and VC-DOP at different concentrations was measured. It was found that at room temperature, the apparent viscosity increased with the increase of the concentration of the polysaccharide solution. Under the same concentration conditions, the apparent viscosity of DOP was greater than that of VC-DOP.

[0075] B. Effect of different solution temperatures on apparent viscosity

[0076] The apparent viscosity of DOP and VC-DOP at different temperatures was measured and it was found that the apparent viscosity value decreased with the increase of temperature. Under the same temperature conditions, the apparent viscosity value of DOP was greater than that of VC-DOP.

[0077] C. Effect of different pH values ​​on apparent viscosity of solution

[0078] The apparent viscosity of DOP and VC-DOP at different pH values ​​was determined according to Figure 7 It can be seen that when the solution is neutral and PH=7, there is no obvious change; when PH=3 and PH=5, other conditions are the same as in Example 1, that is, when the solution is acidic, its apparent viscosity value is slightly reduced; when PH=9 and PH=11, other conditions are the same as in Example 1, that is, when the solution is alkaline, its apparent viscosity value is significantly reduced. In general, the apparent viscosity value of VC-DOP is more affected by PH than DOP.

[0079] In summary, the following conclusions can be drawn from the analysis of the above three single-factor experiments: Under the same concentration conditions, the apparent viscosity of DOP is greater than that of VC-DOP; under the same temperature conditions, the apparent viscosity of DOP is greater than that of VC-DOP. This shows that the decrease in the apparent viscosity of polysaccharides after VC treatment may be due to the decrease in the molecular weight of polysaccharides and the decrease in the degree of polymerization.

[0080] Embodiment 7:

[0081] Evaluation of DOP's anti-aging activity in vitro: DOP and VC-DOP were subjected to an anti-aging activity experiment in vitro. The experiment was designed to measure their secretion-promoting activity on human type I collagen, their elastase inhibitory activity and their collagenase inhibitory activity, and to preliminarily evaluate the potential role of DOP and VC-DOP in skin anti-aging.

[0082] A. Human type Ⅰ collagen secretion-promoting activity test

[0083] The experiment was carried out according to the instruction manual of the Elabscience human type I procollagen amino-terminal propeptide (PINP) enzyme-linked immunosorbent assay kit.

[0084] The steps are as follows:

[0085] a. Add 100L of standard working solution or sample to the corresponding wells and incubate at 37℃ for 90 minutes

[0086] b. After discarding the liquid in the plate, immediately add 100 μL of biotinylated antibody working solution and incubate at 37°C for 60 minutes

[0087] c. Discard the liquid in the plate and wash the plate 3 times

[0088] d. Add 100 μL HRP enzyme conjugate working solution to each well, incubate at 37°C for 30 minutes, discard the liquid in the plate, and wash the plate 5 times

[0089] e. Add 90 μL of substrate solution to each well and incubate at 37°C for about 15 minutes

[0090] f. Add 50 μL of stop solution to each well

[0091] g. Read the wavelength at 450nm immediately and process the data

[0092] Data processing: a. Calculate the average OD value of the standard and sample duplicate wells and subtract the OD value of the blank well as the correction value. With concentration as the horizontal axis and OD value as the vertical axis, fit the standard curve of the four-parameter logistic function on the double logarithmic coordinate axis.

[0093] b. If the sample OD value is higher than the upper limit of the standard curve, it should be appropriately diluted and re-measured and multiplied by the corresponding dilution factor when calculating the sample concentration.

[0094] B. Elastase Inhibition Activity Assay

[0095] Prepare Tris-HCl buffer (0.1M pH = 8.0), and use the buffer to prepare 2mM substrate AAAPVN solution and porcine pancreatic elastase solution (0.171U / mL). Add 50μL sample (DOP and VC-DOP), 50μL porcine pancreatic elastase solution and 100μL reaction substrate to a 96-well plate, shake gently to mix, keep at room temperature for 5 minutes, and measure the absorbance at 420nm on an ELISA reader. Replace the sample with 50μL distilled water in the blank well, and replace the substrate solution with 100μL distilled water in the control well.

[0096] γ1=1-(A2-A1) / A0

[0097] In the formula, γ1 represents the inhibition rate of elastase, A0 represents the OD value of the blank well, A1 represents the OD value of the sample control well, and A2 represents the OD value of the sample test well.

[0098] C. Collagenase Inhibition Activity Assay

[0099] Prepare Tris-HCl buffer (0.1M pH = 8.0), use the buffer to prepare 1.1U / mL type I collagenase solution and 1mM substrate FALGPA solution, the positive control is bifida yeast stock solution (BFL), add 50μL of the sample to be tested (DOP and VC-DOP), 20μL type I collagenase solution and 60μL Tris buffer to the 96-well plate, incubate at 37°C for 20min, then add 50μL substrate FALGPA solution, shake gently to mix, react at 37°C for 30min, and measure the absorbance at 335nm on an enzyme reader. Replace the sample with 50μL distilled water in the blank well, and replace the substrate solution with 50μL distilled water in the control well.

[0100] formula:

[0101] γ2=1-(A5-A4) / A3

[0102] Wherein, γ2 represents the inhibition rate of elastase, A3 represents the OD value of the blank well, A4 represents the OD value of the sample control well, and A5 represents the OD value of the sample measurement well.

[0103] according to Figures 8 to 10 It can be seen that the in vitro anti-aging activity is that both DOP and VC-DOP have a certain effect on promoting collagen secretion, and the VC-DOP group shows better ability to promote collagen secretion than the DOP group at a low concentration of 100μg / mL; both DOP and VC-DOP have a certain effect on inhibiting elastase, and the VC-DOP group shows a higher elastase inhibition rate than the DOP group at a concentration of 200μg / mL. In general, VC-DOP shows a good elastase inhibition effect; both DOP and VC-DOP have a certain effect on inhibiting collagenase, and the VC-DOP group has a higher elastase inhibition rate than the DOP group at a concentration of 100μg / mL, showing a good collagenase inhibition effect.

[0104] In summary, the above-mentioned method for degrading oligosaccharides from Dendrobium officinale has the following advantages:

[0105] 1. Due to the large molecular weight (≥100,000) of Dendrobium officinale polysaccharides, it is difficult to be absorbed through skin cells and mainly acts on the outside of the skin to moisturize. The degraded Dendrobium officinale polysaccharides obtained after enzymatic hydrolysis have a small molecular weight and are easily absorbed through skin cells, playing an anti-aging role.

[0106] 2. The degradation method of Dendrobium officinale oligosaccharides proposed in the present invention can directly obtain degraded Dendrobium officinale polysaccharides, has the advantages of simple operation and low production cost, and the production process does not use any toxic organic solvents, and the technical process involved has no sewage or waste gas emissions, which is safe and environmentally friendly.

[0107] 3. The present invention can degrade Dendrobium officinale polysaccharide by adding an appropriate amount of VC (ascorbic acid), thereby greatly improving the utilization rate and product yield of Dendrobium officinale and having good economic benefits.

[0108] 4. The degraded polysaccharide of Dendrobium officinale prepared by the present invention has a wide range of uses and can be used as an intermediate for the development of a series of Dendrobium officinale products, such as in the fields of medicine, food, and daily necessities development.

[0109] In addition, an embodiment of the present invention further provides a pharmaceutical composition or functional food, comprising the degraded polysaccharide of Dendrobium officinale obtained by degradation in the first embodiment, and the pharmaceutical composition has a good anti-aging effect.

[0110] Illustratively, the pharmaceutical composition described in the embodiments of the present application may be any one of an ointment and a cream.

[0111] In addition, an embodiment of the present application also provides a skin care product, comprising the degraded polysaccharide of Dendrobium officinale obtained by degradation in the first embodiment, and the skin care product has a good anti-aging effect.

[0112] Skin care products can be understood as liquid external skin preparations. For example, the skin care products described in the embodiments of the present application can be any one of lotion, essence, spray, emulsion, cream, mask, gel, sunscreen, and isolation.

[0113] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for degrading oligosaccharides from Dendrobium officinale, characterized in that: The method comprises: Weigh a number of Dendrobium officinale oligosaccharide samples, add deionized water to dissolve, and obtain a crude Dendrobium officinale polysaccharide solution; Prepare ascorbic acid solution; adding the ascorbic acid solution to the crude polysaccharide solution of Dendrobium officinale to carry out an oxidation reaction, and after the reaction is completed, obtaining a degradation crude product solution; The pH of the crude degradation product solution is adjusted to 6-8 to obtain a degradation fine product solution, and the degradation fine product solution is dialyzed, rotary evaporated, and freeze-dried to obtain the degraded polysaccharide of Dendrobium officinale.

2. The method for degrading Dendrobium officinale oligosaccharides according to claim 1, characterized in that: The molecular weight of the Dendrobium officinale polysaccharide in the crude Dendrobium officinale polysaccharide solution is 1×10 4 -1.168×10 7 Da, the molecular weight of the degradable polysaccharide of Dendrobium officinale is 1.49×10 4 Da -9.24×10 5 Da.

3. The method for degrading Dendrobium officinale oligosaccharides according to claim 1, characterized in that: The concentration of the crude polysaccharide solution of Dendrobium officinale is 2-15 mg / mL.

4. The method for degrading Dendrobium officinale oligosaccharides according to claim 1, characterized in that: The concentration of the ascorbic acid solution is 0.01-5 mol / L.

5. The method for degrading Dendrobium officinale oligosaccharides according to claim 1, characterized in that: The volume ratio of the crude polysaccharide solution of Dendrobium officinale to the ascorbic acid solution is 20-60:1-5.

6. The method for degrading Dendrobium officinale oligosaccharides according to claim 1, characterized in that: The reaction temperature of the oxidation reaction is 40-80° C., and the reaction time of the oxidation reaction is 0.5-5 h.

7. The method for degrading Dendrobium officinale oligosaccharides according to claim 2, characterized in that: The Dendrobium officinale polysaccharide comprises component I and component II, and the degradable Dendrobium officinale polysaccharide comprises component II and component III, wherein: Component I: tR = 4.50min, relative molecular weight is 1.168×10 7 Da, component II: tR = 5.92min, relative molecular weight 9.24×10 5 Da, component III: tR = 8.24 min, relative molecular weight 1.49 × 10 4 Da.

8. A method for degrading polysaccharides from Dendrobium officinale, characterized in that: The degradable Dendrobium officinale polysaccharide is prepared according to the degradation method of Dendrobium officinale oligosaccharide according to any one of claims 1-7.

9. The use of the degraded polysaccharide of Dendrobium officinale as claimed in claim 8 in preparing a pharmaceutical composition or functional food with anti-aging effect, characterized in that: The pharmaceutical composition or functional food comprises: Physiologically or pharmaceutically acceptable excipients; The degradable polysaccharide of Dendrobium officinale as claimed in claim 8.

10. The use of the degraded polysaccharide of Dendrobium officinale as claimed in claim 8 in preparing a skin care product with anti-aging effect, characterized in that: The skin care product comprises: Cosmetic base solutions; The degradable polysaccharide of Dendrobium officinale as claimed in claim 8.

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