Degradation method of dendrobium officinale oligosaccharides, degraded dendrobium officinale polysaccharides and uses thereof

Abstract

The present invention provides a degradation method of Dendrobium officinale oligosaccharide, Dendrobium officinale degraded polysaccharide and its use, the method comprising: weighing several Dendrobium officinale oligosaccharide samples, adding deionized water to dissolve, and obtaining a Dendrobium officinale crude polysaccharide solution; configuring ascorbic acid solution; adding the ascorbic acid solution to the Dendrobium officinale crude polysaccharide solution to carry out an oxidation reaction, question reaction terminates, and obtains a degradation crude product solution; the degradation crude product solution is adjusted to pH 6 8 to obtain a degradation fine product solution, the degradation fine product solution is dialyzed, rotary evaporated, and freeze-dried to obtain Dendrobium officinale degraded polysaccharide. The present invention can solve the clarity and transparency problems of Dendrobium officinale polysaccharide, improves the skin permeability problem of Dendrobium officinale water extract, increases the extraction rate and full utilization rate of Dendrobium officinale, and in addition, the Dendrobium officinale degraded polysaccharide obtained by degradation has anti-aging efficacy, and is suitable for skin cosmetics, health foods and medicines with anti-aging efficacy.

Description

Technical Field

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

[0002] Dendrobium officinale Kimura et Migo is a perennial herbaceous plant in the genus Dendrobium of the Orchidaceae family. Also known as black knot grass, it is listed in various pharmacopoeias and is an important source of medicinal materials. It is primarily distributed in Yunnan, Zhejiang, Anhui, and Guangdong provinces in China, and is also found in Myanmar and Vietnam. Dendrobium officinale contains chemical components such as fennel, phenanthrene, polyphenols, and polysaccharides, which exhibit antioxidant, hypoglycemic, immune-modulating, and anti-inflammatory activities. It has the benefits of promoting fluid production and nourishing the stomach; nourishing yin and clearing heat; moistening the lungs and kidneys; and improving eyesight and strengthening the waist. The stems of Dendrobium officinale are used in traditional Chinese medicine for skin moisturizing and treating diabetes.

[0003] Polysaccharides, a series of naturally occurring macromolecular active substances composed of various monosaccharides and their derivatives, have attracted increasing attention due to their diverse functional activities. High-molecular-weight polysaccharides are reportedly less able to cross cell membrane barriers, limiting their absorption and application in vivo. Polysaccharide solutions typically exhibit viscosity and gel formation, a property influenced by polymer concentration, molecular weight, degree of branching, functional groups, and other non-polysaccharide chemical species. Furthermore, the viscosity of polysaccharides significantly influences their physicochemical properties and functionality. In the food industry, product texture and processing are closely related to polymer viscosity. Polysaccharides can also interact with small molecules such as vitamins, polyphenols, and other compounds, thereby affecting their quality and functionality. L-Ascorbic acid (AA), also known as vitamin C (VC), is a naturally occurring organic compound and an important food additive. It exhibits antioxidant, anti-aging, anti-cancer, and immunomodulatory activities. When added as an antioxidant at low concentrations to polysaccharide-rich foods, such as juices, beverages, and pasta, it can promote oxidation, thereby affecting the functional properties of the polysaccharide, such as viscosity. Li et al. used ESR (electron spin trapping) technology to discover 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-assisted), the second is metal ion-assisted, and the third is ultrasound-assisted. When ascorbic acid is added alone to a polysaccharide solution, the viscosity of the polysaccharide solution can be rapidly 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 mainly 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 with different relative molecular masses on cellular immune activity, researchers used ultrasound combined with hydrogen peroxide to degrade Dendrobium officinale polysaccharides and obtained 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 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 white blood cell 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 Dendrobium officinale oligosaccharides, the method comprising:

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

[0011] Furthermore, the molecular weight of the Dendrobium officinale polysaccharide in the Dendrobium officinale crude polysaccharide solution is 1×10 4 -1.168×10 7 Da, the molecular weight of the degraded 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 includes component I and component II, and the degraded Dendrobium officinale polysaccharide includes component II and component III, wherein:

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

[0018] In a second aspect, the present invention further provides a degraded 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 provides a use of degraded polysaccharides from 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 provides a use of degraded polysaccharides from Dendrobium officinale in preparing a skin care product with anti-aging effects, the skin care product comprising:

[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, thus 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 does not use any toxic organic solvents in the production process. The technical process involved also does not emit sewage or waste gas, and 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, which constitute part of the present invention, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

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

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

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

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

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

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

[0037] Figure 7 The apparent viscosity analysis graphs 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] Figure 9 Schematic diagram of the results of the elastase inhibition experiment using DOP (left) and VC-DOP (right);

[0040] Figure 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, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other.

[0042] The following detailed description is an exemplary description and is intended to provide further detailed description of the present invention. Unless otherwise indicated, 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 , shown is a flow chart of a method for degrading oligosaccharides of Dendrobium officinale proposed by the present invention, the method comprising 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 a Dendrobium officinale oligosaccharide sample is as follows:

[0046] Remove excess leaves from the stems of Dendrobium officinale and dry them. Crush the dried stems in a high-speed grinder and degrease them twice with petroleum ether at a solid-liquid ratio of 1:10. Discard the supernatant and spin-dry the residue. Add purified water at a solid-liquid ratio of 1:15, extract at 100°C for 2 hours, and filter through gauze. Repeat the extraction of the residue once. Combine the filtrates, centrifuge, remove the supernatant, and concentrate to an appropriate concentration using 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 refrigerator at 4°C overnight. Centrifuge, dissolve the polysaccharide precipitate in an appropriate amount of water, and concentrate on a rotary evaporator at 50°C until there is no alcohol smell. Adjust the concentration of the polysaccharide extract to approximately 15-20 mg / 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 and discard the upper two layers (organic solvent and protein layer). The lower polysaccharide solution is passed through a rotary evaporator at 50°C to remove the residual organic solvent and concentrated to an appropriate volume. Freeze-drying is performed to obtain the crude polysaccharide after protein removal (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, configuration ascorbic acid solution is in order to join in DOP solution, and it should be pointed out 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 polysaccharide is divided into three categories: the first category is ascorbic acid alone (including hydrogen peroxide auxiliary), the second category is metal ion auxiliary, and the third category is ultrasound-assisted. When ascorbic acid is added separately in Dendrobium officinale polysaccharide solution, the viscosity of Dendrobium officinale polysaccharide solution can be rapidly reduced, and obvious degradation effect is reached. In addition, due to the decomposition of ascorbic acid during the reaction, the degradation product purity of this oxidation system is high, thereby realizing efficient eco-friendly degradation process. Ascorbic acid (VC) can form Fenton system, utilizes the hydroxyl radical oxidation degradation of the Fenton system to degrade Dendrobium officinale polysaccharide, obtains degraded Dendrobium officinale degraded polysaccharide (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 perform an oxidation reaction, and after the reaction is completed, obtaining a degradation crude product solution;

[0052] In this step, the volume ratio of the added amount 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 degraded Dendrobium officinale polysaccharide.

[0055] Furthermore, the study found that Dendrobium officinale polysaccharide (DOP) is composed of components I and II, while chemical degradation of DOP (VC-DOP) consists of components II and III. Component I: tR = 4.50 min, relative molecular weight 1.168 × 107 Da; component II: tR = 5.92 min, relative molecular weight 9.24 × 105 Da; component III: tR = 8.24 min, relative molecular weight 1.49 × 104 Da. Comparison revealed that DOP has a greater molecular weight than VC-DOP, demonstrating that VC is an effective method for degrading DOP into oligosaccharides.

[0056] Example 1

[0057] Remove excess leaves from the stems of Dendrobium officinale and dry them. Crush the dried stems in a high-speed grinder and degrease them twice with petroleum ether at a solid-liquid ratio of 1:10. Discard the supernatant and spin-dry the residue. Add purified water at a solid-liquid ratio of 1:15, extract at 100°C for 2 hours, and filter through gauze. Repeat the extraction of the residue once. Combine the filtrates, centrifuge, remove the supernatant, and concentrate to an appropriate concentration using 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 refrigerator at 4°C overnight. Centrifuge, dissolve the polysaccharide precipitate in an appropriate amount of water, and concentrate on a rotary evaporator at 50°C until there is no alcohol smell. Adjust the concentration of the polysaccharide extract to approximately 15-20 mg / 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 and discard the upper two layers (organic solvent and protein layer). The lower polysaccharide solution is passed through a rotary evaporator at 50°C to remove the residual organic solvent and concentrated to an appropriate volume. Freeze-dry to obtain the deproteinized crude polysaccharide (denoted as DOP).

[0058] Accurately weigh a certain amount of DOP sample into a 50 mL centrifuge tube and dissolve it in deionized water. 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 Dendrobium officinale polysaccharide solution (5 mg / mL) was added 15 mL of 0.05 mol / L VC solution, 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 3500 Da dialysis bag for 48 h, concentrated by rotary evaporation to an appropriate concentration, and freeze-dried to obtain degraded Dendrobium officinale polysaccharide.

[0061] It should be noted that the DOP samples (crude polysaccharide from Dendrobium officinale) and VC-DOP (degraded polysaccharide 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 polysaccharide of Dendrobium officinale, and VC will decompose during the reaction. The degradation products of this oxidation system are of high purity, which can achieve efficient and eco-friendly degradation. The monosaccharide composition analysis of the crude polysaccharide of Dendrobium officinale and the polysaccharide samples treated by VC method was carried out, as shown in the following figure: Figure 3 As shown, the results showed that there was no difference in the types of monosaccharides before and after degradation, all of which were neutral polysaccharides, mainly composed of glucose and mannose, indicating that chemical degradation methods did 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 flaky structures, a small amount of pores, and a rough surface; after VC treatment, the characteristics of the polysaccharide VC-DOP 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 flaky structures is reduced, and there are more network, filamentous and a small amount of spherical structures; this shows 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 4Figures 1 and 2 show the particle distribution (A) and potential (B) of DOP and VC-DOP. Generally, when the particle potential is greater than the electronegativity, close to ±30mV, its stability is relatively strong; when the particle potential is low, its stability is relatively reduced. The potential value of VC-DOP is relatively lower than that of DOP. Particle size measurement revealed that the particle distribution of VC-DOP is significantly smaller 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 decreases, the low molecular weight components increase, and the stability decreases relatively.

[0068] Example 5

[0069] Weigh different polysaccharide samples quantitatively, mix them thoroughly with a certain amount of potassium bromide powder, grind them evenly, and use a tablet press to compress them into tablets. Place the compressed tablets into the instrument for scanning, with a scanning range of 4000-400 cm -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 1540.3 cm is attributed to C=O contraction vibration. -1 The absorption peak at 3433 cm -1 The broad and intense absorption peak is at 1000-1200 cm -1 The absorption peaks in the region are caused by the oxygen bridge COC between sugar rings, while the absorption peaks at 872 cm -1 and 807cm -1 The absorption peak is a typical absorption peak of β-configuration polysaccharide. The characteristic absorption peaks of DOP and degraded polysaccharide are similar, indicating that the degradation treatment has not changed the main functional group structure of polysaccharide.

[0071] Example 6

[0072] DOP apparent viscosity analysis: 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 polysaccharide solution concentration. 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 increasing temperature. Under the same temperature conditions, the apparent viscosity value of DOP was greater than that of VC-DOP.

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

[0078] The apparent viscosity of DOP and VC-DOP at different pH values ​​was determined. Figure 7 It can be seen that when the solution is neutral at 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 decreases slightly; 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 decreases significantly. In general, the apparent viscosity value of VC-DOP is more affected by pH than DOP.

[0079] In summary, analysis of the three single-factor experiments above leads to the following conclusions: at the same concentration, the apparent viscosity of DOP is greater than that of VC-DOP; at the same temperature, the apparent viscosity of DOP is greater than that of VC-DOP. This suggests that the decrease in the apparent viscosity of polysaccharides after VC treatment may be due to a decrease in polysaccharide molecular weight and degree of polymerization.

[0080] Example 7:

[0081] Evaluation of DOP's anti-aging activity in vitro: DOP and VC-DOP were subjected to anti-aging activity experiments in vitro. The experimental design measured their secretion-promoting activity on human type I collagen, elastase inhibitory activity and collagenase inhibitory activity, and preliminarily evaluated 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 instructions 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 100 μL of standard working solution or sample to the corresponding wells and incubate at 37°C 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. Immediately read the wavelength at 450nm and process the data

[0092] Data Processing: a. Calculate the average OD value of the replicate wells containing the standard and sample and subtract the OD value of the blank well as a correction value. Fit a four-parameter logistic function to the standard curve on a double logarithmic scale, using concentration as the horizontal axis and OD value as the vertical axis.

[0093] b. If the sample OD value is higher than the upper limit of the standard curve, it should be diluted appropriately 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 it to prepare a 2mM solution of the substrate AAAPVN and porcine pancreatic elastase solution (0.171U / mL). Add 50μL of sample (DOP and VC-DOP), 50μL of porcine pancreatic elastase solution, and 100μL of reaction substrate to a 96-well plate. Mix thoroughly by gently shaking. After incubating at room temperature for 5 minutes, measure the absorbance at 420nm on a microplate reader. Replace the sample with 50μL of distilled water in the blank wells and replace the substrate solution with 100μL of distilled water in the control wells.

[0096] γ1=1-(A2-A1) / A0

[0097] In the formula, γ1 represents the elastase inhibition rate, 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 (0.1 M, pH 8.0) and use it to prepare a 1.1 U / mL type I collagenase solution and a 1 mM substrate FALGPA solution. A positive control is a stock solution of bifid yeast (BFL). Add 50 μL of the test sample (DOP and VC-DOP), 20 μL of type I collagenase solution, and 60 μL of Tris buffer to a 96-well plate. Incubate at 37°C for 20 min, then add 50 μL of the substrate FALGPA solution. After gentle shaking, incubate at 37°C for 30 min. The absorbance at 335 nm is measured using a microplate reader. Replace the sample with 50 μL of distilled water in the blank wells, and replace the substrate solution with 50 μL of distilled water in the control wells.

[0100] formula:

[0101] γ2=1-(A5-A4) / A3

[0102] Wherein, γ2 represents the elastase inhibition rate, 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 assay well.

[0103] according to Figures 8 to 10 The in vitro anti-aging activity was demonstrated by the fact that both DOP and VC-DOP had a certain effect on promoting collagen secretion, with the VC-DOP group showing a better ability to promote collagen secretion than the DOP group at a low concentration of 100 μg / mL. Both DOP and VC-DOP also had a certain effect on inhibiting elastase, with the VC-DOP group showing a higher elastase inhibition rate than the DOP group at a concentration of 200 μg / mL. Overall, VC-DOP exhibited a better elastase inhibition effect. Both DOP and VC-DOP had a certain effect on inhibiting collagenase, with the VC-DOP group showing a higher elastase inhibition rate than the DOP group at a concentration of 100 μg / mL, demonstrating a better collagenase inhibition effect.

[0104] In summary, the above-mentioned method for degrading Dendrobium officinale oligosaccharides 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, thus 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 does not use any toxic organic solvents in the production process. The technical process involved also does not emit sewage or waste gas, and 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 Dendrobium officinale polysaccharide 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 can 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 Dendrobium officinale polysaccharide 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, lotion, cream, mask, gel, sunscreen, and isolation.

[0113] It is understood from common technical knowledge that the present invention may be implemented by other embodiments that do not depart from its spirit or essential features. Therefore, the embodiments disclosed above are, in all respects, merely illustrative and not exclusive. All modifications within the scope of the present invention or equivalent to the scope of the present invention are intended to be encompassed by 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 and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the 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 by the scope of protection of the claims of the present invention.

Claims

1. A method for degrading Dendrobium officinale oligosaccharides, characterized in that: The method comprises: Weighing a number of crude polysaccharide samples of Dendrobium officinale, adding deionized water to dissolve, and obtaining a crude polysaccharide solution of Dendrobium officinale, wherein the concentration of the crude polysaccharide solution is 2-15 mg / mL; 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, wherein the concentration of the ascorbic acid solution is 0.01-5 mol / L, the volume ratio of the crude polysaccharide solution of Dendrobium officinale to the ascorbic acid solution is 20-60:1-5, the reaction temperature of the oxidation reaction is 40-80° C., and the reaction time of the oxidation reaction is 0.5-5 h; The pH of the crude degradation product solution was adjusted to 6-8 to obtain a degradation product solution, which was dialyzed, rotary evaporated, and freeze-dried to obtain degraded Dendrobium officinale polysaccharide. The molecular weight of the degraded Dendrobium officinale polysaccharide was 1.49×10 4 Da-9.24×10 5 Da.

2. The method for degrading Dendrobium officinale oligosaccharides according to claim 1, wherein The crude polysaccharide of Dendrobium officinale includes component I and component II, and the degraded polysaccharide of Dendrobium officinale includes component II and component III, wherein: Component I: tR = 4.50 min, relative molecular weight is 1.168 × 10 7 Da, component II: tR = 5.92 min, relative molecular weight 9.24 × 10 5 Da, component III: tR = 8.24 min, relative molecular weight 1.49 × 10 4 Da.

3. A method for degrading polysaccharides from Dendrobium officinale, characterized in that: The degraded Dendrobium officinale polysaccharide is prepared according to the method for degrading Dendrobium officinale oligosaccharides according to any one of claims 1-2.

4. The use of the degraded polysaccharide of Dendrobium officinale as claimed in claim 3 in preparing a pharmaceutical composition or functional food with anti-aging effects, 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 3.

5. The use of the degraded polysaccharide of Dendrobium officinale as claimed in claim 3 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 3.

Images