Arundina graminifolia polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses arundina graminifolia polysaccharide as well as a preparation method and application thereof. The method comprises the following steps of: performing degreasing pretreatment on arundina graminifolia rhizomes, extracting with water according to a liquid-material ratio of (5: 1)-(50: 1 mL / g), and leaching twice at the temperature of 60-100 DEG C for 0.5-2.5 hours each time; and after concentration and alcohol precipitation of the extract liquid, deproteinization is carried out by adopting a Sevage method, purification is carried out by adopting a chromatographic column, and the arundina graminifolia polysaccharide is prepared after dialysis. Various novel polysaccharides can be prepared from arundina graminifolia at the same time, the purity is high, an AGP-1 component is novel in structure and uniform in molecular weight, the average molecular weight of the AGP-1 component is 494.3 kDa, a core main chain is composed of alpha-D type mannose connected with (1-> 4) and alpha-D type glucose connected with (1-> 4) according to the molar ratio of 3: 2, the two ends of the AGP-1 component are connected with alpha-D type galactose and beta-D type galactose respectively, and the AGP-1 component and the beta-D type galactose are separated from each other. The AGP-1 can block glycosylation reaction to play a role in inhibiting protein saccharification and delay skin aging, meanwhile, the AGP-1 has good tyrosinase inhibitory activity, melanin accumulation is reduced, and the AGP-1 can be used in whitening and skin care cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of macromolecular natural products, and more specifically to a bamboo orchid polysaccharide and a preparation method and application thereof. Background Art

[0002] Modern research shows that plant polysaccharides have good biological activity and biocompatibility, and have broad application and development prospects in the fields of health products, functional foods and cosmetics. Chinese invention patent CN201010250402.9 discloses a method for preparing Bletilla striata polysaccharide used as a raw material for cosmetics. The patent obtains a Bletilla striata polysaccharide by water extraction enzyme processing and chromatographic refining. In vitro experiments show that the Bletilla striata polysaccharide complex can repair the skin barrier, increase the moisture content of the skin, whiten the skin and delay skin aging. Chinese invention patent CN200610145677.X discloses a method for preparing Dendrobium officinale polysaccharide with enhanced immune function by water extraction and alcohol precipitation. The patent proves through in vivo mouse experiments that the polysaccharide can significantly enhance the immunity of mice and has basically no toxic side effects.

[0003] Glycation reaction is an oxidative stress reaction in the body that causes the oxidation of carbohydrates, which in turn forms highly reactive and electrophilic compounds that attack free amino groups in proteins, causing covalent modification of proteins and leading to the production of advanced glycation end products (AGEs). AGEs products can cause collagen and elastin in the dermis of the skin to lose their support and elasticity for skin tissue, causing the skin to become yellow and dull, with enlarged pores and loss of elasticity. Therefore, glycation reaction is one of the important factors that lead to human aging and dark yellow skin. Therefore, by blocking the occurrence of AGEs reaction, protein glycation can be inhibited, thereby delaying aging.

[0004] Bamboo leaf orchid is an important medicinal plant of the orchid family. It is a unique solution (antidote) in Dai medicine. It has the effects of clearing away heat and detoxifying, diuresis, and removing jaundice. It can be used alone or in Chinese medicine compound. It is often used externally to treat trauma, scalds and burns in clinical practice. Invention patent application CN202311530205.6 discloses a bamboo leaf orchid extract and its preparation method and application in cosmetics. The application uses conventional solvents and general extraction methods to prepare a crude bamboo leaf orchid extract, that is, ultrasonic extraction with 0-80% ethanol aqueous solution, preferably 80% ethanol solution, and the extract is then concentrated and freeze-dried to obtain a crude extract. The whitening and skin care effects of small molecules in the crude extract, such as arbutin, beishengmanin, isoliquiritigenin, kaempferol, luteolin, chlorogenic acid and other components, are studied, but the bamboo leaf orchid crude extract prepared by the existing extraction technology contains almost no high-purity macromolecular polysaccharides. At present, the research on Bamboo Leaf Orchid is mainly focused on small molecule compounds such as stilbenes, flavonoids and phenols. There are no reports in the literature on the structure and functionality of macromolecular polysaccharides of Bamboo Leaf Orchid and its application.

[0005] The purpose of the present invention is to provide a bamboo orchid polysaccharide and an extraction and purification method and application thereof, and to confirm the monosaccharide composition and glycoside connection mode of the separated novel AGP-1 polysaccharide by means of HPLC, GC-MS and the like, and to further investigate the anti-glycosylation activity and tyrosinase inhibitory activity of the bamboo orchid polysaccharide AGP-1 on the basis of structural identification, so as to realize the application of the bamboo orchid polysaccharide in the fields of daily necessities and whitening skin care cosmetics through deep processing. Summary of the invention

[0006] The purpose of the present invention is to provide a polysaccharide of Bamboo Orchid and its preparation method and application. The technical problem to be solved is the effective separation and purification of functional polysaccharides in Bamboo Orchid, the analysis of the novel structure of polysaccharides, the study of anti-glycosylation activity and tyrosinase inhibition activity.

[0007] The present invention uses the rhizome of Bamboo Orchid as a research object, provides a new type of high molecular weight polysaccharide and its preparation method and application, systematically studies the novel structure of the AGP-1 polysaccharide component based on polysaccharide extraction and purification, and also studies its whitening and skin care efficacy. The various new Bamboo Orchid polysaccharides prepared by the present invention have high purity, wherein the AGP-1 polysaccharide component not only has uniform molecular weight and novel structure, but also has a main chain composed of (1→4)-linked α-D-type mannose and (1→4)-linked α-D-type glucose in a molar ratio of 3:2, and the outer end is connected with α-D-galactose and β-D-type galactose; and the polysaccharide can not only effectively inhibit the protein saccharification process, but also exhibits good tyrosinase inhibitory activity to reduce melanin accumulation, so it can be used in cosmetics with whitening and skin care efficacy.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] The invention discloses a bamboo orchid polysaccharide, which is a glucomannan galactan, and comprises (1→4)-linked α-D-type mannose and (1→4)-linked α-D-type glucose.

[0010] Preferably, the cymbidium polysaccharide comprises (1→4)-linked α-D-mannose and (1→4)-linked α-D-glucose connected in a molar ratio of 3:2.

[0011] Preferably, the bamboo orchid polysaccharide, named AGP-1 polysaccharide, has α-D-galactose and β-D-galactose connected at both ends respectively; its glycosidic bonds mainly include →4)-α-D-Glcp-(1→4)-α-D-Manp-(1→, →4)-α-D-Manp-(1→4)-α-D-Glcp-(1→ and →4)-α-D-Manp-(1→4)-α-D-Manp-(1→.

[0012] Preferably, the bamboo orchid polysaccharide has the following structural formula:

[0013]

[0014] The main chain structure of AGP-1 polysaccharide is shown above.

[0015] Preferably, in the structural formula of the Bamboo Orchid Polysaccharide, R1 has the following structural formula:

[0016] α-D-Glcp-(1→4)-α-D-Manp-(1→.

[0017] The fragment structure R1 of AGP-1 polysaccharide is a terminal glucose connected to a (1→4)-linked α-D-mannose, and the structure is shown above.

[0018] Preferably, in the structural formula of the Bamboo Orchid Polysaccharide, R2 has the following structural formula:

[0019]

[0020] The fragment structure R2 of AGP-1 polysaccharide, i.e., the partial structure of the polysaccharide repeating sequence, is shown above.

[0021] A method for preparing the above-mentioned bamboo orchid polysaccharide comprises the following steps:

[0022] A. defatting the Bamboo Leaf Orchid raw material to obtain a defatted mixture;

[0023] B. subjecting the defatted mixture to water extraction to obtain a polysaccharide extract;

[0024] C. Concentrating the polysaccharide extract to obtain a polysaccharide concentrate;

[0025] D. subjecting the polysaccharide concentrate to alcohol precipitation to obtain a crude polysaccharide precipitate;

[0026] E. deproteinizing the crude polysaccharide precipitate to obtain deproteinized polysaccharide;

[0027] F. subjecting the deproteinized polysaccharide to ion exchange column chromatography to obtain a preliminary separated polysaccharide;

[0028] G. Subjecting the preliminarily separated polysaccharides to gel column chromatography to obtain the Bamboo Orchid polysaccharide.

[0029] The invention can simultaneously prepare a plurality of novel high-molecular-weight polysaccharides from the bamboo orchid with high purity. The AGP-1 component has a novel structure and is glucomannan with uniform molecular weight and an average molecular weight of 494.3 kDa. The core main chain is composed of (1→4)-linked α-D-mannose and (1→4)-linked α-D-glucose in a molar ratio of 3:2 to form a glycoside repeating sequence, and the two ends are respectively connected with α-D-galactose and β-D-galactose. The AGP-1 polysaccharide can not only block the glycosylation reaction to exert the protein saccharification inhibitory effect and delay skin aging, but also exert good tyrosinase inhibitory activity and reduce melanin accumulation, and can be used in whitening skin care cosmetics.

[0030] Preferably, in step F, the eluent for the ion exchange column chromatography separation includes a NaCl solution; the concentration of the NaCl solution includes 0 to 0.99 mol / L; the filler for the ion exchange column chromatography separation includes one or more of CM-Sepharose FaseFlow, DEAE-52cellulose, and DEAE-Sepharose Fast Flow; in step G, the eluent for the gel column chromatography separation includes distilled water; the filler for the gel column chromatography separation includes one or more of Sepharose CL-2B, Sephadex G-200, and Bio-Gel P-30.

[0031] Preferably, the method comprises the following steps:

[0032] A. Using dried rhizomes of Bambusa chinensis as raw materials, crushing and sieving, and then adding an organic solvent to perform the degreasing treatment to obtain the degreasing mixture;

[0033] B. adding water to the defatted mixture at a liquid-to-solid ratio of 5 to 50:1 mL / g, performing the water extraction treatment at 60 to 100° C., filtering and combining the extracts to obtain the polysaccharide extract;

[0034] C. performing the concentration treatment on the polysaccharide extract by reduced pressure concentration to obtain the polysaccharide concentrate;

[0035] D. adding ethanol to the polysaccharide concentrate to make the alcohol content range from 65% to 85% by volume, standing overnight at low temperature to perform the alcohol precipitation treatment, filtering out the supernatant, and obtaining the crude polysaccharide precipitate;

[0036] E. Dissolving the crude polysaccharide precipitate in water, adding Sevage reagent at a volume ratio of 3 to 5:1, performing the deproteinization treatment using the Sevage method, centrifuging the upper polysaccharide liquid, evaporating the reagent and freeze-drying to obtain the deproteinized polysaccharide; the Sevage reagent comprises chloroform and n-butanol mixed at a volume ratio of 1 to 5:1;

[0037] F. Redissolving the deproteinized polysaccharide in water, performing the ion exchange column chromatography separation, collecting the polysaccharide eluate and performing the first dialysis to obtain the preliminary separated polysaccharide; the first dialysis uses a dialysis bag with a molecular weight cutoff of less than 10000Da;

[0038] G. Subjecting the preliminarily separated polysaccharide to the gel column chromatography, collecting the polysaccharide eluate and performing a second dialysis to obtain the Bambusa japonica polysaccharide; the second dialysis uses a dialysis bag with a molecular weight cutoff of less than 10,000 Da.

[0039] The impurity problem in the prior art is mainly because small molecules or monosaccharides, oligosaccharides and other substances with properties similar to polysaccharides are obtained at the same time during the extraction process. The present invention obtains most polysaccharide precipitation by setting an alcohol content of 65-85%, preferably 70%-80%, and the supernatant containing small molecules is removed by filtration; the obtained crude polysaccharide is then purified by column chromatography in sections, and the first section uses anion exchange column chromatography (such as cellulose column chromatography). The sample ions will compete with the mobile phase ions for the charge position on the surface of the stationary phase, but due to the difference in competitiveness, the sample components are separated to obtain purified polysaccharides; the second section of column chromatography (such as dextran gel chromatography) uses the molecular sieve principle (the filler is suitable for a wide range of molecular weights) to obtain polysaccharides within the corresponding molecular weight range, thereby removing most monosaccharides and oligosaccharides. The above steps are used to solve the problems of impurities and small molecule adsorption, and obtain purified polysaccharides.

[0040] The deproteinized polysaccharide obtained in step E is redissolved in water to prepare a solution of appropriate concentration, and the polysaccharide is separated by ion exchange column chromatography, and the polysaccharide eluate is collected and dialyzed to obtain the initially separated polysaccharide; the initially separated polysaccharide is further purified by gel column chromatography, and the polysaccharide eluate is collected and dialyzed to obtain the purified Bamboo Cymbidium polysaccharide, i.e., the Bamboo Cymbidium polysaccharide.

[0041] Preferably, in step A, the crushing and screening includes passing through a 24-80 mesh sieve; the amount of the organic solvent added includes a liquid-to-solid ratio of 10-20:1 mL / g; the organic solvent includes one or more of ether, petroleum ether, dichloromethane, chloroform, anhydrous ethanol, and ethyl acetate; in step B, the number of water extraction treatments includes 1-3 times, and the treatment time for each treatment includes 0.5-2.5 hours; in step C, the concentration treatment includes concentrating under reduced pressure to a volume of 1 / 3-1 / 10 of the original volume; in step D, the alcohol content includes 7% by volume 0 to 80%; the low temperature overnight standing includes standing at 4°C overnight; in step E, the Sevage reagent includes chloroform and n-butanol mixed in a volume ratio of 3 to 4:1; in step F, the eluent for ion exchange column chromatography separation includes distilled water; the first dialysis uses a dialysis bag with a molecular weight cutoff of 3500 to 8000; the first dialysis time is greater than 12 hours; in step G, the second dialysis uses a dialysis bag with a molecular weight cutoff of 3500 to 8000; the second dialysis time is greater than 12 hours.

[0042] When preparing AGP-1, the polysaccharide obtained by using pure water as the eluent in both the ion exchange column and the gel column is better.

[0043] An application of the above-mentioned bamboo orchid polysaccharide is used to prepare a product having one or more of the functions of anti-glycation, anti-oxidation and inhibition of tyrosinase activity.

[0044] The bamboo orchid polysaccharide prepared by the invention has high purity and uniform molecular weight, can not only inhibit protein glycation to exert anti-glycation activity and delay aging, but also has good tyrosinase inhibition activity and reduces melanin accumulation, and can be used in whitening and skin care cosmetics.

[0045] The implementation of the present invention has the following beneficial effects:

[0046] 1) There is no research on polysaccharides in bamboo orchid in the prior art, but the present invention can simultaneously prepare a variety of bamboo orchid high-molecular polysaccharides with novel structures and high purity, wherein the AGP-1 component has a uniform molecular weight, the main chain is composed of (1→4)-linked α-D-mannose and (1→4)-linked α-D-glucose in a molar ratio of 3:2, and the outer ends are connected with α-D-galactose and β-D-galactose.

[0047] 2) The polysaccharide prepared by the present invention has a high yield, a simple and feasible process, and is suitable for large-scale industrial production; the purified AGP-1 polysaccharide obtained by the method can block the glycosylation reaction and inhibit the formation of AGEs products, delay skin aging, and also exhibit good tyrosinase inhibitory activity, and can be used as a natural product for whitening and skin care in the field of cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The extraction yield and total soluble sugar content of the crude polysaccharide AGP of Bamboo Leaf Orchid of Examples 1-6.

[0049] Figure 2 The crude polysaccharide of Example 7 was subjected to DEAE-52 ion exchange column chromatography to prepare refined polysaccharides (Fr. AGP-1, Fr. AGP-2, Fr. AGP-3).

[0050] Figure 3 The purified polysaccharide from Example 7 was purified by Sephadex G-200 column chromatography to obtain purified polysaccharides (A: AGP-1; B: AGP-2).

[0051] Figure 4 This is the infrared spectrum of the purified polysaccharides AGP-1 and AGP-2 from the bamboo orchid in Example 7.

[0052] Figure 5 This is the molecular weight determination of the purified polysaccharide from Bamboo Spathiphyllum in Example 7 (A: AGP-1; B: AGP-2).

[0053] Figure 6 The monosaccharide composition of the purified AGP-1 polysaccharide of Example 7 was analyzed by HPLC.

[0054] Figure 7 1D NMR spectrum of the purified AGP-1 polysaccharide of Example 7 (A: 1 H NMR; B: 13 C-NMR).

[0055] Figure 8 2D NMR spectrum of the purified AGP-1 polysaccharide of Example 7 (A: 1 H- 1 H COSY; B: HSQC; C: HMBC).

[0056] Fig. 9 This is a schematic diagram of the structure of the purified AGP-1 polysaccharide of Example 7.

[0057] Fig.10 This is the anti-glycation activity of the purified AGP-1 polysaccharide of Example 7.

[0058] Fig.11 The antioxidant activity of the purified AGP-1 polysaccharide of Example 7 (A: DPPH scavenging; B: ABTS scavenging).

[0059] Fig.12 The tyrosinase inhibitory effect of the purified AGP-1 polysaccharide of Example 7 (A: absorbance change in reaction with tyrosinase; B: tyrosinase inhibitory effect).

[0060] Fig.13GC-MS analysis of the methylated product of AGP-1 purified polysaccharide prepared in Example 11. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with specific embodiments.

[0062] Example 1

[0063] A bamboo orchid polysaccharide and a preparation method thereof, comprising the following steps:

[0064] (1) Raw material pretreatment: The dried rhizomes of Bamboo Leaf Orchid were fully crushed and passed through a 24-mesh sieve. The Bamboo Leaf Orchid powder was then mixed with petroleum ether at a liquid-to-solid ratio of 10:1 mL / g. The mixture was shaken at room temperature for 48 h and filtered to obtain defatted powder.

[0065] (2) Take 5 g of the pretreated bamboo orchid powder, add distilled water for hot water extraction, the extraction temperature is 60°C, the time is 2.5 h, the liquid-to-solid ratio is 40:1 mL / g, and the extraction is repeated twice; filter and combine the polysaccharide extracts, and concentrate under reduced pressure to 1 / 10 of the original volume to obtain a polysaccharide concentrate.

[0066] (3) Add ethanol to the concentrated solution obtained in step 2) to adjust the ethanol volume concentration of the mixed solution to 80%, let it stand overnight at 4°C, filter the precipitate, and freeze-dry to obtain the crude polysaccharide AGP of Bamboo Leaf Orchid. The soluble total sugar content is determined by the phenol-sulfuric acid method, and the crude polysaccharide yield is calculated based on the dry weight of the Bamboo Leaf Orchid raw material.

[0067] Example 2

[0068] A bamboo orchid polysaccharide and a preparation method thereof, comprising the following steps:

[0069] (1) Raw material pretreatment: The dried rhizomes of Bamboo Leaf Orchid were fully crushed and passed through a 45-mesh sieve. The Bamboo Leaf Orchid powder was then mixed with chloroform at a liquid-to-solid ratio of 10:1 mL / g. The mixture was shaken at room temperature for 48 h and filtered to obtain defatted powder.

[0070] (2) Take 5 g of the pretreated bamboo orchid powder, add distilled water for hot water extraction, the extraction temperature is 70°C, the time is 2.0 h, the liquid-to-solid ratio is 5:1 mL / g, and the extraction is repeated twice; filter and combine the polysaccharide extracts.

[0071] (3) Add ethanol to the extract obtained in step 2) to adjust the ethanol volume concentration of the mixed solution to 65%, let it stand overnight at 4°C, filter the precipitate, and freeze-dry to obtain the crude polysaccharide AGP of Bamboo Leaf Orchid. The soluble total sugar content is determined by the phenol-sulfuric acid method, and the crude polysaccharide yield is calculated based on the dry weight of the raw material of Bamboo Leaf Orchid.

[0072] Example 3

[0073] A bamboo orchid polysaccharide and a preparation method thereof, comprising the following steps:

[0074] (1) Raw material pretreatment: The dried rhizomes of Bamboo Leaf Orchid were fully crushed and passed through an 80-mesh sieve. The Bamboo Leaf Orchid powder was then mixed with ether at a liquid-to-solid ratio of 15:1 mL / g. The mixture was shaken at room temperature for 48 h and filtered to obtain defatted powder.

[0075] (2) Take 5 g of the pretreated bamboo orchid powder, add distilled water for hot water extraction, the extraction temperature is 80°C, the time is 0.5 h, the liquid-to-solid ratio is 20:1 mL / g, and the extraction is repeated twice; filter and combine the polysaccharide extracts, and concentrate under reduced pressure to 1 / 4 of the original volume to obtain a polysaccharide concentrate.

[0076] (3) Add ethanol to the concentrated solution obtained in step 2) to adjust the ethanol volume concentration of the mixed solution to 70%, let it stand overnight at 4°C, filter the precipitate, and freeze-dry to obtain the crude polysaccharide AGP of Bamboo Leaf Orchid. The soluble total sugar content is determined by the phenol-sulfuric acid method, and the crude polysaccharide yield is calculated based on the dry weight of the raw material of Bamboo Leaf Orchid.

[0077] Embodiment 4:

[0078] A bamboo orchid polysaccharide and a preparation method thereof, comprising the following steps:

[0079] (1) Raw material pretreatment: The dried rhizomes of Bamboo Leaf Orchid were fully crushed and passed through a 35-mesh sieve. The Bamboo Leaf Orchid powder was then mixed with dichloromethane at a liquid-to-solid ratio of 15:1 mL / g. The mixture was shaken at room temperature for 48 h and filtered to obtain defatted powder.

[0080] (2) Take 5 g of the pretreated bamboo orchid powder, add distilled water for hot water extraction, the extraction temperature is 90°C, the time is 1.0 h, the liquid-to-solid ratio is 10:1 mL / g, and the extraction is repeated twice; filter and combine the polysaccharide extracts, and concentrate under reduced pressure to 1 / 3 of the original volume to obtain a polysaccharide concentrate.

[0081] (3) Add ethanol to the concentrated solution obtained in step 2) to adjust the ethanol volume concentration of the mixed solution to 75%, let it stand overnight at 4°C, filter the precipitate, and freeze-dry to obtain the crude polysaccharide AGP of Bamboo Leaf Orchid. The soluble total sugar content is determined by the phenol-sulfuric acid method, and the crude polysaccharide yield is calculated based on the dry weight of the Bamboo Leaf Orchid raw material.

[0082] Example 5

[0083] A bamboo orchid polysaccharide and a preparation method thereof, comprising the following steps:

[0084] (1) Raw material pretreatment: The dried rhizomes of Bamboo Leaf Orchid were fully crushed and passed through a 50-mesh sieve. The Bamboo Leaf Orchid powder was then mixed with ethyl acetate at a liquid-to-solid ratio of 20:1 mL / g. The mixture was shaken at room temperature for 48 h and filtered to obtain defatted powder.

[0085] (2) Take 5 g of the pretreated bamboo orchid powder, add distilled water for hot water extraction, the extraction temperature is 90°C, the time is 1.5 h, the liquid-to-solid ratio is 50:1 mL / g, and the extraction is repeated twice; filter and combine the polysaccharide extracts, and concentrate under reduced pressure to 1 / 10 of the original volume to obtain a polysaccharide concentrate.

[0086] (3) Add ethanol to the concentrated solution obtained in step 2) to adjust the ethanol volume concentration of the mixed solution to 85%, let it stand overnight at 4°C, filter the precipitate, and freeze-dry to obtain the crude polysaccharide AGP of Bamboo Leaf Orchid. The soluble total sugar content is determined by the phenol-sulfuric acid method, and the crude polysaccharide yield is calculated based on the dry weight of the Bamboo Leaf Orchid raw material.

[0087] Example 6

[0088] A bamboo orchid polysaccharide and a preparation method thereof, comprising the following steps:

[0089] (1) Raw material pretreatment: The dried rhizomes of Bamboo Leaf Orchid were fully crushed and passed through a 65-mesh sieve. Then, the Bamboo Leaf Orchid powder and anhydrous ethanol were mixed at a liquid-to-solid ratio of 20:1 mL / g, shaken at room temperature for 48 h, and filtered to obtain defatted powder.

[0090] (2) Take 5 g of the pretreated bamboo orchid powder, add distilled water for hot water extraction, the extraction temperature is 100°C, the time is 1.5 h, the liquid-to-solid ratio is 30:1 mL / g, and the extraction is repeated twice; filter and combine the polysaccharide extracts, and concentrate under reduced pressure to 1 / 5 of the original volume to obtain a polysaccharide concentrate.

[0091] (3) Add ethanol to the concentrated solution obtained in step 2) to adjust the ethanol volume concentration of the mixed solution to 80%, let it stand overnight at 4°C, filter the precipitate, and freeze-dry to obtain the crude polysaccharide AGP of Bamboo Leaf Orchid. The soluble total sugar content is determined by the phenol-sulfuric acid method, and the crude polysaccharide yield is calculated based on the dry weight of the Bamboo Leaf Orchid raw material.

[0092] The crude polysaccharide yield and soluble total sugar content obtained under the conditions described in Examples 1-6 are as follows: Figure 1 As shown in the figure, it can be seen that the yield of crude polysaccharide and its total sugar content in Example 6 are both high.

[0093] Example 7

[0094] A bamboo orchid polysaccharide and a preparation method thereof, comprising the following steps:

[0095] (1) The crude polysaccharide AGP of Bamboo Leaf Orchid prepared by the method of Example 6 was dissolved in water again, and Sevage reagent was added. The Sevage reagent was prepared by chloroform and n-butanol in a volume ratio of 4:1. The volume ratio of the crude polysaccharide solution to the Sevage reagent was controlled to be 5:1. The mixture was fully shaken for 60 minutes, and the supernatant was obtained by centrifugation. After the organic reagent was evaporated, the supernatant was freeze-dried to obtain the deproteinized polysaccharide.

[0096] (2) The deproteinized polysaccharide obtained in step 1) was dissolved in water to prepare a solution with a concentration of 20 mg / mL, and the polysaccharide was separated and purified using a DEAE-52cellulose ion exchange chromatography column. The sample volume to column volume ratio was 1:5, and distilled water, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, and 0.4 mol / L NaCl solutions were used for gradient elution. The flow rate was controlled at 1.0 mL / min, and 10 mL of the eluate was collected in each tube. The polysaccharide content of each tube was detected by the phenol-sulfuric acid method. The absorbance was measured at 490 nm, and a gradient elution curve was drawn ( Figure 2 ). The fraction eluted with distilled water was named Fr.AGP-1, and the fractions eluted with 0.1mol / L and 0.4mol / L NaCl solutions were named Fr.AGP-2 and Fr.AGP-3, respectively. Among them, the yield of Fr.AGP-1 fraction was the highest, followed by Fr.AGP-2, and Fr.AGP-3 fraction was the least (accounting for only 0.5% to 1% of all purified fractions, lacking practical value).

[0097] (3) The Fr.AGP-1 and Fr.AGP-2 components were dialyzed for 48 h using a 3500Da dialysis bag and then freeze-dried. They were then redissolved in water to prepare a solution with a concentration of 10 mg / mL. The two polysaccharides were further separated and purified using a Sephadex G-200 gel chromatography column. The elution was eluted with distilled water at a flow rate of 0.5 mL / min. 5 mL was collected in each tube. The polysaccharide content in each tube was detected by the phenol-sulfuric acid method. The absorbance was measured at 490 nm, and the elution curve was drawn ( Figure 3 The eluate was collected, dialyzed at 3500Da for 48 hours, and then freeze-dried to obtain the purified polysaccharides AGP-1 and AGP-2 from Rhizoma Coptidis.

[0098] (4) The soluble total sugar contents of the purified components of AGP-1 and AGP-2 were determined by the phenol-sulfuric acid method to be 81.55% and 80.29%, respectively, indicating that the polysaccharide content increased after purification. The protein contents of the two components were determined by the Coomassie Brilliant Blue method to be 1.69% and 1.78%, respectively, indicating that this method has a high deproteinization efficiency.

[0099] (5) Infrared spectral analysis of purified polysaccharides AGP-1 and AGP-2 from Bamboo Leaf Orchid. 5 mg of AGP-1 and AGP-2 were mixed with an appropriate amount of fully dried KBr powder and ground evenly, pressed into tablets using a tablet press, and scanned using a Fourier transform infrared spectrometer (model Nicolet iS10, Thermo Company, USA) in the scanning range of 4000-400 cm -1 The results are as follows Figure 4 As shown, the polysaccharide sample has a peak at 3375 cm -1 The broad peaks around 2931cm are caused by the stretching of OH groups in the polysaccharide molecules. -1 The absorption peaks around 1734cm -1 The absorption peak at 1260 cm is caused by the stretching vibration of C=O in acetyl or carboxylic acid ester; -1 Up to 1000cm -1 The absorption peak of AGP-1 polysaccharide is related to the glycosidic bond vibration of the pyranose ring COC and the stretching vibration of the CO side chain. -1 and 875cm -1 The sharp peak at indicates that both α- and β-glycosidic bonds may exist in the polysaccharide.

[0100] (6) Molecular weight determination of purified polysaccharides AGP-1 and AGP-2 from Bamboo Orchid. The Thermo Ultimate 3000 high performance liquid chromatograph was used, the detector was a RefractoMax 521 differential detector, Ultra hydrogel 1000 (7.8×300mm) and Ultra hydrogel 500 (7.8×300mm) columns were connected in series, the mobile phase was 20mM KH2PO4 buffer solution, the flow rate was set at 0.8mL / min, the injection volume was 20μL, and the column temperature was 35℃. The results are shown in Figure 5 As shown, AGP-1 polysaccharide ( Figure 5 A) has a uniform molecular weight and is a single polysaccharide. Its average molecular weight is calculated to be 494.3 kDa; while AGP-2 polysaccharide ( Figure 5 B) is mainly composed of 39.4kDa, 2.62kDa and 1.57kDa components, not a single polysaccharide. In addition, the yield of AGP-1 polysaccharide is about 85% to 90%, while the yield of AGP-2 polysaccharide is about 10% to 15%. From the perspective of polysaccharide yield and separation purity, the AGP-1 component with the highest polysaccharide yield and separation purity was subsequently selected for characterization and in vitro activity evaluation.

[0101] (7) Analysis of monosaccharide composition of purified polysaccharide AGP-1 from Bamboo Orchid. The monosaccharide composition was determined by high performance liquid chromatography. 1 mL of 5 mg / mL polysaccharide solution was placed in a pressure bottle, 2 mL of trifluoroacetic acid (2 M) was added, the bottle was sealed with a sealing film and placed in a 121°C oil bath for full hydrolysis for 2 h, the bottle was removed and cooled, and then evaporated to dryness under reduced pressure, followed by repeated rinsing with methanol 2-3 times to remove trifluoroacetic acid, and finally 1 mL of deionized water was added to dissolve. 500 μL of polysaccharide hydrolyzate and 500 μL of 0.3 M NaOH solution were mixed evenly, and then 500 μL of 0.5 M PMP-methanol solution was added. After thorough mixing, the bottle was placed in a constant temperature water bath at 80°C for 1 h. After cooling, 500 μL of 0.3 M hydrochloric acid was added to neutralize the reaction. Subsequently, 1 mL of chloroform was added for extraction to remove PMP three times, and the supernatant was filtered through a 0.45 μm filter membrane, and the filtrate was subjected to liquid phase testing. The liquid chromatography detection conditions were as follows: Agilent 1260 high performance liquid chromatography was used, using Infinity Lab C 18 The chromatographic column was (4.6×250mm, 5μm), the column temperature was 30℃, the mobile phase was 78% 0.1M phosphate buffer (pH=6.7) and 22% acetonitrile, the flow rate was set to 1mL / min, the detection wavelength was 245nm, and the injection volume was 10μL. Various monosaccharide standards, namely mannose (Man), rhamnose (Rha), glucuronic acid (GlcUA), galacturonic acid (GalUA), glucose (Glc), galactose (Gal), arabinose (Ara) and fucose (Fuc), were weighed, mixed and dissolved in distilled water, and derivatized and measured according to the above-mentioned derivatization method. According to the retention time of the standard, the monosaccharide composition in the polysaccharide sample can be determined, and the molar percentage of the monosaccharide in the sample can be calculated according to the peak area of ​​each monosaccharide and the molar mass of the monosaccharide. The results are as follows: Figure 6 As shown, the polysaccharide is mainly composed of mannose, glucose and galactose, with molar percentage contents of 56.63%, 36.37% and 5.33% respectively, and a molar ratio of 10:7:1, indicating that AGP-1 polysaccharide is glucomannan.

[0102] (8) NMR analysis of purified polysaccharide AGP-1 from Bamboo Stalk Orchid. Weigh 15 mg of AGP-1 and dissolve it in 0.5 mL of D2O. Take the supernatant and place it in an NMR tube. Use a Bruker AV-500 spectrometer and a 5 mm probe to obtain the NMR spectrum. Record 1 H-NMR, 13 C-NMR, 1 H- 1 H COSY, HSQC and HMBC spectra and processing ( 1 H-NMR and 1 H- 1The incomplete peaks in H COSY, HSQC and HMBC are to avoid the influence of solvent peaks on other signals during the processing. Figure 7 , Figure 8 ). In polysaccharides 1 H NMR spectrum ( Figure 7 A) In H The 4.3-5.5 ppm region is the H-1 signal region of the polysaccharide, where δ H The 5.0-5.8 ppm region is usually the isomeric protons of the polysaccharide α configuration, while the δ H The 3.3-4.2 region corresponds to the other protons of the glycoside. 13 C NMR spectrum ( Figure 7 B), δ C The 95-106 region corresponds to the isomeric carbon of the glycoside, while δ C The 62-82 ppm region corresponds to the other carbons of the glycoside. 1 H and 13 C NMR and HSQC spectra ( Figure 8 In B), five anomeric proton and anomeric carbon signals at δ5.41 / 100.14ppm, 5.42 / 99.70ppm, 5.41 / 101.34ppm, 4.99 / 98.62ppm, and 5.39 / 97.23ppm were measured and assigned to →4)-α-D-Glcp-(1→(A), →4)-α-D-Manp-(1→(B), →3,4)-α-D-Galp-(1→(C), →4,6)-β-D-Galp-(1→(D), and α-D-Glcp-(1→(E), respectively. According to the HSQC spectrum, the obtained anomeric carbon and hydrogen signal peaks were assigned to C-1 / H-1 of the five sugar residues. Then, according to the two-dimensional 1 H- 1 HCOSY spectrum ( Figure 8 A) Signal correlation was used to obtain the H-2 to H-6a signals of each residue, and then the corresponding C-2 to C-6 signals were obtained using the carbon-hydrogen direct connection signal peaks in the HSQC spectrum. The carbon and hydrogen signal shift assignment results of each sugar residue in AGP-1 polysaccharide are shown in Table 1.

[0103] Table 1

[0104]

[0105] “-”: indicates undetermined

[0106] According to Table 1 combined with the HMBC spectrum ( Figure 8C) to determine the order of glycosidic bond connection in the purified polysaccharide AGP-1. The results showed that the correlation peaks of residue A (H-1) and residue C (H-1) with residue B (C-4) were δ5.41 / 71.69ppm, indicating the existence of two glycosidic bond connection modes: →4)-α-D-Glcp-(1→4)-α-D-Manp-(1→ and →3,4)-α-D-Galp-(1→4)-α-D-Manp-(1→). The correlation peaks of residue A (H-4) and residue B (C-1) were δ3.65 / 99.70ppm. pm, indicating the existence of a →4)-α-D-Manp-(1→4)-α-D-Glcp-(1→ connection mode. In addition, there is a correlation peak of δ5.42 / 71.69ppm between residue B(H-1) and its own C-4, which also indicates the existence of mannose related repeating fragments, that is, the existence of a →4)-α-D-Manp-(1→4)-α-D-Manp-(1→ connection mode. In addition, the HMBC spectrum also shows the existence of residue B(H-1) and residue C(C -4), residue B (H-1) and residue D (C-4), residue A (H-4) and residue D (C-6), residue B (H-4) and residue C (C-3), and residue B (C-4) and residue E (H-1) have correlation peaks of δ 5.42 / 76.85 ppm, 5.42 / 76.85 ppm, 3.65 / 60.60 ppm, 3.68 / 74.75 ppm, and 5.39 / 71.69 ppm, respectively, indicating the presence of →4)-α-D -Manp-(1→4)-α-D-Galp-(1,3→、→4)-α-D-Manp-(1→4)-β-D-Galp-(1,6→、→1,4)-β-D-Galp-(6→4)-α-D-Glcp-(1→、→1)-α-D-Manp-(4→3)-α-D-Galp-(1,4→ and α-D-Glcp-(1→4)-α-D-Manp-(1→4) exist in the form of glycosidic bond connection.

[0107] Combined with the results of methylation analysis and one-dimensional and two-dimensional NMR data, it is speculated that the possible structure of the purified polysaccharide AGP-1 from Bamboo Leaf Orchid is mainly connected by mannose and glucose, and →4)-α-D-Glcp-(1→4)-α-D-Manp-(1→) is the main chain of the polysaccharide. The glycoside repeat sequence results are as follows Fig. 9 shown.

[0108] Example 8

[0109] First, AGP crude polysaccharide was prepared according to the conditions of Example 6, and then AGP-1 purified polysaccharide was prepared according to the conditions of Example 7, and their anti-glycation activities were measured.

[0110] Reaction system establishment: A bovine serum albumin-glucose model was established to simulate the Maillard reaction system to evaluate the anti-glycation ability of the sample. The reaction mixture was 10 mL in total, including 5 mL of 20 mg / mL bovine serum albumin, 2 mL of 500 mM glucose solution, 2 mL of 100 mM PBS solution (pH = 7.4) and 1 mL of polysaccharide sample (2 mg / mL). PBS solution was added to replace the sample as the blank group, PBS solution was added to replace the reaction system as the background group, and aminoguanidine (AG) was used as the positive control. All mixtures were stored at 37°C for 28 days, and samples were taken from the entire mixture every 7 days for testing. Determination of the inhibitory ability of glycosylation reaction products (fluorescent AGEs): 50 μL of glycosylation solution was taken from each mixture and added to a 96-well plate, and then 200 μL of PBS solution was added for dilution. The fluorescence intensity of the late glycosylation product was measured by fluorescence spectrometry, and the excitation wavelength was set to 370 nm and the emission wavelength was set to 440 nm to measure the fluorescence intensity of the reaction solution. Three parallel samples were set for each group.

[0111] Inhibition rate (%) = [1-(A1-A2) / A0] × 100%

[0112] In the formula, A1 is the fluorescence intensity of the mixed solution, A2 is the fluorescence intensity of the background group, and A0 is the fluorescence intensity of the blank group. The formation of AGEs products will cause damage to the protein structure and is closely related to skin aging and body damage. Fig.10 As shown, it can be seen that the AGEs inhibitory activity of crude polysaccharide AGP and purified polysaccharide AGP-1 increases with the storage time, while the anti-glycation activity of purified polysaccharide AGP-1 is close to the inhibitory activity of positive control aminoguanidine. The inhibitory activity of AGP-1 polysaccharide increased to 60.75% at 28 days, and the inhibitory effect was more significant, second only to 65.30% of the AG group; at this time, the inhibitory activity of crude polysaccharide AGP was relatively the worst, only 48.26%.

[0113] Example 9

[0114] AGP crude polysaccharide was prepared under the conditions of Example 6, and then AGP-1 purified polysaccharide was prepared according to the conditions of Example 7, and their antioxidant activities were measured.

[0115] The steps of DPPH free radical scavenging experiment are as follows: prepare 0.2mM DPPH solution with anhydrous ethanol and store it in the dark. Take 100μL of the sample solution to be tested (0.5-5mg / mL) and add it to 100μL of DPPH-ethanol solution. After fully mixing, place it at room temperature for 30min, and measure the sample absorbance at 517nm with an enzyme reader. The steps of ABTS free radical scavenging experiment are as follows: prepare 7.4mM ABTS diammonium salt and 2.6mM K2S2O8 solution in a volume ratio of 1:1, then place it in a dark environment at room temperature for 12-16h, then dilute it with ultrapure water until the solution absorbance is 0.7±0.02, and keep it in the dark for later use. Take 50μL of sample solution (0.5-5mg / mL) and mix it evenly with 200μL of ABTS free radical solution, mix it thoroughly, place it in the dark reaction at room temperature for 30min, and measure the absorbance at 734nm with an enzyme reader. The free radical scavenging rate of polysaccharides was calculated according to the following formula:

[0116] Clearance rate (%) = [1-(A s -A0) / A b ]×100%

[0117] In the formula, A s is the absorbance of the sample reaction solution, A0 is the absorbance of the mixture after pure water replaces the free radical solution, and A b It is the absorbance of pure water instead of sample solution. This experiment uses ascorbic acid (Vc) as a positive control, with sample concentration as the horizontal axis and clearance rate as the vertical axis. The test results are as follows Fig.11 As shown in A, the scavenging rate of DPPH free radicals by the purified polysaccharide AGP-1 of Bamboo Leaf Orchid increases with the increase of concentration. When the polysaccharide concentration is 5 mg / mL, the scavenging rate reaches 36.14%. Fig.11 As shown in B, the drug showed a dose-dependent effect at the tested concentration, and the clearance rate reached 90.00% at the maximum tested concentration of 5 mg / mL. Its IC 50 The value was 1.128 mg / mL. The test results showed that AGP-1 polysaccharide had good antioxidant activity.

[0118] Example 10

[0119] The tyrosinase inhibitory activity of the crude AGP polysaccharide prepared under the conditions of Example 6 and the purified AGP-1 polysaccharide prepared under the conditions of Example 7 was measured. The polysaccharides obtained in other examples had similar test results.

[0120] The steps of the polysaccharide tyrosinase inhibition experiment with different reaction times are as follows: 50 μL of polysaccharide solution (0.25, 1.0, 3.0, 5.0 mg / mL), 50 μL of 100 μg / mL tyrosinase solution and 90 μL of PBS solution were added to a 96-well plate and incubated at 37°C for 30 minutes. After taking out, 60 μL of 2.5 mM L-tyrosine solution was quickly added to each well, and the changes in the absorbance value of the mixed solution at 475 nm during the reaction time of 0-60 minutes (0, 1, 3, 5, 7, 9, 11, 13, 15, 17, 20, 25, 35, 45, 60 minutes) were observed, measured and recorded. The experimental steps for the inhibition of tyrosinase by polysaccharides of different concentrations are as follows: 50 μL of polysaccharide solution (0.25-5.0 mg / mL), 50 μL of 100 μg / mL tyrosinase solution and 90 μL of PBS solution were added to a 96-well plate and incubated at 37°C for 30 minutes. After taking out, 60 μL of 2.5 mM L-tyrosine solution was quickly added to each well, and the absorbance value of the mixed solution at 475 nm after 5 minutes of reaction time was observed and recorded as the sample group; the absorbance value at 475 nm was measured by replacing the sample with PBS buffer, which was recorded as the blank group; the absorbance value at 475 nm was measured by replacing the tyrosinase solution with PBS buffer, which was recorded as the background group. Arbutin was used as a positive control, and each sample was tested in triplicate. The inhibition rate of polysaccharides on tyrosinase was calculated by the following formula:

[0121] Inhibition rate (%) = [1-(A1-A2) / A0] × 100%

[0122] In the formula, A1 is the absorbance of the mixed solution, A2 is the absorbance of the background group, and A0 is the absorbance of the blank group. Fig.12 As shown in A, as the concentration of polysaccharides increases, the absorbance of the mixed solution at 475nm gradually decreases, indicating an increase in the tyrosinase inhibitory activity; in addition, the inhibition of tyrosinase by AGP and AGP-1 is concentration-dependent, and the absorbance of AGP-1 polysaccharide at the same concentration is significantly lower than that of AGP polysaccharide, which also indicates that AGP-1 exhibits stronger tyrosinase inhibitory activity. Fig.12 As shown in B, the inhibition rate of AGP and AGP-1 polysaccharide on tyrosinase increased with the increase of concentration. Among them, the inhibition rate of AGP-1 polysaccharide was as high as 68.22% at 5 mg / mL. Its IC 50 The value was 2.19 mg / mL; while the inhibition rate of the positive control arbutin was 54.53% at the maximum test concentration, and its IC 50The value is 5.35 mg / mL; compared with purified polysaccharides, the inhibitory effect of unpurified polysaccharides is weaker, with an inhibition rate of 53.06% at 5 mg / mL. In addition, the purified polysaccharide AGP-1 obtained in this scheme has an inhibitory activity of 39.70% on tyrosinase at 1 mg / mL, which is significantly higher than the 20% tyrosinase inhibitory activity of the water extract of bamboo leaf orchid in patent application CN202311530205.6. Therefore, this also shows that the purified bamboo leaf orchid high molecular weight polysaccharide AGP-1 exhibits good tyrosinase inhibitory activity.

[0123] Embodiment 11

[0124] A bamboo orchid polysaccharide and a preparation method thereof, comprising the following steps:

[0125] (1) The crude polysaccharide AGP of Bambusa japonica was prepared according to Example 6. The obtained crude polysaccharide was re-dissolved in water, and Sevage reagent was added. The Sevage reagent was prepared by chloroform and n-butanol in a volume ratio of 3:1. The volume ratio of the crude polysaccharide to the Sevage reagent was controlled to be 4:1. The mixture was fully shaken for 90 minutes, and the supernatant was obtained by centrifugation. After the organic reagent was evaporated, the deproteinized polysaccharide was freeze-dried to obtain the deproteinized polysaccharide.

[0126] (2) The deproteinized polysaccharide obtained in step 1) was dissolved in water to prepare a 25 mg / mL solution, and the polysaccharide was separated and purified using a DEAE-Sepharose Fast Flow ion exchange chromatography column, with a sample volume to column volume ratio of 1:4, and gradient elution was performed with distilled water, 0.1 mol / L, 0.4 mol / L, 0.6 mol / L, and 0.8 mol / L NaCl solutions, with a flow rate of 1.0 mL / min, and 10 mL of eluate was collected per tube. The polysaccharide content of each tube was detected by the phenol-sulfuric acid method, and the absorbance was measured at 490 nm to draw a gradient elution curve. The ion exchange column chromatography elution curve of this embodiment is similar to that of Example 7, wherein the distilled water elution component is named Fr.AGP-1, and the components eluted with 0.1 mol / L and 0.4 mol / L NaCl solutions are named Fr.AGP-2 and Fr.AGP-3 in sequence. Among them, the yield of Fr.AGP-1 component is the highest, followed by Fr.AGP-2 component, and the yield of Fr.AGP-3 component is the least (accounting for only 0.5% to 1% of all the purified components obtained, lacking practical utilization value).

[0127] (3) The Fr.AGP-1 and Fr.AGP-2 components were dialyzed for 36 hours using an 8000Da dialysis bag and then freeze-dried. They were then redissolved in water to prepare a solution with a concentration of 15 mg / mL. The two polysaccharides were further separated and purified using a Sephadex CL-2B gel column. The elution was eluted with distilled water at a flow rate of 0.5 mL / min. 5 mL was collected from each tube. The polysaccharide content of each tube was detected by the phenol-sulfuric acid method. The absorbance was measured at 490 nm and the elution curve was drawn. The eluate was collected, dialyzed at 8000Da for 36 hours, and freeze-dried to obtain the purified polysaccharides AGP-1 and AGP-2 from the bamboo orchid.

[0128] (4) Methylation analysis of the purified polysaccharide AGP-1 from Bamboo Orchid. Weigh 10 mg of the AGP-1 polysaccharide prepared in Example 11 and place it in a 25 mL reaction bottle. Dry it in a P2O5 dryer for 24 h, then gradually add 5 mL of dimethyl sulfoxide (fully dehydrated with 3A molecular sieves). Then, the polysaccharide is completely dissolved with the assistance of ultrasound, and 20 mg of NaOH powder (dried and anhydrous) is quickly added under nitrogen protection, and ultrasound is continued for 20 min. After completion, the reactor is placed in an ice-water bath, and 3 mL of iodomethane is slowly added under nitrogen protection, and then the reaction is protected from light for 12 h. After the reaction is completed, 3 mL of ultrapure water is added to quench the methylation reaction, and the solution is transferred to a 3500 Da dialysis bag and dialyzed until the reaction solution is clear and transparent (change the water every 8 h), and then freeze-dried to obtain the methylated polysaccharide. If the infrared spectrum of the methylated polysaccharide is 3500 cm -1 If there is still an OH peak around , methylation needs to be repeated until the OH peak disappears completely, that is, the methylation is complete. Then take 5 mg of completely methylated polysaccharide in a reaction bottle, add 5 mL of 2M trifluoroacetic acid, seal it, and place it in a 121°C oil bath for hydrolysis for 3 hours. After the hydrolyzate is cooled, evaporate it under reduced pressure and add methanol to wash it repeatedly 3-5 times to remove excess trifluoroacetic acid. Then dissolve the product in 3 mL of distilled water, add 25 mg of sodium borohydride, react at room temperature for 12 hours, and adjust the pH of the solution to acidic with 0.1 M acetic acid after the reaction is completed, and evaporate it to dryness. Wash it with methanol 3 times and evaporate it under pressure. Then add 3 mL of acetic anhydride to it and react it in an oil bath at 100°C for 2 hours. After taking it out, evaporate it under reduced pressure, repeatedly add methanol 3 times to wash it, and evaporate it to dryness. Then dissolve the acetylated product in 4 mL of chloroform, add ultrapure water to wash 3 times, remove the aqueous phase, add 3A molecular sieves to dry the chloroform layer, concentrate it to about 1 mL under reduced pressure, pass it through a 0.22 μm filter membrane, and perform GC-MS detection.

[0129] GC-MS analysis conditions: Agilent 8890-7000D triple quadrupole gas chromatography-mass spectrometer was used, the chromatographic column was HP-5MS (30m×0.25mm×0.25μm) capillary, high-purity helium was used as the carrier gas, the flow rate was 1mL / min, the injection volume was 1μL, the split ratio was 10:1, and the injection port temperature was 250℃; the heating program was: the starting temperature was 120℃, maintained for 1min; then heated to 210℃ at 3℃ / min, maintained for 2min; heated to 260℃ at 10℃ / min, maintained for 5min. The ion source was electron bombardment, the collision energy was 70eV, and the mass range was 30-600m / z.

[0130] The results are as follows Fig.13 As shown, by analyzing the mass spectrum and ion flow diagram, it can be obtained that the main sugar residues of the AGP-1 polysaccharide prepared in Example 11 include →4)-Manp-(1→(55.84%), →4)-Glcp-(1→(36.49%), →3,4)-Galp-(1→(3.85%), →4,6)-Galp-(1→(2.44%) and Glcp-(1→(1.37%). Compared with Example 7, the molar ratio of mannose (Man), glucose (Glc) and galactose (Gal) in the AGP-1 polysaccharide prepared in Example 11 is also 10:7:1, indicating that the AGP-1 component can be repeatedly obtained under different preparation conditions.

[0131] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A bamboo orchid polysaccharide, characterized in that: The bamboo orchid polysaccharide is glucomannan, which is formed by connecting (1→4)-linked α-D-type mannose and (1→4)-linked α-D-type glucose.

2. The Bamboo Orchid polysaccharide according to claim 1, characterized in that: The bamboo orchid polysaccharide comprises (1→4)-linked α-D-type mannose and (1→4)-linked α-D-type glucose connected in a molar ratio of 3:

2.

3. The Bamboo Orchid polysaccharide according to claim 1, characterized in that: The bamboo orchid polysaccharide has the following structural formula:

4. The Bamboo Orchid polysaccharide according to claim 3, characterized in that: In the structural formula of the Bamboo Orchid polysaccharide, R1 has the following structural formula: aD-Glcp-(1→4)-aD-Manp(1→.

5. The Bamboo Orchid polysaccharide according to claim 3, characterized in that: In the structural formula of the Bamboo Orchid polysaccharide, R2 has the following structural formula:

6. A method for preparing the polysaccharide of Bamboo Orchid as claimed in claim 1, characterized in that: The steps include: A. defatting the Bamboo Leaf Orchid raw material to obtain a defatted mixture; B. subjecting the defatted mixture to water extraction to obtain a polysaccharide extract; C. Concentrating the polysaccharide extract to obtain a polysaccharide concentrate; D. subjecting the polysaccharide concentrate to alcohol precipitation to obtain a crude polysaccharide precipitate; E. deproteinizing the crude polysaccharide precipitate to obtain deproteinized polysaccharide; F. subjecting the deproteinized polysaccharide to ion exchange column chromatography to obtain a preliminary separated polysaccharide; G. Subjecting the preliminarily separated polysaccharides to gel column chromatography to obtain the Bamboo Orchid polysaccharide.

7. The method for preparing Bamboo Orchid Polysaccharide according to claim 6, characterized in that: In step F, the eluent of the ion exchange column chromatography separation includes NaCl solution; the concentration of the NaCl solution includes 0 to 0.99 mol / L; the filler of the ion exchange column chromatography separation includes one or more of CM-Sepharose Fase Flow, DEAE-52cellulose, and DEAE-Sepharose FastFlow; in step G, the eluent of the gel column chromatography separation includes distilled water; the filler of the gel column chromatography separation includes one or more of Sepharose CL-2B, Sephadex G-200, and Bio-Gel P-30.

8. The method for preparing Bamboo Orchid Polysaccharide according to claim 6, characterized in that: The steps include: A. Using dried rhizomes of Bambusa chinensis as raw materials, crushing and sieving, and then adding an organic solvent to perform the degreasing treatment to obtain the degreasing mixture; B. adding water to the defatted mixture at a liquid-to-solid ratio of 5 to 50:1 mL / g, performing the water extraction treatment at 60 to 100° C., filtering and combining the extracts to obtain the polysaccharide extract; C. performing the concentration treatment on the polysaccharide extract by reduced pressure concentration to obtain the polysaccharide concentrate; D. adding ethanol to the polysaccharide concentrate to make the alcohol content range from 65% to 85% by volume, standing overnight at low temperature to perform the alcohol precipitation treatment, filtering out the supernatant, and obtaining the crude polysaccharide precipitate; E. Dissolving the crude polysaccharide precipitate in water, adding Sevage reagent at a volume ratio of 3 to 5:1, performing the deproteinization treatment using the Sevage method, centrifuging the upper polysaccharide liquid, evaporating the reagent and freeze-drying to obtain the deproteinized polysaccharide; the Sevage reagent comprises chloroform and n-butanol mixed at a volume ratio of 1 to 5:1; F. Redissolving the deproteinized polysaccharide in water, performing the ion exchange column chromatography separation, collecting the polysaccharide eluate and performing the first dialysis, and then freeze-drying to obtain the preliminary separated polysaccharide; the first dialysis uses a dialysis bag with a molecular weight cutoff of less than 10000Da; G. Redissolving the initially separated polysaccharide in water, performing the gel column chromatography separation, collecting the polysaccharide eluate and performing a second dialysis to obtain the Bambusa japonica polysaccharide; the second dialysis uses a dialysis bag with a molecular weight cutoff of less than 10,000 Da.

9. The method for preparing Bamboo Orchid Polysaccharide according to claim 8, characterized in that: In step A, the crushing and screening includes passing through a 24-80 mesh sieve; the amount of the organic solvent added includes a liquid-to-solid ratio of 10-20:1 mL / g; the organic solvent includes one or more of ether, petroleum ether, dichloromethane, chloroform, anhydrous ethanol, and ethyl acetate; in step B, the number of water extraction treatments includes 1-3 times, and the treatment time for each treatment includes 0.5-2.5 hours; in step C, the concentration treatment includes concentrating under reduced pressure to a volume of 1 / 3-1 / 10 of the original volume; in step D, the alcohol content includes 70% by volume 80%; the low temperature overnight standing includes standing at 4°C overnight; in step E, the Sevage reagent includes chloroform and n-butanol mixed in a volume ratio of 3 to 4:1; in step F, the eluent for ion exchange column chromatography separation includes distilled water; the first dialysis uses a dialysis bag with a molecular weight cutoff of 3500 to 8000; the first dialysis time is greater than 12 hours; in step G, the second dialysis uses a dialysis bag with a molecular weight cutoff of 3500 to 8000; the second dialysis time is greater than 12 hours.

10. An application of the Bamboo Orchid polysaccharide as claimed in claim 1, characterized in that: Used in preparing products including one or more of anti-glycation, anti-oxidation and inhibition of tyrosinase activity.

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