A polysaccharide of dendrobium officinale and a preparation method and application thereof

Abstract

The application discloses a polysaccharide of arundina griffithii and a preparation method and application thereof. The arundina griffithii rhizome is pretreated by defatting, then extracted by water according to a liquid-material ratio of 5:1-50:1 mL / g, and immersed twice at a temperature of 60-100 DEG C, each time for 0.5-2.5 h; after the immersion liquid is concentrated and alcohol precipitated, the protein is removed by Sevage method, purified by a chromatographic column, and the arundina griffithii polysaccharide is prepared after dialysis. The application can prepare various novel polysaccharides from the arundina griffithii at the same time, and the purity is high; the AGP-1 component is novel in structure, and the molecular weight is uniform; the average molecular weight is 494.3 kDa; the core main chain is composed of (1→4) connected alpha-D type mannose and (1→4) connected alpha-D type glucose in a molar ratio of 3:2; and the two ends are connected with alpha-D type galactose and beta-D type galactose respectively; the arundina griffithii polysaccharide not only can block the glycosylation reaction to play a protein glycosylation inhibition role and delay skin aging, but also has good tyrosinase inhibition activity and reduces melanin accumulation, and 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 particularly to a bamboo orchid polysaccharide and a preparation method and application thereof. Background Art

[0002] Modern research shows that plant polysaccharides have excellent 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 for use as a cosmetic raw material. This patent obtains a Bletilla striata polysaccharide through water extraction, enzyme processing, and chromatographic purification. In vitro experiments show that the Bletilla striata polysaccharide complex can repair the skin barrier, increase skin moisture content, whiten the skin, and delay skin aging. Chinese invention patent CN200610145677.X discloses a method for preparing Dendrobium officinale polysaccharide with immune-enhancing function through water extraction and alcohol precipitation. This patent demonstrates through in vivo mouse experiments that the polysaccharide can significantly enhance mouse immunity and has virtually no toxic side effects.

[0003] Glycation is an oxidative stress reaction in the body that leads to the oxidation of carbohydrates. This in turn forms highly reactive and electrophilic compounds that attack free amino groups in proteins, causing covalent modification and the production of advanced glycation end products (AGEs). AGEs can cause collagen and elastin in the dermis to lose their support and elasticity, resulting in yellowing, dull skin, enlarged pores, and a loss of elasticity. Therefore, glycation is a key factor in aging and dull skin. Therefore, blocking the AGE reaction can inhibit protein glycation and thus slow aging.

[0004] Bamboo orchid is an important medicinal plant of the Orchidaceae family. It is a unique antidote (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 traditional Chinese medicine compound prescriptions. It is often used externally to treat trauma, scalds, and burns in clinical practice. Invention patent application CN202311530205.6 discloses a bamboo orchid extract, its preparation method, and its application in cosmetics. The application uses conventional solvents and a general extraction method to prepare a crude bamboo orchid extract, that is, ultrasonic extraction with a 0-80% ethanol aqueous solution, preferably an 80% ethanol solution, and the extract is then concentrated and freeze-dried to obtain a crude extract. The application also studies 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. However, the bamboo orchid crude extract prepared by existing extraction technology contains almost no high-purity macromolecular polysaccharides. Currently, research on Bamboo Leaf Orchid mostly focuses on small molecule compounds such as stilbenes, flavonoids and phenols. There are no reports in the literature on the structure and functionality of Bamboo Leaf Orchid's macromolecular polysaccharides and their 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 isolated 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 present invention aims to provide a polysaccharide from Bamboo Shoots and its preparation method and application. The technical problems to be solved are the effective separation and purification of functional polysaccharides from Bamboo Shoots, the analysis of the novel structure of polysaccharides, and the study of their anti-glycosylation activity and tyrosinase inhibitory activity.

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

[0010] Preferably, the Bambusa japonica 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, is connected at both ends to α-D-galactose and β-D-galactose 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. Degreasing the Bamboo Herb 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 polysaccharide to gel column chromatography to obtain the Bamboo Herb polysaccharide.

[0029] The present invention can simultaneously prepare multiple novel high-molecular-weight polysaccharides from bamboo orchid with high purity. Among them, the AGP-1 component has a novel structure and is a glucomannan galactan with a uniform molecular weight and an average molecular weight of 494.3 kDa. The core main chain is composed of a glycoside repeating sequence of (1→4)-linked α-D-mannose and (1→4)-linked α-D-glucose in a molar ratio of 3:2, 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 glycation inhibitory effect and delay skin aging, but also exert good tyrosinase inhibitory activity to reduce melanin accumulation, and can be used in whitening and 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 japonica as raw materials, crushing and screening, 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 concentrating under reduced pressure 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 at low temperature overnight 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 a first dialysis to obtain the preliminary separated polysaccharide; the first dialysis uses a dialysis bag with a molecular weight cut-off of less than 10,000 Da;

[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 cut-off of less than 10,000 Da.

[0039] The impurity problem in the existing technology 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 precipitates by setting an alcohol content of 65-85%, preferably 70-80%, for alcohol precipitation, and the supernatant containing small molecules is removed by filtration; the obtained crude polysaccharide is then purified by column chromatography. 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 applicable to a wide range of molecular weights) to obtain polysaccharides within the corresponding molecular weight range, thereby removing most monosaccharides and oligosaccharides. The above steps 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 a preliminary separated polysaccharide; the preliminary separated polysaccharide is further purified by gel column chromatography, and the polysaccharide eluate is collected and dialyzed to obtain the purified Bamboo Cymbidium polysaccharide, namely 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 each treatment time includes 0.5-2.5 hours; in step C, the concentration treatment includes decompression concentration 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 the 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 time of the first dialysis 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 time of the second dialysis 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 elution is better.

[0043] An application of the above-mentioned bamboo orchid polysaccharide is used to prepare a product having one or more 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 inhibitory activity, reduces melanin accumulation, and can be used in cosmetics for whitening and skin care.

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

[0046] 1) There has been no research on polysaccharides in Bamboo Spathiphyllum in the prior art, but the present invention can simultaneously prepare multiple Bamboo Spathiphyllum high-molecular polysaccharides with novel structures and high purity. The AGP-1 component has a uniform molecular weight, and 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 this method can block the glycation reaction and inhibit the formation of AGEs products, thereby delaying skin aging. At the same time, it also exhibits good tyrosinase inhibitory activity and can be used as a natural product for whitening and skin care in the cosmetics field. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The extraction yield and total soluble sugar content of the crude polysaccharide AGP of Bamboo Herb in Examples 1-6 are shown.

[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 Shoots 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] Figure 9 Schematic diagram of the structure of the purified AGP-1 polysaccharide of Example 7.

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

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

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

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

[0061] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to 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. Then, the Bamboo Leaf Orchid powder was mixed with petroleum ether at a liquid-to-solid ratio of 10:1 mL / g, 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) Ethanol was added to the concentrated solution obtained in step 2) to adjust the ethanol concentration of the mixed solution to 80% by volume. The mixture was allowed to stand at 4°C overnight, and the precipitate was filtered and freeze-dried to obtain crude AGP polysaccharide from the herb. The total soluble sugar content was determined by the phenol-sulfuric acid method, and the crude polysaccharide yield was calculated based on the dry weight of the raw material from the herb.

[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) Ethanol was added to the extract obtained in step 2) to adjust the ethanol concentration of the mixed solution to 65% by volume. The mixture was allowed to stand at 4°C overnight, and the precipitate was filtered and freeze-dried to obtain crude AGP polysaccharide from the herb. The total soluble sugar content was determined by the phenol-sulfuric acid method, and the crude polysaccharide yield was calculated based on the dry weight of the raw material.

[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. Then, the Bamboo Leaf Orchid powder was mixed with ether at a liquid-to-solid ratio of 15:1 mL / g, 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) Ethanol was added to the concentrated solution obtained in step 2) to adjust the ethanol concentration of the mixed solution to 70% by volume. The mixture was allowed to stand at 4°C overnight, and the precipitate was filtered and freeze-dried to obtain crude AGP polysaccharide from the herb. The total soluble sugar content was determined by the phenol-sulfuric acid method, and the crude polysaccharide yield was calculated based on the dry weight of the raw material.

[0077] Example 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) Ethanol was added to the concentrated solution obtained in step 2) to adjust the ethanol concentration of the mixed solution to 75% by volume. The mixture was allowed to stand at 4°C overnight, and the precipitate was filtered and freeze-dried to obtain crude AGP polysaccharide from the herb. The total soluble sugar content was determined by the phenol-sulfuric acid method, and the crude polysaccharide yield was calculated based on the dry weight of the raw material from the herb.

[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) Ethanol was added to the concentrated solution obtained in step 2) to adjust the ethanol concentration of the mixed solution to 85% by volume. The mixture was allowed to stand at 4°C overnight, and the precipitate was filtered and freeze-dried to obtain crude AGP polysaccharide from the herb. The total soluble sugar content was determined by the phenol-sulfuric acid method, and the crude polysaccharide yield was calculated based on the dry weight of the 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) Ethanol was added to the concentrated solution obtained in step 2) to adjust the ethanol concentration of the mixed solution to 80% by volume. The mixture was allowed to stand at 4°C overnight, and the precipitate was filtered and freeze-dried to obtain crude AGP polysaccharide from the herb. The total soluble sugar content was determined by the phenol-sulfuric acid method, and the crude polysaccharide yield was calculated based on the dry weight of the 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 Bambusa japonica prepared by the method of Example 6 was dissolved in water again, and Sevage reagent was added. Sevage reagent was prepared by chloroform and n-butanol in a volume ratio of 4:1. The volume ratio of crude polysaccharide solution to Sevage reagent was controlled to be 5:1. The mixture was fully shaken for 60 minutes, centrifuged to obtain the supernatant, evaporated the organic reagent, and freeze-dried to obtain 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. The polysaccharide was separated and purified using a DEAE-52cellulose ion exchange chromatography column. The sample volume to column volume ratio was 1:5. Gradient elution was performed using distilled water, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, and 0.4 mol / L NaCl solutions. The flow rate was controlled at 1.0 mL / min, and 10 mL of 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 0.1 mol / L and 0.4 mol / L NaCl solutions were named Fr.AGP-2 and Fr.AGP-3, respectively. Fr.AGP-1 had the highest yield, followed by Fr.AGP-2, and Fr.AGP-3 had the lowest yield (accounting for only 0.5% to 1% of all purified fractions, lacking practical value).

[0097] (3) The Fr.AGP-1 and Fr.AGP-2 fractions were dialyzed for 48 h using a 3500 Da dialysis bag and then freeze-dried. They were then redissolved in water to prepare a 10 mg / mL solution. The two polysaccharides were further separated and purified using a Sephadex G-200 gel chromatography column. The elution was performed using 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 ( 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 content of the purified components of AGP-1 and AGP-2 was 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 content of the two components was 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 Shoots. 5 mg of AGP-1 and AGP-2 were mixed with an appropriate amount of fully dried KBr powder and ground evenly. The mixture was pressed into tablets using a tablet press and scanned using a Fourier transform infrared spectrometer (Nicolet iS10, Thermo Fisher Scientific, USA) with a 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 appearing around 1734cm may be related to the stretching vibration of the saturated CH bond; -1 The absorption peak at 1260 cm is caused by the stretching vibration of C=O in acetyl or carboxylic acid ester; -1 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; it is worth noting that the absorption peak of AGP-1 polysaccharide is at 810 cm -1 and 875cm -1 The sharp peak at the bottom indicates that both α- and β-type glycosidic bonds may exist in the polysaccharide.

[0100] (6) Molecular weight determination of purified polysaccharides AGP-1 and AGP-2 from Bamboo Shoot. The Thermo Ultimate 3000 high performance liquid chromatograph was used, the detector was a RefractoMax 521 differential detector, and a series of Ultra hydrogel 1000 (7.8×300 mm) and Ultra hydrogel 500 (7.8×300 mm) columns were used. The mobile phase was a 20 mM KH2PO4 buffer solution. The flow rate was set at 0.8 mL / min, the injection volume was 20 μL, and the column temperature was 35°C. The results are shown in Figure 2. 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 primarily composed of 39.4 kDa, 2.62 kDa, and 1.57 kDa fractions, not a single polysaccharide. Furthermore, the yield of AGP-1 polysaccharide is approximately 85%-90%, while the yield of AGP-2 polysaccharide is approximately 10%-15%. Based on the polysaccharide yield and purity, the AGP-1 fraction with the highest yield and 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-resistant bottle, 2 mL of trifluoroacetic acid (2 M) was added, the bottle was sealed with a sealing film, and the solution was fully hydrolyzed in an oil bath at 121°C for 2 h. The solution was removed and allowed to cool, then evaporated to dryness under reduced pressure. Methanol was then added and rinsed 2-3 times to remove the trifluoroacetic acid. Finally, 1 mL of deionized water was added to dissolve the solution. 500 μL of polysaccharide hydrolyzate was mixed with 500 μL of 0.3 M NaOH solution, 500 μL of 0.5 M PMP-methanol solution was added, the mixture was fully mixed, and the solution 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. 1 mL of chloroform was then added to extract and remove PMP three times. The supernatant was filtered through a 0.45 μm filter membrane, and the filtrate was subjected to liquid phase testing. Liquid chromatography detection conditions were as follows: Agilent 1260 high performance liquid chromatography was used, and Infinity Lab C 18 The chromatographic column was (4.6×250mm, 5μm), the column temperature was 30°C, 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 and dissolved in distilled water, and then derivatized and determined 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 based on 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 galactan.

[0102] (8) NMR analysis of the purified polysaccharide AGP-1 from Bamboo Shoots. Weigh 15 mg of AGP-1 and dissolve it in 0.5 mL of D2O. Place the supernatant in an NMR tube and use a Bruker AV-500 spectrometer with a 5 mm probe to obtain the NMR spectrum. 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), δ 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 other protons of glycosides. 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 carbon atoms of the glycoside. 1 H and 13 C NMR and HSQC spectra ( Figure 8 In Figure 5(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. Based on 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. The corresponding C-2 to C-6 signals were then obtained using the direct carbon-hydrogen 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 HMBC spectrum ( Figure 8C) was used to determine the order of glycosidic bond connection in the purified polysaccharide AGP-1. The results showed that the correlation peaks of residues A (H-1) and residues C (H-1) with residues B (C-4) were δ5.41 / 71.69 ppm, respectively, 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 residues A (H-4) and residues B (C-1) were δ3.65 / 99.70 ppm. 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 δ5.42 / 71.69ppm between residue B(H-1) and its own C-4, which also indicates the existence of repeated fragments related to each other by mannose, 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 The glycosidic bond connection modes exist: -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)-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→

[0107] Combined with the results of methylation analysis and one-dimensional and two-dimensional nuclear magnetic resonance data, it is speculated that the possible structure of the purified polysaccharide AGP-1 from Bamboo Herb is mainly connected by mannose and glucose, while →4)-α-D-Glcp-(1→4)-α-D-Manp-(1→) serves as the polysaccharide backbone. The glycoside repeat sequence results are as follows: Figure 9 shown.

[0108] Example 8

[0109] First, crude AGP polysaccharide was prepared according to the conditions of Example 6, and then purified AGP-1 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 samples. The reaction mixture totaled 10 mL and consisted of 5 mL of 20 mg / mL bovine serum albumin, 2 mL of 500 mM glucose, 2 mL of 100 mM PBS (pH 7.4), and 1 mL of polysaccharide sample (2 mg / mL). PBS was added in place of the sample as a blank control, and PBS was added in place of the reaction system as a background control. Aminoguanidine (AG) was used as a positive control. All mixtures were stored at 37°C for 28 days, with samples collected from the entire mixture every 7 days for testing. Determination of the inhibitory ability of glycation reaction products (fluorescent AGEs): 50 μL of the glycosylation solution from each mixture was added to a 96-well plate and diluted with 200 μL of PBS. The fluorescence intensity of the advanced glycation products was measured by fluorescence spectrometry using an excitation wavelength of 370 nm and an emission wavelength of 440 nm. Three replicates 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. Figure 10 As shown, the AGE inhibitory activity of crude and purified AGP-1 polysaccharides increased with storage time, while the anti-glycation activity of purified AGP-1 polysaccharide was close to that of the positive control aminoguanidine. The AGP-1 polysaccharide inhibitory activity increased to 60.75% at 28 days, a significant improvement, second only to the 65.30% of the AG group. At this time, the inhibitory activity of crude AGP polysaccharide was relatively poor, at 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 determined.

[0115] The DPPH free radical scavenging assay was performed as follows: a 0.2 mM DPPH solution was prepared in anhydrous ethanol and stored in the dark. 100 μL of the sample solution (0.5-5 mg / mL) was added to 100 μL of the DPPH-ethanol solution. The solution was mixed thoroughly and allowed to stand at room temperature for 30 minutes. The absorbance of the sample was measured at 517 nm using a microplate reader. The ABTS free radical scavenging assay was performed as follows: a 7.4 mM ABTS diammonium salt solution was mixed with a 2.6 mM K₂S₂O₂ solution in a 1:1 volume ratio. The solution was then allowed to stand at room temperature in the dark for 12-16 hours. The solution was then diluted with ultrapure water to an absorbance of 0.7±0.02 and stored in the dark. 50 μL of the sample solution (0.5-5 mg / mL) was mixed with 200 μL of the ABTS free radical solution. The mixture was mixed thoroughly and allowed to stand in the dark for 30 minutes at room temperature. The absorbance was measured at 734 nm using a microplate reader. The free radical scavenging rate of polysaccharides was calculated according to the following formula:

[0116] Clearance (%) = [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 The absorbance of pure water instead of sample solution is shown in Figure 2. This experiment uses ascorbic acid (Vc) as a positive control, with sample concentration as the horizontal axis and clearance rate as the vertical axis. Figure 11 As shown in A, the scavenging rate of DPPH free radicals by the purified polysaccharide AGP-1 from Bamboo Stalks increased with the increase of concentration. When the polysaccharide concentration was 5 mg / mL, the scavenging rate reached 36.14%. Figure 11 As shown in B, the drug showed a dose-dependent effect at the tested concentrations, 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 has good antioxidant activity.

[0118] Example 10

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

[0120] The following steps were used to perform the polysaccharide tyrosinase inhibition assay with different reaction times: 50 μL of polysaccharide solution (0.25, 1.0, 3.0, and 5.0 mg / mL), 50 μL of 100 μg / mL tyrosinase solution, and 90 μL of PBS were added to a 96-well plate and incubated at 37°C for 30 minutes. After removal, 60 μL of 2.5 mM L-tyrosine solution was quickly added to each well. The absorbance changes at 475 nm were observed, measured, and recorded over the reaction time of 0–60 minutes (0, 1, 3, 5, 7, 9, 11, 13, 15, 17, 20, 25, 35, 45, and 60 minutes). 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 respectively 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, which was recorded as the sample group; the absorbance value at 475 nm was measured using PBS buffer instead of the sample, which was recorded as the blank group; the absorbance value at 475 nm was measured using PBS buffer instead of the tyrosinase solution, which was recorded as the background group. Arbutin was used as a positive control, and each sample was repeated in triplicate. The inhibition rate of polysaccharide on tyrosinase was calculated using 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. Figure 12 As shown in Figure A, as the concentration of polysaccharide increases, the absorbance of the mixture at 475 nm gradually decreases, indicating an increase in 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. Figure 12 As shown in Figure 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, and 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 was 5.35 mg / mL. Compared to the purified polysaccharide, the unpurified polysaccharide had a weaker inhibitory effect, with an inhibition rate of 53.06% at 5 mg / mL. Furthermore, the purified polysaccharide AGP-1 obtained in this protocol exhibited a 39.70% tyrosinase inhibitory activity at 1 mg / mL, significantly higher than the 20% tyrosinase inhibitory activity of the Bamboo Leaf Orchid aqueous extract reported in patent application CN202311530205.6. This also demonstrates that the purified Bamboo Leaf Orchid high-molecular-weight polysaccharide AGP-1 exhibits excellent tyrosinase inhibitory activity.

[0123] Example 11

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

[0125] (1) According to Example 6, crude polysaccharide AGP of Bambusa japonica was prepared. The obtained crude polysaccharide was re-dissolved in water, and Sevage reagent was added. Sevage reagent was prepared by chloroform and n-butanol in a volume ratio of 3:1. The volume ratio of crude polysaccharide to Sevage reagent was controlled to be 4:1. The mixture was fully shaken for 90 minutes, centrifuged to obtain the supernatant, evaporated the organic reagent, and then freeze-dried to obtain 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. The sample volume to column volume ratio was 1:4, and gradient elution was performed using distilled water, 0.1 mol / L, 0.4 mol / L, 0.6 mol / L, and 0.8 mol / L NaCl solutions, respectively. The flow rate was controlled to be 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 least is Fr.AGP-3 component (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 fractions were dialyzed for 36 h using an 8000 Da dialysis bag and freeze-dried. They were then redissolved in water to prepare a 15 mg / mL solution. The two polysaccharides were further separated and purified using a Sephadex CL-2B gel column. The elution was performed 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 determined by the phenol-sulfuric acid method. The absorbance was measured at 490 nm, and an elution curve was drawn. The eluate was collected, dialyzed for 36 h at 8000 Da, 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 of 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 the 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 carried out in the dark 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 polysaccharide after methylation is at 3500 cm -1 If OH peaks are still present, repeat the methylation process until the OH peaks disappear, indicating complete methylation. Next, place 5 mg of the fully methylated polysaccharide in a reaction flask, add 5 mL of 2 M trifluoroacetic acid, seal the flask, and hydrolyze in a 121°C oil bath for 3 h. After cooling, evaporate to dryness under reduced pressure and wash with methanol 3-5 times to remove excess trifluoroacetic acid. The product is then dissolved in 3 mL of distilled water, and 25 mg of sodium borohydride is added. The reaction is continued at room temperature for 12 h. After completion, the pH of the solution is adjusted to acidic with 0.1 M acetic acid and evaporated to dryness. Wash with methanol three times and evaporate to dryness under reduced pressure. Then, add 3 mL of acetic anhydride and react in a 100°C oil bath for 2 h. Remove the flask and evaporate to dryness under reduced pressure. Repeat the washing process with methanol three times and evaporate to dryness. The acetylated product is then dissolved in 4 mL of chloroform and washed three times with ultrapure water. After removing the aqueous phase, the chloroform layer is dried over 3A molecular sieves and concentrated to approximately 1 mL under reduced pressure. The chloroform layer is filtered through a 0.22 μm filter and analyzed by GC-MS.

[0129] GC-MS analysis conditions: An Agilent 8890-7000D triple quadrupole gas chromatography-mass spectrometer was used, with an HP-5MS (30 m × 0.25 mm × 0.25 μm) capillary column. High-purity helium was used as the carrier gas at a flow rate of 1 mL / min, an injection volume of 1 μL, a split ratio of 10:1, and an inlet temperature of 250°C. The temperature program was as follows: initial temperature 120°C, hold for 1 min; then, increase the temperature at 3°C / min to 210°C, hold for 2 min; and then increase the temperature at 10°C / min to 260°C, hold for 5 min. The ion source was electron impact with a collision energy of 70 eV, and the mass range was 30–600 m / z.

[0130] The results are as follows Figure 13 As shown in the figure, by analyzing the mass spectrum and ion current 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 merely a preferred embodiment of the present invention and 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, the main chain of which 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.

2. The Bamboo Herb polysaccharide according to claim 1, characterized in that The bamboo orchid polysaccharide has the following structural formula: 。 3. The Bamboo Herb polysaccharide according to claim 2, characterized in that In the structural formula of the Bamboo Orchid polysaccharide, R1 has the following structural formula: 。 4. The Bamboo Herb polysaccharide according to claim 2, characterized in that In the structural formula of the Bamboo Orchid polysaccharide, R2 has the following structural formula: 。 5. A method for preparing the polysaccharide of Bamboo Herb as claimed in claim 1, characterized in that: The steps include: A. Degreasing the Bamboo Herb 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 polysaccharide to gel column chromatography to obtain the Bamboo Herb polysaccharide.

6. The method for preparing the polysaccharide of Bamboo Herb as claimed in claim 5, characterized in that: In step F, the eluent for the ion exchange column chromatography separation includes a NaCl solution; the concentration of the NaCl solution includes 0~0.99 mol / L; the filler for the ion exchange column chromatography separation includes one or more of CM-Sepharose Fase Flow, DEAE-52 cellulose, 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.

7. The method for preparing Bamboo Herb Polysaccharide according to claim 5, characterized in that: The steps include: A. Using dried rhizomes of Bambusa japonica as raw materials, crushing and screening, 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 5-50:1 mL / g, performing the water extraction treatment at 60-100° C., filtering and combining the extracts to obtain the polysaccharide extract; C. performing the concentration treatment on the polysaccharide extract by concentrating under reduced pressure 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 at low temperature overnight 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 deproteinization using a Sevage method, separating the upper polysaccharide liquid by centrifugation, 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 a first dialysis, followed by freeze-drying to obtain the preliminary separated polysaccharide; the first dialysis uses a dialysis bag with a molecular weight cut-off of less than 10,000 Da; G. Redissolving the preliminarily separated polysaccharide in water, performing the gel column chromatography separation, collecting the polysaccharide eluate and performing a second dialysis to obtain the Bamboo Herb polysaccharide; the second dialysis uses a dialysis bag with a molecular weight cut-off of less than 10,000 Da.

8. The method for preparing the polysaccharide of Bamboo Herb as claimed in claim 7, wherein: 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 each treatment time includes 0.5-2.5 h; 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 70-80% by volume; the low-temperature standing overnight includes standing at 4°C overnight; in step E, the Sevage reagent includes chloroform and n-butanol mixed in a volume ratio of 3-4:1; in step F, the eluent for the ion exchange column chromatography separation includes distilled water; the first dialysis uses a dialysis bag with a molecular weight cutoff of 3500-8000; the time of the first dialysis is greater than 12 h; in step G, the second dialysis uses a dialysis bag including a molecular weight cutoff of 3500~8000; the second dialysis time is greater than 12 h.

9. An application of the Bamboo Herb polysaccharide according to claim 1, characterized in that: Used in preparing one or more products including anti-glycation, anti-oxidation and inhibition of tyrosinase activity.

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