Dendrobium polysaccharide, preparation method and application thereof, and polysaccharide-pigment complex and application thereof

Abstract

This invention provides Dendrobium flower polysaccharides, their preparation methods, and applications, as well as polysaccharide-pigment complexes and their applications, relating to the field of natural pigment technology. The repeating units in the Dendrobium flower polysaccharides provided by this invention are partially acetylated, and the repeating units in the main chain of the Dendrobium flower polysaccharides have specific glycosidic bond linkages, enabling the Dendrobium flower polysaccharides to self-assemble with zeaxanthin and / or anthocyanins to form stable polysaccharide-pigment complexes. Specifically, Dendrobium nobile flower polysaccharides and Dendrobium dentate flower polysaccharides can self-assemble with anthocyanins to form Dendrobium nobile flower polysaccharide-anthocyanin complexes and Dendrobium dentate flower polysaccharide-anthocyanin complexes, respectively. Dendrobium truncatum flower polysaccharides can self-assemble with zeaxanthin to form Dendrobium truncatum flower polysaccharide-zeaxanthin complexes, thereby achieving the stability of zeaxanthin, paeoniflorin-3-O-glucoside, and delphinidin-3-O-glucoside. The self-assembled polysaccharide-zeaxanthin complexes exhibit excellent thermal and light stability.

Description

Technical Field

[0001] This invention relates to the field of natural pigment technology, specifically to Dendrobium flower polysaccharides and their preparation methods and applications, and polysaccharide-pigment complexes and their applications. Background Technology

[0002] Anthocyanins are a class of water-soluble pigments widely found in flowers, fruits, and vegetables. They exist in the form of glycosides (i.e., anthocyanins), giving plants their red, purple, and blue colors. Common anthocyanins include cyanidin, delphinidin, and peonidin. Carotenoids (such as zeaxanthin, ZEA) are typically the main contributors to the yellow color in flowers. They are not only vibrant in color, but studies have shown that their intake is associated with a reduced risk of various chronic diseases, such as cancer, diabetes, cardiovascular disease, and age-related non-communicable diseases. Therefore, anthocyanins and zeaxanthin have broad prospects in the fields of natural pigments and functional food additives. However, anthocyanins and zeaxanthin are extremely sensitive to environmental factors such as temperature and light, and are prone to degradation and fading during processing and storage, which severely limits their industrial application. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide Dendrobium flower polysaccharide, its preparation method and application, and polysaccharide-pigment complex and its application. The Dendrobium flower polysaccharide provided by this invention can self-assemble with paeoniflorin-3-O-glucoside, delphinidin-3-O-glucoside and zeaxanthin to form a polysaccharide-pigment complex, stabilizing the above pigments.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides Dendrobium flower polysaccharides, including one or more of Dendrobium nobile flower polysaccharides, Dendrobium chrysanthum flower polysaccharides, and Dendrobium dentata flower polysaccharides, wherein some of the O atoms in the repeating units that make up the Dendrobium flower polysaccharides are acetylated. The relative molecular weight of the Dendrobium flower polysaccharide is 1×10⁻⁶. 4 ~1.4×10 6 Da; The repeating units in the main chain of the Dendrobium nobile flower polysaccharide contain glucose, mannose and glucuronic acid, with mannose as the non-reducing end, and the repeating units are linked in the manner of 1,4-mannose, 1,4-glucose and 1,4-glucuronic acid. The repeating units in the main chain of the Dendrobium nobile flower polysaccharide contain mannose, galactose, galacturonic acid and arabinose, and the non-reducing ends are arabinose and galactose. The repeating units are linked in the following ways: 1,4-mannose, 1,4,6-mannose, 1,4-galactose, 1,3,4-galactose, 1,4-galacturonic acid, 1,5-arabinose and 1,2-arabinose. The repeating units in the main chain of the Dendrobium nobile flower polysaccharide contain mannose, rhamnose, xylose, arabinose, galactose, and galacturonic acid. The non-reducing ends are arabinose and galactose. The repeating units are linked in the following ways: 1,4-mannose, 1,4,6-mannose, 1,4-rhamnose, 1,3-xylose, 1,5-arabinose, 1,3,6-galactose, 1,3-galactose, and 1,3-galacturonic acid.

[0005] Preferably, the repeating units in the main chain of the Dendrobium nobile flower polysaccharide contain D-glucose, D-mannose and D-glucuronic acid, the non-reducing ends are α-D-mannose, and the repeating units are linked in the manner of β-1,4-D-mannose, β-1,4-D-glucose and β-1,4-D-glucuronic acid. The repeating units in the main chain of the Dendrobium nobile flower polysaccharide contain D-mannose, D-galactose, D-galacturonic acid and L-arabinose, and the non-reducing ends are α-L-arabinose and β-D-galactose. The repeating units are linked in the following ways: β-1,4-D-mannose, β-1,4,6-D-mannose, β-1,4-D-galactose, β-1,3,4-D-galactose, β-1,4-D-galacturonic acid, α-1,5-L-arabinose and α-1,2-L-arabinose. The repeating units in the main chain of the Dendrobium nobile flower polysaccharide contain D-mannose, L-rhamnose, L-arabinose, D-xylose, D-galactose, and D-galacturonic acid. The non-reducing ends are L-arabinose and D-galactose. The repeating units are linked in the following ways: β-1,4-D-mannose, β-1,4,6-D-mannose, α-1,4-L-rhamnose, β-1,3-D-xylose, α-1,5-L-arabinose, β-1,3,6-D-galactose, β-1,3-D-galactose, and β-1,3-D-galacturonic acid.

[0006] Preferably, the relative molecular weight of the *Dendrobium nobile* flower polysaccharide is 1 × 10⁻⁶. 4 ~4×10 4 Da; The relative molecular weight of the Dendrobium nobile flower polysaccharide is 6.5 × 10⁻⁶. 5 ~1.3×10 6 Da; The relative molecular weight of the Dendrobium nobile flower polysaccharide is 7.5 × 10⁻⁶. 5 ~1.4×10 6 Da.

[0007] This invention provides a method for preparing Dendrobium flower polysaccharides as described in the above technical solution, comprising the following steps: The flowers of Dendrobium are decolorized to obtain decolorized Dendrobium flowers; the Dendrobium flowers include Dendrobium nobile, Dendrobium chrysanthum, or Dendrobium dentate. The decolorized Dendrobium flowers were mixed with water and extracted to obtain an aqueous extract. The aqueous extract was subjected to protein removal to obtain a protein-free aqueous solution. The protein-removing aqueous solution and lower alcohol are mixed and subjected to alcohol precipitation to obtain an alcohol precipitate; the volume percentage of lower alcohol in the mixture of protein-removing aqueous solution and lower alcohol is 28-92%; The alcohol precipitate was separated by a cellulose chromatography column. The cellulose chromatography column separation included sequential elution with water, a first sodium chloride aqueous solution, and a second sodium chloride aqueous solution to obtain water eluent, a first sodium chloride aqueous eluent, and a second sodium chloride aqueous eluent, respectively. The concentration of the first sodium chloride aqueous solution was 0.08~0.12 mol / L; the concentration of the second sodium chloride aqueous solution was 0.28~0.32 mol / L. When the Dendrobium flower is Dendrobium nobile flower, the water eluent is subjected to first gel chromatography separation, the first gel chromatography separation includes elution with water to obtain Dendrobium nobile flower polysaccharide; When the Dendrobium flower is Dendrobium nobile flower, the first sodium chloride water eluent is desalted to obtain a first desalted crude purified solution; the first desalted crude purified solution is separated by a second gel chromatography column, the second gel chromatography separation including elution with water to obtain Dendrobium nobile flower polysaccharide. When the Dendrobium flower is Dendrobium dentata flower, the second sodium chloride water eluent is desalted to obtain a second desalted crude purified solution; the second desalted crude purified solution is separated by a third gel chromatography column, the third gel chromatography separation including elution with water to obtain Dendrobium dentata flower polysaccharide.

[0008] Preferably, the lower alcohol includes ethanol; The alcohol precipitation includes gradient alcohol precipitation; the gradient alcohol precipitation includes sequentially performing a first alcohol precipitation, a second alcohol precipitation, a third alcohol precipitation, and a fourth alcohol precipitation; The volume percentage of lower alcohols in the mixture during the first alcohol precipitation process is 28-32%. The volume percentage of lower alcohols in the mixture during the second alcohol precipitation process is 48-52%. The volume percentage of lower alcohols in the mixture during the third alcohol precipitation process is 68-72%. The volume percentage of lower alcohols in the mixture during the fourth alcohol precipitation process is 88-92%. The precipitate is a precipitate obtained from a second or third alcohol precipitation.

[0009] Preferably, the chromatographic column used for the cellulose separation includes a DEAE-52 cellulose chromatographic column; The column packing materials used for the first, second, and third gel chromatography column separations independently include Sephacryl S-300 gel.

[0010] This invention provides the application of Dendrobium flower polysaccharide as described in the above technical solution or Dendrobium flower polysaccharide prepared by the above technical solution in stabilizing pigments; The pigments include anthocyanins and / or zeaxanthin; The anthocyanins are paeoniflorin-3-O-glucoside and / or delphinidin-3-O-glucoside; The Dendrobium flower polysaccharides and pigments form a polysaccharide-pigment complex.

[0011] The present invention also provides a polysaccharide-pigment complex, which is obtained by self-assembly of Dendrobium flower polysaccharide and pigment; The Dendrobium flower polysaccharide is the Dendrobium flower polysaccharide described in the above technical solution or the Dendrobium flower polysaccharide prepared by the preparation method described in the above technical solution; The pigments include anthocyanins and / or zeaxanthin; The anthocyanins are paeoniflorin-3-O-glucoside and / or delphinidin-3-O-glucoside.

[0012] Preferably, when the pigment includes anthocyanins, the polysaccharide-pigment complex includes Dendrobium nobile flower polysaccharide-anthocyanin complex and / or Dendrobium dentata flower polysaccharide-anthocyanin complex; When the pigment includes zeaxanthin, the polysaccharide-pigment complex includes the Dendrobium nobile flower polysaccharide-zeaxanthin complex.

[0013] The present invention also provides the application of the polysaccharide-pigment complex described above in food additives, pharmaceutical excipients or antioxidant health products.

[0014] The Dendrobium flower polysaccharide provided by this invention includes one or more of the following: Dendrobium nobile flower polysaccharide, Dendrobium chrysanthum flower polysaccharide, and Dendrobium dentata flower polysaccharide. A portion of the O atoms in the repeating units constituting the Dendrobium flower polysaccharide are acetylated. The relative molecular weight of the Dendrobium flower polysaccharide is 1 × 10⁻⁶. 4 ~1.4×10 6The repeating units in the main chain of the *Dendrobium nobile* flower polysaccharide contain glucose, mannose, and glucuronic acid, with non-reducing ends being mannose. The repeating units are linked in a manner including 1,4-mannose, 1,4-glucose, and 1,4-glucuronic acid. The repeating units in the main chain of the *Dendrobium nobile* flower polysaccharide contain mannose, galactose, galacturonic acid, and arabinose, with non-reducing ends being arabinose and galactose. The repeating units are linked in a manner including 1,4-mannose, 1,4,6-mannose, 1,4-galactose, and 1,4-glucuronic acid. The polysaccharide contains 3,4-galactose, 1,4-galacturonic acid, 1,5-arabinose, and 1,2-arabinose. The repeating units in the main chain of the Dendrobium nobile flower polysaccharide contain mannose, rhamnose, xylose, arabinose, galactose, and galacturonic acid, with non-reducing ends consisting of arabinose and galactose. The repeating units are linked in the following ways: 1,4-mannose, 1,4,6-mannose, 1,4-rhamnose, 1,3-xylose, 1,5-arabinose, 1,3,6-galactose, 1,3-galactose, and 1,3-galacturonic acid. The present invention provides that some of the O atoms in the repetitive units (the smallest structural segments that appear periodically on the main chain of Dendrobium flower polysaccharide, composed of one or more monosaccharides in a specific linkage mode) of Dendrobium flower polysaccharide are acetylated, and the repetitive units in the main chain of Dendrobium flower polysaccharide have a specific glycosidic bond linkage mode, which enables Dendrobium flower polysaccharide to self-assemble with zeaxanthin and / or anthocyanins to form a stable polysaccharide-pigment complex. The binding of Dendrobium flower polysaccharide and pigment is a spontaneous, enthalpy-driven process. The formation of polysaccharide-pigment complex mainly depends on intermolecular non-covalent interactions, including but not limited to hydrogen bonds, hydrophobic interactions and van der Waals forces. Among them, Dendrobium nobile flower polysaccharide and Dendrobium dentata flower polysaccharide can self-assemble with anthocyanins (paeoniflorin-3-O-glucoside and delphinidin-3-O-glucoside) to form Dendrobium nobile flower polysaccharide-anthocyanin complex and Dendrobium dentata flower polysaccharide-anthocyanin complex, respectively. Dendrobium truncatum flower polysaccharide can self-assemble with zeaxanthin to form Dendrobium truncatum flower polysaccharide-zeaxanthin complex, thereby stabilizing zeaxanthin, paeoniflorin-3-O-glucoside and delphinidin-3-O-glucoside. The self-assembled polysaccharide-pigment complex has excellent thermal stability and light stability.

[0015] The preparation method provided by this invention involves sequentially decolorizing, extracting with water, removing proteins, precipitating with alcohol, separating by cellulose chromatography, desalting, and separating by gel chromatography to obtain Dendrobium flower polysaccharides with specific structures. The obtained Dendrobium flower polysaccharides have a narrow molecular weight distribution and are partially acetylated. They have a stabilizing effect on zeaxanthin, paeoniflorin-3-O-glucoside, and delphinidin-3-O-glucoside, and can form complexes with the above pigments through self-assembly, thereby improving the stability of the pigments. Attached Figure Description

[0016] Figure 1These are chromatograms obtained from HPLC-ELSD online monitoring during the gel chromatography column separation process in Examples 1-3; Figure 2 The results of the analysis of the repeating unit composition of Dendrobium flower polysaccharides obtained in Examples 1-3 are shown. Figure 3 The infrared spectra of the Dendrobium flower polysaccharides obtained in Examples 1-3 are shown below; Figure 4 The ultraviolet spectra of the Dendrobium flower polysaccharides obtained in Examples 1-3 are shown below; Figure 5 The nuclear magnetic resonance spectrum of Dendrobium nobile flower polysaccharide DCFP-1 prepared in Example 1; Figure 6 The nuclear magnetic resonance spectrum of DCFP-2, a polysaccharide from Dendrobium nobile flowers, prepared in Example 1; Figure 7 The nuclear magnetic resonance spectrum of DNFP, a polysaccharide from Dendrobium nobile flowers, prepared in Example 2; Figure 8 The nuclear magnetic resonance spectrum of DDFP-1, a polysaccharide from Dendrobium nobile flowers prepared in Example 3; Figure 9 The nuclear magnetic resonance spectrum of DDFP-2, a polysaccharide from Dendrobium nobile flowers prepared in Example 3; Figure 10 The ITC spectrum of the Dendrobium nobile flower polysaccharide-zeaxanthin complex; Figure 11 ITC spectra of the polysaccharide-anthocyanin complex of Dendrobium nobile and the polysaccharide-anthocyanin complex of Dendrobium dentata; Figure 12 Thermal stability test results for the polysaccharide-zeaxanthin complex, the polysaccharide-anthocyanin complex, and the polysaccharide-anthocyanin complex of Dendrobium nobile flower; Figure 13 UV stability test results for the polysaccharide-anthocyanin complex of Dendrobium nobile and the polysaccharide-anthocyanin complex of Dendrobium dentata. Figure 14 Natural photostability test results for the polysaccharide-zeaxanthin complex, the polysaccharide-anthocyanin complex, and the polysaccharide-anthocyanin complex of Dendrobium nobile. Detailed Implementation

[0017] This invention provides Dendrobium flower polysaccharides, including one or more of Dendrobium nobile flower polysaccharides, Dendrobium chrysanthum flower polysaccharides, and Dendrobium dentata flower polysaccharides, wherein some O atoms in the partial repeating units constituting the Dendrobium flower polysaccharides are acetylated. The relative molecular weight of the Dendrobium flower polysaccharide is 1×10⁻⁶. 4 ~1.4×10 6 Da; The repeating units in the main chain of the Dendrobium nobile flower polysaccharide contain glucose, mannose and glucuronic acid, with mannose at the end. The repeating units are connected in the manner of 1,4-mannose, 1,4-glucose and 1,4-glucuronic acid. The repeating units in the main chain of the Dendrobium nobile flower polysaccharide contain mannose, galactose, galacturonic acid and arabinose, and the non-reducing ends are arabinose and galactose. The repeating units are linked in the following ways: 1,4-mannose, 1,4,6-mannose, 1,4-galactose, 1,3,4-galactose, 1,4-galacturonic acid, 1,5-arabinose and 1,2-arabinose. The repeating units in the main chain of the Dendrobium nobile flower polysaccharide contain mannose, rhamnose, xylose, arabinose, galactose, and galacturonic acid. The non-reducing ends are arabinose and galactose. The repeating units are linked in the following ways: 1,4-mannose, 1,4,6-mannose, 1,4-rhamnose, 1,3-xylose, 1,5-arabinose, 1,3,6-galactose, 1,3-galactose, and 1,3-galacturonic acid.

[0018] In this invention, the relative molecular weight of the Dendrobium flower polysaccharide is 1×10⁻⁶. 4 ~1.4×10 6 Da can be specifically 1×10 4 Da, 5×10 4 Da, 1×10 5 Da, 5×10 5 Da or 1.4×10 6 Da.

[0019] In this invention, the relative molecular weight of the *Dendrobium nobile* flower polysaccharide can be 1 × 10⁻⁶. 4 ~4×10 4 The Da value can specifically be 10 kDa, 13.5 kDa, 20 kDa, 28.2 kDa, 30 kDa, or 40 kDa. In this invention, the repeating units in the main chain of the Dendrobium nobile flower polysaccharide may contain D-glucose, D-mannose, and D-glucuronic acid, with α-D-mannose as the non-reducing terminus. The repeating units are linked in a manner including β-1,4-D-mannose, β-1,4-D-glucose, and β-1,4-D-glucuronic acid.

[0020] In this invention, the relative molecular weight of the Dendrobium nobile flower polysaccharide can be 6.5 × 10⁻⁶. 5 ~1.3×10 6The Da value can specifically be 650 kDa, 800 kDa, 1000 kDa, 1100 kDa, 1269.3 kDa, or 1300 kDa. In this invention, the repeating units in the main chain of the Dendrobium nobile flower polysaccharide may contain D-mannose, D-galactose, D-galacturonic acid, and L-arabinose, with non-reducing ends being α-L-arabinose and β-D-galactose. The repeating units can be linked in the following ways: β-1,4-D-mannose, β-1,4,6-D-mannose, β-1,4-D-galactose, β-1,3,4-D-galactose, β-1,4-D-galacturonic acid, α-1,5-L-arabinose, and α-1,2-L-arabinose.

[0021] In this invention, the relative molecular weight of the Dendrobium nobile flower polysaccharide can be 7.5 × 10⁻⁶. 5 ~1.4×10 6 The Da value can specifically be 750 kDa, 900 kDa, 1000 kDa, 1100 kDa, 1247.9 kDa, 1304.3 kDa, or 1400 kDa. In this invention, the repeating units in the main chain of the Dendrobium nobile flower polysaccharide may contain D-mannose, L-rhamnose, L-arabinose, D-xylose, D-galactose, and D-galacturonic acid, with L-arabinose and D-galactose as the non-reducing ends. The repeating units are linked in the following ways: β-1,4-D-mannose, β-1,4,6-D-mannose, α-1,4-L-rhamnose, β-1,3-D-xylose, α-1,5-L-arabinose, β-1,3,6-D-galactose, β-1,3-D-galactose, and β-1,3-D-galacturonic acid.

[0022] This invention provides a method for preparing Dendrobium flower polysaccharides as described in the above technical solution, comprising the following steps: The flowers of Dendrobium are decolorized to obtain decolorized Dendrobium flowers; the Dendrobium flowers include Dendrobium nobile, Dendrobium chrysanthum, or Dendrobium dentate. The decolorized Dendrobium flowers were mixed with water and extracted to obtain an aqueous extract. The aqueous extract was subjected to protein removal to obtain a protein-free aqueous solution. The protein-removing aqueous solution and lower alcohol are mixed and subjected to alcohol precipitation to obtain an alcohol precipitate; the volume percentage of lower alcohol in the mixture of protein-removing aqueous solution and lower alcohol is 28-92%; The alcohol precipitate was separated by a cellulose chromatography column. The cellulose chromatography column separation included sequential elution with water, a first sodium chloride aqueous solution, and a second sodium chloride aqueous solution to obtain water eluent, a first sodium chloride aqueous eluent, and a second sodium chloride aqueous eluent, respectively. The concentration of the first sodium chloride aqueous solution was 0.08~0.12 mol / L; the concentration of the second sodium chloride aqueous solution was 0.28~0.32 mol / L. When the Dendrobium flower is Dendrobium nobile flower, the water eluent is subjected to first gel chromatography separation, the first gel chromatography separation includes elution with water to obtain Dendrobium nobile flower polysaccharide; When the Dendrobium flower is Dendrobium nobile flower, the first sodium chloride water eluent is desalted to obtain a first desalted crude purified solution; the first desalted crude purified solution is separated by a second gel chromatography column, the second gel chromatography separation including elution with water to obtain Dendrobium nobile flower polysaccharide. When the Dendrobium flower is Dendrobium dentata flower, the second sodium chloride water eluent is desalted to obtain a second desalted crude purified solution; the second desalted crude purified solution is separated by a third gel chromatography column, the third gel chromatography separation including elution with water to obtain Dendrobium dentata flower polysaccharide.

[0023] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0024] This invention decolorizes Dendrobium flowers to obtain decolorized Dendrobium flowers.

[0025] In this invention, the Dendrobium flowers include Dendrobium nobile, Dendrobium chrysanthum, or Dendrobium dentata; the Dendrobium flowers can be fresh Dendrobium petals; the Dendrobium flowers can be from the Longling Dendrobium Research Institute.

[0026] In this invention, the decolorizing agent used for decolorization may include an alcohol-based decolorizing solvent; the alcohol-based decolorizing solvent may include ethanol, specifically anhydrous ethanol; the mass-to-volume ratio of Dendrobium flowers to the alcohol-based decolorizing solvent may be 1g:250~350mL, specifically 1g:250mL, 1g:270mL, 1g:300mL, 1g:320mL, or 1g:350mL. This invention uses anhydrous ethanol and other organic solvents for soaking, dissolving and removing fat-soluble components (such as chlorophyll, carotenoids, and other pigments, as well as some lipids and waxes), which can reduce the burden on subsequent purification steps, avoid interference from colored substances in chromatographic separation and structural identification, and make the obtained Dendrobium flower polysaccharide white or light-colored, meeting the appearance requirements of Dendrobium flower polysaccharide for food and / or pharmaceutical excipients.

[0027] After obtaining the decolorized Dendrobium flowers, the present invention mixes the decolorized Dendrobium flowers with water and performs water extraction to obtain an aqueous extract. In the present invention, the decolorized Dendrobium flowers can also be pulverized before the water extraction.

[0028] In this invention, the water extraction temperature can be 95-100℃, or 97-99℃, specifically boiling water extraction; the water extraction can be reflux extraction; the number of water extractions can be 2-4 times, specifically 2, 3, or 4 times; the mass-to-volume ratio of the decolorized Dendrobium flowers to the water used in a single water extraction can be 1g:20mL; the time for a single water extraction can be 1.5-2.5h, specifically 1.5h, 1.8h, 2h, 2.3h, or 2.5h. This invention utilizes the good water solubility of Dendrobium flower polysaccharides, while cell wall structural components such as cellulose and lignin are insoluble in water. Through hot water extraction, Dendrobium flower polysaccharides are dissolved from plant cells into the aqueous phase, achieving preliminary separation from most insoluble solids.

[0029] After the water extraction is completed, the present invention may further include solid-liquid separation of the water extract and concentration of the resulting liquid to obtain the water extract. In this invention, the solid-liquid separation includes filtration. In this invention, the concentration factor can be 4 to 5 times, specifically 4 times, 4.2 times, 4.5 times, 4.8 times, or 5 times; the concentration can be vacuum concentration; the vacuum concentration temperature can be 55 to 65°C, specifically 55, 58, 60, 62, or 65°C.

[0030] After obtaining the aqueous extract, the present invention removes proteins from the aqueous extract to obtain a protein-free aqueous solution.

[0031] In this invention, the protein removal reagent used includes chloroform-n-butanol. In this invention, the volume ratio of chloroform to n-butanol in the chloroform-n-butanol mixture can be 3.8~4.2:1, specifically 3.8:1, 3.9:1, 4:1, 4.1:1, or 4.2:1. In this invention, the protein removal process can be performed 3~5 times, specifically 3, 4, or 5 times; the volume ratio of the aqueous extract to the protein removal reagent used in a single protein removal process can be 2.5~3.5:1, specifically 2.5:1, 2.8:1, 3:1, 3.2:1, or 3.5:1. This invention utilizes the principle of protein denaturation and precipitation in chloroform-n-butanol. By centrifuging, the protein is removed, leaving Dendrobium flower polysaccharides in the aqueous phase. This process removes water-soluble proteins co-extracted with Dendrobium flower polysaccharides, preventing proteins from interfering with subsequent chromatographic behavior and structural analysis, and improving the purity of the obtained Dendrobium flower polysaccharides.

[0032] After the protein removal is completed, the present invention may further include centrifuging the protein-removed liquid obtained by protein removal, removing the organic solvent from the supernatant, and obtaining the protein-removed aqueous solution.

[0033] In this invention, the centrifugal separation speed can be 3500~4000 rpm, specifically 3500 rpm, 3600 rpm, 3700 rpm, 3800 rpm, 3900 rpm or 4000 rpm; the centrifugal separation time can be 15~25 min, specifically 15 min, 18 min, 20 min, 22 min or 25 min.

[0034] In this invention, the method for removing organic solvents can be vacuum concentration; the temperature for vacuum concentration can be 55~65℃, specifically 55, 58, 60, 62 or 65℃. This invention does not have a particular limitation on the time of vacuum concentration, as long as it is sufficient to completely remove the organic solvents from the solution.

[0035] After obtaining the protein-free aqueous solution, the present invention performs alcohol precipitation with the protein-free aqueous solution and a lower alcohol to obtain an alcohol precipitate.

[0036] In this invention, the lower alcohol may include ethanol, specifically anhydrous ethanol; the volume percentage of the lower alcohol in the mixture of protein aqueous solution and lower alcohol is 28-92%, or 30-90%, specifically 30%, 50%, 70% or 90%.

[0037] In this invention, the alcohol precipitation may include gradient alcohol precipitation; the gradient alcohol precipitation may include sequentially performing a first alcohol precipitation, a second alcohol precipitation, a third alcohol precipitation, and a fourth alcohol precipitation. In this invention, during the first alcohol precipitation process, the volume percentage of lower alcohols in the mixture may be 28-32%, specifically 28%, 29%, 30%, 31%, or 32%; during the second alcohol precipitation process, the volume percentage of lower alcohols in the mixture may be 48-52%, specifically 48%, 49%, 50%, 51%, or 52%; during the third alcohol precipitation process, the volume percentage of lower alcohols in the mixture may be 68-72%, specifically 68%, 69%, 70%, 71%, or 72%; during the fourth alcohol precipitation process, the volume percentage of lower alcohols in the mixture may be 88-92%, specifically 88%, 89%, 90%, 91%, or 92%. In this invention, the time for the first, second, third, and fourth alcohol precipitations can independently be 8-12 hours, or even 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours, specifically overnight. In this invention, the precipitate can be the precipitate obtained from the second or third alcohol precipitation. This invention utilizes the principle that polysaccharides precipitate due to their low solubility in ethanol, while small molecules such as monosaccharides, oligosaccharides, and salts remain in the alcohol-water solution. Through gradient alcohol precipitation, the difference in solubility of different types of polysaccharides at specific ethanol concentrations is utilized. By adjusting the ethanol concentration, the initial separation of the target Dendrobium officinale flower polysaccharides is achieved. The gradient alcohol precipitate with the highest content of target Dendrobium officinale flower polysaccharides during the gradient alcohol precipitation process is used for subsequent purification. This allows for the initial fractionation and precipitation of polysaccharides of different polarities or molecular weights, achieving preliminary enrichment and purification.

[0038] After obtaining the alcohol precipitate, the present invention separates the alcohol precipitate using a cellulose chromatography column. The cellulose chromatography column separation includes sequential elution with water, a first sodium chloride aqueous solution, and a second sodium chloride aqueous solution, yielding water eluent, a first sodium chloride aqueous eluent, and a second sodium chloride aqueous eluent, respectively. The present invention utilizes the principle of ion exchange. Separation is based on the charge difference of the acidic groups (such as glucuronic acid and galacturonic acid) contained in the Dendrobium flower polysaccharides. Neutral Dendrobium nobile flower polysaccharides are eluted with water; acidic Dendrobium chrysanthum flower polysaccharides and Dendrobium dentate flower polysaccharides are eluted with sodium chloride aqueous solution.

[0039] In this invention, the alcohol precipitate is further dissolved in water before being separated by a cellulose chromatography column, and the resulting solution is subsequently concentrated and dried; the concentration may include vacuum concentration; the drying may include freeze drying. In this invention, the freeze-drying temperature can be -53 to -56°C, specifically -53°C, -54°C, -55°C, or -56°C; the freeze-drying time can be 65 to 72 hours, specifically 65 hours, 68 hours, 70 hours, or 72 hours. In this invention, the freeze dryer used for freeze drying may be pre-started before freeze drying; the pre-starting temperature can be -45°C to -55°C, specifically -45°C, -48°C, -50°C, -52°C, or -55°C; the pre-starting time can be 20 to 40 minutes, specifically 20 minutes, 25 minutes, 30 minutes, 35 minutes, or 40 minutes. This invention involves dissolving (redissolving) the alcohol precipitate in water, concentrating under reduced pressure, and freeze-drying it to remove residual ethanol from the protein removal process, prevent ethanol from contaminating the freeze dryer, and ensure the purity of the homogeneous polysaccharide from Dendrobium flowers and the reusability of the experimental equipment.

[0040] In this invention, the chromatographic column used for cellulose separation may include a DEAE-52 cellulose chromatographic column; the concentration of the first sodium chloride aqueous solution may be 0.08~0.12 mol / L, specifically 0.08, 0.09, 0.1, 0.11, or 0.12 mol / L; the concentration of the second sodium chloride aqueous solution may be 0.28~0.32 mol / L, specifically 0.28 mol / L, 0.29 mol / L, 0.3 mol / L, 0.31 mol / L, or 0.32 mol / L. In this invention, the cellulose chromatographic column separation process may further include performing high-performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) on the eluent obtained from the elution, combining components with consistent retention behavior. In this invention, the chromatographic column used in the high performance liquid chromatography-evaporative light scattering detection can be a Shodex OHpak SB-804 column, the mobile phase can be water, and the mobile phase flow rate can be 0.8~1 mL / min, specifically 0.8 mL / min, 0.85 mL / min, 0.9 mL / min, 0.95 mL / min or 1 mL / min.

[0041] When the Dendrobium flower is Dendrobium nobile, the present invention performs a first gel chromatography separation on the obtained water eluent, the first gel chromatography separation including elution with water to obtain Dendrobium nobile flower polysaccharide.

[0042] In this invention, the column packing material used for the first gel chromatography column separation may include Sephacryl S-300 gel; the water may include distilled water.

[0043] In this invention, the gel chromatography column separation process may further include high-performance liquid chromatography-evaporative light scattering (HPLC-ELSD) detection of the eluent obtained from the elution, combining the components with uniform and symmetrical peaks, concentrating, and drying to obtain the Dendrobium flower polysaccharide. In this invention, the HPLC-ELSD detection uses a Shodex OHpak SB-804 column, the mobile phase is water, and the mobile phase flow rate is 0.8~1 mL / min, specifically 0.8 mL / min, 0.85 mL / min, 0.9 mL / min, 0.95 mL / min, or 1 mL / min. In this invention, the drying can be freeze-drying.

[0044] When the Dendrobium flower is Dendrobium nobile flower, the present invention desalts the first sodium chloride water eluent to obtain a first desalted crude purified solution; the first desalted crude purified solution is separated by a second gel chromatography column, the second gel chromatography separation including elution with water to obtain Dendrobium nobile flower polysaccharide. In this invention, the desalination may include dialysis desalination, ultrafiltration desalination, or gel column desalination; the dialysis membrane used for dialysis desalination has a molecular weight cutoff ≥ 500 Da. This invention removes high concentrations of salt ions (Na+) introduced during ion exchange chromatography through desalination. + and Cl - This avoids salt interference with subsequent gel chromatography separation and its impact on polysaccharide freeze-drying and structural analysis.

[0045] In this invention, the separation conditions of the second gel chromatography column can be the same as those of the first gel chromatography column, and will not be repeated here.

[0046] When the Dendrobium flower is Dendrobium dentata flower, the present invention desalts the obtained second sodium chloride water eluent to obtain a second desalted crude purified solution; the second desalted crude purified solution is separated by a third gel chromatography column, the third gel chromatography separation including elution with water to obtain Dendrobium dentata flower polysaccharide.

[0047] In this invention, the desalting process can be the same as described above, and will not be repeated here. In this invention, the separation conditions of the third gel chromatography column can be the same as those of the first gel chromatography column, and will not be repeated here.

[0048] The gel chromatography column separation of this invention utilizes the principle of molecular sieves, separating polysaccharide molecules based on their hydrodynamic volume (related to molecular weight). Large molecules elute first, followed by smaller molecules, to obtain Dendrobium flower polysaccharides with uniform molecular weight.

[0049] This invention provides the application of Dendrobium flower polysaccharide as described in the above technical solution or Dendrobium flower polysaccharide prepared by the above technical solution in stabilizing pigments; The pigments include anthocyanins and / or zeaxanthin; The anthocyanins are paeoniflorin-3-O-glucoside and / or delphinidin-3-O-glucoside; The Dendrobium flower polysaccharides and pigments form a polysaccharide-pigment complex. The Dendrobium flower polysaccharides provided by this invention can specifically bind to anthocyanins or zeaxanthin, and the resulting polysaccharide-pigment complex exhibits excellent thermal and light stability.

[0050] The present invention also provides a polysaccharide-pigment complex, which is obtained by self-assembly of Dendrobium flower polysaccharide and pigment; The Dendrobium flower polysaccharide is the Dendrobium flower polysaccharide described in the above technical solution or the Dendrobium flower polysaccharide prepared by the preparation method described in the above technical solution; The pigments include anthocyanins and / or zeaxanthin; The anthocyanins are paeoniflorin-3-O-glucoside and / or delphinidin-3-O-glucoside.

[0051] In this invention, when the pigment includes anthocyanins, the polysaccharide-pigment complex includes Dendrobium nobile flower polysaccharide-anthocyanin complex and / or Dendrobium dentata flower polysaccharide-anthocyanin complex. When the pigment includes zeaxanthin, the polysaccharide-pigment complex includes the Dendrobium nobile flower polysaccharide-zeaxanthin complex.

[0052] In this invention, the preparation method of the polysaccharide-pigment complex may include the following steps: mixing polysaccharide, pigment and self-assembling solvent to obtain polysaccharide-pigment complex.

[0053] In this invention, mixing the polysaccharide, pigment, and self-assembly solvent can be achieved by: mixing the polysaccharide and the self-assembly solvent to obtain a polysaccharide solution; mixing the pigment and the self-assembly solvent to obtain a pigment solution; or mixing the polysaccharide solution, the pigment solution, and the self-assembly solvent. In this invention, the volume ratio of the polysaccharide solution to the pigment solution can be 1:0.8 to 1.2, specifically 1:0.8, 1:0.9, 1:1, 1:1.1, or 1:1.2.

[0054] In this invention, when the pigment is anthocyanin, the polysaccharide can be Dendrobium nobile flower polysaccharide or Dendrobium dentata flower polysaccharide; the mass ratio of the polysaccharide to the anthocyanin can be 2:0.32~0.48. In this invention, the self-assembly solvent can include PBS buffer; the pH value of the PBS buffer can be 2.8~3.2, specifically 2.8, 2.9, 3, 3.1, or 3.2. In this invention, the concentration of the polysaccharide solution can be 1.8~2.2 mg / mL, specifically 1.8 mg / mL, 1.9 mg / mL, 2 mg / mL, 2.1 mg / mL, or 2.2 mg / mL; the concentration of the anthocyanin solution can be 3.6~4.4 mg / mL, specifically 3.6 mg / mL, 3.8 mg / mL, 4 mg / mL, 4.2 mg / mL, or 4.4 mg / mL.

[0055] In this invention, when the pigment is zeaxanthin, the polysaccharide can be *Dendrobium nobile* flower polysaccharide; the mass ratio of *Dendrobium nobile* flower polysaccharide to zeaxanthin can be 4:0.8~1.2, specifically 4:0.8, 4:0.9, 4:1, 4:1.1, or 4:1.2. In this invention, the self-assembly solvent can include a dimethyl sulfoxide aqueous solution; the volume percentage of dimethyl sulfoxide in the dimethyl sulfoxide aqueous solution can be 5%. In this invention, the concentration of the *Dendrobium nobile* flower polysaccharide solution can be 3~5 mg / mL, specifically 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, or 5 mg / mL; the concentration of the zeaxanthin solution can be 0.5~1.5 mg / mL, specifically 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, or 1.5 mg / mL.

[0056] The present invention also provides the application of the polysaccharide-pigment complex described above in food additives, pharmaceutical excipients or antioxidant health products.

[0057] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0058] In all embodiments of the present invention, paeoniflorin-3-O-glucoside was purchased from Chengdu Zhibiao Chemical Pure Biotechnology Co., Ltd.; delphinidin-3-O-glucoside was purchased from Kunming Shifei Technology Co., Ltd.; zeaxanthin was purchased from Xili Biotechnology Co., Ltd.; the petals of Dendrobium nobile, Dendrobium chrysanthum, and Dendrobium dentata were obtained from Longling Dendrobium Research Institute; the cellulose chromatography column was a DEAE-52 cellulose chromatography column, purchased from Beijing Solarbio Technology Co., Ltd.; the chromatographic column packing used for gel chromatography separation was Sephacryl S-300 gel, purchased from Siterofan Biotechnology Co., Ltd.

[0059] Example 1 Fresh Dendrobium nobile flowers were mixed with anhydrous ethanol and decolorized to obtain decolorized Dendrobium nobile flowers. The decolorized Dendrobium nobile flowers and water were mixed at a mass-volume ratio of 1g:20mL and extracted by boiling water reflux. The above extraction process was repeated twice. The mixture was filtered, the filtrates were combined, and concentrated to 1 / 6 of the volume of water used for water extraction to obtain water extract. The obtained aqueous extract and chloroform-n-butanol were mixed at a volume ratio of 2:1 to remove protein. The resulting protein-removed solution was centrifuged at 3800 rpm for 20 min. The supernatant was concentrated under reduced pressure at 60 °C to remove organic solvent and obtain a protein-removed aqueous solution. The volume ratio of chloroform to n-butanol in the chloroform-n-butanol mixture was 4:1. Anhydrous ethanol was added to the obtained protein-free aqueous solution for gradient alcohol precipitation, resulting in ethanol volume percentages of 30%, 50%, 70%, and 90% in the mixture, respectively. Each concentration was allowed to stand overnight, and the precipitate was collected by centrifugation. The precipitates were reconstituted with hot water, pre-frozen, and finally freeze-dried to obtain the corresponding alcohol precipitates, designated CDCFP30, CDCFP50, CDCFP70, and CDCFP90, with yields (yield = mass of alcohol precipitate / mass of decolorized Dendrobium flowers × 100%) of 1%, 5%, 7%, and 2%, respectively. CDCFP70 was used for subsequent purification.

[0060] The obtained CDFP70 was separated by cellulose chromatography column elution, sequentially using water, a 0.1 mol / L sodium chloride aqueous solution, and a 0.3 mol / L sodium chloride aqueous solution. HPLC-ELSD online monitoring was used to combine components with consistent retention behavior, yielding water eluent, 0.1 mol / L sodium chloride aqueous eluent, and 0.3 mol / L sodium chloride aqueous eluent sequentially. The HPLC-ELSD online monitoring used a Shodex OHpak SB-804 column, with deionized water as the mobile phase and a flow rate of 1 mL / min.

[0061] The obtained water eluent was separated by gel chromatography with water as the elution agent. The chromatographic column was Shodex OHpak SB-804, the mobile phase was deionized water, and the flow rate was 1 mL / min. The components with uniform and symmetrical peaks (smooth HPLC peak curves and symmetrical sides) were combined, concentrated, and freeze-dried to obtain Dendrobium nobile flower polysaccharide (denoted as DCFP).

[0062] Example 2 Fresh Dendrobium nobile flowers were mixed with anhydrous ethanol and decolorized to obtain decolorized Dendrobium nobile flowers. The decolorized Dendrobium nobile flowers and water were mixed at a mass-volume ratio of 1g:20mL and extracted by boiling water reflux. The above extraction process was repeated twice. The mixture was filtered, the filtrates were combined, and concentrated to 1 / 6 of the volume of water used for water extraction to obtain an aqueous extract. The obtained aqueous extract and chloroform-n-butanol were mixed at a volume ratio of 2:1 to remove protein. The resulting protein-removed solution was centrifuged at 3800 rpm for 20 min. The supernatant was concentrated under reduced pressure at 60 °C to remove organic solvent and obtain a protein-removed aqueous solution. The volume ratio of chloroform to n-butanol in the chloroform-n-butanol mixture was 4:1. Anhydrous ethanol was added to the resulting protein-free aqueous solution for gradient alcohol precipitation, resulting in ethanol volume percentages of 30%, 50%, 70%, and 90% in the mixture, respectively. Each concentration was allowed to stand overnight, and the precipitate was collected by centrifugation. The precipitates were reconstituted with hot water, pre-frozen, and finally freeze-dried to obtain the corresponding alcohol precipitates, designated CDNFP30, CDNFP50, CDNFP70, and CDNFP90, with yields of 0.2%, 7%, 2%, and 1%, respectively. CDNFP50 was used for subsequent purification.

[0063] The obtained CDNFP50 was separated by cellulose chromatography, and eluted sequentially with water, a 0.1 mol / L sodium chloride aqueous solution, and a 0.3 mol / L sodium chloride aqueous solution. HPLC-ELSD online monitoring was used to combine components with consistent retention behavior, yielding water eluent, 0.1 mol / L sodium chloride aqueous eluent, and 0.3 mol / L sodium chloride aqueous eluent, respectively. The HPLC-ELSD online monitoring used a Shodex OHpak SB-804 column, with deionized water as the mobile phase and a flow rate of 1 mL / min.

[0064] The obtained 0.1 mol / L sodium chloride water eluent was dialyzed to remove salt using a dialysis membrane with a molecular weight cutoff ≥500 Da, yielding a desalted crude purified solution. The desalted crude purified solution was then separated by gel chromatography using water as elution. HPLC-ELSD online monitoring was performed using a Shodex OHpak SB-804 column, with deionized water as the mobile phase at a flow rate of 1 mL / min. Components with uniform and symmetrical peaks (smooth HPLC peak curves with symmetry on both sides) were combined, concentrated, and freeze-dried to obtain Dendrobium nobile flower polysaccharide (denoted as DNFP).

[0065] Example 3 Fresh Dendrobium dentata flowers were mixed with anhydrous ethanol and decolorized to obtain decolorized Dendrobium dentata flowers. The decolorized Dendrobium dentata flowers and water were mixed at a mass-volume ratio of 1g:20mL and extracted by boiling water reflux. The above extraction process was repeated twice. The mixture was filtered, the filtrates were combined, and concentrated to 1 / 6 of the volume of water used for water extraction to obtain water extract. The obtained aqueous extract and chloroform-n-butanol were mixed at a volume ratio of 2:1 to remove protein. The resulting protein-removed solution was centrifuged at 3800 rpm for 20 min. The supernatant was concentrated under reduced pressure at 60 °C to remove organic solvent and obtain a protein-removed aqueous solution. The volume ratio of chloroform to n-butanol in the chloroform-n-butanol mixture was 4:1. Anhydrous ethanol was added to the obtained protein-free aqueous solution for gradient ethanol precipitation, resulting in ethanol volume percentages of 30%, 50%, 70%, and 90% in the mixtures, respectively. Each concentration was allowed to stand overnight, and the precipitates were collected by centrifugation. The precipitates were reconstituted with hot water, pre-frozen, and finally freeze-dried to obtain the corresponding grades of ethanol precipitates, designated CDDFP30, CDDFP50, CDDFP70, and CDDFP90, with yields of %, 6%, 9%, and 4%, respectively. CDDFP70 was used for subsequent purification.

[0066] The obtained CDDFP70 was separated by cellulose chromatography column elution, sequentially using water, a 0.1 mol / L sodium chloride aqueous solution, and a 0.3 mol / L sodium chloride aqueous solution. HPLC-ELSD online monitoring was used to combine components with consistent retention behavior, yielding water eluent, 0.1 mol / L sodium chloride aqueous eluent, and 0.3 mol / L sodium chloride aqueous eluent sequentially. The HPLC-ELSD online monitoring used a Shodex OHpak SB-804 column, with deionized water as the mobile phase and a flow rate of 1 mL / min.

[0067] The obtained 0.3 mol / L sodium chloride water eluent was dialyzed to remove salt using a dialysis membrane with a molecular weight cutoff ≥500 Da, yielding a desalted crude purified solution. The obtained desalted crude purified solution was then separated by gel chromatography using water as elution. HPLC-ELSD online monitoring was performed using a Shodex OHpak SB-804 column, with deionized water as the mobile phase and a flow rate of 1 mL / min. Components with uniform and symmetrical peaks (smooth HPLC peak curves with symmetry on both sides) were combined, concentrated, and freeze-dried to obtain Dendrobium nobile flower polysaccharide (denoted as DDFP).

[0068] Figure 1 These are chromatograms obtained from HPLC-ELSD online monitoring during the gel chromatography column separation process in Examples 1-3. Figure 1It can be seen that in Example 1, two homogeneous components of Dendrobium nobile flower polysaccharide were prepared, which were denoted as DCFP-1 and DCFP-2, respectively; in Example 2, one homogeneous component of Dendrobium chrysanthum flower polysaccharide DNFP was prepared; and in Example 3, two homogeneous components of Dendrobium dentata flower polysaccharide were prepared, which were denoted as DDFP-1 and DDFP-2, respectively.

[0069] The Dendrobium flower polysaccharides obtained in Examples 1-3 were subjected to repeat unit composition analysis, methylation analysis, infrared spectroscopy analysis, ultraviolet spectroscopy analysis and nuclear magnetic resonance spectroscopy analysis. Figure 2 The results of the repeating unit composition analysis of the Dendrobium flower polysaccharides obtained in Examples 1-3 are as follows: Figure 3 The infrared spectra of the Dendrobium flower polysaccharides obtained in Examples 1-3 are shown below. Figure 4 The UV spectra of the Dendrobium flower polysaccharides obtained in Examples 1-3 are shown below. Figure 5 The nuclear magnetic resonance spectrum of Dendrobium nobile flower polysaccharide DCFP-1 prepared in Example 1; Figure 6 The nuclear magnetic resonance spectrum of DCFP-2, a polysaccharide from Dendrobium nobile flowers, prepared in Example 1; Figure 7 The nuclear magnetic resonance spectrum of DNFP, a polysaccharide from Dendrobium nobile flowers, prepared in Example 2; Figure 8 The nuclear magnetic resonance spectrum of DDFP-1, a polysaccharide from Dendrobium nobile flowers prepared in Example 3; Figure 9 The nuclear magnetic resonance spectrum of DDFP-2, a polysaccharide from Dendrobium nobile flowers prepared in Example 3, is shown. Figures 5-9 In the image above, all images are proton (H) spectra; the images below are carbon (C) spectra. Figures 2-9 The methylation analysis results show that the structural characteristics and relative molecular weights of the Dendrobium flower polysaccharides obtained in Examples 1-3 are as follows: DCFP-1 and DCFP-2 have relative molecular weights of approximately 28.2 kDa and 13.5 kDa, respectively. Their main repeating units are D-glucose, D-mannose, and D-glucuronic acid, with D-mannose as the non-reducing terminus. The repeating units are linked by β-1,4-D-mannose, β-1,4-D-glucose, and β-1,4-D-glucuronic acid.

[0070] DNFP: The relative molecular weight is approximately 1269.3 kDa. The main repeating units are D-mannose, D-galactose, D-galacturonic acid, and L-arabinose. The non-reducing ends are α-L-arabinose and β-D-galactose. The repeating units can be linked in the following ways: β-1,4-D-mannose, β-1,4,6-D-mannose, β-1,3,4-D-galactose, β-1,4-D-galactose, β-1,4-D-galacturonic acid, α-1,5-L-arabinose, and α-1,2-L-arabinose.

[0071] DDFP-1 and DDFP-2 have relative molecular weights of approximately 1247.9 kDa and 1304.3 kDa, respectively. Their main repeating units are D-mannose, L-rhamnose, L-arabinose, D-xylose, D-galactose, and D-galacturonic acid. The non-reducing ends are L-arabinose and D-galactose. The repeating units are linked in the following ways: β-1,4-D-mannose, β-1,4,6-D-mannose, β-1,3-D-xylose, α-1,4-L-rhamnose, α-1,5-L-arabinose, β-1,3,6-D-galactose, β-1,3-D-galactose, and β-1,3-D-galacturonic acid.

[0072] Example 4 Using a 5% (v / v) dimethyl sulfoxide aqueous solution as the self-assembly solvent, 4 mg each of DCFP-1 and DCFP-2 prepared in Example 1 were dissolved in 1 mL of the self-assembly solvent to obtain polysaccharide stock solutions with a concentration of 4 mg / mL. These solutions were then filtered through a 0.45 μm filter membrane for later use. Zeaxanthin was dissolved in the self-assembly solvent to obtain a 1 mg / mL zeaxanthin solution, which was then filtered through a 0.25 μm filter membrane for later use. The obtained polysaccharide stock solution and zeaxanthin solution were mixed evenly in a 1:1 volume ratio in the self-assembly solvent to obtain *Dendrobium nobile* flower polysaccharide-zeaxanthin complex solutions, designated ZEA-DCFP-1 and ZEA-DCFP-2, respectively.

[0073] Example 5 Using PBS buffer (pH=3) as the self-assembly solvent, 2 mg each of DNFP prepared in Example 2, DDFP-1 and DDFP-2 prepared in Example 3 were dissolved in 1 mL of the self-assembly solvent to obtain polysaccharide stock solutions with a concentration of 2 mg / mL. These solutions were then filtered through a 0.45 μm filter membrane for later use. Paeoniflorin-3-O-glucoside and delphinidin-3-O-glucoside were dissolved in the self-assembly solvent to obtain anthocyanin solutions with a concentration of 0.4 mg / mL. These solutions were then filtered through a 0.25 μm filter membrane for later use. In a self-assembly solvent, the obtained polysaccharide stock solution and anthocyanin solution were mixed evenly at a volume ratio of 1:1 to obtain Dendrobium nobile flower polysaccharide-anthocyanin complex solution and Dendrobium dentata flower polysaccharide-anthocyanin complex solution, respectively denoted as P3G-DNFP, D3G-DNFP, P3G-DDFP-1, P3G-DDFP-2, D3G-DDFP-1, and D3G-DDFP-2.

[0074] Test Example 1 The polysaccharide stock solution and zeaxanthin solution prepared in Example 4 were added to the titration needle and titration cell respectively in proportion. The polysaccharide stock solution and anthocyanin solution prepared in Example 5 were added to the titration needle and titration cell respectively in proportion. The formation parameters of the Dendrobium nobile flower polysaccharide-zeaxanthin complex, Dendrobium chrysanthum flower polysaccharide-anthocyanin complex and Dendrobium dentata flower polysaccharide-anthocyanin complex were analyzed by isothermal microcalorimetry (ITC).

[0075] Figure 10 The ITC spectrum of the Dendrobium nobile flower polysaccharide-zeaxanthin complex is shown. Figure 11 The ITC spectra of the *Dendrobium nobile* flower polysaccharide-anthocyanin complex and the *Dendrobium dentata* flower polysaccharide-anthocyanin complex are shown below. Figure 10 and Figure 11 It is known that Dendrobium flower polysaccharides (DNFP, DDFP-1, DDFP-2, DCFP-1 and DCFP-2) and pigments (anthocyanins and zeaxanthin) are spontaneously bound and interact with each other.

[0076] The *Dendrobium nobile* flower polysaccharide-zeaxanthin complex obtained in Example 4, and the *Dendrobium nobile* flower polysaccharide-anthocyanin complex and *Dendrobium dentata* flower polysaccharide-anthocyanin complex (anthocyanin being paeoniflorin-3-O-glucoside) obtained in Example 5 were heated in a water bath at 75°C for 0 h, 0.5 h, 1 h, 2 h, 3 h, and 4 h, respectively. After heating, they were removed and quickly cooled to room temperature in an ice bath. The absorbance of anthocyanins was measured at 520 nm using an ELISA reader, and the absorbance of zeaxanthin was measured at 450 nm to determine the residual amounts of anthocyanins and zeaxanthin after heating at the above time intervals. The degradation rate constant k was obtained through first-order reaction kinetics simulation, and the time required for 50% degradation of anthocyanins and zeaxanthin, i.e., the half-life (t), was determined. 1 / 2 ).

[0077] Table 1. First-order kinetics of the thermal stability of the polysaccharide-zeaxanthin complex, the polysaccharide-anthocyanin complex, and the polysaccharide-anthocyanin complex from *Dendrobium nobile*, *Dendrobium chrysanthemum*, and *Dendrobium dentata*.

[0078] Table 1 shows the first-order kinetics of thermal stability of the Dendrobium nobile flower polysaccharide-zeaxanthin complex, Dendrobium chrysanthum flower polysaccharide-anthocyanin complex, and Dendrobium dentata flower polysaccharide-anthocyanin complex. In the table, rel.TAD represents the total degradation rate of the compounds relative to the initial compound content over a period of time. Figure 12 The thermal stability test results are shown for the polysaccharide-zeaxanthin complex from *Dendrobium nobile*, the polysaccharide-anthocyanin complex from *Dendrobium chrysanthum*, and the polysaccharide-anthocyanin complex from *Dendrobium dentata*. Figure 12As shown in Table 1, compared with the unrecombined paeoniflorin-3-O-glucoside and zeaxanthin, the degradation rates of the Dendrobium nobile flower polysaccharide-zeaxanthin complex, the Dendrobium chrysanthum flower polysaccharide-anthocyanin complex, and the Dendrobium dentata flower polysaccharide-anthocyanin complex were significantly slowed down under high temperature conditions, and the half-life value was significantly increased. This indicates that the Dendrobium flower polysaccharide provided by the present invention has a thermostability effect on anthocyanins and zeaxanthin.

[0079] The *Dendrobium nobile* flower polysaccharide-anthocyanin complex and *Dendrobium dentata* flower polysaccharide-anthocyanin complex obtained in Example 5 were subjected to direct ultraviolet light irradiation at room temperature for 0 h, 0.5 h, 1 h, 2 h, 3 h, and 4 h. The absorbance of anthocyanins was measured at 520 nm using a microplate reader to determine the residual amount of anthocyanins after fixed-time strong light irradiation. The degradation rate constant k was obtained through first-order reaction kinetic simulation, and the time required for 50% degradation of anthocyanins, i.e., the half-life (t), was determined. 1 / 2 ).

[0080] Table 2. First-order kinetics of UV photostable stability of the *Dendrobium nobile* flower polysaccharide-anthocyanin complex and the *Dendrobium dentata* flower polysaccharide-anthocyanin complex.

[0081] Table 2 shows the first-order kinetics of UV photostable stability of the Dendrobium nobile flower polysaccharide-anthocyanin complex and the Dendrobium dentata flower polysaccharide-anthocyanin complex. Figure 13 Images show the UV stability of the polysaccharide-anthocyanin complex from *Dendrobium nobile* and the polysaccharide-anthocyanin complex from *Dendrobium dentata*. Figure 13 As shown in Table 2, compared with paeoniflorin-3-O-glucoside and delphinidin-3-O-glucoside, the half-life of the Dendrobium nobile flower polysaccharide-anthocyanin complex and the Dendrobium dentata flower polysaccharide-anthocyanin complex were significantly increased, and the anthocyanin degradation rate was significantly reduced. This indicates that the Dendrobium nobile flower polysaccharide provided by the present invention has a protective effect on anthocyanins under ultraviolet light.

[0082] The *Dendrobium nobile* flower polysaccharide-zeaxanthin complex obtained in Example 4, and the *Dendrobium nobile* flower polysaccharide-anthocyanin complex and *Dendrobium dentata* flower polysaccharide-anthocyanin complex obtained in Example 5 were exposed to direct natural light at room temperature, simulated by a 20W incandescent lamp. After irradiation for 0h, 3h, 6h, 12h, 24h, and 48h, the samples were collected, and the absorbance of anthocyanins was measured at 520nm and the absorbance of zeaxanthin at 450nm using a microplate reader. The residual amounts of anthocyanins and zeaxanthin after fixed-time strong light irradiation were determined. The degradation rate constant k was obtained through first-order reaction kinetics simulation, and the time required for 50% degradation of anthocyanins and zeaxanthin, i.e., the half-life (t), was calculated. 1 / 2 ).

[0083] Table 3. First-order kinetics of natural photostable properties of the polysaccharide-zeaxanthin complex, the polysaccharide-anthocyanin complex, and the polysaccharide-anthocyanin complex from *Dendrobium nobile*, *Dendrobium chrysanthemum*, and *Dendrobium dentata*.

[0084] Table 3 shows the first-order kinetics of natural photostability of the polysaccharide-zeaxanthin complex, the polysaccharide-anthocyanin complex, and the polysaccharide-anthocyanin complex of Dendrobium nobile. Figure 14 Images show the natural photostability of the polysaccharide-zeaxanthin complex from *Dendrobium nobile*, the polysaccharide-anthocyanin complex from *Dendrobium chrysanthum*, and the polysaccharide-anthocyanin complex from *Dendrobium dentata*. Figure 14 As shown in Table 3, compared with single zeaxanthin and anthocyanins, the half-life values ​​of the Dendrobium nobile flower polysaccharide-zeaxanthin complex, Dendrobium chrysanthum flower polysaccharide-anthocyanin complex, and Dendrobium dentata flower polysaccharide-anthocyanin complex were significantly increased, and the degradation rates of anthocyanins and zeaxanthin were significantly reduced. This indicates that the Dendrobium flower polysaccharides provided by this invention have a protective effect on anthocyanins and zeaxanthin under natural light conditions.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Dendrobium flower polysaccharide, including one or more of Dendrobium nobile flower polysaccharide, Dendrobium chrysanthum flower polysaccharide and Dendrobium dentata flower polysaccharide, wherein some O atoms in the partial repeating units constituting the Dendrobium flower polysaccharide are acetylated; The relative molecular weight of the Dendrobium flower polysaccharide is 1×10⁻⁶. 4 ~1.4×10 6 Da; The repeating units in the main chain of the Dendrobium nobile flower polysaccharide contain glucose, mannose and glucuronic acid, with mannose as the non-reducing end, and the repeating units are linked in the manner of 1,4-mannose, 1,4-glucose and 1,4-glucuronic acid. The repeating units in the main chain of the Dendrobium nobile flower polysaccharide contain mannose, galactose, galacturonic acid and arabinose, and the non-reducing ends are arabinose and galactose. The repeating units are linked in the following ways: 1,4-mannose, 1,4,6-mannose, 1,4-galactose, 1,3,4-galactose, 1,4-galacturonic acid, 1,5-arabinose and 1,2-arabinose. The repeating units in the main chain of the Dendrobium nobile flower polysaccharide contain mannose, rhamnose, xylose, arabinose, galactose, and galacturonic acid. The non-reducing ends are arabinose and galactose. The repeating units are linked in the following ways: 1,4-mannose, 1,4,6-mannose, 1,4-rhamnose, 1,3-xylose, 1,5-arabinose, 1,3,6-galactose, 1,3-galactose, and 1,3-galacturonic acid.

2. The Dendrobium flower polysaccharide according to claim 1, characterized in that, The repeating units in the main chain of the Dendrobium nobile flower polysaccharide contain D-glucose, D-mannose and D-glucuronic acid, and the non-reducing ends are α-D-mannose. The repeating units are linked in the manner of β-1,4-D-mannose, β-1,4-D-glucose and β-1,4-D-glucuronic acid. The repeating units in the main chain of the Dendrobium nobile flower polysaccharide contain D-mannose, D-galactose, D-galacturonic acid and L-arabinose, and the non-reducing ends are α-L-arabinose and β-D-galactose. The repeating units are linked in the following ways: β-1,4-D-mannose, β-1,4,6-D-mannose, β-1,4-D-galactose, β-1,3,4-D-galactose, β-1,4-D-galacturonic acid, α-1,5-L-arabinose and α-1,2-L-arabinose. The repeating units in the main chain of the Dendrobium nobile flower polysaccharide contain D-mannose, L-rhamnose, L-arabinose, D-xylose, D-galactose, and D-galacturonic acid. The non-reducing ends are L-arabinose and D-galactose. The repeating units are linked in the following ways: β-1,4-D-mannose, β-1,4,6-D-mannose, α-1,4-L-rhamnose, β-1,3-D-xylose, α-1,5-L-arabinose, β-1,3,6-D-galactose, β-1,3-D-galactose, and β-1,3-D-galacturonic acid.

3. The Dendrobium flower polysaccharide according to claim 1 or 2, characterized in that, The relative molecular weight of the *Dendrobium nobile* flower polysaccharide is 1×10⁻⁶. 4 ~4×10 4 Da; The relative molecular weight of the Dendrobium nobile flower polysaccharide is 6.5 × 10⁻⁶. 5 ~1.3×10 6 Da; The relative molecular weight of the Dendrobium nobile flower polysaccharide is 7.5 × 10⁻⁶. 5 ~1.4×10 6 Da.

4. The method for preparing Dendrobium flower polysaccharide according to any one of claims 1 to 3, characterized in that, Includes the following steps: The flowers of Dendrobium are decolorized to obtain decolorized Dendrobium flowers; the Dendrobium flowers include Dendrobium nobile, Dendrobium chrysanthum, or Dendrobium dentate. The decolorized Dendrobium flowers were mixed with water and extracted to obtain an aqueous extract. The aqueous extract was subjected to protein removal to obtain a protein-free aqueous solution. The protein-removing aqueous solution and lower alcohol are mixed and subjected to alcohol precipitation to obtain an alcohol precipitate; the volume percentage of lower alcohol in the mixture of protein-removing aqueous solution and lower alcohol is 28-92%; The alcohol precipitate was separated by a cellulose chromatography column. The cellulose chromatography column separation included sequential elution with water, a first sodium chloride aqueous solution, and a second sodium chloride aqueous solution to obtain water eluent, a first sodium chloride aqueous eluent, and a second sodium chloride aqueous eluent, respectively. The concentration of the first sodium chloride aqueous solution was 0.08~0.12 mol / L; the concentration of the second sodium chloride aqueous solution was 0.28~0.32 mol / L. When the Dendrobium flower is Dendrobium nobile flower, the water eluent is subjected to first gel chromatography separation, the first gel chromatography separation includes elution with water to obtain Dendrobium nobile flower polysaccharide; When the Dendrobium flower is Dendrobium nobile flower, the first sodium chloride water eluent is desalted to obtain a first desalted crude purified solution; the first desalted crude purified solution is separated by a second gel chromatography column, the second gel chromatography separation including elution with water to obtain Dendrobium nobile flower polysaccharide. When the Dendrobium flower is Dendrobium dentata flower, the second sodium chloride water eluent is desalted to obtain a second desalted crude purified solution; the second desalted crude purified solution is separated by a third gel chromatography column, the third gel chromatography separation including elution with water to obtain Dendrobium dentata flower polysaccharide.

5. The preparation method according to claim 4, characterized in that, The lower alcohols include ethanol; The alcohol precipitation includes gradient alcohol precipitation; the gradient alcohol precipitation includes sequentially performing a first alcohol precipitation, a second alcohol precipitation, a third alcohol precipitation, and a fourth alcohol precipitation; The volume percentage of lower alcohols in the mixture during the first alcohol precipitation process is 28-32%. The volume percentage of lower alcohols in the mixture during the second alcohol precipitation process is 48-52%. The volume percentage of lower alcohols in the mixture during the third alcohol precipitation process is 68-72%. The volume percentage of lower alcohols in the mixture during the fourth alcohol precipitation process is 88-92%. The precipitate is a precipitate obtained from a second or third alcohol precipitation.

6. The preparation method according to claim 4, characterized in that, The chromatographic column used for the cellulose separation includes the DEAE-52 cellulose chromatographic column; The column packing materials used for the first, second, and third gel chromatography column separations independently include Sephacryl S-300 gel.

7. The use of Dendrobium flower polysaccharide according to any one of claims 1 to 3 or Dendrobium flower polysaccharide prepared by the preparation method according to any one of claims 4 to 6 in stabilizing pigments; The pigments include anthocyanins and / or zeaxanthin; The anthocyanins are paeoniflorin-3-O-glucoside and / or delphinidin-3-O-glucoside; The Dendrobium flower polysaccharides and pigments form a polysaccharide-pigment complex.

8. A polysaccharide-pigment complex, characterized in that, It was obtained by self-assembly of Dendrobium flower polysaccharides and pigments; The Dendrobium flower polysaccharide is the Dendrobium flower polysaccharide according to any one of claims 1 to 3 or the Dendrobium flower polysaccharide prepared by the preparation method according to any one of claims 4 to 6; The pigments include anthocyanins and / or zeaxanthin; The anthocyanins are paeoniflorin-3-O-glucoside and / or delphinidin-3-O-glucoside.

9. The polysaccharide-pigment complex according to claim 8, characterized in that, When the pigment includes anthocyanins, the polysaccharide-pigment complex includes Dendrobium nobile flower polysaccharide-anthocyanin complex and / or Dendrobium dentata flower polysaccharide-anthocyanin complex; When the pigment includes zeaxanthin, the polysaccharide-pigment complex includes the Dendrobium nobile flower polysaccharide-zeaxanthin complex.

10. The use of the polysaccharide-pigment complex according to claim 8 or 9 in food additives, pharmaceutical excipients or antioxidant health products.

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