Preparation method of uric acid-reducing solid microsphere particles containing dendrobium huoshanense leaf extract

Abstract

The invention provides a preparation method of uric acid reducing microsphere particles containing a dendrobium huoshanense leaf extract, and belongs to the technical field of uric acid reducing compositions.The specific method includes the steps that fresh dendrobium huoshanense leaves are subjected to alcohol extraction to obtain a dendrobium huoshanense leaf ethanol extracting solution, and then the dendrobium huoshanense leaf ethanol extracting solution is purified through an AB-8 resin chromatographic column to obtain the uric acid reducing microsphere particles containing the dendrobium huoshanense leaf extract. The brand-new natural dendrobium huoshanense leaf extract microsphere particle for reducing uric acid is obtained by combining a corn polypeptide prolamin mixed solution formed by mixing a corn prolamin solution and a corn polypeptide solution, has the effect of remarkably reducing uric acid of mice, and has the advantages that the stability of effective components is kept after the particle is stored for 1-2 months, and the bioavailability of the particle is improved. The uric acid value of a hyperuricemia mouse can be obviously reduced, and the economic value is very good.

Description

Technical Field

[0001] The invention belongs to the technical field of uric acid lowering compositions and relates to a method for preparing uric acid lowering solid microsphere particles containing a Huoshan dendrobium leaf extract. Background Art

[0002] In recent years, with the development of the economy, people's living standards have been greatly improved, and the dietary structure has changed significantly. Excessive intake of foods rich in protein and purine has led to a significant increase in uric acid levels in the body, becoming the "fourth high" after the "three highs". Surveys show that there are currently a large number of hyperuricemia patients in my country. For example, among the adult physical examination population in Chongming District, Shanghai, the number of hyperuricemia patients has reached about 10.34%, and the prevalence rate has been increasing year by year. Long-term hyperuricemia can cause urate crystals to deposit in joints, soft tissues, cartilage and kidneys, causing hyperuricemia gout, arthritis, urinary stones, kidney damage and fibrosis, etc. Hyperuricemia has become an important pathogenic factor of chronic kidney disease. Although clinical drugs have obvious therapeutic effects in lowering serum uric acid levels, there are adverse reactions such as severe allergic reactions and liver and kidney damage. Natural products have become an important direction for the discovery of new anti-gout drugs due to their wide sources and low toxic side effects. At present, more and more natural products have been found to have good health effects of lowering uric acid or anti-gout, and are highly safe. Therefore, for the large number of hyperuricemia patients in my country, based on the principle of "combining prevention and treatment, prevention first", and based on the huge potential of natural products in the prevention and treatment of hyperuricemia, the development and utilization of natural products is an effective way to prevent and treat hyperuricemia.

[0003] Dendrobium, as a precious Chinese medicinal material, comes from different varieties of Dendrobium in the Dendrobium family of the Orchidaceae family, such as Huoshan Dendrobium, Candidum, and Dendrobium nobile. Dendrobium is rich in polysaccharides, flavonoids, alkaloids, terpenes, tannins, phenols and other compounds. In recent years, the application of Dendrobium in the prevention and treatment of hyperuricemia has gradually received attention. Current research reports are mainly distributed in the research on the prevention and treatment of hyperuricemia by Dendrobium. Huoshan Dendrobium is mainly distributed in Huoshan County, Anhui Province, my country. It has excellent quality. Because it is "shaped like rice" when it first comes out, it is also called "rice dendrobium". Up to now, the stems of Huoshan Dendrobium are mostly used as medicine. Its chemical composition and pharmacological effects have been widely studied and developed, but the leaves of Dendrobium have long been used as waste in the production process of Huoshan Dendrobium and have not been developed and utilized. Previous studies have shown that the content of flavonoids in Huoshan Dendrobium leaves is higher than that in stems, and significantly higher than that in Dendrobium officinale leaves, which suggests that Huoshan Dendrobium leaves may have better health benefits than Dendrobium officinale leaves. However, there are currently no research reports on the use of Huoshan Dendrobium leaves to improve hyperuricemia.

[0004] Corn peptide is a protein extracted from corn, which is a small molecule polypeptide substance obtained through directional enzymatic cleavage and specific small peptide separation technology. Corn peptide is a low-purine food-derived oligomer with good thermal stability and antioxidant activity. Corn peptide is a hydrophobic peptide that can inhibit the activity of angiotensin converting enzyme. As a competitive inhibitor of angiotensin, it reduces vascular tension and produces a hypotensive effect. It also has the functions of lowering cholesterol, promoting cholesterol metabolism in the body, and increasing the excretion of coprostanol. Corn peptide can be used as the main functional ingredient of lipid-lowering foods. The preparation method of corn peptide is simple and the price is cheap. Summary of the invention

[0005] The present invention firstly discovered that certain extracts of Huoshan Dendrobium candidum leaves have the effect of reducing uric acid in mice. On this basis, the inventors purified and extracted the extracts, and processed the functional components with uric acid-lowering effect to make them resistant to digestive juices. Finally, a microsphere composition containing Huoshan Dendrobium candidum leaf extract and a preparation method thereof were obtained, which can significantly reduce the uric acid value of a hyperuricemia mouse model.

[0006] A method for preparing uric acid-lowering microsphere particles containing a Huoshan dendrobium leaf extract comprises the following steps: (1) Fresh Dendrobium leaves were dried to a moisture content of less than 5%, and then crushed to obtain Dendrobium leaf powder. A 65-75% ethanol aqueous solution was added to the Dendrobium leaf powder, and extracted at 85-90°C for 20-50 min. The residue was filtered and the extraction was repeated 1-3 times. The filtrates were combined to obtain the ethanol extract of Dendrobium huoshanense leaves. (2) The ethanol extract of the leaves of Dendrobium huoshanense was passed through an AB-8 resin chromatography column under the condition of a loading volume of 6-8 BV. After passing through the column, it was eluted with a 50-55% volume fraction ethanol solution of 5-7 BV. Finally, the collected solutions were combined, concentrated by evaporation under reduced pressure, and then freeze-dried to obtain a dry powder of the extract of the leaves of Dendrobium huoshanense; (3) dissolving zein powder in a 75-85% ethanol aqueous solution to prepare a 5-10 mg / mL zein solution, adding a neutral protease in an amount of 0.5-2 mg / mL to the solution, subjecting the solution to enzymatic hydrolysis at 28-38°C for 8-15 min, then heating to 80-90°C to inactivate the enzyme, cooling to obtain a corn polypeptide solution, and mixing the zein solution with the corn polypeptide solution to obtain a corn polypeptide and prolamin mixed solution; (4) Prepare a 1.0-2.0 mg / mL sodium carboxymethyl cellulose aqueous solution using ultrapure water; (5) Weigh 30 mg of dried Huoshan Dendrobium leaf extract and dissolve it in 30-90 mL of the prepared corn polypeptide alcohol-soluble protein mixed solution to obtain a Huoshan Dendrobium leaf extract-corn polypeptide alcohol-soluble protein mixed solution, then add the solution dropwise into the above-mentioned sodium carboxymethyl cellulose aqueous solution at a volume ratio of 1:1-1:1.5, and continue stirring until a suspension of corn polypeptide alcohol-soluble protein-sodium carboxymethyl cellulose composite nanoparticles loaded with Huoshan Dendrobium leaf extract is formed; (6) The suspension of corn polypeptide alcohol-soluble protein-sodium carboxymethyl cellulose composite nanoparticles is centrifuged to collect the solid precipitate to obtain uric acid-lowering solid microsphere particles containing the extract of Dendrobium huoshanense leaves.

[0007] Wherein, in the step (1), the method for drying the fresh dendrobium leaves is to dry them naturally in a ventilated place and then place them in a drying oven at 40-70° C. for 6-24 hours.

[0008] Wherein, in the step (1), the dendrobium leaf powder and the 65-75% ethanol aqueous solution are mixed in a solid-liquid ratio of 1:10-1:50.

[0009] Wherein, in the step (2), the conditions for rotary vacuum evaporation and concentration are 50-60°C and 0.05-0.3MPa.

[0010] Wherein, in the step (2), the freeze-drying temperature is -70~-50°C.

[0011] Wherein, in the step (3), the volume ratio of the zein solution to the corn polypeptide solution is 2:1 to 1:2.

[0012] Wherein, in the step (5), the continuous stirring condition is to continuously stir at a rotation speed of 400-600 rpm for 0.5-1 h.

[0013] Wherein, in the step (6), the centrifugation condition is centrifugation at a rotation speed of 10000-12000 rpm for 15-30 min. Beneficial Effects

[0014] After extraction and purification, 33 flavonoids were identified by UPLC-Q-Exactive Orbitrap MS analysis. Comparison with the chemical components of Dendrobium related plants reported previously revealed quercetin-3-O-sophoroside, 5,6,7,8,4'-pentamethoxyflavone, kaempferol-3-O-rutinoside, vitexin glucoside, quercetin 3-O-rutinoside, luteolin-6-C-β-D-glucoside-8-C-β-D-galactoside, vitexin 1, vitexin 2, isovitexin-2"-O-xyloside, vitexin 3, luteolin-6-C-β-D-xyloside-8-C-β-D-glucoside, apigenin-6-C-ɑ-L-arabinoside, and 1,2-dimethoxy-1,2-dimethoxy-1,2-dimethoxy-1,2-dimethoxy-1,2-dimethoxy-1,2-dimethoxy-1,2-dimethoxy-1,2-dimethoxy-1,2-dimethoxy-1,2-dimethoxy-1 Primarinoside-8-C-β-D-xyloside, apigenin-6-C-β-D-xylopyranosyl-8-C-ɑ-L-arabinopyranoside, kaempferol-3-O-glucoside, apigenin-8-C-β-D-glucose-(1→2)-β-D-glucoside, luteolin-6-C-β-D-glucoside-8-C-β-D-xyloside, apigenin-8-O-glucoside, violacein, quercetin-7-O-rutinoside, 4',5,7-trihydroxyflavone, quercetin 3-β-D-glucoside, isovitexin, daidzein, 24 compounds including 4',5,7-trihydroxyisoflavone have been reported to exist in other species of Dendrobium, but this is the first time that they have been confirmed to exist in the leaves of Dendrobium huoshanense. Through relative quantitative analysis, this study first found that schaftoside, violacein, apigenin-6-C-β-D-xylopyranosyl-8-C-ɑ-L-arabinopyranoside and kaempferol-3-O-rutinoside are the main components of flavonoids in the leaves of Dendrobium huoshanense, while Zhou Guifen et al. showed that the main flavonoids in the leaves of Dendrobium officinale are apigenin-6,8-di-C-β-D-pyranoglucoside, apigenin-6-C-β-D -xylose-8-C-β-D-pyranoglucoside, isoschaftoside, schaftoside, apigenin-6-C-β-D-pyranoglucoside-8-C-β-D-xylose-8-C-α-L-arabinoside, apigenin-6,8-di-C-α-L-pyranoglucoside, apigenin-6-C-α-L-arabinoside-8-C-β-D-xylose-8-C-α-L-arabinoside, and the main flavonoid components of the two are significantly different. The results indicate that Huoshan Dendrobium leaves may have potential health benefits and pharmacological effects that are different from those of Dendrobium officinale leaves.

[0015] The applicant found through further experiments that the alcohol extract of Huoshan Dendrobium leaf has a certain effect of lowering uric acid, but its stability is poor and it is not suitable for long-term storage at room temperature. Therefore, the Huoshan Dendrobium leaf extract is made into microsphere particles to improve its stability and conduct uric acid lowering experiments. The reason for choosing zein solution and corn polypeptide solution is that corn peptide is a low-purine food-derived oligopeptide. Corn peptide has good thermal stability and antioxidant activity. Zein will not introduce high purine and can play a good protective and sustained release role on Huoshan Dendrobium leaf extract, which can improve the stability of microsphere particles. Similar wheat peptides and rice peptides cannot form stable microsphere particles.

[0016] In addition, corn peptides may inhibit the effect of gastric acid on the alcohol extract of Dendrobium huoshanense leaves, increase the absorption rate and activity of the alcohol extract of Dendrobium huoshanense leaves in the intestine, and thus improve the uric acid-lowering effect of the alcohol extract of Dendrobium huoshanense leaves. The present invention induces hyperuricemia by intragastric administration of yeast extract (30g / kg / d) to male mice once a day for 14 consecutive days, and intraperitoneally injects potassium salt solution of oxonic acid (200mg / kg) once 1 hour after intragastric administration on the 14th day. Based on this model, the microsphere particles of Dendrobium huoshanense leaf extract were given for intervention at the same time of modeling. The study showed that the microsphere particles of Dendrobium huoshanense leaf extract significantly inhibited the levels of blood uric acid (UA), urea nitrogen (BUN), xanthine oxidase (XOD) and creatinine (Scr), and it was dose-dependent. At the same time, the microsphere particles of Huoshan Dendrobium leaf extract significantly inhibited the levels of inflammatory factors IL-6 and IL-1β caused by high uric acid and the levels of kidney damage factors NGAL and Kim-1. Other slice results showed that the microsphere particles of Huoshan Dendrobium leaf extract significantly improved kidney tissue damage.

[0017] The present invention develops a new natural uric acid-lowering microsphere particle of Huoshan Dendrobium leaf extract, which has good stability and can be stored for 1 to 2 months to maintain the stability of the effective ingredients, can significantly reduce the uric acid level of hyperuricemia mice, and has good economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Total ion chromatograms of the extract of Dendrobium huoshanense leaves in positive (A) and negative (B) ion modes.

[0019] Figure 2 The intervention of microsphere particles of Dendrobium huoshanense leaf extract reduced the levels of UA, BUN, XOD and Scr in mice.

[0020] Figure 3 The intervention of microsphere particles of Dendrobium huoshanense leaf extract reduced the levels of inflammatory factors IL-6 and IL-1β and the levels of kidney injury factors NGAL and Kim-1 in mice.

[0021] Figure 4Intervention with microsphere particles of Dendrobium huoshanense leaf extract improved renal tissue damage in mice.

[0022] Figure 5 The microsphere particles of Huoshan Dendrobium leaf extract inhibited the expression of URAT1 and GLUT9, key proteins in renal uric acid reabsorption. DETAILED DESCRIPTION

[0023] The present invention is described in detail below with reference to specific embodiments.

[0024] In the embodiment, 95% ethanol (analytical grade, Tianjin Yongda Chemical Reagent Co., Ltd.); methanol, acetonitrile (chromatographic grade, Tianjin Biaoshiqi Technology Development Co., Ltd.); ammonium acetate (chromatographic grade; Shanghai Aladdin Biochemical Technology Co., Ltd.); AB-8 macroporous resin (Shanghai MacLean Biochemical Technology Co., Ltd.); neutral protease (enzyme activity ~50000u / g). Example

[0025] This embodiment provides a uric acid-lowering microsphere granule containing a Dendrobium huoshanense leaf extract, which specifically includes the following steps.

[0026] (1) Fresh Dendrobium leaves were naturally dried in a ventilated place and then dried at 60°C for 12 hours to reduce the moisture content to less than 5%. After drying, they were crushed to obtain Dendrobium leaf powder. 70% ethanol was added at a solid-liquid ratio of 1:30 and extracted in a 90°C water bath for 30 min. The residue was filtered and extracted again. The filtrates were combined to obtain the ethanol extract of Dendrobium huoshanense leaves.

[0027] (2) The ethanol extract of Dendrobium huoshanense leaves was passed through an AB-8 resin chromatography column with a loading volume of 7 BV. After passing through the column, it was eluted with 6 BV, 50% volume fraction ethanol solution. Finally, the solutions were combined and concentrated by rotary vacuum evaporation at 55°C and 0.1 MPa. The concentrate was then freeze-dried in a freeze drying oven at -55°C to obtain the dry powder of Dendrobium huoshanense leaf extract.

[0028] (3) Dissolve zein powder in 80% ethanol aqueous solution to prepare an 8 mg / mL zein solution, add neutral protease in an amount of 1 mg / mL to the solution, place it at 35°C for enzymatic hydrolysis for 10 min, then heat it to 80°C to inactivate the enzyme, cool it to obtain a corn polypeptide solution, mix the zein solution with the corn polypeptide solution in a ratio of 1:1 to obtain a corn polypeptide-protein mixed solution.

[0029] (4) Prepare a 1.5 g / mL sodium carboxymethyl cellulose aqueous solution using ultrapure water.

[0030] (5) Weigh 30 mg of dried Huoshan Dendrobium leaf extract and dissolve it in 50 mL of the prepared corn polypeptide alcohol-soluble protein mixed solution to obtain a Huoshan Dendrobium leaf extract-corn polypeptide alcohol-soluble protein mixed solution, then add the solution dropwise into the above-mentioned sodium carboxymethyl cellulose aqueous solution at a volume ratio of 1:1, and continue stirring at a speed of 500 rpm for 0.8 to form a suspension of corn polypeptide alcohol-soluble protein-sodium carboxymethyl cellulose composite nanoparticles loaded with Huoshan Dendrobium leaf extract; (6) The composite nanoparticle suspension obtained in step (5) was centrifuged at a speed of 10,000 rpm for 20 min to obtain uric acid-lowering solid microsphere particles containing the Huoshan Dendrobium officinale leaf extract.

[0031] First, the dry powder of Dendrobium huoshanense leaf extract was analyzed by UPLC-Q-Exactive Orbitrap MS. The specific method is as follows: Chromatographic conditions: Column: ACQUITY UPLC BEH C 18 Column (2.1 mm×100 mm, 1.7 μm); column temperature 40℃; flow rate 0.8 mL / min; injection volume 0.2 μL; mobile phase A was 5 mM ammonium acetate solution, mobile phase B was acetonitrile; gradient elution: 0-10 min, 92%→84%A; 10-25 min, 84%→82%A; 25-60 min, 82%→78%A; 60-75 min, 78%A; 75→85 min, 78%→70%A; 85-100 min, 70%→92%A [4] .

[0032] 1.6.2.2 Mass spectrometry conditions Detection method: heated electrospray ionization (HESI); sheath gas flow rate 45 arb; auxiliary gas flow rate 15 arb; purge gas flow rate 1 arb; capillary temperature 320 °C; auxiliary gas heater temperature 350 °C; positive spray voltage 3.1 kV; negative spray voltage 3.1 kV; MS resolution 70 000; MS / MS resolution 17 500; scanning mode: full scan mode; positive and negative ion modes simultaneous detection; scanning range m / z 100~1 500.

[0033] The qualitative and relative quantitative analysis of compounds was based on the database of the mass spectrometry workstation and the public database of compound information, with reference to existing public mass spectrometry databases such as HMDB, PubChem and METLIN, and compared with the literature to compare and analyze the primary and secondary spectra of mass spectrometry detection. At the same time, the relative quantification of the main flavonoids was performed with reference to the chromatographic peak response value of schaftoside.

[0034] Ion current diagram Figure 1 In the positive ion mode, 3 flavonoids were identified, and in the negative ion mode, 30 flavonoids were identified. The relative contents of other flavonoids were calculated with reference to the chromatographic peak response value of schaftoside. The results showed that schaftoside had the highest content among the flavonoids in the leaves of Dendrobium huoshanense, and the other flavonoids with relative contents greater than 50 included violacein, apigenin-6-C-β-D-xylopyranosyl-8-C-α-L-arabinopyranoside and kaempferol-3-O-rutinoside, while tangerine, apigenin, quercetin 3-O-rutinoside, soybean yellow, naringenin and genistein had the lowest relative contents.

[0035] The following is a validation experiment on the ability of uric acid-lowering solid microsphere particles containing Huoshan Dendrobium leaf extract to reduce blood uric acid in hyperuricemia mice.

[0036] Effect of microspheres of extract from Dendrobium huoshanense leaves on reducing blood uric acid in hyperuricemic mice.

[0037] 60 male Kunming mice, weighing 25-30g, were randomly divided into 6 groups: blank group, model group, benzbromarone (BB), extract microsphere particle low (100mg / kg), medium (200mg / kg), high (400mg / kg) dose group, 10 mice in each group. The extract and positive drug intervention were given by gavage at the same time of modeling, once a day. On the 14th day, 1 hour after gavage, potassium salt solution of oxonic acid (200mg / kg) was injected intraperitoneally. Serum was collected and analyzed 2 hours later. The experimental results are as follows Figure 2 As shown, the results showed that the microsphere particles of Huoshan Dendrobium leaf extract significantly inhibited the levels of blood uric acid (UA), urea nitrogen (BUN), xanthine oxidase (XOD) and creatinine (Scr), and were dose-dependent.

[0038] Microspheres of extract from Dendrobium huoshanense leaves improve renal damage in hyperuricemia mice.

[0039] 60 male Kunming mice, weighing 25-30g, were randomly divided into 6 groups: blank group, model group, benzbromarone (BB), extract microsphere particle low (100mg / kg), medium (200mg / kg), and high (400mg / kg) dose groups, with 10 mice in each group. The extract and positive drug intervention were given by gavage at the same time as modeling, once a day. On the 14th day, potassium salt solution of oxonic acid (200mg / kg) was injected intraperitoneally 1 hour after gavage. Serum and kidney tissue were collected and fixed with 4% paraformaldehyde fixative 2 hours later. The levels of inflammatory factors and kidney damage factors in serum were analyzed, and kidney tissue damage was analyzed by tissue sections. The experimental results are as follows Figure 3 and 4As shown, the results showed that the microsphere particles of Huoshan Dendrobium leaf extract significantly inhibited the levels of inflammatory factors IL-6 and IL-1β and kidney damage factors NGAL and Kim-1 caused by high uric acid. The slice results showed that the microsphere particles of Huoshan Dendrobium leaf extract significantly improved liver tissue damage.

[0040] Microspheres of Dendrobium huoshanense leaf extract reduce the blood uric acid level in hyperuricemic mice by inhibiting uric acid reabsorption.

[0041] 60 male Kunming mice, weighing 25-30g, were randomly divided into 6 groups: blank group, model group, benzbromarone (BB), extract microsphere particle low (100mg / kg), medium (200mg / kg), and high (400mg / kg) dose groups, with 10 mice in each group. The extract and positive drug intervention were given by gavage at the same time as modeling, once a day. On the 14th day, potassium salt solution of oxonic acid (200mg / kg) was injected intraperitoneally 1 hour after gavage, and the kidney tissue was fixed with 4% paraformaldehyde fixative. Immunofluorescence analysis of kidney tissue sections showed the expression levels of URAT1 and GLUT9, key protein molecules in uric acid reabsorption. The experimental results are as follows Figure 5 As shown, the results showed that the microsphere particles of Huoshan Dendrobium leaf extract significantly inhibited the expression of URAT1 and GLUT9 proteins in the kidneys of mice, thereby inhibiting uric acid reabsorption and achieving the health effect of lowering uric acid. Example

[0042] This embodiment provides a uric acid-lowering microsphere granule containing a Dendrobium huoshanense leaf extract, which specifically includes the following steps.

[0043] (1) Fresh Dendrobium officinale leaves were naturally dried in a ventilated place and then dried at 70°C for 6 hours to reduce the moisture content to less than 5%. After drying, they were crushed and extracted with 65% ethanol at a solid-liquid ratio of 1:50 in an 85°C water bath for 20 min. The residues were filtered and extracted twice. The filtrates were combined to obtain the ethanol extract of Dendrobium officinale leaves.

[0044] (2) The ethanol extract of Dendrobium huoshanense leaves was passed through an AB-8 resin chromatography column with a loading volume of 6 BV. After passing through the column, it was eluted with 5 BV, 50% volume fraction ethanol solution. Finally, the solutions were combined and concentrated by rotary vacuum evaporation at 50°C and 0.05 MPa. The concentrate was then freeze-dried in a freeze drying oven at -70°C to obtain the dry powder of Dendrobium huoshanense leaf extract.

[0045] (3) Dissolve zein powder in 75% ethanol aqueous solution to prepare a 5 mg / mL zein solution, add neutral protease at a rate of 0.5 mg / mL to the solution, place it at 28°C for enzymatic hydrolysis for 15 min, then heat to 80°C to inactivate the enzyme, cool it, and obtain a corn polypeptide solution. Mix the zein solution with the corn polypeptide solution at a ratio of 1:2 to obtain a corn polypeptide-protein mixed solution.

[0046] (4) Prepare a 1.0 mg / mL sodium carboxymethyl cellulose aqueous solution using ultrapure water.

[0047] (5) Weigh 30 mg of dried Huoshan Dendrobium leaf extract and dissolve it in 30 mL of the prepared corn polypeptide alcohol-soluble protein mixed solution to obtain a Huoshan Dendrobium leaf extract-corn polypeptide alcohol-soluble protein mixed solution, then add the solution to the above-mentioned sodium carboxymethyl cellulose aqueous solution at a volume ratio of 1:25, and continue stirring for 0.5 h at a speed of 400 rpm to form a suspension of corn polypeptide alcohol-soluble protein-sodium carboxymethyl cellulose composite nanoparticles loaded with Huoshan Dendrobium leaf extract. (6) The composite nanoparticle suspension obtained in step (5) was centrifuged at a speed of 10,000 rpm for 15 min to obtain uric acid-lowering solid microsphere particles containing the Huoshan Dendrobium officinale leaf extract. Example

[0048] This embodiment provides a uric acid-lowering microsphere granule containing a Dendrobium huoshanense leaf extract, which specifically includes the following steps.

[0049] (1) Fresh Dendrobium officinale leaves were dried naturally in a ventilated place and then placed in an oven at 40°C for 24 hours to reduce the moisture content to less than 5%. After drying, they were crushed and extracted in a 90°C water bath with 75% ethanol at a solid-liquid ratio of 1:10 for 50 min. The residues were filtered and extracted three times. The filtrates were combined to obtain the ethanol extract of Dendrobium officinale leaves.

[0050] (2) The ethanol extract of Dendrobium huoshanense leaves was passed through an AB-8 resin chromatography column with a loading volume of 8 BV. After passing through the column, it was eluted with 7 BV, 55% volume fraction ethanol solution. Finally, the solutions were combined and concentrated by rotary vacuum evaporation at 60°C and 0.3 MPa. The concentrate was then freeze-dried in a freeze drying oven at -50°C to obtain the dry powder of Dendrobium huoshanense leaf extract.

[0051] (3) Dissolve zein powder in 85% ethanol aqueous solution to prepare a 10 mg / mL zein solution, add 2 mg / mL neutral protease to the solution, place it at 38°C for 8 min, then heat it to 90°C to inactivate the enzyme, cool it to obtain a corn polypeptide solution, mix the zein solution with the corn polypeptide solution in a ratio of 2:1 to obtain a corn polypeptide-protein mixed solution.

[0052] (4) Prepare a 2.0 mg / mL sodium carboxymethyl cellulose aqueous solution using ultrapure water.

[0053] (5) Weigh 30 mg of dried Huoshan Dendrobium leaf extract and dissolve it in 90 mL of the prepared corn polypeptide alcohol-soluble protein mixed solution to obtain a Huoshan Dendrobium leaf extract-corn polypeptide alcohol-soluble protein mixed solution, then add the solution dropwise into the above sodium carboxymethyl cellulose aqueous solution at a volume ratio of 1:1.5, and continue stirring at a speed of 600 rpm for 1 hour to form a suspension of corn polypeptide alcohol-soluble protein-sodium carboxymethyl cellulose composite nanoparticles loaded with Huoshan Dendrobium leaf extract. (6) The composite nanoparticle suspension obtained in step (5) is centrifuged at a speed of 12000 rpm for 15-30 min to obtain uric acid-lowering solid microsphere particles containing the Huoshan Dendrobium huoshanense leaf extract.

Claims

1. A method for preparing uric acid-lowering microsphere granules containing Huoshan Dendrobium officinale leaf extract, characterized in that: The steps include: (1) Fresh Dendrobium leaves were dried to a moisture content of less than 5%, and then crushed to obtain Dendrobium leaf powder. A 65-75% ethanol aqueous solution was added to the Dendrobium leaf powder, and extracted at 85-90°C for 20-50 min. The residue was filtered and the extraction was repeated 1-3 times. The filtrates were combined to obtain the ethanol extract of Dendrobium huoshanense leaves. (2) The ethanol extract of the leaves of Dendrobium huoshanense was passed through an AB-8 resin chromatography column under the condition of a loading volume of 6-8 BV. After passing through the column, it was eluted with a 50-55% volume fraction ethanol solution of 5-7 BV. Finally, the collected solutions were combined, concentrated by evaporation under reduced pressure, and then freeze-dried to obtain a dry powder of the extract of the leaves of Dendrobium huoshanense; (3) dissolving zein powder in a 75-85% ethanol aqueous solution to prepare a 5-10 mg / mL zein solution, adding a neutral protease in an amount of 0.5-2 mg / mL to the solution, subjecting the solution to enzymatic hydrolysis at 28-38°C for 8-15 min, then heating to 80-90°C to inactivate the enzyme, cooling to obtain a corn polypeptide solution, and mixing the zein solution with the corn polypeptide solution to obtain a corn polypeptide and prolamin mixed solution; (4) Prepare a 1.0-2.0 mg / mL sodium carboxymethyl cellulose aqueous solution using ultrapure water; (5) Weigh 30 mg of dried Huoshan Dendrobium leaf extract and dissolve it in 30-90 mL of the prepared corn polypeptide alcohol-soluble protein mixed solution to obtain a Huoshan Dendrobium leaf extract-corn polypeptide alcohol-soluble protein mixed solution, then add the solution dropwise into the above-mentioned sodium carboxymethyl cellulose aqueous solution at a volume ratio of 1:1-1:1.5, and continue stirring until a suspension of corn polypeptide alcohol-soluble protein-sodium carboxymethyl cellulose composite nanoparticles loaded with Huoshan Dendrobium leaf extract is formed; (6) The suspension of corn polypeptide alcohol-soluble protein-sodium carboxymethyl cellulose composite nanoparticles is centrifuged to collect the solid precipitate to obtain uric acid-lowering solid microsphere particles containing the extract of Dendrobium huoshanense leaves.

2. The method for preparing the uric acid-lowering microsphere granules containing the Huoshan Dendrobium leaf extract according to claim 1, characterized in that: In the step (1), the method for drying the fresh dendrobium leaves is to dry them naturally in a ventilated place and then place them in a drying oven at 40-70° C. for 6-24 hours.

3. The method for preparing the uric acid-lowering microsphere granules containing the Huoshan Dendrobium leaf extract according to claim 1, characterized in that: In the step (1), the dendrobium leaf powder and the 65-75% ethanol aqueous solution are mixed in a solid-liquid ratio of 1:10-1:

50.

4. The method for preparing the uric acid-lowering microsphere granules containing the Huoshan Dendrobium leaf extract according to claim 1, characterized in that: In the step (2), the conditions for rotary vacuum evaporation and concentration are 50-60° C. and 0.05-0.3 MPa.

5. The method for preparing the uric acid-lowering microsphere granules containing the Huoshan Dendrobium leaf extract according to claim 1, characterized in that: In the step (2), the freeze-drying temperature is -70 to -50°C.

6. The method for preparing the uric acid-lowering microsphere granules containing the extract of Dendrobium huoshanense leaves according to claim 1, characterized in that: In the step (3), the volume ratio of the zein solution to the corn polypeptide solution is 2:1 to 1:

2.

7. The method for preparing the uric acid-lowering microsphere granules containing the Huoshan Dendrobium leaf extract according to claim 1, characterized in that: In the step (5), the continuous stirring condition is to continuously stir at a rotation speed of 400-600 rpm for 0.5-1 h.

8. The method for preparing the uric acid-lowering microsphere granules containing the Huoshan Dendrobium leaf extract according to claim 1, characterized in that: In the step (6), the centrifugation condition is to centrifuge at a rotation speed of 10000-12000 rpm for 15-30 min.

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