A eucommia ulmoides extract composition, preparation method and application

Abstract

The application belongs to the field of medicine and particularly relates to a eucommia extract composition, a preparation method and application. Components according to weight fractions include the following components: eucommia polysaccharide 5-10 parts, rutin 5-15 parts, hyperoside 5-15 parts, isochlorogenic acid C 1-5 parts, apigenin 1-5 parts and oleanolic acid 1-5 parts. A preparation method mixes eucommia polysaccharide, rutin, hyperoside, isochlorogenic acid C, apigenin and oleanolic acid or adds a solvent to mix to obtain the composition. The composition has a significant synergistic effect on the treatment of membranous nephropathy, thereby providing a new idea for the prevention and / or treatment of membranous nephropathy related diseases, the preparation method is simple, suitable for large-scale production and popularization and use, and the cost is reduced.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a Eucommia ulmoides extract composition, its preparation method, and its application. Background Technology

[0002] Membranous nephropathy (MN), also known as membranous glomerulonephritis, is pathologically characterized by diffuse deposition of immune complexes under the epithelial cells of the glomerular basement membrane, accompanied by diffuse thickening of the basement membrane. Clinically, it mainly presents as nephrotic syndrome (NS) or asymptomatic proteinuria.

[0003] Eucommia ulmoides is often used as a traditional Chinese medicine in combination with other medicinal herbs to treat diseases such as diabetic nephropathy.

[0004] Chinese invention patent application number 201810071132.1 discloses a traditional Chinese medicine composition for the prevention and treatment of diabetic nephropathy and its preparation method. This traditional Chinese medicine composition is mainly prepared from the following raw materials in parts by weight: Astragalus membranaceus 550-650 parts, Pueraria lobata 150-250 parts, Eucommia ulmoides 150-250 parts, Centella asiatica 150-250 parts, Cicadae periostracum 100-200 parts, Bombyx mori 100-200 parts, Arctium lappa 100-200 parts, Rheum palmatum 40-80 parts, and Curcuma longa 100-200 parts. This invention has a precise formula and significant therapeutic effect. The combined use of these herbs, with their pungent and bitter properties, promotes both tonification and purgation, achieving the effects of tonifying the kidneys, removing blood stasis, and opening the meridians. It has good efficacy in reducing proteinuria and improving renal function. It can improve the general symptoms of diabetic nephropathy, protect renal function, and delay the progression of diabetic nephropathy. This is related to its upregulation of the expression of podocyte-related proteins nephrin and podocin.

[0005] However, as a medicinal composition, the utilization of raw materials is very limited. Therefore, further research on the application of Eucommia ulmoides extract and other traditional Chinese medicine extracts in podocyte injury and membranous nephropathy-related diseases not only provides a new approach for the prevention and / or treatment of these diseases, but also overcomes the drawbacks of conventional traditional Chinese medicine preparations, such as the difficulty in determining side effects due to the large number of components and the complex pharmacological basis of their action, thus greatly improving their therapeutic effects. Summary of the Invention

[0006] To address the aforementioned shortcomings, this invention provides an Eucommia ulmoides extract composition, its preparation method, and its application in doxorubicin-induced human podocyte injury. The Eucommia ulmoides extract composition of this invention exhibits better preventative and / or therapeutic effects on human podocyte injury-related diseases, thus providing a new approach for the prevention and / or treatment of these diseases.

[0007] On one hand, the present invention provides an Eucommia ulmoides extract composition comprising the following components: Eucommia ulmoides polysaccharide, rutin, hyperoside, isochlorogenic acid C, apigenin and oleanolic acid.

[0008] Preferably, the product comprises the following components by weight: 5-10 parts of Eucommia ulmoides polysaccharide, 5-15 parts of rutin, 5-15 parts of hyperoside, 1-5 parts of isochlorogenic acid C, 1-5 parts of apigenin, and 1-5 parts of oleanolic acid.

[0009] Preferably, the product comprises the following components by weight: 5-8 parts of Eucommia ulmoides polysaccharide, 10-15 parts of rutin, 10-15 parts of hyperoside, 3-5 parts of isochlorogenic acid C, 1-2 parts of apigenin, and 3-5 parts of oleanolic acid.

[0010] Preferably, the product comprises the following components by weight: 5 parts Eucommia polysaccharide, 15 parts rutin, 15 parts hyperoside, 3 parts isochlorogenic acid C, 1 part apigenin, and 3 parts oleanolic acid.

[0011] Preferably, the preparation method of the Eucommia ulmoides polysaccharide includes the following steps:

[0012] (1) Add solvent to Eucommia bark and extract to obtain extract;

[0013] (2) The extract was subjected to enzymatic hydrolysis to obtain the hydrolysate;

[0014] (3) The enzyme hydrolysate is obtained by precipitating it with alcohol.

[0015] Preferably, the solvent is an aqueous solution containing 0.05-0.1% lecithin; the mass of the aqueous solution is 5-10 times that of the Eucommia ulmoides bark.

[0016] Preferably, the enzymes used in the enzymatic hydrolysis include saccharifying enzyme, β-glucanase, and cellulase, and the amounts of the three added are 0.1-0.5%, 0.05-0.1%, and 0.1-0.5% of the extract mass, respectively.

[0017] Preferably, the alcohol precipitation is a step precipitation, specifically, the alcohol is precipitated sequentially with a 45-55% aqueous ethanol solution, an 80-90% aqueous ethanol solution, and an 80-90% aqueous ethanol solution containing 5-10% acetone.

[0018] Another object of the present invention is to provide a method for preparing the above composition, comprising the following steps: mixing Eucommia polysaccharide, rutin, hyperoside, isochlorogenic acid C, apigenin and oleanolic acid or mixing them with a solvent.

[0019] In another aspect, the present invention provides the use of the above-mentioned Eucommia ulmoides extract or traditional Chinese medicine composition in the preparation of medicaments for the prevention and / or treatment of membranous nephropathy-related diseases.

[0020] Specifically, the podocyte injury-related diseases include, but are not limited to, minimal change disease, focal segmental nephrosclerosis, membranous nephropathy, infection, lupus nephritis, and poisoning.

[0021] In another aspect, the present invention provides a pharmaceutical preparation for the prevention and / or treatment of membranous nephropathy-related diseases, wherein the pharmaceutical preparation comprises the above-mentioned Eucommia ulmoides extract or traditional Chinese medicine composition.

[0022] Specifically, the drug further includes a pharmaceutically acceptable carrier, which is any one or more of a sustained-release agent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorbent carrier, surfactant, or lubricant.

[0023] Specifically, the drug is any one of a topical preparation, an oral preparation, or an injectable preparation.

[0024] More specifically, the topical preparation is a spray or aerosol.

[0025] More specifically, the oral preparation is any one of granules, capsules, oral tablets, pills, or vesicles.

[0026] More specifically, the injectable formulation is administered via intradermal, subcutaneous, intramuscular, local, or intravenous injection.

[0027] Compared with the prior art, the positive and beneficial effects of the present invention are as follows:

[0028] (1) The Eucommia ulmoides extract of the present invention contains Eucommia ulmoides polysaccharide, rutin, hyperoside, isochlorogenic acid C, apigenin and oleanolic acid, and the components work synergistically to prevent and / or treat membranous nephropathy-related diseases.

[0029] (2) Further research in this invention has found that the combined use of Eucommia ulmoides polysaccharide and hyperoside has a significant synergistic effect in the treatment of membranous nephropathy, thus providing a new approach for the prevention and / or treatment of membranous nephropathy-related diseases.

[0030] (3) The preparation method of the traditional Chinese medicine composition described in this invention is simple, suitable for large-scale production and promotion, and reduces costs. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0032] Example 1

[0033] An extract composition of Eucommia ulmoides, comprising, by weight, the following components: 5 parts Eucommia ulmoides polysaccharide, 15 parts rutin, 15 parts hyperoside, 3 parts isochlorogenic acid C, 1 part apigenin and 3 parts oleanolic acid.

[0034] The preparation method of Eucommia ulmoides polysaccharide is as follows:

[0035] (1) Add 8 times the amount of aqueous solution containing 0.08% lecithin to the bark of Eucommia ulmoides and heat under reflux to extract the extract;

[0036] (2) Add 0.3% saccharifying enzyme, 0.05% β-glucanase and 0.3% cellulase by weight of the extract and carry out enzymatic hydrolysis at 50℃ and pH 5.5 for 1.5h to obtain the hydrolysate;

[0037] (3) Boil the enzyme hydrolysate at 100°C for 5 minutes to inactivate it. Then, precipitate it in sequence with 50% ethanol aqueous solution, 85% ethanol aqueous solution and 85% ethanol aqueous solution containing 8% acetone. Dry it to obtain the final product.

[0038] The preparation method of Eucommia ulmoides extract is as follows:

[0039] The product is obtained by mixing Eucommia polysaccharide, rutin, hyperoside, isochlorogenic acid C, apigenin and oleanolic acid evenly.

[0040] Example 2

[0041] An extract composition of Eucommia ulmoides, comprising, by weight, the following components: 5 parts Eucommia ulmoides polysaccharide, 5 parts rutin, 5 parts hyperoside, 1 part isochlorogenic acid C, 1 part apigenin and 1 part oleanolic acid.

[0042] The preparation method of Eucommia ulmoides polysaccharide is as follows:

[0043] (1) Add 5 times the amount of aqueous solution containing 0.05% lecithin to the bark of Eucommia ulmoides and heat under reflux to extract the extract;

[0044] (2) Add 0.1% saccharifying enzyme, 0.1% β-glucanase and 0.1% cellulase by weight of the extract and carry out enzymatic hydrolysis at 45℃ and pH 6 for 2 hours to obtain the hydrolysate;

[0045] (3) Boil the enzyme hydrolysate at 100°C for 5 minutes, then precipitate it with 55% ethanol aqueous solution, 90% ethanol aqueous solution and 80% ethanol aqueous solution containing 10% acetone in sequence, and dry it to obtain the final product.

[0046] The preparation method of the Eucommia ulmoides extract composition is as follows:

[0047] The product is obtained by mixing Eucommia polysaccharide, rutin, hyperoside, isochlorogenic acid C, apigenin and oleanolic acid evenly.

[0048] Example 3

[0049] An extract composition of Eucommia ulmoides, comprising, by weight, the following components: 10 parts Eucommia ulmoides polysaccharide, 15 parts rutin, 15 parts hyperoside, 5 parts isochlorogenic acid C, 5 parts apigenin and 5 parts oleanolic acid.

[0050] The preparation method of Eucommia ulmoides polysaccharide is as follows:

[0051] (1) Add 10 times the amount of aqueous solution containing 0.1% lecithin to Eucommia bark and heat under reflux to extract the extract;

[0052] (2) Add 0.5% saccharifying enzyme, 0.05% β-glucanase and 0.5% cellulase by weight of the extract and carry out enzymatic hydrolysis at 55℃ and pH5 for 1 h to obtain the hydrolysate;

[0053] (3) Boil the enzyme hydrolysate at 100°C for 5 minutes, then precipitate it with 45% ethanol aqueous solution, 80% ethanol aqueous solution and 80% ethanol aqueous solution containing 5% acetone in sequence, and then dry it to obtain the final product.

[0054] The preparation method of the Eucommia ulmoides extract composition is as follows:

[0055] The product is obtained by mixing Eucommia polysaccharide, rutin, hyperoside, isochlorogenic acid C, apigenin and oleanolic acid evenly.

[0056] Comparative Example 1

[0057] An extract composition of Eucommia ulmoides, comprising, by weight, the following components: 20 parts Eucommia ulmoides polysaccharide, 15 parts rutin, 3 parts isochlorogenic acid C, 1 part apigenin and 3 parts oleanolic acid.

[0058] The preparation method of Eucommia ulmoides polysaccharide is as follows:

[0059] (1) Add 8 times the amount of aqueous solution containing 0.08% lecithin to the bark of Eucommia ulmoides and heat under reflux to extract the extract;

[0060] (2) Add 0.3% saccharifying enzyme, 0.05% β-glucanase and 0.3% cellulase by weight of the extract and carry out enzymatic hydrolysis at 50℃ and pH 5.5 for 1.5h to obtain the hydrolysate;

[0061] (3) Boil the enzyme hydrolysate at 100°C for 5 minutes, then precipitate it with 50% ethanol aqueous solution, 85% ethanol aqueous solution and 85% ethanol aqueous solution containing 8% acetone in sequence, and dry it to obtain the final product.

[0062] The preparation method of the Eucommia ulmoides extract composition is as follows:

[0063] The product is obtained by mixing Eucommia polysaccharide, rutin, isochlorogenic acid C, apigenin and oleanolic acid evenly.

[0064] Comparative Example 2

[0065] An extract composition of Eucommia ulmoides, comprising, by weight, the following components: 15 parts rutin, 20 parts hyperoside, 3 parts isochlorogenic acid C, 1 part apigenin and 3 parts oleanolic acid.

[0066] The preparation method of the Eucommia ulmoides extract composition is as follows:

[0067] The product is obtained by mixing rutin, hyperoside, isochlorogenic acid C, apigenin and oleanolic acid evenly.

[0068] Comparative Example 3

[0069] An extract composition of Eucommia ulmoides, comprising, by weight, the following components: 5 parts Eucommia ulmoides polysaccharide, 15 parts rutin, 15 parts hyperoside, 3 parts isochlorogenic acid C, 1 part apigenin and 3 parts oleanolic acid.

[0070] The preparation method of Eucommia ulmoides polysaccharide is as follows:

[0071] (1) Add 8 times the amount of aqueous solution containing 0.08% lecithin to the bark of Eucommia ulmoides and heat under reflux to extract the extract;

[0072] (2) Add 0.35% saccharifying enzyme and 0.3% cellulase by weight of the extract and carry out enzymatic hydrolysis at 50℃ and pH 5.5 for 1.5 h to obtain the hydrolysate;

[0073] (3) Boil the enzyme hydrolysate at 100°C for 5 minutes, then precipitate it with 50% ethanol aqueous solution, 85% ethanol aqueous solution and 85% ethanol aqueous solution containing 8% acetone in sequence, and dry it to obtain the final product.

[0074] The preparation method of the Eucommia ulmoides extract composition is as follows:

[0075] The product is obtained by mixing Eucommia polysaccharide, rutin, hyperoside, isochlorogenic acid C, apigenin and oleanolic acid evenly.

[0076] Comparative Example 4

[0077] An extract composition of Eucommia ulmoides, comprising, by weight, the following components: 5 parts Eucommia ulmoides polysaccharide, 15 parts rutin, 15 parts hyperoside, 3 parts isochlorogenic acid C, 1 part apigenin and 3 parts oleanolic acid.

[0078] The preparation method of Eucommia ulmoides polysaccharide is as follows:

[0079] (1) Add 8 times the amount of aqueous solution containing 0.08% lecithin to the bark of Eucommia ulmoides and heat under reflux to extract the extract;

[0080] (2) Add 0.3% saccharifying enzyme, 0.05% β-glucanase and 0.3% cellulase by weight of the extract and carry out enzymatic hydrolysis at 50℃ and pH 5.5 for 1.5h to obtain the hydrolysate;

[0081] (3) Boil the enzyme hydrolysate at 100°C for 5 minutes, then add ethanol sequentially until the ethanol concentration in the solution system is 85% to precipitate the alcohol. Let it stand and dry to obtain the final product.

[0082] The preparation method of the Eucommia ulmoides extract composition is as follows:

[0083] The product is obtained by mixing Eucommia polysaccharide, rutin, hyperoside, isochlorogenic acid C, apigenin and oleanolic acid evenly.

[0084] Experiment 1: Pharmacodynamic study of the composition of the present invention on membranous nephropathy

[0085] 1.1 Preparation of experimental reagents:

[0086] The components of the PBS buffer are shown in Table 1 below.

[0087] Table 1

[0088]

[0089] Preparation of pre-immunization solution: Add a small amount of PBS to 0.08g of C-BSA, grind, then add PBS to 40ml, mix well, add 40ml of Freund's incomplete adjuvant, and grind to obtain the solution.

[0090] Preparation of C-BSA solution: Add a small amount of PBS solution to 1.6g of C-BSA, grind, and then add PBS solution until the final concentration of C-BSA is 2.5mg / ml. Mix well.

[0091] 1.2 Preparation of animal models

[0092] 1 mg of C-BSA was added to 0.5 ml of PBS and mixed with an equal volume of Freund's incomplete adjuvant to form a milky white suspension. This suspension was then injected subcutaneously at multiple points in the bilateral axillae and groin of rats. One week after pre-immunization, each rat was injected intravenously with 2.5 mg of C-BSA three times a week for three weeks. 24-hour urinary protein was then quantitatively measured; a 24-hour urinary protein level exceeding 10 mg indicated successful model establishment.

[0093] 1.3 Grouping and Administration

[0094] The model rats were divided into a model group, low-, medium-, and high-dose groups of Example 1, and comparative groups 1, 2, 3, and 4, with 10 rats in each group; another 10 normal rats were used as blank controls.

[0095] Example 1: Rats in the low, medium, and high dose groups, and comparative groups 1, 2, 3, and 4 were administered the corresponding Eucommia ulmoides extract compositions by gavage at a dose of 5 g / kg once a day.

[0096] The model group was administered an equivalent volume of physiological saline by gavage;

[0097] Administer gavage continuously for 4 weeks.

[0098] 1.4 Experimental Results and Statistical Analysis

[0099] The 24-hour urinary protein and plasma albumin levels of rats in each group were measured after 4 weeks. The results are shown in Table 2-3.

[0100] Table 2

[0101] Group N Urine protein / mg / 24h Blank group 10 9.04±0.83 Model group 10 <![CDATA[54.33±7.26 * ]]> Example 1 Low-dose group 10 <![CDATA[17.41±5.58 # ]]> In Example 1, the dosage group 10 <![CDATA[15.80±3.75 # ]]> Example 1 High-dose group 10 <![CDATA[11.27±5.32 # ]]> Comparative Example 1 10 <![CDATA[28.09±7.61 #& ]]> Comparative Example 2 10 <![CDATA[33.48±6.94 #& ]]> Comparative Example 3 Groups 10 <![CDATA[24.57±6.59 #& ]]> Comparative Example 4 Groups 10 <![CDATA[26.04±4.37 #& ]]>

[0102] Note: Comparison between the model group and the normal group. * P < 0.05; compared with the model group, # P < 0.05; compared with the low-dose group in Example 1, & P < 0.05.

[0103] Table 3

[0104] Group N Plasma albumin / g / L Blank group 10 35.78±9.29 Model group 10 <![CDATA[31.02±4.16 * ]]> Example 1 Low-dose group 10 <![CDATA[36.11±5.42 # ]]> In Example 1, the dosage group 10 <![CDATA[36.94±7.80 # ]]> Example 1 High-dose group 10 <![CDATA[38.14±8.37 # ]]> Comparative Example 1 10 <![CDATA[31.05±5.31 & ]]> Comparative Example 2 10 <![CDATA[33.29±6.33 #& ]]> Comparative Example 3 Groups 10 <![CDATA[34.20±3.57 #& ]]> Comparative Example 4 Groups 10 <![CDATA[33.18±4.35 #& ]]>

[0105] Note: Compared with the normal group, *P<0.05; compared with the model group, # P < 0.05; compared with the low-dose group in Example 1, & P < 0.05.

[0106] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A composition of Eucommia ulmoides extract, characterized in that, Based on the weight percentages, it consists of the following components: 5 parts Eucommia polysaccharide, 15 parts Rutin, 15 parts Hyperoside, 3 parts Isochlorogenic acid C, 1 part Apigenin and 3 parts Oleanolic acid. The preparation method of the Eucommia ulmoides polysaccharide includes the following steps: (1) Add solvent to Eucommia bark and extract to obtain extract; (2) The extract was subjected to enzymatic hydrolysis to obtain the hydrolysate; (3) The enzyme hydrolysate is precipitated with alcohol to obtain the final product; The enzymes used in the enzymatic hydrolysis include glucoamylase, β-glucanase, and cellulase; The enzymatic hydrolysis was performed using 0.3% of the extract mass of saccharifying enzyme, 0.05% of β-glucanase, and 0.3% of cellulase, respectively. The alcohol precipitation was performed using a fractional precipitation method, specifically by sequentially using a 50% aqueous ethanol solution, an 85% aqueous ethanol solution, and an 85% aqueous ethanol solution containing 8% acetone.

2. The Eucommia ulmoides extract composition according to claim 1, characterized in that, The solvent is an aqueous solution containing 0.05-0.1% lecithin by mass; the mass of the aqueous solution is 5-10 times that of Eucommia ulmoides bark.

3. A method for preparing the Eucommia ulmoides extract composition according to claim 1 or 2, characterized in that, The process includes the following steps: mixing Eucommia polysaccharides, rutin, hyperoside, isochlorogenic acid C, apigenin, and oleanolic acid, or mixing them with a solvent.

4. The use of the Eucommia ulmoides extract composition according to claim 1 or 2 or the Eucommia ulmoides extract composition prepared by the preparation method according to claim 3 in the preparation of drugs for the prevention and / or treatment of membranous nephropathy.

5. A pharmaceutical preparation for the prevention and / or treatment of membranous nephropathy-related diseases, characterized in that, The pharmaceutical preparation comprises the Eucommia ulmoides extract composition according to claim 1 or 2 or the Eucommia ulmoides extract composition prepared by the preparation method according to claim 3; the pharmaceutical preparation further comprises a pharmaceutically acceptable carrier, wherein the carrier is any one or more of a sustained-release agent, filler, binder, wetting agent, disintegrant, absorption promoter, adsorbent carrier, surfactant or lubricant; the pharmaceutical preparation is any one of a topical preparation, an oral preparation or an injectable preparation.

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